Primaquine-5,6-orthoquinone is directly hemolytic to older G6PD deficient RBCs in a humanized mouse model.

Primaquine and tafenoquine are the only approved drugs that can achieve a radical cure for Plasmodium vivax malaria but are contraindicated in patients who are deficient in glucose 6-phosphate dehydrogenase (G6PDd) due to risk of severe hemolysis from reactive oxygen species (ROS) generated by redox cycling of drug metabolites. 5-hydroxyprimaquine and its quinone-imine cause robust redox cycling in red blood cells (RBCs), but are so labile as to not be detected in blood or urine. Rather, the 5,8-quinoneimine is rapidly converted into primaquine-5,6-orthoquinone (5,6-POQ) that is then excreted in the urine. The extent to which 5,6-POQ contributes to hemolysis remains unclear, although some have suggested that it is a minor toxin that should be used predominantly as a surrogate to infer levels of 5-hydroxyprimaquine. In this report, we describe a novel humanized mouse model of the G6PD Mediterranean variant (hG6PDMed-) that recapitulates the human biology of RBC age dependent enzyme decay, as well as an isogenic matched control mouse with human non-deficient G6PD hG6PDND In vitro challenge of RBCs with 5,6-POQ causes increased generation of superoxide and methemoglobin. Infusion of treated RBCs shows that 5,6-POQ selectively causes in vivo clearance of older hG6PDMed- RBCs. These findings support the hypothesis that 5,6-POQ directly induces hemolysis and challenges the notion that 5,6-POQ is an inactive metabolic waste product. Indeed, given the extreme lability of 5-hydroxyprimaquine and the relative stability of 5,6-POQ, these data raise the possibility that 5,6-POQ is a major hemolytic primaquine metabolite in vivo. Significance Statement These findings demonstrate that 5,6-POQ, which has been suggested to be an inert waste product of active primaquine metabolites, directly induces ROS that lead specifically to removal of older G6PDd RBCs from circulation. As 5,6-POQ is relatively stable compared to other active primaquine metabolites, these data support the hypothesis that 5,6-POQ is a major toxin in primaquine induced hemolysis. In addition, a new model of G6PDd is used to show that young G6PDd RBCs are resistant to primaquine induced hemolysis.

Inter-strain variability in responses to a single administration of the cannabidiol-rich cannabis extract in mice.

Cannabidiol (CBD) has gained widespread popularity; however, its pharmacological and toxicological profiles in the context of human genetic diversity remain largely unexplored. Here, we investigated the variability in metabolism and toxicity of CBD-rich cannabis extract (CRCE) in genetically diverse mouse models: C57BL/6J, B6C3F1/J, and NZO/HlLtJ strains. Mice received a single dose of CRCE containing 57.9% CBD at dosages of 0, 246, 738, and 2460 mg/kg of CBD. At 24 h after treatment, no appreciable histomorphological changes were detected in the liver. Plasma bilirubin levels increased markedly in all strains at the highest CBD dose. Mice in all treatment groups displayed significant but distinct increases in ALT and AST levels. While B6C3F1/J and NZO/HlLtJ mice had negligible plasma CBD levels at 738 mg/kg, C57BL/6J mice exhibited levels exceeding 7000 ng/mL. At 2460 mg/kg, high CBD concentrations were found in B6C3F1/J and C57BL/6J mice, but markedly lower levels were seen in NZO/HlLtJ mice. Gene expression profiling showed significant increases in Cyp2b10 across all strains but varying responses in Cyp1a1 expression, indicating strain-specific CYP dysregulation. Genetically diverse mice exhibited differential pharmacological and toxicological responses to CRCE, suggesting a high potential for inter-individual variability in the pharmacology and toxicology of CBD in humans.

Human quad liver-on-chip system as a tool toward bridging the gap between animals and humans regarding toxicology and pharmacology of a cannabidiol-rich cannabis extract.

