Per- and poly-fluoroalkyl substances (PFAS) are not efficiently degraded and hence cycle through the environment, persist for a very long time, accumulate in living organisms, and cause potential health and ecological risks. PFAS monitoring in the drinking water relies on solid phase extraction (SPE) for sample concentration and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for sensitive and specific detection, but conventional methods suffer from long processing time, inadequate sensitivity for sub-part per trillion (ppt) trace level detection, and high cost. In this study, we aimed to develop and validate an optimized fast flow SPE method to achieve sub-ppt trace level quantification of 40 PFAS compounds that are of environmental concern to the US Environmental Protection Agency. The impact of several key sample preparation parameters, including N2 drying, syringe filtration, SPE elution volume, and SPE flow rate, on the PFAS recovery was determined. These results helped inform the development of the final optimized fast flow SPE method, which was demonstrated using blank water samples spiked with trace levels of PFAS and tap water samples. The new fast flow SPE method substantially reduced the sample loading time (6 min for a 500-mL sample vs. 100 min required for normal flow SPE; 60-70 min for a 4-liter sample vs. 800 min required for normal flow SPE) without compromising the PFAS recovery for 38 out of 40 PFAS compounds and achieved sub-ppt (as low as 0.01 ppt for method detection limit) trace level quantification of PFAS in the drinking water. As a result, the optimized fast flow SPE is a viable strategy to enhance method sensitivity, increase throughput, and reduce cost for PFAS analysis and will positively impact future PFAS monitoring in the drinking water.

The increasing prevalence of diabetes and the aging population have led to a growing market for ophthalmic pharmaceutical drugs. However, the development of ophthalmic drugs is hampered by the poor understanding of drug-metabolizing enzymes (DMEs) and drug transporters (DTs) in complex ocular tissues. The rabbit is the most commonly used preclinical model for human ocular diseases. The purpose of this study was to compare relative gene expression of DMEs and DTs in various rabbit ocular subtissues, including cornea, iris-ciliary body, vitreous humor, and retina-choroid complex. Rabbit liver and duodenum samples were also included to investigate gene expression differences among the eye, liver, and duodenum. The mRNA transcriptome of the 6 tissue types from 6 rabbits (3 males and 3 females) was sequenced in a single run using RNA-seq for cross-tissue gene expression comparisons. A total of 387 DME and 128 DT genes were identified across the 6 tissues, and distinct expression patterns of DME and DT genes were observed between ocular subtissues vs. liver and duodenum and among different ocular subtissues. Furthermore, specific DMEs and DTs that enriched in each tissue type were identified and relative gene expression of DMEs and DTs across the 6 tissue types was measured. These results are expected to aid ophthalmic drug development by providing a basis for ocular tissue-specific drug disposition/response and informing model-based predictions and interspecies extrapolation. SIGNIFICANCE STATEMENT: This study represents the first comprehensive comparative transcriptomic analysis of drug-metabolizing enzymes and drug transporters in rabbit ocular subtissues, with comparisons to key metabolic organs such as liver and duodenum. Our findings highlight critical tissue-specific differences that have significant implications for ophthalmic drug development and ocular drug delivery systems. The quantitative gene expression levels of drug-metabolizing enzymes and drug transporters in the rabbit eye will aid investigations into ocular drug metabolism and disposition in both preclinical and clinical settings.

