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Aspects of the biliary excretion of orphenadrine and its N-demethylated derivative, tofenacin, in the rat
EUROPEAN JOURNAL OF PHARMACOLOGY 13 (1970) 113-122. NORTH-HOLLAND PUBLISHING COMPANY
ASPECTS OF THE BILIARY EXCRETION OF ORPHENADRINE AND ITS N-DE~~YLATED DE~VATI~, TOFENA~IN, IN THE RAT W. HESPE and W.F. KAFOE Research Department N. V. Koninklijke Phannaceutische Fabrieken v/h Br~ades-Sthee~n en Pharmacia, ~oie~~a~ht 27-39, Amsterdam, Ne~~ands
Accepted 29 June 1970
Received 8 April 1970
W, HESPE and W.F. KAFOE, Aspects of the bilia~ excretion ofo~h~ad~e and its N~emethy~ated derivative, tofenacin, in the rat, European J. Pharmacol. 13 (1970) 113-122, A study was made of the excretion of radioactivity in bile of male rats after administration of orphenadrme hydrochloride and tofenachr hydrochloride in labelled forms. In bile fistula rats it was found, particularly after intravenous administration, that biliary excretion was considerable: after five hours 31 to 48% of the radioactivity had been excreted in the bile. In normal rats, total bibary eMination exceeded urinary elimination. A study of the radioactivity in the blood, liver and bile showed two phases of concentration: first, from blood to liver, more than a lOO-fold concen~ation was achieved and second from liver to bile, the concentration increased more than 5 times. When 10 mg/lcg doses of the labelled compounds were administered iv, the approximate points of thne at whkh maximum concentrations were found in liver and bile were 10 and 20 min after administration, while in the blood maximum concentration was reached immediately after the injection. A compartment model is proposed to explain various aspects of the time course of biliary excretion of these drugs in bile fistula rats. Analytical investigations of orphenadrine metabolites in rat bile showed that only minor amounts of the compound were excreted unchanged or in the form of free (nonconjugated) metabolites; evidence was obtained for major excretion in the form of ~ucuro~&s and probably sulfates of aromatic hydroxy~ted o~hen~e metabolites. The bio~~~ormations promoted excretion in the bile, as evidenced by the bile collected in the first fwe minutes after i.v. dosage; in that period conjugates were already dominant and pretreatment with the metabolic inhibitor proadifen produced a significant decrease in biliary excretion of radioactivity over 1 hr after administration - an effect which could not be related to competition. There is strong evidence that compounds of the type studied undergo enterohepatic circulation. Grphenadrine Tofenacin
Proadifen Biliary excretion, rat
1. INTRODUCTION Previous ~vest~tions into the metabolism of labelled o~henad~ne hydroc~o~de in the rat showed that a large amount of the orally administered radioactivity - generally more than 50% of the dose - was eliminated in the faeces (Hespe et al., 1965a). The substance was originally thought to be poorly absorbed, but further expe~ments, inclu~ng some with bile %&la rats, suggested a very consider-
Enterohepatic circufation Glucuronic acid conju~tion
able elimination of radioactivity through the bile. Similar excretion patterns (predominant elimination by the faecal route and considerable biliary excretion) have been demonstrated in the rat for a number of related bi- and tricyclic compounds with a basic side chain (Hespe et al., 1966; 1967; 1968). Since the time course of radioactivity in the rat gastrointestinal tract indicated good absorption after oral administration, it was obvious that the excretion in the faeces was largely due to the high biliary excretion of the
W.Hespe, W.F.Kafoe, Biliary excretion of orphenadrine and tofenacin in the rat
114
compounds in some form. In metabolic studies of various compounds in the rat we collected information on biliary excretion, part of our findings, particularly those on orphenadrine and tofenacin, are presented here. The following points are discussed: (1) quantitative aspects of biliary elimination and its time course; (2) relations between the concentrations in blood, liver and bile at different points of measurement after administration; (3) the form in which the compounds are excreted in the bile; (4) the relationship between biotransformation and excretion in the bile.
2. METHODS
2.1. Compounds investigated Labelled forms of orphenadrine and tofenacin were used, as indicated in fig. 1. The substances are referred to by the numbers given in this figure. These radioactive compounds were prepared in our laboratories;: part of the synthetic work has been published (Hespe et al., 1965b; Hespe and Nauta, 1966). 2.2. Animal studies In all experiments male albino rats (TNO-Wu strain) of 150-250 g were used. Quantitatively, the biliary elimination of radioactive material was studied by two methods, A and B.
. I.lCt
•- . e
.I.ICL
lc
.HCL
H
.HCI.
Fig. 1. Structural formulae of the compounds investigated. Labelled forms of orphenadrine hydrochloride (la, b, c) and tofenacin hydrochloride (2).
A) Rats, fasted for about 20 hr, were anesthetized with phenobarbital sodium (100 mg/kg, i.p.). Bile was continuously collected from a polyethylene cannula inserted into the common bile duct for periods up to 5 hr after administration of the labelled compounds and assayed for radioactivity. B) In order to gain an insight into the amount of radioactive material eliminated through the bile under normal conditions, the elimination pattern of radioactivity in normal rats, was compared with that in rats in which cholestasis had been surgically induced. Urine, faeces and expiratory carbon dioxide were separately collected in metabolic cages. The increase in elimination by the unobstructed routes which resulted from the cholestasis was taken as a measure of the biliary elimination. The excretion experiments were, as a rule, started one day after operation. The time course of the radioactivity in the blood was recorded continuously by passing the blood, through a loop in the carotid artery into a counting unit as described in the next section. The compounds were injected through a cannula inserted into the femoral vein. The animals used in these experiments were anesthetized with phenobarbital sodium (I00 mg/kg, i.p.) and pretreated with heparin (500 I.U., Vitrum, Stockholm).
2.3. Radioactivity measurements The radioactivity of the different excretion products was determined according to procedures described previously (Hespe et al., 1967). In order to determine the radioactivity in liver tissue, samples of about 50 mg were treated directly in counting vials (Mahin and Lofberg, 1966). In earlier experiments a Packard Tri Carb Scintillation Spectrometer 314 EX was used and quench corrections were made by the internal standard method. Subsequently a Packard Tri Carb Scintillation Spectrometer 3375 was used and quench corrections were performed by the external standard method. The time course of radioactivity in the blood was continuously followed by passing the blood through a circuit of plastic scintillating tubing (Nuclear Enterprise NE 102A tubing), placed in a light-tight housing between two photomultipliers (PhilipsPW 4210) connected to additional counting equipment. This method gave good results for the 14C-labelled compounds but its sensitivity was too low for all-labelled substances.
W.Hespe,W.F.Kafoe,Bilinry excretion of orphenadrine and tofenacin in the rat
115
Table 1 Analytical data on orphenadrine and some potential metabolites. Chromatographic assay (Rf value range) orphenadrine Ndemethyl orphenadrine N,N-didemethyl orphenadrine 5-OH orphenadrine 4’-OH orphenadrine 2CHzOH diphenhydramine 2-COOH diphenhydramine
+ denotes that calorimetric determination
Colorim etric assay
0.26-0.34 0.36-0.42 0.45-0.52 0.58-0.66 0.63-0.70 0.60-0.67 0.85-0.95
+ + + + + +
of the compound is possible (methyl orange).
2.4. Analytical procedures
Extraction procedures in combination with alkaline and enzymatic hydrolysis @-glucuronidase and arylsulfatase) and thin-layer chromatography (TLC) were the main analytical methods employed to study the radioactive products in bile. Potential orphenadrine metabolites, synthesized by Den Besten et al. (1970) were used to develop a reversed phase TLC method that proved superior to any other method tested. Typical Rf values found on olive oil-impregnated silica gel, by means of ethanol-25% ammonia as a solvent system, are given in table 1. Although not all compounds were separated completely this TLC technique provided a useful separation into the following groups: Rf range I
0.00-0.50, orphenadrine and N-demethylated analogues; Rf range II 0.50-0.70, hydroxylated metabolites; Rf range III 0.70-0.95,2-carboxydiphenhydramine.
