Thalidomide Metabolism by the CYP2C Subfamily1

1964 Vol. 8, 1964–1973, June 2002 Clinical Cancer Research

Yuichi Ando, Eiichi Fuse, and William D. Figg2 INTRODUCTION Molecular Pharmacology Section, Cancer Therapeutic Branch, Center Thalidomide is an old compound that was originally de- for Cancer Research, National Cancer Institute, NIH, Bethesda, veloped as a sedative and was eventually pulled from the gen- Maryland 20892 eral market because of its catastrophic adverse effect of teratogenesis. It has been reintroduced over the past several years in the treatment of leprosy and, more recently, has been found to ABSTRACT be effective against various diseases including multiple my- Purpose: This research investigated the biotransforma- eloma and prostate cancer, at least in part, through the inhibition tion of thalidomide by cytochrome P-450 (CYP). of angiogenesis (1–3). Experimental Design: We used liver microsomes from There have been numerous investigations of thalidomide, humans and/or animals and the recombinant specific CYP including the molecular target of the pharmacological action (1, isozymes to investigate CYP-mediated metabolism of thalid- 4–8), and it has been known that thalidomide requires micro- omide. somal CYP3-catalyzed biotransformation for its pharmacologi- Results: Thalidomide was biotransformed into 5- cal activity in terms of toxicity to lymphocytes, inhibition of -hydroxythalidomide (5-OH) and diastereomeric 5؅-hydroxy- cellular adhesion, alternation of cell morphology and differen thalidomide (5؅-OH) by liver microsomes. The human liver tiation, and antiangiogenesis (4, 9–13). None of the breakdown microsomes with higher CYP2C19 activity formed more products generated from the nonenzymatic hydrolysis process metabolites than those with lower CYP2C19 activity and are responsible for the activity (14). Because human fetal tissues had less activity in metabolite formations than those from have significant drug-metabolizing enzymatic activity, even at rats. Recombinant human CYP2C19 and rat CYP2C6 early stages of gestation (15, 16), such intrauterine metabolism isozymes were primarily responsible for forming these me- could produce metabolites or reactive intermediates from tha- tabolites, and the male rat-specific CYP2C11 formed only lidomide, causing teratogenic events. Thalidomide has been -5؅-OH. 5-OH was subsequently hydroxylated to 5,6- shown to generate reactive oxygen species through prostag dihydroxythalidomide by CYP2C19, CYP2C9, and CYP1A1 landin H synthase, which may be responsible for the teratoge- in humans and by CYP2C11, CYP1A1, CYP2C6, and nicity of thalidomide using the rabbit model (5, 7). An inter- CYP2C12 in rats. Incubations with S-mephenytoin and species difference in sensitivity to thalidomide has also been omeprazole, substrates of CYP2C19, inhibited metabolism explained by the higher embryonic DNA oxidation from the by human liver microsomes, supporting the involvement of reactive oxygen species in the rabbit model than in the mouse CYP2C19. ␣-Naphthoflavone, an inhibitor of CYP1A, si- model that is believed to be resistant to the thalidomide terato- multaneously stimulated the 5-OH formation and inhibited genicity (7, 17, 18). Because the newborn rabbits as well as -cis-5؅-OH formation catalyzed by human liver microsomes. other rodents, unlike human fetuses, essentially lack CYP en The contribution of the CYP2C subfamily was supported by zyme activities, other enzyme systems, including prostaglandin the immunoinhibition study using human liver microsomes. H synthase, may play an important role in xenobiotic metabo- When we used the microsomes from treated rats, the me- lism in the animal fetus (19, 20). Therefore, even if the pros- tabolite formations did not increase by inducers for CYP1A, taglandin H synthase system would be deeply related to the teratogenicity of thalidomide in the animal fetus, the findings do CYP2B, CYP2E, CYP3A, or CYP4A, suggesting that these not preclude a role of the CYP-mediated metabolism in the could not be involved in the main metabolic pathway in rats. human fetus and adult animal. As a result, we cannot simply Conclusions: We discovered that the polymorphic en- extrapolate the ways in which the mechanisms occur in humans zyme CYP2C19 is responsible for 5- and 5؅-hydroxylation of and adult animals from the experiments using fetal animals. thalidomide in humans. In rats, thalidomide was hydroxy- Despite numerous attempts made over a period of Ͼ40 years to lated extensively by CYP2C6 as well as the sex-specific elucidate how thalidomide could act in humans, the true mech- enzyme CYP2C11. anisms still remain ambiguous and controversial. The main transformation of thalidomide in humans is spontaneous nonenzymatic hydrolysis (21). Indeed, thalidomide undergoes very little metabolism by the CYP system in vitro, and Received 11/29/01; revised: 3/5/02; accepted 3/22/02. as far as CYP3A4, there was little in vivo drug interaction The costs of publication of this article were defrayed in part by the between thalidomide and hormonal contraceptives (22–24). payment of page charges. This article must therefore be hereby marked However, at least two hydroxylated metabolites have been advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work has been funded by the United States Government. 2 To whom requests for reprints should be addressed, at Molecular Pharmacology Section, National Cancer Institute, NIH, Building 10, 3 The abbreviations used are: CYP, cytochrome P-450; 5-OH, 5- Room 5A01, 9000 Rockville Pike, Bethesda, MD 20892. Phone: hydroxythalidomide; 5Ј-OH, 5Ј-hydroxythalidomide; 5,6-dOH, 5,6- (301) 402-3622; Fax: (301) 402-8606; E-mail: [email protected]. dihydroxythalidomide; HPLC, high-performance liquid chromatography.

effective and reliable therapy and a development of more effective new agents.

