noes-srsoosrzaus-o932—939ran2_oo/o
`Dave Mtrrnaotran AND Drrrosmorr
`Copyright 0 I996 lay 'i"h¢ Amerirnn Society for Phtnnueolozy and Exp-rriunentnl ‘therapeutics
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Vol. 24. No. 9
`Printed in -U..5‘.A.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`METABOLISM OF FENTANYL, A SYNTHETIC DPIDID ANALGESIC, BY HUMAN
`LIVER MIGHOSOMES
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ABSTRACT:
`
`Role of CYP3A4
`
`
`
`
`
`
`
`
`
`
`
`
`DENNIS E. FEIERMAN mo JEROME M. LASKEFI
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Dapanments or Ansstltssiology (D.E.F.) and Biochemistry (J.M.L.). Mount Sinai Sehoor or Mecllctne. New York, NY 10029
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`(Received February 29. was: accepted May 23. 1996)
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`The mlerooornal metabolism or fentnnyt, a synthetic opioid oom~
`
`
`
`
`monly used In anesthesia, was Investigated in human liver. Incu-
`
`
`
`
`
`
`bation oi’ lasrtnnyt with human ltapatlo mloroaornna fortified ulrith
`
`
`
`
`
`NADPH resulted in the formation of a single major metabolite.
`namely norfarrtanyl, as determined by GCIMS. No atrlclanoe was
`obtained for the fonnatlon of altltar daaproprtonylfantanyi or N-
`phsnylproplonamide, the latter arising tda N-dealirytadon of the
`
`
`
`
`
`fsnrlsnvl srnitls nltroasn. Kinetic analysis at mlcrosomsl fsntsnlrl
`
`
`
`
`orddatlon revealed a single K... of 117 uM and a ll'..._.. o1'3.8E nmol
`
`
`
`
`
`of norlsntanyi formsdlmlnlnmoi ol cytochrome P450 (P450). Stud-
`
`
`
`
`
`lae using eharnloal lnhlhrlora all human P450 enzymes revealed that
`
`
`
`
`
`
`only agents ltrrown to lnhilrlt GTPEM (rag. ltntooonazoia and eryth-
`
`
`
`romyeln) were oapalrle of strongly inhibiting (290%) mlerosomal
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`tontanyi oxidation. Marked inhibition (2-90%) oi nortsntanyl tonna-
`
`
`
`
`
`
`
`
`tlon by lllllf mlonaaornaa was also obeanrad with poiyeional Inl:l-
`
`
`
`
`
`
`
`
`
`bodiaa to GYF3M. whereas antibodies to other human P4503 were
`
`
`
`
`
`
`
`wlthout attoet. Furthermore. rates at norlonlanyl production by to
`lndhrldual human liver samples were highly oonuiaiaad (r' 2 0.816.
`F = 56.46. p -r 0.001] with Immunochomleally determined levels of
`CYP3A4 present In the samples but not with levels at CYP2C8.
`
`
`
`
`
`¢.‘.YP2t:9, GYP2C‘l9, or CYPEE1. Our manila indicate that CYP3A4 is
`
`
`
`
`the menu eotaiyot Involved In fontanyl oxidation to norlsnlsnyl In
`
`
`
`
`
`human liver. Alterations in CYP3A4 levels or nativity, as wall an the
`
`
`
`
`
`oononmltant administration of other thorapotrlto agents metabo-
`
`
`
`
`Iizacl by this P-I-tit) enzyme. eoutd lead to method perturbations in
`fantasy! sleaosltton and. hoses. snslassla response-
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`<:>/~«~«=-c)\ an
`
`
`
`é
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`cm?‘.9l9Qfll'-‘BOGU0Simmerr=r.4":v112:3“.Io'srrm.rnoErads::‘numruonoo1:nn:nun-ton
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Fenianyl
`is widely used in surgical
`is a synthetic opioid that
`
`
`
`
`procedures requiring analgesia. The organ primarily responsible for
`
`
`
`
`
`fentanyl hiotranstomtation is the liver (I-4). Lehrnrmn er al. (3) first
`
`
`
`
`
`showed that. upon odministmtion Io rout. fentanyl was oxidized to at
`
`
`
`
`
`
`
`variety of polar products and that pretreatment of animals with pitev
`
`
`
`
`
`nobsrbital increased fentanyl oxidation by liver homogenates to one
`
`
`
`
`
`such product. namely phenylseetate. In other in viva studies. nort'en-
`tanyl has been identified as the primary oxidative metabolite of
`featanyl (5-7). although desproprionyllentanyl may also be fanned
`
`
`
`
`
`
`(B). Norfentenyl appears to be the major metabolite produced in
`
`
`
`
`
`
`l).
