STABLE SOLUTION FOR PARENTERAL INJECTION AND MANUFACTURING METHOD OF IT

专利摘要:
stable solution for parenteral injection, its method of manufacture, method of administration, and methods of treating hypoglycemia and diabetes. Stable formulations for the parenteral injection of peptide drugs and methods of using such stable formulations are provided. in particular, the present invention provides stable formulations for the parenteral injection of glucagon and methods of using such glucagon formulations to treat hypoglycemia, especially severe hypoglycemia in emergency situations.
公开号:BR112013023062B1
申请号:R112013023062-2
申请日:2012-03-09
公开日:2022-01-18
发明作者:Steven Prestrelski；John Kinzell
申请人:Xeris Pharmaceuticals, Inc；
IPC主号:

专利说明:

CROSS REFERENCES TO RELATED ORDERS
[001] This application claims the priority and benefit of U.S. Provisional Application No. 61/451,568, filed March 10, 2011, and titled "Stable Formulations for Parenteral Injection of PeptideDrugs"; U.S. Provisional Application No. 61/478,692, filed April 25, 2011, and titled "Stable Formulations for Parenteral Injection of Peptide Drugs"; of US Interim Application No. 61/553,388, filed on October 31, 2011, and entitled "Formulations for the Treatment of Diabetes" and of Interim Application No. 61/609,123, filed on March 9, 2012, and entitled "Formulations for the Treatment of Diabetes", the entire disclosures of which are incorporated by reference herein for all purposes. DECLARATION REGARDING RIGHTS TO INVENTIONS MADE UNDER RESEARCH OR DEVELOPMENT SPONSORED BY THE FEDERAL GOVERNMENT
[002] Not Applicable REFERENCE TO A "SEQUENCE LIST", A TABLE, OR APPENDIX OF THE COMPUTER PROGRAM LIST PRESENTED ON A COMPACT DISK
[003] Not Applicable FIELD OF THE INVENTION
[004] The present invention relates to pharmaceutical formulations, and more particularly to pharmaceutical formulations of peptides which have improved stability, and methods of using such pharmaceutical formulations for the treatment of various diseases, conditions and disorders. BACKGROUND OF THE INVENTION
[005] Diabetes is a serious health problem in modern society. Insulin is a critical treatment for both type I and type II diabetes. Studies over the past two decades have shown that tight metabolic glucose control through the use of insulin not only reduces the incidence, but also delays the development of complications in people with type 1 and type 2 diabetes. Unfortunately, intensive insulin therapy is required to achieve tight glucose control is also associated with a significantly increased risk of developing hypoglycemia, or "low blood sugar".
[006] Symptoms of hypoglycemia vary greatly between patients, but typically include tremor, palpitations, irritability, anxiety, nervousness, hunger, tachycardia, headache, and pallor. Symptoms typically subside once plasma glucose is restored to normal levels. If hypoglycemia is not reversed, a further decrease in plasma glucose can lead to central nervous system glucose depletion and associated neuroglycopenic symptoms such as difficulty concentrating, slurred speech, blurred vision, reduced body temperature, changes in behavior, and if left untreated , unconsciousness, convulsions and possibly death.
[007] In general, hypoglycemia can be defined as mild to moderate hypoglycemia or as severe hypoglycemia, as follows:
[008] Mild to moderate hypoglycemia: Episodes where the patient can self-treat regardless of the severity of symptoms, or any asymptomatic blood glucose measurements where blood glucose levels are less than 70 mg/dl (3 .9mmol/l).
[009] Severe hypoglycemia: Operationally defined as an episode of hypoglycemia in which the patient cannot self-treat, so outside help is required. Typically, neuroglycopenic symptoms and cognitive impairment begin at a blood glucose level around 50 mg/dl (2.8 mmol/l).
[0010] Most episodes of mild to moderate hypoglycemia can be self-treated relatively easily by eating fast-acting carbohydrates such as glucose tablets or foods (juices, sodas, or sugary snacks). Severe hypoglycemia, by definition, cannot be self-treated and thus requires external intervention. If the patient can swallow and is cooperative, it is appropriate to use gels or products such as honey or jelly placed inside the cheek. If the patient is unable to swallow, glucagon, which is injected subcutaneously or intramuscularly, is used to treat severe hypoglycemia.
[0011] Glucagon is a naturally occurring peptide hormone that is 29 amino acids in length and is secreted by the α-cells of the pancreas. The main function of glucagon is to maintain glucose production through both glycogenolysis and gluconeogenesis, mainly mediated through the liver. Glucagon is the primary counter-regulatory hormone to insulin and is used as a first-line treatment of severe hypoglycemia in patients with diabetes.
[0012] Numerous attempts have been made to create a glucagon rescue drug for the treatment of severe hypoglycemia in emergency situations. There are currently two glucagon kits currently available in the United States, manufactured by EliLilly (Glucagon Emergency Kit) and Novo Nordisk (GlucaGen® HypoKit). Both products combine a vial of lyophilized glucagon with a pre-filled syringe of aqueous diluent. Lyophilized glucagon must be reconstituted using a complex procedure that is difficult to use in an emergency situation. These products also provide a large injection volume because glucagon is poorly soluble in water. Recently, attempts have been made to improve the stability of glucagon in an aqueous solution, to create more stable glucagon analogues, and/or to improve glucagon release through powder injection.
[0013] Although some progress has been made, there is still a need for a more user-friendly glucagon rescue medication for treating severe hypoglycemia in emergency situations. Such a glucagon rescue medication would need to be carried continuously by diabetics and/or their caregivers and thus would need to be stable at unrefrigerated temperatures (25 to 30°C) for long periods of time (> 2 years). Ideally, it would also need to be simple to administer to the general population and not require excessive processing/reconstitution before administration to the hypoglycemic patient. Glucagon rescue medication would also need to be functional over a range of temperatures, including temperatures ranging from 0°C to 30°C. BRIEF SUMMARY OF THE INVENTION
[0014] To address these and other needs, the present invention provides a stable glucagon rescue formulation as well as methods of using this stable glucagon formulation for the treatment of severe hypoglycemia. Advantageously, glucagon is stabilized in the formulations of the present invention to allow for long-term storage and/or release over an extended period of time. As such, the glucagon formulations of the present invention are stable at unrefrigerated temperatures for long periods of time, are simple to administer without the need for reconstitution, and are functional over a range of temperatures, including temperatures ranging from 0°C to 30°C. °C
[0015] It is important that the formulation technology of the present invention is widely applicable for the release of numerous other peptides which, such as glucagon, have poor or limited stability and solubility in an aqueous environment. Indeed, it is now clear that the formulation of peptides with a polar aprotic solvent such as DMSO, NMP, ethyl acetate, or a mixture of these in high concentration nonaqueous solutions, is a valuable deliberation platform for this important class of peptide therapeutics. Additionally, the formulation technology of the present invention is widely applicable for delivering two or more peptides in the same solution.
[0016] Thus, in one aspect, the present invention provides a stable formulation for parenteral injection, the formulation comprising: (a) a peptide or a salt thereof, wherein the peptide has been dried in a non-volatile buffer, and wherein the peptide dry has a pH memory that is nearly equal to the pH of the peptide in the non-volatile buffer; and (b) a polar aprotic solvent, wherein the moisture content of the formulation is less than 5%, and wherein the dried peptide maintains a pH memory that is nearly equal to the pH of the peptide in the nonvolatile buffer when the dried peptide is reconstituted in polar aprotic solvent.
[0017] In another aspect, the present invention provides a stable formulation for parenteral injection, the formulation comprising: (a) a first peptide or a salt thereof, wherein the first peptide has been dried in a first non-volatile buffer, and wherein the first dry peptide has a first pH memory that is nearly equal to the pH of the first peptide in the first non-volatile buffer; (b) a second peptide or a salt thereof, wherein the second peptide has been dried in a second non-volatile buffer, and wherein the second dry peptide has a second pH memory that is nearly equal to the pH of the second peptide in the second non-volatile buffer ; and (c) an aprotic polar solvent, where the moisture content of the formulation is less than 5%, where the first dry peptide maintains a first pH memory that is nearly equal to the pH of the first peptide in the first non-volatile buffer when the first The dry peptide is reconstituted in the polar aprotic solvent, and wherein the second dry peptide maintains a second pH memory that is nearly equal to the pH of the second peptide in the second non-volatile buffer when the second dry peptide is reconstituted in the polar aprotic solvent.
[0018] In another aspect, the present invention provides a stable formulation for parenteral injection, the formulation comprising: a peptide or a salt thereof (such as a hydrochloride or its acetate salt); and a polar aprotic solvent, where the moisture content of the formulation is less than 5%.
[0019] The stable formulations described herein are useful for the parenteral injection of any peptide that has limited or poor stability or solubility in an aqueous environment. Thus, in some embodiments, the peptide (or each of the first and second peptides) or its salt is selected from the group consisting of glucagon, pramlintide, insulin, leuprolide, an LHRH agonist, parathyroid hormone (PTH), amylin, botulinum toxin, hematide , an amyloid peptide, cholecystikinin, a conotoxin, a gastric inhibitory peptide, an insulin-like growth factor, a growth hormone releasing factor, an antimicrobial factor, glatiramer, glucagon-like peptide-1 (GLP- 1), a GLP-1 agonist, exenatide, analogues thereof, and mixtures thereof. In a preferred embodiment, the peptide is glucagon or a glucagon analogue or a glucagon peptidomimetic. In another embodiment, the peptide is parathyroid hormone. In yet another embodiment, the peptide is leuprolide. In yet another embodiment, the peptide is glatiramer. In yet another embodiment, the first peptide is pramlintide and the second peptide is insulin. In yet another embodiment, the first peptide is glucagon and the second peptide is exenatide.
[0020] The peptide (or, in embodiments where the formulation comprises two or more peptides, each of the peptides) is mixed with a non-volatile buffer and dried to a dry peptide powder. Suitable non-volatile buffers include, but are not limited to, glycine buffers, citrate buffers, phosphate buffers, and mixtures thereof. In a preferred embodiment, the non-volatile buffer is a glycine buffer. In another preferred embodiment, the non-volatile buffer is a mixture of citrate buffer and phosphate buffer. In some embodiments, where the formulation comprises two or more peptides, the first non-volatile buffer and the second non-volatile buffer are the same. In some embodiments, where the formulation comprises two or more peptides, the first non-volatile buffer and the second non-volatile buffer are different.
[0021] In some formulations of the present invention, the peptide is mixed with a non-volatile buffer and a stabilizing excipient, and then dried to a dry peptide powder. Suitable stabilizing excipients include, but are not limited to, sugars, starches, and mixtures thereof. In some embodiments, the sugar is trehalose. In some embodiments, the starch is hydroxyethyl starch (HES). In some embodiments, the stabilizing excipient is present in the formulation in an amount ranging from about 1% (w/v) to about 60% (w/v), of about 1% (w/v) to about 50% (w/v), from about 1% (w/v) to about 40% (w/v), from about 1% (w/v) ) to about 30% (w/v), from about 1% (w/v) to about 20% (w/v), from about 5% (w/v) to about 60% (w/v) v), from about 5% (w/v) to about 50% (w/v), from about 5% (w/v) to about 40% (w/v), from about 5% (w/v) to about 30% (w/v), from about 5% (w/v) to about 20% (w/v), from about 10% (w/v) to about 60 % (w/v), from about 10% (w/v) to about 50% (w/v), from about 10% (w/v) to about 40% (w/v), from about from 10% (w/v) to about 30% (w/v), or from about 10% (w/v) to about 20% (w/v). In some embodiments, where the formulation comprises two peptides, both the first peptide in the first non-volatile buffer and the second peptide in the second non-volatile buffer further comprise a stabilizing excipient, and the stabilizing excipient with the first peptide in the first non-volatile buffer and the excipient stabilizer with the second peptide in the second non-volatile buffer are the same. In other embodiments, where the formulation comprises two peptides, both the first peptide in the first non-volatile buffer and the second peptide in the second non-volatile buffer further comprise a stabilizing excipient, and the stabilizing excipient having the first peptide in the first non-volatile buffer and the stabilizing excipient with the second peptide in the second non-volatile buffer are different.
[0022] Once the peptide or peptides and the non-volatile peptide buffer, non-volatile buffer and stabilizing excipient are dried to a powder, the dry peptide powder is dissolved or reconstituted in a polar aprotic solvent. Examples of polaresaprotic solvents include, but are not limited to, the following: dimethylsulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate, N-methyl pyrrolidone (NMP), dimethylacetamide (DMA), propylene carbonate, and mixtures thereof . Dimethyl sulfoxide (DMSO), N-methyl pyrrolidone (NMP), ethyl acetate, and mixtures of one or more of DMSO, NMP, and ethyl acetate are particularly preferred polar aprotic solvents. In a preferred embodiment, the polar aprotic solvent is DMSO. In another preferred embodiment, the polar aprotic solvent is a mixture of DMSO and NMP. In yet another preferred embodiment, the polar aprotic solvent is a mixture of DMSO and ethyl acetate.
[0023] In some embodiments, the peptide or peptides are reconstituted in a mixture of a polar aprotic solvent (e.g., dimethylsulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate, n-methyl pyrrolidone (NMP), dimethylacetamide (DMA) ), propylene carbonate, or mixtures thereof) and a co-solvent that lowers the freezing point of the formulation. In some embodiments, the co-solvent reduces the freezing point of the formulation by at least about 5°C, at least about 10°C, at least about 15°C, or at least about 20°C. In some embodiments, the co-solvent reduces the freezing point of the formulation by about 3°C, by about 2°C, by about 1°C, or by about 0°C or below. In some embodiments, the co-solvent is a polar protic solvent. In preferred embodiments, the co-solvent is selected from ethanol, propylene glycol (PG), glycerol and mixtures thereof. In some embodiments, the co-solvent is present in the formulation in an amount ranging from about 10% (w/v) to about 50% (w/v), from about 10% (w/v) to about 40% (w/v), from about 10% (w/v) to about 30% (w/v), from about 10% (w/v) to about 25% (w/v), from about 15% (w/v) to about 50% (w/v), from about 15% (w/v) to about 40% (w/v), from about 15% (w/v) to about 30% (w/v), or from about 15% (w/v) to about 25% (w/v).
[0024] Importantly, the formulations of the present invention have very little residual moisture and thus the peptides in such formulations remain stable for extended periods of time. In preferred embodiments, the moisture content of the formulation of the present invention is less than about 4%, preferably less than about 3%, preferably less than about 2%, and even more preferably less than about 2% 1%, preferably less than about 0.5%, preferably less than 0.25%, preferably less than 0.2%, preferably less than about 0.15%, or preferably less than about 0.1%. In other preferred embodiments, the moisture content of the formulation of the present invention is from about 0.01% to about 4%, preferably from about 0.01% to about 3%, of preferably from about 0.01% to about 2%, preferably from about 0.01% to about 1%, preferably from about 0.1% to about 4%, preferably from about 0.1% to about 0.1% about 3%, preferably from about 0.1% to about 2%, preferably from about 0.1% to about 1%, preferably from about 0.25% to about 4%, most preferably from about 0.25% to about 4% , from about 0.25% to about 3%, of p reference from about 0.25% to about 2%, preferably from about 0.25% to about 1%, or preferably from about 0.5% to about 1%.
[0025] When the peptide is mixed with a non-volatile buffer, the non-volatile buffer is selected such that the peptide has a pH of maximum stability, maximum solubility, and minimum degradation in the aqueous environment. Once dried, the peptide will have a pH memory of maximum stability, maximum solubility, and minimum degradation and will retain this pH memory when dissolved or reconstituted in polar aprotic solvent. As such, in preferred embodiments, the peptide in the formulation will have a pH memory of about 2.0 to about 3.0 to ensure maximum stability, maximum solubility, and minimum degradation. In other embodiments, the peptide in the formulation will have a pH memory of about 3.0 to about 5.0 to ensure maximum stability, maximum solubility, and minimum degradation. In other embodiments, the peptide in the formulation will have a pH memory from about 4.0 to about 5.0 to ensure maximum stability, maximum solubility, and minimum degradation. In still other embodiments, the peptide will have a pH memory of about 4.0 to about 6.0 to ensure maximum stability, maximum solubility, and minimal degradation. In still other embodiments, the peptide will have a pH memory of about 6.0 to about 8.0 to ensure maximum stability, maximum solubility, and minimum degradation. In some embodiments, where the formulation comprises two peptides, the first peptide has a pH memory of about 4.0 to about 6.0 to ensure maximum stability, maximum solubility, and minimum degradation, and the second peptide has a pH memory of from about 1.5 to about 2.5, or from about 6.0 to about 8.0, to ensure maximum stability, maximum solubility, and minimum degradation. In some embodiments, where the formulation comprises two peptides, the first peptide has a pH memory of about 3.0 to about 5.0 to ensure maximum stability, maximum solubility, and minimum degradation, and the second peptide has a pH memory of about 1.5 to about 2.5, or from about 6.0 to about 8.0, to ensure maximum stability, maximum solubility, and minimum degradation. In other embodiments, where the formulation comprises two peptides, the first peptide has a pH memory of about 2.0 to about 3.0 to ensure maximum stability, maximum solubility, and minimum degradation and the second peptide has a memory of about 2.0 to about 3.0. pH from about 4.0 to about 5.0 to ensure maximum stability, maximum solubility, and minimum degradation. It will be readily apparent to one of skill in the art how to determine the ideal pH for obtaining a peptide having maximum stability, maximum solubility, and minimum degradation.
