HK1191230B - Stable formulations for parenteral injection of peptide drugs - Google Patents

Description

Stable formulations of peptide drugs for parenteral injection

Cross Reference to Related Applications

Priority and benefit of us provisional application No. 61/451,568 entitled "stable formulation of parenteral peptide drug" filed on 3/10/2011, us provisional application No. 61/478,692 entitled "stable formulation of parenteral peptide drug" filed on 4/25/2011, us provisional application No. 61/553,388 entitled "formulation for treatment of diabetes" filed on 10/31/2011, and us provisional application No. 61/609,123 entitled "formulation for treatment of diabetes" filed on 3/9/2012, the entire disclosure of which is incorporated herein by reference for all purposes.

Statement regarding rights to inventions made under federally sponsored research or development not applicable

References to "sequence listing", forms or computer program form attachments submitted on optical discs are not applicable

Technical Field

The present invention relates to pharmaceutical formulations, and more particularly to pharmaceutical formulations of peptides having improved stability, and methods of using such pharmaceutical formulations in the treatment of various diseases, disorders, and conditions.

Background

Diabetes is a serious health problem in modern society. Insulin is a key therapeutic for both type I and type II diabetes. Studies over the past two decades have demonstrated that tight metabolic control of glucose by the use of insulin not only reduces incidence, but also delays the development of complications in people with type 1 and type 2 diabetes. Unfortunately, the intensive insulin therapy required to achieve tight glucose control is also associated with a significantly increased risk of developing hypoglycemia or "hypoglycemia".

The symptoms of hypoglycemia vary widely between patients, but generally include tremors, palpitations, irritability, anxiety, nervousness, hunger, tachycardia, headache, and pallor. Once blood glucose returns to normal levels, symptoms typically resolve. If hypoglycemia is not reversed, further lowering of blood glucose can lead to consumption of glucose in the central nervous system associated with symptoms of neuroglycopenia, such as difficulty in concentrating, slurred mouth, blurred vision, reduced body temperature, behavioral changes, and loss of consciousness, epilepsy, and possible death if untreated.

Generally, hypoglycemia can be defined as mild to moderate hypoglycemia or severe hypoglycemia as follows:

mild to moderate hypoglycemia: patients may be self-treated to the onset of symptoms regardless of severity, or any asymptomatic blood glucose measurement in which blood glucose levels are below 70mg/dL (3.9 mmol/l).

Severe hypoglycemia: operationally defined as the onset of hypoglycemia in which the patient is unable to treat himself and needs external assistance. Typically, the symptoms of neuroglycopening and cognitive impairment begin at blood glucose levels of about 50mg/dL (2.8 mmoles/liter).

Most episodes of mild to moderate hypoglycemia can be self-treated relatively easily by ingesting fast-acting sugars such as glucose tablets or foods (juices, soft drinks or sweet snacks). By definition, severe hypoglycemia is not self-treating and requires external intervention. If the patient can swallow and is cooperative, it is appropriate to place a gel or product, such as honey or jelly, inside the cheeks. Glucagon is injected subcutaneously or intramuscularly to treat severe hypoglycemia if the patient cannot swallow it.

Glucagon is a naturally occurring peptide hormone of 29 amino acids in length and secreted by the alpha cells of the pancreas. The main function of glucagon is to maintain glucose production through both glycogenolysis and gluconeogenesis, primarily mediated by the liver. Glucagon is the major insulin counter-regulatory hormone and is used as a first-line treatment of severe hypoglycemia in patients with diabetes.

Many attempts have been made to create glucagon rescue drugs for the treatment of severe hypoglycemia in emergency situations. Currently, currently available in the united statesThere are two glucagon kits, consisting of EliLilly (Glucagon Emergencykit) and NovoNordisk (GlucaGen)HypoKit). Both products combine a vial of freeze-dried glucagon with a syringe pre-filled with aqueous diluent. Freeze-dried glucagon must be reconstituted using complex steps that are difficult to use in emergency situations. These products also provide for large volume injections because glucagon is poorly soluble in water. More recently, attempts have been made to improve the stability of glucagon in aqueous solutions, to open up more stable glucagon analogues and/or to improve the delivery of glucagon via powder injection.

Despite some advances, there remains a need for more user-friendly glucagon rescue medications for the treatment of severe hypoglycemia in emergency situations. Such glucagon rescue medications would need to be continuously carried by the diabetic and/or its caretaker and would therefore need to be stable for long periods (> 2 years) at non-freezing temperatures (25-30 ℃). Ideally, it would also need to be easy to administer to the general population and not require excessive processing/reconstitution prior to administration to hypoglycemic patients. Glucagon rescue medications also need to be available at a range of temperatures, including temperatures from 0 ℃ to 30 ℃.

Summary of The Invention

To address such needs and other problems, the present invention provides a stable glucagon rescue formulation and methods of using the stable glucagon formulation to treat severe hypoglycemia. Advantageously, the glucagon is stabilized in the formulations of the invention, enabling long term storage and/or long term delivery. Likewise, the glucagon formulations of the present invention are stable at non-freezing temperatures for extended periods of time, are easy to administer without reconstitution, and can be used over a range of temperatures, including a temperature range from 0 ℃ to 30 ℃.

Importantly, the formulation techniques of the present invention can be universally applicable to the delivery of many other peptides that have poor or limited stability and solubility in aqueous environments like glucagon. Indeed, it is now clear that formulating peptides into high concentration non-aqueous solutions using aprotic polar solvents such as DMSO, NMP, ethyl acetate or mixtures thereof is a valuable delivery platform for such important peptide therapies. Furthermore, the formulation techniques of the present invention may be generally applicable to the delivery of two or more peptides in the same solution.

Accordingly, in one aspect, the present invention provides a stable formulation for parenteral injection, the formulation comprising: (a) a peptide or salt thereof, wherein the peptide has been dried in a non-volatile buffer, and wherein the dried peptide has a pH memory that is about equal to the pH of the peptide in the non-volatile buffer; and (b) an aprotic polar solvent; wherein the moisture content of the formulation is less than 5%, and wherein when the dried peptide is reconstituted in an aprotic polar solvent, the dried peptide maintains a pH memory approximately equal to the pH of the peptide in a non-volatile buffer.

In another aspect, the present invention provides a stable formulation for parenteral injection, comprising: (a) a first peptide or salt thereof, wherein the first peptide has been dried in a first non-volatile buffer, and wherein the dried first peptide has a first pH memory that is about equal to the pH of the first peptide in the first non-volatile buffer; (b) a second peptide or salt thereof, wherein the second peptide has been dried in a second non-volatile buffer, and wherein the dried second peptide has a second pH memory that is about equal to the pH of the second peptide in the second non-volatile buffer; and (c) an aprotic polar solvent; wherein the moisture content of the formulation is less than 5%, wherein when the dried first peptide is reconstituted in an aprotic polar solvent, the dried first peptide maintains a first pH memory approximately equal to the pH of the first peptide in a first non-volatile buffer, and wherein when the dried second peptide is reconstituted in an aprotic polar solvent, the dried second peptide maintains a second pH memory approximately equal to the pH of the second peptide in a second non-volatile buffer.

In another aspect, the present invention provides a stable formulation for parenteral injection, comprising: a peptide or a salt thereof (e.g., a hydrochloride or acetate salt thereof); and an aprotic polar solvent, wherein the moisture content of the formulation is less than 5%.

The stable formulations described herein are useful for parenteral injection of any peptide having limited or poor stability or solubility in an aqueous environment. Thus, in some embodiments, the peptide (each of the first peptide and the second peptide) or a salt thereof is selected from the group consisting of glucagon, pramlintide, insulin, leuprolide, LHRH agonists, parathyroid hormone (PTH), dextrin, botulinum toxin, hematide, amyloid peptide, cholecystokinin (cholecystokinin), conotoxin, gastric inhibitory peptide, insulin-like growth factor, growth hormone releasing factor, antimicrobial factor, glatiramer, glucagon-like peptide-1 (GLP-1), GLP-1 agonists, exenatide, analogs thereof, and mixtures thereof. In a preferred embodiment, the peptide is glucagon or a glucagon analogue or a glucagon peptide mimetic. 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.

The peptide (or each peptide in embodiments where the formulation comprises two or more 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, wherein the formulation comprises two or more peptides, the first non-volatile buffer and the second non-volatile buffer are the same. In some embodiments, wherein the formulation comprises two or more peptides, the first non-volatile buffer and the second non-volatile buffer are different.

In some formulations of the invention, the peptide is mixed with a non-volatile buffer and stabilizing excipients 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 saccharide is trehalose. In some embodiments, the starch is hydroxyethyl starch (HES). In some embodiments, the stabilizing excipient is present at 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) 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); or, 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 20% (w/v) of the formulation. In some embodiments wherein 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, the stabilizing excipient for the first peptide in the first non-volatile buffer and the stabilizing excipient for the second peptide in the second non-volatile buffer being 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, the stabilizing excipient for the first peptide in the first non-volatile buffer and the stabilizing excipient for the second peptide in the second non-volatile buffer being different.

Once the one or more peptides and the non-volatile buffer or the peptides, non-volatile buffer and stabilizing excipient are dried into a powder, the dried peptide powder is dissolved or reconstituted in an aprotic polar solvent. Examples of aprotic polar solvents include, but are not limited to, the following solvents: dimethyl sulfoxide (DMSO), Dimethylformamide (DMF), ethyl acetate, N-methylpyrrolidone (NMP), Dimethylacetamide (DMA), propylene carbonate, and mixtures thereof. Dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), ethyl acetate and mixtures of one or more of DMSO, NMP and ethyl acetate are particularly preferred aprotic polar solvents. In a preferred embodiment, the aprotic polar solvent is DMSO. In another preferred embodiment, the aprotic polar solvent is a mixture of DMSO and NMP. In yet another preferred embodiment, the aprotic polar solvent is a mixture of DMSO and ethyl acetate.

In some embodiments, one or more peptides are reconstituted in a mixture of aprotic polar solvents (e.g., dimethyl sulfoxide (DMSO), Dimethylformamide (DMF), ethyl acetate, N-methylpyrrolidone (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 lowers the freezing point of the formulation by at least about 5 ℃, at least about 10 ℃, at least about 15 ℃, or at least about 20 ℃. In some embodiments, the co-solvent lowers the freezing point of the formulation to about 3 ℃, about 2 ℃, about 1 ℃ or about 0 ℃ or less. In some embodiments, the co-solvent is a polar protic solvent. In a preferred embodiment, the co-solvent is selected from the group consisting of ethanol, Propylene Glycol (PG), glycerol, and mixtures thereof. In some embodiments, the co-solvent is present in the formulation in an amount of 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), from about 15% (w/v) to about 25% (w/v).

Importantly, the formulations of the present invention have very little residual moisture, and thus the peptides in such formulations remain stable for long periods of time. In preferred embodiments, the moisture content of the formulations of the present invention is less than about 4%, preferably less than about 3%, preferably less than about 2%, even more preferably less than about 1%, preferably less than about 0.5%, preferably less than about 0.25%, preferably less than about 0.2%, preferably less than about 0.15% or preferably less than about 0.1%. In other preferred embodiments, the moisture content of the formulations of the present invention is from about 0.01% to about 4%, preferably from about 0.01% to about 3%, 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 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%, preferably from about 0.25% to about 3%, preferably from about 0.25% to about 2%, preferably from about 0.25% to about 1% or preferably from about 0.5% to about 1%.

