LIQUID WATER FORMULATIONS, PRE-FILLED SYRINGE OR SELF-INJECTOR DEVICE AND USE OF THESE FORMULATIONS

专利摘要:
Liquid aqueous formulations, pre-filled syringe or self-injector device and use of said formulations to treat a disorder associated with impaired tnf alpha activity The invention provides an aqueous liquid pharmaceutical formulation comprising a human anti-tnf alpha antibody, or binding moiety. antigen thereof, which reduces the pain associated with injection in a subject by at least about 50% when compared to an identical injection formulation comprising at least one salt and / or at least one buffer. The invention also provides a liquid aqueous pharmaceutical formulation comprising a human anti-TNF alpha antibody, or an antigen-binding portion thereof, having increased bioavailability following subcutaneous administration to a subject. The formulation may comprise a therapeutic protein, such as a human anti-TNF alpha antibody, or an antigen binding portion thereof, or a biosimilar thereof.
公开号:BR112013011699B1
申请号:R112013011699-4
申请日:2011-11-11
公开日:2019-04-24
发明作者:Michael Neu；Markus Tschoepe；Carsten Weber；Laura Redden；Wolfgang Fraunhofer
申请人:Abbvie Biotechnology Ltd；
IPC主号:

专利说明:

"AQUEOUS LIQUID FORMULATIONS, PRE-FILLED SYRINGE OR SELF-INJECTING DEVICE AND USE OF SUCH FORMULATIONS TO TREAT A DISORDER ASSOCIATED WITH COMMITTED TNFa ACTIVITY"
RELATED APPLICATIONS [001] This application claims priority for Provisional Patent Application no. 61/412728 filed on November 11, 2010 and Provisional Patent Application no. 61/413960 deposited on November 15, 2010, the entire content of the quaos being incorporated herein by this reference.
BACKGROUND OF THE INVENTION [002] The formulation of therapeutic proteins, such as antibodies, is often a challenge due to the numerous desirable properties that the formulation must have to be economically and therapeutically successful, for example, stability, suitability for administration, concentration. During manufacture, storage and distribution, therapeutic proteins have been known to undergo physical and chemical degradation. These instabilities can reduce the potency of the protein and increase the risk of adverse events in patients, and therefore significantly impact regulatory approval (see, for example, Wang et al. Wang et al. J. Pharm. Sci. 96 : 1, 2007). As such, a stable protein formulation is essential for the success of a therapeutic protein.
[003] To be effective, many therapeutic proteins require the administration of high doses, which are preferably formulated in high concentration formulations. High protein formulations are desirable since they can impact the mode (e.g., intravenous versus subcutaneous) and the frequency of administration of the drug to a subject.
[004] Despite the benefits of formulations of high protein concentrations, the formulation of therapeutic proteins of high concentration presents numerous challenges. For example, increased protein concentration often negatively influences protein aggregation, solubility, stability, and viscosity (see, for example, Shire et al. J. Pharm. Sci. 93: 1390, 2004). The increase in viscosity, which is a very common challenge for high-protein solutions, may have negative ramifications on the administration of the formulation, for example, pain and burning sensation syndromes and limitations in manufacturing, processing, device options finishing filling and drug distribution (see, for example, Shire et al. Shire et al. J. Pharm. Sci. 93: 1390, 2004). Even for therapeutic proteins having the common structural characteristics, for example, antibodies, formulations
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2/114 approved until today had different ingredients and concentration ranges. For example, the anti-CD20 Rituxan antibody is formulated for intravenous administration at a concentration of 10 mg / ml, while the anti-RSV Synagis antibody is formulated for intramuscular administration, at a concentration of 100 mg / ml. Thus, formulations with a high protein content, especially antibody formulations, which can be used for therapeutic purposes continue to be a challenge.
[005] Another challenge associated with therapeutic proteins, such as antibodies, is the distribution of drugs. Although self-administration devices allow patients to avoid unnecessary travel to medical facilities to receive treatments, self-awareness and fear of pain associated with self-administration of patients can often affect the distribution of self-administered drug. In addition, formulations containing high concentrations of protein may have high viscosity, resulting in increased pain at the time of delivery, in particular, for subcutaneous administration. Thus, there is a special need for high concentration formulations that reduce the pain associated with the distribution of the drug (eg, self-injection).
[006] Therefore, there is a need for stable, high-protein formulations that provide dosage and administrative benefits, particularly with regard to decreased pain for the patient and / or improved bioavailability.
SUMMARY OF THE INVENTION [007] The present invention is based, at least in part, on the identification of new formulations of high concentration of therapeutic antibodies (including human anti-TNF-α antibodies, or antigen-binding fragments thereof, for example , adalimumab). The formulations of the invention provide a number of surprising characteristics due to the high concentration of the therapeutic antibody. Specifically, the present invention provides pharmaceutical formulations comprising anti-human TNFα antibodies that surprisingly have improved bioavailability, or a decrease in pain after subcutaneous injection.
[008] In particular, the present invention is based, at least in part, on the unexpected and surprising finding that a formulation having a high concentration of antibodies, a surfactant, and a polyol, provides dramatically reduced pain to the patient during drug distribution, especially subcutaneous administration of the antibody through, for example, self-injection. The formulations of the invention are established, at least in part, in the surprising verification that a protein
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3/114 therapy (for example, an anti-TNF-alpha antibody, or the antigen-binding portion thereof), may remain soluble at a high concentration of the protein (for example, at least about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 105, 110 mg / ml, or more) and maintain an adequate viscosity for injection (for example, subcutaneous administration). The formulation of the present invention is even more surprising, in that the formulation does not contain a buffer or a salt, it still has a high concentration of antibody. Notably, the formulation of the invention reduces the pain associated with injection into a patient by at least about 50% (for example, at least about 60%, 65%, 70%, 75%, 80%, 85%, 90 % or more) when purchased with an identical injection formulation, comprising at least one salt and / or at least one buffer.
[009] Thus, in one aspect, the invention provides a liquid aqueous formulation, comprising an anti-TNFα antibody, or antigen binding portion thereof; a surfactant, and a polyol, in which the formulation does not contain a buffer or salt, and reduces pain associated with injection into a patient by at least about 50% (for example, at least about 60%, 65% , 70%, 75%, 80%, 85%, 90% or more), when purchased with injection of an otherwise identical formulation comprising at least one salt and / or at least one buffer.
[0010] In another aspect, the invention provides a liquid aqueous formulation, comprising an isolated human anti-TNFα antibody, or an antigen-binding portion thereof, a surfactant, and less than 50 mg / mL of a polyol, in which the injection of the formulation into a human subject results in a Visual Analogue Pain Scale (VAS) score of less than 1.0. In one embodiment, the invention provides a liquid aqueous formulation consisting essentially of an isolated human anti-TNFα antibody, or antigen-binding portion thereof, a surfactant, and less than 50 mg / mL of a polyol, wherein injection of the formulation into a human subject results in a Visual Analogue Pain Scale (VAS) score of less than 1.0. In one modality, the VAS scale is from 0 (no pain) to 10 (unbearable pain).
[0011] In another aspect, the invention provides a liquid aqueous formulation, comprising an isolated human anti-TNFα antibody, or an antigen-binding portion thereof, a surfactant, and less than 50 mg / mL of a polyol, where the formulation does not contain a buffer or salt, and where the injection of the formulation reduces the pain associated with the injection in a human subject by at least about 50% when compared to the administration of a formulation, otherwise , identical, comprising a salt and / or a buffer. In one embodiment, the formulation in another
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The identical 4/114 comprises a citrate and phosphate buffer and sodium chloride.
[0012] The invention further provides a liquid aqueous formulation comprising an anti-TNFα antibody or antigen-binding portion thereof, at a concentration of at least about 50 mg / ml, a surfactant and a polyol, wherein the formulation has a conductivity of less than about 2 mS / cm. In one embodiment, the formulation has a conductivity of less than 1 mS / cm. In another embodiment, the formulation has a conductivity of less than 0.9 mS / cm.
[0013] The invention also provides, in another embodiment, a liquid aqueous formulation comprising an anti-TNFα antibody or antigen-binding portion thereof, at a concentration of at least about 50 mg / mL, a surfactant and, a polyol, in which the antibody, or antigen-binding portion thereof, has a hydrodynamic diameter of less than 4 nm in the formulation. In one embodiment, the antibody, or antigen binding portion thereof, has a hydrodynamic diameter of less than 3 nm in the formulation.
[0014] The present invention also provides a liquid aqueous formulation, comprising an isolated human anti-TNFα antibody, or an antigen binding portion thereof; a surfactant, and less than 50 mg / ml of a polyol, where the formulation has a characteristic selected from the group consisting of a conductivity of less than about 2 mS / cm, a hydrodynamic diameter (Dh) , which is at least about 50% less than the Dh of the protein in a buffered solution at a given concentration; and a hydrodynamic diameter (Dh) of less than about 4 nm. In one embodiment, the formulation has a conductivity of less than about 1 mS / cm. In another embodiment, the formulation has a conductivity of less than about 0.9 mS / cm. In one embodiment, the antibody, or antigen-binding portion thereof, has a hydrodynamic diameter of less than about 3 nm for the formulation. In another embodiment, the antibody, or antigen-binding portion thereof, has a hydrodynamic diameter of less than about 2 nm in the formulation.
[0015] The invention also provides a liquid aqueous formulation consisting essentially of an anti-TNFα antibody or antigen-binding portion thereof; a surfactant, and a polyol, wherein the concentration of anti-TNFα antibody or antigen-binding portion thereof is at least about 50 mg / mL, 75 mg / mL, 100 mg / mL, or greater than 100 mg / ml.
[0016] In a particular embodiment, the invention provides a liquid aqueous formulation consisting essentially of a concentration of 90-110 mg / ml of an isolated human anti-TNFα antibody, or an antigen-binding portion thereof,
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5/114 having a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5, 7 or 8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, and which has a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single substitution of alanine at position 2, 3, 4, 5, 6, 8, 9.10 or 11, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8, a polysorbate, for example, polysorbate 80, and, to about 38-46 mg / ml of a polyol, for example, mannitol.
[0017] In another aspect, the present invention provides a liquid aqueous formulation, comprising an isolated human anti-TNFα antibody, or an antigen binding portion thereof; a surfactant, and less than 50 mg / mL of a polyol, wherein the formulation is stable at about 30 degrees C for at least about 6 days, about 10 days, or about 14 days, or is stable at about 28 degrees C, for up to about 24 months.
[0018] In another aspect, the invention provides a method of administering an isolated human anti-TNFα antibody, or an antigen-binding portion thereof, to a subject in such a way that pain on injection is reduced after administration , said method comprising administering subcutaneously to the subject a formulation comprising antibody, or antigen binding portion thereof, such that injection pain is reduced after administration, wherein the formulation comprises more than 50 mg / mL of the antibody, or antigen-binding portion of it; a surfactant, and less than 50 mg / ml of a polyol. In one embodiment, pain by injection is determined to be less than 1.0, according to a Visual Analog Pain Scale (VAS).
[0019] In certain modalities, the pain associated with the injection is assessed using a Visual Analog Pain Scale (VAS). In one modality, the VAS scale is from 0 (no pain) to 10 (unbearable pain).
[0020] In certain embodiments, the pain associated with the injection is assessed after the injection (for example, immediately, no more than 1, 2, 3.4, 5, 6, 7, 8, 9.10 minutes, or more than 15 minutes after injection).
[0021] In certain modalities, the formulation reduces the pain associated with the injection in the patient by at least about 60%, 70%, 80% or more, when in
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6/114 purchase with the injection, otherwise, of identical formulation comprising at least one salt and / or at least one buffer.
[0022] The invention further provides a liquid aqueous formulation comprising an anti-TNFα antibody or antigen binding portion thereof, at a concentration of at least about 50, 75, 100 mg / ml, or greater than 100 mg / ml. mL; a surfactant; and, a polyol, in which the formulation does not contain a buffer and a salt.
[0023] In another aspect, the invention provides a liquid aqueous formulation, comprising an isolated human anti-TNFα antibody, or an antigen-binding portion thereof; a surfactant, and less than 50 mg / ml of a polyol, where the formulation is stable at about 30 degrees C for at least about 6 days. In one embodiment, the formulation is stable at room temperature for at least about 7 days. In one embodiment, the formulation is stable at room temperature for at least about 8 days. In one embodiment, the formulation is stable at room temperature for at least about 9 days. In one embodiment, the formulation is stable at room temperature for at least about 10 days. In one embodiment, the formulation is stable at room temperature for at least about 11 days. In one embodiment, the formulation is stable at room temperature for at least about 12 days. In one embodiment, the formulation is stable at room temperature for at least about 13 days. In one embodiment, the formulation is stable at room temperature for at least about 14 days. In one embodiment, the formulation is stable at room temperature for at least about 15 days.
[0024] In one embodiment, the polyol used in the formulation of the invention is mannitol or sorbitol.
[0025] In one embodiment, the formulation of the invention contains about 20-60 mg / ml of mannitol, or, alternatively, about 30-50 mg / ml. In one embodiment, the formulation contains about 38 to 46 mg / mL of mannitol.
[0026] The present invention is also based, at least in part, on the unexpected and surprising finding that a formulation having a high concentration of antibodies and a surfactant provides notably greater bioavailability than similar formulations containing additional excipients, such as a buffer, a polyol and / or a salt.
[0027] Thus, in one aspect, the invention provides a liquid aqueous formulation containing a surfactant and 30-90 mg of an isolated human anti-TNFα antibody or antigen binding portion, wherein the formulation has an antibody concentration of 90 -110 mg / ml; and where the formulation provides increased bioavailability of the
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7/114 antibody, or antigen-binding portion thereof, to a human subject by subcutaneous injection of the formulation with respect to a formulation comprising a citrate phosphate, sodium chloride and mannitol buffer.
[0028] In one aspect, the invention provides a liquid aqueous formulation consisting essentially of a surfactant and 30-90 mg of an isolated human anti-TNFα antibody or antigen binding portion, wherein the antibody concentration, or portion of binding to its antigen, is 90-110 mg / mL.
[0029] In another aspect, the invention provides a liquid aqueous formulation containing a surfactant and 30-90 mg of an isolated human anti-TNFα antibody, or an antigen-binding portion thereof, wherein the formulation has a concentration of antibody of 90-110 mg / mL, and wherein the formulation provides increased bioavailability of the antibody, or antigen-binding portion of it, in a human subject after subcutaneous injection of the formulation, such that the antibody, or portion binding to its antigen, has an AUC0-360 greater than about 1300 pg * hr / ml.
[0030] In another aspect, the invention provides a method for improving the bioavailability of an isolated human anti-TNFα antibody, or an antigen-binding portion thereof, in a human subject, said method comprising administering a formulation comprising an effective amount of the antibody, or antigen-binding portion of the same, and a surfactant for the subject such that the bioavailability of the antibody, or antigen-binding portion of the subject, is improved, where the formulation does not contain a buffer, a polyol, or a salt.
[0031] In another aspect, the invention provides a method for improving the bioavailability of an isolated human anti-TNFα antibody, or an antigen-binding portion thereof, in a subject, said method comprising administering a formulation that comprises an effective amount of the antibody, or antigen-binding portion of the same, and a surfactant for the subject such that the bioavailability of the antibody, or antigen-binding portion of the subject, is improved, at least about 15 % with respect to a second formulation, wherein the formulation does not contain a buffer, a polyol, or a salt, and the second formulation comprises a buffer, a polyol, and its salt. In one embodiment, the bioavailability of the antibody, or antigen-binding portion of it, is improved by at least about 30% over the second formulation. In one embodiment, the bioavailability of the antibody, or antigen-binding portion of it, is improved by at least about 40% over the second formulation.
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The invention further provides a method for improving the bioavailability of an isolated human anti-TNFα antibody, or an antigen binding portion thereof, in a human subject, said method comprising administering a formulation comprising a surfactant and an effective amount of the antibody, or antigen-binding portion of it, with the subject such that the bioavailability of the antibody, or antigen-binding portion of it, is improved, in which the formulation has a characteristic selected from the group consisting of a conductivity of less than about 2 mS / cm; the antibody, or antigen-binding portion of it, has a hydrodynamic diameter (Dh), which is at least about 50% smaller than the antibody's Dh, or antigen-binding portion, in a buffered solution at given concentration, and the antibody, or antigen-binding portion thereof, has a hydrodynamic diameter (Dh) less than about 4 nm. In one embodiment, the formulation has a conductivity of less than about 1 mS / cm. In another embodiment, the formulation has a conductivity of less than about 0.9 mS / cm. In one embodiment, the antibody or antigen-binding portion thereof has a hydrodynamic diameter of less than about 3 nm in the formulation.
[0033] In one embodiment, bioavailability is determined according to either an AUC level or a Cmax. In one embodiment, bioavailability is determined according to either an AUC0-360 or an AUC0-1344. In one embodiment, the bioavailability of the antibody, or antigen-binding portion of it, is an AUC0360 greater than about 1300 ug * hr / ml, when the formulation is injected subcutaneously into the human subject.
[0034] In certain embodiments, the anti-TNFα antibody is an isolated human antibody (for example, a human IgG1 kappa antibody), a humanized antibody, a chimeric antibody, or a murine antibody. For example, the chimeric antibody can be infliximab or a biosimilar to it, and the human antibody can be golimumab or adalimumab, or a biosimilar to it.
[0035] In one embodiment, the human anti-TNFα antibody, or antigen-binding portion thereof, is an IgG1 or an IgG4.
[0036] In one embodiment, the human anti-TNFα antibody, or antigen binding portion thereof, dissociates from human TNFα with a Kd of 1 x 10 ^-8 M or less and has a koff rate constant of 1 x 10 ^-3 s ^-1 or less, both determined by surface plasmon resonance. In certain embodiments, the human anti-TNFα antibody, or antigen-binding portion thereof, dissociates from human TNFα with a Kd of 1 x 10 ^-8 M or less and a koff rate constant of 1 x 10 ^{- 3} s ^-1 or less,
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9/114 both determined by surface plasmon resonance, and neutralizes the cytotoxicity of human TNFα in a standard in vitro L929 assay with an IC 50 of 1 x 10 ^-7 M or less.
[0037] In certain embodiments, the human anti-TNFα antibody, or a binding portion of the antigen thereof, has the following characteristics: it dissociates from human TNFα with a koff rate constant of 1 x 10 ^-3 s ^-1 or less, as determined by surface plasmon resonance; has a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single substitution of alanine at position 1,4, 5, 7 or 8, or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8 and / or 9, and (c) has a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single substitution of alanine in position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or one to five conservative amino acid substitutions in positions 2, 3, 4 , 5, 6, 8, 9, 10, 11 and / or 12.
[0038] In certain embodiments, the human anti-TNFα antibody, or antigen-binding portion thereof, has a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3 , or modified from SEQ ID NO: 3 by a single alanine substitution at position 1,4, 5, 7 or 8 and with a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single substitution of alanine at position 2, 3, 4, 5, 6, 8, 9, 10 or 11.
[0039] In certain embodiments, the human anti-TNFα antibody, or antigen-binding portion thereof, has a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3 , or modified from SEQ ID NO: 3 by a single alanine substitution at position 1,4, 5, 7 or 8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7; and has a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single substitution of alanine at position 2, 3, 4, 5, 6,8, 9,10 or 11, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8.
[0040] In certain embodiments, the human anti-TNFα antibody, or antigen-binding portion thereof, has a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1 and a variable region of
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10/114 heavy chain (HCVR) comprising the amino acid sequence of SEQ ID NO: 2.
[0041] In one embodiment, the human anti-TNFα antibody, or antigen-binding portion thereof, comprises the CDRs corresponding to adalimumab.
[0042] In one embodiment, the human anti-TNFα antibody, or an antigen-binding portion of it, is adalimumab or golimumab, or a biosimilar to it.
[0043] In certain embodiments, the concentration of the anti-human TNFα antibody, or antigen binding portion thereof, in the formulation is at least about 50 mg / mL, about 75 mg / mL, about 100 mg / mL. ml, or greater than 100 mg / ml. In one embodiment, the concentration of the anti-human TNFα antibody, or antigen-binding portion thereof, in the formulation of the invention is 90-110 mg / ml. In one embodiment, the concentration of the anti-human TNFα antibody, or antigen binding portion thereof, in the formulation of the invention is 95-105 mg / ml. In one embodiment, the formulation comprises more than 75 mg / ml of the antibody, or antigen-binding portion of it. In one embodiment, the invention provides a stable, liquid, aqueous formulation, comprising a high concentration, for example, 75-125 mg / ml, of a human anti-hTNFa antibody.
[0044] In certain embodiments, the surfactant used in the formulation of the invention is a polysorbate. In one embodiment, the polysorbate concentration is about 0.11.5 mg / ml, about 0.2-1.4 mg / ml, about 0.3-1.3 mg / ml, about 0 , 4-1.2 mg / mL, about 0.5-1.1 mg / mL, about 0.6-1.0 mg / mL, about 0.6-1.1 mg / mL, about 0.7-1.1 mg / ml, about 0.8-1.1 mg / ml, or about 0.9-1.1 mg / ml. In certain embodiments, the polysorbate has a concentration of about 0.1-10 mg / mL, about 0.5-5 mg / mL, about 0.1-2 mg / mL, or about 1 mg / mL. ml. In one embodiment, the surfactant is polysorbate 80.
[0045] In certain modalities, the patient is human, or a non-human mammal.
[0046] In certain embodiments, the formulation is Formulation 3 or 4 the Formulation described in the Examples.
[0047] In certain embodiments, the formulation, otherwise identical, is the commercially available adalimumab formulation containing adalimumab, sodium chloride, sodium phosphate monobasic dihydrate, diphasic sodium phosphate dihydrate, sodium citrate , citric acid monohydrate, mannitol, polysorbate 80 and water for injection.
[0048] In one embodiment, the formulation, otherwise identical, contains buffer and a salt. In certain embodiments, the salt is a neutral salt or a base salt (for example, NaOH), used for pH adjustment. In certain embodiments, the buffer comprises a phosphate buffer and / or a citrate buffer. For example, the
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11/114 phosphate may contain about 1.35-1.75 mg / mL or about 1.50-1.56 mg / mL Na2HPO4 · 2H2O, and about 0.75-0.95 mg / mL or about 0.83-0.89 mg / mL NaH2PÜ4 • 2H2O). The citrate buffer can contain about 1.15-1.45 mg / mL or about 1.30-1.31 mg / mL citric acid • H2O, and about 0.2-0.4 mg / mL or about 0.30-0.31 mg / mL dehydrated sodium citrate. The at least one salt can be a neutral salt, such as a neutral sodium salt (for example, NaCl).
[0049] In one embodiment, the formulation of the invention is a pharmaceutical formulation.
[0050] In certain embodiments, the formulation of the invention is suitable for subcutaneous injection. In one embodiment, the formulation of the invention is suitable for self-administration subcutaneously by a subject.
[0051] In certain embodiments, the volume of the aqueous formulation is no more than 1.5 ml, 1.0 ml, 0.8 ml, 0.5 ml or 0.4 ml.
[0052] In certain embodiments, the formulation comprises a dose of about 30-90 mg of the antibody, or antigen-binding portion of it. In one embodiment, the formulation comprises about 40 mg of the anti-TNFα antibody, or antigen-binding portion of it. In one embodiment, the formulation comprises about 50 mg of the anti-TNFα antibody, or antigen binding portion thereof. In one embodiment, the formulation comprises about 60 mg of the anti-TNFα antibody, or antigen binding portion thereof. In one embodiment, the formulation comprises about 70 mg of the anti-TNFα antibody, or antigen binding portion thereof. In one embodiment, the formulation comprises about 80 mg of the anti-TNFα antibody, or antigen binding portion thereof. In one embodiment, the formulation comprises about 90 mg of the anti-TNFα antibody, or antigen-binding portion of it. In one embodiment, the formulation comprises 60-85 mg. In another embodiment, the formulation comprises 70-90 mg. In yet another embodiment, the formulation contains 30-110 mg. In one embodiment, the formulation contains 70-110 mg.
[0053] Another aspect of the invention provides a pre-filled syringe or auto-injector device, comprising any of the formulations in question described in the present document. In certain embodiments, the aqueous formulation stored in the pre-filled syringe or auto-injector device contains about 40 mg of adalimumab, or biosimilar to it. In certain embodiments, the aqueous formulation stored in the pre-filled syringe or auto-injector device contains about 80 mg of adalimumab, or a biosimilar thereof.
[0054] Another aspect of the invention provides a method of treating a
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12/114 disorder related to TNFα activity compromised in a patient, comprising administering to the patient any of the formulations described in this document.
[0055] In one embodiment, the formulation, or method, of the invention is used to treat a subject who has rheumatoid arthritis. In one embodiment, the formulation, or method, of the invention is used to treat a subject with Crohn's disease. In one embodiment, the formulation, or method, of the invention is used to treat a subject who has psoriatic arthritis. In one embodiment, the formulation, or method, of the invention is used to treat a subject having psoriasis. In one embodiment, the formulation, or method, of the invention is used to treat a subject who has idiopathic juvenile arthritis (JIA). In one embodiment, the formulation, or method, of the invention is used to treat a subject who has ankylosing spondylitis. In one embodiment, the formulation, or method, of the invention is used to treat a subject who has ulcerative colitis. In one embodiment, the formulation, or method, of the invention is used to treat a subject who has suppurative hidradenitis. In one embodiment, the formulation, or method, of the invention is used to treat a subject who has diabetic retinopathy. In one embodiment, the formulation, or method, of the invention is used to treat a subject who has giant cell arteritis. In one embodiment, the formulation, or method, of the invention is used to treat a subject with Behçet's disease. In one embodiment, the formulation, or method, of the invention is used to treat a subject who has sarcoidosis, for example, cutaneous sarcoidosis. In one embodiment, the formulation, or method, of the invention is used to treat a subject having axial spondyloarthropathy. In one embodiment, the formulation, or method, of the invention is used to treat a subject who has uveitis.
[0056] In one embodiment, the formulation is administered to the subject according to a periodicity selected from the group consisting of weekly, biweekly, every three weeks and monthly. In one embodiment, the formulation of the invention contains 30-90 mg of an anti-human TNFα antibody, or an antigen-binding portion thereof, and is administered on a fortnightly dosing regimen. In another embodiment, the formulation of the invention contains 30-90 mg of an anti-human TNFα antibody, or antigen-binding portion thereof, and is administered according to a monthly dosage regimen. In one embodiment, the formulation of the invention contains 60-85 mg of an anti-human TNFα antibody, or an antigen-binding portion of it, and is administered in a fortnightly dosing regimen. In another embodiment, the formulation of the invention contains 60-85 mg of an anti-human TNFα antibody, or antigen-binding portion of it, and is administered according to a
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13/114 monthly dosing regimen.
[0057] In certain embodiments, the administration of the formulation of the present invention to a subject is through self-administration.
[0058] It is contemplated that any of the modalities described in this document can be combined with one or more other modalities of the invention, including the modalities described only on one aspect of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS [0059] Figure 1 is a graphical panel showing that the administration of high concentration formulations of 1 (F1) and 2 (F2) resulted in a significant decrease in pain assessment at all time points after injection (immediately, 15 minutes, and 30 minutes), compared to the other treatment groups (F4 and the current commercial formulation).
[0060] Figure 2 shows, in a linear flaking, the means and standard deviations of serum concentrations of adalimumab over a period of 56 days after a single dose of 40 mg SC (subcutaneous) of adalimumab.
[0061] Figures 3A and 3B are the graphs showing the stability of the various adalimumab formulations evaluated by the number of aggregates added in the formulations (3A), or added aggregates (3B), over a polysorbate band or a polyol band.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions [0062] In order that the present invention can be more easily understood, certain terms are defined first. In addition, it should be noted that whenever a value or range of values of a parameter is recited, it is intended that the values and intermediate ranges of the values cited are also intended to be part of the present invention.
[0063] The term pain associated with injection (in a patient), as used herein, refers to pain associated with injection of the drug into the tissue of the patient or subject. In one embodiment, the pain is separated from the pain caused by the injection device (if any), such as the needle stick of the injection. In one embodiment, the pain associated with the injection can be caused by the drug formulation to be injected into the patient's tissue.
[0064] The pain associated with the injection can be assessed using a number of means recognized in the technique, such as the Visual Analog Pain Scale (VAS). Pain measurement is, in a modality, quantifiable, in such a way that a percentage of
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14/114 reduction / increase in the pain scale can be directly compared using statistical methods. For example, when the visual analogue pain scale is used, a numerical pain value (for example, mean ± SD) can be assigned to each treatment group, such that a percentage of increase or decrease can be calculated.
[0065] In general, Visual Analogue flaking (VAS) is a measuring instrument that measures a characteristic or attitude that is believed to vary over a continuous series of values (see, for example, Singer and Thods (1998) Academic Emergency Medicine 5 : 1007). For example, the amount of pain a patient feels varies over a continuous series from zero (a count of, for example, 0) to an extreme amount of pain (a count of, for example, 10). From the patient's point of view, this spectrum appears continuous - his pain does not show slight jumps, as a categorization of none, mild, moderate and severe could suggest. Operationally, a VAS is usually a horizontal line, 100 mm long, anchored by word descriptors at each end, such as no pain at one end and extreme pain (or some variation of it) at the other end. The patient's marks on the line at a time point (for example, a score of 0-10) that they feel represent their perception of their current state. The VAS score can be determined by measuring in millimeters from the left side of the line to the point the patient marks.
[0066] There are several ways in which VAS were presented, including vertical lines and lines with additional descriptors. See Wewers & Lowe (“A critical review of visual analogue scales in the measurement of clinical phenomena”. Research in Nursing and Health 13: 227-236, 1990, incorporated by reference) that provide an informative discussion of the benefits and limitations of different styles of VAS.
[0067] The term liquid formulation refers to a formulation in a liquid state and is not intended to refer to resuspended lyophilized formulations. A liquid formulation of the invention is stable during storage, and does not depend on lyophilization (or other methods of changing the state, for example, spray drying) for stability.
[0068] The term liquid aqueous formulation refers to a liquid formulation using water as a solvent. In one embodiment, a liquid aqueous formulation is a formulation that maintains stability (for example, chemical activity and / or physical and / or biological stability) without the need for freeze-drying,
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15/114 spraying and / or freezing.
[0069] The pharmaceutical term as used herein refers to a composition, for example, an aqueous formulation, which is useful for the treatment of a disease or disorder.
[0070] The term subject or patient is intended to include mammalian organisms. Examples of subjects / patients include human and non-human mammals, for example, non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, mice, and transgenic non-human animals. In specific embodiments of the invention, the subject is a human being.
[0071] The term excipient refers to an agent that can be added to a formulation to provide a desired characteristic, for example, consistency, improved stability, and / or adjustment of osmolality. Examples of commonly used excipients include, but are not limited to, sugars, polyols, amino acids, surfactants and polymers.
[0072] A commonly used excipient is a polyol. As used herein, a polyol is a substance with multiple hydroxyl groups, and includes sugars (reducing and non-reducing sugars), sugar alcohols and sugar acids. Non-limiting examples of polyols are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose, glucose, sucrose, trehalose, sorbose, melezitose, raffinose, mannitol, xylitol, erythritol, treitol, sorbitol, glycerol, L- gluconate and metallic salts thereof. In one embodiment, the polyol used in the formulation or methods of the invention is mannitol. In one embodiment, the polyol used in the formulation or methods of the invention is sorbitol.
[0073] A therapeutically active antibody or therapeutic antibody refers to an antibody that can be used for therapeutic purposes, that is, for the treatment of a disorder in a subject. It should be noted that although therapeutic proteins can be used for treatment purposes, the invention is not limited to that use, since said proteins can also be used for in vitro studies.
[0074] As used in this document, buffer is an agent (s) in a solution that allows the solution to resist changes in pH by the action of its acid-base conjugate components. Examples of buffers include acetate (e.g., sodium acetate), succinate (such as sodium succinate), gluconate, histidine, methionine, citrate, phosphate, citrate / phosphate, imidazole, combinations thereof, and other organic acid buffers. In one embodiment, a buffer is not a protein. A buffer can provide a solution with a pH in the range of about 4 to about 8, from about 4.5 to about 7, or between about 5.0 to about 6.5.
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16/114 [0075] Although the formulations of the invention do not contain a buffer (s), otherwise identical formulations containing one or more buffers can be used for the purpose of comparing pain or bioavailability. Examples of such buffers include phosphate, acetate (for example, sodium acetate), succinate (such as sodium succinate), gluconate, glutamate, histidine, citrate and other organic acid buffers. In one embodiment, a representative buffer in the otherwise identical formulation comprises a citrate buffer and / or a phosphate buffer.
[0076] As used herein, the term surfactant generally includes an agent that protects the protein, for example, the antibody, from stresses induced by the air / solution interface, stresses induced by solution / surface, to reduce aggregation of the antibody, or to minimize the formation of particulates in the formulation. Examples of surfactants include, but are not limited to, nonionic surfactants, such as polysorbates (for example, polysorbates 20, 80) or poloxamers (for example, poloxamer 188). The term surfactant or detergent includes nonionic surfactants, such as, but not limited to, polysorbates. In one embodiment, a surfactant includes poloxamers, for example, Poloxamer 188, Poloxamer 407, polyoxyethylene alkyl ethers, for example, Brij35®, Cremophor A25, Sympatens ALM / 230; polysorbates / Tweens, for example, Polysorbate 20 (Tween 20), Polysorbate 80 (Tween 80), Mirj and Poloxamers, for example, Poloxamer 188.
[0077] A stable formulation is one in which the antibody essentially retains its physical stability and / or chemical stability and / or biological activity during the manufacturing process and / or during storage. Various analytical techniques for measuring protein stability are available in the art, and are reviewed in Peptide and Protein Drug Delivery 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991); and Jones, A. (1993) Adv. Drug Delivery Rev. 10: 29-90 (both incorporated by reference). For example, in one embodiment, the stability of a protein is determined according to the percentage of monomer protein in the solution, with a low percentage of degraded (for example, fragmented) and / or aggregated protein. In one embodiment, the formulation can be stable at room temperature, about 25-30 ° C, or at 40 ° C for at least 1 month and / or stable at about 2 to 8 ° C for at least 1 month, 1 year, or alternatively for at least 2 years. In another embodiment, the formulation can be stable up to about 30 degrees C for at least about 6 days, about 10 days, or about 14 days, or it is stable at about 28 degrees C, for up to about 24 months. In one embodiment, the formulation can be stable after freezing (to, for example, -70 ° C) and thawing the formulation,
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17/114 hereinafter referred to as a freeze / thaw cycle.
[0078] An antibody maintains its physical stability in a pharmaceutical formulation if it does not show substantially any sign of, for example, aggregation, precipitation and / or denaturation after visual examination of color and / or clarity, or as measured by dispersion of UV light or by size exclusion chromatography. Aggregation is a process by which individual molecules or complexes associate covalently or non-covalently to form aggregates. Aggregation can proceed to the point where a visible precipitate is formed.
[0079] Stability, like the physical stability of a formulation, can be evaluated by methods well known in the art, including measuring an apparent attenuation of the light of a sample (absorbance, or optical density). Such measurement of light attenuation is related to the turbidity of a formulation. The turbidity of a formulation is partly an intrinsic property of protein dissolved in solution and is usually determined by nephelometry and measured in Nephelometric Turbidity Units (NTU).
[0080] The degree of turbidity, for example, as a function of the concentration of one or more of the components of the solution, for example, the concentration of protein and / or salt, is also referred to as the opalescence or opaque appearance of a formulation. The degree of turbidity can be calculated by reference to a standard curve generated using known turbidity suspensions. Reference standards for determining the degree of turbidity for pharmaceutical compositions can be based on the European Pharmacopoeia criteria (European Pharmacopoeia, fourth Ed. Directorate for the Quality of Medicine of the Council of Europe (EDQM), Strasbourg, France). According to the European Pharmacopoeia criteria, a transparent solution is defined as one with a turbidity less than or equal to a reference suspension that has a turbidity of approximately 3, according to European Pharmacopoeia standards. Nephelometric turbidity measurements can detect Rayleigh dispersion, which typically changes linearly with concentration, in the absence of association or non-ideality effects. Other methods for assessing physical stability are well known in the art.
[0081] An antibody maintains its chemical stability in a pharmaceutical formulation, if the chemical stability for a given time is such that the antibody is considered to still maintain its biological activity, as defined below. Chemical stability can be assessed, for example, by detecting and quantifying chemically altered forms of the antibody. The chemical change may involve modifying the size (for example, cutting), which can be evaluated using exclusion chromatography by
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18/114 size, SDS-PAGE and / or ion assisted by matrix-assisted laser desorption / time-of-flight mass spectrometry (MALDI / TOF MS), for example. Other types of chemical change include charge change (for example, occurring as a result of deamidation or oxidation), which can be evaluated by ion exchange chromatography, for example.
[0082] An antibody maintains its biological activity in a pharmaceutical formulation, if the antibody in a pharmaceutical formulation is biologically active for its intended purpose. For example, biological activity is maintained if the biological activity of the antibody in the pharmaceutical formulation is within about 30%, about 20%, or about 10% (within the assay errors) of the biological activity displayed at the time when that the pharmaceutical formulation has been prepared (for example, as determined in an antigen binding assay).
[0083] In a pharmacological sense, in the context of the present invention, a therapeutically effective amount or effective amount of an antibody refers to an amount effective in preventing or treating or alleviating a symptom of a disorder for the treatment of which the antibody is effective.