Another method used to analyse the bile was that described by Dill and Glazko (1949), as modified by Hespe et al. (1965a). This method, based on double extraction of the biological material followed by calorimetric assay of the complex formed by the N-bases with methyl orange, was used to determine the amounts of the unchanged compounds and their N-demethylated derivatives. However, since unidentified metabolites may be involved, the analytical results were expressed in terms of methyl orange-positive
compounds. The potential orphenadrine metabolites synthesized have been classified accordingly (table 1).
3. RESULTS 3 .l _Quantitative biliary excretion data Two types of experiments, indicated by A and B, were used to elucidate the quantitative importance of the biliary elimination of the labelled compounds in the rat (table 2).
Table 2 Biliary excretion of radioactivity in bile fistula rats over a period of 5 hr after dosage (type A exp.) and in normal rats (type B exp.) after i.v. administration of orphenadrine and tofenacine in labelled forms. Biliary elimination in % of the administered radioactivity (? S.D.) Labelled compound
Type A exp. Type B exp.
la lb lc 2
oral
i.v.
12.5+ 6.4 (2) 16.2f 8.7 (3) 14.6+ 6.7 (5) -
31.3f3.1 (2) 44.1S.l (8) 48.4k5.3 (7)
42.3+ 9.9 36.1k19.0 36.9+ 9.1 42.2+ 7.5
(2) (4) (4) (4)
W.Hespe, W.F.Kafoe, Biliary excretion of orphenadrine and tofenacin in the rat
116
3.2. Time course o f the radioactivity level in blood and bile In experiments with compounds 2 and lc the time course of radioactivity in blood and bile was determined simultaneously (figs. 2 and 3). Initially the bile samples were collected at 5 min, and then at 15 min intervals. The course of the radioactivity in blood was monitored continuously by means of a counting unit (see section 2.3). This was useful in cases where blood sampling at short intervals presented difficulties. The time course of the biliary radioactivity was not only characteristic for the test compounds, but was also observed for a number of structurally related compounds: a rather sharp maximum invariably occurring
"~ 10
180 o
o 0~
Its0
0811\1111 1-1
I~z°~.
°'tllll1"5 lllJ Jo
~s
so
r
"is 9o Ibs
minutes ofter odministrotion
Fig. 2. Time course of the concentration of radioactivity in bile (histogram) and blood of a rat dosed with 10 mg/kg of labelled tofenacin hydrochloride (compound 2), intravenously.
150
10 119
120
08
~o.6
9O o. ~3
as "~ 0.t, 03 02 01 1"5
30
/.5
60 75 90 105 minutes ofter odmlnistrotion
Fig. 3. Time course of the concentration of radioactivity in bile (histogram) and blood of a rat dosed with 10 mg/kg of labelled orphenadrine hydrochloride (compound lc), intravenously.
in the second 15 min period, followed by a prolonged but less marked excretion. The maximal radioactivity level in blood was noted about 15 min prior to that in bile.
3.3. Time course o f the radioactivity level in liver and bile The relation between the course of radioactivity in liver and bile has been studied only for compound 2 (table 3). The maximum concentration of radioactivity in liver was observed 1 0 m in and in the bile 15-20 min after administration. Within 5 min, the concentration of radioactive material in the bile exceeded that in the liver. The ratio between bile and liver levels (table 3) attained a maximum at 20 min. 3.4. Structure o f the products excreted in the bile That orphenadrine and tofenacin were largely excreted in bile as metabolites was easily demonstrated since after extraction with n-heptane of alkalified bile from animals dosed with the labelled compounds, nearly all the radioactivity remained in the aqueous phase. In experiments in which the radioactive compounds were added to rat bile in vitro, about 98% of the radioactivity was extracted into the organic phase. In a more systematic study of these hydrophilic metabolites, two identical samples of pooled bile from rats dosed with compound la were subjected to a series of successive extractions (table 4). One sample underwent various treatments between the extractions, as indicated. It was demonstrated that there are radioactive orphenadrine metabolites in the bile which are thermolabile and/or sensitive to alkaline hydrolysis. An even more striking feature was the presence of metabolites sensitive to/3-glucuronidase/arylsulfatase treatment. After the whole series of extractions has been completed, the biliary residue of both portions was found to contain far less radioactivity than suggested by the amounts extracted. The main cause of this loss, which amounted to 50% of the radioactivity originally present, appeared to be the formation of a foamy interphase in the liquid system. Random foam samples were found to be highly radioactive. Using the analytical methods described in 2.4. a study was carried out into the structures of the various radioactive products in the bite of rats dosed with compound lb (table 5). Without pretreatment
117
W.Ifespe, W.F. Kafoe. Biliary excretion of orphenadrine and tofenacin in the rat
Table 3 Concentration of radioactivity in m/g in a) the liver of rats sacrificed at different points of time after intravenous administration of 10 mg/kg of labelled tofenacin hydrochloride (compound 2), in b) the bile of the same animals collected for 5 mm before death. Bile
Liver
Rat
Period of collection (min after adm.)
Concentration (cleslg)
Min after administration
Mean concentration (ue41g)
1 2 3 4 5 6 7 8 9 10 11 12
o- 5 5-10 10-15 15-20 20-25 25-30 30-35 35 -40 40-45 45-50 50-55 55-60
21.5 105.4 157.8 201.9 118.3 68.5 77.7 101.7 67.9 47.9 40.4 48.4
5 10 15 20 25 30 35 40 45 50 55 60
17.5 37.2 29.4 28.0 25.9 19.9 17.9 24.4 21.3 20.0 18.5 19.1
Ratio concentration bile/liver
1.57 2.83 5.37 7.21 4.57 3.44 2.16 4.17 3.19 2.40 2.18 2.53
Table 4 Yield of successive extractions of two identical samples of pooled bile, collected over a period of 7 hr after administration of an oral 10 mg/kg dose of labelled orphenadrine hydrochloride (compound la) to rats. One sample was subjected to different treatments prior to each extraction.
