GnRH receptor (Type I GnRH receptor). [2]

Alarelin considerably reduced the cell viability when compared to when it wasn't present. A concentration of 10^-5 M was found to have the greatest stimulatory effect on cell viability, and this effect was dose-dependent[1].

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Alarelin significantly decreased the viability of cultured rat gastric smooth muscle cells (GSMC) as measured by MTT assay. At concentrations of 10⁻⁹, 10⁻⁷, and 10⁻⁵ mol/L, the OD values (490 nm) were 0.533±0.073, 0.368±0.029, and 0.243±0.042 respectively, compared to the control group (0.728±0.100). The maximum inhibitory effect was achieved at 10⁻⁵ mol/L, acting in a dose-dependent manner (F=59.083, P<0.01) [1].
- Alarelin inhibited DNA synthesis in GSMC as assessed by ³H-TdR incorporation. After 16 h treatment with alarelin (10⁻⁹, 10⁻⁷, 10⁻⁵ mol/L) followed by 8 h ³H-TdR pulse, the incorporation counts (cpm) were 1448.17±327.72, 945.83±374.32, and 385.83±184.66 respectively, versus control (1936.50±440.99). The antiproliferative effect was most evident at 10⁻⁵ mol/L (F=22.33, P<0.05) and was dose-dependent [1].
- Alarelin reduced the average fluorescent values of proliferating cell nuclear antigen (PCNA) in GSMC. After 24 h treatment with alarelin at 10⁻⁹, 10⁻⁷, and 10⁻⁵ mol/L, the average fluorescent values were 11.37±1.99, 9.35±1.38, and 7.37±1.06 respectively, compared to control (13.48±1.31). The inhibition was dose-dependent (F=15.86, P<0.01) [1].
- Flow cytometric DNA analysis revealed that alarelin significantly increased the proportion of cells in G₁ phase and decreased the proportion in S phase. At alarelin concentrations of 10⁻⁹, 10⁻⁷, and 10⁻⁵ mol/L, the G₁ phase percentages were 80.00±9.42%, 79.60±7.43%, and 80.20±10.03% respectively (control: 70.80±11.30%), and the S phase percentages were 9.93±1.73%, 6.60±1.54%, and 3.20±1.20% respectively (control: 17.50±3.40%). The effect on S phase was dose-dependent (F=32.5, P<0.05) [1].

Alarelin has the ability to directly affect rat parietal cells and also inhibit vagous function, which can both reduce the secretion of gastric acid[2]. Alarelin has the ability to dramatically increase the G1 phase ratio and decrease the S phase ratio in rats' GSMC[1].

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In anaesthetized rats, intra-gastric (chambered stomach) infusion of Alarelin at 2 μg/Kg gradually increased gastric pH in a time-dependent manner from 0 to 45 minutes, reaching a plateau of approximately 5.548 ± 0.237 at 45 minutes, which was significantly higher compared to the control (P < 0.01). [2]
In anaesthetized rats, intravenous (tail vein) administration of Alarelin at 2 μg/Kg gradually increased gastric pH in a time-dependent manner from 0 to 45 minutes, reaching a plateau of approximately 5.244 ± 0.371 at 45 minutes, which was significantly higher compared to the control (P < 0.01). The mechanism of inhibition of gastric acid secretion by intravenous infusion involves a decrease of vagal activity. [2]

The cells are seeded in a 96-well plate after being trypsinized in a 2.5 g/L trypsin solution. Following a 24-hour growth period to a subconfluent state of about 800 g/L, each well is supplemented with 0.1 mL of a medium containing 2.5% calf serum and varying concentrations (0.001, 0.1, and 10 μM) of alarelin. The wells are then incubated for a full day in a CO₂ incubator. At least twelve wells are used to test each concentration. In summary, each well receives 15 μL of MTT solution, which is then incubated for four hours. Once the medium and MTT are taken out, each well receives 150 μL of DMSO, which is added and shaken for 10 minutes to dissolve the crystal. An ELISA reader is used to measure the OD at 490 nm[1].