`humans (6) (fig.
`
`
`
`
`
`
`
`Sulentanil and aifentanil are two newer anesthetics that. like fem-
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`anyl, belong to the anilidopiperidine class of synthetic opioids. In viva
`
`
`
`
`
`and in vltro studies of sufentsnil and slfentanil disposition have shown
`
`
`
`
`that liver mierosomai P450‘ enzymes participate in the metabolism of
`
`
`
`
`both of these compounds (9-12). Sufentanil undergoes deallrylation at
`
`
`
`
`
`
`
`
`
`
`
`
`
`This worlt was supported by National lnatittrtaa of Health Grant .AA07B42
`
`
`
`
`
`
`lJ.M.L). by a Mount Sinai Department at Anesthesiology Research Development.
`
`Award [D.E.F.). and by the Liver Imus Procurement Mel Distribution System
`(National inatltutnll oi Health Grant DK62274}. Portions or this work were pre-
`sentscl at the 22nd Annual Msstlnu of ms Arnsrlesn Society for Ansaihsslflluolslx.
`May 193-5. in Atlanta. GA.
`‘Abbreviations used aria: P450. cytochrome P450; AMX. N-phenylproph
`onarnlds; MSD. mus-selective detector: SD3. sodium dodecyl sullats; PAGE.
`potyaorytamlas gel electrophoresis.
`amt uprrm raql-Isrti to: Dr. Dennis E. Faierrnln. Daplflment or Monaur-
`oioqy. Mount Sinai Medical Center. One Gustavo L. Levy Plane. New York. NY
`1D02tI.
`932
`
`Norllnlanyi
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`N-phanylproplonamldo
`
`[Airlift]
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Fm, 1. Structures affenrarryl and potential oxidative metabolites.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`tom the piper-irline nitrogen and the piperidirre ring. to form norsufon-
`
`
`
`
`
`
`Lani! and SM6, respectively (ill). Aifentanil also undergoes piperidine
`
`nitrogen dealltyiation. wltich gives rise to noraifentanll. and the P450
`responsible has been identified as CYP3A4’ (1 I. I2). Yun 2! Hi. (11)
`used chemical
`inhibitors. CYP3A4 antibodies, and purified P450
`enzymes to demonstrate that CYP3A4 was responsible for most
`noralfentanil formation occurring in human liver. By using Western
`blot analysis. Kharosch and ‘Ihurnn-rel (12) found that alfentanii me-
`
`* The P450 enzymes rsilrred to In this report are designated according to the
`nomenclature oi Nelson at at. (371.
`
`Insys Exhibit 2006
`CFAD v. Insys
`IPR2015-01799
`
`Page 1 of 8
`
`

`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`933
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`FENTANYL OXIDATION BY HUMAN LIVEF-l CYP3A4
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`phosphate buffer, pH 7.4, and then concentrated by uItrafi|u'ation through an
`
`
`
`
`
`
`
`
`
`
`
`tabolism to noralfentanil by human liver microsomes was highly
`
`
`
`Amicon PM-30 membrane. Purified CYP3A4 mignated as E single band (M. =
`
`
`
`
`
`
`
`
`correlated with CYP3A4 levels in the microsomes. A subsequent
`
`
`
`52,000) on SDS-PAGE. and had a specific content of 6.3 nmollmg of protein.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`investigation (13) revealed that. in addition to piperidine nitrogen
`Amino-terminal sequencing of CYP3A4, which was performed after binding
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`deallrylation. CYP3A4 also promoted dealltylatiort of the al.t'entanil
`of the protein to lmmobilon P3” (Nlllllpow.-. Marlboro, MA). using a gas-phase
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`piperidine ring to fortn AMX.
`sequenator/on-line phenylthiohydantoin amino acid analyser (Applied Biosys~
`
`
`
`
`
`
`
`Because fentanyl and alfentanil are structurally similar members of
`tems model 470A), gave the following 16 residues; A-L-l-P-D-l..-A-M-E-T-
`
`
`
`
`
`the anilidopiperidine family of anestltetics, we hypothesized that
`W-L-L-L-A-V. These arrdno-terminal amino acids were identical
`to those
`
`
`
`
`
`
`
`CYP3A4 was also an important detenninant of hepatic fentanyl oxi-
`deduced from the human CYP3.-l4 CDNA sequence (I8) except for a missing
`
`
`
`
`
`
`dation in humans. To assess this, we utilized chemical inhibitors with
`terminal tnetltionine residue.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Immunochemieal Methods. Polyclonal antibodies to human CYP3A4
`known P450 enzyme specificity, inhibitory polyclonal antibodies, and
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`were raised in male New Zealand white rabbits. and the IgG fraction was made
`correlation analyses. The results described herein indicate that the
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`monospecific as described elsewhere (19). Polyclonal anti-human CYPZC9
`
`
`
`
`
`
`
`
`
`
`
`
`
`capacity of human liver rnicrosornes to convert fentanyl to its major
`
`
`
`
`
`
`and anti-human CYP2A6 Igfis were prepared in an identical fashion except
`
`
`
`
`
`
`
`
`
`
`
`oxidative metabolite norfentanyl is indeed due to the presence of
`
`
`
`
`
`
`that these antibody preparations were nearly monospecific as isolated and thus
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`did not require back-adsorption. Prelmmune (control) lgfl was prepared from
`
`
`
`
`
`
`
`
`
`
`
`
`
`rabbit serum obtained before immunization. All lg(i fractions were purified
`
`
`
`
`
`
`
`
`
`
`
`
`front serum by cnprylic acidianunonium sulfate fractionation (20).