[0084] The term human TNF-alpha (here abbreviated as hTNF-alpha, TNFa, or simply hTNF), as used herein, is intended to refer to a human cytokine that exists as a secreted 17 kDa form and a form associated with a 26 kDa membrane, the biologically active form of which being composed of a trimester of 17 kDa molecules covalently linked. The structure of hTNF-alpha is described later, for example, in Pennica, D., et al. (1984) Nature 312: 724-729; Davis, J. M., et al. (1987) Biochem 26: 1322-1326; and Jones, E. Y., et al. (1989) Nature 338: 225-228. The term human TNF-alpha is intended to include recombinant human TNF-alpha (rhTNF-alpha), which can be prepared by conventional recombinant expression methods, or purchased commercially (R & D Systems, Catalog No. 210-TA, Minneapolis, Minn).
[0085] The term antibody, as used herein, is intended to refer to immunoglobulin molecules composed of four polypeptide chains, two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Other naturally occurring antibodies of altered structure, such as, for example, antibodies to camelids, are also included in this definition. Each heavy chain is comprised of a variable heavy chain region (hereinafter abbreviated as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain consists of a variable light chain region (here abbreviated as LCVR or VL) and a region
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19/114 light chain constant. The light chain constant region consists of a CL domain. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDR), interspersed with regions that are more conserved, called structure regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from the amino terminal to the carboxyl terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In one embodiment of the invention, the formulation contains an antibody with CDR1, CDR2 and CDR3 of sequences such as those described in US Patent Nos. 6,090,382 and 6,258,562, each incorporated herein by reference. In certain embodiments, the formulation contains an antibody as claimed in US Patent Nos. 6,090,382 and 6,258,562.
[0086] As used herein, the term CDR refers to the region that determines complementarity within a variable antibody sequence. There are three CDRs in each of the heavy and light chain variable regions, which are designated CDR1, CDR2 and CDR3 for each of the heavy and light chain variable regions. The exact boundaries of these CDRs have been defined differently according to the different systems. The system described by Kabat (Id.) Not only provides an unequivocal residue numbering system applicable to any variable region of an antibody, but also provides precise residue limits that define the three CDRs. These CDRs can be referred to as Kabat CDRs. Chothia et al. found that certain sub-portions within the Kabat CDRs adopt almost identical peptide main chain conformations, despite having a great diversity in terms of the amino acid sequence (Chothia et al. (1987) Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 877-883). These sub-portions were designated as L1, L2 and L3 or H1, H2 and H3, where L and H designate the heavy chain and light chain regions, respectively. These regions can be referred to as Chothia DRCs, which have limits that overlap with the Kabat CDRs. Other limits that define CDRs that overlap with Kabat's CDRs have been described by Padlan (1995) FASEB J. 9: 133-139 and MacCallum (1996) J. Mol. Biol. 262 (5): 732-45. Still other definitions of CDR limits may not strictly follow one of the systems described here, but they still overlap with Kabat CDRs, although they may be shortened or lengthened depending on the prediction or experimental checks that the residues or groups of residues or even particular whole CDRs do not significantly impact antigen binding. The methods used in this document can use CDRs defined according to any of these systems, although certain modalities use CDRs defined by Kabat or Chothia. In one embodiment, the antibody used in the methods and compositions of the
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11/204 invention includes the six CDRs of adalimumab antibody.
[0087] The term antigen-binding portion of an antibody (or simply antibody portion), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (for example, hTNF-alpha). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term antigen binding portion of an antibody include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains, (ii) an F (ab ' ) 2, a divalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region, (iii) an Fd fragment consisting of VH and CH1 domains, (iv) an Fv fragment consisting of domains of a single VL and VH arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546.), which consists of a VH domain and (vi) an isolated complementarity determining region (CDR). In addition, although the two Fv fragment domains, VL and VH are encoded by separate genes, they can be linked, using recombinant methods, by a synthetic linker that allows them to be made up of a single protein chain in which the VL regions and VH pair to form single-stranded monovalent molecules (known as Fv (scFv), see for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85: 5879-5883). Such single chain antibodies are also intended to be included within the term antigen-binding portion of an antibody. Other forms of single chain antibodies, such as diabody, are also included. Diabodies are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains from another chain and create two antigen-binding sites (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, RJ, et al. (1994) Structure 2: 1121-1123). In one embodiment of the invention, the formulation contains antigen-binding portions described in US Patent Nos. 6,090,382 and 6,258,562, each incorporated herein by reference.
[0088] The phrase recombinant antibody refers to antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector, transfected in a host cell, antibodies isolated from a combinatorial library of antibodies
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21/114 recombinants, antibodies isolated from an animal (for example, a mouse) that is transgenic for human immunoglobulin genes (see, for example, Taylor et al. (1992) Nucl. Acids Res. 20: 6287-6295 .) or antibodies prepared, expressed, raised or isolated by any other means involving the splicing of particular immunoglobulin gene sequences (such as human immunoglobulin gene sequences) to other DNA sequences. Examples of recombinant antibodies include humanized antibodies and grafted on recombinant chimeric, human CDRs.
[0089] The term human antibody, as used herein, is intended to include antibodies with variable and constant regions derived from immunoglobulin sequences of the human germline. The human antibodies used in the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (for example, mutations introduced by site-specific or random mutagenesis in vitro or by somatic mutation in vivo), for example, in CDRs and , in particular, on CDR3. However, the term human antibody, as used herein, is not intended to include antibodies in which the CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted into human structure sequences.
[0090] The term chimeric antibody refers to antibodies comprising the sequences of the heavy and light chain variable region of one species and the sequences of the constant region from other species, such as antibodies having light and heavy chain variable regions of murine linked to human constant regions.
[0091] The term antibody grafted with CDR refers to antibodies that comprise the heavy and light chain variable region sequences from one species, but in which the sequences of one or more of the VH and / or CDR regions of VL are replaced by CDR sequences of another species, such as antibodies with variable regions of murine light and heavy chain in which one or more of the murine CDRs (e.g., CDR3) has been replaced with human CDR sequences.
[0092] An isolated antibody, as used herein, is intended to refer to an antibody that is substantially free of other antibodies that have different antigenic specificities (for example, an isolated antibody that specifically binds to hTNF-alpha is substantially free) antibodies that specifically bind to antigens other than hTNF-alpha). An isolated antibody that specifically binds to hTNF-alpha may, however, cross-react with other antigens, such as TNF-alpha from other species of molecules. In addition, an isolated antibody can be substantially free of other cellular material and / or
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22/114 chemicals.
[0093] A neutralizing antibody, as used herein (or an antibody that neutralized hTNF-alpha activity), is intended to refer to an antibody whose binding to hTNF-alpha results in the inhibition of biological activity of hTNF-alpha . This inhibition of biological activity of hTNF-alpha can be assessed by measuring one or more indicators of biological activity of hTNF-alpha, such as hTNFalpha-induced cytotoxicity (in vitro or in vivo), hTNF-alpha-induced cell activation and hTNF binding -alpha to hTNF-alpha receptors. These indicators of the biological activity of hTNF-alpha can be evaluated by one or more of the various in vitro or in vivo assay standards, known in the art and described in US Patent Nos. 6,090,382 and 6,258,562, each incorporated herein by reference. In one embodiment, the ability of an antibody to neutralize hTNF-alpha activity is assessed by inhibition of hTNF-alpha induced by cytotoxicity of L929 cells. As an additional or alternative parameter of hTNF-alpha activity, the ability of an antibody to inhibit ELAM-1-induced hTNF-alpha expression in HUVEC, as a measure of hTNF-alpha-induced cell activation.
[0094] The term surface plasmon resonance, as used in this document, refers to an optical phenomenon, which allows the analysis of biospecific interactions in real time, by detecting changes in protein concentrations within a matrix of biosensor, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For additional descriptions, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51: 19-26; Jonsson, U., et al. (1991) Biotechniques 11: 620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8: 125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198: 268-277.
[0095] The term kon, as used in this document, is intended to refer to the rate constant for the association of a binding protein (eg, an antibody) to the antigen to form, for example, the antibody complex / antigen, as is known in the art.
[0096] The term koff, as used in this document, is intended to refer to the dissociation rate constant for the dissociation of an antibody from the antibody / antigen complex.
[0097] The term Kd, as used in this document, is intended to refer to the dissociation constant of a particular antibody-antigen interaction and refers to the value obtained in a steady-state titration measurement, or dividing the dissociation rate constant (ko) by the association rate constant (kon).
[0098] As used in this document, biosimilar (from a product
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23/114 reference / approved biological drug, such as a therapeutic protein, an antibody, etc.) refers to a biological product, which is similar to the reference product based on data obtained from (a) analytical studies which demonstrate that the biological product is very similar to the reference product, despite minor differences in the clinically inactive components, (b) animal studies (including toxicity assessment), and / or (c) a clinical study or studies (including the assessment of immunogenicity and pharmacokinetics or pharmacodynamics) that are sufficient to demonstrate safety, purity and activity in one or more of the conditions of use suitable for the reference product to be licensed and intended for use and for which licensing is required for the biological product. In one embodiment, the biosimilar biological product and the reference product use the same mechanism or mechanisms of action for the predicted condition or conditions of use, recommended or suggested in the proposed marking, but only to the extent that the mechanism or mechanisms of action are known for the reference product. In one modality, the conditions or conditions of use provided for, recommended or suggested in the proposed marking for the biological product were previously approved for the reference product. In one embodiment, the route of administration, the dosage form, and / or the strength of the biological product are the same as those of the reference product. In one embodiment, the facility in which the biological product is manufactured, processed, packaged, or maintained meets the established standards to ensure that the biological product remains safe, pure and powerful. The reference product can be approved in at least one of the USA, Europe or Japan.
[0099] The term dosage, as used herein, refers to the administration of a substance (for example, an anti-TNF-a antibody) to achieve a therapeutic purpose (for example, the treatment of a disorder associated with TNFa).
[00100] The terms weekly dosing regimen, weekly dosing and weekly administration, as used in this document, refer to a specific course of time (or periodicity) of administration of a substance (for example, an anti-TNFα antibody) ) for a subject to achieve a therapeutic goal (for example, the treatment of a disorder associated with TNFa.). In one embodiment, the antibody, or antigen binding portion thereof, is administered every 6-8 days, or alternatively, every 7 days.
[00101] The terms biweekly dosing regimen, biweekly dosing, and biweekly administration, as used herein, refer to a specific time course (or periodicity) of administration of a substance (for example,
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24/114 example, an anti-TNFα antibody) for a subject to achieve a therapeutic objective (for example, the treatment of a disorder associated with TNFα.). The fortnightly dosing regimen is not intended to include a weekly dosing regimen. In one embodiment, the antibody, or antigen binding portion thereof, is administered every 9-19 days, more preferably, every 11-17 days, even more preferably, every 13-15 days, and with more preferably, every 14 days.
[00102] The terms monthly dosage regimen, monthly dosage, and monthly administration, as used in this document, refer to a specific time course (or periodicity) of administration of a substance (for example, an anti- TNFa) for a subject to achieve a therapeutic goal (for example, the treatment of a disorder associated with TNFa.). In one embodiment, a monthly dosing regimen means that the antibody, or antigen-binding portion of the same, is administered every 28-31 days. In another embodiment, a monthly dosing regimen means that the antibody, or antigen-binding portion of the same, is administered once a month, for example, on the same day of each month, such as, for example, on the first day of each month.
[00103] AUC, Cmax and Tmax are pharmacokinetic parameters that can be used to characterize the pharmacokinetic responses of a given drug product in a human or animal subject. The term AUC means the area under the curve that represents changes in the blood, serum, or plasma concentrations of a substance, for example, an anti-human TNFα antibody, over time. As used herein, the term Cmax refers to the maximum or peak concentration of blood, serum or plasma of the substance observed in a subject, after its administration. The term Tmax refers to the time when Cmax occurred, as measured from the time point of administration.
[00104] The term hydrodynamic diameter or Dh of a particle refers to the diameter of a sphere, which has the density of water and the same speed as the particle. Thus, the term hydrodynamic diameter of an antibody, as used herein, refers to a determination of the size of an antibody, or an antigen-binding portion thereof, for example, a human anti-TNFα antibody, or a fragment of antigen binding thereof, in solution using dynamic light scattering (DLS). A DLS measuring instrument measures time-dependent fluctuation in the intensity of light scattered from the antibody, or antigen-binding fragment thereof, in solution, at a fixed dispersion angle. Dh is determined from the time-dependent fluctuation intensity autocorrelation function. The intensity data
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25/114 dispersion are processed using the DLS instrument software to determine the hydrodynamic diameter value and the size distribution of the dispersion molecules, for example, the human anti-TNFα antibody, or antigen binding fragment thereof, the specimen.
[00105] The term conductivity, as used in this document, refers to the ability of an aqueous solution to conduct an electric current between two electrodes. Generally, electrical conductivity or specific conductivity is a measure of a material's ability to conduct an electric current. In solution, the current flows by transporting ions. Therefore, with an increasing amount of ions present in the aqueous solution, the solution will have greater conductivity. The unit of measurement for conductivity is mmhos (mS / cm), and can be measured using a conductivity meter sold, for example, by Orion Research, Inc. (Beverly, MA). The conductivity of a solution can be altered by changing the concentration of ions in it. For example, the concentration of buffer and / or salt, where the solution can be changed in order to achieve the desired conductivity.
[00106] The conductivity of a solution is measured according to methods known in the art. Conductivity meters and cells can be used to determine the conductivity of the aqueous formulation, and must be calibrated to a standard solution before use. Examples of conductivity meters available in the art include MYRON L Digital (Cole Parmer®), Conductometer (Metrohm AG) and Series 3105/3115 Integrated Conductivity Analyzers (Kemotron).
[00107] Conductivity measurements can be made with any commercially available conductivity meter suitable for conductivity analysis in protein solutions, for example, conductivity meter model SevenMulti, with the ability to expand over a wide pH range (Mettler Toledo , Schwerzenbach, Switzerland). The instrument is operated according to the manufacturers' instructions (for example, if the conductivity sensor is changed on the Mettler Toledo instrument, calibration must be performed again, since each sensor has a different cell constant, refer to to the Operating Instructions for the SevenMulti conductivity meter). If the instructions are followed, conductivity measurements can be made by directly immersing the measurement probe in the sample solution.
[00108] Various aspects of the invention are described in more detail in the following subsections [00109] II. Formulations and Methods of the Invention [00110] The present invention features liquid pharmaceutical formulations
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26/114 stable aqueous solutions comprising an anti-TNFα antibody, or an antigen-binding portion thereof, having improved properties compared to formulations recognized in the art. Although high concentration formulations containing human anti-TNFα antibodies are known in the art (see, for example, US20060153846 and US20100278822), the present invention provides high concentration formulations that have unexpected characteristics, that is, significantly reduced pain or increased pain. bioavailability. The formulations of the invention are based, at least on a combination of only one or two excipients, i.e., a surfactant and a polyol or, alternatively, a surfactant alone. Despite having few excipients, the formulations of the invention contain a high concentration of an antibody, for example, 90-110 mg / ml, and are stable.
[00111] As described in the working examples below, a formulation containing an antibody concentration greater than 50 mg / ml of an isolated human anti-TNFα antibody less than 50 mg / ml of a polyol (such as mannitol), and a surfactant, (such as a polysorbate) has been shown to dramatically reduce pain after injection compared to other high concentration formulations, including the commercial adalimumab formulation described in US20060153846, and the formulation described in US20100278822, each of which is incorporated herein by reference. Thus, in one embodiment, the formulations of the invention are associated with a reduction in pain, despite having a high concentration of antibody (for example, 100 mg / ml) and have no buffer or salt. The low-concentration pain formulations described in this document are based, at least in part, on the surprising finding that by removing or excluding salt (eg, NaCl) and / or a buffer (eg, a phosphate buffer) / citrate) the concentration of a human anti-TNF-alpha antibody in a formulation can be increased, for example, to about 100 mg / ml, while decreasing pain upon delivery to a patient.
[00112] In one embodiment, the formulation of the present invention is surprising, in that the formulation does not contain a buffer or salt, and reduces the pain associated with the injection in a patient by at least about 50% when on purchase with injection of an otherwise identical formulation comprising at least one salt and / or at least one buffer. In one embodiment, the formulation reduces pain associated with injection into a human subject by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75 %, or 80% (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 50, 41, 42, 43, 44 , 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 , 70, 71, 72, 73, 74, 75, 76, 77,
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78, 79, or 80%), when purchased with the injection of an otherwise identical formulation, which further comprises a salt and / or a buffer.
[00113] In one embodiment, the otherwise identical formulation used for the pain comparison assay comprises at least one buffer, such as a citrate buffer and phosphate buffer and / or a salt, for example, NaCl. For example, the buffer (excluded from the formulation of the present invention and present in the reference formulation for comparison of pain) may include citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and / or dihydrate. sodium phosphate. The buffer can include about 1,151.45 mg / ml of citric acid (for example, about 1.15, 1.20, 1.25, 1.30, 1.35, 1.40 or 1.45), about 0.2-0.4 mg / mL dehydrated sodium citrate (for example, about 0.2, 0.25, 0.3, 0.35, or 0.4), about 1.35 -1.75 mg / mL dehydrated disodium phosphate (for example, about 1.35, 1.40, 1.45, 1.50, 1.55, 1.60, 1.65, 1.70, or 1.75), to about 0.750.95 mg / ml of dehydrated sodium dihydrogen phosphate (for example, about 0.75, 0.80, 0.85, 0.9 or 0.95). Intermediate values and ranges in relation to the above mentioned concentrations are also intended to be part of this invention. In addition, ranges of values using a combination of any of the above values as upper and / or lower limits are intended to be included, for example, 0.1 to 0.5 mg / mL or 1.20 to 1, 40 mg / ml. In one embodiment, the pH of the formulation is adjusted with sodium hydroxide.
[00114] In one embodiment, the formulation of the invention includes high concentrations of human anti-TNFα antibodies, or antigen binding portions thereof, for example, 90-110 mg / ml, a polyol at a concentration less than 50 mg / mL, and a surfactant, such that the formulation is suitable for administration, without significant pain, as determined by a visual analog scale score (VAS). In one embodiment, the formulation and methods of the invention include high concentrations of anti-TNFα antibodies, or antigen binding portions thereof, and no buffer or salt, such that they are suitable for administration, for example, subcutaneous administration, without significant pain felt, as determined by a visual analog scale score (VAS). For example, the formulation of the present invention can result in a VAS less than 1, in a scaling of 0 (no pain) to 10 (worst pain imaginable) after subcutaneous injection. As described in Example 1, a formulation with 100 mg / ml adalimumab, polysorbate 80 and mannitol (less than 50 mg / ml) resulted in a VAS less than 1, for example, 0.56, whereas other formulations high concentration of antibodies resulted in a VAS ranging from 1.79 to 4.12.
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28/114 [00115] In one embodiment, the invention provides a liquid aqueous formulation, comprising an isolated human anti-TNFα antibody, or an antigen-binding portion thereof, a surfactant, and less than 50 mg / mL of a polyol, in which subcutaneous injection of the formulation results in a Visual Analogue Pain Score less than 1.0 after injection. In one embodiment, the formulation does not contain a buffer and salt, and results in a pain reduction of at least about 50% after subcutaneous injection when compared to an injection of an otherwise identical formulation, which further comprises a salt and / or a buffer (s).
[00116] Thus, in one aspect of the invention, the liquid formulations of the invention have advantageous tolerability properties in that the formulations produce less pain compared to formulations containing a buffer and a salt. In certain embodiments, the formulation reduces pain associated with injection (or any other form of administration) in a subject. In some embodiments, the pain associated with the injection is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g. at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87 , 88, 89, 90, 91, 92, 93, 94, or 95%). In one embodiment, the pain is reduced by at least about 50%.
[00117] Pain can be assessed using any type of pain assessment known in the art, including, for example, visual analog scales, qualitative pain assessments, or needle pain assessment. For example, the pain at the injection site perceived by the subject can be assessed by means of Visual Analogue Pain flaking (VAS). VAS is a measuring instrument that measures pain, as it varies over a continuum of values, for example, from none to an extreme amount of pain. Operationally a VAS is a horizontal line, about 100 mm long, anchored by numeric and / or word descriptors, for example, 0 or 10, or no unbearable pain or pain, optionally with additional numeric or word descriptors or between extremes, for example, mild, moderate and severe, or from 1 to 9) (see, for example, Lee JS, et al. (2000) Acad Emerg Med 7: 550, or Singer and Thods (1998) AcaPetição 870170080093 , of 10/20/2017, page 34/133
29/114 demic Emergency Medicine 5: 1007). Pain can be assessed once or several times after administration of a formulation of the invention, such as, for example, immediately after injection, at about 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 15, 20, 25, 30, 35, 40, or 45 minutes after the injection.
[00118] In a given modality of the invention, the injection of the formulation in a subject results in a Visual Analogue Scale score of pain less than 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, or 5.0, in a scaling of 0 (no pain) to 10 (unbearable pain).
[00119] Other tools for pain assessment are known in the art, including, for example, the desquamation of Numerical Classification, the desquamation of Verbal Classification and Brief Pain Inventory. Such tools can also be used to assess pain according to the invention.
[00120] Additional indices for skin irritation can be used, including, for example, the Draize scale (hemorrhage, petechiae, erythema, edema, pruritus).
[00121] The formulations of the invention contain a polyol, preferably containing less than about 50 mg of the polyol. In one embodiment, the formulations contain less than about 45 mg / ml of the polyol. In another embodiment, the formulations of the invention contain about 38-46 mg / ml of the polyol (for example, mannitol), for example, about 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 , 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mg / ml of the polyol. In addition, ranges of values using a combination of any of the above recited values as upper and / or lower limits are intended to be included, for example, 39-45 mg / mL, 40-44 mg / mL, or 37 -47 mg / ml. In one embodiment, the formulations of the invention contain about 12-72 mg / ml of polyol, for example, mannitol. In one embodiment, the polyols suitable for use in the formulations and methods of the invention are mannitol or sorbitol.
[00122] In one embodiment, the formulation of the invention contains adalimumab (or a biosimilar thereof), polysorbate 80, mannitol and water for injection. In one embodiment, the formulation contains 80 mg of adalimumab, water for injection, 42 mg / ml of mannitol, and 1 mg / ml of polysorbate 80. In one embodiment, the formulation can contain 20110 mg, alternatively, 20-90 mg adalimumab or, alternatively, 30-80 mg of the antibody. In one embodiment, the formulation contains 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg; 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 mg; 78 mg, 79 mg, 80 mg, 81 mg, 82 mg, 83 mg, 84
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30/114 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89 mg, 90 mg, 91 mg, 92 mg, 93 mg, 94 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99 mg, 100 mg, 101 mg, 102 mg, 103 mg, 104 mg, 105 mg, 106 mg, 107 mg, 108 mg, 109 mg or 110 mg of the antibody. The ranges including the numbers mentioned above are also included in the invention, for example, 70-90 mg, 65-95, or 60-85 mg.
[00123] The present invention is also based, at least in part, on the surprising finding that an aqueous liquid pharmaceutical formulation having a high concentration of a human anti-TNFα antibody, or antigen binding portion thereof, and a surfactant ( that is, in the absence of additional excipients), it has greater bioavailability than other high concentration formulations with additional excipients. As described in the working examples below, a formulation containing more than 50 mg / ml of an isolated human anti-TNFα antibody, and a polysorbate has been shown to have greater bioavailability compared to other high concentration formulations, including the commercial adalimumab formulation described US20060153846.
[00124] As described in Example 2 below, the bioavailability of an anti-TNFα antibody can be increased by combining the antibody with a surfactant, for example, polysorbate 80. The increase in bioavailability is based on the combination of the antibody and the surfactant and the omission or removal of other excipients, including a buffer, polyol and salt. The increase in bioavailability results from either an AUC0360 of the anti-TNFa antibody, or an antigen-binding portion of it, greater than about 1300 pg * h / mL or an AUC0-1344 of anti-TNFa antibody, or a portion of binding to its antigen, greater than about 2600 pg * h / mL, when injected subcutaneously, a human subject.
[00125] Therefore, the present invention provides methods for improving the bioavailability of an isolated anti-TNFα antibody, or an antigen-binding portion thereof, in a pharmaceutical formulation. The methods include combining a therapeutically effective amount of the anti-TNFα antibody, or antigen-binding portion thereof, with a surfactant, and removing or excluding other excipients, for example, a buffer (s), salt and polyol, or combinations thereof, such that the bioavailability of the antibody, or antigen-binding portion of it, is improved. In one embodiment, the formulation is injected subcutaneously into a human subject. The methods can improve bioavailability by providing an AUC0-360 of the antiTNFa antibody, or an antigen-binding portion of it, greater than about 1100, 1125, 1150, 1175, 1200, 1225, 1250, 1275, 1300, 1325, 1350, 1375, 1400, 1425, 1450, 1475, or
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31/114 about 1,500 pg * h / ml, when injected subcutaneously into a human subject.
[00126] The invention further provides a method for improving the bioavailability of an isolated human anti-TNFα antibody, or an antigen-binding portion thereof, in a subject, said method comprising administering a formulation comprising a surfactant and an effective amount of the antibody, or antigen-binding portion of it, for the subject such that the bioavailability of the antibody, or antigen-binding portion of it, in the subject is improved by at least about 15% over a second formulation . In one embodiment, the formulation of the invention does not contain a buffer, a polyol, or a salt, and the second formulation comprises a buffer, a polyol, and a salt. In one embodiment, the bioavailability of the antibody, or antigen-binding portion of it, is improved by at least about 30% during the second formulation. In one embodiment, the bioavailability of the antibody, or antigen-binding portion of it, is improved by at least about 40% during the second formulation. In one embodiment, bioavailability can be determined according to any level of AUC, for example, AUC0-360 or an AUC01344, or a Cmax.
[00127] In one embodiment, the present invention provides a liquid aqueous formulation that includes a surfactant and about 30-90 mg of an isolated human anti-TNFα antibody or antigen binding portion, wherein the formulation has an antibody concentration of about 90-110 mg / ml, and where the formulation provides increased bioavailability of the antibody, or antigen binding portion thereof, to a human subject by subcutaneous injection of the formulation relative to a formulation comprising phosphate buffer of citrate, sodium chloride and mannitol.
[00128] In one embodiment, the present invention provides aqueous liquid formulations that include a surfactant and 30-90 mg of an isolated human anti-TNFα antibody, or an antigen binding portion thereof, wherein the formulation has a concentration of antibody of 90-110 mg / ml, and wherein the formulation provides increased bioavailability of the antibody, or antigen-binding portion of it, to a human subject by subcutaneous injection of the formulation, such that the antibody, or portion of binding to its antigen, has an AUC0-360 greater than about 1100, 1125, 1150, 1175, 1200, 1225, 1250, 1275, 1300, 1325, 1350, 1375, 1400, 1425, 1450, 1475, or about 1500 pg * h / ml.
[00129] In one embodiment, the formulation of the invention contains adalimumab (or a biosimilar thereof), polysorbate 80, and water for injection. In one embodiment, the formulation contains 80 mg of adalimumab, water for injection, and 1 mg / ml of polysorbate 80.
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11/32
The formulation can contain 20-110 mg, alternatively, 20-90 mg adalimumab or, alternatively, 30-80 mg of the antibody. In one embodiment, the formulation contains 30 mg, 31
mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg; 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 mg; 78 mg, 79 mg, 80 mg, 81 mg, 82 mg, 83 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89 mg, 90 mg, 91 mg, 92 mg, 93 mg, 94 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99 mg, 100 mg , 101 m g, 1 02 mg,
103 mg, 104 mg, 105 mg, 106 mg, 107 mg, 108 mg, 109 mg or 110 mg of the antibody. The ranges including the numbers mentioned above are also included in the invention, for example, 70-90 mg, 65-95 mg, or 60-85 mg.
[00130] Thus, formulations of high concentration of antibodies and methods of the invention not only overcome a number of known challenges for pharmaceutical formulations, including high concentrations in a stable formulation, but also have the additional advantage of producing improved bioavailability or providing levels significantly low in pain when injected into patients.
[00131] Another obstacle overcome by the formulations of the invention is the ability to remain stable at room temperature (at about 25 degrees C or until about 30 degrees C). This stability provides advantages for the antibody user, providing more flexible storage options, since the constant need for refrigeration is not necessary. Both the pain reduction formulation and the bioavailability increase formulation (exemplified by formulations F3 and F4, respectively, in the Examples below) are stable for at least 6 days at about 25 degrees C or up to about 30 degrees C. As described in more detail in the Examples, the formulations of the invention are stable at up to 30 degrees C for at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days , at least 12 days, at least 13 days, and at least 14 days. Thus, the invention further provides formulations having expanded shelf life (i.e., at least 6 days, 10 days or 14 days) at room temperature (i.e., about 25 degrees C or up to about 30 degrees C). In one embodiment, the formulation of the invention is stable at 20 to 32 degrees C for at least 6 days. The intermediate temperatures for the concentrations recited above are also intended to be part of the present invention, that is, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32 degrees Celsius (C ). The ranges including the temperatures mentioned above are also included in the invention, for example, 22-26 degrees C, 25-30 degrees C, etc.
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33/114 [00132] The formulations of the invention contain a high concentration of antibody, including, for example, an antibody concentration of about 50 mg / ml, 55 mg / ml, 60 mg / ml. 65 mg / ml, 70 mg / ml, 75 mg / ml, 80 mg / ml, 85 mg / ml, 90 mg / ml, 95 mg / ml 100 mg / ml, 105 mg / ml, 110 mg / ml, 115 mg / ml (or more) of a human anti-TNF-alpha antibody, or antigen-binding fragment thereof. Thus, as described in the examples below, in one aspect of the invention, the liquid pharmaceutical formulations of the present invention contain an anti-human TNF alpha antibody concentration of 50100 mg / ml or more. In one embodiment, the formulations of the invention can comprise an antibody concentration between about 1 mg / ml - 150 mg / ml or about 40 mg / ml-125 mg / ml. In one embodiment, the antibody concentration in the formulation is 50-150 mg / ml, 55-150 mg / ml, 60-150 mg / ml, 65-150 mg / ml, 70-150 mg / ml, 75-150 mg / ml, 80-150 mg / ml, 85-150 mg / ml, 90-150 mg / ml, 90-110 mg / ml, 95-105 mg / ml, 95150 mg / ml, from 100-150 mg / ml, 105-150 mg / ml, 110-150 mg / ml, 115-150 mg / ml, 120150 mg / ml, 125-150 mg / ml, 50-130 mg / ml, 75-125 mg / ml, etc. The concentrations and intermediate ranges in relation to the concentrations recited above are also intended to be part of the present invention (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24,25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38 , 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 , 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 , 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113 , 114, 115, 116 , 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, 150 mg / ml).
[00133] The formulations of the invention may contain an effective amount of the antibody. In one embodiment, an effective amount is about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or about 100 mg of the antibody anti-human TNFα, or antigen-binding portion thereof. In one embodiment, the formulations and methods of the invention comprise about 20-100, about 20-90, about 30-90, about 30-100, about 60-100, about 70-90, about 40-90, about 60-85 mg, or about 40-100 mg of a human anti-TNFα antibody, or antigen-binding portion of it. In one embodiment, the formulation contains 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 mg, 78 mg, 79 mg, 80 mg, 81 mg, 82 mg, 83
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34/114 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89 mg, or 90 mg of the antibody. The ranges including the numbers mentioned above are included in the invention, for example, 70-90 or 75-85 mg or 60-85 mg.
[00134] An important aspect of the formulations and methods of the invention is the omission of a buffer and a salt. Thus, in one embodiment, the formulations and methods of the invention do not contain any buffer (s) (for example, citrate and phosphate) and salts. It should be noted, however, that, although the preferred formulations of the invention do not contain buffers or salts (e.g., NaCl), a small amount of buffer and / or salt may be present in the formulations. Thus, in one embodiment, the formulations of the invention do not contain detectable levels of a buffer (s) and / or a salt.
[00135] In one embodiment, the buffer (s) omitted from the formulations of the invention (or for comparison formulations that include a buffer (s)) may include citric acid (e.g., about 1.3-1 , 31 mg / ml or 1.305 mg / ml). In another embodiment, the buffer system includes dehydrated sodium citrate (for example, about 0.27-0.33 mg / ml or about 0.305 mg / ml). In one embodiment, the buffer system includes dehydrated disodium phosphate (for example, about 1.5-1.56 mg / ml, or about 1.53 mg / ml). In another embodiment, the buffer system includes sodium dihydrogen phosphate dihydrate (for example, about 0.83-0.89 mg / ml or about 0.86 mg / ml).
[00136] In one embodiment of the invention, the conductivity of the formulation can be used to determine whether a formulation has a buffer and / or salt. Both Formulation F3 and F4 (described in the working examples below) were determined to have a conductivity of less than about 2 mS / cm, and for example, about 0.7 qS / cm. Thus, in one embodiment, the pain reduction and increase bioavailability formulations of the invention have a conductivity of less than about 2 mS / cm. In another embodiment, the formulations of the present invention have a conductivity of less than about 1 mS / cm.
[00137] In one embodiment, the formulation of the invention contains an anti-human TNF-alpha antibody, or antigen-binding portion thereof, at a concentration of about 100 mg / ml (or 75-125 mg / ml), a surfactant (for example, polysorbate 80), a polyol (for example, sorbitol or mannitol), and has a conductivity of less than 2 mS / cm. In one embodiment, the formulation of the invention contains an anti-human TNF-alpha antibody, or antigen-binding portion thereof, at a concentration of about 100 mg / ml (or 75-125 mg / ml), about 0 , 8-1.3 mg / mL of a surfactant (for example, polysorbate 80), less than about 50 mg / mL of a polyol (for example, sorbitol or
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35/114 mannitol), and has a conductivity of less than 2 mS / cm.
[00138] In one embodiment, the formulation of the invention contains an anti-human TNF-alpha antibody, or antigen binding portion thereof, at a concentration of about 100 mg / ml (or 75-125 mg / ml), a surfactant (e.g., polysorbate 80), and has a conductivity of less than 2 mS / cm. In one embodiment, the formulation of the invention contains an anti-human TNF-alpha antibody, or antigen-binding portion thereof, at a concentration of about 100 mg / ml (or 75-125 mg / ml), about 0 , 8-1.3 mg / mL of a surfactant (e.g., polysorbate 80), and has a conductivity of less than 2 mS / cm.
[00139] In another embodiment, the invention provides a stable formulation having a high concentration of the antibody, or antigen binding portion thereof, wherein the antibody, or antigen has a hydrodynamic diameter (z-medium), less than that about 4 nm or where the antibody, or antigen has a hydrodynamic diameter (medium), which is at least about 50% less than the hydrodynamic diameter of a buffered solution at the same antibody concentration. In one embodiment, the antibody, or antigen has a hydrodynamic (z-average) diameter of less than about 3 nm.
[00140] In one embodiment, the formulation of the invention contains an anti-human TNF-alpha antibody, or antigen-binding portion thereof, at a concentration of about 100 mg / ml (or 75-125 mg / ml), a surfactant (for example, polysorbate 80), a polyol (for example, sorbitol or mannitol), and has a hydrodynamic diameter of less than 4 nm. In one embodiment, the formulation of the invention contains an anti-human TNF-alpha antibody, or antigen-binding portion thereof, at a concentration of about 100 mg / ml (or 75-125 mg / ml), about 0 , 8-1.3 mg / mL of a surfactant (for example, polysorbate 80), less than about 50 mg / mL of a polyol (for example, sorbitol or mannitol), and has a hydrodynamic diameter of less than 4 nm.
[00141] In one embodiment, the formulation of the invention contains an anti-human TNF-alpha antibody, or antigen binding portion thereof, at a concentration of about 100 mg / ml (or 75-125 mg / ml), a surfactant (for example, polysorbate 80), and has a hydrodynamic diameter of less than 4 nm. In one embodiment, the formulation of the invention contains an anti-human TNF-alpha antibody, or antigen-binding portion thereof, at a concentration of about 100 mg / ml (or 75-125 mg / ml), about 0 , 8-1.3 mg / mL of a surfactant (for example, polysorbate 80), and has a hydrodynamic diameter of less than 4 nm.
[00142] A detergent or surfactant is included in the antibody formulation of the invention. Examples of detergents include non-ionic detergents, such as
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36/114 polysorbates (for example, polysorbates 20, 80, etc.), or poloxamers (for example, poloxamer 188). The amount of detergent added is such that it reduces the aggregation of the formulated antibody and / or minimizes the formation of particles in the formulation and / or reduces adsorption. In a preferred embodiment of the invention, the formulation includes a surfactant that is a polysorbate. In another preferred embodiment of the invention, the formulation contains the detergent polysorbate 80. In one embodiment, the formulation contains between about 0.1 and about 2.0 mg / ml of surfactant (for example, polysorbate), for example, about 1 mg / ml. Other polysorbate ranges that can be included in the formulations of the invention include 0.1 to 1.5 mg / ml, alternatively 0.2-1.4 mg / ml, 0.3-1.3 mg / ml, 0, 4-1.2 mg / mL, 0.5-1.1 mg / mL, 0.6-1.0 mg / mL, 0.6-1.1 mg / mL, 0.7-1.1 mg / ml, 0.8-1.1 mg / ml, or 0.9-1.1 mg / ml. The values and intermediate ranges in relation to the concentrations recited above are also intended to be part of the present invention, for example, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0, 8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9. In addition, ranges of values using a combination of any of the values as upper and / or lower limits are intended to be included, for example, 0.3 to 1.1 mg / mL, recited above.