When the peptide is mixed with a non-volatile buffer, the non-volatile buffer is selected to provide the peptide with the greatest stability, the greatest solubility, and the pH at which degradation is minimized in an 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 an aprotic polar solvent. Likewise, in preferred embodiments, the peptides in the formulation will have a pH memory of about 2.0 to about 3.0 to ensure maximum stability, maximum solubility, and minimal 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 minimal degradation. In other embodiments, the peptide in the formulation will have a pH memory of about 4.0 to about 5.0 to ensure maximum stability, maximum solubility, and minimal degradation. In yet 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 yet other embodiments, the peptide will have a pH memory of about 6.0 to about 8.0 to ensure maximum stability, maximum solubility, and minimal 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 about 1.5 to about 2.5 or 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 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 pH memory of about 4.0 to about 5.0 to ensure maximum stability, maximum solubility, and minimum degradation. It will be apparent to one skilled in the art how to determine the optimal pH for obtaining a peptide with maximum stability, maximum solubility and minimal degradation.

Any suitable dose of one or more peptides may be formulated in the stable formulations of the invention. Typically, the peptide (or each peptide in embodiments comprising two or more peptides) is present in the formulation in an amount of about 0.5mg/mL to about 100 mg/mL. In some embodiments, the peptide is present in the formulation in an amount from about 10mg/mL to about 60 mg/mL. In other embodiments, the peptide is present in the formulation in an amount from about 20mg/mL to about 50 mg/mL. In still other embodiments, the peptide is present in the formulation in an amount from about 5mg/mL to about 15 mg/mL. In yet other embodiments, the peptide is present in the formulation in an amount from about 0.5mg/mL to about 2 mg/mL. In yet other embodiments, the peptide is present in the formulation in an amount from about 1mg/mL to about 50 mg/mL. In addition, the dosage of the peptide may vary depending on the peptide used and the disease, condition or disorder to be treated, as will be apparent to the skilled artisan.

In some embodiments, the formulations of the present invention further comprise an antioxidant. In other embodiments, the formulation further comprises a chelating agent. In yet other embodiments, the formulations of the present invention further comprise a preservative.

In another aspect, the invention provides a method for treating a disease, disorder or condition that can be treated, alleviated or prevented by administering to a subject a stable peptide formulation as described herein in an amount effective for treating, alleviating or preventing the disease, disorder or condition. In some embodiments, the disease, disorder, or condition is hypoglycemia. In some embodiments, wherein the disease, disorder, or condition is hypoglycemia, the method comprises administering an amount of the stabilized glucagon formulation of the invention effective to treat hypoglycemia. In some embodiments, the disease, disorder, or condition is diabetes. In some embodiments, wherein the disease, disorder, or condition is diabetes, the method comprises administering an amount of the stabilized insulin and pramlintide formulations of the present invention effective to treat diabetes.

In yet another aspect, the present invention provides a method for preparing a stable formulation for parenteral injection, the method comprising: drying the peptide and non-volatile buffer to a dry peptide powder; reconstituting the dried peptide powder with an aprotic polar solvent to produce a stable formulation, wherein the stable formulation has a moisture content of less than 5%. In some embodiments, the dried peptide powder has a pH memory about equal to the pH of the peptide in the non-volatile buffer, and when the dried peptide powder is reconstituted in the aprotic polar solvent, the dried peptide powder maintains a pH memory about equal to the pH of the peptide in the non-volatile buffer.

In a further aspect, the present invention provides a kit for treating a disease, disorder or condition, the kit comprising: a stable formulation comprising one or more peptides or salts thereof, wherein the peptides have been dried in a non-volatile buffer, and wherein the dried peptides have a pH memory about equal to the pH of the peptides in the non-volatile buffer; and an aprotic polar solvent; wherein the moisture content of the formulation is less than 5%, and wherein when the dried peptide is reconstituted in an aprotic polar solvent, the dried peptide maintains a pH memory approximately equal to the pH of the peptide in a non-volatile buffer; and a syringe for administering the stable formulation to a subject.

In some embodiments, the kit is for treating hypoglycemia, the stable formulation comprising a glucagon formulation as described herein. In some embodiments, the kit is for use in treating diabetes, and the stable formulation comprises an insulin and pramlintide formulation as described herein. In some embodiments, the injector is some of a pen injection device, an auto-injector device, or a pump. In some embodiments, the syringe is pre-filled with a stable formulation. In some embodiments, the kit further comprises instructions, wherein the instructions direct the administration of the stable formulation to treat a subject in need thereof.

Other objects, features and advantages of the present invention will be apparent to those skilled in the art from the following detailed description and appended claims.

Brief Description of Drawings

Figure 1 shows plasma glucagon levels following injection of lyophilized glucagon-glycine-trehalose dissolved in DMSO or NMP.

Figure 2 shows blood glucose levels after injection of lyophilized glucagon-glycine-trehalose dissolved in DMSO or NMP.

Detailed Description

I. Introduction to the design reside in

Peptides can be degraded by a number of different mechanisms, including deamidation, oxidation, hydrolysis, disulfide interchange, and racemization. In addition, water acts as a plasticizer, which facilitates unfolding of the protein molecules and irreversible molecular aggregation. Thus, 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 typically required.

Concentrating an aqueous peptide formulation into a dry powder formulation is one method of increasing the stability of a pharmaceutical peptide formulation. For example, the peptide formulation may be dried using a variety of techniques, including spray drying, lyophilization or freeze drying and dehydration. The dry powder peptide formulations obtained by such techniques show a significantly increased stability over time at ambient or even physiological temperatures.

The present invention is based in part on the surprising discovery that stable peptide formulations (e.g., stable glucagon rescue formulations) can be readily prepared by first freeze-drying one or more peptides (e.g., glucagon peptides) in a non-volatile buffer to a dry peptide powder. The dried peptide has a defined "pH memory" of the pH of the peptide in the non-volatile buffer in which the peptide is dried. Once dried, the resulting peptide powder, e.g., lyophilized glucagon, is dissolved in an aprotic polar solvent to form a stable formulation, wherein the moisture content of the formulation is less than 5%, 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%. When the dried peptide is reconstituted in an aprotic polar solvent, it retains its defined pH memory, i.e., when reconstituted in an aprotic polar solvent, the pH of the peptide is about equal to the pH of the peptide in the non-volatile buffer in which the peptide is dried. Advantageously, once prepared, the formulations (e.g., glucagon formulations) are stable for extended periods of time, are ready for use without reconstitution, and are available for use over a range of temperatures.

Importantly, the formulation techniques of the present invention can be universally applicable to the delivery of many other peptides that have poor or limited stability and solubility in aqueous environments like glucagon. Indeed, it is now clear that formulating peptides into high concentration non-aqueous solutions using aprotic polar solvents (e.g., DMSO, NMP, ethyl acetate, or mixtures thereof) is a very valuable delivery platform for such important therapeutic agents-therapeutic peptides. The stable formulations described herein advantageously improve the uniform delivery of peptide drugs and provide additional storage stability to prevent aggregation, oxidation and hydrolysis associated with degradation pathways.

In certain preferred embodiments, the stable formulations described herein preserve the peptide drug in a stable form for an extended period of time, e.g., for a period of time sufficient to provide a desired shelf life of the formulation without an unacceptable degree of degradation of the therapeutic agent prior to use. The desirable properties of injectable formulations are that they are non-aqueous and non-reactive with respect to peptides. In such embodiments, the injectable formulation may be stored directly in the injection device itself.

The stable injectable formulations of the invention contain the necessary delivered dose of the therapeutic peptide (e.g., the dose required for drug therapy) and are preferably small in volume. For example, in some embodiments, the volume of the injectable formulation containing a therapeutic dose of a peptide (e.g., glucagon) is at least about 1.0 microliter (which is a lower limit of the functionality of the filling device), more preferably from about 10 milliliters to about 250 microliters. In certain preferred embodiments, the delivery of a therapeutic dose of a peptide in a small volume is accomplished by a dose of the therapeutic peptide (e.g., glucagon) in a concentrated stable form in a suitable aprotic polar solvent for injection according to the invention.

Furthermore, the stable formulations of the present invention are suitable for administration without the need for dilution prior to injection. Many currently available therapeutic peptides and vaccine products are produced in the form of solid particles to improve stability on the shelf. These formulations are diluted in sterile water, phosphate buffered saline or isotonic saline prior to injection. In contrast, in certain preferred embodiments of the invention, the therapeutic peptide is concentrated using particle preparation processing techniques commonly employed in the pharmaceutical industry (e.g., spray drying, lyophilization, and the like) to prepare a formulation for injection. In a preferred embodiment, a therapeutic dose of peptide drug is obtained by dispersing the peptide, which has first been freeze-dried together with a non-volatile buffer (and optionally additional components, such as stabilizing excipients), in a dry powder having very little residual moisture content. Once prepared, the dried peptide powder is dissolved in an aprotic polar solvent, such as DMSO, NMP, ethyl acetate or mixtures of these solvents. Thus, in accordance with the objects of the present invention, the small volume, stable formulations of the present invention are injected, infused or administered into an animal (e.g., a human patient) without first diluting the formulation prior to injection as is required for most reconstituted products. Likewise, in preferred embodiments, the small volume formulations of the present invention are administrable without first being diluted or reconstituted or cooled.

Definition of

For the purposes of this disclosure, the following terms have the following meanings:

the term "therapeutic agent" encompasses peptide compounds as well as pharmaceutically acceptable salts thereof. 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 exert a desirable, beneficial, and generally pharmacological effect upon administration to a human or animal, whether alone or in combination with other pharmaceutical excipients or inert ingredients.

The terms "peptide," "polypeptide," and/or "peptidal compound" refer to polymers of up to about 80 amino acid residues bonded together through amide (CONH) bonds. Analogs, derivatives, agonists, antagonists and pharmaceutically acceptable salts of any of the peptide compounds disclosed herein are included within these terms. The term also includes peptides and/or peptide compounds having as part of their structure modified, derivatized or non-naturally occurring amino acid and/or peptidomimetic units of the D-amino acid, D-or L-configuration.

The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable solvent, suspending agent or vehicle for delivering the peptide compounds of the invention to a mammal, such as an animal or human. In a presently preferred embodiment, the pharmaceutically acceptable carrier is an aprotic polar solvent.

The term "aprotic polar solvent" denotes a polar solvent which does not contain an acidic hydrogen and does not act as a hydrogen bond donor. Examples of aprotic polar solvents include, but are not limited to, dimethyl sulfoxide (DMSO), Dimethylformamide (DMF), ethyl acetate, N-methylpyrrolidone (NMP), Dimethylacetamide (DMA), and propylene carbonate. The term aprotic polar solvent also encompasses mixtures of two or more aprotic polar solvents, for example mixtures of two or more of dimethyl sulfoxide (DMSO), Dimethylformamide (DMF), ethyl acetate, N-methylpyrrolidone (NMP), Dimethylacetamide (DMA) and propylene carbonate.

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.

