XENOBIOTICA,

1992, VOL. 22,

NO.

11, 1229-1237

Fast-track Paper

The metabolism of acitretin and isoacitretin in the in situ isolated perfused rat liver

S. COTLERt, D. CHANG, L. HENDERSON, W. GARLAND and C. TOWN

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Department of Drug Metabolism, Hoffmann-La Roche, Inc., 340 Kingsland Street, Nutley, NJ 07110, USA

Received 1 April 1992; accepted 30 April 1992

1. The metabolism of acitretin and its 1 3 4 s isomer, isoacitretin, has been investigated in the in situ isolated perfused rat liver in order to differentiate the action of the liver from that of the gut on the metabolism of these isomers. 2 . Acitretin undergoes a-oxidation, chain shortening 0-demethylation, and glucuronidation in the perfused rat liver. 3. Isoacitretin undergoes glucuronidation as the major, almost exclusive, route of metabolism in the perfused rat liver.

4. The difference in the hepatic metabolism of the cis and trans isomers of this retinoid may explain the differences in their pharmacokinetics, and may help in understanding the pharmacokinetics of related retinoids.

Introduction The ability of natural and synthetic retinoids to promote normal epithelial differentiation and proliferation can be exploited to treat psoriasis (Brindley 1989). Common side-effects of retinoids in this application are hypervitaminosis A syndrome, e.g. dryness of the lips, and teratogenesis (Jewel1 and McNamara 1990). Etretinate was the first retinoid investigated and marketed particularly for the indication ‘psoriatic diseases’ (Teelman and Bollag 1990). Etretinate is a potent teratogen, and plasma concentrations have been measured in patients 100 days after the last dose of chronically administered drug (Massarella et al. 1985). Acitretin, the metabolite of etretinate, is also a teratogen (Geiger and Brindley 1988), but it does not accumulate in a deep tissue compartment, and plasma levels are not detectable 2 days after the last dose of chronically administered acitretin (McNamara et al. 1988), a significant advantage in treating females of childbearing potential. Acitretin appears similar to etretinate in terms of efficacy (McNamara et at. 1988), and therapeutic index (Teelman and Bollag 1990). One of the metabolites observed after acitretin administration to man is its cisisomer, isoacitretin, which has been shown to be ineffective in psoriasis (Geiger and Brindley 1988). Administration of isoacitretin to man leads to plasma concentrations of both acitretin and isoacitretin which are higher than after administration of an equivalent dose of acitretin. These higher plasma concentration of acitretin were not associated with a clear therapeutic effect in psoriasis (Geiger and Brindley 1988). The elimination half-lives for acitretin and isoacitretin are considerably different, which might reflect differences in their respective routes of

t T o whom correspondence

should be addressed.

0049-8254/92 83.00

0 1992 Taylor & Francis Ltd

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metabolism. Figure 1 shows the structures of etretinate, acitretin and other major metabolites. To better understand the differences in the metabolism of acitretin and isoacitretin, the isolated perfused in situ liver preparation was employed to compare the hepatic metabolism of these two compounds. In situ liver perfusion is a suitable model for studies of metabolic pathways (Pang et al. 1984). T h e organ is maintained in the anatomical position within the rat, but isolated from the rest of the animal by virtue of cannulation of its major blood supply. Full oxygenation is maintained by this route and the liver differs from the ‘normal’ only by lacking its hepatic arterial and nerve supply.

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Methods and materials Liver perfusion Male Sprague-Dawley rats (obtained from Charles River Laboratories) ranging in weight from 368 to 4188 were used for the experiments. The perfusion apparatus used was a Two/Ten perfuser (MX International Aurora, Co., USA). The surgical procedure was a modification of the method of Pang and Roland (Pang and Gillette 1978). The bile duct was cannulated with polyethylene tubing followed by ligation of the inferior vena cava at the level of the right kidney. T h e portal vein was exposed and cannulated for the inflow. The outflow from the liver was collected via a cannula inserted in the vena cava as it enters the heart. The liver was perfused in situ with oxygenated perfusate (5% CO, and 95% 0,) containing 20% washed outdated human red blood cells by volume, 2.5% bovine serum albumin and 3.0 mg/ml of glucose in 200 ml of Krebs-Ringer bicarbonate buffer. A single 5 mg dose of either acitretin or isoacitretin was added to the reservoir and allowed to mix thoroughly with the perfusate. T h e perfusate was delivered at a constant flow of 1ml/min per g of liver weight. The effluent was returned to the reservoir and recirculated. All of the experiments were performed under yellow lights to avoid photoisomerization of the compounds.