[0026] Any suitable dosage of peptide or peptides can be formulated into the stable formulations of the present invention. Generally, the peptide (or, in embodiments comprising two or more peptides, each of the peptides) is present in the formulation in an amount ranging from about 0 .5mg/ml to about 100mg/ml. In some embodiments, the peptide is present in the formulation in an amount ranging from about 10 mg/ml to about 60 mg/ml. In other embodiments, the peptide is present in the formulation in an amount ranging from about 20 mg/ml to about 50 mg/ml. In still other embodiments, the peptide is present in the formulation in an amount ranging from about 5 mg/ml to about 15 mg/ml. In yet other embodiments, the peptide is present in the formulation in an amount ranging from about 0.5 mg/ml to about 2 mg/ml. In yet other embodiments, the peptide is present in the formulation in an amount ranging from about 1mg/ml to about 50mg/ml. Again, it will be readily apparent to those of skill that the dosage of peptide can be varied depending on the peptide used and the disease, disorder or condition being treated.
[0027] In some embodiments, the formulations of the present invention further comprise an antioxidant. In other embodiments, the formulations further comprise a chelator. In still other embodiments, the formulations of the present invention further comprise a preservative.
[0028] In another aspect, the present invention provides a method for treating a disease, condition or disorder that can be treated, alleviated or prevented by administering to a subject a stable peptide formulation as described herein in an amount effective to treat, alleviate or prevent the disease, condition, or disorder. In some embodiments, the disease, condition, or disorder is hypoglycemia. In some embodiments, where the disease, condition or disorder is hypoglycemia, the method comprises administering a stable glucagon formulation of the present invention in an amount effective to treat hypoglycemia. In some embodiments, the disease, condition, or disorder is diabetes. In some embodiments, where the disease, condition or disorder is diabetes, the method comprises administering a stable formulation of epramlintide insulin of the present invention in an amount effective to treat diabetes.
[0029] In yet another aspect, the present invention provides a process for preparing a stable formulation for parenteral injection, the process comprising: drying a peptide and a non-volatile buffer in a dry peptide powder; and reconstituting the dry peptide powder with a polar aprotic solvent, thereby producing the stable formulation, wherein the moisture content of the stable formulation is less than 5%. In some embodiments, the dry peptide powder has a pH memory that is nearly equal to the pH of the peptide in the nonvolatile buffer, and the dry peptide powder maintains a pH memory that is nearly equal to the pH of the peptide in the non-volatile buffer. non-volatile buffer when dry peptide powder is reconstituted in polar aprotic solvent.
[0030] In yet another aspect, the present invention provides kits for treating a disease, condition or disorder, the kit comprising: a stable formulation comprising one or more peptides or their salts, wherein the peptide has been dried in a non-volatile buffer, and wherein the dried peptide has a pH memory that is nearly equal to the pH of the peptide in the non-volatile buffer; and an aprotic polar solvent; wherein the moisture content of the formulation is less than 5%, and wherein the dry peptide maintains a pH memory that is nearly equal to the pH of the peptide in the non-volatile buffer when the dried peptide is reconstituted in the polar aprotic solvent; and a syringe for administering the stable formulation to the subject.
[0031] In some embodiments, the kit is for the treatment of hypoglycemia and the stable formulation comprises a deglucagon formulation as described herein. In some embodiments, the kit is for the treatment of diabetes and the stable formulation comprises an insulin and pramlintide formulation as described herein. In some embodiments, the syringe is part of a pen injection device, an auto-injector device, or a pump. In some embodiments, the syringe is pre-filled with the stable formulation. In some embodiments, the kit further comprises instructions, where the instructions direct the administration of the stable formulation to treat the individual's need.
[0032] Other objects, features and advantages of the present invention will be apparent to a person skilled in the art from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Figure 1 illustrates plasma glucagon levels after injection of lyophilized glucagon-glycine-trehalose dissolved in DMSO or NMP.
[0034] Figure 2 illustrates blood glucose levels after injection of lyophilized glucagon-glycine-trehalose dissolved in DMSO or NMP. DETAILED DESCRIPTION OF THE INVENTIONIntroduction
[0035] Peptides can degrade through several different mechanisms, including deamidation, oxidation, hydrolysis, and disulfide exchange and racemization. In addition, water acts as a plasticizer, which facilitates the unfolding of protein molecules and irreversible molecular aggregation. Therefore, in order to provide a peptide formulation that is stable over time at ambient or physiological temperatures, a non-aqueous or substantially non-aqueous peptide formulation is generally required.
[0036] The reduction of aqueous peptide formulations to dry powder formulations is a way to increase the stability of pharmaceutical peptide formulations. For example, peptide formulations can be dried using various techniques, including spray drying, lyophilization or freeze drying, and desiccation. The dry powder peptide formulations obtained by these techniques have significantly greater stability over a period of time at ambient or physiological temperatures.
[0037] The present invention is based, in part, on the surprising discovery that a stable peptide formulation (e.g., a stable glucagon rescue formulation) can be readily prepared by first lyophilizing one or more peptides (e.g., a peptide of glucagon) in a non-volatile buffer in a dry powdered peptide. The dried peptide has a defined "pH memory" of the pH of the peptide in the non-volatile buffer from which the peptide was added. Once dry, the resulting peptide powder, e.g. lyophilized glucagon, is dissolved in a polaraprotic solvent, thus forming a stable formulation, where the moisture content of the formulation is less than 5% and preferably less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.25%, less than 0.15%, or less than 0.1% . The dried peptide retains its defined pH memory when reconstituted in the polar aprotic solvent, i.e. the pH of the peptide when reconstituted in the polar aprotic solvent is nearly equal to the pH of the peptide in the non-volatile buffer from which it was dried. Advantageously, once prepared, the formulation (eg, the glucagon formulation) is stable for long periods of time, is ready for use without the need for reconstitution, and is functional over a range of temperatures.
[0038] Importantly, the formulation technology of the present invention is widely applicable for the release of numerous other peptides that, like glucagon, have stability and solubility. weak or limited ability in an aqueous environment. Indeed, it is now clear that formulating peptides with a polar aprotic solvent (e.g., DMSO, NMP, ethyl acetate, or a mixture thereof) in high concentration nonaqueous solutions is an invaluable delivery platform for an important class. of therapeutic agents – therapeutic peptides. The stable formulations described herein advantageously promote uniform release of the peptide drugs and provide additional shelf stability against aggregation, oxidation, and hydrolysis-related degradation pathways.
[0039] In certain preferred embodiments, the stable formulations described herein preserve the peptide drugs in a stable form over an extended period of time, for example, for a period of time sufficient to provide a desired shelf life of the formulation without unacceptable levels of agent degradation. therapy before use. A desired property of injectable formulations is that they are non-aqueous and non-reactive towards the peptide. In such embodiments, it is possible to store the injectable formulations directly in the injection device itself.
[0040] The stable injectable formulations of the present invention contain the required delivered dose of therapeutic peptide or peptides (eg, the dose required for drug therapy) and are preferably of low volume. For example, in some embodiments an injectable formulation comprising a therapeutic dose of a peptide (e.g., glucagon) has a volume of at least about 1.0 microliters (the lower limit being a function of the charging equipment), more preferably of about 10 milliliters about 250 microliters. The delivery of a therapeutic dose of the peptide in a low volume is accomplished in certain preferred modalities, by concentrating the dose of the therapeutic peptide or peptides (e.g. glucagon) in a stable form with a polar aprotic solvent suitable for injection in accordance with the invention. .
[0041] In addition, the stable formulations of the present invention are suitable for administration without requiring dilution prior to injection. Many currently available vaccine and therapeutic peptide products are produced in a solid particulate form to promote shelf stability. These formulations are diluted prior to injection with sterile water, phosphate buffered solution, or isotonic saline. In contrast, in certain preferred embodiments of the present invention, the therapeutic peptide is concentrated using the particle preparation processing techniques (eg, spray drying, lyophilization, etc.) routinely used by the pharmaceutical industry to prepare formulations for injection. In preferred embodiments, therapeutic dosages of peptide drugs are achieved by dissolving the peptides, which have first been lyophilized with a non-volatile buffer (and optionally additional components such as a destabilizing excipient) in a dry powder that has very little content of residual moisture. Once prepared, the dry peptide powder is dissolved in a polar aprotic solvent, such as DMSO, NMP, ethyl acetate, or mixtures of these solvents. Thus, in accordance with the objects of the present invention, the stable low volume formulations of the present invention are injected, infused, or otherwise administered to an animal (e.g., human patient), without first diluting the formulation prior to injection as required by most of reconstitution products. As such, in preferred embodiments, the low volume formulations of the present invention are administerable without first being diluted, reconstituted or refrigerated.Definitions
[0042] For the purposes of this disclosure, the following terms have the following meanings:
[0043] The term "therapeutic agent" encompasses peptide compounds together with their pharmaceutically acceptable salts. Useful salts are known to those skilled in the art and include salts with inorganic acids, organic acids, inorganic bases or organic bases. Therapeutic agents useful in the present invention are those peptide compounds that affect a desired beneficial, often pharmacological, effect following administration to a human or animal, alone or in combination with other pharmaceutical excipients or inert ingredients.
[0044] The terms "peptide", "polypeptide" and/or "peptide compound" refer to polymers of up to about 80 amino acid residues linked together by amide bonds (CONH). Analogs, derivatives, agonists, antagonists and pharmaceutically acceptable salts of any of the peptide compounds disclosed herein are included in these terms. The terms also include peptides and/or peptide compounds that have D-amino acids, modified amino acids, derivatives or non-naturally occurring in the D or L configuration and/or peptomimetic units as part of their structure.
[0045] The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable solvent, suspending agent, or vehicle for delivering a peptide compound of the present invention to a mammal such as an animal or human. In a currently preferred embodiment, the pharmaceutically acceptable carrier is a polar aprotic solvent.
[0046] The term "polar aprotic solvent" means a polar solvent that does not contain acidic hydrogen and does not act as a hydrogen bond donor. Examples of polar aprotic solvents include, but are not limited to, dimethylsulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate, n-methyl pyrrolidone (NMP), dimethylacetamide (DMA), and propylene carbonate. The term polaraprotic solvent also encompasses mixtures of two or more polar aprotic solvents, for example a mixture of two or more of dimethylsulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate, n-methyl pyrrolidone (NMP), dimethylacetamide ( DMA), and propylene carbonate.
[0047] The term "pharmaceutically acceptable" ingredient, excipient, or component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.
[0048] The term "chemical stability" means that in relation to the therapeutic agent, an acceptable percentage of degradation products produced by chemical pathways such as oxidation or hydrolysis is formed. In particular, a formulation is considered chemically stable if no more than about 20% of chemical decomposition products are formed after one year of storage at the product's planned storage temperature (eg, room temperature); or storing the product at 30°C/60% relative humidity for one year; or storage of the product at 40°C/75% relative humidity for one month, preferably three months. In some embodiments, a chemically stable formulation has less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% chemical decomposition products formed after an extended period of storage at the product's intended storage temperature.
[0049] The term "physical stability" means that in relation to the therapeutic agent, an acceptable percentage of aggregates (eg, dimers, trimers, and larger forms) are formed. In particular, a formulation is considered to be physically stable if no more than about 15% of aggregates are formed after one year of storage at the product's intended storage temperature (e.g. room temperature), or product storage at 30°C/60°C. % relative humidity for one year; or storage of the product at 40°C/75% relative humidity for one month, preferably three months. In some embodiments, a physically stable formulation with less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of aggregates formed after an extended period of storage at the product's intended storage temperature.
[0050] The term "stable formulation" means that at least about 65% of the chemically and physically stable therapeutic agent remains after two months of storage at room temperature. Particularly preferred formulations are those in which at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% chemical therapeutic agent and physically stable subsist under these storage conditions. Especially preferred stable formulations are those which do not show degradation after sterilization by irradiation (eg gamma, beta or electron beam).
[0051] The term "consisting essentially of" is used herein to exclude any elements that substantially alter the essential properties of the stable formulations to which the term refers.
[0052] The term "bioavailability" is defined for the purposes of the present invention as the extent to which the therapeutic agent, such as a peptide compound, is absorbed from the formulation.
[0053] The term "systemic" means, in connection with the delivery or administration of a therapeutic agent, such as a peptide compound, to a subject, that the therapeutic agent is detectable to a biologically significant level in the subject's blood plasma.
[0054] The term "controlled release" is defined for purposes of the present invention as the release of the therapeutic agent at a rate such that concentrations in blood (eg, plasma) are maintained within the therapeutic range, but below toxic substances for a period of time of about one hour or longer, preferably 12 hours or longer.
[0055] The term "parenteral injection" refers to the administration of therapeutic agents, such as peptide compounds, by injection or through one or more layers of skin or mucosal membranes of an animal, such as a human. Standard parenteral injections are administered intradermally, subcutaneously, or intramuscularly in an animal, eg, a human patient. In some embodiments, a deep site is targeted for injection of a therapeutic agent as described herein.
[0056] The terms "treat" or "treatment" refer to delaying the onset, delaying or reversing progress, or alleviating or preventing the disease or condition to which the term applies, or one or more symptoms of such disease or condition.
[0057] The term "patient", "subject" or "individual", interchangeably, refers to a mammal, e.g. a human or a non-human mammal, e.g. a primate, dog, cat , bovine, sheep, swine, equine, rat, hamster, rabbit or guinea pig.Stable Peptide Formulations
[0058] In one aspect, the present invention provides a stable formulation for parenteral injection. Advantageously, once prepared, the formulation is stable over long periods of time, is ready for use without the need for reconstitution, and is functional over a range of temperatures. Furthermore, the stable formulation of the present invention is useful for the parenteral injection of any peptide that has limited or poor stability or solubility in an aqueous environment. In some embodiments, the formulations of the present invention enhance the physical stability of the peptide or peptides of the formulation, for example, by preventing or decreasing the formation of aggregates of the peptide or peptides.
[0059] In some embodiments, the formulation comprises: (a) a peptide or a salt thereof, wherein the peptide has been dried in a non-volatile buffer, and wherein the dried peptide has a pH memory that is nearly equal to the pH of the peptide in the non-volatile buffer; and (b) a polar aprotic solvent; wherein the moisture content of the formulation is less than 5%, and wherein the dry peptide maintains a pH memory that is nearly equal to the pH of the peptide in the non-volatile buffer when the dried peptide is reconstituted in the polar aprotic solvent.
