OF BIOLOGICAL CHEMISTRY
`THE JOURNAL
`0 1986 by The American Society of Biological Chemists, Inc.
`
`Val. 261, No. 11, Issue of April 15, pp. 5051-5060,1986
`Printed in U.S.A.
`
`Characterization of Rat and Human Liver Microsomal Cytochrome
`P-450 Forms Involved in Nifedipine Oxidation, a Prototype for
`Genetic Polymorphism in Oxidative Drug Metabolism*
`
`(Received for publication, July 25, 1985)
`
`F. Peter GuengerichSS, Martha V. Martin$, Philippe H. BeauneTi, Pierre Kremers(1, Thomas Wolffc*,
`and David J. Waxman$$
`From the $Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,
`Nashville, Tennessee 37232, Wnstitut National de la Sant4 et de la Recherche Medicale U 75, CHU Necker, F75730 Paris Cedex
`15, France, IlLaboratoire de Chimie Medicale B23 Sart-Tilrnan, Liege-I, Belgium, **Gesellschaff fur Strahlen- und
`Umweltforschung-Mu~hen, Znstitut fur Toxikologie, 0-8042 Neuherberg, Federal Republic of Germany, and the $$Department
`of Biological Chemistry and Dana-Farber Cancer Institute, Haruard Medical School, Boston, Massachusetts 021 15
`
`The metabolism of the dihydropyridine calcium an-
`tagonist and vasodilator nifedipine has been reported
`to exhibit polymorphism among individual humans
`(Kleinbloesem, C. H., van Brummelen, P., Faber, H.,
`Danhof, M., Vermeulen, N. P. E., and Breimer, D. D.
`(1984) Biochem. Pharmacol. 33, 3721-3724). Nife-
`dipine oxidation has been shown to be catalyzed by
`cytochrome P-450 (P-450) enzymes. Reconstitution,
`immunoinhibition, and induction studies with rat liver
`indicated that the forms designated P - 4 5 0 ” ~ . ~ and P-
`~ ~ O P C N - E are the major contributors to microsomal
`nifedipine oxidation. The P-450 which oxidizes nife-
`dipine (P-45oNF) was purified to electrophoretic ho-
`mogeneity from several human liver samples. Antibod-
`ies raised to P-45oNF were highly specific as judged by
`immunoblotting analysis and inhibited >90% of the
`nifedipine oxidase activity in human liver microsomes.
`A ’monoclonal antibody raised to the
`human P-450
`
`preparation reacted with both human P - 4 5 0 ~ ~ and rat
`P’450pCN-E. Immunoblotting andysis of 39 human liver
`microsomal samples using anti-P-460NF antibodies re-
`vealed the same 52,000-dalton
`polypeptide, corre-
`sponding to P-450m, with only one of the microsomal
`samples showing an additional immunoreactive pro-
`tein. The level of nifedipine oxidase activity was highly
`correlated with the amount of P-450N~ thus detected
`using either polyclonal (r = 0.78) or monoclonal ( r =
`0.65) antibodies, suggesting that the amount of the P-
`450NF polypeptide may be a major factor in influencing
`the level of catalytic activity in humans as well as rats.
`Cytochrome b6 enhanced the catalytic activity of re-
`constituted P-450NF, and anti-cytochrome b5 inhibited
`nifedipine oxidase activity in human liver microsomes.
`P-450Np also appears to be a major contributor to hu-
`man liver microsomal aldrin epoxidation, d-benzphet-
`amine N-demethylation, 17fl-estradiol 2- and
`4-hy-
`droxylation, and testosterone GB-hydroxylation, the
`major pathway for oxidation of this androgen in human
`liver microsomes.
`
`the oxidative metabolism of
`Interindividual variations in
`* This research was supported in part by Grants CA 30907 and ES
`00267 from the National Institutes of Health (F. P. G.) and BC-462
`from the American Cancer Society (D. J. W.). The costs of publication
`of this article were defrayed in part by the payment of page charges.
