Buffering agent for exendin (9-39)

Abstract

To provide an exendin (9-39) liquid pharmaceutical formulation with improved efficacy, purity, and stability for treating or preventing hyperinsulinic hypoglycemia. [Solution] A liquid pharmaceutical formulation is provided, comprising exendin (9-39) or a pharmaceutically acceptable salt thereof and an isotonic agent in a physiologically acceptable buffer, wherein the isotonic agent comprises mannitol, the physiologically acceptable buffer comprises acetate buffer, and the liquid pharmaceutical formulation has a pH of about 5.1 to about 6.0.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 62 / 424,979, filed on 21 November 2016, and to U.S. Provisional Application No. 62 / 517,065, filed on 8 June 2017. The entire contents of each of these documents are incorporated herein by reference.

[0002] The present invention relates to improved pharmaceutical formulations of exendin(9-39) or its pharmaceutically acceptable salts and isotonic agents in a physiologically acceptable buffer having a pH in the range of about 5 to about 6, and to methods of using formulations of exendin(9-39) for the treatment or prevention of hyperinsulinic hypoglycemia, including post-bariatric hypoglycemia (PBH), and therefore relates to the fields of medicine, medicinal chemistry, pharmacology, chemistry, and biology. [Background technology]

[0003] Insulin is a hormone secreted to control high blood glucose levels. An abnormal increase in insulin secretion can lead to severe hypoglycemia, which can result in seizures, brain damage, and death. Glucagon-like peptide-1 (GLP-1) is a gastrointestinal hormone released from intestinal L-cells after meals. It binds to GLP-1 receptors on pancreatic β-cells, thereby increasing insulin release. In patients with PBH, GLP-1-mediated insulin secretion is increased. [Overview of the project] [Problems that the invention aims to solve]

[0004] In the United States, approximately 150,000 to 200,000 bariatric surgeries are performed annually. As the number of bariatric surgeries to treat severe obesity increases, so does the number of individuals experiencing hyperinsulinic hypoglycemia (PBH). Consequently, there is a growing unmet need for safe and effective treatments to alleviate hyperinsulinic hypoglycemia and PBH.

[0005] Exendin(9-39) is a 31-amino acid peptide that selectively targets and blocks the glucagon-like peptide-1 (GLP-1) receptor, thereby normalizing pancreatic insulin secretion in patients with PBH and reducing hypoglycemia. Exendin(9-39), reconstituted in saline for intravenous or subcutaneous administration, is currently in ongoing human clinical trials as a treatment for PBH (Stanford Clinical Trials, Clinicaltrials.gov, clinical trials identifiers: NCT02771574 and NCT02550145). However, there remains a need for improved liquid pharmaceutical formulations of exendin(9-39) that offer enhanced potency, purity, and stability. [Means for solving the problem]

[0006] In one embodiment, a liquid pharmaceutical formulation is provided comprising exendin(9-39) or a pharmaceutically acceptable salt thereof in a physiological buffer having a pH in the range of about 5 to about 6. In some embodiments, the liquid pharmaceutical formulation comprises exendin(9-39) acetate or exendin(9-39) trifluoroacetate of a pharmaceutically acceptable salt.

[0007] In some embodiments, the physiologically acceptable buffer is an acetate buffer, a citrate buffer, a phosphate buffer, or a histidine buffer. In some embodiments, the physiologically acceptable buffer is sodium acetate or sodium citrate. In some embodiments, the buffer (e.g., sodium acetate or sodium citrate) is present in the formulation at a concentration of about 5 mM to about 30 mM. In some embodiments, the buffer (e.g., sodium acetate or sodium citrate) is present in the formulation at a concentration of about 10 mM to about 30 mM (e.g., 10 mM to 20 mM). In some embodiments, the physiologically acceptable buffer contains sodium acetate at a concentration of about 10 mM, about 20 mM, or about 30 mM. In some embodiments, the physiologically acceptable buffer contains sodium acetate at a concentration of about 10 mM. In some embodiments, the physiologically acceptable buffer contains sodium citrate at a concentration of about 10 mM. In some embodiments, the physiologically acceptable buffer contains sodium acetate at a concentration of at least about 10 mM. In some embodiments, the physiologically acceptable buffer contains sodium citrate at a concentration of at least about 10 mM.

[0008] In some embodiments, the buffer formulation contains an isotonic agent. In some embodiments, the isotonic agent includes mannitol, dextrose, glycerin, lactose, sucrose, trehalose, or a mixture thereof. In some embodiments, the isotonic agent is mannitol. In some embodiments, the isotonic agent is present at a concentration of about 20 to about 60 mg / ml. In some embodiments, the isotonic agent is present at a concentration of about 45 mg / ml. In some embodiments, the isotonic agent is present at a concentration of about 20 mg / ml. In some embodiments, the isotonic agent is added to target an isophysiological osmotic pressure of about 290 mOsm / kg.

[0009] In some embodiments, the buffer formulation has a pH greater than 5, for example, at least pH 5.1 to about pH 6.0. In some embodiments, the buffer formulation includes a buffer having a pH in the range of 5.2 to 5.8. In some embodiments, the buffer has a pH of about 5.5.

[0010] In some embodiments, the buffer formulation contains exendin (9-39) or a pharmaceutically acceptable salt thereof at a concentration (e.g., peptide concentration) of about 10 to about 60 mg / ml (e.g., about 10 mg / ml, about 15 mg / ml, about 20 mg / ml, about 25 mg / ml, about 30 mg / ml, about 35 mg / ml, about 40 mg / ml, about 45 mg / ml, about 50 mg / ml, about 55 mg / ml, or about 60 mg / ml). In some embodiments, the buffer formulation contains exendin (9-39) or a pharmaceutically acceptable salt thereof at a concentration of about 30 to about 180 mg / ml, for example, about 30 mg / ml to about 150 mg / ml, about 30 mg / ml to about 120 mg / ml, about 50 mg / ml to about 150 mg / ml, or about 60 mg / ml to about 120 mg / ml (for example, about 30 mg / ml, about 40 mg / ml, about 50 mg / ml, about 60 mg / ml, about 70 mg / ml, about 80 mg / ml, about 90 mg / ml, about 100 mg / ml, about 110 mg / ml, about 120 mg / ml, about 130 mg / ml, about 140 mg / ml, about 150 mg / ml, about 160 mg / ml, about 170 mg / ml, or about 180 mg / ml). In some embodiments, the buffer formulation contains exendin(9-39) or a pharmaceutically acceptable salt thereof at a concentration of about 15 mg / ml, 30 mg / ml, 45 mg / ml, or 60 mg / ml. In some embodiments, the buffer formulation contains exendin(9-39) or a pharmaceutically acceptable salt thereof at a concentration of at least 15 mg / ml. In some embodiments, the buffer formulation contains exendin(9-39) or a pharmaceutically acceptable salt thereof at a concentration of about 30 mg / ml. In some embodiments, the buffer formulation contains exendin(9-39) or a pharmaceutically acceptable salt thereof at a concentration greater than 30 mg / ml. In some embodiments, the buffer formulation contains exendin(9-39) or a pharmaceutically acceptable salt thereof at a concentration of at least 60 mg / ml. In some embodiments, the buffer formulation contains exendin(9-39) or a pharmaceutically acceptable salt thereof at a concentration of about 60 mg / ml. In some embodiments, the buffer formulation contains exendin (9-39) or a pharmaceutically acceptable salt thereof at a concentration of approximately 90 mg / ml.In some embodiments, the buffer formulation contains exendin (9-39) or a pharmaceutically acceptable salt thereof at a concentration of about 120 mg / ml.

[0011] In some embodiments, a buffer formulation containing exendin(9-39) or a pharmaceutically acceptable salt thereof does not exhibit detectable agglutination of exendin(9-39) or a pharmaceutically acceptable salt thereof. In some embodiments, when the buffer formulation is stored at 50°C for 18, 24, 36, 48, or 72 hours, it does not exhibit detectable agglutination of exendin(9-39) or a pharmaceutically acceptable salt thereof, as determined by whether the buffer formulation remains as a non-gelatinous solution. In some embodiments, when the buffer formulation is stored at 50°C for 18, 24, 36, 48, or 72 hours, it does not exhibit detectable agglutination of exendin(9-39) or a pharmaceutically acceptable salt thereof, as determined by visual or optical microscopy examination of the buffer formulation for agglutination or precipitation.

[0012] In some embodiments, the buffer formulation containing the exendin (9-39) or a pharmaceutically acceptable salt thereof as described herein is formulated for subcutaneous administration. In some embodiments, the buffer formulation is formulated for subcutaneous administration once daily (QD) or twice daily (BID). In some embodiments, the buffer formulation is administered in the morning, evening, or both. In some embodiments, the buffer formulation is administered QD by subcutaneous injection in the morning (e.g., at least 60 minutes before breakfast). In some embodiments, the buffer formulation is administered BID (e.g., morning and evening) by subcutaneous injection.

[0013] In some embodiments, the buffer formulations containing exendin(9-39) or a pharmaceutically acceptable salt thereof described herein exhibit an improved pharmacokinetic profile when administered to human subjects compared to compositions containing the same dose of exendin(9-39) or a pharmaceutically acceptable salt thereof formulated in 0.9% normal saline. In some embodiments, the buffer formulations exhibit a higher C of exendin(9-39) than compositions containing the same dose of exendin(9-39) or a pharmaceutically acceptable salt thereof formulated in 0.9% normal saline. max (For example, by measuring in a plasma sample from a subject who received the formulation) and present the results.

[0014] In another embodiment, a therapeutic method using the buffer exendin (9-39) formulation described herein is provided. In some embodiments, a method for treating or preventing hyperinsulinic hypoglycemia is provided. In some embodiments, a method for treating or preventing hypoglycemia after bariatric surgery is provided. In some embodiments, the buffer exendin (9-39) formulation described herein is administered to the subject twice daily (BID) in doses ranging from about 5 mg to about 30 mg, for example, about 7.5 mg to about 30 mg BID or about 10 mg to about 30 mg BID, for example, about 7.5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, or about 30 mg BID. In some embodiments, the buffered exendin (9-39) formulation described herein is administered to the subject once daily (QD) in doses ranging from about 20 mg to about 75 mg, for example, about 30 mg to about 75 mg QD, 30 to about 60 mg QD, 40 to 70 mg QD, or 30 to 60 mg QD, for example, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, or about 75 mg QD. In some embodiments, the buffered exendin (9-39) formulation described herein is administered to the subject in a dose of about 60 mg QD. In some embodiments, the buffered exendin (9-39) formulation described herein is administered to the subject in a dose of about 30 mg BID. In some embodiments, a method for treating or preventing hyperinsulinic hypoglycemia includes administering a buffered exendin (9-39) preparation to a subject who has previously undergone an upper gastrointestinal procedure, such as bariatric surgery or a metabolic procedure (e.g., gastric bypass surgery). [Brief explanation of the drawing]