Extraction (dichloroethane)
pH on extraction
Extracted radioactivity as a % of the original biliary radioactivity
Successive pretreatments of portion 2
Portion 1
Portion 2
1
12
no pretreatment
2
12
3hrat100°C,pH
3
12
6hrat100°C,pH12
4
12
24 hr incubation with &glucuronidase pH 5 .O
0.7
12.0
5
12
24 hr incubation with p-glucuronidase/arylsulfatase, pH 6.2
0.4
7.0
6
10
no further treatment
0.4
0.9
7
8
no further treatment
0.4
0.1
8
6
no further treatment
0.2
0.1
9
4
no further treatment
0.1
0.2
10
2
no further treatment
0.1
0.1
6.2
29.0
12
Total
1.5
1.6
1.8
4.0
0.6
3.0
118
h/.Hespe, l¢.F.Kafoe, Biliary excretion o f orphenadrine and tofenacin in the rat
Table 5 Analysis of samples of pooled bile (4 ml), collected for 7 hr after administration of an oral 10 mg/kg dose of labelled orphenadrine hydrochloride (compound lb) to rats. Radioactivity as a % of original radioactivity in the bile sample Treatment of separate bile samples
Extracted with dichloroethane at pH = 10
Colorimetric assay of extract
Chromatographic assay of extract Products in Rf range I II III
1) No treatment
1.4
1.4
0.0
0.0
0.0
2) 6 hr at 100°C, pH 12
7.9
1.5
0.0
1.0
6.9
3) 24 hr incubation with &glucuronidase/arylsulfatase, pH 5.0
29.8
19.8
6.8
15.8
7.6
4) treatment (2), followed by (3)
41.3
24.9
7.4
20.8
13.1
hardly any radioactivity could be extracted from the bile and that which was extracted was methyl-orange positive as shown by colorimetric assay of the extract. However, the amount was too small for chromatographic identification. An alkaline hydrolysis procedure liberated products in the Rf ranges II and, especially III. The good agreement between the percentages in Rf range II and in the colorimetric assay of the same extract suggests that the products in Rf range II were methyl-orange positive and those in Rf range III methyl-orange negative. The only potential orphenadrine metabolite synthesized with an Rf value in the latter range was 2-carboxydiphenhydramine; in the form of an ester glucuronide it would certainly be liable to alkaline hydrolysis. Moreover, this product is methyl-orange negative. Recently, in metabolic studies with diphenhydramine in rhesus monkeys, (diphenylmethoxy)acetic acid was reported to be a metabolite, partly conjugated with glutamine (Drach and Howell, 1968). An analogous orphenadrine metabolite, although not synthesized, should be considered an alternative for the unknown product in Rf range III. Enzymatic treatment liberated products in all three Rf ranges. There is reasonable agreement between the percentages of methyl-orange positive compounds and the sum of the percentages in Rf ranges I and II, suggesting that the radioactivity in these Rf ranges may largely originate from methyl-orange positive compounds. Rf range I includes the unchanged
compound and its N-demethylated derivatives. None of the remaining potential metabolites was localized in this range. A marked increase in radioactivity in this range after enzymatic treatment may be related to the presence of N-glucuronides of the N-demethylated products, especially that of N-demethyl olphenadrine. Rf range II includes those orphenadrine metabolites which contain an aromatic hydroxyl group, the pronounced effect of enzymatic treatment suggesting strongly involvement of hydroxylated derivatives of orphenadrine, which are conjugated with glucuronic acid and probably sulfuric acid. The effect of enzymatic treatment on the radioactivity in Rf range III might be related to the presence of an ester glucuronide of the above acids. Further analytical work is in progress to determine more exactly the nature of the different products involved and of the part of the radioactivity which has not been accounted for. 3.5. Relationship between biotransformation and excretion in the bile The presence of only small amounts of unchanged compounds in the bile raised the question of the extent to which biotransformations were essential for excretion in the rat bile. In an initial experimental approach, the excretion in the bile was studied for short periods and the bile samples were analyzed for free and conjugated products. The results are shown in fig. 4.
W.He@e, W.F.Kafoe, Biliory excretion of o~~e~drine
119
and tofe~acin in the rat
200 . 180
N ? 3 iit z
-
‘60 . 160 120 * 100 -
3 &yJ.
“0 80
2 LO . 20
1 minutes after odmmistrotion
Fig. 4. Analysis of bile samples for free and conjugated compounds, collected at successive 5 min intervals from bile-C&la rats intravenously dosed with 10 mg/kg of labelled tofenacin hydr~hlo~de (compo~d 2). Samples from 4 rats were pooled. (1) total amount in samples; (2) amount extractable with dichloroethane after pretreatment with p-glucuronidase/arylsulfatase; (3) amount extxacactabb with dichloroethane without pretreatment.
i
3
hwrs
4
5
of ter admwstration
Fig. 6. Time course of the excretion rate in bile after intravenous administration of 10 mg/kg of labelled tofenati hydrochloride (compound 2) to bile-fistula rats, without (o---o) and with proadifen pre~ea~ent (25 mg/kg, i.p., 30 miu before dosage with compound 2). Each point represents the mean value of 7 rats.
I
1
2
3
L
5
hours after acWinistrotion
Fig. 5. Time course of the excretion rate in bile after intravenous administration of 10 mg/kg of labelled orphenadrine hydrochloride (compound lc) to bilefstula rats, with” out (o---o) and with proadifen pretreatment (25 r&kg, i.p., 30 min before dosage with compound lc). Each point represents the mean value of 8 rats.
hours of tcr administration
Fig. 7. Time course of the excretion rate in bile after intravenous administration of 10 mg/kg of labelled tofenacin hydrochloride (compound 2) to bile-fist& rats, without (o---o) and with pretreatment with the same non-radioactive compound (25 mg/kg, i.p., 30 min before the dosage with compound 2). Each point represents the mean value of 4 rats.
120
W.Hespe, W.F.Kafoe, Biliary excretion o f orphenadrine and tofenacin in the rat
In another experiment the effect of inhibition of biotransformation on biliary excretion was determined. Pretreatment with proadifen *, an inhibitor of microsomal biotransformations, decreased biliary excretion of radioactivity of compounds lc and 2 in the first hour after administration (figs. 5 and 6). Statistical analysis of the data showed the proadifen effect to be highly significant. The higher biliary excretion of radioactivity in pretreated animals seen later, may partly be due to compensation for initial inhibition. It is possible that the inhibitor itself, or a metabolite, was eliminated in the bile, competing with the labelled compounds in biliary excretion. To elucidate this point the experiment was repeated, using rats pretreated with the non-labelled compound, the ideal agent in competition experiments with the labelled form. As illustrated in fig. 7 this pretreatment was devoid of any effect on biliary excretion of radioactivity.
radioactivity absorbed from the bile dose was reexcreted in the bile. After administration of compound lc per se, about 37% of the administered radioactivity was eliminated via the bile (table 2). The figures obtained demonstrate that enterohepatic circulation of radioactive material is efficient, which is not in conflict with the high faecal excretion of radioactivity (net outcome of a number of cycles). A disadvantage of the experiment is that orally administered bile must pass the stomach, whereas, normally, the bile is secreted into the duodenum and therefore not affected by the stomach. However, in experiments in which the bile of donor rats was injected directly into the duodenum of anesthetized acceptor rats excretion of radioactivity in the bile of the acceptor rats was also observed. Thus, the experiment only allowed an approximate calculation and showed that under such conditions part of the radioactivity in the bile dose was absorbed and, to some extent, re-excreted in the bile.
3.6. Enterohepatic circulation
Table 6 shows the results of an excretion experiment, in which bile from rats previously dosed with compound lc, was administered orally to acceptor rats. In several rats of the latter group the bile duct had been ligated. The figures show that reabsorption of radioactive products from the bile occurred. Since absorbed material is responsible for elimination in urine and respiratory air, the percentage absorption must be approximately 65% of the dose administered. Cholestasis in the acceptor rats increased urinary elimination by about 17%, indicating that part of the * SKF 525a, 2-(diethylamino)-2,2-diphenylvalerate.
