Motilin Receptor ( pEC50 = 7.9 )

GSK962040 hydrochloride, also known as camecicinal hydrochloride, exhibits negligible effects on ghrelin and other receptors, ion channels, and enzymes. When Camicinal hydrochloride (GSK962040 hydrochloride) was administered at 300 nmol L 1-10 μmol L 1, the cholinergic-mediated contraction amplitude in the rabbit stomach antrum was prolonged and reached a maximum of 248 ± 47% at 3 μmol L 1. The following were found in the samples: 10.4±0.01 (n=770), 7.3±0.29 (n=4), and 7.9±0.09 (n=17) for motilin, erythromycin, and Camicinal hydrochloride (GSK962040 hydrochloride) [1]. Dog motilin receptor activation is caused by camicinal hydrochloride (GSK962040 hydrochloride) (pEC50 5.79; intrinsic activity 0.72 relative to [Nle13]-motilin) [2]. Because its initial IC50 value on CYP3A4 was much greater than our recommended threshold of 10 μM [3], camicinal hydrochloride (GSK962040 hydrochloride) was preferred.

Camicinal (GSK962040) (5 mg free base·kg 1) also raises the overall weight of the feces after two hours after administration (21.2 ± 4.5 g; P < 0.05). [1]. Camicinal (GSK962040) causes mean plasma concentrations >1.14 μmol L 1 to cause dose-related phasic contractions that last 48 and 173 minutes for 3 and 6 mg kg 1, respectively. Migratory motor complex (MMC) activity returns when the effects of Camicinal (GSK962040) wear off. The 3 mg kg 1 Camicinal (GSK962040) had no effect on the migration of motor complex recovery; however, at the 6 mg kg 1 dose, MMC recovered 253 minutes after delivery as opposed to 101 minutes after saline (n = 5 each).[2]. The oral bioavailability (Fpo) of camicinal (GSK962040) is 48 (13%). In contrast to short-term effects, camicinal (GSK962040) demonstrated long-term effects (T1/2) 46.9 (5.0 min at 3 μM) [Nle13].Motilin (T1/2) 11.4 (at 0.3 μM) for 1.5 minutes [3]. The compound camicinal (GSK962040) is less active in the fundus and small intestine but significantly increases cholinergic activity in the gastric antrum [4].

Motilin Receptor Agonist FLIPR Assay[3]
The potency and efficacy of target compounds at the human motilin receptor were studied using a fluorometric imaging plate reader (FLIPR) and Chinese hamster ovary (CHO-K1) cells which stably express the human motilin receptor. Briefly, the human motilin receptor was cloned (PCR from human genomic DNA) into pCDNA3.1 in the vector, subcloned into pENTR/D-TOPO (using the pENTR D-TOPO directional cloning kit) and then recombined into pCIN1GW vector (LR clonase gateway reaction kit) to give the pCIN1 motilin receptor. CHO-K1 cells (ATCC No. CCl-61) were then stably transfected with the pCIN1 motilin receptor plasmid. These cells were grown as a monolayer in DMEM/HamF-12 supplemented with 10% v/v of FBS, 2 mM GlutaMAX, and 1 mg/mL Geneticin. Stimulation of this cell line with motilin causes intracellular signaling leading to an increase in intracellular calcium which was measured using calcium sensitive fluorescent dyes and quantified using a FLIPR. Briefly, cells were seeded onto 384-well black-walled, clear-bottom microtiter plates (10 000 cells/well) and incubated for 24 h. On the day of assay, media were aspirated from cell plates using a cell washer (leaving 10 μL of media). Cells were immediately loaded with loading buffer (Tyrodes (Elga water + 145 mM NaCl + 5 mM KCl + 20 mM HEPES + 10 mM glucose + 1 mM MgCl2) + 1.5 mM CaCl2 + 0.714 mg/mL Probenicid (predissolved in 1 M NaOH) + 0.5 mM brilliant black + 2.5 μM Fluo 4 dye) and incubated at 37.5 °C for 1 h. Master compound plates were prepared in 100% DMSO. A top concentration of 3 mM was used (giving 12 μM final concentration in assay), and this was serially diluted 1 in 4. Then 1 μL from the master plate was transferred to a daughter plate, to which 50 μL of compound dilution buffer (Tyrodes + 1 mg/mL BSA + 1.5 mM CaCl2) was added. An amount of 10 μL from the compound plates was then added immediately to cell plates using a FLIPR 3 calcium imaging instrument, and changes in fluorescence were measured over a 1 min time frame. Maximum change in fluorescence over baseline was used to determine agonist response, and concentration response curves were constructed using a four-parameter logistic equation. The intrinsic activity of target compounds was calculated by using the maximum asymptote of its concentration−response curve relative to the maximum asymptote of the motilin concentration−response curve.