Sample collection Bile was collected at +-h intervals for 3 h, and a final bile sample was collected from 3 to 4 h after the start of the perfusion. The volumes of the samples were determined gravimetrically (assuming the specific gravity of bile is 1) and the samples were stored at -70°C prior to analysis.

ISOACITRETIN 0

C

Ro 23

- 4293

bH ISORCITRETIN GLUCURONIOE

cHo+eH : HO

CH¶ RO 23

- 3571

ETRETINATE

Figure 1. Structural formula of etritinate, acitretin and major known metabolites.

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Preparation of the bile (free and conjugated) Duplicate 0.02ml aliquots of bile obtained from the in situ perfused rat livers were mixed with 0.25 ml of 1 M acetate buffer (pH 5.5). Water (0.02 ml) was added to one of the aliquots while 0.02 ml of Glusulase@ (Dupont Laboratories) was added to the other aliquot. The samples were incubated in a Dubnoff metabolic shaker for 4 h at 37°C. At the end of the incubation the samples were transferred to a WispTMvial fitted with a low-volume insert and 0.1 ml was injected onto the h.p.1.c. Other aliquots were treated with 0.1 M-NaOH for 30min, neutralized and subjected to h.p.1.c. analysis. H.p.1.c. conditions A h.p.1.c. system consisting of a Waters WisprMAutomatic Injector (Model 712b), a Waters pump (Model 6000), an Alltech Econosphere C18 Column (100mm x 4.6mm), a Kratos variable-wavelength U.V.Detector (Model 757 set at 280nm and 0.02 AUFS) and a Spectrophysics integrator (Model SP4100) was used for the separation of acitretin, isoacitretin and their respective metabolites. The compounds were eluted from the column isocratically using a mobile phase consisting of methanol-0.01 yo aqueous acetic acid (78 : 22 v/v) at a flow of 1.0 ml/min. The U.V.detector was replaced for several samples with a Waters photodiode array detector (Model 990), employing the manufacturer's software to scan peaks from 200 to 400nm.

Mars spectrometry A Nermag Model 1010 mass spectrometer equipped with a thermospray source and the Sidar 8 data system was used for detection and the molecular weight determinations of components in rat bile samples. The h.p.1.c. column effluent (from above) was combined with 0.05M ammonium acetatemethanol (95 : 5 v/v) pumped at 0.5 ml/min and the mixture was directed into the mass spectrometer where the source temp. was set at 223°C and tip temp. at 190°C. The spectra were collected over the mass range of m / z 170-570. Nuclear magnetic resonance Bile samples from a rat liver perfused with isoacitretin were pooled, the pH was adjusted to 4.5 with 1 M acetate buffer, and the pooled sample was extracted with ethyl acetate. The aqueous phase was discarded and the organic phase was brought to dryness at 60°C. The samples were then subjected to h.p.1.c. purification. The column effluent containing the peak of interest was collected and evaporated to dryness at 60°C under a stream of nitrogen. Proton n.m.r. spectra were obtained in deuterochloroform with tetramethylsilane as the internal standard on a Varian XL-400 instrument in the Fouriertransform mode with a flip angle of 35".

Results H.p.l.c. analysis Figure 2 is a chromatogram of a bile sample from an isolated perfused in situ liver following perfusion with acitretin. T h e right panel shows a chromatogram of bile, collected from 3 to 4 h after start of the perfusion, before incubation with Glusulase@ and the left panel shows a chromatogram of the same bile after incubation with Glusulasea. Both panels contain peaks that correspond to the retention times associated with acitretin, Ro 23-4293 and Ro 23-3571. T h e peak heights in the left panel were higher than those in the right panel, indicating that the compounds were conjugated and the conjugates were cleaved by Glusulase@. These chromatograms show evidence of a small amount of conjugated isoacitretin in rat bile following perfusion with acitretin and treatment of the bile with Glusulasea. Figure 3 is a chromatogram of a bile sample from an isolated perfused in situ liver following perfusion with isoacitretin. T h e right panel is a chromatogram of a bile sample collected from 2.5 to 3 h before incubation with Glusulasea and the left panel is a chromatogram after incubation with Glusulasea. There was a peak in the right panel that did not correspond to the retention times of the available authentic standards. Photodiode array analysis confirmed that this peak had an U.V. spectrum with a low 280/365nm ratio, similar to isoacitretin. T h e other significant peak in the chromatogram had a retention time that corresponded to isoacitretin. When incubated with Glusulasea, the unidentified peak was no longer

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Acitretin and isoacitretin in rat liver

