Simultaneous Determination of Levo-tetrahydropalmatine and Naltrexone in Rat Plasma by LC-MS/MS and its Application in a Pharmacokinetic Study.

BACKGROUND: Levo-tetrahydropalmatine and low-dose naltrexone are used in association with reducing cocaine-related cravings, but there are no analytical methods for the quantitative simultaneous analysis of this drug combination. OBJECTIVE: A highly selective and sensitive LC-MS/MS assay was developed and validated to simultaneously quantify l-THP and naltrexone. The analytical method for l-THP offers improved sensitivity compared to previously published methods. METHODS: The product ion transitions of l-THP and naltrexone were 357.0→193.0 and 342.2→324.1, respectively. Chromatographic separations were performed using a BEH-C18 column by an isocratic elution mode with acetonitrile and 0.1% formic acid in water containing 3 mM ammonium acetate. L-THP and naltrexone were extracted from rat plasma using a liquidliquid extraction method. RESULTS: For l-THP and naltrexone, the assay displayed good linear response over a concentration range of 0.5-1000 ng/mL and 0.25-500 ng/mL, respectively. The intra-day accuracy of the method for l-THP and naltrexone was 93.8-101% with a precision (%CV) of 2.43-8.15% and 93.4-108% with a precision of 3.47-8.22%. The inter-day accuracy for l-THP and naltrexone was 91.2-102% with a CV of 2.46-8.06% and 91.5-97.8% with a CV of 3.29-8.92%, respectively. CONCLUSION: The assay has been used for pharmacokinetic studies of l-THP and naltrexone in the rat.

Effects of l-tetrahydropalmatine on different stages of conditioned place preference in ketamine dependent rats and the mechanism of DA / 中国法医学杂志

Objective To observe the effects of Levo-tetrahydropalmatine(l-THP)on the expression,regression and relapse of conditioned place preference(CPP)in ketamine induced rats,and to detect the content of dopamine(DA)in the striatum(caudate putamen,CPu)of the rat brain at different time points.Methods Ketamine addiction rat model was established by CPP.The effects of l-THP on the expression,regression and relapse of ketamine induced rat CPP were investigated using CPP score as the index.The content of DA in CPu of rats was determined by ultra-performance liquid chromatography coupled to tandem mass spectrometry(UPLC-MS/MS)after ketamine administration and l-THP intervention at 30 min,60 min,90 min,120 min and 150 min.Results It indicated that 1-THP could decrease the expression of CPP in ketamine induced rats,promote the process of CPP resolution and inhibit the process of relapse.In addition,l-THP combined with ketamine administration significantly inhibited the ketamine-induced increase in DA content in the CPu of the rats.Conclusion The mechanism of l-THP inhibiting the reward effect of ketamine may be related to blocking DA receptors and reducing the release of DA neurotransmitters.l-THP has potential implications for the treatment of ketamine addiction.

Levo-tetrahydropalmatine ameliorates neuropathic pain by inhibiting the activation of the Clec7a-MAPK/NF-κB-NLRP3 inflammasome axis.

BACKGROUND: Because of the complex pathogenesis of neuropathic pain (NP), the therapeutic efficacy of existing drugs is not satisfactory. Accumulating studies have indicated that neuroinflammation may play a key role in NP onset and progression. Levo-tetrahydropalmatine (l-THP) has been extensively used for relieving chronic pain for decades. However, its potential mechanisms against NP have not yet been fully elucidated. PURPOSE: Exploring and elucidating the therapeutic effect and pharmacological mechanism of l-THP in treating NP. METHODS: RNA-seq and bioinformatics analyses were carried out to identify effective target profiling of I-THP in chronic constrictive injury (CCI) rats. The I-THP related hub targets and signaling pathways were obtained via bioinformatics analysis, then subjected to in-depth analyses through experiments in vivo. A gain-of-function study further confirmed the role of Clec7a in l-THP-mediated pain relief. Finally, the interaction between l-THP and Clec7a was verified through molecular docking and surface plasmon resonance (SPR). RESULTS: l-THP treatment effectively alleviated mechanical and thermal allodynia in NP model rats. Functionally, the I-THP effective targets were mainly enriched in inflammatory response-related pathways. Furthermore, Clec7a-MAPK/NF-κB-NLRP3 inflammasome axis was selected as one of the potential pathways of l-THP against NP. Mechanically, l-THP markedly reduced CCI-induced Clec7a overexpression, significantly inhibited the Clec7a-triggered phosphorylation of MAPK and NF-κB-p65, and decreased the expression of pyroptosis-related protein NLRP3 and Caspase-1-p20. The analgesic effect of l-THP on NP was partly eliminated when transfecting the overexpression vector virus pLVSO5Clec7a. Importantly, molecular docking and SPR data revealed that l-THP directly binds with the Clec7a protein. CONCLUSION: This study is the first to indicate that l-THP may exert an analgesic effect through inhibiting neuroinflammation via the Clec7a-MAPK/NF-κB-NLRP3 inflammasome axis, supporting the clinical utility of l-THP in NP therapy.