MATERIALS AND METHODS Chemicals and Reagents. Racemic thalidomide (100%) was provided by Celgene Corp. (Warren, NJ). Hydroxylated compounds of thalidomide (cis-5Ј-OH, 4,5-dihydroxythalidomide, 5,6-dOH, and N-hydroxythalidomide) were provided by Dr. K. Eger (University of Leipzig, Leipzig, Germany) and Dr. P. K. Li (Ohio State University, Columbus, OH; 5-OH and 4-hydroxythalidomide). S-(ϩ)-Mephenytoin and antihuman CYP antibodies were purchased from Gentest Corp. (Woburn, MA). Other chemicals were purchased through Sigma-Aldrich Co. (St. Louis, MO). Human and Animal Liver Microsomes and Microsomal Fraction Specifically Expressing CYP. Pooled human and individual liver microsomes were purchased from XenoTech, Fig. 1 Structures of thalidomide and two hydroxylated metabolites. ϭ ϭ A, thalidomide. B, 5-OH. C,5Ј-OH. LLC (Kansas City, KS; n 16) and Gentest Corp. (n 10, individual donors H112 and H42). The microsomes from H112 and H42 have higher and lower S-mephenytoin 4Ј-hydroxylation activity, a prototype reaction catalyzed by CYP2C19: 262, found in human urine or plasma, which could be formed at very 8, and 66 pmol/min/mg for H112, H42, and the pooled micro- low concentrations after incubation with human liver microsomes, respectively. The genotype of CYP2C19 from donors somes or 9000 ϫ g supernatant fractions in vitro (22, 23). The H42 and H112 was homozygous for the reference sequences. two metabolites are 5-OH, formed by hydroxylation of the Pooled microsomes prepared from untreated male animals phthalimide ring possibly via arene oxides, and 5Ј-OH, by and those from male Sprague Dawley rats treated with proto- hydroxylation of the glutarimide ring leading to diastereomeric typical CYP inducers were provided by XenoTech, LLC. The products because a new chiral center is formed (Fig. 1). It has male animals included Sprague Dawley rats ( nϭ 200), Fischer also been suggested that at least two distinct metabolites or 344 rats (n ϭ 20), CD1 mice (n ϭ 400), New Zealand rabbits intermediates of thalidomide via a CYP system should exist (n Ն 6), and Beagle dogs (n ϭ 6). The treated rats received with and without an involvement of arene oxide formation, but isoniazid (200 mg/kg; n ϭ 21), dexamethasone (50 mg/kg; n ϭ at present there is no evidence that these are relevant to 5-OH 23), clofibric acid (200 mg/kg; n ϭ 16), or saline (5 ml/kg; n ϭ and 5Ј-OH (25). 42) once/day over a period of 4 days or a single injection of The fundamental questions that remain unanswered are: Aroclor 1254 (500 mg/kg; n ϭ 15) on day 1, followed by does thalidomide undergo CYP metabolism, and if so, what preparations of the liver microsomes on day 5. The anticipated isozymes are involved? Identifying the specific isozyme is crit- induction of each CYP activity was confirmed by the vendor: ical to understanding the pharmacokinetic/pharmacodynamic Aroclor 1254, Ͼ10-fold of CYP1A and CYP2B; isoniazid, variation in thalidomide-treated patients, especially when activ- 2–3-fold of CYP2E1; dexamethasone, 3–5-fold of CYP3A; and ity and/or expression of the enzyme is polymorphic. It is also clofibric acid, Ͼ10-fold of CYP4A. Another batch of pooled useful to predict drug interactions, which may occur with si- microsomes from male and female Sprague Dawley rats (n ϭ multaneously administered drugs. Pharmacogenetics analysis, 15) were purchased from Gentest Corp. the study of genetically determined alternations in a metabolic Recombinant CYP enzymes containing baculovirus- pathway of a drug, could stratify a subpopulation of patients expressed CYP were purchased from Gentest Corp. The micro- with different pharmacokinetic/pharmacodynamic profiles and somal fractions expressing CYP1A1, -1A2, -1B1, -2C18, underlying genetic markers (e.g., single nucleotide polymor- -2D6*1 (-2D6), or -3A5 for human enzymes and CYP1A1, phisms) optimizing drug doses or schedules based on each -1A2, -2D1, or -2D2 for rats were coexpressed with NADPH- patient’s genotype. A few investigators have attempted to find CYP oxidoreductase. Those expressing CYP2A6, -2B6, -2C8, pharmacogenetic risk factors of thalidomide neuropathy in small -2C9*1-Arg144 (-2C9), -2C19, -2E1, -3A4, or -3A7 for humans populations (26, 27). Thus, identifying the pathway should and CYP2A2, -2B1, -2C6, -2C11, -2C12, -2C13, -3A1, or -3A2 provide us with a useful guide in the pharmacogenetic investi- for rats were coexpressed with cytochrome b5 together with gation of thalidomide. NADPH-CYP oxidoreductase. Control microsomes were pre- This research was undertaken to elucidate biotransforma- pared from insect cells infected with wild-type baculovirus. tion of thalidomide by human and/or animal CYP and the Incubation with Microsomes and HPLC Analyses specific CYP isozyme, if any. Because there is in vitro evidence of Thalidomide Metabolites. A typical reaction mixture that thalidomide requires metabolic activation by CYP and the consisted of 100 mM Tris-HCl buffer (pH 7.5), a NADPH- ϩ metabolites of thalidomide can be identified in urine or plasma, generating system (0.2 mM NADP ,8mM MgCl2,4mM understanding the metabolic pathway could indicate the ways in glucose-6-phosphate, and 1.2 units/ml glucose-6-phosphate which thalidomide works, aiding us in our search for a more dehydrogenase), a desired concentration of substrate, and mi-

