Enhanced Cyclophosphamide and Ifosfamide Activation in Primary

ICANCER RESEARCH57. 1946-1954. May 15. 19971 Enhanced Cyclophosphamide and Ifosfamide Activation in Primary Human Hepatocyte Cultures: Response to Cytochrome P-450 Inducers and Autoinduction by Oxazaphosphorines'

Thomas K. H. Chang, Li Yu, Patrick Maurel, and David J. Waxman2

Division of Cell and Molecular Biology. Department of Biology. Boston University, Boston, Massachusetts 02215 fT. K. H. C., L Y., D. .1. W.J; Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, V6T 113 Canada fT. K. H. C.]: and INSERM U-128, Centre National de Ia Recherche Scient(fique, 34033 Montpellier. France (P. M.J

ABSTRACT CYP2A6,4 CYP2B6 (4), CYP3A (4, 5) and all four individual CYP2C enzymes (6). In the case of ifosfamide, a clinically significant bio The anticancer oxazaphosphorine prodrugs cyclophosphamide and if transformation pathway is side-chain N-dechloroethylation (7â€”9), osfamide are activated in human liver by a 4-hydroxylation reaction which is catalyzed by CYP2B and CYP3A enzymes in vitro (5, 10) catalyzed by multiple cytochrome P450 (CYP) enzymes. In the present study, we used a cultured human hepatocyte model to identify possible and generates therapeutically inactive but neurotoxic metabolite(s) inducers of the CYP-catalyzed activation of these two anticancer pro (11, 12). drugs. Treatment of primary cultures of human hepatocytes with pheno Large interpatient differences are found in the clinical pharmaco barbital, dexamethasone, or rifampin elevated hepatocyte microsomal kinetics and biotransformation of cyclophosphamide (13â€”15)and oxazaphosphonne 4-hydroxylation by up to 200â€”400%ofcontrol for both ifosfamide (16â€”18).Genetic factors may account for some of this drug substrates. These inductions were associated with corresponding variability as suggested by in vitro experiments demonstrating that increases in immunoreactive CYP2B6, CYP2C8, CYP2C9, and CYP3A4, allelic variants of CYP2C9 can catalyze cyclophosphamide 4-hy all previously shown to catalyze oxazaphosphorine activation. Rifampin (1 droxylation and ifosfamide 4-hydroxylation with significantly differ gLM, 96-h exposure) was a particularly potent inducer of ifosfamide and ent apparent Km values; moreover, the polymorphically expressed cyclophosphamide 4-hydroxylation, as well as of CYP3A protein levels and CYP3A-dependent testosterone 6@J-hydroxylation. CYP3A4, (S)-mephenytoin 4'-hydroxylase CYP2C19 is a relatively low Km CYP2C8, and CYP2C9 protein levels were also increased by exposure of catalyst of the activation of these prodrugs (6). In addition to genetic the hepatocytes to cyclophosphamide or ifosfamide (50 ELM),which factors, concurrently administered drugs may also contribute to the thereby enhanced their own rates of 4-hydroxylation in the cultured observed interindividual variability in oxazaphosphorine metabolism. hepatocytes. In one human hepatocyte culture that contained the poly For example, phenobarbital (19) and prednisone (20) can decrease the morphically expressed CYP3A5 in addition to the more widely expressed elimination half-life of cyclophosphamide in patients, whereas dexa CYP3A4, only CYP3A4 was induced by cyclophosphamide, ifosfasnide, methasone may increase the total body clearance of this alkylating and rifampin. These studies: (a) demonstrate an underlying metabolic agent (15). The underlying basis for these alterations in the pharma basis for the clinically important oxazaphosphorine autoinduction phar cokinetics of cyclophosphamide is uncertain, however, but could macokinetics seen with these drugs in cancer patients; and (b) Identify involve pharmacodynamic factors in addition to P-450-catalyzed drug rifampin and other CYP inducers as potentially useful for increasing the rates of cyclophosphamide 4-hydroxylation and ifosfamide 4-hydroxyla metabolism. It is important to understand the basis for the large tion in human liver in a manner that could favorably impact the clinical inter-patient variability in the pharmacokinetics and biotransformation pharmacokinetics of these anticancer prodrugs. of cyclophosphamide and ifosfamide because of the potential rela tionship between systemic exposure (as measured by AUC) and the toxicity ofthese agents (18, 21). INTRODUCTION Repeated administration or continuous infusion of cyclophospha mide (13, 22â€”24)or ifosfamide (16, 25, 26) to cancer patients over a Cyclophosphamide and its isomeric analogue, ifosfamide, are period of several consecutive days results in a decreased elimination widely used in the clinical management of a variety of human malig half-life of these drugs. This is accompanied by an increase in total nancies (I, 2). Cyclophosphamide also exhibits immunosuppressive body clearance (16, 26â€”28)but no alteration in the volume of distri properties and is used in organ transplantation protocols and various bution (16, 23, 29) or renal clearance (16, 26, 28), suggesting that autoimmune disorders (I). Cyclophosphamide and ifosfamide are these oxazaphosphorines may enhance their own hepatic clearance. oxazaphosphorine prodrugs that require bioactivation by P-4503 en Although cyclophosphamide (30â€”33)and ifosfamide (3 1) can mod zymes to form a 4-hydroxy metabolite that equilibrates with the ulate P450 levels in the rat liver model, this modulation leads to a ring-opened aldophosphamide. This intermediate can undergo spon decrease rather than an increase in capacity for oxazaphosphorine taneous /3-elimination to yield acrolein and a mustard derivative activation (30, 31). Consequently, it is important to investigate what (phosphoramide mustard or isophosphoramide mustard) or, alterna effects these cancer chemotherapeutic drugs have on human liver tively, it can be inactivated by aldehyde dehydrogenase-catalyzed P-450 expression. Because cancer patients are often prescribed com conversion to carboxyphosphamide (3). Multiple human P-450 en bination chemotherapy, such information would allow clinicians to zymes are capable of activating oxazaphosphorines in vitro, including optimize drug therapy by designing rational drug scheduling and avoiding drug combinations that might compromise therapeutic effi Received I 1/26/96; accepted 3/24/97. The costs of publication of this article were defrayed in part by the payment of page cacy or exacerbate systemic toxicity. charges. This article mutt therefore be hereby marked advertisement in accordance with In the present study, we used a cultured human hepatocyte model: 18 U.S.C. Section 1734 solely to indicate this fact. (a) to examine whether hepatic cyclophosphamide and ifosfamide I Supported in part by Grant CA-49248 from the NIH (to D. J. w.). Presented in part at the Intemational Congress of Toxicology VII. Seattle. Washington, July, 1995. 4-hydroxylation reactions are subject to modulation by P450 inducers 2 To whom requests for reprints should be addressed. at Division ofCell and Molecular in human liver cells; and (b) to investigate whether the oxazaphos Biology, Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215. Phone: (617) 353-7401: Fax: (617) 353-7404: E-mail: [email protected]. 3 The abbreviations used are: P450 or CYP, cytochrome P-450; TCDD, 2,3,7,8- 4 Individual cytochrome P-450 forms are designated according to systematic nomen tetrachlorodibenzo-p-dioxin: AUC. area under the plasma concentration-time curve. clature (52). 1946

