Compositions Comprising Fast-Acting Insulin Analogues

Abstract

[Solution] The pharmaceutical composition of the present invention contains an effective amount of an insulin analog comprising a modified A-chain and B-chain polypeptide. The modified A-chain contains one or more substitutions relative to the wild-type human insulin A-chain selected from a Gln, His, or Glu substitution at position A8, a Glu or Ala substitution at position A14, and an Ala, Gln, Gly, or Thr substitution at position A21. The modified B-chain polypeptide contains one or more modifications relative to the wild-type human insulin B-chain selected from an amino acid or amino acid deletion at positions B1, B1 and B2, or B1-B3, an Ala or Glu substitution at position B2, a Glu or Ala substitution at position B3, an Ala substitution at position B4, and a Glu substitution at position B29. The composition also contains one or more of iloprost, citrate, EDTA, or a polyphosphate compound. The composition can be used to treat diabetes.

Description

[Technical Field]

[0001] This application is an International PCT application claiming the benefit of U.S. Provisional Patent Application No. 62 / 984,165, filed March 2, 2020. The disclosures of the above-referenced applications are incorporated herein by reference.

[0002] This invention was made with government support under Grant Nos. DK040949 and DK074176 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] The present invention relates to a fast-acting insulin analogue and a composition thereof, and a method for treating diabetes or controlling blood glucose levels in a patient using the insulin analogue or a composition thereof.

[0004] Insulin has been used to treat diabetes for over 90 years. Treatment typically requires multiple daily insulin injections. Traditional regimens involve patients receiving one to two daily injections of long-acting insulin to cover basal insulin needs and supplementing them with injections of rapid-acting insulin (or rapid-acting insulin analogs) to cover meal-related insulin needs. However, even when administered appropriately and in a timely manner, insulin injections cannot mimic the natural time-action profile of insulin. For example, commercially available rapid-acting insulin analogs enter the bloodstream and their sites of action too slowly, resulting in too long an overall duration of action. This results in insufficient insulin levels at the start of a meal and too much insulin between meals, especially immediately after. This delayed insulin action leads to early post-meal hyperglycemia and postprandial hypoglycemia.

[0005] In healthy individuals, insulin secretion is tightly coupled to blood glucose levels. When blood glucose levels rise, such as after a meal, insulin is released directly into the bloodstream, providing a responsive surge. During fasting, insulin levels fall to basal levels. The goal of insulin therapy is to replicate this natural time-action profile of insulin in diabetic patients, allowing them to maintain blood glucose levels within the normal range characteristic of healthy individuals. However, current insulin formulations and delivery systems do not adequately meet this goal due to limited absorption of insulin or insulin analogs.

[0006] For example, insulin preparations (or insulin analogue preparations) that contain predominantly monomeric (which is the predominant form of insulin circulating in the blood) and dimeric forms of the protein molecule have a strong tendency to aggregate and form inactive microfibrils. For example, if insulin is solubilized in a zinc-free buffer and stored at room temperature (25–30°C), it will form amyloid microfibrils. To circumvent this problem, currently available insulin preparations generally contain zinc, which forms a complex with insulin called a zinc-insulin hexamer. The zinc-insulin hexamer is stable in solution at room temperature for more than 30 days, which is long enough to meet regulatory requirements for the stability of insulin preparations. However, because the zinc-insulin hexamer is too large to be readily absorbed by capillaries, the hexamer must be degraded in the subcutaneous interstitial fluid after injection before the insulin can be absorbed into the circulation. The hexamerization required for insulin stabilization in the vial prevents these preparations from being absorbed rapidly enough to match physiological insulin secretion.

[0007] Therefore, fast-acting insulin analogs and compositions thereof are needed for better control of blood glucose levels in diabetic patients. The prior art documents relevant to the invention of this application are as follows (including documents cited in the international phase after the international filing date and documents cited when the application entered the national phase in other countries). (Prior art document) (Patent document) (Patent Document 1) International Publication No. 2018 / 094388 (Patent Document 2) International Publication No. 2016 / 057529 (Patent Document 3) International Publication No. 2016 / 064606 (Patent Document 4) International Publication No. 2014 / 015078 Summary of the Invention [Means for solving the problem]

[0008] The present invention relates, in part, to insulin analogs or pharmaceutically acceptable compositions thereof that provide rapid uptake of the analog into the bloodstream, resulting in its rapid onset of action, compared to, for example, existing commercially available insulin products and other similar insulin analogs. Furthermore, in various embodiments, the insulin analogs and compositions have advantages in stability (e.g., as quantified by the rate of fibril formation, the change in chemical stability after 7 and 28 days, and the change in physical stability after 7 and 28 days) and mitogenicity (e.g., as quantified in cell-based proliferation assays). Formulating the analog with optimized excipient selection and concentrations enhances the advantages of the analog's pharmacological and thermodynamic stability. Thus, in some embodiments, the present invention provides insulin analogs or pharmaceutically acceptable compositions thereof that exhibit a more rapid onset of insulin action without compromising stability (compared to commercially available insulin analogs or wild-type human insulin).

[0009] In one embodiment, the present invention provides a pharmaceutical composition comprising an effective amount of an insulin analog comprising a modified A-chain B-chain polypeptide. The modified A-chain comprises one or more substitutions relative to the wild-type human insulin A-chain selected from a Gin, His, or Glu substitution at position A8, a Glu or Ala substitution at position A14, and an Ala, Gin, Gly, or Thr substitution at position A21. The modified B-chain polypeptide comprises one or more modifications relative to the wild-type human insulin B-chain selected from an amino acid or amino acid deletion at positions B1, B1 and B2, or B1-B3, an Ala or Glu substitution at position B2, a Glu or Ala substitution at position B3, an Ala substitution at position B4, and a Glu or Lys substitution at position B29. The composition may further comprise one or more of iloprost, citrate, EDTA, and a polyphosphate compound. The pharmaceutical composition of the present invention may be formulated for use in treating diabetes.

[0010] In another aspect, the present invention provides insulin analogs comprising modified A-chain polypeptides and modified B-chain polypeptides. For example, the A-chain consists of, or consists of, substitutions relative to the wild-type human insulin (HI) A-chain selected from a Glu substitution at position A8, a Glu substitution at position A14, and a Gly substitution at position A21. Furthermore, the B-chain polypeptide consists of, or consists of, modifications relative to the wild-type human insulin B-chain selected from a deletion of the residue at position B1, an Ala or Glu substitution at positions B2 or B3, and a Glu substitution at position B29.

[0011] In some embodiments, the modified A chain polypeptide comprises a Gly substitution at position A21. In some embodiments, the modified B chain polypeptide comprises a Glu substitution at position B3. In some embodiments, the modified A chain polypeptide comprises an Ala substitution at position A21. In some embodiments, the modified B chain polypeptide comprises an Ala substitution at position B3. In one embodiment, the analog is referred to as T-1123, hereinafter, having modifications at GluA8, GluA14, GlyA21, desB1, AlaB2, GluB3, and GluB29.

[0012] Another aspect of the present invention provides pharmaceutical compositions comprising an effective amount of an insulin analog and an excipient to enhance the pharmacological and thermodynamic stability benefits of the insulin analog. For example, the formulation may include, but is not limited to, a tonic, a preservative, a stabilizer, a solubilizer, or an absorption enhancer. In some embodiments, the pharmaceutical composition comprises an effective amount of a fast-acting insulin analog and a polyphosphate compound (e.g., sodium triphosphate). In some embodiments, the pharmaceutical composition comprises an effective amount of a fast-acting insulin analog and iloprost.

[0013] Yet another aspect of the present invention provides a method for treating a subject having diabetes, comprising administering to a subject in need thereof an insulin analogue or pharmaceutical composition of the present disclosure.

[0014] Yet another aspect of the present invention provides a method for determining the profile, or pharmacokinetic (PK) parameters, of an active pharmaceutical ingredient (API) formulation. The method includes administering a composition of multiple inactive or ineffective analogs (nonalogs) of the API to a subject or first subject and determining the concentration of the nonalogs in one or more tissue samples from the subject or first subject. The method also includes determining one or more pharmacokinetic parameters of the API formulation based on the concentration of the nonalogs in the subject or first subject. In one embodiment, the method is used to determine PK parameters of the nonalogs and use them as a surrogate for determining PK parameters of the corresponding API. In some embodiments, the nonalogs are ineffective analogs of peptide hormones such as insulin.

[0015] In some embodiments, the present invention provides a method for preparing a pharmaceutical formulation of an API. The method includes administering multiple nonalogue compositions of the API to a subject and determining the concentration of the nonalogue in one or more samples from the subject. The method also includes determining pharmacokinetic profiles for the nonalogue compositions and formulating a formulation utilizing similar compositions to achieve the desired pharmacokinetic profile of the API as a pharmaceutical formulation. In one embodiment, the method further includes determining the desired pharmacokinetic profile based on the concentration of the nonalogue in one or more samples from the subject. Once the desired pharmacokinetic profile or parameters of the nonalogue composition are achieved, the API can then be formulated to mimic the nonalogue composition, except that the nonalogue in the selected nonalogue composition is replaced with the corresponding API.

[0016] In another aspect, the present invention relates to a non-active analog (nonalogue) composition, which comprises a non-alloy of an API and a pharmaceutically acceptable carrier. The non-alloy composition is used in determining the pharmacokinetic parameters of the corresponding API or a pharmaceutical formulation for the corresponding API.

[0017] In various aspects, the present invention provides methods for improving the efficiency of PK formulation testing. Because multiple, substantially simultaneous administration of clinically relevant doses of an API results in much higher concentrations of the API in a subject's bloodstream than would be achieved with conventional therapy (thus posing potential safety issues and leading to subject morbidity or mortality), the present invention provides for the use of less potent or inactive analogs (nonalogs). The methods and products described herein allow for the evaluation of the PK of potential API formulations without the stress of multiple subjects, while minimizing costs.

[0018] Other aspects and embodiments of the present invention will become apparent from the following detailed description. [Brief explanation of the drawings]

[0019] [Figure 1] Figure 1 summarizes the multicompartment flow model used to evaluate the results of multiple pharmacokinetic studies in pigs. [Figure 2] Figures 2A-2D show results from the statistical analysis of multiplex PK studies for formulation screening. These are graphical depictions of the onset slope of the PK response in pigs subcutaneously injected with a formulation (alternative insulin analog) containing multiple candidate enhancing excipients. Each column represents a unique formulation, with individual animals represented by black dots and the mean represented by blank or filled dots. Filled dots indicate statistically different results from the control (highlighted by arrows at the bottom of the figure and a line in the center of the figure for comparison with each study), whereas empty dots indicate no difference from the control. If the filled dot is to the right of the vertical line that intersects the entire figure, the insulin analog is absorbed statistically faster than the control; if it is to the left of the vertical line, the insulin analog is absorbed statistically slower than the control. [Figure 3]Figure 3 shows the results of a euglycemic clamp study in non-diabetic Yucatan pigs administered subcutaneously with U-100 Fiasp or U-500 T-1123 as a base formulation, showing the glucose infusion rate required to maintain normoglycemia for 360 minutes (n = 7). [Figure 4] Figure 4 shows the results of a euglycemic clamp study showing the glucose infusion rate required to maintain euglycemia (n=7) for 360 minutes in non-diabetic Yucatan pigs administered subcutaneously with U-100 Fiasp or U-500 T-1123 in an accelerated formulation. [Figure 5] Figure 5 shows the results of a euglycemic clamp study in non-diabetic Yucatan pigs administered U-100 Fiasp or U-500 T-1123 subcutaneously as a stability-optimized accelerated formulation, showing the glucose infusion rate required to maintain euglycemia (n=7) for 360 minutes. [Figure 6] Figure 6A-C summarizes the results obtained from euglycemic clamp studies showing the glucose infusion rate required to maintain euglycemia (n = 7) after pigs were treated with either U-100 Fiasp or U-500 T-1123 weight loss formulations. Figure 6A omits citric acid; Figure 6B omits EDTA; and Figure 6C omits iloprost. [Figure 7A] Figure 7A shows results from the accelerated fibrillation assay showing the mean fibrillation lag time for Humalog (n=6), T-1123 in base formulation (n=11), T-1123 in base formulation + iloprost (n=3), T-1123 in citric acid formulation + iloprost (n=6), and citric acid formulation + iloprost + Tris (n=15). The base formulation of U-500T-1123 is 16 mg / mL glycerin, 3.2 mg / mL m-cresol, 50 mM Tris. [Figure 7B] FIG. 7B shows the results from the accelerated fibril assay showing the mean fibril lag time for Humalog formulated with no iloprost (n=3), 15 pg / ml iloprost (n=3), 50 pg / ml iloprost, and 100 pg / ml iloprost (n=3). [Figure 7C] Figure 7C shows results from a 12-month real-time fibril assay showing the average fibril lag time for U-500 T-1123, U-400 Insuman, and U-100 Humalog in accelerated formulations optimized for stability. All samples were placed in vials and then placed on a nutator mixing platform at 30°C for one year. N=3 for all samples. Fibril lag time is measured in days. [Figure 8A] Figure 8A shows the results obtained in chemical degradation studies for the base formulation of U-500 T-1123 (n = 1), commercial U-100 Humalog (n = 2), and commercial U-500 Humulin (n = 2), showing a decrease in purity due to the accumulation of related substances (RS) and covalently bound high molecular weight proteins (HMWPs). [Figure 8B] Figure 8B shows the results obtained in a chemical degradation assay assessing the accumulation of related substances for formulations of U-500 T-1123 with varying concentrations of Mg2+ and Tris. [Figure 8C] Figure 8C shows the results obtained in a chemical degradation assay assessing HMWP accumulation for formulations of U-500 T-1123 with varying Mg 2+ and Tris concentrations. [Figure 8D] FIG. 8D shows the results obtained in a chemical degradation assay evaluating the amount of RS accumulation in U-500 T-1123 formulations containing varying concentrations of glycerol and Tris. [Figure 8E] FIG. 8E shows the results obtained in a chemical degradation assay assessing HMWP accumulation for formulations of U-500 T-1123 with varying concentrations of glycerol and Tris. [Figure 8F] Figure 8F shows the results of a chemical degradation test for evaluating related substance purity when U-500 T-1123 was placed in a vial and placed on a nutator mixing table at 30°C for one year. [Figure 8G] Figure 8G shows the results of a chemical degradation test in which U-500T-1123 in a vial was placed on a Nutator mixing table at 30°C for one year to assess related substance purity. [Figure 9A]Figure 9A shows the results from an intracellular Western blot assay measuring the dephosphorylation of hIR-A in CHO cells over 180 minutes after treatment with either HI (n=1) or T-1123 (n=3). [Figure 9B] Figure 9B shows the results from an intracellular Western blot assay measuring the dephosphorylation of hIR-B in CHO cells over 180 minutes after treatment with either HI (n=1) or T-1123 (n=2). [Figure 10A] FIG. 10A shows the results obtained when rats were monitored for changes in blood glucose concentration over a 300 minute period when treated with either insulin lispro (KP) (n=215) or T-1123 (n=19). [Figure 10B] FIG. 10B shows results from a rat study showing the maximum blood glucose drop (Max BG Drop) when animals were treated with either insulin lispro (KP) (N=215) or T-1123 (n=19). [Figure 11A] FIG. 11A shows results from the accelerated fibril assay showing the mean fibril lag time (n=3) for Humalog® (with or without triphosphate), Novolog® (with or without triphosphate), and T-1123 (formulated with U-500 with or without triphosphate). [Figure 11B] Figure 11B shows the mean fibrillation lag time (n=3) for Humalog® (with or without triphosphate), Novolog® (with or without triphosphate), and T-8602 (formulated in U-100 with or without triphosphate) from an accelerated fibrillation assay. T-8602 is a single-chain insulin analog with the following modifications: EA8, EA14, AA21, AB3, EB29, and an EEGRR linker connecting positions B30 and A1. [Figure 11C]Figure 11C shows the results obtained from the accelerated fibrillation assay, showing the mean fibrillation lag time for Humalog (n=3) and T-1123 in three different formulations: STA-067 (n=3), STA-109 (n=3), and STA-116 (n=3). [Figure 12] FIG. 12 shows the results obtained for RS and HMWP in forced chemical degradation tests based on U-500T-1123 in the absence and presence of TriP04. DETAILED DESCRIPTION OF THE INVENTION

[0020] As used herein, the terms "formulation" and "composition" are used interchangeably. As used herein, the term "formulation" or "composition" encompasses, for example, chemicals or excipients added to an active pharmaceutical ingredient (API) to impart or support desired pharmaceutical properties to the API. These properties may include, but are not limited to, rapid absorption, delayed absorption, sustained release, extended half-life, reduced clearance rate, increased clearance rate, and charge masking. The term "formulation" also encompasses the relative concentrations of the API or nonalogue in the composition, as well as the underlying carrier (e.g., an aqueous solution, a solid powder in a capsule, a coating on a capsule, etc.).