[00143] In one embodiment, the formulation of the present invention consists essentially of an anti-human TNF-alpha antibody, or antigen-binding portion thereof, at a concentration of about 100 mg / ml (or 75-125 mg / mL), a surfactant (for example, polysorbate 80), a polyol (for example, sorbitol or mannitol), do not contain a buffer (s) (for example, citric acid monohydrate, sodium citrate, dihydrate of disodium phosphate, and / or sodium dihydrogen phosphate dihydrate), and which does not contain a salt (eg NaCl).
[00144] In certain embodiments, the otherwise identical formulation with which the formulation of the invention is compared for purposes of pain or bioavailability is a formulation containing adalimumab, sodium chloride, monophasic sodium phosphate dihydrate, dihydrate of diphasic sodium phosphate, sodium citrate, citric acid monohydrate, mannitol, polysorbate 80 and water for injection.
[00145] The present formulation can also be combined with one or more other therapeutic agents, as necessary for the particular indication to be treated. In one embodiment, those with complementary activities that do not adversely affect the antibody in the formulation. Such therapeutic agents are suitably present in combination in amounts that are effective for the intended purpose. Additional therapeutic agents that can be combined with the formulation of the invention are further described in US Patent Nos. 6,090,382 and 6,258,562, each of which is incorporated herein by reference.
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37/114 [00146] All formulations described in this document can be used in the methods of the invention as well.
[00147] III. Antibodies for use in the formulations and methods of the Invention [00148] The formulations and methods of the invention include an antibody, or antigen-binding portion thereof, in particular an anti-TNFα antibody, or antigen-binding portion or fragment the same. Examples of antibodies that can be used in the invention include chimeric antibodies, non-human antibodies, isolated human antibodies, humanized antibodies and domain antibodies (dAbs). All antibodies described herein can be used in the methods of the invention as well.
[00149] In one embodiment, the formulations of the invention comprise an antibody, or antigen-binding portion thereof, which binds to human TNFα, including, for example, adalimumab (also referred to as Humira, adalimumab, or D2E7; Abbott Laboratories ). In another embodiment, the formulation comprises an antibody that binds to the same epitope as adalimumab, such as, but not limited to, a biosimilar adalimumab antibody. In one embodiment, the antibody is a human IgG1 antibody having six CDRs corresponding to adalimumab light and heavy chain.
[00150] In one embodiment, the invention features an isolated human antibody, or antigen-binding portion thereof, which binds to human TNF-alpha with a high affinity and a reduced rate, and also has a high neutralizing capacity. In one embodiment, the human antibodies used in the invention are recombinant human anti-hTNF-alpha neutralizing antibodies.
[00151] In one aspect, the invention relates to adalimumab antibodies and antibody portions, antibodies related to adalimumab and antibody portions and to other human antibodies and antibody portions, with properties equivalent to those of adalimumab, such as high binding affinity to hTNFa with low dissociation kinetics and high neutralization capacity. In one embodiment, the antibody, or antigen-binding fragment thereof, is defined according to the dissociation and binding characteristics similar to adalimumab. For example, the formulation can include a human antibody that dissociates from human TNFα with a Kd of 1 x 10 ⁸ M or less, and a koff rate constant of 1 x 10 ^-3 s ^-1 or less, both determined by resonance of surface plasmons. In another embodiment, the human antibody that dissociates from human TNFα with a Kd of 1 x 10 ^-9 M or less.
[00152] In one embodiment, the antibody, or antigen-binding fragment thereof, is a human antibody that dissociates from human TNFα with a Kd of 1 x 10 ^-8 M or less, and a koff rate constant 1 x 10 ^-3 s ^-1 or less, both
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38/114 determined by surface plasmon resonance, and neutralizes the cytotoxicity of human TNFα in a standard in vitro L929 assay with an IC 50 of 1 x 10 ^-7 M or less. Examples and methods for producing neutralizing human antibodies that have a high affinity for human TNFα, including antibody sequences, are described in US Patent No. 6,090,382 (referred to as D2E7), incorporated herein by reference. The amino acid sequences of D2E7, as described in US 6,090,382 are incorporated in their entirety into this document.
[00153] In one embodiment, the antibody used in the formulation of the invention is D2E7, also referred to as HUMIRA ^TM or adalimumab (the amino acid sequence of the V2 region of D2E7 is shown in SEQ ID NO: 1, the amino acid sequence of V2 region of D2E7 is shown in SEQ ID NO: 2). The properties of D2E7 (adalimumab / HUMIRA®) have been described in Salfeld et al., US Patent Nos. 6,090,382, 6,258,562 and 6,509,015, each of which is incorporated herein by reference.
[00154] In one embodiment, human TNF-alpha, or an antigen-binding portion thereof, dissociates from human TNF-alpha with a Kd of 1 x 10 ^-8 M or less and a Koff Rate constant 1 x 10 ^-3 s ^-1 or less, both determined by surface plasmon resonance, and neutralizes the cytotoxicity of human TNF-alpha in a standard in vitro L929 assay with an IC 50 of 1 x 10 ^-7 M or less . In one embodiment, the isolated human antibody, or antigen-binding portion thereof, dissociates from human TNF-alpha with a Koff of 5 x 10 ^-4 s ^-1 or less, or, in one embodiment, with a Koff of 1 x 10 ^-4 s ^-1 or less. In one embodiment, the isolated human antibody, or antigen-binding portion thereof, neutralizes the cytotoxicity of human TNF-alpha in a standard in vitro L929 assay with an IC 50 of 1 x 10 ^-8 M or less, or, in one embodiment, with an IC50 of 1 x 10 ^-9 M or less, or, in one embodiment, with an IC50 of 1 x 10 ^-10 M or less. In one embodiment, the antibody is an isolated, recombinant human antibody, or antigen-binding portion thereof.
[00155] It is well known in the art that the heavy and light chain CDR3 domains of antibodies play an important role in the specificity / binding affinity of an antibody to an antigen. Thus, in another aspect, the antibody used in the formulation of the present invention has slow dissociation kinetics for association with hTNF-alpha and has light and heavy chain CDR3 domains that are structurally identical or related to those of adalimumab. Position 9 of the adalimumab VL CDR3 can be occupied by Ala or Thr without substantially affecting the Koff. Thus, a consensus reason for adalimumab's VL CDR3 comprises
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39/114 the amino acid sequence: Q-R-Y-N-R-A-P-Y- (T / A) (SEQ ID NO: 3). In addition, the VH CDR3 position 12 of adalimumab can be occupied by Tyr or Asn, without substantially affecting Kof. Thus, a consensus motive for adalimumab VH CDR3 comprises the amino acid sequence: V-S-Y-L-S-T-A-S-S-L-D- (Y / N) (SEQ ID NO: 4). In addition, as shown in Example 2 of US Patent No. 6,090,382, the CDR3 domain of the adalimumab heavy and light chains can be replaced with a single alanine residue (in position 1, 4, 5, 7 or 8 inside the VL CDR3 or in position 2, 3, 4, 5, 6, 8, 9, 10 or 11 inside the VH CDR3) without substantially affecting Koff. In addition, the person skilled in the art will appreciate that, given the receptivity of the CDR3 domains of VL and VH of adalimumab for substitutions by alanine, substitution of other amino acids from the CDR3 domains may be possible, while still maintaining the reduced rate constant of the antibody, in particular substitutions with conservative amino acids. In one embodiment, no more than one to five conservative amino acid substitutions are made within the CDR3 domains of VL and / or VH of adalimumab. In one embodiment, no more than one to three conservative amino acid substitutions are made within the CDR3 domains of VL and / or VH of adalimumab. In addition, conservative amino acid substitutions should not be made at critical amino acid positions for binding to hTNF alpha. Adalimumab VL CDR3 positions 2 and 5 and adalimumab VH CDR3 positions 1 and 7 appear to be critical for interaction with alpha hTNF, and therefore conservative amino acid substitutions are preferably not performed at these positions (although an alanine substitution at position 5 of the VL CDR3 of adalimumab is acceptable, as described above) (see US Patent No. 6,090,382).
[00156] Thus, in one embodiment, the antibody, or antigen-binding portion thereof, used in the formulation of the invention contains the following characteristics:
a) dissociates from human TNFα with a koff rate constant of 1 x 10 ^-3 s ^-1 or less, as determined by surface plasmon resonance;
b) has a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single substitution of alanine at position 1, 4, 5, 7 or 8, or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8 and / or 9;
c) has a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single substitution of alanine at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to five conservative amino acid substitutions in positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and / or 12.
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40/114 [00157] In certain embodiments, the antibody or antigen-binding portion of it dissociates from human TNF-alpha with a Koff of 5 x 10 ^-4 s ^-1 or less. In certain embodiments, the antibody or antigen-binding portion thereof dissociates from human TNF-alpha with a Koff of 1 x 10 ^-4 s ^-1 or less.
[00158] In yet another embodiment, the antibody, or antigen-binding portion thereof, contains a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or modified to from SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5, 7 or 8 and with a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alanine substitution in position 2, 3, 4, 5, 6, 8, 9, 10 or 11. In one embodiment, the LCVR also has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5 (i.e., CDR2 of VL of D2E7) and HCVR further has a domain of CDR2 comprising the amino acid sequence of SEQ ID NO: 6 (i.e., CDR2 of VH of D2E7). In one embodiment, LCVR also has a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7 (i.e., CDR1 of D2E7 VL) and HCVR has a CDR1 domain comprising the amino acid sequence of SEQ ID NO: : 8 (that is, the CDR1 of the VH of D2E7). The VL framework regions can be from the human VkI germline family, or from the human germline Ak Vk gene, or from the adalimumab VL framework sequences shown in Figures 1A and 1B of US Patent No. . 6,090,382. The VH framework regions can be from the human VH3 germline family, or from the human DP-31 germline VH gene, or from the D2E7 VH structure sequences shown in Figures 2A and 2B of the Patent US No. 6,090,382. The nucleic acid sequences corresponding to the variable regions of adalimumab heavy and light chain are described in SEQ ID NOs: 36 and 37, respectively.
Thus, in another embodiment, the antibody, or antigen-binding portion thereof, contains a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1 (i.e., the VL of adalimumab) and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2 (i.e., the VH of adalimumab). In certain embodiments, the antibody comprises a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD. In certain embodiments, the heavy chain constant region is an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. In addition, the antibody may comprise a light chain constant region, either a
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41/114 kappa light chain constant region or lambda light chain constant region. In one embodiment, the antibody comprises a kappa light chain constant region. Alternatively, the antibody portion can be, for example, a Fab fragment or a single chain Fv fragment.
[00160] In still other embodiments, the invention includes the use of an isolated human antibody, or an antigen-binding portion thereof, containing the CDR3 domains of VH and VL related to adalimumab. For example, the antibodies or antigen binding portions thereof may have a light chain variable region (LCVR) having a CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 or with a variable chain region (HCVR) having a CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35.
[00161] In one embodiment, the TNFα antibody used in the present invention includes the chimeric infliximab antibody (Remicade®, Johnson and Johnson; described in US Patent No. 5,656,272, incorporated herein by reference), CDP571 (an IgG4 antibody humanized anti-TNF-alpha monoclonal), CDP 870 (a fragment of humanized anti-TNF-alpha antibody), an anti-TNF dAb (Peptech), or CNTO 148 (golimumab; Medarex and Centocor, see WO 02/12502). Additional TNF antibodies that can be used in the invention are described in US Patent Nos. 6,593,458; 6,498,237, 6,451,983, and 6,448,380, each of which is incorporated herein by reference.
[00162] An antibody, or antibody portion, used in the methods and compositions of the invention, can be prepared by recombinant expression of immunoglobulin light and heavy chain genes from a host cell. To express an antibody recombinantly, a host cell is transfected with one or more recombinant expression vectors that carry fragments of DNA encoding the antibody's immunoglobulin light and heavy chains, such that the light and heavy chains are expressed in the host cell. and, preferably, secreted into the medium in which the host cells are grown, medium from which antibodies can be recovered. Standard recombinant DNA methodologies are used to obtain heavy and light chain antibody genes, incorporate these genes into recombinant expression vectors and
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42/114 introducing the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F.M. et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in US Patent No. 4,816,397 to Boss et al.
[00163] To express an anti-TNF-a antibody, for example, adalimumab (D2E7) or an adalimumab-related antibody (D2E7), the DNA fragments encoding the variable regions of the light and heavy chains are first obtained. These DNAs can be obtained by amplifying and modifying the variable sequences of light chain and heavy chain of the germline using the polymerase chain reaction (PCR). Germline DNA sequences for human heavy and light chain variable region genes are known in the art (see, for example, the Vbase human germline sequence database, see also Kabat, EA, et al. ( 1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, IM, et al. (1992) “The Repertoire of Human Germline VH Sequences Reveals about Fifty Groups of VH Segments with Different Hypervariable Loops "J. Mol. Biol. 227: 776-798; and Cox, JPL et al. (1994)" A Directory of Human Germ-line V78 Segments Reveals a Strong Bias in their Usage "Eur. J. Immunol. 24: 827-836; (the contents of each of which are expressly incorporated herein by reference.) For example, to obtain a DNA fragment encoding the D2E7 heavy chain variable region, or a related antibody with D2E7, a member of the VH3 family of VH genes of the human germline are amplified by conventional PCR. In certain embodiments, the DP-31 germline VH sequence is amplified. To obtain a DNA fragment encoding the D2E7 light chain variable region, or a D2E7-related antibody, a member of the VkI family of the human germline VL genes is amplified by conventional PCR. In certain embodiments, the A20 germline VL sequence is amplified. PCR primers suitable for use in amplifying the DP-31 germline VH and A20 germline VL sequences can be designed based on the nucleotide sequences described in the references cited above, using conventional methods.
[00164] Once the VH and VL fragments of the germline are obtained, these sequences can be mutated to encode the amino acid sequences of anti-TNFα antibodies disclosed herein. The amino acid sequences encoded by the germline VL and VH DNA sequences are compared with the anti-TNFa antibody VH and VL amino acid sequences to identify residues of
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43/114 amino acids in the anti-TNFa antibody sequence that differ from the germline. Then, the appropriate nucleotides for the germline DNA sequences are mutated in such a way that the mutated germline sequence encodes the amino acid sequence of the anti-TNFa antibody, using the genetic code to determine what nucleotide changes should be made. Germline sequence mutagenesis is performed by conventional methods, such as PCR-mediated mutagenesis (in which the mutated nucleotides are incorporated into the PCR primers in such a way that the PCR product contains the mutations), or site-directed mutagenesis.
[00165] In addition, it should be noted that if the germline sequences obtained by PCR amplification encode amino acid differences in the structure regions of the true germline configuration (ie, the differences in the amplified sequence compared to the sequence of the true germline, for example, as a result of a somatic mutation) it may be desirable to change these amino acid differences back to the sequences of true germline strains (i.e., retromutation of structure residues to the germline configuration) .
[00166] Since the DNA fragments encoding the VH and VL segments of the anti-TNFa antibody are obtained (for example, by amplification and mutagenesis of the germline VH and VL genes, as described above) these fragments of DNA can be further manipulated by standard recombinant DNA techniques, for example, to convert the genes of the variable region to full-length antibody chain genes, with the Fab fragment genes, or to an scFv gene. In these manipulations, a fragment of DNA encoding VL or VH is operably linked to another fragment of DNA encoding another protein, such as an antibody constant region or a flexible linker. The term operably linked, as used in this context, is intended to mean that the two DNA fragments are joined in such a way that the amino acid sequences encoded by the two DNA fragments remain in the structure.
[00167] Isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operatively linking the DNA encoding the VH to another DNA molecule encoding the heavy chain constant regions (CH1, CH2 and CH3). Gene sequences of the human heavy chain constant region are known in the art (see, for example, Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health
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44/114 and Human Services, NIH Publication No. 91-3242) and the DNA fragments that encompass these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but more preferably, it is an IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene, the DNA encoding VH can be operably linked to another DNA molecule encoding only CH1 heavy chain constant region.
[00168] Isolated DNA encoding the VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operatively binding DNA that encodes the VL to another molecule of DNA encoding the light chain constant region, CL. The gene sequences of the human light chain constant region are known in the art (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242) and the DNA fragments that encompass these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region. In one embodiment, the light chain constant region is a kappa constant region.
[00169] To create a scFv gene, the DNA fragments that encode VH and VL are operably linked to another fragment that encodes a flexible linker, for example, that encodes the amino acid sequence (Gly4-Ser) 3, in such a way that the VH and VL sequences can be expressed as a contiguous single chain protein, with the VL and VH regions joined by the flexible linker (see, for example, Bird et al. (1988) Science 242: 423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 58795883; McCafferty et al., Nature (1990) 348: 552-554).
[00170] To express the antibodies, or portions of antibodies used in the invention, the DNA encoding full or partial length light or heavy chains, obtained as described above, are inserted into expression vectors such that the genes are operably linked to transcriptional and transductional control sequences. In this context, the term operably linked is intended to mean that an antibody gene is linked in a vector so that the transcription and transduction control sequences within the vector serve its intended function of regulating the transcription and transduction of the gene of the antibody. The expression control and expression vector sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into the separate vector or, more typically, both genes are
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45/114 inserted in the same expression vector. Antibody genes are inserted into the expression vector by standard methods (for example, binding complementary restriction sites on the antibody gene fragment and vector, or binding blunt ends if restriction sites are not present). Before the insertion of the anti-TNFα antibody light and heavy chain sequences, the expression vector can already transport the sequences of the antibody constant region. For example, an approach to convert the VH and VL sequences from the anti-TNFa antibody to the full-length antibody genes is to insert them into the expression vectors that already encode the heavy chain constant regions and the light chain constant regions , respectively, in such a way that the VH segment is operatively linked to the CH segment (s) within the vector and the VL segment is operatively linked to the CL segment in the vector. In addition or alternatively, the recombinant expression vector can encode a signal peptide, which facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in the structure with the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide, or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
[00171] In addition to the antibody chain genes, the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term regulatory sequence is intended to include promoters, enhancers and other elements of expression control (for example, polyadenylation signals) that control the transcription or translation of genes in the antibody chain. Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the desired protein expression level, etc. The regulatory sequences Preferred for mammalian host cell expression include viral elements that drive high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter / enhancer), Viruses Simian 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus (e.g., the main adenovirus late promoter (AdMLP)) and polyoma. For a more detailed description of viral regulatory elements and their sequences, see, for example, US Patent No. 5,168,062 of
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46/114
Stinski, US Patent No. 4,510,245 to Bell et al. and US Patent No. 4,968,615 to Schaffner et al.
[00172] In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors used in the invention can carry additional sequences, such as sequences that regulate vector replication in host cells (e.g., origins of replication) and genes selectable markers. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, for example, US Patent Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, in a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection / amplification) and the neo gene (for selection with G418).
[00173] For expression of the light and heavy chains, the expression vector (s) encoding the light and heavy chains is transfected into a host cell by standard techniques. The various forms of the term transfection are intended to encompass a wide variety of techniques commonly used for introducing exogenous DNA into a eukaryotic or prokaryotic host cell, for example, electroporation, calcium phosphate precipitation, DEAE-dextran transfection and the like . Although it is theoretically possible to express the antibodies of the invention, either in prokaryotic or eukaryotic host cells, the expression of antibodies is preferably in eukaryotic cells. In one embodiment, mammalian host cells are most preferred because these eukaryotic cells, and in particular mammalian cells, are more susceptible than prokaryotic cells to assemble and secrete an immunologically active and properly folded antibody. The prokaryotic expression of antibody genes has been reported to be ineffective for producing high yields of active antibody (Boss, M.A. and Wood, C. R. (1985) Immunology Today 6: 12-13).
Preferred mammalian host cells for expression of the recombinant antibodies of the invention include Chinese hamster ovary cells (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, used with a selectable DHFR marker, for example, as described in RJ Kaufman and PA Sharp (1982) Mol. Biol. 159: 601-621), NS0 myeloma cells, COS cells and cells SP2. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, antibodies are produced by culturing host cells during
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47/114 a period of time sufficient to allow expression of the antibody in the host cells, or more, in one embodiment, the secretion of the antibody in the culture medium in which the host cells are cultured. Antibodies can be recovered from the culture medium using standard protein purification methods.
[00175] Host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. Variations of the process described above are understood to be within the scope of the present invention. For example, it may be desirable to transfect a host cell with DNA that encodes either the light chain or the heavy chain (but not both) of an antibody of the present invention. Recombinant DNA technology can also be used to remove some or all of the DNA encoding either one or both of the light and heavy chains, which is not required for binding to hTNF alpha. Molecules expressed from these truncated DNA molecules are also encompassed by the antibodies of the invention. In addition, bifunctional antibodies can be produced in which one heavy chain and one light chain are one antibody of the invention and the other heavy and light chain are specific for an antigen other than alpha hTNF by crosslinking an antibody of the invention with a second antibody through standard chemical crosslinking methods.
[00176] In a preferred system for recombinant expression of an antibody, or antigen-binding portion thereof, of the present invention, a recombinant expression vector that encodes both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells through calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody's heavy chain and light chain genes are each operably linked to AdMLP promoter / CMV enhancer regulatory elements to drive high levels of gene transcription. The recombinant expression vector also carries a DHFR gene, which allows the selection of CHO cells that have been transfected with the vector using selection / amplification with methotrexate. The host cells of selected transformants are cultured to allow expression of the heavy and light chains of antibody and the intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect host cells, select transformants, cultivate host cells and recover the antibody from the culture medium.
[00177] In view of the above, the compositions of nucleic acid, vectors and host cells that can be used for the recombinant expression of the antibodies and portions of antibodies used in the present invention include the nucleic acids and vectors
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48/114 which comprise said nucleic acids, which comprise the human TNF alpha antibody adalimumab (D2E7). The nucleotide sequence encoding the D2E7 light chain variable region is shown in SEQ ID NO: 36. The CDR1 domain of LCVR includes nucleotides 70-102, the CDR2 domain includes nucleotides 148-168 and the domain of CDR3 encompasses nucleotides 265-291. The nucleotide sequence encoding the D2E7 variable region of the heavy chain is shown in SEQ ID NO: 37. The HCVR CDR1 domain comprises nucleotides 91-105, the CDR2 domain includes nucleotides 148-198 and the domain of CDR3 encompasses nucleotides 295-330. It will be appreciated by the person skilled in the art that nucleotide sequences, which encode D2E7-related antibodies, or portions thereof (for example, a CDR domain, such as a CDR3 domain) can be derived from the nucleotide sequences that encode the D2E7 LCVR and HCVR using the genetic code and standard molecular biology techniques.
[00178] In one embodiment, the liquid pharmaceutical formulation comprises a human TNF alpha antibody, or antigen-binding portion thereof, which is a biosimilar or bioequivalent to the adalimumab antibody. In one embodiment, a biosimilar antibody is an antibody that shows no clinically significant difference when compared to a reference antibody, for example, adalimumab. A biosimilar antibody has equivalent safety, purity and potency as a reference antibody, for example, adalimumab.
[00179] IV. Administration of the Formulations of the Invention for the Treatment of TNFa-Related Disorders [00180] An advantage of the formulations of the invention is that they can be used to deliver a high concentration of an anti-TNF alpha antibody, or antigen binding portion (for example, adalimumab) to a subject subcutaneously, so that any pain after injection is reduced or the bioavailability of the antibody is improved. Thus, in one embodiment, the formulation of the invention is distributed to a subject subcutaneously. In a modality, the subject administers the formulation for himself / herself (self-administration).
[00181] In one embodiment, an effective amount of the formulation is administered. An example of an effective amount of the formulation is an amount sufficient to inhibit harmful TNF-alpha activity or to treat a disorder in which TNF alpha activity is harmful.
[00182] As used herein, the term a disorder in which TNF-alpha dw activity is harmful, is intended to include diseases and other disorders in which
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49/114 the presence of TNF-alpha in a subject suffering from the disorder has been shown to be or is suspected of being responsible for the pathophysiology of the disorder or for a factor that contributes to an aggravation of the disorder. Thus, a disorder in which TNF-alpha activity is harmful is a disorder in which inhibition of TNF-alpha activity is anticipated to alleviate symptoms and / or progression of the disorder. These disorders can be evidenced, for example, by an increase in the concentration of TNF-alpha in a biological fluid of a subject suffering from the disorder (for example, an increase in the concentration of TNF-alpha in serum, plasma, synovial fluid, etc. ., of the subject) that can be detected, for example, using an anti-TNF alpha antibody.
[00183] In one embodiment, the effective amount of the antibody can be determined according to a dosing schedule based strictly on weight (for example, mg / kg) or it can be a total body dose (also referred to as a fixed dose) which is independent of weight. In one embodiment, an effective amount of the antibody is about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or about 100 mg of human anti-TNFα antibody, or antigen-binding portion thereof. In one embodiment, an effective amount of the antibody is about 20-100, about 20-90, about 30-90, about 30-100, about 60-100, about 70-90, about 40 -90, about 60-85 mg, or about 40-100 mg. In one embodiment, the formulation contains an effective amount of the antibody of 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg; 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg , 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg , 76 mg, 77 mg, 78 mg, 79 mg, 80 mg, 81 mg, 82 mg, 83 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89 mg, 90 mg, 91 mg, 92 mg, 93 mg, 94 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99 mg or 100 mg of the antibody. The ranges including the numbers mentioned above are also included in the invention, for example, 70-90 or 75-85 or 60-85 mg mg.
[00184] In one example, an effective amount of the formulation is 0.4 ml or 0.8 ml of the formulation which contains a total body dose of about 80 mg of antibody (ie 0.8 ml of a formulation of 100 mg / ml antibody of the invention). In another example, an effective amount of the formulation is 0.4 ml of the formulation of the invention which contains a total body dose of about 40 mg of antibody (i.e. 0.4 ml of a 100 mg / ml formulation of antibody of the invention). In yet another example, an effective amount of the formulation is twice 0.8 ml of the formulation which contains a total body dose of about 160 mg of antibody (ie two units containing 0.8 ml of
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50/114 each of a 100 mg / ml antibody formulation of the invention). In another example, an effective amount of the formulation is 0.2 ml of the formulation of the present invention containing a total body dose of about 20 mg of antibody (that is, 0.2 ml of a formulation of 100 mg / ml of antibody of the invention). Alternatively, an effective amount can be determined according to a fixed dosage regimen based on weight (see, for example, WO 2008/154543, incorporated herein by reference).
[00185] In one embodiment, TNF-alpha is human TNF-alpha and the subject is a human subject. Alternatively, the subject may be a mammal expressing a TNFalpha, with which an antibody of the present invention cross-reacts. In addition, the subject may be a mammal into which hTNF-alpha has been introduced (for example, through the administration of hTNF-alpha, or by expression of an hTNF-alpha transgene).
[00186] A formulation of the invention can be administered to a human subject for therapeutic purposes (discussed below). In one embodiment of the invention, the liquid pharmaceutical formulation is easily administrable, which includes, for example, a formulation that is self-administered by the patient. In one embodiment, the formulation of the invention is administered via subcutaneous injection, such as single-use subcutaneous injection. In addition, a formulation of the invention can be administered to a non-human mammal expressing TNF-alpha, with which the antibody cross-reacts (for example, a primate, pig or mouse) for veterinary purposes, or as an animal model of human disease. With respect to the latter, these animal models may be useful for assessing the therapeutic effectiveness of the antibodies of the invention (for example, testing dosages and courses of time of administration).
[00187] The formulations of the invention can be administered according to a certain dosage schedule. For example, formulations can be administered according to a weekly, fortnightly or monthly dosing regimen. Alternatively, the formulation can be administered once every three weeks. In one embodiment, the formulations and methods comprise the administration of a human anti-TNFα antibody to the subject according to a periodicity selected from the group consisting of weekly, biweekly, every three weeks and monthly.
[00188] In one embodiment, the liquid aqueous formulation of the invention can be administered to a subject by, for example, a pre-filled syringe, a self-injecting pen, or a needle-free delivery device. Thus, the invention also features a self-injecting pen, a pre-filled syringe, or a needle-free delivery device comprising the liquid aqueous formulation of the invention. In
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51/114 one embodiment, the invention features a delivery device comprising a dose of the formulation comprising 100 mg / ml of a human TNF alpha antibody, or an antigen-binding portion thereof, for example, a self-injecting pen or pre-syringe -filled comprising a dose of about 19 mg; 20, mg, 21 mg, 22 mg, 23
mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg; 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg, 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 mg, 78 mg, 79 mg, 80 mg, 81 mg, 82 mg, 83 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89 mg, 90 mg, 91 mg, 92 mg, 93 mg, 94 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99 mg, 100 mg, 10 1 mg, 1 02 mg; 103 mg, 104 mg, 10 5 mg, and tc.,
formulation. In one embodiment, the syringe or autoinjector contains 60-100 mg, 70-90 mg, or about 80 mg of the antibody.
[00189] In one embodiment, the formulations of the invention can be self-administered using, for example, a pre-filled syringe or an automatic injection device. Automatic injection devices offer an alternative to manually operated syringes for delivering therapeutic agents to patients' bodies and allowing patients to self-administer injections. Automatic injection devices are described, for example, in the following publications, each of which is incorporated herein by reference WO 2008/005315, WO 2010/127146, WO 2006/000785, WO 2011/075524, WO 2005/113039, WO 2011/075524.
[00190] Thus, in one embodiment, the present invention provides the pre-filled syringes or autoinjector devices that contain the formulations of the present invention, as well as the use of pre-filled syringes or autoinjector devices that comprise the formulations described here in the methods of the invention.
[00191] In one embodiment, the formulation of the present invention is used to treat disorders in which TNF alpha activity is harmful. As used herein, the term a disorder in which TNF-alpha activity is harmful is intended to include diseases and other disorders in which the presence of TNF-alpha in a subject suffering from the disorder has been shown to be or is suspected of being responsible for the pathophysiology of the disorder or a factor that contributes to the worsening of the disorder. Thus, a disorder in which TNF-alpha activity is harmful is a disorder in which inhibition of TNF-alpha activity is expected to alleviate the symptoms and / or the progression of the disorder. These disorders can be evidenced, for example, by an increase in the concentration of TNF-alpha in a biological fluid of a subject suffering from the disorder (for example, an increase in the concentration
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52/114 of TNF-alpha in the subject's serum, plasma, synovial fluid, etc.) that can be detected, for example, using an anti-TNF-alpha antibody, as described above.
[00192] There are numerous examples of disorders in which TNFalpha activity is harmful. Examples in which TNF-alpha activity is harmful are also described in US Patent Nos. 6,015,557; 6,177,077, 6,379,666, 6,419,934, 6,419,944, 6,423,321, 6,428,787, and 6,537,549, and PCT Publication No. s. WO 00/50079 and WO 01/49321, the total content of all of which is incorporated herein by reference. The formulations of the invention can also be used to treat disorders in which TNF alpha activity is harmful, as described in US Patent Nos. 6,090,382, 6,258,562 and US Patent Application Publication No. US20040126372, all contents being incorporated by reference.
[00193] The use of the formulations of the invention in the treatment of specific exemplary disorders is discussed below:
A. Sepsis [00194] The formulations and methods of the invention can be used to treat subjects with Sepsis. The tumor necrosis factor has an established role in the pathophysiology of sepsis, with biological effects that include hypotension, myocardial suppression, vascular leak syndrome, organ necrosis, stimulation of the release of toxic secondary mediators and activation of the coagulation cascade (see , for example, Tracey, KJ and Cerami, A. (1994) Annu. Rev. Med. 45: 491-503; Russell, D and Thompson,
R. C. (1993) Curr. Opin. Biotech. 4: 714-721). Consequently, the formulation of the present invention can be used to treat sepsis in any of its clinical situations, including septic shock, endotoxic shock, gram negative sepsis and toxic shock syndrome.
[00195] In addition, to treat sepsis, the formulation of the invention can be co-administered with one or more additional therapeutic agents, which can additionally relieve sepsis, such as an interleukin-1 inhibitor (such as those described in PCT Publication no. WO 92/16221 and WO 92/17583), cytokine interleukin-6 (see, for example, PCT Publication No. WO 93/11793) or a platelet activating factor antagonist (see, for example , European Patent Application Publication No. EP 374 510).
[00196] Furthermore, in one embodiment, the formulation of the invention is administered to a human subject within a subset of sepsis patients with an IL-6 serum or plasma concentration above 500 pg / ml, or, in a modality, 1000 pg / ml; at the time of treatment (see PCT Publication No. WO 95/20978).
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53/114 [00197] B. Autoimmune Diseases [00198] The formulations and methods of the invention can be used to treat subjects having an autoimmune disease. The tumor necrosis factor has been implicated in playing an important role in the pathophysiology of a variety of autoimmune diseases. For example, TNF-alpha has been implicated in the activation of tissue inflammation and causes joint destruction in rheumatoid arthritis (see, for example, Tracey and Cerami, supra; Arend, WP and Dayer, JM. (1995) Arth. Rheum 38: 151-160; Fava, RA, et al. (1993) Clin. Exp. Immunol. 94: 261-266). TNF-alpha has also been implicated in promoting islet cell death and in mediating insulin resistance in diabetes (see, for example, Tracey and Cerami, supra; PCT Publication No. WO 94/08609). TNF-alpha has also been implicated in mediating cytotoxicity to oligodendrocytes and inducing inflammatory plaques in multiple sclerosis (see, for example, Tracey and Cerami, supra). Also included in the group of autoimmune diseases that can be treated using the formulations and methods of the invention are idiopathic juvenile arthritis (JIA) (also referred to as juvenile rheumatoid arthritis) (see Grom et al. (1996) Arthritis Rheum. 39: 1703 ; Mangge et al. (1995) Arthritis Rheum. 8: 211).
[00199] The formulation of the invention can be used to treat autoimmune diseases, in particular those associated with inflammation, including rheumatoid arthritis, rheumatoid spondylitis (also referred to as ankylosing spondylitis), osteoarthritis and gouty arthritis, allergy, multiple sclerosis, autoimmune diabetes , autoimmune uveitis, idiopathic juvenile arthritis (also referred to as juvenile rheumatoid arthritis), and nephrotic syndrome.
[00200] C. Infectious Diseases [00201] The formulations and methods of the invention can be used to treat subjects having an infectious disease. The tumor necrosis factor has been implicated in mediating the biological effects seen in a variety of infectious diseases. For example, TNF-alpha has been implicated in mediating brain inflammation and capillary thrombosis and myocardial infarction (see, for example, Tracey and Cerami, supra). TNF-alpha has also been implicated in mediating brain inflammation, inducing the rupture of the blood-brain barrier, triggering septic shock syndrome and activation of venous infarction in meningitis (see, for example, Tracey and Cerami, supra). TNF-alpha has also been implicated in the induction of cachexia, stimulation of viral proliferation and mediation of central nervous system damage in acquired immunodeficiency syndrome (AIDS) (see, for example, Tracey and Cerami, supra). In this way, the antibodies and antibody portions of the invention can be used in the treatment of infectious diseases, including bacterial meningitis (see, for example, European Patent Application Publication No. EP 585 705),
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54/114 cerebral malaria, AIDS and AIDS-related complex (ARC) (see, for example, European Patent Application Publication No. EP 230 574), as well as cytomegalovirus infection secondary to transplantation (see, for example, Fietze , E., et al. (1994) Transplantation 58: 675-680). The formulation of the present invention can also be used to relieve symptoms associated with infectious diseases, including fever and myalgia due to infection (such as influenza) and cachexia secondary to infection (e.g., secondary to AIDS or ARC).
[00202] D. Transplant [00203] The formulations and methods of the invention can be used to treat subjects with a transplant. The tumor necrosis factor has been implicated as an important mediator of allograft rejection and graft versus host disease (GVHD) and in mediating an adverse reaction, which was observed when the mouse antibody OKT3, directed against the cell receptor complex T CD3 is used to inhibit kidney transplant rejection (see, for example, Tracey and Cerami, supra; Eason, JD, et al. (1995) Transplantation 59: 300-305; Suthanthiran, M. and Strom, TB (1994 ) New Engl. J. Med. 331: 365-375). Consequently, the formulations of the invention can be used to inhibit transplant rejection, including allograft and xenograft rejection and to inhibit GVHD. Although the antibody, or portion of antibody, can be used alone, it can be used in combination with one or more other agents that inhibit the immune response against the allograft, or inhibit GVHD. For example, in one embodiment, formulations of the invention are used in combination with OKT3 to inhibit reactions induced by OKT3. In another embodiment, the formulation of the invention is used in combination with one or more antibodies directed at other targets involved in the regulation of immune responses, such as the CD25 cell surface molecules (interleukin-2 alpha receptor), CD11a (LFA- 1), CD54 (ICAM-1), CD4, CD45, CD28 / CTLA4, CD80 (B7-1) and / or CD86 (B7-2). In yet another embodiment, the formulation of the invention is used in combination with one or more general immunosuppressive agents, such as cyclosporin A or FK506.
