This article is available online at http://dmd.aspetjournals.org ABSTRACT:Midazolam, triazolam (TRZ), testosterone, and nifedipine have all been widely used as probes for in vitro metabolism of CYP3A. We used these four substrates to assess the contributions of CYP3A4 and CYP3A5 to in vitro biotransformation in human liver microsomes (HLMs) and in recombinant enzymes. Recombinant CYP3A4 and CYP3A5 (rCYP3A4 and rCYP3A5) both produced 1-OH and 4-OH metabolites from midazolam and triazolam, 6␤-hydroxytestosterone from testosterone, and oxidized nifedipine from nifedipine. Overall, the metabolic activity of CYP3A5 was less than that of CYP3A4. Ketoconazole potently inhibited midazolam, triazolam, testosterone, and nifedipine metabolite formation in HLMs and in rCYP3A4. The inhibitory potency of ketoconazole in rCYP3A5 was about 5-to 19-fold less than rCYP3A4 for all four substrates. In testosterone interaction studies, testosterone inhibited 1-OH-TRZ formation, but significantly activated 4-OH-TRZ formation in HLMs and rCYP3A4 but not in rCYP3A5. Oxidized nifedipine formation was inhibited by testosterone in rCYP3A4. However, in rCYP3A5, testosterone slightly activated oxidized nifedipine formation at lower concentrations, followed by inhibition. Thus, CYP3A4 and CYP3A5 both contribute to midazolam, triazolam, testosterone, and nifedipine biotransformation in HLMs, with CYP3A5 being metabolically less active than CYP3A4 in general. Because the inhibitory potency of ketoconazole in rCYP3A5 is substantially less than in rCYP3A4 and HLMs, CYP3A5 is probably less important than CYP3A4 in drug-drug interactions involving ketoconazole and CYP3A substrates.In humans, CYP3A represents one of the most important subfamilies of the P450 1 superfamily. The CYP3A subfamily is the most abundantly expressed P450 in human liver, and CYP3A is involved in the biotransformation of approximately 50% of drugs that are metabolized (Komori et al., 1990). As a result, drug-drug interactions associated with modulation of CYP3A-mediated metabolism can be of substantial clinical importance. CYP3A5 is present in significant quantities in 20 to 60% of human liver and is reported to account for at least 50% of total CYP3A in those individuals (Wrighton et al., 1989(Wrighton et al., , 1990Kuehl et al., 2001, Lin et al., 2002. When the expression of CYP3A5 was examined in different age groups, it was observed that CYP3A5 was detected in a significantly higher percentage of children and adolescents (19 years old or under) as compared with the remaining population (47% versus 24%, respectively) (Wrighton et al., 1990). CYP3A5 demonstrates 84% amino acid sequence homology to CYP3A4, and there is substantial overlap between substrate specificities of CYP3A4 and CYP3A5 (Wrighton and Stevens, 1992). Hepatic CYP3A5 content ranges from 2 to 202 pmol/mg of microsomal protein and shows large interindividual variations (Aoyama et al., 1989;Kuehl et al., 2001). The content of CYP3A4 in liver ranges from 47 to 523 pmol/mg of microsomal protein, and also shows ...

Resolution of parasitemia was inadequate with monotherapy with either azithromycin or chloroquine, but combination therapy provided substantially improved clinical and parasitologic outcomes. The combination of azithromycin and chloroquine may be an effective alternative treatment for falciparum malaria and deserves further study.

