Harmaline toxicity on dorsal striatal neurons and its role in tremor.

Harmaline is one of the ß-carboline derivative compounds that is widely distributed in the food chain and human tissues. Harmine, a dehydrogenated form of harmaline, appeared to have a higher concentration in the brain, and appeared to be elevated in essential tremor (ET) and Parkinson's disease. Exogenous harmaline exposure in high concentration has myriad consequences, including inducing tremor, and causing neurodegeneration of Purkinje cells in the cerebellum. Harmaline-induced tremor is an established animal model for human ET, but its underlying mechanism is still controversial. One hypothesis posits that the inferior olive-cerebellum pathway is involved, and CaV3.1 T-type Ca2+ channel is a critical target of action. However, accumulating evidence indicates that tremor can be generated without disturbing T-type channels. This implies that additional neural circuits or molecular targets are involved. Using in vitro slice Ca2+-imaging and patch clamping, we demonstrated that harmaline reduced intracellular Ca2+ and suppressed depolarization-induced spiking activity of medium spiny striatal neurons (MSN), and this effect of harmaline can be partially attenuated by sulpiride (5 µM). In addition, the frequencies of spontaneous excitatory post-synaptic currents (sEPSCs) on MSNs were also significantly attenuated. Furthermore, the induced tremor in C57BL/6 J mice by harmaline injections (i.p. 12.5-18 mg/kg) was also shown to be attenuated by sulpiride (20 mg/kg). This series of experiments suggests that the dorsal striatum is a site of harmaline toxic action and might contribute to tremor generation. The findings also provide evidence that D2 signaling might be a part of the mechanism underlying essential tremor.

Targeting inhibition of microtubule affinity regulating kinase 4 by Harmaline: Strategy to combat Alzheimer's disease.

Microtubule-affinity regulating kinase 4 (MARK4) is linked with the development of cancer, diabetes and neurodegenerative diseases. Due to its direct role in the hyperphosphorylation of tau protein, MARK4 is considered as an attractive target to fight Alzheimer's disease (AD) and neuroinflammation. In the present study, we have selected Harmaline (HAR), an alkaloid of Paganum harmala, to investigate its MARK4 inhibitory potential and its binding mechanism. Molecular docking and fluorescence binding studies were carried out to estimate the binding affinity of the HAR with the MARK4. We observed an excellent binding affinity of HAR to the MARK4 (K = 107 M-1), further complemented by isothermal titration calorimetric measurements. In addition, HAR significantly inhibits the kinase activity of MARK4 (IC50 value of 4.46 µM). Structural investigations suggested that HAR binds to the active site pocket and forms several non-covalent interactions with biologically important residues of MARK4. All-atom molecular dynamics simulation studies further advocated that the MARK4-HAR complex is stabilized throughout the trajectory of 200 ns and causes a little conformational change. All these findings suggest that HAR is a potential MARK4 inhibitor that can be implicated in managing MARK4-associated diseases, including AD.

Heme peroxidases are responsible for the dehydrogenation and oxidation metabolism of harmaline into harmine.

Harmaline and harmine are ß-carboline alkaloids with effective pharmacological effects. Harmaline can be transformed into harmine after oral administration. However, enzymes involved in the metabolic pathway remain unclear. In this study, harmaline was incubated with rat liver microsomes (RLM), rat brain microsomes (RBM), blood, plasma, broken blood cells, and heme peroxidases including horseradish peroxidase (HRP), lactoperoxidase (LPO), and myeloperoxidase (MPO). The production of harmine was determined by a validated UPLC-ESI-MS/MS method. Results showed that heme peroxidases catalyzed the oxidative dehydrogenation of harmaline. All the reactions were in accordance with the Hill equation. The reaction was inhibited by ascorbic acid and excess H2O2. The transformation of harmaline to harmine was confirmed after incubation with blood, plasma, and broken blood cells, rather than RLM and RBM. Harmaline was incubated with blood, plasma, and broken cells liquid for 3 h, and the formation of harmine became stable. Results indicated an integrated metabolic pathway of harmaline, which will lay foundation for the oxidation reaction of dihydro-ß-carboline. Moreover, the metabolic stability of harmaline in blood should not be ignored when the pharmacokinetics study of harmaline is carried out.