[0021] As used herein, the term "subject" or "patient" is intended to include human patients and non-human animals, such as those used in preclinical trials, e.g., mice, rats, rabbits, dogs, pigs, cats, primates, and cows.

[0022] As used herein, the term "thermodynamic stability" is intended to encompass physical and chemical stability as measured by the self-assembly of either amorphous aggregates or insoluble highly structured microfibrillar species, and the loss of purity of the insulin analog composition due to the accumulation of covalently attached high molecular weight proteins (HMWPs) or related substances (RSs), respectively.

[0023] As used herein, the term "active pharmaceutical ingredient (API)" refers to a substance or mixture of substances intended for use in the manufacture of a pharmaceutical (e.g., medicinal) product, which, when used in the manufacture of the pharmaceutical product, becomes the active ingredient of the pharmaceutical product. Such substances are intended to provide pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure or function of the body. Active pharmaceutical ingredients (APIs) include drug substances for use in the diagnosis, cure, mitigation, treatment, or prevention of disease. An API can be, for example, a protein, peptide, small molecule, oligonucleotide, or polymer. An API can also be an insulin analog. As used herein, the term "pharmaceutical product" refers to a finished dosage form, such as a tablet, capsule, or solution, which generally, but not necessarily, contains an active pharmaceutical ingredient in association with inactive ingredients. The term is intended to encompass the final dosage form of the API combined with various pharmaceutically acceptable carriers or excipients to create the API formulation.

[0024] As used herein, the term "administration" is intended to encompass any method by which an API, pharmaceutical agent, or nonalogue is introduced into a subject's body. Exemplary routes of administration include, but are not limited to, oral, subcutaneous, sublingual, intramuscular, intravenous, intradermal, intraperitoneal, buccal, or nasal.

[0025] As used herein, the term "non-analog" is intended to encompass inactive or inactive analogs of an API. These non-analogs have a similar chemical structure, primary structure, secondary structure, tertiary structure, quaternary structure, hydrophobic structure, or similar surface charge pattern as the API. In some examples, a non-analog is sufficiently different from other non-analogs of an API that it can be reliably detected and / or separated from other non-analogs in a sample, yet sufficiently similar that the PK properties of the non-analog are not expected to differ significantly from the corresponding API. As a non-limiting example, different non-analogs of an API can differ from each other by mass.

[0026] The term "tissue sample" or "sample" as used herein is intended to encompass samples of solid, liquid, and gaseous extracts taken from a subject, including, but not limited to, biopsy samples, blood, urine, and cerebrospinal fluid samples, and exhaled breath samples.

[0027] As used herein, unless the context requires otherwise, the term "about" means + or - 10% of the relevant numerical value.

[0028] Insulin has the ability to self-associate (at therapeutic concentrations) into dimers, hexamers, high molecular weight aggregates, and insoluble fibrils. Loss of purity due to the formation of related substances, high molecular weight covalent and noncovalent protein aggregates, or insoluble amyloid fibrils is problematic in the treatment of diabetes, particularly in pharmaceutical compositions intended for storage prior to use. Several types of structural changes can occur in insulin's structure during the fibrillation process. Prior to fibrillation, insulin is thought to form amyloid-like structures or amorphous aggregates. These amyloid fibrils are enriched in b-sheet structures. Fibrillation is thought to occur via amyloidogenic subfolding, independent of native assembly. Zinc-free insulin is particularly susceptible to loss of purity under a wide range of conditions, likely promoted by factors that inhibit native dimerization or higher-order assembly. The storage form of insulin in pancreatic B-cells and most pharmaceutical preparations is stabilized by an axial zinc (Zn) ion coordinated to the insulin amino acid side chains, particularly the HisB10 residue. Formulating insulin or insulin analogs as zinc-stabilized hexamers slows, but does not prevent, fibrillation, especially above room temperature and upon agitation. Storing insulin analogs as zinc-stabilized hexamers has the additional disadvantage of delaying the action of the analogs because insulin must dissociate into monomers to bind to the insulin receptor. This is a particular obstacle in creating fast-acting insulin analogs, which must overcome either the stability issues exhibited by monomeric, zinc-free formulations or the delayed action exhibited by hexameric formulations.

[0029] The present invention in various aspects and embodiments is directed to fast-acting insulin analogs and formulations that provide fast-acting properties over a wide range of protein concentrations and formulation strengths (typically U-100 to U-500, and optionally as high as U-1000). In various embodiments, the mitogenicity of the insulin analog is no greater than that of insulin lispro (abbreviated as KP, a commercially available fast-acting insulin analog). In various embodiments, the thermodynamic stability of the insulin analog or compositions thereof in the absence of zinc ions is equal to or greater than that of commercially available insulin analogs formulated with or without zinc ions.

[0030] In some embodiments, the present invention provides insulin analogs or compositions thereof that do not form fibrils, exhibit delayed fibril formation, or exhibit an increased fibrillation lag time. In some embodiments, the insulin analogs or compositions thereof exhibit high physical and chemical stability, with minimal loss of purity due to accumulation of HMWP and RS. The present invention further provides insulin compositions containing various excipient compounds that provide a high level of thermodynamic stability and a high absorption rate of the insulin analog.

[0031] In some embodiments, the insulin analog or composition thereof does not form insulin fibrils or takes a significantly longer time to form insulin fibrils, e.g., compared to wild-type human insulin or commercially available insulin analog formulations. In some embodiments, the insulin analog or composition thereof exhibits increased thermodynamic stability, e.g., compared to wild-type human insulin or commercially available insulin analog formulations. In embodiments, the insulin analog compositions described herein provide the ability to store the composition for longer periods without unwanted fibril formation and / or without unwanted physical or chemical degradation or aggregation. Methods of using insulin and insulin analog compositions for treating a subject with diabetes (e.g., for managing blood glucose levels) are also provided.

[0032] According to embodiments of the present invention, formulations of fast-acting insulin analogs are provided having excipients that provide unexpectedly superior pharmacodynamic properties and / or physical and / or chemical stability. In various embodiments, the present disclosure is a component of a comprehensive comparative evaluation of the efficacy of various insulin analog formulations. These studies identify component formulations that meet the stability and absorption rate requirements for improved human therapy. According to embodiments of the present invention, insulin analogs and formulations are provided that better mimic the natural time-action profile of insulin without undesirable fibril formation during storage.

[0033] The superior properties of the present invention are due in part to the novel combinations of substitutions within the A and B chains of the insulin analogs, combined with N-terminal deletions of the B chain. The A and B chain substitutions fall into four classes: (i) non-β-branching substitutions at position A8, (ii) helicogen substitutions at position A14 that contain polar, charged, or side chains smaller than native tyrosine, (iii) substitutions at positions B28 and / or B29 to reduce insulin dimerization or increase solubility at neutral pH, and (iv) substitutions near the N-terminus of the B chain combined with N-terminal deletions. Some of these substitutions alone may increase the stability of wild-type insulin, while others alone may impair it. Similarly, some of these substitutions alone may prolong the tail of insulin action (upon intravenous bolus injection), while others alone may moderate or shorten this tail. One aspect of the present invention provides a combination of such substitutions, in combination with an N-terminal deletion of the B chain, which together provide an insulin analog whose formulations retain rapid action upon subcutaneous injection over a wide range of protein concentrations, ranging from 0.6 to 12.0 mM, and exhibit suitable physical and chemical stability to be useful for the treatment of diabetes.

[0034] In some embodiments, the insulin analogs of the invention are two-chain insulin analogs comprising a modified A-chain polypeptide and a modified B-chain polypeptide. See, e.g., WO2018 / 094388, which is incorporated herein by reference in its entirety. In some embodiments, the insulin analogs of the invention comprise or consist of the following variants with respect to wild-type human insulin: EA8, EA14, GA21, desBl, AB2, EB3, and EB29.

[0035] In one embodiment, an insulin analogue of the invention comprises a modified A chain having the following amino acid sequence: GIVEQCC E SICDL E QLENYC G (Sequence ID number: 1)

[0036] In one embodiment, an insulin analogue of the invention comprises a modified B chain having the following amino acid sequence: AE OHLCGSHLVEALYLVCGERGFFYTNETP E T (SEQ ID NO: 2)

[0037] In various embodiments, the insulin analogs of the invention are monomeric or dimeric insulin analogs. As used herein, "monomeric insulin analog" refers to insulin that is formulated substantially free of zinc (e.g., less than about 0.05 moles of zinc per mole of insulin) and is therefore stable at 25°C for at least 30 days when present in solution predominantly in monomeric and / or dimeric form, as opposed to the zinc hexameric form. For example, monomeric insulin analogs can be formulated at high concentrations, such as 100 IU / ml (e.g., about U-100) or higher (e.g., about U-200, about U-300, about U-400, about U-500, about U-1000, about U-1500, or about U-2000), without significant fibril formation or chemical degradation. In some embodiments, the insulin analog is formulated at about U-100 to about U-1000, or at about U-100 to about U-500. In various embodiments, the monomeric insulin is stable in the pharmaceutical composition at 25°C without substantial formation of insulin fibrils (i.e., less than 1% fibril formation) for at least about 1 month, or at least about 2 months, or at least about 3 months, or at least about 4 months, or at least about 5 months, or at least about 6 months, or at least about 9 months, or at least about 12 months.

[0038] In some embodiments, insulin analogs and compositions thereof have greater stability in zinc-free or zinc-containing formulations than wild-type insulin, insulin lispro, or insulin aspart. In various embodiments, insulin analogs or compositions thereof exhibit a lower rate of microfibril formation than Humalog® (Eli Lilly & Co., Indianapolis, Indiana), Novolog® (Novo Nordisk, Bagsvaerd, Denmark), or wild-type human insulin. In various embodiments, insulin analogs or compositions thereof exhibit a lower rate of degradation to HMWP than Humalog®, Novolog®, or wild-type human insulin. In various embodiments, insulin analogs or compositions thereof exhibit a lower rate of degradation to RS than Humalog®, Novolog®, or wild-type human insulin.

[0039] In various embodiments, the insulin analog or composition thereof exhibits a lower rate of microfibril formation compared to Humalog®, Novolog®, or wild-type human insulin in a concentrated formulation of U-500. In various embodiments, the insulin analog or composition thereof exhibits a lower rate of degradation to HMWP compared to Humalog®, Novolog®, or wild-type human insulin in a concentrated formulation of U-500. In various embodiments, the insulin analog or composition thereof exhibits a lower rate of degradation to RS compared to Humalog®, Novolog®, or wild-type human insulin in a concentrated formulation of U-500.

[0040] In various aspects and embodiments, the present invention provides pharmaceutical compositions and / or formulations comprising an effective amount of an insulin analog described herein and a polyphosphate compound. In some embodiments, the formulation is at least U-100 or at least U-500. Polyphosphates are salts or esters of polymeric oxide anions formed from tetrahedral PO (phosphate) structural units linked together by shared oxygen atoms. In some embodiments, the polyphosphate compound is incorporated into the compositions of the present invention in an amount effective to increase the absorption rate of the insulin analog upon administration. In various embodiments, the polyphosphate is selected from one or more of pyrophosphate, triphosphate, trimetaphosphate, and tetraphosphate. Polyphosphates may be used in their acid form or in various salt forms, for example, alkali (e.g., sodium or potassium) salts or alkali metal (e.g., calcium and magnesium) salts. In some embodiments, the polyphosphate comprises sodium triphosphate.

[0041] The concentration of polyphosphate (e.g., sodium triphosphate) in the composition is about 1 mM to about 100 mM, or about 1 mM to about 50 mM, or about 1 mM to about 40 mM, or about 5 mM to about 50 mM. In some embodiments, the concentration of polyphosphate in the composition is about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM. In some embodiments, the concentration of polyphosphate in the composition is about 10 mM to about 100 mM, or about 10 mM to about 50 mM. In some embodiments, the concentration of polyphosphate in the composition is about 15 mM to about 35 mM. In some embodiments, the concentration of polyphosphate is about 10 mM to about 30 mM. In some embodiments, the polyphosphate concentration is about 20 mM, where the polyphosphate is optionally sodium triphosphate. In some embodiments, fibril formation of insulin or its analogs can be assessed using a thioflavin T (ThT) dye assay. Thioflavin T dye is highly sensitive to fibril assembly. ThT dye interacts with amyloid fibrils without altering the protein structure or sequence. Reference: Wang J.-B., Wang Y.-M., Zeng C.-M., Biochem. Biophys. Res. Commun. 2011, 415, 675-679, incorporated herein by reference in its entirety. This dye has emission and excitation bands at 1 = 480 nm and 440 nm, respectively. The mechanism by which ThT dye identifies amyloid fibrils is that, as fibrillation progresses, ThT dye recognizes the beta-sheet structure of the fibrils, resulting in a gradual increase in its emission intensity. The fibrillation lag time is an indicator of the time it takes for insulin or its analogs to begin forming fibrils. For purposes of this disclosure, the microfibril lag time is determined by linear regression of the slope versus time of the fitted curve of the emission spectrum.