`This article must
`therefore be hereby marked “advertisement” in
`accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
`5 Burroughs Wellcome Scholar in Toxicology (1983-1988). T o
`whom correspondence should be addressed.
`
`drugs have been recognized and are probably due at least in
`part to polymorphisms in the cytochrome P-450’ enzymes
`(Kupfer and Preisig, 1984; Guengerich et al., 1986). These
`polymorphisms usually contain a strong genetic component
`and, in addition to predisposing individuals to potential drug
`toxicities, may also contribute to the influence of host factors
`in carcinogenesis, as the P-450s are involved in the biotrans-
`formation of a variety of environmental pollutants, pesticides,
`and cancer-causing agents as well as drugs (Wislocki et al.,
`1980). Such polymorphisms have been characterized in animal
`models (Simmons and Kasper, 1983; Larrey et al., 1984;
`Johnson and Schwab, 1984) but only recently have the mo-
`lecular details been considered in humans. We have recently
`purified human P-450s involved in three of these polymor-
`phisms, namely debrisoquine 4-hydroxylation, phenacetin 0-
`deethylation, and mephenytoin 4-hydroxylation (Distlerath
`et at., 1985; Guengerich et al., 1986; Shimada et at., 1986). In
`each of these polymorphisms about 5% of the individuals in
`Caucasian populations are phenotyically deficient in activity,
`although the fraction varies with race (Kalow, 1984).
`Recently Kleinbloesem et al. (1984) identified an apparently
`bimodal variation in the metabolism of the vasodilator and
`calcium agonist nifedipine. In that study 17% of the individ-
`uals examined were phenotypically deficient in the first step
`
`The abbreviations used are: P-450, liver microsomal cytochrome
`P-45% NaDodSO,, sodium dodecyl sulfate; IgG, immunoglobulin G
`CHAPS, 3-[ (3-cholamidopropyl)dimethylammonio] -1-propanesul-
`fonate; HPLC, high performance liquid chromatogaphy. The termi-
`nology “HL” i s used here to denote human liver microsomal prepa-
`rations, which are identified by code number. The nomenclature used
`for the rat P-450 isozyme and comparison to preparations of others
`has been made elsewhere (Guengerich et al., 1982a; Waxman et al.,
`
`1985). Of particular note here, P - 4 5 0 ~ ~ . ~ (Guengerich et al., 1982a)
`appears to correspond to preparations designated “male-specific P-
`450” (Kamataki et al., 1983), “RLM 5” (Cheng and Schenkman,
`1982), “P-450 2c” (Waxman et al., 1983; Waxman, 19841, “h” (Haniu
`et al., 1984), and “P-450 A” (LeProvost et al., 1983). P-450PCN.E
`(Guengerich et al., 1982a, 198213) appears to correspond to other
`preparations designated ‘‘P-45OPCN’’ (Elshourbagy and Guzelian,
`1980) and “PB-2a” (Waxman, 1984). In some cases where experi-
`ments in this report were done with preparations of these P-450s
`made in Dr. Waxman’s laboratory, a combined designation is used
`(e.g. P-450PCN-E(PB.2a)). The human P-450s are designated P-450NF
`
`
`(this report), P - 4 5 0 ~ ~ (Distlerath et al., 1985), P - 4 5 0 ~ ~ (Distlerath et
`
`al., 1985), and P - 4 5 0 ~ ~ (Guengerich et al., 1986) to signify their
`respective involvements in the genetic polymorphisms of nifedipine
`oxidation (NF), debrisoquine 4-hydroxylation (DB), phenacetin 0-
`deethylation (PA), and S-mephenytoin-4-hydroxylation (MP) activ-
`ities.
