48
J. Das et al. / Bioorg. Med. Chem. Lett. 12 (2002) 45–49
Table 6. Single-dose pharmacokinetic parameters of BMS-189664
Parameters
Monkeys
Beagle dogs
Parameters
Monkeys
Beagle dogs
IV Route
Dose (mmol/kg)
Half-life (h)
Clearance (mL/min/kg)
MRT (h)
Vss (L/kg)
13
(n=3)
6.4
6.4
1.8
12
(n=7)
9a
13.4
1.1
Oral route
Dose (mmol/kg)
Cmax (mM)
Tmax (h)
50
(n=3)
5.6
2.3
6
17
28
(n=7)
3
0.6
4.0
MRT (h)
F (%)
0.7
0.8
15
aValue derived from three dogs that had reliable terminal phase.
the guanidino moiety (3), are nestled in the specifity
pocket and make hydrogen bonding contacts with
Asp189. The hydroxyguanidine function in 30 binds in a
very similar fashion. The d-phenylalanine moieties lie in
the distal d-pocket formed by the side chains of Trp215,
Ile174 and Glu97A-Leu99, while the methyl sulfon-
amido (3) or the benzylsulfonamido (30) groups cover
the specificity pocket of the enzyme. The more pro-
nounced thrombin inhibitory potency of 30 (23-fold
more potent in vitro than 3) may be attributed in part to
the increased hydrophobic interactions between the
benzyl group and the residues at the surface of the
specificity pocket.
antagonist since it does not inhibit [3H]SFFLRR-NH2
binding (IC50> >100 mM) to human platelet mem-
branes. BMS-189664 also did not inhibit SFLLRNP-
stimulated platelet aggregation.
The pharmacokinetic profile of BMS-189664 was deter-
mined in several animal species (Table 6). Single iv and
oral doses of BMS-189664 were given to dogs and
cynomolgus monkeys. BMS-189664 concentrations in
plasma were determined using an LC/MS assay. Plasma
concentrations declined with a mean elimination half-
life of >6 h in both species. The oral bioavailability of
BMS-189664 based on the dose-normalized AUCs after
oral and iv dosing was 15 and 17% in dogs and monkeys,
respectively.
Because of its superior activity in vivo in the anesthe-
tized mouse model BMS-189664 (3) was characterized
further in vitro and in vivo. A modified thrombin time
(TT) was used to determine the direct inhibition of
thrombin activity in a protein-rich plasma environment.
In this assay, BMS-189664 doubled thrombin clotting
time in vitro at 51ꢀ9 nM (n=3). In studies of human
gel-filtered platelet aggregation stimulated by thrombin,
BMS-189664 inhibited thrombin induced platelet
aggregation with a pA2 value of 8.43ꢀ0.04 (n=3) with
a slope near unity (0.89ꢀ0.06). In these studies, BMS-
189664 is not likely to be acting as a thrombin receptor
In pentobarbital-anesthetized cynomolgus monkeys,
BMS-189664 inhibited arterial and venous thrombosis
at doses causing small increases in bleeding time and
more pronounced prolongation of ex vivo clotting time.
Thrombosis was induced in stenotic carotid arteries
using the Folts model9 as detailed in a representative
experiment (Fig. 3). Arterial thrombosis was disrupted
in six out of eight monkeys at a threshold dose of
1.5ꢀ0.2 mg/kg (cumulative iv dose), which increased
the APTT to 3.7ꢀ0.2 times control. Vehicle was with-
out effect in five monkeys. In other monkeys thrombosis
was induced by topical application of filter paper satu-
rated with 25% FeCl2 for 3 min to a vena cava in which
flow was impaired with a stenosis. Thrombus weight
was reduced by 46, 88, and 90% by BMS-189664 doses
of 0.5, 2, and 6 mg/kg, respectively (cumulative doses
achieved through continuous iv infusions for 1 h), while
Figure 3. BMS-189664 inhibited arterial thrombosis in monkeys. Pla-
telet-mediated reductions in blood flow were interrupted by momen-
tary removal of a stenosis on the crush-injured artery. This shaking
loose (SL) of the thrombus was required to prevent occlusion and
resulted in a transient recovery of blood flow. The stenosis was then
replaced to reinstate the cycle. BMS-189664 injections (0.2 mg/kg)
were repeated until spontaneous blood flow restoration (SP) negated
the need for SLto maintain stable blood flow. Once several SP were
observed, the stenosis was reset (RS) in an attempt to reestablish the
cyclic pattern. In this monkey a cumulative iv threshold dose of 1 mg/
kg produced a persistent SP pattern.
Figure 4. Dose-dependent effect of BMS-189664 on venous thrombo-
sis, bleeding time and ex vivo clotting time was determined in anes-
thetized monkeys. BMS-189664 was infused at 9, 25, and 100 mg/kg/
min for 1 h to achieve 0.5, 2, and 6 mg/kg, respectively.