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    • 专利摘要:
    INSULIN CONJUGATE USING AN IMMUNOGLOBULIN FRAGMENTThe present invention relates to an insulin conjugate with improved in vivo duration and stability, which is prepared by covalently binding insulin to an immunoglobulin Fc region by means of a non-peptidyl polymer, a long-acting formulation comprising the same and a method of its preparation. The insulin conjugate of the present invention maintains in vivo activity of the peptide at a relatively high level and notably increases its serum half-life, thereby substantially improving drug adherence to insulin treatment.
    公开号:BR112012025166A2
    申请号:R112012025166-0
    申请日:2011-04-04
    公开日:2020-08-25
    发明作者:Dae Hae Song；Jae Hee Shin；Young Jin Park；Dae Seong In；Sung Min Bae；Se Chang Kwon
    申请人:Hanmi Science Co., Ltd.；
    IPC主号:
    专利说明:

    INSULIN CONJUGATE USING A FRAGMENT OF
    IMMUNOGLOBULIN [Field of the Art] The present invention relates to an insulin conjugate of improved duration and stability in vivo, which is prepared by covalently binding insulin to an immunoglobulin Fc region by means of a non-pepetidyl polymer, a formulation of long action comprising the same and a method of its preparation. The invention provides a method for treating an individual with an insulin deficiency disorder, such as diabetes. The insulin conjugate of the present invention maintains in vivo activity of the peptide at a relatively high level and notably increases its serum half-life, thereby substantially improving drug adherence to insulin treatment. E Ns E [Technical Basics) Insulin, a peptide secreted by pancreatic beta cells, plays a central role in controlling blood glucose levels in the body. When insulin is not secreted properly or the secreted insulin does not work in the body, the blood glucose level is not regulated, and therefore diabetes occurs. This diabetes is called type II diabetes. Type I diabetes occurs when the pancreas does not produce enough insulin to increase the blood glucose level. Type II diabetes is usually treated with oral hypoglycemic agents synthesized by chemical methods, and in some cases, patients are treated with insulin.
    However, type I diabetes requires insulin treatment.
    The insulin treatment method currently used is injection before / after meals.
    However, this injection of insulin must be continuously administered three times a day, which causes pain or discomfort in patients.
    There have been several attempts to overcome the problem.
    One of them is a method of releasing the peptide drug orally or by nasal inhalation by improving its permeability to the membrane.
    Undesirably, the method exhibited very low release efficiency, compared to injectable formulations, and thus there were still many difficulties in maintaining the peptide drug's in vivo activity at the required level.
    However, there was a method of delaying the absorption of the drug after a subcutaneous injection of a large amount of the drug, so that it maintained the blood level with only one daily injection.
    Some of the drugs developed (Lantus, Sanofi-aventis) have been approved and are now used in patients.
    In addition, studies were conducted to prolong the action, resulting in the development of Levemir (Novo Nordisk) prepared by modifying insulin with fatty acid, in which the prolonged action occurs through self-association of insulin molecules at the injection site and through connection reversible to albumin in the blood.
    However, these methods create pain at the injection site, and daily injections also cause considerable discomfort to the patient.
    ] Many efforts have been made to improve the stability of peptide drugs and to keep drugs in the blood at high levels for an extended period of time, thereby maximizing the pharmaceutical efficacy of drugs.
    These long-acting peptide drug formulations need to increase the stability of peptide drugs and maintain titers at sufficiently high levels without causing immune responses in patients.
    For the preparation of long-acting peptide drug formulations, a polymer with high solubility, such as polyethylene glycol (PEG), has been conventionally used to chemically modify the surface of a peptide drug.
    PEG does not specifically bind to a specific site or sites on a target peptide to provide an effect to increase the molecular weight of a peptide,
    : thereby inhibiting kidney loss and preventing hydrolysis, UU without causing side effects.
    For example, WO 2006/076471 describes that a type B natriuretic peptide (BNP), which binds to NPR-A to activate cGMP production and result in a reduction in blood blood pressure and, as a result, is used as a therapeutic agent for congestive heart failure, is linked to PEG, therefore maintaining its physiological activity.
    US Pat.
    No. 6, 924, 264 describes that PEG binds to the lysine residue of an exendin-4 to increase its residence time in vivo.
    This method increases the molecular weight of PEG and thereby increases the in vivo residence time of the peptide drug.
    However, as the molecular weight is increased, the peptide drug titer is remarkably reduced, and the reactivity with the peptide is also reduced.
    As a result, production is undesirably reduced.
    WO 02/46227 describes a fusion protein prepared by coupling GLP-1, an exendin-4, or an analog thereof with human serum albumin or an immunoglobulin fragment (Fc) using a genetic recombination technology, US Pat.