Ultrastructural and hormonal changes related to harmaline-induced treatment in Arabidopsis thaliana (L.) Heynh. root meristem.

Harmaline is an indole alkaloid with demonstrated phytotoxicity and recognized pharmacological applications. However, no information is available concerning its mode of action on plant metabolism. Therefore, the present work evaluated bioherbicide mode of action of harmaline on plant metabolism of Arabidopsis thaliana (L.) Heynh. Harmaline induced a strong inhibitory activity on root growth of treated seedlings, reaching IC50 and IC80 values of 14 and 29 µM, respectively. Treated roots were shorter and thicker than control and were characterized by a shorter root meristem size and an increase of root hairs production. Harmaline induced ultrastructural changes such as increment of cell wall thickness, higher density and condensation of mitochondria and vacuolization, appearance of cell wall deposits, increment of Golgi secretory activity and higher percentage of aberrant nuclei. The ethylene inhibitor AgNO3 reversed high root hair appearance and increment of root thickness, and pTCSn::GFP transgenic line showed fluorescence cytokinin signal in stele zone after harmaline treatment that was absent in control, whereas the auxin signal in the transgenic line DR5 was significantly reduced by the treatment. All these results suggest that the mode of action of harmaline could be involving auxin, ethylene and cytokinin synergic/antagonistic action.

In-silico Prediction of the Beta-carboline Alkaloids Harmine and Harmaline as Potent Drug Candidates for the Treatment of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is a progressive neurodegenerative disease manifested by core symptoms of loss of motor control and postural instability. Loss of dopaminergic neurons is the cause of PD, thus enhancing dopamine level by pharmacological treatment is one of the key treatment strategies for PD. However, the limitations of current treatment strategies open the possibility of novel drug candidates for the treatment of PD. OBJECTIVE: To investigate the anti-PD potential of Harmine and Harmaline. We aim to evaluate the therapeutic potential of Harmine and Harmaline by in-silico approaches; molecular docking, pharmacokinetic and Prediction of Activity Spectra for Substances (PASS) analysis were used for evaluating the therapeutic potential of Harmine and Harmaline and standard drug levodopa (L-DOPA). METHODS: Auto dock vina was used for molecular docking of all three compounds against D2- and D3- dopamine receptors. The pharmacokinetics (PKs) and toxicity profile were predicted by pkCSM, and the pharmacological activity was predicted by PASS analysis. RESULTS: Molecular docking showed a higher binding affinity of Harmine and Harmaline as compared to L-DOPA, and these results were supported by in-silico pharmacokinetic and toxicity profiling. Moreover, PASS analysis showed anti-PD activity of Harmine and Harmaline. CONCLUSION: Harmine and Harmaline exhibit higher binding affinity towards D2- and D3- dopamine receptors compared to L-DOPA, and PKs and toxicity profile support their potential as drug candidates for PD therapy.

Anxiolytic and antidepressant effects of ACPA and harmaline co-treatment.

Depression and anxiety disorders are among the most common illnesses and a close relationship between them has been found. Because the psychotropic effects and abuse liability of cannabis prevent its therapeutic application in depression and anxiety states, we decided to investigate the effects of the combination of ineffective doses of cannabinoid CB1 receptor agonist arachidonylcyclopropylamide (ACPA) and ß-carbolines on anxiety- and depression-related behaviors in male NMRI mice. Anxiety- and depression-related behaviors were assesses using elevated plus maze (EPM) and forced swim test (FST), respectively. Intraperitoneal administration of ACPA (1 mg/kg) decreased the percentage of time spent in the open-arms (%OAT) and the number of entries to the open-arms (OAE) in the EPM, indicating an anxiogenic-like effect. ACPA also decreased immobility time in the FST compared to the control group, suggesting an antidepressant-like effect. ß-carbolines including harmane (5 and 10 mg/kg), norharmane (5 mg/kg) and harmaline (2.5 and 5 mg/kg) produced an anxiogenic-like response, while the highest dose of harmane or harmaline and the middle dose of norharmane induced an antidepressant-like behavior. Furthermore, co-administration of a subthreshold dose of ACPA (0.5 mg/kg) and harmaline (1.25 mg/kg), but not harmane or norharmane (both at the dose of 2.5 mg/kg), caused anxiolytic- and antidepressant-like behaviors and decreased locomotor activity. Our findings suggest a therapeutic potential for combined ineffective doses of ACPA and harmaline on anxiety- and depression-related processes.

