Major Metabolite Research Articles

B-297 Tacrolimus Concentrations in Envarsus vs Prograf Patients: An Evaluation of the Clinical Impact of LC-MS/MS and Immunoassay Methods on Tacrolimus measurement

Abstract Background Tacrolimus is a calcineurin inhibitor used as part of an immunosuppressive regimen in kidney and liver transplant patients to prevent graft-versus-host disease (GvHD) and requires therapeutic drug monitoring due to its narrow therapeutic index. Tacrolimus levels are commonly measured using two different methodologies - immunoassay and liquid chromatography tandem mass spectrometry (LC-MS/MS). A global proficiency study to assess and compare the measurement of trough levels by these two methodologies noted a positive bias with the ARCHITECT immunoassay which is likely due to the presence of tacrolimus metabolites. The aim of our study is to assess the impact of two different formulations of tacrolimus on the accuracy of its analytical measurement. While differences between immunoassay and LC-MS/MS results have been evaluated for immediate-release tacrolimus (Prograf), it has yet to be characterized for extended-release formulations such as Envarsus. The discrepancy between immunoassay and LC-MS/MS has clinical implications, as dosage modifications are based on measured concentrations, analytical variation may cause intervals of subtherapeutic or supratherapeutic exposure, increasing the risk of graft rejection or toxicity. Methods Development of an LC-MS/MS method for the simultaneous quantification of tacrolimus and its major metabolite, desmethyl tacrolimus, was performed in whole blood. Traceable calibrators and quality control material for tacrolimus and specimens supplemented with desmethyl tacrolimus were used to determine accuracy and precision. Tacrolimus concentrations were measured by LC-MS/MS and ARCHITECT immunoassay methods in excess whole blood specimens from patients treated with either Prograf or Envarsus. Measurement biases between LC-MS/MS and immunoassay methodologies were determined for both formulations of tacrolimus. Results External calibration curves for both tacrolimus and desmethyl tacrolimus were linear (R2>0.995), and the analytical measurement range (AMR) for tacrolimus spanned from 1.1 - 31.6 ng/ml. Calibrator and Quality control (QC) biases were within 15% of their target values throughout the AMR and within-run imprecision was less than 10% (n=25) for all calibrators and QCs. Between-run imprecision for Low, Mid, and High QC levels over a period of 2 weeks (n=5 days) was less than 10%. Comparative bias of tacrolimus concentrations between immunoassay and LC-MS/MS was significantly lower (P value=0.0074) for Envarsus (n=20 specimens) relative to Prograf (n=32 specimens). Conclusions The differential bias between immunoassay and LC-MS/MS in the measurement of tacrolimus in patients dosed with immediate-release versus extended-release formulations suggests that their distinct pharmacokinetic profile may impact the accuracy of the measurement. Therefore, applying observed measurement biases from different assays derived from Prograf patients to Envarsus patients and vice versa may result in erroneous estimation of tacrolimus concentrations.

B-281 Performance Characteristics Evaluation of the ARK Diagnostics Immunoassay for Xylazine/4-HydroxyXylazine in Urine

Abstract Background Xylazine exposure is currently common in some US cities, but routine laboratory testing is not available. We evaluated the ARK Diagnostics immunoassay for qualitative detection of xylazine/4-hydroxy Xylazine in urine, prior to its anticipated release as a Forensic Use Only assay. Evaluation of performance characteristics of the ARK assay were conducted at two university hospitals, using either the semi-quantitative application of the assay, as performed on the Roche Cobas 503 analyzer (Institution A), or the qualitative application of the assay, as performed on the Beckman Coulter AU480 analyzer (Institution B). Methods Xylazine (X) and 4-hydroxy Xylazine (4-OHX) were obtained from Cayman Chemical (Ann Arbor, MI). Pooled xylazine-free urine was spiked either with X or 4-OHX in the range of 0-2000 ng/mL. De-identified, to-be-discarded patient urine samples were obtained from among those submitted for routine drugs-of-abuse panel testing. X measurements on all samples were performed by LC-MS/MS; samples were segregated into X-NEGATIVE (X<10 ng/mL) and X-POSITIVE (X>=10 ng/mL). The ARK assay was conducted according to manufacturer’s instructions, either as a semi-quantitative assay (Institution A, Roche analyzer) or as a qualitative assay (Institution B, Beckman Coulter analyzer), with 10 ng/mL as the manufacturer-specified cutoff for ARK POSITIVE, using manufacturer-supplied calibration standards ranging from 0-500 ng/mL X. Results The ARK semi-quantitative analysis (Roche analyzer) showed essentially equivalent curvilinear response curves for either X or 4-OHX at concentrations below 1000 ng/mL; for the semi-quantitative ARK assay, we use “>1200 ng/mL” for X-ARK >1200 ng/mL. By mixing studies (Roche), there were neither positive nor negative interferences observed as assessed for common drugs of abuse or for common therapeutic drugs. The presence of hemoglobin, bilirubin or moderate lipemia were devoid of assay interference. Precision studies for manufacturer-supplied controls were as follows: intra-assay CVs were 6.9% for the NEGATIVE control (5.1±0.35 ng/mL) and 5.9% for the POSITIVE control (15.7±0.53 ng/mL) (n=20); interassay CVs were 7.8% for the NEGATIVE control (5.1±0.40 ng/mL) and 5.7% for the POSITIVE control (15.9±0.90 ng/mL) (n=20). For the qualitative ARK assay as performed on the Beckman analyzer, POSITIVE and NEGATIVE controls ran invariably according to their designations. For the semi-quantitative assay application (Roche analyzer): among 78 X-NEGATIVE samples by LC-MS/MS, there were 0% ARK-POSITIVE results (false-positive rate=0%); among 74 X-POSITIVE samples by LC-MS/MS, there was one ARK-NEGATIVE result (false-negative rate=1.4%); this sample (X=65 ng/mL) was found to have a high amorphous crystalline content, and was unable to be reanalyzed. For the qualitative assay application (Beckman Coulter analyzer): among 78 X-NEGATIVE samples there was one ARK-POSITIVE sample (false-positive rate=1.3%) for a sample with xylazine=8.8 ng/mL by LC-MS/MS, with undetectable 4-OHX (<10 ng/mL); among 74 X-POSITIVE samples, there were no ARK-NEGATIVE results (false-negative rate=0%). Conclusions The ARK xylazine immunoassay demonstrated acceptable analytical performance with respect to detection of xylazine in urine at 10 ng/mL. With additional consideration of the assay’s cross reactivity with the major xylazine metabolite, 4-OH-xylazine, the assay was judged to be highly suitable for routine use as a screening test for detection of xylazine exposure via urine testing.

