Primary Active Metabolite Research Articles

Discovery of a potent, Kv7.3-selective potassium channel opener from a Polynesian traditional botanical anticonvulsant

Plants remain an important source of biologically active small molecules with high therapeutic potential. The voltage-gated potassium (Kv) channel formed by Kv7.2/3 (KCNQ2/3) heteromers is a major target for anticonvulsant drug development. Here, we screened 1444 extracts primarily from plants collected in California and the US Virgin Islands, for their ability to activate Kv7.2/3 but not inhibit Kv1.3, to select against tannic acid being the active component. We validated the 7 strongest hits, identified Thespesia populnea (miro, milo, portia tree) as the most promising, then discovered its primary active metabolite to be gentisic acid (GA). GA highly potently activated Kv7.2/3 (EC50, 2.8 nM). GA is, uniquely to our knowledge, 100% selective for Kv7.3 versus other Kv7 homomers; it requires S5 residue Kv7.3-W265 for Kv7.2/3 activation, and it ameliorates pentylenetetrazole-induced seizures in mice. Structure-activity studies revealed that the FDA-approved vasoprotective drug calcium dobesilate, a GA analog, is a previously unrecognized Kv7.2/3 channel opener. Also an active aspirin metabolite, GA provides a molecular rationale for the use of T. populnea as an anticonvulsant in Polynesian indigenous medicine and presents novel pharmacological prospects for potent, isoform-selective, therapeutic Kv7 channel activation.

Inhibition of HCN1 currents by norquetiapine, an active metabolite of the atypical anti-psychotic drug quetiapine.

Quetiapine is a second-generation atypical antipsychotic drug that has been commonly prescribed for the treatment of schizophrenia, major depressive disorder (depression), and other psychological disorders. Targeted inhibition of hyperpolarization-activated cyclic-nucleotide gated (HCN) channels, which generate Ih, may provide effective resistance against schizophrenia and depression. We investigated if HCN channels could contribute to the therapeutic effect of quetiapine, and its major active metabolite norquetiapine. Two-electrode voltage clamp recordings were used to assess the effects of quetiapine and its active metabolites 7-hydroxyquetiapine and norquetiapine on currents from HCN1 channels expressed in Xenopus laevis oocytes. Norquetiapine, but not quetiapine nor 7-hydroxyquetiapine, has an inhibitory effect on HCN1 channels. Norquetiapine selectively inhibited HCN1 currents by shifting the voltage-dependence of activation to more hyperpolarized potentials in a concentration-dependent manner with an IC50 of 13.9 ± 0.8μM for HCN1 and slowing channel opening, without changing the kinetics of closing. Inhibition by norquetiapine primarily occurs from in the closed state. Norquetiapine inhibition is not sensitive to the external potassium concentration, and therefore, likely does not block the pore. Norquetiapine inhibition also does not dependent on the cyclic-nucleotide binding domain. Norquetiapine also inhibited HCN4 channels with reduced efficacy than HCN1 and had no effect on HCN2 channels. Therefore, HCN channels are key targets of norquetiapine, the primary active metabolite of quetiapine. These data help to explain the therapeutic mechanisms by which quetiapine aids in the treatment of anxiety, major depressive disorder, bipolar disorder, and schizophrenia, and may represent a novel structure for future drug design of HCN inhibitors.

Cultivation optimization promotes ginsenoside and universal triterpenoid production by engineered yeast

Ginseng, a cornerstone of traditional herbal medicine in Asia, garnered significant attention for its therapeutic potential. Central to its pharmacological effects are ginsenosides, the primary active metabolites, many of which fall within the dammarane-type and share protopanaxadiol as a common precursor. Challenges in extracting protopanaxadiol and ginsenosides from ginseng arise due to their low concentrations in the roots. Emerging solutions involve leveraging microbial cell factories employing genetically engineered yeasts. Here, we optimized the fermentation conditions via the Design of Experiment, realizing 1.2 g/L protopanaxadiol in simple shake flask cultivations. Extrapolating the optimized setup to complex ginsenosides, like compound K, achieved 7.3-fold (0.22 g/L) titer improvements. Our adaptable fermentation conditions enable the production of high-value products, such as sustainable triterpenoids synthesis. Through synthetic biology, microbial engineering, and formulation studies, we pave the way for a scalable and sustainable production of bioactive compounds from ginseng.

