2480 Vol. 8, 2480–2487, August 2002 Clinical Cancer Research Advances in Brief Differential Effect of IFN␣-2b on the Cytochrome P450 Enzyme System: A Potential Basis of IFN Toxicity and Its Modulation by Other Drugs1 Mohammed Islam, Reginald F. Frye, Conclusion: These data suggest that strategies to mini- Thomas J. Richards, Ibraham Sbeitan, mize the impairment of CYP enzymes could alter the toxic- Sandra S. Donnelly, Paul Glue, ity profile of HDI and augment its therapeutic utility, and 2 that recognition of these potential interactions is important Sanjiv S. Agarwala, and John M. Kirkwood in the therapeutic application of IFNs. Melanoma Center, University of Pittsburgh Cancer Institute [M. I., T. J. R., I. S., S. S. D., S. S. A., J. M. K.], Department of Medicine, School of Medicine [J. M. K.], and Department of Pharmaceutical Introduction Sciences, School of Pharmacy and Center for Clinical Pharmacology IFNs are cellular proteins of 143–187 amino acids pro- [R. F. F.], University of Pittsburgh, Pittsburgh, Pennsylvania 15213, duced by stimulated immune and nonimmune cells that exhibit and Schering Plough Research Institute, Kennilworth, New Jersey antiviral, differentiating, antiproliferative, and immunomodula- 07033 [P. G.] tory functions. IFN␣-2b exhibits variable therapeutic responses when used in combination with various chemotherapies and Abstract cytokines in metastatic melanoma (1–3). However, no therapy Purpose: High-dose IFN␣-2b therapy (HDI) is the has yet demonstrated unequivocal effects on survival in patients standard of adjuvant therapy for patients with high-risk with metastatic disease. In the adjuvant setting, survival and melanoma, but toxicities of this regimen have limited its relapse-free interval were significantly prolonged in the pivotal application. IFNs affect cytochrome P450 (CYP) enzymes, E1684 trial of the Eastern Cooperative Oncology Group, and in which metabolize many endogenous (e.g., steroids, fatty ac- the most recent intergroup trial E1694 (4, 5). Despite the sig- 3 ids) and exogenous (e.g., drugs) substrates. No systematic nificant therapeutic gain associated with adjuvant HDI therapy, studies have been performed to evaluate the effect of HDI on toxicity and adverse effects have impeded adoption of this CYP enzymes. A significant inhibitory effect of HDI on CYP therapy by physicians and patients. Approximately 78% of enzymes would increase the potential for adverse drug re- patients receiving IFN therapy in E1684 experienced grade 3 actions and altered homeostasis through effects on hormone toxicity, and 24% discontinued therapy because of this toxicity metabolism. (6, 7). The management or prevention of toxicities associated Methods: To evaluate the potential effect of HDI on with HDI therapy is one of the greatest challenges in broadening CYP enzymes, 17 patients with high-risk melanoma were and improving the efficacy of this regimen. treated with HDI, and CYP enzyme activity was measured The CYP enzymes are a superfamily of heme-containing by administration of selectively metabolized probe drugs enzymes distributed widely throughout the body that are in- -over time (days ؊6, ؉1, ؉26, and ؉52 of HDI). Probe drugs volved in the synthesis and metabolism of endogenous sub and/or metabolites were quantified and used to derive in- strates including steroid hormones, fatty acids, and lipids, as dexes of enzyme activity. well as the metabolism of exogenous substrates such as drugs Results: The results indicate that HDI differentially and environmental chemicals. IFNs have been shown to de- impairs CYP-mediated metabolism, having no effect on crease the expression and activity of CYP enzymes in animal some enzymes (CYP2E1) and substantial effects on others models (8–11). Human data are less extensive and primarily (CYP1A2; median 60% decrease). A significant association limited to patients with hepatitis being treated with compara- was found between the magnitude of CYP inhibition and the tively low-dose IFNs (12–15), but the available data indicate a occurrence of side effects including fever and neurological detrimental effect on drug metabolism. However, the selectivity toxicity, which may form a novel basis of the underlying and magnitude of the effect on individual drug metabolizing pathophysiology of some IFN␣-2b-induced toxicity. enzymes is largely unknown. In addition, there are no data on the effect of HDI used to treat patients with melanoma on drug-metabolizing enzymes. A deleterious effect on the CYP enzyme system would have important consequences because of Received 11/16/01; revised 3/23/02; accepted 4/2/02. the increased potential for drug-induced adverse effects related The costs of publication of this article were defrayed in part by the to concomitant drug treatment (i.e., drug-cytokine interactions) payment of page charges. This article must therefore be hereby marked and also the potential for altering homeostasis through effects on advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by NIH/NCRR/GCRC Grant #5M01 RR00056. 