The term "chemical stability" means that an acceptable percentage of degradation products produced by chemical pathways, such as oxidation or hydrolysis, are formed with respect to the therapeutic agent. In particular, a formulation is considered to be chemically stable if it forms no more than about 20% of decomposition products after storage for one year at the expected product storage temperature (e.g., room temperature), or storage for one year at 30 ℃/60% relative humidity, or storage for one month at 40 ℃/75% relative humidity, 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% of decomposition products formed upon prolonged storage at the intended product storage temperature.

The term "physical stability" means that an acceptable percentage of aggregates (e.g., dimers, trimers, and larger forms) are formed with respect to the therapeutic agent. In particular, a formulation is considered physically stable if it forms no more than about 15% aggregates after storage for one year at the intended product storage temperature (e.g., room temperature), or for one year at 30 ℃/60% relative humidity, or for one month, preferably three months, at 40 ℃/75% relative humidity. In some embodiments, the physically 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% aggregates formed after long term storage at the intended product storage temperature.

The term "stable formulation" means that at least about 65% of the chemically and physically stable therapeutic agent remains after storage for two months 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% of the chemically and physically stable therapeutic agent remains under these storage conditions. Particularly preferred stable formulations are those that do not exhibit degradation after disinfecting irradiation (e.g., gamma, beta, or electron beam).

The phrase "consisting essentially of is used herein to exclude any element that would significantly alter the basic properties of the stable formulation to which the phrase refers.

The term "bioavailability" is defined for purposes of the present invention as the degree to which a therapeutic agent, e.g., a peptide compound, is absorbed from a formulation.

The term "systemic" means that with respect to delivery or administration of a therapeutic agent, e.g., a peptide compound, to a subject, the therapeutic agent is detectable at biologically significant levels in the plasma of the subject.

The term "controlled release" is defined herein as the release of the therapeutic agent at a rate such that the blood (e.g., plasma) concentration remains within the therapeutic range, but below the toxic concentration, for a period of about one hour or more, preferably 12 hours or more.

The term "parenteral injection" refers to administration of a therapeutic agent, e.g., a peptide compound, by injection under or across one or more layers of skin or mucosa of an animal, e.g., a human. Standard parenteral injections are performed in areas within the skin, subcutaneously or intramuscularly of an animal, e.g., a human patient. In some embodiments, a deep site is targeted for injection of a therapeutic agent as described herein.

The term "treating" refers to delaying the onset of the disease or condition to which the term applies or one or more symptoms of such disease or condition, arresting or reversing the development of the disease or condition to which the term applies or one or more symptoms of such disease or condition, or alleviating or preventing the disease or condition to which the term applies or one or more symptoms of such disease or condition.

The terms "patient," "subject" or "individual" refer interchangeably to a mammal, e.g., a human or non-human mammal, such as a primate, dog, cat, cow, sheep, pig, horse, mouse, rat, hamster, rabbit, or guinea pig.

Stable peptide formulations

In one aspect, the present invention provides a stable formulation for parenteral injection. Advantageously, once prepared, the formulation is stable for extended periods of time, ready for use without reconstitution, and can be used over a range of temperatures. In addition, the stable formulations of the present invention are useful for parenteral injection of any peptide having limited or poor stability or solubility in an aqueous environment. In some embodiments, the formulations of the invention increase the physical stability of the peptides of the formulation, for example, by preventing or reducing the formation of aggregates of the peptides.

In some embodiments, the formulation comprises: (a) a peptide or salt thereof, wherein the peptide has been dried in a non-volatile buffer, and wherein the dried peptide has a pH memory that is about equal to the pH of the peptide in the non-volatile buffer; and (b) an aprotic polar solvent; wherein the moisture content of the formulation is less than 5%, and wherein when the dried peptide is reconstituted in an aprotic polar solvent, the dried peptide maintains a pH memory approximately equal to the pH of the peptide in a non-volatile buffer.

In some embodiments, the formulation comprises: (a) a first peptide or salt thereof, wherein the first peptide has been dried in a first non-volatile buffer, and wherein the dried first peptide has a first pH memory that is about equal to the pH of the first peptide in the first non-volatile buffer; (b) a second peptide or salt thereof, wherein the second peptide has been dried in a second non-volatile buffer, and wherein the dried second peptide has a second pH memory that is about equal to the pH of the second peptide in the second non-volatile buffer; and (c) an aprotic polar solvent; wherein the moisture content of the formulation is less than 5%, wherein when the dried first peptide is reconstituted in an aprotic polar solvent, the dried first peptide maintains a first pH memory approximately equal to the pH of the first peptide in a first non-volatile buffer, and wherein when the dried second peptide is reconstituted in an aprotic polar solvent, the dried second peptide maintains a second pH memory approximately equal to the pH of the second peptide in a second non-volatile buffer.

In some embodiments, the formulation consists essentially of: (a) a peptide or salt thereof, wherein the peptide has been dried in a non-volatile buffer, and wherein the dried peptide has a pH memory that is about equal to the pH of the peptide in the non-volatile buffer; and (b) an aprotic polar solvent; wherein the moisture content of the formulation is less than 5%, and wherein when the dried peptide is reconstituted in an aprotic polar solvent, the dried peptide maintains a pH memory approximately equal to the pH of the peptide in a non-volatile buffer.

A. Peptides

The stable formulations of the present invention comprise one, two, three, four or more peptides or salts, analogs and/or mixtures thereof. Peptides (and salts thereof) suitable for use in the formulations of the present invention include, but are not limited to, glucagon, pramlintide, insulin, leuprolide, Luteinizing Hormone Releasing Hormone (LHRH) agonists, parathyroid hormone (PTH), dextrins, botulinum toxins, hematides, amyloid peptides, cholecystokinin, gastric inhibitory peptides, insulin-like growth factors, growth hormone releasing factors, antimicrobial factors, glatiramer, glucagon-like peptide-1 (GLP-1), GLP-1 agonists, exenatide, analogs thereof, and mixtures thereof. In some embodiments, the peptide is a hydrochloride or acetate salt.

In a preferred embodiment, the peptide is glucagon or a glucagon analog or a peptide mimetic or a salt thereof (e.g., glucagon acetate). 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 other embodiments, the peptide is a dextrin or dextrin mimetic (e.g., pramlintide). In yet other embodiments, the peptide is insulin or an insulin analog (e.g., Lispro). In some embodiments, the insulin or insulin analog formulation is a low zinc or zinc-free formulation.

In some embodiments, the formulation comprises two peptides, wherein the first peptide is a dextrin or dextrin mimetic and the second peptide is insulin or an insulin analog. 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 or zinc-free insulin formulation.

In some embodiments, the formulation comprises two peptides, wherein the first peptide is glucagon and the second peptide is glucagon-like peptide-1 (GLP-1) or a GLP-1 analog 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.

Any suitable dose of peptide may be administered using the formulations of the invention. The dosage administered will, of course, vary depending on known factors, such as the pharmacodynamic properties of the particular peptide, salt or combination thereof, the age, health or weight of the subject, the nature and extent of the symptoms, the metabolic characteristics of the therapeutic agent and the patient, the type of concurrent treatment, the frequency of treatment, or the desired effect. Typically, the peptide (or each peptide where the stable formulation comprises two or more peptides) is present in the formulation in an amount of from about 0.5mg/mL to about 100mg/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).

In some embodiments, the peptide is present in the formulation in an amount from about 0.5mg/mL to about 60 mg/mL. In some embodiments, the peptide is present in the formulation in an amount from about 10mg/mL to about 50 mg/mL. In other embodiments, the peptide is present in the formulation in an amount from about 20mg/mL to about 50 mg/mL. In still other embodiments, the peptide is present in the formulation in an amount from about 5mg/mL to about 15 mg/mL. In yet other embodiments, the peptide is present in the formulation in an amount from about 0.5mg/mL to about 2 mg/mL. In addition, the dosage of the peptide may vary depending on the peptide used and the disease, condition or disorder to be treated, as will be apparent to the skilled artisan.

In a preferred embodiment, the peptide is mixed with a non-volatile buffer and optionally a stabilizing excipient and then dried to a dry peptide powder. In embodiments where the stable formulation comprises two or more peptides, each peptide is separately mixed with a non-volatile buffer and optionally a stabilizing excipient and then dried to a dry peptide powder. Because peptides are susceptible to hydrolysis at bonds with asparagine residues and oxidation by methionine, the use of non-volatile buffers in the formulations of the present invention beneficially affects chemical stability. As described in further detail below, the charge distribution of a peptide in an aprotic polar solvent can affect its stability when the pH is not relevant in the aprotic polar solvent. The charge distribution of the peptide in the aprotic polar solvent will be a function of the pH of the aqueous solution in which the peptide was previously dried, i.e., there is "pH memory" upon dissolution or reconstitution in the aprotic polar solvent. To obtain the desired charge distribution of the peptide dissolved in the aprotic polar solvent, the peptide is dried in an aqueous buffer solution having a pH that results in optimal stability, optimal solubility, and minimal degradation in the aprotic polar solvent.

Likewise, non-volatile buffers useful in the formulations described herein are those useful for establishing the pH for maximum stability, maximum solubility and minimum degradation, and for removing residual moisture or water content from the dried peptide powder. Non-volatile buffers include those that do not evaporate in a similar manner to water upon 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. Stabilizing excipient

In certain preferred embodiments, the formulations described herein may be further stabilized to ensure stability of the peptides incorporated therein. In some embodiments, the stability of an injectable formulation is enhanced by including one or more stabilizing agents or stabilizing excipients into the formulation prior to drying the peptide. In other embodiments, the stability of the injectable formulation is enhanced by reconstituting the dried peptide with a stabilizing agent or stabilizing excipient in an aprotic polar solvent.

In some embodiments, the stabilizing excipient is a cryoprotectant. As shown in the examples section below, the addition of a cryoprotectant, such as trehalose, protects the peptide formulation of the present invention from the instability associated with freeze-thaw cycling. Furthermore, it has been shown herein that the addition of the cryoprotectant trehalose also promotes enhanced thawing of the frozen peptide preparation. This enhanced thawing property is surprisingly advantageous, especially in emergency medical situations, such as severe hypoglycemic episodes, where the peptide formulation of the invention is frozen and requires rapid administration. Thus, in another aspect of the invention, the stable formulation has improved freeze-thaw stability, increased thawing rate and/or improved thawing morphology.

In some embodiments, the stabilizing excipient is selected from the group consisting of sugars, starches, sugar alcohols, and mixtures thereof. Examples of suitable sugars for the stabilizing excipient include, but are not limited to, trehalose, glucose, sucrose, and the like. Examples of suitable starches for the stabilizing excipient include, but are not limited to, hydroxyethyl starch (HES). Examples of suitable sugar alcohols for the stabilizing excipient include, but are not limited to, mannitol and sorbitol. In some embodiments, at least one stabilizing excipient (e.g., a sugar, starch, sugar alcohol, or mixture thereof) is capable of enhancing the stability of the peptide during freeze-thaw, increasing the thawing rate of the formulation, or improving the thawed morphology of the formulation.

In some embodiments, the stabilizing excipient is present at 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) 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); or, 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 20% (w/v) of the formulation. In some embodiments, the stabilizing excipient is present in the formulation in an amount of about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% (w/v).