Cannabidiol (CBD) is a major phytocannabinoid from Cannabis sativa. It is currently widely available and widely used in the USA, but despite its rapid progress to market, the pharmacology and toxicology of both CBD and cannabidiol-rich cannabis extracts (CRCE) remain largely unknown. The goals of this study were to investigate the potential of a novel human microphysiological system to emulate CRCE-induced hepatotoxicity and pharmacological properties demonstrated in animal models. For this purpose, C57BL6/J male mice were subjected to dosing with either 0, 61.5, 184.5, or 615 mg/kg of CRCE for 10 days. The liver-on-chip system, incorporating human primary hepatocytes, sinusoidal endothelial cells, as well as Kupffer and stellate cells was subjected to 0, 300, 1,200, or 4,400 ng/mL of CRCE (8 h exposure followed by 16 h washout) for 5 days. Administration of CRCE in mice resulted in nearly 4-fold elevations of plasma ALT at 615 mg/kg (p < 0.01) and a dose-dependent decrease in intrahepatic miR-122. Elevated levels of ALT, paralleled by decreased intrahepatic and increased effluent levels of miR-122, were also observed in the liver-on-chip, although these results were not statistically significant. Exposure to CRCE resulted in a robust and dose-dependent induction of key cytochrome P450 enzymes, namely Cyp1a2, Cyp2b6 (CYP2B10), Cyp2e1, and Cyp2c9 (CYP2C19) in both mouse livers and liver-on-chip. The results of this study demonstrate the congruence between the responses observed in mouse and human liver-on-chip experimental systems and provide evidence of the potential microphysiological systems hold for translating animal data into clinical practice.

Population pharmacokinetics of primaquine and its metabolites in African males.

BACKGROUND: Primaquine (PQ) is the prototype 8-aminoquinoline drug, a class which targets gametocytes and hypnozoites. The World Health Organization (WHO) recommends adding a single low dose of primaquine to the standard artemisinin-based combination therapy (ACT) in order to block malaria transmission in regions with low malaria transmission. However, the haemolytic toxicity is a major adverse outcome of primaquine in glucose-6-phosphate dehydrogenase (G6PD)-deficient subjects. This study aimed to characterize the pharmacokinetic properties of primaquine and its major metabolites in G6PD-deficient subjects. METHODS: A single low-dose of primaquine (0.4-0.5 mg/kg) was administered in twenty-eight African males. Venous and capillary plasma were sampled up to 24 h after the drug administration. Haemoglobin levels were observed up to 28 days after drug administration. Only PQ, carboxy-primaquine (CPQ), and primaquine carbamoyl-glucuronide (PQCG) were present in plasma samples and measured using liquid chromatography mass spectrometry. Drug and metabolites' pharmacokinetic properties were investigated using nonlinear mixed-effects modelling. RESULTS: Population pharmacokinetic properties of PQ, CPQ, and PQCG can be described by one-compartment disposition kinetics with a transit-absorption model. Body weight was implemented as an allometric function on the clearance and volume parameters for all compounds. None of the covariates significantly affected the pharmacokinetic parameters. No significant correlations were detected between the exposures of the measured compounds and the change in haemoglobin or methaemoglobin levels. There was no significant haemoglobin drop in the G6PD-deficient patients after administration of a single low dose of PQ. CONCLUSIONS: A single low-dose of PQ was haematologically safe in this population of G6PD-normal and G6PD-deficient African males without malaria. Trial registration NCT02535767.

Juniper Berries Regulate Diabetes and Obesity Markers Through Modulating PPARα, PPARγ, and LXR: In Vitro and In Vivo Effects.

The berries of Juniperus communis have been traditionally used for therapeutic purposes. They have been reported to possess various pharmacological effects such as anti-inflammatory, hypoglycemic and hypolipidemic activities. In this study, a methanolic extract of J. communis berries (JB) was evaluated for its effects on peroxisome proliferator-activated receptors alpha and gamma (PPARα and PPARγ), liver X receptor (LXR), glucose uptake and lipid accumulation using various cellular systems. At a concentration of 25 µg/mL, JB caused 3.77-fold activation of PPARα, 10.90-fold activation of PPARγ, and 4.43-fold activation of LXR in hepatic cells. JB inhibited (11%) the adipogenic effect induced by rosiglitazone in adipocytes and increased glucose uptake (90%) in muscle cells. In high-fat diet (HFD) fed mice, JB at a dose of 25 mg/kg body weight exhibited a 21% decrease in body weight. Fasting glucose levels in mice treated with 12.5 mg/kg of JB were significantly decreased (39%) indicating its efficacy in regulating hyperglycemia and obesity induced by HFD thus ameliorating the symptoms of type 2 diabetes. A series of energy metabolic genes, including Sirt1 (2.00-fold) and RAF1 (2.04-fold), were upregulated by JB, while rosiglitazone regulated the hepatic PPARγ only. Phytochemical analysis of JB indicated presence of a number of flavonoids and biflavonoids which seem to be responsible for the observed activity. It was concluded that JB acted as a multiple agonist of PPARα, PPARγ and LXR without the undesired effect of adipogenesis and exhibited the property of enhancing glucose uptake. The regulation of PPARα, PPARγ and LXR seems to be through Sirt1 and RAF1. In vivo results confirmed the antidiabetic and antiobesity potential of JB and indicated its utility in metabolic disorder and type 2 diabetes.