[0015] [Figure 1] These are optical microscope images showing aliquots of an exendin (9-39) formulation prepared in 0.9% normal physiological saline after standing overnight at 50°C. Left panel: Aggregates formed after 24 hours, appearing gelatinous under the microscope. Right panel: After 36 hours, the solution had the appearance of a jelly-like viscous liquid. [Figure 2A]This shows the efficacy and purity data of exendin (9-39) formulations. (A) A graph showing efficacy data observed over 5 days at 50°C for formulations containing exendin (9-39) at a concentration equivalent to 15 mg / ml, prepared in acetic acid or citrate buffer with varying ionic strengths. [Figure 2B] This shows the efficacy and purity data of exendin (9-39) formulations. (B) A graph showing the purity data observed over 5 days at 50°C for formulations containing exendin (9-39) at a concentration equivalent to 15 mg / ml, prepared in acetic acid or citrate buffer with varying ionic strengths. [Figure 3] This graph shows the effect of pH on the purity and potency of a formulation containing exendin (9-39) at a concentration equivalent to 15 mg / ml, prepared in 10 mM sodium acetate at 50°C for 5 days. [Figure 4] This graph shows the effect of pH on impurities observed in a formulation containing exendin (9-39) at a concentration of 15 mg / ml, prepared in 10 mM sodium acetate. [Figure 5A] This is the plasma pharmacokinetic profile of a formulation containing exendin(9-39). (A) Plasma pharmacokinetic profile of exendin(9-39) acetate powder formulated in 0.9% normal saline, delivered by subcutaneous administration to dogs at a dose of 0.72 mg / kg (total dose of 7.2 mg, equivalent to a human dose of 30 mg). [Figure 5B] This is the plasma pharmacokinetic profile of a formulation containing exendin(9-39). (B) Plasma pharmacokinetic profile of exendin(9-39) acetate (total dose 7.2 mg), formulated in 10 mM sodium acetate at pH 5.5, containing 45 mg / ml of mannitol, and delivered to dogs by subcutaneous administration at exendin(9-39) concentrations equivalent to 15 mg / ml, 30 mg / ml, or 45 mg / ml in humans. [Figure 6]Schematic of the study, including the number of participants, dosing, and oral glucose tolerance test time points for each of the reconstituted lyophilized exendin(9-39) (“Part A”) and buffered liquid exendin(9-39) (“Part B”) formulations. [Figure 7A] Mean metabolic responses to baseline and final day OGTT for treatment with approximately 30 mg of lyophilized exendin(9-39) (“Lyo Ex9-39”) (A, C), n = 6, reconstituted in saline, or 30 mg of buffered liquid exendin(9-39) (“Liq Ex9-39”) (B, D), n = 4, are shown. Plasma concentrations (mean ± SEM) over time and AUC levels (mean ± SEM) in the inset are shown for glucose (A, B) and insulin (C, D). Baseline (BL): solid line with circles, black bar (inset). Lyo Ex9-39: dashed line with squares, red bar (inset). Liq Ex9-39: dashed line with triangles, blue bar (inset). All baseline tests were stopped at glucose <50 mg / dL and IV dextrose was administered. Baseline data shown after 120 minutes represent last observation carried forward (LOCF), thereby underestimating the true difference between treatment and baseline results. P values by paired two-sided paired Student's t test: *≤0.05; **≤0.01. [Figure 7B]The mean metabolic response to baseline and final-day OGTT is shown for treatment with approximately 30 mg of lyophilized exendin (9-39) ("Lyo Ex9-39") (A, C), n=6, reconstituted in saline, or 30 mg of buffered exendin (9-39) ("Liq Ex9-39") (B, D), n=4. Plasma concentration (mean ± SEM) over time and AUC levels (mean ± SEM) in the insets are shown for glucose (A, B) and insulin (C, D). Baseline (BL): Solid line with circles, black bars (inset). Lyo Ex9-39: Dashed line with squares, red bars (inset). Liq Ex9-39: Dashed line with triangles, blue bars (inset). All baseline studies were stopped at glucose <50 mg / dL and IV dextrose was administered. Baseline data shown from 120 minutes onward represent extrapolation by the last observation carried forward (LOCF), thereby underestimating the true difference between the treatment and baseline outcomes. P-values ​​by paired-two-tailed paired-student t-test: *≦0.05;**≦0.01. [Figure 7C]The mean metabolic responses to baseline and day-7 OGTT of treatments using approximately 30 mg of lyophilized exendin(9-39) (“Lyo Ex9-39”) (A, C), n = 6, or 30 mg of buffered liquid exendin(9-39) (“Liq Ex9-39”) (B, D), n = 4, reconstituted in saline are shown. Plasma concentrations (mean ± SEM) over time and AUC levels (mean ± SEM) in the inset are shown for glucose (A, B) and insulin (C, D). Baseline (BL): solid line with circles, black bars (inset). Lyo Ex9-39: dashed line with squares, red bars (inset). Liq Ex9-39: dashed line with triangles, blue bars (inset). All baseline tests were stopped at glucose <50 mg / dL and IV dextrose was administered. Baseline data shown after 120 minutes represent last observation carried forward (LOCF), thereby underestimating the true difference between treatment and baseline results. P values by paired two-sided paired Student's t test: *≦0.05; **≦0.01. [Figure 7D]The mean metabolic response to baseline and final-day OGTT is shown for treatment with approximately 30 mg of lyophilized exendin (9-39) ("Lyo Ex9-39") (A, C), n=6, reconstituted in saline, or 30 mg of buffered exendin (9-39) ("Liq Ex9-39") (B, D), n=4. Plasma concentration (mean ± SEM) over time and AUC levels (mean ± SEM) in the insets are shown for glucose (A, B) and insulin (C, D). Baseline (BL): Solid line with circles, black bars (inset). Lyo Ex9-39: Dashed line with squares, red bars (inset). Liq Ex9-39: Dashed line with triangles, blue bars (inset). All baseline studies were stopped at glucose <50 mg / dL and IV dextrose was administered. Baseline data shown from 120 minutes onward represent extrapolation by the last observation carried forward (LOCF), thereby underestimating the true difference between the treatment and baseline outcomes. P-values ​​by paired-two-tailed paired-student t-test: *≦0.05;**≦0.01. [Figure 8A]The images show the improvement in response to the OGTT between baseline and final day with multiple escalating doses of lyophilized exendin (9-39) ("Lyo Ex9-39") reconstituted in saline (green, yellow, orange, red) and a 30 mg buffered exendin (9-39) preparation ("Liq Ex9-39") (blue). Percentage changes (%) (mean ± SEM) are shown for the lowest glucose point (A), insulin peak (B), and hypoglycemia symptom score (C). Symptoms were graded on a 5-point Likert scale (0=none; 5=severe) according to the Edinburgh Hypoglycemia Symptom Scale: autonomous (sweating, tremors, palpitations, hunger); nervous hypoglycemia (blurred vision, confusion, drowsiness, bizarre behavior, speech difficulties, incoordination, dizziness, inability to concentrate); discomfort (nausea, headache). P-values ​​by paired-two paired-student t-test: *≦0.05;**≦0.01. All baseline studies underestimated the improvement rate (%) because they were stopped at glucose <50 mg / dL followed by administration of IV dextrose. Subjects who received exendin (9-39) <18 mg required rescue with IV dextrose. P-values: *≦0.05;**≦0.01. [Figure 8B]The images show the improvement in response to the OGTT between baseline and final day with multiple escalating doses of lyophilized exendin (9-39) ("Lyo Ex9-39") reconstituted in saline (green, yellow, orange, red) and a 30 mg buffered exendin (9-39) preparation ("Liq Ex9-39") (blue). Percentage changes (%) (mean ± SEM) are shown for the lowest glucose point (A), insulin peak (B), and hypoglycemia symptom score (C). Symptoms were graded on a 5-point Likert scale (0=none; 5=severe) according to the Edinburgh Hypoglycemia Symptom Scale: autonomous (sweating, tremors, palpitations, hunger); nervous hypoglycemia (blurred vision, confusion, drowsiness, bizarre behavior, speech difficulties, incoordination, dizziness, inability to concentrate); discomfort (nausea, headache). P-values ​​by paired-two paired-student t-test: *≦0.05;**≦0.01. All baseline studies underestimated the improvement rate (%) because they were stopped at glucose <50 mg / dL followed by administration of IV dextrose. Subjects receiving exendin (9-39) <18 mg required rescue with IV dextrose. [Figure 8C]The images show the improvement in response to the OGTT between baseline and final day with multiple escalating doses of lyophilized exendin (9-39) ("Lyo Ex9-39") reconstituted in saline (green, yellow, orange, red) and a 30 mg buffered exendin (9-39) preparation ("Liq Ex9-39") (blue). Percentage changes (%) (mean ± SEM) are shown for the lowest glucose point (A), insulin peak (B), and hypoglycemia symptom score (C). Symptoms were graded on a 5-point Likert scale (0=none; 5=severe) according to the Edinburgh Hypoglycemia Symptom Scale: autonomous (sweating, tremors, palpitations, hunger); nervous hypoglycemia (blurred vision, confusion, drowsiness, bizarre behavior, speech difficulties, incoordination, dizziness, inability to concentrate); discomfort (nausea, headache). P-values ​​by paired-two paired-student t-test: *≦0.05;**≦0.01. All baseline studies underestimated the improvement rate (%) because they were stopped at glucose <50 mg / dL followed by administration of IV dextrose. Subjects receiving exendin (9-39) <18 mg required rescue with IV dextrose. [Figure 9A] This is the pharmacokinetic profile of formulations containing exendin (9-39). (A) 12-hour pharmacokinetic profiles by dose and formulation on the final day in 19 PBH subjects of multiple escalating doses of lyophilized exendin (9-39) ("Lyo Ex9-39") (red) reconstituted with saline or a 30 mg buffered exendin (9-39) formulation ("Liq Ex9-39") (blue). Plasma concentration (mean ± SEM) as a function of time for each dose (mg). [Figure 9B] This is the pharmacokinetic profile of formulations containing exendin (9-39). (B) Dose and formulation-specific exendin (9-39) concentrations of Lyo Ex 9-39 (red) or Liq Ex 9-39 (blue) in multiple dose escalations in 19 PBH subjects on the final day. Individual plasma AUC concentrations relative to dose (mg / kg). [Figure 10A]This is the 24-hour pharmacokinetic profile of formulations containing exendin (9-39). (A) PK profile after single subcutaneous administration of buffered exendin (9-39) formulations in doses ranging from 7.5 to 90 mg. Plasma concentration (mean) against time for each dose (mg). S1 = Single dose escalation study, 7.5 mg dose; S2 = Single dose escalation study, 15 mg dose; S3 = Single dose escalation study, 30 mg dose; S4 = Single dose escalation study, 45 mg dose; M1 = Multiple dose escalation study, 60 mg dose; M2 = Multiple dose escalation study, 75 mg dose; M3 = Multiple dose escalation study, 90 mg dose. [Figure 10B] This is the 24-hour pharmacokinetic profile of the formulation containing exendin (9-39). (B) Pharmacokinetic profile of the buffered exendin (9-39) formulation after multiple subcutaneous administrations following 30 mg BID administration for 3 days (Stanford MAD study in Example 3) and 60 mg QD administration (Phase 1 study in Example 4). [Modes for carrying out the invention]

[0016] 1.Overview Exendin (9-39) is a glucagon-like peptide-1 (GLP-1) antagonist that selectively blocks GLP-1 receptors present on pancreatic cells, thereby inhibiting GLP-1-mediated increase in insulin secretion. Exendin (9-39), formulated in normal saline (0.9% sodium chloride, also referred to herein as "0.9% normal saline"), has been administered in animal and human clinical trials for the treatment of hyperinsulinic hypoglycemia. However, in the first clinical trials involving subcutaneous injection of exendin (9-39) reconstituted in normal saline, inverse dose linearity was demonstrated with increasing concentration of the injectable solution, suggesting concentration-dependent peptide aggregation and precipitation, with pharmacokinetic exposure and clinical activity decreasing at higher concentrations. See Example 3 of International Publication No. 2016 / 191395, incorporated herein by reference. Furthermore, as described in Examples 1 and 2 of this specification, it has been revealed that exendin (9-39) in normal physiological saline exhibits aggregation that leads to reduced exposure to exendin (9-39) under specific conditions, such as specific storage conditions or specific exendin (9-39) concentrations.