In formulations comprising two or more peptides, in some embodiments, each peptide is dried in a mixture comprising a non-volatile buffer and a stabilizing excipient. The mixture of non-volatile buffer and stabilizing excipient may be the same for each peptide, or the non-volatile buffer, stabilizing excipient, or both, used to dry 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.

In some embodiments, the formulation further comprises additional stabilizing agents including, for example, antioxidants, chelating agents, and preservatives. Examples of suitable antioxidants include, but are not limited to, ascorbic acid, cysteine, methionine, monothioglycerol, sodium thiosulfate, sulfite, BHT, BHA, ascorbyl palmitate, propyl gallate, N-acetyl-L-cysteine (NAC), and vitamin E. Examples of suitable chelating agents include, but are not limited to, EDTA, tartaric acid and salts thereof, glycerol, and citric acid and salts thereof. Examples of suitable preservatives include, but are not limited to, benzyl alcohol, methyl paraben, propyl paraben, and mixtures thereof.

In some embodiments, the formulation further comprises a stabilizing polyol. Such formulations and materials are described, for example, in U.S. Pat. nos. 6,290,99 and 6,331,310, the contents of each of which are incorporated herein by reference.

C. Reconstitution of dried peptides

In the stabilized formulations of the invention, once the peptide and non-volatile buffer (and optional stabilizing excipient) are dried to a powder, or wherein the formulation comprises two or more peptides, once each peptide and non-volatile buffer (each optionally also comprising a stabilizing excipient) are dried to a powder, the dried peptide powder is dissolved or reconstituted in an aprotic polar solvent. In some embodiments, the aprotic polar solvent is selected from the group consisting of dimethyl sulfoxide (DMSO), Dimethylformamide (DMF), ethyl acetate, N-methylpyrrolidone (NMP), Dimethylacetamide (DMA), propylene carbonate, and mixtures thereof. In some embodiments, the aprotic polar solvent is a mixture of two or more of dimethyl sulfoxide (DMSO), Dimethylformamide (DMF), ethyl acetate, N-methylpyrrolidone (NMP), Dimethylacetamide (DMA), and propylene carbonate. Dimethyl sulfoxide (DMSO), Dimethylformamide (DMF) and ethyl acetate are particularly preferred aprotic polar solvents, each of which is a biocompatible solvent. In some embodiments, the aprotic polar solvent is dimethyl sulfoxide (DMSO). In other embodiments, the aprotic polar solvent is N-methylpyrrolidone (NMP). In other embodiments, the aprotic polar solvent is a mixture of dimethyl sulfoxide (DMSO) and N-methylpyrrolidone (NMP). In yet other embodiments, the aprotic polar solvent is a mixture of dimethyl sulfoxide (DMSO) and ethyl acetate. In some embodiments, the dried peptide powder is reconstituted in an aprotic polar solvent that is "pure," i.e., free of co-solvents. In some embodiments, the dried peptide powder is reconstituted in a solution comprising an aprotic polar solvent but no water as a co-solvent.

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 one embodiment, the co-solvent is selected from the group consisting of 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 of about 10% (w/v) to about 50% (w/v), for example about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% (w/v). In some embodiments, the co-solvent is present in the formulation in an amount 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), 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 a temperature of at least 5 ℃, at least 10 ℃, at least 15 ℃, at least 20 ℃ or more as compared to an otherwise identical formulation that does not comprise the co-solvent. In some embodiments, the at least one co-solvent lowers the freezing point of the formulation to about 3 ℃, about 2 ℃, about 1 ℃ or about 0 ℃ or less.

D. Water content

The formulations of the present invention have very little residual moisture, and therefore the peptides in such formulations remain stable for long periods of time. In some embodiments, the stable formulations of the present invention have a moisture content of 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 3%, from about 0.25% to about 2%, or from about 0.25% to about 1.5%. In yet another preferred embodiment, the moisture content of the formulation is from about 0.5% to about 1%.

pH memory

The "pH memory" of a peptide is the charge distribution (protonated state) that results after drying the peptide in an aqueous buffer (e.g., in a non-volatile buffer). The protonation state of the peptide in very low or zero moisture non-aqueous solvents, and the resulting solubility and stability, are affected by the pH of the aqueous solution of the dried pro-peptide and the drying conditions employed. When a peptide is dried in a buffer substance in which both acidic and basic components are non-volatile, the pH memory of the dried peptide will be about equal to the pH of the peptide in the non-volatile buffer. See, e.g., enzymaticReactionsingOrganic media, Koskinen, A.M.P. and Klibanov, A.M., eds., Springer (1996). Furthermore, the pH of the buffered aqueous solution in which the peptide is dried (e.g., a non-volatile buffer) can be optimized to produce a pH memory for the peptide that results in optimal peptide stability, maximum solubility, and minimal degradation upon reconstitution in an aprotic polar solvent after the dried peptide. Because the aprotic polar solvent has no exchangeable protons, when the dried peptide is reconstituted into the aprotic polar solvent, the reconstituted formulation will retain the solubility and stability characteristics for optimal pH memory.

For stable formulations comprising two, three, four or more peptides, each peptide is dried such that it has its own pH memory optimized for maximum solubility, maximum stability and minimal degradation. In embodiments where two or more peptides are present in the formulation, the pH memory range of the first peptide may partially overlap with the pH memory range of the second peptide (e.g., the pH memory of the first peptide may be from about 4.0 to about 6.0, the pH memory 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 (e.g., the pH memory of the first peptide may be from about 4.0 to about 5.0, the pH memory of the second peptide may be from about 6.0 to about 8.0).

The pH memory of peptides can be measured in several ways. In one method, the pH memory of the peptide is measured by reconstituting the dried peptide into unbuffered water and measuring the pH of the reconstituted peptide using a pH indicator, such as a pH paper or calibrated pH electrode. Alternatively, for peptides that have been reconstituted in an aprotic polar solvent (e.g., DMSO), the pH memory of the peptide can be determined by adding at least 20% water to the aprotic polar solvent (e.g., DMSO) and measuring the pH using a pH indicator. See, for example, Baughman and Kreevoy, "determination of Aciditying in80% Dimethylsulfoxide-20% Water," journal of physical chemistry, 78(4):421-23 (1974). The measurement of pH in aprotic polar solvent-water solutions may require small corrections (i.e. not more than 0.2pH units according to Baughman and Kreevoy above).

In some embodiments, where the deviation of the pH memory of the peptide when the peptide is reconstituted in the aprotic polar solvent from the pH of the peptide in the non-volatile buffer in which the peptide is dried is within a range of one pH unit, the dried peptide has a pH approximately equal to the pH of the peptide in the non-volatile buffer in which the peptide is dried (thus, for example, for a peptide having a pH of 3.0 in the non-volatile buffer in which the peptide is dried, the pH memory of the peptide from 2.0 to 4.0 when the peptide is reconstituted in the aprotic polar solvent will be within one pH unit, such that the pH memory of the dried peptide will be approximately equal to the pH of the peptide in the non-volatile buffer). In some embodiments, where the deviation of the pH memory of the peptide when the peptide is reconstituted in the aprotic polar solvent from the pH of the peptide in the non-volatile buffer in which the peptide is dried is within a half pH unit range, the dried peptide has a pH approximately equal to the pH of the peptide in the non-volatile buffer in which the peptide is dried (thus, for example, for a peptide having a pH of 3.0 in the non-volatile buffer in which the peptide is dried, the pH memory of the peptide from 2.5 to 3.5 when the peptide is reconstituted in the aprotic polar solvent will be within a half pH unit, such that the pH memory of the dried peptide will be approximately equal to the pH of the peptide in the non-volatile buffer).

In some embodiments, the peptide of the stable formulation 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 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 about 2.5 to about 4.0. In some embodiments, the peptide of the stable formulation has a pH memory of about 2.5 to about 3.5. In some embodiments, the peptide of the stable formulation has a pH memory of 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 about 4.0 to about 5.0. In some 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 about 6.0 to about 8.0. In some embodiments, the peptide of the stable formulation has a pH memory of 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. 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 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 some 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 the peptide may be about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, or about 10.0.

F. Exemplary formulations

In some specific embodiments, the present invention provides a stable glucagon formulation comprising: a glucagon peptide or salt thereof (e.g., glucagon acetate), wherein said glucagon has been dried in a non-volatile buffer selected from the group consisting of a glycine buffer, a citrate buffer, a phosphate buffer, and mixtures thereof, and wherein the dried glucagon has a pH memory of from about 2.0 to about 3.0; and an aprotic polar solvent selected from the group consisting of dimethyl sulfoxide (DMSO), ethyl acetate, N-methylpyrrolidone (NMP), and mixtures thereof; wherein the formulation has a moisture content of less than 5% and wherein the dried glucagon maintains a pH memory of about 2.0 to about 3.0 when reconstituted in an aprotic polar solvent. In some embodiments, glucagon is present in the formulation in an amount from about 0.5mg/mL to about 100mg/mL or from about 1mg/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 the group consisting of a sugar (e.g., trehalose), a starch (e.g., hydroxyethyl starch (HES)), and mixtures thereof. The stabilizing excipient may be present in the formulation in an amount from about 1% (w/v) to about 60% (w/v). In some embodiments, the formulation further comprises a co-solvent that lowers the freezing point of the formulation, wherein the co-solvent is selected from the group consisting of ethanol, propylene glycol, glycerol, and mixtures thereof. The co-solvent may be present in the formulation in an amount from about 10% (w/v) to about 50% (w/v).

In other specific embodiments, the present invention provides a stable glucagon formulation comprising: glucagon or a salt thereof (or a glucagon analog or peptide mimetic); and an aprotic polar solvent selected from the group consisting of dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), and mixtures thereof; wherein 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%, or less than 0.25%. In other preferred embodiments, the moisture content is from 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%, more preferably from about 0.5% to about 1%.

In other specific embodiments, the stabilized glucagon formulation further comprises a non-volatile buffer and a stabilizing excipient which is a sugar, starch, or sugar alcohol. For example, in some embodiments, the glucagon formulation further comprises a glycine buffer and mannitol, or a citrate buffer and mannitol, or a phosphate buffer and mannitol. In some embodiments, the glucagon formulation further comprises a glycine buffer and trehalose, or a citrate buffer and trehalose, or a phosphate buffer and trehalose. In these embodiments, the aprotic polar solvent can be DMSO, NMP, ethyl acetate, or mixtures thereof. For example, in a preferred embodiment, the aprotic polar solvent is DMSO and the non-volatile buffer is glycine buffer. In another preferred embodiment, the aprotic polar solvent is DMSO, the non-volatile buffer is citrate buffer, and the stabilizing excipient is mannitol. In another preferred embodiment, the aprotic polar solvent is DMSO, the non-volatile buffer is glycine buffer, and the stabilizing excipient is trehalose. In yet another preferred embodiment, the aprotic polar solvent is DMSO and the non-volatile buffer is citrate buffer. In yet another preferred embodiment, the aprotic polar solvent is NMP and the non-volatile buffer is glycine buffer.