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present, while the peak associated with isoacitretin increased (left panel) indicating that the unknown peak was a conjugate of isoacitretin hydrolysobolized by Glusulase@. There were small but detectable amounts of free and conjugated acitretin in the bile of livers perfused with isoacitretin. Treatment of bile samples with NaOH gave similar results to Glusulasea treatment. Results of mass spectrometry T h e separated and purified metabolite, observed after isoacitretin perfusion, was analysed by h.p.1.c.-mass spectrometry. Figure 4 shows the spectrum from the peak at 6.2min. T h e mass spectrum shows a base peak at mlx 327 which corresponds to a protonated molecule of isoacitretin (aglycone) and peaks at m j z 503 and 520, which correspond to the glucuronide of isoacitretin and the glucuronide ammonia adduct ion, respectively. T h e peaks at mlz 485 and 467 represent the glucuronide with the loss of one and two molecules of water respectively. Fragments were observed at mlz 194 and 177 corresponding to glucuronic acid and glucuronic acid minus water, respectively. Results of n.m.r. spectrometry T h e identification of the glucuronide of isoacitretin ,by h.p.1.c.-mass spectrometry was supported by the n.m.r. spectrum shown in figure 5. Aglycone peaks at 67.70 (H-12 doublet), 67.00 (H-11 doublet of doublet), 66.65 (H-7 doublet), 66.55 (H-4 singlet), 65.70 (H-14 singlet), 83-80 (0-CH, singlet) and five methyl resonances between 62-00 and 62-40 are virtually unchanged from those of the unconjugated material (Vane et al. 1989). Two additional one-proton single resonances at 65-60 and 64.05 can be assigned to H-1' and H-S', respectively. Peaks for H-2', H-3' and H-4' were obscured by impurities in the region of 63.7.

Discussion T h e metabolism of acitretin and isoacitretin was evaluated in the isolated perfused in situ liver preparation. T h e major metabolite that was identified and characterized by mass spectrometry and n.m.r. following isoacitretin perfusion was the ester glucuronide. Following acitretin perfusion the glucuronide of acitretin was one of what appeared to be five metabolites formed in the liver and

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Chromatograms of bile extracts from the in situ liver perfusion with isoacitretin, before Glusulasea treatment (left) and after Glusulasea treatment (right).

S. Cotler et al.

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excreted in the bile. One of the metabolites, Ro 23-4293, which differs from the parent drug by loss of a single carbon and saturation of one double bond, appeared to be formed in the liver, perhaps following a pathway similar to the a-oxidation of branched chain fatty acids, such as phytanic acid (Avigan et al. 1966). Ro 23-4293 was then 0-demethylated in the phenyl ring to form Ro 23-3571. Other, more polar metabolites appeared to be the ester glucuronides of acitretin, isoacitretin, Ro 23-4293 and Ro 23-3571. These identifications were supported by the appearance of the unconjugated metabolites after treatment with glusulase or sodium hydroxide. There appeared to be a very small amount of interconversion between the acitretin and isoacitretin in the isolated perfused rat liver. In comparison, the significant amount of conversion of acitretin to isoacitretin in the whole rat, which has been reported after multiple dosing (McNamara and Blouin 1990), may be due to isomerization occurring in the gut or gut wall. T h e metabolism of a number of retinoids including retinoic acid occurs in both the gastrointestinal tract and the liver (Zile et al. 1982, Cotler et al. 1984, Bornemann et al. 1988). T h e metabolism of retinoids by the gut and gut wall would occur after oral administration or biliary secretion (Cullum and Zile 1985, Cotler et al. 1983). The differences in the hepatic metabolism of acitretin and isoacitretin are significant, i.e. clearly the difference in the cis-trans configuration of the bond joining the p carbon to the rest of the retinoid has a significant effect on the route of metabolism. The trans configuration leads to a-oxidation and further metabolism (including a minimal amount of glucuronidation) while the cis configurations leads exclusively to the molecule being glucuronidated and eliminated. All trans retinoids seem to be metabolized with shorter half-lives than retinoids containing a cis double bond. T h e formation of the glucuronide of isoacitretin and

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enterohepatic recirculation may partially explain the long half-life which is observed with isoacitretin. The metabolisms of 1 3 4 s and all-trans retinoic acid follow a similar pattern. T h e elimination half-life of all-trans-retinoic acid in rats is 20min (Swanson et al. 1981), while the half-life of 13-cis-retinoic acid in rats is 48-60min (Nankevis et al. 1990). The findings in the present study of the difference in the hepatic metabolism of the cis and trans isomers of acitretin may apply to other synthetic and naturally occurring retinoids with cis-trans isomerization at the B-carbon. Oral administration of a cis or trans isomer will lead to isomerization in the gut and gut wall during absorption of the compound. Once in the systemic circulation, all trans isomers may be cleared by the liver by a-oxidation, while cis isomers will be glucuronidated, secreted into the bile, hydrolysed and reabsorbed, leading to longer elimination half-lives for cis isomers than for trans isomers.

