XENOBIOTICA, 1977, VOL. 7, NO. 9, 529-536
`
`The Metabolism of Phenolic Opiates by Rat Intestine
`
`MICHAEL J. RANCE and JOHN S. SHILLINGFORD"
`Drug Metabolism Department, Reckitt and Colman Pharmaceutical Division,
`Dansom Lane, Hull HU8 7DS, U.K.
`
`(Received 11 November 1976)
`
`1. A range of phenolic opiate agonists and antagonists undergo conjugation
`during passage across the rat intestine.
`2. The efficiency of conjugation, mediated by gut UDP-glucuronyltransferase,
`is a function of the lipophilicity of the substrate.
`3. Conjugation of lipophilic substrates such as buprenorphine and etorphine
`in rat intestinal mucosa is such that the gut wall must be regarded as the primary
`site of metabolism of these compounds after oral administration.
`4. N-Dealkylation of the N-cyclopropylmethyl opiate, buprenorphine, has
`been demonstrated in rat gut sacs though dealkylation of N-methyl substrates
`was not detected.
`
`Introduction
`A relative lack of oral activity has long been a problem associated with opiate
`drugs (Houde, Wallenstein & Beaver, 1965), low oral efficacy frequently being
`associated with the presence of a phenolic function. For example, the lowered
`availability of morphine after oral administration in man has been elegantly
`demonstrated in pharmacokinetic terms by Brunk and IUelle (1975), who showed
`that plasma levels of unmetabolized drug following an oral dose of morphine were
`an order of magnitude lower than those obtained after the same dose administered
`by three different parenteral routes.
`It has been postulated that the low oral
`efficacy is due to poor absorption of the drug from the gastro-intestinal tract
`(Way & Adler, 1962), but studies in both animals and man have shown this not
`to be so (Cochin et al., 1954; Brunk & Delle, 1975). It follows therefore that
`the low peripheral availability of morphine after oral administration is probably
`due to its metabolism during a 'first pass ' through gut wall and liver.
`The presence of a phenolic function in morphine-like compounds appears to
`be beneficial to their activity.
`It is, however, this functional group which appears
`to be responsible for the observed first pass effect in many cases, since conjugation
`In the past, the major organ responsible for first
`takes place readily at this site.
`pass metabolism has been assumed to be the liver, and hepatic metabolism of
`opiates has been extensively studied and reviewed (Way & Adler, 1960 ; Scrafani
`& Clouet, 1971). Although the drug-metabolizing capacity of the gut mucosa
`has been known for many years (Hartiala, 1973), it is only recently that the
`involvement of the intestinal wall in the biotransformation of opiates has been
`observed (Del Villar, Sanchez & l'ephly, 1974).
`
`* Present address: Drug Metabolism Department, Allen and Hanbury Research Ltd.,
`Priory Road, Ware, Herts, U.K.
`
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`

`530
`
`M . J. Rance and J. S. Shillingford
`
`The capacity of the intestinal mucosa to carry out phase two metabolism of
`drugs is becoming the subject of an increasing amount of study and recent reports
`of the conjugation of phenol (Powell et al., 1974), isoetharine (Williams et al.,
`1974), isoprenaline (George, Blackwell & Davies, 1974), 1-naphthol (Bock &
`Winne, 1975), paracetamol and morphine (Josting, Winne & Bock, 1976) in
`whole animals have all established the gastro-intestinal tract as a major site of phase
`In the light of this work, a preliminary investigation of the role
`two metabolism.
`of the gut in the metabolism of three phenolic analgesics, dihydromorphine,
`etorphine and buprenorphine, was undertaken and leads to the speculation that
`lipophilicity might be a determining factor with regard to the efficiency of gut
`UDP-glucuronyl transferase activity (Rance & Shillingford, 1976). These
`studies, which have been extended to include a total of five opiates, dihydro-
`morphine (I), naloxone (II), diprenorphine (111), etorphine (IV) and buprenor-
`phine (V) (Fig. 1) are reported here in full.
`
`0
`
`H
`
`O
`
`B
`
`,
`
`
`
`Hl@,
`
`NR
`
`R‘
`
`HO
`
`NR
`
`_--
`---
`X
`
`Me@
`
`,04,Ye
`
`(I) R-Me, R’=H.
`(11) R=allyl, R’=OH, 6=-one.