4. DISCUSSION Two separate processes appear to be responsible for the large excretion of radioactivity from the test compounds into the bile: one governing the passage from the blood into the liver, at a concentration factor of over 100 (found by comparing concurrent liver and blood levels in figs. 2 and 3), and another comprising excretion from the liver into bile, at a concentration factor of more than 5 (table 3). The rapid uptake of this type of compound from the circulation was not specific for liver. Autoradiographic distribution studies have shown that practical-
Table 6 Excretion pattern of radioactivity in rats after oral administration of labelled orphenadrine hydrochloride (lc) or of bile collected from rats dosed with the same compound. Mean excretion as a % of the administered radioactivity Number of rats
Admin. product
Cholestasis
4 2 2
lc bile bile
+
urine (0-48 b_r)
faeces (0-48 hr)
14CO2 (0-7 hr)
total
32.8 47.4 64.6
34.3 40.5 35.7
22.2 2.3 1.3
89.3 90.2 101.6
I¢.Hespe, t¢.F.Kafoe, Biliary excretion o f orphenadrine and tofenacin in the rat
ly all organs concentrate the radioactivity in comparison to blood, several, such as the lungs and various glands, exceed the liver in concentrating capacity (Hespe et al., 1967, 1968; Prins and Hespe, 1968). Labelled substances were also concentrated in vitro by slices and homogenates of various organs; according to the literature, concentration of basic drugs in tissues appears to be mainly due to passive diffusion followed by reversible binding to tissue components (Yamamoto et al., 1968). That many organs concentrated compounds such as orphenadrine, implied that the process of biliary excretion was not necessary related to that of uptake in the liver. It is of interest that the high radioactivity level in the salivary glands of mice and rats did not lead to high excretion in the saliva; in contrast, saliva levels were correlated with blood levels (unpublished findings from our laboratory). The rather sharp decline in excretion rate, which occurred after the maximum had been reached was one of the peculiarities of the time course of biliary excretion (figs. 2-6). This was not simply due to the decrease in radioactivity in the liver, as may appear from table 3. It may be explained by postulating the occurrence of at least two pools for hepatic radioactivity as indicated below. plasma ~ liver1
liver2
bile Introduction of a second liver pool explains the initial short-lasting high excretion rate of biliary excretion; the rate of influx of radioactive material into this second pool is assumed to be relatively high. The subsequent time course will be determined by the rate at which the second pool is emptied, assuming it to have a relatively large capacity. This concept was supported by studies of the subcellular distribution of compounds lb and 2; the presence of at least one free and one bound pool for the radioactivity (unpublished data from our laboratory, provided by R.C. Roozemond) was then demonstrated. With respect to the second phase in the excretion process, i.e. from liver in the bile, the data obtained do not allow definite conclusions as to the mechanism involved. Our investigations suggest that biotransformation favours excretion of this type of compound in the rat bile. Metabolites predominated
121
even in bile collected over the first 5 min after administration (fig. 4). Therefore biotransformation of orphenadrine into these metabolites was rapid. This observation and the relation between the time course of free and conjugated products lend support to the hypothesis that biotransformation to hydrolphilic conjugates promotes excretion in the bile. Moreover, proadifen significantly depressed biliary excretion of radioactivity for about 1 hr (figs. 5 and 6). At first sight, this is in accord with the reported inhibitory effect exerted by this compound on various biotransformations, including glucuronic acid conjugation (Cooper et al., 1954). However, the period in which the effect of proadifen was observed was shorter than, for instance, the 24 hr reported by Rogers (1965) for its effect on drug metabolism in rats. An explanation of this discrepancy might be that under normal circumstances, a certain degree of inhibition of essential biotransformations was required for the decrease in biliary excretion to become manifest. Rogers (1965) observed that the maximum effect of proadifen on different drug biotransformations in male rats occurred within one hour after i.p. administration. Our measurements gave similar resuits. The argument that in our experiments, proadifen competed with the labelled substance in biliary excretion is particularly applicable in that the maximum effect occurred during optimal excretion. However, no competition was observed in experiments with unlabelled substances in a dose equal to that of proadifen (fig. 7). As to the types of biotransformation suggested by our findings, aromatic hydroxylation, followed by conjugation with glucuronic acid and probably sulfuric acid seems to play a major role, while N-glucuronides and ester glucuronides might also be involved. Only minor amounts of the unchanged compounds and N-demethyl derivatives were found. A relatively large part of the radioactivity in the bile cannot be accounted for. Biotransformations favouring biliary excretion seem to share a combination of acidic properties due to pertaining conjugations, to the basic properties of the original compound. Moreover, these biotransformations meet the proposed structural requirements claimed by Williams et al. (1964), Smith (1966), and Millburn (1967) to enhance biliary excretion. These authors state that substances appearing in the bile usually do so in the form of highly polar
122
l¢.Hespe, W.F. Kafoe, Biliary excretion o f orphenadrine and tofenacin in the rat
molecules, while polarity as well as an increase in molecular size is frequently associated with enhanced affinity for hepatic excretion. Speculations on the mechanism c o n c e m e d should also consider the possibility o f passive diffusion. In dialysis experiments, radioactive orphenadrine was concentrated in the bile-containing c o m p a r t m e n t showing that bile constituents bind the drug (unpublished results). It is therefore conceivable that biliary excretion, o f at least the unchanged c o m p o u n d , is due to passive diffusion. Further, we have strong evidence for the enterohepatic circulation o f the test compounds. It should be noted that in rats orally dosed with bile collected from rats treated with c o m p o u n d l c , the respiratory elimination pattern was markedly different from the control pattern: a decrease from 22.2 to 2.3% (table 6) o f the administered radioactivity. This dramatic change is apparently not only related to the decline in absorption from the bile compared to the normal dosage form, b u t also to the more hydrophilic nature of material, probably chiefly consisting o f hydroxylated derivatives, liberated from their conjugates b y hydrolysing enzymes o f the intestinal flora. These hydrophilic products are less likely to penetrate into the microsomal system o f the liver than the original compound. F u r t h e r studies ar in progress to obtain more information about the biliary excretion o f the investigated and structurally related compounds, particular attention is being paid to the consequences o f this elimination with respect to activity and toxicity.
REFERENCES Besten, W. den, D. Mulder, A.B.H. Funcke and W.Th. Nauta, 1970, Synthesis and pharmacology of a number of potential orphenadrine metabolites, Arzneimittel-Forsch. 20, 538. Cooper, J.R., J. Axelrod and B.B. Brodie, 1954, Inhibitory effects of/~-diethylaminoethyl diphenylpropylacetate on a variety of drug metabolic pathways t'n vitro, J. Pharmacol. Exptl. Therap. 112, 55. Dill, W.A. and A.J. Glazko, 1949, Biochemical studies on a
diphenhydramine (Benadryl), I: Chemical determination of diphenhydramine, J. Biol. Chem. 179, 395. Drach, J.C. and J.P. Howell, 1968, Identification of diphenhydramine urinary metabolites in the rhesus monkey, Biochem. Pharmacol. 17, 2125. Hespe, W., A.M. de Roos and W.Th. Nauta, 1965a, Investigation into the metabolic fate of orphenadrine hydrochlofide after oral adminstration to male rats, Arch. Intern. Pharmacodyn. 156,180. Hespe, W., W.J.F. Klopper and W.Th. Nauta, 1965b, The synthesis of [6,7-T]-tropine, Rec. Tray. Chim. 84,476. Hespe, W. and W.Th. Nauta, 1966, The labelling with tritium of 2-(dimethylamino)ethanol and of some drugs containing the 2-(dimethylamino)ethylgroup as a structural part, J. Labelled Compounds 2, 193. Hespe, W., H. Prins, W.F. Kafoe and W.Th. Nauta, 1967, The distribution and metabolic fate of xyloxemine hydrochloride in mice and rats, Arch. Intern. Pharmacodyn. 170, 397. Hespe, W., H. Prins, W.F. Kafoe and W.Th. Nauta, 1968, Investigations into the metabolic fate and distribution of hepzidine maleate in the rat and the mouse, Biochem. Pharmacol. 17,655. Jeffay, H. and J. Alvarez, 1961, Liquid scintillation counting of carbon-14, use of ethanolamine-ethylene glycol monomethyl ether-toluene, Anal. Chem. 33, 612. Mahin, D.T. and R.T. Lofberg, 1966, A simplified method of sample preparation for determination of tritium, carbon-14, or sulfur-35 in blood or tissue by liquid scintillation counting, Anal. Biochem. 16, 500. Millburn, P., R.L. Smith and R.T. Williams, 1967, Biliary excretion of foreign compounds, biphenyl, stilboestrol and phenolphtalein in the rat: molecular weight, polarity and metabolism as factors in biliary excretion, Biochem. J. 105, 1275. Prins, H. and W. Hespe, 1968, Autoradiographic study of the distribution of radioactivity in mice after oral administration of tritinm-labelled orphenadrine hydrochloride, Arch. Intern. Pharmacodyn. 171, 47. Rogers, L.A., 1965, Certain factors which affect the rate of hepatic drug metabolism, Academical thesis, State Univ. of Iowa, Univ. Microfilms, Inc., Ann Arbor, Michigan, USA. Smith, R.L., 1966, in: Progress in Drug Research, Vol 9, ed. E. Jucker (Birkh~iuser, Basel). Williams, R.T., R.L. Smith and P. MiUburn, 1964, in: Therapeutic Agents and the Liver, eds. N. Mclntyre and S. Sherlock (Blackwell, Oxford). Yamamoto, I., R. Inoki and K. Iwatsubo, 1968, Penetration of nicotine- 14C into several rat tissues in vivo and in vitro, Toxicol. Appl. Pharmacol. 12, 560.
