Enzyme Phenotypes Research Articles

CYP3A4-mediated metabolism of artemisinin to 10β-hydroxyartemisinin with comparable anti-malarial potency.

The most widely used anti-malarial drug artemisinin (ART) is metabolized extensively, but the therapeutic capacity of its major metabolite remains unknown. Whether the major metabolite of ART (ART-M) contributes to its antiplasmodial potency was investigated in this study. The metabolite identification and enzyme phenotyping of ART were performed using human liver microsomes (HLMs). The stereostructure of the major metabolite ART-M was elucidated by spectroscopic and X-ray crystallographic analysis. The anti-malarial activity of ART-M against two reference Plasmodium strains (Pf3D7 and PfDd2) was evaluated. The pharmacokinetic profiles of ART and its metabolite ART-M were investigated in healthy Chinese subjects after a recommended two-day oral dose of ART plus piperaquine. Pharmacodynamic parameters based on minimum inhibitory concentration (MIC50) and free plasma concentration were employed to evaluate the therapeutic potency of ART-M, including fAUC0-t/MIC50, fCmax/MIC50 and T > MIC50. A major metabolite 10β-hydroxyartemisinin (ART-M) was found for ART in human, and CYP3A4/3A5 was the major enzymes responsible for ART 10β-hydroxylation. Compared with ART (MIC50, 10.1nM against Pf3D7), weaker antiplasmodial activity was found for ART-M (MIC50, 61.4nM against Pf3D7). However, a 3.5-fold higher maximal free plasma concentration was achieved for ART-M (fCmax, 180.0nM vs. 51.8nM for ART). ART-M displayed comparable antiplasmodial potency to ART, in terms of fAUC0-t/MIC50 (12.5h), fCmax/MIC50 (2.8) and T > MIC50 (5h). The major metabolite 10β-hydroxyartemisinin contributes to the antiplasmodial efficacy of ART, which should be considered when evaluation of ART dosing regimens and/or clinical outcomes.

Association of Angiotensin Converting Enzyme Phenotypes and Polymorphisms with Clinical Outcomes in SARS-CoV2 Patients with Hypertension in an Urban Emergency Department.

The role of Angiotensin-converting enzyme (ACE and ACE2) phenotypes and polymorphisms in modulating severe acute respiratory syndrome coronavirus (SARS-- CoV2) infection in hypertensive patients remains unclear. Our objective was to determine the distribution of ACE and ACE2 receptor phenotypes by patient demographics and correlate ACE and ACE2 levels of activity with SARS-CoV-2 outcomes. Hypertensive patients treated for SARS-CoV-2 at an urban emergency department (ED) were prospectively enrolled in a cohort study between August 2020 and April 2021. Blood samples were collected during ED visits or hospitalization. Outcome measures including hospitalization, intensive care unit (ICU) admission, and 30-day mortality were obtained from electronic health records. Multivariable logistic regression was used. Of the 150 patients enrolled, 60% were Black, 32% Hispanic/Latinx, 4% Non-Hispanic Whites, and 4% others. The mean age was 59 (+/-14) years. The rate of hospitalization was high (86%) and Hispanic/Latinx had a higher likelihood of ICU admission. Patients harboring the rs2285666 genotype TT, AA, and GC alleles were more likely to be admitted to ICU, and those with TT and AA had higher mortality. The ACE level was a significant predictor of hospitalization with a protective effect in both unadjusted and adjusted results. Hispanics/Latinx had a four times higher likelihood of ICU admission compared to all others, and age was significantly associated with 30-day mortality. Our results show that even after adjusting for age, race, and sex, ACE levels remained a predictor of hospitalization. ACE/ACE2 phenotypes and genotypes potentially play an important role in disease progression in SARS-CoV-2 patients.

Predictive Model for Opioid Use Disorder in Chronic Pain: A Development and Validation Study.

