Intracellular Disposition of Fludarabine Triphosphate in Human Natural Killer Cells

Purpose. Fludarabine is a key component of several reduced-intensity conditioning regimens for hematopoietic cell transplantation (HCT). Shortly after reduced-intensity conditioning, the percent of donor natural killer (NK) cells has been associated with progression-free survival. Insufficient suppression of the recipient’s NK cells by fludarabine may lead to lower donor chimerism; however, the effect of fludarabine upon NK cells is poorly understood. Thus, in purified human NK cells we evaluated the uptake and activation of fludarabine to its active metabolite, fludarabine triphosphate (F-ara-ATP), and assessed the degree of interindividual variability in F-ara-ATP accumulation. Methods. Intracellular F-ara-ATP was measured in purified NK cells isolated from healthy volunteers (n = 6) after ex vivo exposure to fludarabine. Gene expression levels of the relevant transporters and enzymes involved in fludarabine uptake and activation were also measured in these cells. Results. F-ara-ATP accumulation (mean ± s.d.) was 6.00 ± 3.67 pmol/1x106 cells/4 hours, comparable to average levels previously observed in CD4+ and CD8+ T-lymphocytes. We observed considerable variability in F-ara-ATP accumulation and mRNA expression of transporters and enzymes relevant to F-ara-ATP accumulation in NK cells from different healthy volunteers. Conclusions. Human NK cells have the ability to form F-ara-ATP intracellularly and large interindividual variability was observed in healthy volunteers. Further studies are needed to evaluate whether F-ara-ATP accumulation in NK cells are associated with apoptosis and clinical outcomes

Genetic and seasonal determinants of vitamin D status in Confederated Salish and Kootenai Tribes (CSKT) participants

Background: Vitamin D is a hormone produced in the skin upon ultraviolet B (UVB) radiation. Vitamin D is a crucial regulator of calcium and phosphate levels for bone mineralization and other physiological roles. Vitamin D levels vary globally in human populations due to genetics, geography, and other demographic factors. It is estimated that 20-85 % of the variability in vitamin D levels is driven by genetic variation. To improve our understanding of contributors to vitamin D levels, we conducted a candidate-gene study in partnership with the Confederated Salish and Kootenai Tribes (CSKT). Methods: We recruited 472 CSKT study participants on the Flathead Reservation in Montana. Demographic factors included age, BMI, and gender (185 male and 287 female; ≥ 18 years old). Genomic DNA and plasma were isolated from whole blood. We sequenced 14 vitamin D regulatory candidate genes: CASR, CUBN, CYP2R1, CYP3A4,CYP24A1, CYP27B1, DHCR7, GC, RXRA, RXRB, RXRG, SULT2A1, UGT1A4, and VDR. We also measured plasma levels of vitamin D and vitamin D metabolites by liquid chromatography/mass-spectrometry (LC/MS), including the clinical marker of vitamin D status, 25-hydroxyvitamin D3 [25(OH)D3]. We tested demographic factors as well as common and rare genetic variants for statistical associations with vitamin D levels using bioinformatics software and R statistical programming language code. Results: We identified 7,370 total genetic variants with 8% (n = 585) of them being novel. We identified 60 genetic variants that may be of clinical significance (disease associated or predicted to influence medication response). Vitamin D levels were below sufficiency [25(OH)D3 + 25(OH)D2 levels \u3c 20 ng/mL] in 56 % of CSKT participants across the year. We observed seasonal vitamin D and metabolite level fluctuations in a seasonal, sinusoidal statistical model with peak concentrations in June – August and trough concentrations in December – February. In linear regression analysis, we found that age, BMI, season, and 5 variants in CUBN and CYP3A4 were significantly associated with 25(OH)D3 concentration (p-value\u3c 0.05). In logistic regression, we found that 4 variants in CUBN, CYP3A4, and UGT1A4 were associated with 25(OH)D sufficiency status [25(OH)D3 + 25(OH)D2 levels of 20 ng/mL] (p-value\u3c 0.05). Multivariate linear regression analysis revealed that genetic variation alone explained ~13% of the variability in 25(OH)D3 concentration in CSKT participants. Genetic variation and environmental factors together explained ~23 % of the variability in 25(OH)D3 concentration in CSKT participants. It is likely that genetic variation in additional genes and other environmental factors (e.g., dietary vitamin D intake) that were not included in this study explain the remaining variability in 25(OH)D3 concentration. Conclusion: This research addresses the need for increased inclusion of American Indian and Alaska Natives in precision medicine health research. We are the first to describe the contribution of season and genetics to vitamin D levels in an American Indian population. Our next steps will be to use these findings to perform mechanistic studies and develop interventional strategies for the CSKT people