Effects of Glycine on epigenetic modification and early embryonic development in porcine oocytes exposed to monobutyl phthalate

Monobutyl phthalate (MBP) is the primary active metabolite of dibutyl phthalate (DBP), the key plasticizer component. A substantial body of evidence from studies conducted on both animals and humans indicates that MBP exposure could result in harmful impacts on toxicity pathways. In addition, it can seriously affect human and animal reproductive health. In our present study, we showed that exposure to MBP causes abnormal epigenetic modifications in porcine oocytes and failure of early embryonic development. However, glycine (Gly) can protect oocytes and early embryos from damage caused by MBP. Our study indicated a significant decrease in the percentage of porcine oocytes that reached the metaphase II (MII) phase when exposed to MBP. SET-domain-containing 2(SETD2)-mediated H3K36me3 histone methylation was detected, and the results showed that MBP significantly decreased the protein expression of H3K36me3 and SETD2. Moreover, the expression of the DNA break markers γH2AX and the mRNA expression of Asf1a, and Asf1b increased in the MBP group. The detection of DNA methylation marker proteins showed that MBP significantly increased the fluorescence intensity of 5-methylcytosine (5mC). The results from our RT-qPCR analysis demonstrated a significant decrease in the mRNA expression of the DNA methylation-related genes Dnmt1 and Dnmt3a, as well as the embryonic developmental potential-related genes Oct4 and Nanog, in porcine oocytes following exposure to MBP. Additionally, the mRNA expression of p53 significantly increased. Subsequently, the effects of MBP on early embryonic development were examined via parthenogenesis activation (PA) and in vitro fertilization (IVF). Exposure to MBP significantly impacted the development of embryos in both PA and IVF processes. The TUNEL staining data showed that MBP significantly increased embryonic apoptosis. However, Gly can ameliorate MBP-induced defects in oocyte epigenetic modifications and early embryonic development.

Mechanism of action of Taohong Siwu decoction in the alleviation of primary dysmenorrhea.

As one of the most common gynecological disorders, PD significantly impacts the quality of life for women. TSD, a well-known traditional Chinese medical prescription, has gained popularity for its use in treating gynecological cold coagulation and blood stasis syndromes such as PD. However, the lack of comprehensive data hinders our understanding of its molecular mechanism. The objective of the present study is to investigate the therapeutic effects of TSD on PD and elucidate its plausible mechanism. HPLC was employed to confirm the presence of the principal metabolites of TSD. The rat model of PD was induced by OT exposure following IWM and EB pretreatment, and subsequently treated with TSD via gastric gavage. The effects and potential mechanisms of TSD on PD rats were explored, encompassing general behavior, morphological alterations in the uterus and ovaries, biochemical indicators in the uterus and serum, and levels of proteins related to the PI3K/AKT signaling pathway. Gallic acid, hydroxysafflower yellow A, albiflorin, paeoniflorin, and ferulic acid were determined to be the primary active metabolites of TSD. The pharmacological studies yielded results indicating the successful establishment of the PD model in rats. Additionally, TSD demonstrated its ability to protect PD rats by ameliorating general behavior, mitigating pathological damage to uterine and ovarian tissues, and modulating the expression levels of correlated factors (PGE2, PGF2α, Ca2+, TXB2, IL-6, TNF-α, NO, and COX-2) as well as p-PI3K/PI3K and p-AKT/AKT proteins. TSD exhibited protective effects against PD in rats through its interaction with multiple targets including P13K/AKT signaling pathway, indicating that TSD holds therapeutic potential for PD treatment and providing evidence supporting the rational utilization of TSD.

The pharmacokinetics of single-dose oral atorvastatin and its metabolites support therapeutic use in cockatiels (Nymphicus hollandicus).

