Pharmacomicrobiomics: Immunosuppressive Drugs and Microbiome Interactions in Transplantation.

The human microbiome is associated with human health and disease. Exogenous compounds, including pharmaceutical products, are also known to be affected by the microbiome, and this discovery has led to the field of pharmacomicobiomics. The microbiome can also alter drug pharmacokinetics and pharmacodynamics, possibly resulting in side effects, toxicities, and unanticipated disease response. Microbiome-mediated effects are referred to as drug-microbiome interactions (DMI). Rapid advances in the field of pharmacomicrobiomics have been driven by the availability of efficient bacterial genome sequencing methods and new computational and bioinformatics tools. The success of fecal microbiota transplantation for recurrent Clostridioides difficile has fueled enthusiasm and research in the field. This review focuses on the pharmacomicrobiome in transplantation. Alterations in the microbiome in transplant recipients are well documented, largely because of prophylactic antibiotic use, and the potential for DMI is high. There is evidence that the gut microbiome may alter the pharmacokinetic disposition of tacrolimus and result in microbiome-specific tacrolimus metabolites. The gut microbiome also impacts the enterohepatic recirculation of mycophenolate, resulting in substantial changes in pharmacokinetic disposition and systemic exposure. The mechanisms of these DMI and the specific bacteria or communities of bacteria are under investigation. There are little or no human DMI data for cyclosporine A, corticosteroids, and sirolimus. The available evidence in transplantation is limited and driven by small studies of heterogeneous designs. Larger clinical studies are needed, but the potential for future clinical application of the pharmacomicrobiome in avoiding poor outcomes is high.

Medical Cannabis Received by Patients According to Qualifying Condition in a US State Cannabis Program: Product Choice, Dosing, and Age-Related Trends.

Background: Little is known about the distribution of cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC) to patients participating in state medical cannabis programs. The Minnesota cannabis program requires third-party testing of products with limited formulations of cannabis for distribution to patients. Objective: To characterize the distribution of cannabis products, their CBD/THC content, and dosing among patients with qualifying conditions. Methods: This is a retrospective analysis of ∼50% of registered users receiving medical cannabis in Minnesota (June 16, 2016, to November 15, 2019). Data included formulation, CBD/THC prescribed doses, and qualifying conditions. The primary end points were calculated using daily dose and duration of use. Comparisons were made for CBD and THC total daily dose dispensed, patient age, and approved product. Nonparametric statistical tests were used (significance was set at p < 0.05). Results: A total of 11,520 patients were listed with 1 qualifying condition. The most common condition was intractable pain (60.0%). Median dispensation duration varied from 53 days (cancer) to 322 days (muscle spasms). Most (≥62.8%) patients across all qualifying conditions received both CBD and THC. Median THC dose was lower in older (≥65 years) compared with younger adults with intractable pain (p < 0.0001) and cancer patients (p = 0.0152), and the same pattern was found CBD dose with seizure (p = 0.0498) patients. For commercial products with Food and Drug Administration indications, the median CBD total daily dose was 86.9% lower than the recommended doses for patients with seizures (Epidiolex: Jazz Pharmaceuticals, Palo Alto CA) and median THC total daily dose was 65.3% (Syndros: Benuvia Manufacturing, Round Rock, TX) or 79.3% lower (Marinol: Banner Pharmacaps, Inc., High Point, NC) for cancer patients. Conclusions: A majority of patients received products containing both CBD and THC. Dosages varied by age group and were lower than recommended for conditions with Food and Drug Administration-approved products. Complex pharmacokinetics of THC and CBD, possible age-related changes in physiology, unknown efficacy, and potential for drug interactions all increase the need for monitoring of patients receiving cannabis products. (Curr Ther Res Clin Exp. 2023; 84:XXX-XXX).

Pharmacokinetics and safety of prolonged paracetamol treatment in neonates: An interventional cohort study.