`
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`5052
`
`P-450 Nifedipine Oxidase
`
`TABLE IV
`NifediDine oxidase activities of rat liver microsomes
`
`Sex of
`rats
`
`Treatment of rats
`
`0.25
`
`Phenobarbital 0.46
`
`
`
`
`Phenobarbital
`P-Naphthoflavone
`Isosafrole
`
`Aroclor
`
`3.79
`
`
`
`Female
`Female
`Male
`Male
`Male
`Male
`1254
`Male
`Male
`Male
`
`Nifedipine oxidase activity
`
`nmol product
`formedjminjmg
`microsomal protein
`0.25
`1.63
`5.34
`13.2
`1.80
`7.96
`
`nmol product
`formed/min/
`nmol P-450
`
`5.51
`3.91
`1.60
`
`1.65 (+ 0.21) 2.71
`
`2.34 (+ 0.57)
`
`(+ 0.35)
`2.52 (+ 0.61)
`
`Experiment I“
`
`1.76
`
`
`
`8.03
`
`Experiment IIb
`
`Pregnenolone 16a-car-
`bonitrile
`Dexamethasone
`(+ 1.05)
`4.16 (+ 0.98) 4.24
`
`
`Male
`(+ 0.45)
`Triacetvloleandomvcin
`2.28 (& 0.32) 3.25
`
`
`Male
`~~ ’ Results are expressed as means of duplicate assays.
`* Results are expressed as means (+ S.D.) of triplicate assays. In these studies the rats weighed only 75-100 g,
`and the lower catalytic activity (compared to Experiment I) is attributed to lack of development of P-45Om.~
`(Waxman et al., 1985).
`
`of metabolism, the formal 2-electron oxidation of the nifedi-
`pine dihydropyridine ring (Scheme 1). Such an oxidation
`could in principle be catalyzed by a number of different
`oxidoreductases, but some precedent exists for the involve-
`ment of P-450s (August0 et al., 1982). The primary metabolite
`is further transformed by saponification of the ester and
`hydroxylation of the ring methyls (Raemsch and Sommer,
`1983).
`In this report we identify the P-450 forms in rat liver which
`are responsible for nifedipine oxidation. The human liver P-
`450 involved in this activity has also been identified, purified
`to apparent homogeneity, and partially characterized with
`regard to its role in the interindividual variation. Preliminary
`reports of these findings have been presented in abstract form
`(Guengerich et al., 1985).
`
`EXPERIMENTAL PROCEDURES AND RESULTS~
`Nifedipine Oxidase Activity in Rat Liver Microsomes-In
`order to define which rat P-450s contribute to microsomal
`nifedipine oxidation, the effects of sex and monooxygenase
`induction agents on rat liver microsomal nifedipine metabo-
`lism were examined (Table IV). Microsomes prepared from
`untreated males oxidized nifedipine 20-fold faster than did
`those prepared from untreated females. On a per mg protein
`basis, the activity could be increased by treatment with phen-
`
`CH,OOC
`
`COOCH, “b C . ; O O C ~ C O O C H .
`
`H
`SCHEME 1. Oxidation of nifedipine to its pyridine metabolite.
`
`CH, N’
`
`CH,
`
`Portions of this paper (including “Experimental Procedures,” part
`of “Results,” Tables 1-111, Figs. 1-9, and Footnotes 3-6) are presented
`in miniprint at the end of this paper. Miniprint is easily read with
`the aid of a standard magnifying glass. Full size photocopies are
`available from the Journal of Biological Chemistry, 9650 Rockville
`Pike, Bethesda, MD 20814. Request Document No. 85M-2478, cite
`the authors, and include a check or money order for $11.60 per set of
`photocopies. Full size photocopies are also included in the microfilm
`edition of the Journal that is available from Waverly Press.
`
`obarbital in either sex or with isosafrole or the polychlorinated
`biphenyl mixture Aroclor 1254. Administration of p-na-
`phthoflavone (which both induces P-450i(NF.B and P-45OIsp.~
`and suppresses the male-specific P-45oUT.A (Guengerich et al.,
`1982a)) decreased nifedipine oxidase activity. Pregnenolone
`16a-carbonitrile, dexamethasone, and triacetyloleandomycin,
`which all induce P-450P~~.E, all elevated nifedipine oxidase
`activity (Table IV, Experiment 11). Of the phenobarbital-
`inducible P-450s, only P-450pCN.E is male specific in untreated
`rats (Waxman et al., 1985).