To evaluate the plasma concentrations and determine the pharmacokinetic parameters of atorvastatin and its primary active metabolites (para- and orthohydroxyatorvastatin) after administration of a single oral dose in cockatiels (Nymphicus hollandicus). 14 adult cockatiels (7 male, 7 female) around 2 years of age. A compounded oral suspension of atorvastatin 10 mg/mL made with an oral suspending agent and an oral sweetener was administered via oral gavage at 20 mg/kg to each bird. Blood samples were collected at 7 different time points from 0.5 to 24 hours postadministration in a balanced incomplete block design with 3 blood samples per bird and 6 replicates per time point. Plasma concentrations of atorvastatin, parahydroxyatorvastatin, and orthohydroxyatorvastatin were determined by liquid chromatography-tandem mass spectrometry. Pharmacokinetic analysis was performed using noncompartmental analysis. The estimated time to maximum concentration (tmax) for atorvastatin, parahydroxyatorvastatin, and orthohydroxyatorvastatin was 3 hours for each. The estimated maximum plasma concentration (Cmax) for atorvastatin, parahydroxyatorvastatin, and orthohydroxyatorvastatin was 152.6, 172.4, and 68.8 ng/mL, respectively. The terminal half-lives were 4, 6.8, and 4.6 hours, respectively. These results support the therapeutic use of atorvastatin at the dose evaluated in this species based on human pharmacokinetic data. A starting dose of 20 mg/kg PO every 12 to 24 hours could be used to treat lipid disorders in cockatiels pending more data on multidose use and hypolipidemic efficacy.

Pharmacokinetics of single-dose oral atorvastatin and its metabolites support therapeutic use in orange-winged Amazon parrots (Amazona amazonica).

To evaluate the plasma concentrations and determine pharmacokinetic parameters of atorvastatin and its primary active metabolites (para- and ortho-hydroxyatorvastatin) after administration of a single oral dose in orange-winged Amazon parrots (Amazona amazonica). 8 adult orange-winged Amazon parrots (4 male, 4 female) of varying ages. A compounded oral suspension of atorvastatin 10 mg/mL was administered via oral gavage at 20 mg/kg to each bird. Blood samples were collected at 10 different time points from 0 to 30 hours postadministration to evaluate plasma levels of atorvastatin, para-hydroxyatorvastatin, and ortho-hydroxyatorvastatin. Pharmacokinetic analysis was performed using noncompartmental analysis and commercially available software. Mean ± SD atorvastatin half-life, tmax, and Cmax were 5.96 ± 11.50 hours, 1.60 ± 0.80 hours, and 82.60 ± 58.30 ng/mL, respectively. For para-hydroxyatorvastatin, the half-life, tmax, and Cmax were 6.46 ± 54.20 hours, 5.00 ± 2.51 hours, and 34.10 ± 16.00 ng/mL, respectively, and 5.58 ± 9.92 hours, 3.38 ± 2.10 hours, and 7.35 ± 3.96 ng/mL for ortho-hydroxyatorvastatin. The plasma concentrations and pharmacokinetic profile shown support the therapeutic use of atorvastatin at the dose evaluated in this species based on human pharmacokinetic data. While 20 mg/kg PO q24 hours could be used as a starting dosage until further studies evaluating multiple dose administration and efficacy in this species become available, the high interindividual variability results warrant monitoring of the treatment response to make dosing adjustments if needed.

Investigations on the electrochemical behavior of sunitinib and metabolites N-desethyl-sunitinib and sunitinib-N-oxide and its selective determination using molecularly imprinted polymer-based sensor

Sunitinib (SUN) is an orally administered tyrosine kinase inhibitor and is mainly metabolized via N-deethylation to form the primary active metabolite N-desethyl-sunitinib (M1) and via oxidation to sunitinib-N-oxide (M2). In this study, we first investigated the electrochemical behavior of sunitinib and its N-desethyl and N-oxide metabolites on a bare glassy carbon electrode (GCE) by cyclic voltammetry (CV) technique. Based on the results, a plausible electrooxidation mechanism is proposed, yielding the oxo-desfluoro-sunitinib (quinoneimine metabolite) via oxidative defluorination of sunitinib. After this phase of the study, due to the low sensitivity and selectivity results obtained from the bare GCE, we aimed to develop the first molecularly imprinted polymer (MIP)-based electrochemical sensor for the sensitive and selective determination of SUN. The MA@SDS/MIP-GCE sensor was prepared by thermal polymerization of methacrylic acid (MA) as a functional monomer in the presence of SUN on a GCE surface under the optimized parameters such as template/monomer ratio, drop amount, polymerization temperature and time, removal solution and time, and rebinding time, which affect the structure and response of the sensor. The surface characterization of the developed sensor was performed by scanning electron microscopy (SEM), and the electrochemical characterization was performed by CV and electrochemical impedance spectroscopy (EIS). The electroanalytical performance of the MA@SDS/MIP-GCE sensor was tested by differential pulse voltammetry (DPV) under the optimized conditions in comparison with the NIP-based sensor. The linearity of the developed sensor for the determination of SUN was investigated in the concentration range of 0.5 – 5.0 nM for standard and serum solutions with low LOD and LOQ values. The application of the MIP sensor for the determination of SUN in pharmaceutical dosage form (capsule) and serum samples was demonstrated with excellent recoveries. The results showed that the MA@SDS/MIP-GCE sensor is a sensitive, specific, selective, rapid, and economical option for the quantitative determination of SUN.