Acknowledgements T h e authors wish to thank Patricia Seymore for her word-processing assistance, and T. Williams and G. Sasso for performing nuclear-magnetic resonance analysis.

References AVIGAN, J . , STEINBERG, D., and GUTMAN, A., 1966, Alpha-decarboxylation, an important pathway for degradation of phytanic acid in animals. Biochemical and Biophysical Research Communications, 24, 838-844. BORNEMANN, L. D., COTLER, S., KHOO,K. C., CARBONE, J . J . , and COLBORN, W. A,, 1988, Effect of the route of administration on the pharmacokinetics of etretinate in the dog. Bzopharmaceutics and Drug Disposition, 9, 119-126. BRINDLEY,C. J . , 1989, Overview of recent clinical pharmacokinetic studies with acitretin (Ro 10-1670, Etretin). Dermatologia, 178, 79-87. COTLER, S., BUGGE, C. L. J . , and COLBURN, W. A,, 1983, Role of gut contents, intestinal wall, and liver on the first pass metabolism and absolute bioavailability of isotretinoin in the dog. Drug Metabolism and Disposition, 11, 458-462. COTLER, S., CHEN,S., MACASIEB, T., and COLBURN, W. A,, 1984, Effect of route administration and biliary excretion on the pharmacokinetics of isotretinoin in the dog. Drug Metabolism and Disposition, 12, 143-147. CULLUM, M. E., and ZILE,M. H., 1985, The metabolism of all-trans retinoic acid and all-trans retinyl acetate. Journal of Biological Chemistry, 260, 10590-10596. GEIGER, J . M., and BRINDLEY, C. J . , 1988, Cis-trans interconversion of acitretin in man. Skin Pharmacology, 1, 230-236. JEWELL, R. C., and MCNAMARA, P. J . , 1990, Glutathione catalysis of interconversion of acitretin and its 13-cis isomer, isoacitretin. Journd of Pharmaceutical Science, 79, 444446. MASSARELLA, J . , VANE,F., BUGGE, C., RODRIGUEZ, L., CUNNINGHAM, W. J . , FRANZ, T., and COLBURN, C., 1985, Etretinate kinetics during chronic dosing in severe psoriasis. Clinical Pharmacology and Therapeutics, 37, 439-466. MCNAMARA, P. J . , and BLOUIN,R. A., 1990, Pharmacokinetic profile of two aromatic retinoids (etretinate and acitretin) in the obese Zucker rat. Journal of Pharmaceutical Science, 79, 301 -304. MCNAMARA, P. J., JEWELL, R. C., JENSEN, B. K.. and BRINDLEY, C. J . , 1988, Food increases the bioavailability of acitretin. Journal of Clinical Pharmacology, 28, 1051 -1055. NANKEVIS, R. CHEEMA, M. S., DAVIS,S. S., DAY,M. H., and SHAW,I. P. N., 1990. Pharmacokinetics of isotretinoin in the anesthetized rat following intravenous administration of an emulsion. Journal of Pharmacy and Pharmacology, 40S, 35P. PANG,K. S., and GILLETTE, J . R., 1978, Kinetics of metabolite formation and elimination in the perfused rat liver prepration: differences between the elimination of preformed acetaminophen and acetaminophen formed from phenacetin. Journal of Pharmacology and Experimental Therapeutics, 207, 178-194. PANG,K. S., HUANG, J . C., FINKLE, C., KONG,P., CHERRY, W. F., and FAYZ,S., 1984, Kinetics of procainamide N-acetylation in the rat in-wiwo and in the perfused rat liver preparation. Drug Metabolism and Disposition, 12, 314-322.

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SWANSON, B. N., FROLIK, C. A,, ZAHAREVITY, D . W., ROLLER,P. R., and SPORN,M. B., 1981, Dose-dependent kinetics of all-tram-retinoic acid in rats. Biochemical Pharmacology, 30, 107- 11 3. TEELMANN, K., and BOLLAG,W., 1990, The relevance of the mouse papilloma test as a predictor of retinoid activity in human psoriasis. Dermatologia, 180, 30-35. VANE,F. M., BUGGE,C. L. J., and RODRIGUEZ, L. C., 1989, Identification of etretinate metabolites in human blood. Drug Metabolism and Disposition, 17, 280-285. ZILE,M. H., INHORN, R. C., and DELUCA, H. F., 1982, Metabolism in vivo of all-trans retinoic acid. Journal of Biological Chemistry, 2576, 3544-3550.

The metabolism of acitretin and isoacitretin in the in situ isolated perfused rat liver.

1. The metabolism of acitretin and its 13-cis isomer, isoacitretin, has been investigated in the in situ isolated perfused rat liver in order to diffe...
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