N, N-dimethyltryptamine forms oxygenated metabolites via CYP2D6 - an in vitro investigation.

N, N-dimethyltryptamine (DMT) is a psychedelic compound that has shown potential in the treatment of depression. Aside from the primary role of monoamine oxidase A (MAO-A) in DMT metabolism, the metabolic pathways are poorly understood. Increasing this understanding is an essential aspect of ensuring safe and efficacious use of DMT.This work aimed to investigate the cytochrome 450 (CYP) mediated metabolism of DMT by incubating DMT with recombinant human CYP enzymes and human liver microsomes (HLM) followed by analysis using high-resolution mass spectrometry for metabolite identification.DMT was rapidly metabolised by CYP2D6, while stable with all other investigated CYP enzymes. The metabolism of DMT in HLM was reduced after inclusion of harmine and SKF-525A whereas quinidine did not affect the metabolic rate, likely due to MAO-A residues present in HLM. Analysis of the CYP2D6 incubates showed formation of mono-, di- and tri-oxygenated metabolites, likely as a result of hydroxylation on the indole core.More research is needed to investigate the role of this metabolic pathway in vivo and any pharmacological activity of the proposed metabolites. Our findings may impact on safety issues following intake of ayahuasca in slow CYP2D6 metabolizers or with concomitant use of CYP2D6 inhibitors.

Development and application of a highly sensitive LC-MS/MS method for simultaneous quantification of N,N-dimethyltryptamine and two of its metabolites in human plasma.

A highly sensitive LC-MS/MS method for the quantification of N,N-dimethyltryptamine (DMT) and its metabolites indole-3-acetic acid and DMT N-oxide in human plasma has been developed and validated. Chromatography was performed using a diphenyl column with gradient elution (0.1% formic acid in methanol/water). The mass spectrometer was operated in multiple reaction monitoring mode. A methanolic solution containing internal standards 2-methylindole 3-acetic acid and deuterated DMT, was added to plasma samples, followed by protein precipitation with acetonitrile. The samples were centrifuged and supernatants transferred to new tubes and evaporated to dryness before reconstitution in aqueous mobile phase. The method was validated with regards to accuracy, precision, sensitivity, selectivity, recovery, matrix effects, stability, carry-over and dilution integrity. The validated linear range was 0.25-200 nM for DMT and 15-250 nM for DMT N-oxide. For the endogenous compound indole-3-acetic acid a different approach was taken due to its significant presence in blank samples. The change in signal response from a blank sample was used when constructing the calibration curve with linearity demonstrated between elevations of 500-5000 nM above the blank. Applicability of the described method was demonstrated through analysis of plasma samples from healthy volunteers having received intravenous injections of DMT. The presented method for rapid and sensitive quantification of DMT and its metabolites in human plasma can be applied to future studies aiming to characterize DMT disposition and its relationship to immediate psychedelic or long-term antidepressive effects.

Factors Affecting the Pharmacokinetics of Pyrazinamide and Its Metabolites in Patients Coinfected with HIV and Implications for Individualized Dosing.

Pyrazinamide is a first-line drug used in the treatment of tuberculosis. High exposure to pyrazinamide and its metabolites may result in hepatotoxicity, whereas low exposure to pyrazinamide has been correlated with treatment failure of first-line antitubercular therapy. The aim of this study was to describe the pharmacokinetics and metabolism of pyrazinamide in patients coinfected with tuberculosis and HIV. We further aimed to identify demographic and clinical factors which affect the pharmacokinetics of pyrazinamide and its metabolites in order to suggest individualized dosing regimens. Plasma concentrations of pyrazinamide, pyrazinoic acid, and 5-hydroxypyrazinamide from 63 Rwandan patients coinfected with tuberculosis and HIV were determined by liquid chromatography-tandem mass spectrometry followed by nonlinear mixed-effects modeling. Females had a close to 50% higher relative pyrazinamide bioavailability compared to males. The distribution volumes of pyrazinamide and both metabolites were lower in patients on concomitant efavirenz-based HIV therapy. Furthermore, there was a linear relationship between serum creatinine and oral clearance of pyrazinoic acid. Simulations indicated that increasing doses from 25 mg/kg of body weight to 35 mg/kg and 50 mg/kg in females and males, respectively, would result in adequate exposure with regard to suggested thresholds and increase probability of target attainment to >0.9 for a MIC of 25 mg/liter. Further, lowering the dose by 40% in patients with high serum creatinine would prevent accumulation of toxic metabolites. Individualized dosing is proposed to decrease variability in exposure to pyrazinamide and its metabolites. Reducing the variability in exposure may lower the risk of treatment failure and resistance development.

