Identification of the active metabolite of ticlopidine from rat in vitro metabolites

Abstract

1 Ticlopidine is a well-known anti-platelet agent, but is not active by itself in vitro. We identified a metabolite with anti-platelet activity, which was generated after incubation of 2-oxo-ticlopidine with phenobarbital-induced rat liver homogenate in vitro. 2 An active moiety (UR-4501) was isolated by high-performance liquid chromatography after large-scale preparation of metabolites. 3 The chemical structure of UR-4501 was determined by a combination of liquid chromatography mass/mass spectrometry (LC/MS/MS) and nuclear magnetic resonance (NMR) analysis. 4 UR-4501 produced a concentration-dependent inhibition (3-100 microm) of ADP (10 microm)-induced human platelet aggregation, whereas 2-oxo-ticlopidine (3-100 microm) did not elicit inhibitory responses. 5 UR-4501 (10-100 microm) strongly inhibited ADP- and collagen-induced aggregation and slightly inhibited thrombin-induced aggregation. 6 The inhibition of rat washed platelet aggregation by UR-4501 (100 microm) persisted, even after the platelets had been washed twice. 7 These results suggest that UR-4501 is the molecule responsible for the in vivo activities of ticlopidine.

Figure 1

Chemical structure of ticlopidine and 2-oxo-ticlopidine.

Figure 2

HPLC chromatogram and anti-platelet activity of 2-oxo-ticlopidine metabolites. The lyophilized mixture containing metabolites of 2-oxo-ticlopidine was fractionated by HPLC. While monitoring the absorbance at 240 nm, 1-min fractions were collected and lyophilized. To assess the anti-platelet activity of the metabolites, human PRP was pre-incubated with each fraction solution for 5 min and aggregation was induced by ADP (10 μM).

Figure 3

LC/MS/MS spectra of active metabolite. (a) LC-ESI(+)/MS/MS spectrum of m/z 298 ion (MH⁺ containing ³⁵Cl isotope). (b) LC-ESI(+)/MS/MS spectrum of m/z 300 ion (MH⁺ containing ³⁷Cl isotope). (c) Primary structure of the active metabolite of ticlopidine (UR-4501) and MS fragmentation pathway. The numbers in the parenthesis indicate the m/z measured by LC-ESI(+)/MS/MS.

Figure 4

In vitro effects of the active metabolite (UR-4501) of ticlopidine on human platelet aggregation. Human platelets were pre-incubated with UR-4501, 2-oxo-ticlopidine, ticlopidine and vehicle for 1 h and aggregation was induced by ADP (10 μM). Results were expressed as the percentage of aggregation inhibition relative to vehicle control (100%). Each point is the mean ±s.e.m. (n=4). ^**P<0.01 vs control.

Figure 5

In vitro effects of the active metabolite of ticlopidine (UR-4501) on platelet aggregation agonists. Rat washed platelets were pre-incubated with UR-4501 for 5 min and aggregation was induced by ADP (a), collagen (b) and thrombin (c). Results were expressed as the mean ±s.e.m. (n=4). ^*P<0.05, ^**P<0.01 vs each control.

Figure 6

In vitro duration of anti-aggregation effects. Rat platelet-rich plasma was pre-incubated with UR-4501 (100 μM) or PGE₁ (2 μM) for 1 min, and then ADP (10 μM)-induced platelet aggregation was measured before and after washing the platelets. Results are presented as the mean ±s.e.m. (n=4). ^**P<0.01 vs each control.