[0060] In some embodiments, the formulation comprises: (a) a first peptide or a salt thereof, wherein the first peptide has been dried in a first non-volatile buffer, and wherein the first dried peptide has a first pH memory which is nearly equal to the pH of the first peptide in the first non-volatile buffer; (b) a second peptide or a salt thereof, wherein the second peptide has been dried in a second non-volatile buffer, and wherein the second dried peptide has a second pH memory that is nearly equal to the pH of the second peptide in the second non-volatile buffer; and (c) a polaraprotic solvent; where the moisture content of the formulation is less than 5%, where the first dry peptide maintains a pH memory that is nearly equal to the pH of the first peptide in the first non-volatile buffer when the first dry peptide is reconstituted in a polar aprotic solvent, and wherein the second dried peptide maintains a second pH memory that is nearly equal to the pH of the second peptide in the second non-volatile buffer when the second dried peptide is reconstituted in the polar aprotic solvent.
[0061] In some embodiments, the formulation essentially consists of: (a) a peptide or a salt thereof, wherein the peptide has been dried in a non-volatile buffer, and wherein the dried peptide has a pH memory that is nearly equal to that of the peptide. pH of the peptide in the non-volatile buffer; and (b) a polar aprotic solvent; where the moisture content of the formulation is less than 5%, and where the dry peptide maintains a pH memory that is nearly equal to the pH of the peptide in the non-volatile buffer, when the dry peptide is reconstituted in the polar aprotic solvent.Peptides
[0062] The stable formulations of the present invention comprise one, two, three, four, or more peptides or their salts, analogues and/or mixtures. Peptides (as well as their salts) suitable for use in the formulations of the present invention include, but are not limited to, glucagon, pramlintide, insulin, leuprolide, a luteinizing hormone releasing hormone (LHRH) agonist, parathyroid hormone (PTH) , amylin, botulinum toxin, hematide, an amyloid peptide, cholecystikinin, gastric inhibitory peptide, an insulin-like growth factor, growth hormone releasing factor, antimicrobial factor, glatiramer, glucagon-like peptide-1 (GLP-1), a GLP-1 agonist, exenatide, its analogues, and mixtures thereof. In some embodiments, the peptide is a hydrochloride salt or an acetate salt.
[0063] In a preferred embodiment, the peptide is glucagon or a glucagon analogue or peptidomimetic, or a salt thereof (eg, glucagon acetate). In another embodiment, the peptide is parathyroid hormone. In yet another embodiment, the peptide is eleuprolide. In yet another embodiment, the peptide is glatiramer. In other embodiments, the peptide is amylin or an amylinomimetic (eg, pramlintide). In still other embodiments, the peptide is insulin or an insulin analogue (e.g., Lispro). In some embodiments, the insulin or insulin analogue preparation is a low-zinc or zinc-free preparation.
[0064] In some embodiments, the formulation comprises two peptides, wherein the first peptide is amylin or an amylinomimetic and the second peptide is insulin or an insulin analogue. In some embodiments, the first peptide is pramlintide and the second peptide is insulin. In some embodiments, the first peptide is pramlintide and the second peptide is a low-zinc insulin preparation or a zinc-free insulin preparation.
[0065] In some embodiments, the formulation comprises two peptides, wherein the first peptide is glucagon and the second peptide is a glucagon-like peptide-1 (GLP-1) or a GLP-1 analogue or agonist (e.g., exenatide). In some embodiments, the first peptide is glucagon and the second peptide is GLP-1. In some embodiments, the first peptide is glucagon and the second peptide is exenatide.
[0066] Any suitable dosage of peptide or peptides can be administered using the formulations of the present invention. The dosage administered will, of course, vary depending on known factors, such as the pharmacodynamic characteristics of the particular peptide, the salt, or a combination thereof; the age, health, or weight of the individual; the nature and extent of symptoms; the metabolic characteristics of the therapeutic agent and the patient, the type of concurrent treatment; the frequency of treatment; or the desired effect. Generally, the peptide (or, where the stable formulation comprises two or more peptides, each of the peptides) is present in the formulation in an amount ranging from about 0.5 mg/mL to about 100 mg/mL (e.g. , about 0.5, 1, 2, 3, 4, 5, 6, 7, 8.9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 , 75, 80, 85, 90, 95, or 100 mg/ml).
[0067] In some embodiments, the peptide is present in the formulation in an amount ranging from about 0.5 mg/ml to about 60 mg/ml. In some embodiments, the peptide is present in the formulation in an amount ranging from about 10 mg/mL to about 50 mg/mL. In other embodiments, the peptide is present in the formulation in an amount ranging from about 20 mg/mL to about 50 mg/mL. In still other embodiments, the peptide is present in said formulation in an amount ranging from about 5 mg/ml to about 15 mg/ml. In yet other embodiments, the peptide is present in the formulation in an amount ranging from about 0.5 mg/ml to about 2 mg/ml. Again, it will be readily apparent to those of skill that the dosage of peptide can be varied depending on the peptide used and the disease, disorder, or condition being treated.
[0068] In preferred embodiments, the peptide is mixed with a non-volatile buffer, and optionally a stabilizing excipient, and then dried into a dry peptide powder. In embodiments where the stable formulation comprises two or more peptides, each of the peptides is separately mixed with a non-volatile buffer, and optionally a stabilizing excipient, and then dried into a dry peptide powder. Peptides are susceptible to hydrolysis in bonds with asparagine residues and methionine oxidation, so the use of non-volatile buffers in the formulations of the present invention beneficially affects chemical stability. As described in more detail below, although pH is not relevant in a polar aprotic solvent, the charge profile of a peptide in a polar aprotic solvent will affect its stability. The charge profile of a peptide in a polar aprotic solvent will be a function of the pH of the aqueous solution from which it was previously dried, ie there is a "pH memory" after dissolution or reconstitution in a polar aprotic solvent. To achieve the desired charge profile for a peptide dissolved in a polar aprotic solvent, the peptide is dried from a buffered aqueous solution at a pH that produces optimal stability, optimal solubility, and minimal degradation in the polar aprotic solvent. .
[0069] As such, the non-volatile buffers that are useful in the formulations described herein are those that are useful in establishing a pH of maximum stability, maximum solubility, and minimum degradation, as well as those that are useful in removing residual moisture or water content. of water of the dry peptide powder. Non-volatile buffers include those buffers that will not evaporate in a similar way to water after drying/lyophilization. Suitable non-volatile buffers include, for example, glycine buffers, citrate buffers, phosphate buffers, and mixtures thereof. In some embodiments, the non-volatile buffer is a glycine buffer or a citrate buffer. In some embodiments, the non-volatile buffer is a glycine buffer. In some embodiments, the non-volatile buffer is a mixture of glycine buffer and citrate buffer. In some embodiments, the non-volatile buffer is a mixture of citrate buffer and phosphate buffer.B. Stabilization Excipients
[0070] In certain preferred embodiments, the formulations described herein may be further stabilized to ensure the stability of the peptide incorporated therein. In some embodiments, the stability of the injectable formulation is increased by including one or more stabilizing agents or stabilizing excipients in the formulation prior to drying the peptide or peptides. In other embodiments, the stability of the injectable formulation is enhanced by reconstituting the dried peptide or peptides with a stabilizing agent or stabilizing excipient in the polar aprotic solvent.
[0071] In some embodiments, the stabilization excipient is a cryoprotectant. As shown below in the Examples section, the addition of a cryoprotectant, such as trehalose, protects the peptide formulations of the present invention against instability associated with freeze-thaw cycles. Furthermore, it was shown here that the addition of cryoprotective trehalose also promotes sharp thawing of a frozen peptide formulation. This sharp thaw property is surprisingly advantageous, particularly in medical emergencies, such as a severe hypoglycemic episode, where a peptide formulation of the present invention is frozen and must be administered rapidly. Thus, in another aspect of the present invention, the stable formulation has an improved freeze-thaw stability, an improved thaw rate, and/or an enhanced thaw profile.
[0072] In some embodiments, the stabilizing excipient is selected from sugars, starches, sugar alcohols, and mixtures thereof. Examples of suitable sugars for stabilizing excipients include, but are not limited to, trehalose, glucose, sucrose, etc. Examples of suitable starches for stabilizing excipients include, but are not limited to, hydroxyethyl starch (HES). Examples of suitable sugar alcohols for stabilizing excipients include, but are not limited to, mannitol and sorbitol. In some embodiments, the at least one stabilizing excipient (e.g., a sugar, a starch, a sugar alcohol, or a mixture thereof) is capable of increasing the stability of the peptide during a freeze-thaw process by increasing the thaw rate. formulation, or enhancing the formulation's thaw profile.
[0073] In some embodiments, the stabilization excipient is present in the formulation in an amount ranging from about 1% (w/v) to about 60% (w/v), from about 1% (w/v) to about 50% (w/v), from about 1% (w/v) to about 40% (w/v), from about 1% (w/v) to about 30% (w/v) , from about 1% (w/v) to about 20% (w/v), from about 5% (w/v) to about 60% (w/v), from about 5% (w/v) /v) to about 50% (w/v), from about 5% (w/v) to about 40% (w/v), from about 5% (w/v) to about 30% ( w/v), from about 5% (w/v) to about 20% (w/v), from about 10% (w/v) to about 60% (w/v), from about 10% (w/v) to about 50% (w/v), from about 10% (w/v) to about 40% (w/v), from about 10% (w/v) to about 30% (w/v), or from about 10% (w/v) to about 20% (w/v). In some embodiments, the stabilizing excipient is present in the formulation in an amount that is around 1%, around 5%, around 10%, around 15%, around 20%, around 25%, around 30%, around 35%, around 40%, around 45%, around 50%, around 55%, or around 60% (p/v).
[0074] In formulations comprising two or more peptides, in some embodiments each of the peptides is dried in a mixture comprising a non-volatile buffer and a stabilizing excipient. The mixtures of the non-volatile buffer and the stabilizing excipient may be the same for each peptide, or the non-volatile buffer, the stabilizing excipient, or both the non-volatile buffer and the stabilizing excipient that are used for drying each peptide may be different. In other embodiments, some, but not all, of the peptides may be dried in a mixture comprising a non-volatile buffer and a stabilizing excipient, while other peptides may be dried in a non-volatile buffer in the absence of a stabilizing excipient.
[0075] In some embodiments, the formulation further comprises additional stabilizing agents including, for example, antioxidants, chelators and preservatives. Examples of suitable antioxidants include, but are not limited to, ascorbic acid, cysteine, methionine, monothioglycerol, sodium thiosulfate, sulfites, BHT, BHA, ascorbyl palmitate, propyl gallate, N-acetyl-L-cysteine (NAC) and vitamin E. Examples of suitable chelators include, but are not limited to, EDTA, tartaric acid and its salts, glycerin, and citric acid and its salts. Examples of suitable preservatives include, but are not limited to, benzyl alcohols, methyl parabens, propyl parabens and mixtures thereof.
[0076] In some embodiments, the formulation further comprises a stabilizing polyol. Such formulations and materials are described, for example, in U.S. Patent Nos. 6,290,991 and 6,331,310, the contents of which are each incorporated herein by reference.C. Reconstitution of Dry Peptides
[0077] In the stable formulations of the present invention, once the peptide and non-volatile buffer (and optionally the destabilizing excipient) are dried to a powder, or where the formulation comprises two or more peptides, once each of the peptide and buffer non-volatile (each optionally also comprising a stabilizing excipient) is dried to a powder, the dry peptide powder, or the dry powder peptides, are dissolved or reconstituted in a polar aprotic solvent. In some embodiments, the polaraprotic solvent is selected from dimethylsulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate, n-methyl pyrrolidone (NMP), dimethylacetamide (DMA), propylene carbonate, and mixtures thereof. In some embodiments, the polar aprotic solvent is a mixture of two or more of dimethylsulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate, nmethylpyrrolidone (NMP), dimethylacetamide (DMA), and propylene carbonate. Dimethyl sulfoxide (DMSO), ethyl acetate, and n-methyl pyrrolidone (NMP), are particularly preferred polar aprotic solvents, each of which is a biocompatible solvent. In some embodiments, the polar aprotic solvent is dimethyl sulfoxide (DMSO). In other embodiments, the polar aprotic solvent is n-methyl pyrrolidone (NMP). In other embodiments, the polar aprotic solvent is a mixture of dimethyl sulfoxide (DMSO) and n-methyl pyrrolidone (NMP). In still other embodiments, the polar aprotic solvent is a mixture of dimethylsulfoxide (DMSO) and ethyl acetate. In some embodiments, the dry powdered peptide is reconstituted in a polar aprotic solvent that is "pure", ie, does not contain a co-solvent. In some embodiments, the dry peptide powder is reconstituted in a solution comprising a polar aprotic solvent and which does not contain water as a co-solvent.
[0078] In some embodiments, the formulations of the present invention further comprise at least one co-solvent that lowers the freezing point of the formulation. The co-solvent is a polar protic solvent. In some embodiments, the co-solvent is selected from ethanol, propylene glycol (PG), glycerol and mixtures thereof. In some embodiments, the co-solvent is ethanol or propylene glycol (PG). The co-solvent may be present in the formulation in an amount ranging from about 10% (w/v) to about 50% (w/v), for example, around 10%, around 15%, around 20 %, around 25%, around 30%, around 35%, around 40%, around 45%, or around 50% (w/v). In some embodiments, the co-solvent is present in the formulation in an amount ranging from about 10% (w/v) to about 50% (w/v), from about 10% (w/v) to about 40 %(w/v), from about 10% (w/v) to about 30% (w/v), from about 10%(w/v) to about 25% (w/v), of about 15% (w/v) to about 50% (w/v), from about 15% (w/v) to about 40% (w/v), from about 15% (w/v) ) to about 30% (w/v), or from about 15% (w/v) to about 25% (w/v). In some embodiments, at least one co-solvent lowers the freezing point of the formulation by at least 5°C, at least 10°C, at least 15°C, at least 20°C or more compared to a formulation of another identical way that does not comprise the co-solvent. In some embodiments, at least one co-solvent lowers the freezing point of the formulation by around 3°C, by around 2°C, by around 1°C, or by around 0°C or below.D. Moisture Content
[0079] The formulations of the present invention have very little residual moisture and thus the peptides in such formulations remain stable over long periods of time. In some embodiments, the stable formulations of the present invention have a moisture content that is less than 5%. In some embodiments, the moisture content is less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.25%, less than 0.2%, less than 0.15%, less than 0.1%, less than 0.075%, less than 0.05% , less than 0.025%, or less than 0.01%. In some preferred embodiments, the moisture content of the formulations of the present invention is from about 0.01% to about 5%, from about 0.01% to about 4%, from about 0.01% to about 3% , from about 0.01% to about 2%, from about 0.01% to about 1.5%, or from about 0.01% to about 1%. In other preferred embodiments, the moisture content of the formulations of the present invention is from about 0.1% to about 5%, from about 0.1% to about 4%, from about 0.1% to about 3% %, from about 0.1% to about 2%, from about 0.1% to about 1.5%, or from about 0.1% to about 1%. In other preferred embodiments, the moisture content of the formulations of the present invention is from about 0.25% to about 5%, from about 0.25% to about 4%, from about 0.25% to about from 3%, from about 0.25% to about 2%, or from about 0.25% to about 1.5%. In still other preferred embodiments, the moisture content of the formulations is from about 0.5% to about 1%.E. pH memory
[0080] The "pH memory" of a peptide is the resulting charge profile (protonation state) after drying the peptide from a buffered aqueous solution (eg from a non-volatile buffer). The protonation state, and thus the solubility and stability of the peptides, in non-aqueous solvents of very low or zero moisture are affected by the aqueous pH of the peptide solution prior to drying and by the drying conditions employed. When the peptide is dried in a kind of buffer in which both acidic and basic components are non-volatile, the pH memory of the dried peptide will be nearly equal to the pH of the peptide in the non-volatile buffer. See, for example, Enzymatic Reactions in Organic Media, Koskinen, A.M.P., and Klibanov, A.M., eds., Springer (1996). In addition, the pH of the aqueous buffered solution (e.g., non-volatile buffer) in which the peptide is dried can be optimized to produce a pH memory for the peptide that results in optimal peptide stability, maximum solubility, and minimal degradation when the dried peptide is subsequently reconstituted in a polar aprotic solvent. Since polar aprotic solvents do not have exchangeable protons, when the dry peptide is reconstituted in a polar aprotic solvent, the reconstituted formulation will maintain the solubility and stability characteristics of optimal pH memory.
[0081] For stable formulations comprising two, three, four or more peptides, each peptide is dried so that it has its own pH memory that is optimized with respect to maximum solubility, maximum stability and minimum degradation. where there are two or more peptides in the formulation, the pH memory range of the first peptide may partially overlap with the pH memory range of the second peptide (for example, the pH memory range of the first peptide may be from about 4.0 to about 6.0, and the pH memory range of the second peptide may be from about 6.0 to about 8.0), or the pH memory range of the first peptide may not overlap with the pH memory range of the second peptide (for example, the pH memory of the first peptide can be from about 4.0 to about 5.0, and the pH memory of the second peptide can be from about 6.0 to about 8.0).
