Human fiver microsomes

Powell PK, Wolf I, Lasker JM. Identification of CYP4A11 as the major lauric acid omega-hydroxylase in human fiver microsomes. Arch Biochem Biophys 1996 335( 1 ) 219—226. 

However, it has also been reported that quinidine increased the 4 -hydroxylation of Awarfarin and the 10-hydroxylation of 7 -warfarin in human fiver microsomes and intact hepatocytes by stimulation of CYP3A4 (83). The increases were concentration-dependent and respectively maximized at about three and five times control values. In contrast, warfarin did not affect the 3-hydroxylation of quinidine. These results are consistent with previous findings suggesting that there is more than one binding site on CYP3A4 through which interactions can occur. 

Table 3 lists the dose and incidence of the hepatotoxicity for various drugs, some of which have been withdrawn as a result of these findings. Some of the drugs show effects on liver function earlier than the actual onset of liver toxicity, with serum aminotransferase levels raised by threefold the upper limit of normal (ULN). The incidence of this is much higher and can show a classical dose response for instance, tolcapone, a catechol-O-methyltransferase inhibitor used as an adjunct to levodopa in Parkinson s disease, produces threefold the ULN in 1-3% of patients receiving 100 mg TID and 3.7% of patients receiving 200 mg TID (Olanow and Watkins 2007). Tolcapone, an o-nitrocatechol, is metabohzed to reactive intermediates - o-quinone or quinoneimine species - by human fiver microsomes (Smith et al. 2003). 

Enzyme concentrations in in vitro incubations will determine rate of turnover. Typical concentrations for human fiver microsomes range between 0.5 and l.Omg/mL protein. At concentrations above 2mg/mL the nonspecific binding of parent drug and metabolites may be too great to yield useful information. Typical cell concentrations for hepatocytes range from 0.5 to 1.0 million cells/mL (Williams et al., 2003). 

Iwatsubo T, Suzuki H, Shimada N, Chiba K, Ishizaki T, Green CE, Tyson CA, Yokoi T, Kamataki T, Sugiyama Y. Prediction of in vivo hepatic metabolic clearance of YM796 from in vitro data by use of human fiver microsomes and recombinant P-450 isozymes. J Pharmacol Exp Ther 1997a 282 909—919. 

Gyoubu, K. and M. Miyazawa, 2007. In vitro metabolism of (-)-camphor using human fiver microsomes and CYP2A6. Biol. Pharm. Bull., 30 230-233. 

With this focus on CYP and fiver metabolism, most companies have established high throughput assays to measure compound stability in the presence of human (or preclinical species) fiver microsomes [49]. Disappearance of starting compound from an incubation with microsomes is monitored. Measurement at a single time point enables a rank-ordering of compounds for stability based on percent of parent compound remaining acquisition of data at multiple time points allows determination of half-life, intrinsic clearance, and extrapolation to a predicted in vivo clearance [50]. 

T rani -nonachlor, a major component of technical chlordane, was frequently found as the major chlordane residue in humans, whereas oxychlordane was the major component in rats fed technical chlord e. Trans-nonachlor is converted efficiently by rat fiver microsomes to frans-chlordane, but this ability is lacking in humans, resulting in the accumulation of frans-nonachlor in humans. 

Strychnine is rapidly metabolized by the liver microsomal enzyme system requiring NADPH and O2. Five metabolites formed in vitro by rabbit fiver were isolated and identified as 2 -hydroxystrycfinine, 11,12-defiydrostrychnine, strychnine-21, 22-epoxide, 21,22-dihydroxy-22-hydrostrychnine, and strychnine-N-oxide which was the major metabolite and accounted for approximately 15% of the metabolized strychnine. AU other metabolites accounted for less than 1% (Mishimaetfl/., 1985). Similar metabolites were identified in rat urine where the major metabolite was strychnine-21,22-epoxide (Oguri et a/., 1989). The metabolic fate of strychnine in humans is unknown. 

Similar to the techniques used for calculation of chemical disposition parameters, in vivo biotransformation kinetic parameters of a substrate can be estimated from in vitro systems such as microsomes, freshly isolated hepatocytes, fiver slices, and isolated perfused livers (24). Intrinsic clearance or Michaelis-Menten parameters for the whole liver can also be obtained by scaling in vitro parameters based on the cytochrome P450 enzyme content (25-27). These parameters can also be estimated from in vitro data obtained from recombinant human CYP systems, and also through allometric scaling of clearance estimates from animal PBPK models. 

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