Can oral midazolam predict oral cyclosporine disposition?
Introduction
Cyclosporine A (cyclosporine) is a cyclic undecapeptide that has revolutionized the area of immunosuppressant therapy for solid organ transplantation since its introduction in the early 1980s (Beveridge and Calne, 1995; Ponticelli et al., 1996). Although we have gained considerable clinical experience in the use of cyclosporine over the years, management of therapy is still not without its difficulties. Not only does the drug have a narrow therapeutic window, it is notorious for exhibiting large interindividual variation in its oral bioavailability. That for the conventional formulation, Sandimmune®, ranges from less than 5 to 89% (Ptachcinski et al., 1986). Consequently, suboptimal dosing is common and can lead to either graft rejection or nephrotoxicity. Factors that can contribute to cyclosporine’s unpredictable oral bioavailability include low aqueous solubility and poor intestinal permeability, variable hepatic and intestinal first-pass metabolism, and intestinal P-glycoprotein-mediated active efflux (Benet et al., 1996). Although the microemulsion formulation, Neoral®, has improved absorption properties over Sandimmune® (Noble and Markham, 1995), it still appears to suffer from significant and variable first-pass metabolism and active intestinal efflux, as evidenced by an average oral bioavailability that ranges from 21 to 73% (Wallemacq et al., 1997; Chueh and Kahan, 1998; Ku et al., 1998). Since cyclosporine is eliminated almost entirely by metabolism — less than 1% of an administered dose appears in the bile and urine as unchanged drug (Ptachcinski et al., 1986) — many investigators have focused on this contributing factor to interindividual variability in cyclosporine oral bioavailability.
Cyclosporine is biotransformed to more than 20 metabolites, all of which retain the cyclic structure (Christians and Sewing, 1993). Kronbach et al. (1988), using human liver microsomes, identified cytochrome P450 3A (CYP3A)1 as the principal enzyme subfamily catalyzing the formation of the three primary metabolites observed in vivo (AM1, AM9, and AM4N). In vivo and in vitro investigations later revealed that CYP3A in the small intestine, in addition to the liver, participates in the first-pass metabolism of cyclosporine (Kolars et al., 1991; Webber et al., 1992). Moreover, CYP3A in the small intestine may be more susceptible to the effects of inhibitors (Watkins, 1992; Gomez et al., 1995; Lown et al., 1997; Gorski et al., 1998) and inducers (Hebert et al., 1992; Wu et al., 1995; Fromm et al., 1996; Holtbecker et al., 1996) compared to CYP3A in the liver. Therefore, a priori assessment of combined hepatic and intestinal CYP3A activity could aid in the optimization of cyclosporine dosage regimens as well as in the identification of individuals most at risk to interactions involving cyclosporine and known (e.g., ketoconazole, itraconazole, diltiazem, erythromycin, grapefruit juice, rifampin, phenytoin) (Watkins, 1992; Compana et al., 1996; Ku et al., 1998; Foradori et al., 1998) or as yet unknown CYP3A activity modulators.