[0082] The pH memory of a peptide can be measured in several ways. In one method, the pH memory of a peptide is measured by reconstituting the dried peptide in unbuffered water and measuring the pH of the reconstituted peptide with a pH indicator such as pH paper or a calibrated pH electrode. Alternatively, the pH memory of a peptide can be determined by a peptide that has been reconstituted in the polar aprotic solvent (eg DMSO) by adding at least 20% water to the polar aprotic solvent (eg DMSO) and measuring the pH common pH indicator. See, for example, Baughman and Kreevoy, "Determination of Acidity in 80% Dimethyl Sulfoxide-20% Water," Journal of Physical Chemistry, 78(4):421-23 (1974). Measuring pH in a polar aprotic solvent-water solution may require a small correction (ie, no more than 0.2 pH unit as per Baughman and Kreevoy, supra).
[0083] In some embodiments, a dried peptide has a pH memory that is nearly equal to the pH of the peptide in the nonvolatile buffer from which it was dried when the pH memory of the peptide when reconstituted in a polar aprotic solvent is within one unit of pH of the pH of the peptide in the non-volatile buffer from which it is dried (so, for example, for a peptide having a pH of 3.0 in the non-volatile buffer from which the peptide is dried, a pH memory for the peptide of 2.0 0 to 4.0, when reconstituted in the polar aprotic solvent, should be within one pH unit, and thus the pH memory of the dry peptide would be nearly equal to the pH of the peptide in the non-volatile buffer). In some embodiments, a dried peptide has a pH memory that is nearly equal to the pH of the peptide in the nonvolatile buffer from which it was dried when the pH memory of the peptide is reconstituted in a polar aprotic solvent is within half of a pH unit of the peptide's pH in the non-volatile buffer from which it is dried (so, for example, for a peptide having a pH of 3.0 in the non-volatile buffer from which the peptide is dried, a pH memory for the peptide 2.5 to 3.5, when reconstituted in the polar aprotic solvent, should be within half a pH unit, and thus the pH memory of the dry peptide would be nearly equal to the pH of the peptide in the non-volatile buffer).
[0084] In some embodiments, the stable formulation peptide has a pH memory of about 1.5 to about 2.5. In some embodiments, the peptide of the stable formulation has a pH memory of from about 2.0 to about 3.0. In some embodiments, the peptide of the stable formulation has a pH memory of about 2.0 to about 4.0. In some embodiments, the peptide of the stable formulation has a pH memory of from about 2.5 to about 4.0. In certain embodiments, the peptide of the stable formulation has a pH memory of about 2.5 to about pH 3.5. In certain embodiments, the peptide of the stable formulation has a pH memory of from about 3.0 to about 5.0. In some embodiments, the peptide of the stable formulation has a pH memory of about 3.0 to about 4.5. In some embodiments, the peptide of the stable formulation has a pH memory of from about 4.0 to about 5.0. In certain embodiments, the peptide of the stable formulation has a pH memory of about 4.0 to about 6.0. In some embodiments, the peptide of the stable formulation has a pH memory of from about 6.0 to about 8.0. In certain embodiments, the peptide of the stable formulation has a pH memory of from about 6.5 to about 8.0. In some embodiments, the peptide of the stable formulation has a pH memory of about 6.5 to about 7.5 pH. In some embodiments, the peptide of the stable formulation has a pH memory of about 6.5 to about 9.0. In some embodiments, the peptide of the stable formulation has a pH memory of from about 7.0 to about 9.0. In some embodiments, the peptide of the stable formulation has a pH memory of about 7.5 to about 9.0. In certain embodiments, the peptide of the stable formulation has a pH memory of about 8.0 to about 10.0. In some embodiments, the peptide of the stable formulation has a pH memory of about 8.5 to about 10.0. In some embodiments, the pH memory of a peptide can be around 1.5, around 2.0, around 2.5, around 3.0, around 3.5, around 4 .0, around 4.5, around 5.0, around 5.5, around 6.0, around 6.5, around 7.0, around 7.5, around 8.0, around 8.5, around 9.0, around 9.5, or around 10.0.F. Exemplary Formulations
[0085] In some particular embodiments, the present invention provides a stable glucagon formulation, the glucagon formulation comprising: a glucagon peptide or a salt thereof (e.g. glucagon acetate), wherein the glucagon has been dried in a selected non-volatile buffer of a glycine buffer, a citrate buffer, a phosphate buffer, and mixtures thereof, and wherein the dry glucagon has a pH memory that is from about 2.0 to about 3.0; and a polar aprotic solvent selected from group consisting of dimethyl sulfoxide (DMSO), ethyl acetate, n-methyl pyrrolidone (NMP), and mixtures thereof; wherein the moisture content of the formulation is less than 5%, and wherein the dry glucagon maintains a pH memory of about 2.0 to about 3.0 when the dry glucagon is reconstituted in the polar aprotic solvent. In some embodiments, the glucagon is present in the formulation in an amount ranging from about 0.5 mg/ml to about 100 mg/ml, or from about 1 mg/ml to about 50 mg/ml. In some embodiments, the moisture content of the formulation is less than about 2%, less than about 1%, less than about 0.5%, or less than about 0.01%. In some embodiments, the moisture content of the formulation is from about 0.01% to about 3%. In some embodiments, the formulation further comprises a stabilizing excipient selected from sugars (e.g. trehalose), starches (e.g., hydroxyethyl starch (HES)), and mixtures thereof. The stabilizing excipient may be present in the formulation in an amount ranging from about 1% (w/v) to about 60% (w/v). In some embodiments, the formulation further comprises a co-solvent which lowers the freezing point of the formulation, wherein the co-solvent is selected from ethanol, propylene glycol, glycerol, and mixtures thereof. The co-solvent may be present in the formulation in an amount ranging from about 10% (w/v) to about 50% (w/v).
[0086] In other particular embodiments, the present invention provides a stable glucagon formulation, the glucagon formulation comprising: a glucagon peptide or a salt thereof (or the glucagon analogue or peptidomimetic); and a polar aprotic solvent selected from the group consisting of dimethyl sulfoxide (DMSO), nmethyl pyrrolidone (NMP) and mixtures thereof; where the moisture content of the formulation is less than 3%. In preferred embodiments, the moisture content of the formulation is less than 2%, less than 1%, less than 0.5%, and less than 0.25%. In other preferred embodiments, the moisture content is from about 0.25% to about 3%, preferably from about 0.25% to about 2%, more preferably from about 0.25% to about 1.5% , more preferably from about 0.25% to about 1%, most preferably from about 0.5% to about 1%.
[0087] In other particular embodiments, the stable glucagon formulation further comprises a non-volatile buffer and a stabilizing excipient which is a sugar, a starch, or a sugar alcohol. For example, in some embodiments, the glucagon formulation further comprises a glycine and mannitol buffer, or a decitrate and mannitol buffer, or a phosphate and mannitol buffer. In some embodiments, the glucagon formulation further comprises a glycine and trehalose buffer, or a citrate and trehalose buffer, or a phosphate and trehalose buffer. In these embodiments, the polar aprotic solvent can be DMSO, NMP, ethyl acetate, or a mixture thereof. For example, in a preferred embodiment, the polar aprotic solvent is DMSO, and the non-volatile buffer is a glycine buffer. In another preferred embodiment, the polar aprotic solvent is DMSO, the nonvolatile buffer is a citrate buffer, and the stabilizing excipient is mannitol. In other preferred embodiments, the polar aprotic solvent is DMSO, the nonvolatile buffer is a glycine buffer, and the stabilizing excipient is trehalose. In yet another preferred embodiment, the polar aprotic solvent is DMSO, and the non-volatile buffer is a citrate buffer. In yet another preferred embodiment, the polar aprotic solvent is NMP, and the non-volatile buffer is a glycine buffer.
[0088] In other particular embodiments, the present invention provides a stable formulation comprising: glucagon, or a salt (e.g., glucagon acetate), wherein the glucagon has been dried in a non-volatile buffer, and wherein the dried glucagon has a memory that is nearly equal to the pH of glucagon in the nonvolatile buffer selected from a glycine buffer, a citrate buffer, a phosphate buffer, and mixtures thereof, where the pH memory of dry glucagon is about 2.0 to about 3.0; and a polaraprotic solvent selected from dimethyl sulfoxide (DMSO), n-methyl pyrrolidone (NMP), ethyl acetate, and mixtures thereof; wherein the moisture content of the formulation is less than 1%, and wherein the dry glucagon maintains a pH memory that is nearly equal to the pH of the glucagon in the non-volatile buffer when the dry glucagon is reconstituted in the polaraprotic solvent. In some embodiments, the glucagon formulation further comprises a co-solvent which lowers the freezing point of the formulation, wherein the co-solvent is selected from ethanol, propylene glycol, glycerol and mixtures thereof. In some embodiments, the glucagon formulation further comprises a stabilizing excipient selected from sugars, starches and mixtures thereof. In some embodiments, the glucagon is present in the formulation in an amount ranging from about 1 mg/ml to about 50 mg/ml.
[0089] In other particular embodiments, the present invention provides a stable glucagon formulation, the glucagon formulation essentially consisting of: a glucagon peptide or a salt thereof (e.g., glucagon acetate), wherein the glucagon has been dried in a non-toxic buffer. volatiles selected from a glycine buffer, a citrate buffer, a phosphate buffer, and mixtures thereof, wherein the dried glucagon has a pH memory that is from about 2.0 to about 3.0; and a polar aprotic solvent selected from the group consisting of dimethylsulfoxide (DMSO), ethyl acetate, n-methylpyrrolidone (NMP), and mixtures thereof; wherein the moisture content of the formulation is less than 5%, and wherein the dry glucagon maintains a pH memory of about 2.0 to about 3.0 when the dry glucagon is reconstituted in the polar aprotic solvent.
[0090] In still other particular embodiments, the present invention provides a stable glucagon formulation, the glucagon formulation essentially consisting of: a glucagon peptide or a salt thereof (e.g., glucagon acetate), wherein the glucagon has been dried in a buffer nonvolatile selected from a glycine buffer, a citrate buffer, a phosphate buffer, and mixtures thereof, and wherein dry glucagon has a pH memory that is from about 2.0 to about 3.0, and a mixture of a polar aprotic solvent and a co-solvent which lowers the freezing point of the formulation, wherein the polar aprotic solvent is selected from the group consisting of dimethyl sulfoxide (DMSO), ethyl acetate, n-methyl pyrrolidone (NMP), and mixtures thereof and wherein the co-solvent is selected from ethanol, propylene glycol, glycerol and mixtures thereof; wherein the moisture content of the formulation is less than 5%, and wherein the dry glucagon maintains a pH memory of about 2.0 to about 3.0 when the dry glucagon is reconstituted in the polar aprotic solvent.
[0091] In other particular embodiments, the present invention provides a stable glucagon formulation, the glucagon formulation essentially consisting of: a glucagon peptide or a salt thereof (e.g., glucagon acetate), wherein the glucagon has been dried in a mixture of a non-volatile buffer and a stabilizing excipient, wherein the non-volatile buffer is selected from a glycine buffer, a citrate buffer, a phosphate buffer, and mixtures thereof, and the stabilizing excipient is selected from sugars (e.g., trehalose ), starches (e.g., hydroxyethyl starch (HES)), and mixtures thereof, and wherein the dry glucagon has a pH memory that is from about 2.0 to about 3.0, and a selected polaraprotic solvent from the group consisting of dimethyl sulfoxide (DMSO), ethyl acetate, n-methyl pyrrolidone (NMP), and mixtures thereof; in which the moisture content of the formulation is less than 5%, and in which the dry glucagon maintains the memory of pH from about 2.0 to about 3.0 when glu dry cagon is reconstituted with the polar aprotic solvent.
[0092] In yet other particular embodiments, the present invention provides a stable formulation comprising: insulin, wherein the insulin has been dried in a first non-volatile buffer selected from a glycine buffer, a citrate buffer, a phosphate buffer, and mixtures thereof , and where the dry insulin has a first pH memory that is nearly equal to the pH of insulin in the first nonvolatile buffer, where the first pH memory is from about 1.5 to about 2.5, or from about 6.0 to about 8.0; pramlintide, in which opramlintide has been dried in a second non-volatile buffer selected from a glycine buffer, a citrate buffer, a phosphate buffer, and mixtures thereof, and in which the dried pramlintide has a second pH memory that is nearly equal to the pH of pramlintide in the second non-volatile buffer, wherein the second pH memory is from about 3.0 to about 5.0, or from about 4.0 to about 6.0, and an aprotic polar solvent selected from dimethyl sulfoxide (DMSO ), n-methyl pyrrolidone (NMP), ethyl acetate, and mixtures thereof; wherein the moisture content of the formulation is less than 1%, wherein the dry insulin maintains a first pH memory that is nearly equal to the pH of insulin in the first non-volatile buffer when the dry insulin is reconstituted in a polar aprotic solvent, and wherein the dry pramlintide maintains a second pH memory that is nearly equal to the pH of the pramlintide in the second non-volatile buffer when the dried pramlintide is reconstituted in a polar aprotic solvent. In some embodiments, the insulin and pramlintide formulation further comprises a co-solvent that lowers the freezing point of the formulation, wherein the co-solvent is selected from ethanol, propylene glycol, glycerol and mixtures thereof. In some embodiments, one or both of the insulin in the first non-volatile buffer and the pramlintide in the second non-volatile buffer further comprise a stabilizing excipient selected from sugars, starches, and mixtures thereof. In certain embodiments, the first non-volatile buffer and the second non-volatile buffer are the same. In some embodiments, the first non-volatile buffer and the second non-volatile buffer are different. In some embodiments, each of the epramlintide insulin is present in the formulation in an amount ranging from about 1 mg/ml to about 50 mg/ml. In some embodiments, the first pH memory is from about 1.5 to about 2.5. In some embodiments, the first pH memory is from about 6.0 to about 8.0. In certain embodiments, the second pH memory is from about 3.0 to about 5.0. In some embodiments, the second pH memory is from about 4.0 to about 6.0. In certain embodiments, the first pH memory is from about 1.5 to about 2.5 and the second pH memory is from about 3.0 to about 5.0.IV. Methods of Producing Stable Peptide Formulations
[0093] In yet another aspect, the present invention provides a process for preparing a stable formulation for parenteral injection. In some embodiments, the process comprises: drying a peptide and a non-volatile buffer to a dry peptide powder, and reconstituting the dry peptide powder with an aprotic polar solvent, rendering the formulation stable, where the moisture content of the formulation stable is less than 5%. In some embodiments, the dry peptide powder has a pH memory that is nearly equal to the pH of the peptide in the nonvolatile buffer, and the dry powder peptide maintains a pH memory that is nearly equal to the pH of the peptide in the nonvolatile buffer when the of dry peptide powder is reconstituted in the polar aprotic solvent.
[0094] The process for producing stable peptide formulations can be used to formulate any peptide that has limited or poor stability or solubility in an aqueous environment. Peptides (or salts thereof) suitable for use in the formulations of the present invention include, but are not limited to, glucagon, insulin, leuprolide, a luteinizing hormone releasing hormone (LHRH), agonists, pramlintide, parathyroid hormone (PTH) ), amylin, botulinum toxin, a conotoxin, hematide, an amyloid peptide, cholecystikinin, gastric inhibitory peptide, an insulin-like growth factor, growth hormone releasing factor, antimicrobial factor, glatiramer, glucagon-like peptide-1 (GLP) -1), a GLP-1 agonist, exenatide, and its analogues. In a preferred embodiment, the peptide is glucagon or a glucagon analogue or peptidomimetic. In another embodiment, the peptide is parathyroid hormone. In yet another embodiment, the peptide is leuprolide. In yet another embodiment, the peptide is glatiramer.
[0095] In some embodiments, two, three, four or more peptides are formulated into a stable formulation. In embodiments where two or more peptides are formulated into the stable formulation, each peptide is separately dried with a non-volatile buffer on a dry peptide powder, and each dry peptide powder has a pH memory that is nearly equal to the pH of the peptide in the non-volatile buffer ( that is, the first peptide has a first pH buffer that is nearly equal to the pH of the first peptide in the first nonvolatile buffer, and the second peptide has a second pH buffer that is nearly equal to the pH of the second peptide in the second nonvolatile buffer). The two or more dry powder peptides are reconstituted with a polar aprotic solvent, thus making the formulation stable, where the moisture content of the stable formulation is less than 5%, and where each dry powder peptide maintains a pH memory that is nearly equals the pH of the peptide in the non-volatile buffer when the dry peptide powder is reconstituted in a polar aprotic solvent (i.e., the first dry peptide maintains the first pH memory when the first dry peptide is reconstituted in the polar aprotic solvent, and the second dry peptide maintains the second pH memory when the second dry peptide is reconstituted in a polar aprotic solvent).
[0096] In the process for preparing stable peptide formulations, suitable non-volatile buffers include, for example, glycine buffers, citrate buffers, phosphate buffers, and mixtures thereof. In some embodiments, the non-volatile buffer is a glycine buffer or a citrate buffer. In certain embodiments, the non-volatile buffer is a mixture of a citrate buffer and a phosphate buffer. In some embodiments, the peptide is mixed with either the non-volatile buffer or a stabilizing excipient (such as a sugar, a starch, or mixtures thereof) and then dried into a dry peptide powder. In other embodiments, the stabilizing excipient (such as a sugar, a starch, a sugar alcohol, or mixtures thereof) is added to the peptide reconstituted in the polaraprotic solvent. In certain embodiments, the stabilizing excipient is present in the formulation in an amount ranging from about 1% (w/v) to about 60% (w/v), for example, around 1%, around 5% , around 10%, around 15%, around 20%, around 25%, around 30%, around 35%, around 40%, around 45%, around 50%, around 55%, or around 60% (w/v). In some embodiments, the stabilizing excipient is trehalose. In some embodiments, the stabilizing excipient is HES. In some embodiments, the stabilization excipient is a mixture of trehalose and HES.