Active metabolites and potential mechanisms of Notopterygium incisum against obstructive sleep apnea Syndrome (OSAS): network analysis and experimental assessment.

Background: Notopterygium incisum K.C. Ting ex H.T. Chang, a synonym of Hansenia weberbaueriana (Fedde ex H. Wolff) Pimenov & Kljuykov, is an anti-inflammatory medicinal plant. Although abrnotopterol has been reported to be its primary active metabolite, the other metabolites and their mechanisms of action remain unclear. This study aims to investigate the potential mechanisms by which its active metabolites treat Obstructive Sleep Apnea Syndrome (OSAS) through network analysis and experimental assessment. Methods: The metabolites and potential targets of Notopterygium incisum were extracted from public databases. We searched for OSAS-related genes in the Genecards, OMIM, PharmGkb, TTD, and DrugBank databases. Cytoscape 3.9.0 was used to construct the drug-target-disease network and screen for hub genes. Human bronchial epithelial (HBE) cells were cultivated in normoxia and chronic intermittent hypoxia (CIH) medium for 24h. Interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and prostaglandin E2 (PGE2) were quantified using enzyme-linked immunosorbent assay (ELISA). Prostaglandin-endoperoxide synthase 2(PTGS2) mRNA was detected using RT-qPCR, while PTGS2 and nuclear factor-kappa B (NF-κB) proteins were identified using Western blot analysis. Co-Immunoprecipitation (CoIP) and Western blotting were utilized to evaluate the ubiquitination of PTGS2 in HBE cells. Results: Pterostilbene and notopterol, isolated from Notopterygium incisum, had potential therapeutic effects on OSAS. The PTGS2 and estrogen receptor alpha (ESR1) hub genes were associated with OSAS. The pathway enrichment analysis focuses on the NF-κB, apoptosis, and HIF-1A pathways. In response to CIH, pterostilbene and notopterol decreased IL-6, TNF-α, and PGE2 levels. The NF-κB pathway was activated by an increase in PTGS2 levels. Pterostilbene promoted proteasome-mediated ubiquitination of PTGS2 protein and reduced PTGS2 levels, inhibiting the NF-κB pathway. Conclusion: This study reveals the active metabolites of Notopterygium incisum and hub genes involved in treating OSAS, which provide a basis for the follow-up development and exploitation of the botanical drug.

The antipsychotic medications aripiprazole, brexpiprazole and cariprazine are off-target respiratory chain complex I inhibitors

Antipsychotic drugs are the mainstay of treatment for schizophrenia and provide adjunct therapies for other prevalent psychiatric conditions, including bipolar disorder and major depressive disorder. However, they also induce debilitating extrapyramidal syndromes (EPS), such as Parkinsonism, in a significant minority of patients. The majority of antipsychotic drugs function as dopamine receptor antagonists in the brain while the most recent ‘third’-generation, such as aripiprazole, act as partial agonists. Despite showing good clinical efficacy, these newer agents are still associated with EPS in ~ 5 to 15% of patients. However, it is not fully understood how these movement disorders develop. Here, we combine clinically-relevant drug concentrations with mutliscale model systems to show that aripiprazole and its primary active metabolite induce mitochondrial toxicity inducing robust declines in cellular ATP and viability. Aripiprazole, brexpiprazole and cariprazine were shown to directly inhibit respiratory complex I through its ubiquinone-binding channel. Importantly, all three drugs induced mitochondrial toxicity in primary embryonic mouse neurons, with greater bioenergetic inhibition in ventral midbrain neurons than forebrain neurons. Finally, chronic feeding with aripiprazole resulted in structural damage to mitochondria in the brain and thoracic muscle of adult Drosophila melanogaster consistent with locomotor dysfunction. Taken together, we show that antipsychotic drugs acting as partial dopamine receptor agonists exhibit off-target mitochondrial liabilities targeting complex I.