Combined Levo-tetrahydropalmatine and diphenyleneiodonium chloride enhances antitumor activity in hepatocellular carcinoma.

Metabolic dysregulation is a hallmark of hepatocellular carcinoma (HCC). AMPK is a crucial hub of metabolic regulation during cancer progression. We show that phytochemical Levo-tetrahydropalmatine (THP) activates AMPK-dependent autophagy to downregulate the mitochondrial respiration and glycolysis. Consequently, THP significantly decreased cell viability in two HCC cell lines, BEL-7402 and SMMC-7721. Similarly, NOX4 inhibitor diphenyleneiodonium chloride (DPI) induces concomitant downregulation of the mitochondrial and glycolytic metabolism. In contrast to THP, cells are less sensitive to proliferation inhibition induced by DPI treatment as compared to THP treatment did. Combined treatment of THP and DPI was found to be more efficacious in killing cancer cells than either of the agents treated individually. Indeed, the co-operative effect by the THP-DPI combination improves the pro-apoptotic activity in response to the energy depletion as outlined by a drastic decrease in ATP levels. Therapeutic regime significantly reduced the tumor growth in mice. Importantly, this is realized without causing systemic toxicity to other organs. Collectively, our work shows that the combinatorial therapy of autophagy activator THP and NOX4 inhibitor DPI may be considered as a therapeutic avenue against HCC.

[Levo-tetrahydropalmatine promotes apoptosis of human hepatocellular carcinoma through switching energy metabolism phenotype via upregulation of mitochondrial reactive oxygen species].

Mitochondrion is an important organelle that maintains cellular homeostasis and plays a crucial role in determining cell fate. The present study investigated the effect of levo-tetrahydropalmatine(THP) on autophagic flux and energy metabolism phenotype of human hepatocellular carcinoma(HCC) SMMC-7721 and BEL-7402 cells. SMMC-7721 and BEL-7402 cells were treated with THP(100 µmol·L~(-1)) with or without N-acetyl-L-cysteine(NAC, 10 µmol·L~(-1)) for 24 h. The mitochondrial reactive oxygen species(mtROS) was detected by flow cytometry(FCM) with MitoSOX probe and fluorescence microscopy, respectively. Thereafter, autophagic flux was detected by FCM with CYTO-ID probe, and the protein levels of microtubule-associated protein 1 A/1 B-light chain 3-Ⅰ(LC3Ⅰ), LC3Ⅱ, and phosphorylated AMP-activated protein kinase(p-AMPK)/AMPK were measured by Western blot. Mitochondrial respiration was examined by Seahorse XFp assay and cell proliferation by a system. Annexin V-FITC and PI/RNase staining was employed to detect apoptosis of SMMC-7721 and BEL-7402 cells treated with THP and/or NAC. Subsequently, membrane potential was measured with MitoTracker Red CMXRos. Compared with the control group, THP promoted mtROS production and THP combined with NAC attenuated the autophagic flux increase induced by THP alone in SMMC-7721 and BEL-7402 cells. When cells were co-treated with THP and chloroquine(CQ, an autophagy inhibitor), THP further increased mtROS and apoptosis. In addition, THP significantly reduced mitochondrial respiration in terms of mitochondrial basal respiration, ATP production, and maximal respiration. Meanwhile, THP significantly reduced the proliferation index and mitochondrial membrane potential of HCC cells accompanied by the increased apoptosis. This study demonstrates that the up-regulation of mtROS by THP significantly promotes HCC cell autophagy(protective autophagy) and impairs mitochondrial respiration through reprogramming energy metabolism, ultimately inducing the mitochondria-mediated apoptosis of SMMC-7721 and BEL-7402 cells.