[0097] As explained above, when the peptide is mixed with the non-volatile buffer, the non-volatile buffer is selected such that the peptide has a pH of maximum stability/minimum degradation in the aqueous environment. Once dry, the peptide will have a pH memory of maximum stability/minimum degradation and will retain this pH memory when dissolved or reconstituted in the polar aprotic solvent. As such, in one embodiment, the pH of the non-volatile buffer is such that the dry peptide powder has a pH memory of about 2 to about 3. In another embodiment, the pH of the non-volatile buffer is such that the peptide dry powder has a memory pH of about 4 to about 6. In yet another embodiment, the pH of the non-volatile buffer is such that the dry powder peptide has a memory pH of about 4 to about 5. In yet another embodiment, the pH of the non-volatile buffer is such that the dry peptide powder has a pH memory of about 6 to about 8.
[0098] Once the peptide and non-volatile buffer (and optionally other components, such as a stabilizing excipient, which are added to the peptide and non-volatile buffer before drying) are dried to a powder, the dry peptide powder is dissolved or reconstituted in a polar aprotic solvent as described herein (eg, dimethyl sulfoxide (DMSO), n-methyl pyrrolidone (NMP), ethyl acetate, and mixtures thereof). In some embodiments, the polaraprotic solvent is dimethyl sulfoxide (DMSO). In other embodiments, the polar aprotic solvent is n-methyl pyrrolidone (NMP).
[0099] In some embodiments, the step of reconstituting the dry peptide powder comprises diluting or reconstituting the dry peptide with a mixture comprising a polaraprotic solvent and a co-solvent that lowers the freezing point of the formulation. In some embodiments, the co-solvent is selected from ethanol, propylene glycol, glycerol and mixtures thereof. In some embodiments, the co-solvent is present in the formulation in an amount ranging from about 10% (w/v) to about 50% (w/v), for example, around 10%, around 15% , around 20%, around 25%, around 30%, around 35%, around 40%, around 45%, or around 50% (p/v).
[00100] The formulations of the present invention have very little residual moisture and thus the peptides in such formulations remain stable over extended periods of time. In preferred embodiments, the moisture content of the stable formulation that is prepared by the process of the present invention is less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than than 0.4%, less than 0.3%, less than 0.25%, less than 0.2%, less than 0.15%, less than 0.1%, less than 0.075% , less than 0.05%, less than 0.025%, or less than 0.01%.
[00101] In the above process, the drying of the peptide compound with the non-volatile buffer (and optionally the stabilizing excipient) is carried out using spray drying techniques, freeze drying techniques or lyophilization techniques. Spray drying techniques are well known to those skilled in the art. Spray drying includes the steps of atomizing a solution containing one or more solids (e.g., a therapeutic agent) through a rotating disk, mouthpiece, or other device, followed by evaporating the solvent from the droplets. The nature of the powder that results is a function of several variables including the initial solute concentration, the size distribution of droplets produced, and the rate of solute removal. The particles produced may comprise aggregates of the primary particles consisting of crystals and/or amorphous solids, depending on the data rate and solvent removal conditions.
[00102] A spray drying process for preparing ultra-fine powders of biological macromolecules such as proteins, oligopeptides, high molecular weight polysaccharides, and nucleic acids is described, for example, in US Patent No. 6,051,256. Freeze-drying procedures are well known in the art, and are described, for example, in US Patent No. 4,608,764 and US Patent No. 4,848,094. Spray-freeze-drying processes are described, for example, in U.S. Patent No. 5,208,998. Other spray drying techniques are described, for example, in U.S. Patent Nos. 6,253,463, 6,001,336, 5,260,306; and International PCT Publication Nos. WO 91/16882 and WO96/09814.
[00103] Lyophilization techniques are well known to those skilled in the art. Lyophilization is a dehydration technique that occurs while a product is in a frozen state (sublimation of ice under a vacuum) and under a vacuum (drying by gentle heat). These conditions stabilize the product. and minimize oxidation and other degradative processes. Freeze-drying conditions allow the process to be carried out at low temperatures, so thermally unstable products can be preserved. The steps in freeze drying include pretreatment, freezing, primary drying, and secondary drying. Pretreatment includes any method of treating the product before freezing. This may include concentration of the product, review of the formulation (i.e., the addition of components to increase stability and/or improve processing), decreasing a solvent with high vapor pressure, or increasing surface area. . Pretreatment methods include: freeze concentration, solution phase concentration, and specific formulation to preserve product appearance or to provide lyoprotection for reactive products, and are described, for example, in U.S. Patent No. 6,199,297. "Standard" lyophilization conditions are described, for example, in US Patent No. 5,031,336, and in "Freeze Drying of Pharmaceuticals" (DeLuca, Patrick P., J. Vac. Sci. Technol., Vol. 14, No. 1, January/February 1977); and "The Lyophilization of Pharmaceuticals: Literature Review" (Williams, N.A., and G.P. Polli, Journal of Parenteral Science and Technology, Vol. 38, No. 2, March/April 1984).
[00104] In certain preferred embodiments, the lyophilization cycle is partially performed above the glass transition temperature (Tg) of the therapeutic agent formulation to induce a mass collapse to form a dense cake containing residual moisture. In other modes, the freeze-drying cycle is carried out below the glass transition temperature in order to avoid collapse in order to achieve complete drying of the particles.V. Therapeutic Methods
[00105] In another aspect, the present invention provides methods of treating diseases or conditions by administering to a subject a stable formulation as described herein in an amount effective to treat, alleviate or prevent the disease, condition or disorder. In some embodiments, the disease, condition or disorder to be treated with a stable formulation of the present invention is a diabetic condition. Examples of diabetic conditions include, but are not limited to, type 1 diabetes, type 2 diabetes, gestational diabetes, prediabetes, hyperglycemia, hypoglycemia and metabolic syndrome. In some embodiments, the disease, condition, or disorder is hypoglycemia. In some embodiments, the disease, condition, or disorder is diabetes.
[00106] In some embodiments, a therapeutic method of the present invention comprises treating hypoglycemia by administering to a subject having hypoglycemia a stable formulation as described herein, in an amount effective to treat hypoglycemia. In some embodiments, the subject is administered a stable formulation comprising glucagon.
[00107] In some embodiments, a therapeutic method of prediction 870210098166, of 10/25/2021, p. This invention comprises treating diabetes by administering to a subject having diabetes a stable formulation as described herein, in an amount effective to treat diabetes. In some embodiments, the subject is administered a stable formulation comprising insulin. In some embodiments, the subject is administered a stable formulation comprising pramlintide. In some embodiments, the subject is administered a stable formulation comprising insulin and pramlintide. In certain embodiments, the subject is administered a stable formulation comprising exenatide. In certain embodiments, the subject is administered a stable formulation comprising glucagon and exenatide.
[00108] Dosages administered for the peptide drugs as described herein for the treatment of a disease, condition, disorder (e.g., a diabetic condition, e.g., hypoglycemia or diabetes) are in accordance with the planned dosages and regimens practiced by those of skill in technique. General guidance for appropriate dosages of all pharmacological agents used in the present methods is provided in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 11th Edition, 2006, supra, and in a Physicians' Desk Reference (PDR), for example, in the 65th (2011) or 66th (2012) Eds., PDR Network, LLC, each of which is hereby incorporated by reference. The appropriate dosage of a peptide drug for the treatment of a disease, condition, or disorder as described herein will vary depending upon a number of factors, including the formulation of the composition, the patient's response, the severity of the condition, the individual's weight, and the judgment of the treating physician. prescribe. Effective doses of the described formulations deliver a clinically effective amount of a peptide drug. The dosage may be increased or decreased over time as required by an individual patient.
[00109] Determining an effective amount or dose is well within the ability of those skilled in the art, especially in light of the detailed disclosure provided herein. Generally, formulations to deliver these doses may contain one, two, three, four, or more peptides or peptide analogues (collectively "peptide" unless peptide analogues are expressly excluded), where each peptide is present in a concentration of about 0.1 mg/ml up to the solubility limit of the peptide in the formulation. This concentration is preferably from about 1mg/ml to about 100mg/ml, for example around 1mg/ml, around 5mg/ml, around 10mg/ml, around 15mg /ml, around 20 mg/ml, around 25 mg/ml, around 30 mg/ml, around 35 mg/ml, around 40 mg/ml, around 45 mg/ml , around 50 mg/ml, around 55 mg/ml, around 60 mg/ml, around 65 mg/ml, around 70 mg/ml, around 75 mg/ml, around 80 mg/ml, around 85 mg/ml, around 90 mg/ml, around 95 mg/ml, or around 100 mg/ml.
[00110] The formulations of the present invention may be for subcutaneous, intradermal or intramuscular administration (eg, by injection or by infusion). In some embodiments, the formulation is administered subcutaneously.
[00111] The formulations of the present disclosure are administered by infusion or by injection using any suitable device. For example, a formulation of the present invention may be placed within a syringe, a pen injection device, an auto-injector device, or an injection device. bomb. In some embodiments, the injection device is a multi-dose injection pump device or a multi-dose auto-injector device. The formulation is presented in the device in such a way that the formulation is easily able to flow out of the needle after actuation of an injection device, such as an autoinjector, in order to deliver the peptide drugs. Suitable pen/auto-injector devices include, but are not limited to, those pen/auto-injector devices manufactured by Becton-Dickenson, Swedish Healthcare Limited (SHL Group), YpsoMed Ag, and others. Suitable pump devices include, but are not limited to, those pump devices manufactured by Tandem Diabetes Care, Inc., Delsys Pharmaceuticals and others.
[00112] In some embodiments, the formulations of the present invention are provided ready for administration in a vial, cartridge, or pre-filled syringe.
[00113] In another aspect, the present invention provides the use of a stable formulation as described herein for formulating a drug for the treatment of any disease, condition or disorder that can be treated with the peptide of the formulation. In some embodiments, the stable formulation is used to formulate a drug for the treatment of a diabetic condition, for example, type 1 diabetes, type 2 diabetes, gestational diabetes, prediabetes, hyperglycemia, hypoglycemia, or metabolic syndrome.
[00114] In some embodiments, the stable formulation is used for the formulation of a drug for the treatment of hypoglycemia. In some embodiments, the stable formulation comprises glucagon or a salt thereof (eg, glucagon acetate). In some embodiments, the stable formulation comprises glucagon and exenatide.
[00115] In some embodiments, the stable formulation is used to formulate a drug for the treatment of diabetes. In some embodiments, the stable formulation comprises insulin. In some embodiments, the stable formulation comprises exenatide. In certain embodiments, the stable formulation comprises pramlintide. In some embodiments, the stable formulation comprises insulin and pramlintide.VI. kits
[00116] In another aspect, the present invention provides kits for treating a disease, condition or disorder as described herein. In some embodiments, the kit comprises: a stable formulation comprising one, two, three, four or more peptides or their salts, wherein the peptide has been dried in a non-volatile buffer, and wherein the dried peptide has a pH memory that is nearly equal to the pH of the peptide in the non-volatile buffer; and a polar aprotic solvent; wherein the moisture content of the formulation is less than 5%, and wherein the dry peptide maintains a pH memory that is nearly equal to the pH of the peptide in the non-volatile buffer when the dried peptide is reconstituted in the polar aprotic solvent ; and a syringe for administering the stable formulation to the subject.
[00117] In some embodiments, the kit comprises a stable formulation of glucagon as described herein for use in treating hypoglycemia in a subject. In some embodiments, the kit comprises a glucagon formulation comprising: glucagon or a salt thereof (e.g., glucagon acetate), wherein the glucagon has been dried in a non-volatile buffer; and wherein dry glucagon has a pH memory that is nearly equal to the pH of glucagon in the nonvolatile buffer selected from a glycine buffer, a decitrate buffer, a phosphate buffer, and mixtures thereof, wherein the pH memory of glucagon dry is about 2.0 to about 3.0; and an aprotic polar solvent selected from dimethyl sulfoxide (DMSO), n-methyl pyrrolidone (NMP), ethyl acetate, and mixtures thereof; wherein the moisture content of the formulation is less than 1%, and wherein the glucagon maintains a pH memory that is nearly equal to the pH of the glucagon in the non-volatile buffer when the dry glucagon is reconstituted in the polar aprotic solvent. In certain embodiments, the glucagon formulation further comprises a co-solvent which lowers the freezing point of the formulation, wherein the co-solvent is selected from ethanol, propylene glycol, glycerol and mixtures thereof. In some embodiments, the glucagon formulation further comprises a stabilizing excipient selected from sugars, starches and mixtures thereof. In some embodiments, glucagon is present in the formulation in an amount ranging from about 1 mg/ml to about 50 mg/ml.
[00118] In some embodiments, the kit comprises a stable formulation of insulin and pramlintide as described herein for use in treating diabetes in an individual. In some embodiments, the kit comprises an insulin and pramlintide formulation comprising: insulin, wherein the insulin has been dried in a first non-volatile buffer selected from a glycine buffer, a citrate buffer, a phosphate buffer, and mixtures thereof, and in that dry insulin has a first pH memory that is nearly equal to the pH of insulin in the first non-volatile buffer, where the first pH memory is from about 1.5 to about 2.5 or from about 6.0 to about 6.0 to about 8.0; pramlintide, in which the pramlintide has been dried in a second non-volatile buffer selected from a glycine buffer, a citrate buffer, a phosphate buffer, and mixtures thereof, and in which the dried pramlintide has a second pH memory that is nearly equal the pH of pramlintide in the second non-volatile buffer, wherein the second pH buffer is from about 3.0 to about 5.0 or from about 4.0 to about 6.0; a polar aprotic solvent selected from dimethyl sulfoxide (DMSO), nmethyl pyrrolidone (NMP), ethyl acetate, and mixtures thereof; where the moisture content of the formulation is less than 1%, where the dry insulin maintains a first pH memory that is nearly equal to the pH of insulin in the first non-volatile buffer when the dry insulin is reconstituted in the polar aprotic solvent, and where dry pramlintide retains a second pH memory that is nearly equal to the pH of pramlintide in the second non-volatile buffer when dry pramlintide is reconstituted in a polar aprotic solvent. In some embodiments, the insulin and pramlintide formulation further comprises a co-solvent which lowers the freezing point of the formulation, wherein the co-solvent is selected from ethanol, propylene glycol, glycerol and mixtures thereof. In some embodiments, one or both of the insulin in the first non-volatile buffer and the pramlintide in the second non-volatile buffer further comprises a stabilizing excipient selected from sugars, starches and mixtures thereof. In some embodiments, the first non-volatile buffer and the second non-volatile buffer are the same. In some embodiments, the first non-volatile buffer and the second non-volatile buffer are different. In some embodiments, each of the insulin and pramlintide is present in the formulation in an amount ranging from about 1 mg/ml to about 50 mg/ml. In certain embodiments, the first pH memory is about 1.5 about 2.5. In certain embodiments, the first pH memory is from about 6.0 to about 8.0. In some embodiments, the second pH memory is from about 3.0 to about 5.0. In some embodiments, the second pH memory is from about 4.0 to about 6.0. In certain embodiments, the first pH memory is from about 1.5 to about 2.5 and the second pH memory is from about 3.0 to about 5.0.
[00119] In some embodiments, the kit comprises a syringe that is part of a pen injection device, an auto-injector device, or a pump. In some embodiments, the syringe is pre-filled with the stable formulation. In certain embodiments, the kitinda comprises instructions, wherein the instructions direct the administration of the stable formulation to treat the subject with its need (eg, the subject having hypoglycemia or diabetes).VII. Examples
[00120] The present invention will be described in more detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any way. Those of skill in the art will readily recognize a variety of non-critical parameters that can be altered or modified to produce essentially the same results.Example 1: Preparation of Glucagon Solutions for Use in Freeze Drying
[00121] Various solutions have been prepared to contain glucagon at a concentration of 10 mg/ml. The solutions alternatively contained 5 mM glycine, citrate or phosphate, usually providing a buffer that establishes a pH of 3. The solution also contained a sugar, alone or in combination, in amounts equal to the w/v amount of glucagon (1:1) or 200% (2:1) of the amount of glucagon. The sugars were trehalose, HES and β-cyclodextrin (β-CD). Some solutions also contained 0.10% w/v Tween-20 as a surfactant. The various formulations were mixed to substantial homogeneity in amounts as described in Table 1 below.


