Stereoselectivity And Gender-related Differences In The Pharmacokinetics Of Trans Tramadol And Its Active Metabolite, Trans O-demethyltramadol

Tramadol (T) has two chiral carbons and thus has four stereoisomers. Trans T, a racemic mixture of 1R, 2R-T [(+)-trans T] and 1S, 2S-T [(-)-trans T], is used as a centrally acting analgesic. The enantiomers of trans T take as the action in different mechanisms. (+)-Trans T preferentially inhibits serotonin reuptake and enhances basal sterotonin release, whereas (-)-trans T preferentially inhibits norepinephrine reuptake and enhances stimulation-evoked norepinephrine release. Trans O-demethyltramadol (M1) is the only pharmacologically active metabolite. (+)-M1 has a high affinity to μ-opioid receptor, whereas (-)-M1 inhibits monoamine reuptake. This dual model of action of trans T, opioid and nonopioid, may contribute to its efficiency in certain pain, little or no respiratory depression and tolerance after repeated administration.Trans T is mainly metabolized in liver, and eliminated by kidney into urine. In liver, trans T is changed to 5 phase I metabolites through O-demethylation and N-demethylation. Three metabolites formed through O-demethylation may be further conjugated with glucuronic acid and sulfuric acid to form 6 phase II metabolites. Until now, the stereoselectivity in pharmacokinetics of trans T and/or M1 has been discussed only in 3 papers. It was proved by Paar et al that M1 formation was catalyzed by CYP2D6, and was of stereoselectivity in human liver microsomes. By measuring the enantiomers of trans T and its metabolites in the urine, Elsing et al found that the (+)/(-)-enantiomeric ratios of trans T and M1 were different among healthy volunteers.In order to further know the pharmacokinetic characteristics of trans T and Ml, to use trans T more properly, and to further develop trans T or Mland its pure enantiomers, we carried out detail studies on the stereoselectivity in pharmacokinetics of trans T and Ml, the relative mechanisms, and the influent factors. In this paper, it was presented that the stereoselectivity and gender-related differences in pharmacokinetics of trans T and Ml, and the mechanism on absorption and metabolism.1 Stereoselectivity and gender-related differences in the pharmacokinetic of trans T and Ml in ratsFollowing a single oral dose of 10 mg-kg'1 trans T hydrochloride to SD rats (8 male and 8 female), the plasma concentrations of the enantiomers of trans T and Ml were determined at certain sampling times by a high performance capillary electrophoresis method (HPCE). The pharmacokinetic parameters of. the enantiomers of trans T and Ml were determined by a method of noncompartmental analysis. The maximum plasma concenerations (Cmax) and their corresponding times (Tmax) were recorded as observed. The elimination rate constant {Xz) was estimated as the absolute value of the slope of a least-square linear regression of the terminal phase of the logarithmic plasma concentration-time profile. The plasma terminal half-life (Tm) was calculated as 0.693/Az. The area under the plasma concentration-time curve (AUC0.t) from time zero to the time of last quantifiable concentration (Ct) was calculated using the linear trapezoidal method. The area under the plasma concentration-time profile from time zero to the infinite time (AUCq.^) was calculated as the sum of corresponding AUC0.t and Ct/Xz values. The plasma oral clearance (CL/F) was calculated as Dose/AUCo.^,. The apparent volume of distribution (F/F) was obtained by using the equation: F/F=(Dose/AUC0.oo)/ylz. As a measure for the rate of absorption, the Cmax/AUC0.oo ratio was also calculated. Paired Mest was used to compare the pharmacokinetic parameters between the enantiomers of trans T or Ml in male or female rats. To compare the pharmacokinetic parameters of each enantiomer of trans T or Ml between male and female rats, unpaired Mest was used to all parameters except Tmax, to which nonparametric Wilcoxon two-sample test was used.Following an oral dose of trans T hydrochloride to SD rats, the values of Qiax> AUCo.^, and Tm for (+)-trans T were significantly higher than those for {-)-trans T; the values of CL/F, V/F, and CmJAVC0^ for (+)-trans T were significantly lower than those for {-)-trans T. Both in the males and in the females, all pharmacokinetic parameters but 7^ of {+)-trans T were significantly different from those of {-)-trans T. It was indicated that the pharmacokinetics of trans T was stereoselective in both sex rats. The value of Cmax for (+)-trans T, the values of Tmax, AUC0^, and Tm for (+)-trans T and (+)-trans T were significantly higher in the females than in the males. Meanwhile, the values of CL/F, V/F, and Cmax/AUCo.oo for the enantiomers of trans T were significantly lower in the females than in the males. It was indicated that the pharmacokinetics of each enantiomer of trans T was different in male and female rats. The (+)/(-)-enantiomeric ratios were not different in the males and the females, it was indicated that the stereoselectivity in pharmacokinetics of trans T was similar in male and female rats.Following an oral dose of trans T hydrochloride to SD rats, the values of Cmax and AUCo.