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Loperamide in rat intestines: A unique disposition
Life Sciences, Vol. 30, pp. 2203-2206 Printed in the U.S.A.
Pergamon Press
LOPERAMIDE IN RAT INTESTINES: A UNIQUE DISPOSITION H.Miyazaki, K.Nambu and M.Hashimoto Research Laboratories, Dainippon Pharmaceutical Co., Ltd. Enoki 33-94, Suita, Osaka, Japan (Received in final form April 12, 1982)
Summary When everted sacs of rat duodenum, jejunum and ileum were incubated with [14C]loperamide in v i t r o , unchanged drug and its metabolites were found not only in tissues but also in media of the mucosal side with v i r t u a l l y no radioactivity in media of the serosal side. The amounts of metabolites fnund in media of the mucosal side were comparable to or larger than those in tissues. Di-desmethyl loperamide was more predominant in the media as compared with mono-demethylatedone than in the tissues. Therefore, a portion of loperamide absorbed in intestines can be metabolized there and directly secreted back into lumen. Oral loperamide thus undergoes a unique disposition, l i k e l y constituting one of mechanisms for its distinct dissociation of central and antidiarrheal activities. Loperamide hydrochloride, 4-(p-chlorophenyl)-4-hydroxyl-N,N-dimethyl-~,~diphenyl-l-piperidine butyramide hydrochloride, is an oral antidiarrheal agent (1). I t directly acts on the intestinal walls to suppress their peristaltic reflex (2). Our previous studies on the disposition and metabolism of the drug in rats (3) suggested that, when given per os, a considerable portion of the drug was metabolized in intestinal walls, not in their lumen, and then secreted back directly into lumen. The in vitro study given in this paper indicates that the presumptive above disposition of loperamide is actually present fn intestines. This unique disposition of the drug in the gut (the target organ) can be a major mechanism accounting for i t s distinct dissociation of peripheral and central effects, i . e . , potent antidiarrheal a c t i v i t y and few effects on systemic tissues (1,4). Experimental Male Wistar rats weighing about 200 g were sacrificed by decapitation. The duodenum, jejunum and ileum of about 5 cm length were excised, flushed with physiological saline and everted as described previously (5). The everted intestine was f i l l e d with about 2 ml of 0.3 M glucose-supplementedKrebs-Ringer phosphate buffer (6) as a medium of the serosal side and ligated at both ends. Three everted sacs thus prepared from different rats of each region were incubated with 0.3 ~Ci/6 ~g of [carbonyl-14C]loperamide (3) in 30 ml of the buffer at 37°C for l hr with bubbling of 02/C02 (95/5, v/v). Scraping of the mucosa during incubation was negligible under the present conditions. After incubation, each sac was isolated from the incubation system and the medium of the serosal side was ~emoved by puncture with a syringe. Tissues were homogenized in cold water. A part of the homogenate was dissolved in Soluene 350T (Packard Instruments, Inc., I l l . ) and mixed with toluene s c i n t i l l a t i o n 0024-3205/82/252203-04503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
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Loperamide in Intestines
Vol. 30, No. 25, 1982
cocktail. A portion of the medium of the serosal side and incubation medium (of the mucosal side) were mixed with Bray's solution (7). Radioactivity was measured in TriCarb liquid s c i n t i l l a t i o n spectrometer Model 3380. Remainings of the tissue homogenate and media were shaken with chloroform at pHlO with extraction efficiency of v i r t u a l l y I00%. The organic layer was chromatographed on thin layer of s i l i c a gel 60 F254 of 0.25 mm thickness (Merck precoated plates) with the solvent system, chloroform/iso-propanol/ammonium hydroxide (70/30/I) (3). Plates were scanned to localize radioactivity in a Packard radiochromatogram scanner. Radioactivity peaks appeared on the radiochromatogram were identified by co-chromatography with the authentic loperamide and i t s metabolites, mono- and di-desmethyl loperamide (8). Silica gel of each peak was scraped o f f and suspended in Bray's solution for radioactivity measurement for the estimation of the unchanged drug and its metabolites. Results Table I summarizes results of three incubations. Considerable radioa c t i v i t y was found to be accumulated in intestinal tissues of each region from the medium of the mucosal side, whereas serosal radioactivity was v i r t u a l l y null in all regions of intestines. The results indicate that [14C]loperamide penetrated from the mucosal side into tissues but further penetration of the drug into the serosal side hardly occurred under the present in vitro conditions.