[0042] In some embodiments, an insulin analogue or pharmaceutical composition thereof described herein exhibits a fibril formation lag time (at 40°C with rapid stirring) of about 5 hours or more, or about 10 hours or more, or about 15 hours or more, or about 20 hours or more, or about 25 hours or more, or about 30 hours or more, or about 40 hours or more, or about 45 hours, or about 50 hours or more, or about 55 hours or more, or about 60 hours or more, or about 65 hours or more, or about 70 hours or more, or about 75 hours or more, or about 80 hours or more, or about 85 hours or more, or about 90 hours or more, or about 95 hours or more, or about 100 hours or more, or about 125 hours or more, or about 150 hours or more, or about 200 hours or more, or about 250 hours or more.

[0043] In various embodiments, the insulin analogs or compositions described herein do not exhibit any reduction or substantial reduction in fibril formation lag time upon formulation with polyphosphates (e.g., sodium triphosphate).

[0044] In some embodiments, the insulin analogs described herein are less mitogenic than commercially available insulin lispro.

[0045] In some aspects and embodiments, the present invention provides methods for determining the pharmacokinetic parameters or pharmacokinetic profile of an API, for example, using a nonalogue. The present invention also provides methods for determining an appropriate or optimal API formulation, for example, using a nonalogue composition. The present invention further includes a nonalogue or composition thereof that can be used to determine the pharmacokinetic parameters or pharmacokinetic profile of an API. The methods described herein for determining the PK characteristics of API formulations minimize stress and toxicity in test subjects, reduce costs, reduce the impact of inter-animal variability, and increase throughput. In one embodiment, the methods allow multiple formulations to be studied in a single subject in a single experiment.

[0046] In some embodiments, the present invention provides a method for determining the pharmacokinetic (PK) parameters or profile of an API formulation. The method includes administering to a subject a composition of multiple inactive or non-potent analogs (nonalogs) of the API and determining the concentrations of the nonalogs in one or more tissue samples from the subject. The method also includes determining one or more pharmacokinetic parameters of the API formulation based on the concentrations of the nonalogs in the subject. In one embodiment, the method is used to determine the PK parameters of the nonalogs and use them as a surrogate for determining the PK parameters of the corresponding API.

[0047] Thus, the present disclosure provides a method for preparing a pharmaceutical formulation of an API. The method includes administering multiple nonalogue compositions of the API to a subject and determining the concentration of the nonalogue in one or more tissue samples from the subject. The method also includes determining the pharmacokinetic profile of the nonalogue compositions and formulating the API as a pharmaceutical formulation having the desired pharmacokinetic profile. In one embodiment, the method further includes determining the desired pharmacokinetic profile based on the concentration of the nonalogue in one or more tissue samples from the subject. Once the desired pharmacokinetic profile or parameters of the nonalogue composition are achieved, the API can then be formulated according to the selected nonalogue composition, except that the nonalogue in the selected nonalogue composition is replaced with the corresponding API. Other changes to the API formulation may be made without substantially affecting the pharmacokinetic profile.

[0048] In another aspect, the present invention provides a non-potent analog (nonalog) or multiple nonalogs of a given API (e.g., insulin) that have substantially the same interactions with non-human subjects but are chemically or physically distinguishable. This aspect includes compositions of nonalogs with different pharmaceutically acceptable carriers that affect the pharmacokinetics in a subject. These non-analog compositions are useful for determining the pharmacokinetic parameters of various formulations of the corresponding API (e.g., insulin). In some embodiments, the present invention provides kits of at least three, at least four, at least five, or at least six nonalogs that can be differentially formulated according to the present disclosure.

[0049] In drug discovery, candidate active pharmaceutical ingredients (APIs) (also called "drug substances") are typically designed and evaluated for the purpose of diagnosing, curing, mitigating, treating, or preventing specific diseases or conditions. For example, peptides may be evaluated for their ability to bind to and inhibit the action of cellular receptors, and small molecules may be evaluated for their antibiotic properties. When a specific drug substance is selected for further development, several methods or tools are typically used to optimize its activity. One of these methods or tools is drug substance formulation. A drug substance formulation consists of chemicals that are mixed with the drug substance to impart optimized pharmacological properties. The formulated drug substance or API is called the drug product. The substances included in the formulation are called excipients. For example, weakly acidic excipients may be included in a drug substance to make it more soluble in liquid. Ionic salts may also be incorporated into the API to mask the charge in subcutaneous tissue, preventing the API's charged surface from being "trapped" by charged elements in the tissue and thereby allowing the API to move more quickly toward the bloodstream. Formulation optimization plays an important role in ensuring that the API has the designed time-action profile. Therefore, optimizing the formulation of a drug substance is a critical step in drug development.

[0050] Pharmacokinetics (PK) is the study of the time courses of absorption, distribution, metabolism, and excretion of substances in animals, including humans and non-human mammals. Absorption refers to the process by which a substance (e.g., a drug substance) moves from the site of administration into the bloodstream. Distribution describes how a substance (e.g., a drug substance) reaches various parts of a subject's body. Metabolism refers to the process by which a substance administered to a subject is broken down and converted into metabolites. Excretion refers to the process by which a substance is eliminated from a subject's body, often via the kidneys into urine or via bile into feces. Examples of PK data (or parameters) include, but are not limited to, the rate of absorption into the bloodstream, bioavailability, half-life, rate of metabolite formation, rate of clearance from the bloodstream, and rate of excretion from a subject. A pharmacokinetic profile can include all or a subset of these parameters, depending on the purpose or need of the PK study.

[0051] PK studies are useful for optimizing drug substance formulations, and the importance of efficient and effective PK studies in animals is well recognized. PK data is essential in the evaluation of any drug product or API because determining how a drug enters, interacts with, and exits the subject's body is critical to determining dosage forms, dosage concentrations, and dosing intervals. PK influences the pattern of appearance and disappearance of a drug substance in the blood following administration of the drug substance formulation. This pattern determines the drug substance concentration that target tissues are exposed to over time, and is therefore important in developing a drug's pharmacodynamic (PD) or time-action profile.

[0052] However, animal testing is often a bottleneck in the drug screening process due to its cost and time-consuming nature, which leads to high inter-animal variability. To reduce this variability, the solution is often to use many animals to obtain relevant yet redundant data.

[0053] The costs of maintaining animal colonies and conducting the resulting numerous studies can be prohibitive, especially in large animals like pigs. However, studies in pigs are particularly desirable because their response to insulin is similar to that of humans. Furthermore, the space required to maintain a large colony limits the number of studies that can be conducted simultaneously. The high cost of testing formulations in animal models often leads to little evaluation of formulations during drug development, potentially leading to the overlooking of formulations that may be optimally beneficial for patients. Animal testing, including PK studies, also aims to minimize stress on test animals. Studies in large animal models often require surgery to install catheters in animals to allow repeated blood sampling from the same site throughout the study. These catheters are prone to infection and must be kept clean and easily accessible throughout the study. Catheter installation and maintenance increase costs and represent an external stressor for research animals. Even with small animals, performing repeated experiments on the same animals can be a continuous and often recurring source of stress. Therefore, more efficient methods of conducting PK studies will reduce stress on test animal colonies.

[0054] The effects of the drug substance being tested create additional costs and stress for the test animals. For example, pigs treated with insulin analogs must have their blood glucose levels monitored, and these animals must be given additional glucose to avoid hypoglycemic events. Drug substances have other toxicities at high doses, limiting the frequency with which individual animals can be tested.

[0055] Furthermore, the economic and medical impacts of variability in drug absorption and bioavailability are immeasurable. One of the most significant consequences of drug bioavailability issues is the inability to identify promising and problematic drug candidates during drug discovery and preclinical testing. Therefore, there is a need for the development of comprehensive physiologically based pharmacokinetic test systems capable of assessing drug bioavailability and its variability in humans. Furthermore, given the urgent need to provide new therapeutics to the medical community and the current use of high-throughput drug screening to select lead drug candidates, comprehensive biopharmaceutical tools that can rapidly provide pharmacokinetic parameters or profiles are needed.

[0056] Therefore, there is a need for more efficient, cost-effective methods for optimizing drug substance formulations that minimize stress and harm to test animals.

[0057] Broadly, the present invention, in some aspects, provides a method for evaluating the effect of formulation on PK parameters in an animal study in which non-analogs of an API with different formulations are administered to the animals substantially simultaneously. In one embodiment, blood is collected from the animals during the study at predetermined time points after administration. The PK characteristics of each nonalogue formulation are determined by assessing the concentration of each nonalogue in plasma samples over the course of the study. Because all nonalogues are closely related and pharmacologically inactive, variations in PK parameters are largely or entirely attributable to formulation differences. This information can be used to screen formulations that do not meet the desired PK criteria or to refine the formulation to improve the PK parameters. Formulations with desirable properties can then be used as drug substances.

[0058] In one embodiment, samples are extracted from subjects at predetermined time points after administration of the nonalogue composition. The sample (e.g., a blood sample) contains all of the nonalogues used in the study. The number and frequency of time points at which samples are taken can be determined according to the desired resolution of the PK data. Generally, the more time points taken during the same period, the higher the resolution of the PK data obtained at the end of the study. The number and frequency of time points can also be determined depending on the level of stress imposed on the subject each time a sample is taken. The concentrations of different nonalogues obtained from samples taken over time are used to evaluate the effects of different formulations using a selected detection method. Samples are taken from test animals at predetermined time points after administration, and the concentrations of each nonalogue are determined to evaluate the PK characteristics of the different formulations used in the study. When using a selected detection method, each relevant nonalogue has a different signature, so the concentrations of each nonalogue over the course of the study can be evaluated from samples from a single study and used to determine the PK characteristics of each administered nonalogue composition. After samples are evaluated for the concentration of each nonalogue over time, the data is evaluated. Formulations may or may not confer beneficial PK properties to each nonalogue, as indicated by the concentration of each nonalogue in each sample over time, and formulations are either selected to proceed to more rigorous testing or eliminated from the screening process.

[0059] In some embodiments, the method includes administering multiple nonalogue compositions to a second subject and determining the concentrations of the nonalogues in one or more tissue samples from the second subject. A nonalogue is then selected to be administered to both the first and second subjects, and its clearance rate is determined in the second subject. The absorption rate of the selected nonalogue in the first subject is then determined based on the clearance rate of the selected nonalogue in the second subject. In this embodiment, the clearance rate of the nonalogue is calculated by administering the nonalogue(s) to one or more subjects, e.g., two, three, or more subjects. In one example of this embodiment, one or more nonalogues are administered to the subjects (e.g., the first and second subjects). One or more tissue or other (e.g., blood) samples are then obtained from the subjects, and the concentration of each nonalogue (or selected nonalogues) in each sample is measured. The clearance rate of each (or selected) nonalogue from a second subject can then be determined to calculate the absorption rate of that nonalogue formulation of the drug substance administered to the first subject. In one embodiment, the nonalogue compositions are administered simultaneously or nearly simultaneously to the first and second subjects. In another embodiment, the nonalogue compositions are administered sequentially to the second subject. Samples from the second subject can be collected, for example, from blood, liver, kidney, lung, spleen, heart, or brain. Tissue or other samples collected from the second subject can be the same as those from the first subject or from a different tissue or source. In one embodiment, a subject (including the first subject or the second subject) can be administered multiple nonalogues simultaneously, e.g., 1 to 25 nonalogues. In one embodiment, a subject is administered 2, 3, 4, 5, 6, 7, 8, 9, or 10 nonalogue compositions.

[0060] Active drug substances, or APIs, suitable for use in the formulations and methods described herein are therapeutically, prophylactically, and / or diagnostically active drug substances (also abbreviated herein as "active drug substances"). For example, in various embodiments, the API is a protein, peptide, antibody or portion thereof, small molecule, oligonucleotide, or polymer. In one embodiment, the API is insulin or a drug used to treat diabetes. Alternative APIs include analgesics, opioids, antipyretics, anesthetics, antimigraine drugs, antiepileptic drugs, antiparkinsonian drugs, dopaminergic drugs, anticonvulsants, anxiolytics, sedatives, antidepressants, psychostimulants, dopamine, noradrenaline, nicotine, alpha-adrenergic drugs, serotonin, H3 antagonists used in ADHD, and nootropics used in addictive disorders. In still further embodiments, the active agent is selected from therapeutic drug classes such as centrally acting analgesics, sedative-hypnotics, appetite suppressants, decongestants, antitussives, antihistamines, antiemetics, antidiarrheals, and agents used in the treatment of narcolepsy and attention deficit hyperactivity disorder. In certain embodiments, the active drug substance is associated with an abuse syndrome and may be selected from, for example, opioids, CNS depressants, CNS stimulants, cannabinoids, nicotinic compounds, glutamate antagonists, and N-methyl-D-aspartate (NMDA) antagonists.

[0061] The use of inactive or non-active analogs allows multiple non-analog compositions to be administered simultaneously or sequentially to subjects. Simultaneous administration of clinically relevant or effective concentrations of fully active APIs almost always results in very high concentrations of the API in the subject, potentially causing toxicity. Even if high doses of a particular API do not result in toxicity, the effects of the active API on animals must be addressed, increasing the cost of the study. Furthermore, administering high doses of the API may affect the subject or their physiology in a way that prevents reliable data from being obtained in PK studies. Similarly, administering low concentrations of the fully active API to counteract toxicity may alter the PK parameters of the combined API or result in analytically undetectable blood concentrations of the API. Inactive or non-active analogs allow for multiple, near-simultaneous, or sequential administration of clinically relevant doses to subjects while reducing or eliminating the risk of toxicity. This reduced risk of toxicity can result in cost savings, efficiency, and subject welfare.

[0062] Non-analogs can have a wide variety of designs. In one embodiment, the non-analogs are inactive, i.e., have no pharmacological / biological / chemical activity or only a subset of the pharmacological / biological / chemical activity of the API. In another embodiment, non-analogs of an API are sufficiently different from one another so that multiple non-analogs can be detected in a single tissue sample (e.g., a plasma sample). In yet another embodiment, the non-analogs are sufficiently similar to the parent API so that no change in PK is expected from changes or modifications of the non-analogs. In a preferred embodiment, the non-analogs have no pharmacological / biological / chemical activity, are sufficiently different from one another so that multiple non-analogs can be detected in a single tissue sample, and are sufficiently similar to the parent API so that little or no change in PK is expected from changes or modifications of the non-analogs.

[0063] In various embodiments, nonalogues are produced in recombinant DNA expression systems or chemically synthesized. In one embodiment, nonalogues are isotopically labeled with different isotopes to enhance specificity for detection using mass spectrometry detection methods. In another embodiment, unrelated analogs are designed to have different hydrophobicities to enhance specificity using HPLC detection methods. In another embodiment, amino acids with similar chemical properties but different masses are substituted within a peptide or protein sequence to enhance specificity using ligand-bound liquid chromatography tandem mass spectrometry (LBA-LC-MS / MS). This flexibility in molecular design provides a method for synthesizing sufficient amounts of material for statistically relevant population sizes.