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1997 American Association for Cancer Research. INDUCTION OF OXAZAPHOSPHORINE ACTIVATION phorine autoinduction response seen in cancer patients reflects 106 cells and by measuring the rate of de novo protein synthesis after labeling changes in liver P-450-dependent drug metabolism. Our findings human hepatocytes with tritiated leucine. No sign of cytotoxicity was observed establish that cyclophosphamide and ifosfamide activation can be at the oxazaphosphorine concentrations tested (50â€”250 SM). Moreover, con enhanced in human hepatocytes by inducers of CYP2B, CYP2C, and centrations of cyclophosphamide and ifosfamide as high as 1 msi did not CYP3A. Furthermore, cyclophosphamide and ifosfamide are shown decrease the yield of isolated microsomal protein (data not shown). Preparation of Hepatocyte Microsomes. At various times after drug to enhance their own activation by a mechanism that involves induc treatment (as indicated in each table or figure legend), microsomes were tion of liver P-450 enzymes. isolated from the cultured hepatocytes by differential ultracentrifugation (35). Microsomal protein was measured by the Bradford method with BSA as MATERIALS AND METHODS standard. Western Blotting Using Anti-P450 Antibodies. Polyclonalrabbitanti-rat Materials. Cyclophosphamide and ifosfamide were obtained from the CYP2B (36), anti-rat CYP2C (37), and anti-rat CYP3A antibodies (38) were Drug Synthesis and Chemistry Branch, National Cancer Institute (Bethesda, prepared as detailed elsewhere. These antibody preparations have been shown MD). Authentic4-hydroperoxyifosfamidemetabolitestandardwaskindlypro to cross-react with human liver microsomal CYP2B, CYP2C, and CYP3A vided by Dr. J. Pohi (ASTA Pharma, Bielefeld, Germany). Collagenase, cell proteins (4), respectively. Polyclonal goat anti-rabbit CYPIAI and goat anti culture reagents, and NADPH were purchased from Sigma Chemical Co. (St. rabbit CYP1A2 antibodies were purified as described (35). These latter two Louis, MO). FCS (Life Technologies, Inc., Paisley, Scotland), collagen-coated antibodies have been shown to cross-react with the corresponding cDNA culture dishes (Coming, Iwaki Glass, Japan), and [4-'4Cltestosterone expressed human P-450 (39). Microsomes isolated from cultured hepatocytes (Amersham Corp., Arlington Heights, IL) were obtained from the sources were analyzed for expression of individual microsomal human P-450 proteins indicated. cDNA-expressed human P-450s used as standards for Western by Western blot analysis (40). Each blot was probed with the P-450 subfamily blotting were obtained from Gentest Corp. (Woburn, MA). specific polyclonal antibodies indicated above. Source of Human Liver Specimens. Approval for the scientific use of Oxazaphosphorine Activation Assay. Microsomal cyclophosphamide human liver tissues was obtained from the French National Ethics Committee. 4-hydroxylation and ifosfamide 4-hydroxylation reactions were assayed by a Liver specimens were obtained as discarded surgical material excised from fluorometric method (41) as described previously (4). Briefly, 100 @gof patients undergoing hepatic lobectomy for medical reasons unrelated to the microsomal protein was preincubated with 0.25 or 2 mt@icyclophosphamide or present study. The clinical characteristics of the liver tissue donors are given ifosfamide (as indicated in each figure legend) at 37Â°C for 4 mm in a buffer in Table 1. containing 100 mM potassium phosphate (pH 7.4), 5 mM semicarbazide HCI, Isolation and Primary Culture of Hepatocytes. Human hepatocytes were and 0. 1 mM EDTA. Substrate oxidation was initiated by the addition of isolated and grown as primary cultures as described previously (34). Briefly, NADPH (final concentration, 1 mM)and stopped 60 mm later by the addition hepatic tissues were extensively washed with Eurocollins (2.05 g/liter of 80 pAof ice-cold 5.5% zinc sulfate solution, followed by 80 p1 of saturated K'@2@[email protected],I.12g/literKC1,0.84g/literNaHCO1,and35 barium hydroxide and 40 pJ of 0.01 M HCI. After centrifugation, 300 pJ of the gfliter glucose, pH 7.3) shortly after resection to eliminate erythrocytes and supernatant was derivatized with a solution containing aminophenol (6 mg/mI) then