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Aim: To establish the rat model of morphine-induced conditioned place preference (CPP) and to investigate the effects of morphine psychical dependence on the levels of neurosteroids in rat brain., Methods: Rats were ip administered morphine 5 mg x kg(-1) for 10 days to induce CPP in morphine group. The concentrations of dehydroepiandrosterone (DHEA), pregnenolone (PREG), allopregnanolone (AP), dehydroepiandrosterone sulfate (DS) and pregnenolone sulfate (PS) in nucleus accumbens (Nac), hypothalamus (Ht), amygdale (A) and plasma of rats were determined with liquid chromatography-negative atmospheric pressure ionization mass spectrometry (LC-MS)., Results: Trained with morphine for 10 days resulted in the acquisition of CPP in morphine group with the time that the rats spent in drug-pairing room was longer than that of control group. Compared with control group, morphine treatment could significantly decrease the contents of DHEA in Nac and plasma, decrease that of PREG in Ht., Conclusion: Morphine could induce the CPP in rats and affected the contents of some neurosteroids in rat brain, which suggests that endogenous neurosteroids might he related to the development of morphine dependence.

Aim: To prepare rabbit anti-rat P450scc antibody, and to detect the expression of rP450scc in rat brain tissue and nerve cells., Methods: An eight-branched polypeptide, rP450scc-16 was synthesized using solid phase synthesis (Fmoc) method. A Newzealand rabbit was immunized with rP450scc-16 and the serum was separated from the whole blood 5 days after the last immunization. The titer and specificity of the antiserum were evaluated, and the expression of rP450scc in normal rat brain and primary rat astrocytes was detected by using ELISA, Western blot and immunocytochemcal staining., Results: The titer of the antiserum was 1:6,400. Western blot analysis showed that the rP450scc protein in rat brain, testis and adrenal gland homogenate was recognized by the antiserum as a single band at M(r) being 50,000, which indicated a high specificity of the antiserum. Hypothalamus, cerebral cortex, and hippocampus of rat brain and the cytoplasm of cultured rat astrocytes were positively stained by the antiserum., Conclusion: A highly specific anti-rP450scc antibody was prepared. The antibody can be used to study the expression and distribution of rP450scc in rat brain.

Aim: To simultaneously determine three unconjugated neurosteroids, dehydroepiandrosterone (DHEA) , pregnenolone (PREG), allopregnenolone (AP), from several brain regions of the rat., Methods: Neurosteroids were isolated separately in a two steps procedure by using ethyl acetate-n-hexane (90:10) as the first step to extract the unconjugated steroids, then the steroid fractions were further purified by SPE. All steroids were derivatized with 2-nitro-4-trifluoromethylphenylhydrazine (2-NFPH) and analyzed by HPLC-MS ( APCI) using selected-ion monitoring. Methyltestosterone was chosen as the internal standard. Results The linear calibration curve of DHEA was obtained in the concentration range of 0.030-2.00 microg x L(-1). The linear calibration curves of PREG and AP were obtained in the concentration range of 0.025-2.00 microg x L(-1). The concentrations of DHEA, PREG and AP in male rat brain regions were (0.70 +/- 0.23), (4.8 +/- 1.9), (1.1 +/- 0.6) ng x g(-1) for frontal cortex, (0.57 +/- 0.28), (6 +/- 3), (0.5 +/- 0.3) ng x g(-1) for hippocampus, (1.5 +/- 1.0), (9 +/- 5), (1.4 +/- 0.9) ng x g(-1) for amygdale, (0.52 +/- 0.14), (7.7 +/- 2.8), (0.5 +/- 0.6) ng x g(-1) for striatum, (2.9 +/- 1.6), (18 +/- 9), (1.6 +/- 1.3) ng x g(-1) for nucleus accumbens, (4.0 +/- 2.0), (27 +/- 12), (0.8 +/- 0.5) ng x g(-1) for pituitary gland, (1.7 +/- 1.2), ( 16 +/- 10), and (0. 8 +/- 0.7) ng x g(-1) for hypothalamus, respectively., Conclusion: Good linearity and accuracy were observed for each steroid. The procedure was suitable for measuring concentrations of the unconjugated steroids in rat brain simultaneously.

Aim: To investigate the effects of morphine dependence and withdrawal on the concentrations of neurosteroids in rat brain., Methods: A method of simultaneous quantification of neurosteroids by gas chromatography-mass spectrometry (GC-MS) had been established., Results: The chronic morphine administration (ip) resulted in a marked decrease in the brain concentrations of pregnenolone (PREG), progesterone (PROG), and pregenenolone sulfate (PREGS) in rats killed 6 h after the last treatment. In contrast, there were no significant effects of morphine dependence on the brain concentrations of allopregnanolone (AP), dihydroepiandrosterone (DHEA), and dihydroepiandrosterone sulfate (DHEAS). Naloxone-induced withdrawal produced a significant increase in the concentrations of PREG, PROG, AP, DHEA, PREGS, and DHEAS as compared with the control group., Conclusion: Morphine dependence and withdrawal affected the concentrations of neurosteroids in rat brain, which suggests that endogenous neurosteroids in brain might be related to the development of morphine dependence and withdrawal., (Copyright 2004 Acta Pharmacologica Sinica)

Aim: To investigate the stereoselectivity in biliary excretion of trans tramadol (trans T) and trans O-demethyltramadol (M1) in rats., Methods: After a single intravenous dose of trans T hydrochloride (10 mg.kg-1) or M1 (2.5 mg.kg-1) to rats, the bile was collected for 30 min, then, blood was obtained from the heart. The enantiomers of trans T, M1 and M1 conjugated with glucuronic acid (M1c) in the bile and plasma were analyzed by high performance capillary electrophoresis (HPCE)., Results: After the rats were given trans T, the bile concentrations of (+)-trans T were higher than those of (-)-trans T, and the (+)/(-)-trans T ratios were lower compared with those in the plasma. After the rats were given M1, the bile concentrations of (+)-M1 were higher than those of (-)-M1, and the bile concentrations of (+)-M1c were lower than those of (-)-M1c. The glucuronidation rate of (+)-M1 was lower than that of (-)-M1 in the bile., Conclusion: The biliary excretion of trans T and M1 was stereoselective, (+)-trans T and (-)-M1 being preferentially excreted.

