Formation of multiple ion types during MALDI imaging mass spectrometry analysis of Mitragyna speciosa alkaloids in dosed rat brain tissue.

Mitragyna speciosa, more commonly known as kratom, has emerged as an alternative to treat chronic pain and addiction. However, the alkaloid components of kratom, which are the major contributors to kratom's pharmaceutical properties, have not yet been fully investigated. In this study, matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry was used to map the biodistribution of three alkaloids (corynantheidine, mitragynine, and speciogynine) in rat brain tissues. The alkaloids produced three main ion types during MALDI analysis: [M + H]+, [M - H]+, and [M - 3H]+. Contrary to previous reports suggesting that the [M - H]+ and [M - 3H]+ ion types form during laser ablation, these ion types can also be produced during the MALDI matrix application process. Several strategies are proposed to accurately map the biodistribution of the alkaloids. Due to differences in the relative abundances of the ions in different biological regions of the tissue, differences in ionization efficiencies of the ions, and potential overlap of the [M - H]+ and [M - 3H]+ ion types with endogenous metabolites of the same empirical formula, a matrix that mainly produces the [M + H]+ ion type is optimal for accurate mapping of the alkaloids. Alternatively, the most abundant ion type can be mapped or the intensities of all ion types can be summed together to generate a composite image. The accuracy of each of these approaches is explored and validated.

Pharmacokinetic and Pharmacodynamic Consequences of Cytochrome P450 3A Inhibition on Mitragynine Metabolism in Rats.

Mitragynine, an opioidergic alkaloid present in Mitragyna speciosa (kratom), is metabolized by cytochrome P450 3A (CYP3A) to 7-hydroxymitragynine, a more potent opioid receptor agonist. The extent to which conversion to 7-hydroxymitragynine mediates the in vivo effects of mitragynine is unclear. The current study examined how CYP3A inhibition (ketoconazole) modifies the pharmacokinetics of mitragynine in rat liver microsomes in vitro. The study further examined how ketoconazole modifies the discriminative stimulus and antinociceptive effects of mitragynine in rats. Ketoconazole [30 mg/kg, oral gavage (o.g.)] increased systemic exposure to mitragynine (13.3 mg/kg, o.g.) by 120% and 7-hydroxymitragynine exposure by 130%. The unexpected increase in exposure to 7-hydroxymitragynine suggested that ketoconazole inhibits metabolism of both mitragynine and 7-hydroxymitragynine, a finding confirmed in rat liver microsomes. In rats discriminating 3.2 mg/kg morphine from vehicle under a fixed-ratio schedule of food delivery, ketoconazole pretreatment increased the potency of both mitragynine (4.7-fold) and 7-hydroxymitragynine (9.7-fold). Ketoconazole did not affect morphine's potency. Ketoconazole increased the antinociceptive potency of 7-hydroxymitragynine by 4.1-fold. Mitragynine (up to 56 mg/kg, i.p.) lacked antinociceptive effects both in the presence and absence of ketoconazole. These results suggest that both mitragynine and 7-hydroxymitragynine are cleared via CYP3A and that 7-hydroxymitragynine is formed as a metabolite of mitragynine by other routes. These results have implications for kratom use in combination with numerous medications and citrus juices that inhibit CYP3A. SIGNIFICANCE STATEMENT: Mitragynine is an abundant kratom alkaloid that exhibits low efficacy at the µ-opioid receptor (MOR). Its metabolite, 7-hydroxymitragynine, is also an MOR agonist but with higher affinity and efficacy than mitragynine. Our results in rats demonstrate that cytochrome P450 3A (CYP3A) inhibition can increase the systematic exposure of both mitragynine and 7-hydroxymitragynine and their potency to produce MOR-mediated behavioral effects. These data highlight potential interactions between kratom and CYP3A inhibitors, which include numerous medications and citrus juices.

Determination of five positive control drugs in hERG external solution (buffer) by LC-MS/MS to support in vitro hERG assay as recommended by ICH S7B.