There are several questionnaires for the challenge of anticipating opioid use disorder (OUD). However, many are not specific for chronic non-cancer pain (CNCP) or have been developed in the American population, whose sociodemographic factors are very different from the Spanish population, leading to scarce translation into clinical practice. Thus, the aim of this study is to prospectively validate a predictive model for OUD in Spanish patients under long-term opioids. An innovative two-stage predictive model was developed from retrospective (n = 129) and non-overlapping prospective (n = 100) cohorts of real-world CNCP outpatients. All subjects used prescribed opioids for 6 or more months. Sociodemographic, clinical and pharmacological covariates were registered. Mu-opioid receptor 1 (OPRM1, A118G, rs1799971) and catechol-O-methyltransferase (COMT, G472A, rs4680) genetic variants plus cytochrome P450 2D6 (CYP2D6) liver enzyme phenotypes were also analyzed. The model performance and diagnostic accuracy were calculated. The two-stage model comprised risk factors related to OUD (younger age, work disability and high daily opioid dose) and provided new useful information about other risk factors (low quality of life, OPRM-G allele and CYP2D6 extreme phenotypes). The validation showed a satisfactory accuracy (70% specificity and 75% sensitivity) for our predictive model with acceptable discrimination and goodness of fit. Our study presents the results of an innovative model for predicting OUD in our setting. After external validation, it could represent a change in the paradigm of opioid treatment, helping clinicians to better identify and manage the risks and reduce the side effects and complications.

Phenotype-Genotype Correlation Applying a Cocktail Approach and an Exome Chip Analysis Reveals Further Variants Contributing to Variation of Drug Metabolism.

Although great progress has been made in the fine-tuning of diplotypes, there is still a need to further improve the predictability of individual phenotypes of pharmacogenetically relevant enzymes. The aim of this study was to analyze the additional contribution of sex and variants identified by exome chip analysis to the metabolic ratio of five probe drugs. A cocktail study applying dextromethorphan, losartan, omeprazole, midazolam, and caffeine was conducted on 200 healthy volunteers. CYP2D6, 2C9, 2C19, 3A4/5, and 1A2 genotypes were analyzed and correlated with metabolic ratios. In addition, an exome chip analysis was performed. These SNPs correlating with metabolic ratios were confirmed by individual genotyping. The contribution of various factors to metabolic ratios was assessed by multiple regression analysis. Genotypically predicted phenotypes defined by CPIC discriminated very well the log metabolic ratios with the exception of caffeine. There were minor sex differences in the activity of CYP2C9, 2C19, 1A2, and CYP3A4/5. For dextromethorphan (CYP2D6), IP6K2 (rs61740999) and TCF20 (rs5758651) affected metabolic ratios, but only IP6K2 remained significant after multiple regression analysis. For losartan (CYP2C9), FBXW12 (rs17080138), ZNF703 (rs79707182), and SLC17A4 (rs11754288) together with CYP diplotypes, and sex explained 50% of interindividual variability. For omeprazole (CYP2C19), no significant influence of CYP2C:TG haplotypes was observed, but CYP2C19 rs12777823 improved the predictability. The comprehensive genetic analysis and inclusion of sex in a multiple regression model significantly improved the explanation of variability of metabolic ratios, resulting in further improvement of algorithms for the prediction of individual phenotypes of drug-metabolizing enzymes.

In vitro pharmacokinetic behavior in lung of harringtonine, an antagonist of SARS-CoV-2 associated proteins: New insights of inhalation therapy for COVID-19

BackgroundRecent studies have shown that harringtonine (HT) could specifically bind with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein and host cell transmembrane serine protease 2 (TMPRSS2) to block membrane fusion, which is an effective antagonist for SARS-CoV-2. PurposeOur study focused on in-depth exploration of in vitro pharmacokinetic characteristics of HT in lung. MethodsHPLC-fluorescence detection method was used to detect changes of HT content. Incubation systems of lung microsomes for phase I metabolism and UGT incubation systems for phase II metabolism were performed to elucidate metabolites and metabolic mechanisms of HT, and then the metabolic enzyme phenotypes for HT were clarified by chemical inhibition method and recombinant enzyme method. Through metabolomics, we comprehensively evaluated the physiological dynamic changes in SD rat and human lung microsomes, and revealed the relationship between metabolomics and pharmacological activity of HT. ResultsHPLC-fluorescence detection method showed strong specificity, high accuracy, and good stability for rapid quantification of HT. We confirmed that HT mainly underwent phase I metabolism, and the metabolites of HT in different species were all identified as 4′-demethyl HT, with metabolic pathway being hydrolysis reaction. CYP1A2 and CYP2E1 participated in HT metabolism, but as HT metabolism was not NADPH dependent, the esterase HCES1 in lung also played a role. The main KEGG pathways in SD rat and human lung microsomes were cortisol synthesis and secretion, steroid hormone biosynthesis and linoleic acid metabolism, respectively. The downregulated key biomarkers of 11-deoxycortisol, 21-deoxycortisol and 9(10)-EpOME suggested that HT could prevent immunosuppression and interfere with infection and replication of SARS-CoV-2. ConclusionHT was mainly metabolized into 4′-demethyl HT through phase I reactions, which was mediated by CYP1A2, CYP2E1, and HCES1. The downregulation of 11-deoxycortisol, 21-deoxycortisol and 9(10)-EpOME were key ways of HT against SARS-CoV-2. Our study was of great significance for development and clinical application of HT in the treatment of COVID-19.