Background: Glucuronide recycling in the gut and liver profoundly affects the systemic and/or local exposure of drugs and their glucuronide metabolites, impacting both clinical efficacy and toxicity. This recycling also alters drug exposure in the colon, making it critical to establish local concentration for drugs targeting colon (e.g., drugs for colon cancer and inflammatory bowel disease). Methods: In this study, a parent–metabolite middle-out physiologically based pharmacokinetic (PBPK) model was built for genistein and its glucuronide metabolite to estimate the systemic and local exposure of the glucuronide and its corresponding aglycone in rats by incorporating UDP-glucuronosyltransferase (UGT)-mediated metabolism and transporter-dependent glucuronide disposition in the liver and intestine, as well as gut microbial-mediated deglucuronidation that enables the recycling of the parent compound. Results: This parent–metabolite middle-out rat PBPK model utilized in vitro-to-in vivo extrapolated (IVIVE) metabolic and transporter clearance values based on in vitro kinetic parameters from surrogate species, the rat tissue abundance of relevant proteins, and saturable Michaelis–Menten mechanisms. Inter-system extrapolation factors (ISEFs) were used to account for transporter protein abundance differences between in vitro systems and tissues and between rats and surrogate species. Model performance was evaluated at multiple dose levels for genistein and its glucuronide. Model sensitivity analyses demonstrated the impact of key parameters on the plasma concentrations and local exposure of genistein and its glucuronide. Our model was applied to simulate the quantitative impact of glucuronide recycling on the pharmacokinetic profiles in both plasma and colonocytes. Conclusions: Our study underlines the importance of glucuronide recycling in determining local drug concentrations in the intestine and provides a preliminary modeling tool to assess the influence of transporter-mediated drug–drug interactions on glucuronide recycling and local drug exposure, which are often misrepresented by systemic plasma concentrations.

INTRODUCTION:Longitudinal curriculum has been suggested for improving pharmacogenomics education, however the outcome of such curriculum design has yet to be reported. Here we evaluated the effectiveness of a simple longitudinal curriculum consisting of didactic lecturing and laboratory-based teaching in two sequential semesters towards pharmacogenomics education. METHODS:Four pharmacogenomics lectures were offered to professional year 3 (PY3) pharmacy students during the fall semester. During the following spring semester, students participated in two laboratories followed by an implementation project. Knowledge attainment was assessed through an exam following the fall lectures. Students' perception about their clinical pharmacogenomics skills were collected by electronic questionnaire before, immediately after, and 3 months after the fall lectures and the spring laboratories. Statistical analysis was performed using one-way ANOVA followed by pairwise t-test. RESULTS:The average exam score in Fall 2023 was 79 % (54 %-96 %). Students' perception in a 1-5 Likert scale improved from 1.35 to 3.63 immediately following the lectures (p < 0.0001) but dropped to 1.94 after three months (p < 0.0001). In contrast, after two laboratories in Spring 2024, students' perception improved from 1.94 to 3.67 immediately following the laboratories (p < 0.0001), and importantly, remained high at 3.55 three months later (p = 0.36). CONCLUSIONS:Combination of didactic lecturing and laboratory-based teaching offered in two sequential semesters is conducive to maintaining student's positive perception about their clinical pharmacogenomics skills. Our curriculum design is simple to implement and has the potential to improve long-term retention of pharmacogenomics knowledge.

Background: Despite progress in access to family planning services in many sub-Saharan 11 African countries in recent decades, advances in early postpartum contraceptive adoption remain 12 low, and the unmet need for early postpartum contraceptives is high. According to the Ethiopia 13 Demographic and Health Survey report, early postpartum modern contraceptive method uptake is 14 still unacceptably low in Ethiopia. Objectives: This study aimed to determine the magnitude of 15 intention to adoption of early postpartum modern contraceptive methods and its associated factors 16 among pregnant women in Dessie and Kombolcha town zones, northeast Ethiopia. Methods: A 17 community-based cross-sectional study was deployed from January 15–February 15, 2023, in the 18 Dessie and Kombolcha zones, north-east Ethiopia among pregnant women. The study involved 780 19 pregnant women using the cluster sampling technique. A census was conducted in 20 randomly 20 selected clusters to identify eligible pregnant women. Actual data were collected home-to-home in 21 the community through face-to-face interviews. Data were collected by Open Data Kit (ODK) and 22 exported to STATA 17 for analysis. A multivariable logistic regression analysis was done and the 23 goodness of the model was checked by Hosmer-Lemeshow's test statistic and rock curve. An ad- 24 justed odds ratio with a 95% confidence interval and P-value < 0.05 was considered statistically sig- 25 nificant. Result: The study revealed that 49.6% of pregnant women lack autonomy and 50% lack 26 knowledge about early postpartum contraception, with participants' wealth index status ranging 27 from rich (36.6%) to poor (33.2%). The study found that 75.8% of pregnant women intended to adopt 28 early postpartum modern contraceptive methods early after childbirth. After controlling the poten- 29 tial confounder’s mothers age (AOR = 6.2 [2.6–14.6], birth interval (AOR = 2.5 [1.6–3.7]), have paid 30 work (AOR = 1.9 [1.3–2.8]), health facility from home (AOR=2.6[1.5-4.4]), last delivery 31 Place(AOR=2.4[1.1-5.7]), knowledge on (AOR = 1.5 [1.1-2.1]), and antenatal care follow-up (AOR = 32 1.9 [1.2-3.3]) were significant associated factors of intention to uptake early postpartum modern con- 33 traceptive methods among pregnant women. Conclusion: The study found that 75% of the partici- 34 pants had the intention to adoption of contraceptive methods during the early postpartum period. 35 Identified factors influencing this intention were age, birth interval, women's employment status, 36 area of residence, distance to health facilities, last delivery place, knowledge of early postpartum 37 modern contraception, gravidity, and antenatal care follow-up. These findings highlight the need 38 for targeted interventions to address these factors, framing the intended users and enabling access 39 to early adoption of postpartum contraceptive methods.