ICH S7B recommends screening for hERG channel block using patch clamp recordings to assess a drug's proarrhythmic risk. Block of the hERG channel has been associated with clinical QTC prolongation as well as the rare, but potentially fatal ventricular tachyarrhythmia Torsade de Pointes (TdP). During recording, drug concentrations perfused to the cells can deviate from nominal concentrations due to molecule-specific properties (such as non-specific binding), thereby introducing error when assessing drug potency. To account for this potential source of error, both the original ICH S7B and the newly released ICH E14/S7B Q&As guidelines call for verifying drug solutions' concentrations. Dofetilide, cisapride, terfenadine, sotalol and E-4031 are hERG blockers commonly used as positive controls to illustrate hERG assay sensitivity. The first four compounds are also clinical drugs associated with high TdP risk; therefore, their safety margins may be useful comparators to better understand an investigational product's TdP risk. Having analytical methods to quantify these five compounds in the hERG external solution that will be used for patch clamp recordings is important from a regulatory science research perspective. However, a literature search revealed no analytical methods or stability information for these molecules in the high salt, serum-free matrix that constitutes the hERG external solution. This study was conducted to develop and validate LC-MS/MS methods to quantify these 5 molecules in hERG external solution. The bioanalytical methods for these positive controls were validated as per the FDA's bioanalytical method validation guidance along with various stabilities.

Metabolism of Speciociliatine, an Overlooked Kratom Alkaloid for its Potential Pharmacological Effects.

Speciociliatine, a diastereomer of mitragynine, is an indole-based alkaloid found in kratom (Mitragyna speciosa). Kratom has been widely used for the mitigation of pain and opioid dependence, as a mood enhancer, and/or as an energy booster. Speciociliatine is a partial µ-opioid agonist with a 3-fold higher binding affinity than mitragynine. Speciociliatine has been found to be a major circulating alkaloid in humans following oral administration of a kratom product. In this report, we have characterized the metabolism of speciociliatine in human and preclinical species (mouse, rat, dog, and cynomolgus monkey) liver microsomes and hepatocytes. Speciociliatine metabolized rapidly in monkey, rat, and mouse hepatocytes (in vitro half-life was 6.6 ± 0.2, 8.3 ± 1.1, 11.2 ± 0.7 min, respectively), while a slower metabolism was observed in human and dog hepatocytes (91.7 ± 12.8 and > 120 min, respectively). Speciociliatine underwent extensive metabolism, primarily through monooxidation and O-demethylation metabolic pathways in liver microsomes and hepatocytes across species. No human-specific or disproportionate metabolites of speciociliatine were found in human liver microsomes. The metabolism of speciociliatine was predominantly mediated by CYP3A4 with minor contributions by CYP2D6.

UPLC-MS/MS method for the quantification of MCI-77, a novel sigma-1 receptor ligand, and its application to pharmacokinetic studies.

Sigma-1 receptors are involved in pain modulation, particularly in cases of nerve injury and neuropathic pain. High-affinity ligands with improved pharmacokinetic profiles are needed to further investigate the properties of these receptors and their potential as a therapeutic target. The novel compound MCI-77 is one such selective sigma-1 receptor ligand, and the purpose of this study was to characterize its preclinical pharmacokinetic parameters. An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to quantify MCI-77 in mouse plasma and brain homogenate. The method was validated for sensitivity, selectivity, linearity, accuracy, precision, stability, and dilution integrity. The method has a linearity range of 2-200 ng/mL, a short run-time of 3.2 min, and requires a low sample volume of 25 µL. A simple protein precipitation procedure was used for compound extraction. Samples were run on an Acquity UPLC BEH C18 column (1.7 µm, 2.1 × 50 mm) following a gradient elution method using a mobile phase consisting of water containing 0.1% (v/v) formic acid and acetonitrile. The method was applied to the analysis of plasma and brain homogenate samples from preclinical pharmacokinetic studies in CD-1 mice. MCI-77 exhibited high systemic clearance (8.5 ± 0.3 L/h/kg) and extensive tissue distribution indicated by a high volume of distribution (20.1 ± 0.3 L/kg). The concentration levels were consistently higher in brain samples than in plasma (brain/plasma AUC ratio 2.9), indicating its ability to cross the blood-brain barrier.

