0022-:156S/ H2/222 1~:U l4$02.00/ 0
`TNt:. ,JOURN"L or PHAltMACOJ.OCY A.ND F.iJCPEIUMl-:.NTAL 1'HP:RAPEU"MC8
`Copyright© 1982 hy TI,e American Socioty for Phannarology ond F.J<porimental Therapoutia,
`
`Vol. 2:22, No. I
`Printed in V .S.A .
`
`Metabolite Kinetics: Formation of Acetaminophen from
`Deuterated and Nondeuterated Phenacetin and Acetanilide on
`Acetaminophen Sulfation Kinetics in the Perfused Rat Liver
`Preparation 1
`
`K. SANDY PANG.2 LEWIS WALLER. MARJORIE G. HORNING and KENNETH K. CHAN
`Department of Pharmaceutics, University of Houston (K.S.P., L. W.), Institute for Lipid Research, Baylor College of Medicine, Texas Medical
`Center, Houston, Texas (M.G.H.) and Department of Pharmaceutics, University of Southern California, Los Angeles, California (K.K.C.J
`Accepted for publication March 22, 1982
`
`ABSTRACT
`The role of hepatic intrinsic clearances for metabolite forma(cid:173)
`tion from various precursors on subsequent metabolite elimi(cid:173)
`nation was investigated in the once-through perfused rat liver
`preparation. Two pairs of acetaminophen precursors: [1'C]
`phenacetin-d 5 and (3H]phenacetin-do, [1'C]acetanilide and [3H]
`phenacetin were delivered by constant flow (10 ml/min/liver)
`either by normal or retrograde perfusion to the rat liver prepa(cid:173)
`rations. The extents of acetaminophen sulfation were com(cid:173)
`pared within the same preparation. The data showed that the
`higher the hepatocellular activity (intrinsic clearance) for acet(cid:173)
`aminophen formation , the greater the extent of subsequent
`acetaminophen sulfation . The findings were explained on the
`
`basis of blood transit time and metabolite "duration time."
`Because of blood having only a finite transit time in liver, the
`longer the drug requires for metabolite formation, the less lime
`will remain for metabolite sulfation and the less will be the
`degree of subsequent sulfation. Conversely, when the drug
`forms the primary metabolite rapidly, a longer time will remain
`for the metabolite to be sulfated in liver to result in a greater
`degree of metabolite sulfation. Finally, the effects of hepatic
`intrinsic clearances for metabolite formation and zonal distri(cid:173)
`bution of enzyme systems for metabolite formation and elimi(cid:173)
`nation in liver are discussed.
`
`Conjugation reactions in drug biotransfonnation are impor(cid:173)
`tant as the addition of polar functional groups renders the
`molecules more suitable for renal excretion. Normally, sulfate
`conjugation presents this characteristic in terms of drug detox(cid:173)
`ification (Williams, 1959). It was discovered recently, however,
`that sulfation of some compounds (De Baun et al., 1970; Kad(cid:173)
`lubar et al., 1976; Mulder et al., 1977) can induce drug-mediated
`toxicity. The manner in which the kinetics of sulfation for
`xenobiotics, that undergo Phase II (conjugation) reactions sub(cid:173)
`sequent to Phase I reactions, remain mostly unknown.
`The liver is the major organ for drug biotransformation and
`conjugation reactions and much work has been done on specific
`compounds (Villeneuve and Sourkes, 1966; Slotkin et al., 1970;
`Minck et al., 1973; Wiebkin et al., 1978; Reinke et al., 1981)
`that are initially oxidized (Phase I reaction) and subsequently
`conjugated (Phase II reaction). A specific example on oxidative
`metabolism and subsequent sulfation, however, is provided by
`
`Received for publication October 22, 198 I.
`' Thi& work was supported by U.S. Public Health Service Grant GM 27323.
`' Recipient of Research Career Development Award Grant AM 01208 from the
`U.S. Public Health Service.