ASPECTS OF THE BILIARY EXCRETION OF ORPHENADRINE AND ITS N-DE~~YLATED DE~VATI~, TOFENA~IN, IN THE RAT W. HESPE and W.F. KAFOE Research Department N. V. Koninklijke Phannaceutische Fabrieken v/h Br~ades-Sthee~n en Pharmacia, ~oie~~a~ht 27-39, Amsterdam, Ne~~ands
Accepted 29 June 1970
Received 8 April 1970
W, HESPE and W.F. KAFOE, Aspects of the bilia~ excretion ofo~h~ad~e and its N~emethy~ated derivative, tofenacin, in the rat, European J. Pharmacol. 13 (1970) 113-122, A study was made of the excretion of radioactivity in bile of male rats after administration of orphenadrme hydrochloride and tofenachr hydrochloride in labelled forms. In bile fistula rats it was found, particularly after intravenous administration, that biliary excretion was considerable: after five hours 31 to 48% of the radioactivity had been excreted in the bile. In normal rats, total bibary eMination exceeded urinary elimination. A study of the radioactivity in the blood, liver and bile showed two phases of concentration: first, from blood to liver, more than a lOO-fold concen~ation was achieved and second from liver to bile, the concentration increased more than 5 times. When 10 mg/lcg doses of the labelled compounds were administered iv, the approximate points of thne at whkh maximum concentrations were found in liver and bile were 10 and 20 min after administration, while in the blood maximum concentration was reached immediately after the injection. A compartment model is proposed to explain various aspects of the time course of biliary excretion of these drugs in bile fistula rats. Analytical investigations of orphenadrine metabolites in rat bile showed that only minor amounts of the compound were excreted unchanged or in the form of free (nonconjugated) metabolites; evidence was obtained for major excretion in the form of ~ucuro~&s and probably sulfates of aromatic hydroxy~ted o~hen~e metabolites. The bio~~~ormations promoted excretion in the bile, as evidenced by the bile collected in the first fwe minutes after i.v. dosage; in that period conjugates were already dominant and pretreatment with the metabolic inhibitor proadifen produced a significant decrease in biliary excretion of radioactivity over 1 hr after administration - an effect which could not be related to competition. There is strong evidence that compounds of the type studied undergo enterohepatic circulation. Grphenadrine Tofenacin
Proadifen Biliary excretion, rat
1. INTRODUCTION Previous ~vest~tions into the metabolism of labelled o~henad~ne hydroc~o~de in the rat showed that a large amount of the orally administered radioactivity - generally more than 50% of the dose - was eliminated in the faeces (Hespe et al., 1965a). The substance was originally thought to be poorly absorbed, but further expe~ments, inclu~ng some with bile %&la rats, suggested a very consider-
Enterohepatic circufation Glucuronic acid conju~tion
able elimination of radioactivity through the bile. Similar excretion patterns (predominant elimination by the faecal route and considerable biliary excretion) have been demonstrated in the rat for a number of related bi- and tricyclic compounds with a basic side chain (Hespe et al., 1966; 1967; 1968). Since the time course of radioactivity in the rat gastrointestinal tract indicated good absorption after oral administration, it was obvious that the excretion in the faeces was largely due to the high biliary excretion of the
W.Hespe, W.F.Kafoe, Biliary excretion of orphenadrine and tofenacin in the rat
114
compounds in some form. In metabolic studies of various compounds in the rat we collected information on biliary excretion, part of our findings, particularly those on orphenadrine and tofenacin, are presented here. The following points are discussed: (1) quantitative aspects of biliary elimination and its time course; (2) relations between the concentrations in blood, liver and bile at different points of measurement after administration; (3) the form in which the compounds are excreted in the bile; (4) the relationship between biotransformation and excretion in the bile.
2. METHODS
2.1. Compounds investigated Labelled forms of orphenadrine and tofenacin were used, as indicated in fig. 1. The substances are referred to by the numbers given in this figure. These radioactive compounds were prepared in our laboratories;: part of the synthetic work has been published (Hespe et al., 1965b; Hespe and Nauta, 1966). 2.2. Animal studies In all experiments male albino rats (TNO-Wu strain) of 150-250 g were used. Quantitatively, the biliary elimination of radioactive material was studied by two methods, A and B.
. I.lCt
•- . e
.I.ICL
lc
.HCL
H
.HCI.
Fig. 1. Structural formulae of the compounds investigated. Labelled forms of orphenadrine hydrochloride (la, b, c) and tofenacin hydrochloride (2).
A) Rats, fasted for about 20 hr, were anesthetized with phenobarbital sodium (100 mg/kg, i.p.). Bile was continuously collected from a polyethylene cannula inserted into the common bile duct for periods up to 5 hr after administration of the labelled compounds and assayed for radioactivity. B) In order to gain an insight into the amount of radioactive material eliminated through the bile under normal conditions, the elimination pattern of radioactivity in normal rats, was compared with that in rats in which cholestasis had been surgically induced. Urine, faeces and expiratory carbon dioxide were separately collected in metabolic cages. The increase in elimination by the unobstructed routes which resulted from the cholestasis was taken as a measure of the biliary elimination. The excretion experiments were, as a rule, started one day after operation. The time course of the radioactivity in the blood was recorded continuously by passing the blood, through a loop in the carotid artery into a counting unit as described in the next section. The compounds were injected through a cannula inserted into the femoral vein. The animals used in these experiments were anesthetized with phenobarbital sodium (I00 mg/kg, i.p.) and pretreated with heparin (500 I.U., Vitrum, Stockholm).
2.3. Radioactivity measurements The radioactivity of the different excretion products was determined according to procedures described previously (Hespe et al., 1967). In order to determine the radioactivity in liver tissue, samples of about 50 mg were treated directly in counting vials (Mahin and Lofberg, 1966). In earlier experiments a Packard Tri Carb Scintillation Spectrometer 314 EX was used and quench corrections were made by the internal standard method. Subsequently a Packard Tri Carb Scintillation Spectrometer 3375 was used and quench corrections were performed by the external standard method. The time course of radioactivity in the blood was continuously followed by passing the blood through a circuit of plastic scintillating tubing (Nuclear Enterprise NE 102A tubing), placed in a light-tight housing between two photomultipliers (PhilipsPW 4210) connected to additional counting equipment. This method gave good results for the 14C-labelled compounds but its sensitivity was too low for all-labelled substances.
W.Hespe,W.F.Kafoe,Bilinry excretion of orphenadrine and tofenacin in the rat
115
Table 1 Analytical data on orphenadrine and some potential metabolites. Chromatographic assay (Rf value range) orphenadrine Ndemethyl orphenadrine N,N-didemethyl orphenadrine 5-OH orphenadrine 4’-OH orphenadrine 2CHzOH diphenhydramine 2-COOH diphenhydramine
+ denotes that calorimetric determination
Colorim etric assay
0.26-0.34 0.36-0.42 0.45-0.52 0.58-0.66 0.63-0.70 0.60-0.67 0.85-0.95
+ + + + + +
of the compound is possible (methyl orange).
2.4. Analytical procedures
Extraction procedures in combination with alkaline and enzymatic hydrolysis @-glucuronidase and arylsulfatase) and thin-layer chromatography (TLC) were the main analytical methods employed to study the radioactive products in bile. Potential orphenadrine metabolites, synthesized by Den Besten et al. (1970) were used to develop a reversed phase TLC method that proved superior to any other method tested. Typical Rf values found on olive oil-impregnated silica gel, by means of ethanol-25% ammonia as a solvent system, are given in table 1. Although not all compounds were separated completely this TLC technique provided a useful separation into the following groups: Rf range I
0.00-0.50, orphenadrine and N-demethylated analogues; Rf range II 0.50-0.70, hydroxylated metabolites; Rf range III 0.70-0.95,2-carboxydiphenhydramine.