[00204] E. Malignancy [00205] The formulations and methods of the invention can be used to treat subjects with cancer or a malignant tumor. The tumor necrosis factor has been implicated in the induction of cachexia, stimulation of tumor growth, increased metastatic potential and mediation of cytotoxicity, in malignancies (see, for example, Tracey and Cerami, supra). Thus, the formulations of the invention can be used in the treatment of malignancies, to inhibit tumor growth or metastases and / or to
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55/114 relieve cachexia secondary to malignancy. The formulation of the invention can be administered systemically or locally to the tumor site.
[00206] F. Lung Diseases [00207] The formulations and methods of the invention can be used to treat subjects with a lung disease. The tumor necrosis factor has been implicated in the pathophysiology of adult respiratory distress syndrome, including stimulating leukocyte-endothelial activation, targeting cytotoxicity to pneumocytes, and inducing vascular leak syndrome (see, for example, Tracey and Cerami, supra ). Thus, the formulations of the invention can be used to treat various lung disorders, including adult respiratory distress syndrome (see, for example, PCT Publication No. WO 91/04054), shock lung, chronic inflammatory lung disease, pulmonary sarcoidosis, fibrosis and pulmonary silicosis. The formulation of the invention can be administered systemically or locally to the surface of the lung, for example, as an aerosol.
[00208] G. Intestinal Disorders [00209] The formulations and methods of the invention can be used to treat subjects having an intestinal disease. The tumor necrosis factor has been implicated in the pathophysiology of inflammatory bowel diseases (see, for example, Tracy, KJ, et al. (1986) Science 234: 470-474; Sun, XM., Et al. (1988) J Clin Invest 81: 1328-1331; MacDonald, TT, et al. (1990) Clin Exp Immunol 81: 301-305). Murine chimeric anti-hTNF-alpha antibodies have undergone clinical tests for the treatment of Crohn's disease (Dullemen, H. M., et al. (1995) Gastroenterology 109: 129-135). The formulation of the present invention can also be used to treat intestinal disorders, such as idiopathic inflammatory bowel disease, which includes two syndromes, Crohn's disease and ulcerative colitis. In one embodiment, the formulation of the invention is used to treat Crohn's disease. In one embodiment, the formulation of the invention is used to treat ulcerative colitis.
[00210] H. Cardiac Disorders [00211] The formulations and methods of the invention can also be used to treat various cardiac disorders, including ischemia of the heart (see, for example, European Patent Application Publication No. EP 453 898) and heart failure (weakness of the heart muscle) (see, for example, PCT Publication No. WO 94/20139).
I. Spondyloarthropathies [00212] The formulations and methods of the invention can also be used to treat subjects who have a spondyloarthropathy, including, for example, a
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56/114 axial spondyloarthropathy. TNF α has been implicated in the pathophysiology of a wide variety of disorders, including inflammatory diseases such as spondyloartopathies (see, for example, Moeller, A., et al. (1990) Cytokine 2: 162-169; US Patent No. 5,231,024 to Moeller et al.; European Patent Publication No. 260 610 B1 to Moeller, A). In one embodiment, spondyloarthropathy is an axial spondyloarthropathy. Other examples of spondyloarthropathies that can be treated with the TNFα antibody of the invention are described below:
1. Psoriatic arthritis [00213] The formulations and methods of the invention can also be used to treat subjects who have psoriatic arthritis. The tumor necrosis factor has been implicated in the pathophysiology of psoriatic arthritis (Partsch et al. (1998) Ann Rheum Dis. 57: 691; Ritchlin et al. (1998) J Rheumatol. 25: 1544). As mentioned here, psoriatic arthritis (PsA) or psoriasis associated with the skin, refers to chronic inflammatory arthritis, which is associated with psoriasis. Psoriasis is a common chronic skin condition that causes red spots on the body. About 1 in 20 individuals with psoriasis will develop arthritis, along with the skin condition, and in about 75% of cases, psoriasis precedes arthritis. PsA comes in a variety of forms, ranging from moderate to severe arthritis in which arthritis typically affects the fingers and spine. When the spine is affected, the symptoms are similar to those of ankylosing spondylitis, as described above.
[00214] PsA is sometimes associated with mutilating arthritis. Mutilating arthritis refers to a disorder that is characterized by excessive bone erosion, which results in a gross, erosive deformity that cripples the joint. In one embodiment, the formulations and methods of the invention can be used to treat mutilating arthritis.
[00215] 2. Reiter's Syndrome / Reactive Arthritis [00216] The formulations and methods of the invention can also be used to treat subjects suffering from Reiter's syndrome and reactive arthritis. The tumor necrosis factor has been implicated in the pathophysiology of reactive arthritis, which is also referred to as Reiter's syndrome (Braun et al. (1999) Arthritis Rheum. 42 (10): 2039). Reactive arthritis (ReA) refers to arthritis that complicates an infection in other parts of the body, often after enteric or urogenital infections. ReA is often characterized by certain clinical symptoms, including inflammation of the joints (arthritis), urethritis, conjunctivitis and lesions of the skin and mucous membranes. In addition, ReA can occur after infection with a sexually transmitted disease or dysenteric infection, including Chlamydia, Campylobacter, Salmonella and Yersinia.
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57/114 [00217] 3. Undifferentiated spondyloarthropathies [00218] The formulations and methods of the invention can also be used to treat subjects who have undifferentiated spondyloarthropathy (see Zeidler et al. (1992) Rheum Dis Clin North Am. 18: 187 ). Other terms used to describe undifferentiated spondyloarthropathies include seronegative oligoarthritis and undifferentiated oligoarthritis. Undifferentiated spondyloarthropathies, as used herein, refer to a disorder in which the subject shows only some of the symptoms associated with a spondyloarthropathy. This condition is usually seen in young adults who do not have IBD, psoriasis, or classic symptoms of Reiter's syndrome or AS. In some cases, undifferentiated spondyloarthropathies may be an early indication of AS.
[00219] J. Skin and nail disorders [00220] In one embodiment, The formulations and methods of the invention are used to treat a skin and / or nail disorder. As used herein, the term skin and nail disorder in which TNFa activity is harmful is intended to include skin and / or nail disorders and other disorders in which the presence of TNF-alpha in a subject who suffering from the disorder has been shown to be or is suspected of being responsible for the pathophysiology of the disease or a factor that contributes to the worsening of the disease, for example, psoriasis. An example of a skin disorder that can be treated using the formulation of the invention is psoriasis. In one embodiment, the formulation of the invention is used to treat plaque psoriasis. The tumor necrosis factor has been implicated in the pathophysiology of psoriasis (Takematsu et al. (1989) Arch Dermatol Res. 281: 398; Victor and Gottlieb (2002) J Drugs Dermatol. 1 (3): 264).
1. Psoriasis [00221] The formulations and methods of the invention can be used to treat subjects with psoriasis, including subjects with plaque psoriasis. The tumor necrosis factor has been implicated in the pathophysiology of psoriasis (Takematsu et al. (1989) Arch Dermatol Res. 281: 398; Victor and Gottlieb (2002) J Drugs Dermatol. 1 (3): 264). Psoriasis is described as an inflammation of the skin (irritation and redness), characterized by frequent episodes of redness, itching and thick skin, dryness, silvery scales on the skin. In particular, lesions are formed which involve primary and secondary changes in the proliferation of the epidermis, inflammatory skin responses and expression of regulatory molecules such as lymphokines and inflammatory factors. Psoriatic skin is morphologically characterized by an increase in epidermal cell renewal, thick epidermis, abnormal keratinization, inflammatory cell infiltrates for
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58/114 epidermis and leukocyte and polymorphonuclear lymphocyte infiltration into the epidermis layer, resulting in an increase in the basal cell cycle. Psoriasis often involves the nails, which often exhibit corrosion, separation of the nail, thickening, and discoloration. Psoriasis is often associated with other inflammatory diseases, for example, arthritis, including rheumatoid arthritis, inflammatory bowel disease (IBD) and Crohn's disease.
[00222] Evidence of psoriasis is most commonly seen on the torso, elbows, knees, scalp, skin folds or finger nails, but can affect any or all parts of the skin. This usually takes about a month for new skin cells to move upward from the lower layers of the surface. In psoriasis, this process takes only a few days, resulting in an accumulation of dead skin cells and the formation of thick scales. Symptoms of psoriasis include: patches on the skin, which are dry or red, covered with silvery scales, raised patches of the skin, accompanied by red edges, which may crack and become painful, and which are usually located on the elbows, knees, scalp, trunk, and hands, skin lesions, including pustules, cracking of the skin and redness of the skin, joint pain or pain that can be associated with arthritis, for example, psoriatic arthritis.
[00223] Psoriasis treatment often includes a topical corticosteroid, vitamin D analogs, and topical or oral retinoids, or combinations thereof. In one embodiment, the TNF-alpha inhibitor of the invention is administered in combination with, or in the presence of, one of these common treatments.
[00224] The diagnosis of psoriasis is usually based on the appearance of the skin. In addition, a skin biopsy, or scraping and culture of spots on the skin may be necessary to rule out other skin diseases. An x-ray can be used to check for psoriatic arthritis, if joint pain is present and persistent.
[00225] In one embodiment of the invention, a TNF alpha inhibitor is used for the treatment of psoriasis, including chronic plaque psoriasis, guttate psoriasis, reverse psoriasis, pustular psoriasis, pemphigus vulgaris, erythrodermic psoriasis, psoriasis associated with inflammatory disease of the intestine (IBD) and psoriasis associated with rheumatoid arthritis (RA). The specific types of psoriasis included in the treatment methods of the invention are described in detail below:
[00226] a. Chronic Plaque Psoriasis [00227] The formulations and methods of the invention can be used to treat subjects with chronic plaque psoriasis. The tumor necrosis factor has been
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59/114 involved in the pathophysiology of psoriasis with chronic plaques (Asadullah et al. (1999) Br J Dermatol. 141: 94). Chronic plaque psoriasis (also referred to as psoriasis vulgaris) is the most common form of psoriasis. Chronic plaque psoriasis is characterized by raised reddish patches of skin, ranging from small to much larger in size. In chronic plaque psoriasis, plaques can be single or multiple, which can vary in size from a few millimeters to several centimeters. The plates are usually red with a scaly surface, and reflect light when gently scraped, creating a silvery effect. Psoriasis lesions with chronic plaques (which are often symmetrical) occur over the entire body, but preferably on extensor surfaces, including the knees, elbows, lumbosacral regions, scalp and nails. Occasionally, chronic plaque psoriasis can occur on the penis, vulva and flexors, but flaking is usually absent. The diagnosis of patients with chronic plaque psoriasis is generally based on the clinical features described above. In particular, the typical silvery distribution, color and peeling of the lesion in psoriasis with chronic plaques are characteristic of psoriasis with chronic plaques.
[00228] b. Guttate psoriasis [00229] The formulations and methods of the invention can be used to treat subjects with guttate psoriasis. Guttate psoriasis refers to a form of psoriasis with a characteristic drop of water in the form of scaly plaques. Seizures of guttate psoriasis usually follow an infection, especially a strep throat infection. The diagnosis of guttate psoriasis is usually based on the appearance of the skin, and the fact that there is often a recent history of sore throat.
[00230] c. Inverse Psoriasis [00231] The formulations and methods of the invention can be used to treat subjects with reverse psoriasis. Reverse psoriasis is a form of psoriasis in which the patient usually has smooth moist areas of the skin that are red and inflamed, which are different from the flaking related to plaque psoriasis. Reverse psoriasis is also referred to as intertiginous psoriasis or flexural psoriasis. Inverse psoriasis occurs mainly in the armpits, groin, under the breasts and in other skin folds around the genitals and buttocks, and, as a result of the presentation sites, friction and sweat can irritate these affected areas.
[00232] d. Pustular Psoriasis [00233] The formulations and methods of the invention can be used to treat subjects with pustular psoriasis. Pustular psoriasis is a form of psoriasis, which causes pus-filled blisters, which vary in size and location, but occur
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60/114 often on the hands and feet. The blisters can be located, or spread over large areas of the body. Pustular psoriasis can be both mild and painful, and can cause fevers.
[00234] e. Other Psoriasis Disorders [00235] Other examples of psoriasis disorders that can be treated with the formulations and methods of the invention include erythrodermic psoriasis, vulgaris, psoriasis associated with IBD, and psoriasis associated with arthritis, including rheumatoid arthritis.
[00236] 2. Pemphigus Vulgaris [00237] The formulations and methods of the invention can be used to treat subjects with pemphigus vulgaris. Pemphigus vulgaris is a severe autoimmune systemic dermatological disease that often affects the oral mucosa and skin. The pathogenesis of pemphigus vulgaris is believed to be an autoimmune process that is directed at the skin and desmosomes of the oral mucous membranes. Consequently, the cells do not adhere to each other. The disorder manifests itself the more it is filled with fluid, prone to rupture of bubbles and has a distinct histological appearance. Anti-inflammatory agents are the only effective treatment for this disease that has a high mortality rate. The complications that arise in patients suffering from pemphigus vulgaris are intractable pain, interference with nutrition and fluid loss, and infections.
[00238] 3. Atopic Dermatitis / Eczema [00239] The formulations and methods of the invention can be used to treat subjects with atopic dermatitis. Atopic dermatitis (also referred to as eczema) is a chronic skin disorder categorized by scaly plaques and itching. People with eczema often have a family history of allergic conditions like asthma, hay fever or eczema. Atopic dermatitis is a hypersensitivity reaction (similar to an allergy) that occurs on the skin, causing chronic inflammation. Inflammation causes the skin to itch and peel. Chronic itching and irritation can make the skin thick and leathery. Exposure to environmental irritants can worsen symptoms, as can skin dryness, exposure to water, temperature changes and stress.
[00240] Subjects with atopic dermatitis can be identified by some symptoms, which often include severe itching, blisters with swelling and exfoliation, redness or inflammation of the skin around the blisters, rash, dryness, leather-like areas, sore areas of the skin itching, and discharge / bleeding from the ear.
[00241] 4. Sarcoidosis [00242] The formulations and methods of the invention can be used to treat subjects with sarcoidosis. Sarcoidosis is a disease in which inflammation
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Granulomatous 61/114 occurs in the lymph nodes, lungs, liver, eyes, skin, and / or other tissues. Sarcoidosis includes cutaneous sarcoidosis (sarcoidosis of the skin) and nodular sarcoidosis (sarcoidosis of the lymph nodes). Sarcoidosis patients can be identified by symptoms that often include general discomfort, malaise, or a feeling of discomfort, skin lesions.
[00243] 5. Erythema Nodosum [00244] The formulations and methods of the invention can be used to treat subjects with erythema nodosum. Erythema nodosum refers to an inflammatory disorder that is characterized by red, soft nodules under the skin, usually in the lower anterior part of the legs. The lesions associated with erythema nodosum often begin as painful, red, but firm, flat nodules (about an inch in diameter). Within a few days, the lesions can turn purple, and then over several weeks it can turn into a flat brownish stain.
[00245] In some cases, erythema nodosum may be associated with infections, including, by streptococci, coccidioidomycosis, tuberculosis, hepatitis B, syphilis, cat scratch disease, tularemia, yersinia, leptospirosis, histoplasmosis, infectious mononucleosis (EBV) . In other cases, erythema nodosum may be associated with sensitivity to certain drugs, including oral contraceptives, penicillins, sulfonamides, sulfones, barbiturates, hydantoin, phenacetin, salicylates, iodides, and progestin. Erythema nodosum is often associated with other diseases, including leukemia, sarcoidosis, rheumatic fever, and ulcerative colitis.
[00246] Symptoms of erythema nodosum usually present on the legs, but injuries can also occur on other areas of the body, including the buttocks, calves, ankles, thighs, and upper extremities. Other symptoms in patients with erythema nodosum may include fever and malaise.
[00247] 6. Hidradenitis Suppurativa [00248] The formulations and methods of the invention can be used to treat subjects with hidradenitis suppurativa. Suppurative hidradenitis refers to a skin disorder in which painful, swollen, inflamed nodules or lesions develop in the groin and sometimes under the arms and under the breasts. Suppurative hidradenitis occurs when exits from the apocrine glands become blocked by sweating or are unable to flow normally because of incomplete development of the gland. Secretions trapped in the glands force perspiration and bacteria into the surrounding tissue, causing subcutaneous hardening, inflammation and infection. Suppurative hidradenitis is confined to areas of the body that contain apocrine glands. These areas are the armpit regions,
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62/114 areola of the nipple, groin, perineum, circurnanal and periumbilical.
[00249] 7. Lichen Planus [00250] The formulations and methods of the invention can be used to treat subjects with lichen planus. The tumor necrosis factor has been implicated in the pathophysiology of lichen planus (Sklavounou et al. (2000) J Oral Pathol Med. 29: 370). Lichen planus refers to a disorder of the skin and mucous membranes, resulting in inflammation, irritation and distinctive skin lesions. Lichen planus may be associated with hepatitis C or certain medications.
[00251] 8. Sweet's Syndrome [00252] The formulations and methods of the invention can be used to treat subjects with Sweet's syndrome. Inflammatory cytokines, including tumor necrosis factor, have been implicated in the pathophysiology of Sweet's syndrome (Reuss-Borst et al. (1993) Br. J. Haematol. 84: 356). Sweet's syndrome, which was described by R. D. Sweet in 1964, is characterized by the sudden onset of fever, leukocytosis; and rash. The rash consists of erythematous, well-demarcated, soft papules and plaques that show microscopically dense neutrophilic infiltration. Lesions can appear anywhere, but prefer the upper body, including the face. Individual lesions are often described as pseudovesicular or pseudopustular, but they can be frankly pustular, bullous or ulcerative. Oral and eye involvement (conjunctivitis or episcleritis) have also been reported in patients with Sweet's syndrome. Leukemia was also associated with Sweet's syndrome.
[00253] 9. Vitiligo [00254] The formulations and methods of the invention can be used to treat subjects with vitiligo. Vitiligo refers to a skin condition in which there is loss of pigment from areas of skin, resulting in irregular white patches, with normal skin texture. The characteristic lesions of vitiligo appear as flat depigmented areas. The edges of the lesions are well defined, but irregular. Areas frequently affected in individuals with vitiligo include the face, elbows and knees, hands and feet, and genitals.
[00255] 10. Scleroderma [00256] The formulations and methods of the invention can be used to treat subjects with scleroderma. The tumor necrosis factor has been implicated in the pathophysiology of scleroderma (Tutuncu Z et al. (2002) Clin Exp Rheumatol. 20 (6 Suppl 28): S146-51; Mackiewicz Z et al. (2003) Clin Exp Rheumatol. 21 ( 1): 41-8; Murota H et al. (2003) Arthritis Rheum. 48 (4): 1117-25). Scleroderma refers to a tissue disease
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63/114 diffuse connective characterized by changes in the skin, blood vessels, skeletal muscles, and internal organs. Scleroderma is also referred to as CREST syndrome or progressive systemic sclerosis and usually affects people between the ages of 30-50. Women are more affected than men.
[00257] The cause of scleroderma is unknown. The disease can produce local or systemic symptoms. The course and severity of the disease vary widely in people affected. Excess collagen is deposited on the skin and other organs produce symptoms. Damage to the small blood vessels inside the affected skin and organs also occurs. On the skin, ulcerations, calcification, and changes in pigmentation can occur. Systemic features may include fibrosis and degeneration of the heart, lungs, kidneys and gastrointestinal tract.
[00258] Patients suffering from scleroderma have certain clinical features, including whitening, bluish color or redness of the fingers and toes, in response to heat and cold (Raynaud's phenomenon), pain, stiffness and swelling of the fingers and joints, thickening of the skin and shiny hands and forearms, reflux or heartburn, difficulty in swallowing and shortness of breath. Other clinical symptoms used to diagnose scleroderma include, a high erythrocyte sedimentation rate (ESR), a high rheumatoid factor (RF), a positive antinuclear antibody test, a urine test showing microscopic protein and blood, a ray Chest X that may show fibrosis, and lung function studies that have restrictive lung disease.
[00259] 11. Nail Disorders [00260] The formulations and methods of the invention can be used to treat subjects with a nail disorder. Nail disorders include any abnormality of the nail. Specific nail disorders include, but are not limited to, by point corrosion, koilonychia, Beau lines, spoon nails, onycholysis, yellow nails, pterygium (seen in lichen planus), and leukonychia. Point corrosion is characterized by the presence of small depressions on the nail surface. Ridges or linear elevations may develop along the nail occurring in a cross-shaped length or direction. Beau lines are linear depressions that occur crosswise (crosswise) in the nail. Leukonychia describes streaks or white spots on the nails. Koilonychia is an abnormal shape of the nail, where the nail has high ridges and is thin and concave. Koilonychia is often associated with iron deficiency.
[00261] Nail disorders that can be treated with the TNFalpha antibody
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64/114 of the invention also include psoriatic nails. Psoriatic nails include nail changes that are attributable to psoriasis. In some cases, psoriasis can occur only on the nails and nowhere else on the body. Changes due to psoriasis in the nails vary from mild to severe, usually reflecting the degree of psoriatic involvement of the nail plate, nail matrix, that is, tissue from which the nail grows, the nail bed, that is, the tissue under the nail and skin at the base of the nail. Damage to the nail bed by the pustular type of psoriasis can result in nail loss. Nail changes due to psoriasis fall into general categories that can occur alone or together. In a category of psoriatic nails, the nail plate is deeply corroded, probably due to defects in nail growth caused by psoriasis. In another category the nail has a yellow to pink-yellow discoloration, probably due to psoriatic involvement of the nail bed. A third subtype of psoriatic nails is characterized by white areas that appear under the nail plate. The white areas are actually the air bubbles that mark the points where the nail plate is increasingly separated from the nail bed. There may also be reddish skin around the nail. The fourth category is evidenced by the nail plate exfoliating in yellowish spots, that is, onychodystrophy, probably due to psoriatic involvement in the nail matrix. A fifth category is characterized by the loss of the nail in its entirety, due to psoriatic involvement of the nail matrix and nail bed.
[00262] The formulations and methods of the invention can also be used to treat nail disorders often associated with lichen planus. The nails in individuals with lichen planus often show thinning and roughness of the nail plate surface, with longitudinal ridges or pterygium.
[00263] The formulations and methods of the invention can be used to treat disorders of the nails, such as those described herein. Nail disorders are often associated with skin disorders. In one embodiment, the invention includes a method of treating nail disorders with a TNF alpha antibody. In another embodiment, the nail disorder is associated with another disorder, including a skin disorder such as psoriasis. In another embodiment, the disorder associated with a nail disorder is arthritis, including psoriatic arthritis.
[00264] 12. Other Skin and Nail Conditions [00265] The formulations and methods of the invention can be used to treat other skin and nail disorders, such as chronic actinic dermatitis, bullous pemphigoid, and alopecia areata. Chronic actinic dermatitis (CAD) is also
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65/114 known as photosensitivity dermatitis / reticuloid actinic syndrome (PD / RA). CAD is a condition in which the skin becomes inflamed, particularly in areas that have been exposed to sunlight or artificial light. Generally, CAD patients are allergic to certain substances that come into contact with their skin, particularly various types of flowers, woods, perfumes, sunscreens and rubber compounds. Bullous pemphigoid refers to a skin disease characterized by the formation of large blisters on the trunk and extremities. Alopecia areata refers to hair loss characterized by round patches of complete baldness on the scalp or beard.
[00266] K. Metabolic Disorders [00267] The formulations and methods of the invention can be used to treat a metabolic disease. TNF α has been implicated in the pathophysiology of a wide variety of disorders, including metabolic disorders, such as diabetes and obesity (Spiegelman and Hotamisligil (1993) Cell 73: 625; Chu et al. (2000) Int J Obes Relat Metab Disord. 24: 1085; Ishii et al. (2000) Metabolism. 49: 1616).
[00268] Metabolic disorders affect the way the body processes the substances necessary to perform physiological functions. A number of metabolic diseases of the invention share certain characteristics, that is, they are associated with insulin resistance, lack of ability to regulate blood sugar, weight gain and increased body mass index. Examples of metabolic disorders include diabetes and obesity. Examples of diabetes include type 1 diabetes mellitus, type 2 diabetes mellitus, diabetic neuropathy, peripheral neuropathy, diabetic retinopathy, diabetic ulcerations, retinopathy ulcerations, diabetic macrovasculopathy and obesity. Examples of metabolic disorders that can be treated with the formulations and methods of the invention are described in more detail below:
1. Diabetes [00269] The formulations and methods of the invention can be used to treat diabetes. The tumor necrosis factor has been implicated in the pathophysiology of diabetes. (See, for example, Navarro JF, Mora C., Maca, Am J Kidney Dis. 2003 Jul; 42 (1): 53-61; Daimon M et al., Diabetes Care. 2003 Jul; 26 (7): 2015 -20; Zhang M et al., J Tongji Med Univ. 1999; 19 (3): 203-5, Barbieri M et al., Am J Hypertens. 2003 Jul; 16 (7): 537-43.). For example, TNF α is implicated in the pathophysiology of insulin resistance. Serum TNF levels in patients with gastrointestinal cancer have been found to correlate with insulin resistance (see, for example, McCall, J. et al. Br. J. Surg. 1992; 79: 1361-3).
[00270] Diabetes includes the two most common types of the disease, ie type I diabetes and type II diabetes, both of which result from the body's inability to
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66/114 regulate insulin. Insulin is a hormone released by the pancreas in response to increased blood sugar (glucose) levels in the blood.
[00271] The term type 1 diabetes, as used in this document, refers to a chronic disease that occurs when the pancreas produces too little insulin to properly regulate blood sugar levels. Type 1 diabetes is also referred to as insulin-dependent diabetes mellitus, IDMM, juvenile diabetes and type I diabetes. Type 1 diabetes represents the result of progressive self-destruction of β cells in the pancreas with subsequent insulin deficiency.
[00272] The term type 2 diabetes refers to a chronic disease that occurs when the pancreas does not produce enough insulin to maintain normal blood glucose levels, often because the body does not respond well to insulin. Type 2 diabetes is also referred to as non-insulin-dependent diabetes mellitus, NDDM and type II diabetes.
[00273] Diabetes is can be diagnosed by administering a glucose tolerance test. Clinically, diabetes is often divided into several basic categories. Primary examples of these categories include, autoimmune diabetes mellitus, non-insulin dependent diabetes mellitus (NDDM type 1), insulin dependent diabetes mellitus (IDDM type 2), non-autoimmune diabetes mellitus, non-insulin dependent diabetes mellitus (NIDDM type 2) and youth-onset diabetes (MODY). Another category, often referred to as secondary, refers to diabetes caused by some identifiable condition that causes or allows a diabetic syndrome to develop. Examples of secondary categories include, diabetes caused by disease in the pancreas, hormonal changes, drug-induced or chemical diabetes, diabetes caused by abnormalities of insulin receptors, diabetes associated with genetic syndromes, diabetes from other causes. (See, for example, Harrison (1996) 14th ed., New York, McGraw-Hill).
[00274] Diabetes manifests in the previous categories and can cause several complications that are discussed in the following sections. Thus, the antibody, or antigen-binding fragment thereof can be used to treat diabetes. In one embodiment, the TNFα antibody, or an antigen-binding fragment thereof, is used for the treatment of diabetes associated with the categories identified above.
[00275] Diabetes is also treated with diet, insulin dosages, and various drugs described here. Consequently, the formulations of the invention can also be administered in combination with agents commonly used in the treatment
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67/114 of metabolic disorders and pain usually associated with diabetes.
[00276] Diabetes manifests itself in many complications and conditions associated with diabetes, including the following categories:
The. Diabetic Neuropathy and Peripheral Neuropathy [00277] The formulations and methods of the invention can be used to treat diabetic neuropathy or peripheral neuropathy. The tumor necrosis factor has been implicated in the pathophysiology of diabetic neuropathy and peripheral neuropathy. (See Benjafield et al. (2001) Diabetes Care. 24: 753; Qiang, X. et al. (1998) Diabetologia.41: 1321-6; Pfeiffer et al. (1997) Horm Metab Res. 29: 111).
[00278] The term neuropathy, also referred to as diabetic nerve damage, as used in this document, refers to a common complication of diabetes, in which the nerves are damaged as a result of hyperglycemia (high blood sugar levels) . A variety of diabetic neuropathies are recognized, such as distal sensory motor polyneuropathy, focal motor neuropathy, and autonomic neuropathy.
[00279] The term peripheral neuropathy also known as peripheral neuritis and diabetic neuropathy, as used in this document, refers to the deficiency of the nerves to carry information to the brain, and the brain and spinal cord. Peripheral neuropathy produces symptoms such as pain, loss of sensation, and an inability to control muscles. In some cases, failure of nerves to control blood vessels, bowel function, and other organs results in abnormal blood pressure, digestion, and loss of other basic involuntary processes. Peripheral neuropathy can involve damage to a single nerve or group of nerves (mononeuropathy) or it can affect multiple nerves (polyneuropathy).
[00280] Neuropathies that mainly affect the small myelinated and unmyelinated fibers of the sympathetic and parasympathetic nerves are known as peripheral neuropathies. In addition, the disorder related to peripheral neuropathy, also known as peripheral neuritis and diabetic neuropathy, refers to the deficiency of nerves to carry information to the brain and the brain in the spinal cord. This produces symptoms such as pain, loss of sensation, and an inability to control muscles. In some cases, the failure of nerves that control blood vessels, bowel function, and other organs results in abnormal blood pressure, digestion, and loss of other basic involuntary processes. Peripheral neuropathy can involve damage to a single nerve or group of nerves (mononeuropathy) or it can affect multiple nerves (polyneuropathy).
[00281] The term diabetic neuropathy refers to a common complication of
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68/114 diabetes, in which the nerves are damaged as a result of hyperglycemia (high blood sugar levels). Diabetic neuropathy is also referred to as diabetic neuropathy and nerve damage. A variety of diabetic neuropathies are recognized, such as distal sensory motor polyneuropathy, focal motor neuropathy, and autonomic neuropathy.
[00282] b. Diabetic Retinopathy [00283] The formulations and methods of the invention can be used to treat diabetic retinopathy. The tumor necrosis factor has been implicated in the pathophysiology of diabetic retinopathy (Scholz et al. (2003) Trends Microbiol. 11: 171). The term diabetic retinopathy, as used herein, refers to progressive damage to the retina of the eye caused by long-term diabetes. Diabetic retinopathy includes proliferative retinopathy. Proliferative neuropathy includes, in turn, neovascularization, peretinal hemorrhage and retinal detachment.
[00284] In advanced retinopathy, small vessels proliferate on the surface of the retina. These blood vessels are fragile, tend to bleed and can cause peretinal bleeding. Hemorrhage can obscure vision, and as the bleeding is reabsorbed it forms fibrous tissue, predisposing to retinal detachments and loss of vision. In addition, diabetic retinopathy includes proliferative retinopathy that includes neovascularization, peretinal hemorrhage and retinal detachment. Diabetic retinopathy also includes background retinopathy that involves changes that occur with the layers of the retina.
[00285] c. Diabetic Ulcerations and Retinopathy Ulcerations [00286] The formulations and methods of the invention can be used to treat diabetic ulcers or retinopathy ulcerations. The tumor necrosis factor has been implicated in the pathophysiology of diabetic ulcers, (see Lee et al. (2003) Hum Immunol. 64: 614; Navarro et al. (2003) Am J Kidney Dis. 42:53; Daimon et al ( 2003) Diabetes Care. 26: 2015; Zhang et al. (1999) J Tongji Med Univ. 19: 203; Barbieri et al. (2003) Am J Hypertens. 16: 537; Venn et al. (1993) Arthritis Rheum. 36: 819; Westacott et al. (1994) J Rheumatol. 21: 1710).
[00287] The term diabetic ulcerations as used in this document, refers to an ulcer that results as a complication of diabetes. An ulcer is a crater-like lesion in the mucous membrane or skin caused by an infectious, malignant inflammatory condition and metabolic disease. Typically diabetic ulcers can be found on the limbs and extremities, most typically the feet. These ulcers caused by diabetic conditions such as neuropathy and an insufficiency
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69/114 can lead to ischemia and poor healing. The most extensive ulcerations can progress to osteomyelitis. Once osteomyelitis develops, it can be difficult to eradicate with antibiotics and amputation may be necessary.
[00288] The term ulcerations of retinopathy, as used in this document refers to an ulcer that causes or results in damage to the eyes and the retina of the eyes. Retinoic ulcerations can include conditions such as retinal bleeding.
[00289] d. Diabetic Macrovasculopathy [00290] The formulations and methods of the invention can be used to treat diabetic macrovasculopathy. The tumor necrosis factor has been implicated in the pathophysiology of diabetic macrovasculopathy (Devaraj et al. (2000) Circulation. 102: 191; Hattori Y et al. (2000) Cardiovasc Res. 46: 188; Clausell N et al. (1999) Cardiovasc Pathol.8: 145). The term diabetic macrovasculopathy, also known as macrovascular disease, as used herein, refers to a disease of the blood vessels that results from diabetes. The complication of diabetic macrovasculopathy occurs when, for example, fat and blood clots accumulate in the large blood vessels and adhere to the vessel walls. Diabetic macrovasculopathies include diseases such as coronary heart disease, cerebrovascular disease and peripheral vascular disease, hyperglycemia and cardiovascular disease and strokes.
[00291] 2. Obesity [00292] The formulations and methods of the invention can be used to treat obesity. The tumor necrosis factor has been implicated in the pathophysiology of obesity (see, for example, Pihlajamaki J et al. (2003) Obes Res.11: 912; Barbieri et al. (2003) Am J Hypertens. 16: 537; Tsuda et (2003) J Nutr. 133: 2125). Obesity increases a person's risk of illness and death due to diabetes, stroke, coronary artery disease, hypertension, high cholesterol and kidney and gallbladder diseases. Obesity can also increase the risk of some types of cancer, and can be a risk factor for the development of osteoarthritis and sleep apnea. Obesity can be treated with the antibody of the invention, alone or in combination with other metabolic diseases, such as diabetes.
[00293] l. vasculitis [00294] The formulations and methods of the invention can be used to treat a subject having a vasculitis. TNF α has been implicated in the pathophysiology of a variety of vasculitis, (see, for example, Deguchi et al. (1989) Lancet. 2: 745). As used herein, the term vasculitis in which TNFa activity is
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Harmful 70/114 is intended to include vasculitis in which the presence of TNFa in a subject suffering from the disorder has shown to be or is suspected of being responsible for the pathophysiology of the disorder or a factor that contributes to the worsening of the disease. These disorders can be evidenced, for example, by an increase in the concentration of TNFa in a biological fluid of an individual suffering from the disorder (for example, an increase in the concentration of TNFa in the subject's serum, plasma, synovial fluid, etc.) , which can be detected, for example, using an anti-TNFα antibody as described above.
[00295] There are numerous examples of vasculitis in which TNFa activity is harmful, including Behçet's disease. The use of the formulations and methods of the invention in the treatment of specific vasculitis is discussed below. In certain embodiments, the antibody, or antibody portion, is administered to the subject in combination with another therapeutic agent, as described below.
[00296] The formulations and methods of the invention are used for the treatment of vasculitis in which TNFa activity is harmful, in which inhibition of TNFa activity is believed to alleviate the symptoms and / or progression of vasculitis or prevent vasculitis . Subjects suffering from, or at risk of developing, vasculitis can be identified through symptoms and clinical tests. For example, subjects with vasculitis often develop antibodies to certain proteins in the neutrophil cytoplasm, antineutrophil cytoplasmic antibodies (ANCA). Thus, in some cases, vasculitis can be evidenced by means of assays (for example, ELISA), which measure the presence of ANCA.
[00297] Vasculitis and its consequences may be the only manifestation of the disease or may be a secondary component of another primary disease. Vasculitis can be limited to a single organ or can affect multiple organs simultaneously and depending on the syndrome, arteries and veins of all sizes can be affected. Vasculitis can affect any organ in the body.
[00298] In vasculitis, the lumen of the vessel is usually compromised, which is associated with ischemia of the tissues provided by the vessels involved. The wide variety of disorders that can result from this process is due to the fact that any type, size and location of the container (for example, artery, vein, arterioles, venule, capillary) may be involved. Vasculitides are usually classified according to the size of the affected vessels, as described below. It should be noted that some small and large vessels with vasculitis can involve medium-sized arteries, but large and medium-sized vessels with vasculitis do not involve smaller vessels than arteries. Large vessel disease includes, but is not limited to, giant cell arteritis, also
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71/114 known as temporal arteritis or cranial arteritis, polymyalgia rheumatica and Takayasu's disease or arteritis, which is also known as aortic arch syndrome, young female arteritis and pulseless disease. Middle vessel disease includes, but is not limited to, classic polyarthritis nodosa and Kawasaki disease, also known as mucocutaneous lymph node syndrome. Non-limiting examples of small vessel disease are Behçet's syndrome, Wegner's granulomatosis, microscopic polyangitis, hypersensitivity vasculitis, also known as cutaneous vasculitis, small vessel vasculitis, Henoch-Schonlein purpura, vasculitis and allergic granulamotosis, also known as Churg-Strauss syndrome. Other vasculitis include, but are not limited to, isolated vasculitis of the central nervous system, and thromboangitis obliterans, also known as Buerger's disease. Classical polyarteritis nodosa (PAN), microscopic PAN and allergic granulomatosis are also grouped and are called systemic necrotizing vasculitis. A more detailed description of vasculitis is described below:
1. Large vessel vasculitis [00299] In one embodiment, the formulations and methods of the invention are used to treat subjects who have large vessel vasculitis. The term large vessel (s), as used in this document, refers to the aorta and the larger branches directed towards the regions of the main body. The great vessels include, for example, the aorta, and its corresponding branches and veins, for example, the subclavian artery, the brachioencephalic artery, the common carotid artery, the nameless vein; internal and external jugular veins, pulmonary arteries and veins, vena cava, renal arteries and veins, femoral arteries and veins, and carotid arteries. Examples of large vessel vasculitis are described below.
The. Giant Cell Arteritis (GCA) [00300] The formulations and methods of the invention can be used to treat giant cell arteritis. The tumor necrosis factor has been implicated in the pathophysiology of giant cell arteritis (Sneller, MC (2002) Cleve. Clin. J. Med. 69: SII40-3; Schett, G., et al. (2002) Ann. Rheum Dis. 61: 463). Giant cell arteritis (GCA) refers to a vasculitis involving inflammation and damage to blood vessels, particularly the large or medium arteries that branch out from the external carotid artery in the neck. GCA is also referred to as temporal arteritis or cranial arteritis, and is the most common primary vasculitis in the elderly. It affects almost exclusively individuals over 50 years of age, however, there are well-documented cases of patients aged 40 or under. GCA usually affects extracranial arteries. THE
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GCA can affect the branches of the carotid arteries, including the temporal artery. GCA is also a systemic disease that can involve arteries in several places.