`
`the O-deethylation of phenacetin in the formation of acetamin(cid:173)
`ophen, which, under tracer dose/concentration conditions, is
`primarily metabolized to acetaminophen sulfate conjugate in
`vivo (Pang et al., 1979) and in vitro (Pang and Gillette, 1978) in
`the rat. The kinetics of sulfation were examined by the com(cid:173)
`parison of the extent of acetaminophen sulfation under these
`tracer conditions, when [3H]acetaminophen, a preformed me(cid:173)
`
`tabolite, and e4C)phenacetin, a precursor of [ 14C]acetamino(cid:173)
`
`phen, were delivered simultaneously once-through the perfused
`rat liver preparation. The preformed metabolite was sulfated to
`a larger extent (67 ± 8%) than the derived metabolite (59 ±
`10%) (Pang and Gillette, 1978). But, when directional flow was
`reversed with retrograde perfusion to the liver (Trowell, 1942),
`discrepancies in acetaminophen sulfation for the preformed
`metabolite and derived metabolite disappeared (Pang and Ter(cid:173)
`rell, 198 la) . These findings suggest a heterogeneous distribution
`of drug metabolizing enzymes in the rat liver; O-deethylation
`as mediated by the cytochrome P-450 system may be more
`abundant in the centrilobular region and sulfation as mediated
`by the sulfotransferases may be preponderant in the periportal
`region.
`The role of hepatocellular activities for oxidative metabolism
`or intrinsic clearance to form a primary metabolite on the
`
`ABBREVIATIONS: TLC, thin-layer chromatography; HPLC, high-perlormance liquid chromatography.
`
`14
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`1982
`
`extents of sulfation of this metabolite, however, is unknown.
`The present paper is an examination of acetaminophen sulfa(cid:173)
`tion kinetics when acetaminophen is formed from a pair of
`precursors ([ 14C]phenacetin-ds and [3H]phenacetin-do; fig. l,
`Structures I and II, respectively) of similar hepatic extraction
`ratios and from a pair of precursors ([3H]phenacetin and [ 14C]
`acetanilide; fig. 1, Structures II and III, respectively) of different
`hepatic extraction ratios. The first pair of precursors is chosen
`because Garland et al. (1976) had reported a slight kinetic
`isotope effect ( 1.61) on the O-deethylation of phenacetin in the
`formation of acetaminophen for the deuterated analog in rab(cid:173)
`bits. But, because the values of the extraction ratio of phenace(cid:173)
`tin were very high (-0.9), the kinetic isotope effect, if present
`to the same degree in the perfused rat liver preparation, may
`not perturb the values of the hepatic extraction ratios. The
`second pair of precursors is chosen because preliminary studies
`had shown that the hepatic extraction ratio of acetanilide (0.38-
`0.52) was about half the value for phenacetin (0.84-0.98) . These
`two pairs of precursors of acetaminophen will bring out differ(cid:173)
`ences of acetaminophen sequential sulfation when their hepatic
`extraction ratios are 1) very similar and 2) drastically different.
`
`Materials and Methods
`Liver perfusion. The surgical procedure and the perfusion appa(cid:173)
`ratus for normal perfusion were identical to a previously described
`method (Pang and Gillette, 1978) . Modification of the perfusion appa·
`ratus and the surgical procedure for retrograde perfusion was identical
`to those described in an earlier report (Pang and Terrell, 1981a).
`Male Sprague-Dawley rats (300-367 g) which were supplied by
`TIMCO Laboratories (Houston, TX) were used as liver donors in all
`studies. Only single-pass (nonrecirculating) experiments were per(cid:173)
`formed. Perfusate containing tracer and constant concentrations of the
`precursors were delivered under constant flow (10 ml/min/liver) either
`by normal or retrograde tlow into the rat liver preparation.
`Preliminary experiments were performed to identify a system that
`would be devoid of drug-drug interactions when each pair of precursors
`was delivered simultaneously into the rat liver preparation. For exam(cid:173)
`ple, for precursors I and II, the first-period of perfusion (40 min)
`entailed the perfusion of I singly and the second period (40 min)
`involved the simultaneous perfusion of both I and II, followed by the
`third and last perfusion period (40 min) ofl singly. The experiment was
`repeated with the interchange of substrates; the three perfusion periods
`were: II only, I and II and II only. The criterion of constancy in drug
`hepatic extraction was used. These experiments were repeated at
`various concentrations of I and II until the metabolism of both I and II
`(as estimated by the hepatic extraction ratios) in the presence and
`absence of each other would remain a constant.
`Such a system was identified for the dual delivery of [ "C]phenacetin(cid:173)
`d ~ and ["H]phenacetin-do simultaneously to the same rat liver prepa(cid:173)
`ration. Studies were to be carried out by three perfusion periods (40
`min each): normal, retrograde and normal perfusions at these tracer
`concentrations. But drug-drug interactions had reaulted between ["CJ
`
`11111
`1111
`Ill
`Fig. 1. Structures of ["C]phenacetin-ds (I). (3H]phenacetin-do (Ill and
`( 1•c]acetanilide (Ill).