^ for (+)-Ml were significantly different from those for (-)-Ml both in the males and in the females. It was indicated that the pharmacokinetics of Ml was also stereoselective in male and female rats. The value of Cmax, AUCq^, and Tm for (+)-Ml was significantly higher in the females than in the males. It was indicated that the pharmacokinetics of (+)-Ml was different in male and female rats. In the males, the values of Cmax and AUC0^ for (+)-Ml were significantly lower than those for (-)-Ml, and the (+)/(_)_enantiomeric ratios of Cmax and AUCo.*, were lower than 1. In the females, the values of Cmax and AUC^ for (+)-Ml were significantly higher than those for (-)-Ml, and the (+)/(-)-enantiomeric ratios of Cmax and AUCq.^ were larger than 1. Therefore, the stereoselectivity in pharmacokinetics of Ml was different in male and female rats. 2 Absorption characteristics of the enantiomers of trans T in rat intestineSixty SD rats, both sex, were anesthetized with pentobarbital. The smallintestine was exposed via a midline incision. The bile duct was ligated. The duodenum, jejunum and ileo, about 10 cm each, were cannulated at the proximal and distal ends. After being washed, the intestinal sections were separately connected with a peristaltic pump, and recirculated with the perfusate at a rate of 1 mL-min"1. The perfusate consisted of trans T dissolved in Krebs-Ringer buffer (pH 7.4). The enantiomers of trans T in the perfusate were analyzed by using a HPCE method at different times after the recirculation. The absorbed fraction and absorption rate were calculated. The absorption rates at different concentrations of trans T were also checked to investigate the kinetics of absorption by using the equation: J=Jmax*CQ/(Kt+C0)+Kd*C0.When the concentration of trans T in the perfusate was set to 20 umol-L"1, the absorbed .fractions of the enantiomers of trans T were similar among different segments of the rat intestine. The absorbed fraction of (+)-trans T was lower than that of {-)-trans T. Therefore, the enantiomers of trans T could be absorbed at different parts in the intestine; and the absorption was of stereoselectivity, with (-)-trans T being preferentially absorbed.As the concentration of trans T in the perfusate to jejunum increased, the absorbed fractions of the enantiomers of trans T were reduced and the difference in absorbed fractions between the enantiomers of trans T became not significant at the concentration of 40 umol-L"1. The absorption rates for the enantiomers of trans T were fitted to the equation: J=Jm!>x*C(J(Kt+C0)+Kd*C0. The values of/max for (+)-trans T and (-)-trans T were 546.47 nmol-h"'-g"! and 525.60 nmol-h'^g"1, respectively. The values of Kt for (+)-trans T and (-)-trans T were 42.54 umol-L"1 and 35.64 umol-L"1, respectively. The values ofKd for both (+)-trans T and {-)-trans T were 0.03 uL-rf'-g"1. It was indicated that both active and passive transport manners were involved in the intestinal absorption of the enantiomers of trans T.When the concentration of trans T in the perflisate was 20 umol-L"1, the absorbed fractions of each enantiomer of trans T were similar in the male and female rat jejunum. It was indicated that the intestinal absorption of each enantiomer of trans T was not different based on gender. There were nodifferences in the (+)/(-)-enantiomeric ratios of trans T absorbed fraction in the male and female rat jejunum. Therefore, the stereoselectivity in absorption of trans T was similar in male and female rat intestinal. 3 Stereoselectivity and gender-related differences in the metabolism of trans T and Ml in rat liver microsomesSD rats (9 male and 9 female) were used to prepare the rat liver microsomes by method of differential ultracentrifugation. Trans T or Ml, at different concentrations, were separately incubated with rat liver microsomes in vitro. After incubation at 37 °C with constant shaking for 90 min or 60 min, respectively, the incubation products were removed out and extracted with ethyl acetate. The concentrations of the enantiomers of trans T and Ml were determined by a HPCE method. In the trans T metabolism test, the metabolic rates of the enantiomers of trans T were obtained by dividing their depleted concentrations with the incubation times. The formation rates of the enantiomers of Ml were obtained by dividing their concentrations in the incubation products with the incubation times. In the Ml conjunction test, the glucuronidation rates of the enantiomers of Ml were obtained by dividing their depleted concentrations with the incubation times. For the formation and glucuronidation of the enantiomers of Ml, the enzyme kinetic parameters including apparent Michaelis-Menten constant (Km), maximal velocity (Fmax), and intrinsic clearance (C/int) were assayed by