Table 1 HPLC gradient 50-min incubations of reaction mixture without a NADPH- Ј A mobile phase consisted of 0.1 M NaH2PO4 buffer (pH 3.0) and generating system. However, because the rate of cis-5 -OH acetonitrile. formation was linear, only tentative Kms were obtained using Method Acetonitrile (v/v, %) Lineweaver-Burk plots measured at thalidomide concentrations A25(0–8 min), 80 (8–9 min), 25 (9–13 min) between 50 and 600 ␮M. B20–25 (0–10 min), 25 (10–11 min), 80 (11–12 min), All incubations and determinations were performed sepa- 20 (12–16 min) rately using each metabolite. Results are presented as means C6(0–5 min), 6 to 25 (5–11 min), 25 (11–19 min), 80 with SD from three independent experiments performed in (19–20 min), 6 (20–23 min) duplicate or as representative means of duplicate. All paired or unpaired t tests were two-tailed. Inhibition Studies. Thalidomide was incubated alone or with a CYP-specific chemical inhibitor to estimate the compet- crosomal protein in a final volume of 200 ␮l. Thalidomide and itive inhibitory effect on 5-OH and cis-5Ј-OH formations (29, the metabolite were dissolved in DMSO (final concentration of 30). The chemicals were dissolved in methanol (orphenadrine, DMSO was Ͻ1%), and the concentrations at the beginning of quercetin, sulfaphenazole, S-mephenytoin, omeprazole, and ke- incubations were usually 400 ␮M for thalidomide and 20 ␮M for toconazole) or in DMSO (␣-naphthoflavone). The final concen- the metabolites. In the preliminary experiments, the substrate trations of all organic solvents in the incubation mixture were concentration of 400 ␮M for thalidomide and of 20 ␮M for 5-OH uniformly 0.2%. The metabolite formations with the inhibitors was decreased by Ͻ10% after a 50-min incubation at 37°Cina were compared with the controls of methanol or DMSO as the shaking water bath. The microsomal amount was usually 0.8 vehicle. The effect of omeprazole on cis-5Ј-OH formation by mg/ml for liver microsomes and 100 pmol CYP/ml for the human liver microsomes could not be evaluated because the recombinant enzymes. Polypropylene tubes were used for the possible metabolite peak from omeprazole in human liver mi- incubation with recombinant enzymes instead of glass as rec- crosomes, as well as recombinant CYP3A4 enzyme, overlaid ommended by the vendor. with that of cis-5Ј-OH. The concentrations of the inhibitor were After a 5-min preincubation at 37°C in a shaking water determined to cover the Ki for each prototype reaction in human bath, the reaction was initiated by adding a NADPH-generating liver microsomes (29). system for incubation with liver microsomes or by adding The immunoinhibition of 5-OH and cis-5Ј-OH formations enzyme for incubation with recombinant CYP. After a subse- was examined by preincubation of the human liver microsomes quent 50-min incubation, the reaction was terminated by adding from donor H112 with preimmune IgG or anti-CYP IgG (0.5 200 ␮l of 10% trichloroacetic acid containing phenacetin as an and 4.0 mg IgG/nmol CYP) under the conditions recommended internal standard and was vortexed and then centrifuged by the vendor, using antiserums for CYP2C13 (polyclonal), (9000 ϫ g) for 10 min to precipitate protein (28). Hydrolytic CYP1A1/1A2 (polyclonal), or CYP2D6 (monoclonal). Accord- degradation could be prevented during analysis by the acidifi- ing to the vendor’s assay, the antiserum for CYP2C13 nonse- cation of the samples, all of which were assayed immediately lectively inhibits all human CYP2C isozymes. after the incubations. For experiments including incubation for 0 min, the trichloroacetic acid was added just after preincubation, followed by the addition of a NADPH-generating system RESULTS or an enzyme. Supernatant (320 ␮l) was injected into a Waters Metabolite Formations from Thalidomide and 5-OH by ϫ ␮ Nova-Pak C18 HPLC column (3.9 300-mm, 4 m), separated Human and Rat Liver Microsomes. 5-OH, diastereomeric at room temperature at a flow rate of 1 ml/min, and detected at cis- and trans-5Ј-OH, and unknown metabolite(s) were found UV wavelengths of 220 nm for cis-5Ј-OH, 236 nm for 5-OH, after incubating thalidomide (400 ␮M) for 50 min with 0.8 and 248 nm for the internal standard and 5,6-OH (Hewlett mg/ml human or rat liver microsomes in the presence of a Packard 1100 Series). A mobile phase consisted of 0.1 M NADPH-generating system (Table 2; Fig. 2). The final concen- Ͻ ␮ NaH2PO4 buffer (pH 3.0) and acetonitrile (Table 1): method A tration of 5-OH was 0.02 M in the human liver microsomes was used for quantification of 5-OH; method B for cis-5Ј-OH; from donor H112. The peaks of 5-OH and cis-5Ј-OH were and method C for 5,6-dOH and other possible metabolites. corroborated by the same migration in the chromatograms and Average retention times were 5.1 min for 5-OH in method A, the equivalent UV spectrums as the authentic standards. The 6.0 min for cis-5Ј-OH in method B, and 15.1 min for 5,6-dOH metabolite trans-5Ј-OH had the same migration as an epimer- in method C. The limits of quantification were 8 nM for 5-OH, ized product converted from cis-5Ј-OH after hydrolysis (31). cis-5Ј-OH, and 5,6-dOH. The intra- and interassay coefficients The retention time (14.2 min in method C) of the unknown of variation at 10 nM were 1.2% (n ϭ 3) and 12.0% (n ϭ 9) for metabolite(s) differed from that of N-hydroxythalidomide (18.8 5-OH, 2.2% (n ϭ 3) and 10.5% (n ϭ 8) for cis-5Ј-OH, and 1.0% min in method C), 4-hydroxythalidomide (16.0 min in method (n ϭ 3) and 3.5% (n ϭ 4) for 5,6-dOH, respectively. C), 4,5-dihydroxythalidomide (14.6 min in method C), or po- Exact kinetic parameters for the metabolite formations tential metabolites from incubations of these compounds as were not calculated because of hydrolysis of the generated substrates. A structural elucidation of the unknown metabo- metabolites, epimerization between cis-5Ј-OH and trans-5Ј-OH, lite(s) was not attempted. and the subsequent 6-hydroxylation of 5-OH. In preliminary The formations of 5-OH and cis-5Ј-OH from thalidomide experiments, 5-OH, cis-5Ј-OH, and 5,6-dOH at concentrations by human liver microsomes were less than one-tenth of those by of 0.01 ␮M were decreased by 3, 32, and 19%, respectively, after rat microsomes (Table 2). Microsomes from donor H112, who