[0017] Accordingly, in one embodiment, the present disclosure provides an improved liquid pharmaceutical formulation of exendin(9-39) that exhibits lower aggregation compared to a composition containing the same concentration of exendin(9-39) formulation in 0.9% normal saline. In another embodiment, the present invention provides an exendin(9-39) formulation that exhibits an improved pharmacokinetic profile compared to a composition containing the same dose of exendin(9-39) formulated in 0.9% normal saline. In some embodiments, as shown, for example, in Example 2, the buffered exendin(9-39) formulation of the present disclosure, when administered to a subject, exhibits a higher C of exendin(9-39) than a composition containing the same dose of exendin(9-39) or a pharmaceutically acceptable salt thereof formulated in 0.9% normal saline. maxThe following is presented. Furthermore, as described in Example 3, the buffered exendin (9-39) formulation of this disclosure was found to provide a longer duration of action and higher pharmacokinetic exposure compared to exendin (9-39) formulated in 0.9% normal saline. Thus, the liquid pharmaceutical formulation of exendin (9-39) described herein also offers the advantage of improved pharmacokinetics. In addition, the liquid pharmaceutical formulation of exendin (9-39) described herein can also support lower doses and / or lower frequency administration for the treatment or prevention of hyperinsulinic hypoglycemia.

[0018] II. Definition The terms used herein are intended to describe, and not limit, specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. In this specification and the subsequent claims, certain terms are defined to have the following meanings unless otherwise clearly intended. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not be construed as representing a substantial difference from the commonly understood definitions of terms in the art.

[0019] Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods and materials are described below. All technical and patent publications referenced herein are incorporated herein by reference in their entirety.

[0020] All numerical values, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximations that may vary (+) or (-) by increments of 0.1 or 1.0 as appropriate (e.g., pH 5.4 or 5.5). While not always explicitly stated, it is understood that all numerical values ​​are preceded by the term "approximately." Ranges indicate the endpoint unless otherwise specified. For example, a dose of exendin (9-39) in the range of 15 mg / ml to 45 mg / ml includes doses of 15 mg / ml or 45 mg / ml.

[0021] The singular forms "a," "an," and "the" refer to multiple objects unless the context clearly requires a different interpretation. Therefore, for example, when we say "one compound," it refers to multiple compounds.

[0022] The term "contains" is intended to mean that the compounds, compositions, and methods include the elements described but do not exclude others. "Consists of" (or "composed of"), when used to define compounds, compositions, and methods, means that it does not exclude other elements that may substantially affect the fundamental and novel features of the claimed invention. "Consists of" (or "composed of") means that any elements, steps, or components not described in the claims are excluded. Embodiments defined by each of these transformation terms are included within the scope of the invention.

[0023] "Excendin (9-39)" or "Ex (9-39)" is C 149 H 234 N 40 O 47S refers to the empirical formula and a 31-amino acid peptide with a molecular weight of 3369.8 Daltons. Exendin (9-39) contains residues 9-39 of the GLP-1 receptor agonist exendin-4 and is a GLP-1 receptor antagonist. See Montrose-Rafizadeh et al., Journal of Biological Chemistry, 272:21201-21206 (1997). The amino acid sequence of exendin (9-39) is as follows: H-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 (SEQ ID NO: 1). Exendin(9-39) has a predicted isoelectric point of 4.69 and a net charge of -1 at pH 6, which increases to a net charge of +4 at pH 3.0. As used herein, the term “Exendin(9-39)” also includes pharmaceutically acceptable salts of exendin(9-39), including, but not limited to, sulfates, hydrochlorides, phosphates, sulfamates, acetates, citrates, lactates, tartrates, methanesulfons, ethanesulfons, benzenesulfons, p-toluenesulfons, cyclohexylsulfamates, and quinates. In some embodiments, exendin(9-39) is in the form of exendin(9-39) acetate or exendin(9-39) trifluoroacetate. Unless otherwise specified herein, exendin(9-39) acetate is used. Exendin (9-39) and its pharmaceutically acceptable salts are commercially available (e.g., Bachem (Clinalfa, Laeufelfingen, Switzerland)).

[0024] As used herein, the term “isotonic agent” refers to a compound or agent that modulates the tonicity (osmotic gradient) of a solution to prevent adverse effects that may occur when a solution with a significantly different tonicity from that of a physiological fluid is administered. In some embodiments, the isotonic agent includes mannitol, dextrose, glycerin, lactose, sucrose, trehalose, or a mixture thereof.

[0025] As used herein, the term “physiologically acceptable buffer” refers to a solution suitable for use in a formulation for administration to a subject and having the effect of maintaining or controlling the pH of the formulation within the pH range required for that formulation. In some embodiments, the physiologically acceptable buffer maintains the pH of the formulation within a pH range of about 5 to about 6. In some embodiments, the physiologically acceptable buffer maintains the pH of the formulation at a pH greater than 5. Acceptable buffers include, but are not limited to, acetate buffers, citrate buffers, phosphate buffers, and mixtures thereof.

[0026] As used herein, the terms “pharmaceutical formulation” or “pharmaceutical formulation” refer to a composition suitable for administration to a subject. Generally, pharmaceutical formulations are sterile and preferably free from contaminants that could induce an undesirable response in the subject (for example, the compounds in the pharmaceutical formulation are pharmaceutical grade). Pharmaceutical formulations can be designed for administration to subjects or patients in need via several different routes of administration, including oral, intravenous, oral cavity, rectal, parenteral, intraperitoneal, intradermal, intramuscular, subcutaneous, and inhalation. In some embodiments, the pharmaceutical formulations described herein are formulated for subcutaneous administration.

[0027] As used herein, “therapeutic dose” is the amount of the active ingredient (e.g., exendin(9-39) or a pharmaceutically acceptable salt thereof) that eliminates, improves, alleviates, reduces or brings about a clinical outcome for the condition to which it is administered.

[0028] The terms “treatment,” “to treat,” and “to treat” as used herein in relation to the administration of exendin (9-39) for the treatment of hyperinsulinic hypoglycemia, encompass all treatments of the disease in human subjects, including: (a) reducing the risk, frequency or severity of hypoglycemic episodes in patients with a history of hyperinsulinic hypoglycemia; (b) reducing the risk of hypoglycemia occurring in subjects judged to be susceptible to the disease, e.g., persons who have undergone bariatric surgery but have not yet been diagnosed with the disease; (c) preventing the onset of the disease; and / or (d) alleviating the disease, i.e., causing disease regression and / or alleviating one or more symptoms of the disease.

[0029] As used herein, the terms “administer,” “dosing,” and “administer” refer to introducing a compound (e.g., exendin (9-39)), composition, or drug into a subject or patient, such as a human. As used herein, this term encompasses both direct administration (e.g., self-administration or administration to a patient by a healthcare professional) and indirect administration (e.g., the act of prescribing a compound or composition to a subject).

[0030] "QD" and "BID" have their usual meanings, respectively, of administering the exendin (9-39) buffer formulation once or twice daily. In some embodiments, once daily (QD) administration means that at least 20 hours, at least 22 hours, or about 24 hours elapse between administrations. In some embodiments, once daily administration means administration every 24 hours. In some embodiments, twice daily (BID) administration means that at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 11 hours, or about 12 hours elapse between administrations. In some embodiments, twice daily administration means administration every 12 hours.

[0031] As used herein, the terms “patient” and “subject” are interchangeable to an individual (e.g., human or non-human mammal) having or susceptible to a condition that can be treated or prevented by administration of the exendin (9-39) formulations described herein. In some embodiments, the patient or subject has hyperinsulinic hypoglycemia. In some embodiments, the patient or subject has previously undergone an obesity procedure (e.g., gastric bypass surgery).

[0032] As used herein, the terms “aggregates,” “aggregation,” and “precipitation” are interchangeable to refer to physical interactions between exendin(9-39) polypeptides in a formulation that result in oligomer formation, thereby causing large aggregates to form and precipitate from the solution. In some embodiments, large exendin(9-39) aggregates may be visible to the naked eye or can be detected using detection methods known in the art, such as an optical microscope. In some embodiments, aggregation by polypeptides may adversely affect the biological activity of the polypeptides, for example, during storage of the formulation, potentially leading to a loss of therapeutic effect of the pharmaceutical formulation. In some embodiments, formulations containing exendin(9-39) as described herein do not exhibit “detectable aggregation,” for example, during storage or administration to a subject, and the aggregates are not visible by optical microscopy (for example, after a period of time such as 24, 36, or 48 hours).

[0033] As used herein, the terms “stored” or “storage” refer to the storage of a formulation, for example, a buffer formulation containing exendin (9-39) or a pharmaceutically acceptable salt thereof as described herein, at a specified temperature for a specified period of time. In some embodiments, the formulation is stored for a long period of time (e.g., one month, two months, three months, four months, five months, six months or longer). In some embodiments, the formulation is stored at a temperature of approximately 5°C, 25°C, 30°C, 37°C, 40°C, or 50°C. In some embodiments, the formulation is stored at a specified temperature (e.g., 50°C) for a specified period of time (e.g., 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or 72 hours) for the purpose of testing one or more properties of the formulation, for example, whether the formulation exhibits agglutination.

[0034] III. Exendin (9-39) preparations In one embodiment, a buffer formulation is provided comprising exendin (9-39) or a pharmaceutically acceptable salt thereof in a physiologically acceptable buffer with a pH greater than 5.0. In some embodiments, a buffer formulation is provided comprising exendin (9-39) or a pharmaceutically acceptable salt thereof in a physiologically acceptable buffer with a pH in the range of about 5 to about 6. In some embodiments, the formulation further comprises an isotonic agent.

[0035] As described herein, it has been found that, surprisingly, certain properties and pharmacokinetic parameters of buffer formulations containing exendin(9-39) or a pharmaceutically acceptable salt thereof can be adjusted by selecting an appropriate exendin(9-39) concentration, isotonic agent, physiologically acceptable buffer, and pH. For example, as described in Example 1 below, buffer formulations containing exendin(9-39) or a pharmaceutically acceptable salt thereof can be improved with respect to properties such as exendin(9-39) aggregation after a predetermined period of storage, the potency of exendin(9-39) after a predetermined period of storage, and the purity of exendin(9-39) after a predetermined period of storage, by selecting an exendin(9-39) concentration, isotonic agent, physiologically acceptable buffer, and pH.

[0036] Furthermore, as described in Examples 2 and 3 below, formulations containing exendin (9-39) in a physiologically acceptable buffer having a pH greater than 5, for example, in the range of approximately 5 to approximately 6, were found to exhibit improved pharmacokinetic properties compared to reconstituted lyophilized exendin (9-39) known in the art. For example, in Example 2, subcutaneous injection of the buffered exendin (9-39) formulation showed higher plasma exendin (9-39) levels compared to a composition containing the same dose of exendin (9-39) or a pharmaceutically acceptable salt thereof formulated in 0.9% normal saline. max This shows that the following was achieved. See Figures 5A and 5B. Example 3 shows that subcutaneous injection of a buffered exendin (9-39) formulation resulted in a higher C of exendin (9-39) compared to reconstituted lyophilized exendin (9-39). max This indicates that a high 12-hour AUC and high trough plasma concentration were achieved.