`Catalytic Activities of Purified Rat P-450 Isozymes-Ten
`different purified rat P-450s were assayed for nifedipine oxi-
`dase activity after reconstitution with rat NADPH-P-450
`reductase and phospholipid (Table v). P-45o”T.A clearly ex-
`hibited the highest catalytic activity but P-450pCN.E and P-
`450pB.B also had some activity. P-450pCN.E, apparently alone
`among the rat P-450s studied to date,
`is known to lose
`catalytic activity during purification (Elshourbagy and Guz-
`elian, 1980; Guengerich et al., 1982a; Shimada and Guenger-
`ich, 1985; Waxman et al., 1985).
`Immunochemical Inhibition of Catalytic Activity in Liver
`Microsomes-The studies with microsomes and reconstituted
`P-450 systems described above suggested that P-~~OIJT-A, P-
`
`4 5 0 p ~ ~ . ~ , and P-450pB.B might be the major forms of P-450
`contributing to nifedipine oxidation in rat liver. In order to
`examine the roles of these enzymes more closely, inhibitory
`antibodies specific for each of these P-450s were incubated
`with microsomes prepared from untreated male rats (Fig. 10).
`Both anti-P-450uT.A and anti-P-450pcN.E showed extensive
`inhibition, but
`anti-P-450pB.n did not. Moreover, anti-P-
`450pB.B did not inhibit nifedipine oxidase activity in liver
`microsomes prepared from phenobarbital-treated adult female
`rats (which contain P-450PCN-E and P-450pB.B but not P-
`450UT.A (Waxman et al., 1985)) (data not shown).
`Purification, Properties, and Reconstitution of Human Liver
`P-450NF”P-45oNF was isolated from several human liver sam-
`ples as described in the Miniprint using a high performance
`liquid chromatography assay to monitor nifedipine oxidase
`activity. Also detailed in the Miniprint are the electrophoretic,
`spectral, and immunochemical properties of P-450N~ and
`studies on its catalytic specificity.
`Anti-P-450~~ was found to almost completely inhibit nife-
`dipine oxidation in human liver microsomes (Fig. 11). Anti-
`
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`
`P-450 Nifedipine Oxidase
`
`5053
`
`TABLE V
`Nifedipine oxidase activities of purified rat P-450 preparations
`Nifedipine oxidase activity
`P-450 isozyme
`nmol product formed/min/nmol P-450
`3.5
`0.39
`0.15
`<0.03
`0.15
`0.66
`0.06
`<0.03
`0.13
`<0.03
`
`n
`
`pre-immune
`
`
`
`o /
`0
`
`I
`5
`
`I
`10
`
`I
`15
`
`I
`20
`
`I
`
`mg IgG/nrnol MICROSOMAL P - 4 5 0
`FIG. 11. Immunoinhibition of human liver microsomal ni-
`fedipine oxidase activity. Human liver microsomes (sample HL92)
`were assayed for nifedipine oxidase activity after preincubation with
`IgG fractions prepared from rabbit preimmune sera (O), anti-rat P-
`~ ~ O U T - A (V), anti-rat P"i50PCN.E (m), or P-45oNF (0). The IgG prepa-
`rations were the same used in Fig. 10. Incubations contained 50 pmol
`of microsomal P-450, and the
`incubation time was 10 min. The
`uninhibited activity was 6.7 nmol of product formed/min/nmol of P-
`450. The indicated points represent means of duplicate experiments.
`Similar patterns of inhibition with anti-P-450NF have been observed
`in several other human
`liver microsomal preparations (data not
`shown).