[00301] Histopathologically, GCA is a panarteritis with infiltrates of inflammatory mononuclear cells inside the vessel wall with frequent formation of giant Langhans type cells. There is a proliferation of inflammation and granulomatous, intimate fragmentation of the internal elastic lamina. Pathological findings in organs are the result of ischemia related to the vessels involved.
[00302] Patients suffering from GCA have some clinical symptoms, including fever, headache, anemia and high erythrocyte sedimentation rate (ESR). Other typical indications for GCA include lameness of the jaw or tongue, softness of the scalp, constitutional symptoms, edema of the pale optic disc (particularly edema of the 'white chalk' disc), and vision disorders. The diagnosis is confirmed by biopsy of the temporal artery.
[00303] b. Polymyalgia Rheumatica [00304] The formulations and methods of the invention can be used to treat polymyalgia rheumatica. The tumor necrosis factor has been implicated in the pathophysiology of polymyalgia rheumatica (Straub, RH, et al. (2002) Rheumatology (Oxford) 41: 423; Uddhammar, A., et al. (1998) Br. J. Rheumatol.37 : 766). Polymyalgia rheumatica refers to a rheumatic disorder, which is associated with moderate to severe pain and muscle stiffness in the neck, shoulder, and hip, most visible in the morning. IL-6 and IL-1 β expression was also detected in most circulating monocytes in patients with polymyalgia rheumatica. Polymyalgia rheumatica can occur independently, or it can coexist with, or precede, GCA, which is an inflammation of blood vessels.
[00305] c. Takayasu arteritis [00306] The formulations and methods of the invention can be used to treat Takayasu arthritis. The tumor necrosis factor has been implicated in the pathophysiology of Takayasu's arteritis (Kobayashi, Y. and Numano, F. (2002) Intern. Med. 41:44; Fraga, A. and Medina F. (2002) Curr. Rheumatol. Rep.4: 30). Takayasu's arteritis refers to a vasculitis characterized by an inflammation of the aorta and its main branches. Takayasu's arteritis (also known as aortic arch syndrome, young female arteritis and pulseless disease) affects the thoracic and abdominal aorta and its main branches or pulmonary arteries. Fibrotic thickening of the aortic wall and its branches (for example, innate and subclavian carotid arteries) can lead to a reduction in the lumen size of the vessels that arise from the aortic arch. This condition also generally affects the renal arteries.
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73/114 [00307] Takayasu's arteritis mainly affects young women, usually aged between 20-40 years of age, mainly of Asian origin, and can manifest itself through malaise, arthralgia and the gradual appearance of lameness in the extremity . Most patients have asymmetrically reduced pulses, usually together with a blood pressure differential in their arms. Coronary and / or renal artery stenosis may occur.
[00308] The clinical features of Takayasu's arteritis can be divided into the features of early inflammatory disease and the features of late disease. The clinical features of the early inflammatory stage of Takayasu's disease are: malaise, low fever, weight loss, myalgia, arthralgia and erythema multiforme. The advanced stages of Takayasu's disease are characterized by fibrotic stenosis of the arteries and thrombosis. The main clinical characteristics resulting are ischemic phenomena, for example, weak and asymmetric arterial pulse, discrepancy in blood pressure between the arms, visual disturbances, for example, scotomas and hemianopia, other neurological characteristics, including vertigo and syncope, hemiparesis or stroke . The clinical features result from ischemia due to arterial stenosis and thrombosis.
[00309] 2. Medium vessel disease [00310] The formulations and methods of the invention can be used to treat subjects who have medium vessel vasculitis. The term medium vessel (s) is used to refer to blood vessels, which are the main visceral arteries. Examples of middle vessels include the mesenteric arteries and veins, the iliac arteries and veins, and the maxillary arteries and veins. Examples of medium vessel vasculitis are described below.
The. Polyarteritis Nodosa [00311] The formulations and methods of the invention can be used to treat polyarthritis nodosa. The tumor necrosis factor has been implicated in the pathophysiology of polyarthritis nodosa (DiGirolamo, N., et al. (1997) J. Leukoc. Biol. 61: 667). Nodular polyarthritis, or periarteritis nodosa, refers to vasculitis, which is a serious blood vessel disease in which small and medium-sized arteries become swollen and damaged because they are attacked by dishonest immune cells. Polyarthritis nodosa usually affects adults more often than children. It damages the tissues supplied by the affected arteries because they do not receive enough oxygen and food without an adequate blood supply.
[00312] The symptoms that are exhibited in patients with polyarteritis nodosa usually result from damage to the affected organs, often the skin, heart, kidneys and nervous system. Generalized symptoms of polyarthritis nodosa include fever, fatigue,
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74/114 weakness, loss of appetite and weight loss. Muscle pain (myalgia) and joint pain (arthralgia) are common. The skin of subjects with polyarthritis nodosa may also have rashes, swelling, ulcers and nodules (nodular lesions).
[00313] Classic PAN (polyarthritis nodosa) is a systemic arthritis of small to medium muscular arthritis in which involvement of the renal and visceral arteries is common. Abdominal vessels have aneurysms or occlusions in 50% of patients with PAN. Classical PAN does not involve the pulmonary arteries, although bronchial vessels may be involved. Granulomas, significant eosinophilia and an allergic diathesis are not part of the syndrome. Although any organ system may be involved, the most common manifestations include peripheral neuropathy, multiplex mononeuritis, intestinal ischemia, renal ischemia, testicular pain and livedo reticularis.
[00314] b. Kawasaki disease [00315] The formulations and methods of the invention can be used to treat Kawasaki disease. The tumor necrosis factor has been implicated in the pathophysiology of Kawasaki disease (Sundel, R.P. (2002) Curr. Rheumatol. Rep. 4: 474; Gedalia, A. (2002) Curr. Rheumatol. Rep. 4:25). Although the cause of Kawasaki's disease is unknown, it is associated with acute inflammation of the coronary arteries, which suggests that tissue damage associated with this disease can be mediated by pro-inflammatory agents, such as TNFa. Kawasaki's disease refers to a vasculitis that affects mucous membranes, lymph nodes, lining of blood vessels and the heart. Kawasaki's disease is also often referred to as lymph node mucocutaneous syndrome, lymph node mucocutaneous disease and childhood polyarteritis. Subjects affected with Kawasaki's disease develop vasculitis often involving the coronary arteries that can lead to myocarditis and pericarditis. Often, as the acute inflammation subsides, the coronary arteries can develop an aneurysm, thrombosis and lead to myocardial infarction.
[00316] Kawasaki disease is a febrile systemic vasculitis associated with edema on the palms and soles of the feet, with enlargement of the cervical lymph nodes, chapped lips and strawberry tongue. Although the inflammatory response is found in vessels throughout the body, the most common site of damage to the final organ is the coronary arteries. Kawasaki disease predominantly affects children under 5 years of age. The highest incidence is in Japan, but it is becoming increasingly recognized in the West and is now the leading cause of heart disease acquired in American children. The most serious complication of Kawasaki's disease is coronary arteritis and aneurysm formation, which occurs in one third of untreated patients.
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75/114 [00317] 3. Small vessel disease [00318] The formulations and methods of the invention can be used to treat small vessel disease. In one embodiment, the TNFα antibody of the invention is used to treat subjects who have small vessel vasculitis. The term small vessel (s) is used to refer to arterioles, venules and capillaries. Arterioles are arteries that contain only 1 or 2 layers of smooth muscle cells and are terminal and continuous with the capillary network. Venules transport blood from the capillary network to veins and capillaries connect arterioles and venules. Examples of small vessel vasculitis are described below.
The. Behçet's disease [00319] The formulations and methods of the invention can be used to treat Behçet's disease. The tumor necrosis factor has been implicated in the pathophysiology of Behçet's disease (Sfikakis, PP (2002) Ann. Rheum. Dis. 61: ii51-3; Dogan, D. and Farah, C. (2002) Ophthalmology. 52:23 ). Behçet's disease is a chronic disease that involves inflammation of blood vessels throughout the body. Behçet's disease can also cause different types of skin lesions, arthritis, inflammation of the intestine, and meningitis (inflammation of the membranes of the brain and spinal cord). As a result of Behçet's disease, the subject with the disorder may have inflammation in the tissues and organs throughout the body, including the gastrointestinal tract, central nervous system, vascular system, lungs and kidneys. Behçet's disease is three times more common in men than in women and is more common in the eastern Mediterranean and Japan.
[00320] b. Wegener's granulomatosis [00321] The formulations and methods of the invention can be used to treat Wegener's granulomatosis. The tumor necrosis factor has been implicated in the pathophysiology of Wegener's granulomatosis (Marquez, J., et al. (2003) Curr. Rheumatol. Rep. 5: 128; Harman, LE and Margo, CE (1998) Surv. Ophthalmol. 42: 458). Wegener's granulomatosis refers to a vasculitis that causes inflammation of the blood vessels in the upper respiratory tract (nose, sinuses, ears), lungs and kidneys. Wegener's granulomatosis is also referred to as midline granulomatosis. Wegener's granulomatosis includes granulomatous inflammation involving the respiratory tract and necrotizing vasculitis that affects small to medium-sized vessels. Subjects who have Wegener's granulomatosis often also have arthritis (inflammation of the joints). Glomerulonephritis can also be present in affected individuals, but virtually all organs can be involved.
[00322] c. Churg-Strauss syndrome
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76/114 [00323] The formulations and methods of the invention can be used to treat Churg-Strauss syndrome. The tumor necrosis factor has been implicated in the pathophysiology of Churg-Strauss syndrome (Gross, W.L (2002) Curr. Opin. Rheumatol. 14:11; Churg, W.A. (2001) Mod. Pathol. 14: 1284). Churg-Strauss syndrome refers to a vasculitis that is systemic and shows signs of early asthma and eosinophilia. Churg-Strauss syndrome is also referred to as allergic angeitis and granulomatosis and occurs in the establishment of allergic rhinitis, asthma and eosinophilia. Sinusitis and pulmonary infiltrates can also occur in Churg-Strauss syndrome, which mainly affect the lung and the heart. Peripheral neuropathy, coronary arthritis and gastrointestinal involvement are common.
[00324] M. Other Diseases [00325] The formulations and methods of the invention can be used to treat various other disorders in which TNF-alpha activity is harmful. Examples of other diseases and disorders in which TNF-alpha activity has been implicated in pathophysiology and which can therefore be treated using an antibody or antibody portion of the invention, include bone inflammatory disorders and bone resorption disorder (see, for example, example, Bertolini, DR, et al. (1986) Nature 319: 516-518; Konig, A .. et al. (1988) J. Bone Miner. Res. 3: 621-627; Lerner, UH and Ohlin, A (1993) J. Bone Miner. Res. 8: 147155; and Shanlar. G. and Stem, PH (1993) Bone 14: 871-876), hepatitis, including alcoholic hepatitis (see, for example, McClain, CJ and Cohen, DA (1989) Hepatology 9: 349-351; Felver, ME, el al. (1990) Alcohol. Clin. Exp. Res. 14: 255-259; and Hansen, J., el al. (1994) Hepatology 20: 461-474), viral hepatitis (Sheron, N., et al. (1991) J. Hepatol. 12: 241-245; and Hussain, MJ, et al. (1994) J. Clin. Pathol. 47: 1112-1115), and fulminant hepatitis; coagulation disorders (see, for example, van der Poll, T., el al. (1990) N. Engl. J. Med. 322: 16221627; and van der Poll, T., et al. (1991) Prog. Clin. Biol. Res. 367: 55-60), burns (see, for example, Giroir, BP, el al. (1994) Am. J. Physiol. 267: H 118-124; and Liu. XS, el al (1994) Burns 20: 40-44), reperfusion injury (see, for example, Scales. WE, et al. (1994) Am. J Physiol. 267: G1122-1127; Serrick, C., el al. (1994) Transplantation 58: 1158-1162; and Yao, YM, et al. (1995) Resuscitation 29: 157-168), keloid formation (see, for example, McCauley, RL, et al. (1992) J. Clin Immunol 12: 300-308), scar tissue formation, pyrexia, periodontal disease, obesity and radiation toxicity.
[00326] Examples of other diseases that can be treated with the formulations and methods of the invention are described in documents US6258562 and US20040126372, each of which is incorporated herein by reference.
[00327] In one embodiment, the formulation and methods of the invention are
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77/114 used to treat rheumatoid arthritis, psoriatic arthritis, or ankylosing spondylitis. The formulation of the invention comprising an isolated human TNF alpha antibody, or antigen binding portion thereof, (for example, adalimumab) can be administered to a human subject according to a dosage schedule and the effective dose amount for the treatment of rheumatoid arthritis, psoriatic arthritis, or ankylosing spondylitis. In one embodiment, a dose of about 40 mg of a human TNF alpha antibody, or an antigen-binding portion of it, (for example, adalimumab) (for example, 0.4 ml of a 100 mg / ml of the invention) in the formulation of the invention are administered to a human subject every two weeks for the treatment of rheumatoid arthritis, psoriatic arthritis, or ankylosing spondylitis. In one embodiment, a dose of about 80 mg of a human TNF alpha antibody, or antigen-binding portion of it, (for example, adalimumab) (for example, 0.8 ml of a 100 mg / ml of the invention) in the formulation of the invention are administered to a human subject monthly for the treatment of rheumatoid arthritis, psoriatic arthritis, or ankylosing spondylitis. In one embodiment, the formulation is administered subcutaneously, every two weeks (also referred to as fortnightly, see methods of administration described in document US20030235585, incorporated herein by reference) for the treatment of rheumatoid arthritis, ankylosing spondylitis, or psoriatic arthritis. In one embodiment, the formulation is administered subcutaneously, monthly for the treatment of rheumatoid arthritis, ankylosing spondylitis, or psoriatic arthritis.
[00328] In one embodiment, the formulation of the invention is used to treat Crohn's disease or ulcerative colitis. The formulation of the invention comprising an isolated human TNF alpha antibody, or antigen binding portion thereof, (for example, adalimumab) can be administered to a human subject according to a dosage schedule and the effective dose amount for the Crohn's disease treatment. In one embodiment, a dose of about 160 mg of a human TNF alpha antibody, or antigen-binding portion of it, (for example, adalimumab) (for example, 1.6 ml of a 100 mg / ml of the invention) in the formulation of the invention are administered to a human subject, initially about 1 day, followed by a subsequent dose of 80 mg of the antibody (for example, 0.8 ml of a 100 mg / ml formulation of the invention ), two weeks later, followed by administration of about 40 mg (for example, 0.4 ml of a 100 mg / ml formulation of the invention) every two weeks for the treatment of Crohn's disease. In one embodiment, the formulation is administered subcutaneously, according to a multiple variable dose regimen comprising an induction dose (s) and maintenance dose (s) (see, for example,
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US patent no. US20060009385 and US20090317399, incorporated herein by reference) for the treatment of Crohn's disease or ulcerative colitis, each of which is incorporated herein by reference) for the treatment of Crohn's disease or ulcerative colitis. In one embodiment, the formulation is administered subcutaneously, biweekly or monthly to treat Crohn's disease or ulcerative colitis. In one embodiment, a dose of about 80 mg of a human TNF alpha antibody, or antigen-binding portion of it, (for example, adalimumab) (for example, 0.8 ml of a 100 mg / ml of the invention) in the formulation of the invention are administered to a human subject monthly for the treatment of Crohn's disease or ulcerative colitis.
[00329] In one embodiment, the formulation of the invention is used to treat psoriasis. The formulation of the invention comprising an isolated human TNF alpha antibody, or antigen binding portion thereof, (for example, adalimumab) can be administered to a human subject according to a dosage regimen and the effective dose amount for the psoriasis treatment. In one embodiment, an initial dose of about 80 mg of a human TNF alpha antibody, or an antigen-binding portion of it, (for example, adalimumab) (for example, 0.8 ml of a 100 mg formulation / ml of the invention) in the formulation of the invention are administered to a human subject, followed by a subsequent 40 mg dose of the antibody (eg 0.4 ml of a 100 mg / ml formulation of the invention) every two weeks starting one week after the initial dose. In one embodiment, the formulation is administered subcutaneously, according to a multiple variable dose regimen comprising an induction dose (s) and a maintenance dose (s) (see, for example, US 20060009385 and WO 2007/120823, each of which is incorporated here as a reference) for the treatment of psoriasis. In one embodiment, the formulation is administered subcutaneously, fortnightly or monthly for the treatment of psoriasis. In one embodiment, a dose of about 80 mg of a human TNF alpha antibody, or antigen-binding portion of it, (for example, adalimumab) (for example, 0.8 ml of a 100 mg / ml of the invention) in the formulation of the invention are administered to a human subject monthly for the treatment of psoriasis.
[00330] In one embodiment, the formulation of the invention is used to treat idiopathic juvenile arthritis (JIA). The formulation of the invention comprising an isolated human TNF alpha antibody, or antigen binding portion thereof, (for example, adalimumab) can be administered to a human subject according to a dosage regimen and the effective dose amount for the JIA treatment. In one modality, 20
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79/114 mg of a human TNF alpha antibody, or antigen-binding portion thereof, in the formulation of the present invention (for example, 0.2 ml of a 100 mg / ml formulation of the invention) are administered to a subject weighing 15 kg (about 33 pounds) to less than 30 kg (66 pounds) every two weeks for the treatment of JIA. In another embodiment, 40 mg of a human TNF alpha antibody, or antigen binding portion thereof, in the formulation of the present invention (for example, 0.4 ml of a 100 mg / ml formulation of the invention) are administered to a subject weighing more than, or equal to, 30 kg (66 pounds) every two weeks for the treatment of JIA. In one embodiment, the formulation is administered subcutaneously, according to a fixed dose based on weight (see, for example, US Patent Publication No. 20090271164, incorporated herein by reference) for the treatment of JIA. In one embodiment, the formulation is administered subcutaneously, fortnightly or monthly for the treatment of JIA.
[00331] In one embodiment, an isolated human TNF alpha antibody, or antigen-binding portion thereof, (for example, adalimumab) can be administered to a human subject for the treatment of a disorder associated with impaired TNFα activity of according to a monthly dosing schedule, in which the antibody is administered once a month or once every four weeks. As described above, examples of disorders that can be treated according to a monthly dosing schedule using the formulations and methods of the invention include, but are not limited to, rheumatoid arthritis, ankylosing spondylitis, JIA, psoriasis, Crohn's disease, ulcerative colitis, suppurative hidradenitis, giant cell arteritis, Behçet's disease, sarcoidosis, diabetic retinopathy or psoriatic arthritis. Thus, the formulation of the present invention comprising an isolated human TNF alpha antibody, or antigen-binding portion thereof (for example, adalimumab) can be administered to a human subject for the treatment of a disorder associated with impaired activity of TNFa according to a monthly dosing schedule. In one embodiment, 80 mg of a human TNF alpha antibody, or antigen binding portion thereof, in the formulation of the present invention (for example, 0.8 ml of a 100 mg / ml formulation of the invention) are administered to a subject with a disorder associated with impaired TNFα activity. In one embodiment, 80 mg of a human TNF alpha antibody, or antigen binding portion thereof, in the formulation of the present invention (for example, 0.8 ml of a 100 mg / ml formulation of the invention) are administered monthly or twice a week to a subject for the treatment of a disorder associated with impaired TNFα activity.
[00332] The dose quantities described in this document can be
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80/114 presented as a single dose (for example, a single dose of 40 mg in 0.4 ml or a dose of 80 mg in 0.8 ml), or, alternatively, can be distributed as multiple doses (for example , four 40 mg doses or two 80 mg doses for the distribution of a 160 mg dose).
[00333] The formulation of the invention comprising an isolated human TNF alpha antibody, or antigen binding portion thereof, (for example, adalimumab), can also be administered to a subject in combination with an additional therapeutic agent. In one embodiment, the formulation is administered to a human subject for the treatment of rheumatoid arthritis, in combination with methotrexate or other disease-modifying antirheumatic drugs (DMARDs). In another embodiment, the formulation is administered to a human subject for the treatment of JIA in combination with methotrexate or other disease-modifying antirheumatic drugs (DMARDs). Other combination therapies are described in US Patent Nos. 6,258,562 and 7,541,031, and US Patent Publication No. US20040126372, all content of which is incorporated herein by reference.
[00334] The formulation of the invention, which comprises a human TNF alpha antibody, or an antigen binding portion thereof, can also be used to treat a subject who has not been successful with previous TNF inhibitor therapy, for example, a subject who has lost a response to, or is intolerant of, infliximab.
[00335] The invention is further illustrated in the following examples, which should not be interpreted as additional limitations.
[00336] EXAMPLES [00337] EXAMPLE 1: ANTIBODY FORMULATION OF HIGH CONCENTRATION REDUCES INJECTION PAIN [00338] There have been reports of pain associated with subcutaneous administration of a human anti-TNFa antibody, for example, adalimumab. In placebo-controlled trials, 20% of patients treated with adalimumab developed reactions at the injection site (erythema and / or irritation, bleeding, pain or swelling), compared with 14% of patients who received placebo. Most reactions at the injection site are mild and generally did not require discontinuation of the drug.
[00339] There are two main components of the pain associated with the injection of adalimumab: the pain associated with the needle stick, and the pain associated with the injection of drug into the tissue. Injection-related pain may be related to the formulation of Adalimumab and / or the volume of medication. The present study examined
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81/114 whether multiple formulations have an impact on pain by injection after subcutaneous distribution of adalimumab.
[00340] Materials and Methods [00341] Study Design [00342] The main objectives of this study were to compare pain related to the injection of three formulations of high concentration of adalimumab (100 mg / mL) in the PHYSIOLIS ™ pre-filled syringe with current commercial formulation of adalimumab (50 mg / ml) in the current pre-filled syringe, and to assess the bioavailability of three high concentration formulations of adalimumab (100 mg / ml), compared to the current commercial formulation of adalimumab (50 mg / ml). The secondary objective of this study was to assess the safety and tolerability of all four adalimumab formulations.
[00343] Two hundred (200) healthy male and female adults who met the study's eligibility criteria were recruited to participate in the study. In general, the study was conducted according to a randomized parallel group project. Pain assessment data were preferably obtained from all 200 subjects. The pharmacokinetic (PK) assessment was made only for the first 100 or more subjects.
[00344] The subjects in each treatment group were programmed to receive a subcutaneous injection of 40 mg of adalimumab through a pre-filled syringe. There were four treatment groups, one for each of the four formulations as defined in Table 1 below. After meeting the selection criteria, the subjects were randomly assigned in numbers approximately equal to one of the four treatment groups shown in Table 1.
[00345] The three high concentration formulations (F1, F3 and F4) each contained 40 mg of adalimumab in 0.4 ml of solution in the PHYSIOLIS ™ pre-filled syringe. F1, F3 and F4 were compared with the commercial formulation of real adalimumab 40 mg adalimumab in 0.8 ml of the solution in the current pre-filled syringe. The ingredients for each of the formulations are described below in Table 1. The formulations described in Table 1 also refer to the formulations described in Examples 2-7 below.
Table 1 Treatment Groups
Treatment group No. of Subjects Sc Injection on Day 1 of Studies Formulation THE 50 High conc. Formulation 1 (F1) (40 mg / 0.4 mL in the PHYSIOLIS ™ pre-filled syringe) adalimumab,Mannitol,Citric acid monohydrate,Sodium citrate,Disodium phosphate dihydrate, Polysobarto 80,Water for injection,Sodium hydroxide added as needed
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to adjust the pH. B 50 High conc. Formulation 3 (F3)(40 mg / 0.4 mL in the PHYSIOLIS ™ pre-filled syringe) adalimumab,Mannitol,Polysobarto 80,Water for injection Ç 50 High conc. Formulation 4 (F4)(40 mg / 0.4 mL in the PHYSIOLIS ™ pre-filled syringe) adalimumab,, Polysobarto 80,Water for injection D 50 Current Commercial Formulation(40 mg / 0.8 mL in the current pre-filled seriga) adalimumab,Mannitol,Citric acid monohydrate,Sodium citrate,Disodium phosphate dihydrate,Sodium dihydrogen phosphate dihydrate, Sodium chloride,Polysobarto 80,Water for injection,Sodium hydroxide added as needed to adjust the pH
[00346] The first 100 subjects (approximately) who met all entry criteria and enrolled in the study were randomized to the four treatment groups in approximately equal numbers in each group and participated in both Cohort 1 and Cohort 2. The second 100 subjects (approximately) who met all entry criteria and enrolled in the study were randomized to the four treatment groups, in approximately equal numbers in each group, and participated as Cohorts 3-5. It is the Cohort number that specifies whether a subject has pharmacokinetic (PK) and pain assessments or just pain assessments, as described in Table 2 below.
Table 2 Designation of Study Subjects
No. of Subjects Cohort Total N Assessments THE B Ç D 1 50 PK and Pain 13 12 13 12 2 44 PK and Pain 11 12 10 11 3 38 Ache 9 9 10 10 4 39 Ache 10 10 10 9 5 29 Ache 7 7 7 8
[00347] The collection of pharmacokinetic samples and pain assessment was done for all subjects in the first two cohorts of about 100 patients (Cohorts 1 and 2). Subjects in Cohorts 3-5 participated only in pain assessments, and no pharmacokinetic samples were collected for these subjects. Safety and tolerability were assessed in all subjects in all five groups. Each subject was randomly assigned to receive an injection of adalimumab on Day 1 of the Study. Each dose of the study drug should be administered subcutaneously by an appropriate local team member using a pre-filled syringe according to the appropriate injection method. The injection was administered subcutaneously in the abdomen with 2 inches to
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83/114 to the right of the navel. The questionnaires were administered by a member of the study team other than the individual who administered the injection, as quickly as possible.
[00348] The subjects in Cohorts 1 and 2 (pharmacokinetic and pain assessments) were confined to the study site and supervised for approximately 10 days (9 nights). The confinement for each subject started on Day -1 of Study (1 day before the day of dosing) and ended after the collection of blood samples of 192 hours and study procedures scheduled on Study Day 9. Serial blood samples were collected on Day 57 of the Study after dosing with subjects who return for outpatient care. Safety and tolerability were assessed throughout the study. Subjects in Cohorts 3-5 (pain assessment only) were confined to the study site and supervised for approximately 3 days (2 nights). The confinement for each subject started on Day -1 of the Study (1 day before the dosing day) and ended after the study procedures were completed on Day 2 of the Study. Safety and tolerability were assessed throughout the study.
[00349] In addition, for bioavailability and AAA tests, tolerability was preferably assessed as follows:
1) Immediately after the injection on Day 1 of the study: Pain Assessment Module was completed by the subject.
2) Approximately 10 minutes after injection on Day 1 of the study: Draize scale (bleeding, petechiae, erythema, edema and itching) was assessed by a qualified local staff member.
3) Approximately 15 minutes after injection on Day 1 of the study: Pain Assessment Module was completed by the subject.
4) Approximately 30 minutes after injection on day Study 1: Pain Assessment Module and Draize Scale Assessment were completed by the subject and a qualified local team member, respectively.
[00350] The demographic data of the subjects in the treatment groups are as follows, shown in Table 3, below.
Table 3 Demographic data of patients
Variable High conc. Formulation 1 (N = 50) High conc. Formulation 3 (N = 50) High conc. Formulation 4 (N = 50) Commercial Formulation (N = 50) Age years) 29.6 ± 8.7 29.5 ± 9.4 30.0 ± 8.9 30.3 ± 9.7 Weight (kg) 68.3 ± 13.9 69.6 ± 9.6 67.0 ± 8.4 68.5 ± 10.0 Sex 31 F (62%), 19M (38%) 25 F (50%),25M (50%) 31 F (62%),19 M (38%) 30 F (60%),20M (40%)
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Breed 37 white (74%),9 black (18%),4 others (8%) 45 white (90%),3 black (6%),2 others (4%) 36 white (72%), 10 black (20%),4 others (8%) 40 white (80%),8 black (16%),2 others (4%)
Formulations [00351] Three new high concentration Formulations (referred to herein as Formulation 1, 3, or 4, or F1, F3 and F4, respectively) have been studied in relation to the commercial Formulation of 50 mg / ml adalimumab. The compositions for each of these Formulations are listed below in Tables 4-7.
Table 4: Formulation 1 (F1)
COMPOSITION OF THE MASS SOLUTION (BULK) mL of mass solution contains
Ingredient name Concentration[bad] Active substance adalimumab (A-765865) * 100.00 Excipients Mannitol 42.00 Citric acid monohydrate 1.31 Sodium citrate 0.31 Disodium phosphate dihydrate 1.53 Sodium dihydrogen phosphate dihydrate 0.86 Polysobarto 80 1.00 Sodium hydroxide q.s. Water for injections ad 1,041.00 Nitrogen ...
Solution density: 1.041 g / mL * Used as a concentrate.
Table 5: Formulation 3 (F3)
COMPOSITION OF THE MASS SOLUTION (BULK) 1 mL bulk solution contains * Used as a concentrate. Density of solution: 1.040 g / mL
Ingredient name ConcentrationJma] Active substance adalimumab (A-765865) * 100 Excipients Mannitol 42.00 Polysobarto 80 1.00 Water for injections ad 1,041.00 Nitrogen ...
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Table6: Formulation 4 (F4)
COMPOSITION OF THE MASS SOLUTION (BULK) 1 mL bulk solution contains
Ingredient name Concentration[mg] Active substance adalimumab (A-765865) * 100.00 Excipients Polysobarto 80 1.00 Water for injections ad 1,026.00 Nitrogen ___
* Used as a concentrate.
Density of solution: 1.026 g / mL
Table 7: Commercial 50 mg / mL adalimumab Formulation
COMPOSITION OF THE MASS SOLUTION (BULK) mL bulk solution contains
Ingredient name Concentration[bad] Active substance adalimumab (A-765865) * 50.00 Excipients Mannitol 12.00 Citric acid monohydrate 1.30 Sodium citrate 0.30 Disodium phosphate dihydrate 1.53 Sodium dihydrogen phosphate dihydrate 0.86 Polysobarto 80 1.00 Sodium hydroxide q.s. Water for injections about 1,000 Sodium chloride 6.16 Nitrogen ___
[00352] Study Drug Administration [00353] The study drug (adalimumab) in the various formulations was administered in the morning at Hour 0 on Day 1 of the study. The four treatment groups are shown in Table 1 above, as Groups A, B, C and D. The subjects in each treatment group were injected subcutaneously with a single formulation of adalimumab through a pre-filled syringe.
[00354] Two types of syringes were used in this study, the pre-filled glass syringe, currently commercially available (current pre-filled syringe) was used for the current commercial reference formulation of adalimumab (40 mg adalimumab in 0.8 mL
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86/114 solution), and PHYSIOLIS ™ pre-filled syringe for the three high concentration test formulations (40 mg adalimumab in 0.4 ml solution). The pre-filled PHYSIOLIS ™ syringe has a gauge of needle 29 (current pre-filled syringe has a 27 gauge needle and ¹ / 2inch fixed length) needle protection of latex-free, and a plunger which is coated to minimize leachables.
[00355] Pain test: Pain Scale [00356] The Visual Analog Pain Scale was used to quantitatively assess the sensation of pain. The following instructions were followed for the assessment of the Visual Analogue Pain Scale (VAS):
[00357] The pain scale was administered to the subject at three different times after the injection: immediately after the injection, 15 minutes after the injection, and 30 minutes after the injection on Day 1 of the study. The pain scale was shown and read to the subject, who was asked to place a vertical straight mark along the line on the pain scale to indicate his current level of pain at the injection site (for example, see below). An indication of 0 means no pain, while the maximum score (10) indicates the worst pain imaginable. An illustrative pain scale used in the study is shown below:
[00358] Qualitative Pain Assessment
What is your current level of pain at the injection site
0 ................................................. ......................... 10
Painless................................................ ..... the worst pain imaginable [00359] Qualitative Pain Assessment [00360] After the pain scale was completed, the qualitative pain assessment was applied three times after the injection: immediately after the injection, 15 minutes after the injection. injection, 30 minutes after injection on Day 1 of the study. An exemplary qualitative assessment of the pain used in the study is shown below:
[00361] Select everything that describes your current pain level at the injection site:
Extreme pain
Acute pain
Stinging pain
Attenuated
Uncomfortable
Pressure
Hurting
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Sore
Localized burning
Others_
OR [00362] Currently I have no discomfort at my injection site [00363] Needle Pain Assessment [00364] After the qualitative pain assessment was completed, the needle pain assessment was applied immediately after the injection. An evaluation of the exemplary needle pain used in the study is shown below:
[00365] Were you able to tell the difference between needle pain since the needle entered your skin and pain from the solution that was injected
Yes
No
The. If so, was most of the pain caused by the needle entering your skin or was most of your pain caused by the solution that was injected
Most of my pain was caused by the needle entering
Most of my pain was caused by the solution that was injected [00366] Draize scale [00367] The qualified local team completed this assessment for each subject in approximately 10 minutes and 30 minutes after the injection on Day 1 of the study.
[00368] Hemorrhage / petechiae at the injection site:
• 0: None • 1: Isolated, up to 5 petechiae • 2: Isolated, but> 5 petechiae • 3: Many petechiae, some coalescence • 4: Bloodstains on the surface • 5: Generous hemorrhage
Erythema at the injection site:
• 0: No erythema • 1: Very mild erythema (almost imperceptible) • 2: Well-defined erythema • 3: Moderate to severe erythema • 4: Severe erythema (beet-like redness)
Edema at the injection site:
• 0: No edema
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88/114 • 1: Very mild edema (almost imperceptible) • 2: Slight edema (edges of the area well defined by the slight increase) • 3: Moderate edema (flush of ~ 1mm) • 4: Severe edema (elevation of> 1 mm , extending beyond the injection area)
Itching at the injection site:
• 0: No itching • 1: Occasional itching • 2: Constant itching [00369] Results [00370] To determine whether the distribution of adalimumab could be improved, new high-concentration formulations have been developed. Formulations F1, F3 and F4, as shown below, are half the volume (ie 0.4 ml versus 0.8 ml) and twice the protein concentration (100 mg / ml vs 50 mg / ml) in comparison with the current commercial formulation of adalimumab, and also have different excipient compositions. The experiments described in this document were designed to assess whether any of the new formulations are superior to the current commercial formulation of adalimumab.
[00371] The Visual Analog Pain Scale was chosen to assess pain at the site, and was used to assess the impact of the composition of the formulation on pain sensations. In addition, the tolerability of several new adalimumab 100 mg / ml formulations has been compared with the current commercial formulation (50 mg / ml adalimumab formulation). The data in this example supports the surprising finding that the new formulations, in particular Formulation 3 (F3), have significantly lessened the pain compared to the current commercial formulation. Surprisingly, F3 also significantly reduced pain compared to formulations F1 and F4.
[00372] Specifically, Figure 1 shows that the administration of formulations 1 and 3 of high concentration resulted in a significant decrease in pain assessment at all time points after injection (immediately, 15 minutes, and 30 minutes), compared to the other treatment groups (F4 and the current commercial formulation). Table 8 shown below summarizes the individual data and shows a comparison of formulations F1, F3 and F4 with 0.8 ml, 50 mg / ml of the commercial formulation.
[00373] As described in Table 8, immediately after the injection, subjects who received the current Humira formulation reported an average pain score (SD) of 3.29 (2.57) cm. The mean pain scores for Formulation 1 and Formulation 3 were statistically significantly lower than for
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89/114 current Humira formulation (p <0.001). The estimated differences with the current Humira formulation were -1.50 (95% CI: -2.31 - 0.69 cm) for Formulation F1, and -2.70 (95% CI: -3.52 - 1.89 cm) for Formulation F3. Thus, Treatments A and B (high concentration formulations 1 and 3) resulted in 45.6% and 82.7% reductions in pain at the site, respectively. Statistical tests were not performed for pain scores assessed at 15 minutes and 30 minutes after injection, because most individuals did not report pain at all times. As described in Figure 1, the minimum / maximum VAS scores immediately after injection were as follows: Formulation F1, 0.00-8.3; Formulation F3, 0.00-2.20; Formulation F4, 0.20-8.70 and current commercial formulation, 0.00-10.00.
[00374] It was evident that the pain associated with the injection of Formulation 3 was drastically reduced compared to the same for the current commercial formulation. Specifically, the average pain value, as assessed by the visual analogue pain scale (VAS), immediately after injection, decreased from an average of 3.29 in the current commercial formulation to 0.56 in Formulation 3, a notable reduction of 83 %. In fact, the pain reduction associated with Formulation 3 was so significant that it was reduced by 69% from the level of the next best formulation (in terms of pain) - Formulation 1 (1.79).
[00375] Likewise, the average pain dimension, for Formulation 1, reduced to 1.79, a 45% reduction from the 3.29 pain scale associated with the current commercial formulation.
Table 8 Visual Analog Pain Scales (VAS)
Immediately After Injection
Treatment N Mean (SD) Mean LS & Comparison with Current Formulation & Estimate P-Value # 95% CI High conc. Formulation 1 50 1.79 (2.08) 1.79 -1.50 <0.001 -2.31, -0.69 High conc. Formulation 3 50 0.56 (0.56) 0.58 -2.71 <0.001 -3.52, -1.90 High conc. Formulation 4 50 4.12 (2.50) 4.11 0.82 0.976 0.01, 1.63 Current wording 50 3.29 (2.57) 3.29
# starting from a unilateral test with the null hypothesis being that the average for the test formulation are> the average for the Current Formulation & based on ANOVA.
[00376] Qualitative pain assessment was also administered to subjects immediately after injection, 15 minutes after injection and 30 minutes after injection for all four adalimumab treatments. Immediately after the injection, a
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90/114 evaluation of no discomfort was reported more frequently by 31 subjects (31/50, 62.0%) who received Formulation 3, followed by 19 subjects (19/50, 38.0%) who received Formulation 1 , 7 subjects (7/50, 14.0%) who received the current Humira formulation, and one subject (1/50, 2.0%) who received Formulation 4. Of those subjects who reported discomfort immediately after the injection, acute pain was the sensation most frequently reported with 30 individuals (30/50, 60%) for each of Formulation 4 and the current Formulation, 16 subjects (16/50, 32.0%) for Formulation 1 and 4 subjects (4/50, 8.0%) for Formulation 3. At 15 minutes after injection, the vast majority of individuals who received each treatment reported no discomfort at the injection site.
[00377] The local Study team also used the Draize scale to assess hemorrhage / petechiae, erythema, edema and itching at the injection site of each subject. Ten minutes after the injection, most subjects in all treatment groups had no bleeding or petechiae, edema or itching at the injection site.
[00378] All four formulations were well tolerated throughout the study. A summary of preliminary adverse event (AE) data is shown in Table 9 below.
Table 9 Preliminary Adverse Events (EA)
Formulation 1highconc. (N = 50) Formulation 3highconc. (N = 50) Formulation 4highconc. (N = 50) Formulationcurrent (N = 50) Any AE 7 (14%) 7 (14%) 6 (12%) 3 (6%) Any AE at least possibly related to the drug 3 (6%) 3 (6%) 2 (4%) 1 (2%) Any serious AE 0 0 0 0 Any serious AE 0 0 0 0 Any AE that leads to discontinuationstudy continuation 0 0 0 0 Deaths 0 0 0 0
[00379] Thus, the data demonstrate that the new 100 mg / mL formulations, especially Formulations 1 and 3, are well tolerated and were effective in reducing pain at the injection site after subcutaneous injection of similar therapeutic doses in comparison with the currently marketed formulation of adalimumab. Formulation F3 had a particularly low VAS score compared to other tested formulations.
[00380] Pain reduction using the VAS scale was not related to the difference in needle size (27G needle was used to administer the current commercial formulation of adalimumab and a 29G needle was used to administer As
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91/114 formulations F1, F3 and F4). In particular, a needle stick is responsible for an immediate pain response, while the pain response measured by the VAS scale indicated persistent pain lasting several minutes, demonstrating that the injected solution alone contributes to the most of the answer. In addition, all test formulations (F1, F3 and F4) were injected using the same size needle, however, F1, F3 and F4 had very different VAS scores. This result further demonstrates that this was the formulation that contributed to the pain effect and that it can be separated from the size of the needle used to administer the formulations.
[00381] EXAMPLE 2: HIGH CONCENTRATION ANTI-TNFA ANTIBODY FORMULATIONS INCREASE HUMAN BIOAVAILABILITY [00382] The following example describes a Phase 1, double-blind, randomized, single-dose study in healthy volunteers (same study described above in Example 1). The main objectives of this study were to compare pain related to the injection of three formulations of high concentration of adalimumab (100 mg / mL) in PFS Physiolis with the formulation (Humira) of current adalimumab (50 mg / mL) in the current PFS (see Example 1), and to assess the bioavailability of three formulations of high concentration of adalimumab (100 mg / mL), compared to the current commercial formulation (Humira) of adalimumab (50 mg / mL). The secondary objective of this study was to assess the safety and tolerability of all four adalimumab formulations.