Pharmacokinetics, disposition, and biotransformation of the cardiac myosin inhibitor aficamten in humans.

Aficamten, a cardiac myosin inhibitor, is being developed for the treatment of patients with symptomatic hypertrophic cardiomyopathy (HCM). The purpose of this study was to determine the absorption, metabolism, and excretion of aficamten. Eight healthy male participants received a single oral dose of 20 mg aficamten (containing approximately 100 μCi of radiocarbon). Blood, urine, and feces samples were collected up to a maximum of Day 26. The pharmacokinetics of aficamten were characterized by moderate absorption, with a median tmax of 2.0 h postdose. The median t1/2 of aficamten was 99.6 h with similar t1/2 observed for metabolites and total radioactivity in plasma and whole blood. The overall total recovery of administered total radioactivity was 89.7% with 57.7% of the dose recovered in feces and 32.0% in urine. The main circulating metabolites in plasma included monohydroxylated metabolites M1a (CK-3834282) and M1b (CK-3834283) accounting for 10.5% and 36.4% of the total radioactivity AUC both with a median tmax of 5 h. The other major plasma metabolite was M5 (an oxygen-linked glucuronide conjugate of M1a), which accounted for 10.3% of the total plasma radioactivity exposure, with a tmax of 24 h. In urine, M5 was the most abundant metabolite with 8.02% total radioactive dose (TRD), followed by M1a and M1b with 6.16% and 2.85% TRD, respectively; however, there were no metabolites in urine observed at >10% of dose. The major metabolite in feces was M18 representing 44.1% of the radioactive dose. These findings indicated that aficamten was eliminated by metabolism, and to a minor extent, by fecal excretion of unchanged aficamten with renal excretion playing a minor role. Feces were the principal route of excretion of the radioactive dose.

Effects of thermal and UV stress on the polar and non-polar metabolome of photosymbiotic jellyfish and sea anemones

Recently, the impacts of climate change, notably ocean warming and solar ultraviolet radiation, have led to significant stress and mortality in cnidarians. The objective of this study is to decode the metabolic responses of sea anemones Entacmaea quadricolor and upside-down jellyfish Cassiopea andromeda upon exposure to thermal and ultraviolet stress. Gas chromatography-mass spectrometry and ultraperformance liquid chromatography coupled with high-resolution mass spectrometry targeting polar and non-polar metabolites were applied. In total, 72 polar and 242 lipophilic metabolites were detected in jellyfish and sea anemones, respectively. Amino acids are the major metabolite class in jellyfish, and triacylglycerides are the predominant lipids in jellyfish and anemones. Exposure to stressors led to metabolic alterations, marked by elevated amino acids in jellyfish and increased amino acids and sugar alcohols in sea anemones at 34 °C and after four days of ultraviolet radiation. Non-polar metabolome analysis indicated distinct responses to ultraviolet radiation and thermal stress in both species.