[0037] Excendin (9-39) In some embodiments, the buffer formulation comprises exendin(9-39). In some embodiments, the formulation comprises a pharmaceutically acceptable salt of exendin(9-39). In some embodiments, the formulation comprises a pharmaceutically acceptable salt, exendin(9-39) acetate, or exendin(9-39) trifluoroacetate.

[0038] In some embodiments, the formulation is approximately 4-90 mg / ml, approximately 4-60 mg / ml, approximately 4-45 mg / ml, approximately 10-90 mg / ml, approximately 10-60 mg / ml, approximately 10-45 mg / ml, approximately 10-40 mg / ml, approximately 10-35 mg / ml, approximately 10-30 mg / ml, approximately 12-25 mg / ml, approximately 12-20 mg / ml, approximately 12-15 mg / ml, approximately 30-90 mg / ml, approximately 30-60 mg / ml, approximately 30-70 mg / ml, approximately 45-90 mg / ml, approximately 45-75 mg / ml, approximately 60-90 mg / ml, or approximately 30-70 mg / ml (for example, approximately 4 mg / ml, approximately 5 mg / ml, approximately 6 mg / ml, approximately 7 mg / ml, approximately The formulation contains exendin (9-39) or a pharmaceutically acceptable salt thereof at a concentration of 8 mg / ml, approximately 9 mg / ml, approximately 10 mg / ml, approximately 11 mg / ml, approximately 12 mg / ml, approximately 13 mg / ml, approximately 14 mg / ml, approximately 15 mg / ml, approximately 16 mg / ml, approximately 17 mg / ml, approximately 18 mg / ml, approximately 19 mg / ml, approximately 20 mg / ml, approximately 25 mg / ml, approximately 30 mg / ml, approximately 35 mg / ml, approximately 40 mg / ml, approximately 45 mg / ml, approximately 50 mg / ml, approximately 55 mg / ml, approximately 60 mg / ml, approximately 65 mg / ml, approximately 70 mg / ml, approximately 75 mg / ml, approximately 80 mg / ml, approximately 85 mg / ml, or approximately 90 mg / ml. In some embodiments, the formulation contains exendin (9-39) at a concentration of approximately 15 mg / ml to approximately 45 mg / ml. In some embodiments, the formulation contains exendin (9-39) at a concentration of about 15 mg / m³. In some embodiments, the formulation contains exendin (9-39) at a concentration ranging from about 25 mg / ml to about 35 mg / ml. In some embodiments, the formulation contains exendin (9-39) at a concentration of about 30 mg / m³. In some embodiments, the formulation contains exendin (9-39) at a concentration ranging from about 40 mg / ml to about 50 mg / ml. In some embodiments, the formulation contains exendin (9-39) at a concentration of about 45 mg / m³. In some embodiments, the formulation contains exendin (9-39) at a concentration ranging from about 30 mg / ml to about 60 mg / ml. In some embodiments, the formulation contains exendin (9-39) at a concentration ranging from about 30 mg / ml to about 90 mg / ml.In some embodiments, the formulation contains exendin(9-39) at a concentration ranging from about 45 mg / ml to about 90 mg / ml. In some embodiments, the formulation contains exendin(9-39) at a concentration of about 60 mg / ml. In some embodiments, the formulation contains exendin(9-39) at a concentration of about 75 mg / ml.

[0039] Physiologically acceptable buffer In some embodiments, the buffer formulation contains exendin(9-39) or a pharmaceutically acceptable salt thereof in a physiologically acceptable buffer having a pH in the range of about 5 to about 6. In some embodiments, the buffer is suitable for subcutaneous administration. In some embodiments, the physiologically acceptable buffer is a buffer that yields a liquid formulation having a pH at or near the physiological pH, or within a relatively narrow pH range near the physiological pH (e.g., about 5.0 to about 8.0). In some embodiments, the physiologically acceptable buffer has a pH that prevents, limits, or reduces the formation of exendin(9-39) aggregates in the liquid pharmaceutical formulation during storage or administration to a subject. In some embodiments, the physiologically acceptable buffer has a pH greater than 5.0.

[0040] In one embodiment, the physiologically acceptable buffer comprises a solution having a stable pH over a long period of time (e.g., about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 12 hours, about 3 days, about 5 days, about 7 days, about 10 days, about 14 days, about 1 month or longer). In some embodiments, the physiologically acceptable buffer comprises a solution that stabilizes the functionality of exendin (9-39) during long-term storage. In one embodiment, storage may include about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 12 hours, about 3 days, about 5 days, about 7 days, about 10 days, about 14 days, about 1 month or longer.

[0041] In one embodiment, the buffer formulation includes a physiologically acceptable buffer having a pH in the range of about 5 to about 6 (e.g., a range including 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, and 6.0). In one embodiment, the buffer formulation includes a physiologically acceptable buffer having a pH greater than about 5.0 to about 6. In one embodiment, the physiologically acceptable buffer has a pH greater than about 5.0 and up to about 5.5. In one embodiment, the physiologically acceptable buffer has a pH in the range of about 5.2 to 5.8 (e.g., 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, or 5.8). In one embodiment, the physiologically acceptable buffer has a pH in the range of about 5.0 to 5.5 (e.g., 5.1, 5.2, 5.3, 5.4, or 5.5). In one embodiment, the physiologically acceptable buffer has a pH in the range of about 5.5 to about 6. In one embodiment, the physiologically acceptable buffer has a pH of about 5.5.

[0042] In one embodiment, the physiologically acceptable buffer comprises an acetate buffer, a citrate buffer, a phosphate buffer, a histidine buffer, or a mixture thereof. In one embodiment, the physiologically acceptable buffer comprises sodium acetate, potassium acetate, trisodium citrate, magnesium citrate, potassium citrate, potassium phosphate, or a mixture thereof. In one embodiment, the physiologically acceptable buffer comprises a buffer (e.g., sodium acetate) at concentrations of about 5 mM to about 30 mM, about 10 mM to about 30 mM, about 15 mM to about 30 mM, about 20 mM to about 30 mM, or about 25 mM to about 30 mM (e.g., about 5 mM, about 8 mM, about 10 mM, about 12 mM, about 15 mM, about 18 mM, about 20 mM, about 22 mM, about 25 mM, about 28 mM, or about 30 mM). In some embodiments, the physiologically acceptable buffer contains a buffer (e.g., sodium acetate) at a concentration of at least 10 mM.

[0043] In some embodiments, the physiologically acceptable buffer contains an acetate buffer. In some embodiments, the buffer is sodium acetate. In some embodiments, the buffer is potassium acetate. In some embodiments, the physiologically acceptable buffer contains an acetate buffer (e.g., sodium acetate or potassium acetate) at a concentration of about 5 mM to about 30 mM, for example, about 10 mM to about 20 mM. In some embodiments, the physiologically acceptable buffer contains an acetate buffer (e.g., sodium acetate or potassium acetate) at a concentration of about 10 mM.

[0044] In some embodiments, the physiologically acceptable buffer contains a citrate buffer. In some embodiments, the buffer is trisodium citrate. In some embodiments, the buffer is magnesium citrate. In some embodiments, the buffer is potassium citrate. In some embodiments, the physiologically acceptable buffer contains a citrate buffer (e.g., sodium citrate, magnesium citrate, or potassium citrate) at a concentration of about 5 mM to about 30 mM, for example, about 10 mM to about 20 mM. In some embodiments, the physiologically acceptable buffer contains a citrate buffer (e.g., sodium citrate, magnesium citrate, or potassium citrate) at a concentration of about 10 mM.

[0045] In one embodiment, the physiologically acceptable buffer contains a phosphate buffer. In some embodiments, the physiologically acceptable buffer contains potassium phosphate. In some embodiments, the physiologically acceptable buffer contains potassium phosphate at a concentration of about 5 mM to about 30 mM, for example, about 10 mM to about 20 mM. In some embodiments, the physiologically acceptable buffer contains a phosphate buffer (e.g., potassium phosphate) at a concentration of about 10 mM.

[0046] Isotonic agent In some embodiments, the buffering formulation includes an isotonic agent. In some embodiments, the isotonic agent includes mannitol, dextrose, glycerin, lactose, sucrose, trehalose, or a mixture thereof. In some embodiments, the isotonic agent is mannitol. The use of isotonic agents is well known in the medical field, and those skilled in the art will be able to use one or more of the isotonic agents disclosed herein to provide liquid pharmaceutical formulations suitable for subcutaneous administration. See, for example, Pramanick et al., Pharma Times, Vol 45, No. 3, (2013); and also see Formulating Poorly Water Soluble Drugs, Williams, Watts, and Miller, eds., Springer Science and Business Media (2011).

[0047] In some embodiments, the isotonic agent or combination of isotonic agents is approximately 20-75 mg / ml, approximately 20-60 mg / ml, approximately 25-55 mg / ml, approximately 30-75 mg / ml, approximately 30-50 mg / ml, approximately 35-45 mg / ml, approximately 40-45 mg / ml, approximately 45-75 mg / ml, or approximately 45-60 mg / ml (for example, approximately 20 mg / ml, approximately 22 mg / ml, approximately 25 mg / ml, approximately It is present in the formulation at concentrations of approximately 28 mg / ml, approximately 30 mg / ml, approximately 32 mg / ml, approximately 35 mg / ml, approximately 38 mg / ml, approximately 40 mg / ml, approximately 42 mg / ml, approximately 45 mg / ml, approximately 48 mg / ml, approximately 50 mg / ml, approximately 52 mg / ml, approximately 55 mg / ml, approximately 58 mg / ml, approximately 60 mg / ml, approximately 65 mg / ml, approximately 70 mg / ml, or approximately 75 mg / ml. In some embodiments, the formulation contains an isotonic agent in a concentration range of approximately 30 to approximately 60 mg / ml.

[0048] In some embodiments, an isotonic agent or combination of isotonic agents is present in the formulation in an amount that results in a formulation having an isophysiological osmotic pressure. In some embodiments, an isotonic agent or combination of isotonic agents is present in the formulation in an amount that results in a formulation having an osmotic pressure of about 275–300 mOsm / kg (e.g., about 275 mOsm / kg, about 280 mOsm / kg, about 285 mOsm / kg, about 290 mOsm / kg, about 295 mOsm / kg, or about 300 mOsm / kg). In some embodiments, an isotonic agent or combination of isotonic agents (e.g., mannitol, dextrose, glycerin, lactose, sucrose, trehalose, or a combination thereof) is present in the formulation in an amount that results in a formulation having an osmotic pressure of about 290 mOsm / kg.

[0049] In some embodiments, the isotonic agent includes mannitol. In some embodiments, mannitol is present at a concentration of about 40–50 mg / ml. In some embodiments, mannitol is present at a concentration in the range of about 40 mg / ml to about 45 mg / ml. In some embodiments, mannitol is present at a concentration of about 45 mg / ml. In some embodiments, mannitol is present at a concentration of at least 45 mg / ml.

[0050] In one embodiment, the isotonic agent includes dextrose. In one embodiment, the dextrose is present at a concentration of about 20 mg / ml to about 60 mg / ml (for example, about 20 mg / ml, about 40 mg / ml, about 45 mg / ml, or about 60 mg / ml).

[0051] In one embodiment, the isotonic agent includes glycerin. In some embodiments, glycerin is present at a concentration of about 20 mg / ml to about 60 mg / ml (for example, about 20 mg / ml, about 40 mg / ml, about 45 mg / ml, or about 60 mg / ml).

[0052] In one embodiment, the isotonic agent includes lactose. In one embodiment, lactose is present at a concentration of about 20 mg / ml to about 60 mg / ml (for example, about 20 mg / ml, about 40 mg / ml, about 45 mg / ml, or about 60 mg / ml).