Table 2 Metabolite formations by human and rat microsomes Metabolites were formed after 50-min incubations of thalidomide (400 ␮M) for 5-OH and cis-5Ј-OH, or 5-OH (20 ␮M) for 5,6-dOH with human or rat liver microsomes (0.8 mg/ml). Metabolites (pmol/min/mg)b Total CYPa Microsomes (nmol/mg) 5-OH cis-5Ј-OH 5,6-dOH Human Pooled 420 Ͻ0.20 0.61 Ϯ 0.10 0.55 Ϯ 0.07 H112 651 0.36 Ϯ 0.06 0.87 Ϯ 0.26 0.56 Ϯ 0.09 H42 371 Ͻ0.20 0.50 Ϯ 0.20 0.41 Ϯ 0.05 Rats Pooled male 550 2.09 Ϯ 0.57 9.23 Ϯ 1.33 11.70 Ϯ 1.44 Pooled female 670 2.39 Ϯ 0.54 5.45 Ϯ 0.81 1.92 Ϯ 0.31 a The CYP contents and CYP2C19 activity (S-mephenytoin 4Ј- hydroxylation) were provided from the vendor. The CYP2C19 activity was 66, 262, and 8 (pmol/min/mg) for the microsomes from pooled human, individual donors H112 and H4, respectively. b Results are presented as mean with SD from independent three experiments performed in duplicate.

has higher CYP2C19 activity, formed measurable amounts of 5-OH, but those from the pooled and donor H42 did not. The 5-OH formation by female rats was higher than that found in the male rats (P Ͻ 0.01, paired t test; n ϭ 3), and the cis-5Ј-OH formation by female rats was lower than male rats (P ϭ 0.02, t test; n ϭ 3). The rates of cis-5Ј-OH formation from thalidomide increased linearly with microsomes up to 1.0 mg/ml liver mi- Fig. 2 HPLC chromatograms of metabolites after 50-min incubations crosomes from rats (data not shown) and with time up to 50 min; of thalidomide (400 ␮M) or 5-OH (20 ␮M) with human liver microsomes those of 5-OH formation were not linear (Fig. 3). The tentative (0.8 mg/ml, H112). Amounts of the metabolites are close to the quan- Ј Ϯ ϭ Ϯ Kmsof5-OH formation were 380 40 (n 3) and 310 10 tifiable limits. A, metabolites from thalidomide in the chromatogram of ␮M (n ϭ 3) for male and female rats, respectively (Fig. 4). method C. An unknown metabolite was seen at 14.2 min just before the Ј Formation of 5,6-dOH from 5-OH was subsequently con- hydrolysis product of thalidomide. Peaks of trans-5 -OH and 5-OH ␮ were overlapped. B, metabolites from thalidomide in method A. C, firmed after a 50-min incubation of 5-OH (initially 20 M)asa metabolites from thalidomide in method B. D, metabolites from 5-OH in substrate with 0.8 mg/ml human or rat liver microsomes (Table method C. I.S., internal standard; mAU, milli arbitrary unit(s). 2, Fig. 2). Similar to the metabolites from thalidomide, the 5,6-dOH formation by human liver microsomes was less than one-tenth of that of the male rats. The 5,6-dOH formation by male rats was Ͼ5 times higher than by female rats (P Ͻ 0.01, Metabolite Formations by Recombinant CYP Enzymes. paired t test; n ϭ 3). A small peak with the same retention time When thalidomide was incubated with the human CYP as 4,5-dihydroxythalidomide was formed from 5-OH by the isozymes, CYP2C19 formed both 5-OH and cis-5Ј-OH (Table male rat liver microsomes but was not quantifiable. No apparent 5). A lesser amount of cis-5Ј-OH was formed by CYP2B6. The metabolites were formed from cis-5Ј-OH after incubations with cis-5Ј-OH formation by CYP2C9 was detected, although this human or rat liver microsomes. was below the quantification limit. Among the rat CYP Metabolite Formations by Induced Rats and Interspe- isozymes, CYP2C6 showed high activity of both metabolite cies Comparison. The metabolite formations were compared formations. CYP2C11, the male-specific isozyme (32, 33), using the microsomes from rats treated with the CYP-specific formed a large amount of cis-5Ј-OH but did not form the inducer to assess the isozyme responsible for metabolism (Table measurable 5-OH. Moderate activities for both metabolite for- 3). The treatment with each CYP inducer did not increase the mations by CYP2D2 were found. When 5-OH was incubated as formations of 5-OH, cis-5Ј-OH, and 5,6-dOH as much as marker a substrate, CYP2C19, followed by CYP1A1 and CYP2C9 in activities of the induction indicated. These three metabolite human isozymes, had activity for 5,6-dOH formation. Among formations by the microsomes from isoniazid- and dexametha- the rat isozymes, CYP2C11 had the highest activity, followed sone-induced rats and the 5,6-dOH formation by Aroclor 1254- by CYP1A1, CYP2C6, and CYP2C12. The isozymes involved treated rats were less than those found in the controls. in the 5,6-dOH formation were less specific, and the formations Among the animals evaluated, the microsomes from hu- by CYP1A2 (human and rat), -2D2, -2D6, -2C18, -3A1, -3A2, mans formed the least amounts of 5-OH and cis-5Ј-OH. The and -3A4 were detectable but under the quantification limit. All 5-OH formation by CD1 mice microsomes was Ͼ20 times after of the remaining isozymes showed similarly low activities of the a 50-min incubation compared with that by humans (Table 4; metabolite formations as those of the control microsomes. Fig. 3). The increase of 5-OH formation with 100 pmol/ml micro-