AIMS: To investigate the pharmacokinetics and safety of prolonged paracetamol use (>72 h) for neonatal pain. METHODS: Neonates were included if they received paracetamol orally or intravenously for pain treatment. A total of 126 samples were collected. Alanine aminotransferase and bilirubin were measured as surrogate liver safety markers. Paracetamol and metabolites were measured in plasma. Pharmacokinetic parameters for the parent compound were estimated with a nonlinear mixed-effects model. RESULTS: Forty-eight neonates were enrolled (38 received paracetamol for >72 h). Median gestational age was 38 weeks (range 25-42), and bodyweight at inclusion was 2954 g (range 713-4750). Neonates received 16 doses (range 4-55) over 4.1 days (range 1-13.8). The median (range) dose was 10.1 mg/kg (2.9-20.3). The median oxidative metabolite concentration was 14.6 µmol/L (range 0.12-113.5) and measurable >30 h after dose. There was no significant difference (P > .05) between alanine aminotransferase and bilirubin measures at <72 h or >72 h of paracetamol treatment or the start and end of the study. Volume of distribution and paracetamol clearance for a 2.81-kg neonate were 2.99 L (% residual standard error = 8, 95% confidence interval 2.44-3.55) and 0.497 L/h (% residual standard error = 7, 95% confidence interval 0.425-0.570), respectively. Median steady-state concentration from the parent model was 50.3 µmol/L (range 30.6-92.5), and the half-life was 3.55 h (range 2.41-5.65). CONCLUSION: Our study did not provide evidence of paracetamol-induced liver injury nor changes in metabolism in prolonged paracetamol administration in neonates.

Higher number of tacrolimus dose adjustments in kidney transplant recipients who are extensive and intermediate CYP3A5 metabolizers.

Kidney transplant recipients carrying the CYP3A5*1 allele have lower tacrolimus troughs, and higher dose requirements compared to those with the CYP3A5*3/*3 genotype. However, data on the effect of CYP3A5 alleles on post-transplant tacrolimus management are lacking. The effect of CYP3A5 metabolism phenotypes on the number of tacrolimus dose adjustments and troughs in the first 6 months post-transplant was evaluated in 78 recipients (64% Caucasians). Time to first therapeutic concentration, percentage of time in therapeutic range (TTR), and estimated glomerular filtration rate (eGFR) were also evaluated. Fifty-five kidney transplant recipients were CYP3A5 poor metabolizers (PM), 17 were intermediate metabolizers (IM), and 6 were extensive metabolizers (EM). Compared to PMs, EMs/IMs had significantly more dose adjustments (6.1 vs. 8.1, p = .015). Overall, 33.82% of trough measurements resulted in a dose change. There was no difference in the number of tacrolimus trough measurements between PMs and EM/IMs. The total daily tacrolimus dose requirements were higher in EMs and IMs compared to PMs (<.001). TTR was ∼50% in the PMs and EMs/IMs groups. CYP3A5 EM/IM metabolizers have more tacrolimus dose changes and higher dose requirements which increases clinical management complexity. Larger studies are needed to assess the cost and benefits of including genotyping data to improve clinical management.

Precision Dosing for Tacrolimus Using Genotypes and Clinical Factors in Kidney Transplant Recipients of European Ancestry.

Genetic variation in the CYP3A4 and CYP3A5 (CYP3A4/5) genes, which encode the key enzymes in tacrolimus metabolism, is associated with tacrolimus clearance and dose requirements. Tacrolimus has a narrow therapeutic index with high intra- and intersubject variability, in part because of genetic variation. High tacrolimus clearance and low trough concentration are associated with a greater risk for rejection, whereas high troughs are associated with calcineurin-induced toxicity. The objective of this study was to develop a model of tacrolimus clearance with a dosing equation accounting for genotypes and clinical factors in adult kidney transplant recipients of European ancestry that could preemptively guide dosing. Recipients receiving immediate-release tacrolimus for maintenance immunosuppression from 2 multicenter studies were included. Participants in the GEN03 study were used for tacrolimus model development (n = 608 recipients) and was validated by prediction performance in the DeKAF Genomics study (n = 1361 recipients). Nonlinear mixed-effects modeling was used to develop the apparent oral tacrolimus clearance (CL/F) model. CYP3A4/5 genotypes and clinical covariates were tested for their influence on CL/F. The predictive performance of the model was determined by assessing the bias (median prediction error [ME] and median percentage error [MPE]) and the precision (root median squared error [RMSE]) of the model. CYP3A5*3, CYP3A4*22, corticosteroids, calcium channel blocker and antiviral drug use, age, and diabetes significantly contributed to the interindividual variability of oral tacrolimus apparent clearance. The bias (ME, MPE) and precision (RMSE) of the final model was good, 0.49 ng/mL, 6.5%, and 3.09 ng/mL, respectively. Prospective testing of this equation is warranted.

Pharmacogenomics in kidney transplant recipients and potential for integration into practice.