Miltefosine (MTS) is the only approved oral drug for treating leishmaniasis caused by intracellular Leishmania parasites that localize in macrophages of the liver, spleen, skin, bone marrow, and lymph nodes. MTS is extensively distributed in tissues and has prolonged elimination half-lives due to its high plasma protein binding, slow metabolic clearance, and minimal urinary excretion. Thus, understanding and predicting the tissue distribution of MTS help assess therapeutic and toxicologic outcomes of MTS, especially in special populations, e.g., pediatrics. In this study, a whole-body physiologically-based pharmacokinetic (PBPK) model of MTS was built on mice and extrapolated to rats and humans. MTS plasma and tissue concentration data obtained by intravenous and oral administration to mice were fitted simultaneously to estimate model parameters. The resulting high tissue-to-plasma partition coefficient values corroborate extensive distribution in all major organs except the bone marrow. Sensitivity analysis suggests that plasma exposure is most susceptible to changes in fraction unbound in plasma. The murine oral-PBPK model was further validated by assessing overlay of simulations with plasma and tissue profiles obtained from an independent study. Subsequently, the murine PBPK model was extrapolated to rats and humans based on species-specific physiological and drug-related parameters, as well as allometrically scaled parameters. Fold errors for pharmacokinetic parameters were within acceptable range in both extrapolated models, except for a slight underprediction in the human plasma exposure. These animal and human PBPK models are expected to provide reliable estimates of MTS tissue distribution and assist dose regimen optimization in special populations.

Sterol biosynthesis requires the oxidative removal of two methyl groups from the C-4 position by sterol C-4-demethylase and one methyl group from the C-14 position by sterol C-14-demethylase. In Leishmania donovani, a CYP5122A1 (Cytochrome P450 family 5122A1) protein was recently identified as the bona fide sterol C-4 methyl oxidase catalyzing the initial steps of C-4-demethylation. Besides CYP5122A1, Leishmania parasites possess orthologs to ERG25 (ergosterol pathway gene 25), the canonical sterol C-4 methyl oxidase in Saccharomyces cerevisiae. To determine the contribution of CYP5122A1 and ERG25 in sterol biosynthesis, we assessed the essentiality of these genes in Leishmania major, which causes cutaneous leishmaniasis. Like in L. donovani, CYP5122A1 in L. major could only be deleted in the presence of a complementing episome. Even with strong negative selection, L. major chromosomal CYP5122A1-null mutants retained the complementing episome in both promastigote and amastigote stages, demonstrating its essentiality. In contrast, the L. major ERG25-null mutants were fully viable and replicative in culture and virulent in mice. Deletion and overexpression of ERG25 did not affect the sterol composition, indicating that ERG25 is not required for C-4-demethylation. These findings suggest that CYP5122A1 is the dominant and possibly only sterol C-4 methyl oxidase in Leishmania, and inhibitors of CYP5122A1 may have strong therapeutic potential against multiple Leishmania species.