Activity of Mitragyna speciosa ("Kratom") Alkaloids at Serotonin Receptors.

Kratom alkaloids have mostly been evaluated for their opioid activity but less at other targets that could contribute to their physiological effects. Here, we investigated the in vitro and in vivo activity of kratom alkaloids at serotonin receptors (5-HTRs). Paynantheine and speciogynine exhibited high affinity for 5-HT1ARs and 5-HT2BRs, unlike the principal kratom alkaloid mitragynine. Both alkaloids produced antinociceptive properties in rats via an opioid receptor-independent mechanism, and neither activated 5-HT2BRs in vitro. Paynantheine, speciogynine, and mitragynine induced lower lip retraction and antinociception in rats, effects blocked by a selective 5-HT1AR antagonist. In vitro functional assays revealed that the in vivo 5-HT1AR agonistic effects may be due to the metabolites 9-O-desmethylspeciogynine and 9-O-desmethylpaynantheine and not the parent compounds. Both metabolites did not activate 5-HT2BR, suggesting low inherent risk of causing valvulopathy. The 5-HT1AR agonism by kratom alkaloids may contribute to the mood-enhancing effects associated with kratom use.

Oral Pharmacokinetics in Beagle Dogs of the Mitragynine Metabolite, 7-Hydroxymitragynine.

BACKGROUND AND OBJECTIVES: 7-Hydroxymitragynine (7-HMG) is an oxidative metabolite of mitragynine, the most abundant alkaloid in the leaves of Mitragyna speciosa (otherwise known as kratom). While mitragynine is a weak partial µ-opioid receptor (MOR) agonist, 7-HMG is a potent and full MOR agonist. It is produced from mitragynine by cytochrome P450 (CYP) 3A, a drug-metabolizing CYP isoform predominate in the liver that is also highly expressed in the intestine. Given the opioidergic potency of 7-HMG, a single oral dose pharmacokinetic and safety study of 7-HMG was performed in beagle dogs. METHODS: Following a single oral dose (1 mg/kg) of 7-HMG, plasma samples were obtained from healthy female beagle dogs. Concentrations of 7-HMG were determined using ultra-performance liquid chromatography coupled with a tandem mass spectrometer (UPLC-MS/MS). Pharmacokinetic parameters were calculated using a model-independent non-compartmental analysis of plasma concentration-time data. RESULTS: Absorption of 7-HMG was rapid, with a peak plasma concentration (Cmax, 56.4 ± 1.6 ng/ml) observed within 15 min post-dose. In contrast, 7-HMG elimination was slow, exhibiting a mono-exponential distribution and mean elimination half-life of 3.6 ± 0.5 h. Oral dosing of 1 mg/kg 7-HMG was well tolerated with no observed adverse events or significant changes to clinical laboratory tests. CONCLUSIONS: These results provide the first pharmacokinetic and safety data for 7-HMG in the dog and therefore contribute to the understanding of the putative pharmacologic role of 7-HMG resulting from an oral delivery of mitragynine from kratom.

Pharmacokinetics of Eleven Kratom Alkaloids Following an Oral Dose of Either Traditional or Commercial Kratom Products in Rats.