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`0
`k
`w
`n
`
`0 1
`
`I
`
`I
`10
`
`anti-P-450
`
`f
`PCN-E
`
`J
`
`30
`
`I
`25
`
`
`l
` 5
`o
`I
`I
`15
`20
`m g I g G / n m o i MICROSOMAL P-450
`FIG. 10. Immunoinhibition of rat liver microsomal nifedi-
`pine oxidase activity. Liver microsomes prepared from untreated
`male rats were assayed for nifedipine oxidase activity after preincu-
`bation with IgG fractions prepared from rabbit anti-P-450pe.~ (O),
`(m). Preimmune IgG was added
`(V), or anti-P-450pc~.~
`a n t i - P - 4 5 0 ~ ~ . ~
`to each incubate to give a total IgG level of 25 mg/nmol P-450 in
`each case. Incubations contained 52 pmol of microsomal P-450, and
`the incubation time was 15 min. The uninhibited activity was 4.4
`nmol of product formed/min/nmol of P-450. The indicated points
`represent means of duplicate experiments.
`
`P-450PCN-E was
`rat P-450uT.A did not inhibit, but anti-rat
`somewhat inhibitory in human as well as rat liver microsomes
`(Fig. 11).
`Role of Cytochrome b5 in Human Microsomal Nifedipine
`Oxidase Activity-Preliminary studies indicated that the in-
`clusion of either purified rat or human liver cytochrome b5 in
`the reconstituted P-45oNF resulted in a variable enhancement
`of nifedipine oxidase activity. The influence of cytochrome b5
`on three distinct purified P-45oNF preparations was, therefore,
`examined in greater detail (Fig. 12). While a positive effect
`was observed with each P-450NF preparation, the extent of
`stimulation and the amount of cytochrome b5 required for the
`maximal effect were rather variable.
`In order to gain more insight into the role of cytochrome
`b5, antibodies were raised against purified cytochrome b5 iso-
`lated from liver sample HL91. The antibodies recognized a
`single polypeptide in each of three human microsomal prep-
`arations as demonstrated
`by immunoblotting analysis and
`also inhibited cytochrome b5-dependent (Omura and Takesue,
`1970) NADH-cytochrome-c reductase activity in human liver
`microsomes. Although the inhibition of microsomal nifedipine
`oxidase activity required large amounts of anti-cytochrome b5
`IgG, parallel inhibition of cytochrome b5-dependent NADH-
`cytochrome c reduction was evident (Fig. 13).
`
`!
`
`I
`
`I
`30C
`
`!
`I
`!
`100
`200
`pmol CYTOCHROME p,
`FIG. 12. Enhancement of nifedipine oxidase activity of pu-
`rified human P - 4 5 0 ~ ~
`by human liver cytochrome bs. Recon-
`stituted systems containing 50 pmol of P-45oNF isolated from liver
`sample HL37 (0), HL92 (O), or HL93 (A), 250 pmol of rabbit
`NADPH-P-450 reductase, and 15 nmol of L-a-1,z-dilauroyl-sn-gly-
`cero-3-phosphocholine were fortified with the indicated amounts of
`purified human liver cytochrome b, (added as a 1 ~ L M solution 30 min
`after mixing the previous components at 23 "C). After 10 min, other
`reaction components were added (total volume, 0.5 ml), and the
`reactions were incubated for 5 min.
`
`Correlation of Nifedipine Oxidase Activity with Immuno-
`
`chemically Determined Concentrations of P - 4 5 0 ~ ~ " T h e levels
`of nifedipine oxidase activity in individual human liver mi-
`crosomal preparations were compared to the immunochemi-
`(Fig. 14). When the compar-
`cally estimated levels of P - 4 5 0 ~ ~
`ison was made using rabbit anti-P-450NF, the correlation was
`highly significant (r = 0.78, n = 32, p < 0.005). When a similar
`comparison was made using a monoclonal antibody raised to
`human liver P-4505 (Wang et al., 1983; Beaune et al., 1985),
`the correlation was only slightly less significant (r = 0.65, n
`= 39, p < 0.005).