Clinical prediction of opioid use disorder in chronic pain patients: a cohort-retrospective study with a pharmacogenetic approach.

Opioids are widely used in chronic non-cancer pain (CNCP) management. However, they remain controversial due to serious risk of causing opioid use disorder (OUD). Our main aim was to develop a predictive model for future clinical translation that include pharmacogenetic markers. An observational study was conducted in 806 pre-screened Spanish CNCP patients, under long-term use of opioids, to compare cases (with OUD, N.=137) with controls (without OUD, N.=669). Mu-opioid receptor 1 (OPRM1, A118G, rs1799971) and catechol-O-methyltransferase (COMT, G472A, rs4680) genetic variants plus cytochrome P450 2D6 (CYP2D6) liver enzyme phenotypes were analyzed. Socio-demographic, clinical and pharmacological outcomes were also registered. A logistic regression model was performed. The model performance and diagnostic accuracy were calculated. OPRM1-AA genotype and CYP2D6 poor and ultrarapid metabolizers together with three other potential predictors: 1) age; 2) work disability; 3) oral morphine equivalent daily dose (MEDD), were selected with a satisfactory diagnostic accuracy (sensitivity: 0.82 and specificity: 0.85), goodness of fit (P=0.87) and discrimination (0.89). Cases were ten-year younger with lower incomes, more sleep disturbances, benzodiazepines use, and history of substance use disorder in front of controls. Functional polymorphisms related to OPRM1 variant and CYP2D6 phenotypes may predict a higher OUD risk. Established risk factors such as young age, elevated MEDD and lower incomes were identified. A predictive model is expected to be implemented in clinical setting among CNCP patients under long-term opioids use.

Enantioselective in vitro metabolism of the herbicide diclofop-methyl: Prediction of toxicokinetic parameters and reaction phenotyping

Human exposure to contaminants of emerging concern, like pesticides, has increased in the past decades. Diclofop-methyl (DFM) is a chiral herbicide that is employed as a racemic mixture (rac-DFM) in soybean and other crops against wild oats. Studies have shown that DFM has enantioselective action (higher for R-DFM), degradation (faster for S-DFM), and metabolism, producing diclofop (DF) which is also a pesticide. Although toxic effects have been reported for DFM, information regarding how DFM affects humans is lacking, especially when its chirality is concerned. In this study, the in vitro metabolism of rac-DFM and its isolated enantiomers was assessed by using a human model based on human liver microsomes. The kinetic model and parameters were obtained, and the hepatic clearance (CLH) and hepatic extraction ratio (EH) were estimated. Enzyme phenotyping was carried out by employing carboxylesterase isoforms (CES 1 and CES 2). DFM was metabolized through positive homotropic cooperativity with slight preference for (−)-DFM metabolism to (−)-DF. CLH and EH were above 19.60 mL min−1 kg−1 and 98 % for all the monitored reactions, respectively, and CES 1 was the main enzyme underlying the metabolism. These findings point out that liver contributes to DFM metabolism, which is fast, resulting in nearly complete conversion to DF after exposition to DFM.

Detection of KPC and VIM Genes in Carbapenem-resistant Klebsiella pneumoniae Isolates from Blood Culture in Southern Anhui, China

Background: Klebsiella pneumoniae is one of the main pathogens of lower respiratory tract infections. Carbapenems are considered the last line of defense for the treatment of Gram-negative bacteria with multidrug resistance. In recent years, with the increase of bacteria producing carbapenemase, the resistance rate of carbapenems has increased gradually. Objectives: The main objective of this study was to detect the blaKPC and blaVIM genes in K. pneumoniae isolates from blood culture specimens. Methods: Within September 2020 to August 2022, 1033 bacterial strains were isolated from blood cultures in Yijishan Hospital of Wannan Medical College, Wuhu, Anhui province, China, including 141 strains of K. pneumoniae. All K. pneumoniae strains were processed for antimicrobial susceptibility testing (AST) using the minimum inhibitory concentration method. Meanwhile, the isolates were phenotypically identified for carbapenemase production by the colloidal gold method. Finally, the confirmed carbapenem enzyme phenotype was further verified for the production of blaKPC and blaVIM by polymerase chain reaction (PCR). Results: Regarding the rate of isolated strains in blood culture, positivity was 11.16% (1033/9255), and the proportion of K. pneumoniae was 13.65% (141/1033). Overall, according to AST results, 7.80% (11/141) of the isolates demonstrated resistance to carbapenems, such as ertapenem, imipenem, and meropenem; nevertheless, they showed sensitivity to colistin and ceftazidime/avibactam. Colloidal gold phenotypically confirmed 81.82% (9/11) of the isolates as carbapenemase producers. Subsequently, nine isolates’ strains were verified to be positive for blaKPC and blaVIM by PCR; the proportions of the blaKPC and blaVIM genes were 88.89% (8/9) and 11.11% (1/9), respectively. Conclusions: The identification of carbapenemase phenotype and genotype is helpful for the accurate understanding of drug resistance and management of the disease.