Interspecies metabolic diversity of harmaline and harmine in in vitro 11 mammalian liver microsomes.

The ß-carboline alkaloids harmaline and harmine are widely present in hallucinogenic plants with great potential for treating depression, Parkinson's disease, and Alzheimer's disease. The present study was to elucidate metabolic difference of harmaline and harmine in 11 mammalian liver microsomes in order to quantitate species-specific metabolic profiles. Using the probe substrate reaction, the enzymatic activities for 8 CYP450 isozymes of 11 liver microsomes were characterized. Combining ultra performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q/TOF-MS) and ultra performance liquid chromatography combined with electrospray ionization quadrupole tandem mass spectrometry (UPLC-ESI-MS/MS) methods, 18 metabolites for harmaline and 11 for harmine were identified. The metabolism patterns differences of them presented discrepancy in the quality and quantity of metabolites. It was notable that O-sulfate conjugation was detected in all species except sheep. The intrinsic clearance CLint, LM values for the metabolites harmine and harmol in rabbits (37.5 and 42.4 µL/min/mg) were higher than those in other animals, while dogs (16.2 and 16.7 µL/min/mg) and humans (16.0 and 16.3 µL/min/mg) exhibited similar in vitro metabolic clearance. These observations suggested that harmaline and harmine were rapidly metabolized in liver microsomes of rat, mouse, and rabbit; moderately metabolized in human and dog; while weakly metabolized in sheep. Comprehensive analysis of the metabolism indicated that dogs and humans showed considerable similarity in the elimination of parent drugs, metabolic profiles, and catalytic processes. To summarize, these findings illustrated that in vitro studies of harmaline and harmine metabolic profiles in different species are helpful for the proper selection and interpretation of animal models for pharmacological and toxicological evaluation, and will ultimately provide useful guidance for the development of ß-carboline alkaloids. Copyright © 2016 John Wiley & Sons Ltd.

DNA binding and apoptotic induction ability of harmalol in HepG2: Biophysical and biochemical approaches.

Harmalol administration caused remarkable reduction in proliferation of HepG2 cells with GI50 of 14.2 µM, without showing much cytotoxicity in embryonic liver cell line, WRL-68. Data from circular dichroism (CD) and differential scanning calorimetric (DSC) analysis of harmalol-CT DNA complex shows conformational changes with prominent CD perturbation and stabilization of CT DNA by 8 °C. Binding constant and stoichiometry was calculated using the above biophysical techniques. The Scatchard plot constructed from CD data showed cooperative binding, from which the cooperative binding affinity (K'ω) of 4.65 ± 0.7 × 10(5) M(-1), and n value of 4.16 were deduced. The binding parameter obtained from DSC melting data was in good agreement with the above CD data. Furthermore, dose dependent apoptotic induction ability of harmalol was studied in HepG2 cells using different biochemical assays. Generation of ROS, DNA damage, changes in cellular external and ultramorphology, alteration of membrane, formation of comet tail, decreased mitochondrial membrane potential and a significant increase in Sub Go/G1 population made the cancer cell, HepG2, prone to apoptosis. Up regulation of p53 and caspase 3 further indicated the apoptotic role of harmalol.