In other specific embodiments, the present invention provides a stable formulation comprising: glucagon or a salt thereof (e.g., glucagon acetate), wherein the glucagon has been dried in a non-volatile buffer, and wherein the dried glucagon has a pH about equal to the glucagon in a non-volatile buffer selected from the group consisting of glycine buffer, citrate buffer, phosphate buffer, and mixtures thereof, wherein the pH memory of the dried glucagon is from about 2.0 to about 3.0; and an aprotic polar solvent selected from the group consisting of dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), ethyl acetate, and mixtures thereof; wherein the moisture content of the formulation is less than 1%, and wherein when the dried glucagon is reconstituted in an aprotic polar solvent, the dried glucagon maintains a pH memory approximately equal to the pH of the glucagon in the non-volatile buffer. In some embodiments, the glucagon formulation further comprises a co-solvent that lowers the freezing point of the formulation, wherein the co-solvent is selected from the group consisting of ethanol, propylene glycol, glycerol, and mixtures thereof. In some embodiments, the glucagon formulation further comprises a stabilizing excipient selected from the group consisting of a sugar, a starch, and mixtures thereof. In some embodiments, glucagon is present in the formulation in an amount from about 1mg/mL to about 50 mg/mL.

In other specific embodiments, the present invention provides a stable glucagon formulation consisting essentially of: a glucagon peptide or salt thereof (e.g., glucagon acetate), wherein said glucagon has been dried in a non-volatile buffer selected from the group consisting of a glycine buffer, a citrate buffer, a phosphate buffer, and mixtures thereof, and wherein the dried glucagon has a pH memory of from about 2.0 to about 3.0; and an aprotic polar solvent selected from the group consisting of dimethyl sulfoxide (DMSO), ethyl acetate, N-methylpyrrolidone (NMP), and mixtures thereof; wherein the formulation has a moisture content of less than 5% and wherein the dried glucagon maintains a pH memory of about 2.0 to about 3.0 when reconstituted in an aprotic polar solvent.

In yet other specific embodiments, the present invention provides a stable glucagon formulation consisting essentially of: a glucagon peptide or salt thereof (e.g., glucagon acetate), wherein said glucagon has been dried in a non-volatile buffer selected from the group consisting of a glycine buffer, a citrate buffer, a phosphate buffer, and mixtures thereof, and wherein the dried glucagon has a pH memory of from about 2.0 to about 3.0; and an aprotic polar solvent and a co-solvent that lowers the freezing point of the formulation, wherein the aprotic polar solvent is selected from the group consisting of dimethyl sulfoxide (DMSO), ethyl acetate, N-methylpyrrolidone (NMP), and mixtures thereof, wherein the co-solvent is selected from the group consisting of ethanol, propylene glycol, glycerol, and mixtures thereof. Wherein the formulation has a moisture content of less than 5% and wherein the dried glucagon maintains a pH memory of about 2.0 to about 3.0 when reconstituted in an aprotic polar solvent.

In other specific embodiments, the present invention provides a stable glucagon formulation consisting essentially of: a glucagon peptide or salt thereof (e.g., glucagon acetate), wherein the glucagon has been dried in a non-volatile buffer selected from the group consisting of a glycine buffer, a citrate buffer, a phosphate buffer, and mixtures thereof, a stabilizing excipient selected from the group consisting of a saccharide (e.g., trehalose), a starch (e.g., hydroxyethyl starch (HES)), and mixtures thereof, and wherein the dried glucagon has a pH memory of from about 2.0 to about 3.0; and an aprotic polar solvent selected from the group consisting of dimethyl sulfoxide (DMSO), ethyl acetate, N-methylpyrrolidone (NMP), and mixtures thereof; wherein the formulation has a moisture content of less than 5% and wherein the dried glucagon maintains a pH memory of about 2.0 to about 3.0 when reconstituted in an aprotic polar solvent.

In yet other specific embodiments, the present invention provides a stable formulation comprising: insulin, wherein the insulin has been dried in a first non-volatile buffer selected from the group consisting of glycine buffer, citrate buffer, phosphate buffer, and mixtures thereof, and wherein the dried insulin has a first pH memory that is about equal to the pH of the insulin in the first non-volatile buffer, wherein the first pH memory is from about 1.5 to about 2.5 or from about 6.0 to about 8.0; pramlintide, wherein the pramlintide has been dried in a second non-volatile buffer selected from the group consisting of glycine buffer, citrate buffer, phosphate buffer, and mixtures thereof, and wherein the dried pramlintide has a second pH memory that is about 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 the group consisting of dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), ethyl acetate, and mixtures thereof; wherein the moisture content of the formulation is less than 1%, wherein when the dried insulin is reconstituted in said aprotic polar solvent, the dried insulin maintains a first pH memory approximately equal to the pH of the insulin in the first non-volatile buffer, and wherein when the dried pramlintide is reconstituted in said aprotic polar solvent, the dried pramlintide maintains a second pH memory approximately equal to the pH of the pramlintide in the second non-volatile buffer. In some embodiments, the insulin and pramlintide formulations further comprise a co-solvent that lowers the freezing point of the formulation, wherein the co-solvent is selected from the group consisting of 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 the group consisting of a sugar, a starch, 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 from about 1mg/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 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 some 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.

Method for preparing stable peptide formulations

In yet another aspect, the present invention provides a method for preparing a stable formulation for parenteral injection. In some embodiments, a method comprises: drying the peptide and non-volatile buffer to a dry peptide powder; reconstituting the dried peptide powder with an aprotic polar solvent to produce a stable formulation, wherein the stable formulation has a moisture content of less than 5%. In some embodiments, the dried peptide powder has a pH memory about equal to the pH of the peptide in the non-volatile buffer, and when the dried peptide powder is reconstituted in the aprotic polar solvent, the dried peptide powder maintains a pH memory about equal to the pH of the peptide in the non-volatile buffer.

The methods for preparing stable formulations may be used to formulate any peptide having 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, Luteinizing Hormone Releasing Hormone (LHRH) agonists, pramlintide, parathyroid hormone (PTH), dextrin, botulinum toxin, conotoxin, hematide, amyloid peptide, cholecystokinin, gastric inhibitory peptide, insulin-like growth factor, growth hormone releasing factor, antimicrobial factor, glatiramer, glucagon-like peptide-1 (GLP-1), GLP-1 agonists, exenatide, and analogs thereof. In a preferred embodiment, the peptide is glucagon or a glucagon analogue or peptide mimetic. 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 some embodiments, two, three, four or more peptides are formulated into a stable formulation. In embodiments where two or more peptides are formulated into a stable formulation, each peptide is separately dried with a non-volatile buffer into a dried peptide powder, each dried peptide powder having a pH memory that is about equal to the pH of the peptide in the non-volatile buffer (i.e., a first peptide has a first pH memory that is about equal to the pH of the first peptide in a first non-volatile buffer, and a second peptide has a second pH memory that is about equal to the pH of the second peptide in a second non-volatile buffer). Two or more dried peptide powders are reconstituted with an aprotic polar solvent to produce a stable formulation, wherein the stable formulation has a moisture content of less than 5% and wherein each dried peptide powder retains a pH memory that is about equal to the pH of the peptide in the non-volatile buffer when the dried peptide powder is reconstituted in the aprotic polar solvent (i.e., the dried first peptide retains a first pH memory when the dried first peptide is reconstituted in the aprotic polar solvent and the dried second peptide retains a second pH memory when the dried second peptide is reconstituted in the aprotic polar solvent).

In the methods 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 some embodiments, the non-volatile buffer is a mixture of citrate buffer and phosphate buffer. In some embodiments, the peptide is mixed with both a non-volatile buffer and a stabilizing excipient (e.g., a sugar, starch, or mixture thereof) and then dried into a dry peptide powder. In other embodiments, stabilizing excipients (e.g., sugars, starches, sugar alcohols, or mixtures thereof) are added to the peptide reconstituted in an aprotic polar solvent. In some embodiments, the stabilizing excipient is present in the formulation in an amount from about 1% (w/v) to about 60% (w/v), e.g., about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% (w/v). In some embodiments, the stabilizing excipient is trehalose. In some embodiments, the stabilizing excipient is HES. In some embodiments, the stabilizing excipient is a mixture of trehalose and HES.

As explained above, when the peptide is mixed with a non-volatile buffer, the non-volatile buffer is selected to be a pH that allows the peptide to have maximum stability/minimal degradation in an aqueous environment. Once dried, the peptide will have a pH memory of maximum stability/minimum degradation and will retain that pH memory when dissolved or reconstituted in an aprotic polar solvent. Likewise, in one embodiment, the pH of the non-volatile buffer is such that the dried peptide powder has a pH memory of from about 2 to about 3. In another embodiment, the pH of the non-volatile buffer is such that the dried peptide powder has a pH memory of from about 4 to about 6. In yet another embodiment, the pH of the non-volatile buffer is such that the dried peptide powder has a pH memory of from about 4 to about 5. In yet another embodiment, the pH of the non-volatile buffer is such that the dried peptide powder has a pH memory of from about 6 to about 8.

Once the peptide and non-volatile buffer (and optional other components, such as stabilizing excipients added to the peptide and non-volatile buffer prior to drying) are dried into a powder, the dried peptide powder is dissolved or reconstituted in an aprotic polar solvent as described herein (e.g., dimethyl sulfoxide (DMSO), N-methyl pyrrolidone (NMP), ethyl acetate, and mixtures thereof). In some embodiments, the aprotic polar solvent is dimethyl sulfoxide (DMSO). In other embodiments, the aprotic polar solvent is N-methylpyrrolidone (NMP).

In some embodiments, the step of reconstituting the dried peptide powder comprises diluting or reconstituting the dried peptide with a mixture comprising an aprotic polar solvent and a co-solvent that lowers the freezing point of the formulation. In some embodiments, the co-solvent is selected from the group consisting of ethanol, propylene glycol, glycerol, and mixtures thereof. In some embodiments, the co-solvent is present in the formulation in an amount from about 10% (w/v) to about 50% (w/v), e.g., about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% (w/v).

The formulations of the present invention have very little residual moisture, and therefore the peptides in such formulations remain stable for long periods of time. In preferred embodiments, the moisture content of the stable formulation 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 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 the foregoing methods, drying of the peptide compound with the non-volatile buffer (and optional stabilizing excipient) is accomplished 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 step of atomizing a solution containing one or more solids (e.g., therapeutic agents) after evaporation of the solvent from the droplets through a nozzle carousel or other device. The properties of the resulting powder are a function of several variables including the initial solute concentration, the size distribution of the droplets produced, and the rate of solute removal. The resulting particles may comprise aggregates of primary particles composed of crystalline and/or amorphous solids, depending on the rate and conditions of solvent removal.

Spray drying processes for the preparation of ultra-fine powders of biological macromolecules such as proteins, oligopeptides, high molecular weight polysaccharides and nucleic acids are described, for example, in U.S. Pat. No. 6,051,256. Freeze-drying procedures are well known in the art and are described, for example, in U.S. patent No. 4,608,764 and U.S. patent No. 4,848,094. The spray-freeze drying process is 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 PCT international publications nos. WO91/16882 and WO 96/09814.