[00122] To prepare the mixtures, glucagon was dissolved in the respective buffers (phosphate, citrate and/or glycine buffers, 5 mM, pH 3.0) with 10 mg/ml. The solution was then mixed in a 1:1 (v/v) ratio with various solutes, which were prepared at twice the desired concentration using the corresponding buffer, in order to obtain a final glucagon concentration of 5 mg/ml and the of final desired solute. The solutions were then filtered through a 0.2 μm Millipore PES membrane to remove insoluble materials. Sample preparations were carried out in a cold environment at 4°C. Glucagon concentration and purity were determined by RP HPLC and size exclusion (SE).Example 2: Preparation of Dry Powder Glucagon by Freeze Drying
[00123] The above formulations from Table 1 were pipetted (0.3 ml) into 3 ml lyophilization vials (13 mm ID). The formulations were lyophilized in an FTS Durastop freeze dryer (Stoneridge, NY). Samples were frozen at -40°C with an elevation of 2.5°C/min and held for 2 hours (h) to allow sufficient freezing. The sample temperature was then increased to -5°C at an elevation of 2°C/min and held for 2 h as an annealing step. The temperature was then lowered to 30°C at an elevation of 1.5°C/min and the vacuum was turned on at 60 mTorr. Primary drying was determined for 24 h. The temperature was gradually increased to 40°C with a rise of 0.5°C/min and held for an additional 10 h. Once drying was complete, the vials were capped under vacuum using XX stoppers from West Pharmaceutical company (product # 10123524). None of the formulations showed any evidence of cake collapse after freeze drying. The moisture content of the final dry product was less than 1% w/w.Example 3: Preparation of Glucagon Formulations in Polar Aprotic Solvents
[00124] Six of the dry powders manufactured from the solutions in Table 1 were selected for formulation in polar aprotic solvents:
[00125] Buffer (glycine) + trehalose (200% relative to glucagon) (formulation #3)
[00126] Buffer (glycine) + HES (200% relative to glucagon) (formulation #4)
[00127] Buffer (glycine) + trehalose (100% versus glucagon) + HES (100% versus glucagon) (formulation #5)
[00128] Buffer (glycine) + Tween-20 (0.01% w/v) + trehalose (200% relative to glucagon) (formulation #19)
[00129] Buffer (glycine) + Tween-20 (0.01% w/v) + HES (200% relative to glucagon) (formulation #20)
[00130] Buffer (glycine) + Tween-20 (0.01% w/v) + trehalose (100% relative to glucagon) + HES (100% relative to glucagon) (formulation # 21)Example 4: Preparation of a Glucagon Solution with a pH Memory of 4 to 5
[00131] Solutions were prepared to contain glucagon in a concentration of 10 to 20 mg/ml. The solutions contained a citrate buffer that sets the pH to 4 to 5. The solution also contained a sugar alcohol, mannitol, at a concentration of 50 to 100mg/ml. The formulation was mixed to substantial homogeneity and freeze-dried through the drying cycle described in Example 2 to a residual moisture of less than 0.5% w/w. The dry powder is dissolved in DMSO to a concentration of 10 to 20mg/ml of glucagon and of 50 to 100mg/ml of mannitol.Example 5: Preparation of a solution of PTH(1-34) with low moisture content and low boiling point. freezing
[00132] Solutions were prepared to contain PTH (1-34) in a concentration of 10 to 20 mg/ml. The solutions contained a citrate buffer that sets a pH of 4 to 5. The solution also contained a sugar alcohol, mannitol, at a concentration of 50 mg/ml. The formulation was mixed to substantial homogeneity and freeze-dried through the drying cycle described in Example 2 to a residual moisture of less than 0.5% w/w. The dry powder is dissolved in DMSO to a concentration of 10 to 20 mg/ml of PTH (1-34) and 50 to 100 mg/ml of mannitol.Example 6: Increase in Blood Glucagon Levels and Blood Glucose After Administration of Glucagon formulation
[00133] Two non-aqueous glucagon formulations in polar aprotic solvents, based on glucagon-glycine-trehalose powders dissolved in NMP or DMSO, were tested in a pharmacokinetic and pharmacodynamic study in rats and compared with an aqueous formulation. Mice were all dosed at a rate of 10 μg glucagon/mouse. Non-aqueous glucagon solutions were given as 10 μl subcutaneous injections, as was the aqueous control solution. All formulations tested demonstrated a rapid rise in blood glucagon concentrations (see Figure 1).
[00134] Pharmacokinetic parameters (PK) were analyzed in relation to the four treatment groups plus the aqueous control. A non-compartmental PK analysis was performed for each rat. Cmax and Tmax were calculated from the observed data. Area under the curve (AUC) estimates were computed without extrapolation. Data were analyzed using a five-group ANOVA to compare PK parameters across groups. No significant differences in Cmax, Tmax or AUC between the three groups were observed. The relative bioavailability of the NMP and DMSO formulations relative to the aqueous control group were all close to 100% (76% and 92%, respectively). Thus, the non-aqueous formulations are essentially bioequivalent to the aqueous formulation of glucagon based on the results of these mouse PK studies.
[00135] As predicted from the pharmacokinetic results, non-aqueous glucagon formulations produced pharmacodynamic profiles essentially equivalent to an aqueous formulation of reconstituted glucagon at the same dose level (see, Figure 2).Example 7: Enhanced Solubility of Glucagon in Polar Aprotic Solvents in relation to Aqueous Solutions
[00136] Glucagon was prepared at 1.0 mg/ml by dissolving in one of the following buffers:
[00137] 2 mM citric acid, pH 2.0 (titrated with concentrated HCl)("C2.0")
[00138] 2 mM citric acid, pH 3.0 (titrated with concentrated HCl)("C3.0")
[00139] Each formulation was placed in sterile 2 cc vials in 1 ml loading volume. Samples were freeze-dried to low residual moisture and reconstituted at various nominal concentrations in DMSO, NMP, or a 50/50 DMSO/NMP co-solvent. Reconstitution concentrations ranged from 1 to 30 mg/ml. Solubility was measured by visual inspection for clarity, turbidity through A630, and RP-HPLC.
[00140] As shown in Table 2 below, glucagon lyophilized with a citrate buffer at buffers of pH 2.0 and 3.0 was readily soluble at concentrations of 30 mg/ml. The same formulations were only fully soluble in H2O at lower concentrations. For the pH memory of 3.0, complete reconstitution was only achieved at 5 mg/ml with H2O. Furthermore, glucagon solubilized in H2O was only metastable, i.e. it only remained soluble for a few hours and then began to gel or fibrillate at rates that depend on pH and concentration, whereas glucagon solubilized in polar solvents aprotics/co-solvents was stable indefinitely.Table 2. Glucagon solubility in memory pH of 2.0 and 3.0
Example 8: Effect of pH on the Solubility of Glucagon in Polar Aprotic Solvents
[00141] When the data presented in Example 8 and Table 2 are viewed from a pH memory perspective, it is evident that higher glucagon solubilities can be achieved in polar aprotic solvents at a lower pH memory (e.g., pH 2.0) than at a higher pH. In addition, although the recoveries in Table 2 indicate essentially 100% of the nominal concentration, A630 measurements showed increased turbidity of the 30 mg/ml glucagon solutions at pH memory of 3.0 (C3.0) in pure NMP and the co -DMSO/NMP solvent, while the C2.0 formulations with a memory of 2.0 pH remained essentially free of turbidity.
[00142] In another example, the effect of pH on the solubility of glucagon in polar aprotic solvents was measured with respect to glucagon acetate dissolved in 2 mg/ml H2O with 2 ml of 2 mM glycine or citrate buffer and pH adjusted to the desired value. Samples were lyophilized and reconstituted at various nominal concentrations in DMSO, NMP, or a DMSO/NMP50/50 co-solvent. Solubility was measured by visual inspection for clarity, turbidity through A630, and RP-HPLC.
[00143] It was observed that "pH memory" of lyophilization had an important effect on glucagon stability. Glucagon was soluble in up to 30 mg/ml reconstitution for lyophilizates "G2.5" (memory pH 2.5) DMSO, DMSO/NMP and NMP. Significantly reduced solubility was observed for "G3.5" lyophilizates (memory pH 3.5). The G3.5 lyophilizates were all cloudy and recoveries were less than complete, even at a nominal reconstitution concentration of 10 mg/ml. DMSO and the DMSO/NMP co-solvent showed around 95% recovery, while NMP showed only around 60% recovery.Example 9: Effect of Buffer Species on Stability of Glucagon in DMSO
[00144] Glucagon acetate was prepared at 1.0 mg/ml by dissolving in one of the following buffers:
[00145] 2 mM L-glycine, pH 3.0 (titrated with concentrated HCl)
[00146] 2 mM Citric Acid, pH 3.0 (titrated with concentrated HCl)
[00147] These formulations were lyophilized and reconstituted with DMSO at a nominal concentration of 5 mg/ml glucagon. Formulations were placed in stability incubators at 5°C, 25°C and 40°C. Glucagon purity was determined with a reversed-phase HPLC method.
[00148] The stability of the formulation in glycine buffer was significantly higher after 1 month of incubation at various temperatures. Table 3 below shows purity by RP-HPLC at various times of incubation at 40°C. Table 3. Effect of buffer species on glucagon stability in DMSO
Example 10: Effect of Moisture on Stability of Glucagon in DMSO
[00149] Glucagon acetate was prepared at 1.0 mg/ml by dissolving in one of the following buffers:
[00150] 2 mM L-glycine, pH 3.0 (titrated with concentrated HCl)
[00151] 2 mM L-glycine, pH 3.0 (titrated with concentrated HCl)
[00152] These formulations were lyophilized and reconstituted with DMSO at a nominal concentration of 5 mg/ml glucagon. Additional moisture was added to the second formulation. Moisture content was measured using the Karl Fisher method. The first formulation had a moisture content of 0.13% (w/w), while the second formulation had a moisture content of 0.54% (w/w). The formulations were placed in stability incubators at 5°C, 25°C and 40°C. The purity of glucagon was determined with a reverse-phase HPLC method.
[00153] The stability of the formulation at lower humidity was significantly higher after 1 month of incubation at various temperatures. Table 4 below shows the purity by RP-HPLC at various times of incubation at 40°C. Even at moisture contents below 1%, a significant difference in stability can be detected.Table 4. Effect of residual moisture on glucagon stability in DMSO
Example 11: Freezing Point Depression of DMSO Solutions
[00154] Using PerkinElmer Instruments PYRIS Diamond Differential Scanning Calorimetry ("DSC"), samples were cooled to -40°C and heated to 40°C at 8°C per minute for screening purposes.
[00155] DMSO/NMP mixtures
[00156] Various mixtures of DMSO and NMP were tested, including:
[00157] 90% DMSO + 10% NMP
[00158] 80% DMSO + 20% NMP
[00159] 70% DMSO + 30% NMP
[00160] 60% DMSO + 40% NMP
[00161] 50% DMSO + 50% NMP
[00162] DSC examinations showed that the solvent crystallization temperature progressively reduced from ~18°C with respect to pure DMSO to -5.7°C with respect to a mixture of 50% NMP/50% DMSO . Addition of glucagon acetate, lyophilized glycine at a glucagon concentration of 5 mg/ml resulted in a further ~1°C reduction in freezing point.
[00163] DMSO/Ethyl Acetate Mixtures
[00164] Various mixtures of DMSO and ethyl acetate were tested, including:
[00165] 80% DMSO + 20% ethyl acetate (Tc = 16°C)
[00166] 70% DMSO + 30% ethyl acetate
[00167] 60% DMSO + 40% ethyl acetate (Tc = 6.5°C)
[00168] 50% DMSO + 50% ethyl acetate (Tc = 2.9°C)
[00169] 40% DMSO + 60% ethyl acetate (Tc = none observed)
[00170] DSC examinations showed that the solvent crystallization temperature progressively reduced from ~18°C with respect to pure DMSO to 2.9°C with respect to a mixture of 50% NMP/50% DMSO. No crystallization peaks were observed for a 40% DMSO/60% ethyl acetate mixture. Additionally, these formulations were stored at refrigerated temperature (4°C) for several days and visually observed for evidence of freezing. All formulations with 30% ethyl acetate or more in the co-solvent remained liquid and did not freeze. This is slightly different from the Tc seen in DSC studies.
[00171] DMSO Solutions with Alcohol Co-Solvents
[00172] Various DMSO solutions to which an alcohol co-solvent (ethanol, glycerol or propylene glycol) was added were tested, including:
[00173] 95% DMSO + 5% alcohol
[00174] 90% DMSO + 10% alcohol
[00175] 80% DMSO + 20% alcohol
[00176] 70% DMSO + 30% alcohol
[00177] 60% DMSO + 40% alcohol
[00178] 50% DMSO + 50% alcohol
[00179] 40% DMSO + 60% alcohol
[00180] 30% DMSO + 70% alcohol
[00181] 20% DMSO + 80% alcohol
[00182] 10% DMSO + 90% alcohol
[00183] These formulations were stored at refrigerated temperature (4°C) for several days and visually observed for evidence of freezing. All formulations with 20% alcohol co-solvents or more remained liquid and did not freeze. DSC examinations showed the freezing point of 20% alcohol co-solvents to be 2.3°C, 0.6°C and 3 .3°C for ethanol, glycerol and propylene glycol, respectively.Example 12: Freeze-Thaw Stability of Glucagon
[00184] Glucagon acetate was prepared at 1.0 mg/ml by dissolving in 2 mM L-glycine, pH 3.0 (titrated with concentrated HCl). The glucagon formulations were lyophilized and reconstituted with DMSO at a nominal concentration of 5 mg/ml glucagon. The solution samples were divided and trehalose was added to the solution at a concentration of 5%. These formulations were aliquoted into vials and placed in a stability incubator at 5°C. At 5°C, these solutions were observed to freeze. Glucagon solutions were thawed at various intervals and turbidity was determined using absorbance at 630 nm.
[00185] Table 5 below shows the turbidity of glucagon solutions at various incubation periods at 5°C. Solutions without trehalose showed increases in turbidity at various times of incubation. Solutions containing trehalose, however, showed no increase in turbidity. Turbidity measurements were confirmed by visual observation. Frozen samples incubated without trehalose were cloudy or hazy upon observation.Table 5. Cloudiness of glucagon solutions after incubation at 5°C