[0053] In one embodiment, the isotonic agent includes sucrose. In some embodiments, sucrose is present at a concentration of about 20 mg / ml to about 60 mg / ml (for example, about 20 mg / ml, about 40 mg / ml, about 45 mg / ml, or about 60 mg / ml).

[0054] In one embodiment, the isotonic agent includes trehalose. In one embodiment, the trehalose is present at a concentration of about 20 mg / ml to about 60 mg / ml (for example, about 20 mg / ml, about 40 mg / ml, about 45 mg / ml, or about 60 mg / ml).

[0055] In some embodiments, the buffering formulation comprises two or more isotonic agents. In some embodiments, the buffering formulation comprises two or more isotonic agents selected from the group consisting of mannitol, dextrose, glycerin, lactose, sucrose, and trehalose. In some embodiments, the buffering formulation comprises mannitol and at least one other isotonic agent.

[0056] Another excipient In some embodiments, the formulation further includes one or more other excipients, such as preservatives, surfactants (e.g., polysorbates or polyoxomers), or colorants (e.g., pharmaceutically acceptable dyes, inorganic pigments, and natural colorants). A wide variety of pharmaceutically acceptable excipients are known in the art. Pharmaceutically acceptable excipients are described in detail in various publications, such as A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. Ansel et al., eds., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc., each of which is incorporated herein by reference.

[0057] IV. Treatment method In another embodiment, a therapeutic method is provided comprising the step of administering a buffer formulation containing exendin(9-39) or a pharmaceutically acceptable salt thereof as described herein. In some embodiments, the method comprises the step of administering a buffer formulation of exendin(9-39) as described herein in an amount effective in preventing or reducing the symptoms of hyperinsulinic hypoglycemia. In some embodiments, the method comprises the step of administering a buffer formulation of exendin(9-39) as described herein in an amount effective in preventing or reducing the symptoms, metabolic outcomes, and / or clinical outcomes of hypoglycemia after bariatric surgery.

[0058] Patient group In some embodiments, the subjects to be treated according to the methods described herein are subjects with hyperinsulinic hypoglycemia (HH). In certain embodiments, subjects with hyperinsulinic hypoglycemia have previously undergone bariatric surgery (e.g., Roux-en-Y gastric bypass) and / or related metabolic procedures. In certain embodiments, subjects who have previously undergone bariatric surgery (e.g., Roux-en-Y gastric bypass) and / or related metabolic procedures are at risk of developing hyperinsulinic hypoglycemia. In some embodiments, subjects with hyperinsulinic hypoglycemia have previously undergone upper gastrointestinal procedures such as gastrectomy or esophagectomy.

[0059] As used herein, “hyperinsulinic hypoglycemia” includes dumping syndrome, late dumping syndrome, islet cell disease, non-insulinoma hypoglycemia (NIPHS), and / or postprandial reactive hypoglycemia. Hyperinsulinic hypoglycemia may be caused by gastric, obese, or metabolic procedures such as Roux-en-Y gastric bypass (RYGB) or vertical sleeve gastrectomy (VSG), or may have congenital, acquired, or induced causes.

[0060] Subjects with hyperinsulinic hypoglycemia can be identified by any preferred method. In some embodiments, hyperinsulinic hypoglycemia is diagnosed by the presence of Whipple's triad, which has the following criteria: (1) the occurrence of symptoms of hypoglycemia; (2) low plasma glucose levels recorded for the type of symptoms; and (3) the fractionation of symptoms after an increase in plasma glucose. In some embodiments, hyperinsulinic hypoglycemia is defined by the occurrence of tubular glucose ≤ 50 mg / dL at least once a month, as reported by the subject or recorded in medical records. In some embodiments, hyperinsulinic hypoglycemia is defined by a plasma glucose concentration < 54 mg / dL detected by self-monitoring of plasma glucose, continuous glucose monitoring for at least 20 minutes, or laboratory measurement of plasma glucose. In some embodiments, hyperinsulinic hypoglycemia is defined by a plasma glucose concentration of ≤55 mg / dL during an oral glucose tolerance test or meal tolerance test, associated with an inappropriately elevated plasma insulin (≥3 uU / mL) or c-peptide (>0.3 mg / dL) when glucose is ≤55 mg / dL. In some embodiments, hyperinsulinic hypoglycemia is defined by a plasma glucose concentration of ≤60 mg / dL during an oral glucose tolerance test or meal tolerance test, associated with an inappropriately elevated plasma insulin (≥3 uU / mL) or c-peptide (>0.3 mg / dL) when glucose is ≤60 mg / dL. In some embodiments, hyperinsulinic hypoglycemia is diagnosed by a provocation test, such as an oral glucose tolerance test (OGTT) or a mixed meal tolerance test (MMTT). See also Eisenberg et al., Surgery for Obesity and Related Diseases, 2017, 13:371-378; and Diabetes Care, 2016, doi:10.2337 / dc16-2215.

[0061] In one embodiment, the patient to be treated has previously undergone bariatric surgery and / or related metabolic procedures, such as Roux-en-Y gastric bypass. Bariatric surgery and / or related metabolic procedures include, but are not limited to, Roux-en-Y gastric bypass, vertical sleeve gastrectomy, placement of an endo-sleeve device, such as the EndoBarrier Gastrointestinal Liner System, also known as an "intracavitary liner," duodenal mucosal ablation, partial duodenal bypass with duodenoileal or duodenojejunal anastomosis, vagus nerve block and / or pyloroplasty.

[0062] Bariatric treatment (i.e., bariatric surgery) typically involves one of the following: partial or complete bypass formation of the duodenum and / or reduction of nutrient exposure to the duodenum; increased speed of nutrient transport to the lower part of the intestine (often particularly the ileum); and / or otherwise increased nutrient exposure to the ileum. Bariatric surgery may aim at weight loss, metabolic benefits (e.g., diabetes relief), or both. Such weight loss or metabolic treatments, referred herein as “bariatric treatments,” may increase the secretion of GLP-1 from the distal small intestine, particularly the ileum, which can lead to increased insulin secretion and, in some subjects, hypoglycemia. In some embodiments, subjects may also be referred to as “post-bariatric surgery” patients or “post-RYGB.”

[0063] In another embodiment, the subject to be treated has previously undergone a relevant metabolic procedure. For example, in one embodiment, the subject to be treated has previously undergone non-obesity surgery relating to the gastrointestinal tract (including, but not limited to, esophagectomy for the treatment of esophageal cancer, Nissen fundoplication for the treatment of gastroesophageal reflux disease, or gastrectomy for the treatment or prevention of gastric cancer), and is therefore sometimes referred to herein as “post-gastrointestinal surgery.”

[0064] In another embodiment, the subject to be treated is prediabetes and / or insulin resistance and may benefit from the prevention of pancreatic overstimulation caused by oral intake of carbohydrates leading to postprandial hypoglycemia. In yet another embodiment, the subject to be treated has congenital, acquired, or inducible hyperinsulinic hypoglycemia, such as congenital hyperinsulinism (or sometimes also called congenital islet cell dystrophy).

[0065] A suitable patient population and a method for identifying patients are also described in PCT Patent Application No. PCT / US2016 / 033837, which is incorporated herein by reference.

[0066] In some embodiments, the patient is a human patient. In some embodiments, the patient is an adult. In some embodiments, the patient is a young adult. In some embodiments, the patient is an adult who has previously undergone an obesity treatment (e.g., gastric bypass surgery).

[0067] Route of administration and administration regimen In some embodiments, the buffer formulation containing exendin (9-39) described herein is administered subcutaneously (e.g., by subcutaneous injection). The injection site is not limited to, but may include, the thigh, abdomen, upper arm region, or upper buttock region.

[0068] In some embodiments, the buffered exendin (9-39) formulation of the present disclosure is formulated for subcutaneous administration. In one embodiment, the buffered exendin (9-39) formulation of the present invention is formulated for subcutaneous administration according to a once-daily (QD) or twice-daily (BID) regime.

[0069] Injectable solution In some embodiments, the buffered exendin(9-39) formulation is formulated as single-use prefill syringes in a kit containing, for example, a number of single-use prefill syringes (e.g., 10, 20, 30, 40, 50, or 60 prefill syringes). In some embodiments, the single-use prefill syringe contains an exendin(9-39) liquid pharmaceutical formulation comprising approximately 5 to 75 mg of exendin(9-39), an isotonic agent, and a buffer having a pH in the range of 5.0 to 6.0.

[0070] In some embodiments, the buffered exendin (9-39) formulation is formulated as an isotonic solution stored in sterile, multi-dose glass vials or ampoules for administration using a syringe, similar to a glucagon emergency kit. In some embodiments, the buffered exendin (9-39) formulation is provided as an injectable solution in a single-dose tray containing vials, vial connectors, syringes, and one or more needles of the buffered exendin (9-39) formulation described herein (e.g., a formulation comprising about 5 to 75 mg of exendin (9-39), an isotonic agent, and a buffer having a pH of about 5.5, and optionally an appropriate amount of antimicrobial preservative).

[0071] In some embodiments, the buffered exendin (9-39) formulation is formulated as an isotonic solution that is sterile and stored in a glass cartridge pen-type syringe device. In non-limiting examples, the formulation comprises about 5 to 75 mg of exendin (9-39) (e.g., about 5 to 45 mg or about 30 to 75 mg of exendin (9-39)), an isotonic agent, and a buffer having a pH in the range of about 5.5, and optionally an appropriate amount of antimicrobial preservative.

[0072] In some embodiments, each dose is administered as a total volume of injection solution ranging from 0.25 to 2 ml, while most subjects receive injection volumes ranging from 0.5 to 1.5 ml, for example, from 0.7 to 1 ml.

[0073] Treatment parameters In some embodiments, a composition comprising a therapeutically effective amount of a liquid pharmaceutical formulation containing exendin (9-39) or a pharmaceutically acceptable salt thereof and an isotonic agent in a physiologically acceptable buffer having a pH in the range of about 5 to about 6 is administered to a subject in need for the treatment or prevention of hyperinsulinic hypoglycemia.

[0074] In some embodiments, the method includes administering (e.g., subcutaneously) a therapeutically effective amount of exendin (9-39) or a pharmaceutically acceptable salt thereof and an isotonic agent in a physiologically acceptable buffer having a pH in the range of about 5 to about 6 to a subject requiring it. In some embodiments, a therapeutically effective dose of exendin (9-39) (or a pharmaceutically acceptable salt thereof) is in the range of about 15 mg / ml to about 180 mg / ml, for example, about 15 mg / ml to about 60 mg / ml, about 18 mg / ml to about 50 mg / ml, about 20 mg / ml to about 30 mg / ml, about 30 mg / ml to about 60 mg / ml, about 30 mg / ml to about 45 mg / ml, about 45 mg / ml to about 90 mg / ml, about 45 mg / ml to about 60 mg / ml, about 30 mg / ml to about 180 mg / ml, about 30 mg / ml to about 150 mg / ml, about 30 mg / ml to about 90 mg / ml, about 30 mg / ml to about 120 mg / ml, about 45 mg / ml to about 150 mg / ml, or about 60 mg / ml to about 180 mg / ml. In some embodiments, a therapeutically effective dose of exendin (9-39) (or a pharmaceutically acceptable salt thereof) is approximately 15 mg / ml, approximately 18 mg / ml, approximately 20 mg / ml, approximately 25 mg / ml, approximately 30 mg / ml, approximately 35 mg / ml, approximately 40 mg / ml, approximately 42 mg / ml, approximately 45 mg / ml, approximately 48 mg / ml, approximately 50 mg / ml, approximately 52 mg / ml, approximately 55 mg / ml, approximately 58 mg / ml, approximately 60 mg / ml, approximately 65 mg / ml, approximately 70 mg / ml, approximately 75 mg / ml, approximately 80 mg / ml, approximately 85 mg / ml, or approximately 90 mg / ml.