WHAT IS KNOWN AND OBJECTIVE: Pharmacogenomic biomarkers are now used in many clinical care settings and represent one of the successes of precision medicine. Genetic variants are associated with pharmacokinetic and pharmacodynamic changes leading to medication adverse effects and changes in clinical response. Actionable pharmacogenomic variants are common in transplant recipients and have implications for medications used in transplant, but yet are not broadly incorporated into practice. METHODS: From the Clinical Pharmacogenetics Implementation Consortium and Dutch Pharmacogenetics Working Group guidelines, and PharmGKB databases, 12 pharmacogenomic genes with 30 variants were selected and used to create diplotypes and actionable pharmacogenomic phenotypes. A total of 853 kidney allograft recipients who had genomic information available from a genome-wide association study were included. RESULTS: Each recipient had at least one actionable pharmacogenomic diplotype/phenotype, whereas the majority (58%) had three or four actionable diplotypes/phenotypes and 17.4% had five or more among the 12 genes. The participants carried actionable diplotypes/phenotypes for multiple medications, including tacrolimus, azathioprine, clopidogrel, warfarin, simvastatin, voriconazole, antidepressants and proton-pump inhibitors. WHAT IS NEW AND CONCLUSION: Pharmacogenomic variants are common in transplant recipients, and transplant recipients receive medications that have actionable variants. CLINICAL TRIAL: Genomics of Transplantation, clinicaltrials.gov (NCT01714440).

Food effect on pharmacokinetics of cannabidiol oral capsules in adult patients with refractory epilepsy.

OBJECTIVE: To evaluate the pharmacokinetics of a purified oral cannabidiol (CBD) capsule administered with and without food in adults with refractory epilepsy. METHODS: Adult patients who were prescribed CBD for seizures, had localization-related intractable epilepsy with ≥4 seizures per month, and qualified for Minnesota cannabis were enrolled. A single dose of 99% pure CBD capsules was taken under both fasting (no breakfast) and fed (high fat 840-860 calorie) conditions. Blood sampling for CBD plasma concentrations was performed under each condition between 0 and 72 hours post-dose and measured by a validated liquid chormatography-mass spectometry assay. CBD pharmacokinetic profiles including maximum concentration (Cmax ), area-under-the-curve from zero to infinity (AUC0-∞ ), and time-to-maximum concentration (Tmax ) were calculated. The confidence intervals (CIs) for log-transformed Cmax and AUC0-∞ ratios between fed and fasting states were calculated. Seizure and adverse events information was collected. RESULTS: Eight patients completed the study. On average Cmax was 14 times and AUC0-∞ 4 times higher in the fed state. The 90% CI for the ratio of fed versus fast conditions for Cmax and AUC0-∞ were 7.47-31.86 and 3.42-7.82, respectively. No sequence or period effect for Cmax and AUC0-∞ was observed. No adverse events were reported. SIGNIFICANCE: Administering CBD as a capsule rather than a liquid allows for more precise determination of pharmacokinetics parameters and is more representative of CBD swallowed products. The fat content of a meal can lead to significant increases in Cmax and AUC0-∞ and can account for variability in bioavailability and overall drug exposure within patients with oral products.

Tacrolimus troughs and genetic determinants of metabolism in kidney transplant recipients: A comparison of four ancestry groups.

Tacrolimus trough and dose requirements vary dramatically between individuals of European and African American ancestry. These differences are less well described in other populations. We conducted an observational, prospective, multicenter study from which 2595 kidney transplant recipients of European, African, Native American, and Asian ancestry were studied for tacrolimus trough, doses, and genetic determinants of metabolism. We studied the well-known variants and conducted a CYP3A4/5 gene-wide analysis to identify new variants. Daily doses, and dose-normalized troughs were significantly different between the four groups (P < .001). CYP3A5*3 (rs776746) was associated with higher dose-normalized tacrolimus troughs in all groups but occurred at different allele frequencies and had differing effect sizes. The CYP3A5*6 (rs10264272) and *7 (rs413003343) variants were only present in African Americans. CYP3A4*22 (rs35599367) was not found in any of the Asian ancestry samples. We identified seven suggestive variants in the CYP3A4/5 genes associated with dose-normalized troughs in Native Americans (P = 1.1 × 10-5 -8.8 × 10-6 ) and one suggestive variant in Asian Americans (P = 5.6 × 10-6 ). Tacrolimus daily doses and dose-normalized troughs vary significantly among different ancestry groups. We identified potential new variants important in Asians and Native Americans. Studies with larger populations should be conducted to assess the importance of the identified suggestive variants.