Levo-tetrahydropalmatine inhibits α4ß2 nicotinic receptor response to nicotine in cultured SH-EP1 cells.

Nicotine, a major component of tobacco, is highly addictive and acts on nicotinic acetylcholine receptors (nAChRs) to stimulate reward-associated circuits in the brain. It is well known that nAChRs play critical roles in mediating nicotine reward and addiction. Current FDA-approved medications for smoking cessation are the antidepressant bupropion and the nicotinic partial agonist varenicline, yet both are limited by adverse side effects and moderate efficacy. Thus, development of more efficacious medications with fewer side effects for nicotine addiction and smoking cessation is urgently needed. l-Tetrahydropalmatine (l-THP) is an active ingredient of the Chinese medicinal herb Corydalis ambigua that possesses rich neuropharmacological actions on dopamine (DA) receptors in the mesocorticolimbic dopaminergic reward pathway. L-THP has been explored as anti-addiction treatments for drug abuse including nicotine. However, the targets and mechanisms of l-THP-caused anti-nicotine effects are largely unknown. In this study we address this question by elucidating the effects of l-THP on human neuronal nAChRs using patch-clamp recordings. Human neuronal α4ß2-nAChRs were heterologously expressed in SH-EP1 human epithelial cells. Bath application of nicotine (0.1-100 µM) induced inward currents, co-application of l-THP (3 µM) inhibited nicotine-induced currents in the transfected cells. L-THP-caused inhibition was concentration-dependent (the EC50 values for inhibiting the peak and steady-state current were 18 and 2.1 µM, respectively) and non-competitive. Kinetic analysis of the whole-cell currents showed that l-THP slowed rising time and accelerated decay time constants. L-THP specifically modulated α4ß2-nAChRs, as it did not affect α7-nAChRs or α1*-nAChRs (muscle type). Interestingly, two putative α4ß2-nAChR isoforms, namely sazetidine A-activated, high-sensitive one (α42ß23-nAChR) and cytisine-activated, low-sensitive one (α43ß22-nAChR) were pharmacologically separated, and the low-sensitive one was more susceptible to l-THP inhibition than the high-sensitive one. In conclusion, we demonstrate that l-THP blocks neuronal α4ß2-nAChR function, which may underlie its inhibition on nicotine addiction.

AMPK-Mediated Metabolic Switching Is High Effective for Phytochemical Levo-Tetrahydropalmatine (l-THP) to Reduce Hepatocellular Carcinoma Tumor Growth.

Targeting cancer cell metabolism has been an attractive approach for cancer treatment. However, the role of metabolic alternation in cancer is still unknown whether it functions as a tumor promoter or suppressor. Applying the cancer gene-metabolism integrative network model, we predict adenosine monophosphate-activated protein kinase (AMPK) to function as a central hub of metabolic landscape switching in specific liver cancer subtypes. For the first time, we demonstrate that the phytochemical levo-tetrahydropalmatine (l-THP), a Corydalis yanhusuo-derived clinical drug, as an AMPK activator via autophagy-mediated metabolic switching could kill the hepatocellular carcinoma HepG2 cells. Mechanistically, l-THP promotes the autophagic response by activating the AMPK-mTOR-ULK1 and the ROS-JNK-ATG cascades and impairing the ERK/AKT signaling. All these processes ultimately synergize to induce the decreased mitochondrial oxidative phosphorylation (OXPHOS) and mitochondrial damage. Notably, silencing AMPK significantly inhibits the autophagic flux and recovers the decreased OXPHOS metabolism, which results in HepG2 resistance to l-THP treatment. More importantly, l-THP potently reduces the growth of xenograft HepG2 tumor in nude mice without affecting other organs. From this perspective, our findings support the conclusion that metabolic change is an alternative approach to influence the development of HCC.

Levo-tetrahydropalmatine: A new potential medication for methamphetamine addiction and neurotoxicity.