`
`Acetaminophen Formation and Su"atlon Kinetics
`
`15
`
`acetanilide and ['H]phenacetin, as the hepatic extraction ratios of both
`compounds were decreased in the presence of each other. For studies
`to be carried out with ["C]acetanilide and [3H]phenacetin, only normal
`perfusion of the compounds was employed. The three perfusion periods
`(40 min) of the compounds singly by normal flow were: {"C)acetanilide,
`[ 3H]phenacetin and ["C]acetanilide.
`Sampling tlmea. The mean of two determinations of the input
`medium taken before and after the perfusion period was used as the
`steady-state input drug concentration. The mean of the determinations
`of five consecutive 4-min samples of the outflow medium taken at 24
`min after the commencement of the perfusion period was used as the
`steady-state output concentration (steady-state conditions were ap(cid:173)
`proached around 20 min after the commencement of the period for the
`drug and metabolites).
`Bile was collected at 10-min intervals after the start of the experi(cid:173)
`ment. The volumes were noted for each collection.
`All88y. [' 4C)phenacetin-d&. {3H}phenacetin, [ 1•CJacetanilide, radio(cid:173)
`labeled acetaminophen and acetaminophen conjugates in bile and
`perfusate were quantified as previously described (Pang and Gillette,
`1978) . The sulfate, glucuronide or glutathione conjugatea in plasma and
`in bile were determined by spotting plasma (100 ,d) or bile (IO µI) on
`Avicel F (250 µM, Analtech, Inc., Newark, DE) thin-layer plates which
`contained carriers (acetaminophen, acetaminophen sulfate and glucu(cid:173)
`ronide conjugates) at the origin. The plates were developed in n(cid:173)
`propanolol-0.4 N NH3 (80 :20 v/v) and the radiolabeled fractions of the
`sulfate, glucuronide and glutathione were scraped into scintillation
`vials. After the addition of 1.0 ml of water and 10 ml of Aquasol (New
`England Nuclear, Boston, MA), radioactivity in each fraction was
`quantified by liquid scintillation spectrometry (Beckman LS 7500, Palo
`Alto, CA). ["C]Phenacetin-ds. [3H]phenacetin-do and ["C]acetanilide
`in bile were determined by spotting an aliquot (10 ,u.l) on silica gel GF
`TLC plates (250 p.m, Analtech) that contained carriers and developing
`in a system of ether. The R, values were: acetanilide, 0.53; phenacetin,
`0.43; and acetaminophen, 0.29.
`[ 04C]Phenacetin-d., ["H]phenacetin-do, ["C]acetanilide and radio(cid:173)
`labeled acetaminophen in blood perfuaate (l.0 ml of input and output
`blood) were quantified by extraction into ethyl acetate (6 ml) (Burdick
`and Jackson, Muskegon, Ml). The extracts were evaporated to dryness
`under a stream of nitrogen. The residue wu redi88olved in ethyl acetate
`(200 µI) and JOO id was spotted on the origin of silica gel TLC plates
`which contained unlabeled acetanilide, phenacetin and acetaminophen
`as carriers and developed in a system of ether. A set of standards which
`contained varying amounts of ( "C]acetanilide. [ 1•C]phenacetin-d ,. [3H]
`phenacetin-du and [3H)acetaminophen in blank perfusate (l.O ml) were
`extracted by the same procedure to correct for loues due to the
`extraction procedure.
`A HPLC 88118.Y method (Wilaon et al., 1982) was used to validate the
`TLC procedure. A mobile phase of methanol: 0.1 M KH2PO, in 0.75%
`acetic acid (7 :93, v/v) was utilized at a flow of 1.0 ml/min through a
`guard column (Waters Associates, Milford, MA) of ODS packing, 10
`µ.m (Whatman, Inc., Clifton. NJ). and an Ultraaphere column (Altex
`Scientific, Inc., Palo Alto, CA) in a liquid chromatograph (Laboratory
`Data Control, Riviera Beach, Fl) that was equipped with a Hheodyne
`injector, a Constametric III pump and a UV detector (Spectro Monitor
`III) at 254 nm and monitored on a linear recorder (Chart speed 20 cm/
`hr, Linear Instruments Corp., Irvine. CA) . The retention times for the
`authentic standards were: glucuronide, 5 min 45 sec; sulfate, 10 min 10
`sec; acetaminophen, 15 min 55 sec; glutathione conjugate, 19 min 50
`sec; and acetanilide, 48 min. Phenacetin was not eluted from the
`column. Perfuaate samples were prepared by removing the plasma by
`centrifugation and an aliquot (200 µI of plasma) was precipitated with
`methanol (500 ,d). Bile samples were prepared by similar dilutions. A
`volume (40-50 µI) of the supernatant was injected into the HPLC
`system. All HPLC eluants were collected for 60 min at 1-min intervals
`by a fraction collector (FOXY, ISCO, Lincoln, NB). The peaks of
`radioactivity collection were compared to the peaks by UV detection of
`the authentic compounds.