2 To whom requests for reprints should be addressed, at Department of Medicine, Division of Hematology/Oncology, University of Pittsburgh 3 The abbreviations used are: HDI, high-dose IFN␣-2b; AUC, area Medical Center, 200 Lothrop Street, Montefiore Hospital N-755, Pitts- under the plasma concentration-time curve; Cmax, maximum observed burgh, PA 15216. Phone: (412) 648-6571; Fax: (412) 648-6599; E-mail: serum concentration; CYP, cytochrome P450; IL, interleukin; Tmax, [email protected]. time to achieve maximum serum concentration. Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2002 American Association for Cancer Research. Clinical Cancer Research 2481 Table 1 Schema of study designed to evaluate the differential effect of HDI therapy on the CYP enzyme system Table 2 Five probe drugs regimen or “Pittsburgh mixture” and their doses, corresponding CYP, and activity measure Probe drug (dose) Enzyme Specimen Trait measure Reference Caffeine (100 mg) CYP1A2 Plasma, 8 hours Paraxanthine/caffeine 16 Mephenytoin (100 mg) CYP2C19 Urine, 0–8 hours 4ЈOH-mephenytoin recovery 47 Debrisoquine (10 mg) CYP2D6 Urine, 0–8 hours DBRRa 18 Chlorzoxazone (250 mg) CYP2E1 Plasma, 4 hours 6-OH-chlorzoxazone/chlorzoxazone 17 Dapsone (100 mg) CYP2C8/9b Urine, 0–8 hours DPRRc 22 NATd Plasma, 8 hours MAD/DDSe a Debrisoquine recovery ratio, calculated as the urinary recovery of 4-Hydroxydebrisoquine divided by the sum of 4-Hydroxydebrisoquine and debrisoquine. b May also involve CYP3A4/5 and CYP2E1. c Dapsone recovery ratio calculated as the urinary recovery of dapsone hydroxylamine divided by the sum of dapsone hydroxylamine and dapsone. d NAT, N-acetyltransferase. e MAD, monoacetyldapsone; DDS, dapsone. endogenous substrates such as hormones. Thus, this study was of s.c. IFN␣-2b) as shown in Table 1. CYP enzyme activities conducted to examine the effects of acute and chronic high-dose were estimated in vivo using the “Pittsburgh mixture” approach, IFN␣-2b monotherapy on several important drug-metabolizing which involves the simultaneous oral administration of five CYP enzymes in vivo using known enzyme-selective probe drugs for which the metabolism has been well characterized. drugs (16–18). Each patient received the mixture of five drugs during each visit as shown in Table 2. We have shown previously that there is no Patients and Methods interaction between the mixture drugs at the doses given (20). Study Design. Seventeen patients with high-risk, re- Blood samples (20 ml) were obtained at 0, 4, and 8 h, and urine sected melanoma who were scheduled to undertake adjuvant was collected from 0 to8htomeasure the concentration of therapy with high-dose IFN␣-2b participated in this study after probe drugs and/or their metabolites. Plasma harvested by cen- providing written informed consent. Eligibility criteria included trifugation, and urine aliquots were stored frozen at Ϫ20°C until biopsy-proven high-risk melanoma, normal renal (serum creat- analyzed. The probe drugs and their metabolites were analyzed inine Ͻ2.0 mg/dl) and liver function (total bilirubin Ͻ1.5 mg/ by high-performance liquid chromatography for the determina- dl), and no history of allergy to sulfa drugs. All of the subjects tion of caffeine and paraxanthine, (21), 4-hydroxymephenytoin, were instructed to abstain from alcohol, caffeine, barbecued (20), debrisoquine, and 4-hydroxydebrisoquine (18), chlorzoxa- meat, and grapefruit or grapefruit juice consumption for at least zone and 6-hydroxychlorzoxazone (17), dapsone and N- 2 days before each visit (19). Patients with other underlying hydroxydapsone in urine, and dapsone and monoacetyldap- medical diseases taking drugs known to affect the CYP enzyme sone in plasma (22). Probe drug analyses were conducted system that could not be safely interrupted were excluded from such that all of the samples for a given patient were analyzed the study. within the same run so as to minimize within-subject varia- All of the patients received i.v. IFN-␣2b (INTRON A; tion because of analytical procedures. The interday coeffi- Schering Plough, Kenilworth, NJ) at a dose of 20 million units cients of variation for each of these assays were Ͻ15%. In (MU)/m2/day for 5 days/week ϫ 4 weeks (induction phase) addition, all of the assay procedures were cross-validated to followed by s.c. IFN-␣2b at a dose of 10 MU/m2/day for 3 ensure that no analytical interference would occur with si- days/week ϫ 48 weeks (maintenance