    No. 6, 756, 480 describes an Fc fusion protein prepared by coupling a parathyroid hormone (PTH) and an analog thereof with the region
    Fc.
    These methods can generate problems such as low pegylation production and non-specificity, but they are still endowed with a problem since the blood half-life increase effect is not as noticeable as expected, and, in some cases, the titers are also low.
    To maximize the effect of increasing blood half-life, several species of peptide linkers have been used, but there is a
    - - - - possibility of.-causing an immune response.
    In addition, if a peptide with disulfide bonds, such as BNP, is used, there is a high probability of folding error,
    and, if a peptide with non-naturally occurring amino acid residues is used, it can be produced by genetic recombination only with great difficulty. [Discovery]
    [Technical problem]
    In this regard, resulting in the present invention, intensive and extensive research was carried out to develop a method capable of simultaneously maximizing serum half-life and in vivo insulin activity, which culminated in the finding that an immunoglobulin Fc region, a non-peptidyl polymer and insulin are selectively linked to the site by a covalent bond, therefore notably increasing the serum half-life compared to the known infamous fusion method. [Technical Solution] It is a purpose of the present invention to provide an excellent insulin conjugate that maintains insulin activity in vivo and remarkably prolongs its serum half-life, a long-acting formulation comprising the same and a method of preparing these.
    [Advantageous Effects] The insulin conjugate of the present invention maintains the in vivo activity of the peptide at a remarkably high level and dramatically increases its serum half-life, thereby improving the drug adherence of patients in need of insulin treatment. [Description of Drawings] 'FIG.-1 is the result of pharmacokinetic analysis of —— =: insulin-PEG-conjugate immunoglobulin Fc fragment; FIG. 2 is the result of comparing in vivo efficacies between the insulin derivative-PEG conjugates-immunoglobulin Fc fragment; and FIG. 3 is the result of analysis of 90% or more phenylalanine pegylation (BIF) of the beta chain of the insulin-PEG-Fc fragment of immunoglobulin using a size exclusion column.
    FIGs. 4a to 4c are the result of analysis of specific binding of the beta chain of the insulin-PEG-Fc immunoglobulin fragment.
    [Best Mode]
    In one aspect to achieve the above objectives, the present invention provides an insulin conjugate that is prepared by binding insulin to an immunoglobulin Fc region by means of a non-peptidyl polymer, in which the non-peptidyl polymer is bound to the amino- end of the beta insulin chain.
    In the present invention, insulin is a peptide that is secreted by the pancreas in response to high levels of glucose in the blood so that there is glucose uptake in the liver, muscle and adipose tissue and its transformation into glycogen and to stop the use of fat as source of energy, therefore functioning as a control of the level of glucose in the blood. This peptide includes agonists, precursors, derivatives, fragments and variants thereof, and preferably natural, short-acting or long-acting insulin.
    “Om 7 7 - + - Natural insulin is a hormone that is secreted by = = pancreas to promote glucose absorption and inhibit fat breakdown, therefore acting to control the blood glucose level. Insulin is formed from a precursor with no function of regulating the level of glucose in the blood, known as proinsulin, by processing. The amino acid sequences of insulin are as follows: Alpha chain: Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu- Tyr-Gln-Leu-Glu-Asn-Tyr -Cys-Asn (SEQ ID NO. 1) Beta chain:
    Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu- Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Pro-Lys-Thr (SEQ ID NO. 2) The insulin agonist means a compound that binds to the insulin receptor to exhibit insulin-like biological activity, which is irrelevant to the structure of insulin.
    The insulin derivative means a peptide with at least 80% homology in the amino acid sequence with the natural insulin, which may have some groups in the chemically substituted amino acid residues (eg, alpha-methylation, alpha-hydroxylation), deleted (eg, deamination) or modified (eg, N-methylation) and has a function of regulating the level of blood glucose in the body.
    The insulin fragment means a fragment with a B or more amino acids added or deleted at the N-terminal 5 or the C-terminal of natural insulin, in which non-naturally occurring amino acids (for example, type D amino acid) can be added and have a function of regulating the level of blood glucose in the body.
    Insulin variant means a peptide with one or more amino acid sequences different from those of natural insulin and with a function of regulating the level of blood glucose in the body.
    Each of the preparation methods for agonists, derivatives, fragments and variants of insulin can be used individually or in combination. For example, the present invention includes a peptide with one or more amino acids different from those of the natural peptide and deamination of the N-terminal amino acid residue and its function is to regulate the blood glucose level in the body.
    In a specific embodiment, the insulin used in the present invention can be produced by recombinant technology and can also be synthesized using a solid phase synthesis method.
