GLP-1 receptor
Avexitide (Exendin-(9-39)) is a specific glucagon-like peptide-1 receptor (GLP-1R) antagonist. [1]

GLP-1 is involved in the regulation of fasting glucose. Avexitide (also known as Exendin (9-39)) is a particular GLP-1 receptor antagonist that is derived from a shortened version of the GLP-1 agonist exendin-4[1].

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In isolated pancreatic islets from neonates with KATPHI, Avexitide (at 100 nmol/L) significantly inhibited insulin secretion stimulated by a physiologic mixture of amino acids (4 mmol/L). Amino acid stimulation alone caused a fourfold increase in insulin secretion, and this increase was significantly reduced by pretreatment with Avexitide. [1]

In SUR-1 -/- mice, continuous subcutaneous infusion of Avexitide (Exendin (9-39)) dramatically increases fasting blood glucose levels without compromising glucose tolerance[2].

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In a randomized, crossover pilot study involving nine subjects with KATPHI, continuous intravenous infusion of Avexitide during a prolonged fast significantly elevated fasting blood glucose levels compared to vehicle infusion.
The mean nadir blood glucose concentration was significantly higher during Avexitide infusion (4.9 ± 1.1 mmol/L) compared to vehicle (3.5 ± 0.3 mmol/L).
The mean blood glucose area under the curve (AUC) from 0 to 360 minutes was significantly greater during Avexitide infusion (2096 ± 454 mmol·min/L) than during vehicle infusion (1678 ± 281 mmol·min/L).
Hypoglycemia (blood glucose <3.9 mmol/L) occurred in eight subjects during vehicle infusion, with three requiring dextrose rescue. No subject developed hypoglycemia during Avexitide infusion.
The insulin-to-glucose AUC ratio was significantly lower during infusion of Avexitide at the higher doses (300 and 500 pmol/kg/min) compared to vehicle, indicating a relative reduction in insulin secretion relative to glucose levels.
Plasma concentrations of glucagon and intact GLP-1 were not significantly affected by Avexitide treatment. [1]

cAMP Content Determination—Islets were isolated as above, hand-picked, and cultured for 3 days. Cultured islets were preincubated in glucose-free Krebs-Ringer bicarbonate buffer for 60 min, and 100 nm exendin-(9–39) was added 30 min into the preincubation period. Then, islets were exposed to different treatments for an additional 30 min in the presence of 0.1 mm isobutylmethylzanthine. After incubation, islets were washed two times by cold glucose-free Hanks' buffer. cAMP was measured in islet lysates by an enzyme-linked immunosorbent assay[2].

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Cytosolic Free Ca2+ Measurements—Mouse islets were isolated and cultured on poly-l-lysine-coated glass coverslips under the same conditions as described above. The perifusion procedure and cytosolic-free Ca2+ ([Ca2+]i) measurement were described previously. In brief, the coverslip with attached islets was incubated with 15 μm Fura-2 acetoxymethylester in Krebs-Ringer bicarbonate buffer with 5 mm glucose for 35 min at 37 °C. Islets were then perifused with Krebs-Ringer bicarbonate buffer with 0.25% bovine serum albumin at 37 °C at a flow rate of 2 ml/min while various agents were applied. [Ca2+]i was measured with a dual wavelength fluorescence microscope as previously described[2].

Islet Studies—Islets were isolated by collagenase digestion and cultured for 3 days in RPMI 1640 medium containing 10 mm glucose. The culture medium was supplemented with 10% fetal bovine serum, 2 mm glutamine, 100 units/ml penicillin, and 50 μg/ml streptomycin. Islets were incubated at 37 °C in a 5% CO2, 95% air-humidified incubator. Batches of 100 cultured mouse islets were loaded onto a nylon filter in a chamber and perifused with Krebs-Ringer bicarbonate buffer (115 mm NaCl, 24 mm NaHCO3, 5 mm KCl, 1 mm MgCl2, 2.5 mm CaCl2, 10 mm HEPES, pH 7.4) with 0.25% bovine serum albumin at a flow rate of 2 ml/min. Perifusate solutions were gassed with 95% O2, 5% CO2 and maintained at 37 °C. Islets were stimulated with a ramp of amino acids. The mixture of 19 amino acids when used at a maximum concentration of 12 mm (about 3 times physiological concentration) had the following composition: 2 mm glutamine, 1.25 mm alanine, 0.53 mm arginine, 0.11 mm aspartate, 0.27 mm citrulline, 0.35 mm glutamate, 0.85 mm glycine, 0.22 mm histidine, 0.27 mm isoleucine, 0.46 mm leucine, 1.06 mm lysine, 0.14 mm methionine, 0.20 mm ornithine, 0.23 mm phenylalanine, 1 mm proline, 1.62 mm serine, 0.77 mm threonine, 0.21 mm tryptophan, 0.57 mm valine. Samples were collected every minute for insulin assays. Insulin was measured by radioimmunoassay[2].