Another method used to analyse the bile was that described by Dill and Glazko (1949), as modified by Hespe et al. (1965a). This method, based on double extraction of the biological material followed by calorimetric assay of the complex formed by the N-bases with methyl orange, was used to determine the amounts of the unchanged compounds and their N-demethylated derivatives. However, since unidentified metabolites may be involved, the analytical results were expressed in terms of methyl orange-positive
compounds. The potential orphenadrine metabolites synthesized have been classified accordingly (table 1).
3. RESULTS 3 .l _Quantitative biliary excretion data Two types of experiments, indicated by A and B, were used to elucidate the quantitative importance of the biliary elimination of the labelled compounds in the rat (table 2).
Table 2 Biliary excretion of radioactivity in bile fistula rats over a period of 5 hr after dosage (type A exp.) and in normal rats (type B exp.) after i.v. administration of orphenadrine and tofenacine in labelled forms. Biliary elimination in % of the administered radioactivity (? S.D.) Labelled compound
Type A exp. Type B exp.
la lb lc 2
oral
i.v.
12.5+ 6.4 (2) 16.2f 8.7 (3) 14.6+ 6.7 (5) -
31.3f3.1 (2) 44.1S.l (8) 48.4k5.3 (7)
42.3+ 9.9 36.1k19.0 36.9+ 9.1 42.2+ 7.5
(2) (4) (4) (4)
W.Hespe, W.F.Kafoe, Biliary excretion of orphenadrine and tofenacin in the rat
116
3.2. Time course o f the radioactivity level in blood and bile In experiments with compounds 2 and lc the time course of radioactivity in blood and bile was determined simultaneously (figs. 2 and 3). Initially the bile samples were collected at 5 min, and then at 15 min intervals. The course of the radioactivity in blood was monitored continuously by means of a counting unit (see section 2.3). This was useful in cases where blood sampling at short intervals presented difficulties. The time course of the biliary radioactivity was not only characteristic for the test compounds, but was also observed for a number of structurally related compounds: a rather sharp maximum invariably occurring
"~ 10
180 o
o 0~
Its0
0811\1111 1-1
I~z°~.
°'tllll1"5 lllJ Jo
~s
so
r
"is 9o Ibs
minutes ofter odministrotion
Fig. 2. Time course of the concentration of radioactivity in bile (histogram) and blood of a rat dosed with 10 mg/kg of labelled tofenacin hydrochloride (compound 2), intravenously.
150
10 119
120
08
~o.6
9O o. ~3
as "~ 0.t, 03 02 01 1"5
30
/.5
60 75 90 105 minutes ofter odmlnistrotion
Fig. 3. Time course of the concentration of radioactivity in bile (histogram) and blood of a rat dosed with 10 mg/kg of labelled orphenadrine hydrochloride (compound lc), intravenously.
in the second 15 min period, followed by a prolonged but less marked excretion. The maximal radioactivity level in blood was noted about 15 min prior to that in bile.
3.3. Time course o f the radioactivity level in liver and bile The relation between the course of radioactivity in liver and bile has been studied only for compound 2 (table 3). The maximum concentration of radioactivity in liver was observed 1 0 m in and in the bile 15-20 min after administration. Within 5 min, the concentration of radioactive material in the bile exceeded that in the liver. The ratio between bile and liver levels (table 3) attained a maximum at 20 min. 3.4. Structure o f the products excreted in the bile That orphenadrine and tofenacin were largely excreted in bile as metabolites was easily demonstrated since after extraction with n-heptane of alkalified bile from animals dosed with the labelled compounds, nearly all the radioactivity remained in the aqueous phase. In experiments in which the radioactive compounds were added to rat bile in vitro, about 98% of the radioactivity was extracted into the organic phase. In a more systematic study of these hydrophilic metabolites, two identical samples of pooled bile from rats dosed with compound la were subjected to a series of successive extractions (table 4). One sample underwent various treatments between the extractions, as indicated. It was demonstrated that there are radioactive orphenadrine metabolites in the bile which are thermolabile and/or sensitive to alkaline hydrolysis. An even more striking feature was the presence of metabolites sensitive to/3-glucuronidase/arylsulfatase treatment. After the whole series of extractions has been completed, the biliary residue of both portions was found to contain far less radioactivity than suggested by the amounts extracted. The main cause of this loss, which amounted to 50% of the radioactivity originally present, appeared to be the formation of a foamy interphase in the liquid system. Random foam samples were found to be highly radioactive. Using the analytical methods described in 2.4. a study was carried out into the structures of the various radioactive products in the bite of rats dosed with compound lb (table 5). Without pretreatment
117
W.Ifespe, W.F. Kafoe. Biliary excretion of orphenadrine and tofenacin in the rat
Table 3 Concentration of radioactivity in m/g in a) the liver of rats sacrificed at different points of time after intravenous administration of 10 mg/kg of labelled tofenacin hydrochloride (compound 2), in b) the bile of the same animals collected for 5 mm before death. Bile
Liver
Rat
Period of collection (min after adm.)
Concentration (cleslg)
Min after administration
Mean concentration (ue41g)
1 2 3 4 5 6 7 8 9 10 11 12
o- 5 5-10 10-15 15-20 20-25 25-30 30-35 35 -40 40-45 45-50 50-55 55-60
21.5 105.4 157.8 201.9 118.3 68.5 77.7 101.7 67.9 47.9 40.4 48.4
5 10 15 20 25 30 35 40 45 50 55 60
17.5 37.2 29.4 28.0 25.9 19.9 17.9 24.4 21.3 20.0 18.5 19.1
Ratio concentration bile/liver
1.57 2.83 5.37 7.21 4.57 3.44 2.16 4.17 3.19 2.40 2.18 2.53
Table 4 Yield of successive extractions of two identical samples of pooled bile, collected over a period of 7 hr after administration of an oral 10 mg/kg dose of labelled orphenadrine hydrochloride (compound la) to rats. One sample was subjected to different treatments prior to each extraction.