    In addition, the insulin used in the present invention is characterized since a non-peptidyl polymer is attached to the amino-terminus of the insulin beta chain. This non-peptidyl polymer is used as a coupler in the present invention. The modification of the insulin alpha chain results in reduced activity and safety. In the present invention, therefore, the non-peptidyl polymer as a coupler is linked to the amino-terminus of the beta insulin chain, so that it maintains insulin activity and improves safety.
    The term "activity", as appropriate. if used here, it means the UU's insulin's ability to bind to the insulin receptor and it means that the insulin exhibits its action by binding to the insulin receptor.
    Such binding of the non-peptidyl polymer to the amino-terminus of the beta insulin chain can be achieved by controlling the pH, and preferably in the range of 4.5 to 7.5.
    The term "N-terminal", as used here, can be used interchangeably with "N-terminal region".
    In a specific Example, the present inventors prepared an insulin-PEG-immunoglobulin Fc fragment conjugate by binding PEG to the N-terminus of an immunoglobulin Fc region and coupling the N-terminus of the insulin beta chain to that complex .
    The serum half-life of the insulin-PEG-immunoglobulin Fc fragment prepared in the present invention was notably increased to approximately 18 hours and exhibited a hypoglycemic effect in animal models with the disorder.
    Therefore, a new, long-acting insulin formulation that maintains insulin activity in vivo can be prepared.
    The immunoglobulin Fc region is safe for use as a drug carrier because it is a biodegradable polypeptide that is metabolized in vivo.
    In addition, the immunoglobulin Fc region has a relatively low molecular weight when compared to the total immunoglobulin molecules, and therefore is advantageous in the preparation, purification and production of the conjugate.
    The immunoglobulin Fc region does not contain a Fab fragment, which is highly. non-homogeneous due to different UU amino acid sequences according to the antibody subclasses, and therefore it is possible to expect that the immunoglobulin Fc region can greatly increase the homogeneity of substances and be less antigenic.
    The term "immunoglobulin Fc region", as used herein, refers to a protein that contains the heavy chain constant region 2 (CH2) and the heavy chain constant region (CH3) of an immunoglobulin, excluding the variable regions of the light and heavy chains, the heavy chain constant region 1 (CH1) and the light chain constant region (CL1) of the immunoglobulin.
    It may also include a hinge region in the heavy chain constant region.
    In addition, the immunoglobulin Fc region of the present invention may contain part or all of the Fc region including the heavy chain constant region 1 (CH1) and / or the light chain region 1 (CL1), with the exception of the variable regions heavy and light chains, as long as it has a physiological function substantially similar to or better than natural protein.
    In addition, it can be a fragment with a deletion in a relatively long portion of the CcH2 and / or CH3 amino acid sequence. That is, the immunoglobulin Fc region of the present invention can comprise 1) a CH1 domain, a CH2 domain, a CH3 domain and a CH4, 2 domain) a CH1l domain and a CH2 domain, 3) a CH1 domain and a CH3 domain , 4) a CH2 domain and a CH3 domain, 5) a combination of one or more domains with an immunoglobulin hinge region (or a portion of the hinge region), and 6) a dimer from each domain of the chain constant regions heavy and light chain constant region. In addition, the immunoglobulin Fc region of the present invention includes a derivative of its (mutant) sequence as well as an original amino acid sequence.
    A derivative of the amino acid sequence has a sequence that is different from the original amino acid sequence due to a deletion, an insertion, a non-conservative or conservative substitution or combinations thereof of one or more amino acid residues.
    For example, in an IgG Fc region, amino acid residues that are proven to be important in binding, at positions 214 to 238, 297 to 299, 318 to 322 or 327 to 331, can be used as a suitable target for modification.
    In addition, several other derivatives are possible, including derivatives with an deletion of a region capable of forming a disulfide bond, a deletion of various amino acid residues at the N-terminus in an original Fc form or an addition of methionine residue to the N -terminal in an original Fc form. In addition, to eliminate effector functions, a deletion can occur at a complement binding site, such as a Clq binding site and an ADCC site. Preparation techniques as sequence derivatives of the immunoglobulin Fc region are shown in WO 97/34631 and WO 96/32478. Amino acid exchanges in proteins and peptides, which generally do not alter the activity of molecules, are known in practice (H.Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979). The most common exchanges are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro , Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly, in 'both directions. - MNA The Fc region, if desired, can be modified by phosphorylation, sulfatization, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation and the equivalent.
    The aforementioned Fc derivatives are derivatives that have identical biological activity to those of the Fc region of the present invention or improved structural stability, for example, against heat, pH or the equivalent.
    In addition, these Fc regions can be obtained from natural forms isolated from humans and other animals including cattle, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs, or they can be recombinant or derived from them, acquired from animal cells or of transformed microorganisms.
    Here, they can be obtained from a natural "immunoglobulin by isolating the total immunoglobulins from human or animal organisms and treated with a proteolytic enzyme.
