P2Y12 Receptor

Pharmacokinetic Parameters of Clopidogrel Thiolactone after Oral Administration of Clopidogrel or 9a to Rats The dose of clopidogrel or 9a at 24 μmol kg–1 was orally administered to SD male rats. Blood was collected at 0 h (before dosing) and 0.25, 0.5, 1, 2, 4, 6, 8, 24 h postdose. The dose of clopidogrel thiolactone at 8 μmol kg–1 was intravenous administration to SD male rats to determine the conversion rate or bioavailability of clopidogrel or 9a to clopidogrel thiolactone. Blood was collected at 0 h (before dosing) and 0.083, 0.167, 0.5, 1, 2, 4, 6, 8, 24 h postdose. After sample cleanup, the plasma samples were subjected to LC–MS/MS analysis to determine the plasma concentrations of clopidogrel thiolactone (Figure 5). After oral dosing of clopidogrel, the Cmax, Tmax, t1/2, and AUC0–∞ of clopidogrel thiolactone in plasma were 6.93 ± 3.36 μg L–1, 0.583 ± 0.382 h, 2.48 ± 0.466 h, and 32.2 ± 10.9 μg·h L–1, respectively. After oral dosing of 9a, the Cmax, Tmax,t1/2, and AUC0–∞ of clopidogrel thiolactone in plasma were 67.2 ± 42.3 μg L–1, 1.17 ± 0.764 h, 2.19 ± 1.68 h, and 211 ± 119 μg·h L–1, respectively. The aforementioned data proved that after oral administration, 9a could be readily converted into clopidogrel thiolactone, and bioavailability of clopidogrel thiolactone generated from 9a was 6-fold higher than that generated from clopidogrel at the same dose.[1]

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Clopidogrel (2mg and 10mg/kg/day) reduces the expression of the EGF receptor, phosphorylated extracellular signal-regulated kinase (PERK), and ulcer-induced gastric epithelial cell proliferation at the rat ulcer margin significantly. In congestive heart failure-ridden rats, clopidogrel increases NO bioavailability and endothelial function. Rats with congestive heart failure (CHF) treated with clopidogrel exhibit increased phosphorylation of AKT and eNOS. The rabbit bleeding time prolongation caused by ear transection is only affected in an additive manner by the clopidogrel/aspirin combination, indicating that the combined inhibition of cyclooxygenase and ADPs effects results in a markedly enhanced antithrombotic efficacy.

Clopidogrel could be metabolically activated to form its active metabolite (AM, 5) via the thiolactone intermediate (4a, Clopidogrel thiolactone) under in vitro incubation conditions with liver microsomes.The formed 2-hydroxytetrahydrothienopyridine is a tautomer of thiolactone 4a, which is the metabolic intermediate resulting from the first oxidative activation of clopidogrel. The metabolic activation pathways of clopidogrel and 9a converge after the first steps through this 2-hydroxytetrahydrothienopyridine–thiolactone tautomerization and share the subsequent pathways including the second step of NAPDH-dependent oxidative ring-opening of 4a that eventually lead to active metabolite formation[1].

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Prasugrel and clopidogrel are antiplatelet prodrugs that are converted to their respective active metabolites through thiolactone intermediates (e.g. Clopidogrel thiolactone). Prasugrel is rapidly hydrolysed by esterases to its thiolactone intermediate, while clopidogrel is oxidized by cytochrome P450 (CYP) isoforms to its thiolactone. The conversion of both thiolactones to the active metabolites is CYP mediated. This study compared the efficiency, in vivo, of the formation of prasugrel and clopidogrel thiolactones and their active metabolites. The areas under the plasma concentration versus time curve (AUC) of the thiolactone intermediates in the portal vein plasma after an oral dose of prasugrel (1 mg kg(-1)) and clopidogrel (0.77 mg kg(-1)) were 15.8 +/- 15.9 ng h ml(-1) and 0.113 +/- 0.226 ng h ml(-1), respectively, in rats, and 454 +/- 104 ng h ml(-1) and 23.3 +/- 4.3 ng h ml(-1), respectively, in dogs, indicating efficient hydrolysis of prasugrel and little metabolism of clopidogrel to their thiolactones in the intestine. The relative bioavailability of the active metabolites of prasugrel and clopidogrel calculated by the ratio of active metabolite AUC (prodrug oral administration/active metabolite intravenous administration) were 25% and 7%, respectively, in rats, and 25% and 10%, respectively, in dogs. Single intraduodenal administration of prasugrel showed complete conversion of prasugrel, resulting in high concentrations of the thiolactone and active metabolite of prasugrel in rat portal vein plasma, which demonstrates that these products are generated in the intestine during the absorption process. In conclusion, the extent of in vivo formation of the thiolactone and the active metabolite of prasugrel was greater than for clopidogrel's thiolactone and active metabolite.[2]

