Mephenytoin hydroxylation in the Cuna Amerindians of

Transcription

1 Br. J. clin. Pharmac. (8), 25, Mephenytoin hydroxylation in the Cuna Amerindians of Panama T. INABA1, L. F. JORGE2 & T. D. ARIAS2 1Department of Pharmacology, Faculty of Medicine, University of Toronto, Toronto M5S 1A8, Canada and 2The World Health Organization Collaborating Center for Drug Quality Control, Instituto Especializado de Analisis, and the College of Pharmacy, University of Panama, Panama, Panama. 1 Mephenytoin p(4')-hydroxylation, which is deficient in 3-5% of Caucasians, was examined in 96 Cuna Amerindians of Panama. 2 Attempts were made to exclude poor compliance with urine collection and ingestion of the drug dose since the assignment of phenotype was based upon urinary recovery of the metabolite. These involved the measurement of the urinary recovery of sparteine, added to the ingested capsule, and of the renal excretion of creatinine. 3 Of the 90 Cunas deemed to be reasonably compliant, none of them appeared to be deficient in p(4')-hydroxylation of mephenytoin. Keywords mephenytoin genetic polymorphism Cuna Amerindians Introduction Mephenytoin, an active anticonvulsant, undergoes polymorphic oxidation (Kupfer & Preisig, 4; Inaba, 6; Kalow, 6). Kupfer et al. (1979) first noted a deficiency in the p(4')- hydroxylation of the S-enantiomer of the racemic drug in a Swiss family. It was later determined that the frequency of this defect was 5% in Swiss subjects (Kupfer & Preisig, 4) and that its mode of inheritance was autosomal recessive (Inaba et al., 6). The mephenytoin polymorphism has also been reported in Caucasian populations of the United States and Canada with a frequency of 34% (Jurima et al., 5; Nakamura et al., 5; Wedlund et al., 4), in Japanese with a frequency of 18-23% (Jurima etal., 5; Nakamura et al., 5) and in Chinese with a frequency of 5% (Jurima et al., 5). This report describes a study of the oxidative metabolism of mephenytoin in Amerindians, an ethnic group whose population is estimated to be about 30 million and which constitutes the main ethnic stock of mixed groups in Latin America (Rodriguez & Soubie, 1978; Salzano, 4). Panama is unique in that its population includes five groups of genetically distinct Amerindians. One of these, the Cunas, comprising about 40,000 people, represent the second most numerous Amerindian group in Panama (Torres de Arauaz, 0) and one of the largest showing negligible racial heterogeneity in Latin America (Barrantes et al., 2). We have also studied the polymorphism of sparteine oxidation in the same group (Arias et al., 6, 7a). Methods Subjects, drug administration and urine collection Ninety six healthy Cuna Amerindians volunteered for the study. None of them was related in the fourth degree of consanguinity or closer. Most subjects were inhabitants of the Amerindian villages of Rio Azucar (Comarca de San Blas subjects ) and Chivo-Chivo (Province of Panama, subjects ) and twelve were from Correspondence: Dr T. Inaba, Department of Pharmacology, Faculty of Medicine, University of Toronto, Toronto M5S 1A8, Canada. 75