    Papain digests natural immunoglobulin in Fab and Fc regions, and treatment with pepsin results in the production of pF'c and F (ab) 2 fragments. These fragments can be subjected, for example, to size exclusion chromatography to isolate Fc or pF'c.
    Preferably, a human-derived Fc region is a recombinant immunoglobulin Fc region that is obtained from a microorganism.
    In addition, the immunoglobulin Fc region of the present invention can be in the form of chains with natural sugar, chains with increased sugar in comparison with a natural form or chains with reduced sugar in comparison with the natural form, .or.can be in a deglycosylated form.
    It is possible to increase, reduce or remove the sugar chains of the immunoglobulin Fc region by common methods in practice, such as a chemical method, an enzymatic method and a genetic engineering method using a microorganism.
    Removal of sugar chains from an Fc region results in a marked decrease in complement binding affinity (Clgq) and a reduction or loss in antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity, therefore not inducing immune responses unnecessary in vivo.
    In this regard, an immunoglobulin Fc region in a deglycosylated or aglycosylated form may be more suitable for the purpose of the present invention as a drug carrier.
    The term "deglycosylation", as used here, means to enzymatically remove functional groups of sugar from an Fc region, and the term "aglycosylation" means that an Fc region is produced in a non-glycosylated form by a prokaryote, preferably E. coli.
    In addition, the immunoglobulin Fc region can be an Fc region that is derived from IgG, IgA, IgD, IgE and IgM or that is made by combinations of them or hybrids of them.
    Preferably, it is derived from IgG or IgM, which are among the most abundant proteins in human blood, and preferably from IgG, which has been shown to increase the half-life of ligand-binding proteins.
    The term "combination", as used here, means that polypeptides encoding 'single-stranded immunoglobulin Fc regions of the same origin are linked = "to a single-stranded polypeptide of a different origin to form a dimer or a multimer.
    That is, a dimer or a multimer can be formed from two or more fragments selected from the group consisting of fragments of IgG Fc, IgA Fc, IgM Fc, IgD Fc and IgE Fc.
    The term "hybrid", as used here, means that sequences encoding two or more immunoglobulin Fc regions of different origin are present in a single chain immunoglobulin Fc region.
    In the present invention, several types of hybrids are possible.
    That is, domain hybrids can be composed of one to four domains selected from the group consisting of CHl, CH2,
    It is CH3 and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc and may include the hinge region. On the other hand, IgG is divided into subclasses IgGl, IgG2, IgG3 and IgG4, and the present invention includes combinations or hybrids thereof. The preferred ones are the IgG2 and IgG4 subclasses, and the most preferred is the IgG4 Fc region, rarely having effector functions such as CDC (complement-dependent cytotoxicity).
    As the drug carrier of the present invention, the most preferable immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4. The human-derived Fc region is more preferable than a non-human-derived Fc region, which can act as an antigen on the human body and cause undesirable immune responses such as the production of a new antibody against the antigen.
    The term "non-peptidyl polymer", as used herein, refers to a biocompatible polymer including two = or more repeating units linked together by any covalent bond with the exception of a peptide bond.
    The non-peptidyl polymer with the possibility of being used in the present invention can be selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol and propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether (as polymers, PLA poly (lactic)) and PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid and combinations thereof, and preferably polyethylene glycol. Derivatives of these well known in practice and easily prepared within practical experience are also included in the field of the present invention.
    The peptide coupler that is used in the fusion protein obtained by a conventional inframe fusion method was disadvantageous since it is easily cleaved in vivo by a proteolytic enzyme, and thus a sufficient effect to increase the serum half-life of the active drug by a carrier cannot be obtained as expected.
    However, in the present invention, the polymer that is resistant to the proteolytic enzyme can be used to maintain the serum peptide similar half-life to that of the carrier.
    Therefore, any non-peptidyl polymer can be used without any limitation, as long as it is a polymer with the function mentioned above, that is, a polymer with resistance to the proteolytic enzyme in vivo.
    The non-peptidyl polymer has a molecular weight in the range of 1 to 100 kDa, and "- preferably. Of 1. to 20 kDa.
    The non-peptidyl polymer of === the present invention, linked to the immunoglobulin Fc region, can be a polymer or a combination of different types of polymer.
    The non-peptidyl polymer used in the present invention has a reactive group capable of binding the immunoglobulin Fc region and the protein drug.
    The non-peptidyl polymer has a reactive group at both ends, which is preferably selected from the group consisting of a reactive aldehyde group, a propionaldehyde group, a butyraldehyde group, a maleimide group and a succinimide derivative.
    The succinimide derivative can be succinimidyl propionate, hydroxy-succinimidyl, succinimidyl-carboxymethyl or succinimidyl carbonate.