Azithromycin has demonstrated activity in a prevention of Plasmodium vivax infection, but no controlled treatment studies have been performed. We conducted a double-blinded trial in P. vivax malaria in which patients were randomized to either azithromycin 1,000 mg q.d. x 3 or chloroquine 600 mg q.d. x 2 then 300 mg on Day 3 followed by primaquine on Days 7 through 20. Eighty-five of 97 (88%) of those on azithromycin and 101 of 102 (99%) of those on chloroquine [difference 11%; 95% CI: -18, -4] were clinically cured at Day 7. The Day 28 results were similar [89% versus 99%, azithromycin versus chloroquine, respectively]. Parasitologic success was seen in 81 of 97 (84%) on azithromycin and 100 of 102 (98%) on chloroquine [difference 14%; 95% CI: -22, -6]. The median parasite clearance time was 55 hours on azithromycin and 20 hours on chloroquine (P < 0.001). Drug-related adverse events were seen in 13 of 98 (13%) on azithromycin and 24 of 102 (24%) on chloroquine (P = 0.062). Resolution of parasitemia was significantly faster with chloroquine compared with azithromycin, but azithromycin was better tolerated. These data provide support for further study of azithromycin to better define its role in the treatment of P. vivax malaria, either alone as second-line treatment or in combination with other active therapies.

We studied age-related changes in enzyme kinetic parameters in human liver microsomes (HLMs) in vitro, using triazolam (TRZ), an index of CYP3A activity. HLMs were prepared from male livers from four age groups, n ϭ 5 per group: A (14 -20 years), B (21-40 years), C (41-60 years), and D (61-72 years). Mean V max values in groups B and C for both 1-hydroxytriazolam (1-OH-TRZ) and 4-hydroxy-triazolam (4-OH-TRZ) formation were significantly greater as compared with groups A and D individually, as well as the net intrinsic clearance (sum of the two pathways). The mean net intrinsic clearance (Cl int ) values were 25.2, 89.8, 78, and 20.6 nl/min/mg protein in A, B, C, and D, respectively. TRZ Cl int correlated well with total CYP3A content (r s ϭ 0.84; P Ͻ 0.0001). Testosterone (TST) inhibited 1-OH-TRZ formation and activated 4-OH-TRZ formation in all age groups, with no significant differences among the groups; this suggests that the drug-drug interaction potential using TRZ and TST as index CYP3A substrates may not change with age. Reduced V max and Cl int for TRZ hydroxylation and CYP3A protein in livers from elderly men suggest reduced CYP3A gene expression in this group.There is considerable pharmacokinetic evidence suggesting that age-related changes occur in drug disposition (Schmucker, 1985;Greenblatt et al., 1989;von Moltke et al., 1995a). However, in some clinical studies, no age-related changes were demonstrated in the biotransformation of CYP3A substrates (see reviews by Greenblatt et al., 1982;Cotreau et al., 2004). Factors that could influence drug clearance in the elderly include the expression, content, and function of catalytically active enzymes, as well as liver mass, hepatic blood flow, and renal function (Vestal, 1982;Greenblatt et al., 1986). It has been shown that triazolam (TRZ) clearance is reduced in the elderly (Greenblatt et al., 1983a(Greenblatt et al., , 1991.In vitro studies could be helpful in examining the role of age on the metabolic activity of CYP3A and liver CYP3A content. However, the results of such studies have been inconsistent. Some studies using human liver microsomes (HLMs) have shown an age-related decline in CYP3A content (George et al., 1995;Sotaniemi et al., 1997) or total P450 activity (Sotaniemi et al., 1997), whereas others have found no change associated with age in content (Shimada et al., 1994;Transon et al., 1996) or activity of CYP3A (Schmucker et al., 1990;Hunt et al., 1992;Shimada et al., 1994;Transon et al., 1996).Previous in vitro studies have evaluated the effect of age on metabolite formation rate via CYP3A-mediated biotransformation, but without consideration for enzyme affinity (Hunt et al., 1992;Transon et al., 1996), HLMs were classified into one or more classes over a wide age range, and these studies did not evaluate the possible contribution of CYP3A5 to agerelated effects on CYP3A metabolism (Hunt et al., 1992;Shimada et al., 1994).To address these issues, we examined the effect of age on TRZ metabolism in HLMs from male donors ranging in ag...

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