Levo-tetrahydropalmatine (l-THP) is mainly derived from the dried tuber of the Papaveraceae plant Corydalis, also called Corydalis B, which is a drug with analgesic, hypnotic, sedative and other effects. Methamphetamine (METH) belongs to the central nervous stimulant and is a highly addictive drug. It is an urgent problem to study the mechanism of methamphetamine neurotoxicity and to search for the therapeutic targets of the METH addiction. This review is aimed to discuss the pharmacological mechanism and the protective effects of l-THP on METH-induced neurotoxicity, and to explore the therapeutic prospects of l-THP for METH addiction to provide an innovative application of l-THP in clinic. It was found that exposure to METH leads to the compulsive drug-seeking and drug-taking behavior, which is ultimately resulted in METH addiction and neurotoxicity. L-THP has the inhibitory effects on the incidence, maintenance and relapse of METH addiction. L-THP can effectively enhance the plasticity of nerve cells and improve the function of nerve cells where brain-derived neurotrophic factor (BDNF) and its pathways play a protective role. Therefore, l-THP has the potential to become an important therapeutic drug for METH addiction and neurotoxicity.

Levo-tetrahydropalmatine attenuates the acquisition of fentanyl-induced conditioned place preference and the changes in ERK and CREB phosphorylation expression in mice.

Levo-tetrahydropalmatine (L-THP) is the main active ingredient of Corydalis and Stephania and is widely used for its sedative, analgesic, and neuroleptic effects. Though L-THP is an antagonist of dopamine receptors and has been proven to be effective in treating drug addiction, its effect on fentanyl-induced reward learning still remains unclear. This experiment was designed to investigate the effects of L-THP on fentanyl-induced rewarding behavior through conditioned place preference (CPP) in mice. Western blot assays were used to dissect the accompanying changes in the phosphorylation of extracellular signal-regulated kinase (ERK) and cAMP response element binding protein (CREB) in related brain regions, including the hippocampus (Hip), caudate putamen (CPu), prefrontal cortex (PFC), and nucleus accumbens (NAc), which may mediate the effects of L-THP on fentanyl-induced CPP. The results revealed that fentanyl could induce CPP in mice at doses of 0.025 mg/kg, 0.05 mg/kg, 0.1 mg/kg, and 0.2 mg/kg, and L-THP could attenuate the acquisition of fentany-induced CPP at a dose of 10.0 mg/kg. The levels of p-ERK and p-CREB of the saline+fentanyl group (0.05 mg/kg) increased significantly in the Hip, NAc, and PFC compared to the saline+saline group. Furthermore, L-THP (10.0 mg/kg) co-administered with fentanyl during conditioning prevented the enhanced phosphorylation of ERK and CREB in the Hip, NAc, and PFC. Our research revealed that L-THP could suppress the rewarding properties of fentanyl-induced CPP, the inhibitory effect may be related to the suppression of ERK and CREB phosphorylation in the Hip, NAc, and PFC of mice. Thus, L-THP may have therapeutic potential for fentanyl addiction.

PPARγ/NF-κB and TGF-ß1/Smad pathway are involved in the anti-fibrotic effects of levo-tetrahydropalmatine on liver fibrosis.

Liver fibrosis is a necessary stage in the development of chronic liver diseases to liver cirrhosis. This study aims to investigate the anti-fibrotic effects of levo-tetrahydropalmatine (L-THP) on hepatic fibrosis in mice and cell models and its underlying mechanisms. Two mouse hepatic fibrosis models were generated in male C57 mice by intraperitoneal injection of carbon tetrachloride (CCl4) for 2 months and bile duct ligation (BDL) for 14 days. Levo-tetrahydropalmatine was administered orally at doses of 20 and 40 mg/kg. An activated LX2 cell model induced by TGF-ß1 was also generated. The results showed that levo-tetrahydropalmatine alleviated liver fibrosis by inhibiting the formation of extracellular matrix (ECM) and regulating the balance between TIMP1 and MMP2 in the two mice liver fibrosis models and cell model. Levo-tetrahydropalmatine inhibited activation and autophagy of hepatic stellate cells (HSCs) by modulating PPARγ/NF-κB and TGF-ß1/Smad pathway in vivo and in vitro. In conclusion, levo-tetrahydropalmatine attenuated liver fibrosis by inhibiting ECM deposition and HSCs autophagy via modulation of PPARγ/NF-κB and TGF-ß1/Smad pathway.