Simultaneous quantification of four first line antitubercular drugs and metabolites in human plasma by hydrophilic interaction chromatography and tandem mass spectrometry.

Co-infection of tuberculosis in HIV-patients is a major health concern worldwide and especially so in Sub-Saharan Africa. To enhance the study of potential drug-drug interactions when simultaneously treating the two infections, a liquid chromatography tandem mass spectrometry method was developed for the quantitation of the four first line anti-tuberculosis drugs isoniazid, rifampicin, pyrazinamide, ethambutol and four of their major metabolites in human plasma. Analytes were extracted from 200 µL of plasma using a sequential liquid-liquid extraction with ethyl acetate at neutral and acidic pH. The combined extracts were analyzed by liquid chromatography with mass spectrometric detection in a multiple reaction monitoring mode. The chromatographic separation was performed on a hydrophilic interaction column using a stepwise gradient with two mobile phases consisting of water with 0.3% formic acid and methanol with 0.3% formic acid, respectively. The total run time of each analysis was 4 min. The lower limit of quantification applied was 40 ng/mL for ethambutol, acetylisoniazid and 25-desacetylrifampicin, 60 ng/mL for 5-hydroxypyrazinamide, 80 ng/mL for isoniazid and isonicotinic acid, 200 ng/mL for rifampicin and 320 ng/mL for pyrazinamide. The method was validated according to US Food and Drug Administration guidance. The method exhibited adequate accuracy (87.1-114.9%), precision (CV < 12.8%) and specificity. Recovery and matrix effect were consistent (CV < 11.9%). The extracted samples were stable in the autosampler at 8 °C for up to 24 h as well as after three freeze-thaw cycles (recovery > 86.3%). The method has been shown to be robust for the analysis of the stated drugs and metabolites in human plasma obtained from 73 patients receiving these four first line anti-tuberculosis drugs.

Inhibition of CYP3A by Antimalarial Piperaquine and Its Metabolites in Human Liver Microsomes With IVIV Extrapolation.

The potential of the antimalarial piperaquine and its metabolites to inhibit CYP3A was investigated in pooled human liver microsomes. CYP3A activity was measured by liquid chromatography-tandem mass spectrometry as the rate of 1'-hydroxymidazolam formation. Piperaquine was found to be a reversible, potent inhibitor of CYP3A with the following parameter estimates (%CV): IC50 = 0.76 µM (29), Ki = 0.68 µM (29). In addition, piperaquine acted as a time-dependent inhibitor with IC50 declining to 0.32 µM (28) during 30-min pre-incubation. Time-dependent inhibitor estimates were kinact = 0.024 min-1 (30) and KI = 1.63 µM (17). Metabolite M2 was a highly potent reversible inhibitor with estimated IC50 and Ki values of 0.057 µM (17) and 0.043 µM (3), respectively. M1 and M5 metabolites did not show any inhibitory properties within the limits of assay used. Average (95th percentile) simulated in vivo areas under the curve of midazolam increased 2.2-fold (3.7-fold) on the third which is the last day of piperaquine dosing, whereas for its metabolite M2, areas under the curve of midazolam increased 7.7-fold (13-fold).

LC-MS/MS quantitation of antimalarial drug piperaquine and metabolites in human plasma.

PURPOSE: This study aimed to develop a sensitive, quantitative assay for the antimalarial piperaquine (PQ) and its metabolites M1 and M2 in human plasma. RESULTS: Analytes were gradiently separated on a C18 column and detected with a Sciex API 4000 MS/MS with an ESI source operated in the positive ion mode with deuterated PQ as internal standard. The response was linear in the range 3.9-2508nM with a runtime of 7.0min per sample. The method was applied to clinical samples from healthy volunteers. CONCLUSION: This LC-MS/MS method for the simultaneous quantitation of PQ and two of its metabolites in plasma may prove helpful for assessment of metabolite safety issues in vivo.

A high-throughput LC-MS/MS assay for quantification of artesunate and its metabolite dihydroartemisinin in human plasma and saliva.

AIM: Saliva is an alternative sampling matrix to plasma, offering a noninvasive technique, but requires a highly sensitive bioanalytical method. MATERIALS & METHODS: An API 3000 triple quadrupole mass spectrometer with an electrospray ionization source operated in the positive ion mode was used for the analysis. RESULTS: A high-throughput LC-MS/MS method using SPE for the quantification of artesunate and dihydroartemisinin in plasma and saliva has been optimized and validated according to US FDA guidelines. For both analytes the LLOQ was determined to 5 ng/ml and the calibration range was 5-1000 ng/ml for artesunate and 5-2000 ng/ml for dihydroartemisinin. CONCLUSION: For the first time, a bioanalytical method for determination of artesunate and dihydroartemisinin in human saliva has been described, showing possible applicability in clinical saliva samples in addition to plasma samples.