Sprague-Dawley male rats
8 μm/kg; orally clopidogrel (24 μm/kg)
Intravenous injection; collected blood at 0 h (before dosing) and 0.083, 0.167, 0.5, 1, 2, 4, 6, 8, 24 h postdose.

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Pharmacokinetic Studies[1]
Sprague–Dawley male rats (SPF grade) were used to determine oral bioavailability and PK parameters of the clopidogrel and 9a following oral and intravenous administration. In this study, Clopidogrel Thiolactone (4a) was measured by LC/MS/MS analysis to determine the PK parameters for both clopidogrel and 9a. PK parameters were calculated using a noncompartmental model. Solutions of clopidogrel and 9a in N,N-dimethylacetamide (DMA)/polyethylene glycol 400 (PEG 400) (v/v 5:95) at 1.26 and 1.14 mg/mL were prepared for oral dosing. A solution of clopidogrel thiolactone in DMA/PEG 400/saline (v:v:v 5:15:80) at 0.54 mg/mL was prepared for intravenous administration to determine the oral bioavailability of both clopidogrel and 9a to be converted to clopidogrel thiolactone. The solution formulations of clopidogrel thiolactone along with clopidogrel and 9a were administered separately by bolus injection into a tail vein and by oral gavage. Blood samples (about 0.20 mL) were collected from the retro-orbital plexus from three animals in each treatment group at 0 (predose), 0.083, 0.167. 0.5, 1, 2, 4, 6, 8, and 24 h for iv and 0 (predose), 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h for po. Calibration standards with clopidogrel thiolactone concentrations from 0.5 to 1000 ng/mL were prepared by serial dilution in pretreated naive rat plasma. A portion (25 μL) of each calibration standard and unknown study sample was mixed with 25 μL of acetonitrile containing the internal standard followed by addition of 200 μL of MTBE into each sample. All samples were vortex-mixed for 3 min. The mixture was then centrifuged at 15700g at 4 °C for 3 min. The supernatants containing the organic component for each sample were used for analysis. The lower limit of quantitation was 0.5 ng/mL.

[1]. Overcoming clopidogrel resistance: discovery of vicagrel as a highly potent and orally bioavailable antiplatelet agent. J Med Chem. 2012 Apr 12;55(7):3342-52.

[2]. Comparison of formation of thiolactones and active metabolites of prasugrel and clopidogrel in rats and dogs. Xenobiotica. 2009 Mar;39(3):218-26.

2-oxoclopidogrel is an organic molecular entity.

C16H16CLNO3S

337.82

337.053

C, 56.89; H, 4.77; Cl, 10.49; N, 4.15; O, 14.21; S, 9.49

1147350-75-1

Clopidogrel; 113665-84-2; Clopidogrel hydrogen sulfate; 120202-66-6

56848893

Brown to reddish brown solid powder

1.4±0.1 g/cm3

488.1±45.0 °C at 760 mmHg

249.0±28.7 °C

0.0±1.2 mmHg at 25°C

1.640

2.96

0

5

4

22

496

1

ClC1=C([H])C([H])=C([H])C([H])=C1[C@@]([H])(C(=O)OC([H])([H])[H])N1C([H])([H])C2=C([H])C(=O)SC2([H])C([H])([H])C1([H])[H]

JBSAZVIMJUOBNB-WUJWULDRSA-N

InChI=1S/C16H16ClNO3S/c1-21-16(20)15(11-4-2-3-5-12(11)17)18-7-6-13-10(9-18)8-14(19)22-13/h2-5,8,13,15H,6-7,9H2,1H3/t13?,15-/m0/s1

methyl (2S)-2-(2-chlorophenyl)-2-(2-oxo-4,6,7,7a-tetrahydrothieno[3,2-c]pyridin-5-yl)acetate

2934.99.03.00

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.40 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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 (Please use freshly prepared in vivo formulations for optimal results.)