[0064] A non-analog, in some embodiments, is an inactive or inactive analog of an API that has about 75% or less activity compared to the fully active form of the API. In one embodiment, a non-analog has about 10% or less activity compared to the fully active form of the API. The activity of an API or non-analog can be, for example, biological, pharmacological, pharmaceutical, or chemical activity. In some embodiments, an API or non-analog that has activity is based on an in vitro or in vivo assay. The assay for measuring activity can be, for example, an enzyme-linked immunosorbent assay, an immunohistochemistry assay, flow cytometry, a biochemical assay that measures the binding affinity of the API / non-analog to a target (e.g., a receptor), or an assay that measures inhibition of enzymatic activity.

[0065] In one embodiment, a non-analog has a similar physical, chemical, or biological structure to an API. For example, a non-analog has a similar chemical structure, a similar primary structure, a similar secondary structure, a similar tertiary structure, or a similar quaternary structure to an API. In another embodiment, a non-analog has similar hydrophobic properties or a similar surface charge pattern to an API. The term "hydrophobicity" includes the overall hydrophobicity of an API or non-analog, as well as the hydrophobicity of portions of an API or non-analog. For example, hydrophobicity includes the hydrophobicity of the protein as a whole, as well as the hydrophobicity of portions exposed on the surface of the protein and portions buried within the protein's structure.

[0066] In various embodiments, a non-analog of a peptide or protein (e.g., insulin) consists of an amino acid sequence with one or more amino acid mutations compared to the API. In some embodiments, the one or more amino acid mutations can be independently selected from substitutions, insertions, deletions, and truncations. A non-analog can have, for example, one mutation, two mutations, three mutations, four mutations, five mutations, six mutations, seven mutations, eight mutations, nine mutations, or ten mutations. In certain embodiments, the mutations include amino acid substitutions, such as conservative and / or non-conservative substitutions. In some embodiments, a non-analog is a protein or peptide having 10-100 amino acids in which one or two amino acids have been substituted, inserted, or deleted, resulting in an inactive or non-potent analog. In some embodiments, a protein may have more than 100 amino acids and one or two additional substitutions, deletions, or insertions. In some embodiments, each non-analog in a set of non-analogs has a modification(s) at the same position(s) relative to the API. In some embodiments, a non-analog has an amino acid sequence that is within 90% identity to the amino acid sequence of the API, or within 95% identity to the amino acid sequence of the API, or within 98% or 99% identity to the amino acid sequence of the API. "Conservative substitutions" include substitutions that are made within a group of amino acids having similar side chains. For example, neutral and hydrophobic amino acids include glycine (Gly or G), alanine (Ala or A), valine (Val or V), leucine (Leu or L), isoleucine (Lie or I), proline (Pro or P), tryptophan (Trp or W), phenylalanine (Phe or F), and methionine (Met or M); neutral polar amino acids include serine (Ser or S), threonine (Thr or T), tyrosine (Tyr or Y), cysteine ​​(Cys or C), glutamine (Glu or Q), and asparagine (Asn or N); basic amino acids include lysine (Lys or K), arginine (Arg or R), and histidine (His or H); and acidic amino acids include aspartic acid (Asp or D) and glutamic acid (Glu or E).Furthermore, standard amino acids may also be substituted with non-standard amino acids, e.g., those belonging to the same chemical class. As a non-limiting example, a basic side chain lysine may be substituted with a basic amino acid with a shorter side chain length (ornithine, diaminobutyric acid, or diaminopropionic acid). Lysine may also be substituted with the neutral aliphatic isostere norleucine (Nle), which may then be substituted with an analog containing a shorter aliphatic side chain (aminobutyric acid or aminopropionic acid). In some embodiments, insulin analogs have one to five mutations relative to the sequence of insulin lispro, insulin aspart, or a fluorolog (Asp BIO, orthomonofluorophenylalanine-B24, lispro insulin). In some embodiments, these mutations are conservative, with no more than one, two, or three non-conservative or non-standard mutations.

[0067] In one embodiment, the mutation results in a decrease in the activity of the nonalogue, e.g., a decrease in the binding affinity or activity of the nonalogue at the receptor or biological target of the API. In one embodiment, the non-analog can activate the receptor of the corresponding API with reduced efficacy. In other embodiments, the mutation results in the non-analog having reduced or eliminated activation activity at the receptor or biological target of the corresponding API.

[0068] In another embodiment, the nonalogues described herein are modified to reduce affinity or activity for one or more of their receptors or biological targets, allowing for attenuated activity (including agonism or antagonism). In some embodiments, the mutations allow the nonalogue to have attenuated activity, such as reduced binding affinity to a target, reduced enzymatic activity, or reduced inhibition or activation of a target (e.g., a receptor), compared to the unmutated nonalogue, i.e., API. In some embodiments, the activity of the nonalogue can be determined using in vitro and in vivo assays known to those skilled in the art.

[0069] The methods described herein relate to the pharmacokinetic-based design of API formulations using one or more non-analogs administered to a subject as a surrogate for the API. In certain embodiments, a non-analog composition may be administered to a subject to determine the formulation to be used with the corresponding API to achieve a desired absorption profile for the API, a desired release or liberation profile of the API from the composition, a desired distribution profile of the API in the subject, a desired metabolic profile of the API in the subject, or a desired excretion profile of the API in the subject. In some embodiments, the methods disclosed herein measure the absorption effect of one or more formulations. Because metabolism of an API produces predictable metabolites, the concentration of one or more non-analog metabolites over time may be determined via LC-MS / MS or another detection method to calculate metabolic rates. The methods described herein also facilitate predicting the fate of an API in a mammal based on the absorption of the non-analog and one or more additional bioavailability parameters, including distribution, metabolism, excretion, and, optionally, toxicity.

[0070] In this embodiment, the method allows for optimizing an API formulation using a nonalog composition as a surrogate, which can have several iterations in which an excipient (or concentration thereof) is administered to a subject and a nonalog composition is selected based on specific desired properties, and this selected nonalog composition is then used to design / optimize a formulation of the corresponding API.

[0071] In some embodiments, the excretion rate is calculated by detecting the presence of one or more unrelated substances and their metabolites in the urine or feces of a subject.

[0072] In some embodiments, one or more PK parameters can be measured to generate a PK profile. This PK profile, generated using the nonalogue compositions and methods of the present invention, can be a one-dimensional or multi-dimensional output reflecting one or more PK parameters of interest for the API. The results can be used to profile or rank nonalogue compositions by selected PK parameters, and optionally absorption and one or more additional bioavailability parameters and toxicity.

[0073] In some embodiments, the nonalog composition mimics a desired or existing API formulation. In some embodiments, the API formulation mimics a non-analog formulation. The term "mimic," as used herein in the context of a formulation or composition, means that two or more compositions have similar components. For example, an API formulation that mimics a nonalog composition will have similar components in similar concentrations as the nonalog composition, except that the nonalog in the composition has been replaced with the API. Similarly, a nonalog composition that mimics an API formulation will have similar components in similar concentrations as the API formulation, except that the API in the formulation has been replaced with the nonalog.

[0074] In some embodiments, the reason the nonalog composition mimics an API formulation is to use the nonalog formulation as a control or to test an existing API formulation to assess any variations in PK parameters in a multiplexed study environment in which multiple nonalog compositions are administered to subjects. In one embodiment, for example, a known API formulation is administered with the nonalog composition to serve as a control, to correct for errors that may be introduced during the study, or to compare the known PK parameters of the API formulation with the PK parameters obtained after administering the API formulation in combination with the nonalog composition.

[0075] In another embodiment, several nonalogues with different compositions are administered to a subject, and a nonalogue composition is selected based on the desired PK profile or response in the subject, and the API formulation is then formulated to mimic the selected nonalogue composition.

[0076] The nonalogue compositions disclosed herein may be administered to a subject simultaneously, nearly simultaneously, or sequentially. For example, in one embodiment, multiple nonalogue compositions are administered to a subject simultaneously, i.e., together with other nonalogues. In one embodiment, the nonalogues are administered to a subject via the same route of administration or via different routes of administration. For example, one nonalogue may be administered orally and another may be administered intravenously. Note that in the context of this embodiment, simultaneous administration can mean that all nonalogues are administered together or within a short time span of each other (nearly simultaneously). For example, in near-simultaneous administration, all nonalogues are administered within a time span ranging from about 1 minute to about 30 minutes.

[0077] Simultaneous or near-simultaneous administration of a nonalcohol or each of the compositions to a single subject can increase the efficiency and reduce costs of PK formulation studies because fewer subjects are required. Another advantage is that multiple nonalcohols can be detected simultaneously in a single tissue sample. This increases throughput for evaluating PK parameters of different formulations and reduces the time and cost required to conduct these studies. Another advantage of this method is that it reduces the stress level of the subject. Furthermore, another advantage of this method is that multiple formulations can be evaluated in the same subject in a single study, reducing inter-animal variability.

[0078] In some embodiments, the nonalogue or each composition thereof is administered sequentially to a subject. Sequential administration minimizes the number of animals required to conduct a PK study and allows for PK studies to be conducted without overloading the subject with several nonalogues in a given time span. Sequential administration can be performed in such a way that a second batch of nonalogue is administered after the first batch of nonalogue has been removed from the subject. Sequential administration can also be performed to study the absorption rate of a nonalogue, such that the second batch of nonalogue is administered after complete absorption of the second batch of nonalogue. The second or subsequent batch of nonalogue can be administered, for example, about 35 minutes to about 24 hours after administration of the first batch of nonalogue or composition thereof. Note that these time spans are exemplary, and the time span between sequential administrations of a nonalogue or composition thereof can be calculated based, for example, on the excretion rate of the nonalogue, the in vivo half-life of the nonalogue, or the absorption rate of the nonalogue composition. For example, in one embodiment, if the nonalogue or composition thereof is fast-acting or rapidly absorbed, the second administration can be administered shortly (e.g., within about 35 minutes) after the first administration, such that the second administration avoids the maximum plasma concentration of the first fast-acting nonalogue composition in the subject, thereby reducing any potential side effects due to high concentrations of the nonalogue in the subject.

[0079] In various embodiments, tissue samples are collected at multiple time intervals before or after administration of the nonalog composition to the subject.

[0080] The routes by which nonalcohols and their formulations are administered to subjects vary. If the sample collected from the subject is blood, the drug must ultimately be present in the bloodstream so that blood samples can be collected from the animal to measure nonalcohol concentrations over time to assess PK differences resulting from the different formulations being tested. It is expected that the route of administration will vary not only between studies, but also between nonalcohols within the same study in the same subject.

[0081] In some embodiments, the nonalogue composition is administered to a subject using one or more of the following routes: enteral (e.g., buccal or sublingual, oral (PO), rectal (PR)), parenteral (e.g., intravenous, intravenous bolus, intravenous infusion, intramuscular, subcutaneous injection), inhalation or nasal routes, transdermal (transdermal), intradermal, intraperitoneal, or intrahepatic. In one embodiment, the selected route of administration determines the type and / or source of assays employed to obtain PK parameters. For example, in the case of oral administration, assays can be used to determine the release rate or dissolution of the nonalogue composition in tablets or capsules. In another embodiment, multiple routes of administration are used to determine the distribution of the nonalogue to specific tissues. For example, oral, hepatic, systemic, and blood-brain barrier assays can be used to obtain distribution parameters for a compound targeted to brain tissue. In some embodiments, pharmacokinetic parameters are determined using the concentration of the nonalogue in one or more tissues or other samples, such as, without limitation, blood, bone, liver, kidney, lung, spleen, heart, brain, gastrointestinal tract, spinal cord, spinal fluid, cerebrospinal fluid, eye, mouth, or muscle. In a preferred embodiment, the tissue sample is a blood sample.

[0082] In some embodiments, tissue samples collected from a subject before or after administration of a nonalog composition are used to measure the concentration of the nonalog, API, or a metabolite of the nonalog. In one embodiment, the concentration of the nonalog, API, or a metabolite thereof is measured using a single assay or analytical method. For example, in one example, multiple tissue samples are collected from a subject and evaluated in a single step using a single assay, such as LC-MS. In another embodiment, multiple tissue samples are analyzed using multiple assays or analytical methods. For example, one set of tissue samples may be analyzed using liquid chromatography and ultraviolet detection, while another set of samples from the same subject may be analyzed using LC-MS. The choice of analytical method depends on the nonalog being analyzed and can be determined based on the physical or chemical properties of the nonalog or API. Analytical methods that can be used in the methods described herein include, but are not limited to, LC-MS / MS, LC-MS, immunoassay, radioimmunoassay, or chromatography. There are various methods for detecting blood concentrations of an API over time to determine PK characteristics. Immunoassays that quantitatively measure APIs using specific antibodies that bind to them are useful, but require the availability of suitable antibodies. Chromatography, which separates a test solution into a gaseous or liquid phase by partitioning with a solid or liquid phase, is also useful for quantifying APIs. Liquid chromatography coupled with mass spectrometry (LC-MS) or tandem mass spectrometry (LC-MS / MS) is a very useful method for accurately quantifying the concentration of APIs in solution. Ligand-binding assays coupled with LC-MS / MS can extract APIs from biological matrices, improving quantitation in urine, serum, plasma, or tissue samples.

[0083] In some embodiments, an immunoassay is performed to determine the concentration of a nonalogue in a tissue sample. In another embodiment, U / HPLC is performed to determine the concentration of a nonalogue in a tissue sample. In another embodiment, LC-MS / MS is performed to determine the concentration of a nonalogue in a tissue sample. In another embodiment, LBA-LC-MS / MS is used to quantify a nonalogue in a biological matrix. This method allows for accurate calculation of the peptide concentration in a solution, even when the solution contains many components. This is particularly beneficial for the present invention, where the formulations being evaluated are potentially very complex and may contain multiple components. In some embodiments, the nonalogues described herein are labeled for easy detection in analytical methods. In some embodiments, labeling does not affect the pharmacokinetic, physical, chemical, or pharmacological behavior of the nonalogue. In other embodiments, labeling causes the nonalogue to lose its activity compared to the API. The nonalogues described herein can be labeled with radioactive labels, fluorescent labels, e.g., heavy isotopes of carbon, oxygen, hydrogen, sulfur, or nitrogen.

[0084] The unanalogous API and methods described herein are useful for evaluating PK parameters in a wide range of formulations or compositions. The compositions or formulations may contain different concentrations of the API. The concentrations may be, for example, high, medium, or low. In this example, the methods of the present invention may be used to evaluate the effect of API concentration and formulation volume on the pharmacokinetic properties of the formulation. The compositions or formulations may also contain a wide range of excipients.