Fig. 3 Metabolite formations from thalidomide (400 ␮M) after 50-min incubations by rat or mouse liver microsomes (0.8 mg/ml; A) and by the recombinant CYP enzymes (100 pmol/ml; B). Closed symbols, 5-OH; open symbols, 5Ј-OH. Fig. 4 Representative Lineweaver-Burk plots of thalidomide 5Ј- hydroxylation by rat liver microsomes (A) and by the recombinant CYP2C6 and CYP2C11 (B). The concentrations of thalidomide were 50, 100, 200, 400, and 600 ␮M. Each plot represents the means of duplicate somes of CYP2C19 was linear with time until 50 min (Fig. 3). determinations. The 5-OH formation by CYP2C6 increased with time; however, it did not demonstrate clear linearity. The cis-5Ј-OH formation from thalidomide was increased linearly with microsomes up to 125 pmol/ml and with time up to 50 min with 100 pmol/ml 6). When we used the H112 microsomes, 10 ␮M ␣-naphthofla- microsomes of CYP2C19, CYP2C6, or CYP2C11. The tentative vone increased the rate of 5-OH formation from 0.24 Ϯ 0.02 K sofcis-5Ј-OH formations were 300 Ϯ 60 (n ϭ 3) and 310 Ϯ m (DMSO as a vehicle) to 0.67 Ϯ 0.06 pmol/min/mg (P Ͻ 0.01, 70 ␮M (n ϭ 3) for CYP2C6 and CYP2C11, respectively (Fig. 4). paired t test; n ϭ 3). The pooled human liver microsomes Inhibition Studies. Incubation with S-mephenytoin, a prototype substrate of CYP2C19 in humans (29, 34, 35), inhib- (Gentest Corp.) formed quantifiable amounts of 5-OH with 10 ␮ ␣ Ϯ ϭ ited 5-OH and cis-5Ј-OH formations by the human and rat liver M -naphthoflavone (0.22 0.03 pmol/min/mg; n 3) as did microsomes, which were consistent with the results of the inhi- the recombinant CYP3A4 enzyme (with NADPH-CYP oxi- bition studies using recombinant enzymes CYP2C19, CYP2C6, doreductase and cytochrome b5, 4.1 pmol/min/nmol CYP). ␣ and CYP2C11 (Table 6). Omeprazole, potent inhibitor for There was no metabolite from -naphthoflavone that had a CYP2C19 (29), also strongly inhibited 5-OH formation of hu- similar retention time as 5-OH on the HPLC chromatogram after man liver microsomes, Ͻ59% of the control by omeprazole of incubating the chemical alone with human liver microsomes or 2.5 ␮M. Orphenadrine, a relatively specific inhibitor of the CYP3A4 enzyme. These increased 5-OH formations were CYP2B6, also inhibited the metabolite formations, especially by inhibited to the levels below quantification by the addition of 1 the rat liver microsomes (30). The inhibitory effects of or- ␮M ketoconazole, an inhibitor for CYP3A4. The rat microsomes phenadrine on the formations of 5-OH and cis-5Ј-OH by and the recombinant CYP2C19 and CYP2C6 enzymes were CYP2C19, CYP2C6, and CYP2C11 were validated using the moderately inhibited from 5-OH formations by ␣-naphtho- recombinant enzymes. The inhibitory effects of sulfaphenazole flavone. and quercetin, inhibitors of CYP2C9 and CYP2C8, respectively, In the presence of the anti-CYP2C antibodies of 4.0 mg were little or weak. IgG/nmol CYP, both 5-OH and cis-5Ј-OH formations were In contrast, ␣-naphthoflavone stimulated 5-OH formation inhibited by 21 and 14%, respectively, whereas the influences of but not cis-5Ј-OH formation by human liver microsomes (Table anti-CYP2D6 and anti-CYP1A were slight (Table 7).