Lyophilization techniques are well known to those skilled in the art. Lyophilization is a dehydration technique in which the product is subjected to freezing conditions (ice sublimation under vacuum) and to vacuum conditions (drying by slight heat). These conditions stabilize the product, minimizing oxidation and other degradation processes. The conditions of freeze-drying allow the process to be carried out at low temperatures, and therefore thermally unstable products can be protected. The freeze drying step comprises pretreatment, freezing, primary drying and secondary drying. Pretreatment includes any method of treating the product prior to freezing. This may include concentrating the product, formulation modifications (i.e., adding components to increase stability and/or improve processing), reducing high vapor pressure solvents, or increasing surface area. The pretreatment method comprises the following steps: freeze concentration, solution phase concentration and in particular formulation to preserve the product appearance or to provide freeze-drying protection for reactive products and is described, for example, in U.S. patent No. 6,199,297. "Standard" lyophilization conditions are described, for example, in U.S. Pat. No. 5,031,336 and "FreezeDryingofpharmaceuticals" (Deluca, Patrick P., J.Vac.Sci.Technol., Vol.14, No.1, January/February 1977) and "the Lyophilizaiton pharmaceuticals: Aliterurea review" (Williams, N.A. and G.P.Polli, journal of Parenteral sciences and technology, Vol.38, No.2, March.1984).

In certain preferred embodiments, the lyophilization cycle is conducted, in part, above the glass transition temperature (Tg) of the therapeutic agent formulation to cause the bolus to rapidly collapse, thereby forming a dense cake containing residual moisture. In other embodiments, the lyophilization cycle is performed below the glass transition temperature to avoid collapse in order to obtain complete drying of the particles.

Methods of treatment

In another aspect, the invention provides a method of treating a disease or disorder by administering to a subject an amount of a stable formulation as described herein effective to treat, ameliorate, or prevent the disease, disorder, or condition. In some embodiments, the disease, disorder, or condition to be treated with the stable formulations of the present invention is a diabetic disorder. 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, disorder, or condition is hypoglycemia. In some embodiments, the disease, disorder, or condition is diabetes.

In some embodiments, the treatment methods of the invention comprise treating hypoglycemia by administering to a subject having hypoglycemia an effective amount of a stable formulation as described herein to treat hypoglycemia. In some embodiments, a stable formulation comprising glucagon is administered to a subject.

In some embodiments, the treatment methods of the present invention comprise treating diabetes by administering to a diabetic patient a stable formulation as described herein in an amount effective to treat diabetes. In some embodiments, a stable formulation comprising insulin is administered to a subject. In some embodiments, a stable formulation comprising pramlintide is administered to a subject. In some embodiments, a stable formulation comprising insulin and pramlintide is administered to a subject. In some embodiments, a stable formulation comprising exenatide is administered to the subject. In some embodiments, a stable formulation comprising glucagon and exenatide is administered to the subject.

The dosage of administration of a peptide drug as described herein for the treatment of a disease, disorder, condition (e.g., a diabetic disorder, such as hypoglycemia or diabetes) is consistent with dosages and planned courses of treatment practiced by those of skill in the art. General guidelines for suitable dosages of all pharmaceutical agents used in the present invention are provided in the aforementioned pharmacological basis of therapeutics, 11 th edition, 2006, and in Physicians' desk reference (pdr), e.g., 65 th edition (2011) or 66 th edition (2012), PDRNetwork, LLC, each of which is incorporated herein by reference. Suitable dosages of peptide drugs for treating a disease, disorder, or condition as described herein will vary depending on several factors, including the formulation of the composition, the patient's response, the severity of the disorder, the weight of the subject, and the judgment of the prescribing physician. An effective dose of the formulation delivers a medically effective amount of the peptide drug. The dosage may be increased or decreased over time according to the needs of the individual patient.

Determination of an effective amount or dosage is well within the ability of those skilled in the art, especially in light of the detailed disclosure provided herein. Generally, formulations for delivering these doses may contain one, two, three, four, or more peptides or peptide analogs (collectively "peptides" unless peptide analogs are expressly excluded), wherein each peptide is present at a concentration of from about 0.1mg/mL up to the solubility limit of the peptide in the formulation. The concentration is preferably from about 1mg/mL to about 100mg/mL, such as about 1mg/mL, about 5mg/mL, about 10mg/mL, about 15mg/mL, about 20mg/mL, about 25mg/mL, about 30mg/mL, about 35mg/mL, about 40mg/mL, about 45mg/mL, about 50mg/mL, about 55mg/mL, about 60mg/mL, about 65mg/mL, about 70mg/mL, about 75mg/mL, about 80mg/mL, about 85mg/mL, about 90mg/mL, about 95mg/mL, or about 100 mg/mL.

The formulations of the invention may be used for subcutaneous, intradermal or intramuscular administration (e.g. by injection or by infusion). In some embodiments, the formulation is administered subcutaneously.

The formulations of the present disclosure are administered by infusion or by injection using a suitable device. For example, the formulations of the present invention may be placed into a syringe, pen injection device, auto-injector device, or pump device. In some embodiments, the injection device is a multi-dose syringe pump device or a multi-dose autoinjector device. The formulation is presented in the device in a manner such that the formulation can easily flow out of the needle upon actuation of the drive injection device, e.g., an auto-injector, to deliver the peptide drug. Suitable pen/auto injector devices include, but are not limited to, those manufactured by Becton-Dickenson, Swedish healthcare Limited (SHLGroup), YpsoMedAg, and the like. Suitable pump devices include, but are not limited to, those manufactured by tandemdiabetes care, inc.

In some embodiments, the formulations of the invention are provided ready for administration in a vial, syringe or prefilled syringe.

In another aspect, the invention provides the use of a stable formulation as described herein for the formulation of a medicament for the treatment of any disease, disorder or condition that can be treated with the peptide of the formulation. In some embodiments, the stable formulation is used to formulate a medicament for treating a diabetic condition, such as type 1 diabetes, type 2 diabetes, gestational diabetes, prediabetes, hyperglycemia, hypoglycemia, or metabolic syndrome.

In some embodiments, the stable formulation is used to formulate a medicament for treating hypoglycemia. In some embodiments, the stable formulation comprises glucagon or a salt thereof (e.g., glucagon acetate). In some embodiments, the stable formulation comprises glucagon and exenatide.

In some embodiments, the stable formulation is used to formulate a medicament for treating diabetes. In some embodiments, the stable formulation comprises insulin. In some embodiments, the stable formulation comprises exenatide. In some embodiments, the stable formulation comprises pramlintide. In some embodiments, the stable formulation comprises insulin and pramlintide.

VI. kit

In another aspect, the invention provides a kit for treating a disease, disorder, or condition as described herein. In some embodiments, a kit comprises: a stable formulation comprising one, two, three, four or more peptides or salts thereof, wherein the peptides have been dried in a non-volatile buffer, and wherein the dried peptides have a pH memory that is about equal to the pH of the peptides in the non-volatile buffer; and an aprotic polar solvent; wherein the moisture content of the formulation is less than 5%, and wherein when the dried peptide is reconstituted in an aprotic polar solvent, the dried peptide maintains a pH memory approximately equal to the pH of the peptide in a non-volatile buffer; and a syringe for administering the stable formulation to a subject.

In some embodiments, the kit comprises a stable glucagon formulation 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 the dried glucagon has a pH about equal to the glucagon in a non-volatile buffer selected from the group consisting of glycine buffer, citrate buffer, phosphate buffer, and mixtures thereof, wherein the pH memory of the dried glucagon is from about 2.0 to about 3.0; and an aprotic polar solvent selected from the group consisting of dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), ethyl acetate, and mixtures thereof; wherein the moisture content of the formulation is less than 1%, and wherein when the dried glucagon is reconstituted in an aprotic polar solvent, the dried glucagon maintains a pH memory approximately equal to the pH of the glucagon in the non-volatile buffer. In some embodiments, the glucagon formulation further comprises a co-solvent that lowers the freezing point of the formulation, wherein the co-solvent is selected from the group consisting of ethanol, propylene glycol, glycerol, and mixtures thereof. In some embodiments, the glucagon formulation further comprises a stabilizing excipient selected from the group consisting of a sugar, a starch, and mixtures thereof. In some embodiments, glucagon is present in the formulation in an amount from about 1mg/mL to about 50 mg/mL.

In some embodiments, the kit comprises a stable insulin and pramlintide formulation as described herein for use in treating diabetes in a subject. In some embodiments, a kit includes an insulin and pramlintide formulation comprising: insulin, wherein the insulin has been dried in a first non-volatile buffer selected from the group consisting of glycine buffer, citrate buffer, phosphate buffer, and mixtures thereof, and wherein the dried insulin has a first pH memory that is about equal to the pH of the insulin in the first non-volatile buffer, wherein the first pH memory is from about 1.5 to about 2.5 or from about 6.0 to about 8.0; pramlintide, wherein the pramlintide has been dried in a second non-volatile buffer selected from the group consisting of glycine buffer, citrate buffer, phosphate buffer, and mixtures thereof, and wherein the dried pramlintide has a second pH memory that is about 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 the group consisting of dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), ethyl acetate, and mixtures thereof; wherein the moisture content of the formulation is less than 1%, wherein when the dried insulin is reconstituted in the aprotic polar solvent, the dried insulin maintains a first pH memory approximately equal to the pH of the insulin in the first non-volatile buffer, and wherein when the dried pramlintide is reconstituted in the aprotic polar solvent, the dried pramlintide maintains a second pH memory approximately equal to the pH of the pramlintide in the second non-volatile buffer. In some embodiments, the insulin and pramlintide formulations further comprise a co-solvent that lowers the freezing point of the formulation, wherein the co-solvent is selected from the group consisting of 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 the group consisting of a sugar, a starch, 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 from about 1mg/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 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 some 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.

In some embodiments, the kit includes a syringe as part of a pen injection device, an auto-injector device, or a pump. In some embodiments, the syringe is pre-filled with a stable formulation. In some embodiments, the kit further comprises instructions, wherein the instructions direct the administration of the stable formulation to treat a subject in need thereof (e.g., a patient with hypoglycemia or diabetes).

VII. examples

The present invention will be described in more detail by way of specific examples. The following examples are provided for illustrative purposes and are not intended to limit the invention in any way. Those skilled in the art will readily recognize a variety of noncritical parameters that may be varied or altered to produce substantially the same results.

Example 1: preparation of glucagon solutions for use in freeze-drying

Various solutions were prepared to contain glucagon at a concentration of 10 mg/mL. The solution contains 5mM glycine, citrate or phosphate, typically providing a buffer establishing a pH of 3. The solution also contains a sugar, either alone or in combination, in an amount equal to the w/v amount of glucagon (1: 1) or in an amount of 200% (2: 1) glucagon. The saccharide is trehalose, HES or beta-cyclodextrin (beta-CD). Some solutions also contained 0.10% tween-20 as a surfactant. The various formulations were mixed to substantial homogeneity in the amounts as described in table 1 below.

TABLE 1 glucagon mixtures for subsequent lyophilization

To prepare the mixture, glucagon was dissolved at 10mg/mL in each buffer (phosphate, citrate and/or glycine buffer, 5mM, ph 3.0). The solution was then mixed at a ratio of 1: 1 (v/v) was mixed with various solutes prepared at twice the desired concentration using the corresponding buffers to obtain a final glucagon concentration of 5mg/mL and a final desired solute concentration. The solution was then filtered through a 0.2 μm Millipore PES membrane to remove insoluble material. Sample preparation was performed in a cold room at 4 ℃. The concentration and purity of glucagon was determined by RP-HPLC and Size Exclusion (SE) -HPLC.