transported to the laboratory in a sterile bag on ice. The tissue was then and hydroxylamine hydrochloride (6 mg/mI) in I M HCI (31). Authentic sequentially perfused with calcium-free HEPES, 0.5 mM EGTA, in the same 4-hydroperoxyifosfamide was used as a standard for acrolein and measured buffer, and finally with 0.05% collagenase (Sigma; type IV) in HEPES buffer fluorometrically (350-nm excitation wavelength, 5 15-nm emission wave containing 7 mM calcium chloride at 37Â°C. After dissociation, hepatocytes length). were filtered through nylon gauze (250 g@m),andcells were washed three times Testosterone 6@3-Hydroxylation Assay. Microsomal testosterone 6/3-hy in culture medium by centrifugation at 50 X g. Cell viability, as assessed by droxylase activity was determined by a TLC method (42). Each 200 pi of trypan blue exclusion, was generally between 70 and 90%. The cells were incubation mixture, containing 100 m@i HEPES (pH 7.4), 0.1 mM EDTA, 20 plated into 60-mm plastic dishes precoated with collagen at 3 X 106 cells/plate !.Lgofmicrosomal protein, and 50 ,LM14-'4Cltestosteronewas preincubated at in a total volume of 3 ml of a hormonally and chemically defined medium 37Â°C for 4 mm. Reactions were initiated by the addition of NADPH (final consisting of a mixture of Williams' E and Ham's F-l2 (1:1 in volume) and concentration, 1 mM) and stopped 20 mm later with 1 ml of ethyl acetate. The incubated at 37Â°Cina humidified atmosphere of air containing 5% CO2. FCS incubation mixture was then extracted with ethyl acetate and chromatographed (5%) was present in the medium to enhance cell attachment during the first 4 h. on silica gel TLC plates developed with dichloromethane:acetone (4: 1) fol The medium was replaced with fresh serum-free medium at that time and every lowed by chloroform:ethyl acetate:absolute ethanol (4: 1:0.7). Metabolites were 24 h thereafter. localized by autoradiography and quantitated by liquid scintillation counting. Treatment of Hepatocytes in Culture. By 12â€”24hafter plating, primary cultures of hepatocytes were exposed to (3-naphthoflavone (25 or 50 j.@M), TCDD (1 nM),phenobarbital(2mM),dexamethasone(50 ,.LM),rifampin(1â€”50 RESULTS MM), cyclophosphamide (0.05â€”1 mM), or ifosfamide (0.05â€”I mM) for times up to 144 h as specified in each table or figure legend. Control cells were treated Enhanced Cyclophosphamide 4-Hydroxylation and Ifosfamide with the vehicle (DMSO; final concentration, 0. 1%). In cultures where a time 4-Hydroxylation following Treatment with Phenobarbital, course of inducer treatment was carried out, similar inductions were observed Rifampin, or Dexamethasone. Primary cultures of human hepato at 96 h and at 120 h (data not shown). The possible cytotoxic effects of cytes were treated with @3-naphthoflavone (25 or 50 .LM), TCDD (I oxazaphosphorines was assessed by determining the yield of microsomes per nM), phenobarbital (2 mM), rifampin ( 1â€”50 iM), dexamethasone (50 LM), or DMSO (0. 1%, vehicle control) for 96â€”I 20 h, and hepatocyte microsomes were then prepared. As shown in Table 2, phenobarbital donorsLiverOrigin Table 1Liver tissue and rifampin elevated cyclophosphamide activation by at least 2â€”3- liveridentificationSexAgeDiagnosisspecimenHTL92F60Metastatic of fold when measured at 2 m@visubstrate concentration. At 0.25 mr@i cyclophosphamide substrate, a more modest induction (average of lobeFf43F61Metastatic colon cancerRight 30â€”35% for three to four independent cultures) was obtained. Simi lobeFF52M73Metastatic colon cancerRight larly, phenobarbital and rifampin treatment increased ifosfamide 4-hy lobeFT59F46Nodular colon cancerLeft droxylation to a greater extent at 2 m@isubstrate concentration (â€”4- IIIFF60M71Metastatic hyperplasia of II, normal liverSegments fold) than at 0.25 mM (50â€”60% average increase; Table 3). In Iâ€”lVFf69M52Metastatic colon cancerSegments addition, dexamethasone treatment increased these two drug activa lobeFF80M60Metastatic gastric cancerLeft lobeFHIO5M57Pancreas colon cancerLeft tion reactions (2 mM substrate) by â€”3-foldin culture HTL92 (data not liverFl'l08F29AdenomaLeft cancerWhole shown). By contrast, the P-450 inducers @-naphthoflavone (Tables 2 lobe and 3) and TCDD (data not shown) did not increase 4-hydroxylation 1947