`
`
`
`
`
`
`
`
`Protein blotting of liver microsomal proteins to nitrocellulose was per-
`
`
`
`
`
`
`
`
`fcnned as described previously (19. 21). After transfer. the blots were reacted
`
`
`
`
`
`
`
`
`
`with anti-I-IA4 IgG (5 pg of IgG!ml) or,
`in the case of partially purified
`
`
`
`
`
`
`
`CYP3A4 fractions, with anti-l-ll... IgG (2 _u.glml) and were then imrnanocltemi-
`
`
`
`
`
`
`
`
`cally stained using a streptavidin-biotinylated horseradish peroxidase system
`
`
`
`
`
`
`
`
`
`
`
`(19, 21). lmmunoresction intensity was assessed by first scanning the blots
`
`
`
`
`
`
`
`
`with :1 Sharp JX-325 flat-bed scanner interfaced to a computer and then using
`
`
`
`
`
`
`
`
`
`
`
`
`lmagefluant software (Molecular Dynarnlcs. Sunnyvale. CM ID qllfifllilfllt
`
`
`
`
`
`
`
`
`
`
`anti—3A4—imn1unoreactive areas on the image. lmmunochemical staining was
`
`
`
`
`
`
`
`
`
`
`
`
`performed under conditions where reaction density on the blots was directly
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`proportional to the amount of rnicrosomal protein originally applied to the
`
`
`
`
`
`
`
`
`
`polyacrylamide gel.
`
`
`
`
`
`
`
`
`
`
`
`Micros-omal Fentanyl Oxidation. Reaction mixtures contained human
`
`
`
`
`
`
`
`
`
`
`
`
`liver microsomes (protein equivalent to 100-200 pmol of P450) and I03 mM
`
`
`
`
`
`
`
`
`
`potassium phosphate buffer. pH 14. in a final volume of 2.0 rttl. The concen-
`
`
`
`
`
`
`
`
`
`
`
`uation of fentanyl used was 250 phi except in ltinetic analysis experiments.
`
`
`
`
`
`
`
`
`
`
`
`
`where the substrate concentration was varied front 5 to 500 MM. lncubations
`
`
`
`
`
`
`
`
`
`
`
`were initiated with 1 mM NADPH and were terminated after ID min at 37°C
`
`
`
`
`
`
`
`
`
`
`
`
`with 1.0 ml of 10% N_aOH. Afier addition of 2 pg of the internal standard
`
`
`
`
`
`
`
`
`
`
`
`R031930 (a compound strucmr-ally similar to fentanyl). the reaction mitturres
`
`
`
`
`
`
`
`
`
`
`
`
`
`were extracted either with methylene chloride (22) for assessment of norfenw
`
`
`
`
`
`
`
`
`
`
`
`tanyl fonnation or with two 3.0-ml portions of ethyl acetate for assessment of
`
`
`
`
`
`
`
`
`
`
`AMI formation. The organic extracts Went: dried under it st1‘cal‘tt of H300.