TABLE I Radioactivity Found in Tissues and Media and in Metabolites after Incubation of Everted Sacs of Rat Intestines with [14C]Loperamide for l hr Metabolites Region
Fraction
Radioactivity L
DML
DDML
Recovery (% of Radioactivity in the incubation system) Duodenum Tissue
38 ± 3
30 ± 2
7 ± l
Medium, Mucosal
62 ± 3
44 ± 4
9 ± O
lO ± l
Serosal
0 ± 0
-
-
-
Jejunum Tissue
Ileum
l ± 0
44 ± 3
32 ± 2
lO ± 2
I ± 0
Medium, Mucosal
56 ± 3
41 ± 5
9 ± 1
6 ± 1
Serosal
0 ± 0
-
-
-
Tissue
30 ± 2
23 ± l
6 ± 0
l ± 0
Medium, Mucosal
70 ± 2
49 ± 3
II ± 0
I0 ± 2
Serosal
0 ± 0
-
-
-
Values are means of three experiments ±S.E. -: not determined due to the extremely low radioactivity. L: unchanged loperamide, DML: mono-desmethyl loperamide, DDML: di-desmethyl Ioperamide.
Vol. 30, No. 25, 1982
Loperamide in Intestines
2205
Table I also reveals that tissues of all regions contained three radioactive materials: unchanged loperamide, mono- and di-desmethyl metabolites. This indicates that loperamide penetrated into intestinal walls and underwent demethylation there regardless of the region. On the other hand, the medium of the mucosal side of each region were found to contain not only unchanged loperamide but also mono- and di-desmethyl loperamide. As evident in the table, the amounts of the two metabolites found in the medium were comparable to or larger than those in tissues. I t was already shown (3) that loperamide is stable when incubated both with pre-heated tissues and in intestinal contents under similar conditions to the present study or even for longer period. Therefore, the results indicate that a portion of loperamide i n i t i a l l y present in the mucosal medium once penetrated into tissues, where i t was demethylated, and was efluxed back mostly into the medium of the mucosal side under the present conditions. Furthermore, metabolite composition was different from each other in tissues and media, and di-desmethyl loperamide was more predominant in the medium than in the tissues as seen in the table. The results suggest that the di-desmethyl metabolite was eliminated from the gut tissues more rapidly than mono-demethylated one. Discussion Gastrointestinal absorption of a drug can be estimated by various measures. In our previous studies (3), the absorption of loperamide was estimated by two measures. The f i r s t was urinary excretion ratio of radioactivity after oral to parenteral administration of [14C]loperamide, which gave about 40% of oral dose being absorbed in the circulation. The second was the difference between administered dose (I00%) and the amount of unchanged [14C]loperamide excreted in feces (30%). I f the latter was assumed to be the drug unabsorbed, the results gave about 70% being absorbed. The discrepancy in the above absorption extents suggests that the difference is derived from metabolic change of the drug caused by either intestinal microflora or intestinal tissue itself. In our recent unpublished studies a similar discrepancy in absorption extents of [14C]loperamide estimated by the same methods was also seen in rats treated with oral antibiotic ampicillin. Based on the observation (3) that the drug was metabolized in vitro by intestinal tissue segments but not by intestinal contents, we have suggested that the discrepancy between the estimates of absorption may be due to the metabolism of a fraction of orally given loperamide in intestinal walls and subsequent direct elimination of metabolites in the lumen; the disposition and metabolism not involved in the circulation. The present in vitro studies clearly presented evidence that there is such disposition for loperamide as above: [l~C~loperamide present in the medium of the mucosal side was partly converted to its desmeth9 metabolites after incubation in the presence of everted intestines. The findings lead to the above conclusion since demethylation of loperamide hardly occurs spontaneously (3). I t seems that a portion, not a l l , of loperamide absorbed in the gut follows this route in vivo since low plasma levels were observed after oral administration of the drug (3,9). Although biotransformation by intestinal walls is known as a mechanism of the f i r s t pass effects for many drugs, the disposition found here for loperamide is rather novel. I t is reported that paminobenzoate undergoes acetylation in the intestinal tissues and subsequent release into serosal and mucosal side (lO), a partly similar disposition to that for loperamide. Disposition found in the present studies provides sufficient availability in the qut (the target oragan) of the drug which directly acts on the tissues to suppress their motility (2) possibly by interacting with their intramural ganglia and nerve endings ( l l ) . Demethylation of loperamide in the gut and
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Loperamlde in Intestines
Vol. 30, No. 25, 1982
release of the formed metabolites into lumen would serve for reducing a v a i l a b i l i t y of the drug in the systemic circulation possibly related with undesirable side-effects on central nervous systems and others. The d i s t i n c t dissociation of loperamide (l,12) between central and antidiarrheal effects have been partly explained by i t s kinetic properties, less e f f i c i e n t passage of blood brain barrier (13,14) and e f f i c i e n t hepatic uptake followed by b i l i a r y excretion after metabolic change (3,15). However, we believe that the unique disposition found here is a major mechanism underlying the d i s t i n c t dissociation of loperamide which has high a f f i n i t y to both central and peripheral opiate receptors (13,14,16). References I. 2. 3. 4.
5. 6. 7. 8. 9. lO. If. 12. 13. 14. 15. 16.
C.J.E. NIEMEGEERS, F.M. LENAERTS and P.A.J. JANSSEN, Arzneim.-Forsch. 24 1633-1636 (1974) J.M.V. NUETEN, P.A.J. JANSSEN and J. FONTAINE, Arzneim.-Forsch. 24 16411645 (1974) H. MIYAZAKI, K. NAMBU, Y. MATSUNAGAand M. HASHIMOTO, Eur. J. Drug Metab. Pharmacokinet. 4 199-206 (1979) R. MARSBOOM,V.-HERIN, A. VERSTRAETEN, R. VANDESTEENEand J. FRANSEN, Arzneim.-Forsch. 24 1645-1649 (1974) W.T. AGAR, F.J.R. HIRD and G.S. SIDHU, Biochim. Biophys. Acta. 14 80-84 (1954) R.M.C. DAWSON, D.C. ELLIOTT, W.H. ELLIOTT and K.M. JONES, Data for Biochemical Research, p. 507, Oxford University Press, London (1969) G.A. BRA'Y, Anal. Biochem. Z 279-285 (1960) K. YOSHIDA, K. NAMBU, S. ARAKAWA, H. MIYAZAKI and M. HASHIMOTO, Biomed. Mass Spectrom. 6 253-259 (1979) H.S. WEINTRAUB,-J.M. KILLINGER, J. HEYKANTS, M. KANZLERand J.H. JAFFE, Curr. Ther. Res. 21 867-876 (1977) M. YASUHARA, H. K~AYASHI, S. MURANISHI, H. SEZAKI and T. KIMURA, J. Pharm Dyn. l ll4-121 (1978) J.M.VTNUETEN, L. HELSEN, M. MICHIELS, J.J.P. HEYKANTS, Biochem. Pharmacol 28 1433-1434 (1979) C.J.E. NIEMEGEERS, J.L. McGUIRE, J.J.P. HEYKANTSand P.A.J. JANSSEN, J. Pharmacol. Exp. Ther. 210 327-333 (1979) L. TERENIUS, Acta pharmacol, et toxicol. 37 211-221 (1975) M. WUSTERand A. HERZ, Naunyn-Schmiedeberg's Arch. Pharmacol. 301 187-194 (1978) J.J.P. HEYKANTS, W.E.G. MEULDERMANS,A.G. KNAEPSand L.J.M. MICHIELS, Eur. J. Drug Metab. Pharmacokinet. 2 81-91 (1977) C.R. MACKERER, G.A. CLAY and E.Z. DAJANI, J. Pharmacol. Exp. Ther. 199 131-140 (1976)
Pergamon Press
LOPERAMIDE IN RAT INTESTINES: A UNIQUE DISPOSITION H.Miyazaki, K.Nambu and M.Hashimoto Research Laboratories, Dainippon Pharmaceutical Co., Ltd. Enoki 33-94, Suita, Osaka, Japan (Received in final form April 12, 1982)