Biochemical and Bioinformatic Studies of Mutations of Residues at the Monomer-Monomer Interface of Human Ornithine Aminotransferase Leading to Gyrate Atrophy of Choroid and Retina.

Deficit of human ornithine aminotransferase (hOAT), a mitochondrial tetrameric pyridoxal-5'-phosphate (PLP) enzyme, leads to gyrate atrophy of the choroid and retina (GA). Although 70 pathogenic mutations have been identified, only few enzymatic phenotypes are known. Here, we report biochemical and bioinformatic analyses of the G51D, G121D, R154L, Y158S, T181M, and P199Q pathogenic variants involving residues located at the monomer-monomer interface. All mutations cause a shift toward a dimeric structure, and changes in tertiary structure, thermal stability, and PLP microenvironment. The impact on these features is less pronounced for the mutations of Gly51 and Gly121 mapping to the N-terminal segment of the enzyme than those of Arg154, Tyr158, Thr181, and Pro199 belonging to the large domain. These data, together with the predicted ΔΔG values of monomer-monomer binding for the variants, suggest that the proper monomer-monomer interactions seem to be correlated with the thermal stability, the PLP binding site and the tetrameric structure of hOAT. The different impact of these mutations on the catalytic activity was also reported and discussed on the basis of the computational information. Together, these results allow the identification of the molecular defects of these variants, thus extending the knowledge of enzymatic phenotypes of GA patients.

First Report of Root-Knot Nematode, Meloidogyne incognita, Infecting Buckwheat, Fagopyrum esculentum, in State of São Paulo, Brazil.

Buckwheat (Fagopyrum esculentum Moench) belongs to the Polygonaceae family and has been widely cultivated due to its high nutritional, nutraceutical, and medicinal properties. Brazil ranks seventh-largest producer, with 66,000 tons produced in 2018. Buckwheat is also valued for its adaptability as a cover crop, in grain fields of soybean (Glycine max (L.) Merr., maize (Zea mays L.), and sorghum (Sorghum bicolor (L.) Moench) (Görgen et al. 2016, Babu et al. 2018) especially in fields highly infested with plant-parasitic nematodes (PPN). PPN cause severe root damage, suppressing plant development and yield production. In October 2018, six samples of roots and soil were collected in symptomatic patches of buckwheat, in Guaíra SP (20° 19' 32"S 48° 13' 15.4"W). Samples were analyzed in the Nematology Laboratory (LabNema), UNESP, Jaboticabal, SP, BR. Plants presented symptoms of yellow leaves and galled and volume-reduced roots. Meloidogyne sp. was found, comprising 6,320 eggs and second-stage juveniles (J2s) from 10 g of root and 1,628 J2s in 100 cm³ of soil. Adult morphological characteristics, isoenzyme phenotype of esterase, and molecular analysis were performed to identify the Meloidogyne species. The perineal patterns presented high and trapezoidal dorsal arch (n=15), and the males showed a trapezoidal labial region, including a high head cap formed by a large round labial disc that is raised above the medial lips and centrally concave (n=15) (Eisenback and Hirscmann 1981). These characteristics are typical in Meloidogyne incognita (Kofoid and White, 1912) Chitwood, 1949 (Nascimento et al., 2020; Eisenback and Hirschmann 1981; Netscher and Taylor 1974). The enzymatic phenotype was performed with females (n=8), and the phenotype I1 was verified, described by Esbenshade and Triantaphyllou (1985) as typical for M. incognita. To confirm the species DNA samples were extracted from individual females (n=6) and PCR with specific primers for M. incognita (Mi-F 5'- GTGAGGATTCAGCTCCCCAG-3' and Mi-R 5'-ACGAGGAA CATACTTCTCCGTCC-3') and M. javanica (Treub) Chitwood 1949 (Fjav 5'-GGTGCGCGATTGAACTGAGC-3' and Rjav 5'-CAG GCCCTTCAGTGGAACTATAC-3') that amplify SCAR markers described by Meng et al. (2004) and Zijlstra et al. (2000), respectively, and specific primers for M. enterolobii Yang & Eisenback 1983 that amplify rDNA-IGS2 region (Me-F 5'-AACTTTTG TGAAAGTGCCGCTG-3' and Me-R 5'-TCAGTTCAGGCAGG ATCAACC-3') described by Long et al. (2006) were tested. A fragment of 955 pb DNA size was amplified in Mi-F/R primer, which confirmed the M. incognita identification (Meng et. al., 2004). The original population was used to execute pathogenicity test. In a greenhouse, single buckwheat seeds (cv. IPR 91 Baili) were sown in six 5L pots filled with autoclaved-soil and inoculated with 3,000 eggs and J2s per pot (n=6) and control (n=6). After 60 days, the nematodes were extracted from roots and the M. incognita was confirmed. An average of 15,738 eggs and J2s were recovered, (reproductive factor = 5.24), which confirmed buckwheat as a host to M. incognita. The inoculated plants showed symptoms as those observed in the field. No symptom or nematode was noted on the control. Meloidogyne incognita has been reported causing high damage to the F. esculentum in California (Gardner and Caswell-Chen 1994) plus several crops in Brazil (Nascimento et al., 2020). However, this is the first report of this nematode infecting buckwheat in Brazil. Given the importance of buckwheat in Brazil, with extensive use as forage, cover crop, and its nutritional properties, this report is essential to specific management measures are adopted to avoid further losses.