`
`
`
`
`
`
`
`
`
`
`
`
`
`into
`followed by rescluhilization in 30 pl of ecetonitrile and placement
`
`
`
`
`
`
`
`
`
`
`microvial inserts. The samples (2.0 id) were injected automatically onto a
`
`
`
`
`
`
`
`
`
`
`
`
`Hewlett-Packard model 5890 Series ll gas chromatograph. with the purge
`
`
`
`
`
`
`
`
`
`
`valve closed for the first i rrtin (splidess mode), and were resolved by using
`
`
`
`
`
`
`
`
`
`
`
`either a DB -5 capillary column (30 m X 0.25 mm; 0.25-um film thicltness) or
`
`
`
`
`
`
`
`
`
`
`
`
`
`a DB-l7 capillary column (15 In K 0.25 mm: 0.25-pm fihn thickness) U 3i W
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Scientific, Poison, CA). A l-lewlett~Packart:l model 5972. MSD was used in the
`
`
`
`
`
`
`
`
`
`
`
`selective ion mode to quantitste fentsnyl metabolites, using the following ml:
`
`
`
`
`
`
`
`
`
`
`
`
`ratios for detection: E3. 159. and 115 for norfentanyl: 44, 118. 146. and 159 for
`
`
`
`
`
`
`
`
`
`desproprionylfentanyl; 5'1, 93. and 149 for AMJC: and H-0. lB7. and 351 forthe
`
`
`
`
`
`
`
`
`
`
`
`internal standard. Helium was used as the carrier gas. at a flow rate of 0.5
`
`
`
`
`
`
`
`
`
`
`mlfmin (DB-5) or 2.1 mllmin (D347). Initial temperature settings for the
`
`
`
`
`
`
`
`
`
`
`
`
`oven. injector, and M8!) were 100°C, 225°C, and 280°C, respectively. The
`
`
`
`
`
`
`
`
`
`
`even tentpersttrre was increased at a rate of l0°Clrnin to 30D"C over the first
`
`
`
`
`
`
`
`
`
`20 min and was held constant for the remaining ll min of the analysis: the
`
`
`
`
`
`
`
`
`
`
`MSD was usually turned off between 16 min and 21 min to protect the filarttont
`
`
`
`
`
`
`
`
`
`
`
`from the very large fentanyl peak. Rates of fentanyl metabolite fonnation were
`
`
`
`
`
`
`
`
`
`
`
`
`deterrrrined by comparison with standard curves constructed using lrnown
`amounts of authentic norfentanyl or AMX (see below).
`Reagents. Fentanyl. norferttanyl. and desproprionylfentanyl were purchased
`from Alltech lac. (Darfield, IL). The internal standard R03l9B0 was front
`Research Diagnostics Inc. (Flanders, NI). 1'-tabbitantibndies to human HL, {r'.e.
`
`
`
`
`
`Methods
`
`
`
`
`
`
`
`
`
`
`Human Liver Specimens. Human liver samples were obtained front the
`
`
`
`
`
`
`Liver Transplant. Procurement. and Distribution System (University of Min-
`
`
`
`
`
`
`nesota, Minneapolis, MN). None of the subjects had a known history of alcohol
`
`
`
`
`
`or dntg abuse. The livers were removed within 30 min of death, frozen in
`
`
`
`
`
`
`
`liquid nitrogen. and stored at -80°C until microsomes could be isolated.
`
`
`
`
`
`
`
`
`
`Preparation of Microaomea. Liver samples were thawed in ice-cold I00
`
`
`
`
`
`
`mM Tris-I-lCl buffer, pH 7.4, containing 100 mM KCI,
`l mM EDTA, and 1
`
`
`
`
`
`
`
`mM pbenyhnethylsulfonyl fluoride. and were then homogenized in 4 volumes
`
`
`
`
`
`of the same buffer with two 40-soc bursts in a Waring blender. The mixture
`
`
`
`
`
`
`
`
`was further homogenized with a motoodriven Teflonfglass tissue grinder.
`
`
`
`
`
`
`Microsomes were then prepared as described elsewhere (I4). The pyrophos-
`
`
`
`
`
`
`phatewashed microsomes were resuspended at a protein concentration of
`
`
`
`
`
`
`20-40 ruglrni in 10 mivt potassium phosphate buffer. pH 7.4, containing 0.25
`
`
`
`
`
`M sucrose. and were frozen at -80°C until use. Protein and P450 contents
`
`
`
`
`
`
`were determined using the bicinchoninie acid procedure (15) and according to
`
`
`
`
`
`
`
`the method of Omura and Sam (16), respectively.