Visceral leishmaniasis is a life-threatening parasitic disease, but current antileishmanial drugs have severe drawbacks. Antifungal azoles inhibit the activity of cytochrome P450 (CYP) 51 enzymes which are responsible for removing the C14 alpha-methyl group of lanosterol, a key step in ergosterol biosynthesis in Leishmania. However, they exhibit varying degrees of antileishmanial activities in culture, suggesting the existence of unrecognized molecular targets. Our previous study reveals that, in Leishmania, lanosterol undergoes parallel C4- and C14-demethylation to form 4 alpha,14 alpha-dimethylzymosterol and T-MAS, respectively. In the current study, CYP5122A1 is identified as a sterol C4-methyl oxidase that catalyzes the sequential oxidation of lanosterol to form C4-oxidation metabolites. CYP5122A1 is essential for both L. donovani promastigotes in culture and intracellular amastigotes in infected mice. CYP5122A1 overexpression results in growth delay, increased tolerance to stress, and altered expression of lipophosphoglycan and proteophosphoglycan. CYP5122A1 also helps to determine the antileishmanial effect of antifungal azoles in vitro. Dual inhibitors of CYP51 and CYP5122A1 possess superior antileishmanial activity against L. donovani promastigotes whereas CYP51-selective inhibitors have little effect on promastigote growth. Our findings uncover the critical biochemical and biological role of CYP5122A1 in L. donovani and provide an important foundation for developing new antileishmanial drugs by targeting both CYP enzymes. CYP5122A1 is a sterol C4-methyl oxidase that catalyzes the sequential oxidation of lanosterol, is essential for both Leishmania donovani promastigotes in culture and intracellular amastigotes in infected mice, and provides a new target for antileishmanial drug discovery.

CYP5122A1, an enzyme involved in sterol biosynthesis in Leishmania, was recently characterized as a sterol C4-methyl oxidase. Screening of a library of compounds against CYP5122A1 and CYP51 from Leishmania resulted in the identification of two structurally related classes of inhibitors of these enzymes. Analogs of screening hit N-(3,5-dimethylphenyl)-4-(pyridin-4-ylmethyl)piperazine-1-carboxamide (4a) were generally strong inhibitors of CYP51 but were less potent against CYP5122A1 and typically displayed weak inhibition of L. donovani promastigote growth. Analogs of screening hit N-(4-(benzyloxy)phenyl)-4-(2-(pyridin-4-yl)ethyl)piperazine-1-carboxamide (18a) were stronger inhibitors of both CYP5122A1 and L. donovani promastigote proliferation but also remained selective for inhibition of CYP51. Two compounds in this series, N-(4-((3,5-bis(trifluoromethyl)benzyl)oxy)phenyl)-4-(2-(pyridin-4-yl)ethyl)piperazine-1-carboxamide (18e) and N-(4-((3,5-di-tert-butylbenzyl)oxy)phenyl)-4-(2-(pyridin-4-yl)ethyl)piperazine-1-carboxamide (18i) showed modest selectivity for inhibiting L. donovani promastigote proliferation compared to J774 macrophages and were effective against intracellular L. donovani with EC50 values in the low micromolar range. Replacement of the 4-pyridyl ring present in 18e with imidazole resulted in a compound (4-(2-(1H-imidazol-1-yl)ethyl)-N-(4-((3,5-bis(trifluoromethyl)benzyl)oxy)phenyl)piperazine-1-carboxamide, 18p) with approximately fourfold selectivity for CYP5122A1 over CYP51 that inhibited both enzymes with IC50 values ≤ 1 µM, although selective potency against L. donovani promastigotes was lost. Compound 18p also inhibited the proliferation of L. major promastigotes and caused the accumulation of 4-methylated sterols in L. major membranes, indicating that this compound blocks sterol demethylation at the 4-position in Leishmania parasites. The molecules described here may therefore be useful for the future identification of dual inhibitors of CYP51 and CYP5122A1 as potential antileishmanial drug candidates and as probes to shed further light on sterol biosynthesis in Leishmania and related parasites.

