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.

A peptidomimetic modulator of the CaV2.2 N-type calcium channel for chronic pain.

Transmembrane Cav2.2 (N-type) voltage-gated calcium channels are genetically and pharmacologically validated, clinically relevant pain targets. Clinical block of Cav2.2 (e.g., with Prialt/Ziconotide) or indirect modulation [e.g., with gabapentinoids such as Gabapentin (GBP)] mitigates chronic pain but is encumbered by side effects and abuse liability. The cytosolic auxiliary subunit collapsin response mediator protein 2 (CRMP2) targets Cav2.2 to the sensory neuron membrane and regulates their function via an intrinsically disordered motif. A CRMP2-derived peptide (CBD3) uncouples the Cav2.2-CRMP2 interaction to inhibit calcium influx, transmitter release, and pain. We developed and applied a molecular dynamics approach to identify the A1R2 dipeptide in CBD3 as the anchoring Cav2.2 motif and designed pharmacophore models to screen 27 million compounds on the open-access server ZincPharmer. Of 200 curated hits, 77 compounds were assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons. Nine small molecules were tested electrophysiologically, while one (CBD3063) was also evaluated biochemically and behaviorally. CBD3063 uncoupled Cav2.2 from CRMP2, reduced membrane Cav2.2 expression and Ca2+ currents, decreased neurotransmission, reduced fiber photometry-based calcium responses in response to mechanical stimulation, and reversed neuropathic and inflammatory pain across sexes in two different species without changes in sensory, sedative, depressive, and cognitive behaviors. CBD3063 is a selective, first-in-class, CRMP2-based peptidomimetic small molecule, which allosterically regulates Cav2.2 to achieve analgesia and pain relief without negative side effect profiles. In summary, CBD3063 could potentially be a more effective alternative to GBP for pain relief.

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.

Sustained Morphine Delivery Suppresses Bone Formation and Alters Metabolic and Circulating miRNA Profiles in Male C57BL/6J Mice.

Opioid use is detrimental to bone health, causing both indirect and direct effects on bone turnover. Although the mechanisms of these effects are not entirely clear, recent studies have linked chronic opioid use to alterations in circulating miRNAs. Here, we developed a model of opioid-induced bone loss to understand bone turnover and identify candidate miRNA-mediated regulatory mechanisms. We evaluated the effects of sustained morphine treatment on male and female C57BL/6J mice by treating with vehicle (0.9% saline) or morphine (17 mg/kg) using subcutaneous osmotic minipumps for 25 days. Morphine-treated mice had higher energy expenditure and respiratory quotient, indicating a shift toward carbohydrate metabolism. Micro-computed tomography (µCT) analysis indicated a sex difference in the bone outcome, where male mice treated with morphine had reduced trabecular bone volume fraction (Tb.BV/TV) (15%) and trabecular bone mineral density (BMD) (14%) in the distal femur compared with vehicle. Conversely, bone microarchitecture was not changed in females after morphine treatment. Histomorphometric analysis demonstrated that in males, morphine reduced bone formation rate compared with vehicle, but osteoclast parameters were not different. Furthermore, morphine reduced bone formation marker gene expression in the tibia of males (Bglap and Dmp1). Circulating miRNA profile changes were evident in males, with 14 differentially expressed miRNAs associated with morphine treatment compared with two differentially expressed miRNAs in females. In males, target analysis indicated hypoxia-inducible factor (HIF) signaling pathway was targeted by miR-223-3p and fatty acid metabolism by miR-484, -223-3p, and -328-3p. Consequently, expression of miR-223-3p targets, including Igf1r and Stat3, was lower in morphine-treated bone. In summary, we have established a model where morphine leads to a lower trabecular bone formation in males and identified potential mediating miRNAs. Understanding the sex-specific mechanisms of bone loss from opioids will be important for improving management of the adverse effects of opioids on the skeleton. © 2022 American Society for Bone and Mineral Research (ASBMR).

hERG block potencies for 5 positive control drugs obtained per ICH E14/S7B Q&As best practices: Impact of recording temperature and drug loss.

According to the ICH S7B guideline, drug candidates are screened for hERG block prior to first-in-human testing to predict the likelihood of delayed repolarization associated with a rare, but life-threatening, ventricular tachyarrhythmia. The new ICH E14 Q&As guideline allows hERG results to be used in later clinical development for decision-making (Q&As 5.1 and 6.1). To pursue this path, the hERG assay should be conducted following the new ICH S7B Q&A 2.1 guideline, which calls for best practice considerations of the recording temperature, voltage protocol, stimulation frequency, recording/data quality, and concentration verification. This study investigated hERG block by cisapride, dofetilide, terfenadine, sotalol, and E-4031 - positive controls commonly used to demonstrate assay sensitivity - using the manual whole cell patch clamp method and an action potential-like voltage protocol presented at 0.2 Hz. Recordings were conducted at room and near physiological temperature. Drug concentrations were measured using samples collected during real patch clamp experiments and satellite experiments. Results showed temperature effects for E-4031, terfenadine, and sotalol, but not cisapride and dofetilide. Cisapride and terfenadine showed substantial concentration losses, largely due to nonspecific binding to the perfusion apparatus. Using concentrations measured from the real and satellite experiments to assess block potencies yielded comparable results, indicating that satellite sample collection may be viable for drugs with nonspecific binding concerns only. In summary, this study provides block potencies for 5 hERG positive controls, and serves as a case study for hERG assays conducted, and results illustrated in accordance with the new ICH E14/S7B Q&As.