    In particular, when the non-peptidyl polymer has a reactive aldehyde group at both ends, it is effective at binding at both ends a physiologically active polypeptide and an immunoglobulin with minimal non-specific reactions. A final product generated by reductive alkylation through an aldehyde bond is much more stable than one that is bound by an amidic bond. The reactive aldehyde group binds selectively to an N-terminal at low pH and binds to lysine residues to form a covalent bond at high pH, such as pH 9.0.
    The reactive groups at both ends of the non-peptidyl polymer can be the same or different. For example, the non-peptide polymer may have a maleimide group at one end and an aldehyde group, a propionaldehyde group or a butyraldehyde group at the other end. When a polyethylene glycol containing a reactive hydroxy group at both ends is used as one.
    non-peptidyl polymer, the hydroxy group can be activated in various reactive groups by known chemical reactions, or a polyethylene glycol containing a commercially available modified reactive group can be used in this way to prepare the protein conjugate of the present invention.
    In another aspect of the present invention, the present invention provides a long-acting insulin formulation comprising the insulin conjugate of the present invention.
    The term "administration", as used here, means introducing a predetermined amount of a substance to a patient by a certain appropriate method. The conjugate can be administered via any of the common routes, as long as it is able to reach a desired tissue.
    A variety of modes of administration are considered, including intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary and intrarectal routes, but the present invention is not limited to such exemplified modes of administration.
    However, since peptides are digested when administered orally, active ingredients in a composition for oral administration must be coated or formulated to provide protection against degradation in the stomach.
    Preferably, The conjugate can be administered in an injectable form.
    In addition, the long-acting formulation can be administered using a mechanism capable of transporting the active ingredient to a target cell.
    The long-acting formulation comprising the conjugate of the present invention can include carriers. : pharmaceutically acceptable.
    For oral administration, The pharmaceutically acceptable carrier can include a coupler, a lubricant, a disintegrator, an excipient, a solubilizer, a dispersing agent, a stabilizer, a suspending agent, a coloring agent and a fragrance.
    For injectable preparations, the pharmaceutically acceptable carrier may include a buffering agent, a preservative, an analgesic, a solubilizer, an isotonic agent and a stabilizer.
    For topical administration preparations, the pharmaceutically acceptable carrier can include a base, an excipient, a lubricant and a preservative.
    The long-acting formulation of the present invention can be formulated in a variety of dosage forms in combination with the aforementioned pharmaceutically acceptable carriers. For example, for oral administration, the long-acting formulation can be formulated into tablets, lozenges, capsules, elixirs, suspensions, syrups or wafers. For injectable preparations, the long-acting formulation can be formulated in a single dose ampoule or multiple dose container. The long-acting formulation can also be formulated in solutions, suspensions, tablets, pills, capsules and slow-release preparations.
    Examples of the appropriate carrier, excipient and diluent for the formulations include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose , microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oils. In addition, formulations may also include fillers, anticoagulants, lubricants, humectants, fragrances and antiseptics.
    The long-acting formulation of the present invention can be determined by several related factors including the types of diseases to be treated, the routes of administration, the age of the patient, the sex, the weight and severity of the disease, and also by the types of the disease. drug as an active component. Since the pharmaceutical composition of the present invention has an excellent duration and titer in vivo, it can remarkably reduce the frequency of administration and the dose of the drugs of the present invention.
    The long-acting formulation of the present invention maintains insulin duration and stability in vivo at a very high level and is therefore effectively used for the treatment of insulin-dependent diabetes.
    In yet another aspect, the present invention provides a method for preparing an insulin conjugate, comprising the steps of: (1) covalently bonding a non-peptidyl polymer containing a reactive group of aldehyde, maleimide or succinimide derivatives in each of their ends to an amino group or a thiol group of the immunoglobulin Fc region; (2) isolating a conjugate from the reaction mixture of (1), wherein the conjugate comprises the immunoglobulin Fc region covalently linked to the non-peptidyl polymer; and (3) covalent binding of insulin to the other UU end of the non-peptidyl polymer of the isolated conjugate to produce a peptide conjugate comprising the immunoglobulin and insulin Fc region, which are attached to each end of the non-peptidyl polymer.
    Preferably, the non-peptidyl polymer of step (1) has a reactive aldehyde derivative at its end and, more preferably, three reactive aldehyde groups.
    In yet another aspect, the present invention provides a method for preparing an insulin conjugate, comprising the steps of: (1) covalent bonding of a non-peptidyl polymer containing a reactive aldehyde group in each of its
    Ê ends at the N-terminus of the immunoglobulin Fc region at pH 6.0; (2) isolation of a conjugate from the reaction mixture (1), in which the conjugate comprises the immunoglobulin Fc region covalently linked to the non-peptidyl polymer at its N-terminus; and (3) covalent binding of insulin to the other end of the non-peptidyl polymer of the isolated conjugate to produce a peptide conjugate comprising the immunoglobulin and insulin Fc region, which are attached to each end of the non-peptidyl polymer.