Feasibility, Acceptability, and Preliminary HIV Care and Psychological Health Effects of iTHRIVE 365 for Black Same Gender Loving Men.

OBJECTIVES: This uncontrolled pilot study examined the feasibility, acceptability, and preliminary HIV and psychological health effects of iTHRIVE 365, a multicomponent intervention designed by and for Black same gender loving men (SGLM) to promote: health knowledge and motivation, Black SGLM social support, affirming health care, and housing and other economic resources. DESIGN METHODS: We conducted a 14-day daily diary study with 32 Black SGLM living with HIV connected to THRIVE SS in Atlanta, GA. Daily surveys assessed intervention engagement, antiretroviral medication (ART) use, depressive symptoms, anxiety symptoms, and emotion regulation difficulties. App paradata (ie, process data detailing app usage) assessed amount of intervention engagement via page access. Participants began receiving access to the intervention on day 7. After the 14-day daily diary period, participants responded to follow-up items on the user-friendliness, usefulness, helpfulness, and whether they would recommend iTHRIVE 365 to others. Chi-square analyses examined associations between intervention engagement and ART use, and dynamic structural equation modelling assessed longitudinal associations from intervention engagement to next-day psychological health. This intervention trial is registered on ClinicalTrials.gov (NCT05376397). RESULTS: On average, participants engaged with iTHRIVE 365 over once every other day and accessed intervention pages 4.65 times per day. Among participants who engaged with the intervention, 78% reported it was helpful to extremely helpful, 83% reported it was moderately to extremely useful, and 88% reported it was user-friendly and they would recommend it to others. On intervention engagement days, participants had higher odds of ART use, χ 2 (1) = 4.09, P = 0.04, than intervention nonengagement days. On days after intervention engagement, participants showed non-null decreases in depressive symptoms (τ = -0.14; 95% CI : = [-0.23, -0.05]) and emotion regulation difficulties (τ = -0.16; 95% CI : = [-0.24, -0.02]). CONCLUSIONS: Findings suggest iTHRIVE 365 is feasible, acceptable, and positively affects daily ART use, depressive symptoms, and emotion regulation difficulties.

iTHRIVE 365: A Community-Led, Multicomponent Health Promotion Intervention for Black Same Gender Loving Men.

Although health inequities among Black same gender loving men (SGLM) are well documented (e.g., chronic psychological disorders, HIV, suicide), there are few accessible, culturally affirming, and community-led interventions designed to reduce these inequities. The present manuscript describes the process through which we developed iTHRIVE 365, a multicomponent health-promotion intervention designed by Black SGLM for Black SGLM. We utilized a community-based participatory research approach (CBPR) that included collaboration between THRIVE SS, a Black SGLM-run community-based organization, and a multisectoral team of public health, research, and digital design professionals to develop the intervention. A five-phase development process included four phases of focus groups and a technical pilot to assess community priorities and incorporate input on each feature of the intervention. Directed content analysis indicated that participants wanted a multicomponent and technology-mediated intervention that promotes health knowledge and motivation, Black SGLM social support, access to affirming healthcare, and housing and economic resources. iTHRIVE 365 combines multilevel and culturally affirming intervention features to combat the effects of oppression and ultimately promote Black SGLM's biopsychosocial health.

Reducing Stigma and HIV-related Disparities through a Peer-led Support Network for Black Gay Men Who are Living with HIV.