Summary When everted sacs of rat duodenum, jejunum and ileum were incubated with [14C]loperamide in v i t r o , unchanged drug and its metabolites were found not only in tissues but also in media of the mucosal side with v i r t u a l l y no radioactivity in media of the serosal side. The amounts of metabolites fnund in media of the mucosal side were comparable to or larger than those in tissues. Di-desmethyl loperamide was more predominant in the media as compared with mono-demethylatedone than in the tissues. Therefore, a portion of loperamide absorbed in intestines can be metabolized there and directly secreted back into lumen. Oral loperamide thus undergoes a unique disposition, l i k e l y constituting one of mechanisms for its distinct dissociation of central and antidiarrheal activities. Loperamide hydrochloride, 4-(p-chlorophenyl)-4-hydroxyl-N,N-dimethyl-~,~diphenyl-l-piperidine butyramide hydrochloride, is an oral antidiarrheal agent (1). I t directly acts on the intestinal walls to suppress their peristaltic reflex (2). Our previous studies on the disposition and metabolism of the drug in rats (3) suggested that, when given per os, a considerable portion of the drug was metabolized in intestinal walls, not in their lumen, and then secreted back directly into lumen. The in vitro study given in this paper indicates that the presumptive above disposition of loperamide is actually present fn intestines. This unique disposition of the drug in the gut (the target organ) can be a major mechanism accounting for i t s distinct dissociation of peripheral and central effects, i . e . , potent antidiarrheal a c t i v i t y and few effects on systemic tissues (1,4). Experimental Male Wistar rats weighing about 200 g were sacrificed by decapitation. The duodenum, jejunum and ileum of about 5 cm length were excised, flushed with physiological saline and everted as described previously (5). The everted intestine was f i l l e d with about 2 ml of 0.3 M glucose-supplementedKrebs-Ringer phosphate buffer (6) as a medium of the serosal side and ligated at both ends. Three everted sacs thus prepared from different rats of each region were incubated with 0.3 ~Ci/6 ~g of [carbonyl-14C]loperamide (3) in 30 ml of the buffer at 37°C for l hr with bubbling of 02/C02 (95/5, v/v). Scraping of the mucosa during incubation was negligible under the present conditions. After incubation, each sac was isolated from the incubation system and the medium of the serosal side was ~emoved by puncture with a syringe. Tissues were homogenized in cold water. A part of the homogenate was dissolved in Soluene 350T (Packard Instruments, Inc., I l l . ) and mixed with toluene s c i n t i l l a t i o n 0024-3205/82/252203-04503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
2204
Loperamide in Intestines
Vol. 30, No. 25, 1982
cocktail. A portion of the medium of the serosal side and incubation medium (of the mucosal side) were mixed with Bray's solution (7). Radioactivity was measured in TriCarb liquid s c i n t i l l a t i o n spectrometer Model 3380. Remainings of the tissue homogenate and media were shaken with chloroform at pHlO with extraction efficiency of v i r t u a l l y I00%. The organic layer was chromatographed on thin layer of s i l i c a gel 60 F254 of 0.25 mm thickness (Merck precoated plates) with the solvent system, chloroform/iso-propanol/ammonium hydroxide (70/30/I) (3). Plates were scanned to localize radioactivity in a Packard radiochromatogram scanner. Radioactivity peaks appeared on the radiochromatogram were identified by co-chromatography with the authentic loperamide and i t s metabolites, mono- and di-desmethyl loperamide (8). Silica gel of each peak was scraped o f f and suspended in Bray's solution for radioactivity measurement for the estimation of the unchanged drug and its metabolites. Results Table I summarizes results of three incubations. Considerable radioa c t i v i t y was found to be accumulated in intestinal tissues of each region from the medium of the mucosal side, whereas serosal radioactivity was v i r t u a l l y null in all regions of intestines. The results indicate that [14C]loperamide penetrated from the mucosal side into tissues but further penetration of the drug into the serosal side hardly occurred under the present in vitro conditions.