using an enzyme kinetic analysis method. Paired Mest was used to compare the enzyme kinetic parameters between the enantiomers of trans T or Ml in male or female rats, and unpaired /-test was used to compare the enzyme kinetic parameters of each enantiomer of trans T or Ml between the male and female rats.When trans T was incubated with the rat liver microsermers, the metabolic rates of (+)-trans T were significantly lower than those of {-)-trans T. The formation rates of (+)-Ml were significantly lower than those of (-)-Ml. The formation of the enantiomers of Ml was found to fit a single-enzyme Michaelis-Menten model. The values of Km for (+)-Ml and (-)-Ml were similar, the values of Vmax, C/int for (+)-Ml were significantly lower thanthose for (-)-Ml. Thus, trans T metabolism and Ml formation were stereoselective in male and female rat liver microsomes, with {-)-trans T being preferentially metabolized and (-)-Ml preferentially forming.The metabolic rates of the enantiomers of trans T in the male rat liver microsomes were significantly higher than those in the female rat liver microsomes at every substrate concentrations. The values of Fmax and C/int for the enantiomers of Ml in the male rat liver microsomes were significantly higher than those in the female rat liver microsomes. It was indicated the metabolism of the enantiomers of trans T and the formation of the enantiomers of Ml were different based on gender, the activities of metabolic enzyme(s) being higher in male rats.The (+)/(.-)-enantiomeric ratios of trans T metabolic rates (1.2-1.5) in the male rat liver microsomes were similar with those in the female rat liver microsomes (1.1-1.3). The (+)/(-)-enantiomeric ratios of Vmax and Clml for Ml formation (6.9 and 6.3, respectively) in male rat liver microsomes were lower than those in the female rat liver microsomes (8.4 and 8.7, respectively). Therefore, the stereoselectivity in metabolism of trans T was not significantly different in male and female rat liver microsomes. While, the stereoselectivity in formation of Ml was significantly higher in female rat liver microsomes than in male rat liver microsomes.When Ml was incubated with the rat liver microsermers, the glucuronidation rates of (+)-Ml were significantly lower than those of (-)-Ml in the male and female rat liver microsomes. The glucuronidation of Ml was found to fit a single-enzyme Michaelis-Menten model. The value of Km for (+)-Ml was significantly higher than that for (-)-Ml, the value of C7int for (+)-Ml was significantly lower than that for (-)-Ml, and the values of Vmax for (+)-Ml and (-)-Ml were different in the rat liver microsomes. It was indicated that the glucuronidation of Ml was stereoselective in rat liver microsomes. Compared with (-)-Ml, (+)-Ml had lower affinity to the enzyme and a lower glucuronidation rate.In the glucuronidation of Ml, the values of Fmax and C/int for (+)-Ml inthe male rat liver microsomes were significantly higher than the corresponding values in the female rat liver microsomes. While, the values of Vmax and C/int for (-)-Ml in the male rat liver microsomes were significantly lower than the corresponding values in the female rat liver microsomes. It was indicated that the glucuronidations of each enantiomer of Ml were different based on gender in rat liver microsomes.The (+)/(-)-enantiomeric ratios of Vmax and C/int for Ml glucuronidation (0.9 and 1.8, respectively) in the male rat liver microsomes were lower than those in the female rat liver microsomes (1.4 and 2.3, respectively). Therefore, the stereoselectivity in glucuronidation of Ml was significantly different between male and female rat liver microsomes, being higher in female rat liver microsomes. 4 ConclusionAfter an oral dose of trans T, the pharmacokinetics of trans T is stereoselective, and the pharmacokinetics of the enantiomers of trans T is different based on gender in rats. In male and female rats, the system exposure of (+)-trans T is higher than that of {-)-trans T. This might be attributed to the preferential metabolism oi{-)-trans T in the liver microsomes. The elimination rate of trans T is lower, and system exposure of trans T is higher in female rats than in male rats. This could be attributed to the lower metabolic rate of trans T in female liver microsomes.After an oral dose of trans T, the pharmacokinetics of Ml is stereoselective, and the pharmacokinetics of the enantiomers of Ml is different based on gender in rats. The system exposure of (+)-Ml is lower than that of (-)-Ml in male rats. But, the system exposure of (+)-Ml is higher than that of (-)-Ml in female rats. The stereoselectivity in pharmacokinetics of Ml could be thought to be related with the stereoselectivity in pharmacokinetics of trans T, the preferential formation and the preferential glucuronation of Ml in the liver microsomes.After an oral dose of trans T hydrochloride, the stereoselectivity in pharmacokinetics of trans T is not obviously different in male and female rats.