    In yet another aspect, the present invention provides a method for preparing an insulin conjugate, comprising the steps of: (1) covalent bonding of a non-peptidyl polymer - containing a reactive group of aldehyde derivatives, - - - maleimide or succinimide at each of its ends to an amine group or an insulin thiol group; (2) isolating a conjugate from the reaction mixture of (1), wherein the conjugate comprises insulin covalently linked to the non-peptidyl polymer; and (3) covalent attachment of an immunoglobulin Fc region to the other end of the isolated conjugate non-peptidyl polymer to produce a peptide conjugate comprising the immunoglobulin and insulin Fc region, which are attached to each end of the non-peptidyl polymer.
    In yet another aspect, the present invention provides a method for preparing an insulin conjugate, comprising the steps of: (1) covalently bonding a non-peptidyl polymer containing a reactive aldehyde group at one end to an insulin amine group ; (2) isolating an insulin conjugate from the reaction mixture of (1), wherein the conjugate comprises insulin covalently bound to the non-peptidyl polymer; and (3) covalent attachment of an immunoglobulin Fc region to the other end of the isolated conjugate non-peptidyl polymer to produce a peptide conjugate comprising the immunoglobulin Fc region and insulin, which are attached to each end of the non-peptidyl polymer.
    In yet another aspect, the present invention provides a method for treating an individual with an insulin deficiency disorder, the method comprising administering to the individual an effective amount of the long-acting formulation. Preferably, the insulin deficiency disorder is diabetes.
    As used here, an individual can be a mammal, such as a human being, a non-human primate, a horse, a sheep, a cat, a dog, an ox or a pig. [Mode for the Invention]
    Thereafter, a better understanding of the present invention can be achieved based on the following Examples which are shown to illustrate, but are not to be construed as the limit of the present invention.
    Example 1. Purification of the pegylated immunoglobulin Fc region For pegylation of the immunoglobulin Fc region at its N-terminal, PEG 5K PropionALD (3) (PEG containing three propylaldehyde groups, NOF, Japan) was used to carry out pegylation by reacting the Fc region immunoglobulin with PEG at 4ºC for 4.5 hours and in a 1: 2 molar ratio, with an immunoglobulin Fc concentration of 10 mg / ml.
    During this period, the reaction was carried out in a 100 mM potassium phosphate buffer solution at pH 6.0, and 20 mM SCB (NaCNBH;) as a reducing agent was also added ... A mono-PEGylated immunoglobulin Fc was purified from the solution of reaction using a Source column | 15Q (GE Healthcare). Example 2, Preparation of the immunoglobulin Insulin-PEG-Fc conjugate To prepare an immunoglobulin insulin-PEG-Fc conjugate with 90% or more pegylation in phenylalanine (B1F) of the beta insulin chain, the immunoglobulin mono- Fc PEGylated obtained in Example 1 and insulin were subjected to reaction in a molar ratio of 4: 1 and at 4ºC for 20 h, with a total protein concentration of 20 mg / ml.
    During this period, the reaction was carried out in a 100 mM potassium phosphate buffer solution at pH h 6.0, and 20 mM SCB as a reducing agent was also added.
    After completion of the reaction, the reaction solution was subjected to primary purification using a Source 15Q column.
    Subsequently, secondary purification was performed using a Source 151SO column to obtain an insulin-PEG-Fc immunoglobulin conjugate.
    A size exclusion column was used to analyze 90% or more of B1F pegylation of the immunoglobulin insulin-PEG-Fc conjugate, and the results are shown in FIG. 3. Example 3. Preparation of immunoglobulin Insulin lispro (Humalog) -PEG-Fc conjugate The mono-PEGylated immunoglobulin Fc obtained in Example 1 and insulin lispro were subjected to reaction in a molar ratio of 4: 1 and at 4 ° C for 20 h, with a total concentration of 20 mg / ml.
    During this period, the reaction was - carried out in a buffer solution of 100 mM potassium phosphate at pH 6.0, and 20 mM SCB as a reducing agent was also ”added.
    After completion of the reaction, purification was carried out in the same manner as in Example 2. Example 4. Preparation of immunoglobulin Glargine (Lantus) -PEG-Fc conjugate The mono-PEGylated immunoglobulin Fc obtained in example 1 and insulin glargine were subjected to reaction in a molar ratio of 4: 1 and at 4ºC for 20 h, with a total protein concentration of 20 mg / ml.
    During this period, the reaction was carried out in a buffer solution of 100 mM potassium phosphate at pH 6.0, and 20 mM SCB as a reducing agent was also added.