`SoW'ce of Materials. Unlabeled acetaminophen. phenacetin and
`acetanilide were purchased from Eastman Kodak Co. (Rochester, NY).
`
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`16
`
`Pang etal.
`
`Unlabeled acetaminophen su1£ate and glucuronide conjugates were
`generously and kindly supplied by Dr. Josiah N. Tam, McNeil.s Con(cid:173)
`sumers Company (Fort Washington, PA). Acetaminophen glutathione
`conjugate was supplied by Dr. Bernhard Lauterberg, Baylor College of
`Medicine (Houston, TX).
`[ "C]Phenacetin-d • (I, N •[ acetyl• l • "C]-p-ethoxy-d •-aniline; specific
`activity, 21.4 mCi/mmol) and ("H]phenacetin-do (II, N-[ acetyl-3H)•p(cid:173)
`ethoxyaniline; specific activity, 500 mCi/mmol) were synthesized as
`described in a previous report (Chan and Pang, 1982). Essentially, ['H]
`phenacetin-do was prepared by direct acylation of p-phenetidine with
`[3H)acetic anhydride under the Schotten-Bauman condition and ["CJ
`phenacetin-do was obtained by similar acylation of p-phenetidine-d;
`which was synthesized by ethylation of acetaminophen with perdeuter(cid:173)
`ated ethyl iodide (Garland et al., 1977). [ 1•C]Acetanilide (Ill) was
`synthesized by reacting a 5-fold excess of aniline (Eastman Kodak)
`with [I·"C)acetyl chloride (New England Nuclear; specific activity,
`57.7 mCi/nunol). The resultant [1•cJacetanilide was purified by acidic,
`basic and aqueous washed before the final extraction into ethyl acetate
`and was stored over sodium sulfate. The acetanilide obtained was
`greater than 99% radiochemically pure, as determined by TLC. [3H]
`Acetanimophen (specific activity, 937.5 mCi/nunol) was purchased
`from New England Nuclear.
`
`Results
`Assay. The radiometric HPLC procedure confirmed the
`TLC assay that radiolabeled acetaminophen and acetamino(cid:173)
`phen sulfate conjugate were the only major metabolites in
`effluent perfusate resulting from the perfusion of[ 14C]phenace(cid:173)
`tin-d5 and CH]phenacetin-do into the rat liver preparation.
`["C]Acetanilide was biotransformed to [ 14C]acetaminophen,
`[ 14C]acetaminophen sulfate and glucuronide conjugates. No
`other radioactive metabolic species were detected by the HPLC
`system. In bile, acetaminophen glutathione conjugate was the
`only other metabolite present that accompanied acetamino(cid:173)
`phen sulfate and acetaminophen glucuronide conjugates. More(cid:173)
`over, the amounts of radioactivity of each component in the
`selected samples as measured by HPLC and TLC were virtually
`identical.
`[1(cid:141) C]Phenacetin-da and [1H]Phenacetin-de. Constant
`concentrations of [ 14C]phenacetin-ds (range, 80-300 x 102
`dpm/ml or 0.18-0.68 µ.M) and CH]phencetin (range, 49-62 x
`104 dpm/ml or 0.45-0.56 µ.M) were delivered simultaneously
`by normal (40 min), retrograde (40 min) and normal (40 min)
`perfusions into the same rat liver preparation. The sampling
`schedule denoted that steady-state conditions were approached
`during the collecting interval as identified by the constancy in
`hepatic venous concentrations of phenacetin and its major
`metabolites, acetaminophen and acetaminophen sulfate conju(cid:173)
`gate. The paired steady-state extraction ratios for ds (0.81-
`0.978) were slightly smaller than those of the nondeuterated
`counterpart (0.83-0.995). The report of Garland et al (1976),
`however, discussed a kinetic isotope effect of 1.61 in rabbit
`microsomes. Although the V "'"' and Km values in the liver
`perfusion studies were not explored, the ratio of the hepatic
`intrinsic clearances for d.o/d5 ranged from 1.1 to 1.2 according
`to the "venous-equilibration" or "well stirred" model on hepatic
`clearance (Rowland et al; 1973) and for highly extracted com(cid:173)
`pound such as phenacetin-do and phenacetin•d5, these differ(cid:173)
`ences in hepatic intrinsic clearances will not affect the values of
`the steady-state hepatic extraction ratio by much.