Effects of artemisinin antimalarials on Cytochrome P450 enzymes in vitro using recombinant enzymes and human liver microsomes: potential implications for combination therapies.

1. Cytochrome P450 enzyme system is the most important contributor to oxidative metabolism of drugs. Modification, and more specifically inhibition, of this system is an important determinant of several drug-drug interactions (DDIs). 2. Effects of the antimalarial agent artemisinin and its structural analogues, artemether, artesunate and dihydroartemisinin, on seven of the major human liver CYP isoforms (CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6 and 3A4) were evaluated using recombinant enzymes (fluorometric assay) and human liver microsomes (LC-MS/MS analysis). Inhibitory potency (IC50) and mechanisms of inhibition were evaluated using nonlinear regression analysis. In vitro-in vivo extrapolation using the [I]/Ki ratio was applied to predict the risk of DDI in vivo. 3. All compounds tested inhibited the enzymatic activity of CYPs, mostly through a mixed type of inhibition, with CYP1A2, 2B6, 2C19 and 3A4 being affected. A high risk of interaction in vivo was predicted if artemisinin is coadministrated with CYP1A2 or 2C19 substrates. 4. With respect to CYP1A2 inhibition in vivo by artemisinin compounds, our findings are in line with previously published data. However, reported risks of interaction may be overpredicted and should be interpreted with caution.

A rapid and selective HPLC-UV method for the quantitation of efavirenz in plasma from patients on concurrent HIV/AIDS and tuberculosis treatments.

Owing to heterogeneity in therapeutic response, efavirenz is of research and clinical interest. There is a need to quantitate it using noncostly and selective methods. A method for efavirenz quantitation in plasma containing HIV and tuberculosis drugs was developed. Chromatographic separation was carried out using a C18 column. The mobile phase consisted of 0.1% formic acid and acetonitrile, and was pumped at a flow rate of 0.3 mL/min. Efavirenz and ritonavir (internal standard) were monitored at 247 nm. Plasma proteins were precipitated by centrifugation. The analysis time was 6 min. The response was linear (r = 0.9997). The accuracy ranged between 98 and 115% (intraday) and between 99 and 117% (interday). The precision ranged from 1.670 to 4.087% (intraday) and from 3.447 to 13.347% (interday). Recovery ranged from 98 to 132%. Stability ranged between 99 and 123%. The selectivity was proven by analysis of drugs used for the management of HIV/AIDS and tuberculosis. Plasma sample analysis showed an efavirenz retention time of 5.57 min and a peak plasma concentration of 2.4 µg/mL occurring at 2 h. This method is rapid and selective, and thus suitable for monitoring efavirenz in patients with HIV/AIDS alone or co-infected with tuberculosis in a less resourced setting.

Biliary excretion of ximelagatran and its metabolites and the influence of erythromycin following intraintestinal administration to healthy volunteers.

The biliary excretion of the oral thrombin inhibitor ximelagatran and its metabolites was investigated by using duodenal aspiration in healthy volunteers following intraintestinal dosing. In the first investigation, radiolabeled [(14)C]ximelagatran was administered, enabling quantification of the biliary excretion and identification of metabolites in the bile. In the second study, the effect of erythromycin on the biliary clearance of ximelagatran and its metabolites was investigated to clarify the reported ximelagatran-erythromycin interaction. Approximately 4% of the intraintestinal dose was excreted into bile with ximelagatran and its active form, melagatran, being the most abundant compounds. Four novel ximelagatran metabolites were identified in bile (<0.1% of dose). Erythromycin changed the pharmacokinetics of ximelagatran and its metabolites, with an elevated ximelagatran (78% increase), OH-melagatran (89% increase), and melagatran (86% increase) plasma exposure and higher peak plasma concentrations of the compounds being measured. In parallel, the biliary clearance was moderately reduced. The results suggest that inhibition of hepatobiliary transport is a likely mechanism for the interaction between erythromycin and ximelagatran. Furthermore, the study demonstrated the value of direct bile sampling in humans for the identification of primary biliary metabolites.

Influence of erythromycin on the pharmacokinetics of ximelagatran may involve inhibition of P-glycoprotein-mediated excretion.