`
`
`
`
`
`
`Enzyme Purification. CYP3A4 was purified from human liver microsomes
`
`
`
`
`
`
`
`
`from subject 920908 with a modification of the methods used for isolating
`
`
`
`
`
`
`
`CYP2E.l. CYPZCB. and CYPQCQ (14, i7). Potassium phosphate buffers used
`
`
`
`
`
`
`
`
`
`for the purification contained I mM dithiothreitol,
`1 mM EDTA, and 20%
`
`
`
`
`
`
`glycerol unless noted otherwise. As reported elsewhere (l4. l7). CYP2E1-
`
`
`
`
`
`
`enriched fractions eluted from tryptninine CH-Sepharose 43 with 10 mM
`
`
`
`
`potassium phosphate buffer. pH 7.4. containing 0.5% cholate and l.0% Lubrol
`
`
`
`
`
`
`
`
`PX (buffer B]. were subjected to hydroxylapatite chromatography on Hypatite
`
`
`
`
`
`
`
`
`C (Clarksoa Chemical Co... Williamsport. PA). The P450 recovered (75 nrnol)
`
`
`
`
`
`during elutlon of the Hypatite C column with 500 rttl of 50 mlvt potassium
`
`
`
`
`
`
`phosphate buffer. pH 7.4. containing 0.596 Lubrol PX. was treated with XAD-2
`
`
`
`
`
`
`
`
`resin (75 mg of resinlml) to reduce the detergent content, concentrated 15-fold
`
`
`
`
`by ultrafiluation through an Amicon PM-30 membrane, and exhaustively
`
`
`
`
`
`
`
`dialyzed against 5 mM potassium phosphate buffer. pH 7.7. The sample was
`
`
`
`
`
`
`then subjected to anion-exchange chromatography on DE-53 cellulose (1.5 X
`
`
`
`
`
`
`
`28 cm) as described for C'i'P2E1 (17). except that the column was developed
`
`
`
`
`
`
`
`
`with at linear gradient of 0-0.15 M KC] prepared in 5 column volumes of
`
`
`
`
`
`
`equilibration buffer. Analysis of column fractions by SDS—PA(iE and immu~
`
`
`
`
`
`
`
`noblotting with anti—l-III...-. (see below) revealed that the first A,” peak. which
`
`
`
`
`
`
`eluted at 30 mlvl KC], contained a single major hemoprotein that reacted
`
`
`
`
`
`
`
`strongly with anti-I-lL,... These CYP3A4-enriched fractions were pooled (ll
`
`
`
`
`
`
`
`nmol of P450 total). dialyzed against 20 volumes of 10 rah‘! potassium
`
`
`
`
`
`
`phosphate buffer. pH 6.5. containing 0.5% Lubrol PX. for 3 hr at room
`
`
`
`
`
`
`temperature. and then applied to a l.5— X Gem C'M—Sepharose column equil-
`
`
`
`
`
`ibrated with the same buffer used for dialysis. Upon charging, the column was
`
`
`
`
`
`washed consecutively with 5 volumes of equilibration buffer containing 50
`111M KCl and 5 volumes of equilibration buffer containing 100 mM KCL
`CYP3A4 was then eluted from the catiottrexchsnge resin as a broad peak with
`a linear gradient of 100-300 mM KC1 prepared in 15 column volumes of
`equilibration buffer. A flow rate of 0.5 Inlimin was used during sample
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ctn?‘IJoontcaerrno§|nu.tn0t‘t¢t.t5?.‘rtlsEJORIEUJTI-0ll3ti9l212tlII[}tt.to.t:rnsnsotnmon
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Page 2 of 8
`
`

`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`FElEFlMAN AND LASKEFI
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`to remove unreacted aniline. The resulting AM}-t was harvested by filtration
`
`
`
`
`
`
`onion. Fig. 5 shows that anti-CYP3A4 IgG reacted primarily with two
`
`
`
`
`
`
`
`
`
`
`and washed well with water. The compound was then recrystallized from a l:l
`
`
`
`
`
`proteins present in human liver microsomes. The lower of the two
`mirtttue of methanol and water. The melting point (llJ5°C) of recrystallized
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`immunoreactive proteins had essentially the same molecular weight as
`ANIX ccnespcnded to literature values. and mass spectra were identical to
`
`
`
`
`
`
`
`
`
`
`
`purified CYP3A4 (fig. 5, compare lanes if and L), whereas the upper
`those published in the literature.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`protein was putatively identified as CY‘P3A5.3 CYP3A4 was found in
`
`
`
`
`
`
`
`
`
`
`
`
`
`all 11 of the samples shown here, albeit at varying levels (see below).
`
`
`
`
`
`
`
`
`
`whereas CYP3A5 was found in only four of these specimens.
`
`
`
`
`
`
`
`
`
`‘The addition of anti-CYP3A4 IgG to human liver microscmes
`
`
`
`
`
`
`
`
`
`resulted in a marked (.7:-90%) inhibition of norfentanyl fonnation
`
`
`
`
`
`
`
`
`(table 2). Such immunoinhibition of fentanyl oxidation was achieved
`
`
`
`
`
`
`
`
`
`using an anti-CYP3A4 IgCl to microsomal P450 ratio of 7.5 rnglmnol.