Table 3 Metabolite formations by the induced rat microsomes Metabolites were formed after 50-min incubations of thalidomide (400 ␮M) for 5-OH and cis-5Ј-OH, or 5-OH (20 ␮M) for 5,6-dOH with liver microsomes from the induced rats (0.8 mg/ml). Metabolites (pmol/min/mg)b Agent Induced for Total CYPa (nmol/mg) 5-OH cis-5Ј-OH 5,6-dOH Saline 0.80 2.25 6.82 7.89 Aroclor 1254 CYP1A, -2B 4.00 3.59 (160) 7.79 (114) 6.60 (84) Isoniazid CYP2E1 1.13 1.68 (75) 3.72 (55) 2.87 (36) Dexamethasone CYP3A 1.94 1.79 (80) 5.95 (87) 6.10 (77) Clofibric acid CYP4A 1.91 3.73 (166) 8.92 (131) 9.25 (117) a The CYP contents were provided from the vendor. b Results are presented as means of duplicate determinations. Values in parentheses indicate the percentage of the control activities treated with saline.

Table 4 Metabolite formations by human or animal liver Table 5 Metabolite formations by recombinant CYP isozymes microsomes Metabolites were formed after 50-min incubations of thalidomide Metabolites were formed after 50-min incubations of thalidomide (400 ␮M) for 5-OH and 5Ј-OH, or 5-OH (20 ␮M) for 5,6-dOH with the (400 ␮M) with liver microsomes from human or animals (0.8 mg/ml). recombinant enzymes (100 pmol/ml). Metabolites (pmol/min/mg)b Metabolites (pmol/min/nmol CYP)a Total CYPa Microsomes (nmol/mg) 5-OH cis-5Ј-OH Isozyme 5-OH cis-5Ј-OH 5,6-dOH Human 0.43 0.36 (1.0) 0.51 (1.0) Control Ͻ1.6 Ͻ1.6 Ͻ1.6 Sprague Dawley rats 0.73 2.16 (6.1) 7.64 (14.9) Human CYPs Fischer 344 rats 1.22 2.67 (7.5) 10.35 (20.1) CYP1A1 human Ͻ1.6 Ͻ1.6 2.8 Ϯ 1.2 CD1 mice 1.04 8.26 (23.2) 8.70 (16.9) CYP2B6 Ͻ1.6 3.5 Ϯ 0.9 Ͻ1.6 New Zealand rabbits 1.34 2.17 (6.1) 2.49 (4.8) CYP2C19 10.0 Ϯ 3.2 7.6 Ϯ 1.9 3.4 Ϯ 0.4 Beagle dogs 0.90 4.06 (11.4) 9.81 (19.1) CYP2C9 Ͻ1.6 Ͻ1.6 2.0 Ϯ 0.7 Rat CYPs a The CYP contents were provided from the vendor. CYP1A1 rat Ͻ1.6 Ͻ1.6 18.2 Ϯ 4.0 b Results are presented as means of duplicate determinations. Val- CYP2B1 Ͻ1.6 2.3 Ϯ 0.6 Ͻ1.6 ues in parentheses indicate the relative activities compared with the CYP2C6 18.9 Ϯ 8.1 34.6 Ϯ 1.5 5.5 Ϯ 2.6 human liver microsomes. CYP2C11 Ͻ1.6 40.1 Ϯ 2.4 63.7 Ϯ 7.1 CYP2C12 Ͻ1.6 Ͻ1.6 3.8 Ϯ 1.0 CYP2D2 7.7 Ϯ 3.8 9.3 Ϯ 3.4 Ͻ1.6 DISCUSSION a Results are presented as means with SD from independent three experiments performed in duplicate. Thalidomide was biotransformed into 5-OH and 5Ј-OH by human and animal liver microsomes. Recombinant human CYP2C19 and rat CYP2C6 isozymes were primarily responsible for forming these metabolites, and the male rat-specific 5Ј-OH (Table 5). Because the amount of CYP2B6 (Ͻ1% of the CYP2C11 formed only 5Ј-OH. The 5-OH was subsequently total CYP) is not more than that of CYP2C19 (around 1%; Refs. hydroxylated to 5,6-dOH by CYP2C19, CYP2C9, and CYP1A1 35, 37, 38), the most important CYP isozyme for thalidomide in humans and by CYP2C11, CYP1A1, CYP2C6, and metabolism in human liver should be CYP2C19. The tentative

CYP2C12 in rats. A nonlinear increase of the 5-OH formation in Kms of rat microsomes were comparable with those of recom- liver microsomes would be attributable to the subsequent me- binant CYP2C6 and CYP2C11. The chemical inhibition studies tabolism of 5-OH into 5,6-dOH in addition to the spontaneous showed that S-mephenytoin and omeprazole, the substrates of hydrolysis of the metabolite. Because the male rat-specific CYP2C19, inhibited the metabolite formations by human liver CYP2C11 isozyme has extensive activities in both cis-5Ј-OH microsomes, supporting the involvement of CYP2C19 in tha- and 5,6-dOH formations, it is reasonable to assume that male lidomide metabolism (Table 6). Limited involvement of rats could form these metabolites at a greater rate than females. CYP2C8 and CYP2C9 in the thalidomide metabolism was sup- In contrast, the 5-OH formation was slightly greater in female ported by a lack of the effects by their inhibitors quercetin and rats than males, which reflects less activity of the subsequent sulfaphenazole, respectively. Although the inhibitory effects of 6-hydroxylation of 5-OH in females. Phenytoin received atten- these chemicals on the activities of rat CYP isozyme are not tion for its toxicity, including teratogenicity, possibly via a generally characterized, the inhibitory effects observed in rat reactive intermediate. Interestingly, phenytoin is also a substrate liver microsomes were consistent with those in the recombinant of rat CYP2C6 and CYP2C11, resulting in the sex differences in CYP2C6 and CYP2C11. The contribution of the CYP2C sub- the metabolite formations (36), whereas the drug is metabolized family to thalidomide metabolism in human liver microsomes by CYP2C19 (minor) as well as CYP2C9 (major) in humans. was also supported by the immunoinhibition of anti-CYP2C Thus, there appear to be partially common mechanisms of the antibodies (Table 7). According to the vendor, the microsomes metabolic activation between phenytoin and thalidomide. from H112 have higher catalytic activities than those from H42 The recombinant CYP2B6 formed lesser amounts of cis- in S-mephenytoin 4Ј-hydroxylase (CYP2C19) and diclofenac