Visceral leishmaniasis, caused by Leishmania donovani, is a life-threatening parasitic disease, but current antileishmanial drugs are limited and have severe drawbacks. There have been efforts to repurpose antifungal azole drugs for the treatment of Leishmania infection. Antifungal azoles are known to potently inhibit the activity of cytochrome P450 (CYP) 51 enzymes which are responsible for removing the C14α-methyl group of lanosterol, a key step in ergosterol biosynthesis in Leishmania. However, they exhibit varying degrees of antileishmanial activities in culture, suggesting the existence of unrecognized molecular targets for these compounds. Our previous study reveals that, in Leishmania, lanosterol undergoes parallel C4- and C14-demethylation reactions to form 4α,14α-dimethylzymosterol and T-MAS, respectively. In the current study, CYP5122A1 is identified as a sterol C4-methyl oxidase that catalyzes the sequential oxidation of lanosterol to form C4-oxidation metabolites. CYP5122A1 is essential for both L. donovani promastigotes in culture and intracellular amastigotes in infected mice. Overexpression of CYP5122A1 results in growth delay, differentiation defects, increased tolerance to stress, and altered expression of lipophosphoglycan and proteophosphoglycan. CYP5122A1 also helps to determine the antileishmanial effect of antifungal azoles in vitro. Dual inhibitors of CYP51 and CYP5122A1, e.g., clotrimazole and posaconazole, possess superior antileishmanial activity against L. donovani promastigotes whereas CYP51-selective inhibitors, e.g., fluconazole and voriconazole, have little effect on promastigote growth. Our findings uncover the critical biochemical and biological role of CYP5122A1 in L. donovani and provide an important foundation for developing new antileishmanial drugs by targeting both CYP enzymes.

BACKGROUND:Clinical pharmacogenomics is an expanding area in healthcare that relies heavily on pharmacists for advocacy and implementation. To support pharmacists' significant roles in clinical pharmacogenomics, pharmacy schools and colleges in the United States (US) have strived to incorporate pharmacogenomics education into their curricula, and various teaching strategies have been employed in recent years to meet pharmacogenomics educational outcomes. The six major strategies reported in the literature are described and compared in this review, which culminates in a proposed longitudinal curriculum design for pharmacogenomics education.METHODS:Publications focused on pharmacogenomics education to pharmacy students within the US in the past decade were evaluated and summarized.RESULTS:The major education strategies that have been studied are didactic lecture, personal genotyping or personal genomic testing, simulation laboratory activity, interprofessional education, practice-based activity such as clinical rotation, and combinational courses. Strengths and limitations of each teaching strategy are summarized and discussed.IMPLICATIONS:Based upon each education strategy's strengths and weaknesses, the authors propose a longitudinal curriculum design to ensure that pharmacogenomics is taught multiple times to pharmacy students with diverse formats and teaching objectives conducive to long-term knowledge retention and practice readiness. Through this longitudinal curriculum design, pharmacy graduates will be well equipped to lead clinical pharmacogenomics in practice.

PDF file - 38K, This provides a supplemental table of gene expression data and background on the data set. The GEO accesssion number and hyperlink are included.

Human leishmaniasis is an infectious disease caused by Leishmania protozoan parasites. Current chemotherapeutic options against the deadly disease have significant limitations. The ergosterol biosynthetic pathway has been identified as a drug target in Leishmania. However, remarkable differences in the efficacy of antifungal azoles that inhibit ergosterol biosynthesis have been reported for the treatment of leishmaniasis. To better understand the sterol biosynthetic pathway in Leishmania and elucidate the mechanism underlying the differential efficacy of antifungal azoles, we developed a new LC-MS/MS method to study sterol profiles in promastigotes of three Leishmania species, including two L. donovani, one L. major and one L. tarentolae strains. A combination of distinct precursor ion masses and LC retention times allowed for specific detection of sixteen intermediate sterols between lanosterol and ergosterol using the newly developed LC-MS/MS method. Although both posaconazole and fluconazole are known inhibitors of fungal lanosterol 14α-demethylase (CYP51), only posaconazole led to a substantial accumulation of lanosterol in azole-treated L. donovani promastigotes. Furthermore, a key intermediate sterol accumulated by 40- and 7-fold when these parasites were treated with posaconazole and fluconazole, respectively, which was determined as 4α,14α-dimethylzymosterol by high resolution mass spectrometry and NMR spectroscopy. The identification of 4α,14α-dimethylzymosterol supports a branched ergosterol biosynthetic pathway in Leishmania, where lanosterol C4- and C14-demethylation reactions occur in parallel rather than sequentially. Our results suggest that selective inhibition of leishmanial CYP51 is insufficient to effectively prevent parasite growth and dual inhibitors of both CYP51 and the unknown sterol C4-demethylase may be required for optimal antiparasitic effect.