Elimination of tucatinib, a small molecule kinase inhibitor of HER2, is primarily governed by CYP2C8 enantioselective oxidation of gem-dimethyl.

Tucatinib, a small molecule for the treatment of metastatic HER2-positive breast cancer, was extensively metabolized in humans to multiple oxidative metabolites. To fully understand the elimination and biotransformation pathways of tucatinib, we investigated the in vitro and in vivo metabolism of tucatinib, and also conducted a Phase I trial using [14C]tucatinib. To identify the responsible enzymes for tucatinib clearance, we investigated the in vitro metabolism of tucatinib including enzyme phenotyping, which facilitated the discovery of several metabolites in human and monkey plasma and excreta, in particular M1 (ONT-993, an aliphatic hydroxylated metabolite). Stereoselective formation of M1 was further investigated in vitro, in vivo, and in silico. In humans, approximately 86% of the total radiolabeled dose was recovered in feces and 4% in urine; in plasma, approximately 76% of radioactivity circulated as parent drug, with 19% attributed to multiple metabolites. The primary isoforms responsible for the elimination of tucatinib were CYP2C8 and CYP3A4/5. CYP2C8 was shown to possess sole catalytic activity for the formation of M1, whereas CYP3A4/5 and aldehyde oxidase catalyzed the formation of the remaining metabolites. Subsequent investigation revealed that M1 was formed in a stereoselective manner. Examination of the enantiomeric ratio of M1 stereoisomers observed in humans relative to cynomolgus monkeys revealed comparable results, suggesting that the enantiomers that comprise M1 were not considered to be unique or disproportionately high in human. CYP2C8 and CYP3A4/5 are the primary drug-metabolizing enzymes involved in the in vitro metabolism of tucatinib, which provided the basis to describe human disposition of tucatinib and formation of the observed metabolites.

Identification of Metabolites and Study on Enzyme Phenotype of Compound P109, Analogues of Valbenazine in Human Liver Microsomes

Identification of Metabolites and Study on Enzyme Phenotype of Compound P109, Analogues of Valbenazine in Human Liver Microsomes

Novel Bruton's Tyrosine Kinase inhibitor remibrutinib: Drug-drug interaction potential as a victim of CYP3A4 inhibitors based on clinical data and PBPK modeling.