Effects of Yuanhu- Zhitong tablets on alcohol-induced conditioned place preference in mice.

OBJECTIVES: This study aimed at determining the synergistic effects of Yuanhu Zhitong tablets (YHZTP) on alcohol-induced conditioned place preference (CPP) in mice, in addition, the intervention mechanism was preliminarily explored based on traditional Chinese Medicine (TCM) network pharmacology on alcohol addiction. METHODS: Alcohol-induced CPP mice were used to evaluate the effects of either YHZTP or levo-tetrahydropalmatine (l-THP) plus imperatorin (IMP) administration on animal behavior. The network pharmacological strategy was used to establish the "compound-target" and "disease-drug-target" network. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed on the shared targets between the compound and the disease. Twelve algorithms on CytoHubba were used to find the hub genes that were verified by qPCR. RESULTS: Systemic administration (2 g/kg, i.p.) of ethanol (EtOH) to mice was used to induce CPP. YHZTP On its own did not induce CPP or conditioned place aversion (CPA) at the doses of 0.3 g/kg or 0.6 g/kg (i.g.), but attenuated the acquisition and expression of EtOH-induce CPP in mice. In addition, YHZTP (0.3 or 0.6 g/kg) did not exhibit any effect on the motor activity of mice. Acquisition of alcohol-induced CPP was blocked by a combination of l-THP (5 mg/kg, i.g.) + IMP (2.5 mg/kg, i.g.) or l-THP (10 mg/kg, i.g.) + IMP (5 mg/kg, i.g.). However, the combination of l-THP (2.5 mg/kg, i.g.) + IMP (1.25 mg/kg, i.g.) or mono-administration of l-THP and IMP did not exhibit any effect on alcohol-induced CPP. YHZTP was also shown to reverse the up-regulation of Gabra1, Ptgs2, Mapk1, Mapk8, Mapk14, Nr3c, Prkca and Sirt1 genes and the down-regulation of Hhtr2a and Drd2 genes in the prefrontal cortex of EtOH induced CPP mice. These genes were associated with neuroactive ligand-receptor interactions, activation of the sphingolipid, calcium, cAMP, ErbB, NF-kappa B and MAPK signaling pathways. CONCLUSION: YHZTP inhibits EtOH-induced CPP behavior in mice while a combination of l-THP and IMP exerts a synergistic effect on the reduction of EtOH-induced CPP. Possible pharmacological mechanisms include inhibition of the expression of inflammatory factors and regulation of neurotransmitter receptor levels. Therefore, YHZTP is a novel candidate for the treatment of alcohol addiction.

Levo-tetrahydropalmatine attenuates methamphetamine reward behavior and the accompanying activation of ERK phosphorylation in mice.

Levo-tetrahydropalmatine (L-THP) is the main active ingredient of traditional Chinese herbal medicine Corydalis and Stephania, which have been used for sedative, neuroleptic, and analgesic purposes. Previous studies have demonstrated that L-THP has antagonistic activation on dopamine receptors. Despite its effectiveness on treating drug addiction, L-THP's underlying molecular mechanisms in modulating methamphetamine (METH) reward behavior remain unclear. In order to clarify the mechanisms behind, we designed an experiment of conditioned place preference (CPP) to investigate the effects of L-THP on METH-induced CPP in mice. We then dissected the underlying molecular mechanisms of L-THP in modulating METH-induced CPP by evaluating accompanying changes in expression of phosphorylated extracellular signal-regulated kinase (p-ERK) in reward-relevant brain regions, including nucleus accumbens (NAc), prefrontal cortex (PFc), caudate putamen (CPu), and hippocampus (Hip), which may mediate the effects of L-THP on METH-induced CPP. The results showed that 1.0 mg/kg METH could induce obvious CPP in mice; 10.0 mg/kg L-THP could significantly attenuate METH-induced CPP in mice, though it could not induce CPP or conditioned place aversion by itself. Moreover, the levels of p-ERK in NAc and PFc of the METH group were significantly higher than that of the saline group. Although there was no evident difference between the levels of p-ERK of the L-THP group with that of the saline group, the levels of p-ERK in NAc and PFc of the M + T group were significantly lower than that of the METH group. There was no striking difference among the levels of p-ERK in CPu and Hip of all experimental groups. Our research suggested that NAc and PFc function as circuits contributing to METH addiction, and the activation of the ERK phosphorylation plays an important role in the mechanisms of METH addiction. Besides, L-THP significantly decreased ERK phosphorylation's high expression induced by METH, which suggested that the inhibitory effect of L-THP on modulating METH reward behavior may be related to the suppression of ERK phosphorylation in NAc and PFc of mice. In conclusion, L-THP could suppress the reward properties of METH, therefore, it may be a promising candidate for the treatment of METH addiction.