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Primary cultured rat gastric smooth muscle cells (GSMC) were obtained from 1-3-day-old male Sprague-Dawley rats. Stomachs were removed, mucus was scraped off, and smooth muscle tissues were minced into 1.0 mm³ pieces. The tissue pieces were digested in DMEM containing collagenase I at 37°C for 30-60 min, and cells were collected every 20 min. The cell suspension was centrifuged at 500 r/min for 5 min, washed twice with DMEM, and then cultured in DMEM with 100 mL/L calf serum, penicillin G (100 U/mL), and streptomycin (100 U/mL) under 50 mL/L CO₂ humidified atmosphere. To remove fibroblasts, cells were pre-attached for 90 min. The medium was changed every 3 days and subcultured every 4-5 days using trypsin (2.5 g/L). Experiments were performed on passages 3-5 [1].
- MTT assay: GSMC were seeded in 96-well plates at 10⁴ cells per 0.1 mL well. After 24 h growth to ~80% subconfluence, 0.1 mL medium containing 2.5% calf serum and alarelin (10⁻⁹, 10⁻⁷, 10⁻⁵ mol/L) was added and incubated for 24 h. Then 15 mL of MTT solution (5 g/L in PBS, pH 7.2) was added to each well and incubated for 4 h. The medium and MTT were removed, 150 mL DMSO was added to each well and shaken for 10 min to dissolve crystals. OD was measured at 490 nm using an ELISA reader [1].
- ³H-TdR incorporation assay: GSMC were plated in 96-well plates at 10⁴ cells per 0.1 mL well. After 24 h, 0.1 mL medium containing 2.5% calf serum and alarelin (10⁻⁹, 10⁻⁷, 10⁻⁵ mol/L) was added and incubated for 16 h. Then ³H-TdR (2 mCi/mL) was added to each well and incubated for 8 h. Incubation was stopped by adding 1 volume of cold 100 mL/L trichloroacetic acid (TCA) to disrupt cells and precipitate macromolecules. After washing with methanol, the precipitate was collected on fiberglass filter paper, dried, mixed with 0.5 mL scintillant liquid, and radioactivity was counted with a scintillation counter [1].
- PCNA immunofluorescence assay: GSMC were seeded on coverglasses in 6-well plates. After 24 h growth to ~80% subconfluence, 2 mL medium containing 25 mL/L calf serum and alarelin (10⁻⁹, 10⁻⁷, 10⁻⁵ mol/L) was added and incubated for another 24 h. Cells were fixed in 40 g/L paraformaldehyde for 30 min, washed with PBS, treated with methanol-H₂O₂ for 30 min to remove endogenous peroxidase, then incubated with mouse anti-PCNA antibody (1:200 dilution) at 4°C overnight, followed by biotin-labeled goat anti-rabbit IgG antibody (1:200 dilution) for 2 h at room temperature, and then with SABC-fluo Cy3 (1:100 dilution) for 1 h at room temperature. Average fluorescent values of PCNA were scanned using laser confocal microscopy [1].
- Cell cycle analysis by flow cytometry: GSMC were seeded in 12-well plates at 10⁷ cells per 1.5 mL well. After 24 h growth to ~80% subconfluence, 2 mL medium containing 25 mL/L calf serum and alarelin (10⁻⁹, 10⁻⁷, 10⁻⁵ mol/L) was added and incubated for 48 h. Cells were trypsinized (2.5 g/L trypsin), fixed in 700 mL/L ethanol, and stained with propidium iodide (PI) for DNA analysis. DNA content and cell cycle phase distribution were analyzed using a flow cytometer (EPICS Profile II) [1].

Rats: There are two groups of male Sprague-Dawley rats. Group I: A chambered stomach is used to measure the amount of gastric acid secreted. In short, the abdomen is cut, the stomach and duodemun are exposed and tied, respectively, and 1.5 mL of 0.9% sodium chloride (containing 2 μg/kg of Alarelin) is infused into each of the stomach's chambers. The pH is measured in the ABL-500 after 15, 30, 45, and 60 minutes, respectively, and the gastric juice is extracted from the chambered stomach. Saline is infused in place of alarelin as the control. Group II: Following anesthesia, 2 mL of Alarelin (2 μg/kg) is injected into the vein of the tail. Saline is injected as the control rather than alarelin. After that, the duodenum and stomach are tied off and given an instant 1.5 mL saline infusion.