`
`(111) R=cyclopropylmethyl, R’-Me, X=CH2CH2.
`(IV) R==Me, R’-n-Pr, X=CII=CH.
`(V) R == cyclopropylmethyl, R’= t-Bu, X = CH,CH,.
`Fig. 1. Structures of opiates studied.
`
`Materials and methods
`Radio chemical methods
`[1,7,8(n)-3H]Dihydromorphine (70 Ci/mmol) was supplied by the Radio-
`[15, 16(n)-3H]Diprenorphine (5.68 Ci/mmol),
`chemical Centre, Amersham.
`[15, 16(n)-3H]etorphine (30 Ci/mmol) and [15, 16(n)-3H]buprenorphine (28
`Ci/mmol) were synthesized by the method of Lewis, Rance and Young, (1974).
`[15-3H]Naloxone (4 Ci/mmol) was generously given by Professor H. W.
`Kosterlitz.
`The radiochemical purity of all the labelled compounds was shown to be
`> 95% by t.1.c. on Kieselgel F,,, plates using ( a ) n-butanolLacetic acid-water
`(85 : 15) as developing solvents.
`(20 : 5 : 8), and ( h ) ethyl acetate-methanol
`Samples of drug solutions and biological fluids were diluted to 1.0 ml with water
`before addition to NE 260 (10 ml ; Nuclear Enterprises Ltd.) for counting.
`Counting efficiencies were determined by internal standardization with [3H]-
`hexadecane. All samples were counted in a Packard 2450 or a Packard 3003
`scintillation spectrometer.
`
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`

`Phenolic Opiate Metabolism by Intestine
`
`53 1
`
`Chromatographic methods
`Plasma samples were diluted (SO:/, v/v) with methanol to precipitate proteins
`which were removed by centrifugation. Samples of serosal fluids and plasma
`supernatants were applied to t.1.c. plates (0.25 mm Kieselgel HF254, Merck Ag)
`and developed with solvents ( a ) or (b).
`Authentic compounds and the N-dealkylated products, noretorphine and
`norbuprenorphine, were also applied to plates and visualized under U.V. light.
`Bands (1 cm) of silica gel were removed and counted in suspension in water
`(2 ml) and an aliquot ( 5 ml) ofasolutionof 2-(4’-t-butylphenyl)-5-(4”-biphenylyl)-
`1,3,4-oxadiazole (7.5 g) in Triton X-100 (334ml) and toluene (666 ml).
`Samples containing conjugated species were diluted with citrate buffers
`(pH 5.0; pH 6.S), ca. 100 units of P-glucuronidase (Type HI ; Type 1 ; Sigma)
`or sulphatase (Sigma) added and the mixture incubated overnight. Saccharo-
`1,4-lactone was added in some cases to inhibit ,8-glucuronidase activity. The
`hydrolysates were extracted with chloroform ( 5 ml), the organic layer taken to
`small volume and applied to silica plates which were analysed for radioactivity
`as above.
`
`Everted gut studies
`The general procedure used was that of Wilson and Wiseman (1954).
`The
`rat was killed by cervical dislocation and a length of small intestine removed,
`everted and cut into segments (5 cm) which were kept in isotonic saline gassed
`with 0,-CO, (95 : 5 ) during subsequent manipulations. The intestinal segments
`were rinsed with isotonic saline to remove food and bacterial contaminants.
`Sacs 4cm in length were filled with citrate-phosphate buffer (0.154~1, pH7.4,
`1.0 ml) containing glucose (0.5% w/v) and incubated at 37” in an aliquot ( 5 ml)
`of the same buffer solution containing the radiolabelled drug. Drug and metab-
`olites in serosal fluid were separated by t.1.c. and assayed radiochemically as
`described above.
`
`Absovption studies in situ
`A modification of the method of Doluisio et al. (1970) was used. Albino male
`rats (Sprague-Dawley, Bantin and Kingman ca. 200 g), fasted overnight but
`allowed water ad libitum, were anaesthetized with urethane (2.5% W/V in saline).