Table 6 Effects of CYP chemical inhibitors on metabolite formations by human and rat liver microsomes and recombinant enzymes Metabolites were formed after 50-min incubations of thalidomide (400 ␮M) with liver microsomes (0.8 mg/ml) or with the recombinant enzymes (100 pmol/ml) alone or together with each inhibitor. 5-OHb cis-5Ј-OHb

Inhibitors (␮M)a H112c CYP2C19 Rat CYP2C6 H112c CYP2C19 Rat CYP2C6 CYP2C11 S-Mephenytoin (300) Ͻ59 Ͻ22 29 23 56 Ͻ23 49 33 37 Orphenadrine (100) Ͻ59 40 18 NDd 65 Ͻ23 8 ND ND Orphenadrine (10) 95 81 25 15 77 40 28 Ͻ3 Ͻ6 Sulfaphenazole (10) 105 91 88 105 73 93 89 108 90 Quercetin (10) 99 117 74 108 73 89 82 97 110 ␣-Naphthoflavone (10) 287 54 58 46 67 Ͻ42 72 35 74 a The 5-OH formations in the control were: 0.33 (pmol/min/mg) by H112, 9.9 (pmol/min/nmol CYP) by CYP2C19, 2.18 (pmol/min/mg) by rat microsomes, and 22.3 (pmol/min/nmol CYP) by CYP2C6 against S-mephenytoin, orphenadrine, sulfaphenazole, and quercetin (dissolved in methanol); and 0.26 by H112, 11.3 by CYP2C19, 2.75 by rat, and 27.4 by CYP2C6 against ␣-naphthoflavone (dissolved in DMSO). The cis-5Ј-OH formations in the control were: 0.80 by H112, 6.4 by CYP2C19, 7.35 by rat, 39.5 by CYP2C6, and 31.9 (pmol/min/nmol CYP) by CYP2C11 against S-mephenytoin, orphenadrine, sulfaphenazole, and quercetin; and 0.56 by H112, 3.9 by CYP2C19, 6.40 by rat, 43.4 by CYP2C6, and 29.5 by CYP2C11 against ␣-naphthoflavone. b Results are presented as means of duplicate determinations. Values indicate the percentage of the control activities with vehicle. c Microsomes from the individual donor H112 with higher S-mephenytoin 4Ј-hydroxylation activity than the pooled human liver microsomes. d ND, not done.

Table 7 Immunoinhibition study on metabolite formations Thalidomide undergoes slight CYP-mediated metabolism Thalidomide (400 ␮M) was incubated for 50 min with human liver in this research, substantiating the results found in previous microsomes (HG112, 0.10 nmol CYP) in the absence or presence of studies, although formations should be underestimated because anti-CYP antibodies. of the hydrolysis, the subsequent metabolisms, and the epimer- Metabolites (pmol/min/nmol CYP)a ization. The lower activity of thalidomide metabolism by hu- Antibody (mg IgG/nmol CYP) 5-OH cis-5Ј-OH mans is attributable to CYP enzymes, not to a lack of cofactors or epoxide hydrolase activity. The recombinant CYP2C19, Anti-CYP2C Preimmune 0.91 1.26 CYP2C6, and CYP2C11 enzymes were coexpressed with cyto- 0.5 0.83 (91) 1.30 (103) chrome b5 combined with NADPH-CYP oxidoreductase. In our 4.0 0.72 (79) 1.09 (86) preliminary experiments, 5-OH and cis-5Ј-OH formations on the Anti-CYP1A chromatogram did not change with or without the microsomes Preimmune 0.78 1.04 0.5 0.78 (100) 1.02 (98) containing human microsomal epoxide hydrolase (0.4 mg/ml 4.0 0.93 (119) 1.14 (109) styrene oxide hydrolase activity of 13,830 nmol/min/mg, pre- Anti-CYP2D6 pared from a human lymphoblast-expressed cell line; Gentest Preimmune 0.55 0.63 Corp.). Although mice and rats are generally considered insen- 0.5 0.56 (102) 0.61 (97) sitive to thalidomide teratogenesis (17, 18), the microsomes 4.0 0.72 (131) 0.61 (97) a from mice as well as rats could form the metabolites from Results are presented as means of duplicate determinations. Val- thalidomide through a CYP system more extensively than in ues in parentheses indicate the percentage of control activities incubated with the preimmune IgG. humans and rabbits. This result is rather consistent with previous findings that thalidomide acts as an angiogenesis inhibitor in mice when administered i.p (41). Some previous reports indicated that rat microsomes did not confer pharmacological ac- 4Ј-hydroxylase (CYP2C9; 1720 versus 3560 pmol/min/mg) and tivity on thalidomide by CYP-mediated metabolism and that have lower activities in all other prototype reactions by thalidomide had no antiangiogenic effects in mice implanted CYP1A2, -2A6, -2B6, -2C8, -2D6, -2E1, -3A4, and -4A. Thus, with tumor cells (4, 12, 42). This discrepancy may be explained the higher metabolite formations by the microsomes having by the differences in the experimental methods, especially in the higher CYP2C19 activity agreed with the involvement of the strain or pretreatment of the animals (43). A recent study sug- isozyme in thalidomide metabolism. In the experiments using gested that male rats would be more sensitive to thalidomide in the microsomes from treated rats, the metabolite formations terms of neurobehavioral teratogenicity (44). were not increased by the inducers for CYP1A, -2B, -2E, -3A, The polymorphic enzyme CYP2C19 catalyzes the biotrans- or -4A, suggesting that these CYP subfamilies are not involved formation of thalidomide in humans. The interindividual varia- in the main metabolic pathway in rats. Although Aroclor 1254, tion of the enzyme activity caused by its polymorphism could a commercial preparation of polychlorinated biphenyls, was affect the efficacy and toxicity of therapeutic agents catalyzed primarily used as a CYP1A and CYP2B inducer and was ex- by this enzyme, such as mephenytoin, certain barbiturates, di- pected to increase 5,6-dOH formation from 5-OH, we found a azepam, imipramine, omeprazole, and proguanil (34, 45). Poly- slight decrease of the activity in Aroclor 1254-treated rats. The morphism of CYP2C19 has been studied extensively since the concomitant suppression of CYP2C11 by Aroclor 1254 would first report on poor metabolizers of mephenytoin, and the fre- compensate the CYP1A1 activity (39, 40). quencies reported were 2–5% in Caucasians and 13–23% in