[00383] Study Project [00384] Two hundred healthy volunteers were included in this study (Table 10). Pain assessment data were obtained from all 200 subjects. The pharmacokinetics of Adalimumab were evaluated in the first 100 subjects. A description of the formulations is provided in Table 1 above.
Table 10. Treatment group
Group oftreatment Number of subjects for the Study Number of subjects for Pharmacokinetic Data SC Injection on Study Day 1 THE 50 24 Formulation No. 1 of high concentration of Adalimumab (40 mg / 0.4 mL in PFS Physiolis) B 50 24 Formulation No. 3 of high concentration of Adalimumab (40 mg / 0.4 mL in PFS Physiolis) Ç 50 23 Formulation No. 4 of high concentration of Adalimumab (40 mg / 0.4 mL in PFS Physiolis) D 50 23 Current Commercial Humira Formulation (40 mg / 0.8 mL in the current PFS) * See Tables 4-7 for Compositions of Formulation
[00385] Subjects in each treatment group received a subcutaneous injection of 40 mg of adalimumab through PFS on Day 1 of the study. Each dose of the study drug was administered subcutaneously by an appropriate local team member according to the appropriate injection method, as described in the protocol. THE
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92/114 injection was administered subcutaneously in the abdomen with 2 inches to the right of the navel. The questionnaires were administered by a member of the study team other than the individual who administered the injection, as quickly as possible.
[00386] Results [00387] Conclusions and Pharmacokinetic Results [00388] After a single subcutaneous dose of adalimumab, the central values of the pharmacokinetic parameters, Tmax, Cmax, AUC0-360 and AUC0-1344 were similar between treatments A, B (Formulations 1 and 3 of high concentration adalimumab, respectively) and D (Current Commercial Humira Formulation). After single dose administration, Tmax was lower for Treatment C (Formulation 4 of high concentration adalimumab) compared to Treatment D (Figures 2 and 3). Central Cmax values and AUC0-360 values were higher (p <0.05) for Treatment C versus Treatment D.
[00389] Bioavailability / Bioequivalence for Treatments A, B and C (High Concentration Adalimumab Formulations) in Relation to Treatment D (Commercial Humira Formulation) [00390] For Treatment Group A versus D, point estimates for the reasons for the central values of Cmax, AUC0-360 and AUC0-1344 for treatments A and B were close to the unit, and the 90% confidence intervals were within the range of 0.80 to 1.25. For treatment B against D, point estimates for the Cmax and AUC0-360 central value ratios were close to the unit and the 90% confidence intervals were within the range of 0.80 to 1.25. For AUC0-1344, the upper limit of the 90% confidence interval for treatments B versus D was above 1.25. For Treatment C versus D, the point estimates for the ratio between the central values of Cmax, AUC0-360 and AUC0-1344 were 1.429, 1.309 and 1.170, respectively, which indicates that the relative bioavailability of Treatment C (Formulation 4 ) It was bigger.
Table 11. Relative bioavailability and 90% confidence intervals for the
Bioequivalence Assessment
Relative Bioavailability
^£ Test vs. Treatments Reference ParameterPK Central Valuetest Reference EstimatePunctual 90% Confidence interval A vs. D Cmax 4.47 4.39 1,018 0.859 - 1.207 AUC0-360 1192.14 1192.23 1,000 0.860 - 1.163 AUC0-1344 2306.91 2387.28 0.966 0.814 - 1.147 B vs. D Cmax 4.52 4.39 1,029 0.868 - 1.219 AUC0-360 1222.24 1192.23 1,025 0.882 - 1.192 AUC0-1344 2547.95 2387.28 1,067 0.899 - 1.266 C vs. D Cmax 6.28 4.39 1,429 1.202 - 1.699 AUC0-360 1561.05 1192.23 1.309 1.123 - 1.527 AUC0-1344 2794.29 2387.28 1,170 0.983 - 1.394
[00391] £ Treatments A, B or C: a single dose of Formulation 1, 3 or 4 of
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93/114 high concentration adalimumab, respectively, administered as a single sc injection using a PFS Physiolis (40 mg / 0.4 mL).
[00392] Treatment D: a single dose of the current Humira Formulation administered as a single sc injection using the current available glass PFS (40 mg / 0.8 mL).
PK = Pharmacokinetics.
Pharmacokinetic Conclusions [00393] Based on the pharmacokinetic results, the relative bioavailability of Treatments A and B were similar to Treatment D, the Humira formulation currently marketed. The relative bioavailability of Treatment C was greater when compared to Treatment D. The unexpected increase in bioavailability for Treatment C suggests that the amount of effective dose administered to a subject may be reduced.
[00394] Immunogenicity Findings [00395] Twelve subjects had AAA positive samples at any time during the study, with only two subjects determined to be AAA positive according to the predefined definition. Due to the small sample size and the relatively similar numbers of positive AAA samples, no conclusions can be drawn about the immunogenicity between treatments.
[00396] Safety Conclusions [00397] The tested treatments were generally well tolerated by the subjects. None of the clinically significant vital signs, ECG or laboratory measurements were observed during the course of the study. Most adverse events were assessed by the investigator as probably unrelated or unrelated to the study of drugs and mild in severity. None of the adverse events was rated as serious.
[00398] No deaths, serious adverse events or discontinuities due to adverse events occurred during the study.
[00399] The results of other safety analyzes, including changes in individual subjects and potentially clinically significant values of vital signs, ECG and laboratory measurements, were normal in all treatment groups.
[00400] Tolerability [00401] The tolerance assessments that were conducted included the completion of a Pain Assessment Module (Visual Analog Pain Scale [VAS]), Qualitative Pain Assessment and Needle Pain Assessment and the Draize (see Example 1).
[00402] EXAMPLE 3: FORMULATION OF ANTI-TNF α ANTIBODY
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HIGH CONCENTRATION INCREASES BIOAVAILABILITY IN THE PRECLINICAL MODEL [00403] The aim of the present study was to evaluate the pharmacokinetic profiles of Formulation F4 of adalimumab in contrast to the commercial formulation of adalimumab (see Table 7 above for a description of the formulation).
[00404] The pharmacokinetic profiles of HUMIRA (Adalimumab) were studied in male and female Beagle dogs (sc / 2 / sex administration and iv / 2 male administration, Marshall Bio Resources USA, Inc., North Rose, NY 14516) after a single subcutaneous injection (sc) of the commercial Humira formulation (adalimumab) and a HUMIRA test formulation corresponding to the F4 formulation of the previous examples (adalimumab), as well as an intravenous injection (iv) of the commercial HUMIRA formulation as a control. The dose administered was 40 mg / dog (at 100 mg / ml for F4 and 50 mg / ml of the commercial formulation).
[00405] For the determination of serum exposure levels of Adalimumab, blood samples were collected at 0.083, 4, 24, 48, 96, 168, 240, 312, 384, 456, 528 and 864 hours post-administration (pa ). The parameters analyzed were clinical signs (twice a week) and mortality.
[00406] With the exception of the mucous feces of a male animal in the control group, no relevant clinical sign was observed. The incidence of clinical signs is summarized in Tables 14 and 15 below.
[00407] The pharmacokinetic results (described in Table 12 below) from this study indicated that bioavailability after sc dosing was about 80% and exposure levels appeared to be higher in females than in males after sc dosing. trend towards higher exposure levels after sc dosing the test formulation compared to sc dosing the commercial formulation in males.
Table 12: Pharmacokinetic Results
Treatment Sex Animal Number AUC0-528 h^ g * Hours / mL) AUC / Dose(gg * Hours / mL / mg / kg) Vdss (mL) T1 / 2 (Hours) Test formulation, s.c. male 1001 9020 226 708 39.3 1003 11400 286 870 187.2 Average 10200 ± 1680 256 ± 42.4 789 ± 115 113.3 ± 104.6 female 1002 15400 384 388 55.5 1004 15800 395 469 54.5 Average 15600 ± 283 390 ± 7.78 429 ± 57.3 55 ± 0.7 Commercial Formulation, s.c. male 2001 8010 200 692 21.4 2003 8230 206 695 72.7 Average 8120 ± 156 203 ± 4.24 694 ± 2.12 47.1 ± 36.3 female 2002 12700 319 385 34.0 2004 17200 431 477 119.0
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Average 15000 ± 3180 375 ± 79.2 431 ± 65.1 76.5 ± 60.1 Commercial Formulation, i.v. male 3001 9360 234 548 45.5 3003 11900 298 407 22.2 Average 10600 ± 1800 266 ± 45.3 478 ± 99.7 33.9 ± 16.5 Tabe to 13: Animal Identification
No. OfAnimal No. givesmarking. Sex 1001 1246730 Male 1003 1230230 Male 1002 1282302 Female 1004 1288688 Female 2001 1284879 Male 2003 1298951 Male 2002 1297237 Female 2004 1280491 Female 3001 1285514 Male 3003 1290143 Male
Table14: Summary of Clinical Observations in Males
Dosage Group: 2 3 1
Examined Animals: 4 4 2
Normal Number: _3_3_2
Category, Note a b a b
Excretion, faeces 14 0 0
Note: a = Number of Observed Animals b = Number of Observation Days Viewed
Table 15: Summary of Clinical Observations in Females
Dosage Group: 2 3 1
Examined Animals: 2 2 2
Normal Number: _2_2_2_
Category, Note
Note: a = Number of Observed Animals b = Number of Observation Days Seen [00408] EXAMPLE 4: STABILITY OF HIGH ANTI-TNF α ANTIBODY FORMULATIONS AGAINST FREEZING / DEFROSTING STRESS [00409] The following example compares to stability of high concentration formulations of F1, F3 and F4 of adalimumab with the commercial formulation. Stability was analyzed using freeze / thaw tests.
[00410] Experimental Configuration [00411] High concentration formulations of human anti-TNF α antibodies were prepared as described in Example 1, Table 1 above.
[00412] The composite solutions were sterilized by filtration and aliquoted with 8 x 30 ml PETG bottles at 20 ml, respectively. The solutions were practically free of particles in the visual inspection.
[00413] The samples for T0 were placed directly in a refrigerator
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96/114 at 2-8 ° C. The remaining vials were placed in the cube at -80 ° C to freeze.
[00414] The following day, the bottles were thawed in a water bath with a temperature of 25 ° C or 37 ° C, respectively.
[00415] The freeze / thaw cycles were repeated 5 times. At T0 (before all freeze-thaw cycles), T1 (after one freeze-thaw cycle), T3 (after three freeze-thaw cycles) and T5 (after five freeze-thaw cycles), samples were taken for analysis and stored in a refrigerator at 2 -8 ° C.
n = 1 per pullpoint of the four samples Sample volume: 20 mL Freeze / thaw: -80 ° C / 25 ° C 37 ° C Freeze / thaw cycles: 5 [00416] After the cycles, the samples were analyzed in the laboratory using each of the following measures: optical appearance (at each time point); absorption at 340 nm; subvisible particles (in the GGDDA); Photo-correlation spectroscopy (PCS); Size Exclusion Chromatography (SEC), and Ion Exchange Chromatography (IEC).
[00417] Subvisible Particles [00418] The measurement of subvisible particles was made using the Klotz particle measurement device. The results are shown in Table 16.
Table 16: Particle counts> = 1 um,> = 10 um, and> = 25um
Particles> = Time point Sample Temperature Charge 1 pm 10 pm 25 pm T0 HC F1 (25 ° C) E161118001CL 9 1 0 T0 HC F1 (37 ° C) E161118001CL 7 2 1 T1 HC F1 25 ° C E161118001CL 3 0 0 T1 HC F1 37 ° C E161118001CL 33 1 0 T3 HC F1 25 ° C E161118001CL 3 0 0 T3 HC F1 37 ° C E161118001CL 20 1 0 T5 HC F1 25 ° C E161118001CL 4 0 0 T5 HC F1 37 ° C E161118001CL 94 0 0 T0 HC F3 (25 ° C) E161119001CL 6 3 1 T0 HC F3 (37 ° C) E161119001CL 12 2 0 T1 HC F3 25 ° C E161119001CL 4 1 0 T1 HC F3 37 ° C E161119001CL 7 2 0 T3 HC F3 25 ° C E161119001CL 3 1 0 T3 HC F3 37 ° C E161119001CL 9 2 1 T5 HC F3 25 ° C E161119001CL 7 0 0 T5 HC F3 37 ° C E161119001CL 5 0 0 T0 HC F4 (25 ° C) E161120001CL 5 1 1 T0 HC F4 (37 ° C) E161120001CL 7 1 0 T1 HC F4 25 ° C E161120001CL 6 1 0
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T1 HC F4 37 ° C E161120001CL 5 1 0 T3 HC F4 25 ° C E161120001CL 12 1 1 T3 HC F4 37 ° C E161120001CL 60 0 0 T5 HC F4 25 ° C E161120001CL 13 0 0 T5 HC F4 37 ° C E161120001CL 22 1 0 T0 commercial (25 ° C) E161121001CL 464 2 1 T0 commercial (37 ° C) E161121001CL 198 0 0 T1 commercial 25 ° C E161121001CL 143 1 0 T1 commercial 37 ° C E161121001CL 285 0 0 T3 commercial 25 ° C E161121001CL 108 0 0 T3 commercial 37 ° C E161121001CL 224 0 0 T5 commercial 25 ° C E161121001CL 39 0 0 T5 commercial 37 ° C E161121001CL 151 0 0
[00419] Particular data of> = 1 pm showed a clear trend towards a higher particle load in the formulation of commercial Humira and high concentration F1 (HC) in T5, reflecting a characteristic behavior of adalimumab formulations containing buffer salt or sodium chloride.
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Sample Name Sum of Aggregates Monomer Sum of Fragments T0, HC F1.25 ° C 0.42 99.50 0.09 T0, HC F1.37 ° C 0.43 99.46 0.11 T0, HC F3, 25 ° C 0.39 99.54 0.07 T0, HC F3, 37 ° C 0.41 99.50 0.09 T0, HC F4, 25 ° C 0.43 99.46 0.11 T0, HC F4, 37 ° C 0.42 99.48 0.11 T0, commercial, 25 ° C 0.36 99.55 0.09 T0, commercial, 37 ° C 0.35 99.56 0.09 T1, HC F1.25 ° C 0.43 99.47 0.10 T1, HC F1.37 ° C 0.44 99.48 0.08 T1, HC F3, 25 ° C 0.38 99.53 0.09 T1, HC F3, 37 ° C 0.37 99.54 0.09 T1, HC F4, 25 ° C 0.44 99.47 0.09 T1, HC F4, 37 ° C 0.44 99.46 0.10 T1, commercial, 25 ° C 0.35 99.56 0.08 T1, commercial, 37 ° C 0.35 99.56 0.10 T3, HC F1.25 ° C 0.42 99.47 0.10 T3, HC F1.37 ° C 0.42 99.48 0.11 T3, HC F3, 25 ° C 0.40 99.48 0.12 T3, HC F3, 37 ° C 0.40 99.52 0.08 T3, HC F4, 25 ° C 0.48 99.41 0.11 T3, HC F4, 37 ° C 0.44 99.48 0.08 T3, commercial, 25 ° C 0.36 99.54 0.10 T3, commercial, 37 ° C 0.34 99.55 0.11 T5, HC F1.25 ° C 0.43 99.48 0.09 T5, HC F1.37 ° C 0.45 99.45 0.10 T5, HC F3, 25 ° C 0.41 99.48 0.11 T5, HC F3, 37 ° C 0.39 99.48 0.13 T5, HC F4, 25 ° C 0.47 99.43 0.10 T5, HC F4, 37 ° C 0.49 99.40 0.11 T5, commercial, 25 ° C 0.36 99.56 0.08 T5, commercial, 37 ° C 0.40 99.47 0.13
[00420] Subvisible particle counts for> = 10 pm and> = 25 were very low. The freeze / thaw cycle did not lead to an increase in the number of subvisible particles, indicating that the tested formulations showed favorable stability.
[00421] Size Exclusion Chromatography (SEC) [00422] SEC results are shown in Table 17. Table 17 shows the percentages of SEC aggregates, monomers, and fragments in each of the T0 solutions (before all freeze-thaw cycles), T1 (after one freeze-thaw cycle), T3 (after three freeze-thaw cycles) and T5 (after five freeze-thaw cycles). These results indicate that each of Formulations 1, 3 and 4 showed similar stability to that of the commercial Formulation.
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99/114 [00423] Table 17: Percentage of aggregates, monomers, and fragments before and after freezing and thawing cycles, as assessed by SEC [00424] Ion Exchange Chromatography (IEC) [00425] Ion exchange chromatography did not reveal sensitivity different from the tested solutions. No significant degradation can be observed.
[00426] However, with the increase in the number of freeze / thaw, samples that were thawed at 25 ° C showed a higher amount in the second acidic region after 5 cycles.
[00427] IEC results are shown in Table 18.
[00428] Table 18: IEC measurements before and after freezing cycles
Sample name Sum of Plant Peaks area1 .ac area2.ac Lysine0 Lysine1 Peakintermediate Lysine2 T0, HC F1.25 ° C 86.74 2.09 10.51 68.63 16.72 0.99 0.40 T0, HC F3, 25 ° C 87.29 1.89 10.22 66.35 16.16 0.91 3.87 T0, HC F4, 25 ° C 87.35 1.85 10.20 66.46 16.15 0.89 3.85 T0, commercial, 25 ° C 87.18 1.93 10.22 66.25 16.12 0.94 3.88 T1, HC F1.25 ° C 87.17 1.98 10.18 66.24 16.11 0.94 3.87 T1, HC F3, 25 ° C 87.30 1.81 10.24 66.39 16.13 0.90 3.88 T1, HC F4, 25 ° C 87.21 1.88 10.25 66.31 16.11 0.90 3.88 T1, commercial, 25 ° C 87.19 2.01 10.20 66.27 16.11 0.93 3.88 T3, HC F1.25 ° C 87.23 1.94 10.20 66.34 16.12 0.91 3.87 T3, HC F3, 25 ° C 87.27 1.86 10.25 66.37 16.14 0.88 3.88 T3, HC F4, 25 ° C 87.26 1.82 10.27 66.34 16.15 0.88 3.88 T3, commercial, 25 ° C 87.20 1.88 10.28 66.29 16.11 0.91 3.89 T5, HC F1.25 ° C 87.39 1.74 10.21 66.43 16.18 0.89 3.88 T5, HC F3, 25 ° C 87.27 1.79 10.32 66.42 16.15 0.84 3.86 T5, HC F4, 25 ° C 87.33 1.69 10.32 66.49 16.14 0.85 3.85 T5, commercial, 25 ° C 87.05 1.95 10.38 66.17 16.10 0.89 3.88 T0, HC F1.37 ° C 87.25 1.94 10.19 66.36 16.13 0.90 3.86 T0, HC F3, 37 ° C 87.42 1.82 10.19 66.54 16.16 0.85 3.87 T0, HC F4, 37 ° C 87.38 1.89 10.11 66.52 16.14 0.86 3.86 T0, commercial, 37 ° C 87.25 1.86 10.24 66.34 16.12 0.91 3.88 T1, HC F1.37 ° C 87.21 1.98 10.17 66.27 16.11 0.95 3.88 T1, HC F3, 37 ° C 87.32 1.91 10.12 66.40 16.13 0.90 3.88 T1, HC F4, 37 ° C 87.27 1.98 10.13 66.36 16.14 0.91 3.86 T1, commercial, 37 ° C 87.28 1.97 10.14 66.34 16.12 0.93 3.88 T3, HC F1.37 ° C 87.27 1.95 10.15 66.36 16.11 0.94 3.86 T3, HC F3, 37 ° C 87.18 2.03 10.11 66.29 16.13 0.90 3.87 T3, HC F4, 37 ° C 87.21 1.95 10.15 66.35 16.09 0.92 3.85 T3, commercial, 37 ° C 87.31 1.95 10.21 66.50 16.09 0.91 3.81 T5, HC F1.37 ° C 87.29 2.01 10.06 66.40 16.11 0.93 3.85 T5, HC F3, 37 ° C 87.25 2.07 10.06 66.37 16.10 0.92 3.87 T5, HC F4, 37 ° C 87.28 2.04 10.02 66.42 16.11 0.93 3.83 T5, commercial, 37 ° C 87.53 1.91 10.02 66.72 16.11 0.88 3.83
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100/114 [00429] EXAMPLE 5: STABILITY OF ANTIBODY FORMULATIONS
HIGH CONCENTRATION ANTI-TNF α AGAINST AGRICULTURE STRESS [00430] The following example describes a study that examined the stability of formulations F1, F3 and F4 using the agitation stress test. Each formulation was tested at a variety of pH levels.
Materials
Humira HC pH 4.2
100 mg / mL pH 4.7 pH 5.7 pH 6.2
Humira F3 pH pH 4.2
100 mg / mL pH 4.7 pH 5.7 pH 6.2
Humira F4 pH pH 4.2
100 mg / mL pH 4.7 pH 5.7 pH 6.2 [00431] Procedure [00432] The bottles, stir bars and stoppers were steam sterilized before use.
[00433] The stirring experiment was carried out with the following experimental configuration:
* Protein solutions: F1, F3, F4 from Humira HC, each at a pH of 4.2, 4.7, 5.7, 6.2 to 100mg / mL, F3 from Humira HC pH 5.2 to 100 mg / ml, from 50 mg / ml Humet of Vetter;
* 5 mL filling volume per 6R bottle * n = 3 2x stirred (with 7x2 mm magnetic bar), 1 unstirred control (without magnetic bar) * Multipoint HP with magnetic stirrer: 550rpm * Ambient temperature * Sample withdrawal: t = 0, t = 1 h, t = 4 h, t = 24 h, t = 48 h [00434] Three 6R vials were filled with 5 mL for each protein solution and closed with stoppers. Two of them were equipped with a magnetic stir bar.
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101/114 [00435] The bottles were kept at 5 ° C overnight. The next morning, the samples (one per protein solution, as they were all the same at the beginning) were measured with the turbidity meter. The measured solutions were refilled into the flasks before starting the experiment. After 1, 4, 24 and 48 h, samples were collected and turbidity was determined.
[00436] Samples without stirring were only measured at 0 and 48 h time points.
[00437] For Humira HC F3 at pH 5.2 the subvisible particles were also determined for all time points.
[00438] Results [00439] Turbidity [00440] Turbidity results for samples subjected to 0, 1,4, 24, or 48 hour stress under agitation, as well as a 48 hour control without agitation, are shown in the Table 19.
Table 19: Turbidity (NTU) of samples subjected to agitation stress
Sample 0 h 1 h 4 h 24 h 48 h Adalimumab Commercial 20.90 23.90 31.20 98.05 176.00 Humira HC F3 pH 5.2 6.13 6.69 8.92 18.05 29.50 Humira HC 4.2 F1 8.62 8.89 9.40 6.48 15.05 Humira HC 4.7 F1 14.00 15.50 20.05 10.88 81.40 Humira HC 5.7 F1 30.70 33.25 36.40 23.40 100.40 Humira HC 6.2 F1 38.00 40.95 52.60 32.65 168.00 Humira HC 4.2 F3 3.20 3.35 3.72 4.88 6.69 Humira HC 4.7 F3 4.81 5.20 6.09 9.54 18.70 Humira HC 5.7 F3 8.75 10.03 11.30 25.90 46.10 Humira HC 6.2 F3 9.24 - 13.05 22.60 37.30 Humira HC 4.2 F4 3.44 3.74 3.80 6.48 9.79 Humira HC 4.7 F4 5.13 5.67 6.60 10.88 17.00 Humira HC 5.7 F4 9.23 10.15 12.50 23.40 32.20 Humira HC 6.2 F4 10.30 11.65 15.55 32.65 56.75
[00441] The increase in pH correlated with the increase in turbidity for all tested formulations, in both unstirred and stirred samples / T0. This effect was more pronounced for Formulation 1. In addition, Formulation 1 showed the greatest increase in turbidity after 48 hours at all pH values except 4.2. Formulations 3 and 4 showed similar behaviors and turbidity values were comparable at all time points.
[00442] The formulation of Humira HC (100 mg / mL), F3, pH 5.2 showed only a slight increase in turbidity over time. In contrast, the commercial Humira solution showed both a significantly higher initial value and increased turbidity over time. Thus, Formulation 3 showed less turbidity than
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[00443] The stirred samples showed a higher turbidity compared to the unstirred controls. The turbidity of the unstirred controls remained almost constant, compared to the 0 h samples, indicating that the execution of the experiment at room temperature did not influence the results obtained.
[00444] Subvisible particles [00445] Table 20 shows the results for the numbers of subvisible particles.
[00446] Table 20: Subvisible particle counts before and after agitation stress
Subvisible particles Humira HC F3 pH 5.2 > = 1 pm > = 10 pm > = 25 pm 0 h 103 3 1 1 h 194 4 0 4 h 202 4 0 24 h 262 2 0 48 h 192 3 0 48 h unstirred 80 1 0
Particles> 1 um
Stirring induced a slight increase in subvisible particle count> 1 pm. The unstirred control was comparable to the 0 h sample
Particles> 10 um
Stirring had no significant effect on particle counts of> 10 pm. The unstirred control was comparable to the 0 h sample.
Particles> 25 µm [00447] Stirring had no significant effect on particle counts> 25 pm. The unstirred control was comparable to the 0 h sample.
[00448] In general, the results of the experiments presented in Example 5 showed that formulation 3, when subjected to stirring stress, was surprisingly stable compared to a commercial Humira solution. Formulation 3 was robust to stress under agitation according to the turbidity measure, and the agitation of Formulation 3 also had little or no effect on the formation of subvisible particles.
[00449] EXAMPLE 6: HIGH CONCENTRATION ANTI-TNF α LONG-TERM STORAGE STABILITY [00450] The following example describes a study examining the
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103/114 long-term storage stability (up to 24 months) of formulations F1, F3 and F4.
[00451] Formulations F1, F3 and F4 were tested before long-term (initial) storage, and after 3, 6, 9, 12, 18, and 24 months of storage. The following storage conditions were used: (1) 5 ° C, (2) 25 ° C / 60% relative humidity (RH), and (3) 40 ° C / 75% RH. During storage, the samples were packed in a 1 ml pre-filled syringe (colorless, type I glass, European Pharmacopoeia.); BD 260 BD HYPAK syringe with a Fluorotec DB Hypak 4023/50 stopper. The following measures were used to assess storage stability: particulate contamination: visible particles; clarity and opalescence; solution color (visual); neutralization of TNF-α in vitro; Cation Exchange Chromatography (CEX-HPLC), Size Exclusion Chromatography (SE-HPLC); contamination of particulate subvisible particles; integrity of container closure, pH, and microbiological quality.
[00452] All formulations tested were stable under storage conditions from 2-8 ° C and up.
[00453] RESULTS [00454] The results for the F1 Formulation are shown in the Table
21.
Table 21: Stability Summary Report for Formulation F1
\ Test item Component Specification Test duration Storage Conditions [° C /% of R.H.] 5 ° C 25 ° C / 60% R.H. 40 ° C / 75% R.H. Particulate contamination: visible particles Visible particles NMT 4.5 Start 0.0 0.0 0.0 3 months 0.0 0.0 0.0 6 months 0.0 0.1 0.0 9 months 0.0 - - 12 months 0.0 - - 18 months 0.0 - - 24 months 0.0 - - Clarity and opalescence Evaluation No more opaque than the reference suspension IV Start <= RS III <= RS III <= RS III 3 months <= RS III <= RS III <= RS IV 6 months <= RS III <= RS III <= RS IV 9 months <= RS IV - - 12 months <= RS III - - 18 months <= RS III - - 24 months <= RS III - - Ropesolution (visual) BY scale Reported value Start <= BY 7 <= BY 7 <= BY 7 3 months <= BY 7 <= BY 7 <= BY 7 6 months <= BY 7 <= BY 7 <= BY 6 9 months <= BY 7 - - 12 months <= BY 7 - -
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\ Test item Component Specification Test duration Storage Conditions [° C /% of R.H.] 5 ° C 25 ° C / 60%R.H. 40 ° C / 75% R.H. 18 months <= BY 7 - - 24 months <= BY 7 - - TNF neutralization in vitro (cytotoxicity test) [%] 80% to 125% neutralization capacity of the reference standard Start 99 99 99 3 months 97 110 97 6 months 87 81 68 9 months 88 - - 12 months 110 - - 18 months 97 - - 24 months 111 - - Fiducial error limit (p = 0.95) lower limit [%] NLT 64 Start 96.1 96.1 96.1 3 months 91.1 104.7 93.2 6 months 84.2 76.3 64.4 9 months 84.3 - - 12 months 105.2 - - 18 months 95.3 - - 24 months 108.9 - - Fiducial error limit (p = 0.95) upper limit [%] NMT 156 Start 101.3 101.3 101.3 3 months 101.9 115.5 100.1 6 months 90.8 85.9 71.2 9 months 90.8 - - 12 months 114.2 - - 18 months 98.8 - - 24 months 113.5 - - Cation Exchange Chromatography (CEX-HPLC) Sum of lysine variants [%] NLT 75 Start 86.8 86.8 86.8 3 months 86.2 74.7 26.0 6 months 85.9 65.0 11.9 9 months 85.2 - - 12 months 85.2 - - 18 months 84.1 - - 24 months 83.9 - - Size Exclusion Chromatography (SEHPLC) Main peak (monomer) [%] NLT 98 Start 99.6 99.6 99.6 3 months 99.5 99.0 96.4 6 months 99.4 98.5 92.9 9 months 99.4 - - 12 months 99.4 - - 18 months 99.3 - - 24 months 99.3 - - Particulate contamination Subvisible particles * Particles> = 10 pm [/ container] NMT 6000 Start 11 11 11 3 months 8 37 55 6 months 33 102 98 9 months 32 - - 12 months 58 - - 18 months 44 - - 24 months 11 - - Particles> = 25 pm [/ container] NMT 600 Start 0 0 0 3 months 0 0 1 6 months 0 2 2 9 months 0 - -
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\ Test item Component Specification Test duration Storage Conditions [° C /% of R.H.] 5 ° C 25 ° C / 60% R.H. 40 ° C / 75% R.H. 12 months 0 - - 18 months 1 - - 24 months 0 - - Container Closure Integrity Evaluation Firm Start Answer Answer Answer 6 months Answer Answer Answer 12 months Answer - - 18 months Answer - - 24 months Answer - - pH Unique values 4.7 to 5.7 Start 5.3 5.3 5.3 3 months 5.2 5.2 5.2 6 months 5.3 5.3 5.3 9 months 5.3 - - 12 months 5.3 - - 18 months 5.3 - - 24 months 5.3 - - Microbial quality Sterility of the drug product No evidence of microbial growth is verified Start Answer Answer Answer
[00455] The results described above show that, when stored for 24 months at 5 ° C, Formulation F1 did not show any visible particulate contamination, clarity and opalescence <= RS III and visual color <= BY7 (brownish yellow 7). Formulation F1 also demonstrated 111% of the TNF neutralizing capacity of the reference standard, 83.9% of lysine variants, 99.3% of monomers, particles> = 11 = 10 qm, and no particles> = 25 qm. In addition, F1 maintained a stable pH of 5.3 and showed no evidence of microbial growth. When stored for 6 months at 25 ° C / 60% RH, Formulation F1 showed 0.1 visible particles, clarity and opalescence <= RS III, visual color <= BY7, 81% of the TNF neutralizing ability of the reference, 65% lysine variants, 98.5% monomers, 102 particles> = 10 qm, 2 particles> = 25 qm, a stable pH of 5.3 and no evidence of microbial growth. When stored for 6 months at 45 ° C / 75% RH, Formulation F1 showed no visible particles, clarity and opalescence <= RS IV, visual color <= BY6, 68% of the TNF neutralizing capacity of the reference standard, 11.9% of lysine variants, 92.9% of monomers, 98 particles> = 10 qm, 2 particles> = 25 qm, and no evidence of microbial growth.
[00456] The results for Formulation F3 are shown in Table 22. Table 22: Stability Summary Report for Formulation F3
Test item Component Specification Test duration Storage Conditions [° C /% of R.H.] 5 ° C 25 ° C / 60%R.H. in 40 ° C / 75%R.H. in Contamination of Visible particles NMT 4.5 Start 0.0 0.0 0.0
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Test item Component Specification Test duration Storage Conditions [° C /% of R.H.] 5 ° C 25 ° C / 60%R.H. 40 ° C / 75%R.H. particulate: visible particles 3 months 0.0 0.0 0.2 6 months 0.2 0.1 0.0 9 months 0.0 - - 12 months 0.0 - - 18 months 0.0 - - 24 months 0.0 - - Clarity andopalescence Evaluation No more opaque than the reference suspension IV Start <= RS II <= RS II <= RS II 3 months <= RS II <= RS II <= RS II 6 months <= RS II <= RS II <= RS II 9 months <= RS II - - 12 months <= RS II - - 18 months <= RS II - - 24 months <= RS II - - Ropesolution (visual) BY scale Reported value Start <= BY 7 <= BY 7 <= BY 7 3 months <= BY 7 <= BY 7 <= BY 7 6 months <= BY 7 <= BY 7 <= BY 6 9 months <= BY 7 - - 12 months <= BY 7 - - 18 months <= BY 7 - - 24 months <= BY 7 - - TNF neutralization in vitro (cytotoxicity test) [%] 80% to 125% neutralization capacity of the reference standard Start 87 87 87 3 months 101 106 89 6 months 100 101 90 9 months 98 - - 12 months 96 - - 18 months 96 - - 24 months 98 - - Fiducial error limit (p = 0.95) lower limit [%] NLT 64 Start 85.4 85.4 85.4 3 months 92.9 88.1 80.6 6 months 98.3 97.4 86.5 9 months 97.0 - - 12 months 93.3 - - 18 months 93.9 - - 24 months 96.7 - - Fiducial error limit (p = 0.95) upper limit [%] NMT 156 Start 88.5 88.5 88.5 3 months 110.5 122.2 97.9 6 months 101.7 103.8 92.6 9 months 99.8 - - 12 months 99.2 - - 18 months 98.1 - - 24 months 99.6 - - Cation Exchange Chromatography (CEX-HPLC) Sum of lysine variants [%] NLT 75 Start 86.8 86.8 86.8 3 months 86.6 77.8 32.8 6 months 86.4 70.1 16.5 9 months 86.0 - - 12 months 86.2 - - 18 months 85.2 - - 24 months 85.1 - -
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Test item Component Specification Test duration Storage Conditions [° C /% of R.H.] 5 ° C 25 ° C / 60%R.H. 40 ° C / 75%R.H. in Chromatography Main peak NLT 98 Start 99.7 99.7 99.7 Exclusion bySize (SEHPLC) (monomer) [%] 3 months 99.6 99.2 96.9 6 months 99.5 98.8 93.8 9 months 99.5 - - 12 months 99.5 - - 18 months 99.4 - - 24 months 99.4 - - Contamination of Particles> = 10 NMT 6000 Start 10 10 10 particulate -Subvisible particles * pm [/ container] 3 months 12 45 73 6 months 22 157 275 9 months 50 - - 12 months 54 - - 18 months 45 - - 24 months 14 - - Particles> = 25 NMT 600 Start 0 0 0 pm [/ container] 3 months 0 0 1 6 months 0 2 9 9 months 0 - - 12 months 1 - - 18 months 0 - - 24 months 0 - - Integrity of Evaluation Firm Start Answer Answer Answer Container Closure 6 months Answer Answer Answer 24 months Answer - - pH Unique values 4.7 to 5.7 Start 5.2 5.2 5.2 3 months 5.3 5.3 5.3 6 months 5.2 5.2 5.3 9 months 5.3 - - 12 months 5.4 - - 18 months 5.2 - - 24 months 5.1 - - Microbial quality Sterility of the drug product No evidence of microbial growth is verified Start Answer Answer Answer
[00457] The results provided in Table 22 indicate that, when stored for 24 months at 5 ° C, Formulation F3 did not show any visible particulate contamination, clarity and opalescence <= RS II and visual color <= BY7. Formulation F3 showed 98% of the TNF neutralizing capacity of the reference standard, 85.1% of lysine variants, 99.4% of monomers, 14 particles> = 10 pm, and no particles> = 25 pm. The pH showed little change and there was no evidence of microbial growth.
[00458] When stored for 6 months at 25 ° C / 60% RH, Formulation F3 showed no visible particles, clarity and opalescence <= RS II, and visual color <= BY7. In addition, Formulation F3 showed 101% of the TNF neutralizing capacity of the
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108/114 reference standard, 97.4% lysine variants, 70.1% monomers, 157 particles> = μm, and 2 particles> = 25 μm. The pH remained stable and there was no evidence of microbial growth.
[00459] When stored for 6 months at 45 ° C / 75% RH, Formulation F3 showed no visible particles, clarity and opalescence <= RS II, and visual color <= BY6. In addition, Formulation F3 showed 90% of the TNF neutralizing capacity of the reference standard, 16.5% of lysine variants, 93.8% of monomers, 275 particles> = 10 μm, and 9 particles> = 25 μm . The pH was very stable, and there was no evidence of microbial growth.
[00460] The results for Formulation F4 are shown in Table 23. Table 23: Summary Stability Report for Formulation F4
Test item Component Specification Test duration Storage Conditions [° C /% of R.H.] 5 ° C 25 ° C / 60% R.H. 40 ° C / 75% R.H. Appearance Visual particles NMT 4.5 Start 0.0 0.0 0.0 3 months 0.0 0.0 0.0 6 months 0.0 0.0 0.0 9 months 0.0 - - 12 months 0.0 - - 18 months 0.0 - - Color Evaluation No more opaque than the reference suspension IV Start <= RS II <= RS II <= RS II 3 months <= RS II <= RS II <= RS II 6 months <= RS II <= RS II <= RS II 9 months <= RS II - - 12 months <= RS II - - 18 months <= RS II - - Color BY scale Start <= BY 7 <= BY 7 <= BY 7 3 months <= BY 6 <= BY 6 <= BY 6 6 months <= BY 7 <= BY 7 <= BY 6 9 months <= BY 7 - - 12 months <= BY 7 - - 18 months <= BY 7 - - TNF neutralization in vitro (cytotoxicity test) (cytotoxicity test) [%] 80% to 125% neutralization capacity of the reference standard Start 111 111 111 3 months 105 101 80 6 months 97 101 76 9 months 112 - - 12 months 97 - - 18 months 104 - - Fiducial error limit (p = 0.95) lower limit [%] NLT 64 Start 105.2 105.2 105.2 3 months 103.2 100.1 79.2 6 months 92.9 97.5 74.7 9 months 109.3 - - 12 months 90.2 - - 18 months 101.2 - - Fiducial error limit (p = 0.95) NMT 156 Start 116.3 116.3 116.3 3 months 106.2 102.7 80.4
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Test item Component Specification Test duration Storage Conditions [° C /% of R.H.] 5 ° C 25 ° C / 60% R.H. 40 ° C / 75% R.H. upper limit [%] 6 months 101.1 103.5 78.1 9 months 113.9 - - 12 months 104.9 - - 18 months 107.5 - - Cation Exchange Chromatography (CEX-HPLC) Sum of lysine variants [%] NLT 75 Start 85.5 85.5 85.5 3 months 85.8 76.8 31.6 6 months 85.4 68.7 15.7 9 months 85.2 - - 12 months 84.5 - - 18 months 84.4 - - Size Exclusion Chromatography (SE-HPLC) Main peak (monomer) [%] NLT 98 Start 99.7 99.7 99.7 3 months 99.6 99.1 96.5 6 months 99.6 98.8 93.1 9 months 99.5 - - 12 months 99.5 - - 18 months 99.4 - - Particulate contamination - Subvisible particles Particles> = 10 pm [/ container] NMT 6000 Start 17 17 17 3 months 51 174 207 6 months 39 144 218 9 months 82 - - 12 months 57 - - Particles> = 25 pm [/ container] NMT 600 Start 0 0 0 3 months 0 1 5 6 months 0 1 1 9 months 1 - - 12 months 2 - - Container Closure Integrity Evaluation Need to meet (no blue tint) Start Answer Answer Answer 6 months Answer Answer Answer pH Unique values 4.7 to 5.7 Start 5.1 5.1 5.1 3 months 5.2 5.2 5.1 6 months 5.2 5.1 5.2 9 months 5.2 - - 12 months 5.2 - - 18 months 5.1 - - Microbial quality Sterility of the drug product No evidence of microbial growth is verified Start Answer Answer Answer
[00461] The results provided in Table 23 indicate that, when stored for 18 months at 5 ° C, Formulation F4 did not show visible particulate contamination, clarity and opalescence <= RS II and visual color <= BY7. Formulation F4 showed 104% of the TNF neutralizing capacity of the reference standard, 84.4% of lysine variants and 99.4% of monomers. In addition, the pH remained stable and there was no evidence of microbial growth.
[00462] When stored for 6 months at 25 ° C / 60% RH, Formulation F4
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110/114 showed no visible particles, clarity and opalescence <= RS II, and visual color <= BY7. THE
Formulation F4 showed 101% of the TNF neutralizing capacity of the reference standard, 68.7% of lysine variants, 98.8% of monomers, 144 of particles> = 10 pm, and 1 particles> = 25 pm. In addition, the pH was very stable and there was no evidence of microbial growth.
[00463] When stored for 6 months at 45 ° C / 75% RH, Formulation F4 showed no visible particles, clarity and opalescence <= RS II, and visual color <= BY6. The F4 Formulation showed 76% of the TNF neutralizing capacity of the reference standard, 15.7% of lysine variants, 93.1% of monomers, 218 particles> = 10 pm, and 1 particle> = 25 pm. In addition, the pH was very stable and there was no evidence of microbial growth.
[00464] In summary, the results of long-term stability experiments, as presented in Tables 21-23, show that the high concentration formulations F1, F3 and F4 were surprisingly stable when subjected to long-term storage. The stability of these formulations was similar to that of the commercial formulation. Formulations F1 and F3 showed stability similar to the commercial formulation after long-term storage for at least 24 months. Formulation F4 showed stability similar to that of the commercial formulation after long-term storage for at least 18 months.
[00465] EXAMPLE 7: STORAGE STABILITY AT HIGH CONCENTRATION ANTIBODY ANTIBODY AMBIENT TEMPERATURE [00466] Liquid pharmaceutical products containing therapeutic antibodies often require storage at 2-8 ° C until the end of shelf life . Cooling is therefore also necessary for patients between the purchase of the medications and the use. Depending on the proposed dosage regimen, this can result in storage times, under the responsibility of the patient in the case of self-administration of drugs for up to several weeks.
[00467] Therefore, drugs that do not require storage in refrigerated conditions present both a significant increase in patient comfort for home care products and a reduction in concerns about the quality of the drug, in the case of improper storage, thereby reducing , complaint rates and investigations of temperature excursions.
[00468] The product currently containing Adalimumab (Humira), has been successfully reformulated to a higher protein concentration with the
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Formulation F3, as described above in Examples 1-6. The following stability data for Formulation F3 resulted in checks for greater stability against protein degradation. The resulting degradation kinetics measured at 25 ° C met the requirements for storage at room temperature for up to 3 months.
[00469] For general long-term stability data related to storage at 25 ° C of Formulation F3, see Example 6 above.
[00470] The following data describes the long-term storage characteristics for Formulation F3. The data show that even after 18 months and 24 months of long-term storage at 2-5 ° C, additional storage at 25 ° C / 30 ° C is acceptable.
Table 24: 24-month storage of F3 at 2-8 ° C, followed by 7 days / 14 days under accelerated conditions (25 ° C, 30 ° C)
Test criteria Specification Feature to(24M5 ° C) + 7 Days + 14 Days 25 ° C 30 ° C /65% R.H. 25 ° C 30 ° C / 65% R.H. Appearance colorless to slightly yellow solution answer answer answer answer answer Particlesvisible * single bottle <2 => practically free of particulate matter visible in the bottle 1>10 => 0.0 Fee per analyst: 3 x0; single bottle <2 => practically free of visible particulate matter Fee per analyst: 3 x0; single bottle <2 => practically free of visible particulate matter Fee per analyst: 3 x0; single bottle <2 => practically free of visible particulate matter Fee per analyst: 3 x0; single bottle <2 => practically free of visible particulate matter Size exclusion HPLC % purity of the 98% NLT monomer aggregate 0.4 0.4 0.4 0.4 0.4 monomer 99.4 99.3 99.3 99.3 99.2 fragments 0.2 0.2 0.3 0.3 0.3 Cation exchange HPLC NMT 8% first acid reaction 2.7 2.8 3.0 2.9 3.3 NMT 16% second acid reaction 10.5 10.9 11.4 11.6 12.7 NLT 75% sum of lysine variants 85.1 84.2 83.4 83.0 81.2 NMT 4% peak between Lysine 1 and Lysine 2 1.5 1.7 1.9 1.6 1.8 reported value[%] peaks after Lysine 2 0.2 0.4 0.4 0.9 1.0 PCS reported value[%] Z-medium 1,390 1,365 1,395 1.397 1,420 Pdl 0.193 0.176 0.188 0.175 0.210
Table 25: Storage of 18 months at 2-8 ° C of F3, followed by 7 Days / 14
Days in accelerated conditions (25 ° C, 30 ° C)
Test criteria Specification Feature to(18M + 7 Days + 14 Days
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5 ° C) 25 ° C 30 ° C / 65% R.H. 25 ° C 30 ° C / 65% R.H. Appearance colorless to slightly yellow solution answer answer answer answer Particlesvisible * single bottle <2 => practically free of particulate matter visible in bottle 1> 10 => inform the laboratory manager Fee per analyst: 3 x0; single bottle <2 => practically free of visible particulate matter Fee per analyst: 3 x0; single bottle <2 => practically free of visible particulate matter Fee per analyst: 3 x0; single bottle <2 => practically free of visible particulate matter Fee per analyst: 3 x0; single bottle <2 => practically free of visible particulate matter Particulate contamination, subvisible particles * particles <10 pm:<6000 particles / particles from container <25 pm: <600 particles / container <1 pm / 1ml 2250 5623 9355 10252 <10 pm / 1ml 6 7 39 45 <25 pm / 1ml 0 0 0 1 PCS reported value [%] Z-medium 2,378 2,344 2,353 2,358 Pdl 0.102 0.077 0.077 0.077
[00471] EXAMPLE 8: CONDUCTIVITY OF HIGH CONCENTRATION ANTI-TNF α ANTIBODY FORMULATIONS [00472] The conductivity of Formulations F3 and F4 of high concentration anti-TNFa antibody (see Examples 1-6, supra) was determined using a InoLab Cond Level2 WTW device standardized at 25 ° C. Table 26 shows the influence of non-ionic excipients on the conductivity of Formulations F3 and F4 of adalimumab. Table 26. Conductivity of Formulations F3 and F4
Sample Temperature[° C] Conductivity[S / cm] Adalimumab DP F3 22.4 663 | 2 22.4 651 Γ 3 23.8 660 | 4 2Ϊ4 715 Γ 5 2Ϊ7 691 Γ 6 23J 680 | 7 23.3 644 | 8 22.9 647 Γ Adalimumab DP F4 22.0 797 | 2 22.9 746 Γ
[00473] As described above in Table 26, the average conductivity for both Formulations F3 and F4 was less than 2 mS / cm.
[00474] EXAMPLE 9: DYNAMIC LIGHT DISPERSION (DLS) OF
HIGH CONCENTRATION ANTI-TNF α ANTIBODY FORMULATIONS [00475] Dynamic light scattering analysis of diluted solutions was used
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113/114 to evaluate the hydrodynamic diameter (reported as the average or Z-average size, calculated by cumulative analysis with intensity autocorrelation function measured by DLS and polydispersity index, PDI, of the particle size distribution). DLS measurements were specifically used to detect small amounts of higher molecular weight species, for example, aggregates, in a size distribution, since these species have a greater dispersion intensity (proportional to d6) and, therefore, will influencing the Z-mean and the polydispersion index (PDI) as an indicator of the Z-mean size distribution significantly.
[00476] A 150 pl sample of each of Formulations F3 and F4 (see examples 1-6, above) was measured to analyze the average particle size (Z-mean) and the polydispersity index (PDI), an indicator amplitude of the particle size distribution using DLS. The results are shown below. DLS data did not show any signs of development of higher molecular weight aggregates, since the polydispersity index, a sensitive indicator for low levels of higher molecular weight subpopulations did not increase significantly.
Formulation F3
No. Sample Z-Mean (nm) PDI 1 2.4 at. 2 2.3 0.08 3 2.3 0.14 4 2.3 0.09 Formulation F4 No. Sample Z-Mean (nm) PDI 1 1.3 at. 2 2.5 at.
[00477] As described above, the measurement of z-mean for both F3 and F4 was less than 4 nm. This smaller hydrodynamic diameter is representative of the fact that both Formulations F3 and F4 do not contain additional excipients other than a polysorbate and a polyol or polysorbate.
[00478] EXAMPLE 10: FACTORS THAT INFLUENCE THE STABILITY OF HIGH CONCENTRATION ANTI-TNF α ANTIBODY FORMULATIONS [00479] The effect of various concentrations of mannitol and polysorbate concentrations on the stability of adalimumab in water was examined.
[00480] Formulations containing 100 mg / mL of adalimumab in water were prepared. Subsequently, at various concentrations, either mannitol or polysorbate
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114/114 were added over a range of concentrations to determine the impact of each excipient on the stability of the formulation, as measured by aggregation and fragmentation. The concentrations of polysorbate and mannitol varied from 0.1 to 1.0 mg / ml and 072 mg / ml, respectively, as shown in Figures 3A and 3B. As shown in Figure 3A, varying the concentration of mannitol from about 12 to about 72 mg / ml had a minimal effect on the stability of adalimumab. Likewise, varying the concentration of polysorbate 80 between about 0.1 and about 1.0 mg / ml had no effect on the stability of adalimumab.
[00481] INCORPORATION BY REFERENCE [00482] The contents of all references cited (including, for example, literature references, patents, patent applications, websites) that may be cited throughout this application are hereby expressly incorporated by reference in the its entirety for any purpose. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of protein formulations, which are well known in the art.
[00483] EQUIVALENTS [00484] The invention can be incorporated in other specific forms without departing from the spirit or its essential characteristics. The foregoing embodiments should therefore be considered in all illustrative and non-limiting aspects of the invention described herein. The scope of the invention is therefore indicated by the appended claims instead of the previous description, and any changes that are part of the meaning and equivalence of the claims are therefore intended to be covered in this document.
Petition 870170080093, of 10/20/2017, p. 120/133

