Study On Metabolic Treatment Of L - Tetrahydropalmatine In Vitro And In

Levo-tetrahydropalmatine(l-THP), the levo isomer of tetrahydropalmatine, is a neuroactive alkaloid naturally occurred in traditional herb plant species of Stephania rotunda Lour(Qian jin teng) and Corydalis ambigua(Yan hu suo). Based on its excellent sedative, neuroleptic and analgesic properties, l-THP has been approved, under the tradename Rotundine, for clinical use in China for about 50 years. In recent years, a large body of evidence suggests that l-THP is effective in inhibiting the drug addiction-producing, addiction-sustaining and preventing relapse, which makes it a promising clinical medication for management of drug addiction. However, little is known concerning the detailed knowledge of metabolism and disposition of l-THP in animals and humans because of the early approval for clinical use in the country. In spite of the early reports describing the preclinical ADME studies with 3H-labeled dl-tetrahydropalmatine, the complete ADME data of non-labeled l-THP still hangs in the air. The recent studies indicated that l-THP underwent extensive metabolism in animals and the major phase I metabolites were pharmacologically active demethylated l-THP. But the relevance of metabolism and disposition of l-THP to the exposure level in vivo remains unclear. Besides, the metabolism data of l-THP in human is scarce. Hence the present study aimed to investigate systematically the ADME profiles of l-THP and its metabolites in rats, the metabolism and clearance pathways in human, and the in vitro inter-species differences of l-THP metabolism between human and rat. Furthermore, the pharmacokinetics of l-THP in special populations and metabolic DDI potentials are predicted by establishing a PBPK model of l-THP in the healthy asian population based on the in vitro and rat data. The study enhances the understanding of the pharmacological mechanism of the drug, and also provides experimental data and scientific base for the clinical development of the drug for the new indication effectively and safely.The major results of the study are summarized as follows:1. A sensitive LC-MS/MS method for the simultaneous analysis of l-THP and its six demethylated metabolites(2-DM, 3-DM, 9-DM, 10-DM, 2,3-DM and 2,10-DM) was developed and fully validated. By incorporating an enzymatic hydrolysis into sample preparation, the corresponding conjugates of the six metabolites could be quantitatively determined. The newly developed quantitative method could cover the parent drug, its demethylated metabolites and the corresponding conjugates, which was more robust, rapid, and reliable and satisfied the requirement of disposition and metabolism studies of l-THP and its metabolites.2. The absorption and metabolism of l-THP were extensively investigated in rats after an oral dose of 9 mg·kg-1. The drug could be absorbed rapidly and the elimination half life was found to be 6.5 h. l-THP underwent remarkable first-pass metabolism following oral administration and the absolute bioavailability was 29.7%. The major components detected in plasma were l-THP, three demethylated metabolites(2-DM, 3-DM and 10-DM) and their glucuronidated conjugates. 2-DM-Glu and 10-DM were the dominant free and conjugated metabolites, respectively. Metabolites were eliminated slowly and showed significant double peaks in the concentration-time curves.3. After oral administration, l-THP could widely distributed into tissues, and the peak level was observed at 1 h post dose in most of the tissues. The concentrations of l-THP in tissues showed the order of intestine>liver>testis>kidney>lung>brain>heart>spleen>plasma. The major metabolites in tissues were 2-DM, 3-DM, 10-DM and their glucuronides, whose tissue distribution profiles were similar to that of the parent drug. l-THP and its demethylated metabolites could penetrate BBB readily. Abundant amounts of 10-DM and 3-DM and minor amounts of their glucuronides were detected in the brain. The binding rates of l-THP and its demethylated metabolites with plasma protein of rats or humans were ranging from 78% to 92%, and their total brain bounding fractions were higher than 91%.4. The excretion of l-THP was minor(0.22% of the oral dose) in rat urine and feces, and the majority of the dose were excreted as metabolites. Within 72 h post dose, 28.5% of the dosed drug was recovered as glucuronides and sulfates in urine and 6.12% as the demethylated in feces.5. The recovery of l-THP and its metabolites in bile within 48 h was up to 70.1% of the oral dose applied to bile-duct cannulated rats. Only trace amounts of the parent drug were recovered( < 0.05%), and the predominant components in bile were the demethylated glucuronides and sulfates. Utilizing the paired rats model, it was proved that the demethylated metabolites underwent significant enterohepatic circulation in rats, with the percentage of enterohepatic circulation of the metabolites ranging from 30% to 66%. Almost 50% of the biliary excreted metabolites were deconjugated and reabsorbed in intestine, which explained the double peak phenomenon in the concentration-time curves and slow elimination of