TABLE I Radioactivity Found in Tissues and Media and in Metabolites after Incubation of Everted Sacs of Rat Intestines with [14C]Loperamide for l hr Metabolites Region
Fraction
Radioactivity L
DML
DDML
Recovery (% of Radioactivity in the incubation system) Duodenum Tissue
38 ± 3
30 ± 2
7 ± l
Medium, Mucosal
62 ± 3
44 ± 4
9 ± O
lO ± l
Serosal
0 ± 0
-
-
-
Jejunum Tissue
Ileum
l ± 0
44 ± 3
32 ± 2
lO ± 2
I ± 0
Medium, Mucosal
56 ± 3
41 ± 5
9 ± 1
6 ± 1
Serosal
0 ± 0
-
-
-
Tissue
30 ± 2
23 ± l
6 ± 0
l ± 0
Medium, Mucosal
70 ± 2
49 ± 3
II ± 0
I0 ± 2
Serosal
0 ± 0
-
-
-
Values are means of three experiments ±S.E. -: not determined due to the extremely low radioactivity. L: unchanged loperamide, DML: mono-desmethyl loperamide, DDML: di-desmethyl Ioperamide.
Vol. 30, No. 25, 1982
Loperamide in Intestines
2205
Table I also reveals that tissues of all regions contained three radioactive materials: unchanged loperamide, mono- and di-desmethyl metabolites. This indicates that loperamide penetrated into intestinal walls and underwent demethylation there regardless of the region. On the other hand, the medium of the mucosal side of each region were found to contain not only unchanged loperamide but also mono- and di-desmethyl loperamide. As evident in the table, the amounts of the two metabolites found in the medium were comparable to or larger than those in tissues. I t was already shown (3) that loperamide is stable when incubated both with pre-heated tissues and in intestinal contents under similar conditions to the present study or even for longer period. Therefore, the results indicate that a portion of loperamide i n i t i a l l y present in the mucosal medium once penetrated into tissues, where i t was demethylated, and was efluxed back mostly into the medium of the mucosal side under the present conditions. Furthermore, metabolite composition was different from each other in tissues and media, and di-desmethyl loperamide was more predominant in the medium than in the tissues as seen in the table. The results suggest that the di-desmethyl metabolite was eliminated from the gut tissues more rapidly than mono-demethylated one. Discussion Gastrointestinal absorption of a drug can be estimated by various measures. In our previous studies (3), the absorption of loperamide was estimated by two measures. The f i r s t was urinary excretion ratio of radioactivity after oral to parenteral administration of [14C]loperamide, which gave about 40% of oral dose being absorbed in the circulation. The second was the difference between administered dose (I00%) and the amount of unchanged [14C]loperamide excreted in feces (30%). I f the latter was assumed to be the drug unabsorbed, the results gave about 70% being absorbed. The discrepancy in the above absorption extents suggests that the difference is derived from metabolic change of the drug caused by either intestinal microflora or intestinal tissue itself. In our recent unpublished studies a similar discrepancy in absorption extents of [14C]loperamide estimated by the same methods was also seen in rats treated with oral antibiotic ampicillin. Based on the observation (3) that the drug was metabolized in vitro by intestinal tissue segments but not by intestinal contents, we have suggested that the discrepancy between the estimates of absorption may be due to the metabolism of a fraction of orally given loperamide in intestinal walls and subsequent direct elimination of metabolites in the lumen; the disposition and metabolism not involved in the circulation. The present in vitro studies clearly