Analysis of 75 Candidate SNPs Associated With Acute Rejection in Kidney Transplant Recipients: Validation of rs2910164 in MicroRNA MIR146A.

BACKGROUND: Identifying kidney allograft recipients who are predisposed to acute rejection (AR) could allow for optimization of clinical treatment to avoid rejection and prolong graft survival. It has been hypothesized that a part of this predisposition is caused by the inheritance of specific genetic variants. There are many publications reporting a statistically significant association between a genetic variant, usually in the form of a single-nucleotide polymorphism (SNP), and AR. However, there are additional publications reporting a lack of this association when a different cohort of recipients is analyzed for the same single-nucleotide polymorphism. METHODS: In this report, we attempted to validate 75 common genetic variants, which have been previously reported to be associated with AR, using a large kidney allograft recipient cohort of 2390 European Americans and 482 African Americans. RESULTS: Of those variants tested, only 1 variant, rs2910164, which alters the expression of the microRNA MIR146A, was found to exhibit a significant association within the African American cohort. Suggestive variants were found in the genes CTLA and TLR4. CONCLUSIONS: Our results show that most variants previously reported to be associated with AR were not validated in our cohort. This shows the importance of validation when reporting the associations with complex clinical outcomes such as AR. Additional work will need to be done to understand the role of MIR146A in the risk of AR in kidney allograft recipients.

Genetic Variants Associated With Immunosuppressant Pharmacokinetics and Adverse Effects in the DeKAF Genomics Genome-wide Association Studies.

BACKGROUND: The immunosuppressants tacrolimus and mycophenolate are important components to the success of organ transplantation, but are also associated with adverse effects, such as nephrotoxicity, anemia, leukopenia, and new-onset diabetes after transplantation. In this report, we attempted to identify genetic variants which are associated with these adverse outcomes. METHODS: We performed a genome-wide association study, using a genotyping array tailored specifically for transplantation outcomes containing 722 147 single nucleotide polymorphisms, and 2 cohorts of kidney allograft recipients-a discovery cohort and a confirmation cohort-to identify and then confirm genetic variants associated with immunosuppressant pharmacokinetics and adverse outcomes. RESULTS: Several genetic variants were found to be associated with tacrolimus trough concentrations. We did not confirm variants associated with the other phenotypes tested although several suggestive variants were identified. CONCLUSIONS: These results show that adverse effects associated with tacrolimus and mycophenolate are complex, and recipient risk is not determined by a few genetic variants with large effects with but most likely are due to many variants, each with small effect sizes, and clinical factors.

Identification of genetic variants associated with tacrolimus metabolism in kidney transplant recipients by extreme phenotype sampling and next generation sequencing.

An extreme phenotype sampling (EPS) model with targeted next-generation sequencing (NGS) identified genetic variants associated with tacrolimus (Tac) metabolism in subjects from the Deterioration of Kidney Allograft Function (DeKAF) Genomics cohort which included 1,442 European Americans (EA) and 345 African Americans (AA). This study included 48 subjects separated into 4 groups of 12 (AA high, AA low, EA high, EA low). Groups were selected by the extreme phenotype of dose-normalized Tac trough concentrations after adjusting for common genetic variants and clinical factors. NGS spanned > 3 Mb of 28 genes and identified 18,661 genetic variants (3961 previously unknown). A group of 125 deleterious variants, by SIFT analysis, were associated with Tac troughs in EAs (burden test, p = 0.008), CYB5R2 was associated with Tac troughs in AAs (SKAT, p = 0.00079). In CYB5R2, rs61733057 (increased allele frequency in AAs) was predicted to disrupt protein function by SIFT and PolyPhen2 analysis. The variants merit further validation.

Simultaneous Quantification of 13 Cannabinoids and Metabolites in Human Plasma by Liquid Chromatography Tandem Mass Spectrometry in Adult Epilepsy Patients.