Microwave-assisted extraction of three bioactive alkaloids from Peganum harmala L. and their acaricidal activity against Psoroptes cuniculi in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Peganum harmala L. is a perennial herbaceous, glabrous plant that grows in semi-arid conditions, steppe areas and sandy soils. It is used to treat fever, diarrhoea, subcutaneous tumours, arthralgia, rheumatism, cough, amnesia and parasitic diseases in folk medicines. In this paper, we aimed to develop a simpler and faster method for the extraction of three alkaloids from Peganum harmala L. than other conventional methods by optimizing the parameters of a microwave-assisted extraction (MAE) method, and to investigate the acaricidal activities of three compounds against Psoroptes cuniculi. MATERIALS AND METHODS: After optimizing the operating parameters with the single factor experiment and a Box-Behnken design combined with a response-surface methodology, a MAE method was developed for extracting the alkaloids from the seeds, and a high-performance liquid chromatography was used to quantify these compounds. An in vitro experiments were used to study the acaricidal activities. RESULTS: The optimal conditions of MAE method were as follows: liquid-to-solid ratio 31.3:1mL/g, ethanol concentration 75.5%, extraction time 10.1min, temperature 80.7°C, and microwave power 600W. Compared to the heat reflux extraction (HRE, 60min) and the ultrasonic-assisted extraction (UAE, 30min) methods, MAE method require the shortest time (10min) and obtain the highest yield of three compounds (61.9mg/g). Meanwhile, the LT50 values for the vasicine (1.25 and 2.5mg/mL), harmaline (1.25 and 2.5mg/mL), harmine (1.25 and 2.5mg/mL) and MAE extract (100mg/mL) against Psoroptes cuniculi were 12.188h, 9.791h, 11.994h, 10.095h, 11.293h, 9.273h and 17.322h, respectively. CONCLUSIONS: The MAE method developed exhibited the highest extraction yield within the shortest time and thus could be used to extract the active compounds from Peganum harmala L. on an industrial basis. As the active compounds of Peganum harmala L., vasicine, harmalin and harmine presented the marked acaricidal activities against Psoroptes cuniculi, and could be widely applied for the treatments of acariasis in animals.

Sequence specific binding of beta carboline alkaloid harmalol with deoxyribonucleotides: binding heterogeneity, conformational, thermodynamic and cytotoxic aspects.

BACKGROUND: Base dependent binding of the cytotoxic alkaloid harmalol to four synthetic polynucleotides, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) was examined by various photophysical and calorimetric studies, and molecular docking. METHODOLOGY/PRINCIPAL FINDINGS: Binding data obtained from absorbance according to neighbor exclusion model indicated that the binding constant decreased in the order poly(dG-dC).poly(dG-dC)>poly(dA-dT).poly(dA-dT)>poly(dA).poly(dT)>poly(dG).poly(dC). The same trend was shown by the competition dialysis, change in fluorescence steady state intensity, stabilization against thermal denaturation, increase in the specific viscosity and perturbations in circular dichroism spectra. Among the polynucleotides, poly(dA).poly(dT) and poly(dG).poly(dC) showed positive cooperativity where as poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT) showed non cooperative binding. Isothermal calorimetric data on the other hand showed enthalpy driven exothermic binding with a hydrophobic contribution to the binding Gibbs energy with poly(dG-dC).poly(dG-dC), and poly(dA-dT).poly(dA-dT) where as harmalol with poly(dA).poly(dT) showed entropy driven endothermic binding and with poly(dG).poly(dC) it was reported to be entropy driven exothermic binding. The study also tested the in vitro chemotherapeutic potential of harmalol in HeLa, MDA-MB-231, A549, and HepG2 cell line by MTT assay. CONCLUSIONS/SIGNIFICANCE: Studies unequivocally established that harmalol binds strongly with hetero GC polymer by mechanism of intercalation where the alkaloid resists complete overlap to the DNA base pairs inside the intercalation cavity and showed maximum cytotoxicity on HepG2 with IC50 value of 14 µM. The results contribute to the understanding of binding, specificity, energetic, cytotoxicity and docking of harmalol-DNA complexation that will guide synthetic efforts of medicinal chemists for developing better therapeutic agents.

Metabolic pathways of the psychotropic-carboline alkaloids, harmaline and harmine, by liquid chromatography/mass spectrometry and NMR spectroscopy.

The ß-carboline alkaloids, harmaline and harmine, are present in hallucinogenic plants Ayahuasca and Peganum harmala, and in a variety of foods. In order to establish the metabolic pathway and bioactivities of endogenous and xenobiotic bioactive ß-carbolines, high-performance liquid chromatography, coupled with mass spectrometry, was used to identify these metabolites in human liver microsomes (HLMs) in vitro and in rat urine and bile samples after oral administration of the alkaloids. Three metabolites of harmaline and two of harmine were found in the HLMs. Nine metabolites for harmaline and seven metabolites for harmine, from the rat urine and bile samples, were identified. Among them, four in vivo metabolites were isolated and fully characterised by NMR analysis. For the first time, harmaline is shown transforming to harmine by oxidative dehydrogenation in rat. Five metabolic pathways were therefore proposed, namely, oxidative dehydrogenation, 7-O-demethylation, hydroxylation, O-glucuronide conjugation and O-sulphate conjugation.