Undetectables Atlanta (UA), a peer support network for Black gay men living with HIV, conducted an exploratory evaluation to begin identifying outcomes of UA membership. Th is initial evaluation suggested decreased HIV stigma and increased comfort with disclosure, treatment adherence, and other areas of well-being that warrant prospective evaluation.

Quantitative analysis of primaquine and its metabolites in human urine using liquid chromatography coupled with tandem mass spectrometry.

Primaquine (PQ), a prototype 8-aminoquinoline (8-AQ) drug used to treat malaria, is rapidly metabolized into different inactive and active metabolites. Due to the hemolytic toxicity, the uses of PQ have been confined. To understand its overall metabolism and its relation to drug efficacy and toxicity, profiling of urine for the parent drug and its metabolites is important. The current study presents a convenient and rapid method for simultaneously quantifying primaquine (PQ) and its metabolites in human urine. A simple liquid-liquid extraction followed by chromatographic separation and quantification through ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed and validated to quantify PQ and its eleven metabolites in the urine of healthy human volunteers who received a single oral dose of PQ. The developed method separated fourteen analytes, including internal standards, within nine minutes of run time. The linearity of all analytes was suitable in the range of 1-500 ng/mL. The extraction recovery for all concentrations of analytes from urine was ranged from 90.1 to 112.9 %. The relative standard deviation for intra- and inter-day precision were < 9.8 and < 10.7 %, respectively. Along with PQ, its different metabolites were detected in urine. Primaquine-5,6-orthoquinone, the N-carbamoylglucuronide conjugate of PQ and carboxyprimaquine were the major metabolites found in urine. Significant enantiomeric differences in the urinary excretion profiles for PQ and metabolites were observed. This analytical method can be implemented in the pharmacokinetic analysis of PQ to explain its toxicity and clinical decision making.

Comparative single dose pharmacokinetics and metabolism of racemic primaquine and its enantiomers in human volunteers.

Primaquine (PQ) is a racemic drug used in treatment of malaria for six decades. Recent studies suggest that the two enantiomers of PQ are differentially metabolized in animals, and this results in different pharmacological and toxicological profiles. The current study characterizes the pharmacokinetic (PK) properties, metabolism and tolerability of the individual enantiomers of PQ in healthy human volunteers with normal glucose-6-phosphate dehydrogenase (G6PD) activity. Two cohorts (at two dose levels), each with 18 subjects, participated in three study arms in a crossover fashion: a single dose of the (-)-R enantiomer (RPQ), a single dose of the (+)-S enantiomer (SPQ), and a single dose of racemic PQ (RSPQ). PQ and its key metabolites carboxyprimaquine (cPQ) and PQ-N-carbamoyl glucuronide (PQ-N-CG) were analyzed. Clear differences were observed in PK and metabolism of the two enantiomers. Relative PQ exposure was higher with SPQ as compared to RPQ. PQ maximum plasma concentration (Cmax) and area under the plasma concentration-time curve were higher for SPQ, while the apparent volume of distribution and total body clearance were higher for RPQ. Metabolism of the two enantiomers showed dramatic differences: plasma PQ-N-CG was derived solely from SPQ, while RPQ was much more efficiently converted to cPQ than was SPQ. Cmax of cPQ and PQ-N-CG were 10 and 2 times higher, respectively, than the parent drugs. The study demonstrates that the PK properties of PQ enantiomers show clear differences, and metabolism is highly enantioselective. Such differences in metabolism suggest potentially distinct toxicity profiles in multi-dose regimens, especially in G6PD-deficient subjects.

Mechanisms of 8-aminoquinoline induced haemolytic toxicity in a G6PDd humanized mouse model.