Kratom, Mitragyna speciosa Korth., is being widely consumed in the United States for pain management and the reduction of opioid withdrawal symptoms. The central nervous system (CNS) active alkaloids of kratom, including mitragynine, 7-hydroxymitragynine, and numerous additional compounds, are believed to derive their effects through opioid receptor activity. There is no literature describing the systemic exposure of many of these alkaloids after the consumption of kratom. Therefore, we have developed and validated a bioanalytical method for the simultaneous quantitation of 11 kratom alkaloids (mitragynine, 7-hydroxymitragynine, corynantheidine, speciogynine, speciociliatine, paynantheine, corynoxine, corynoxine-B, mitraphylline, ajmalicine, and isospeciofoline) in rat plasma. The validated method was used to analyze oral pharmacokinetic study samples of lyophilized kratom tea (LKT) and a marketed product, OPMS liquid shot, in rats. Among the 11 alkaloids, only mitragynine, 7-hydroxymitragynine, speciociliatine, and corynantheidine showed systemic exposure 8 h postdose, and the dose-normalized systemic exposure of these four alkaloids was higher (1.6-2.4-fold) following the administration of the commercial OPMS liquid. Paynantheine and speciogynine levels were quantifiable up to 1 h postdose, whereas none of the other alkaloids were detected. In summary, the method was successfully applied to quantify the exposure of individual kratom alkaloids after an oral dose of traditional or commercial products. This information will contribute to understanding the role of each alkaloid in the overall pharmacology of kratom and elucidating the pharmacokinetic differences between traditional and commercial kratom products.

Preclinical pharmacokinetic study of speciociliatine, a kratom alkaloid, in rats using an UPLC-MS/MS method.

Speciociliatine is a minor indole alkaloid found in kratom, a southeast Asian medicinal plant, used for centuries to increase energy, enhance mood, and mitigate pain and opioid dependence. An ultra-performance liquid chromatography tandem mass spectrometry method was developed and validated to quantify speciociliatine in rat plasma. The quantitation range was 3-600 ng/mL. The validated method was applied to a preclinical pharmacokinetic study in male Sprague-Dawley rats after 2.5 mg/kg intravenous (I.V.) and 20 mg/kg oral (P.O.) dosing. The plasma was analyzed to obtain concentration-time profiles and results were subjected to non-compartmental analysis to determine pharmacokinetic parameters including volume of distribution (6.2 ± 2.3 L/kg I.V.), clearance (0.7 ± 0.2 L/hr/kg), and absolute oral bioavailability (20.7 %). Speciociliatine had higher systemic exposure and lower clearance compared to the other kratom alkaloids mitragynine and corynantheidine. The speciociliatine pharmacokinetic parameters described here will help to better understand the overall effects reported with kratom product use.

Metabolism of a Kratom Alkaloid Metabolite in Human Plasma Increases Its Opioid Potency and Efficacy.

Kratom is widely consumed in the United States for self-treatment of pain and opioid withdrawal symptoms. Mitragynine is the most abundant alkaloid in kratom and is a µ-opioid receptor agonist. 7-Hydroxymitragynine (7-HMG) is a mitragynine metabolite that is a more potent and efficacious opioid than its parent mitragynine. 7-HMG contributes to mitragynine's antinociceptive effects in mice, but evidence suggests it may also have a higher abuse potential. This in vitro study demonstrates that 7-HMG is stable in rodent and monkey plasma but is unstable in human plasma. Surprisingly, in human plasma 7-HMG is converted to mitragynine pseudoindoxyl, an opioid that is even more potent than either mitragynine or 7-HMG. This novel metabolite is formed in human plasma to a much greater extent than in the preclinical species tested (mouse, rat, dog, and cynomolgus monkey) and due to its µ-opioid potency may substantially contribute to the pharmacology of kratom in humans to a greater extent than in other tested species.

Bioanalytical method development and pharmacokinetics of MCI-92, a sigma-1 receptor ligand.