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Male Sprague-Dawley rats (250-300 g, postnatal 30-60 days) were deprived of food for 24 hours but had free access to tap water. After anaesthetization with Sodium Pentobarbital (40 mg/kg, intraperitoneal injection), animals were randomly divided into two groups (n=12 per group).
Group I: Gastric acid secretion was measured in a chambered stomach. The abdomen was incised, the stomach and duodenum were exposed and tied; then 1.5 ml of 0.9% sodium chloride containing Alarelin (2 μg/Kg) was infused into the chambered stomach. After 15, 30, 45, and 60 minutes, gastric juice was drawn out and pH was measured. Controls received saline instead of Alarelin. [2]
Group II: After anaesthetization, 2 ml of Alarelin (2 μg/Kg) was administered into the tail vein. Controls received saline instead of Alarelin. Then the stomach and duodenum were tied and infused immediately with 1.5 ml saline. After 15, 30, 45, and 60 minutes, gastric juice was drawn out and pH was measured. [2]

[1]. Expression of gonadotropin-releasing hormone receptor and effect of gonadotropin-releasing hormone analogue on proliferation of cultured gastric smooth muscle cells of rats. World J Gastroenterol. 2004 Jun 15;10(12):1780-4.

[2]. Distribution, cloning and sequencing of GnRH, its receptor, and effects of gastric acid secretion of GnRH analogue in gastric parietal cells of rats. Life Sci. 2005 Feb 4;76(12):1351-65.

See also: LHRH, Ala(6)-Gly(10)-acetamide- (note moved to); Surfagon (note moved to).

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Alarelin is a GnRH-1 analogue that directly acts on gastric parietal cells via GnRH receptors. The binding of GnRH to its receptor activates signal transduction pathways involving the Giα subunit, which inhibits adenylyl cyclase, leading to inhibition of gastric acid secretion. It also inhibits gastric acid secretion by decreasing vagal activity. [2]

C60H86N16O16

1287.42

1286.64

C, 55.98; H, 6.73; N, 17.41; O, 19.88

79561-22-1

(Des-Gly10,D-Ala6,Pro-NHEt9)-LHRH; 52435-06-0

9855027

White to off-white solid powder

1.482 g/cm3

1.694

-0.5

15

14

30

84

2320

9

C(=O)(O)C.C(C1=CNC2C=CC=CC1=2)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(N1CCC[C@H]1C(=O)NCC)=O)CC1C=CC(O)=CC=1)NC(=O)[C@@H](NC([C@@H]1CCC(=O)N1)=O)CC1NC=NC=1

DPWSRXJWCYEGIV-PFHUABGLSA-N

InChI=1S/C56H78N16O12.2C2H4O2/c1-5-60-54(83)45-13-9-21-72(45)55(84)39(12-8-20-61-56(57)58)66-50(79)40(22-30(2)3)67-47(76)31(4)64-49(78)41(23-32-14-16-35(74)17-15-32)68-53(82)44(28-73)71-51(80)42(24-33-26-62-37-11-7-6-10-36(33)37)69-52(81)43(25-34-27-59-29-63-34)70-48(77)38-18-19-46(75)65-38;2*1-2(3)4/h6-7,10-11,14-17,26-27,29-31,38-45,62,73-74H,5,8-9,12-13,18-25,28H2,1-4H3,(H,59,63)(H,60,83)(H,64,78)(H,65,75)(H,66,79)(H,67,76)(H,68,82)(H,69,81)(H,70,77)(H,71,80)(H4,57,58,61);2*1H3,(H,3,4)/t31-,38+,39+,40+,41+,42+,43+,44+,45+;;/m1../s1

acetic acid;(2S)-N-[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2R)-1-[[(2S)-1-[[(2S)-5-(diaminomethylideneamino)-1-[(2S)-2-(ethylcarbamoyl)pyrrolidin-1-yl]-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-3-(1H-imidazol-5-yl)-1-oxopropan-2-yl]-5-oxopyrrolidine-2-carboxamide

LHRH-A; larelin Acetate; Pyr-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt; 6-D-Ala-10-D-gly-LHRH-ethylamide; 6-D-Ala-10-D-gly-LHRH-ethylamide

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)

DMSO: ~62.5 mg/mL (~48.6 mM)
H2O: ~100 mg/mL (~77.7 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (1.62 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (1.62 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (1.62 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (77.67 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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