[0085] In various aspects and embodiments, pharmaceutical compositions of insulin analogs described herein include one or more pharmaceutically acceptable excipients or carriers suitable for subcutaneous or intradermal administration. A particular excipient may have more than one function in the formulation. Table 1 below provides a list of exemplary excipients and their exemplary functions in the formulation. [Table 1]

[0086] In various embodiments, the pharmaceutical composition comprises one or more of a pharmaceutically acceptable buffer, stabilizer, surfactant, solubilizer, anti-aggregant, diffusion enhancer, absorption enhancer, and preservative. These agents can be used in combination and function synergistically to, for example, enhance insulin absorption, promote more rapid insulin pharmacokinetics, and / or improve insulin stability.

[0087] In certain embodiments, pharmaceutical compositions may include one or more agents that maintain or adjust the tonicity of the formulation. Such agents include, but are not limited to, glycerol, mannitol, sorbitol, lactose, dextrose, trehalose, sodium chloride, and propylene glycol (see, e.g., U.S. Patent Publication No. 2012 / 0225810, the entire contents of which are incorporated herein by reference). In various embodiments, the formulation may be hypertonic or hypotonic. For example, the pharmaceutical composition may include one or more agents designed to render the formulation hypertonic. Exemplary agents include any agent that is soluble in the formulation and does not freely permeate the cell membrane, such as glycerin, dextrose, mannitol, NaCl, and KCl. In some embodiments, the pharmaceutical composition includes about 1 mg / ml to about 100 mg / ml of glycerin. In various embodiments, the pharmaceutical composition comprises from about 1 mg / mL to about 50 mg / mL, or from about 8 mg / mL to about 25 mg / mL of glycerin (eg, about 16 mg / mL).

[0088] In certain embodiments, pharmaceutical compositions may contain one or more buffering agents to maintain a particular pH of the formulation. Exemplary buffering agents include, but are not limited to, sodium phosphate, arginine, TRIS (2-amino-2-hydroxymethyl-1,3-propanediol), glycylglycine, L-histidine, HEPES, bicine, sodium acetate, sodium carbonate, sodium citrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, bicine, tricine, malic acid, succinic acid, fumaric acid, tartaric acid, aspartic acid, ethylenediamine, or mixtures thereof. See U.S. Patent No. 6,906,028 and U.S. Patent Publication No. 2012 / 0225810, the entire contents of which are incorporated herein by reference. In some embodiments, pharmaceutical compositions of the present invention contain about 5 mM to about 100 mM Tris. In various embodiments, the pharmaceutical compositions of the present invention comprise about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM Tris. In one embodiment, the pharmaceutical composition comprises about 50 mM Tris.

[0089] In certain embodiments, the pharmaceutical composition may include one or more stabilizers to stabilize the insulin formulation. Exemplary stabilizers include zinc (e.g., in a molar ratio of less than 0.05 relative to the insulin in the formulation), phenol, m-cresol, benzoate, TRIS, non-reducing carbohydrates (e.g., mannitol or dextran), surfactants (e.g., polysorbates such as TWEEN®, bile salts, salts of fatty acids, or phospholipids, polyhydric alcohol moieties and fatty acid esters and ethers, glycerol or sorbitol moieties and sucrose, and polyols, such as SPAN polysorbates, MYRJ, BRIJ®, TRITON®, etc.). Polyhydric alcohol moieties such as CREMOPHOR (trademarks), and CREMOPHOR (fatty acid esters and ethers, polyoxyethylene ethers, and polyethylene glycol ethers), amino acids (e.g., L-arginine, L-glutamic acid, L-histidine, or L-methionine), alkyl saccharides (e.g., dodecyl-bD-maltoside, tridecyl maltoside, tetradecyl maltoside, sucrose monododecanoate, sucrose monotridecanoate, sucrose monotetradecanoate), ALSLA compounds, chromium salts, acetone, methyl ethyl ketone, pyruvic acid, glyoxylic acid, α-ketobutyric acid, α-ketoglutaric acid, acetoacetic acid, pyridoxal, pyridoxal pyrophosphate, iloprost, and the like may be used alone or in combination.

[0090] In certain embodiments, the pharmaceutical composition may include one or more solubilizing agents to prevent precipitation of the insulin or insulin analog within the formulation and to increase the solubility of the insulin or insulin analog. Exemplary solubilizing agents include, but are not limited to, L-arginine, L-arginine analogs or arginine, tripeptides, guanidine, magnesium, alcohol, alcohol esters of organic acids, nitrogen-containing solvents, phospholipids, acetic acid, ascorbic acid, citric acid, glutamic acid, aspartic acid, succinic acid, fumaric acid, maleic acid, adipic acid, agmatine, 4-guanidinobenzoic acid, guanidoacetic acid, guanidinosuccinic acid, and copolyamino acids, alone or in combination.

[0091] In certain embodiments, the pharmaceutical composition may include one or more anti-aggregating agents to prevent aggregation of insulin in solution. Exemplary anti-aggregating agents include, but are not limited to, arginine, polysorbate 20, histidine, proline or proline derivatives, sulfobutylether-P-cyclodextrin, tripeptide HTD, arginium ions or lysine, and propylene glycol, citric acid, and nicotinamide.

[0092] In certain embodiments, the pharmaceutical composition may include one or more membrane-penetrating agents to facilitate penetration and diffusion of insulin or insulin analogs through membranes. Exemplary membrane-penetrating agents include, but are not limited to, antennapedia protein, HSV type 1 protein VP22, and HIV Tat protein, alone or in combination.

[0093] In certain embodiments, the pharmaceutical compositions may include one or more absorption enhancers to facilitate absorption of insulin or insulin analogs by any of a variety of mechanisms. Exemplary absorption enhancers include surfactants (e.g., bile salts, salts of fatty acids, or phospholipids), nicotinic acid agents (e.g., nicotinamide, nicotinic acid, niacin, niacinamide, vitamin B3, and any salts thereof), pancreatic trypsin inhibitors, magnesium salts, polyunsaturated fatty acids, didecanoylphosphatidylcholines, aminopolycarboxylates, tolmetin, and sodium caprate. Salicylic acid, oleic acid, linoleic acid, EPA, DHA, benzilic acid, NO donors (3-(2-hydroxy-1-(1-methylethyl)-2-nitrosohydrazino)-1-propanamine, N-ethyl-2-(L-ethylhydroxy-2-1-nitrosohydrazino)-ethanolamine, S-nitroso-N-acetylpenitylamine, etc.), bile acids, glycine conjugates of bile acids, sodium ascorbate, potassium ascorbate, sodium salicylate, potassium salicylate, acetylsalicylic acid, salicylsalicylic acid, aluminum acetylsalicylate, choline salicylate, salicylic acid amide. Examples of suitable oleic acid salts include, but are not limited to, lysine salicylate, exalamide, diflunisal, EDTA, acetic acid, ascorbic acid, citric acid, glutamic acid, aspartic acid, succinic acid, fumaric acid, maleic acid, adipic acid, polyphosphoric acid, and ethenzamid, either alone or in combination.

[0094] In certain embodiments, agents that minimize degradation of the active agent (e.g., insulin) may be included in the pharmaceutical composition. Without wishing to be bound by theory, such agents are believed to inhibit the activity of neutrophils, monocytes, macrophages, lymphocytes, and platelets, which release proteases, lipases, oxygen radicals, IL-1, IL-6, IL-8, MCP-1, and TNF, which degrade insulin that accumulates in granulation tissue after tissue trauma and surrounds the infusion catheter. These medications include, but are not limited to, glucocorticoids such as dexamethasone, cortisol, solumedrol, and medrol; anesthetics such as lidocaine, bupivacaine, procaine, etidocaine, ropivacaine, mepivacaine, isoflurane, and halothane; anesthetics such as sevoflurane, desflurane, and enflurane; aprotinin or trasylol; aspirin and nonsteroidal anti-inflammatory drugs (NSAIDs); chromolysodium; and immunosuppressants such as cyclosporine, tacrolimus, and sirolimus, either alone or in combination.

[0095] In certain embodiments, the pharmaceutical composition may include one or more diffusion enhancers, such as basic diffusion enhancers. Exemplary diffusion enhancers include, but are not limited to, glycosaminoglycanases (e.g., hyaluronidase).

[0096] In certain embodiments, pharmaceutical compositions may contain one or more preservatives to prevent microbial growth. Exemplary preservatives include, but are not limited to, phenol, meta-cresol, methylparaben, propylparaben, and sodium benzoate. In various embodiments, pharmaceutical compositions contain about 0.1 mg / mL to about 10 mg / mL of m-cresol. In embodiments, pharmaceutical compositions contain about 0.1 mg / mL, about 0.5 mg / mL, about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, or about 10 mg / mL of m-cresol. In one embodiment, the pharmaceutical composition contains about 3.2 mg / mL of m-cresol.

[0097] In various embodiments, the pharmaceutical composition may include one or more vasodilators, anti-inflammatory agents, antithrombotic agents, antidegradative agents, insulin-binding antagonists, antifibrotic agents, antioxidants, antiproliferative agents, neuropathy agents, and antibiotics. See U.S. Pat. No. 9,901,622, the entire contents of which are incorporated herein by reference. These agents can be used in combination with any of the other excipients and agents described herein and can function synergistically to, for example, enhance insulin absorption, promote more rapid insulin pharmacokinetics, and increase the stability of insulin or insulin analogs. In some embodiments, the composition includes a prostacyclin PGL analog, such as iloprost or treprostinil.

[0098] In some embodiments, iloprost is present in the pharmaceutical composition at a concentration of about 1 μg to about 100 μg / mL, or optionally at a concentration of about 5 μg / mL to about 50 μg / mL, or optionally at a concentration of about 10 μg / mL to about 25 μg / mL.

[0099] In some embodiments, the pharmaceutical composition comprises one or more polyphosphate compounds. In various embodiments, the polyphosphate is selected from one or more of pyrophosphate, triphosphate, trimetaphosphate, and tetraphosphate. The polyphosphates may be used in their acidic form or in various salt forms, such as alkali (e.g., sodium or potassium) salts or alkali metal (e.g., calcium and magnesium) salts. In some embodiments, the polyphosphate comprises sodium triphosphate. In these embodiments, the concentration of the polyphosphate (e.g., sodium triphosphate) in the composition is about 1 mM to about 100 mM, or about 1 mM to about 50 mM, or about 1 mM to about 40 mM, or about 5 mM to about 50 mM. In some embodiments, the concentration of polyphosphate in the composition is about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM. In some embodiments, the concentration of polyphosphate in the composition is about 10 mM to about 100 mM, or about 10 mM to about 50 mM. In some embodiments, the concentration of polyphosphate in the composition is about 15 mM to about 35 mM. In some embodiments, the concentration of polyphosphate is about 10 mM to about 30 mM. In some embodiments, the concentration of polyphosphate is about 20 mM, where the polyphosphate is optionally sodium triphosphate.

[0100] In certain embodiments, the pharmaceutical composition may include agents that increase the diffusion of an active agent (e.g., insulin), increase lymphatic flow by increasing muscle movement and microventilation, increase the flow of an active agent (e.g., insulin) into lymphatic vessels, increase absorption from capillary and venous walls into plasma, and / or increase blood flow through adipose tissue capillaries and veins (see U.S. Patent No. 9,901,622, the entire contents of which are incorporated herein by reference).

[0101] In various embodiments, the pharmaceutical compositions of the present invention have an onset of insulin activity (e.g., time to first positive glucose infusion rate in a euglycemic clamp, or T) in less than about 40 minutes, or less than about 30 minutes, or less than about 20 minutes, or less than about 15 minutes, or less than about 10 minutes, or less than about 5 minutes after administration. onset ) is provided.

[0102] In various embodiments, the pharmaceutical composition comprises, for example, C max and / or T max As used herein, C provides a rapid insulin absorption profile as measured by max is the maximum or peak concentration of a drug observed after its administration. max is the maximum concentration (C max In certain embodiments, the pharmaceutical composition exhibits ½T in less than about 120 minutes, or less than about 90 minutes, or less than about 60 minutes, or less than about 50 minutes, or less than about 40 minutes, or less than about 30 minutes, or less than about 20 minutes, or less than about 15 minutes after administration. max Reach Early.

[0103] In various embodiments, the pharmaceutical composition provides a short duration of insulin activity. In certain embodiments, the pharmaceutical composition provides a duration of insulin activity of about 6 hours or less, about 5 hours or less, about 4 hours or less, about 3 hours or less, or about 2 hours or less after administration. In some embodiments, the duration of activity is measured as the time for insulin action to subside to less than half of its maximum activity.

[0104] In various embodiments, the pharmaceutical composition provides enhanced storage stability, hi certain embodiments, the pharmaceutical composition is stable at 25° C. for at least about 1 month, or at least about 3 months, or at least about 6 months, or at least about 12 months, or at least about 18 months, or longer, without substantial formation of insulin fibrils.

[0105] In a further embodiment, the pharmaceutical composition maintains at least about 60% potency, about 70% potency, about 80% potency, about 90% potency, or about 95% potency after 6 months at 25° C. In another embodiment, the pharmaceutical composition maintains at least about 60% potency, about 70% potency, about 80% potency, about 90% potency, or about 95% potency after 9 months at 30° C. In a further embodiment, the pharmaceutical composition maintains at least about 60% potency, about 70% potency, about 80% potency, about 90% potency, or about 95% potency after 12 months at 25° C.

[0106] In some embodiments, the pharmaceutical composition is formulated for subcutaneous administration and may be formulated as an aqueous formulation. In some embodiments, the formulation is provided in an infusion device, a prefilled insulin pump, or a prefilled injection pen for subcutaneous administration, and / or a single- or multi-dose vial or cartridge. For example, the formulation may be provided in a vial containing 1 to about 1000 bolus doses, or 1 to about 100 bolus doses, or 1 to about 50 bolus doses, or 1 to about 25 bolus doses. In some embodiments, the vial or cartridge is sufficient to provide a patient's daily, weekly, or monthly insulin needs. The vial or cartridge can contain doses, for example, in a total volume of about 3 mL to about 50 mL, e.g., about 3 mL to about 10 mL. Bolus doses will generally have an injectable volume of 1 mL or less, such as about 0.5 mL or less, about 0.2 mL or less, or about 0.1 mL. For example, the formulation may be provided in a pre-filled dispenser in a disposable insulin pump containing a volume of about 0.5 mL, a volume of about 1.0 mL, a volume of about 1.2 mL, a volume of about 1.5 mL, a volume of about 2 mL, or a volume of about 2.5 mL.

[0107] It is to be understood that the actual dosage of insulin or insulin analogues administered in accordance with the present invention will vary depending, for example, on the particular dosage form and mode of administration, as well as the disease and weight of the individual patient.

[0108] The pH of insulin compositions of the invention is typically 7.0 to 7.8, adjusted with physiologically appropriate acids and bases, typically 10% hydrochloric acid and 10% sodium hydroxide. In some embodiments, the pH ranges from about 7.2 to about 7.6, with 7.4±0.1 being a common target pH.