[0075] In some embodiments, the method includes administering (e.g., subcutaneously) an exendin (9-39) buffer formulation described herein in a total daily dose of about 10 mg to about 90 mg of exendin (9-39), for example, about 10 mg to about 75 mg, about 10 mg to about 60 mg, about 15 mg to about 90 mg, about 15 mg to about 75 mg, about 15 mg to about 60 mg, about 20 mg to about 90 mg, about 20 mg to about 75 mg, about 25 mg to about 75 mg, about 25 mg to about 60 mg, about 30 mg to about 90 mg, about 30 mg to about 75 mg, about 30 mg to about 60 mg, or about 40 mg to about 90 mg of exendin (9-39). In some embodiments, the buffering agent is administered in a total daily dose of at least about 20 mg, at least about 30 mg, at least about 40 mg, at least about 50 mg, or at least about 60 mg of exendin (9-39). In some embodiments, the buffering agent is administered in a total daily dose of about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, or about 90 mg of exendin (9-39).

[0076] The dose ranges described above are exemplary adult doses and, as is well known to those skilled in the field of pharmaceutical formulation, may vary depending on the patient's age and weight. It should be understood that in some embodiments, the dose may be increased or decreased during the course of treatment. For example, some physicians may choose to treat with a low or initial dose and gradually increase it to a higher dose if the initial dose does not provide sufficient therapeutic benefit, or maintain the initial dose if it does provide sufficient therapeutic benefit.

[0077] In some embodiments, a therapeutically effective dose of exendin (9-39) (or a pharmaceutically acceptable salt thereof) is administered once daily (QD) as a buffer formulation. QD administration is well known in the medical field. In some embodiments, the QD dose is administered at intervals of about 24 hours (e.g., 7 a.m. daily) (e.g., self-administered). However, shorter (e.g., 8 a.m. and 6 a.m. daily) or longer (e.g., 7 a.m. and 9 a.m. daily) administration intervals are also possible, provided that administrations are given at least about 18 hours apart. Preferably, administrations are given at intervals of at least about 20, 21, 22, 23, or 24 hours. Preferably, administrations are given at intervals of 30 hours or less. Once daily administration is preferred, but the dose may be administered more frequently (e.g., twice daily) or less frequently (e.g., every other day).

[0078] In some embodiments, the buffer formulation is administered twice daily (BID). BID (twice daily) administration is well known in the medical field. The buffer formulation can be administered at specific points in the day or schedule of the subject, for example, in the morning, afternoon, evening, night, before or during or after meals, or before bedtime. In some embodiments, the liquid pharmaceutical formulation is administered approximately once every 12 hours. In some embodiments, the BID dose is administered (e.g., self-administered) at intervals of about 12 hours (e.g., 7 a.m. and 7 p.m.). However, shorter (e.g., 8 a.m. and 6 p.m.) or longer (e.g., 7 a.m. and 10 p.m.) administration intervals are also possible. In some embodiments, administrations are spaced at least 4, 6, 7, 8, 9, 10, or 11 hours apart. Preferably, administrations are spaced at intervals of 15 hours or less. A method for determining the timing of BID dose administration is described, for example, in PCT Patent Application No. PCT / US2016 / 033837, which is incorporated herein by reference.

[0079] In some embodiments, the buffer formulation is administered (e.g., subcutaneously) in doses of exendin (9-39) ranging from 5 mg to 30 mg BID or 10 mg to 45 mg BID, for example, about 7.5 to 30 mg BID, about 10 to 30 mg BID, about 15 to 45 mg BID, about 25 to 45 mg BID, or about 30 to 45 mg BID. In some embodiments, the buffer formulation is administered in a dose of about 7.5 mg BID. In some embodiments, the buffer formulation is administered in a dose of about 10 mg BID. In some embodiments, the buffer formulation is administered in a dose of about 15 mg BID. In some embodiments, the buffer formulation is administered in a dose of about 20 mg BID. In some embodiments, the buffer formulation is administered in a dose of about 30 mg BID. In some embodiments, the buffer formulation is administered in a dose of about 45 mg BID. In some embodiments, a buffer formulation containing exendin (9-39) at a concentration of 30 mg / ml or higher is administered in doses of approximately 30-45 mg BID, for example, in doses of approximately 30 mg BID.

[0080] In some embodiments, the buffer formulation is administered (e.g., subcutaneously) in doses of exendin (9-39) ranging from 20 mg to 90 mg QD, for example, about 30 to 90 mg QD, about 30 to 75 mg QD, about 45 to 90 mg QD, or about 45 to 75 mg QD. In some embodiments, the buffer formulation is administered in a dose of about 20 mg QD. In some embodiments, the buffer formulation is administered in a dose of about 30 mg QD. In some embodiments, the buffer formulation is administered in a dose of about 45 mg QD. In some embodiments, the buffer formulation is administered in a dose of about 60 mg QD. In some embodiments, the buffer formulation is administered in a dose of about 75 mg QD. In some embodiments, the buffer formulation is administered in a dose of about 90 mg QD. In some embodiments, a buffer formulation containing exendin (9-39) at a concentration of 60 mg / ml or higher is administered in doses of at least 45 mg QD, for example, 45-90 mg QD, about 60 mg QD, or about 75 mg QD.

[0081] In some embodiments, the buffer preparation is administered twice daily (BID) within approximately 60 minutes before breakfast and dinner (or before the two main meals of the day) (e.g., subcutaneously). In some embodiments, the administration before breakfast and dinner (or before the two main meals of the day) is spaced at least approximately 6 hours apart. In some embodiments, the administration of the buffer preparation is not timed with meals.

[0082] In some embodiments, the buffer formulation is administered once daily (QD) in the morning or evening (e.g., subcutaneously) to provide maximum coverage for breakfast and dinner. For example, in some embodiments, the formulation is administered at night, after dinner, or early in the morning, before breakfast (e.g., at least 60 minutes before breakfast).

[0083] In some embodiments, the buffer formulation is administered twice daily (BID) in different doses (e.g., subcutaneously). In some embodiments, the formulation is administered in different doses in the morning and afternoon. In some embodiments, the formulation is administered in different doses in the morning and evening or night. For example, in some embodiments, the formulation is administered in the morning and evening or night, in which case the evening or night dose is lower than the morning dose.

[0084] Patients receiving the buffer formulations containing exendin (9-39) described herein may receive treatment for a planned period, an unspecified period, or until an endpoint is reached. Treatment may be continued continuously on a daily or weekly basis for at least two to three months, six months, one year, or longer. In some embodiments, treatment may last for at least 30 days, at least 60 days, at least 90 days, at least 120 days, at least 150 days, or at least 180 days. In some embodiments, treatment may continue for at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, or at least one year. In some embodiments, treatment may continue for the lifetime of the patient or until administration is no longer effective in providing a meaningful therapeutic benefit.

[0085] Improvement of pharmacokinetic properties In some embodiments, a buffered liquid formulation comprising exendin(9-39) or a pharmaceutically acceptable salt thereof and an isotonicity agent, in a physiologically acceptable buffer having a pH in the range of about 5 to about 6, results in an improved absorption profile of exendin(9-39) as compared to a composition comprising the same dose of exendin(9-39) or a pharmaceutically acceptable salt thereof formulated in 0.9% normal saline. In some embodiments, the buffered liquid formulation results in a higher plasma concentration of exendin(9-39) over about 1 to 6 hours (e.g., about 1 hour, about 2 hours, about 3 hours, or about 4 hours, over 1 to 4 hours) after administration to a subject as compared to the plasma concentration of exendin(9-39) over the same time period in a subject administered the same dose of exendin(9-39) formulated in 0.9% normal saline.

[0086] In some embodiments, the buffered liquid exendin(9-39) formulation of the present disclosure results in a greater increase in the plasma concentration of exendin(9-39) over about 1 to 12 hours (e.g., about 1 hour, about 2 hours, about 3 hours, or about 4 hours, over 1 to 10 hours, 6 to 12 hours, 1 to 8 hours, or 4 to 12 hours) after administration to a subject as compared to the increase in plasma concentration over the same time period in a subject administered a composition comprising the same dose of exendin(9-39) formulated in 0.9% normal saline. In some embodiments, the change in the plasma concentration of exendin(9-39) is measured about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours after administering the exendin(9-39) formulation to the subject.

[0087] In some embodiments, subcutaneous injection of the buffered liquid formulation comprising exendin(9-39) described herein results in a higher C max of exendin(9-39) as compared to a composition comprising the same dose of exendin(9-39) formulated in 0.9% normal saline.

[0088] In some embodiments, subcutaneous injection of a buffer formulation containing exendin (9-39) as described herein results in a higher AUC of exendin (9-39) compared to a composition containing the same dose of exendin (9-39) formulated in 0.9% normal saline. For example, in some embodiments, subcutaneous injection of a buffer formulation containing exendin (9-39) as described herein results in a higher 12-hour AUC of exendin (9-39) compared to a composition containing the same dose of exendin (9-39) formulated in 0.9% normal saline.

[0089] In some embodiments, subcutaneous injection of a buffer formulation containing exendin (9-39) as described herein provides a slower T of exendin (9-39) compared to a composition containing the same dose of exendin (9-39) formulated in 0.9% normal saline. max It brings about.

[0090] In some embodiments, subcutaneous injection of the buffer formulation containing exendin(9-39) described herein results in a higher trough concentration of exendin(9-39) compared to a composition containing the same dose of exendin(9-39) formulated in 0.9% normal saline (for example, when measured after repeated administration such as administration for at least 3 days, at least 1 week, at least 2 weeks, or at least 1 month). [Examples]

[0091] V. Examples The following examples are provided for illustrative purposes only and are not intended to limit the claimed invention.

[0092] Example 1: Characteristics of exendin (9-39) reconstituted in 0.9% normal physiological saline and buffered exendin (9-39) formulations. This example describes the physical and chemical properties of lyophilized exendin (9-39) acetate reconstituted in 0.9% normal physiological saline, compared to the properties of an exendin (9-39) buffering preparation.

[0093] Characteristics of exendin (9-39) formulated in normal physiological saline Lyophilized exendin (9-39) acetate at various concentrations (5, 10.24, 15.3, 25.22, and 44.99 mg / ml) was prepared by reconstitution in 0.9% normal physiological saline and stored at 5°C. The pH of each reconstituted exendin (9-39) preparation was recorded on day 0 and monitored for up to 14 days. All concentrations of the reconstituted exendin (9-39) preparations yielded an initial pH of 4.4–4.5. Visual inspection of each reconstituted exendin (9-39) preparation was also recorded on day 0 and monitored for up to 14 days while maintained at 5°C. The results are shown in Table 1 below. No aggregation, gelation, or precipitation of the reconstituted exendin (9-39) preparations was observed in any of the samples maintained at 5°C for 14 days.

[0094] [Table 1]

[0095] Aliquots of reconstituted exendin (9-39) formulations (5, 10.24, 15.3, 25.22, and 44.99 mg / ml) maintained at 5°C for 7 days were left overnight at 50°C. All reconstituted exendin (9-39) formulations showed aggregation within 24 hours at 50°C, and the aggregates appeared gelatinous under a light microscope (Figure 1, left panel). After 36 hours at 50°C, the reconstituted exendin (9-39) formulations aggregated further (Figure 1, right panel).