Enhanced biodegradation of trinitrotoluene in rhizosphere soil by native grasses

Soil contamination by the munition explosive residues of 2,4,6-trinitrotoluene (TNT) and its metabolites resulting primarily from military operations has been identified as a threat to human health and ecosystems. Biodegradation by native plants to remove this hazardous compound or reduce its toxicity is considered a cost-effective and environmentally sound approach for the cleanup or restoration of TNT-contaminated soils. This study aims to investigate the TNT biodegradation and kinetics by two selected native grasses in the species-specific rhizosphere soils through growth chamber experiments. Native eastern gamma grass (Tripsacum dactyloides) and switchgrass (Panicum virgatum L.) were grown in soil spiked with 14C-TNT for 8 weeks. The 14C-TNT degradation and degradative metabolite profile in the rhizosphere soils were determined by liquid scintillation counter and high-performance liquid chromatography, respectively. The results indicated that both native grass species significantly enhanced the TNT degradation in the rhizosphere soils as compared with the control rhizosphere soils. More than 95% of the applied 14C-TNT was degraded in the first 7 days, and the rate then reached a steady state afterward, but less than 10% of the TNT applied was completely mineralized and transformed into CO2. The degradative reaction was found to follow second-order kinetics. Six major TNT degradative metabolites have been detected and identified in the rhizosphere soils. Overall, switchgrass appeared more effective for biodegrading TNT than eastern gamma grass. This research demonstrated that the native grass species, especially switchgrass, has the potential to mitigate the adverse human health and ecological risks of TNT-contaminated sites and can be considered an environmentally friendly, sustainable approach to safeguarding human health from TNT contamination.

The major biogenic amine metabolites in mood disorders.

Mood disorders, including major depressive disorder and bipolar disorder, have a profound impact on more than 300 million people worldwide. It has been demonstrated mood disorders were closely associated with deviations in biogenic amine metabolites, which are involved in numerous critical physiological processes. The peripheral and central alteration of biogenic amine metabolites in patients may be one of the potential pathogeneses of mood disorders. This review provides a concise overview of the latest research on biogenic amine metabolites in mood disorders, such as histamine, kynurenine, and creatine. Further studies need larger sample sizes and multi-center collaboration. Investigating the changes of biogenic amine metabolites in mood disorders can provide biological foundation for diagnosis, offer guidance for more potent treatments, and aid in elucidating the biological mechanisms underlying mood disorders.

Differential Selectivity of Human and Mouse ABCC4/Abcc4 for Arsenic Metabolites.

Millions of people globally are exposed to the proven human carcinogen arsenic at unacceptable levels in drinking water. In contrast, arsenic is a poor rodent carcinogen, requiring >100-fold higher doses for tumour induction, which may be explained by toxicokinetic differences between humans and mice. The human ATP-binding cassette (ABC) transporter hABCC4 mediates the cellular efflux of a diverse array of metabolites, including the GSH conjugate of the highly toxic monomethylarsonous acid (MMAIII), MMA(GS)2, and the major human urinary arsenic metabolite dimethylarsinic acid (DMAV). Our objective was to determine if mouse Abcc4 (mAbcc4) protected against and/or transported the same arsenic species as hABCC4. The anti-ABCC4 antibody M4I-10 epitope was first mapped to an octapeptide (411HVQDFTA418F) present in both hABCC4 and mAbcc4, enabling quantification of relative amounts of hABCC4/mAbcc4. mAbcc4 expressed in HEK293 cells did not protect against any of the six arsenic species tested [arsenite, arsenate, MMAIII, monomethylarsonic acid, dimethylarsinous acid or DMAV], despite displaying remarkable resistance against the antimetabolite 6-mercaptopurine (>9-fold higher than hABCC4). Furthermore, mAbcc4-enriched membrane vesicles prepared from transfected HEK293 cells did not transport MMA(GS)2 or DMAV, despite a >3-fold higher transport activity than hABCC4-enriched vesicles for the prototypic substrate 17β-estradiol-17-(β-D-glucuronide). Abcc4(+/+) mouse embryonic fibroblasts (MEFs) were ~3-fold more resistant to arsenate than Abcc4(-/-) MEFs; however, further characterization indicated this was not mAbcc4 mediated. Thus, under the conditions tested, arsenicals are not transported by mAbcc4, and differences between the substrate selectivity of hABCC4 and mAbcc4 seem likely to contribute to differences in human and mouse arsenic toxicokinetics. Significance Statement Toxicokinetics of the carcinogen arsenic differ among animal species. Arsenic methylation is known to contribute to this, whereas arsenic transporters have not been considered. The human ATP-binding cassette transporter hABCC4 is a high affinity transporter of toxicologically important arsenic metabolites. Here we used multiple cell models to demonstrate that mouse Abcc4 does not protect cells against, or transport, any arsenic species tested. Thus, differences between hABCC4 and mAbcc4 substrate selectivity likely contribute to differences in human and mouse arsenic toxicokinetics.

Absorption, Distribution, Metabolism, and Excretion of Icenticaftor (QBW251) in Healthy Male Volunteers at Steady State and In Vitro Phenotyping of Major Metabolites.