[0109] Individual doses of insulin or insulin analogs described herein can be, for example, from about 1 U / mL (1 insulin unit / mL) to about 2000 U / mL, or from about 1 U / mL to about 1000 U / mL, or from about 1 U / mL to about 500 U / mL, or from about 1 U / mL to about 400 U / mL, or from about 1 U / mL to about 300 U / mL, or from about 1 U / mL to about 200 U / mL, or from about 1 U / mL to about 100 U / mL, or from about 1 U / mL to about 50 U / mL, or from about 1 U / mL to about 10 U / mL. In some embodiments, formulations can be formulated to contain from about 100 U / mL to about 1000 U / mL, or from about 100 U / mL to about 500 U / mL.

[0110] In various embodiments, the pharmaceutical composition contains no added zinc or less than 0.05 moles of zinc per mole of insulin, hi various embodiments, the pharmaceutical composition does not contain magnesium.

[0111] In some embodiments, the pharmaceutical composition comprises one or more anti-inflammatory agents and / or one or more anti-fibrotic agents. In various embodiments, the pharmaceutical composition consists of or consists essentially of pharmaceutically acceptable buffers, solubilizers, vasodilators, absorption enhancers, tonics, preservatives, and stabilizers.

[0112] For example, an exemplary fast-acting formulation for the analog designated T-1123 is 5-100 mM citrate, 1-10 mM EDTA, 0.25-30 pg / mL iloprost, 0.5-10 mM Mg ++, 1-50 mM Tris (pH 7.4), and 1-25 mg / ml glycerin. For example, an exemplary fast-acting formulation of the analog designated T-1123 contains about 45 mM citrate, about 6.2 mM EDTA, about 15 pg / mL iloprost, about 4 mM Mg ++ , about 10 mM Tris (pH 7.4), and about 16 mg / mL glycerin. For example, another exemplary fast-acting formulation of an analog designated T-1123 consists of about 50 mM Tris buffer (pH 7.4), about 3.2 mg / mL m-cresol, about 16 mg / mL glycerin, and about 20 mM sodium triphosphate.

[0113] In some aspects, the present invention provides methods for treating or preventing a condition in a patient by administering an insulin analog or pharmaceutical composition described herein. In some embodiments, the present invention provides methods for treating a subject with diabetes or other conditions treated with insulin or its analogs using any of the pharmaceutical compositions or formulations comprising insulin or an insulin analog described herein. In embodiments, the subject has type 1 diabetes or type 2 diabetes. In some embodiments, the patient exhibits insulin resistance. In further embodiments, the subject has gestational diabetes or pre-diabetes.

[0114] Optionally, the subject may suffer from a metabolic disease that may benefit from insulin administration, such as obesity or metabolic syndrome. As used herein, the term "metabolic disease" refers to a group of identified disorders that result in metabolic errors, metabolic imbalances, or suboptimal metabolic homeostasis.

[0115] In embodiments, the metabolic disorder is obesity. For example, the subject may suffer from central obesity. In some embodiments, the obesity is one of simple obesity (digestive obesity; usually resulting from consuming more calories than the body can utilize), secondary obesity (usually resulting from an underlying condition such as Cushing's syndrome or polycystic ovary syndrome), and childhood obesity. In some embodiments, obesity is classified as follows: Class I includes a BMI of 30 to 34.99, Class II includes a BMI of 35 to 39.99, and Class III includes a BMI of over 40. Furthermore, the present invention provides for obesity of any of Classes I, II, and III, which are further classified into severe obesity, morbid obesity, and extreme obesity.

[0116] In another embodiment, metabolic disease is lipotrophic diabetes.For example, subject may lack fat anywhere or in specific body regions, and require a very large amount of exogenous insulin to maintain euglycemia.In another embodiment, metabolic disease is gestational diabetes, latent autoimmune diabetes of adults (LADA) and maturity-onset diabetes of the young (MODY).

[0117] In further embodiments, the present invention provides methods for treating a subject with pre-diabetes using any of the analogs and pharmaceutical compositions described herein. Pre-diabetes, also known as impaired fasting glucose (IFG) or impaired glucose tolerance (IGT), is a precursor to type 2 diabetes. Pre-diabetes is diagnosed when a fasting blood glucose level is 100-125 mg / dL (5.56-6.94 mmol / L), a 2-hour blood glucose level after a 75 g glucose load is 140-199 mg / dL (7.78-11.06 mmol / L), or an HbAlc level of 5.7-6.4%. Without intervention and appropriate treatment, individuals with pre-diabetes are at risk of developing type 2 diabetes.

[0118] The pharmaceutical compositions and / or formulations of the present invention can be used to administer insulin before or during meals. Due to rapid absorption, the administered insulin can block the conversion of glycogen to glucose in the liver, thereby preventing hyperglycemia. In embodiments, the pharmaceutical compositions and / or formulations are used to administer fast-acting insulin less than about 30 minutes, less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes before a meal, or around the time a meal begins. In embodiments, the pharmaceutical compositions and / or formulations are used to administer insulin within about 10 to about 20 minutes after the start of a meal (e.g., before or after the start of a meal). In certain embodiments, the subject receives a regimen of a basal insulin formulation or a basal insulin analog formulation. The basal insulin can be administered as a bolus injection one to three times daily.

[0119] In various embodiments, the pharmaceutical composition or formulation is administered at least once, at least twice, at least three times, at least four times, at least five times, or more times daily, with or without meals. In embodiments, the pharmaceutical composition or formulation is administered at least three times daily in connection with the consumption of meals. In certain embodiments, the pharmaceutical composition or formulation is administered (or delivery is activated) during hyperglycemia. In embodiments, the pharmaceutical composition is administered with an insulin pump, such as a tube-based pump or patch pump. In embodiments, the insulin pump administers the pharmaceutical composition according to direct human input or according to an algorithm that calculates the dose based on input from one or more sensors, such as glucose or activity. In embodiments, the pharmaceutical composition is delivered in both variable-sized basal and bolus doses.

[0120] In certain embodiments, the pharmaceutical composition or formulation is administered as a bolus subcutaneous injection.For example, administration can be achieved by a single bolus subcutaneous injection.In some embodiments, the pharmaceutical composition and / or formulation is automatically administered upon detection of hypoglycemia or a decreasing blood glucose level.In some embodiments, the pharmaceutical composition is administered as a single bolus subcutaneous injection or infusion.

[0121] In some embodiments, the volume of the administered pharmaceutical composition or formulation varies. In some embodiments, the volume of the composition delivered varies. In various embodiments, the volume of the injection solution is less than about 3 ml, less than about 2.9 ml, less than about 2.8 ml, less than about 2.7 ml, less than about 2.6 ml, less than about 2.5 ml, less than about 2.4 ml, less than about 2.3 ml, less than about 2.2 ml, less than about 2.1 ml, less than about 2 ml, less than about 1.9 ml, less than about 1.8 ml, less than about 1.7 ml, less than about 1.6 ml, less than about 1.5 ml, less than about 1.4 ml, less than about 1.3 ml, less than about 1.2 ml, less than about 1.1 ml, less than about 1.0 ml, less than about 0.9 ml, less than about 0.8 ml, less than about 0.7 ml, less than about 0.6 ml, less than about 0.5 ml. less than about 0.4 ml, less than about 0.3 ml, less than about 0.2 ml, less than about 0.1 ml, or about 90 μl or less, or about 80 μl or less, or about 70 μl or less, or about 60 μl or less, or about 50 μl or less, or about 40 μl or less, or about 30 μl or less, or about 20 μl or less, or about 10 μl or less, or about 9 μl or less, about 8 μl or less, about 7 μl or less, about 6 μl or less, about 5 μl or less, about 4 μl or less, about 3 μl or less, about 2 μl or less, about 1 μl or less, about 0.5 μl or less, or about 0.1 μl or less, including all values ​​and ranges therebetween.

[0122] The present invention is further illustrated by the following non-limiting examples. [Example]

[0123] Example 1. Discovery of a stability-optimized two-chain insulin US2019 / 0322719, incorporated herein by reference, describes fast-acting insulin analogs with specific mutations at approximately 16 sites that can potentially be mutated to confer beneficial effects (see Table 1 therein). Taken together, all of the potential mutations define a vast space of potential analogs (see Table 2). [Table 2]

[0124] The number of candidate analogs to be considered was reduced by narrowing down mutations at specific positions based on differences in charge, size, and hydrophobicity from the native amino acid at each position.

[0125] A8 and A14 positions. Compared to alternative substitutions, the selection of glutamic acid at both the A8 and A14 positions was favored to (a) optimize the net negative charge of the insulin monomer or dimer at neutral pH, thereby enhancing solubility and electrostatic repulsion between monomers; (b) thermodynamic stability by avoiding the unfavorable β-branched amino acid at A8 and the unfavorable reverse hydrophobic effect at A14; and (c) prolong the lag time for fibrillation in zinc-free solution due to electrostatic repulsion between native or partially unfolded monomers, increased thermodynamic stability, and attenuated structural fluctuations. Glu A8 and Glu A14 Both are located in the α-helical segment, where this amino acid has a high intrinsic propensity. The two negative charges introduced into the A chain complementarily increase the three negative charges in the B chain (i.e., glutamic acids at positions B3 and B29, the latter replacing the basic side chain lysine in wild-type human insulin).

[0126] "S / ie-2 Substitution" In the absence of insulin, the free insulin receptor resides on the cell surface like an inverted horseshoe (P), with the open end (IR b-subunit) penetrating the cell membrane and entering the cell. Upon insulin binding to the receptor, a major change in insulin's quaternary structure occurs, bringing the two insulin legs together. The transmembrane beta subunit α-helix of insulin then propagates to the intracellular tyrosine kinase domain, inducing its autophosphorylation. WO 2013 / 110069, incorporated herein by reference, proposed that disrupting the site 2-intersection residues of insulin could disrupt the duration of this autophosphorylation signal. To evaluate this, over 1,000 insulin tolerance tests were performed in rats to assess the effect of mutations to site 2 residues on the duration of insulin activity in vivo.

[0127] Male Lewis rats (average weight ~300 g) were treated with streptozotocin to induce diabetes. Blood glucose levels averaged 100 mg / dL in control rats and 360 mg / dL in diabetic rats. Rats (weight 250-350 g) were housed two per cage and were allowed to restrain throughout the assay. To examine the in vitro potency of representative insulin analogs relative to wild-type insulin, wild-type insulin, the analog being evaluated, or a protein solution containing buffer (protein-free sterile diluent obtained from Lilly and Company, consisting of 16 mg glycerol, 1.6 mg meta-cresol, 0.65 mg phenol, and 3.8 mg sodium phosphate (pH 7.4) per ml) were intravenously infused, and blood glucose changes were monitored by continuous glucometer measurements. Assays were performed under fasting conditions (starting late in the morning after a 2-hour fast). Rats were injected via the tail vein at time t = 0 with 10 pg of insulin in 100 pI of buffer per 300 g rat. Dose-response studies of wild-type insulin showed that this dose produced a near-maximal glucose excretion rate within 1 hour of injection. Blood was collected from the tip of the tail at time 0 and every 10 minutes for up to 90 minutes. Over 115 analogs (average n = 8 rats) containing one (over 35), two (65), or three (16) site 2 mutations were studied.

[0128] The blood glucose time course for each animal was fitted using (1) a Loessian model and (2) a "four-phase" model consisting of consecutive linear segments: an "onset" segment, a horizontal "peak" segment (at or near the maximum blood glucose drop), a "first recovery" segment in which blood glucose levels rapidly recover, and a "second recovery" segment in which blood glucose levels slowly return to pre-test levels. From the fitted data, the following values ​​were calculated for each animal and for the average fitted curve across animals exposed to the same test substance: slope of the onset curve, peak effect (the difference between the mean pre-injection blood glucose level and the lowest measured blood glucose level), duration of peak effect (for the four-phase fit), time to peak effect (the time from injection to the observation of peak effect), time from peak effect to 50% recovery, and slope of the first recovery curve. Statistical calculations were performed to determine which mutations or sets of mutations may have an effect on shortening the duration of insulin action. Based on these studies, the following mutations were eliminated as likely to have the desired effect: [Table 3]

[0129] Although studies in rats have shown that classic "site 2" substitutions (defined by DeMeyts and Schaffer in 1994) can, in some cases, lead to a decrease in the duration of insulin signaling once insulin receptors are engaged in target tissues (see WO 2014 / 145593, incorporated herein by reference in its entirety), such substitutions tended to reduce insulin stability and promote microfibrillation in vitro. When the tendency toward PD observed in rats was not observed in pigs, this category of substitutions was eliminated. An important advantage of such elimination was that advantageous features elsewhere in the molecule (such as those conferred by glutamic acids at positions A8 and A14 described above) could be realized without offsetting the penalty at site 2 (which may arise from potential substitutions of suboptimal side chains with respect to the packing of adjacent side chains, reduced α-helical propensity, exposed nonpolar surfaces, or reduced electrostatic repulsion between insulin monomers).

[0130] N-terminal B chain segment. The N-terminal segment of human insulin (residues B1-B3) contributes to the foldability of proinsulin but is not required for the biological activity of the mature hormone. Phe B1 -Val B2 -Asn B3 The native sequence is compatible with the in vivo biosynthesis and subsequent steps: storage, secretion, and hormonal regulation of the zinc hexamer in pancreatic β-cells. However, the same sequence element is also present in Phe B1 and Val B2 The pendant, disordered nonpolar side chains of Asn are responsible for fibrillation. B3 The side chain of Phe is prone to chemical degradation and is therefore not optimal for pharmaceutical use. To avoid these problems, insulin analogs are made by replacing the Phe with a combination of deletions. B1 Removal of Asn eliminates the pendant aromatic ring, while the substitution of valine for alanine in B2 replaces a β-sheet-preferring residue with an α-helix-preferring residue. B3 Substitution of glutamic acid at positions A8 and A14 increases the net negative charge provided by glutamic acid, further enhancing solubility at neutral pH and strengthening electrostatic repulsion between monomers, avoiding the potential chemical degradation pathway to Asp or Asn at B3. Deletion of residue B1 was preferred over deletion of residues [Bl, B2] or B1-B3 to minimize changes from wild-type insulin and avoid the potential chemical degradation pathway to glutamic acid or glutamine at the neo-N-terminus. The alanine at position A2 provides a neutral spacer element between the α-amino group and the functionalized side chains of B3 and B4. Furthermore, if residues B1 and B3 are deleted, residue B3 can be Ala, and if residues B1-B3 are deleted, residue B4 can be Ala.

[0131] Acidic B-chain tail. The two-residue extension of the B-chain, consisting of glutamic acids at the extended positions B31 and B32, was conceived as a means of introducing two negative charges in an effort to increase solubility at neutral pH and increase the net negative charge of the protein, whether in the monomeric, dimeric, or hexamer form. The acidic C-terminal B-chain tail reduces cross-binding of insulin with the mitogen type 1 IGF-1 receptor (IGF-1R). However, such a tail could potentially serve as a neoepitope, eliciting anti-insulin antibodies, whose complexation could delay absorption from subcutaneous depots or inhibit insulin action. Because five negative charges were introduced elsewhere in the A- and B-chains (A8, A14, B3, and B29), the additional negative charge at B31-B32 was not necessary to optimize solubility, physical stability, or chemical stability. Furthermore, the glutamic acid at position B29 was sufficient to render cross-binding with IGF-1R unfavorable. Thus, the inclusion of an acidic B-chain tail poses an antigenic risk without any compensating benefit.