    After completion of the reaction, purification was carried out in the same manner as in Example 2. Example 5. Preparation of immunoglobulin insulin detemir (Levemir) -PEG-Fc conjugate The monoPEGylated immunoglobulin Fc obtained in Example 1 and insulin detemir were subjected to reaction in a molar ratio of 4: 1 and at 4ºC for 20 h, with a total protein concentration of 20 mg / ml.
    During this period, the reaction was carried out in a 10 mM potassium phosphate buffer solution at pH 6.0, and 20 mM SCB as a reducing agent was also added.
    After the completion of the reaction, purification was carried out in the same manner as in Example 2. Example 6. Determination of the in vivo elimination half-life of the long-acting insulin conjugate ENS L: Basa Para, in vivo analysis of the duration of the insulin, normal male SD rats were used rec in order to perform pharmacokinetic analysis.
    Normal male SD rats underwent subcutaneous injection of natural insulin and long-acting insulin conjugate at a dose of 100 1pg / kg (based on insulin) once, and then time-dependent changes in serum level were determined using an ELISA kit, and pharmacokinetic parameters were calculated from the values determined using Winnolin 5.0 software. The in vivo elimination half-life of the long-acting insulin conjugate was 17.67 h, which is about 30 times that of 0.58 h natural insulin (FIG. 1).
    Example 7. In vivo efficacy test on the Insulin Conjugate derivative To compare the in vivo efficacy between insulin conjugate derivatives, rats with streptozotocin-induced diabetes were used to analyze their hypoglycemic effects.
    Normal rats were fasted for 16 h and intraperitoneal injection of streptozotocin in a 10 mM citric acid buffer solution (pH 4.5) at a dose of 60 mg / kg to induce diabetes.
    When the blood glucose level of the rats reached 500 mg / dl or more, the rats were subjected to subcutaneous injection of the insulin conjugate, the insulin detemir conjugate or the insulin lispro conjugate at a dose of 0.5 mg / kg once, and then their hypoglycemic effects were compared.
    The hypoglycemic effects of the insulin conjugate and the insulin lispro conjugate were maintained for about 4 "- days after injection, and, 5 days after injection, the level of 7 VUTTX blood glucose increased.
    The insulin conjugate detemir also exhibited the hypoglycemic effects, but the effects were less than those of the insulin conjugate or the insulin lispro conjugate at an equal dose (FIG. 2). Example 8. Identification of the binding site of the immunoglobulin 5K PEG-Fc insulin-conjugate To identify the insulin binding site for immunoglobulin 5K PEG-Fc, W a Glu-c mapping was performed. 20 µg of Glu-c endoproteinase (1 mg / ml) was added to 100 µg of insulin-5 K-PEG-Fc immunoglobulin (1 mg / ml). The reaction solution was 50 mM HEPES at pH 7.5, and the mixture was subjected to reaction at 25ºC for
    8 hours.
    Subsequently, 10 up7l of 1N HCl were added to complete the reaction.
    The mapping was performed by reverse HPLC chromatography.
    The results showed the maximum change in the N-terminal of the beta insulin chain, indicating that 5 K immunoglobulin PEG-Fc binds to the N-terminal of the beta insulin chain (FIGsS. 4da-c). Column: Jupiter C18 4.6x250 mm, 5 µm (Phenomenex) Mobile Phase A: 20% NaSO, 0.1 M (pH 2.0), 10% CAN Mobile Phase B: 20% NaSO, 0.1 M (pH 2.0), 40% CAN Gradient O% B in 10 min> 0-10% B in 5 min> 10-70% B in 60 min
    权利要求:
    Claims (21)
    [1]
    1. Insulin conjugate characterized by the fact that it is prepared by binding insulin to an immunoglobulin Fc region by means of a non-peptidyl polymer selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol and propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipid polymers, chitins, hyaluronic acid and combinations of these, in which the non-peptidyl polymer is linked to the amino-terminus of the insulin beta chain.
    [2]
    2. Insulin conjugate according to claim 1, characterized by the fact that insulin is natural insulin, or a variant, or a fragment of it that is prepared by any of the replacement methods, - deletion and modification or combinations of these to amino acids of natural insulin.
    [3]
    Insulin conjugate according to claim 1, characterized in that each end of the non-peptidyl polymer is linked to an amino group or a thiol group of the immunoglobulin and insulin Fc region.
    [4]
    4, Insulin conjugate according to claim 1, characterized by the fact that the immunoglobulin Fc region is non-glycosylated.
    [5]
    5. Insulin conjugate according to claim 1, characterized by the fact that the immunoglobulin Fc region is composed of one to four domains selected from the group consisting of the CH1, CH2,
    a t CcH3 and CH4.