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Human Islet Insulin Secretion Assay: Fresh pancreatic islets were isolated from surgical specimens of three neonates with KATPHI. Islets were cultured for 3 days prior to experiments.
Batches of 50 islets were preincubated in glucose-free Krebs-Ringer bicarbonate buffer for 60 minutes. Avexitide (100 nmol/L) was added 30 minutes into the preincubation period.
Islets were then exposed to stimulation with either 10 mmol/L glucose or a 4 mmol/L mixture of amino acids for a defined period.
The culture media were collected, and insulin concentration was measured by radioimmunoassay to determine the level of insulin secretion. [1]

Mice: Avexitide (Exendin (9-39)) or a vehicle (0.9% NaCl, 1% bovine serum albumin) is administered subcutaneously via Alzet miniosmotic pumps for a period of two weeks[2].

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Exendin-(9–39) Administration—Alzet miniosmotic pumps were implanted subcutaneously to deliver exendin-(9–39) at a rate of 150 pmol/kg/min or vehicle (0.9% NaCl, 1% bovine serum albumin) for 2 weeks.[2]
Glucose Homeostasis—For determination of fasting blood glucose levels, mice were fasted for 12–16 h. Oral glucose tolerance testing was carried after a 12–16-h fast by administering 2 g/kg of dextrose by oral gavage (feeding needles). For insulin tolerance testing, mice received 0.5 units/kg of insulin intraperitoneally after a 4-h fast. Blood glucose levels were measured using a hand-held glucose meter. Insulin and glucagon were measured by ELISA.[2]

[1]. GLP-1 receptor antagonist exendin-(9-39) elevates fasting blood glucose levels in congenital owing to inactivating mutations in the ATP-sensitive K+ channel. Diabetes. 2012 Oct;61(10):2585-91.

[2]. Exendin-(9-39) corrects fasting hypoglycemia in SUR-1-/- mice by lowering cAMP in pancreatic beta-cells and inhibiting secretion. J Biol Chem. 2008 Sep 19;283(38):25786-93.

Avexitide is being investigated in the clinical trial NCT02996812 (evaluating the efficacy of a single subcutaneous injection of escalating doses of exenatide 9-39 in patients with post-barbiturative hypoglycemia). Avexitide is a truncated form of the glucagon-like peptide-1 receptor (GLP-1R) agonist exenatide-4, possessing both GLP-1 receptor (GLP-1R) antagonistic and GLP-1R-mediated signal transduction inhibitory activities. After administration, avexitide competitively binds to and inhibits the activity of GLP-1R, thereby inhibiting GLP-1/GLP-1R-mediated signal transduction. This antagonizes the glucagon-inhibiting and insulin-secreting effects of GLP-1. By inhibiting GLP-1-mediated insulin release and reducing postprandial glucagon secretion, exenatide 9-39 can be used to investigate the potential effects of excessive GLP-1 production on food intake, weight loss, and blood glucose levels. GLP-1 receptors are located on pancreatic β cells and are overexpressed in certain tumor cell types. GLP-1 is a gastrointestinal insulin secretion hormone that is released after meals and plays a key role in the regulation of blood glucose levels. See also: Avicivide (note moved to). Avicivide (exenatide-(9-39)) is a truncated form of the GLP-1 agonist exenatide-4 and acts as a competitive GLP-1 receptor antagonist. It may also act as an inverse agonist, inhibiting the ligand-independent basal activity of the GLP-1 receptor. [1] This study aimed to evaluate its potential as a novel therapy for congenital hyperinsulinemia (KATPHI) caused by mutations in the inactivation of the KATP channel, a disease for which there are currently limited effective treatments. [1]
The clinical dosing regimen was a stepwise intravenous infusion: 100 pmol/kg/min for 2 hours; followed by 300 pmol/kg/min for 2 hours; and finally 500 pmol/kg/min for 2 hours. The peptide was dissolved in a 0.9% sodium chloride solution containing 0.25% human serum albumin (final concentration 0.1 mg/mL). [1]
The results indicate that endogenous GLP-1 receptor signaling plays a role in the regulation of fasting blood glucose in KATPHI patients, and GLP-1 receptor antagonists are expected to become a new therapeutic target for the prevention of hypoglycemia in this disease. [1]