Implementing community-engaged pharmacogenomics in Indigenous communities

Innovative pharmacogenomic approaches (genetic variation related to medication response) are needed to reduce disease and disparities in Indigenous communities. We support community-based pharmacogenomics research, inclusive of Indigenous values and priorities, to improve the health and well-being of Indigenous peoples

Absence of P-Glycoprotein Transport in the Pharmacokinetics and Toxicity of the Herbicide Paraquat

Genetic variation in the multidrug resistance gene ABCB1, which encodes the efflux transporter P-glycoprotein (P-gp), has been associated with Parkinson disease. Our goal was to investigate P-gp transport of paraquat, a Parkinson-associated neurotoxicant. We used in vitro transport models of ATPase activity, xenobiotic-induced cytotoxicity, transepithelial permeability, and rhodamine-123 inhibition. We also measured paraquat pharmacokinetics and brain distribution in Friend leukemia virus B-type (FVB) wild-type and P-gp-deficient (mdr1a(-/-)/mdr1b(-/-)) mice following 10, 25, 50, and 100 mg/kg oral doses. In vitro data showed that: 1) paraquat failed to stimulate ATPase activity; 2) resistance to paraquat-induced cytotoxicity was unchanged in P-gp-expressing cells in the absence or presence of P-gp inhibitors GF120918 [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide] and verapamil-37.0 [95% confidence interval (CI): 33.2-41.4], 46.2 (42.5-50.2), and 34.1 µM (31.2-37.2)-respectively; 3) transepithelial permeability ratios of paraquat were the same in P-gp-expressing and nonexpressing cells (1.55 ± 0.39 and 1.39 ± 0.43, respectively); and 4) paraquat did not inhibit rhodamine-123 transport. Population pharmacokinetic modeling revealed minor differences between FVB wild-type and mdr1a(-/-)/mdr1b(-/-) mice: clearances of 0.47 [95% confidence interval (CI): 0.42-0.52] and 0.78 l/h (0.58-0.98), respectively, and volume of distributions of 1.77 (95% CI: 1.50-2.04) and 3.36 liters (2.39-4.33), respectively; however, the change in clearance was in the opposite direction of what would be expected. It is noteworthy that paraquat brain-to-plasma partitioning ratios and total brain accumulation were the same across doses between FVB wild-type and mdr1a(-/-)/mdr1b(-/-) mice. These studies indicate that paraquat is not a P-gp substrate. Therefore, the association between ABCB1 pharmacogenomics and Parkinson disease is not attributed to alterations in paraquat transport

Transfer learning enables prediction of CYP2D6 haplotype function.

Cytochrome P450 2D6 (CYP2D6) is a highly polymorphic gene whose protein product metabolizes more than 20% of clinically used drugs. Genetic variations in CYP2D6 are responsible for interindividual heterogeneity in drug response that can lead to drug toxicity and ineffective treatment, making CYP2D6 one of the most important pharmacogenes. Prediction of CYP2D6 phenotype relies on curation of literature-derived functional studies to assign a functional status to CYP2D6 haplotypes. As the number of large-scale sequencing efforts grows, new haplotypes continue to be discovered, and assignment of function is challenging to maintain. To address this challenge, we have trained a convolutional neural network to predict functional status of CYP2D6 haplotypes, called Hubble.2D6. Hubble.2D6 predicts haplotype function from sequence data and was trained using two pre-training steps with a combination of real and simulated data. We find that Hubble.2D6 predicts CYP2D6 haplotype functional status with 88% accuracy in a held-out test set and explains 47.5% of the variance in in vitro functional data among star alleles with unknown function. Hubble.2D6 may be a useful tool for assigning function to haplotypes with uncurated function, and used for screening individuals who are at risk of being poor metabolizers