Comparative activity of the enantiomers of fenfluramine and norfenfluramine in rodent seizure models, and relationship with their concentrations in plasma and brain.

To investigate the comparative antiseizure activity of the individual enantiomers of fenfluramine and its major active primary metabolite norfenfluramine in rodent seizure models, and its relationship with the pharmacokinetics of these compounds in plasma and brain. The antiseizure potency of d,l-fenfluramine (racemic fenfluramine) was compared with the respective potencies of its individual enantiomers and the individual enantiomers of norfenfluramine using the maximal electroshock (MES) test in rats and mice, and the 6-Hz 44 mA test in mice. Minimal motor impairment was assessed simultaneously. The time course of seizure protection in rats was compared with the concentration profiles of d-fenfluramine, l-fenfluramine, and their primary active metabolites in plasma and brain. All compounds tested were active against MES-induced seizures in rats and mice after acute (single-dose) administration, but no activity against 6-Hz seizures was found even at doses up to 30 mg/kg. Estimates of median effective doses (ED50 ) in the rat-MES test were obtained for all compounds except for d-norfenfluramine, which caused dose-limiting neurotoxicity. Racemic fenfluramine had approximately the same antiseizure potency as its individual enantiomers. Both d- and l-fenfluramine were absorbed and distributed rapidly to the brain, suggesting that seizure protection at early time points (≤2 h) was related mainly to the parent compound. Concentrations of all enantiomers in brain tissue were >15-fold higher than those in plasma. Although there are differences in antiseizure activity and pharmacokinetics among the enantiomers of fenfluramine and norfenfluramine, all compounds tested are effective in protecting against MES-induced seizures in rodents. In light of the evidence linking the d-enantiomers to cardiovascular and metabolic adverse effects, these data suggest that l-fenfluramine and l-norfenfluramine are potentially attractive candidates for a chiral switch approach leading to development of a novel, enantiomerically-pure antiseizure medication.

Susceptibility of cervical cancer to dihydroartemisinin-induced ferritinophagy-dependent ferroptosis.

The clinical therapeutics of cervical cancer is limited due to the drug resistance and metastasis of tumor. As a novel target for antitumor therapy, ferroptosis is deemed to be more susceptible for those cancer cells with resistance to apoptosis and chemotherapy. Dihydroartemisinin (DHA), the primary active metabolites of artemisinin and its derivatives, has exhibited a variety of anticancer properties with low toxicity. However, the role of DHA and ferroptosis in cervical cancer remained unclear. Here, we showed that DHA could time-dependently and dose-dependently inhibit the proliferation of cervical cancer cells, which could be alleviated by the inhibitors of ferroptosis rather than apoptosis. Further investigation confirmed that DHA treatment initiated ferroptosis, as evidenced by the accumulation of reactive oxygen species (ROS), malondialdehyde (MDA) and liquid peroxidation (LPO) levels and simultaneously depletion of glutathione peroxidase 4 (GPX4) and glutathione (GSH). Moreover, nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy was also induced by DHA leading to subsequent increases of intracellular labile iron pool (LIP), exacerbated the Fenton reaction resulting in excessive ROS production, and enhanced cervical cancer ferroptosis. Among them, we unexpectedly found that heme oxygenase-1 (HO-1) played an antioxidant role in DHA-induced cell death. In addition, the results of synergy analysis showed that the combination of DHA and doxorubicin (DOX) emerged a highly synergistic lethal effect for cervical cancer cells, which was related also to ferroptosis. Overall, our data revealed the molecular mechanisms that DHA triggered ferritinophagy-dependent ferroptosis and sensitized to DOX in cervical cancer, which may provide novel avenues for future therapy development.

Review of 5-FU resistance mechanisms in colorectal cancer: clinical significance of attenuated on-target effects.