Extraction (dichloroethane)
pH on extraction
Extracted radioactivity as a % of the original biliary radioactivity
Successive pretreatments of portion 2
Portion 1
Portion 2
1
12
no pretreatment
2
12
3hrat100°C,pH
3
12
6hrat100°C,pH12
4
12
24 hr incubation with &glucuronidase pH 5 .O
0.7
12.0
5
12
24 hr incubation with p-glucuronidase/arylsulfatase, pH 6.2
0.4
7.0
6
10
no further treatment
0.4
0.9
7
8
no further treatment
0.4
0.1
8
6
no further treatment
0.2
0.1
9
4
no further treatment
0.1
0.2
10
2
no further treatment
0.1
0.1
6.2
29.0
12
Total
1.5
1.6
1.8
4.0
0.6
3.0
118
h/.Hespe, l¢.F.Kafoe, Biliary excretion o f orphenadrine and tofenacin in the rat
Table 5 Analysis of samples of pooled bile (4 ml), collected for 7 hr after administration of an oral 10 mg/kg dose of labelled orphenadrine hydrochloride (compound lb) to rats. Radioactivity as a % of original radioactivity in the bile sample Treatment of separate bile samples
Extracted with dichloroethane at pH = 10
Colorimetric assay of extract
Chromatographic assay of extract Products in Rf range I II III
1) No treatment
1.4
1.4
0.0
0.0
0.0
2) 6 hr at 100°C, pH 12
7.9
1.5
0.0
1.0
6.9
3) 24 hr incubation with &glucuronidase/arylsulfatase, pH 5.0
29.8
19.8
6.8
15.8
7.6
4) treatment (2), followed by (3)
41.3
24.9
7.4
20.8
13.1
hardly any radioactivity could be extracted from the bile and that which was extracted was methyl-orange positive as shown by colorimetric assay of the extract. However, the amount was too small for chromatographic identification. An alkaline hydrolysis procedure liberated products in the Rf ranges II and, especially III. The good agreement between the percentages in Rf range II and in the colorimetric assay of the same extract suggests that the products in Rf range II were methyl-orange positive and those in Rf range III methyl-orange negative. The only potential orphenadrine metabolite synthesized with an Rf value in the latter range was 2-carboxydiphenhydramine; in the form of an ester glucuronide it would certainly be liable to alkaline hydrolysis. Moreover, this product is methyl-orange negative. Recently, in metabolic studies with diphenhydramine in rhesus monkeys, (diphenylmethoxy)acetic acid was reported to be a metabolite, partly conjugated with glutamine (Drach and Howell, 1968). An analogous orphenadrine metabolite, although not synthesized, should be considered an alternative for the unknown product in Rf range III. Enzymatic treatment liberated products in all three Rf ranges. There is reasonable agreement between the percentages of methyl-orange positive compounds and the sum of the percentages in Rf ranges I and II, suggesting that the radioactivity in these Rf ranges may largely originate from methyl-orange positive compounds. Rf range I includes the unchanged
compound and its N-demethylated derivatives. None of the remaining potential metabolites was localized in this range. A marked increase in radioactivity in this range after enzymatic treatment may be related to the presence of N-glucuronides of the N-demethylated products, especially that of N-demethyl olphenadrine. Rf range II includes those orphenadrine metabolites which contain an aromatic hydroxyl group, the pronounced effect of enzymatic treatment suggesting strongly involvement of hydroxylated derivatives of orphenadrine, which are conjugated with glucuronic acid and probably sulfuric acid. The effect of enzymatic treatment on the radioactivity in Rf range III might be related to the presence of an ester glucuronide of the above acids. Further analytical work is in progress to determine more exactly the nature of the different products involved and of the part of the radioactivity which has not been accounted for. 3.5. Relationship between biotransformation and excretion in the bile The presence of only small amounts of unchanged compounds in the bile raised the question of the extent to which biotransformations were essential for excretion in the rat bile. In an initial experimental approach, the excretion in the bile was studied for short periods and the bile samples were analyzed for free and conjugated products. The results are shown in fig. 4.
W.He@e, W.F.Kafoe, Biliory excretion of o~~e~drine
119
and tofe~acin in the rat
200 . 180
N ? 3 iit z
-
‘60 . 160 120 * 100 -
3 &yJ.
“0 80
2 LO . 20
1 minutes after odmmistrotion
Fig. 4. Analysis of bile samples for free and conjugated compounds, collected at successive 5 min intervals from bile-C&la rats intravenously dosed with 10 mg/kg of labelled tofenacin hydr~hlo~de (compo~d 2). Samples from 4 rats were pooled. (1) total amount in samples; (2) amount extractable with dichloroethane after pretreatment with p-glucuronidase/arylsulfatase; (3) amount extxacactabb with dichloroethane without pretreatment.
i
3
hwrs
4
5
of ter admwstration
Fig. 6. Time course of the excretion rate in bile after intravenous administration of 10 mg/kg of labelled tofenati hydrochloride (compound 2) to bile-fistula rats, without (o---o) and with proadifen pre~ea~ent (25 mg/kg, i.p., 30 miu before dosage with compound 2). Each point represents the mean value of 7 rats.
I
1
2
3
L
5
hours after acWinistrotion
Fig. 5. Time course of the excretion rate in bile after intravenous administration of 10 mg/kg of labelled orphenadrine hydrochloride (compound lc) to bilefstula rats, with” out (o---o) and with proadifen pretreatment (25 r&kg, i.p., 30 min before dosage with compound lc). Each point represents the mean value of 8 rats.
hours of tcr administration
Fig. 7. Time course of the excretion rate in bile after intravenous administration of 10 mg/kg of labelled tofenacin hydrochloride (compound 2) to bile-fist& rats, without (o---o) and with pretreatment with the same non-radioactive compound (25 mg/kg, i.p., 30 min before the dosage with compound 2). Each point represents the mean value of 4 rats.
120
W.Hespe, W.F.Kafoe, Biliary excretion o f orphenadrine and tofenacin in the rat
In another experiment the effect of inhibition of biotransformation on biliary excretion was determined. Pretreatment with proadifen *, an inhibitor of microsomal biotransformations, decreased biliary excretion of radioactivity of compounds lc and 2 in the first hour after administration (figs. 5 and 6). Statistical analysis of the data showed the proadifen effect to be highly significant. The higher biliary excretion of radioactivity in pretreated animals seen later, may partly be due to compensation for initial inhibition. It is possible that the inhibitor itself, or a metabolite, was eliminated in the bile, competing with the labelled compounds in biliary excretion. To elucidate this point the experiment was repeated, using rats pretreated with the non-labelled compound, the ideal agent in competition experiments with the labelled form. As illustrated in fig. 7 this pretreatment was devoid of any effect on biliary excretion of radioactivity.
radioactivity absorbed from the bile dose was reexcreted in the bile. After administration of compound lc per se, about 37% of the administered radioactivity was eliminated via the bile (table 2). The figures obtained demonstrate that enterohepatic circulation of radioactive material is efficient, which is not in conflict with the high faecal excretion of radioactivity (net outcome of a number of cycles). A disadvantage of the experiment is that orally administered bile must pass the stomach, whereas, normally, the bile is secreted into the duodenum and therefore not affected by the stomach. However, in experiments in which the bile of donor rats was injected directly into the duodenum of anesthetized acceptor rats excretion of radioactivity in the bile of the acceptor rats was also observed. Thus, the experiment only allowed an approximate calculation and showed that under such conditions part of the radioactivity in the bile dose was absorbed and, to some extent, re-excreted in the bile.
3.6. Enterohepatic circulation
Table 6 shows the results of an excretion experiment, in which bile from rats previously dosed with compound lc, was administered orally to acceptor rats. In several rats of the latter group the bile duct had been ligated. The figures show that reabsorption of radioactive products from the bile occurred. Since absorbed material is responsible for elimination in urine and respiratory air, the percentage absorption must be approximately 65% of the dose administered. Cholestasis in the acceptor rats increased urinary elimination by about 17%, indicating that part of the * SKF 525a, 2-(diethylamino)-2,2-diphenylvalerate.