Sigma-1 receptors are found throughout the nervous system and play a role in regulating nociception. They are highly expressed in nerve injury, making them a potential target for the treatment of neuropathic pain. Although sigma-1 receptor antagonists have been shown to have anti-nociceptive and anti-allodynic effects, improved selectivity of these ligands is needed to further investigate their potential to treat neuropathic pain. MCI-92 is a novel, selective sigma-1 receptor ligand developed to address this need. A sensitive and rapid ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantification of MCI-92 in mouse plasma and brain homogenate. A structural analog of the analyte, MCI-147, was used as the internal standard (IS). The chromatographic separation was achieved on an Acquity UPLC BEH C18 column using a mobile phase consisting of water acidified with 0.1 % v/v formic acid and acetonitrile with gradient elution over 3.2 min. The method was linear over a concentration range of 1-200 ng/mL. Multiple reaction monitoring in the positive ionization mode was used for the mass spectrometric quantitation using m/z transitions 369.2 > 126.0 for MCI-92 and 448.9 > 350.1 for the IS. The method was successfully applied to the analysis of plasma and brain samples obtained in the course of oral and intravenous pharmacokinetic studies in CD-1 mice. MCI-92 showed a high volume of distribution (11.3 ± 0.6 L/kg) and rapid clearance (6.1 ± 0.8 L/h/kg) from systemic circulation. The concentration of the MCI-92 was higher in the brain than in plasma throughout all terminal time points, indicating high blood-to-brain partitioning and slow brain clearance.

Discovery of a Highly Selective Sigma-2 Receptor Ligand, 1-(4-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-3-methyl-1H-benzo[d]imidazol-2(3H)-one (CM398), with Drug-Like Properties and Antinociceptive Effects In Vivo.

The sigma-2 receptor has been cloned and identified as Tmem97, which is a transmembrane protein involved in intracellular Ca2+ regulation and cholesterol homeostasis. Since its discovery, the sigma-2 receptor has been an extremely controversial target, and many efforts have been made to elucidate the functional role of this receptor during physiological and pathological conditions. Recently, this receptor has been proposed as a potential target to treat neuropathic pain due to the ability of sigma-2 receptor agonists to relieve mechanical hyperalgesia in mice model of chronic pain. In the present work, we developed a highly selective sigma-2 receptor ligand (sigma-1/sigma-2 selectivity ratio > 1000), 1-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-3-methyl-1H- benzo[d]imidazol-2(3H)-one (CM398), with an encouraging in vitro and in vivo pharmacological profile in rodents. In particular, radioligand binding studies demonstrated that CM398 had preferential affinity for sigma-2 receptor compared with sigma-1 receptor and at least four other neurotransmitter receptors sites, including the norepinephrine transporter. Following oral administration, CM398 showed rapid absorption and peak plasma concentration (Cmax) occurred within 10 min of dosing. Moreover, the compound showed adequate, absolute oral bioavailability of 29.0%. Finally, CM398 showed promising anti-inflammatory analgesic effects in the formalin model of inflammatory pain in mice. The results collected in this study provide more evidence that selective sigma-2 receptor ligands can be useful tools in the development of novel pain therapeutics and altogether, these data suggest that CM398 is a suitable lead candidate for further evaluation.

Pharmacokinetics and Safety of Mitragynine in Beagle Dogs.

Mitragynine is the most abundant psychoactive alkaloid derived from the leaves of Mitragyna speciosa (kratom), a tropical plant indigenous to regions of Southeast Asia. Mitragynine displays a moderate affinity to opioid receptors, and kratom is often self-prescribed to treat pain and/or opioid addiction. The purpose of this study was to investigate the safety and pharmacokinetic properties of mitragynine in the dog. Single dose oral (5 mg/kg) and intravenous (0.1 mg/kg) pharmacokinetic studies of mitragynine were performed in female beagle dogs. The plasma concentrations of mitragynine were measured using ultra-performance liquid chromatography coupled with a tandem mass spectrometer, and the pharmacokinetic properties were analyzed using non-compartmental analysis. Following intravenous administration, mitragynine showed a large volume of distribution (Vd, 6.3 ± 0.6 L/kg) and high clearance (Cl, 1.8 ± 0.4 L/h/kg). Following oral mitragynine dosing, first peak plasma (Cmax, 278.0 ± 47.4 ng/mL) concentrations were observed within 0.5 h. A potent mu-opioid receptor agonist and active metabolite of mitragynine, 7-hydroxymitragynine, was also observed with a Cmax of 31.5 ± 3.3 ng/mL and a Tmax of 1.7 ± 0.6 h in orally dosed dogs while its plasma concentrations were below the lower limit of quantification (1 ng/mL) for the intravenous study. The absolute oral bioavailability of mitragynine was 69.6%. Administration of mitragynine was well tolerated, although mild sedation and anxiolytic effects were observed. These results provide the first detailed pharmacokinetic information for mitragynine in a non-rodent species (the dog) and therefore also provide significant information for allometric scaling and dose predictions when designing clinical studies.