Primaquine (PQ) and Tafenoquine (TQ) are clinically important 8-aminoquinolines (8-AQ) used for radical cure treatment of P. vivax infection, known to target hepatic hypnozoites. 8-AQs can trigger haemolytic anaemia in individuals with glucose-6-phosphate dehydrogenase deficiency (G6PDd), yet the mechanisms of haemolytic toxicity remain unknown. To address this issue, we used a humanized mouse model known to predict haemolytic toxicity responses in G6PDd human red blood cells (huRBCs). To evaluate the markers of eryptosis, huRBCs were isolated from mice 24-48 h post-treatment and analysed for effects on phosphatidylserine (PS), intracellular reactive oxygen species (ROS) and autofluorescence. Urinalysis was performed to evaluate the occurrence of intravascular and extravascular haemolysis. Spleen and liver tissue harvested at 24 h and 5-7 days post-treatment were stained for the presence of CD169+ macrophages, F4/80+ macrophages, Ter119+ mouse RBCs, glycophorin A+ huRBCs and murine reticulocytes (muRetics). G6PDd-huRBCs from PQ/TQ treated mice showed increased markers for eryptosis as early as 24 h post-treatment. This coincided with an early rise in levels of muRetics. Urinalysis revealed concurrent intravascular and extravascular haemolysis in response to PQ/TQ. Splenic CD169+ macrophages, present in all groups at day 1 post-dosing were eliminated by days 5-7 in PQ/TQ treated mice only, while liver F4/80 macrophages and iron deposits increased. Collectively, our data suggest 8-AQ treated G6PDd-huRBCs have early physiological responses to treatment, including increased markers for eryptosis indicative of oxidative stress, resulting in extramedullary haematopoiesis and loss of splenic CD169+ macrophages, prompting the liver to act as the primary site of clearance.

Comparative pharmacokinetics and tissue distribution of primaquine enantiomers in mice.

BACKGROUND: Primaquine (PQ) has been used for the radical cure of relapsing Plasmodium vivax malaria for more than 60 years. PQ is also recommended for prophylaxis and prevention of transmission of Plasmodium falciparum. However, clinical utility of PQ has been limited due to toxicity in individuals with genetic deficiencies in glucose 6-phosphate dehydrogenase (G6PD). PQ is currently approved for clinical use as a racemic mixture. Recent studies in animals as well as humans have established differential pharmacological and toxicological properties of the two enantiomers of PQ. This has been attributed to differential metabolism and pharmacokinetics of individual PQ enantiomers. The aim of the current study is to evaluate the comparative pharmacokinetics (PK), tissue distribution and metabolic profiles of the individual enantiomers in mice. METHODS: Two groups of 21 male Albino ND4 Swiss mice were dosed orally with 45 mg/kg of S-(+)-PQ and R-(-)PQ respectively. Each of the enantiomers was comprised of a 50:50 mixture of 12C- and 13C- stable isotope labelled species (at 6 carbons on the benzene ring of the quinoline core). Three mice were euthanized from each group at different time points (at 0, 0.5, 1, 2, 4, 8, 24 h) and blood was collected by terminal cardiac bleed. Liver, spleen, lungs, kidneys and brain were removed, extracted and analysed using UPLC/MS. The metabolites were profiled by tandem mass (MS/MS) fragmentation profile and fragments with 12C-13C twin peaks. Non-compartmental analysis was performed using the Phoenix WinNonLin PK software module. RESULTS: The plasma AUC0-last (µg h/mL) (1.6 vs. 0.6), T1/2 (h) (1.9 vs. 0.45), and Tmax (h) (1 vs. 0.5) were greater for SPQ as compared to RPQ. Generally, the concentration of SPQ was higher in all tissues. At Tmax, (0.5-1 h in all tissues), the level of SPQ was 3 times that of RPQ in the liver. Measured Cmax of SPQ and RPQ in the liver were about 100 and 40 times the Cmax values in plasma, respectively. Similar observations were recorded in other tissues where the concentration of SPQ was higher compared to RPQ (2× in the spleen, 6× in the kidneys, and 49× in the lungs) than in the plasma. CPQ, the major metabolite, was preferentially generated from RPQ, with higher levels in all tissues (> 10× in the liver, and 3.5× in the plasma) than from SPQ. The PQ-o-quinone was preferentially formed from the SPQ (> 4× compared to RPQ), with higher concentrations in the liver. CONCLUSION: These studies show that in mice, PQ enantiomers are differentially biodistributed and metabolized, which may contribute to differential pharmacologic and toxicity profiles of PQ enantiomers. The findings on higher levels of PQ-o-quinone in liver and RBCs compared to plasma and preferential generation of this metabolite from SPQ are consistent with the higher anti-malarial efficacy of SPQ observed in the mouse causal prophylaxis test, and higher haemolytic toxicity in the humanized mouse model of G6PD deficiency. Potential relevance of these findings to clinical use of racemic PQ and other 8-aminoquinolines vis-à-vis need for further clinical evaluation of individual enantiomers are discussed.