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`
`5054
`
`P-450 Nifedipine Oxidase
`
`O
`
`I
`40
`
`I
`
`1
`80
`
`I
`
`120
`
`DISCUSSION
`In this report we have identified and characterized the P-
`450 isozymes involved in the oxidation of the drug nifedipine.
`Two rat P-450s, designated as P-45oUT.A and P-450pCN.E, were
`identified as having significant roles in the reaction. The
`human liver enzyme active in nifedipine oxidation, designated
`P-45oNF, was purified on the basis of catalytic activity and
`found to be apparently indistinguishable from several prepa-
`rations of human liver P-450 which had previously been
`isolated in this laboratory (Wang et al., 1983). Immunochem-
`ical inhibition studies strongly suggest that this enzyme is
`responsible for most of the primary oxidation of nifedipine,
`with the immunochemically determined levels of P-45oNF
`being well correlated with nifedipine oxidase activity in hu-
`man liver microsomes.
`In rats, evidence for the roles of P - 4 5 0 ~ ~ . * and P-450pCN.E
`in nifedipine oxidation comes from both reconstitution and
`immunoinhibition studies. The 20-fold sex difference in un-
`treated rats is consistent with the demonstration that in adult
`animals both of the isozymes mentioned are male specific
`(Waxman et al., 1985). P-450pCN.E can be induced in males or
`females with pregnenolone 16a-carbonitrile, phenobarbital,
`dexamethasone, isosafrole, or triacetyloleandomycin (Guen-
`gerich et al., 1982a; Heuman et al., 1982; Wrighton et al., 1985;
`Waxman et al., 1985). P-45OUT.* levels are decreased by the
`above compounds and particularly by P-naphthoflavone and
`other chemicals which induce P-450ONF.B (Dannan et al., 1983;
`Waxman, 1984). The rat P-450pCN.E activity may have some
`relevance to the human situation, but caution is advised; the
`P-450uT.A activity is probably not useful in making compari-
`sons to humans.
`In humans evidence for the role of P-45oNF in nifedipine
`oxidation comes from reconstitution and
`immunochemical
`inhibition studies. The correlation between catalytic activity
`and P-45oNF levels also supports this conclusion. The question
`arises as to the identity and number of P-450s in humans
`which cross-react immunochemically with P-45oNF. Anti-P-
`450m reacted with a minor protein(s) which was resolved
`using DEAE-cellulose but did not exhibit nifedipine oxidase
`activity (Fig. 2B). Furthermore, in previous work human liver
`P--450 NF, n m o l / m g protein
`P-450s that appeared to be immunochemically similar to each
`FIG. 14. Correlation of nifedipine oxidase activity with im-
`other could be separated chromatographically (Wang et al.,
`munochemically determined P-45ONP in human liver micro-
`1983). Recently Watkins et al. (1985) isolated a P-450 from
`somes. Microsomal samples prepared from 32 individual livers were
`human liver on the basis of its strong cross-reactivity with
`assayed for nifedipine oxidase activity (using 50 pmol of total P-450,
`what appears to be rat P-450pCN.E or a homolog (catalytic
`incubation time 10 min, substrate concentration, 0.20 mM) and for
`activity was not reported). At this point we do not know the
`P-45oNF using immunoblotting: 10 pg of microsomal protein were
`electrophoresed in each well along with 0.1 pg of equine liver alcohol
`
`extent of microheterogeneity in the structure of P - 4 5 0 ~ ~ and
`dehydrogenase (Steward et al., 1985). Each gel also contained lanes
`related enzymes. The possibility certainly exists that this is a
`with 0.4, 1.0, 2.0, 5.0, and 10.0 pmol of purified P-45oNF (from sample
`multigene family, but no further evidence is available.