权利要求:
Claims (7)
[1]
(1) 100 mg / ml adalimumab having a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2;
(1) 100 mg / ml adalimumab having a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2;
1. Liquid aqueous formulation CHARACTERIZED by the fact that it comprises:
[2]
(2) 1.0 mg / ml of polysorbate 80; and (3) 42 mg / ml of mannitol;
wherein the formulation has a pH of 4.7 to 5.7 and does not contain a buffer or salt.
2. Liquid aqueous formulation CHARACTERIZED by the fact that it consists of:
(2) 1.0 mg / ml of polysorbate 80; and (3) 42 mg / ml of mannitol;
wherein the formulation has a pH of 4.7 to 5.7 and does not contain a buffer or salt.
[3]
3. Pre-filled syringe or auto-injector device CHARACTERIZED by the fact that it comprises the formulation as defined in claim 1 or 2.
[4]
4. Use of the formulation as defined in claim 1 or 2, CHARACTERIZED by the fact that it is for the manufacture of a drug to treat a disorder associated with compromised TNFα activity.
[5]
5. Use according to claim 4, CHARACTERIZED by the fact that the disorder is selected from the group consisting of rheumatoid arthritis, Crohn's disease, psoriatic arthritis, psoriasis, juvenile idiopathic arthritis, ankylosing spondylitis, ulcerative colitis and hydradenitis suppurative.
[6]
6. Use, according to claim 4, CHARACTERIZED by the fact that the formulation is formulated to be administered to the individual according to a periodicity selected from the group consisting of weekly, fortnightly, every three weeks and monthly.
[7]
7. Use, according to claim 6, CHARACTERIZED by the fact that the administration is self-administration.
Petition 870180049269, of 06/08/2018, p. 12/13