Identification of an Orally Bioavailable, Brain-Penetrant Compound with Selectivity for the Cannabinoid Type 2 Receptor.

Modulation of the endocannabinoid system (ECS) is of great interest for its therapeutic relevance in several pathophysiological processes. The CB2 subtype is largely localized to immune effectors, including microglia within the central nervous system, where it promotes anti-inflammation. Recently, a rational drug design toward precise modulation of the CB2 active site revealed the novelty of Pyrrolo[2,1-c][1,4]benzodiazepines tricyclic chemotype with a high conformational similarity in comparison to the existing leads. These compounds are structurally unique, confirming their chemotype novelty. In our continuing search for new chemotypes as selective CB2 regulatory molecules, following SAR approaches, a total of 17 selected (S,E)-11-[2-(arylmethylene)hydrazono]-PBD analogs were synthesized and tested for their ability to bind to the CB1 and CB2 receptor orthosteric sites. A competitive [3H]CP-55,940 binding screen revealed five compounds that exhibited >60% displacement at 10 µM concentration. Further concentration-response analysis revealed two compounds, 4k and 4q, as potent and selective CB2 ligands with sub-micromolar activities (Ki = 146 nM and 137 nM, respectively). In order to support the potential efficacy and safety of the analogs, the oral and intravenous pharmacokinetic properties of compound 4k were sought. Compound 4k was orally bioavailable, reaching maximum brain concentrations of 602 ± 162 ng/g (p.o.) with an elimination half-life of 22.9 ± 3.73 h. Whether administered via the oral or intravenous route, the elimination half-lives ranged between 9.3 and 16.7 h in the liver and kidneys. These compounds represent novel chemotypes, which can be further optimized for improved affinity and selectivity toward the CB2 receptor.

Comparative metabolism and tolerability of racemic primaquine and its enantiomers in human volunteers during 7-day administration.

Primaquine (PQ) is an 8-aminoquinoline antimalarial, active against dormant Plasmodium vivax hypnozoites and P. falciparum mature gametocytes. PQ is currently used for P. vivax radical cure and prevention of malaria transmission. PQ is a racemic drug and since the metabolism and pharmacology of PQ's enantiomers have been shown to be divergent, the objectives of this study were to evaluate the comparative tolerability and metabolism of PQ with respect to its two enantiomers in human volunteers in a 7 days' treatment schedule. Fifteen subjects with normal glucose-6-phosphate dehydrogenase (G6PDn) completed four arms, receiving each of the treatments, once daily for 7 days, in a crossover fashion, with a 7-14 days washout period in between: R-(-) enantiomer (RPQ) 22.5 mg; S-(+) enantiomer (SPQ) 22.5 mg; racemic PQ (RSPQ) 45 mg, and placebo. Volunteers were monitored for any adverse events (AEs) during the study period. PQ and metabolites were quantified in plasma and red blood cells (RBCs) by UHPLC-UV-MS/MS. Plasma PQ was significantly higher in SPQ treatment group than for RPQ. Carboxy-primaquine, a major plasma metabolite, was much higher in the RPQ treated group than SPQ; primaquine carbamoyl glucuronide, another major plasma metabolite, was derived only from SPQ. The ortho-quinone metabolites were also detected and showed differences for the two enantiomers in a similar pattern to the parent drugs. Both enantiomers and racemic PQ were well tolerated in G6PDn subjects with the 7 days regimen; three subjects showed mild AEs which did not require any intervention or discontinuation of the drug. The most consistent changes in G6PDn subjects were a gradual increase in methemoglobin and bilirubin, but these were not clinically important. However, the bilirubin increase suggests mild progressive damage to a small fraction of red cells. PQ enantiomers were also individually administered to two G6PD deficient (G6PDd) subjects, one heterozygous female and one hemizygous male. These G6PDd subjects showed similar results with the two enantiomers, but the responses in the hemizygous male were more pronounced. These studies suggest that although the metabolism profiles of individual PQ enantiomers are markedly different, they did not show significant differences in the safety and tolerability in G6PDn subjects.