Harmine is a potent antimalarial targeting Hsp90 and synergizes with chloroquine and artemisinin.

Previous studies have shown an antimalarial effect of total alkaloids extracted from leaves of Guiera senegalensis from Mali in West Africa. We independently observed that the beta-carboline alkaloid harmine obtained from a natural product library screen inhibited Plasmodium falciparum heat shock protein 90 (PfHsp90) ATP-binding domain. In this study, we confirmed harmine-PfHsp90-specific affinity using surface plasmon resonance analysis (dissociation constant [K(d)] of 40 µM). In contrast, the related compound harmalol bound human Hsp90 (HsHsp90) (K(d) of 224 µM) more tightly than PfHsp90 (K(d) of 7,010 µM). Site-directed mutagenesis revealed that Arg98 in PfHsp90 is essential for harmine selectivity. In keeping with our model indicating that Hsp90 inhibition affords synergistic combinations with existing antimalarials, we demonstrated that harmine potentiates the effect of chloroquine and artemisinin in vitro and in the Plasmodium berghei mouse model. These findings have implications for the development of novel therapeutic combinations that are synergistic with existing antimalarials.

Metabolism and disposition of N,N-dimethyltryptamine and harmala alkaloids after oral administration of ayahuasca.

Ayahuasca is an Amazonian psychotropic plant tea obtained from Banisteriopsis caapi, which contains ß-carboline alkaloids, chiefly harmine, harmaline and tetrahydroharmine. The tea usually incorporates the leaves of Psychotria viridis or Diplopterys cabrerana, which are rich in N,N-dimethyltryptamine (DMT), a psychedelic 5-HT(2A/1A/2C) agonist. The ß-carbolines reversibly inhibit monoamine-oxidase (MAO), effectively preventing oxidative deamination of the orally labile DMT and allowing its absorption and access to the central nervous system. Despite increased use of the tea worldwide, the metabolism and excretion of DMT and the ß-carbolines has not been studied systematically in humans following ingestion of ayahuasca. In the present work, we used an analytical method involving high performance liquid chromatography (HPLC)/electrospray ionization (ESI)/selected reaction monitoring (SRM)/tandem mass spectrometry(MS/MS) to characterize the metabolism and disposition of ayahuasca alkaloids in humans. Twenty-four-hour urine samples were obtained from 10 healthy male volunteers following administration of an oral dose of encapsulated freeze-dried ayahuasca (1.0 mg DMT/kg body weight). Results showed that less than 1% of the administered DMT dose was excreted unchanged. Around 50% was recovered as indole-3-acetic acid but also as DMT-N-oxide (10%) and other MAO-independent compounds. Recovery of DMT plus metabolites reached 68%. Harmol, harmalol, and tetrahydroharmol conjugates were abundant in urine. However, recoveries of each harmala alkaloid plus its O-demethylated metabolite varied greatly between 9 and 65%. The present results show the existence in humans of alternative metabolic routes for DMT other than biotransformation by MAO. Also that O-demethylation plus conjugation is an important but probably not the only metabolic route for the harmala alkaloids in humans.

Modulation of NMDA receptor function by inhibition of D-amino acid oxidase in rodent brain.

Observations that N-Methyl-D-Aspartate (NMDA) antagonists produce symptoms in humans that are similar to those seen in schizophrenia have led to the current hypothesis that schizophrenia might result from NMDA receptor hypofunction. Inhibition of D-amino acid oxidase (DAAO), the enzyme responsible for degradation of D-serine, should lead to increased levels of this co-agonist at the NMDA receptor, and thereby provide a therapeutic approach to schizophrenia. We have profiled some of the preclinical biochemical, electrophysiological, and behavioral consequences of administering potent and selective inhibitors of DAAO to rodents to begin to test this hypothesis. Inhibition of DAAO activity resulted in a significant dose and time dependent increase in D-serine only in the cerebellum, although a time delay was observed between peak plasma or brain drug concentration and cerebellum D-serine response. Pharmacokinetic/pharmacodynamic (PK/PD) modeling employing a mechanism-based indirect response model was used to characterize the correlation between free brain drug concentration and D-serine accumulation. DAAO inhibitors had little or no activity in rodent models considered predictive for antipsychotic activity. The inhibitors did, however, affect cortical activity in the Mescaline-Induced Scratching model, produced a modest but significant increase in NMDA receptor-mediated synaptic currents in primary neuronal cultures from rat hippocampus, and resulted in a significant increase in evoked hippocampal theta rhythm, an in vivo electrophysiological model of hippocampal activity. These findings demonstrate that although DAAO inhibition did not cause a measurable increase in D-serine in forebrain, it did affect hippocampal and cortical activity, possibly through augmentation of NMDA receptor-mediated currents.