[00186] Surprisingly, the use of a carbohydrate additive such as trehalose in a peptide solution in DMSO increases the stability of the peptide during the freeze-thaw process.Example 13: Thawing Rate Enhanced with Trehalose
[00187] Glucagon acetate was prepared at 1.0 mg/ml by dissolving in 2 mM L-glycine, pH 3.0 (titrated with concentrated HCl) as described above for Example 13. After removal from storage at 5° C, samples of the trehalose-containing glucagon solutions were observed to thaw completely in a much shorter time than the solutions without trehalose. Samples containing trehalose were observed to thaw completely in less than 30 seconds, in contrast to glucagon solutions without trehalose, which were typically observed to thaw completely over several minutes. The ability to quickly thaw a peptide formulation can be particularly advantageous in an emergency medical setting, in the event that a solution has been frozen and has to be injected quickly.Example 14: Effect of pH on Insulin Solubility
[00188] Insulin was dissolved with H2O at 10 mg/ml with a buffer of 10 mM phosphate/citrate-1 Mn EDTA at pH 2 or pH 7. These solutions were lyophilized to dryness (> 1% residual moisture) using a conservative cycle and reconstituted at various nominal concentrations with DMSO. Solubility was assessed by visual inspection for clarity and turbidity using A630.
[00189] At a pH of 2, insulin has been observed to be soluble in concentrations of at least 100 mg/ml. However, at a memory pH of 7, even at the lowest concentration tested, 10 mg/ml, poor insulin solubility was observed as cloudy or misty solutions with increasing light scattering (A630). At some lower concentrations, eg 10 mg/ml, insulin solutions with a pH memory of 7 have been observed to slowly dissolve in a clear solution over a period of about 24 hours.Example 15: Effect of pH on Solubility of pramlintide
[00190] Pramlintide acetate was dissolved with H2O at 2 mg/ml with a 10 mM citrate buffer, pH 4, or 10 mM phosphate buffer, pH 7. These solutions were lyophilized to dryness (>1% residual moisture) using a preservative cycle and reconstituted at various nominal concentrations in DMSO. Solubility was measured by visual inspection for clarity and turbidity using A630.
[00191] At no concentration was pramlitide with a pH memory of 7 soluble in DMSO. However, a low concentration of pramlintide with a pH memory of 4 was soluble in DMSO.Example 16: Peptide Co-Formulations in Polar Aprotic Solvents
[00192] Preparation of co-formulations is prepared by separately drying individual compound formulations from an aqueous solution that provides optimal solubility/stability after reconstitution in the polar aprotic solvent. Solution pH is a property that affects peptide solubility, and a dry peptide, when reconstituted in a polar aprotic solvent, will retain a "pH memory" of the aqueous formulation from which it was dried when a non-volatile buffer is used. Since polaresaprotic solvents do not have exchangeable protons, the individual peptides will maintain the solubility and stability characteristics of optimal pH memory.
[00193] Current formulations of pramlintide and insulin are in conflict with their buffering systems, making compatibility of a mixed formulation difficult. Most insulins and insulin analogues have an isoelectric point in the range of 5 to 6 and are thus formulated at a pH around 7 or a lower pH around 2. Pramlintide has an isoelectric point of > 10.5 and is formulated at a pH around 4 where it is ideally stable. The interaction of pramlintide and insulin formulations at different pHs and different buffering capacities often results in precipitation of soluble insulin components or solubilization of crystalline insulin components. In vitro studies with both short- and long-acting formulations of pramlintide and insulin observed substantial variability in insulin solubility when varying amounts of insulin were mixed with fixed amounts of pramlintide.
[00194] Thus, the present invention provides a formulation whereby both a fast acting insulin species and an amylin analogue are stable and can be administered simultaneously from a single injection or formulation. This formulation more closely mimics the natural physiological response to the postprandial elevation of blood glucose than the prior art.
[00195] Examples of peptides that can be co-formulated include, but are not limited to: (1) insulin-amylin (insulin at a pH memory of about 2.0 or about 7.0, and amylin or an analogue of amylin (eg pramlintide) in a memory of pH around 4.0); and (2) glucagon-GLP-1 (glucagon in a memory pH of about 3.0 or below, and glucagon-like peptide-1 (GLP-1) or an analogue thereof (eg, exenatide) in a memory of pH around 4.0 to 5.0).
A co-formulation of insulin and pramlintide was prepared as follows: A 100 mg/ml insulin formulation of insulin, pH 2 memory, was prepared as described above in Example 14. A 1 mg/ml pramlintide formulation was prepared. ml of pramlintide, memory pH 4, was prepared as described above in Example 15. 5 µl of the insulin formulation was mixed with 95 ml of the pramlintide solution. The resulting solution was observed to be clear and thus a soluble co-formulation of insulin and pramlinitide was created with the respective memory of pH 2 and 4, respectively.
[00197] It should be understood that the above description is intended to be illustrative and not restrictive. Many embodiments will be evident to those of skill in the art after reading the above description. The scope of the invention should therefore be determined not with reference to the above description, but should instead be determined with reference to the appended claims, together with the full scope of equivalents to which such claims are entitled. The disclosures of all articles and references, including patent applications, patents and PCT publications are hereby incorporated by reference for all purposes.