`HL92) and the same amount of alcohol dehydrogenase. The nitrocel-
`Human P-45oNF appears to share immunochemical homol-
`lulose sheets were treated with a 1:lOO dilution of rabbit anti-P-450~~
`ogy (Figs. 6 and 11) as well as catalytic activity with rat P-
`(and a 1500 dilution of anti-alcohol dehydrogenase), and amounts of
`P-45oNF were estimated using the ratio of peaks (obtained after
`450PCN.E, insofar as P-450pCN.E contributes partially to nife-
`
`densitometry) of P - 4 5 0 ~ ~ and alcohol dehydrogenase (Steward et al.,
`dipine oxidation in rat liver microsomes. The regulation of P-
`1985). The line through the points was drawn using linear regression
`450NF and P-450pCN-E is, however, quite distinct. Thus, P-
`analysis.
`450PCN.E is a male-specific enzyme in untreated adult rats,
`although present in young rats of both sexes before puberty.
`Neonatal gonadectomy and hormone replacement can be used
`to alter the patterns of P-450pCN-E expression in rats, as can
`xenobiotic administration (Waxman et al., 1985). In humans
`no sex or age dependence of nifedipine metabolism has been
`observed (Kleinbloesem et al., 1984). Interestingly, the liver
`sample which had the highest levels of both nifedipine oxidase
`activity and immunochemically detectable P-45oNF (detected
`with both rabbit anti-P-450NF and the monoclonal antibody)
`(Fig. 14) was obtained from a patient who had been adminis-
`
`b 5
`rng ANTI-CYTOCHROME b5/nrnol CYTOCHROME
`FIG. 13. Immunoinhibition of human liver microsomal ni-
`fedipine oxidase activity by anti-human liver cytochrome b5.
`Human liver microsomes (sample HL92) were assayed for NADH-
`cytochrome c reductase activity (0) and nifedipine oxidase activity
`(V) after 60 min of preincubation at 23 "C with the indicated amount
`of the IgG fraction of rabbit-anti-human liver cytochrome b,. The
`mixtures were adjusted with the IgG fraction from preimmune anti-
`serum such that all contained 110 mg of total IgG/nmol of microsomal
`b,. The indicated points represent means of duplicate experiments.
`
`Since a role for cytochrome b5 in nifedipine oxidase activity
`could also be demonstrated (uide supra), we also considered
`the effects of variation of this enzyme as well. Among 20
`individual samples examined the specific content of cyto-
`chrome b5 (estimated spectrally) varied from 0.25-1.19 nmol
`(mg protein)" (mean 0.82 f 0.24 (S.D.)). When nifedipine
`oxidase activity was compared to the amount of cytochrome
`b5 in these samples, the r value was only 0.25 (not significant,
`p > 0.10).
`
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`
`5055
`
`P-450 Nifedipine Oxidase
`Kaminsky, L. S. (1982a) Biochemistry 21, 6019-6030
`tered de~amethasone.~ This observation suggests that the
`Guengerich, F. P., Wang, P., and Davidson, N. K. (1982b) Biochem-
`enzyme may be inducible (e.g. by glucocorticoids), but com-
`istu 21,1698-1706
`parison with the liver before treatment is not possible and
`Guengerich, F. P., Shimada, T., and Martin, M. V. (1985) Fed. Proc.
`conclusions regarding “induction” should be considered hy-
`44,1467
`pothetical. The correlation between nifedipine oxidase activ-
`Guengerich, F. P., Distlerath, L. M., Reilly, P. E. B., Wolff, T.,
`Shimada, T., Umbenhauer, D. R., and Martin, M. V. (1986) Xe-
`ity and the immunochemically determined levels of P - 4 5 0 ~ ~
`nobiotica, in press
`suggests that the amount of the enzyme may be important in
`Haniu, M., Ryan, D. E., Iida, S., Lieber, C. S., Levin, W., and Shively,
`determining the catalytic activity. If this is the case, then the
`J. E. (1984) Arch. Biochem. Biophys. 235,304-311
`locus in determining whether a person is a phenotypic defi-
`Heuman, D. M., Gallagher, E. J., Banvick, J. C., Elshourbagy, N.,
`cient metabolizer may be at the level of factors involved in
`and Guzelian, P. S. (1982) Mol. Pharrnacol. 21, 753-760
`transcription rather than the structural gene. Further exper-
`Johnson, E. F., and Schwab, G. E. (1984) Xenobiotica 14,3-18
`imentation will be required to address these hypotheses, but
`Kalow, W. (1984) Fed. Proc. 43, 2326-2331
`Kamataki, T., Maeda, K., Yamazoe, Y., Nagai, T., and Kato, R.