`The small intestine was exposed by a midline abdominal incision and a poly-
`ethylene cannula (2.5 mmi.d., 3.5 mm0.d.) was inserted into duodenal and ileal
`ends. A syringe (SO ml) fitted with a threc way tap was attached to the duodenal
`cannula and the intestinal lumen was cleared of particulate matter by slow intro-
`duction of solution from the syringe. A duplicate syringe was affixed to the ileal
`cannula and 0.15 M Smensens phosphate buffer (pH 7.4, 10 ml) containing the
`radiolabelled drug was introduced into the small intestine. Portal venous blood
`was sampled by the introduction of a fine injection needle connected to a polythene
`cannula (Portex P P 25). Samples (0.2ml) of portal blood were taken every
`10 min and plasma separated by centrifugation. Plasma was assayed radio-
`chemically for drug and metabolites as described above.
`Results
`The rate of transfer of the five opiate drugs across the intestinal mucosa of the
`rat everted gut sac is shown in Table 1, together with the percentage of the free
`
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`5 32
`
`M . J. Rance and J . S. Shillingford
`
`Table 1. Absorption of "-opiates in aitro in the rat everted gut sac preparation.
`
`Drug
`
`Drug luminal
`concentration
`(pgiml)
`
`Rate of absorption
`of drug-related
`material
`(pg drug equivalent/h)
`
`o/
`/ 0
`Free drug O,/,Dealkylation
`absorbed
`
`Bupren orphine
`
`Etorphine
`
`Diprenorphine
`
`Naloxone
`
`1
`5
`10
`
`1
`5
`10
`
`1
`5
`10
`
`1
`5
`10
`
`0.32 f 0.04
`2.68 fO.10
`4.25 0.3 1
`
`0.39 i 0.07
`2.85 f 0.08
`4.25 f 0,26
`
`0.91 k 0.20
`3.70 f 0.31
`5.92 & 0.36
`
`0.70 i 0.04
`3.33 k 0.34
`4.70 k 0.68
`
`3.4k0.3
`15.2f0.4
`20.0 k 1.9
`
`3.3 & 0.2
`6.2 k 0.4
`30.1 f 4.3
`
`19.3 f 2.3
`25.9 f 0.9
`51.3 24.7
`
`56.4 k 5-1
`62.4 +- 1.6
`61,Ok 5.0
`
`11.5k1.8
`22.2k3.6
`24.6 i: 1 *7
`
`-
`-
`-
`
`5.2 k 0.7"
`8.3 f 3.4"
`11.6 k 1.9"
`
`~~ -
`-
`-
`
`0.22 f 0.03
`1.60 k 0.11
`Dihydromorphine
`3.22 f 0.1 7
`____-__
`__
`Results are means S.E., n>4.
`"Unidentified metabolite, possibly the nor compound.
`
`1
`5
`10
`
`~
`
`~
`
`~
`
`85.5 i 0 . 8
`-
`-
`86.8 i 2.4
`90.7 i 1.9
`-
`___-______
`
`drug found in the serosal fluid in each case. T h e remainder of the radioactivity
`in the serosal fluid was present as polar conjugates in all cases. T h e conjugates
`were readily cleaved by P-glucuronidase and the hydrolysis was inhibited by
`saccharo-l,4-lactone indicating that the major conjugating species was glucuronic
`acid. The principle conjugated species formed in the intact rat after oral
`administration of buprenorphine is also the glucuronic acid conjugate (Jordan
`and Rance, unpublished). Hydrolysis by the sulphatase enzyme was low
`showing minimal sulphate ester formation with the substrates used. Chromato-
`graphic examination of the products of /3-glucuronidase hydrolysis indicated the
`presence of the N-dealkylation product of buprenorphine (norbuprenorphine).
`No N-dealkylation was detected with the N-methyl compounds, dihydromorphine
`and etorphine, nor with naloxone which possesses an N-ally1 function. With
`diprenorphine, which has, in common with buprenorphine, an N-cyclopropyl-
`methyl group, an unidentified metabolite was detected in low amounts which
`might be the nor compound derived from this substrate. Table 2 presents the
`results of the studies carried out in situ using the two drugs (dihydromorphine
`and buprcnorphine) of most widely different lipophilicity (Table 3). T h e results
`are expressed as total plasma levels of drug-related material present in the portal
`system together with the percentages of that material which were present as the
`unchanged drug.
`
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`

`

`w
`w
`ul
`
`m
`x
`w. N.
`2
`ir 2
`
`(b
`
`f?.
`0 %.
`
`-___
`
`-f No free drug detected
`* Total volume introduced into the small intestine was 10.0 ml in each case.