The emergence of chemoresistant disease during chemotherapy with 5-Fluorouracil-based (5-FU-based) regimens is an important factor in the mortality of metastatic CRC (mCRC). The causes of 5-FU resistance are multi-factorial, and besides DNA mismatch repair deficiency (MMR-D), there are no widely accepted criteria for determining which CRC patients are not likely to be responsive to 5-FU-based therapy. Thus, there is a need to systematically understand the mechanistic basis for 5-FU treatment failure and an urgent need to develop new approaches for circumventing the major causes of 5-FU resistance. In this manuscript, we review mechanisms of 5-FU resistance with an emphasis on: (1) altered anabolic metabolism limiting the formation of the primary active metabolite Fluorodeoxyuridylate (5-Fluoro-2'-deoxyuridine-5'-O-monophosphate; FdUMP); (2) elevated expression or activity of the primary enzymatic target thymidylate synthase (TS); and (3) dysregulated programmed cell death as important causes of 5-FU resistance. Importantly, these causes of 5-FU resistance can potentially be overcome through the use of next-generation fluoropyrimidine (FP) polymers (e.g., CF10) that display reduced dependence on anabolic metabolism and more potent TS inhibitory activity.

Formation of CYP3A-specific metabolites of ibrutinib in vitro is correlated with hepatic CYP3A activity and 4β-hydroxycholesterol/cholesterol ratio.

Ibrutinib is an orally administered Bruton's tyrosine kinase inhibitor approved for the treatment of B-cell malignancies, including chronic lymphocytic leukemia. Ibrutinib is metabolized primarily via oxidation by cytochrome P450 (CYP) 3A4/5 to M37 (the primary active metabolite), M34, and M25. The objectives of this study were to assess the relationship between formation of the major CYP3A-specific ibrutinib metabolites in vitro and hepatic CYP3A activity and protein abundance, and to evaluate the utility of the endogenous CYP3A biomarker, plasma 4β-hydroxycholesterol (4β-HC) to cholesterol ratio, to predict ibrutinib metabolite formation in individual cadaveric donors with matching hepatocytes. Ibrutinib (5μM) was incubated with single-donor human liver microsomes (n=20) and primary human hepatocytes (n=15), and metabolites (M37, M34, and M25) were measured by liquid chromatography-tandem mass spectrometry analysis. CYP3A4/5 protein concentrations were measured by quantitative targeted absolute proteomics, and CYP3A activity was measured by midazolam 1'-hydroxylation. Ibrutinib metabolite formation positively correlated with midazolam 1'-hydroxylation in human liver microsomes and hepatocytes. Plasma 4β-HC and cholesterol concentrations were measured in plasma samples obtained at the time of liver harvest from the same 15 donors with matching hepatocytes. Midazolam 1'-hydroxylation in hepatocytes correlated with plasma 4β-HC/cholesterol ratio. When an infant donor (1 year old) was excluded based on previous ontogeny studies, M37 and M25 formation correlated with plasma 4β-HC/cholesterol ratio in the remaining 14 donors (Spearman correlation coefficients [r] 0.62 and 0.67, respectively). Collectively, these data indicate a positive association among formation of CYP3A-specific ibrutinib metabolites in human hepatocytes, hepatic CYP3A activity, and plasma 4β-HC/cholesterol ratio in the same non-infant donors.

Effects of genotype and food on naltrexone exposure in adolescents.

Naltrexone (NTX), an opioid antagonist metabolized by aldo-keto reductase 1C4 (AKR1C4), is prescribed for psychiatric conditions like eating disorders with variable response. Systemic exposure is highly variable in adults, yet no data exist in children. The purpose of this study was to evaluate NTX exposure in adolescents with eating disorders. Adolescents aged 12-21 years with eating disorders underwent postdose blood sampling in the fasted and/or fed state. NTX and primary active metabolite, 6-β-naltrexol, were determined by ultra-high performance liquid chromatography tandem mass spectrometry. Pharmacokinetic parameters were determined by noncompartmental analysis. DNA was genotyped for AKR1C4 missense mutations associated with decreased activity (rs3829125 and rs17134592). Linear mixed effects modeling was performed. In 21 participants, aged 16.9 ± 1.9 years (15-21 years), 81% female participants, maximum concentration (Cmax ) was 90.4 ± 129 nM/mg/kg, area under the concentration-time curve from zero to infinity (AUC0-∞ ) was 166 ± 154 nM h/mg/kg, and varied 63-fold and 21-fold, respectively. Compared with wildtype, those with AKR1C4 allelic variations (n=7) displayed 3.2-fold higher AUC0-∞ , four-fold higher Cmax and delayed time to Tmax . Linear mixed effects modeling demonstrated a large effect of genotype on AUC0-∞ (Cohen's d -2.3) and Cmax (Cohen's d -1.4). Food effect was large for AUC0-∞ (Cohen's d 2.6), but highly variable and failed to reach significance for Cmax. The respective model accounted for 82% of the variance in NTX AUC0-∞ and 46% of the variance in Cmax . NTX systemic exposure is highly variable in adolescents with eating disorders and modulated, in part, by AKR1C4 genotype and food intake. These findings may, in part, explain the large degree of interindividual variability observed response to NTX.