2 76 T. Inaba, L. F. Jorge & T. D. Arias Panama City. They were instructed in Spanish or the Cuna language of their rights, as set down in the Helsinki Declaration, and were specifically asked not to take any alcoholic drinks or drugs during the study. Interviews and physical examination were performed and written informed consent was obtained. The Ethics Committee of the University of Panama approved the study. Only healthy subjects of Cuna ancestry, as established through anthropometric criteria, medical examination and interviews, were accepted as volunteers. The subject ingested a gelatine capsule containing 50 mg of racemic mephenytoin and 50 mg of sparteine sulphate at about h. Urine was collected for the subsequent 12 h and small aliquots were kept in plastic vials at 4 C for 1-4 days before being transferred to a -20 C freezer. The addition of sparteine to the capsules containing mephenytoin and its analysis in the urine helped to establish complete ingestion of the mephenytoin dose. The measurement of urinary creatinine also served as a check on the completeness of urinary recovery. Non-compliance or incomplete urine collection were assumed when less than 10% of the dose was recovered as sparteine plus metabolites in the urine sample and urinary creatinine was less than 50 mg. Analytical methods The analysis of urine for p(4')-hydroxymephenytoin was by the method of Kupfer et al. (4) with minor modifications (Inaba et al., 6b). Briefly, an aliquot of urine was extracted with ethyl acetate after the addition of HC1. The unconjugated metabolite was derivatized using 1-iodopropane as an alkylating agent under anhydrous conditions. The perpropylated derivative was measured by gas chromatography using nitrogen-selective detection and 5-methyl- 5-phenylhydantoin (Aldrich, Milwaukee, Wis.) as internal standard. The results of duplicate assays did not vary by more than 5% of the mean values. The detection limit was 0.2% of the dose excreted in a 0-12 h urine. The metabolites of mephenytoin were found to be stable at 4 C for 3 weeks. Sparteine and dehydrosparteines were measured as described previously (Arias et al., 7a). Urinary creatinine was measured using alkaline-picric acid (Faulkner & King, 1970). Results The results from six Cuna subjects were excluded since they eliminated less than 10% of sparteine plus metabolites and less than 50 mg of creatinine. In the remainder sparteine recovery was 43 ± 20 s.d.% (in Caucasians 47 ± 10%) and creatinine excretion was 358 ± 168 s.d. mg-1 12 h (range mg). Incomplete urine collection by a number of other subjects, though of a lesser magnitude, was suspected from low urine volumes (Table 1). The individual results from 90 subjects are shown in Table 1. The frequency histogram of p(4')-hydroxymephenytoin urinary recovery in the Cunas (Figure 1) did not indicate a subgroup of deficient metabolizers, as was evident in data from a sample of the Canadian population (Jurima et al., 5). A greater spread of p(4')-hydroxymephenytoin recoveries in the Cunas compared with that in Caucasian EMs (Figure 1, Table 2) was possibly due to incomplete urine collection by many of the Cuna subjects. Normalisation by creatinine recovery reduced the spread of the Cuna data but it still exceeded that in EM Caucasians (Table 2). Discussion Our data emphasize the difficulties of carrying out pharmacogenetic studies in culturally distinct populations. Problems of poor compliance are especially important when the method of assigning phenotype is based upon the absolute urinary recovery of metabolite. Normalisation for creatinine excretion removes some of the doubt but, ideally, an index based upon the ratio of drug to metabolite is preferable. In the case of mephenytoin hydroxylation, measurement of the ratio of stereoisomers would obviate the need for complete urine collection (Wedlund et al., 4). Assuming that normalisation of urinary recoveries by creatinine excretion is valid, we did not detect any PMs of mephenytoin in our sample of the Cuna population. In view of the mongoloid ancestry of Amerindians, the data appeared contradictory to the high frequency of PMs among Japanese. However, this finding does not establish the absence of the poor hydroxylator phenotype in this ethnic group since, based on a frequency of 4% from Caucasian data, about 200 Cuna subjects would be necessary to establish a zero percent incidence with a 5% probability of type 1 error. This work was supported by the Medical Research Council of Canada and the Regional Program for Scientific and Technological Development, Organization of American States.

3 Mephenytoin hydroxylation in Cuna Amerindians 77 Table 1 p(4')-hydroxymephenytoin excretion in 90 Cuna Amerindians of Panama Subject Urine HM Creatinine Subject Urine HM Creatinine m/f volume (ml) (% dose) (mg) m/f volume (ml) (% dose) (mg) 79m 85f 86f 88f 91f 92f 93f 95f 96f 97f 99f loom lo1f 102f 104f 107f 108f 11Of ilif 112f 113f 117m 118m 119m m 121f 123m 124m 125f 126f 128f 129f 130f 132f 134m 136f 137f 142m 143m 144m 145m 146m 147f 148m 150m m 152f 153m 154m 155m 156m 157m 158m 159m 163m 164m 165m 166m 167m 168m 169f 170f 171m 172m 173f 174m 175f 176m 177m 178m 179f 180m 181m 183f 184m 185f 186m 187m 189f 190m 191f 193m 194m 195f m 199f 200m 201f 202m 203f