[0096] To further investigate the temperature at which the reconstituted exendin (9-39) formulation in 0.9% physiological saline begins to precipitate and / or aggregate, aliquots of 15 mg / ml reconstituted exendin (9-39) formulation were stored overnight at 5°C, 25°C, 30°C, 37°C, and 40°C. As shown in Table 2 below, visual inspection of the reconstituted exendin (9-39) formulations revealed that none of the 15 mg / ml reconstituted exendin (9-39) formulations stored overnight up to 40°C precipitated from the solution.

[0097] [Table 2]

[0098] Characteristics of the buffer formulation of exendin (9-39) Exendin (9-39) acetate was formulated in one of two ion buffers (acetate buffer or citrate buffer) with varying ionic strengths (10 mM to 30 mM) and different pH levels (pH 3.5 to 6.0). Each sample was stored at 5°C or 50°C for up to 5 days. The formulations were visually inspected for aggregation, and their purity and potency were tested by strong cation exchange (SCX)-HPLC.

[0099] As shown in Table 3 below, none of the tested buffered exendin (9-39) formulations precipitated from the solution at 5°C. Within 48 hours at 50°C, the formulations with pH 4.5 and 5.0 showed visible agglutination, but none of the other formulations showed agglutination.

[0100] [Table 3]

[0101] The purity and potency of the formulations were analyzed over a 5-day period using strong cation exchange-high-performance liquid chromatography (SCX-HPLC). The effects of storage at 50°C for 5 days on the purity and potency of the exendin (9-39) buffer formulations are shown in Figures 2A and 2B.

[0102] As shown in Figure 2A and Table 4 below, citrate and acetate buffers were equivalent in terms of exendin (9-39) efficacy at the end of a 5-day storage period. The smallest exendin (9-39) loss (i.e., the last effect on efficacy) was observed at pH 3.5 and pH 6.0. Higher buffering strengths were accompanied by more exendin (9-39) loss at the end of the 5-day period. As shown in Figure 2B and Table 4, the smallest purity loss was observed for the pH 4.0 formulation, while the largest purity loss was observed for the pH 6.0 formulation. Purity loss increased with increasing ion buffering strength (e.g., from 10 mM to 20 mM or 30 mM). Citrate and acetate buffers exhibited equivalent activity.

[0103] [Table 4]

[0104] Figure 3 shows the efficacy and purity of exendin (9-39) formulated in 10 mM acetate buffer at various pH levels after storage at 50°C for 5 days. As shown in Figure 3, the minimum decrease in exendin (9-39) was observed at pH levels below 4.0 or above 5.5. The loss of efficacy was greatest when aggregation was observed (pH 4.5 and 5.0).

[0105] The contribution of purity to pH was plotted for exendin (9-39) formulated in 10 mM acetate buffer. Figure 4 shows the effect of pH on purity, which was measured as the % area contribution.

[0106] While not intended to be bound by any particular theory, it has been suggested that it is advantageous to formulate exendin(9-39) in a buffer solution above its predicted isoelectric point (pH 4.7). This is because, once the formulation is administered (e.g., by injection), it does not pass through the isoelectric pH during its transition to the physiological pH of approximately 7.4. Furthermore, formulation of exendin(9-39) in a buffer solution of this pH is expected to result in improved properties such as increased potency, chemical stability, and reduced susceptibility to aggregation. In contrast, formulating exendin(9-39) in typical physiological saline, which usually has a pH of approximately 4.5, may increase the likelihood of precipitation or aggregation because the formulation must pass through the isoelectric pH of exendin(9-39) on its way to the physiological pH.

[0107] Example 2: Plasma concentration profile of buffered exendin (9-39) formulation This example demonstrates that exendin(9-39) formulated in a buffer with a pH in the range of approximately 5–6 exhibits improved pharmacokinetic properties compared to exendin(9-39) reconstituted in normal physiological saline. For this example, a dose of 7.2 mg of exendin(9-39) acetate (equivalent to a 30 mg dose in humans) was formulated in 10 mM sodium acetate and 45 mg / ml mannitol. The formulation had a pH of 5.5. The formulation was administered to dogs (3 male dogs per group) by subcutaneous injection at one of three concentrations: 15 mg / ml, 30 mg / ml, or 45 mg / ml. Plasma concentrations of exendin(9-39) were monitored over a period of 0–24 hours after administration (Figure 5B).

[0108] As a control, a 7.2 mg dose of exendin(9-39) acetate (equivalent to a 30 mg dose in humans) was reconstituted in 0.9% normal saline with a pH of approximately 4.5 and administered subcutaneously to two dogs (one male and one female). Figure 5A shows the plasma concentration profiles obtained after administration of exendin(9-39) reconstituted in 0.9% normal saline.

[0109] As can be seen from the comparison of Figures 5A and 5B, the buffer formulation (containing exendin (9-39) and 45 mg / ml mannitol in 10 mM sodium acetate with a pH of 5.5) resulted in higher plasma concentrations of exendin (9-39) over the first 4 hours after administration, compared to lyophilized exendin (9-39) reconstituted in normal saline, at all concentrations tested.

[0110] Example 3: Repeated subcutaneous administration of exendin 9-39 reduces hyperinsulinic hypoglycemia and neurohypoglycemic symptoms in patients with hypoglycemia after bariatric surgery. summary Post-barre surgery hypoglycemia (PBH) is a rare but serious complication of bariatric surgery, manifesting as a common episode of symptomatic postprandial hypoglycemia, for which there is no approved drug therapy. The significant role of increased meal-induced secretion of the incretin hormone, glucagon-like peptide-1 (GLP-1), accompanied by dysregulation of insulin secretion, has made GLP-1 receptor antagonism an attractive targeted therapy. A study evaluating the use of a single intravenous (IV) or subcutaneous (SC) injection of the GLP-1 receptor antagonist exendin (9-39) demonstrated that a single dose of exendin (9-39) could prevent postprandial hypoglycemia, normalize β-cell function, and alleviate neurohypoglycemic symptoms in PBH patients during oral glucose tolerance tests (OGTTs).

[0111] This multiple dose escalation (MAD) study evaluated the efficacy, tolerability, and pharmacokinetic profiles of two exendin (9-39) formulations administered subcutaneously via BID for up to 3 days in participants with PBH. In Part A of this two-part study, 14 participants with PBH received multiple dose escalations of lyophilized exendin (9-39) ("Lyo") reconstituted in 0.9% normal saline for up to 3 days via BID, following a baseline oral glucose tolerance test (OGTT), and underwent a repeated OGTT on the final day of administration. In Part B of this study, 5 participants received a 3-day BID treatment with a buffer formulation ("Liq") containing 30 mg of exendin (9-39) and 45 mg / ml of mannitol in 10 mM sodium acetate at pH 5.5. Repeated administration of both SC exendin (9-39) formulations demonstrated good tolerability and dose-dependently improved hyperinsulinic hypoglycemia and reduced associated symptoms in PBH patients. The buffered exendin (9-39) formulation was found to improve postprandial metabolism and clinical parameters with efficiency equivalent to or higher than lyophilized exendin (9-39) reconstituted in saline, and to provide higher exposure and duration of action. In conclusion, the buffered exendin (9-39) formulation provides a promising and convenient formulation for subcutaneous administration of exendin (9-39), enabling lower doses and / or lower frequency administration.

[0112] background Postprandial hypoglycemia (PBH) is a rare but serious complication of bariatric surgery, manifesting as a common episode of symptomatic postprandial hypoglycemia, for which there is no approved drug therapy. The significant role of increased meal-induced secretion of the incretin hormone glucagon-like peptide-1 (GLP-1), accompanied by dysregulation of insulin secretion, has been established, making GLP-1 receptor antagonism an attractive and targeted therapeutic approach. A study evaluating the IV infusion (Salehi et al., Gastroenterology, 2014, 146:669-680; Craig et al., Diabetologia, 2017, 60:531-540) or SC injection (see International Publication No. 2016 / 191395) of the GLP-1 receptor antagonist Ex-9-39 demonstrated that a single dose of exendin (9-39) prevented postprandial hypoglycemia, normalized β-cell function, and reduced neurohypoglycemic symptoms in PBH patients during an OGTT. This study represents the first human evaluation of subcutaneously administered exendin (9-39) formulated in a buffer. The present inventors present interim results (19 out of 20 participants) obtained from this study, which aimed to evaluate the efficacy, tolerability, and kinetic profile of multiple dose escalations of two subcutaneously administered exendin (9-39) formulations (lyophilized exendin (9-39) reconstituted in saline, or exendin (9-39) in a buffer formulation) administered via BID to PBH participants for up to 3 days.

[0113] method Test design and procedure: This Phase II MAS trial was conducted in two parts, Part A and Part B. In Part A, 14 participants received a BID of reconstituted lyophilized exendin (9-39) in the range of 2.5–32 mg for up to 3 days, following a baseline OGTT, and underwent a repeated OGTT on the last day of treatment. In Part B, 5 participants received a 30 mg BID of exendin (9-39) ("Liq") formulated in a buffer containing 10 mM sodium acetate and 45 mg / ml mannitol at pH 5.5, following a baseline OGTT, and underwent a repeated OGTT on the third day of treatment (see Figure 6). Metabolic, clinical, and pharmacokinetic responses were evaluated in both parts, and tolerability and safety were monitored. Dose level and frequency determination was based on interim assessments of PK, PD, and safety data. Symptoms of hypoglycemia were assessed during each oral glucose tolerance test (OGTT) using the Edinburgh Hypoglycemia Symptom Scale (EHSS) (4, 5). OGTT was stopped by investigator rescue with IV dextrose when plasma glucose levels were ≤50 mg / dL.

[0114] Test participants: Eligible participants were males or females aged 18–65 years who had undergone Roux-en-Y gastric bypass (RYGB) surgery at least 12 months prior, had a history of Whipple's triad, had inappropriately elevated insulin levels (>3 μU / mL) during hypoglycemia (≤55 mg / dL), and reported at least one symptomatic episode per month. The characteristics of the 14 study participants are described in Table 5 below.

[0115] [Table 5]

[0116] result Metabolism and clinical responses: Part A: Treatment with the lyophilized reconstituted exendin (9-39) reduced the presence and severity of hypoglycemia at all dose levels. Participants receiving doses ≥18 mg did not require IV dextrose rescue. With reconstituted lyophilized exendin (9-39), a dose-response relationship was observed with gradually increasing improvements in lowest glucose point, insulin peak, and symptom scores (Figures 8A-8C and Table 6 below). The top two dose cohorts that received an average of approximately 30 mg of reconstituted lyophilized exendin (9-39) BID over 3 days showed a mean 37% increase in lowest glucose point, a 50% decrease in peak insulin concentration, a 50% decrease in overall hypoglycemic symptom score, and a 50% decrease in neurogenic hypoglycemic symptoms (Figures 7A and 7C and Table 6 below). All doses were well tolerable, with only mild headache or nausea reported, and no drug-related adverse events (DRAEs) were observed.

[0117] Based on interim efficacy, safety, and tolerability results, a fixed dose of 30 mg BID of exendin (9-39) buffer formulation was selected for Part B.