Regulatory sampling of industrial hemp plant samples (Cannabis sativa L.) using UPLC-MS/MS method for detection and quantification of twelve cannabinoids.

BACKGROUND: In 2018, the Farm Bill mandated the United States Department of Agriculture to develop regulations governing the cultivation, processing, and marketing of industrial hemp. Industrial hemp is defined as Cannabis sativa L. with a total Δ-9-tetrahydrocannabinol (Δ-9-THC) content ≤0.3%. Therefore, for hemp to become an agricultural commodity, it is important to regulate production by developing standard methods for sampling and testing of the plant material. METHODS: An ultra-performance liquid chromatography-tandem mass spectrometry analytical method for the quantification of twelve cannabinoids was developed. The method was applied to a regulatory sampling trial of three hemp varieties cultivated for cannabidiol (CBD) production. Two samples were taken from 28 plants with one sample being flower only while the other was a composite sample that included flowers, leaves, and stems. RESULTS: The assay method was validated for specificity, range, repeatability, reproducibility, and recovery in accordance with all applicable standards for analytical methods. The results of the regulatory study indicated a significant decrease in the concentration of total Δ-9-THC and total CBD of 0.09% and 1.32%, respectively, between a flower only and a composite sample. CONCLUSIONS: There are many factors that may influence reported total Δ-9-THC content in industrial hemp. A robust analytical method was developed to analyze hemp samples in a trial regulatory study. The results indicate that the way hemp is sampled and analyzed may influence the legality of a crop, which could have negative economic and legal consequences.

Bioanalytical method development and validation of corynantheidine, a kratom alkaloid, using UPLC-MS/MS, and its application to preclinical pharmacokinetic studies.

Corynantheidine, a minor alkaloid found in Mitragyna speciosa (Korth.) Havil, has been shown to bind to opioid receptors and act as a functional opioid antagonist, but its unique contribution to the overall properties of kratom remains relatively unexplored. The first validated bioanalytical method for the quantification of corynantheidine in rat plasma is described. The method was linear in the dynamic range from 1-500 ng/mL, requires a small plasma sample volume (25 µL), and a simple protein precipitation method for extraction of the analyte. The separation was achieved with Waters BEH C18 2.1 × 50 mm column and the 3-minute gradient of 10 mM ammonium acetate buffer (pH = 3.5) and acetonitrile as mobile phase. The method was validated in terms of accuracy, precision, selectivity, sensitivity, recovery, stability, and dilution integrity. It was applied to the analysis of the male Sprague Dawley rat plasma samples obtained during pharmacokinetic studies of corynantheidine administered both intravenously (I.V.) and orally (P.O.) (2.5 mg/kg and 20 mg/kg, respectively). The non-compartmental analysis performed in Certara Phoenix® yielded the following parameters: clearance 884.1 ±â€¯32.3 mL/h, apparent volume of distribution 8.0 ±â€¯1.2 L, exposure up to the last measured time point 640.3 ±â€¯24.0 h*ng/mL, and a mean residence time of 3.0 ±â€¯0.2 h with I.V. dose. The maximum observed concentration after a P.O. dose of 213.4 ±â€¯40.4 ng/mL was detected at 4.1 ±â€¯1.3 h with a mean residence time of 8.8 ±â€¯1.8 h. Absolute oral bioavailability was 49.9 ±â€¯16.4 %. Corynantheidine demonstrated adequate oral bioavailability, prolonged absorption and exposure, and an extensive extravascular distribution. In addition, imaging mass spectrometry analysis of the brain tissue was performed to evaluate the distribution of the compound in the brain. Corynantheidine was detected in the corpus callosum and some regions of the hippocampus.