`Under these tracer concentrations, total radioactivity in bile
`accounted for <5% of this infused dose and the rate of the total
`acetaminophen species in perfusate leaving the liver at steady(cid:173)
`state represented the rate of formation of acetaminophen; both
`
`Vol. 222
`
`phenacetin species were converted primarily to acetaminophen
`(0.92-1.05), as measured by the ratio of the concentration of
`acetaminophen formed in liver (total acetaminophen species in
`effluent perfusate) to the drop in concentration of phenacetin
`across the rat liver. This ratio also denoted the fraction of total
`hepatic elimination of phenacetin that was converted to the
`metabolite, acetaminophen. No trend was demonstrable, how(cid:173)
`ever, from the data on the formation of acetaminophen from
`either perdeuterated or nondeuterated phenacetin.
`The extent of acetaminophen sulfation was calculated by
`dividing the hepatic venous concentration of acetaminophen
`sulfate by the sum of all acetaminophen species (unconjugated
`acetaminophen and conjugated acetaminophen) in hepatic ve(cid:173)
`nous blood, all measured at steady-state. The paired compari(cid:173)
`son of the extents of subsequent sulfation of acetaminophen,
`derived from two phenacetin precursors of almost identical
`hepatocellular activities (hepatic intrinsic clearances) in acet•
`aminophen formation, were quite similar (P < .25) . Larger
`extents of acetaminophen sulfation were observed during ret•
`rograde flow that were significantly higher ( P < .0005) than
`during normal flow and the observations (table I) were consist(cid:173)
`ent with the findings reported earlier (Pang and Terrell, 1981a).
`[1 4C]Acetanilide and ('H]pbenacetin. Because of dual de(cid:173)
`livery of [ 14C]acetanilide and [3H]phenacetin mutually inhibited
`the metabolism of each other, the simultaneous delivery of
`these precursors was avoided. Three perfusion periods (40 min
`each) with the successive and single delivery by normal flow of
`[ 14C]acetanilide (range 40-61 X lOl dpm/ml or 0.89-1 .23 µM) ,
`['1H]phenacetin (range, 450-610 x 10~ dpm/ml or 0.41-0.55 µM)
`and [ 1•C]acetanilide were examined. For these studies, the
`steady-state extraction ratios were: ( 14C]acetanilide, 0.38 to 0.52;
`and (3H]phenacetin, 0.84 to 0.98. Biliary excretion of total
`radioactivity accounted for <5% of the total infused dose for
`both precursors and very little appeared as unchanged [ 14C]
`acetanilide (6-10% of excreted amount), ['1H]phenacetin and
`[aH]acetaminophen (<2% of excreted amount). The ratio of
`( 1 •C]acetaminophen sulfate/[ 14C]acetaminophen glucuronide in
`bile was 0.48 to 1.1:1, the ratio of CH]acetaminophen sulfate/
`( 3H]acetaminophen glucuronide in bile was usually 2:1. The
`metabolic conversion of[ 14C]acetanilide to [ .. C]acetaminophen
`accounted for only 68 to 80% of the total disappearance of [ 1•q
`acetanilide. The possibility that N-deacylation of{ 14C]acetani(cid:173)
`lide occurred to form aniline that accompanied loss of radiolabel
`in the rat, a metabolic pathway that was suggested by Brodie
`and Axelrod (1948) in humans, was not explored. By contrast,
`biotransformation ofCH]phenacetin to (3H]acetaminophen was
`almost complete (91-100%). Therefore, the hepatocellular ac(cid:173)
`tivity or hepatic intrinsic clearance (ratio of V ma.!Km) for the
`formation of acetaminophen from phenacetin was greater than
`that from acetanilide.