Table 2 Effect of P-450 enzyme inducers on @â€˜clophosphamide4-hydroxvlationin primary cultures of human hepatocvtes Primary cultures of human hepatocytes were treated with dimethylsulfoxide (vehicle control, 0. 1%), @3-naphthoflavone(25@sM),phenobarbital(2 mM),or rifampin (50 @M)for96 h, and microsomes were prepared. Microsomal cyclophosphamide 4-hydroxylation activity was determined as described in â€œMaterialsandMethods.â€•Resultsare shown as relative activity (control = 100). Control activities (expressed as pmol/min/mgcontrols.0.25 microsomal protein) are shown in parentheses for the uninduced

cyclophosphamideCulture mr@icyclophosphamide 2 msi

RIFHTL92no. Control BNFÂ° PB RIF Control BNF PB NA NA NA NA (995) NA 314 238 FF43 (97) 45h 189 123 (119) 118â€• 676 477 FTS2 (395) NA 75 112 (630) NA 193 250 FTS9 (494) 89 NA 106c (598) 129 NA 244c FF60 (406) 147 142 I72@ (487) 111 210 216' Mean (348) 94 135 128 (566) 119 348 285 SD (173) 51 57 30 (314) 9 225 108

a BNF, @3-naphthoflavone; PB. phenobarbital; RIF. rifampin: NA, data not available owing to the limited amount of hepatocytes available from these individual donors. bCulturetreatedwith 50 .LMBNF. CCulturetreatedwith25 @LMRIF. of either cyclophosphamide or ifosfamide. These data demonstrate pressed constitutively in the lymphoblast cell line cDNA expression that the rate of activation of both oxazaphosphorines can be enhanced system (Fig. IC, band marked by asterisk, Lanes 1â€”3).In control by select P450 inducers, albeit to an extent that may vary between experiments, TCDD was shown to induce CYPI A2 in these same individual livers. Analysis of the data from nine individual human cultures (39). hepatocyte cultures (Table 1) suggests that the extent to which ri Concentration and Time Dependence of Rifampin Induction fampin induces oxazaphosphorine 4-hydroxylation activity, expressed Response. To investigate the potency of rifampin with respect to as a percentage of uninduced control, is inversely related to the basal induction of cyclophosphamide and ifosfamide 4-hydroxylation, pri activity (i.e., uninduced activity) of the culture (data not shown). mary cultures of human hepatocytes were treated with 1, 2, 5, 25, or Induction of Multiple P-450 Proteins by Phenobarbital, Dexa 50 @LMrifampinfor 96 h. A near-maximal increase in cyclophospha methasone, and Rifampin. Phenobarbital, dexamethasone and ri mide 4-hydroxylation and ifosfamide 4-hydroxylation was achieved fampin have been shown to induce CYP3A mRNA (43â€”45),protein, with 1 @LMrifampin, both when the 4-hydroxylation assays were and activity (46) in primary cultures of human hepatocytes. To con performed at 0.25 mM substrate (Fig. 2A) and at 2 mr@tsubstrate (Fig. firm the effect of these drugs on CYP3A and to determine whether 2B). The rifampin-induced increase in ifosfamide 4-hydroxylation they also induce other P-450 forms (e.g., CYP2B6 and CYP2C) in our was, however, greater than the increase in cyclophosphamide 4-hy experiments, Western blot analysis was carried out using heterologous droxylation at both substrate concentrations. A similar concentration P.450 subfamily-specific polyclonal antibodies (4, 35). As shown in dependence was observed for induction of immunoreactive CYP3A Fig. 1A, phenobarbital induced an immunoreactive CYP2B protein protein (Fig. 3A) and CYP3A-mediated testosterone 6@3-hydroxylase (Lane 6 versus Lane 4), which had an electrophoretic mobility cor activity (Fig. 3B). A time course study demonstrated that CYP3A responding to that of cDNA-expressed CYP2B6 (Lane 3). This protein was detectably elevated as early as 72 h and was sustained at CYP2B protein was also induced by dexamethasone and rifampin an elevated level from 96â€”192h (Fig. 3A, Lanes 5 and 7â€”11).The (Lanes 7 and 8 versus Lane 4). Phenobarbital, dexamethasone, and preferential induction by rifampin of ifosfamide 4-hydroxylase activ rifampin also induced two major CYP2C proteins (Fig. 1B, Lanes ity seen in this experiment (Fig. 2) was also observed in hepatocyte 6â€”8).The top band corresponds in electrophoretic mobility to cDNA cultures from other individuals, albeit to different extents (e.g., Fig. 4; expressed CYP2C9, whereas the lower band has a mobility equivalent see below). This induction of ifosfamide 4-hydroxylation may in part to cDNA-expressed CYP2C8 (Fig. IB, Lane I; Ref. 4). Whereas the reflect the preferential role of CYP3A enzymes in catalyzing this same three inducers were each effective in inducing CYP3A pro reaction (4). tein(s) (Fig. IC, Lanes 6â€”8), none of these compounds altered Autoinduction of Cyclophosphamide and Ifosfamide Activation CYPIAI or CYPIA2 protein content in these cells (data not shown). in Human Hepatocytes. Repeated treatment of cancer patients with The CYP1A inducer TCDD (Lane 5) did not induce CYP2B, 2C, or cyclophosphamide (27, 28) or ifosfamide (16, 26, 47) results in a 3A proteins in the human hepatocytes, although it did increase an decrease in elimination half-life and an increase in total body clear unidentified CYP3A-immunoreactive protein (Fig. lC, Lane 5) with a ance of the alkylating agent. To determine whether these oxazaphos mobility similar to a CYP3A-immunoreactive protein that is ex phorines enhance their own activation, primary cultures of human

hepatocytesPrimary Table 3 Effect of P-450 enzyme inducers on afosfamide 4-hydroxylation in prinwr@' cultures of human h,and cultures of human hepatocytes were treated with dimethylsulfoxide (vehicle control, 0.1%), f3-naphthoflavone(25 SM),phenobarbital (2 mM),or rifampin (50 @M)for96 activity(controlmicrosomes were prepared. Microsomal ifosfamide 4-hydroxylation activity was determined as described in â€œMaterialsand Methods.â€•Results are shown as relative = 100). Absolute activity values, expressed ascontrols.0.25 pmol/min/mg microsomal protein, are shown in parentheses for the uninduced

ifosfamideCulture m@iifosfamide 2 msi

RIFHTL92no. Control BNF'@ PB RIF Control BNF PB NA NA NA NA (I090) NA 277 286 FF43603FT52 (76) 33h 297 213 (119) 64â€• 819 375F1@59 (385) NA 71 120 (561) NA 221 304'Ff60 (405) 101 NA 145' (559) 92 NA 244'Mean (467) 86 110 116' (467) 90 207 362SD (333) 77 159 149 (559) 82 381 143a (175) 30 121 45 (348) 16 294

BNF, @3-naphthoflavone: PB. phenobarbital: RIF, rifampin; NA. data not available. I,BNF.â€˜. Culture treated with 50 @.LM