[0132] Based on this reasoning, the following mutations were eliminated: [Table 4] As a result, the number of possible permutations was reduced to about 2 million.

[0133] Expression, Purification and Stability Insulin analogs are commercially produced through biofermentation. Yeast or bacteria are genetically engineered to produce insulin, and these cells are fermented in large tanks. The resulting medium is then processed to extract and purify the insulin analog. Because insulin is a necessary drug for many people, production cost is an important consideration. Therefore, a key criterion for selecting candidates for clinical development is whether the analog can be produced with high yield. Initial efforts to ferment or purify certain two-chain insulin analogs led to the elimination of analogs containing leucine or valine at A21 or arginine at A17. This resulted in a reduction in the number of analogs to approximately 162,000.

[0134] Physical and chemical stability testing using USP standards excluded analogs with leucine at A14, leaving approximately 86,400 analogs.

[0135] Gradient Descent The remaining set of analogs represents a 33-dimensional discrete state space (11 positions with 5, 3, 5, 2, 5, 3, 3, 2, 3, and 2 possible discrete states, respectively). An experimental design was implemented to rapidly search this space for analogs that maximize utility along various metrics, including stability, mitogenesis, cell signaling, and pharmacodynamic performance. For each of the 11 positions, there existed at least three pairs of analogs (a) where each pair differed from the other by the same pair of residues but was otherwise identical (i.e., had the same "context"), and (b) where no pair had a similar context. These 56 analogs were synthesized and evaluated, respectively, as follows: [Table 5]

[0136] As a result, the following findings were obtained: [Table 6]

[0137] Based on these findings, the research team ruled out the following mutations: [Table 7] These studies have identified approximately 160 possible analogs.

[0138] Design Optimization Optimization of the molecular design of T-1123 expanded on the favorable sequence obtained in the gradient descent study, focusing on five mutations at position A21 and two mutations at position B13 and evaluating their impact on chemical and physical stability. Forty analogs were evaluated, including two B1-B3 and B29-B30 designs and a limited re-evaluation of four mutations at position A8. Stricter stability and yield criteria led to the selection of Gly at A21, maintaining the native residue at B13, selecting desBl AlaB2 and GluB3 as the optimal N-terminal combination, and selecting GluB29 over desB30 for the C-terminus. This narrowed the optimal sequence down to the amino acid substitutions: GluA8 GluA14 GlyA21 desBl AlaB2 GluB3 GluB29 (referred to as T-1123). The runner-up was: [Table 8] [Example]

[0139] Example 2: Multiplex testing of insulin analogs in male Yucatan miniature pigs A study was conducted using male Yucatan miniature pigs to determine the pharmacokinetic (PK) profiles of various formulations of up to five inactive insulin analogs after single intravenous (IV) or multiple subcutaneous (SC) administration in castrated male Yucatan miniature pigs. The insulin analogs used in this example are "dead" analogs (previously defined as a type of non-analog) that do not bind to the IR due to modifications of LeuA3 known in the art to almost completely inhibit binding to the IR.

[0140] In the IV phase of the study, a single formulation containing all five inactive insulin analogs was administered intravenously (Table 9). This took place in the animals' normal housing. Administration was via one VAP, which was not used for PK blood sampling during the IV phase, after being flushed with 6 ml of heparinized saline. [Table 9]

[0141] In the SC phase of the study, dosing was subcutaneous, with individual co-injections of analog formulations. Each injection was a different analog, but the formulation of each analog was the same in some cases and the same in others. The animals were removed from their housing and restrained in a "V-trough" in the supine position to avoid pressure on the injection site.

[0142] For both routes of administration, 3 ml of blood was serially drawn at designated time points to measure blood glucose levels and plasma concentrations of each analog. Time 0 = time of administration. -Intravenous administration phase: before administration, 1, 2, 5, 10, 15, 30, 60, 120, 180, 240, 480, 720 minutes after administration -SC phase: Before administration, 1, 3, 5, 7, 10, 15, 20, 30, 45, 60, 90, 120, 180 minutes after administration

[0143] Blood samples were collected in tubes containing the anticoagulant K2EDTA. Whole blood samples were processed within 30 minutes of collection. Plasma was processed by centrifugation at -3,000 RPM for -15 minutes at -4°C. The resulting plasma was divided equally into two pre-labeled polypropylene cryotubes (primary and backup), placed on dry ice, and then frozen (approximately -70°C). [Example]

[0144] Example 3: Detection and quantification of nonalogues in plasma samples Nonalcoholic insulin was isolated from miniature pig plasma by coating Sepharose paramagnetic beads with streptavidin and derivatizing biotinylated anti-insulin monoclonal antibodies onto the magnetic beads. The biological samples were thawed on ice, vortexed, and transferred to a deep-well plate spiked with lispro (USP), an internal standard (IS). The nonalcoholic plasma samples spiked with lispro calibration, quality control, internal standard, and IS were mixed with biotinylated anti-insulin antibody-derivatized magnetic beads, centrifuged, capped, and incubated. The antibody-derivatized beads were washed using a liquid handling device, and the internal standard and unrelated compounds were eluted from the beads. The eluate was transferred to a round-bottom plate and stored at 4°C until separation and detection using a hybrid liquid chromatography-mass spectrometry system. Separation was performed using an ultra-high-performance liquid chromatograph (UPLC) system equipped with a thermostatic column compartment and an autoinjector. All samples were separated at 60°C using a C4 chemistry column with aqueous mobile phase A and organic mobile phase B. Separation was performed with a gradient of 10% to 40% mobile phase B over 3 minutes. Nonalogues were detected using a triple quadrupole mass spectrometer in positive ion mode. T-0068 was detected by the ion transitions m / z 954.40 → 136.10, T-1069 by the ion transitions m / z 941.60 → 136.10, T-1072 by the ion transitions m / z 946.40 → 136.10, T-1071 by the ion transitions m / z 939.30 > 136.10, and Lispro internal standard by the ion transition m / z 968.8 → 217.20. Samples were analyzed by peak area ratio method, weighted 1 / x, using Lispro as the internal standard. 2 Quantitation was performed from a calibration curve constructed using linear regression. [Example]

[0145] Example 4: Pilot Study Pilot studies performed in rats showed that the inactive insulin analog of Example 3 was cleared from the bloodstream within 8 hours, demonstrating that the inactive insulin analog can be efficiently cleared from the bloodstream without binding to the insulin receptor. [Example]

[0146] Example 5: Analysis of multiplex PK studies using nonalogues The data from the IV and SC studies in Example 2 were combined into a single database and analyzed as follows. A multicompartment model of insulin action was developed (Figure 1), identifying five distinct compartments: (1) the subcutaneous (SC) space, where the initial injection occurs; (2) the lymphatic system (LY), which takes up a portion of the insulin and ultimately pumps it into the bloodstream; (3) the bloodstream, also known as the central compartment (CT), which takes up the remainder of the insulin from the SC; (4) a large processing compartment (PB), which receives and metabolizes insulin but returns a portion to the CT; and (5) a small processing compartment (PA), which also receives, metabolizes, and returns insulin to the CT. The model also includes a non-metabolic excretion pathway (OU) (i.e., renal excretion from the CT). The model is described by a set of partial differential equations (based on standard intercompartmental flow equations) for variables that describe insulin concentration within the compartments and flow between compartments as a function of time.

[0147] First, we analyzed the pharmacokinetics of Nonalog after absorption into CT using the data from the IV study. The parameters of a reduced model (excluding SC and LY) after CT absorption were fitted to the IV study data by numerical calculation. As a result, we found that the half-life of Nonalog in pigs was approximately 8 hours (compared to approximately 5 minutes for potent insulin analogs).

[0148] It should be noted that the above analysis is not the only possible analysis approach. Although the above elimination parameters are fixed by prior estimation of IV and are assumed to be constant between subjects and between analogs, in reality, the elimination parameters likely vary between subjects (and possibly between analogs). Also, this design provides only one or two time points to characterize the tail of the PK curve. If more sampling time points in the tail of the PK curve had been included, the assumption that the elimination parameters are constant could have been removed.

[0149] Once these post-CT parameters were determined, they were fixed as constants in the full model, and the data from each SC experiment were fitted to this large model to determine the SC→LY, SC→CT, and LY→CT flow parameters for each SC experiment. An absorption model for each SC experiment was then determined by utilizing the calculated parameters for pre-CT flow and setting post-CT flow to zero. From this absorption model, specific empirical absorption metrics were obtained (e.g., absorption rate over the first 30 minutes, maximum slope of absorption, and area under the absorption curve (AUC)). These empirical absorption metrics obtained from each SC experiment were then used to evaluate the effect of different excipients on absorption.

[0150] Mixed-effects models were used to isolate the effect of specific excipients on absorption measures, controlling for variables such as nonalcohol excretion, pig variability, and different experimental days. The results revealed the magnitude and significance of the effect of excipients or excipient cocktails on absorption. These results were used to identify the most promising excipients and cocktails to evaluate in the euglycemic clamp. [Example]

[0151] Example 6: Formulation design A series of tests based on the methods described in Examples 2, 3, 4 and 5 were carried out to determine the optimum excipients and concentrations to use in insulin compositions.

[0152] Figure 2 shows the results of statistical analysis of multiple PK studies for formulation screening.

[0153] The pharmacodynamic properties of various formulations of T-1123 were investigated in the Yucatan Minipig model. On the day of the test, pigs received an intravenous bolus of octreotide acetate (7.2 pg / kg) approximately 60 minutes before the start of the clamp test to inhibit endogenous pancreatic a- and b-cell secretion, followed immediately by a maintenance intravenous infusion at 3.6 pg / kg / h until the end of the clamp. After establishing baseline blood glucose levels with a 10% dextrose infusion, 1.35 nmol / kg of T-1123 formulation, 1.35 nmol / kg of Fiasp, or diluent was administered subcutaneously to initiate the clamp. To quantify peripheral abdominal insulin-mediated glucose uptake, a variable-rate glucose infusion was administered to maintain blood glucose levels at approximately 85 mg / dL for the duration of insulin action (typically 4–6 hours). Glucose consumption (GC) is a measure of insulin action during the euglycemic clamp. It differs from the glucose infusion rate (GIR) because it accounts for changes in blood glucose (BG). This is important because clamp protocols are imperfect and BG will fluctuate if actual insulin action differs from that predicted at the beginning of the GIR window. GC between time tl and time t2 is calculated as GIR(between tl and t2) - (BG(t2) - BG(tl)) * Vd / (t2 - tl), where Vd is the "volume of distribution" (i.e., blood volume in the subject). GIR is in mg / min. BG in the US is in mg / dL. Vd is in dL. tl and t2 are in minutes.

[0154] The fitted GC curves were used to calculate the following parameters: time to half-effect (early), time to half-effect (late), time to maximum effect, and area under the curve (AUC) relative to baseline.

[0155] U-500T-l123 was comparable in potency and onset of action to U-100 Fiasp in a base formulation consisting of a neutral buffer, preservative, and tonic (Table 10). However, the base of U-500T-l123 had a longer duration of action than U-100 Fiasp (Figure 3).

[0156] Several absorption-enhancing excipients identified in the multiplex PK study in Figure 2 were tested in the U-500 T-l 123 formulation and compared with U-100 Fiasp. The combination of three active excipients (citrate, EDTA, and iloprost) in U-500 T-l 123 (accelerated formulation) showed a time-of-action profile and pharmacodynamic properties consistent with those of U-100 Fiasp, with no significant differences in the onset and end of action (Figure 1). 4) A further improvement of this formulation, U-500T-l 123 (an accelerated formulation with optimized stability), showed comparable potency in terms of AUC, onset, and Tmax, with a slightly longer tail of action (Figure 5). [Table 10]

[0157] After determining the optimal excipient set, PD studies were conducted in pigs using variant T-1123 formulations subtracted from specific excipients (Table 11). These subtractive analysis studies confirmed that each active ingredient in the formulation was necessary to achieve optimal PD parameters. Figures 6A, 6B, and 6C show the replacement of citrate with Tris, the removal of EDTA, and the removal of iloprost, respectively. Replacing citrate with Tris (Figure 6A) increased the time to maximum glucose consumption in pigs and prolonged the tail of increased glucose consumption toward the end of the study. Removing EDTA from the formulation (Figure 6B) resulted in a tail of increased glucose consumption toward the end of the study. Removing iloprost from the formulation (Figure 6C) increased the time to maximum glucose consumption in pigs and significantly prolonged the tail of increased glucose consumption toward the end of the study. PD metrics are summarized in Table 12. Taken together, these studies demonstrate that Tris, EDTA, and iloprost provide a favorable PD profile for the T-1123 composition. [Table 11] [Table 12] [Example]

[0158] Example 7: Effect of Iloprost on Microfibril Lag Time Insulin analogs (Humalog®, T1 123) formulated according to Table 13 were evaluated for fibrillation lag time, as determined through an accelerated thioflavin T (ThT) dye assay. Five formulations were tested in triplicate in the accelerated fibrillation assay at 40°C with agitation. Briefly, 250 pL of formulation sample was added to a 96-well plate, with each well containing 5 pM thioflavin T (ThT). Fluorescence data were acquired every 20 minutes at an emission wavelength of 480 nm after excitation at 440 nm. The plate reader was maintained at 40°C for 7 days, with continuous cycling of 30 seconds of linear agitation (1000 10 cpm) followed by 30 seconds of rest. The mechanism by which ThT dye identifies amyloid fibrils is that, as fibrillation progresses, ThT dye binds to the beta-sheet structure of the fibrils, resulting in a steady increase in its emission intensity. The fibrillation lag time is an indicator of the time it takes for insulin or its analogs to begin forming fibrils. For the purposes of this disclosure, microfibrillation lag time is determined by linear regression of the slope of the fitted curve of the 15 emission spectra against the X-axis (time). [Table 13]

[0159] When Tl 123 was formulated with buffer, preservative, and tonic ("base formulation"), the fibril formation lag time (25.1 hours) was significantly longer than that of Humalog (7.47 hours) (Table 14, Figure). 7A). Surprisingly, the addition of iloprost to the same formulation dramatically increased the fibril formation lag time of Tl 123 by more than six-fold to 168+ hours. This increase in fibril formation time was unchanged when Tris was replaced with the buffering agent citric acid or when Tris and citric acid were used together. Prior to this observation, there had been no indication that iloprost had any noteworthy properties as a stabilizing excipient. [Table 14]

[0160] The protective effect of iloprost against fibrillogenesis was then tested with commercially available insulin analogs. Fibrillogenesis lag time was assessed with Humalog using three increasing concentrations of iloprost according to the method described above. Iloprost had only a small effect on the fibrillogenesis lag time of Humalog at the iloprost concentrations tested (Table 15). [Table 15]

[0161] The T-1123 formulation (Example 6, Table 10), which exhibits a time-action profile comparable to Fiasp, was evaluated for physical stability performance in an accelerated fibrillation assay using ThT fluorescence. The U-500T-1123 formulation containing 15 μg / mL iloprost was non-fibrillable at 168 hours (Table 14), whereas the base formulation U-500T-1123 without iloprost formed fibrils in 25.10±5.59 hours, and the marketed Humalog formed fibrils in 7.47±1.89 hours (Table 15). [Example]

[0162] Example 8: Chemical and physical stability of T-1123 The chemical stability of U-500 T-1123 formulated in a base formulation (Table 10) was compared with that of commercially available U-100 Humalog and U-500 Humulin using RS and HMWP, as well as microfibrillation, as described in Example 7. To determine RS and HMWP, the insulin analog U-500 (17.5 mg / ml) zinc-free formulation, U-100 Humalog, and U-500 Humulin were subjected to heat stress at 40°C for 28 days. RS was characterized by reversed-phase UPLC and LC-MS. HMWP formation as covalent dimers and oligomers was quantified by size-exclusion high-performance liquid chromatography (SEC-HPLC). After the incubation period, T-1123 in the base formulation showed chemical degradation by RS and HMWP at levels similar to those of U-100 Humalog and U-500 Humulin (Figure 8A). Across all three compositions tested, no loss of purity greater than 3% was observed during the study period. U-500T-1123 showed slightly improved RS compared to U-100Humalog and slightly improved HMWP compared to U-100Humalog and U-500Humulin.