4 78 T. Inaba, L. F. Jorge & T. D. Arias C0 X log HM (as percentage of dose) Figure 1 Frequency histogram of the urinary recovery of p(4')-hydroxymephenytoin (HM) as log % of dose in 90 Cuna Amerindians of Panama (n). The data for 118 Caucasians (Jurima et al., 5) are shown for comparison. Table 2 Urinary recovery ofp(4')-hydroxymephenytoin in Cuna Amerindians in comparison with Canadian Caucasians: descriptive statistics Cuna Amerindian Caucasians (a) Caucasians (a) Cuna Amerindian (n = 90) (n = 113, EM) (n = 118, EM+ PM) (n = 90) log (HM) log (HM) log (HM) log (HM/gCR) Median Min; Max 0.55; ; ; ; 2.43 Mean ± s.d ± ± ± 0.24 Relative s.d. 18.8% 5.7% 18.4% 13.3% Skewness (ns) (b) (b) 0.16 (ns) Kurtosis 3.73 (ns) 4.22 (c) (d) 2.81 (ns) Mephenytoin: 50 mg (R + S) p.o. EM and PM: extensive and poor metabolizer HM: p-hydroxymephenytoin as % of dose in 12 h urine HMIgCR: HM normalized to 1 g creatinine Relative s.d.: 100 x s.d./mean (a) Ref. Jurima et al. (5) (b) Negative (lower tail, < 0.01) (c) Positive (peaked distribution < 0.05) (d) Positive (peaked distribution < 0.01) References Arias, T. D., Hernandez, J. N. & Lachman, D. G. (0). Estudios sobre la velocidad de acetilaci6n en los indigenas Cunas del caserio de Paya, en la provincia de Darien. Rev. Med. de Panama, 5, Arias, T. D., Jorge, L. F. & Inaba, T. (6). No evidence for the presence of poor metabolizers of sparteine in an Amerindian group: the Cunas of Panama. Br. J. clin. Pharmac., Arias, T. D., Jorge, L. F, Lee, D., Barrantes, R. & Inaba, T. (7a). Absence of the Caucasian-type polymorphism in the oxidative metabolism of sparteine in the Cuna Amerindian group of Panama. Clin. Pharmac. Ther. (in press). Arias, T. D., Jorge, L. F., Nuniez, G. & Inaba, T. (7b). Estudio preliminar sobre el fenotipo de acetilador de los amerindios teribes. Rev. Med. de Panama, 12,

5 Mephenytoin hydroxylation in Cuna Amerindians 79 Barrantes, R., Smouse, P. E., Neel, J. V., Mohrenweiser, H. W. & Gershowitz, H. (2). Migration and genetic infrastructure of the Central American Guaymi and their affinities with other tribal groups. Am. J. Phys. Anthrop., 58, Faulkner, W. R. & King, J. W. (1970). Renal function In Fundamentals of Clinical Chemistry, ed. Tiez, N. W., p 997. Philadelphia: Saunders Co. Inaba, T. (6). Genetic polymorphism of mephenytoin metabolism. In: Ethnic differences in reaction to drugs and xenobiotics, eds Kalow, W. & Goedde, H. W., pp New York: Alan R. Liss Inc. Inaba, T., Jurima, M. & Kalow, W. (6b). Family studies of mephenytoin hydroxylation deficiency. Am. J. Human Gen., 38, Jurima, M., Inaba, T., Kadar, D. & Kalow, W. (5). Genetic polymorphism of mephenytoin p(4')- hydroxylation: Difference between Orientals and Caucasians. Br. J. clin. Pharmac., 19, Kalow, W. (6). The genetic defect of mephenytoin hydroxylation. Xenobiotica, 16, Kuipfer, A., Desmond, P., Schenker, S. & Branch, R. (1979). Family study of a genetically determined deficiency of mephenytoin hydroxylation in man. Pharmacologist, 21, 173 (Abstract). Kupfer, A. & Preisig, R. (4). Pharmacogenetics of mephenytoin: A new drug hydroxylation polymorphism in man. Eur. J. Pharmac., 26, Nakamura, K., Goto, F., Ray, W. A., McAllister, C. B., Jacqz, E., Wilkinson, G. R. & Branch, R. A. (5). Interethnic differences in genetic polymorphism of debrisoquine and mephenytoin hydroxylation between Japanese and Caucasian populations. Clin. Pharmac. Ther., 38, Rodriguez, N. & Soubie, E. (1978). La poblaci6n indigena actual en America Latina. Nueva Antropologia, 3, 49. Salzano, F. M. (4). The peopling of the Americas as viewed from South America. Acta Anthropogenetica, 8, Torres de Arauz, R. (0). Panama Indigena, pp 105. Panama: Instituto Nacional de Cultura. Wedlund, P. J., Aslanian, W. S., McAllister, C. B., Wilkinson, G. R. & Branch, R. A. (4). Mephenytoin hydroxylation deficiency in Caucasians: Frequency of a new oxidative drug metabolism polymorphism. Clin. Pharmac. Ther., 36, (Received 28 April 7, accepted 6 August 7)