Cytochromes P450 2C8 and 3A Catalyze the Metabolic Activation of the Tyrosine Kinase Inhibitor Masitinib.

Masitinib is a small molecule tyrosine kinase inhibitor under investigation for the treatment of amyotrophic lateral sclerosis, mastocytosis, and COVID-19. Hepatotoxicity has been reported in some patients while taking masitinib. The liver injury is thought to involve hepatic metabolism of masitinib by cytochrome P450 (P450) enzymes to form chemically reactive, potentially toxic metabolites. The goal of the current investigation was to determine the P450 enzymes involved in the metabolic activation of masitinib in vitro. In initial studies, masitinib (30 μM) was incubated with pooled human liver microsomes in the presence of NADPH and potassium cyanide to trap reactive iminium ion metabolites as cyano adducts. Masitinib metabolites and cyano adducts were analyzed using reversed-phase liquid chromatography-tandem mass spectrometry. The primary active metabolite, N-desmethyl masitinib (M485), and several oxygenated metabolites were detected along with four reactive metabolite cyano adducts (MCN510, MCN524, MCN526, and MCN538). To determine which P450 enzymes were involved in metabolite formation, reaction phenotyping experiments were conducted by incubation of masitinib (2 μM) with a panel of recombinant human P450 enzymes and by incubation of masitinib with human liver microsomes in the presence of P450-selective chemical inhibitors. In addition, enzyme kinetic assays were conducted to determine the relative kinetic parameters (apparent Km and Vmax) of masitinib metabolism and cyano adduct formation. Integrated analysis of the results from these experiments indicates that masitinib metabolic activation is catalyzed primarily by P450 3A4 and 2C8, with minor contributions from P450 3A5 and 2D6. These findings provide further insight into the pathways involved in the generation of reactive, potentially toxic metabolites of masitinib. Future studies are needed to evaluate the impact of masitinib metabolism on the toxicity of the drug in vivo.

Fatal Intoxication with a mixture of para-methoxyamphetamine, N-ethylpentylone, and ketamine

New psychoactive substances (NPS) have had a serious impact worldwide, and their determination in postmortem samples has been challenging. Here, we present a fatal case of poisoning involving the combined ingestion of para-methoxyamphetamine (PMA), N-ethylpentylone (NEP), and ketamine, and quantitative analysis of these drugs and para-hydroxyamphetamine (PHA), a primary metabolite of PMA. Case history: A man in his 20s was found dead in his room, wearing only underwear. Pink star-shaped tablets were found adjacent to the decedent. The tablets were submitted to a police forensic science laboratory and identified to contain PMA, NEP, and ketamine. An autopsy was performed in our laboratory, and samples of femoral vein blood and urine were collected for quantitative analysis. Quantitative analyses of PMA, NEP, ketamine, and PHA were performed using an ultra-high-performance liquid chromatograph coupled with a triple quadrupole tandem mass spectrometer (LCMS8030, Shimadzu). Blood samples were extracted using a Micro Volume QuEChERS kit (Shimadzu). Urine samples were prepared by diluting 100-fold with water prior to hydrolysis. Hydrolysis conditions were investigated using β-glucuronidase (Helix pomatia, type HP-2, ≥ 100,000 units/mL) or hydrochloric acid (HCl), as PHA was expected to form conjugates. The method was validated by assessing linearity, intra- and inter-day accuracy and precision, recovery, and matrix effect. Quantitative analyses revealed that the concentrations of PMA, NEP, and ketamine were 2.2 μg/mL, 0.21 μg/mL, and 0.22 μg/mL in femoral vein blood and 133 μg/mL, 9.5 μg/mL, and 13 μg/mL in urine, respectively. The concentration of PHA in urine was 24 μg/mL. PHA concentration was not affected by hydrolysis with β-glucuronidase, but was significantly increased with HCl. This result suggests that PHA is predominantly conjugated as sulfate, rather than glucuronide. The linearity (R2) of the calibration curves was > 0.99, and the accuracy and precision were < 15% for all the target drugs. Based on autopsy findings and toxicology results, the forensic pathologist concluded that the cause of death was intoxication by the simultaneous ingestion of PMA, NEP, and ketamine. Previous reports showed that the toxic blood levels for PMA and NEP ranged above 0.5 μg/mL and 0.1 μg/mL, respectively. The blood levels of PMA and NEP in the present case exceeded the toxic levels, which may have caused acute intoxication and led to death. Ketamine concentration was at the therapeutic level. As in this case, it is not unusual for several substances with different pharmacological effects, such as excitation, sedation, and hallucinations, to be detected simultaneously in a single NPS-related product. Products with multiple pharmacological actions may have unexpected effects on the human body. This report describes a fatal case of poisoning due to PMA, NEP, and ketamine. In addition, PHA, the primary active metabolite of PMA, was quantified in the postmortem samples. There have been no reports examining the hydrolysis conditions for PHA. These validated quantitative values would be helpful in interpreting the cases in which these drugs were ingested.