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.

Voluntary exercise blocks ongoing pain and diminishes bone remodeling while sparing protective mechanical pain in a rat model of advanced osteoarthritis pain.

ABSTRACT: Exercise is the most common treatment recommended by healthcare providers for the treatment of musculoskeletal pain. We examined whether voluntary running wheel exercise improves pain and bone remodeling in rats with monosodium iodoacetate-induced unilateral knee joint pain. During acquisition of wheel running before osteoarthritis (OA) treatment, rats separated into 2 groups characterized by either high or low levels of voluntary wheel running as indicated by distance and peak speed. After the induction of knee joint OA, all rats showed diminished voluntary wheel running throughout the study. Voluntary wheel running failed to alter evoked nociceptive responses evaluated as weight asymmetry or hind paw tactile thresholds at any timepoint of the study. By contrast, relief of ongoing pain was demonstrated by conditioned place preference produced by lidocaine injection into the monosodium iodoacetate-treated knee in high but not low-running rats. Both high and low voluntary runners showed diminished trabecular bone loss compared with sedentary controls. These observations indicate that both high-intensity and low-intensity exercise is beneficial in protecting against bone remodeling in advanced OA. The data suggest that similar to clinical observation, bone remodeling does not correlate with pain. In addition, these results suggest that higher intensity exercise may relieve persistent ongoing OA pain while maintaining movement-evoked nociception. The relief of ongoing pain can potentially offer significant improvement in quality of life, whereas preservation of responses to movement-evoked pain may be especially important in protecting the joint from damage because of overuse.

Results of the patient report of intermittent catheterization experience (PRICE) study.

AIMS: Patient satisfaction is paramount to health-related quality of life (HR-QoL) outcomes. High quality, quantitative data from the US describing patients' actual experiences, difficulties, and HR-QoL while on an intermittent self-catheterization (ISC) regimen is very scarce. Our objective was to better understand patient practices with and attitudes towards ISC. METHODS: This is a cross-sectional, multi-centered, clinical study of adult men and women performing ISC in the United States. Data collected included demographics, medical history, catheter characteristics, specific self-catheterization habits and two validated HR-QoL questionnaires: The Intermittent Self-Catheterization Questionnaire (ISC-Q) and the Intermittent Catheterization Difficulty Questionnaire (ICDQ). RESULTS: Two hundred participants were recruited from six sites; 70.0% were male, 73.5% were Caucasian with a median age was 51.0 years (range 19-90 years). The ISC-Q showed that the vast majority of participants reported ease with ISC (82.0% satisfaction score) had confidence in their ability to perform ISC (91.9% satisfaction score); yet, many felt self-conscious about doing so (58.3% satisfaction score) and had concerns about long-term adverse effects (58.1% satisfaction score). The ICDQ indicated little to no difficulty for most participants with all routine ISC practices. A small minority of participants reported some difficulty with a "blocking sensation" during initiation of catheterization, leg spasticity, and painful catheterization. Multivariate linear regression results are also reported. DISCUSSION/CONCLUSION: Participants are confident with ISC and have little overall difficulty, which may be a product of successful education and/or catheter design. urinary tract infections (UTIs) were common (yet variable) and may contribute to the noted long-term ISC concerns. Limitations exist including various selection biases leading to concerns of external validity. Future educational interventions in this population may further improve HR-QoL, optimize UTIs prevention, and diminish concerns with long-term ISC.

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.

Effects of Vancomycin on Persistent Pain-Stimulated and Pain-Depressed Behaviors in Female Fischer Rats With or Without Voluntary Access to Running Wheels.

The present experiments determined the effects of the narrow-spectrum antibiotic vancomycin on inflammatory pain-stimulated and pain-depressed behaviors in rats. Persistent inflammatory pain was modeled using dilute formalin (0.5%). Two weeks of oral vancomycin administered in drinking water attenuated Phase II formalin pain-stimulated behavior, and prevented formalin pain-depressed wheel running. Fecal microbiota transplantation produced a non-significant trend toward reversal of the vancomycin effect on pain-stimulated behavior. Vancomycin depleted Firmicutes and Bacteroidetes populations in the gut while having a partial sparing effect on Lactobacillus species and Clostridiales. The vancomycin treatment effect was associated with an altered profile in amino acid concentrations in the gut with increases in arginine, glycine, alanine, proline, valine, leucine, and decreases in tyrosine and methionine. These results indicate that vancomycin may have therapeutic effects against persistent inflammatory pain conditions that are distal to the gut. PERSPECTIVE: The narrow-spectrum antibiotic vancomycin reduces pain-related behaviors in the formalin model of inflammatory pain. These data suggest that manipulation of the gut microbiome may be one method to attenuate inflammatory pain amplitude.

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.