A clinically applicable method to assess an individual’s ability to metabolize CYP3A substrates requires an enzyme-selective probe. Several are described in the literature and include the intravenous erythromycin breath test (Watkins et al., 1989); the oral dapsone recovery ratio (May et al., 1992, May et al., 1994); the oral dextromethorphan/3-methoxymorphinan urinary ratio (Jones et al., 1996); and the urinary ratio of endogenous 6β-hydroxycortisol to free cortisol (Bienvenu et al., 1991; Horsmans et al., 1992). Each has applications and recognized limitations (Watkins, 1994; Streetman et al., 2000). For example, although the erythromycin breath test has been used successfully to assess hepatic CYP3A levels (Cheng et al., 1997; Lown et al., 1997; Hirth et al., 2000), it cannot be used to assess intestinal levels as intravenous administration avoids significant metabolism by the gut. Dapsone is administered orally, thus formation of its hydroxylamine metabolite is likely a reflection of both intestinal and hepatic CYP3A activities. However, another CYP isoform, CYP2E1, was found to account for the majority of dapsone hydroxylamine formation at concentrations observed in vivo, raising questions of CYP3A selectivity (Mitra et al., 1995). Dextromethorphan is also administered orally, but in addition to the polymorphically expressed CYP2D6 being the major enzyme responsible for the overall metabolism of this drug, at least a 72-h urine collection is required (11-day if the subject is a CYP2D6 poor metabolizer) (Jones et al., 1996), rendering this test impractical (Kashuba et al., 1999). Finally, for reasons not well understood, the urinary ratio of 6β-hydroxycortisol to free cortisol appears to only be a sensitive marker for CYP3A induction and is a poor measure of basal CYP3A activity (Ged et al., 1989). Collectively, these findings indicate the need for a versatile, yet specific, CYP3A probe.
Our laboratory and others have investigated the utility of midazolam as an in vitro and in vivo probe for both intestinal and hepatic CYP3A metabolic activity (Gorski et al., 1994; Lown et al., 1994; Thummel et al., 1994, Thummel et al., 1996; Paine et al., 1996, Paine et al., 1997). Like cyclosporine, midazolam is eliminated almost entirely by metabolism (Dundee et al., 1984) and principally by CYP3A (Fabre et al., 1988; Kronbach et al., 1989). In addition, orally administered midazolam undergoes significant intestinal and hepatic first-pass metabolism (Thummel et al., 1996; Paine et al., 1996). Hence, midazolam has the desirable characteristics of a CYP3A phenotyping probe that should predict intravenous and oral cyclosporine disposition. Indeed, midazolam clearance was found to correlate strongly with cyclosporine clearance after the two CYP3A substrates were administered intravenously to the same group of liver transplant recipients (r=0.81, P<0.001) (Thummel et al., 1994).
Based on these earlier findings, the presystemic and systemic clearance characteristics of orally administered midazolam could parallel those of orally administered cyclosporine. Accordingly, we conducted a study in kidney transplant recipients to determine whether the oral clearance of midazolam would predict the oral clearance of cyclosporine.
Section snippets
Materials
Midazolam, 15N3-midazolam, 1′-hydroxymidazolam, and 1′-[2H2]-1′-hydroxymidazolam were gifts from Roche Laboratories (Nutley, NJ). N-Methyl-N-(t-butyl-dimethylsilyl)trifluoroacetamide was purchased from Pierce Chemical (Rockford, IL). Cyclosporine A, NADPH (reduced form, tetrasodium salt), and β-glucuronidase from Helix pomatia were purchased from Sigma (St. Louis, MO). All other chemicals were of reagent grade or better.
Human subject study
This study was approved by the University of Washington Human Subjects
Cyclosporine disposition
Of the 15 subjects who recorded their cyclosporine administration times for the 3 days prior to the study day (nos. 2–7, 9–12, 15–19), all but one took their doses within 1.5 h of the scheduled times. Subject 17 stated she had been traveling and her administration times were not always consistent, as much as 3 h from the scheduled times. For four subjects (nos. 7, 8, 18, and 20), the 0- and 12-h trough levels differed by more than 25% (Table 3). This variation was attributed to diurnal
Discussion
The aim of this study was to determine whether midazolam oral clearance would predict cyclosporine oral clearance under a routine clinical setting. If favorable, effective cyclosporine dosing regimens could be predicted and potential adverse events prevented. However, in contrast to previous findings where the systemic clearance of midazolam correlated strongly with the systemic clearance of cyclosporine in liver transplant recipients (Thummel et al., 1994), we observed a weak correlation
Acknowledgements
This work was supported in part by grants from the National Institutes of Health, GM48349 and GM32165 (K.E.T.) and the American Foundation for Pharmaceutical Education (M.F.P.).
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