Aim: To study the stereoselectivity in trans-tramadol [(+/-)-trans-T] metabolism and trans-O-demethyltramadol (M1) formation., Methods: (+)-, (-)-, Or (+/-)-trans-T was separately incubated with rat liver microsomes in vitro. The concentrations of (+/-)-trans-T and M1 enantiomers were determined by high performance capillary electrophoresis (HPCE)., Results: When each enantiomer of (+/-)-trans-T was incubated with rat liver microsomes, the metabolic rate of (+)-trans-T was lower than that of (-)-trans-T. The kinetics of (+)-, (-)-M1 formation was found to fit the single-enzyme Michaelis-Menten model. The Vmax and CLint of (+)-M1 formation were lower than those of (-)-M1 formation. When (+/-)-trans-T was used as the substrate, the metabolic rates of (+)-, (-)-trans-T, and the formation rates of (+)-M1, (-)-M1 decreased to different extents. Dextromethorphan (Dex), propafenone (Pro), and fluoxetine (Flu) could inhibit both the metabolism of (+/-)-trans-T enantiomers and the formation of M1 enantiomers. Pro and Flu were shown to enhance the stereoselectivity in both (+/-)-trans-T metabolism and M1 formation, and Dex could only enhance that in M1 formation., Conclusion: (+/-)-Trans-T metabolism and M1 formation were stereoselective, (-)-trans-T being preferentially metabolized and (-)-M1 being preferentially formed. There was interaction in metabolism between (+/-)-trans-T enantiomers. Dex, Pro, and Flu had different effects on the stereoselectivity.

Aim: To study the stereoselectivity in O-demethylation of trans tramadol., Methods: With or without quinine and quinidine as inhibitors, rat liver microsomes were incubated in vitro with the enantiomers or the racemate of trans tramadol. The concentrations of the enantiomers of trans tramadol and O-demethyltramadol in the incubates were determined by high performance capillary electrophoresis. The O-demethylation processes were assayed by using the enzyme kinetic analysis method., Results: After incubation, the concentrations of (-)-O-demethyltramadol were higher than those of (+)-enantiomer in all rat liver microsomal incubates. Enzyme kinetic analysis showed that the Km of the formation of the enantiomers of O-demethyltramadol were similar; The Vmax and Clint of the formation of (-)-O-demethyltramadol were significantly higher than those of the formation of (+)-enantiomer. When the racemate of trans tramadol was used as the substrate, there was interaction between the two enantiomers. The Km of the formation of the enantiomers of O-demethyltramadol increased, the Vmax of the formation of (+)-O-demethyltramadol decreased, the Vmax of the formation of (-)-O-demethyltramadol increased slightly. The O-demethylation of the enantiomers of trans tramadol was shown to be inhibited competitively by quinine and quinidine. The Ki of quinine and quinidine were 1.6 and 10.8 mumol.L-1 to the formation of (-)-O-demethyltramadol, 0.8 and 3.4 mumol.L-1 to the formation of (+)-O-demethyltramadol, respectively. Furthermore, quinine and quinidine were found to have stereoselective inhibition on the formation of O-demethyltramadol, both mainly inhibited the formation of (+)-O-demethyltramadol., Conclusion: The O-demethylation of trans tramadol was found to be stereoselective in rat liver microsomes in vitro, preferentially metabolized (-)-enantiomer. The stereoselectivity could be influenced by the interaction between the two enantiomers and the enzyme selective inhibitors.

Aim: To study the stereoselectivity in renal clearance of trans-tramadol and its active metabolite, trans-O-demethyltramadol., Methods: The right kidneys were isolated from male SD rats and perfused with 100 mL of perfusate medium containing trans-tramadol 300 microg/L or trans-O-demethyltramadol 50 microg/L. After perfusion, the concentrations of the enantiomers of trans-tramadol and trans-O-demethyltramadol in the perfusate and urine were determined by high performance capillary electrophoresis. The enantiomeric ratios were calculated., Results: After the kidneys being perfused with trans-tramadol hydrochloride, the concentration of (+)-trans-tramadol was higher than that of (-)-trans-tramadol, and the concentration of (+)-trans-O-demethyltramadol was lower than that of (-)-trans-O-demethyltramadol in the perfusate; meanwhile, (+)-trans-tramadol was more than (-)-trans-tramadol, and (+)-trans-O-demethyltramadol was less than (-)-trans-O-demethyltramadol in the urine. After the kidneys being perfused with trans-O-demethyltramadol, the concentration of (+)-trans- O-demethyltramadol was lower than that of (-)-trans-O-demethyltramadol in the perfusate, and (+)-trans-O-demethyltramadol was more than (-)-trans-O-demethyltramadol in the urine., Conclusion: The renal clearance of trans-tramadol was stereoselective. The O-demethylation of trans-tramadol was stereoselective in the kidneys, (-)-trans-tramadol being preferentially metabolized. The renal clearance of trans-O-demethyltramadol was also stereoselective, the (+)-enantiomer being preferentially cleared into the urine.