Subcellular organelles have long been an interest in biochemical research and drug development as the isolation of those organelles can help to probe protein functions and elucidate drug disposition within the cell. Usually, the purity of isolated subcellular organelle fractions was determined using immunoblot analysis of subcellular organelle marker proteins, which can be labor-intensive and lack reproducibility due to antibody batch-to-batch variability. As such, a higher throughput and more robust method is needed. Here, a UPLC-MRM-based targeted proteomic method was developed for a panel of human organelle marker proteins and used to profile a series of sucrose fractions isolated from the protein extract of human liver tissues. The method was validated by comparing to the traditional immunoblot and determining subcellular localization of three case study proteins (CYP3A4, FcRn, and β2M) pertaining to the disposition of small molecule and biologic drugs. All three case study proteins were co-enriched with their corresponding subcellular protein marker, and complete recoveries were achieved from isolated fractions. This newly developed MRM method for the panel of human organelle marker proteins can potentially accelerate future intracellular drug disposition analysis and facilitate subcellular organelle quality assessment.

Trypanosoma cruzi, the agent of Chagas disease, probably infects tens of millions of people, primarily in Latin America, causing morbidity and mortality. The options for treatment and prevention of Chagas disease are limited and underutilized. Here we describe the discovery of a series of benzoxaborole compounds with nanomolar activity against extra- and intracellular stages of T. cruzi. Leveraging both ongoing drug discovery efforts in related kinetoplastids, and the exceptional models for rapid drug screening and optimization in T. cruzi, we have identified the prodrug AN15368 that is activated by parasite carboxypeptidases to yield a compound that targets the messenger RNA processing pathway in T. cruzi. AN15368 was found to be active in vitro and in vivo against a range of genetically distinct T. cruzi lineages and was uniformly curative in non-human primates (NHPs) with long-term naturally acquired infections. Treatment in NHPs also revealed no detectable acute toxicity or long-term health or reproductive impact. Thus, AN15368 is an extensively validated and apparently safe, clinically ready candidate with promising potential for prevention and treatment of Chagas disease.

Due to the limitations of existing medications, there is a critical need for new drugs to treat visceral leishmaniasis. Since arylimidamides and antifungal azoles both show oral activity in murine visceral leishmaniasis models, a molecular hybridization approach was employed where arylimidamide and azole groups were separated by phenoxyalkyl linkers in an attempt to capitalize on the favorable antileishmanial properties of both series. Among the target compounds synthesized, a greater antileishmanial potency against intracellular Leishmania donovani was observed as the linker length increased from two to eight carbons and when an imidazole ring was employed as the terminal group compared to a 1,2,4-triazole group. Compound 24c (N-(4-((8-(1H-imidazol-1-yl)octyl)oxy)-2-isopropoxyphenyl) picolinimidamide) displayed activity against L. donovani intracellular amastigotes with an IC50 value of 0.53 μM. When tested in a murine visceral leishmaniasis model, compound 24c at a dose of 75 mg/kg/day p.o. for five consecutive days resulted in a modest 33% decrease in liver parasitemia compared to the control group, indicating that further optimization of these molecules is needed. While potent hybrid compounds bearing an imidazole terminal group were also strong inhibitors of recombinant CYP51 from L. donovani, as assessed by a fluorescence-based assay, additional targets are likely to play an important role in the antileishmanial action of these compounds.