Icenticaftor (QBW251) is a potentiator of the CFTR protein and is currently in clinical development for the treatment of chronic obstructive pulmonary disease and chronic bronchitis. An absorption, distribution, metabolism, and excretion (ADME) study was performed at steady state to determine the pharmacokinetics, mass balance, and metabolite profiles of icenticaftor in humans. In this open-label study, six healthy men were treated with unlabeled oral icenticaftor (400 mg b.i.d.) for 4 days. A single oral dose of [14C]icenticaftor was administered on Day 5, and unlabeled icenticaftor was administered b.i.d. from the evening of Day 5 to Day 12. Unchanged icenticaftor accounted for 18.5% of plasma radioactivity. Moderate to rapid absorption of icenticaftor was observed (median Tmax: 4 hours), with 93.4% of the dose absorbed. It exhibited moderate distribution (Vz/F: 335 L) and was extensively metabolized, principally through N-glucuronidation, O-glucuronidation, and/or O-demethylation. The metabolites M8 and M9, formed by N-glucuronidation and O-glucuronidation of icenticaftor, respectively, represented the main entities detected in plasma (35.3% and 14.5%, respectively) in addition to unchanged icenticaftor (18.5%). The apparent mean T1/2 of icenticaftor was 15.4 hours in blood and 20.6 hours in plasma. Icenticaftor was eliminated from the body mainly through metabolism followed by renal excretion, and excretion of radioactivity was complete after 9 days. In vitro phenotyping of icenticaftor showed that cytochrome P450 and uridine diphosphate glucuronosyltransferase were responsible for 31% and 69% of the total icenticaftor metabolism in human liver microsomes, respectively. This study provided invaluable insights into the disposition of icenticaftor. Significance Statement The ADME of a single radioactive oral dose of icenticaftor was evaluated at steady state to investigate the nonlinear pharmacokinetics observed previously with icenticaftor. [14C]Icenticaftor demonstrated good systemic availability after oral administration and was extensively metabolized and moderately distributed to peripheral tissues. The most abundant metabolites, M8 and M9, were formed by N-glucuronidation and O-glucuronidation of icenticaftor, respectively. Phenotyping demonstrated that [14C]icenticaftor was metabolized predominantly by UGT1A9 with a remarkably low Km value.

Molecular docking of major secondary metabolites of Pteris vittata L. against OmpA protein

Molecular docking of major secondary metabolites of Pteris vittata L. against OmpA protein

Antibacterial Compounds and Draft Genome Sequence of Streptomyces sp. PA5.6 Isolated from Deciduous Dipterocarp Forest Soils, University of Phayao, Thailand

Antibiotic resistance is a major problem in the control of infectious diseases. To overcome this obstacle, a common approach is to search for novel antimicrobial drugs from natural sources, especially microorganisms such as Streptomyces. Streptomyces has long been demonstrated to produce bioactive secondary metabolites with antimicrobial activities. The objectives of this research were to isolate and characterize Streptomyces strains from the deciduous dipterocarp forest soils in the University of Phayao, and to evaluate their metabolites for antibacterial activities. The results show that the morphological and physiological characteristics of PA5.6 are consistent with those of the genus Streptomyces. Furthermore, a comparative analysis of the whole genome sequence verified that PA5.6 represents a novel species within the genus Streptomyces. The genome sequence analysis of Streptomyces sp. PA5.6 revealed a total length of 9,146,340 bp, a G + C content of 71.1 %, and an N50 scaffold of 320,345 bp. The antiSMASH analysis revealed putative secondary metabolite Biosynthetic Gene Clusters (BGCs) involved in the biosynthesis of antimicrobial metabolites, including terpene, Type I PKS, NRPS and lassopeptide gene clusters. Functional prediction of these gene clusters indicates that they are involved in the biosynthesis of several antimicrobial-associated metabolites, such as albaflavenone, monensin and citrulassin D. Streptomyces sp. PA5.6 culture supernatant and its crude ethyl acetate extract exhibited antibacterial activity against several drug-resistant, bacterial pathogens, including S. aureus, E. faecalis, E. coli, P. aeruginosa, A. baumannii, MRSA, VREF, ESBL-E. coli and CRPA. The GC-MS analysis of the crude ethyl acetate extracts revealed benzeneacetic acid, benzeneacetic acid, 4-hydroxy and benzeneacetamide as major active compounds metabolites. In conclusion, Streptomyces sp. PA5.6 exhibited promising potential for producing bioactive compounds against bacterial pathogens. HIGHLIGHTSThe Streptomyces sp. PA5.6 isolated from the soil in the deciduous dipterocarp forest and was identified as a new species within the genus Streptomyces by comparative analysis of the whole genome sequence. The crude ethyl acetate extract of Streptomyces sp. PA5.6 exhibited antibacterial activity against bacterial pathogens. GC-MS analysis of crude ethyl acetate extract showed major and minor compounds that exhibit antimicrobial activity. GRAPHICAL ABSTRACT

An Investigational Study on the Role of CYP2D6, CYP3A4 and UGTs Genetic Variation on Fesoterodine Pharmacokinetics in Young Healthy Volunteers.