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CYP 3A4 Time Dependent Inhibition (TDI) Assay[3]
Inhibition was determined by quantifying the production of fluorescent metabolite following incubation of CYP3A4 specific profluorescent probe substrate diethoxyfluorescein (DEF), with heterologously expressed CYP3A4 in E. coli (Cypex) and the test compound or positive control (troleandomycin). A NADPH-regenerating system (cofactor) was prepared as follows: 7.8 mg/mL glucose 6-phosphate (27.65 mM), 1.7 mg/mL NADP (2.22 mM), and glucose 6-phosphate dehydrogenase at 6 enzyme units/mL were made up in 2% w/v sodium bicarbonate solution. Incubation mixtures containing enzyme, probe substrate, and 50 mM potassium phosphate buffer (at pH 7.4) were prepared, and 220 μL was added to each well of a 96-well plate. An amount of 5 μL of the serially diluted test compounds was added, and the plate was incubated at 37 °C for 10 min. To start the reaction, 25 μL of cofactor was added. The production of fluorescence was then measured every minute over a 30 min time frame at 37 °C. A summary of the assay conditions is given in Table 5.

There is an urgent clinical need for a safe, efficacious stimulant of gastric emptying; current therapies include erythromycin (an antibiotic with additional properties which preclude chronic use) and metoclopramide (a 5-hydroxytryptamine type 4 receptor agonist and an antagonist at brain D2 receptors, associated with movement disorders). To move away from the complex motilide structure of erythromycin, a small molecule motilin receptor agonist, GSK962040, was identified and characterized. The compound was evaluated using recombinant human receptors, rabbit and human isolated stomach preparations known to respond to motilin and in vivo, by measuring its ability to increase defecation in conscious rabbits. At the human motilin receptor, the pEC50 (the negative logarithm to base 10 of the EC50 value, the concentration of agonist that produces 50% of the maximal response) values for GSK962040 and erythromycin as agonists were, respectively, 7.9 and 7.3; GSK962040 had no significant activity at a range of other receptors (including ghrelin), ion channels and enzymes. In rabbit gastric antrum, GSK962040 300 nmol L(-1)-10 micromol L(-1) caused a prolonged facilitation of the amplitude of cholinergically mediated contractions, to a maximum of 248 +/- 47% at 3 micromol L(-1). In human-isolated stomach, GSK962040 10 micromol L(-1), erythromycin 10 micromol L(-1) and [Nle13]-motilin 100 nmol L(-1), each caused muscle contraction of similar amplitude. In conscious rabbits, intravenous doses of 5 mg kg(-1) GSK962040 or 10 mg kg(-1) erythromycin significantly increased faecal output over a 2-h period. Together, these data show that GSK962040, a non-motilide structure, selectively activates the motilin receptor. Simplification of the structural requirements to activate this receptor greatly facilitates the design of potentially new medicines for gastroparesis[1].

Rat Pharmacokinetic Studies[3]
\nThe pharmacokinetics and oral bioavailability of the HCl salt of compound 12/Camicinal (GSK962040) were investigated in the male Sprague−Dawley rat (n = 3). The study was carried out on 2 study days with a period of 2 days between each study day. On study day 1, compound 12 was dissolved in 0.9% (w/v) saline at a target concentration of 0.2 mg of free base/mL. Compound 12 was administered as a 1 h intravenous infusion at 5 (mL/kg)/h to three rats to achieve a target dose of 1 mg of free base/kg. Serial blood samples were taken from each rat up to 12 h after the start of the infusion. On study day 2, compound 12 was suspended in 1% (w/v) methylcellulose at a target concentration of 1 mg of free base/mL. Three rats received an oral gavage dose of compound 12 administered at 5 mL/kg to achieve a target dose of 5 mg of free base/kg. Serial blood samples were taken from each rat up to 12 h after dosing. Diluted blood samples were analyzed for compound 12 by LC/MS/MS (LLQ was 5 ng/mL, 0.012 μM).
\n\nThe systemic exposure of the HCl salt of compound 16 following oral suspension administration was investigated the male Sprague−Dawley rat. Compound 16 was dosed to three rats orally by gavage at a target dose of 5 mg of free base/kg. Compound 16 was prepared on the day of dosing in 1% (w/v) aqueous methylcellulose at a concentration of 1 mg of free base/mL and administered at 5 mL/kg. Serial blood samples were taken from each rat up to 8 h after dose administration. Diluted blood samples were analyzed for parent compound by LC/MS/MS.\n