Culture treated with 25 @sM RIF. 1948

INDUCTION OF OXAZAPHOSPHORINEACTIVATION

cDNAs HumanHepatocytes HumanLivers famide (4). Therefore, Western blot analysis was performed to deter 2C8 3A4 2B6 UT TCDD PB DEX RIF 2 4 7 8 mine whether the autoinduction of oxazaphosphorine activation in A. cultured hepatocytes seen in Fig. 4 is accompanied by induction of any of these enzymes. As is shown in Figs. S and 6, both oxazaphos

@..â€˜ phorines stimulated increases in CYP3A, CYP2C8, and CYP2C9 proteins that were detectable at 50 p.Moxazaphosphorine inducer and @ weremaximalat250p.M.Noincreasesormarginalincreaseswere detected in the case of CYP2B6 (Fig. 6A). The enhanced CYP3A protein expression could also be monitored by the associated increase in CYP3A-catalyzed hepatocyte microsomal testosterone 6j3-hydrox ylase activity (Table 4). Interestingly, oxazaphosphorine induction of B CYP3A protein and activity could be detected (albeit to a small extent) in hepatocyte culture FF59 (Fig. 6; Table 4), although in this case the increase did not lead to a major elevation of microsomal CYP cyclophosphamide or ifosfamide 4-hydroxylase activity (Fig. 4A). @ 2C82C9 -@ ___ 2C Together, these findings demonstrate that cyclophosphamide and if osfamide can enhance their own activation by inducing P-450 en zymes such as CYP2C8, CYP2C9, and CYP3A. Differential Effect of Oxazaphosphonnes on Hepatocyte Micro somal CYP3A4 and CYP3A5 Protein Levels. As shown in Fig. 5A, C. cyclophosphamide and ifosfamide induced an immunoreactive CYP3A protein that displayed an electrophoretic mobility indistin guishable from that of cDNA-expressed CYP3A4. In addition to CYP CYP3A4, the polymorphically expressed CYP3A5 is found at signif 3A icant levels in â€”20â€”25%of adult human livers (49â€”51). We, there fore, investigated whether CYP3A5 is also subject to oxazaphospho rime induction. Fig. 6B shows that cyclophosphamide, ifosfamide, and rifampin each induced CYP3A4 (lower band of doublet in Lanes 4â€”8) but not CYP3A5 (upper band) in one culture of human hepatocytes 1 2 3 4 5 6 7 8 9 10 11 12 (Ff59) where both of these proteins were detected by immunoblot Fig. 1. Induction of immunoreactive CYP2B, CYP2C, and CYP3A proteins by phe assay of uninduced cells. nobarbital, dexamethasone, and rilampin. Primary cultures of human hepatocytes (culture HTL92) were treated for 96 h with DMSO (vehicle control, 0.1%; Lane 4), TCDD (1 nM; Lane 5), phenobarbital (2 ross; Lane 6), dexamethasone (50 p@s;Lane 7), or rifampin (50 (A) 400 ELM; LWW 8). Microsomes were prepared and subjected to SDS-PAGE. Shown are CPAâ€”0.25 mM immunoblots probed with anti-rat CYP2B (A), anti-rat CYP2C (B), or anti-rat CYP3A C 0.25mMIFA antibodies(C).Includedfor comparisonarecDNA-expressedhumanP-450standards 0 300 (Lanes 1â€”3)and human liver microsomes (samples HLS2, 4, 7, and 8; Lanes 9â€”12). 0. Anti-CYP3A antibody cross-reacts with an unidentified protein (*) present in the cDNA expressed CYP standard samples (C, Lanes 1-3) that runs higher than bonafide CYP3A4 200 (Lane 2). A cross-reactive protein of the same electrophoretic mobility that appears to be inducible by TCDD is seen in Lane 5.

0 100 E 0. hepatocytes were treated for 96 h with 50 @.tMcyclophosphamide or 0 @ 0@I 1IiiRI 2 5 25 50 ifosfamide. This corresponds to a concentration of oxazaphosphorine r that is clincally relevant and is without apparent toxicity to the cells RIFAMPINCONCENTRATION(j@tM) (see â€œMaterialsand Methodsâ€•).As shown in Fig. 4, cyclophospha mide treatment of human hepatocytes increased microsomal cyclo (B) phosphamide 4-hydroxylation or ifosfamide 4-hydroxylation activity __%___ @2mMCPA by as much as 140%. Ifosfamide (50 p.M) increased its own 4-hy .@ 2mMIFA droxylation activity, as well as cyclophosphamide 4-hydroxylation 0 activity, in a similar manner. Clear evidence for significant autoin 0. 800 duction of oxazaphosphorine metabolic activity was obtained in three of five human hepatocyte cultures examined (FF60, FH1O5, and <@Q400 FF108) but not in cultures FF59 or Ff80 (Fig. 4 and data not shown), despite the responsiveness of the latter two cultures to rifampin. This variability in oxazaphosphorine mducibility is reminiscent of the @@@ interindividual differences in the autoinduction response seen in the I 2 5 2550 clinic (13, 48) and is analogous to the variability in responsiveness of RIFAMPINCONCENTRATION(riM) cultured human hepatocytes to classic P.450 inducers seen in Tables Fig. 2. Concentration-dependence of rifampin induction of cyclophosphamide 4-hy 2 and 3. droxylation and ifosfamide 4-hydroxylation activities. Primary human hepatocytes (cul Effect of Cyclophosphainide and Ifosfamide Treatment on ture Ff69) were treated for 96 h with 0.1% DMSO (vehicle control) or I, 2, 5, 25, or 50 CYP2B, CYP2C, and CYP3A Protein Expression in Cultured @.LMrifampin. Microsomal cyclophosphamide (CPA) 4-hydroxylation and ifosfamide (IFA) 4-hydroxylation activities were determined at 0.25 msi (A) and 2 msi (B) concen Hepatocytes. CYP2B6, CYP2C8, CYP2C9, and CYP3A4 are each trations as described in â€œMaterialsandMethods.â€•Resultsare expressed as means; bars, catalytically competent in activating cyclophosphamide and ifos one-half the range for duplicate determinations. 1949