Introduction: Fesoterodine is one of the most widely used antimuscarinic drugs to treat an overactive bladder. Fesoterodine is extensively hydrolyzed by esterases to 5-hydroxymethyl tolterodine (5-HMT), the major active metabolite. CYP2D6 and CYP3A4 mainly metabolize 5-HMT and are, therefore, the primary pharmacogenetic candidate biomarkers. Materials and Methods: This is a candidate gene study designed to investigate the effects of 120 polymorphisms in 33 genes (including the CYP, COMT, UGT, NAT2, and CES enzymes, ABC and SLC transporters, and 5-HT receptors) on fesoterodine pharmacokinetics and their safety in 39 healthy volunteers from three bioequivalence trials. Results: An association between 5-HMT exposure (dose/weight corrected area under the curve (AUC/DW) and dose/weight corrected maximum plasma concentration (Cmax/DW)), elimination (terminal half-life (T1/2) and the total drug clearance adjusted for bioavailability (Cl/F)), and CYP2D6 activity was observed. Poor/intermediate metabolizers (PMs/IMs) had higher 5-HMT AUC/DW (1.5-fold) and Cmax/DW (1.4-fold) values than the normal metabolizers (NMs); in addition, the normal metabolizers (NMs) had higher 5-HMT AUC/DW (1.7-fold) and Cmax/DW (1.3-fold) values than the ultrarapid metabolizers (UMs). Lower 5-HMT exposure and higher T1/2 were observed for the CYP3A4 IMs compared to the NMs, contrary to our expectations. Conclusions: CYP2D6 might have a more important role than CYP3A4 in fesoterodine pharmacokinetics, and its phenotype might be a better predictor of variation in its pharmacokinetics. An association was observed between different genetic variants of different genes of the UGT family and AUC, Cmax, and CL/F of 5-HMT, which should be confirmed in other studies.

Headspace aroma and secondary metabolites profiling in 3 Pelargonium taxa using a multiplex approach of SPME‐GC/MS and high resolution‐UPLC/MS/MS coupled to chemometrics

AbstractBACKGROUNDThe present study focuses on the aroma and secondary metabolites profiling of three Pelargonium graveolens cultivars, baladi (GRB), sondos (GRS) and shish (GRSH), grown in Egypt. Utilizing a multiplex approach combining high resolution‐ultraperformance liquid chromatography (HR‐UPLC)/tandem mass spectrometry (MS/MS) and gas chromatography (GC)‐MS coupled with chemometrics, the study aims to identify and profile various secondary metabolites and aroma compounds in these cultivars.RESULTSHR‐UPLC/MS/MS analysis led to the annotation of 111 secondary metabolites, including phenolics, flavonoids, terpenes and fatty acids, with several compounds being reported for the first time in geranium. Multivariate data analysis identified vinylanisole, dimethoxy‐flavonol, and eicosadienoic acid as discriminatory metabolites among the cultivars, particularly distinguishing the GRS cultivar in its phenolics profile. In total, 34 aroma compounds were detected using headspace solid‐phase microextraction coupled with GC‐MS, including alcohols, esters, ketones, ethers and monoterpene hydrocarbons. The major metabolites contributing to aroma discrimination among the cultivars were β‐citronellol in GRB, α‐farnesene in GRS and isomenthone in GRSH.CONCLUSIONThe study provides a comprehensive profiling of the secondary metabolites and aroma compounds in the three Pelargonium graveolens cultivars. The GRS cultivar was identified as particularly distinct in both its phenolics and aroma profiles, suggesting its potential as a premium variety for cultivation and use. Future studies should focus on isolating and investigating the newly detected metabolites and exploring the biological effects of these compounds in food applications and other uses. © 2024 Society of Chemical Industry.

Polymorphism in the Drug Transporter Gene ABCB1 as a Potential Disease Modifier in Cortisol-Producing Adrenal Adenomas.

Endogenous hypercortisolism presents with variable phenotypes. Etiological factors accounting for the level of hypercortisolism or varying severity of associated comorbidities are lacking. Recently, the adrenal ATP-binding cassette B1 (ABCB1) gene was identified as a modulator of glucocorticoid secretion. To evaluate the effect of ABCB1 polymorphism rs2032582 on steroid metabolome and clinical phenotypes in patients with endogenous hypercortisolism. In this cross-sectional cohort study, 137 patients prospectively enrolled in the German Cushing's registry were included (41 with ACTH-producing pituitary adenoma, 21 with cortisol-producing adrenal adenoma, and 75 with excluded hypercortisolism). In all patients, ABCB1 polymorphism was analyzed using a TaqMan genotyping assay, glucocorticoid metabolite excretion in 24-hour urine samples was analyzed by gas chromatography-mass spectrometry, and the clinical phenotype was assessed systematically. In patients with cortisol-producing adrenal adenomas, but not in patients with ACTH-producing pituitary adenomas, homozygous major allele GG of ABCB1 polymorphism rs2032582 was associated with higher overall cortisol metabolite secretion (median 13515 [IQR 10347; 25669] µg/24h vs. 9645 [6146; 10732] µg/24h in minor homo- and heterozygotes, p=0.036) and elevated major cortisol metabolites αTHF, THF and THE (9339 [6929; 17789] µg/24h vs. 6288 [4184; 7455] µg/24h, p=0.045). Moreover, these patients showed higher mean arterial pressure (116 [111; 131] mmHg in major homozygotes vs. 105 [96; 112] mmHg in minor homo- and heterozygotes, p=0.036). The genotype of drug transporter gene ABCB1 rs2032582 polymorphism is associated with the degree of cortisol metabolite secretion in cortisol-producing adrenal adenomas and could, therefore, represent a modifier of disease severity in this context.