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\n\nDog Pharmacokinetic Studies[3]
\nThe pharmacokinetics and oral bioavailability of the HCl salt of compound 12/Camicinal (GSK962040) were investigated in the male beagle dog (n = 3). The study was carried out on 2 study days with a period of 7 days between each study day. On study day 1, compound 12 was dissolved in 0.9% (w/v) saline at a concentration of 0.4 mg of free base/mL. Compound 12 was administered as a 1 h intravenous infusion at 5 (mL/kg)/h to three dogs to achieve a target dose of 2 mg of free base/kg. Serial blood samples were taken from each dog up to 30 h after the start of the infusion. On study day 2, compound 12 was suspended in 1% (w/v) methylcellulose at a concentration of 1 mg of free base/mL. The same three dogs each received an oral gavage dose of compound 12 administered at 5 mL/kg to achieve a target dose of 5 mg of free base/kg. Serial blood samples were taken from each dog up to 30 h after dosing. Diluted blood samples were analyzed for compound 12 by LC/MS/MS (LLQ was 5 ng/mL, 0.012 μM).

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\n\n\nBackground: GSK962040, a small molecule motilin receptor agonist, was identified to address the need for a safe, efficacious gastric prokinetic agent. However, as laboratory rodents lack a functional motilin system, studies in vivo have been limited to a single dose, which increased defecation in rabbits. Motilin agonists do not usually increase human colonic motility, so gastric prokinetic activity needs to be demonstrated.\n
\nMethods: The effect of intravenous GSK962040 on gastro-duodenal motility was assessed in fasted dogs implanted with strain gauges. Activity was correlated with blood plasma concentrations of GSK962040 (measured by HPLC-MS/MS) and potency of GSK962040 at the dog recombinant receptor [using a Fluorometric Imaging Plate Reader (Molecular Devices, Wokingham, UK) after expression in HEK293 cells].\n
\nKey results: GSK962040 activated the dog motilin receptor (pEC(50) 5.79; intrinsic activity 0.72, compared with [Nle(13) ]-motilin). In vivo, GSK962040 induced phasic contractions, the duration of which was dose-related (48 and 173 min for 3 and 6 mg kg(-1) ), driven by mean plasma concentrations >1.14 μmol L(-1) . After the effects of GSK962040 faded, migrating motor complex (MMC) activity returned. Migrating motor complex restoration was unaffected by 3 mg kg(-1) GSK962040 but at 6 mg kg(-1) , MMCs returned 253 min after dosing, compared with 101 min after saline (n=5 each).\n
\nConclusions & inferences: The results are consistent with lower potency for agonists at the dog motilin receptor, compared with humans. They also define the doses of GSK962040 which stimulate gastric motility. Correlation of in vivo and in vitro data in the same species, together with plasma concentrations, guides further studies and translation to other species.[2]

The study found that the presence of a more polar cyano group was unfavorable to the oral pharmacokinetics of compound 16. In male Sprague-Dawley rats, the oral exposure of compound 16 was low and unstable, while the exposure of the fluorinated analog 12 was higher and more stable. After determining its intravenous pharmacokinetics, the oral bioavailability (Fpo) of compound 12 was found to be 48 ± 13%. This promising result prompted us to determine the pharmacokinetics of compound 12 in male beagle dogs, and we were pleased to find that its oral bioavailability was 51 ± 16%. [1] Compound 12 was also evaluated for its inhibitory effects on other major human CYP isoenzymes, and it was found to have good inhibitory properties (1A2, 2C19, 2C9 IC50 > 100 μM, 2D6 IC50 = 34 μM). In addition, no TDI interaction was observed on these isoenzymes on CYP3A4 with 7BQ as a substrate. It exhibits high selectivity for the closely associated human ghrelin receptor (pEC50 < 6.0) and no interaction was observed on hERG channels (pIC50 = 4.8 in binding assay). In vitro plasma protein binding is acceptable (83% in humans, 63% in rats), and it is highly soluble in water and a range of simulated gastrointestinal fluids (hydrochloride, >1 mg/mL). [1] Furthermore, the duration of action of compound 12 in native tissue assays of the rabbit antrum has been determined. Full results will be reported elsewhere, but in brief, compound 12 showed a sustained effect (T1/2 = 46.9 ± 5.0 min at 3 μM) compared to the short-acting effect of [Nle13]motilin (0.3 μM, T1/2 = 11.4 ± 1.5 min). Its duration of action is also longer than that of erythromycin 1 (T1/2 = 24.0 ± 5.6 min at 3 μM), which has been successfully used clinically for low-dose repeated administration to improve gastric emptying. Therefore, these data may suggest that compound 12 is less likely to develop rapid tolerance when administered appropriately in vivo. The overall selectivity of compound 12, its efficacy in human native gastric tissue, and its prokinetic activity in a rabbit whole intestinal transport model have also been determined, and these data will be reported in full elsewhere. [1]