presented evidence that there is such disposition for loperamide as above: [l~C~loperamide present in the medium of the mucosal side was partly converted to its desmeth9 metabolites after incubation in the presence of everted intestines. The findings lead to the above conclusion since demethylation of loperamide hardly occurs spontaneously (3). I t seems that a portion, not a l l , of loperamide absorbed in the gut follows this route in vivo since low plasma levels were observed after oral administration of the drug (3,9). Although biotransformation by intestinal walls is known as a mechanism of the f i r s t pass effects for many drugs, the disposition found here for loperamide is rather novel. I t is reported that paminobenzoate undergoes acetylation in the intestinal tissues and subsequent release into serosal and mucosal side (lO), a partly similar disposition to that for loperamide. Disposition found in the present studies provides sufficient availability in the qut (the target oragan) of the drug which directly acts on the tissues to suppress their motility (2) possibly by interacting with their intramural ganglia and nerve endings ( l l ) . Demethylation of loperamide in the gut and
2206
Loperamlde in Intestines
Vol. 30, No. 25, 1982
release of the formed metabolites into lumen would serve for reducing a v a i l a b i l i t y of the drug in the systemic circulation possibly related with undesirable side-effects on central nervous systems and others. The d i s t i n c t dissociation of loperamide (l,12) between central and antidiarrheal effects have been partly explained by i t s kinetic properties, less e f f i c i e n t passage of blood brain barrier (13,14) and e f f i c i e n t hepatic uptake followed by b i l i a r y excretion after metabolic change (3,15). However, we believe that the unique disposition found here is a major mechanism underlying the d i s t i n c t dissociation of loperamide which has high a f f i n i t y to both central and peripheral opiate receptors (13,14,16). References I. 2. 3. 4.
5. 6. 7. 8. 9. lO. If. 12. 13. 14. 15. 16.
C.J.E. NIEMEGEERS, F.M. LENAERTS and P.A.J. JANSSEN, Arzneim.-Forsch. 24 1633-1636 (1974) J.M.V. NUETEN, P.A.J. JANSSEN and J. FONTAINE, Arzneim.-Forsch. 24 16411645 (1974) H. MIYAZAKI, K. NAMBU, Y. MATSUNAGAand M. HASHIMOTO, Eur. J. Drug Metab. Pharmacokinet. 4 199-206 (1979) R. MARSBOOM,V.-HERIN, A. VERSTRAETEN, R. VANDESTEENEand J. FRANSEN, Arzneim.-Forsch. 24 1645-1649 (1974) W.T. AGAR, F.J.R. HIRD and G.S. SIDHU, Biochim. Biophys. Acta. 14 80-84 (1954) R.M.C. DAWSON, D.C. ELLIOTT, W.H. ELLIOTT and K.M. JONES, Data for Biochemical Research, p. 507, Oxford University Press, London (1969) G.A. BRA'Y, Anal. Biochem. Z 279-285 (1960) K. YOSHIDA, K. NAMBU, S. ARAKAWA, H. MIYAZAKI and M. HASHIMOTO, Biomed. Mass Spectrom. 6 253-259 (1979) H.S. WEINTRAUB,-J.M. KILLINGER, J. HEYKANTS, M. KANZLERand J.H. JAFFE, Curr. Ther. Res. 21 867-876 (1977) M. YASUHARA, H. K~AYASHI, S. MURANISHI, H. SEZAKI and T. KIMURA, J. Pharm Dyn. l ll4-121 (1978) J.M.VTNUETEN, L. HELSEN, M. MICHIELS, J.J.P. HEYKANTS, Biochem. Pharmacol 28 1433-1434 (1979) C.J.E. NIEMEGEERS, J.L. McGUIRE, J.J.P. HEYKANTSand P.A.J. JANSSEN, J. Pharmacol. Exp. Ther. 210 327-333 (1979) L. TERENIUS, Acta pharmacol, et toxicol. 37 211-221 (1975) M. WUSTERand A. HERZ, Naunyn-Schmiedeberg's Arch. Pharmacol. 301 187-194 (1978) J.J.P. HEYKANTS, W.E.G. MEULDERMANS,A.G. KNAEPSand L.J.M. MICHIELS, Eur. J. Drug Metab. Pharmacokinet. 2 81-91 (1977) C.R. MACKERER, G.A. CLAY and E.Z. DAJANI, J. Pharmacol. Exp. Ther. 199 131-140 (1976)