12-HETE/GPR31 induces endothelial dysfunction in diabetic retinopathy.

12-hydroxyeicosatetraenoic acid (12-HETE), a major metabolite of arachidonic acid, is converted by 12/15-lipoxygenase and implicated in diabetic retinopathy (DR). Our previous study demonstrated a positive correlation between 12-HETE and the prevalence of DR. However, reasons for the increased production of 12-HETE are unclear, and the underlying mechanisms through which 12-HETE promotes DR are unknown. This study aimed to elucidate the correlation between 12-HETE and DR onset, investigate potential mechanisms through which 12-HETE promotes DR, and seek explanations for the increased production of 12-HETE in diabetes. We conducted a prospective cohort study, which revealed that higher serum 12-HETE levels could induce DR. Additionally, G protein-coupled receptor 31 (GPR31), a high-affinity receptor for 12-HETE, was expressed in human retinal microvascular endothelial cells (HRMECs). 12-HETE/GPR31-mediated HRMEC inflammation occurred via the p38 MAPK pathway. 12-HETE levels were significantly higher in the retina of mice with high-fat diet (HFD)- and streptozotocin (STZ)-induced diabetes than in those with only STZ-induced diabetes and healthy controls. They were positively correlated with the levels of inflammatory cytokines in the retina, indicating that HFD could induce increased 12-HETE synthesis in patients with diabetes in addition to hyperglycemia. Conclusively, 12-HETE is a potential risk factor for DR. The 12-HETE/GPR31 axis plays a crucial role in HRMEC dysfunction and could be a novel target for DR prevention and control. Nevertheless, further research is warranted to provide comprehensive insights into the complex underlying mechanisms of 12-HETE in DR.

An in vitro evaluation on metabolism of mitragynine to 9-O-demethylmitragynine

Kratom (Mitragyna Speciosa Korth.) is an indigenous tree native to Southeast Asia whose leaves have been traditionally ingested as a tea and has seen its popularity increase in the United States. Although kratom and its constituents presently have no approved uses by the Food and Drug Administration, its major alkaloids (e.g., mitragynine) have psychoactive properties that may hold promise for the treatment of opioid cessation, pain management, and other indications. 9-O-demethylmitragynine is a major metabolite formed from mitragynine metabolism (36 % total metabolism) and displays similar pharmacologic activity. Cytochrome P450 (CYP) 3A4 has been identified as a major enzyme involved in mitragynine metabolism; however, the in vitro metabolism parameters of 9-O-demethylmitragynine formation are not well defined and a risk of potential drug interactions exists. Using human liver S9 fractions, 9-O-demethylmitragynine formation was generally linear for enzyme concentrations of 0–0.25 mg/mL and incubation times of 5–20 min. 9-O-demethylmitragynine displayed a Km 1.37 μM and Vmax of 0.0931 nmol/min/mg protein. Known CYP inhibitors and compounds that might be concomitantly used with kratom were assessed for inhibition of 9-O-demethylmitragynine formation. Ketoconazole, a CYP3A index inhibitor, demonstrated a significant effect on 9-O-demethylmitragynine formation, further implicating CYP3A4 as a major metabolic pathway. Major cannabinoids (10 μg/mL) displayed minor inhibition of 9-O-demethylmitragynine formation, while all other compounds had minimal effects. Mixtures of physiological achievable cannabinoid concentrations also displayed minor effects on 9-O-demethylmitragynine formation, making a metabolic drug interaction unlikely; however, further in vitro, in vivo, and clinical studies are necessary to fully exclude any risk.