Remibrutinib, a novel oral Bruton’s Tyrosine Kinase inhibitor (BTKi) is highly selective for BTK, potentially mitigating the side effects of other BTKis. Enzyme phenotyping identified CYP3A4 to be the predominant elimination pathway of remibrutinib. The impact of concomitant treatment with CYP3A4 inhibitors, grapefruit juice and ritonavir (RTV), was investigated in this study in combination with an intravenous microtracer approach. Pharmacokinetic (PK) parameters, including the fraction absorbed, the fractions escaping intestinal and hepatic first‐pass metabolism, the absolute bioavailability, systemic clearance, volume of distribution at steady‐state, and the fraction metabolized via CYP3A4 were evaluated. Oral remibrutinib exposure increased in the presence of RTV 4.27‐fold, suggesting that remibrutinib is not a sensitive CYP3A4 substrate. The rich PK dataset supported the building of a robust physiologically‐based pharmacokinetic (PBPK) model, which well‐described the therapeutic dose range of 25–100 mg. Simulations of untested scenarios revealed an absence of drug‐drug interaction (DDI) risk between remibrutinib and the weak CYP3A4 inhibitor fluvoxamine (area under the concentration‐time curve ratio [AUCR] <1.25), and a moderate effect with the CYP3A4 inhibitor erythromycin (AUCR: 2.71). Predictions with the moderate and strong CYP3A4 inducers efavirenz and rifampicin, suggested a distinct remibrutinib exposure decrease of 64% and 89%. Oral bioavailability of remibrutinib was 34%. The inclusion of an intravenous microtracer allowed the determination of all relevant remibrutinib PK parameters, which facilitated construction of the PBPK model. This will provide guidance on the selection or restriction of comedications and prediction of DDI risks.

Study on metabolic dynamics,metabolic enzyme phenotype and species difference of hepatic and intestinal microsome of psoralidin

This study aims to investigate metabolic activities of psoralidin in human liver microsomes( HLM) and intestinal microsomes( HIM),and to identify cytochrome P450 enzymes( CYPs) and UDP-glucuronosyl transferases( UGTs) involved in psoralidin metabolism as well as species differences in the in vitro metabolism of psoralen. First,after incubation serial of psoralidin solutions with nicotinamide adenine dinucleotide phosphate( NADPH) or uridine 5&apos;-diphosphate-glucuronic acid( UDPGA)-supplemented HLM or HIM,two oxidic products( M1 and M2) and two conjugated glucuronides( G1 and G2) were produced in HLM-mediated incubation system,while only M1 and G1 were detected in HIM-supplemented system. The CLintfor M1 in HLM and HIM were 104. 3,and57. 6 μL·min~(-1)·mg~(-1),respectively,while those for G1 were 543. 3,and 75. 9 μL·min~(-1)·mg~(-1),respectively. Furthermore,reaction phenotyping was performed to identify the main contributors to psoralidin metabolism after incubation of psoralidin with NADPH-supplemented twelve CYP isozymes( or UDPGA-supplemented twelve UGT enzymes),respectively. The results showed that CYP1 A1( 39. 5 μL·min~(-1)·mg~(-1)),CYP2 C8( 88. 0 μL·min~(-1)·mg~(-1)),CYP2 C19( 166. 7 μL·min~(-1)·mg~(-1)),and CYP2 D6( 9. 1 μL·min~(-1)·mg~(-1)) were identified as the main CYP isoforms for M1,whereas CYP2 C19( 42. 0 μL·min~(-1)·mg~(-1)) participated more in producing M2. In addition,UGT1 A1( 1 184. 4 μL·min~(-1)·mg~(-1)),UGT1 A7( 922. 8 μL·min~(-1)·mg~(-1)),UGT1 A8( 133. 0 μL·min~(-1)·mg~(-1)),UGT1 A9( 348. 6 μL·min~(-1)·mg~(-1)) and UGT2 B7( 118. 7 μL·min~(-1)·mg~(-1)) played important roles in the generation of G1,while UGT1 A9( 111. 3 μL·min~(-1)·mg~(-1)) was regarded as the key UGT isozyme for G2. Moreover,different concentrations of psoralidin were incubated with monkey liver microsomes( MkLM),rat liver microsomes( RLM),mice liver microsomes( MLM),dog liver microsomes( DLM) and mini-pig liver microsomes( MpLM),respectively. The obtained CLintwere used to evaluate the species differences.Phase Ⅰ metabolism and glucuronidation of psoralidinby liver microsomes showed significant species differences. In general,psoralidin underwent efficient hepatic and intestinal metabolisms. CYP1 A1,CYP2 C8,CYP2 C19,CYP2 D6 and UGT1 A1,UGT1 A7,UGT1 A8,UGT1 A9,UGT2 B7 were identified as the main contributors responsible for phase Ⅰ metabolism and glucuronidation,respectively. Rat and mini-pig were considered as the appropriate model animals to investigate phase Ⅰ metabolism and glucuronidation,respectively.