Example 2: preparation of dried glucagon powder by freeze drying

The formulation of Table 1 above was pipetted into a 3-mL lyophilization flask (13-mmID). The formulation was lyophilized in an ftsdurstop lyophilizer (Stoneridge, NY). The samples were frozen at-40 ℃ with a gradient of 2.5 ℃/min and held for 2 hours to enable adequate freezing. The sample temperature was then raised to-5 ℃ with a gradient of 2 ℃/min and held for 2h as a renaturation step. The temperature was then reduced to 30 ℃ in a gradient of 1.5 ℃/min and the vacuum was turned on to 60 millitorr. Primary drying was set for 24 h. The temperature was gradually raised to 40 ℃ in a gradient of 0.5 ℃/min and maintained for a further 10 h. After drying was complete, the vial was capped under vacuum using the XX stopper from WestPharmaceutical corporation (product # 10123524). All formulations did not show any sign of lump collapse after freeze-drying. The final dried product has a moisture content of less than 1% w/w.

Example 3: preparation of glucagon formulations in aprotic polar solvents

Six of the dry powders prepared from the solutions in table 1 were selected for formulation in polar, aprotic solvents:

1. buffer (glycine) + trehalose (200% relative to glucagon%) (formulation # 3)

2. Buffer (glycine) + HES (200% relative to glucagon%) (formulation # 4)

3. Buffer (glycine) + trehalose (100% relative to glucagon) + HES (100% relative to glucagon) (formulation # 5)

4. Buffer (glycine) + tween-20 (0.01% w/v) + trehalose (200% relative to glucagon) (formulation # 19)

5. Buffer (glycine) + tween-20 (0.01% w/v) + HES (200% relative to glucagon) (formulation # 20)

6. Buffer (glycine) + tween-20 (0.01% w/v) + trehalose (100% relative to glucagon) + HES (100% relative to glucagon) (formulation # 21)

Example 4: preparation of glucagon solution with pH memory of 4-5

The solution is prepared to contain glucagon at a concentration of 10-20 mg/mL. The solution contains citrate buffer establishing a pH of 4-5. The solution also contains sugar alcohol, mannitol, with a concentration of 50-100 mg/mL. The formulation was mixed to substantial homogeneity and freeze dried by the drying cycle described in example 2 to a residual moisture of less than 0.5% w/w. Dissolving the dry powder into DMSO until the concentration of glucagon is 10-20mg/mL and the concentration of mannitol is 50-10 mg/mL.

Example 5: preparation of PTH (1-34) solution having low moisture and low freezing point

Solutions were prepared to contain PTH (1-34) at concentrations of 10-20 mg/mL. The solution contains citrate buffer establishing a pH of 4-5. The solution also contained sugar alcohol, mannitol, at a concentration of 50 mg/mL. The formulation was mixed to substantial homogeneity and freeze dried by the drying cycle described in example 2 to a residual moisture of less than 0.5% w/w. Dissolving the dry powder in DMSO until the concentration of PTH (1-34) is 10-20mg/mL and the concentration of mannitol is 50-100 mg/mL.

Example 6: increase in blood glucagon levels and blood glucose levels following glucagon administration

Two non-aqueous glucagon formulations based on glucagon-glycine-trehalose powder dissolved in NMP or DMSO in aprotic polar solvents were tested in pharmacokinetic and pharmacodynamic studies in rats and compared to aqueous formulations. Rats were dosed at a rate of 10g glucagon/rat. The non-aqueous glucagon solution was administered as a subcutaneous injection of 10 μ L, as was the aqueous control solution. All tested formulations demonstrated a rapid rise in blood glucagon concentration (see figure 1).

Pharmacokinetic (PK) parameters were analyzed for the four treatment groups plus an aqueous control. Non-atrioventricular PK analyses were performed for each rat. C_{Maximum of}And T_{Maximum of}Calculations are made from the observed data. The area under the curve (AUC) estimate was calculated without extrapolation. Data were analyzed using five-group ANOVA to compare PK parameters between groups. C between three groups_{Maximum of}、T_{Maximum of}Or no significant difference in AUC was observed. Relative bioavailability of NMP and DMSO formulations were all close to 100% (76% and 92%, respectively) relative to the aqueous control. Thus, based on the results of these rat PK studies, the non-aqueous formulation was essentially bioequivalent to the aqueous glucagon formulation.

As predicted from pharmacokinetic results, the non-aqueous glucagon formulations produced pharmacodynamic profiles that were substantially equivalent to the aqueous reconstituted glucagon formulations at the same dosage levels (see figure 2).

Example 7: enhanced solubility of glucagon in aprotic polar solvents compared to aqueous solutions

Glucagon was prepared at 1.0mg/mL by dissolution in one of the following buffers:

1.2mM citric acid, pH2.0 (titrated with concentrated hydrochloric acid) ("C2.0")

2.2mM citric acid, pH3.0 (titrated with concentrated HCl) ("C3.0")

Each formulation was placed in a sterile 2cc vial at a fill volume of 1 mL. Samples were freeze dried to reduce residual moisture and reconstituted to various nominal concentrations in DMSO, NMP or 50/50 DMSO/NMP cosolvent. Reconstituted concentrations range from 1 to 30 mg/mL. Solubility by passing through A₆₃₀The transparency, turbidity was visually measured and measured by RP-HPLC.

As shown in table 2 below, the pH memory of 2.0 and 3.0 glucagon lyophilized with citrate buffer could be readily dissolved to a concentration of 30 mg/mL. The same preparation can be completely dissolved in H only at a lower concentration₂And O. For pH memory of 3.0, at H₂Only a complete reconstitution in O was obtained at a concentration of 5 mg/mL. In addition, dissolved in H₂Glucagon of O is only metastable, i.e. it remains soluble for only a few hours and then begins to gel or fibrillate at a rate dependent on pH and concentration, whereas glucagon dissolved in aprotic polar solvents/co-solvents is stable indefinitely.

TABLE 2 solubility of glucagon at pH memory of 2.0 and 3.0

Example 8: effect of pH on solubility of glucagon in aprotic polar solvents.

When looking at the data shown in example 8 and table 2 from a pH memory perspective, it is clear that higher glucagon solubility can be obtained at lower pH memory (e.g., pH 2.0) than at higher pH in aprotic polar solvents. Furthermore, although the reconstitution in table 2 indicates a nominal concentration of substantially 100%, a₆₃₀Measurements showed an increase in turbidity of 30mg/mL glucagon (C3.0) solution with pH memory of 3.0 in pure NMP and DMSO/NMP cosolvent, while the C2.0 formulation with pH memory of 2.0 remained essentially haze-free.

In another example, for solubilization in H at 2mg/mL with 2mL glycine or 2mM citrate buffer₂Glucagon O acetate measures the effect of PH on the solubility of glucagon in aprotic polar solvents, with the PH adjusted to the desired value. Samples were freeze-dried and reconstituted to various nominal concentrations in DMSO, NMP or 50/50 in DMSO/NMP co-solvent. Solubility by passing through A₆₃₀The transparency, turbidity was visually measured and measured by RP-HPLC.

The "pH memory" from lyophilization was found to have a major effect on glucagon stability. For the "G2.5" (pH memory 2.5) lyophiles DMSO, DMSO/NMP and NMP, glucagon is soluble up to 30mg/mL reconstitution. A significantly reduced solubility was observed for the lyophile of "G3.5" (pH memory 3.5). The lyophilic of G3.5 was all turbid and the reconstitution was incomplete, even at a nominal reconstitution concentration of 10 mg/mL. DMSO and DMSO/NMP co-solvents showed about 95% recovery, whereas NMP only showed about 60% recovery.

Example 9: effect of buffer species on glucagon stability in DMSO

Glucagon acetate was prepared at 1.0mg/mL by dissolution in one of the following buffers:

1.2 mML-Glycine, pH3.0 (titration with concentrated HCl)

2.2mM citric acid, pH3.0 (titration with concentrated hydrochloric acid)

These formulations were lyophilized and reconstituted in DMSO at a nominal concentration of 5mg/mL glucagon. The preparation was placed in a stability incubator at 5 deg.C, 25 deg.C and 40 deg.C. The purity of glucagon was determined using a reverse phase HPLC method.

After incubation at various temperatures for one month, the stability of the formulation in glycine buffer was significantly better. Table 3 below shows the RP-HPLC purity at various periods of incubation at 40 ℃.

TABLE 3 Effect of buffer species on glucagon stability in DMSO

Preparation	Time =0	1 week	2 weeks	4 weeks
					Glycine, pH3.0	99.4	99.1	99.0	96.6
Citrate, pH3.0	98.6	97.7	97.3	92.7

Example 10: effect of moisture on glucagon stability in DMSO

Glucagon acetate was prepared at 1.0mg/mL by dissolution in one of the following buffers:

1.2 mML-Glycine, pH3.0 (titration with concentrated HCl)

2.2 mML-Glycine, pH3.0 (titration with concentrated HCl)

These formulations were lyophilized and reconstituted in DMSO at a nominal concentration of 5mg/mL glucagon. Additional moisture was added to the second formulation. The moisture content was measured using the karl fisher method. The moisture content of the first formulation was 0.13% (w/w), while the moisture content of the second formulation was 0.54% (w/w). The preparation was placed in a stability incubator at 5 deg.C, 25 deg.C and 40 deg.C. The purity of glucagon was determined using a reverse phase HPLC method.

After one month incubation at various temperatures, the stability of the formulation with less moisture was significantly higher. Table 4 below shows the RP-HPLC purity at various periods of incubation at 40 ℃. Even at moisture levels below 1%, significant stability differences could be detected.

TABLE 4 Effect of residual moisture on glucagon stability in DMSO

Preparation	Time =0	1 week	2 weeks	4 weeks
					Less water content	99.4	99.1	99.0	96.6
Additional moisture	99.2	98.9	98.8	95.6

Example 11: freezing point depression of DMSO solutions

For screening, the samples were cooled to-40 ℃ per minute at 8 ℃ and heated to 40 ℃ using a PerkinElmer Instrument instrumentation sYRISIS Diamond differential scanning calorimeter ("DSC").

DMSO/NMP mixtures

Various DMSO and NMP mixtures were tested, including:

1.90%DMSO+10%NMP

2.80%DMSO+20%NMP

3.70%DMSO+30%NMP

4.60%DMSO+40%NMP

5.50%DMSO+50%NMP

the DSC scan showed that the temperature of the solvent crystallization gradually decreased from-18 ℃ for pure DMSO to-5.7 ℃ for a 50% NMP/50% DMSO mixture. Glucagon acetate, glycine lyophile were added to a glucagon concentration of 5mg/mL resulting in a freezing point depression of-1 ℃ again.

DMSO/ethyl acetate mixtures

Various DMSO and ethyl acetate mixtures were tested, including:

1.80% DMSO +20% ethyl acetate (Tc =16 ℃ C.)

2.70% DMSO +30% ethyl acetate

3.60% DMSO +40% ethyl acetate (Tc =6.5 ℃ C.)

4.50% DMSO +50% ethyl acetate (Tc =2.9 ℃ C.)