Special Issue: "James D. McChesney, Vision, Passion and Leadership in the Development of Plant-Derived Natural Products".

It is a distinct pleasure for me to offer something in recognition of and tribute to Dr [...].

Enantioselective Interactions of Anti-Infective 8-Aminoquinoline Therapeutics with Human Monoamine Oxidases A and B.

8-Aminoquinolines (8-AQs) are an important class of anti-infective therapeutics. The monoamine oxidases (MAOs) play a key role in metabolism of 8-AQs. A major role for MAO-A in metabolism of primaquine (PQ), the prototypical 8-AQ antimalarial, has been demonstrated. These investigations were further extended to characterize the enantioselective interactions of PQ and NPC1161 (8-[(4-amino-1-methylbutyl) amino]-5-[3, 4-dichlorophenoxy]-6-methoxy-4-methylquinoline) with human MAO-A and -B. NPC1161B, the (R)-(-) enantiomer with outstanding potential for malaria radical cure, treatment of visceral leishmaniasis and pneumocystis pneumonia infections is poised for clinical development. PQ showed moderate inhibition of human MAO-A and -B. Racemic PQ and (R)-(-)-PQ both showed marginally greater (1.2- and 1.6-fold, respectively) inhibition of MAO-A as compared to MAO-B. However, (S)-(+)-PQ showed a reverse selectivity with greater inhibition of MAO-B than MAO-A. Racemic NPC1161 was a strong inhibitor of MAOs with 3.7-fold selectivity against MAO-B compared to MAO-A. The (S)-(+) enantiomer (NPC1161A) was a better inhibitor of MAO-A and -B compared to the (R)-(-) enantiomer (NPC1161B), with more than 10-fold selectivity for inhibition of MAO-B over MAO-A. The enantioselective interaction of NPC1161 and strong binding of NPC1161A with MAO-B was further confirmed by enzyme-inhibitor binding and computational docking analyses. Differential interactions of PQ and NPC1161 enantiomers with human MAOs may contribute to the enantioselective pharmacodynamics and toxicity of anti-infective 8-AQs therapeutics.

First In Class (S,E)-11-[2-(Arylmethylene)Hydrazono]-PBD Analogs As Selective CB2 Modulators Targeting Neurodegenerative Disorders.

Newly designed pyrrolo[2,1-c][1,4]benzodiazepines tricyclic skeleton has shown potential clusters of cannabinoid receptors CB1/CB2 selective ligands. CB2 plays a critical role in microglial-derived neuroinflammation, where it modulates cell proliferation, migration, and differentiation into M1 or M2 phenotypes. Beginning with computer-based docking studies accounting the recently discovered X-ray crystal structure of CB2, we designed a series of PBD analogs as potential ligands of CB2 and tested their binding affinities. Interestingly, computational studies and theoretical binding affinities of several selected (S,E)-11-[2-(arylmethylene)hydrazono]-PBD analogs, have revealed the presence of potential selectivity in binding attraction towards CB1 and CB2. Reported here is the discovery of the first representatives of this series of selective binding to CB2. Preliminary data showed that this class of molecules display potential binding efficacy towards the cannabinoid receptors tested. Intriguingly, initial cannabinoid binding assay showed a selective binding affinity of 4g and 4h showed K i of 0.49 and 4.7 µM towards CB2 receptors while no binding was observed to CB1. The designed leads have shown remarkable stability pattern at the physiological pH magnifying their therapeutic values. We hypothesize that the PBD tricyclic structure offers the molecule an appropriate three-dimensional conformation to fit snugly within the active site of CB2 receptors, giving them superiority over the reported CB2 agonists/inverse agonists. Our findings suggested that the attachment of heterocyclic ring through the condensation of diazepine hydrazone and S- or N-heterocyclic aldehydes enhances the selectivity of CB2 over CB1.

Therapeutic Intervention of COVID-19 by Natural Products: A Population-Specific Survey Directed Approach.