`
`
`
`
`
`
`
`
`
`
`which was optimized in preliminary experiments; ratios greater than
`
`
`
`
`
`
`
`
`
`this gave no additional inhibition of the reaction (data not shown). In
`
`
`
`
`
`
`
`
`
`
`contrast, antibodies against two other human P430 enzymes, namely
`
`
`
`
`
`
`
`
`CYP2A6 and C'YP2C9, had essentially no effect on microsomal
`
`
`
`
`
`
`
`
`
`fentanyl metabolism.
`It should he noted that the failure of anti-
`
`
`
`
`
`
`
`
`
`
`
`CYPZCQ to inhibit fentanyl oxidation allows us to also rule out
`
`
`
`
`
`
`
`
`
`
`participation of CYPZCB and CYPZCI9 in the reaction, because the
`
`
`
`
`
`
`
`
`CYP2C9 antibodies used here not only cross-react with CYPZCB and
`
`
`
`
`
`
`
`
`
`
`CYP2Cl9 but also inhibit their catalytic activity (23).
`
`
`
`
`
`
`
`
`
`
`Correlation of Mlcrosornal Fentanyl Diddation with CYP3A4
`
`
`
`
`
`
`
`
`
`
`
`Content. Protein blots similar to that shown in fig. 5 were used to
`
`
`
`
`
`
`
`
`
`
`quantitate CYP3A4 levels in 10 individual human liver samples.
`
`
`
`
`
`
`
`CYP3A4 content was found to vary ‘:-*7-fold among the specimens
`
`
`
`
`
`
`
`(fig. 6). These data were then compared widt rates of fentanyl oxida-
`
`
`
`
`
`
`
`
`
`
`tion in the same samples. which also exhibited considerable variation
`
`
`
`
`
`
`
`
`
`
`
`
`
`(0.2B—l.48 nmol of norfentanyl producedlminlmg of protein). Nev-
`
`
`
`
`
`
`
`
`
`
`ertheless, a strong correlation (r1 = 0.876, F = 56.46, p sf. 0.001) was
`
`
`
`
`
`
`
`
`
`
`
`obtained between these two parameters (fig. 6). In fact, in the one
`
`
`
`
`
`
`
`
`
`subject (UC94|J2) where the level of CYP3A4 was below detection.
`
`
`
`rates of norfentanyl formation were also near the limits of detection
`
`
`(0.01 nmol of norfentanyl producedfmin/mg of protein). No signifi-
`
`
`
`cant correlation was found between rates of microsomal fcntanyl
`
`
`
`
`
`
`
`
`
`
`
`oxidation and the levels of CYP2El (P = 0.092), cvrzcs (P =
`
`
`
`
`
`
`0.285), and CYP2C9 (r2 = 0.197) in human liver microsomes: con-
`
`
`
`
`
`
`
`tents of these latter P4503 were quantitatcd by immunoblotting with
`
`
`
`
`
`
`
`
`
`the corresponding antibodies in a manner similar to that used for
`
`
`
`
`
`
`
`
`
`
`
`
`CYP3A4 (data not shown).
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Discussion
`
`
`
`
`
`
`
`
`
`
`
`In the present study, we have shown that fentanyl is metabolized in
`
`
`
`
`
`
`
`virro to a single major metabolite, namely norfentanyl. by human liver
`
`
`
`
`
`
`
`
`
`
`microsomes. The kinetic parameters associated with fentanyl deal]cy-
`
`
`
`
`
`
`
`
`
`
`lation at the piperidine nitrogen were consistent with participation of
`
`
`
`
`
`
`
`
`a single P450 enzyme in the reaction. This P450 enzyme was subse-
`
`
`
`
`
`
`
`
`quently identified as CYP3A4. Chemical inhibitors of CYP3A4 ac-
`
`
`
`tivity, including keloconazole and 7,8-benzoflavone (24). as well as
`
`
`
`
`
`
`
`
`
`
`
`the CY‘P3A4 substrate erythromycin (25), proved to potently inhibit
`
`
`
`
`
`
`
`
`
`
`microsomal fentanyl oxidation to norfcntanyl. Antibodies to CYP3A4
`
`
`
`
`
`
`
`
`
`
`
`
`
`also markedly inhibited norfentanyl formation by human liver micro-
`
`
`
`
`
`
`
`somes. whereas antibodies to other human P450 enzymes were with-
`
`
`
`
`
`
`
`
`
`
`
`
`
`out effect. In addition, we found an excellent correlation (:9 = 0.876)
`
`
`
`
`
`
`
`
`between microsomal CYP3A4 content and rates of fentanyl oxidation.‘
`
`
`
`
`is the synthetic opioid analgesic most often used by
`Fcntanyl
`
`
`
`Results
`
`
`
`
`
`
`
`
`
`
`
`
`Metabolism of Fentanyt by Human Liver Microsotnes. We
`
`
`
`
`
`initially studied fentanyl oxidation by human liver microsomcs to
