TABLE 1

Increase in CYP3A activity in microsomes from human hepatocytes dosed once daily for 3 days with N-hydroxyformamide TACE/MMP inhibitors

Microsomes were prepared from hepatocytes collected 24 h after the last daily dose of vehicle or compound. Production of the CYP3A-specific metabolite, 6β-hydroxy testosterone, was measured by LC/MS-MS after incubation of microsomes with testosterone as described under Materials and Methods. CYP3A activity is reported as the rate of 6β-hydroxy testosterone formed per milligram of protein. A smooth spline curve was fit to the fold induction data for each TACE/MMP inhibitor, and a significant elevation in CYP3A activity was assessed as described under Materials and Methods. The minimum concentrations that caused a CYP3A fold induction different from control were 0.5, 0.9, and 7.7 μM for GW3333, GW6495, and GI4023, respectively.

Treatment
6β-Hydroxy Testosterone Formation Rate
Human Liver 1a
 Human Liver 2
pmol/min/mg protein fold induction pmol/min/mg protein fold induction
Controlb 519 1.0 3394 1.0
Rifampicin, 10 μM 3558 6.9 17287 5.1
Phenobarbital, 100 μM NM NM 4867 1.4
Phenobarbital 200 μM NM NM NM NM
GW3333, 0.1 μM NM NM 3358 1.0
GW3333, 0.5 μM NM NM 5866 1.7
GW3333, 1.0 μM 1498 2.9 8769 2.6
GW3333, 2.5 μM NM NM 14125 4.2
GW3333, 5.0 μM* 3899 7.5 17587 5.2
GW3333, 10.0 μM* NM NM 18420 5.4
GW3333, 25.0 μM 3934 7.6 NM NM
GW3333, 50.0 μM 3208 6.2 NM NM
GW3333, 100 μM 3683 7.1 NM NM
GW6495, 1.0 μM 1051 2.0 8122 2.4
GW6495, 5.0 μM 2048 3.9 9829 2.9
GW6495, 10.0 μM NM NM 10527 3.1
GW6495, 25.0 μM 2479 4.8 10303 3.0
GW6495, 100 μM 1414 2.7 7696 2.3
GI4023, 1.0 μM 658 1.3 3373 1.0
GI4023, 5.0 μM 808 1.6 2642 0.8
GI4023, 25.0 μM 1470 2.8 3314 1.0
GI4023, 100 μM 2477 4.8 6599 1.9
GI4023, 250 μM
NM
NM
5703
1.7

  • NM, not measured.

  • ↵* Significantly different (p < 0.05) CYP3A fold induction by GW3333 relative to GI4023 and GW6495 at the same concentration using a Welch two-sample t test.

  • ↵ a Hepatocytes obtained from cirrhotic liver tissue.

  • ↵ b Control hepatocytes were treated with media containing 0.1% DMSO, except for liver 1 hepatocytes, which were treated with 1% DMSO.