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WO2019246317A1|2018-06-20|2019-12-26|Progenity, Inc.|Treatment of a disease or condition in a tissue originating from the endoderm|

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法律状态:
2016-01-26| B65X| Notification of requirement for priority examination of patent application|

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2016-02-23| B65Y| Grant of priority examination of the patent application (request complies with dec. 132/06 of 20061117)|

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2016-05-10| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2017-02-21| B15W| Others matters related to applications: legal action concerning application|

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2017-03-01| B15N| Others concerning applications: notification of judicial decision|

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2017-07-25| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2018-03-13| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2019-02-12| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-03-12| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2019-04-24| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 11/11/2011, OBSERVADAS AS CONDICOES LEGAIS. (CO) 20 (VINTE) ANOS CONTADOS A PARTIR DE 11/11/2011, OBSERVADAS AS CONDICOES LEGAIS |

优先权:

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申请号 | 申请日 | 专利标题

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US41272810P| true| 2010-11-11|2010-11-11|

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US61/412,728|2010-11-11|

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US41396010P| true| 2010-11-15|2010-11-15|

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US61/413,960|2010-11-15|

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PCT/US2011/060388|WO2012065072A2|2010-11-11|2011-11-11|IMPROVED HIGH CONCENTRATION ANTI-TNFα ANTIBODY LIQUID FORMULATIONS|

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