`
`
`
`
`
`detennine the profile of metabolites formed. The GCIMS method used
`
`
`
`
`
`here allowed for sensitive detection of at least three potential fentanyl
`
`
`
`
`
`
`derivatives, including norfentanyl. desproprionylfentanyl, and AMI
`
`
`
`
`
`
`(fig. 2A). The total-ion chromalogram in fig. 2C shows the presence
`
`
`
`
`
`
`of two prominent peaks, with retention times of 5.0 and 1D.B min, that
`
`
`
`were formed upon incubation of fcntanyl with liver microsomes in the
`
`
`
`
`
`
`presence of NADPH. Mass spectra of these peaks were essentially
`
`
`
`
`
`identical (97-9996 similarity) to the published mass spectra of AMX
`
`
`
`
`
`
`
`
`and norfentanyl, respectively. In contrast to norfentanyl formation,
`
`
`
`
`
`
`
`however, AMX formation was not dependent upon microsomal P450
`
`
`
`
`
`
`enzymes, because similar amounts of this compound were found in
`
`
`
`
`
`
`incubations performed in the absence of NADPH (fig. 2B) or with
`rnicrosornes omitted (data not shown). in addition. no evidence for
`
`
`
`
`
`
`
`
`
`
`
`desproprionylfcntanyl fomtation by human liver rnicrosomcs was
`
`
`
`
`
`
`found. at least under the experimental conditions used here.
`
`
`
`
`
`
`Norfentanyl production by human liver microsomes was linear with
`
`
`
`
`respect to time of incubation (up to 15 min) and rnicrosomal P450
`
`
`
`
`concentration (up to 200 pmol) (fig. 3). Formation of this metabolite,
`
`
`
`
`
`
`which proceeds via deallrylation of the fentanyl piperidine nitrogen,
`
`
`
`was strictly dependent upon the presence of NADPH as well as
`
`
`
`
`
`
`
`
`substrate in the reaction mixtures. Although some AMX was found in
`
`
`
`
`
`
`
`all incubations with hepatic microsomes, amounts of the compound
`
`
`
`
`formed bore no relationship to incubation time, microsomal protein
`
`
`
`
`
`
`
`
`
`concentration. or the presence or absence of NADPH.
`
`
`
`
`
`
`Kinetic parameters of microsomal fentanyl oxidation were deter-
`
`mined using substrate concentrations ranging from 5 to 500 p.M (fig.
`
`
`
`
`4). The conversion of fentanyl
`to norfentanyl exhibited typical
`
`
`
`
`
`
`
`Michaelis-Menton kinetics. The Eadie-Hofstee plot shown in fig. 4.
`
`
`
`
`
`right. was used to derive the apparent Km and Vm“ for the reaction,
`
`
`
`
`
`which were I17 ttltd fcntanyl and 3.86 nmol of nor-fentanyl formed!
`
`
`
`
`
`
`
`
`minlnmol of microsomal P450, respectively. Regression analysis.
`
`
`
`
`
`which was used to fit the data to a straight line. assuming involvement
`
`
`
`
`of a single enzymatic component, gave a correlation coefficient of
`
`
`
`
`
`
`
`
`Inhibition Studies. P450 chemical inhibitors and specific antibod-
`
`
`
`
`
`
`ies were used to identify the human P450 enzyrne(s) involved in
`
`
`
`
`
`hepatic fentanyl oxidation to norfentanyl. The effects of various
`
`
`
`
`
`chemical inhibitors on fentanyl oxidation by liver microsomcs are
`
`
`
`
`presented in table 1. Of the different agents tested. only those com-
`
`
`
`
`
`
`pounds ltnown to inhibit CYP3A4 activity, namely ketoconazole,
`
`
`
`
`
`7,8-beozoflavone, and erythromycin, were capable of blocking fant-
`
`
`
`
`
`anyl conversion to norfentanyl. Ketoconaznle was the most potent.
`
`
`
`
`
`
`inhibiting essentially all norfentanyl formation at a concentration of5
`
`
`
`
`
`p.M, whereas the weaker inhibition noted with erythromycin was
`
`
`
`
`
`
`the CYP1A2 inhibitor furafylline, the
`dose-dependent. In contrast,
`CYPZDG inhibitor quinidine. the CYl’2C9 inhibitor tolbutamide, and
`the CYPi2El inhibitors 4-methylpyrazole and ethanol either had no
`effect on fentanyl oxidation or slightly stimulated the reaction.