Mechanism of Klebsiella pneumoniae resistance to carbapenem antibiotics

Purpose: To investigate the mechanism of action of Klebsiella pneumoniae (Kpn) resistance to carbapenem.&#x0D; Methods: The susceptibility of six Klebsiella pneumoniae strains to antibiotics was determined using KB assay. Six isolated strains which were resistant to carbapenem were identified and collected using modified Hodge test. The phenotypes of metal enzyme were evaluated by ethylene diamine tetraacetic acid (EDTA) disk diffusion method. The genes for beta-lactamases, including KPC gene, were examined.&#x0D; Results: The six carbapenem-resistant strains of Klebsiella pneumoniae were resistant to imipenem, meropenem and aztreonam, but were sensitive to amikacin, fosfomycin, minocycline, and polymyxin. The six pathogens did not produce metal enzyme, but they produced carbapenemases. Moreover, the six strains partially carried blaTEM or blaSHV gene, but all had blaKPC-2 gene.&#x0D; Conclusion: These results suggest that the pathogens that contain blaKPC-2 gene may be involved in the mechanism of Klebsiella pneumoniae (Kpn) resistance to carbapenem.

Chronological Metabolic Response to Intensive Phase TB Therapy in Patients with Cured and Failed Treatment Outcomes.

Despite the arguable success of the standardized tuberculosis (TB) treatment regime, a significant number of patients still present with treatment failure. To improve on current TB treatment strategies, we sought to gain a better understanding of the hosts' response to TB therapy. A targeted metabolomics approach was used to compare the urinary acylcarnitine and amino acid profiles of eventually cured TB patients with those of patients presenting with a failed treatment outcome, comparing these patient groups at the time of diagnosis and at weeks 1, 2, and 4 of treatment. Among the significant metabolites identified were histidine, isoleucine, leucine, methionine, valine, proline, tyrosine, alanine, serine, and γ-aminobutyric acid. In general, metabolite fluctuations in time followed a similar pattern for both groups for most compounds but with a delayed onset or shift of the pattern in the successfully treated patient group. These time-trends detected in both groups could potentially be ascribed to a vitamin B6 deficiency and fluctuations in the oxidative stress levels and urea cycle intermediates, linked to the drug-induced inhibition and stimulation of various enzymes. The earlier onset of observed trends in the failed patients is proposed to relate to genotypic and phenotypic variations in drug metabolizing enzymes, subsequently leading to a poor treatment efficiency either due to the rise of adverse drug reactions or to insufficient concentrations of the active drug metabolites. This study emphasizes the need for a more personalized TB treatment approach, by including enzyme phenotyping and the monitoring of oxidative stress and vitamin B6 levels, for example.

Aromatic Amino Acid Decarboxylase Deficiency: The Added Value of Biochemistry.

Aromatic amino acid decarboxylase (AADC) deficiency is a rare, autosomal recessive neurometabolic disorder caused by mutations in the DDC gene, leading to a deficit of AADC, a pyridoxal 5′-phosphate requiring enzyme that catalyzes the decarboxylation of L-Dopa and L-5-hydroxytryptophan in dopamine and serotonin, respectively. Although clinical and genetic studies have given the major contribution to the diagnosis and therapy of AADC deficiency, biochemical investigations have also helped the comprehension of this disorder at a molecular level. Here, we reported the steps leading to the elucidation of the functional and structural features of the enzyme that were useful to identify the different molecular defects caused by the mutations, either in homozygosis or in heterozygosis, associated with AADC deficiency. By revisiting the biochemical data available on the characterization of the pathogenic variants in the purified recombinant form, and interpreting them on the basis of the structure-function relationship of AADC, it was possible: (i) to define the enzymatic phenotype of patients harboring pathogenic mutations and at the same time to propose specific therapeutic managements, and (ii) to identify residues and/or regions of the enzyme relevant for catalysis and/or folding of AADC.

Metabolic Retroversion of Piperaquine (PQ) via Hepatic Cytochrome P450-Mediated N-Oxidation and Reduction: Not an Important Contributor to the Prolonged Elimination of PQ.