`[ 3H]Acetaminophen sulfate was the only conjugated metab(cid:173)
`olite detected in the hepatic venous blood with the perfusion of
`( 3H]phenacetin. But [ .. C]acetaminophen and glucuronide con•
`jugates were both detected (ratio of sulfate/glucuronide con(cid:173)
`jugate was 4: 1) in hepatic venous blood with the perfusion of
`{ 14C]acetanilide. The extents of acetaminophen sulfation, again
`measured by the ratio of the steady-state output concentrations
`of acetaminophen sulfate conjugate to the sum of total acet•
`aminophen species (unconjugated acetaminophen, and acet(cid:173)
`aminophen sulfate and glucuronide conjugates) were compared
`for this pair of precursors (table 2). Differences on the extents
`of acetaminophen sulfation between the precursors [ .. C]acet•
`anilide and (3H]phenacetin were highly significant ( P < .0005).
`
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`1982
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`Acetaminophen Formation and Sulfation Kinetics
`
`17
`
`TABLE l
`Conversion of acetaminophen (derived from phenacetin do and phenacetln•d5) to Its aulfate conjugates
`A three-way analysis of variance was performed on the data. Significant differences exist between the liver preparations and tor the extents of acetaminophen
`between normal vs. retrograde flows (P < .0005), whereas differences of the extents of acetaminophen sulfation between r•cp11enacetin-d, and ('H]phenacetin-do
`were significant only at P < .25.
`
`Conversion ol Acetaminophen 10 its Sulfate Conjugate (%)
`
`Study No.
`
`(I)
`Normal per1uslon
`
`(tt)
`Retrograde perfusion
`
`(Ill)
`Normal perfusion
`
`From
`phenacetilH10
`42.5
`43.3
`49.7
`48.1
`46.5
`
`From
`phenacelilH1,
`
`40.7
`40.4
`48.9
`49.1
`42 .8
`
`From
`phenacetin-do
`64.7
`45.8
`65.3
`56.0
`61 .7
`
`From
`phenacetin-d,
`
`62.2
`40.6
`62.0
`55.7
`58.0
`
`From
`pl,enacetilH1 o
`43.3
`39.0
`51.0
`44.2
`45 .6
`
`From
`pl,enacehlH1,
`41.4
`35.3
`52.1
`46.8
`41 .0
`
`1
`2
`3
`4
`5
`
`TABLE 2
`Conversion of acetaminophen (derived from acetanllide and
`phenacetin to lta aulfate conjugate
`A two-way analysis of variance was performed on the data. Significant differences
`exist between the liver preparations and tor the extents of acetaminophen
`sulfation between ('HJphenacetin-do and (' 4C)acetanilide (P < .0005).
`% Sultatlon of Acetamlnophen
`
`Stlldy No.
`
`1
`2
`3
`4
`5
`
`(I)
`From acetaolude
`36.6
`30.2
`27.5
`23.2
`35.1
`
`(»)
`From phenacelin
`51 .1
`44 .0
`32.2
`37 .4
`53.3
`
`(Ill)
`From acetanilioe
`43.2
`25.4
`22.0
`25.9
`38.7
`
`[ 3H)Phenacetin, the precursor with high hepatocellular activity
`(intrinsic clearance) for the acetaminophen formation was ac(cid:173)
`companied by a greater extent in sulfation for acetaminophen.
`
`Discussion
`
`Previous reports have alluded to the kinetic disposition of
`acetaminophen and phenacetin under tracer conditions in the
`perfused rat liver preparation; uneven distribution of enzymes
`within the liver may be responsible for differences in the extent
`of sulfation of acetaminophen when acetaminophen is formed
`by oxidative metabolism from a precursor or as preformed
`acetaminophen (Pang and Gillette, 1978; Pang and Terrell,
`1981a). The present findings imply that in addition to the
`possible existence of zonal localization of drug metabolizing
`enzymes, the oxidative hepatocellular activity for metabolite
`formation also appears to influence the degree of subsequent
`sulfation of a metabolite.
`For the precursor pairs of [1'C]phenacetin-d5 and [3H]phen(cid:173)
`acetin-do, which differ only slightly in enzymic capabilities in
`0-deethylation, differences in the extent of acetaminophen
`sulfation were very similar (table 1). Also, consistent with our
`earlier data (Pang and Terrell, 1981a), the extent of acetamin(cid:173)
`ophen sulfation is greater during retrograde than during normal
`directional flow . For the precursor pair of [ 14C]acetanilide and
`and [3H]phenacetin which differ quite markedly in enzymic
`capabilities in the formation of acetaminophen, however, statis(cid:173)
`tically significant differences (P < .0005) were observed in the
`extents of acetaminophen sulfation; the faster the formation of
`acetaminophen, the greater the extent of acetaminophen sul(cid:173)
`fation (table 2). Indeed, the phenomenon is not due to a
`nonlinearity in sulfation reactions either resulting from a
`saturation of enzyme capacity or a depletion of the cosubstrate
`
`3' -phosphoadenosine 5' -phosphosulfate, as found in other sys(cid:173)
`tems (Grafstom et al., 1979), because the concentration range
`whereby the system is linear (Q µg/ml or 6.7 14M) with respect
`to acetaminophen sulfation (Pang and Terrell, 1981b) has not
`been exceeded by the sum (1.24 ~) of the tracer concentrations
`used in this experiment. Moreover, linearity for the oxidative
`pathways has been checked out by our preliminary experi(cid:173)
`ments.