[0118] Part B: Treatment with a BID dose of 30 mg of exendin (9-39) buffer resulted in elevated postprandial glucose lows during the OGTT on day 3 of administration in all evaluated participants, with no need for IV dextrose rescue. On average, participants achieved a 49% increase in glucose lows, a 58% decrease in peak insulin concentration, a 56% decrease in overall hypoglycemic symptom scores, and a 12% decrease in neurogenic hypoglycemic symptoms (Figures 7B, 7D, 8A-8C). All doses were well-tolerated, and no DRAEs were observed.

[0119] Pharmacokinetic response: Part A: By increasing the dose of reconstituted lyophilized exendin (9-39), C max Excendin (9-39) exposure gradually increased, as indicated by the 12-hour AUC concentration (Figures 9A-9B and Table 6).

[0120] Part B: Exendin (9-39) buffer preparations administered at equivalent doses based on mg / kg standards showed a higher C than reconstituted lyophilized exendin (9-39). max , higher 12-hour AUC, and slower T max The exendin (9-39) buffer formulation resulted in higher trough plasma concentrations on the final day of administration and exhibited a more sustained absorption profile than equivalent doses of reconstituted lyophilized exendin (9-39) (Figures 9A-9B and Table 6).

[0121] [Table 6]

[0122] conclusion In patients with refractory PBH, repeated subcutaneous administration of exendin (9-39) resulted in the following outcomes during OGTT induction: (1) dose-dependent improvement of postprandial hyperinsulinic hypoglycemia and substantial reduction of associated symptoms; (2) prevention of neurohypoglycemia without rescue therapy at doses ≥ 18 mg; and (3) no drug-related adverse events or tolerability concerns. Exendin (9-39) buffer formulations, ready-to-use formulations, provide at least equivalent protection against symptomatic hyperinsulinic hypoglycemia and may confer higher pharmacokinetic exposure along with a longer duration of action.

[0123] Example 4: Pharmacokinetic profiles of single and multiple dose-escalating subcutaneous buffered exendin (9-39) formulations A Phase 1 study was conducted to investigate the safety, tolerability, pharmacokinetics, and pharmacodynamic profiles of subcutaneous buffer formulations of single and multiple escalating doses of exendin (9-39). For this study, exendin (9-39) was formulated at a concentration of 30 mg / ml in a liquid buffer containing 10 mM sodium acetate and 45 mg / ml mannitol, with a pH of 5.5. In this single-center study with healthy volunteers, 32 subjects received either a single escalating dose of exendin (9-39) ranging from 7.5 mg to 45 mg (24 volunteers) or placebo (8 volunteers) subcutaneously (Part A); 16 subjects received one of three escalating doses (60, 75, or 90 mg of exendin (9-39) once daily for three consecutive days).

[0124] In Part A, the single-dose escalation group, 32 healthy subjects were enrolled in four consecutive cohorts of eight subjects each, as follows: six subjects received exendin (9-39) at doses of 7.5, 15, 30, or 45 mg, and two subjects in each cohort received placebo. In Part B, the multiple-dose escalation group, 16 healthy subjects were enrolled in three consecutive cohorts of six, six, and four subjects each, and received one of three dose levels of exendin (9-39) of 60, 75, or 90 mg once daily for three consecutive days.

[0125] As shown in Table 7 and Figure 10A below, mean whole-body exposure (C) max AUC 0-tau and AUC 0-inf ) increased almost proportionally with the dose.

[0126] [Table 7]

[0127] As shown in Figure 10B, the pre-administration trough plasma concentration on day 3 of 30 mg BID administration was close to the plasma concentration of exendin (9-39) considered therapeutic (>220 ng / ml), and relatively sustained plasma concentrations were observed throughout the daytime (T=12 hours before the next administration). Within 60 minutes of administration of the 60 mg AM dose, the target expected therapeutic concentration (>220 ng / ml) was achieved, and higher peak and sustained therapeutic concentrations were observed throughout the daytime.

[0128] The dose levels and dosing intervals for exendin (9-39) for the planned Phase 2 trial were selected based on the results of this Phase 1 trial (e.g., as shown in Table 7 and Figures 10A-10B) and the results of the completed MAD trial conducted at Stanford University in patients with PBH. In the Stanford MAD trial, patients with refractory PBH experienced significant improvement in the lowest glucose threshold and neurogenic hypoglycemia symptoms during the oral glucose tolerance test after administration of 30 mg BID of exendin (9-39), and postprandial metabolism and clinical parameters were improved after the buffered formulation compared to lyophilized exendin (9-39) reconstituted in saline. In the Stanford MAD trial, the optimal pharmacodynamic effect (lowest postprandial glucose threshold > 50 mg / dl and at least a 50% reduction in peak insulin) was observed with a peak plasma concentration of at least 220 ng / ml (C max This was achieved in [specific example]. In contrast, in the studies described herein (as shown in this example and Example 3, for example), the buffered formulations showed higher pharmacokinetic and pharmacodynamic effects compared to reconstituted lyophilized exendin (9-39) formulated in normal saline.

[0129] In summary, as shown in Table 7 and Figures 10A-10B, the PK data from this Phase 1 trial in healthy volunteers, as well as the PK and PD data from the completed Stanford MAD trial, as described in Example 3, suggest that repeated subcutaneous administration of the 30 mg BID buffer formulation may provide sufficient plasma concentrations of exendin (9-39) to confer protection from postprandial hypoglycemia without the need to wait for a meal after administration. The data also suggest that longer and higher exposure conferred by higher doses, such as 60 mg QD, may provide therapeutic plasma concentrations over approximately 16 hours for patients requiring higher systemic exposure and / or those prioritizing the convenience of a once-daily regimen. Under a once-daily administration regimen, it would be preferable to administer the drug before breakfast, allowing at least 60 minutes between administrations, and to avoid eating late at night. Therefore, a total daily dose of 60 mg (administered subcutaneously as either 30 mg every 12 hours or 60 mg every morning) was selected for the next Phase 2 trial in patients with refractory PBH.

[0130] While the above-described invention has been explained in some detail as an example for clarity and ease of understanding, it will be apparent to those skilled in the art that many modifications and changes to the invention can be implemented without departing from the spirit and scope of the invention. The specific embodiments described herein are provided for illustrative purposes only and are not intended to be limiting in any way. This specification and the examples are for illustrative purposes only, and the true scope and spirit of the invention are intended to be shown by the following claims.

[0131] All publications, patents, patent applications, or other documents referenced herein are incorporated herein by reference in their entirety for any purpose to the same extent as each publication, patent, patent application, or other document is individually stated to be incorporated by reference for any purpose.

Claims

1. A liquid pharmaceutical preparation comprising exendin (9-39) or a pharmaceutically acceptable salt thereof and an isotonic agent in a physiologically acceptable buffer having a pH in the range of approximately 5 to approximately 6.

2. The liquid pharmaceutical formulation according to claim 1, wherein the physiologically acceptable buffer comprises an acetate buffer or a citrate buffer.

3. The liquid pharmaceutical formulation according to claim 1 or 2, wherein the physiologically acceptable buffer comprises sodium acetate or sodium citrate.

4. The liquid pharmaceutical preparation according to any one of claims 1 to 3, wherein the physiologically acceptable buffer solution contains sodium acetate or sodium citrate at a concentration of about 5 mM to about 30 mM.

5. The liquid pharmaceutical preparation according to any one of claims 1 to 4, wherein the physiologically acceptable buffer solution contains sodium acetate at a concentration of about 10 mM.

6. The liquid pharmaceutical preparation according to any one of claims 1 to 5, wherein the buffer solution has a pH in the range of 5.2 to 5.

8.

7. The liquid pharmaceutical preparation according to claim 6, wherein the pH is approximately 5.

5.

8. The liquid pharmaceutical preparation according to any one of claims 1 to 7, wherein the isotonic agent comprises mannitol, dextrose, glycerin, lactose, sucrose, or trehalose.

9. The liquid pharmaceutical preparation according to claim 8, wherein the isotonic agent comprises mannitol.

10. The liquid pharmaceutical preparation according to claim 8 or 9, wherein the isotonic agent is present in an amount of about 20 to about 60 mg / ml.

11. The liquid pharmaceutical preparation according to claim 10, wherein the isotonic agent is present in an amount that achieves an osmotic pressure of approximately 290 mOsm / kg.

12. The liquid pharmaceutical preparation according to any one of claims 1 to 11, wherein the pharmaceutically acceptable salt of exendin(9-39) is exendin(9-39) acetate or exendin(9-39) trifluoroacetate.

13. The liquid pharmaceutical preparation according to any one of claims 1 to 12, wherein the exendin (9-39) or a pharmaceutically acceptable salt thereof is present at a concentration of about 10 mg / ml to about 120 mg / ml.

14. The liquid pharmaceutical preparation according to claim 13, wherein the exendin (9-39) or a pharmaceutically acceptable salt thereof is present in a concentration of at least 15 mg / ml.

15. The liquid pharmaceutical preparation according to claim 13, wherein the exendin (9-39) or a pharmaceutically acceptable salt thereof is present at a concentration of about 30 mg / ml.

16. The liquid pharmaceutical preparation according to claim 13, wherein the exendin (9-39) or a pharmaceutically acceptable salt thereof is present at a concentration of about 60 mg / ml.

17. The liquid pharmaceutical formulation according to any one of claims 1 to 16, wherein the exendin (9-39) or a pharmaceutically acceptable salt thereof does not exhibit detectable aggregation in the formulation.

18. A liquid pharmaceutical preparation according to any one of claims 1 to 17, formulated for subcutaneous administration.

19. The liquid pharmaceutical formulation according to any one of claims 1 to 18, wherein when administered to a human subject, the liquid pharmaceutical formulation has an improved pharmacokinetic profile compared to a composition containing the same dose of exendin (9-39) or a pharmaceutically acceptable salt thereof formulated in 0.9% normal physiological saline.

20. When the aforementioned liquid pharmaceutical formulation is administered to a human subject, it contains a higher C content of exendin (9-39) than a composition containing the same dose of exendin (9-39) or a pharmaceutically acceptable salt thereof formulated in 0.9% normal physiological saline. ｍａｘ A liquid pharmaceutical preparation according to claim 19, which presents the following.

21. The liquid pharmaceutical formulation according to claim 19, wherein when administered to a human subject, the liquid pharmaceutical formulation exhibits a higher 12-hour AUC of exendin (9-39) than a composition containing the same dose of exendin (9-39) or a pharmaceutically acceptable salt thereof formulated in 0.9% normal physiological saline.

22. The liquid pharmaceutical formulation according to claim 19, wherein when administered to a human subject, the liquid pharmaceutical formulation exhibits a higher trough plasma concentration of exendin (9-39) than a composition containing the same dose of exendin (9-39) or a pharmaceutically acceptable salt thereof formulated in 0.9% normal physiological saline.

23. A method for treating or preventing hyperinsulinic hypoglycemia of a subject, comprising the step of administering a liquid pharmaceutical preparation according to any one of claims 1 to 22 to the subject.

24. The method according to claim 23, wherein the subject has previously undergone upper gastrointestinal treatment.

25. The method according to claim 24, wherein the subject has previously received treatment for obesity.

26. The method according to any one of claims 23 to 25, wherein the method comprises the step of subcutaneously administering a liquid pharmaceutical formulation containing exendin (9-39) to the subject once daily (QD) at a dose of about 45 mg to about 75 mg.

27. The method according to claim 26, wherein the method comprises the step of subcutaneously administering a liquid pharmaceutical formulation containing exendin (9-39) to the subject at a dose of approximately 60 mg QD.

28. The method according to any one of claims 23 to 25, wherein the method comprises the step of subcutaneously administering a liquid pharmaceutical formulation containing exendin (9-39) to the subject at a dose of about 30 mg twice daily (BID).

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