Comparison of mibefradil and derivative NNC 55-0396 effects on behavior, cytochrome P450 activity, and tremor in mouse models of essential tremor.

NNC 55-0396 [(1S,2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2, 3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride], is a mibefradil derivative that retains potent in vitro T-type calcium channel antagonist efficacy. We compared the two compounds for behavioral toxicity, effects on cytochrome P450 activity, and efficacy against tremor in the γ-aminobutyric acid type A (GABAA) receptor subunit α1-null mouse, and the harmaline tremor model of essential tremor in wild-type mice. NNC 55-0396 was better tolerated than mibefradil in the horizontal wire test of sedation/motor function, with 3/6 failing at 300 and 30mg/kg respectively. To assess for a potential interaction with harmaline, mice were given the drugs, followed by harmaline or vehicle, and tested 30min later in the inverted wire grid test. Mibefradil exacerbated, whereas NNC 55-0396 ameliorated harmaline-induced test deficits. In mouse liver microsomes, NNC 55-0396 was a less potent inhibitor of harmaline O-demethylation than mibefradil (Ki: 0.95 and 0.29µM respectively), and also less potent at inhibiting testosterone 6-ß-hydroxylation (Ki: 0.71 and 0.12µM respectively). In the GABAA α1-null model, NNC 55-0396 but not mibefradil, (each at 20mg/kg), suppressed tremor while NNC 55-0396 at 12.5mg/kg suppressed harmaline-induced tremor by half by 20-100min, whereas mibefradil at the same dose did not significantly affect tremor. In contrast to mibefradil, NNC 55-0396 is well tolerated and suppresses tremor, and exerts less cytochrome P450 inhibition. These results suggest potential clinical utility for NNC 55-0396 or similar derivatives as a T-type calcium antagonist.

Mitochondrial dysfunction and biotransformation of ß-carboline alkaloids, harmine and harmaline, on isolated rat hepatocytes.

The cytotoxic effects and biotransformation of harmine and harmaline, which are known ß-carboline alkaloids and potent hallucinogens, were studied in freshly isolated rat hepatocytes. The exposure of hepatocytes to harmine caused not only concentration (0-0.50mM)- and time (0-3h)-dependent cell death accompanied by the formation of cell blebs and the loss of cellular ATP, reduced glutathione, and protein thiols but also the accumulation of glutathione disulfide. Of the other analogues examined, the cytotoxic effects of harmaline and harmol (a metabolite of harmine) at a concentration of 0.5mM were less than those of harmine. The loss of mitochondrial membrane potential and generation of oxygen radical species in hepatocytes treated with harmine were greater than those with harmaline and harmol. In the oxygen consumption of mitochondria isolated from rat liver, the ratios of state-3/state-4 respiration of these ß-carbolines were decreased in a concentration-dependent manner. In addition, harmine resulted in the induction of the mitochondrial permeability transition (MPT), and the effects of harmol and harmaline were less than those of harmine. At a weakly toxic level of harmine (0.25mM), it was metabolized to harmol and its monoglucuronide and monosulfate conjugates, and the amounts of sulfate rather than glucuronide predominantly increased with time. In the presence of 2,5-dichloro-4-nitrophenol (50µM; an inhibitor of sulfotransferase), harmine-induced cytotoxicity was enhanced, accompanied by decrease in the amount of harmol-sulfate conjugate, due to an increase in the amount of unconjugated harmol and the inhibition of harmine loss. Taken collectively, these results indicate that (a) mitochondria are target organelles for harmine, which elicits cytotoxicity through mitochondrial failure related to the induction of the MPT, mitochondrial depolarization, and inhibition of ATP synthesis; and (b) the toxic effects of harmine are greater than those of either its metabolite harmol or its analogue harmaline, suggesting that the onset of harmine-induced cytotoxicity may depend on the initial and/or residual concentrations of harmine rather than on those of its metabolites.