Asian populations (45). Because of very low amounts of bio- formations of 5-OH and cis-5Ј-OH are similar to those observed transformation to the metabolites from thalidomide, the contri- in aflatoxin B1, stimulation in 8,9-epoxidation and inhibition in bution of the polymorphic metabolism to the total pharmacoki- 3-hydroxylation (54). The mechanism of stimulation of 5- netics of parent thalidomide would be insignificant. However, hydroxylation of thalidomide by ␣-naphthoflavone is unclear; the polymorphic activity to form the active metabolites and/or however, the finding implies that 5-OH formation could be intermediates could segregate the patient population into sub- increased by dietary flavonoids contained in fruits, vegetables, groups that differ in their metabolic ability. The biotransforma- and grain products. tion would be impaired in poor metabolizers, resulting in very We discovered definitively that the polymorphic enzyme low or absent concentrations of these metabolites. If the clinical CYP2C19 is responsible for 5- and 5Ј-hydroxylation of thalid- impact of the CYP2C19 polymorphism on thalidomide treat- omide in humans. The 5-OH formation could be environmen- ment were revealed, a poor metabolizer might require relatively tally modulated by dietary flavonoid. In rats, thalidomide was large doses of thalidomide for therapeutic purposes; a normal hydroxylated extensively by CYP2C6 as well as the sex-specific “extensive” metabolizer might have a higher risk of adverse enzyme CYP2C11. Although there is in vitro evidence that effects. A small foresighted study phenotyped CYP2C19 using thalidomide requires metabolic activation by CYP, clinical sig- mephenytoin as a probe in three patients suffering from thalid- nificance of CYP-mediated metabolism of the drug needs to be omide neuropathy (27). Although the study was far from con- further evaluated in future clinical studies. We are in the process clusive, none of the patients were poor metabolizers. CYP1A1 of clinical research to see whether polymorphism of the levels are very low in the adult liver, and this enzyme is mainly CYP2C19 gene is associated with the pharmacokinetics of the expressed in extrahepatic tissues such as lung and intestine, metabolites and clinical outcomes of thalidomide. Additionally, where it is highly induced by cigarette smoking and exoge- when a patient’s genetic markers, including the CYP2C19 gen- nous compounds such as polycyclic aromatic hydrocarbons otype, are related to the therapeutic effects of thalidomide, the and 2,3,7,8-tetrachlorodibenzo-p-dioxin (16, 46). Increased pharmacogenetic assessment of a patient would be imperative CYP1A1 activity by exposure to these inducers might modulate before the administration of thalidomide. pharmacological effects of thalidomide. It seems unlikely that a CYP system in fetal liver would ACKNOWLEDGMENTS form the metabolites or intermediates responsible for teratogenicity. The CYP2C expression and S-mephenytoin 4Ј-hydroxy- We thank Dr. Kristian Riesbeck (Malmo¨ University Hospital, Swe- den) and Dr. Katsunori Nakamura (Vanderbilt University, Nashville, lase activity (CYP2C19 activity) are significantly lower in hu- TN) for helpful advice. We also thank Dr. Kurt Eger (University of man fetal livers than in adults (47). Additionally, CYP3A7, the Leipzig, Leipzig, Germany) and Dr. Pui-Kai Li (Ohio State University, major isozyme in fetal liver (48), did not form 5-OH or cis- Columbus, OH) for providing hydroxylated compounds of thalidomide. 5Ј-OH at a quantifiable level in this research. Nonetheless, the CYP2C subfamily has been detected recently in extrahepatic tissues such as human endothelial cells and the intestine; the REFERENCES metabolism of thalidomide in the target organ or cells in the 1. D’Amato, R. J., Loughnan, M. S., Flynn, E., and Folkman, J. Tha- fetus remains to be investigated (49, 50). lidomide is an inhibitor of angiogenesis. Proc. 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Yuichi Ando, Eiichi Fuse and William D. Figg

Clin Cancer Res 2002;8:1964-1973.

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