Motilin Receptor ( pEC50 = 7.9 )

Camicinal (GSK962040) did not exhibit any noteworthy effects at other receptors, ion channels, or enzymes, such as ghrelin. Camicinal (GSK962040) 300 nmol L 1-10 μmol L 1 prolongedly facilitated the amplitude of cholinergically mediated contractions in the rabbit gastric antrum, reaching a maximum of 248 ± 47% at 3 μmol L 1. Motilin, erythromycin, and Camicinal (GSK962040) had pEC50 values of 10.4 ± 0.01 (n = 770), 7.3 ± 0.29 (n = 4), and 7.9 ± 0.09 (n = 17), respectively [1]. Camicinal (GSK962040) compared with [Nle13]-motilin, activated the dog motilin receptor (pEC50 5.79; intrinsic activity 0.72)[2]. Camicinal (GSK962040) was the drug of choice because, at CYP3A4, its initial IC50 values were much higher than our recommended threshold of 10 μM [3].

Camicinal (GSK962040) (5 mg free base kg 1) additionally caused a rise in total faecal weight (21.2 ± 4.5 g; P < 0.05) during the 2-hour postdose period[1]. Camicinal (GSK962040) caused dose-dependent phasic contractions, lasting 48 and 173 minutes for 3 and 6 mg kg1, respectively, and were triggered by mean plasma concentrations greater than 1.14 μmol L 1. Migration of the migrating motor complex (MMC) resumed after GSK962040's effects subsided. 3 mg kg 1 GSK962040 had no effect on the restoration of the migrating motor complex, but at 6 mg kg 1, MMCs returned 253 min after dosing, as opposed to 101 min after saline (n = 5 each)[2]. It was discovered that Camicinal (GSK962040) had an oral bioavailability (Fpo) of 48 (13%). When compared to the short-lived effect of [Nle13]motilin (T1/2 ) 11.4 ( 1.5 min at 0.3 μM), camecicinal (GSK962040) exhibits a long-lasting effect (T1/2 ) 46.9 ( 5.0 min at 3 μM) [3]. Only the fundus and small intestine showed reduced cholinergic activity when camicinal (GSK962040) was administered [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]
The 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).
The 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.

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Dog Pharmacokinetic Studies[3]
The 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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Background: 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.
Methods: 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].
Key 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).
Conclusions & 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]

It was found that the presence of the more polar cyano group was detrimental to the oral pharmacokinetics of compound 16. In the male Sprague−Dawley rat, 16 showed low and variable oral exposure whereas the fluoro analogue 12 gave higher and more consistent levels. Following determination of its intravenous pharmacokinetics, the oral bioavailability (Fpo) of 12 was found to be 48 ± 13%. This highly promising result led us to determine the pharmacokinetic profile of 12 in the male beagle dog, and we were pleased to find that its oral bioavailability was 51 ± 16%.[1]
Compound 12 has also been assessed for its inhibition of the other major human CYP isoforms, and it was found to possess a favorable profile (1A2, 2C19, 2C9 IC50 > 100 μM, 2D6 IC50 = 34 μM). Furthermore, there were no TDI liabilities at any of these isoforms or at CYP3A4 with 7BQ as the substrate. Selectivity at the closely related human ghrelin acceptor was high (pEC50 < 6.0), and there were no liabilities at the hERG channel (binding assay pIC50 = 4.8). In vitro plasma protein binding levels were acceptable (human 83%, rat 63%), and solubility in water and a range of simulated gastrointestinal fluids was high (HCl salt, >1 mg/mL).[1]
Additionally, the duration of action of 12 in the rabbit gastric antrum native tissue assay has been determined. This will be reported in full elsewhere, but in summary, 12 shows a long lasting effect (T1/2 = 46.9 ± 5.0 min at 3 μM) when compared with the short-lived effect of [Nle13]motilin (T1/2 = 11.4 ± 1.5 min at 0.3 μM). Its duration of action is also longer than that of erythromycin 1 (T1/2 = 24.0 ± 5.6 min at 3 μM), which is used successfully in the clinic to improve gastric emptying when dosed repeatedly at a low level. Therefore, these data may indicate a low potential for 12 to cause tachyphylaxis when dosed appropriately in vivo. General selectivity, efficacy in human native stomach tissue, and prokinetic activity in a rabbit model of whole gut transit have also been determined for 12, and these data will also 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 is a member of acetamides.
Camicinal has been used in trials studying the treatment of Gastroparesis.

C25H33FN4O

424.55412

424.264

C, 70.73; H, 7.83; F, 4.47; N, 13.20; O, 3.77

923565-21-3

Camicinal hydrochloride; 923565-22-4

15984937

Solid powder

3.542

2

5

6

31

560

1

C(N1CCC(NC2C=CC=C(F)C=2)CC1)(=O)CC1C=CC(CN2CCN[C@@H](C)C2)=CC=1

RZKDEGZIFSJVNA-IBGZPJMESA-N

InChI=1S/C25H33FN4O/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/t19-/m0/s1

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

Camicinal; GSK-962040; GSK962040; 923565-21-3; Camicinal free base; Camicinal [USAN:INN]; UNII-3C8348951H;GSK 962040

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO: ~100 mg/mL (~235.5 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.89 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 (5.89 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 (5.89 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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 (Please use freshly prepared in vivo formulations for optimal results.)