`
`Results are means of 3 experiments. Ranges in parentheses.
`
`0.094 (0'090-0'098)
`40.2 (40.045.9)
`0.086 (0.082-0.094) 41.6 (38.046'7)
`0.079 (0.072-0.084) 39.9 (37.643.1)
`35.4 (27'342.9)
`0.044 (0.040-0.048)
`0'034 (0'028-0.036)
`41.8 (32.6-53.5)
`
`9.3 (6.6-15.5)
`9.5 (7.8-15.0)
`13.5 (9.6-20.1)
`14.2 (8.2-20.2)
`10.1 (3.3-13.4)
`
`0.55 (0.32-0.78)
`0.61 (0.42-0.73)
`0.54 (0.40-0.65)
`0.55 (0.45-0.73)
`0.37 (0'304.66)
`
`0'036 (0.028-0.040)
`39.9 (37.045.7)
`0.048 (0.039-0.052) 40.2 (33.132.0)
`0,043 (0.036-0.050) 36.7 (31.042.1)
`0.024 (0.021-0.027) 38.8 (36443.4)
`41.2 (34.1-50.1)
`0.014 (0'009-0.016)
`
`-
`-
`-
`--t
`
`0.08 (0.06-0.09)
`0.12 (0.09-0.15)
`0.1 1 (0.09-0.14)
`0.06 (0.04-0.07)
`
`50
`40
`30
`20
`10
`
`50
`40
`30
`20
`10
`
`10.0
`
`1 .o
`
`free drug
`
`04
`
`(pg dihydromorphine
`
`equivalent/ml)
`
`total radioactivity
`Plasma level of
`
`yo free drug
`
`(pg buprenorphine
`total radioactivity
`Plasma level of
`
`equivalent/ml)
`
`(min)
`Time
`
`Dihydromorphine
`
`Bupren orphine
`
`concentration
`
`Drug
`
`(pg/ml)
`intestine"
`in small
`
`Table 2. Portal vein plasma levels of [3H]buprenorphine, [3H]dihydromorphine and conjugates
`
`after absorption of the drugs from the rat isolated small intestine in situ
`
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`

`534
`
`M . J. Rance und J. S. Shillingford
`
`Table 3. Log Partition coefficients ( N ) of phenolic opiates
`
`Drug
`
`Log N ;
`heptane /phosphate buffer
`pH 7.4(20")
`
`~
`
`~
`
`~~
`
`1.78"
`Buprenorphine
`o.1st
`Etorphine
`- 0.381
`Diprenorphine
`- 1.761
`Naloxone
`- 5.00"
`Dihydromorphine
`* Personal communication P. H. McNally.
`t Data from Herz and Teschemacher (1971)
`1 Determined by standard techniques using radiolabelled drugs.
`
`Discussion
`The results in Table 1 demonstrate that all the drugs examined were con-
`jugated to some extent by the gut sac preparation. For the lipophilic compounds
`buprenorphine and etorphine, the efficiency of conjugation was such that, at low
`initial mucosal concentrations of drug, essentially all the drug-related material
`reaching the serosal fluid was present as polar conjugates. The degree of con-
`jugation of these lipophilic substrates decreased with increasing mucosal drug
`concentration but remained significant at the highest concentration examined
`(10 pgiml).
`In contrast, dihydromorphine, the most hydrophilic substrate studied, was
`subject to only minimal metabolism on crossing the intestinal barrier. Table 3
`presents the partition coefficients between heptane and phosphate buffer (pN 7.4)
`for each drug and using this partition coefficient ( N ) as a measure of lipophilicity,
`a regression analysis was carried out using the in vitro data obtained at a luminal
`drug concentration of 10 pg/ml.
`The following equation was computed :
`D = 39.96-10'55 logN (n = 25, Y = - 0.96)
`where D is the percentage of the total radioactivity in the serosa present as un-
`changed drug, n is the number of data points and r the correlation coefficient.
`The correlation is illustrated in Fig. 2.
`It appears therefore that the availability of opiates to the portal system is
`significantly influenced by intestinal conjugation and that the efficiency of the
`conjugation, mediated by mucosal UDP-glucuronyl transferase activity, is a
`function of the lipophilicity of the substrate.