4. DISCUSSION Two separate processes appear to be responsible for the large excretion of radioactivity from the test compounds into the bile: one governing the passage from the blood into the liver, at a concentration factor of over 100 (found by comparing concurrent liver and blood levels in figs. 2 and 3), and another comprising excretion from the liver into bile, at a concentration factor of more than 5 (table 3). The rapid uptake of this type of compound from the circulation was not specific for liver. Autoradiographic distribution studies have shown that practical-
Table 6 Excretion pattern of radioactivity in rats after oral administration of labelled orphenadrine hydrochloride (lc) or of bile collected from rats dosed with the same compound. Mean excretion as a % of the administered radioactivity Number of rats
Admin. product
Cholestasis
4 2 2
lc bile bile
+
urine (0-48 b_r)
faeces (0-48 hr)
14CO2 (0-7 hr)
total
32.8 47.4 64.6
34.3 40.5 35.7
22.2 2.3 1.3
89.3 90.2 101.6
I¢.Hespe, t¢.F.Kafoe, Biliary excretion o f orphenadrine and tofenacin in the rat
ly all organs concentrate the radioactivity in comparison to blood, several, such as the lungs and various glands, exceed the liver in concentrating capacity (Hespe et al., 1967, 1968; Prins and Hespe, 1968). Labelled substances were also concentrated in vitro by slices and homogenates of various organs; according to the literature, concentration of basic drugs in tissues appears to be mainly due to passive diffusion followed by reversible binding to tissue components (Yamamoto et al., 1968). That many organs concentrated compounds such as orphenadrine, implied that the process of biliary excretion was not necessary related to that of uptake in the liver. It is of interest that the high radioactivity level in the salivary glands of mice and rats did not lead to high excretion in the saliva; in contrast, saliva levels were correlated with blood levels (unpublished findings from our laboratory). The rather sharp decline in excretion rate, which occurred after the maximum had been reached was one of the peculiarities of the time course of biliary excretion (figs. 2-6). This was not simply due to the decrease in radioactivity in the liver, as may appear from table 3. It may be explained by postulating the occurrence of at least two pools for hepatic radioactivity as indicated below. plasma ~ liver1
liver2
bile Introduction of a second liver pool explains the initial short-lasting high excretion rate of biliary excretion; the rate of influx of radioactive material into this second pool is assumed to be relatively high. The subsequent time course will be determined by the rate at which the second pool is emptied, assuming it to have a relatively large capacity. This concept was supported by studies of the subcellular distribution of compounds lb and 2; the presence of at least one free and one bound pool for the radioactivity (unpublished data from our laboratory, provided by R.C. Roozemond) was then demonstrated. With respect to the second phase in the excretion process, i.e. from liver in the bile, the data obtained do not allow definite conclusions as to the mechanism involved. Our investigations suggest that biotransformation favours excretion of this type of compound in the rat bile. Metabolites predominated
121
even in bile collected over the first 5 min after administration (fig. 4). Therefore biotransformation of orphenadrine into these metabolites was rapid. This observation and the relation between the time course of free and conjugated products lend support to the hypothesis that biotransformation to hydrolphilic conjugates promotes excretion in the bile. Moreover, proadifen significantly depressed biliary excretion of radioactivity for about 1 hr (figs. 5 and 6). At first sight, this is in accord with the reported inhibitory effect exerted by this compound on various biotransformations, including glucuronic acid conjugation (Cooper et al., 1954). However, the period in which the effect of proadifen was observed was shorter than, for instance, the 24 hr reported by Rogers (1965) for its effect on drug metabolism in rats. An explanation of this discrepancy might be that under normal circumstances, a certain degree of inhibition of essential biotransformations was required for the decrease in biliary excretion to become manifest. Rogers (1965) observed that the maximum effect of proadifen on different drug biotransformations in male rats occurred within one hour after i.p. administration. Our measurements gave similar resuits. The argument that in our experiments, proadifen competed with the labelled substance in biliary excretion is particularly applicable in that the maximum effect occurred during optimal excretion. However, no competition was observed in experiments with unlabelled substances in a dose equal to that of proadifen (fig. 7). As to the types of biotransformation suggested by our findings, aromatic hydroxylation, followed by conjugation with glucuronic acid and probably sulfuric acid seems to play a major role, while N-glucuronides and ester glucuronides might also be involved. Only minor amounts of the unchanged compounds and N-demethyl derivatives were found. A relatively large part of the radioactivity in the bile cannot be accounted for. Biotransformations favouring biliary excretion seem to share a combination of acidic properties due to pertaining conjugations, to the basic properties of the original compound. Moreover, these biotransformations meet the proposed structural requirements claimed by Williams et al. (1964), Smith (1966), and Millburn (1967) to enhance biliary excretion. These authors state that substances appearing in the bile usually do so in the form of highly polar
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l¢.Hespe, W.F. Kafoe, Biliary excretion o f orphenadrine and tofenacin in the rat
molecules, while polarity as well as an increase in molecular size is frequently associated with enhanced affinity for hepatic excretion. Speculations on the mechanism c o n c e m e d should also consider the possibility o f passive diffusion. In dialysis experiments, radioactive orphenadrine was concentrated in the bile-containing c o m p a r t m e n t showing that bile constituents bind the drug (unpublished results). It is therefore conceivable that biliary excretion, o f at least the unchanged c o m p o u n d , is due to passive diffusion. Further, we have strong evidence for the enterohepatic circulation o f the test compounds. It should be noted that in rats orally dosed with bile collected from rats treated with c o m p o u n d l c , the respiratory elimination pattern was markedly different from the control pattern: a decrease from 22.2 to 2.3% (table 6) o f the administered radioactivity. This dramatic change is apparently not only related to the decline in absorption from the bile compared to the normal dosage form, b u t also to the more hydrophilic nature of material, probably chiefly consisting o f hydroxylated derivatives, liberated from their conjugates b y hydrolysing enzymes o f the intestinal flora. These hydrophilic products are less likely to penetrate into the microsomal system o f the liver than the original compound. F u r t h e r studies ar in progress to obtain more information about the biliary excretion o f the investigated and structurally related compounds, particular attention is being paid to the consequences o f this elimination with respect to activity and toxicity.
REFERENCES Besten, W. den, D. Mulder, A.B.H. Funcke and W.Th. Nauta, 1970, Synthesis and pharmacology of a number of potential orphenadrine metabolites, Arzneimittel-Forsch. 20, 538. Cooper, J.R., J. Axelrod and B.B. Brodie, 1954, Inhibitory effects of/~-diethylaminoethyl diphenylpropylacetate on a variety of drug metabolic pathways t'n vitro, J. Pharmacol. Exptl. Therap. 112, 55. Dill, W.A. and A.J. Glazko, 1949, Biochemical studies on a
diphenhydramine (Benadryl), I: Chemical determination of diphenhydramine, J. Biol. Chem. 179, 395. Drach, J.C. and J.P. Howell, 1968, Identification of diphenhydramine urinary metabolites in the rhesus monkey, Biochem. Pharmacol. 17, 2125. Hespe, W., A.M. de Roos and W.Th. Nauta, 1965a, Investigation into the metabolic fate of orphenadrine hydrochlofide after oral adminstration to male rats, Arch. Intern. Pharmacodyn. 156,180. Hespe, W., W.J.F. Klopper and W.Th. Nauta, 1965b, The synthesis of [6,7-T]-tropine, Rec. Tray. Chim. 84,476. Hespe, W. and W.Th. Nauta, 1966, The labelling with tritium of 2-(dimethylamino)ethanol and of some drugs containing the 2-(dimethylamino)ethylgroup as a structural part, J. Labelled Compounds 2, 193. Hespe, W., H. Prins, W.F. Kafoe and W.Th. Nauta, 1967, The distribution and metabolic fate of xyloxemine hydrochloride in mice and rats, Arch. Intern. Pharmacodyn. 170, 397. Hespe, W., H. Prins, W.F. Kafoe and W.Th. Nauta, 1968, Investigations into the metabolic fate and distribution of hepzidine maleate in the rat and the mouse, Biochem. Pharmacol. 17,655. Jeffay, H. and J. Alvarez, 1961, Liquid scintillation counting of carbon-14, use of ethanolamine-ethylene glycol monomethyl ether-toluene, Anal. Chem. 33, 612. Mahin, D.T. and R.T. Lofberg, 1966, A simplified method of sample preparation for determination of tritium, carbon-14, or sulfur-35 in blood or tissue by liquid scintillation counting, Anal. Biochem. 16, 500. Millburn, P., R.L. Smith and R.T. Williams, 1967, Biliary excretion of foreign compounds, biphenyl, stilboestrol and phenolphtalein in the rat: molecular weight, polarity and metabolism as factors in biliary excretion, Biochem. J. 105, 1275. Prins, H. and W. Hespe, 1968, Autoradiographic study of the distribution of radioactivity in mice after oral administration of tritinm-labelled orphenadrine hydrochloride, Arch. Intern. Pharmacodyn. 171, 47. Rogers, L.A., 1965, Certain factors which affect the rate of hepatic drug metabolism, Academical thesis, State Univ. of Iowa, Univ. Microfilms, Inc., Ann Arbor, Michigan, USA. Smith, R.L., 1966, in: Progress in Drug Research, Vol 9, ed. E. Jucker (Birkh~iuser, Basel). Williams, R.T., R.L. Smith and P. MiUburn, 1964, in: Therapeutic Agents and the Liver, eds. N. Mclntyre and S. Sherlock (Blackwell, Oxford). Yamamoto, I., R. Inoki and K. Iwatsubo, 1968, Penetration of nicotine- 14C into several rat tissues in vivo and in vitro, Toxicol. Appl. Pharmacol. 12, 560.