metabolites.6. It was observed in incubations with HLM and RLM that O-demethylation was the predominant metabolic reaction of l-THP, and generated 2-DM, 3-DM and 10-DM as major metabolites in both microsomes. Results showed that, with the use of substrate depletion and product formation approaches, the Km and Vmax obtained from the 2-DM formation in HLM was comparable to those obtained from the l-THP depletion, which suggested that 2-O-demethylation reaction was the predominant metabolic pathway of l-THP in HLM. For demethylated metabolites, glucuronidation appeared to be the major secondary metabolic pathway in microsomes.7. The CYP reaction phenotyping of l-THP was studied in both recombinant human CYP supersomes and human liver microsomes with CYP specific inhibitors. The metabolism of l-THP was mediated by several CYP isoforms, including CYP3A4, 2D6, 1A2, 2C9 and 2C19. Among of these isoforms, CYP3A4 was identified as the major isoform that had predominant contribution(54.0%) to metabolism of l-THP, and next was CYP2D6 with contribution of 11.3%. Formation of 10-DM and 3-DM was mainly mediated by CYP3A4, and the generation of 2-DM was predonminantly by CYP2D6. The results suggested that much attention should be paid to the CYP3A4 and 2D6-mediated drug-drug interactions when l-THP is used clinically.8. Metabolites of l-THP in HLM, human plasma, urine and feces were screened using LC/UV and UPLC-QTOF-MS/MS. A total of 20 metabolites were detected and identified. Among them, 15 metabolites were newly reported. Demethylation, didemethylation, tridemethylation, mono-oxidation, dehydrogenation and alkenes to dihydrodiol were identified as the phase I metabolic pathways of l-THP. The major phase II reactions observed were glucuronidation and sulfation of desmethyls.9. The exposure level and excretion routes of the parent drug and metabolites were investigated in healthy volunteers after an oral administration of Rotundine tablets(90mg). 2-DM-Glu, 10-DM-Sul and 3-DM-Sul were detected as the dominant drug components in plasma. The 72 h accumulative urinary excretion of l-THP and its metabolites was 38% of the dose and the fecal excretion was 6.12%. However, the excretion of l-THP was minor, amounting to only less than 0.7% of the dose. Demethylations followed by glucuronide and sulfate conjugations and renal excretion were the major drug clearance pathways of l-THP in human.10. Inter-species differences of the metabolism and disposition of l-THP were observed between human and rat. Sulfates were detected as the more extensive in human plasma, while the desmethyls and glucuronides were the predominant components in rat plasma and no sulfates were found. Regarding the excretion profile, the accumulative urinary excretion of the demethylation related components in human was significantly higher than that in rats.11. Using Gastro PlusTM software, the PBPK models of l-THP in rat and human were successfully established and used to predict the PK behaviors of special populations, as well as DDI potentials. Results showed that the exposure of l-THP was significantly elevated in the populations with severely impaired hepatic functions, while apparently decreased in CYP2D6 ultra-rapid metabolizers. The plasma exposure of l-THP in the concomitant groups with ketoconazole and quinidine was significantly increased with the AUC being 2.78-fold and 1.57-fold of the l-THP alone group, respectively. It is necessary to monitor plasma exposure of the drug when it is used in special populations and concomitant with the CYP inhibitors or substrates.The novel points or new findings of this study are illustrated as follows:1. A novel quantitative method was established for simultaneously quantitation of l-THP, demethylated metabolites and conjugates in biological samples by LC-MS/MS.2. The systematic studies on rat pharmacokinetics and tissue distribution revealed that the demethylated metabolites, particularly 3-DM and 10-DM, were the drug related components in plasma and brain. It is proposed that these active metabolites might contribute to the pharmacological effects of l-THP. This finding provided new insights and essential scientific basis for understanding the molecular mechanism of l-THP.3. For the first time, the metabolism and clearance of l-THP were characterized in human. Results showed that major components in plasma were 2-DM-Glu and 10-DM-Sul, and their exposure levels were much higher than that of the parent drug. Glucuronide and sulfate conjugation of desmethyls followed by renal excretion was one of the major drug clearance pathways of l-THP in human. The inter-species differences in metabolism and disposition between human and rat were also explored.4. With newly established human PBPK model of l-THP, it was predicted that the plasma exposure of l-THP were significantly increased in patients with severe hepatic impairment due to the reduced metabolic clearance, and the reverse result was obtained in CYP2D6 ultra-rapid metabolizers. The DDI prediction indicated the risk of CYP3A4 and 2D6 inhibition based DDI. The information is useful for facilitating clinical development of the drug, as well as for the safe use in clinic.