`The apparent correlation of a faster formation and a faster
`sulfation of a primary metabolite may be explained by blood
`transit time and time required for drug conversion to primary
`metabolite. The blood transit time in liver is a measure of its
`transit in that organ and is assessed by the ratio of volume of
`blood in liver to hepatic blood flow (1.5 ml/(10 ml/min) in our
`case or 0.15 min]. The distance that a drug molecule has
`traveled within the liver is proportional to the time elapsed
`after the entry of this molecule into the liver. Because the
`transit time is finite, for metabolic reactions that occur sequen(cid:173)
`tially, that is, a drug undergoes oxidative metabolism to form a
`metabolite before the metabolite is ultimately sulfated, the
`more time required for the Phase I biotransformation process,
`the less time will remain and the less likelihood there will be
`for subsequent metabolite sulfation. Contrastingly, the less time
`for metabolite formation, the more time will remain for further
`sulfation of the metabolite.
`This concept that an oxidative product has a "duration time"
`within the liver that may be governed by the time required by
`the biotransformation process for its formation is illustrated by
`a simulation. Simple mass-balance equations were written to
`denote the rate of change drug (equation 1) and its primary
`(equation 2) and terminal (sulfated) metabolites (equation 3) in
`liver:
`
`·°' N
`
`0
`N
`
`d[D]
`Vr.<l>t dt = QCrn - cr....,.,,_M,, [D]
`
`d[Mr)
`V L, 14 , - d t = CL;n,,.,_M , [D] - Cl.;0 , 114 _ 14
`
`I
`
`I
`
`I
`
`, [Mi]
`
`II
`
`(1)
`
`(2)
`
`(3)
`
`d[Mn] _
`V
`L,M.,, "'cit - CL;n,,M,-Mo• [Mi)
`
`where Vr. denotes the volume of distribution of drug in liver, Q
`the hepatic venous blood and C1n the input drug (D) concentra(cid:173)
`tion. The square brackets denote liver concentrations of drug
`(D), primary metabolite (Mr) or terminal (sulfated) metabolite
`(Mu); CL;n, denotes the hepatic intrinsic clearance and sub(cid:173)
`scripts (D -+ Mr) and (M, -+ Mn), respectively, denote the
`metabolic conversions of drug to primary metabolite and pri-
`
`Auspex Exhibit 2016
`Apotex v. Auspex
`IPR2021-01507
`Page 4
`
`

`

`Vol. 222
`
`distance" for the enzyme systems for primary metabolite for(cid:173)
`mation and metabolite sulfation remain constant (fig. 4). This
`median distance is conceptually shown by the distribution and
`the medians of two enzymic systems, A and B; the median is a
`measure of the "center" of enzymic distribution, the point on
`the scale of observations on each side of which there are equal
`areas of the enzyme system, and the median distance for either
`enzymes A or B is described as the distance between the median
`of the enzymic system and the point of entry of blood to the
`liver (fig. 4). For different precursors that form a common
`primary metabolite with different hepatocellular activities and
`different enzymic systems, and the primary metabolite is elim(cid:173)
`inated via a common conjugation pathway, for example, sulfa.
`
`80
`
`,
`,
`,
`/
`• /
`,' I
`,' I
`,' /
`,;
`20 V
`
`40
`
`/
`
`/
`
`,,
`
`/
`
`----
`----
`--
`
`0.4
`0.1
`0.025
`
`120
`
`,..,
`LL.
`I-z
`0
`z w
`0 0
`j:: a: 100
`w
`z ....
`< Q.
`~ a:
`w
`..J >
`w ::;
`~ 60
`..J
`~
`I- w
`z I-w ::;
`::, 0
`0 w al
`Cf) <
`I-
`LL. w
`0 ~
`I- >
`z a: w <
`I- :E
`X cc
`w
`
`0
`0
`
`Q.