Transcriptional Regulatory Mechanism of Differential Metabolite Formation in Root and Stem of Ephedra sinica.

Ephedra sinica, a well-known Chinese medicinal plant, is characterized as having the opposite medicinal effect among its root and stem. However, there is a lack of understanding to differentiate the active components present in the root and stem of E. sinica, as well as the molecular mechanisms underlying the formation of the differential compounds, which has significantly hampered the further development and utilization of E. sinica resource. In this study, forty-five differential metabolic markers are affiliated to alkaloids, flavonoids, terpenoids, and organic acids between root and stem of E. sinica, and sixty genes of key enzymes are involved in their biosynthesis distributed in metabolic pathway branches such as phenylalanine metabolism, flavonoid biosynthesis and phenylpropane biosynthesis, based on combination non-targeted metabolome with transcriptome technologies. The finding revealed that the expression activity changes of these enzyme genes had a direct impact on the distinction of differential metabolic markers in the root and stem of E. sinica. This study will help to understand the molecular mechanism of the differentiation and biosynthesis of the primary active metabolites in the root and stem of E. sinica, providing a theoretical foundation for its quality control and promotion in cultivation.

Pleiotropic Effects of Icariside II on the Cardiovascular System: Novel Applications of Ethnopharmacology in Targeting Vascular Remodeling.

Pleiotropic Effects of Icariside II on the Cardiovascular System: Novel Applications of Ethnopharmacology in Targeting Vascular Remodeling.

Qualitative and quantitative determination of the primary active components and metabolites in human plasma after oral administration of Shuanghuanglian liquid.

Shuanghuanglian oral liquid is a common traditional Chinese medicine used to treat respiratory tract infections. Its major components are baicalin, chlorogenic acid, and forsythin. In this study, the main drug-related components in human plasma after oral administration of Shuanghuanglian were initially identified using ultra-performance liquid chromatography-ultraviolet detector/quadrupole time-of-flight mass spectrometry. Thirteen components from baicalin were identified, including the parent drug baicalin and aglycone baicalein. Only one metabolite related to chlorogenic acid, a sulfate conjugate formed after hydrolysis, and one metabolite related to forsythin, a sulfate conjugate of forsythin aglycone, were detected. Subsequently, a liquid chromatography-tandem mass spectrometry method was established and validated to simultaneously determine baicalin and baicalein, the primary active components. After simple protein precipitation, the analytes were separated on a BEH C18 column using a 5min-gradient elution to avoid interference from baicalin isomers and their in-source dissociation. Excellent linearity was observed over the concentration ranges of 5.00-2000ng/ml for baicalin and 1.00-100ng/ml for baicalein. The validated method was successfully applied to a pharmacokinetic study of an oral administration of 60ml Shuanghuanglian in healthy subjects. This study provided a foundation to investigate the clinical efficacy and safety of Shuanghuanglian further.