`we feel that we have established a basis for such studies.
`(1983) Arch. Biochem. Biophys. 225, 758-770
`Various correlation, reconstitution, and immunochemical
`Kaminsky, L. S., and Guengerich, F. P. (1985) Eur. J. Biochem. 149,
`inhibition data support
`
`the view that P - 4 5 0 ~ ~ is a major
`479-489
`enzyme involved in human liver nifedipine oxidation, aldrin
`Kleinbloesem, C. H., van Brummelen, P., Faber, H., Danhof, M.,
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`Vermeulen, N. P. E., and Breimer, D. D.
`(1984) Biochem. Phar-
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`Kremers, P., Beaune, P., Cresteil, T., de Graeve, J., Columelli, S.,
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`Kupfer, A., and Preisig, R. (1984) Eur. J. Clin. Pharrnacol. 26, 753-
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`759
`This result has
`been confirmed with anti-P-450NF in the
`Laemmli,. U. K. (1970) Nature 227, 680-685
`present study (data not shown). Thus, P-45oNF or a closely
`Larrey, D., Distlerath, L. M., Dannan, G. A., Wilkinson, G. R., and
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`Guengerich, F. P. (1984) Biochemistry 23, 2787-2795
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`J. Biol. Chem. 193, 265-275
`uiuo deficiency of this activity, the implications are unknown.
`Merck Index (1983) pp. 936-937, Merck and Co., Inc., Rahway, NJ
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`Metzger, H., Shapiro, M. B., Mosimann, J. E., and Viaton, J. E.
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`Park, S. S., Fujino, T., West, D., Guengerich, F. P., and Gelboin, H.
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`
`’Kindly provided by Dr. P. S. Guzelian, Medical College of Vir-
`ginia, Richmond, VA.
`
`Continued on next page.
`
`Vanda Exhibit 2007 - Page 5
`
`

`
`5056
`
`P-450 Nifedipine Oxidase
`
`Supplemental Material to:
`
`Characterization of Rat and Human Liver Microsomal Cytoehrome
`P-450 Poms Involved in Nifedipine Oxidation. A Pmtotype
`for Genetic Polymorphism in Oxidative Drug Metabolism
`
`F. Peter Guengerich, Martha V. Martin, Philippe H. Beaune,
`Pierre Klemers, Thomas Wolff, and David J. Waxman
`
`EXPERIMENTAL PROCEDURFX
`
`"
`
`Chemicals - Nifedipine (3,5-dimethmycarbonyl-2,6-dimethyl-4-LZ-nitrophenyll-l,4-dihydropyri-
`dine) was a gift of Dr. N. Beleher, Pfiner
`Inc., Groton, CT. The pyridine derivative
`of nifedipine
`(3,5-dimethoxycarbo~yl-2,6-dimethyi-4-[ 2-nitrophenyll-pyridine) was a gift of Dr. Jeffrey Idle, St.
`Mary's Hospital Medical School, London, U.K.; a procedure for the Synthesis of this latter compound from
`1965). Nitrendipine was B gift of Dr. A.
`nifedipine has been desoribed elsewhere
`(Low and Snader,
`Scriabqe, Miles Laboratmies, New Haven, CT. CHAPS was purchased from Sigma Chemical
`Co., St.
`Louis, MO. Othel chemic& were prepared as described 01 pwehased fmm sowees listed elsewhere
`(Guengerich and Martin, 1980; Guengwichetg., 1982a, 1962b).
`m- P-450 concentrations were determined speetrelly (Omura and SatO, 19641 using a stand&
`ern-' a t 450 nm. Protein concentrations were estimated as
`difference extincti