`Because we had hypothesized that fentanyl, like alfentanil, was
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`“At least thrao different but highly homologous enzymes belonging to the
`GYPSA subfamily are expressed In human llvar. C-YP3M, CYP3A5. and CYP3A7
`are found in adults. whereas only the latter two P4503 are expressed ln fatal and
`
`
`
`CInt’.‘Jxaotuaosnno-snmrnorI’-l.-Vile-‘12€.1o's1eumofisdse‘onIowoncaoecrumon
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Page 3 of 8
`
`

`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`FENTANYL OXIDATION BY HUMAN LIVEFI CYPSA4
`
`
`
`
`
`935
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`RETENTION TIME (min)
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`RETENTION TIME (min)
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ctnz‘rlaoulaaaflT10StuumorIClJ9:"u'tsEm-'sI‘eLI.1t1of1adse'numtnonmat:-eoru.-norl
`
`
`
`
`
`3J'lI..'I.V'I3H
`SONVGNIIEVHUI
`
`3I'tl.I.‘H"EIH
`SSNVGNTIEVNO!
`3i‘I.I.l.V'EH
`33N‘U‘flNf'lE‘H'NOI
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`RETENTION 11:45 (min)
`
`
`
`
`
`
`
`
`
`
`
`FIG. 2. GOMS analysis offentanyl metabolism by humtm liver micrusantes.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`A. Representative total-ion citnuntatogram obtained upon GCIMS analysis of a mlttture containing authentic fentanyl (PEN). AMX. norfentanyl (NOR).
`‘ii’-$PI0I1|'i0fl¥IfEI1lflfl)fI (DES). flfltl the internal standard E03191?-0 (STD). 3. Typical total-ion CI|l'Dl1l.BlD5l'tflI| obtained upon incubation of human liver mictusomes
`with fentanyl in the absence of NADPH. C. Analogous incubation to which NADPH was added. The fentanyl peak cannot be observed in E and C‘. because the
`MS!) was shut off during the time period (16.5-20 min) when this compound eluted from the CIC column (see Methods).
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Page 4 of 8
`
`

`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`FEIEFIMAN AND LASKEF-'l
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`15
`
`- NI
`
`
`
`
`
`NORFENTANYLFORMEDtnmolf10mm]
`
`
`
`
`
`
`
`
`
`
`
`
`P450)
`NORFENTANYLF0RMED(nmcktnmoi
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`B o
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`D
`
`5
`
`IO
`
`1!
`
`0
`
`
`
`TIM E
`
`(min)
`
`we
`too
`so
`M ICROSOMAL P450 ADDED
`
`(Pine!)
`
`
`
`
`
`
`
`
`FIG. 3. Hetabaiism offertfanyi by human liver microsomes: relationship to time of incubation (A) and P450 concentration (B),
`
`
`
`zoo
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Fentanyl (250 ,u.M} was incubated with human liver microsomes (protein equivalent of 0.05—0.2 nmol of P450) for 5-15 min at 37"C, in the presence of
`
`
`NADPH. Norfentnnyl formation was then assessed by GCJMS, as described in Methods.
`
`5'9o
`
`toID‘oin
`
`1.5
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`K.“ = 117 pm
`V3“ I
`l‘l‘lIi'l“
`
`
`
`
`
`
`
`
`
`
`
`1.0
`
`0.5
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`‘\fl[FENTAN‘rL]
`
`
`
`1.5
`
`
`
`
`
`
`2.0
`
`
`
`2.5
`
`
`
`
`
`
`5.0
`
`
`
`
`
`
`
`
`
`
`
`
`0.0
`
`0
`
`
`
`Hit)
`
`
`200
`
`
`
`
`
`
`300
`
`
`
`
`
`
`
`400
`
`500
`
`
`
`
`
`
`
`
`
`
`
`0.0
`
`
`0.5
`
`
`1.0
`
`
`V
`FENTANYL
`(nmol rtorfenlanyl lonnodtntinfn moi P450)
`(HM)
`
`
`
`
`
`
`
`
`
`
`
`
`Pro. 4. Kinetic artaiysis affentanyi oxidation by human iiver microscntes.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Fentnnyl metabolism was determined in incubation mixtures (2.0 ml) containing 100 mM Tris-HCl buffer, pH 7.4, liver microsomcs front subject UC94l(l
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`(protein equivalent to 100 pmol of P450), 0.5 mM NADPH. and fentanyl in concentrations ranging from 5 to Silt} ,..M. Reactions were initiated with NADPH
`
`
`
`
`
`
`
`
`
`and terminated after 10 min at 37°C. Norientanyl formation was quantitated by GCIMS, as described in Methods. Lefi, plot of reaction velocity vs. featanyl
`
`
`
`
`
`concentration: right, Eadie-i-Iofstee transi'on'natioa oi‘ the same data. The apparent K," and Vmu were d