A pharmacokinetic interaction between erythromycin and ximelagatran, an oral direct thrombin inhibitor, was demonstrated in this study in healthy volunteers. To investigate possible interaction mechanisms, the effects of erythromycin on active transport mediated by P-glycoprotein (P-gp) in vitro in Caco-2 and P-gp-over-expressing Madin-Darby canine kidney-human multidrug resistance-1 cell preparations and on biliary excretion of melagatran in rats were studied. In healthy volunteers (seven males and nine females; mean age 24 years) receiving a single dose of ximelagatran 36 mg on day 1, erythromycin 500 mg t.i.d. on days 2 to 5, and a single dose of ximelagatran 36 mg plus erythromycin 500 mg on day 6, the least-squares mean estimates (90% confidence intervals) for the ratio of ximelagatran with erythromycin to ximelagatran given alone were 1.82 (1.64-2.01) for the area under the concentration-time curve and 1.74 (1.52-2.00) for the maximum plasma concentration of melagatran, the active form of ximelagatran. Neither the slope nor the intercept of the melagatran plasma concentration-effect relationship for activated partial thromboplastin time statistically significantly differed as a function of whether or not erythromycin was administered with ximelagatran. Ximelagatran was well tolerated regardless of whether it was administered with erythromycin. Erythromycin inhibited P-gp-mediated transport of both ximelagatran and melagatran in vitro and decreased the biliary excretion of melagatran in the rat. These results indicate that the mechanism of the pharmacokinetic interaction between oral ximelagatran and erythromycin may involve inhibition of transport proteins, possibly P-gp, resulting in decreased melagatran biliary excretion and increased bioavailability of melagatran.

Characterisation of the human liver in vitro metabolic pattern of artemisinin and auto-induction in the rat by use of nonlinear mixed effects modelling.

AIMS: The aims of the study were to characterise the metabolic pattern of artemisinin in human and rat liver microsomes and to assess the magnitude of auto-induction in the rat. METHODS: (14)C-artemisinin was incubated with human liver microsomes and with liver microsomes from rats pretreated with oral artemisinin or placebo. The metabolic fate of (14)C-artemisinin in microsomes from human B-lymphoblastoid cell lines transformed with CYP2A6, CYP2B6 and CYP3A4 was also investigated. The human liver microsome data and the rat liver microsomes data were analysed by nonlinear mixed effects modelling and naïve pooling using NONMEM, respectively. RESULTS: Four metabolites were radiometrically detected in experiments with rat liver microsomes. The model that best described the data involved three primary metabolites of which one metabolite was further metabolised to a secondary metabolite. The formation of the four metabolites was induced 2.8, 7.2, 4.8 and 2.5-fold, respectively, in liver microsomes from rats pre-treated with artemisinin. Three metabolites were formed in human liver microsomes; having the same retention times as three of the metabolites formed in the rat. The final model consisted of two primary metabolites and a secondary metabolite with CYP2B6 and CYP2A6 influencing the formation rates of the major and minor primary metabolites, respectively. CONCLUSIONS: CYP2B6 and CYP2A6 activities described variability in the formation of the major and minor primary metabolites, respectively, in human liver microsomes. All artemisinin metabolic pathways in rat liver microsomes were induced in artemisinin pretreated animals. We suggest modelling as a method for the discrimination and detection of more complex metabolic patterns from in vitro metabolism rate data.

Absorption, distribution, metabolism, and excretion of ximelagatran, an oral direct thrombin inhibitor, in rats, dogs, and humans.

The absorption, metabolism, and excretion of the oral direct thrombin inhibitor, ximelagatran, and its active form, melagatran, were separately investigated in rats, dogs, and healthy male human subjects after administration of oral and intravenous (i.v.) single doses. Ximelagatran was rapidly absorbed and metabolized following oral administration, with melagatran as the predominant compound in plasma. Two intermediates (ethyl-melagatran and OH-melagatran) that were subsequently metabolized to melagatran were also identified in plasma and were rapidly eliminated. Melagatran given i.v. had relatively low plasma clearance, small volume of distribution, and short elimination half-life. The oral absorption of melagatran was low and highly variable. It was primarily renally cleared, and the renal clearance agreed well with the glomerular filtration rate. Ximelagatran was extensively metabolized, and only trace amounts were renally excreted. Melagatran was the major compound in urine and feces after administration of ximelagatran. Appreciable quantities of ethyl-melagatran were also recovered in rat, dog, and human feces after oral administration, suggesting reduction of the hydroxyamidine group of ximelagatran in the gastrointestinal tract, as demonstrated when ximelagatran was incubated with feces homogenate. Polar metabolites in urine and feces (all species) accounted for a relatively small fraction of the dose. The bioavailability of melagatran following oral administration of ximelagatran was 5 to 10% in rats, 10 to 50% in dogs, and about 20% in humans, with low between-subject variation. The fraction of ximelagatran absorbed was at least 40 to 70% in all species. First-pass metabolism of ximelagatran with subsequent biliary excretion of the formed metabolites account for the lower bioavailability of melagatran.