One-Week Kava Dietary Supplementation Increases Both Urinary N- and O-Glucuronides of NNAL, a Lung Carcinogen Major Metabolite, among Smokers.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (commonly known as NNK) is one of the most prevalent and potent pulmonary carcinogens in tobacco products that increases the human lung cancer risk. Kava has the potential to reduce NNK and tobacco smoke-induced lung cancer risk by enhancing urinary excretion of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, the major metabolite of NNK) and thus reducing NNK-induced DNA damage. In this study, we quantified N-glucuronidated NNAL (NNAL-N-gluc), O-glucuronidated NNAL (NNAL-O-gluc), and free NNAL in the urine samples collected before and after 1-week kava dietary supplementation. The results showed that kava increased both NNAL-N-glucuronidation and O-glucuronidation. Since NNAL-N-glucuronidation is dominantly catalyzed by UGT2B10, its representative single-nucleotide polymorphisms (SNPs) were analyzed among the clinical trial participants. Individuals with any of the four analyzed SNPs appear to have a reduced basal capacity in NNAL-N-glucuronidation. Among these individuals, kava also resulted in a smaller extent of increases in NNAL-N-glucuronidation, suggesting that participants with those UGT2B10 SNPs may not benefit as much from kava with respect to enhancing NNAL-N-glucuronidation. In summary, our results provide further evidence that kava enhances NNAL urinary detoxification via an increase in both N-glucuronidation and O-glucuronidation. UGT2B10 genetic status has not only the potential to predict the basal capacity of the participants in NNAL-N-glucuronidation but also potentially the extent of kava benefits.

Postmortem distribution of ropivacaine and its metabolite in human body fluids and solid tissues by GC-MS/MS using standard addition method.

An analytical method was developed for determining ropivacaine and its main metabolite, 3-hydroxyropivacaine in biomedical samples using gas chromatography-tandem mass spectrometry (GC-MS/MS). Then, this established method was applied to investigate the distribution of ropivacaine and its metabolite in human fluids and solid tissues obtained from an authentic case ropivacaine involved. The fluid sample was added acetonitrile, and solid tissue was homogenized using a freezer mill and then added into acetonitrile. Then, an internal standard solution was added to the mixtures. The mixture was centrifuged at 12,000 × g for 5min, and the upper layer of acetonitrile was transferred to magnesium sulfate and octadecyl silica (C18) gel for cleaning up the sample. After centrifugation, the upper layer was then evaporated to dryness with nitrogen, and dissolved with methanol, then injected into the GC-MS/MS system. The coefficients of determination (r2) of constructed calibration curves were all greater than 0.999. The limits of detection for ropivacaine and 3-hydroxyropivacaine in target samples were 15ng/mL and 10ng/mL, respectively. The recovery rates of ropivacaine and 3-hydroxyropivacaine ranged from 97.6% to 103% and from 96.5% to 104%, respectively. The inter-day precision values of ropivacaine and 3-hydroxyropivacaine were not greater than 6.25% and 7.98%, respectively, and the inter-day trueness values were not greater than 6.90% and 8.33%, respectively; the intra-day precision and trueness values of ropivacaine and 3-hydroxyropivacaine were not greater than 3.20%, 6.78%, 7.84% and 8.99%, respectively. GC-MS/MS method for simultaneous detection and quantification of ropivacaine and 3-hydroxyropivacaine in biological samples was successfully developed. The method could also be applied to samples obtained from an authentic case; their distribution among tested fluids and solid tissues were also measured. This is the first report on the distribution of ropivacaine and its major metabolite 3-hydroxyropivacaine in a human case.

Biological monitoring of exposure to benzene in water pipe smokers of rural Western Uttar Pradesh (India)

Aim: Present study was performed to investigate the possible human exposure to benzene through water pipe smoking, if any, in the selected rural population of Western Uttar Pradesh. Methodology: Urine samples from water pipe smokers of both the sexes were collected and analyzed for urinary concentration of phenol, creatinine and malondialdehyde employing standard methods. Results: The results showed that tobacco used by selected rural population contained traces of benzene in the range of 2.75 to 9.682µl ml-1. Analysis of urine samples for phenol, a major metabolite of benzene, showed its increased concentrations in water pipe smokers than non smokers. Further, the effects of age and gender differences on the excretion of phenol were also recorded. Female water pipe smokers were found to be more vulnerable to benzene exposure than male smokers. Intriguingly, alcoholic water pipe smokers were less vulnerable than the non-alcoholic smokers. Interpretation: It is concluded that respiratory, cardiac and haematological disorders caused by water pipe smoking might be attributed to long term benzene exposure amongst water pipe smokers. Large scale epidemiological studies are warranted in Indian rural population considering WHO (Framework Convention on Tobacco Control) regulations of water pipe smoking and tobacco products. Key words: Biological monitoring, Benzene, Oxidative stress, Phenol, Tobacco, Waterpipe smoking

Abstract C042: Identification of a non-canonical pathway used by CAF to deliver gamma-aminobutyric acid in pancreatic cancer