Dopamine D1 and D2 receptors mediate analgesic and hypnotic effects of l-tetrahydropalmatine in a mouse neuropathic pain model.

RATIONALE: Levo-tetrahydropalmatine (l-THP), an active ingredient of Corydalis yanhusuo, has been reported to be a partial agonist for dopamine D1 receptors (D1R) and an antagonist for D2R. Although it has been safely used clinically in China for decades as an analgesic with sedative/hypnotic properties, there are few studies that address the mechanisms by which l-THP exerts its beneficial effects in chronic pain-induced sleep disturbance. OBJECTIVES: To investigate the effects and mechanisms of l-THP on sleep disturbance in a neuropathic pain-like condition. METHODS: A mouse model of chronic neuropathic pain induced by partial sciatic nerve ligation (PSNL) was employed. The antinociceptive and hypnotic effects of l-THP were evaluated by measurement of mechanical allodynia, thermal hyperalgesia, and electroencephalogram (EEG) recordings in PSNL mice. Pharmacological approaches and c-Fos expression were used to clarify the mechanisms of l-THP. RESULTS: Intraperitoneal injection of l-THP at 5 and 10 mg/kg not only significantly increased the mechanical threshold by 134.4% and 174.8%, and prolonged the thermal latency by 49.4% and 69.2%, but also increased non-rapid eye movement sleep by 17.5% and 29.6%, and decreased sleep fragmentation in PSNL mice, compared with the vehicle control. Moreover, the antinociceptive effect of l-THP was prevented by D1R antagonist SCH23390 or D2R agonist quinpirole; meanwhile, the hypnotic effect of l-THP was blocked by quinpirole rather than by SCH23390. Immunohistochemistry demonstrated that l-THP inhibited c-Fos overexpression induced by PSNL in the cingulate cortex and the periaqueductal gray. CONCLUSIONS: These findings indicated that l-THP exerted analgesic effects by agonism D1R and antagonism D2R, and the antagonism of D2R mediated the hypnotic effect of l-THP in PSNL mice.

Protective effects of levo-tetrahydropalmatine on hepatic ischemia/reperfusion injury are mediated by inhibition of the ERK/NF-κB pathway.

BACKGROUND: Hepatic ischemia/reperfusion (IR) injury is a common medical phenomenon that occurs during a number of clinical conditions, such as liver transplantation, severe injuries, and shock. In our study, we determined the protective functions of levo-tetrahydropalmatine (L-THP) on hepatic IR injury in mice by inhibiting the ERK/NF-κB signaling pathway. METHOD: BALB/c mice were randomly divided into six groups as follows: normal control (NC); sham; L-THP (40 mg/kg); IR; L-THP (20 mg/kg) + IR; and L-THP (40 mg/kg) + IR. Liver tissues and sera were collected at three time points after reperfusion (2, 8, and 24 h). The liver enzyme, inflammatory factor, and other protein levels in the serum and liver tissues were detected. RESULTS: L-THP pretreatment alleviated hepatocyte injury caused by IR and reduced the production of proinflammatory cytokines, such as IL-6 and TNF-α. Furthermore, L-THP could inhibit the ERK/NF-κB signaling pathway to attenuate hepatocyte apoptosis and autophagy. And the protective effect of L-THP is positively correlated with its dose. CONCLUSION: L-THP protects the liver from IR injury by inhibiting the release of inflammatory factors and alleviating liver cell apoptosis and autophagy. The protective functions of L-THP may be partly based on the downregulation of the ERK/NF-κB pathway.

Levo-tetrahydropalmatine inhibits the acquisition of ketamine-induced conditioned place preference by regulating the expression of ERK and CREB phosphorylation in rats.