5.40% DMSO +60% ethyl acetate (Tc = not observed)

The DSC scan showed that the crystallization temperature of the solvent gradually decreased from-18 ℃ for pure DMSO to 2.9 ℃ for a 50% NMP/50% DMSO mixture. No crystallization peak was observed for the 40% DMSO/60% ethyl acetate mixture. In addition, these formulations were stored at refrigeration temperature (4 ℃) for several days and observed visually for signs of solidification. All formulations with 30% or more ethyl acetate in the co-solvent remained liquid without solidification. This is slightly different from the Tc observed in the DS study.

DMSO solutions containing alcohol co-solvents

Various DMSO solutions were tested with addition of alcohol (ethanol, glycerol or propylene glycol) co-solvents, including

1.95% DMSO +5% alcohol

2.90% DMSO +10% alcohol

3.80% DMSO +20% alcohol

4.70% DMSO +30% alcohol

5.60% DMSO +40% alcohol

6.50% DMSO +50% alcohol

7.40% DMSO +60% alcohol

8.30% DMSO +70% alcohol

9.20% DMSO +80% alcohol

10.10% DMSO +90% alcohol

These formulations were stored at refrigeration temperature (4 ℃) for several days and observed by eye for signs of solidification. All formulations containing 20% or more alcohol co-solvent remained liquid without solidification. DSC scans showed freezing points for 20% alcohol co-solvent at 2.3 ℃, 0.6 ℃ and 3.3 ℃ for ethanol, glycerol and propylene glycol, respectively.

Example 12: freeze-thaw stability of glucagon

Glucagon acetate was prepared at 1.0mg/mL by dissolving 2mM L-glycine at ph3.0 (titrated with concentrated hydrochloric acid). The glucagon formulations were lyophilized and reconstituted in DMSO at a nominal concentration of 5mg/mL glucagon. The solution samples were divided and trehalose was added to one portion of the solution to a concentration of 5%. These formulations were aliquoted into vials and placed into a stability incubator at 5 ℃. These solutions were observed to solidify at 5 ℃. The glucagon solution was thawed at various time intervals and turbidity was determined using absorbance at 630 nm.

Table 5 below shows the turbidity of the glucagon solution incubated at 5 ℃ for various periods of time. The solution without trehalose showed an increase in turbidity at each time point of incubation. However, the solution containing trehalose showed no increase in turbidity. Turbidity measurements were confirmed by visual inspection. The frozen and incubated samples without trehalose were cloudy or opaque when observed.

TABLE 5 turbidity of glucagon solution after incubation at 5 deg.C

Preparation	Time =0	1 week	2 weeks	4 weeks
					Has no trehalose	0.024	0.142	0.130	0.160
5% trehalose	0.016	0.029	0.028	0.035

Surprisingly, the use of carbohydrate additives, such as trehalose, in DMSO solutions of peptides enhances the stability of the peptides during the freeze-thaw process.

Example 13: enhancing thawing rate with trehalose

For example 13, glucagon acetate was prepared as described above at 1.0mg/mL by dissolving in 2mM L-glycine at pH3.0 (titrated with concentrated HCl). After removal from storage at 5 ℃, it was observed that the sample of glucagon solution containing trehalose was completely thawed in much shorter time than the solution without trehalose. The trehalose containing samples were observed to thaw completely in less than 30 seconds, unlike glucagon solutions without trehalose, which were observed to thaw completely within typically minutes. The ability to thaw peptide preparations quickly is particularly advantageous in emergency care situations if the solution is frozen and must be injected quickly.

Example 14: effect of pH on insulin solubility

Insulin was dissolved in H at 10mg/mL with 10mM phosphate/citrate-1 mM EDTA buffer, pH2 or pH7₂And O. These solutions were lyophilized to dryness using a conservative cycle (>1% residual moisture) and reconstituted in DMSO to various nominal concentrations. Solubility by passing through A₆₃₀The transparency and the turbidity were measured visually.

At a pH of 2, it was observed that insulin could be dissolved to a concentration of at least 100 mg/mL. However, at a pH memory of 7, increased light scattering was utilized even at the lowest test concentration of 10mg/mL (A)₆₃₀) Poor solubility of insulin was observed as a cloudy or opaque solution. Some lower concentration, e.g., 10mg/mL, insulin solution with pH memory of 7 was observed to slowly dissolve into a clear solution over a period of about 24 hours.

Example 15: effect of pH on Pramlintide solubility

Pramlintide acetate was dissolved in H at 2mg/mL with 10mM citrate buffer pH4 or 10mM phosphate buffer pH7₂And O. These solutions were lyophilized to dryness using a conservative cycle (>1% residual moisture) and reconstituted in DMSO to various nominal concentrations. Solubility by passing through A₆₃₀The transparency and the turbidity were measured visually.

pramlintide with a pH memory of 7 is not soluble in DMSO at any concentration. However, low concentrations of pramlintide with a pH memory of 4 are soluble in DMSO.

Example 16: co-formulation of peptides in aprotic polar solvents

The preparation of co-formulations is prepared by separately drying the formulations of the individual compounds in aqueous solution which provides optimal solubility/stability upon reconstitution into an aprotic polar solvent. Solution pH is a property that affects the solubility of peptides, which when the dried peptide is reconstituted into an aprotic polar solvent, will maintain the "pH memory" of the aqueous formulation in which the peptide is dried, with the use of a non-volatile buffer. Because aprotic polar solvents have no exchangeable protons, a single peptide will retain the solubility and stability characteristics of optimal pH memory.

Current pramlintide and insulin formulations conflict in their buffer systems, making compatibility of the mixed formulation difficult. Most insulin and insulin analogues have isoelectric points between 5 and 6 and are therefore formulated at a pH of about 7 or at a lower pH of about 2. Pramlintide has an isoelectric point of >10.5 and is formulated at a pH of about 4 at which it is optimally stable. The interaction of pramlintide formulations and insulin formulations at different pH and different buffering capacity often results in precipitation of the soluble insulin component or dissolution of the crystalline insulin component. In vitro studies using formulations of pramlintide and short and long acting insulins found a significant change in insulin solubility when varying amounts of insulin were mixed with fixed amounts of pramlintide.

Thus, the present invention provides a formulation whereby both the fast acting insulin class and the dextrin analogue are stable and can be administered simultaneously from a single formulation for injection or formulation. The formulation more closely mimics the natural physiological response to postprandial blood glucose elevation than the prior art.

Examples of peptides that may be co-formulated include, but are not limited to: (1) insulin-dextrin (insulin with a pH memory of about 2.0 or about 7.0, and dextrin or dextrin analogs (e.g., pramlintide) with a pH memory of about 4.0); and (2) glucagon-GLP-1 (glucagon with a pH memory of about 3.0 or less, and glucagon-like peptide-1 (GLP-1) or an analog thereof (e.g., exenatide) with a pH memory of about 4.0-5.0).

A co-formulation of insulin and pramlintide was prepared as follows: an insulin preparation with pH memory of 2, 100mg/mL insulin was prepared as described in example 14 above. Pramlintide formulations with pH memory of 4, 1mg/mL pramlintide were prepared as described in example 15 above. Mu.l of the insulin preparation was mixed with 95mL of pramlintide solution. The resulting solution was observed to be clear, thus producing soluble co-formulations of insulin and pramlintide with respective pH memories of 2 and 4, respectively.

It is to be understood that the above description is intended to be illustrative, and not restrictive. Many embodiments will be apparent to those of skill in the art upon reading the above description. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims, along 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 applications, are incorporated herein by reference for all purposes.

Claims (20)

1. A stable solution for parenteral injection comprising:

(a) glucagon or a salt thereof that has been dried by a non-volatile buffer selected from a glycine buffer, a citrate buffer, a phosphate buffer, or mixtures thereof, wherein the dried glucagon has a pH memory of 2 to 3.5; and

(b) an aprotic polar solvent selected from the group consisting of dimethyl sulfoxide, N-methylpyrrolidone, ethyl acetate and mixtures thereof, wherein said dried glucagon or salt thereof reconstitutes and dissolves in said aprotic polar solvent in an amount of 0.1mg/mL up to the solubility limit of said glucagon or salt thereof;

wherein the dried glucagon maintains the pH memory when the dried glucagon is reconstituted and solubilized in the aprotic polar solvent; and

wherein the moisture content of the solution is less than 5%.

2. The solution of claim 1, wherein the pH memory is equal to the pH of the glucagon in the non-volatile buffer.

3. The solution of claim 1, wherein the pH memory of the dried glucagon is from 2 to 3.

4. The solution of claim 1, further comprising a co-solvent that lowers the freezing point of the solution, wherein the co-solvent is selected from the group consisting of ethanol, propylene glycol, glycerol, and mixtures thereof.

5. The solution of claim 1, further comprising a stabilizing excipient selected from the group consisting of a sugar, a starch, and mixtures thereof.

6. The solution of claim 1, comprising 0.1mg/mL to 100mg/mL of said dried glucagon.

7. The solution of claim 1, comprising 1mg/mL to 30mg/mL of said dried glucagon.

8. The solution of claim 1, wherein the buffer is glycine buffer and the aprotic polar solvent is dimethyl sulfoxide.

9. The solution of claim 8, wherein the solution further comprises a stabilizing excipient selected from the group consisting of a sugar, a starch, and mixtures thereof.

10. The solution of claim 9, wherein the stabilizing excipient is trehalose.

11. A method of preparing a stable solution for parenteral injection, the method comprising:

(a) drying a mixture comprising glucagon or a salt thereof in a non-volatile buffer selected from a glycine buffer, a citrate buffer, a phosphate buffer, or mixtures thereof, to dry glucagon, wherein the dry glucagon has a pH memory of 2 to 3.5; and

(b) reconstituting 0.1mg/mL up to the solubility limit of said dried glucagon in an aprotic polar solvent selected from the group consisting of dimethyl sulfoxide, N-methylpyrrolidone, ethyl acetate, and mixtures thereof, wherein said dried glucagon is dissolved in said aprotic polar solvent;

wherein the dried glucagon maintains the pH memory when the dried glucagon is reconstituted and solubilized in the aprotic polar solvent; and

wherein the moisture content of the solution is less than 5%.

12. The method of claim 11, comprising adding 0.1mg/mL to 100mg/mL of said dried glucagon to said aprotic polar solvent.

13. The method of claim 11, comprising adding 1mg/mL to 30mg/mL of said dried glucagon to said aprotic polar solvent.

14. The method of claim 11, wherein the pH memory of the dried glucagon is from 2 to 3.

15. The method of claim 11, wherein the buffer is a glycine buffer and the aprotic polar solvent is dimethyl sulfoxide.

16. The method of claim 15, wherein the mixture further comprises a stabilizing excipient selected from the group consisting of a sugar, a starch, and mixtures thereof.

17. The method of claim 16, wherein the stabilizing excipient is trehalose.

18. Use of the stable solution of any one of claims 1-10 for the preparation of a pharmaceutical composition for parenteral administration.

19. Use of the stable solution of any one of claims 1-10 for the preparation of a pharmaceutical composition for the treatment of hypoglycemia, wherein the pharmaceutical composition is administered parenterally.

20. Use of the stable solution of any one of claims 1-10 for the preparation of a pharmaceutical composition for the treatment of diabetes, wherein the pharmaceutical composition is administered parenterally.