To date very few promising leads from natural products (NP) secondary metabolites with antiviral and immunomodulatory properties have been identified for promising/potential intervention for COVID-19. Using in-silico docking studies and genome based various molecular targets, and their in vitro anti-SARS CoV-2 activities against whole cell and/or selected protein targets, we select a few compounds of interest, which can be used as potential leads to counteract effects of uncontrolled innate immune responses, in particular those related to the cytokine storm. A critical factor for prevention and treatment of SARS-CoV-2 infection relates to factors independent of viral infection or host response. They include population-related variables such as concurrent comorbidities and genetic factors critically relevant to COVID-19 health disparities. We discuss population risk factors related to SARS-CoV-2. In addition, we focus on virulence related to glucose-6-phosphate dehydrogenase deficiency (G6PDd), the most common human enzymopathy. Review of data on the response of individuals and communities with high prevalence of G6PDd to NP, prompts us to propose the rationale for a population-specific management approach to rationalize design of therapeutic interventions of SARS-CoV-2 infection, based on use of NP. This strategy may lead to personalized approaches and improve disease-related outcomes.

Quantitative determination of primaquine-5,6-ortho-quinone and carboxyprimaquine-5,6-ortho-quinone in human erythrocytes by UHPLC-MS/MS.

The antimalarial drug primaquine (PQ) causes methemoglobinemia and hemolysis in individuals with a genetic deficiency of glucose 6-phosphate dehydrogenase. Reactive oxygen species (ROS) generated by redox cycling of the metabolite primaquine-5,6-orthoquinone (POQ) in erythrocytes has been attributed to be responsible for the toxicity of PQ. Carboxyprimaquine (CPQ), the major human plasma metabolite of PQ, can also form the analogous carboxyprimaquine-5,6-orthoquinone (CPOQ) metabolite, which can also generate ROS in erythrocytes by redox cycling, thus contributing to the hematotoxicity of this drug. In order to study these pathways and characterize such effects in vivo, methods are needed for characterization and quantification of POQ and CPOQ in human erythrocytes. The purpose of this work was to develop a validated method for the quantitative determination of CPOQ and POQ metabolites in human erythrocytes, suitable for clinical studies of PQ metabolism. Several liquid-liquid extraction methods using different organic solvents had been investigated. The solvent mixture of water-methanol-acetonitrile (9:9:5, v/v) was shown to yield the best results for the two analytes. Chromatographic analysis of POQ and CPOQ in human erythrocytes was achieved on a high strength silica (HSS) column and gradient elution (water and acetonitrile, both containing 0.1% formic acid) by ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Quantitative estimation of POQ and CPOQ was executed by monitoring ion pairs of m/z 260.23 > 175.03 and m/z 275.19 > 175.04, respectively. The method, which was validated for precision, accuracy, selectivity, and linearity, was successfully applied for the quantitative determination of POQ and CPOQ, the key metabolites of PQ in human erythrocytes in PQ clinical study.

Safety and Molecular-Toxicological Implications of Cannabidiol-Rich Cannabis Extract and Methylsulfonylmethane Co-Administration.

Cannabidiol (CBD) is a biologically active, non-psychotropic component of Cannabis sativa whose popularity has grown exponentially in recent years. Besides a wealth of potential health benefits, ingestion of CBD poses risks for a number of side effects, of which hepatotoxicity and CBD/herb-drug interactions are of particular concern. Here, we investigated the interaction potential between the cannabidiol-rich cannabis extract (CRCE) and methylsulfonylmethane (MSM), a popular dietary supplement, in the mouse model. For this purpose, 8-week-old male C57BL6/J mice received MSM-containing water (80 mg/100 mL) ad libitum for 17 days. During the last three days of treatment, mice received three doses of CRCE administered in sesame oil via oral gavage (123 mg/kg/day). Administration of MSM alone did not result in any evidence of liver toxicity and did not induce expression of mouse cytochrome P450 (CYP) enzymes. Administration of CRCE did produce significant (p < 0.05) increases in Cyp1a2, Cyp2b10, Cyp2c29, Cyp3a4, Cyp3a11, Cyp2c65, and Cyp2c66 messenger RNA, however, this effect was not amplified by MSM/CRCE co-treatment. Similarly, no evidence of liver toxicity was observed in MSM/CRCE dosed mice. In conclusion, short-term MSM/CRCE co-administration did not demonstrate any evidence of hepatotoxicity in the mouse model.