`The consequences of this detoxication system in vivo is illustrated by the
`results of the in situ studies shown in Table 2. As was found in vitro, buprenor-
`phine was efficiently metabolized at low mucosal concentrations (1.0 pg/ml), all
`drug-related material being recovered from the portal blood as conjugates.
`Dihydromorphine was conjugated to a greater extent in vivo than was found in
`the gut sac preparation though considerable levels of free drug were detected even
`It should be noted that during the in situ
`at low mucosal drug concentrations.
`reported studies here, portal blood was assayed using a sampling technique. Thus,
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`

`Phenolic Opiate Metabolism by Intestine
`
`535
`
`hepatic and peripheral metabolism might be expected to make a contribution to
`the metabolic pattern at later times after drug administration. The results
`obtained are however consistent with those of Josting, Bock and VC'inne (1976)
`who showed considerable conjugation of morphine with glucuronic acid in rat
`intestine using an in vivo preparation in which the total venous output of an
`intestinal loop was collected and analysed for metabolites.
`
`u
`8 C
`
`6 C
`
`4c
`
`2c
`
`-30
`
`4 0
`
`-30 -23 -10 0 +I0
`Log N
`
`Fig. 2. Relationship between the percentage of free drug absorbed and the partition coefficient
`in the rat everted gut sac preparation.
`The partition coefficients ( N ) were determined between heptane and 0.1 M phosphate
`The initial luminal drug concentration was 10 pg/ml.
`Results represent
`buffer.
`means & S.E. (n 3 4). The percentage of free drug absorbed (D) was determined
`radiochemically after separation of the metabolites by t.1.c.
`0 , dihydromorphine; 0, naloxone; A, diprenorphine; A , etorphine; w , buprenor-
`phine.
`Regression equation: D=39.96- 10.55 log N ; r = -0.96 (P< 0.001), n=25.
`
`Diprenorphine, a compound of intermediate lipophilicity (Table 3), showed
`the highest overall rate of absorption in the gut sac preparation, with both the more
`polar and less polar compounds being absorbed at a slower rate. This result is
`consistent with the theory that an optimal partition coefficient exists for the
`intestinal absorption of xenobiotics (Houston, Upshall & Bridges, 1974).
`The pharmacological implication of gut wall metabolism of buprenorphine is
`illustrated by its antinociceptive potency after oral and intraperitoneal admin-
`istration in rat (A. Cowan, personal communication). Buprenorphine is much
`more potent intraperitoneally despite the fact that the majority of the drug
`administered by this route is absorbed via the mesenteric system and is subject
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`536
`
`Phenolic Opiate Metabolism by Intestine
`
`to a first pass through the liver. These pharmacological results thus provide
`further supportive evidence for the importance of the gut in the metabolism of
`orally administered phenolic opiates.
`During this study, the loss of the N-cyclopropylmethyl function from
`buprenorphine (and possibly from diprenorphine) in the rat gut sac preparation
`has been demonstrated (Table 1 ) though no N-demethylation of dihydromorphine
`or etorphine was detected. Very little is known of the capacity of the intestinal
`mucosa for N-dealkylation of opiate substrates. T h e existence of ethylinorphine
`N-demethylase activity in intestinal microsomes from rabbit and guinea-pig has
`been recently demonstrated (Chhabra, Pohl & Fouts, 1974), though these workers
`failed to find similar activity in rat, mouse or hamster. T h e existence of significant
`N-dealkylation capacity in the gut would certainly contribute to any route-
`dependence of biotrarisformation that might be found with opiates, and Brunk
`and Delle (1975) have, in fact, shown increased N-demethylation of morphine in
`man after oral administration of the drug. There is some evidence that oxidative
`N-dealkylation of narcotics with antagonist N-substituents, such as the ally1 and
`cyclopropylmethyl groups, is more facile than N-demethylation. For instance,
`nalorphine is more rapidly dealkylated than morphine in liver preparations
`(Axelrod & Cochin, 1957).
`Increased N-dealkylation during a first pass through
`gut wall and liver after oral administration may well have particular significance
`with narcotic antagonist analgesics as the N-alkyl function has a particularly
`important role to play in determining the pharmacological profile of this class of
`compounds.
`
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`J. T. & CLourr, D. H. (1971).
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`WAY, E. L. & ADLER, T. K. (1960).
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`In Analgesits, Ed. de
`
`Xenobiotica,
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