Levo-tetrahydropalmatine (l-THP) is an alkaloid purified from the Chinese herbs Corydalis and Stephania and has been used in many traditional Chinese herbal preparations for its sedative, analgesic and hypnotic properties. Previous studies demonstrated that l-THP has antagonistic activity on dopamine receptors; thus, it may have potential therapeutic effects on drug abuse. However, whether l-THP affects ketamine-induced conditioned place preference (CPP) remains unclear. Therefore, the present study was designed to evaluate the effects of l-THP on the rewarding behavior of ketamine through CPP. Results revealed that ketamine (5, 10 and 15mg/kg) induced CPP in rats. Furthermore, Ketamine (10mg/kg) promoted the phosphorylation of extracellular-regulated kinase (ERK) and cAMP responsive element binding protein (CREB) in the hippocampus (Hip) and caudate putamen (CPu), but not in the prefrontal cortex (PFc). l-THP (20mg/kg) co-administered with ketamine during conditioning inhibited the acquisition of ketamine-induced CPP in rats. Furthermore, l-THP (20mg/kg) prevented the enhanced phosphorylation of ERK and CREB in CPu and Hip. These results suggest that l-THP has potential therapeutic effects on ketamine-induced CPP. The underlying molecular mechanism may be related to its inhibitory effect on ERK and CREB phosphorylation in Hip and CPu. The present data supports the potential use of l-THP for the treatment of ketamine addiction.

l-tetrahydropalmatine reduces nicotine self-administration and reinstatement in rats.

BACKGROUND: The negative consequences of nicotine use are well known and documented, however, abstaining from nicotine use and achieving abstinence poses a major challenge for the majority of nicotine users trying to quit. l-Tetrahydropalmatine (l-THP), a compound extracted from the Chinese herb Corydalis, displayed utility in the treatment of cocaine and heroin addiction via reduction of drug-intake and relapse. The present study examined the effects of l-THP on abuse-related effects of nicotine. METHODS: Self-administration and reinstatement testing was conducted. Rats trained to self-administer nicotine (0.03 mg/kg/injection) under a fixed-ratio 5 schedule (FR5) of reinforcement were pretreated with l-THP (3 or 5 mg/kg), varenicline (1 mg/kg), bupropion (40 mg/kg), or saline before daily 2-h sessions. Locomotor, food, and microdialysis assays were also conducted in separate rats. RESULTS: l-THP significantly reduced nicotine self-administration (SA). l-THP's effect was more pronounced than the effect of varenicline and similar to the effect of bupropion. In reinstatement testing, animals were pretreated with the same compounds, challenged with nicotine (0.3 mg/kg, s.c.), and reintroduced to pre-extinction conditions. l-THP blocked reinstatement of nicotine seeking more effectively than either varenicline or bupropion. Locomotor data revealed that therapeutic doses of l-THP had no inhibitory effects on ambulatory ability and that l-THP (3 and 5 mg/kg) significantly blocked nicotine induced hyperactivity when administered before nicotine. In in-vivo microdialysis experiments, l-THP, varenicline, and bupropion alone elevated extracellular dopamine (DA) levels in the nucleus accumbens shell (nAcb). CONCLUSIONS: Since l-THP reduces nicotine taking and blocks relapse it could be a useful alternative to varenicline and bupropion as a treatment for nicotine addiction.

Characterization of human metabolism and disposition of levo-tetrahydropalmatine: Qualitative and quantitative determination of oxidative and conjugated metabolites.

Levo-tetrahydropalmatine (l-THP) is a tetrahydroprotoberberine isoquinoline alkaloid and has been used as an analgesic agent in China for over 50 years. Recent studies revealed that l-THP was effective in the treatment of drug addiction. However, the plasma metabolic profile, mass balance and clearance pathways of l-THP in human remain unknown. In the present study, an analytical strategy was developed for qualitative and quantitative investigation of metabolism and disposition of l-THP in human. Detection and structural characterization of l-THP metabolites were performed using liquid chromatography-quadrupole time-of-flight mass spectrometry. Selected major metabolites in plasma, urine and feces determined by liquid chromatography with UV detection were further quantified using a triple quadruple mass spectrometry and reference standards. A total of 20 metabolites were identified, most of which were formed via demethylation, mono-hydroxylation, and glucuronidation and sulfonation of desmethyl metabolites. Five major metabolites accounted for over 10% of the parent drug concentration in plasma. Major urinary and fecal metabolites and the parent drug that were monitored for 72h accounted for 46.3% of the dose, while only 0.16% of the dose was the unchanged drug. Multiple demethylations followed by glucuronide and sulfate conjugations and renal excretion were the major drug clearance pathways of l-THP in human.