Abstract Cancer-associated fibroblasts (CAFs) are cells responsible for deposition and remodeling of the extracellular matrix, and modification of the stromal microenvironment, which facilitates tumor growth. Moreover, CAFs are extremely immunosuppressive because they produce large amounts of inhibitory cytokines, growth factors and metabolites. Thus, understanding how CAFs impart severe immunosuppression in the tumor microenvironment is critical to make PDAC amenable to immunotherapies. The link between metabolism and immunosuppression in tumors has become clearer in recent years, and we postulate that one such metabolite, gamma-aminobutyric acid (GABA), a key inhibitory neurotransmitter, can act as a major immunosuppressive metabolite in PDAC. Our preliminary data shows that CAFs produce high levels of glutamate (Glu) and that metabolites derived from glutamate, such as GABA, are immunosuppressive to NK cells. Moreover, we found that CAFs could produce GABA, de novo, as determined by heavy isotope tracing. Thus, our goal is to uncover the mechanisms used by CAFs to produce GABA, and to evaluate the immunosuppressive mechanisms behind CAF- derived GABA. GABA production was measured in the conditioned media of CAFs grown in 3D culture, where it was observed that inhibition of glutaminase (GLS) and glutamine synthetase (GS) enzymes was not able to decrease the GABA levels. In addition, CAFs did not express glutamate decarboxylase (GAD), the canonical enzyme responsible for the conversion of Glu to GABA, which led us to explore the expression of a non-canonical GABA synthesis pathways in CAFs. Intriguingly, we found that CAFs derived from patient tumors expressed the enzymes of non-canonical pathway (ODC1, DAO, ALDH1A1 and ALDH9A1), and to a greater degree when compared to normal fibroblasts isolated from donor pancreata (Gift of Life Program). CRISPRi mediated knockdown (KD) of aldehyde dehydrogenase 1 (ALDH1a1) in CAFs decreased GABA production compared to control CAFs. On the other hand, CAFs knocked down for diamine oxidase (DAO), showed increased GABA production, suggesting a compensatory mechanism for GABA production upstream of ALDH1a1. Overall, our preliminary data demonstrate that CAFs produce GABA de novo, via a putative alternative GABA synthesis pathway. Our next steps are to identify the cytokine profile of KDs CAFs and perform functional assays utilizing our 3D co-culturing system, uncovering natural killer cell responses to tumor cells in the presence of CAFs with and without the enzymes necessary for GABA production. Translationally, we are analyzing the tumor interstitial fluid from PDAC patients to uncover metabolites/cytokines (secreted factors) that can be correlated with patient parameters (ex. overall survival) and immune cell activation and functionality. It is our hope that the insight provided by this study can lead to a novel set of prognostic or diagnostic factors to improve clinical outcomes in this devasting disease. Citation Format: Ariana Musa de Aquino, Myree Graves, Farrah Ali Ali, David A. Rangel, Langley Kyra, Julie Clark, David Kwon, Guillaume Cognet, Alex Muir, Débora Barbosa Vendramini Costa, Ralph Francescone. Identification of a non-canonical pathway used by CAF to deliver gamma-aminobutyric acid in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C042.

Disruption of the PGE2 synthesis / response pathway restrains atherogenesis in programmed cell death-1 (Pd-1) deficient hyperlipidemic mice.

Immune checkpoint inhibitors (ICIs) that target programmed cell death 1 (PD-1) have revolutionized cancer treatment by enabling the restoration of suppressed T-cell cytotoxic responses. However, resistance to single-agent ICIs still limits their clinical utility. Combinatorial strategies enhance the antitumor effects, but may also enhance the risk of immune related adverse effects of ICIs. Prostaglandin (PG) E2, formed by the sequential action of the cyclooxygenase (COX) and microsomal PGE synthase (mPGES-1) enzymes, acting via its E prostanoid (EP) receptors, EPr2 and EPr4, promotes lymphocyte exhaustion, revealing an additional target for ICIs. Thus, COX inhibitors and EPr4 antagonists are currently being combined with ICIs potentially to enhance antitumor efficacy in clinical trials. However, given the cardiovascular (CV) toxicity of COX inhibitors, such combinations may increase the risk particularly of CV AEs. Here, we compared the impact of distinct approaches to disruption of the PGE2 synthesis /response pathway- global or myeloid cell specific depletion of mPges-1 or global depletion of Epr4- on the accelerated atherogenesis in Pd-1 deficient hyperlipidemic (Ldlr KO) mice. All strategies restrained the atherogenesis. While depletion of mPGES-1 suppresses PGE2 biosynthesis, reflected by its major urinary metabolite, PGE2 biosynthesis was increased in mice lacking EPr4, consistent with enhanced expression of aortic Cox-1 and mPges-1. Deletions of mPges-1 and Epr4 differed in their effects on immune cell populations in atherosclerotic plaques; the former reduced neutrophil infiltration, while the latter restrained macrophages and increased the infiltration of T-cells. Consistent with these findings, chemotaxis by bone-marrow derived macrophages from Epr4 KO mice as impaired. in Epr4 depletion also resulted in extramedullary lymphoid hematopoiesis and inhibition of lipoprotein lipase activity (LPL) with coincident spelenomegaly, leukocytosis and dyslipidemia. Targeting either mPGES-1 or EPr4 may restrain lymphocyte exhaustion while mitigating CV irAEs consequent to PD-1 blockade.