As a partner antimalarial with an extremely long elimination half-life (∼30 days), piperaquine (PQ) is mainly metabolized into a pharmacologically active N-oxide metabolite [piperaquine N-oxide (PN1)] in humans. In the present work, the metabolic retroversion of PQ and PN1, potentially associated with decreased clearance of PQ, was studied. The results showed that interconversion existed for PQ and its metabolite PN1. The N-oxidation of PQ to PN1 was mainly mediated by CYP3A4, and PN1 can rapidly reduce back to PQ via cytochrome P450 (P450)/flavin-containing monooxygenase enzymes. In accordance with these findings, the P450 nonselective inhibitor (1-ABT) or CYP3A4 inhibitor (ketoconazole) inhibited the N-oxidation pathway in liver microsomes (>90%), and the reduction metabolism was inhibited by 1-ABT (>90%) or methimazole (∼50%). Based on in vitro physiologic and enzyme kinetic studies, quantitative prediction of hepatic clearance (CLH) of PQ was performed, which indicated its negligible decreased elimination in humans in the presence of futile cycling, with the unbound CLH decreasing by 2.5% (0.069 l/h per kilogram); however, a minor decrease in unbound CLH (by 12.8%) was found in mice (0.024 l/h per kilogram). After an oral dose of PQ (or PN1) to mice, the parent form predominated in the blood circulation, and PN1 (or PQ) was detected as a major metabolite. Other factors probably associated with delayed elimination of PQ (intestinal metabolism and enterohepatic circulation) did not play a key role in PQ elimination. These data suggested that the metabolic interconversion of PQ and its N-oxide metabolite contributes to but may not significantly prolong its duration in humans. SIGNIFICANCE STATEMENT: This paper investigated the interconversion metabolism of piperaquine (PQ) and its N-oxide metabolite in vitro as well as in mice. The metabolic profiles of PQ were reestablished by this futile cycling, which contributes to but may not significantly prolong its elimination in humans. Enzyme phenotyping indicated a low possibility of interaction of PQ during artemisinin drug-based combination therapy treatment.

Integrative taxonomy of Meloidogyne graminicola populations with different esterase phenotypes parasitising rice in Brazil

Summary The rice root-knot nematode, Meloidogyne graminicola, has been reported in Southeast Asia, China, India, South Africa, USA, Brazil, and other countries. Recent surveys in Southern Brazil showed that M. graminicola was widespread in irrigated rice in Rio Grande do Sul, Santa Catarina and Paraná states, and the presence of a species complex with a predominance of M. graminicola (Est VS1 = G1) and other variants showing similar esterase phenotypes (Est G2 = R2, G3 = R3). Meloidogyne oryzae (Est O1) and M. ottersoni (Est Ot0) were also part of this complex and were recently re-described and detected on rice. The present study provides an integrative taxonomy approach of the typical and atypical populations of M. graminicola on the basis of morphological, morphometric and molecular data. Considering morphological and morphometric features, the two atypical populations (Est G2 and G3) are in close agreement with the description of M. graminicola. Based on the molecular characterisation, populations G1, G2 and G3 were successfully amplified by M. graminicola SCAR markers, although the specificity of these markers was questioned. Phylogenetic relationships complemented and confirmed the other studies. In maximum likelihood analysis of ITS, D2-D3 rRNA and COXII-16S rRNA sequences, all populations of M. graminicola from different esterase phenotypes clustered together with other M. graminicola populations, thus confirming that these enzyme phenotypes (G1, G2 and G3) are related to the same species. A high level of intraspecific variability was detected among all populations, but no correlation between genetic variability and geographic origins occurred.

In vitro and in vivo studies of triacetone triperoxide (TATP) metabolism in humans

PurposeTriacetone triperoxide (TATP) is a volatile but powerful explosive that appeals to terrorists due to its ease of synthesis from household items. For this reason, bomb squad, canine (K9) units, and scientists must work with this material to mitigate this threat. However, no information on the metabolism of TATP is available.MethodsIn vitro experiments using human liver microsomes and recombinant enzymes were performed on TATP and TATP-OH for metabolite identification and enzyme phenotyping. Enzyme kinetics for TATP hydroxylation were also investigated. Urine from laboratory personnel collected before and after working with TATP was analyzed for TATP and its metabolites.ResultsWhile experiments with flavin monooxygenases were inconclusive, those with recombinant cytochrome P450s (CYPs) strongly suggested that CYP2B6 was the principle enzyme responsible for TATP hydroxylation. TATP-O-glucuronide was also identified and incubations with recombinant uridine diphosphoglucuronosyltransferases (UGTs) indicated that UGT2B7 catalyzes this reaction. Michaelis–Menten kinetics were determined for TATP hydroxylation, with Km = 1.4 µM and Vmax = 8.7 nmol/min/nmol CYP2B6. TATP-O-glucuronide was present in the urine of all three volunteers after being exposed to TATP vapors showing good in vivo correlation to in vitro data. TATP and TATP-OH were not observed.ConclusionsSince scientists working to characterize and detect TATP to prevent terrorist attacks are constantly exposed to this volatile compound, attention should be paid to its metabolism. This paper is the first to elucidate some exposure, metabolism and excretion of TATP in humans and to identify a marker of TATP exposure, TATP-O-glucuronide in urine.