Levo-tetrahydropalmatine, a natural, mixed dopamine receptor antagonist, inhibits methamphetamine self-administration and methamphetamine-induced reinstatement.

Despite the high prevalence of methamphetamine (METH) use, no FDA-approved pharmacological treatment is currently available for individuals with a METH addiction. Levo-tetrahydropalmatine (l-THP) is an alkaloid substance derived from corydalis and stephania that has been used in traditional Asian medicine for its analgesic, sedative and hypnotic properties. Previous pharmacological studies of l-THP indicated that it not only binds to D1 and D2 receptors but also has a low affinity for D3 receptors and may function as an antagonist. The unique pharmacological profile of l-THP suggests that it may have potential therapeutic effects on drug addiction; however, the effects of l-THP in individuals with METH addictions are largely unknown. In this study, we investigated the effects of l-THP on METH self-administration and METH-induced reinstatement. In our experiments, l-THP (1.25, 2.50 and 5.00 mg/kg, i.p.) decreased METH self-administration under the fixed-ratio 1 schedule. l-THP (2.50 and 5.00 mg/kg, i.p) also prevented the METH-induced reinstatement of METH-seeking behaviors. Interestingly, l-THP (1.25 and 2.50mg/kg, i.p) did not affect locomotor activity following METH injection (1mg/kg) suggesting that the observed effects of l-THP (2.50mg/kg) on METH-induced reinstatement were not due to motor impairments. Thus, l-THP (a natural, mixed dopamine (DA) receptor antagonist) attenuates METH self-administration and METH-induced reinstatement.

Pharmacokinetic analysis of levo-tetrahydropalmatine in rabbit plasma by rapid sample preparation and liquid chromatography-tandem mass spectrometry.

A rapid extraction method was developed and validated for levo-tetrahydropalmatine (l-THP) determination in rabbit plasma by liquid chromatography tandem-mass spectrometry (LC-MS/MS). The sample preparation included a single-step acetonitrile extraction and salting out liquid-liquid partitioning from the water in plasma with MgSO4. Berberine was used as internal standard. The mass spectrometry source was negative electrospray ionization. The method showed good performance in the concentration range from 5 to 200ngmL(-1). The limit of quantification (LOQ) was 1ngmL(-1). The method was successfully applied to a pharmacokinetic study in rabbit comparing the two drug formulation of l-THP including the raw material and the self-microemulsifying drug delivery system pellet.

Inhibition of acid-sensing ion channels by levo-tetrahydropalmatine in rat dorsal root ganglion neurons.

Levo-tetrahydropalmatine (l-THP), a main bioactive Chinese herbal constituent from the genera Stephania and Corydalis, has been in use in clinical practice for years in China as a traditional analgesic agent. However, the mechanism underlying the analgesic action of l-THP is poorly understood. This study shows that l-THP can exert an inhibitory effect on the functional activity of native acid-sensing ion channels (ASICs), which are believed to mediate pain caused by extracellular acidification. l-THP dose dependently decreased the amplitude of proton-gated currents mediated by ASICs in rat dorsal root ganglion (DRG) neurons. l-THP shifted the proton concentration-response curve downward, with a decrease of 40.93% ± 8.45% in the maximum current response to protons, with no significant change in the pH0.5 value. Moreover, l-THP can alter the membrane excitability of rat DRG neurons to acid stimuli. It significantly decreased the number of action potentials and the amplitude of the depolarization induced by an extracellular pH drop. Finally, peripherally administered l-THP inhibited the nociceptive response to intraplantar injection of acetic acid in rats. These results indicate that l-THP can inhibit the functional activity of ASICs in dissociated primary sensory neurons and relieve acidosis-evoked pain in vivo, which for the first time provides a novel peripheral mechanism underlying the analgesic action of l-THP.