Exploration of cytochrome P450 inhibition mediated drug-drug interaction potential of kratom alkaloids.

In vitro cytochrome P450 inhibition of major kratom alkaloids: mitragynine (MTG), speciogynine (SPG), speciocilliatine (SPC), corynantheidine (COR), 7-hydroxymitragynine (7HMG) and paynantheine (PAY) was evaluated using human liver microsomes (HLMs) to understand their drug-drug interaction potential. CYP450 isoform-specific substrates of CYP1A2, 2C8, 2C9, 2C19, 2D6, and 3A4/5 were incubated in HLMs with or without alkaloids. Preliminary CYP450 inhibition (IC50) data were generated for each of these isoforms. In addition, the type of inhibition and estimation of the inhibition constants (Ki) of MTG and COR were determined. Among the tested alkaloids, MTG and COR were potent inhibitors of CYP2D6 (IC50, 2.2 and 4.2 µM, respectively). Both MTG and COR exhibited competitive inhibition of CYP2D6 activity and the Ki were found to be 1.1 and 2.8 µM, respectively. SPG and PAY showed moderate inhibition of CYP2D6 activity. Additionally, moderate inhibitory effects by SPC, MTG, and SPG were observed on CYP2C19 activity. Interestingly, inhibition of only midazolam hydroxylase CYP3A4/5 activity by COR, PAY, and MTG was observed while no inhibitory effect was observed when testosterone was used as a probe substrate. In conclusion, MTG and COR may lead to clinically significant adverse drug interactions upon coadministration of drugs that are substantially metabolized by CYP2D6.

Bioanalytical method development and validation of MES207, a neuropeptide FF receptor antagonist, and its application in preclinical pharmacokinetics.

The nonpeptide small molecule, MES207, exhibits 17-fold preferential binding to the neuropeptide FF receptor 1 (NPFFR1) over NPFFR2 and shows antagonist functionality at NPFF receptors. In order to further the development of MES207 as a NPFFR1 probe, an UPLC-MS/MS bioanalytical method was developed and validated to quantify MES207 in rat plasma for a linearity range of 3-200 ng/mL. The method was applied in the analysis of the plasma, brain, and urine samples collected during pharmacokinetic studies in healthy male and female Sprague Dawley rats. The animals were dosed through oral gavage (50 mg/kg) and intravenously (2.5 mg/kg). Test samples were analyzed using the validated bioanalytical method to generate plasma concentration-time profiles. The results were further subjected to non-compartmental analysis using Phoenix 6.4®. MES207 exhibits a large volume of distribution (1.2 ±â€¯0.6 L), high clearance (0.8 ±â€¯0.1 L/h), and a poor oral bioavailability (1.7 ±â€¯0.4%). The compound also showed a multiple peak phenomenon with a very short absorption phase. It appears that gender does not significantly influence the differences in pharmacokinetic parameters of this NPFF probe.

Simultaneous quantification of ten key Kratom alkaloids in Mitragyna speciosa leaf extracts and commercial products by ultra-performance liquid chromatography-tandem mass spectrometry.

Kratom (Mitragyna speciosa) is a psychoactive plant popular in the United States for the self-treatment of pain and opioid addiction. For standardization and quality control of raw and commercial kratom products, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantification of ten key alkaloids, namely: corynantheidine, corynoxine, corynoxine B, 7-hydroxymitragynine, isocorynantheidine, mitragynine, mitraphylline, paynantheine, speciociliatine, and speciogynine. Chromatographic separation of diastereomers, or alkaloids sharing same ion transitions, was achieved on an Acquity BEH C18 column with a gradient elution using a mobile phase containing acetonitrile and aqueous ammonium acetate buffer (10mM, pH 3.5). The developed method was linear over a concentration range of 1-200 ng/mL for each alkaloid. The total analysis time per sample was 22.5 minutes. The analytical method was validated for accuracy, precision, robustness, and stability. After successful validation, the method was applied for the quantification of kratom alkaloids in alkaloid-rich fractions, ethanolic extracts, lyophilized teas, and commercial products. Mitragynine (0.7%-38.7% w/w), paynantheine (0.3%-12.8% w/w), speciociliatine (0.4%-12.3% w/w), and speciogynine (0.1%-5.3% w/w) were the major alkaloids in the analyzed kratom products/extracts. Minor kratom alkaloids (corynantheidine, corynoxine, corynoxine B, 7-hydroxymitragynine, isocorynantheidine) were also quantified (0.01%-2.8% w/w) in the analyzed products; however mitraphylline was below the lower limit of quantification in all analyses.