INDUCTION OF OXAZAPHOSPHORINEACTIVATION A RIF(@sM),96h RIF(hr),25I@M contrast to phenobarbital and rifampin, the CYP1A inducers (3-naph thoflavone and TCDD did not affect cyclophosphamide 4-hydroxyla . 0 1 2 5 25 50 0 24 48 72 192 tion or ifosfamide 4-hydroxylation activities in cultured human hepa tocytes. This is in accord with the finding that cDNA-expressed CYP1A1 and CYP1A2 do not catalyze these reactions (4). Therefore, tobacco smoke, which induces CYP1A, is unlikely to influence cy CYP clophosphamide and ifosfamide activation in humans. At least two P-450s belonging to the CYP3A subfamily may be expressed in adult human liver (52). Whereas CYP3A4 is inducible, the polymorphically expressed CYP3A5 may not respond to typical CYP3A inducers (Fig. 6; Ref. 44). The present study confirms pre vious findings that the antitubercular drug rifampin elevates CYP3A protein and activity levels in primary cultures of human hepatocytes 1 2 3 4 5 6 7 8 9 10 11 (39, 44, 46, 53). This is in agreement with the clinical observation that rifampin accelerates the elimination pharmacokinetics of drugs such

> as quinidine (54) and 17a-ethinylestradiol (55), which are metabo I. lized by hepatic CYP3A (56, 57). Rifampin was presently found to be @ 1500 L'jâ€”.. a relatively potent inducer of hepatocyte oxazaphosphorine 4-hydrox Inc ylase activity, as well as CYP3A protein and enzyme activity, with 1 >-0 @ 0. 1000 .LM rifampin stimulating near maximal increases in each of these re activities (Figs. 2 and 3). Rifampin at I p@ was also effective in enhancing CYP3A-mediated lidocaine N-deethylation activity (58) @.E500 and CYP3A mRNA levels (59) in cultured human hepatocytes. Al though the present studies identify rifampin as a particularly potent ZE inducer of ifosfamide 4-hydroxylation and cyclophosphamide 4-hy @ 0 In droxylation in human liver cells, further studies, including a con 0 In trolled clinical trial, will be needed to ascertain whether rifampin w RIFAMPINCONCENTRATION(NM) effects corresponding changes in the pharmacokinetics and pharma Fig. 3. Concentration-dependence and time course for induction of immunoreactive codynamics of these alkylating agents and whether this translates into CYP3A proteins (A) and testosterone 6@3-hydroxylaseactivity(B) by rifampin. Human an enhanced therapeutic effect. The typical serum drug concentration hepatocytes (culture Ff69) were treated for 96 h with 0.1% DMSO (vehicle control) or I, 2, 5, 25, or 50 p.Mrifampin (RIF), and microsomes were then isolated. A, immunoblots of achieved in patients after standard dosages of rifampin is 2â€”30p@M hepatocyte microsomes probed with anti-rat CYP3A antibodies. Lane 1, vehicle control; (60), which corresponds to the range of concentrations (1â€”50pM)that Lanes 2â€”6,1â€”[email protected] 96 h, as indicated. Lanes 7â€”11,25MMrifampin for t = 0 to 192 h, as indicated. B, microsomal testosterone 6@3-hydroxylaseactivitiesdetermined enhance oxazaphosphorine activation in cultured human hepatocytes. using the same microsomal samples shown in A. Lanes 1â€”6.with the results expressed as In a previous study, prednisone was found to decrease the elimination pmol product formed per mm per mg of microsomal protein. half-life of cyclophosphamide in patients (20). This may be related to the fact that this corticosteroid increases CYP3A4 mRNA, protein, and activity levels (43). DISCUSSION CYP2B6 is the only CYP2B protein that has been isolated and Clinical pharmacokinetic studies have suggested that hepatic clear purified from human liver (61, 62). The present study provides the ance of cyclophosphamide in cancer patients can be increased by first demonstration that an immunoreactive CYP2B protein, likely concurrent administration of drugs such as phenobarbital (19), pred CYP2B6, is inducible by several xenobiotics, such as phenobarbital, nisone (20), and dexamethasone (15), which are inducers of human rifampin, and dexamethasone in cultured human hepatocytes. That p-450 (46). The present study shows that cyclophosphamide 4-hy CYP2B6 is inducible in human cells is consistent with studies in droxylation and ifosfamide 4-hydroxylation are both increased in animal models where one or more liver CYP2B proteins are subject to cultured human hepatocytes treated with select P450 inducers, and barbiturate induction (63). In the case of human hepatocytes, pheno that these activity increases are associated with induction of the barbital increased not only CYP2B6 but also CYP2C8, CYP2C9, and human P450 enzymes CYP2B6, CYP2C8, CYP2C9, and CYP3A4. CYP3A. The previous finding that phenobarbital decreases the elim Phenobarbital and rifampin, which induced all four P-450 proteins in ination half-life of cyclophosphamide in human subjects (19) may these cells, increased cyclophosphamide and ifosfamide activation to now be explained by the phenobarbital induction of both the high a greater extent at 2 msi than at 0.25 mr@isubstrate concentration. This capacity (high Vmax) CYP2B6 (4) and the lower capacity CYP2C is consistent with our finding that CYP2B6 and CYP3A4 are high Km enzymes (6) that contribute to hepatic activation of this anticancer catalysts of these two drug activation reactions (4), whereas CYP2C drug. From the present study, it appears likely that phenobarbital will forms such as the wild-type CYP2C9-!1e359 allele are lower Km also impact on the clinical pharmacokinetics of ifosfamide. oxazaphosphorine 4-hydroxylases (6). We have observed previously Treatment of primary human hepatocyte cultures with rifampin or that in human liver, CYP2B6 preferentially contributes to cyclophos phenobarbital was previously shown to increase CYP2C mRNA and phamide 4-hydroxylation, whereas CYP3A4 is a major catalyst of protein levels (53). Using SDS-PAGE conditions that can resolve ifosfamide 4-hydroxylation (4). In several hepatocyte cultures, phe CYP2C8 from CYP2C9 and using polyclonal anti-rat CYP2C anti nobarbital and nfampin enhanced ifosfamide 4-hydroxylation to a bodies that cross-react with both of these human CYP2C forms, we greater extent than cyclophosphamide 4-hydroxylation (Tables 2 and presently demonstrate that rifampin, phenobarbital, and dexametha 3; Fig. 4). This suggests that although CYP2B6 and CYP3A are sone elevate both CYP2C8 and CYP2C9 in human hepatocytes. That increased by both P-450 inducers, the CYP3A induction may domi CYP2C9 is inducible in these liver cells is consistent with the clinical nate the metabolic profile with these anticancer drug substrates. In finding that rifampin enhances the total body clearance of tolbutamide 1950