Effects of CYP2D6 status on harmaline metabolism, pharmacokinetics and pharmacodynamics, and a pharmacogenetics-based pharmacokinetic model.

Harmaline is a beta-carboline alkaloid showing neuroprotective and neurotoxic properties. Our recent studies have revealed an important role for cytochrome P450 2D6 (CYP2D6) in harmaline O-demethylation. This study, therefore, aimed to delineate the effects of CYP2D6 phenotype/genotype on harmaline metabolism, pharmacokinetics (PK) and pharmacodynamics (PD), and to develop a pharmacogenetics mechanism-based compartmental PK model. In vitro kinetic studies on metabolite formation in human CYP2D6 extensive metabolizer (EM) and poor metabolizer (PM) hepatocytes indicated that harmaline O-demethylase activity (V(max)/K(m)) was about 9-fold higher in EM hepatocytes. Substrate depletion showed mono-exponential decay trait, and estimated in vitro harmaline clearance (CL(int), microL/min/10(6)cells) was significantly lower in PM hepatocytes (28.5) than EM hepatocytes (71.1). In vivo studies in CYP2D6-humanized and wild-type mouse models showed that wild-type mice were subjected to higher and longer exposure to harmaline (5 and 15mg/kg; i.v. and i.p.), and more severe hypothermic responses. The PK/PD data were nicely described by our pharmacogenetics-based PK model involving the clearance of drug by CYP2D6 (CL(CYP2D6)) and other mechanisms (CL(other)), and an indirect response PD model, respectively. Wild-type mice were also more sensitive to harmaline in marble-burying tests, as manifested by significantly lower ED(50) and steeper Hill slope. These findings suggest that distinct CYP2D6 status may cause considerable variations in harmaline metabolism, PK and PD. In addition, the pharmacogenetics-based PK model may be extended to define PK difference caused by other polymorphic drug-metabolizing enzyme in different populations.

Various alkaloid profiles in decoctions of Banisteriopsis caapi.

Twenty nine decoctions of Banisteriopsis caapi from four different sources and one specimen of B. caapi paste were analyzed for N,N-dimethyltryptamine (DMT), tetrahydroharmine (THH), harmaline and harmine. Other plants were also used in the preparation of these products, typically Psychotria viridis, which provides DMT. There were considerable variations in alkaloid profiles, both within and between sample sources. DMT was not detected in all samples. Additional THH may be formed from both harmine and harmaline during the preparation of these products. The alkaloid composition of one decoction sample did not change significantly after standing at room temperature for 80 days, but the initial acidic pH was neutralized by natural fermentation after 50 days.

Beta-carboline and quinoline alkaloids in root cultures and intact plants of Peganum harmala.

Alkaloid profiles of root and shoot cultures, seedlings and mature plants were analysed by capillary GLC and GLC-MS. beta-Carboline alkaloids, such as harmine, harmaline dominate in normal and root cultures transformed by Agrobacterium rhizogenes, as well as in roots and fruits of the plant. In shoots, flowers and shoot cultures quinoline alkaloids such as peganine, deoxypeganine, vasicinone and deoxyvasicinone widely replace the beta-carboline alkaloids. In root cultures, the formation of beta-carboline alkaloids can be induced by methyljasmonate and several other elicitors indicating that these alkaloids are part of the reactive chemical defence system of Peganum harmala.

Microbial metabolites of harman alkaloids.

Several microorganisms showed the ability to transform the harman alkaloids, harmaline (1), harmalol (2) and harman (5). Harmaline (1) and harmalol (2) were converted by Rhodotorula rubra ATCC 20129 into the tryptamines, 2-acetyl-3-(2-acetamidoethyl)-7-methoxyindole (3) and 2-acetyl-3-(2-acetamidoethyl)-7-hydroxyindole (4), respectively. Harman (5) was biotransformed by Cunninghamella echinulata NRRL 3655 into 6-hydroxyharman (6) and harman-2-oxide (7).