C₁₄₉H₂₃₄N₄₀O₄₇S

3369.76

3367.687

C, 53.11; H, 7.00; N, 16.63; O, 22.31; S, 0.95

133514-43-9

2051593-46-3 (acetate); 133514-43-9

16198321

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

DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2

Off-white to pale purple solid powder

1.5±0.1 g/cm3

1.671

-5.88

45

52

109

237

8000

29

[DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2]

WSEVKKHALHSUMB-MVNVRWBSSA-N

InChI=1S/C149H234N40O47S/c1-14-78(10)120(185-139(227)98(62-81-29-16-15-17-30-81)177-136(224)97(61-76(6)7)175-129(217)88(35-24-53-158-149(156)157)172-144(232)119(77(8)9)184-122(210)79(11)164-126(214)90(41-46-114(199)200)168-131(219)91(42-47-115(201)202)169-132(220)92(43-48-116(203)204)170-134(222)94(50-58-237-13)171-130(218)89(40-45-109(153)194)167-127(215)86(33-20-22-51-150)166-140(228)103(72-192)182-137(225)95(59-74(2)3)174-123(211)84(152)64-118(207)208)145(233)173-93(44-49-117(205)206)133(221)178-99(63-82-66-159-85-32-19-18-31-83(82)85)138(226)176-96(60-75(4)5)135(223)165-87(34-21-23-52-151)128(216)179-100(65-110(154)195)124(212)161-67-111(196)160-69-113(198)186-54-25-36-105(186)142(230)183-104(73-193)141(229)181-102(71-191)125(213)162-68-112(197)163-80(12)146(234)188-56-27-38-107(188)148(236)189-57-28-39-108(189)147(235)187-55-26-37-106(187)143(231)180-101(70-190)121(155)209/h15-19,29-32,66,74-80,84,86-108,119-120,159,190-193H,14,20-28,33-65,67-73,150-152H2,1-13H3,(H2,153,194)(H2,154,195)(H2,155,209)(H,160,196)(H,161,212)(H,162,213)(H,163,197)(H,164,214)(H,165,223)(H,166,228)(H,167,215)(H,168,219)(H,169,220)(H,170,222)(H,171,218)(H,172,232)(H,173,233)(H,174,211)(H,175,217)(H,176,226)(H,177,224)(H,178,221)(H,179,216)(H,180,231)(H,181,229)(H,182,225)(H,183,230)(H,184,210)(H,185,227)(H,199,200)(H,201,202)(H,203,204)(H,205,206)(H,207,208)(H4,156,157,158)/t78-,79-,80-,84-,86-,87-,88-,89-,90-,91-,92-,93-,94-,95-,96-,97-,98-,99-,100-,101-,102-,103-,104-,105-,106-,107-,108-,119-,120-/m0/s1

(4S)-4-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-3-carboxypropanoyl]amino]-4-methylpentanoyl]amino]-3-hydroxypropanoyl]amino]hexanoyl]amino]-5-oxopentanoyl]amino]-4-methylsulfanylbutanoyl]amino]-4-carboxybutanoyl]amino]-4-carboxybutanoyl]amino]-5-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S,3S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-4-amino-1-[[2-[[2-[(2S)-2-[[(2S)-1-[[(2S)-1-[[2-[[(2S)-1-[(2S)-2-[(2S)-2-[(2S)-2-[[(2S)-1-amino-3-hydroxy-1-oxopropan-2-yl]carbamoyl]pyrrolidine-1-carbonyl]pyrrolidine-1-carbonyl]pyrrolidin-1-yl]-1-oxopropan-2-yl]amino]-2-oxoethyl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]carbamoyl]pyrrolidin-1-yl]-2-oxoethyl]amino]-2-oxoethyl]amino]-1,4-dioxobutan-2-yl]amino]-1-oxohexan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-4-carboxy-1-oxobutan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-1-oxopropan-2-yl]amino]-5-oxopentanoic acid

Exendin 9-39; Avexitide Free Base; Exendin (9-39); Avexitide; Exendin-3 (9-39); Asp-LEU-SER-LYS-GLN-MET-GLU-GLU-GLU-ALA-VAL-ARG-LEU-PHE-ILE-GLU-TR; p; -LEU-LYS-ASN-GLY-GLY-PRO-SER-SER-GLY-ALA-PRO-PRO-PRO-SER-NH2; 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-; Exendin-(9-39)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: ~50 mg/mL (~14.8 mM)

Solubility in Formulation 1: 100 mg/mL (29.68 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)