[1]. GSK962040: a small molecule, selective motilin receptor agonist, effective as a stimulant of human and rabbit gastrointestinal motility. Neurogastroenterol Motil, 2009. 21(6): p. 657-64, e30-1.

[2]. GSK962040: a small molecule motilin receptor agonist which increases gastrointestinal motility in conscious dogs. Neurogastroenterol Motil, 2011. 23(10): p. 958-e410.

[3]. Discovery of N-(3-fluorophenyl)-1-[(4-([(3S)-3-methyl-1-piperazinyl]methyl)phenyl)acetyl]-4-pi peridinamine (GSK962040), the first small molecule motilin receptor agonist clinical candidate. J Med Chem, 2009. 52(4): p. 1180-9.

[4]. Regional- and agonist-dependent facilitation of human neurogastrointestinal functions by motilin receptor agonists. Br J Pharmacol, 2012. 167(4): p. 763-74.

Camicinal belongs to the acetamide class of compounds. Camicinal has been used in clinical trials for the treatment of gastroparesis. N-(3-fluorophenyl)-1-[(4-([(3S)-3-methyl-1-piperazinyl]methyl)phenyl)acetyl]-4-piperidinamine 12 (GSK962040) is a novel small molecule motilin receptor agonist. It has excellent activity against both recombinant human motilin receptor and native rabbit motilin receptor, and its agonist activity can enhance the amplitude of nerve-mediated contractions in isolated gastric antrum tissue. Compound 12 has shown promising pharmacokinetic characteristics in rats and dogs, and these results, combined with further analysis in native human tissue and rabbit gastrointestinal transport models, make it a candidate for further development. [1] In summary, compound 12 exhibits very desirable overall properties and has been selected as a candidate for further development. Therefore, compound 12 is a novel selective small molecule motilin receptor agonist (molecular weight 424), which was developed through a broad structure-activity relationship study based on the activity of recombinant human motilin receptor and is the first compound of its kind to enter Phase I clinical trials, and is expected to be used to treat diseases associated with delayed gastric emptying. Therefore, compound 12 is a novel selective small molecule motilin receptor agonist (molecular weight 424), which was developed through a broad SAR program based on the activity of recombinant human motilin receptor and is the first compound of its kind to enter Phase I clinical trials, and is expected to be used as a drug to treat diseases associated with delayed gastric emptying. [1]

C25H33N4OF.HCL

461.01506

460.24

C, 65.13; H, 7.43; Cl, 7.69; F, 4.12; N, 12.15; O, 3.47

923565-22-4

Camicinal;923565-21-3

25222897

Typically exists as solid at room temperature

3.542

3

5

6

32

560

1

O=C(N1CCC(NC2=CC=CC(F)=C2)CC1)CC3=CC=C(CN4C[C@H](C)NCC4)C=C3.Cl

MZEVMNJFEUATKJ-FYZYNONXSA-N

InChI=1S/C25H33FN4O.ClH/c1-19-17-29(14-11-27-19)18-21-7-5-20(6-8-21)15-25(31)30-12-9-23(10-13-30)28-24-4-2-3-22(26)16-24;/h2-8,16,19,23,27-28H,9-15,17-18H2,1H3;1H/t19-;/m0./s1

1-[4-(3-fluoroanilino)piperidin-1-yl]-2-[4-[[(3S)-3-methylpiperazin-1-yl]methyl]phenyl]ethanone;hydrochloride

Camicinal hydrochloride; 923565-22-4; GSK962040 (HCl salt); GSK962040 (hydrochloride); GSK962040 hydrochloride; 1-[4-(3-fluoroanilino)piperidin-1-yl]-2-[4-[[(3S)-3-methylpiperazin-1-yl]methyl]phenyl]ethanone;hydrochloride; CHEMBL489679;

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 (e.g. under nitrogen), 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 : ~100 mg/mL (~216.91 mM)
DMSO : ~100 mg/mL (~216.91 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.42 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 25.0 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: ≥ 2.5 mg/mL (5.42 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 25.0 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: ≥ 2.5 mg/mL (5.42 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 25.0 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 (216.91 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.)