A: FF59 B: FF60 1@D4-OHCPAU4-OH

0 50â€¢- @.]@â€˜@[email protected] T CPA IFA RIF CYTOCHROME P450 INDUCER C

C: FH1O5 D:FI'108

@ 4-OH CPA C U 4-OHIF

@ Sc 1 I CPA IFA RIF

CYTOCHROME P450 INDUCER

Fig. 4. Enhanced cyclophosphamide and ifosfamide activation in human hepatocytes treated with cyclophosphamide or ifosfamide. Four independent human hepatocyte cultures were treated for 96 h with 0.1% DMSO (vehicle control), cyclophosphamide (CPA; 50 @sM,A,C, and D; 250 @sM,B),ifosfamide (JFA; 50 @sM,A,C, and D; 250 MM,B),or nfampin (RIF; 25 @u.i,Aand B; I psi, C and D). Microsomal cyclophosphamide 4-hydroxylation and ifosfamide 4-hydroxylation activities were determined at 2 m@isubstrate as described in â€œMaterials andMethods.â€•ResultsareexpressedasthepercentageofincreaseinenzymeactivityabovetheDMSOcontrol(means;bars.one-halftherangeforduplicatedeterminations).Control (uninduced) enzyme activities were 0.60, 0.49, 1.40, and 0.31 nmol 4-hydroxycyclophosphamide/mi&mg and 0.56, 0.47. 1.36, and 0.46 nmol 4-hydroxyifotfamide/minlmg for hepatocyte cultures Ff59, Ff60, FH1O5, and Fl' 108. respectively.

(64) and warfarin (65), both of which are metabolized to a large extent been suggested that these alkylating agents undergo autoinduction by CYP2C9 (66â€”68). (17, 29, 47, 69). In principle, autoinduction of cyclophosphamide and Repeated administration of cyclophosphamide (27, 28) and ifos ifosfamide biotransformation could occur via enhanced 4-hydroxyla famide (16, 26) can shorten the plasma half-life and increase the total tion (activation) and/or N-dechloroethylation (inactivation). Clinical body clearance of the parent oxazaphosphorine. Consequently, it has pharmacokinetic studies have shown that fractionated ifosfamide ther apy is associated with increased formation of the N-dechloroethylated metabolites ( I7, 70, 7 1). In patients administered cyclophosphamide @ 0 CD C) 0 LO 0 0 0 < @O by i.v. infusion on each of two consecutive days, the AUC for the A Sc) 04 â€˜- 00 If) 0 C') C'J csj @@@ ,-@_ < < < C'JLt) csJ i-. parent drug is lower on the second day than on the first day, but the -J I- 0- 0@ 0. L@:< < < >- >->- @ I 000 @Y:o 00 AUCs for the metabolites 4-hydroxycyclophosphamide and phosphor amide mustard are not altered (13). A potential explanation for the lack of a change in the AUC of the activated metabolites is that @ CYP3A â€˜ . ______repeated oxazaphosphorine administration induces not only the mi crosomal P-450s that catalyze the 4-hydroxylation reaction but also B. the cytosolic aldehyde dehydrogenase enzymes that oxidize aldophos @@@ @yp2C9 -- phamide to the carboxy metabolite and thereby inactivate it. Consist @ 2C8â€”@@ ..â€”@. -.-@ ent with this proposal, repeated administration of ifosfamide is asso ciated with elevated urinary levels of carboxyifosfamide (70, 7 1). In the present study, we used microsomes isolated from primary human 1 2 3 4 5 6 7 8 9 10 11 12 hepatocytes treated with cyclophosphamide or ifosfamide to demon Fig. 5. Effect of cyclophosphamide and ifosfamide on immunoreactive CYP2C8. strate directly that autoinduction of oxazaphosphorine biotransforma CYP2C9, and CYP3A protein levels in microsomes from cultured human hepatocytes. Primary cultures of human hepatocytes (culture FF108; Lanes 2â€”9)weretreated for 120h tion involves enhanced 4-hydroxylation. It has been suggested that with 0.1% DMSO (vehicle control), cyclophosphamide (CPA), or ifosfamide (JFA) at 50, induction of P-450 enzymes may not impact on the therapeutic effi 250, or 1000 p@, as indicated above each lane. Rifampin treatment (20 @sMfor96 h) was included as a positive control in Lane 6. Shown is an immunoblot of isolated hepatocyte cacy of cyclophosphamide because of the unaltered AUCs for 4-hy microsomes probed with anti-rat CYP3A antibodies (A) and anti-rat CYP2C antibodies droxycyclophosphamide and phosphoramide mustard (13), which is (B). Lane 1, human liver microsomes HLS9. Lane 2. vehicle control. Lanes 3â€”9,induction thought to be the therapeutically active metabolite (3). However, with CPA, WA, or RIF at the concentrations indicated. Lanes 10â€”12.cDNA-expressed CYP3A4, CYP2B6, and CYP2C9-I1e359standards. induction of P450 enzymes such as CYP3A could potentially impact 1951