权利要求:
Claims (19)
[0001]
1. Stable solution for parenteral injection, characterized in that it comprises: (a) glucagon or a salt thereof which has been dried in a non-volatile buffer, and wherein the dried glucagon has a pH memory that is equal to the pH of the glucagon in the buffer non-volatile; and (b) a polar aprotic solvent; wherein the dry glucagon maintains the pH memory when the dry glucagon is reconstituted and solubilized in the polar aprotic solvent; and wherein the moisture content of the solution is less than 5%.
[0002]
2. Solution according to claim 1, characterized in that the non-volatile buffer is selected from a glycine buffer, a citrate buffer, a phosphate buffer, or mixtures thereof.
[0003]
3. Solution according to claim 1, characterized by the fact that the pH memory of dry glucagon is from 2 to 3.
[0004]
4. Solution according to claim 1, characterized in that the polar aprotic solvent is selected from dimethyl sulfoxide (DMSO), N-methyl pyrrolidone (NMP), ethyl acetate, and mixtures thereof.
[0005]
5. Solution according to claim 1, characterized in that it further comprises a co-solvent that depresses the freezing point of the formulation, in which the co-solvent is selected from ethanol, propylene glycol, glycerol and their mixtures.
[0006]
6. Solution according to claim 1, characterized in that it comprises from 0.1 mg/mL up to the solubility limit of dry glucagon.
[0007]
7. Solution according to claim 1, characterized in that it comprises from 0.1 mg/mL to 100 mg/mL of dry glucagon or from 1 mg/mL to 30 mg/mL of dry glucagon.
[0008]
8. Solution according to claim 1, characterized in that the buffer is a glycine buffer and the aprotic solvent is dimethyl sulfoxide (DMSO).
[0009]
9. Solution according to claim 8, characterized in that the mixture further comprises a stabilization excipient selected from sugars, starches and their mixtures.
[0010]
10. Solution according to claim 9, characterized by the fact that the stabilization excipient is trehalose.
[0011]
11. A method of manufacturing a stable solution for parenteral injection, as defined in claim 1, the method comprising: (a) drying a mixture comprising a glucagon or its salt in a dry glucagon buffer, wherein dry glucagon has a pH memory; and (b) reconstituting the dry glucagon in a polar aprotic solvent; wherein the moisture content of the formulation is less than 5%, and wherein the dry glucagon maintains the pH memory when the dry glucagon is reconstituted and solubilized in the solvent aprotic polar.
[0012]
12. Method according to claim 11, characterized in that it comprises adding 0.1 mg/mL up to the limited solubility of dry glucagon in the polar aprotic solvent.
[0013]
13. Method according to claim 11, characterized in that it comprises adding from 0.1 mg/mL to 100 mg/mL of dry glucagon in the polar aprotic solvent or from 1 mg/mL to 30 mg/mL of dry glucagon in the solvent aprotic polar.
[0014]
14. Method according to claim 11, characterized in that the buffer is a non-volatile buffer selected from a glycine buffer, a citrate buffer, a phosphate buffer, and their mixtures.
[0015]
15. Method according to claim 11, characterized in that the pH memory of dry glucagon is from 2 to 3.
[0016]
16. Method according to claim 11, characterized in that the polar aprotic solvent is selected from dimethyl sulfoxide (DMSO), n-methyl pyrrolidone (NMP), ethyl acetate, and their mixtures.
[0017]
17. Method according to claim 11, characterized in that the buffer is a glycine buffer and the aprotic solvent is dimethylsulfoxide (DMSO).
[0018]
18. Method according to claim 17, characterized in that the mixture further comprises a stabilization excipient selected from sugars, starches and mixtures thereof.
[0019]
19. Method according to claim 18, characterized in that the stabilization excipient is trehalose

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-03-26| 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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2019-05-28| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2019-11-19| B25G| Requested change of headquarter approved|Owner name: XERIS PHARMACEUTICALS, INC. (US) |

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2021-07-27| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-11-09| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2022-01-18| 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 09/03/2012, 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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US201161451568P| true| 2011-03-10|2011-03-10|

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US61/451,568|2011-03-10|

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US201161478692P| true| 2011-04-25|2011-04-25|

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US61/478,692|2011-04-25|

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US201161553388P| true| 2011-10-31|2011-10-31|

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US61/553,388|2011-10-31|

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US201261609123P| true| 2012-03-09|2012-03-09|

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PCT/US2012/028621|WO2012122535A2|2011-03-10|2012-03-09|Stable formulations for parenteral injection of peptide drugs|

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US61/609,123|2012-03-09|

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