`
`100
`60
`40
`20
`80
`TIME ELAPSED AFTER DRUG
`ENTRY IN LIVER (SEC)
`Fig. 3. The extent of subsequent elimination of a primary metabolite as
`a function of the time elapsed after drug entry into the liver and the
`hepatocellular activities for primary metabolite formation (0.4. 0 .1 and
`0 .025 ml/sec); the hepatocellular activity for primary metabolite sul(cid:173)
`lation is kept constant (0.05 ml/sec). The simulations are transformed
`data from figure 2.
`
`~
`
`"' 1-z
`> a:
`< a:
`I-m a:
`<
`
`~
`'1i -g
`-s
`o ID
`.i-o O
`f
`
`Q.
`
`ENZYME A
`
`ENZYME B
`
`·°' N
`
`0
`N
`
`..
`
`..
`
`Median Distance
`
`Median Distance
`
`LENGTH OF LIVER
`Ftg. 4 . The distribution of enzymic activities tor enzyme systems A and
`B and their median values along the length of the liver. The distance
`between the median and the point of entry of blood is the median
`distance.
`
`18
`
`Pang etal.
`
`1 I_. 11 •
`
`mary metabolite to terminal (sulfated) metabolite. For the sake
`of convenience, V L and Q were designated as unity and M1 and
`Mn were the only metabolites formed in the sequence of D --+
`M1 ..... Mn. The intrinsic clearance for metabolite formation,
`CL;.,,,. _M1, , were assigned values of 0.4, 0.1 and 0.025 ml/sec.
`The intrinsic clearance for terminal (sulfated) metabolite for(cid:173)
`mation, CL;,,, ..
`., was fixed at 0.05 ml/sec.
`The concentrations of drug and metabolite species in liver
`against time (time elapsed after drug entry) were simulated by
`use of a computer program, MLAB (Knott and Reece, 1977).
`Differing profiles (concentration vs. time in liver) for drug,
`primary metabolite and sulfated metabolite result along the
`flow path (time) in the liver (fig. 2). H the liver were of infinite
`length and the blood transit time infinite, the sulfated metab(cid:173)
`olite (Mu) will be the only species detected in hepatic venous
`blood for a single passage of drug. But the liver is of finite
`length and the blood of finite transit time. The concentrations
`of drug and metabolite species at the end of the flow path in
`liver (the value of blood transition time) becomes the effluent
`concentrations and will have different concentrations, depend(cid:173)
`ing on the intrinsic clearance for metabolite formation (fig. 2).
`This simulation can be presented in an alternate fashion by
`expressing the ratio of the concentration of the terminal metab(cid:173)
`olite to the sum of all metabolites, that is, [M11]/([M1] +
`[Mu]) at various times (fig. 3). It can be seen from this plot,
`which expresses the extent of sequential sulfation of the primary
`metabolite vs. the time elapsed after the entry of drug into the
`liver, that the faster the formation of the primary metabolite
`(higher intrinsic clearance for metabolite formation), the
`greater the extent of subsequent metabolite sulfation.
`The simulations (figs. 2 and 3) are indeed oversimplistic views
`of the sequential sulfation of a primary metabolite which is
`itself formed via oxidative metabolism from various precursors
`because uneven distribution of drug metabolizing enzymes may
`exist in the rat liver lobule. The real trend for metabolite
`sulfation, however, will not deviate qualitatively from the pre(cid:173)
`dicted curves (fig. 2 and 3) as long as the relative "median
`
`--
`
`- -
`
`- 0.4
`- - • • · 0.1
`-
`-
`0.025
`
`,.
`
`~ - -, , ,
`II,,,
`,
`, ,
`
`,,
`
`/
`
`/
`
`/
`
`100
`
`\
`\
`~
`
`80
`
`60
`
`'
`
`40
`
`20
`
`o~:;.:::.......,. _ _;_;-;..:--.....,._...::::;::::::::::ii,,i~---....;;;..;::..
`20
`80
`120
`0
`40
`140
`60
`100
`TIME ELAPSED AFTER DRUG ENTRY IN LIVER (SEC)
`
`~
`~
`< cc
`1-z w
`(.) z
`0
`(..)
`
`I-i
`!
`LL
`0
`
`1-z w
`(..) cc
`~
`
`Ftg. 2. A simulation of the concentrations of drug (D) and its primary
`(M 1) and terminal (M1) metabolites in liver. The hepatic intrinsic clear(cid:173)
`ances for M, formation from Dare varied (0.4, 0 .1 and 0 .025 ml/sec),
`whereas th