[0163] Long-term studies were also conducted to evaluate the chemical and physical stability of T-1123. In these studies, U500 T-1123 was formulated in a citrate formulation containing Tris as described in Table 13, placed in vials, and placed in a nutrient return tube at 30 °C for one year. The RS of T-1123 decreased by only 3.14% after one year under these conditions (Figure 8F). Furthermore, in contrast to the commercially available formulations U400 Insuman (which developed fibrillation in 81 days) and U100 Humalog (which developed fibrillation in 32.75 days), none of the test samples developed fibrillation (Figure 7D). [Example]

[0164] Example 9: Optimization of Chemical Stability of T-1123 Ultra-Rapid Formulations. Excipient replicate titrations were performed to determine the optimal combination and concentration for use in T-1123 formulation development. All test formulations contained 45 mM sodium citrate, 6.2 mM EDTA (various salts), 15 pg / mL iloprost, and 0.32% m-cresol at U-500 T-1123 concentrations. Formulations of insulin analogs were heat-stressed at 45°C for 7 days, and RS and HMWP were characterized as described in Example 8. An exemplary study is shown in Figures 8B-8E. Figures 8B and 8C summarize the effect of magnesium and Tris on the chemical stability of T-1123. EDTA / Mg 2+ At non-equimolar concentrations of 4 mM Mg, there is a direct correlation between API purity and increasing Tris concentration, indicating improved stability. 2+ This figure summarizes the effect of titrating glycerol and Tris in HCl. Increasing the concentration of Tris and glycerol decreases HMWP formation, with this effect being greatest at 20 mM Tris and 20 mg / mL glycerol.

[0165] Further chemical degradation test results are provided in Figure 8F. T-123 compositions were formulated as shown in Table 16. Figure 8F shows the loss of purity due to accumulation of related substances (RS) and covalently bound high molecular weight proteins (HMWP) for selected formulations STA-0067 (n=3), STA-0109 (n=3), and STA-0116 (n=2). Table 16: Formulations of U500T-1123 with varying concentrations of Mg+, Glycerin, and Tris [Table 16] [Example]

[0166] The self-association state of T-1123 was investigated by dynamic light scattering (DFS) using a Wyatt Dynapro plate reader III (Wyatt Technology) at a protein concentration of 3.0 mM in a Zn-free formulation containing m-cresol and glycerin in Tris buffer, pH 7.4. U-500 T-1123 Zn-free formulation samples, U-100 Humalog, and U-500 Humulin R commercial samples were monitored for changes in aggregation state at 40°C for 28 days. For in situ measurements, the autocorrelation function of scattered light was acquired 10 times (5 seconds each) and converted to a particle size distribution using a "regularized" particle size distribution and the Rayleigh spheres model. The aggregation state of Zn-free U-500 T-1123 did not change during the test period, regardless of heat stress. Analysis of the scattering data revealed that the estimated average molecular weights of U-100 Humalog and U-500 Humulin R were those expected for insulin hexamers at 10 min, and that U-500 Humulin R showed a significant further increase in average molar mass (i.e., beyond hexamers) over the course of the study, whereas T-1123 was predominantly dimeric under these conditions. The ultra-rapid formulation of T-1123 described in Table 17 was assessed for changes in aggregation state in response to heat stress (40°C, 28 days) as described above, and demonstrated no change over the course of the study. [Table 17] [Example]

[0167] The activity of T-1123 against insulin was measured in a cell-based receptor activation assay using CHO cells overexpressing human insulin type A (hIR-A) or type B (hIR-B), quantifying tyrosine phosphorylation involving the insulin receptor. The increase in tyrosine phosphorylation after insulin stimulation was measured using an In Cell Western kit (LICOR) with anti-pTyr primary antibody 4G10 (Millipore). Results were normalized to cell number using DNA quantification. The dose-response data were fitted with a four-parameter logistic model to determine the EC50 value. The activation EC50 values ​​of T-l123 and HI for human insulin receptor (isoform A; hIR-A) were 23.2 ± 1.28 and 10.5 ± 2.28 nM, respectively; similarly, the hIR-B activation EC50 values ​​were 3.7 ± 0.13 and 2.6 ± 0.11 nM, respectively. The relative potency of T-l123 in the IR activation assay is consistent with its full in vivo potency against HI.

[0168] The dephosphorylation kinetics of T-l123 and HI were assessed by quantifying the decrease in tyrosine phosphorylation after washout of insulin stimulation using In-Cell Western (LICOR). Dephosphorylation assays were performed in CHO cells overexpressing hIR-A or hIR-B receptors. CHO cells were stimulated with T-l123 or HI at a fixed concentration of 100 nM for 5 minutes and then washed to remove insulin. Tyrosine phosphorylation was measured at five post-wash time points (0, 30, 60, 120, and 180 minutes) using the anti-pTyr primary antibody 4G10 (Millipore) with an In-Cell Western kit (LICOR). Results were normalized to cell number by DNA quantification. The duration of signaling in hIR-A and hIR-B cells was less than that of HI (Figure 9A and Figure 9B), indicating no greater mitigation risk potential than HI.

[0169] The metabolic potential of T-1123 was evaluated in an anti-lipolysis assay using differentiated human preadipocytes. Preadipocytes were differentiated for 14 days in 96-well plates using DM-2 subcutaneous adipocyte differentiation medium (Zenbio). Differentiated cells were stimulated with isoproterenol (0.5 nM) to promote lipolysis, which could be measured as glycerol released into the cell culture medium. Lipolysis was inhibited for 4 hours with a dilution series of HI or T-1123. The cell culture medium was transferred to a new 96-well plate, and glycerol release was measured in a three-step process, culminating in the production of a quinoline dye with absorbance at 540 nm. The dose-response data for glycerol release were fitted with a four-parameter logistic model, and EC50 values ​​for inhibition were determined to be 0.54 ± 0.14 nM and 0.56 ± 0.15 nM for T-1123 and HI, respectively. The indistinguishable potency of T-l123 and HI in the antilipolysis assay is consistent with the full metabolic potency of T-l123.

[0170] The mitogenic potential of T-l123 was demonstrated in a cell proliferation assay using human breast cancer-derived MCF-7 cells [2- 14 Serum-starved cells were stimulated with a dilution series of T-1123 or HI control from 10 pM to 1500 nM, incubated overnight, and then [2- 14 The cells were incubated with a 2-C-thymidine solution for 6 hours. 14 C-thymidine uptake was measured using a scintillation counter, and a dose-response curve was calculated from eight technical replicates. The EC50 values ​​of T-l123 were 80.5 nM and 12.9 nM, respectively, indicating lower activity than HI. Furthermore, even after correcting for the lower intrinsic IR activation capacity of T-l123 compared with HI, its activity remained lower than that of HI. [Example]

[0171] The efficacy of T-l123 and insulin lispro formulated with a neutral buffer, preservatives, and tonic was investigated in diabetic Lewis rats. T-l123 and insulin lispro (KP) were administered intravenously to rats at 10 pg / 300 g doses after a 2-hour fast during the light phase. Blood glucose levels were measured for 5 hours after administration using an EasyMax V glucometer, and a time-course fitted curve of BG was obtained. The time-course fitted BG curve is shown in Figure 10A. Efficacy in Figure 10B is expressed as the mg / dL decrease in BG per pg administered and is calculated from the maximum drop in the PD curve. The measured efficacy indicates that T-l123 is at least as effective as insulin lispro in this diabetic rat model. [Example]

[0172] The insulin analogs T-1123 (EA8, EA14, GA21, desBl, AB2, EB3, and EB29 relative to wild-type human insulin) and T-8602 (EA8, EA14, AA21, AB3, EB29, and EEGRR linker relative to wild-type human insulin, SEQ ID NO: 14) were evaluated for fibril formation lag time determined through an accelerated thioflavin T (ThT) dye assay as described in Example 7. Samples were prepared for testing in 50 mM Tris buffer (pH 7.4) with or without 3.2 mg / mL m-cresol, 16 mg / mL glycerin, and 20 mM sodium triphosphate. See Table 18. Additionally, Humalog® (Eli Lilly, with or without triphosphate) and Novolog® (Novo Nordisk, with or without triphosphate) were also tested for lag time before the onset of ThT-positive fibril formation. See Table 19. [Table 18] [Table 19] [Example]

[0173] Example 14: Accelerated fibril formation assay to test the stability of insulin analogues Triplicate samples of insulin analogs (Humalog®, Novolog®, T-1123, and T-8602) were dispersed in microplates in a synergy HI spectrofluorometer (BioTek Instruments, Inc., Winooski, VT) at 40°C with rapid agitation for up to 7 days, and fibrils were detected by thioflavin T (ThT) fluorometry. ThT fluorescence measurements were performed every 20 minutes using an excitation wavelength of 440 nm and an emission wavelength of 485 nm.

[0174] T-1123, formulated with U-500 in a buffer, preservative, and tonic base formulation, demonstrates that the addition of 20 mM sodium triphosphate does not affect the fibril formation lag time (Table 20, Figure 11A). This contrasts sharply with Humalog® and Novolog®, where the addition of 20 mM sodium triphosphate rapidly decreases the fibril formation lag time (Table 20). It is also noteworthy that the fibril formation lag time for T-1123 U-500 base formulations with and without added sodium triphosphate is more than four times longer than that for Humalog® or Novolog® commercial formulations without added sodium triphosphate. T-8602, in a buffer, preservative, and tonic base formulation, demonstrates a fibril formation lag time that is unaffected by the addition of 20 mM sodium triphosphate (Table 20, Figure 11B). This is in stark contrast to Humalog® and Novolog®, where the addition of 20 mM sodium triphosphate rapidly reduces the fibril formation lag time. Also noteworthy was that the fibril formation lag time for the base formulations of T-8602U-100 with and without sodium triphosphate was more than 20-fold longer than that of the marketed Humalog® or Novolog® formulations without added sodium triphosphate. [Table 20] Figure 11C provides additional accelerated fibril formation assay results. T-123 compositions were formulated as provided in Table 16 and tested against Humalog (n=3 in each example). Each T-1123 formulation exhibited a fibril formation lag time of over 168 days, while Humalog exhibited a mean fibril formation lag time of 6.96 days. [Example]

[0175] Example 15: Chemical stability of T-1123 in polyphosphate formulations T-1123 was formulated using base formulations with and without TriP04, as shown in Table 21. These formulations were heat stressed at 40°C for 28 days to determine RS and HMWP. RS and HMWP were characterized as described in Example 8. After the incubation period, the presence of TriP04 did not affect the chemical stability of T-1123, as both RS and HMWP remained similar upon addition of 20 mM TriP04 (Figure 12). [Table 21]

[0176] equipment While the invention has been described in relation to specific embodiments thereof, it will be understood that it is capable of further modifications, and this application is generally intended to cover any variation, use, or adaptation of the invention in accordance with its principles and including departures from the present disclosure which are within known or customary practice in the art to which this invention pertains and which may apply to the essential features hereinafter defined and within the scope of the appended claims.

[0177] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments specifically described herein which equivalents are intended to be encompassed by the following claims.

[0178] Incorporation by Reference All patents and publications referred to herein are incorporated by reference in their entirety.

Claims

1. 1. A pharmaceutical composition comprising an effective amount of an insulin analogue comprising a modified A-chain polypeptide and a modified B-chain polypeptide, said insulin analogue consisting of the following modifications relative to wild-type human insulin: EA8, EA14, GA21, desB1, AB2, EB3, and EB29; and A pharmaceutical composition, wherein the composition comprises iloprost.

2. 2. The pharmaceutical composition of claim 1, wherein the insulin analogue is a monomer or a dimer when formulated with U-500.

3. 10. The pharmaceutical composition of claim 1, wherein the iloprost is present at a concentration of about 1 μg / mL to about 100 μg / mL.

4. 4. The pharmaceutical composition of claim 3, wherein the insulin analogue is further formulated with EDTA and citrate.

5. 10. The pharmaceutical composition of claim 1, wherein the insulin analogue is formulated with less than 0.05 moles of zinc per mole of insulin.

6. 10. The pharmaceutical composition of claim 1, wherein the insulin analogue is further formulated with one or more of about 10 to about 100 mM Tris, about 0.1 mg / mL to about 10 mg / mL m-cresol, and about 0.1 mg / mL to about 25 mg / mL glycerin.

7. 3. The pharmaceutical composition of claim 2, wherein the insulin analogue further comprises magnesium.

8. 10. The pharmaceutical composition of claim 1, comprising iloprost at a concentration of about 5 μg / mL to about 50 μg / mL.

9. The pharmaceutical composition according to any one of claims 1 to 8 for the treatment of diabetes or pre-diabetes.

10. 10. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition is administered within 30 minutes before a meal.

11. 1. An insulin analogue comprising a modified A-chain polypeptide and a modified B-chain polypeptide, said insulin analogue consisting of the following modifications relative to wild-type human insulin: EA8, EA14, GA21, desB1, AB2, EB3, and EB29.

12. 10. The pharmaceutical composition of claim 1 for use as a medicament for the treatment of diabetes.

13. 10. Use of the pharmaceutical composition of claim 1 for the manufacture of a medicament for the treatment of diabetes.

14. 10. The pharmaceutical composition of claim 1, comprising iloprost at a concentration of about 10 μg / mL to about 25 μg / mL.

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