HL HL FF59 FF60 HL 2@râ€”â€˜@ 8 2B6 RIFBNFCPAIFA - RIF BNF PB CPA IFA 9 Fig. 6. Differential effect of cyclophosphamide and ifosfamide on immu A. noreactive CYP2B6, CYP3A4, and CYP3A5 protein levels in microsomes 3_up - .@ ;; : from cultured human hepatocytes. Human hepatocytes (Lanes 4â€”8forcul @.- â€” ture FF59 and Lanes 9â€”14forculture VI'60) were treated for 96 h with 0.1% DMSO (vehicle control), 25 pM f3-naphthoflavone(BNF), 25 ,sMrifampin (RiP), 2 mM phenobarbital (PB), 50 @i.si(FF59)or 250 @Mcyclophosphamide (CPA; FT6O), or 50 @LM(FF59)or 250 @.LMifosfamide(IFA; FT6O). Shown are immunoblots of hepatocyte microsomes probed with anti-rat CYP2B antibodies (A) and anti-CYP3A antibodies (B). Lanes I, 2, and IS, human liver microsomes HLS2, HLS8, and HLS9, respectively. Lane 3, cDNA expressed CYP2B6 (A) or CYP3A4 (B). Lanes 4 and 9. vehicle control. Two FF60 HL CYP3A immunoreactive bands are seen in samples from hepatocyte culture HL HL FF59 F-r59 (Lanes 4â€”8).Theupper band is the dominant band present in Lane 4 2 8 3A4- RIP BNFCPAIFA RIP BNF PB CPA IFA 9 and corresponds in mobility to cDNA-expressed CYP3A.5and is not induced B.

@@@@@ by RIF (Lane 5 versus Lane 4) or by BNF, CPA, or IFA (Lanes 6â€”8versus --- a@lI@ @0-@3â€•@A@ Lane 4). The lower band corresponds in mobility to cDNA-expressed CYP3A4. Induction of CYP3A4 was found with RIF (Lanes 5 and 10), PB (Lane 12), CPA (Lanes 7 and 13), and IFA (Lanes 8 and 14) but not BNF (Lanes 6 and 11). Corresponding testosterone 6(3-hydroxylase activities (CYP3A-catalyzed) are shown in Table 4.

2 3 4 5 6 7 8 9 10 11 12 13 14 15

on the host toxicity of ifosfamide because this compound is exten sponding protein in cultured human hepatocytes (73). The lack of an sively metabolized by N-dechloroethylation (7â€”9),which is at least in effect of the oxazaphosphorines on CYP3A5 protein levels is consist part catalyzed by CYP3A enzymes in human liver (5) and leads to the ent with previous findings that the expression of this P.450 is not production of neurotoxic metabolite(s) (1 1, 12). Recent studies mdi subject to modulation by known CYP3A4 inducers in cultured human cate, however, that the subset of P-450 enzymes that catalyzes oxaza hepatocytes (44) or in vivo (51). Our observation that oxazaphospho phosphorine N-dechloroethylation is distinct from that which cata rime anticancer drugs are inducers of human P-450 in cultured liver lyzes drug activation via 4-hydroxylation, suggesting possible cells suggests the potential for a pharmacokinetic drug interaction in strategies for improving the balance between these two competing patients given combination cancer chemotherapy regimens that in metabolic pathways based on the use of P-450 inducers in combina dude other drugs subject to CYP2C8, CYP2C9, or CYP3A metabo tion with P-450 form-selective inhibitors (10). The present identifi lism. Future clinical studies will be required to investigate this cation of several effective inducers of oxazaphosphorine activation in possibility. human liver cells is an important first step leading to testing of the In summary, the treatment of primary cultures of human hepato therapeutic utility of such strategies. cytes with rifampin, phenobarbital, or dexamethasone, which induced The present study establishes that cyclophosphamide and ifosf CYP2B, CYP2C, and CYP3A, increased cyclophosphamide 4-hy amide are modulators of human P-450 expression and have the droxylation and ifosfamide 4-hydroxylation. These two oxazaphos potential to induce P-450 enzymes such as CYP2C8, CYP2C9, and phorines enhanced their own activation by inducing P-450 enzymes CYP3A4. That cyclophosphamide induces CYP3A4 in primary cul such as CYP2C8, CYP2C9, and CYP3A4. tures of human hepatocytes is consistent with the finding in bone marrow transplant patients that this drug increases total body clear ACKNOWLEDGMENTS ance of dexamethasone (27), which is metabolized by CYP3A in human liver (72). Although cyclophosphamide and ifosfamide were We thank Dr. J. Pohl (ASTA Pharma, Bielefeld, Germany) for kindly both effective in inducing CYP3A4 protein in cultured human hepa providing authentic 4-hydroperoxyifosfamide metabolite standard and Dr. tocytes, the present study showed that these drugs are ineffective in Lydiane Pichard for providing some of the primary hepatocyte microsomes modulating the protein levels of the polymorphically expressed used in this study. CYP3A5. However, these alkylating anticancer agents might affect CYP3A5 mRNA expression because in the case of another CYP3A, REFERENCES rifampin increases the levels of CYP3A7 mRNA but not the corre I. 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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1997 American Association for Cancer Research. Enhanced Cyclophosphamide and Ifosfamide Activation in Primary Human Hepatocyte Cultures: Response to Cytochrome P-450 Inducers and Autoinduction by Oxazaphosphorines

Thomas K. H. Chang, Li Yu, Patrick Maurel, et al.

Cancer Res 1997;57:1946-1954.

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