    [6]
    6. Insulin conjugate according to claim 5, characterized in that the immunoglobulin Fc region further comprises a hinge region.
    [7]
    7. Insulin conjugate according to claim 1, characterized by the fact that the immunoglobulin Fc region is an Fc region derived from IgG, IgA, IgD, IgE or IgM.
    [8]
    8. Insulin conjugate according to claim 7, characterized in that each domain of the immunoglobulin Fc region is a hybrid of a different region domain derived from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE and IgM.
    [9]
    Insulin conjugate according to claim 7, characterized by the fact that the immunoglobulin 7 Fc region is a UU dimer or a multimer composed of single chain immunoglobulins of the same origin.
    [10]
    Insulin conjugate according to claim 7, characterized by the fact that the immunoglobulin Fc region is an IgG4 Fc region.
    [11]
    11. Insulin conjugate according to claim 10, characterized by the fact that the immunoglobulin Fc region is a non-glycosylated human IgG4 Fc region.
    [12]
    12. Insulin conjugate according to claim 1, characterized in that the reactive group of the non-peptidyl polymer is selected from the group consisting of an aldehyde group, a propionaldehyde group, a butyraldehyde group, a maleimide group and a derivative the ”succinimide.
    [13]
    Insulin conjugate according to claim 12, characterized in that the succinimide derivative is succinimidyl propionate, succinimidyl carboxymethyl, hydroxy succinimidyl or succinimidyl carbonate.
    [14]
    Insulin conjugate according to claim 12, characterized in that the non-peptidyl polymer has a reactive aldehyde group at both ends.
    [15]
    15. Long-acting insulin formulation with improved in vivo duration and stability, characterized in that it comprises the insulin conjugate of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13,
    14.
    [16]
    16. Long-acting insulin formulation, according to claim 15, characterized by the fact that the formulation is used for the treatment of diabetes.
    [17]
    Method for preparing the insulin conjugate of claim 1, characterized in that it comprises the steps of: (1) covalent bonding of a non-peptidyl polymer containing a reactive group of aldehyde, maleimide or succinimide derivatives at each of its ends to a amino group or a thiol group of an immunoglobulin Fc region; (2) isolating a conjugate from the reaction mixture of (1), wherein the conjugate comprises the immunoglobulin Fc region covalently linked to the non-peptidyl polymer; and K (3) covalent binding of insulin to the other end of the non-peptidyl polymer of the isolated conjugate to produce a peptide conjugate comprising the immunoglobulin and insulin Fc region, which are attached to each end of the non-peptidyl polymer.
    [18]
    Method for preparing the insulin conjugate of claim 1, characterized in that it comprises the steps of: (1) covalent bonding of a non-peptidyl polymer containing a reactive aldehyde group at each of its ends to the N-terminus of an immunoglobulin Fc in pH
    6.0; (2) isolation of a conjugate from the mixture of the reaction of (1), characterized by the fact that the conjugate comprises the immunoglobulin Fc region covalently linked to the non-peptidyl polymer at its N-terminal; and: Ú (3) ”covalent insulin bond. to the other UU end of the non-peptidyl polymer of the isolated conjugate to produce a peptide conjugate comprising the immunoglobulin and insulin Fc region that are attached to each end of the non-peptidyl polymer.
    [19]
    Method for preparing the insulin conjugate of claim 1, characterized in that it comprises the steps of: (1) covalent bonding of a non-peptidyl polymer containing a reactive group of aldehyde, maleimide or succinimide derivatives at each of its ends to a amine group or an insulin thiol group; (2) isolation of a conjugate from the mixture of the reaction of (1), characterized by the fact that the conjugate to
    RN comprises insulin covalently linked to the non-peptidyl polymer; and (3) covalent attachment of an immunoglobulin Fc region to the other end of the isolated conjugate non-peptidyl polymer to produce a peptide conjugate comprising the immunoglobulin and insulin Fc region, which are attached to each end of the non-peptidyl polymer.
    [20]
    20. Method for treating an individual with an insulin deficiency disorder, the method comprising administering to the individual an effective amount of the long-acting formulation of claim 15.
    [21]
    21. Method according to claim 20, characterized by the fact that the insulin deficiency disorder is diabetes.
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    法律状态:
    2020-11-17| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |
    2021-05-04| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|
    2021-05-25| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2021-09-21| B11B| Dismissal acc. art. 36, par 1 of ipl - no reply within 90 days to fullfil the necessary requirements|
    2021-11-23| B350| Update of information on the portal [chapter 15.35 patent gazette]|
    优先权:
    申请号 | 申请日 | 专利标题
    KR1020100030575|2010-04-02|
    KR20100030575|2010-04-02|
    PCT/KR2011/002331|WO2011122921A2|2010-04-02|2011-04-04|An insulin conjugate using an immunoglobulin fragment|
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