Metabolite profiling and identification of enzymes responsible for the metabolism of mitragynine, the major alkaloid of Mitragyna speciosa (kratom).

1. Mitragynine is the major indole-based alkaloid of Mitragyna speciosa (kratom). Decoctions (teas) of the plant leaves have been used traditionally for cough, diarrhoea, pain, hypertension and for the treatment of opioid addiction. In the West, kratom has become increasingly utilized for mood elevation, pain treatment and as a means of self-treating opioid addiction. 2. Metabolic pathways of mitragynine were identified in human liver microsomes (HLM) and S9 fractions. A total of thirteen metabolites were identified, four oxidative metabolites and a metabolite formed by demethylation at the 9-methoxy group were the major metabolites of mitragynine. 3. The cytochrome P450 enzymes involved in the metabolism of mitragynine were identified using selective chemical inhibitors of HLM and recombinant cytochrome P450. The metabolism of mitragynine was predominantly carried out through the CYP3A4 with minor contributions by CYP2D6 and CYP2C9. The formation of five oxidative metabolites (Met2, Met4, Met6, Met8 and Met11) was catalyzed by the CYP3A4. 4. In summary, mitragynine was extensively metabolized in HLM primarily to O-demethylated and mono-oxidative metabolites. The CYP3A4 enzyme plays a predominant role in the metabolic clearance of mitragynine and also in the formation of 7-hydroxymitragynine (Met2), a known active minor alkaloid identified in the leaf material.

Calculating the 2D motion of lumbar vertebrae using splines.

In this study we investigate the use of splines and the ICP method [Besl, P., McKay, N., 1992. A method for registration of 3d shapes. IEEE Transactions on Pattern Analysis and Machine Intelligence 14, 239-256.] for calculating the transformation parameters for a rigid body undergoing planar motion parallel to the image plane. We demonstrate the efficacy of the method by estimating the finite centre of rotation and angle of rotation from lateral flexion/extension radiographs of the lumbar spine. In an in vitro error study, the method displayed an average error of rotation of 0.44 +/- 0.45 degrees, and an average error in FCR calculation of 7.6 +/- 8.5 mm. The method was shown to be superior to that of Crisco et al. [Two-dimensional rigid-body kinematics using image contour registration. Journal of Biomechanics 28(1), 119-124.] and Brinckmann et al. [Quantification of overload injuries of the thoracolumbar spine in persons exposed to heavy physical exertions or vibration at the workplace: Part i - the shape of vertebrae and intervertebral discs - study of a yound, healthy population and a middle-aged control group. Clinical Biomechanics Supplement 1, S5-S83.] for the tests performed here. In general, we believe the use of splines to represent planar shapes to be superior to using digitised curves or landmarks for several reasons. First, with appropriate software, splines require less effort to define and are a compact representation, with most vertebra outlines using less than 30 control points. Second, splines are inherently sub-pixel representations of curves, even if the control points are limited to pixel resolutions. Third, there is a well-defined method (the ICP algorithm) for registering shapes represented as splines. Finally, like digitised curves, splines are able to represent a large class of shapes with little effort, but reduce potential segmentation errors from two dimensions (parallel and perpendicular to the image gradient) to just one (parallel to the image gradient). We have developed an application for performing all the necessary computations which can be downloaded from http://www.claritysmart.com.