BACKGROUND: A sensitive, robust method was developed and validated to quantitate 13 major natural cannabinoid parent and metabolite compounds in human plasma at or below 0.5 ng/mL. METHODS: A liquid chromatography tandem mass spectrometry method was developed and validated to measure 13 cannabinoid compounds: cannabidiol (CBD), cannabidiolic acid, cannabidivarin, cannabinol, cannabigerol, cannabigerolic acid, cannabichromene, Δ-tetrahydocannabinol (THC), Δ-tetrahydrocannabinolic acid A (THCA), Δ-tetrahydrocannabivarin (THCV), 11-hydroxy-Δ-tetrahydrocannbinol (11-OH-THC), 11-nor-9-carboxy-Δ-tetrahydrocannbinol (THC-COOH), and 11-nor-9-carboxy-Δ-tetrahydrocannabinol glucuronide (THC-COOH-glu). Samples (200 µL) were extracted through protein precipitation and separated with a Kinetex EVO C18 column and a 65%-95% gradient of methanol and 0.2% ammonium hydroxide/H2O at a flow rate of 0.4 mL/min. Samples were obtained from patients with epilepsy receiving cannabis for the treatment of seizures. RESULTS: The extracted lower limit of quantification was 0.05 ng/mL for CBD, cannabidivarin, cannabinol, and 11-OH-THC; 0.10 ng/mL for cannabidiolic acid, cannabigerol, cannabichromene, cannabigerolic acid, THC, THCA, and THCV; and 0.50 ng/mL for THC-COOH and THC-COOH-glu. Mean quality control intraday accuracy and precision for all analytes ranged 96.5%-104% and 2.7%-4.9%, respectively, whereas interday accuracy and precision ranged 98%-103.3% and 0.2%-3.6%, respectively. An absolute matrix effect was observed for some analytes, however, with minimal relative matrix effect. Lack of nonspecific drug binding to extraction glass and plasticware was verified. Patient CBD levels ranged from 0.135 to 11.13 ng/mL. CONCLUSIONS: The validated method met FDA guidelines for bioanalytical assays precision and accuracy criteria. The assay reliably confirmed the use of particular medical cannabis formulations in patient samples as well as reliably measured low CBD concentrations from single-dose CBD exposure.

Tacrolimus trough and dose intra-patient variability and CYP3A5 genotype: Effects on acute rejection and graft failure in European American and African American kidney transplant recipients.

BACKGROUND: Suboptimal immunosuppression after kidney transplantation contributes to toxicity and loss of efficacy. Little is known regarding the impact of intra-patient variability of tacrolimus (TAC) doses and troughs in the early post-transplant period or the influence of genetic variants on variability. METHODS: Coefficients of variation (CV) of TAC troughs and doses of 1226 European American (EA) and 246 African American (AA) adult recipients enrolled in DeKAF Genomics were compared for association with acute rejection and graft failure. Additionally, the influence of recipients' number of CYP3A5 loss-of-function alleles was assessed. RESULTS: Acute rejection was associated with greater CV of dose in AA (P < 0.001) and EA recipients (P = 0.012). Graft failure was associated with a greater CV of dose (P = 0.022) and trough (P < 0.001) in AA, and higher CV of trough (P = 0.024) in EA recipients. In EA, CYP3A5 loss-of-function alleles were associated with decreased CV of trough (P = 0.0042) and increased CV of dose (P < 0.0001). CONCLUSION: CYP3A5 loss-of-function alleles influence intra-patient TAC trough and dose variability. High variability of TAC dose increases risk of acute rejection. High variability of TAC trough increases risk of graft failure. Early clinical recognition of TAC dose and trough variability may improve patient management and outcomes.

Urinary microbiome associated with chronic allograft dysfunction in kidney transplant recipients.

BACKGROUND: We performed a study to identify differences in the urinary microbiome associated with chronic allograft dysfunction (CAD) and compared the urinary microbiome of male and female transplant recipients with CAD. METHODS: This case-control study enrolled 67 patients within the Deterioration of Kidney Allograft Function (DeKAF) Genomics cohort at two transplant centers. CAD was defined as a greater than 25% rise in serum creatinine relative to a 3 month post-transplant baseline. Urine samples from patients with and without CAD were analyzed using 16S V4 bacterial ribosomal DNA sequences. RESULTS: Corynebacterium was more prevalent in female and male patients with CAD compared to non-CAD female patients (P = 0.0005). A total 21 distinct Operational Taxonomic Unit (OTUs) were identified as significantly different when comparing CAD and non-CAD patients using Kruskal-Wallis (P < 0.01). A subset analysis of female patients with CAD compared to non-CAD females identified similar differentially abundant OTUs, including the genera Corynebacterium and Staphylococcus (Kruskal-Wallis; P = 0.01; P = 0.004, respectively). Male CAD vs female CAD analysis showed greater abundance of phylum Proteobacteria in males. CONCLUSION: There were differences in the urinary microbiome when comparing female and male CAD patients with their female non-CAD counterparts and these differences persisted in the subset analysis limited to female patients only.

Lamotrigine pharmacokinetics following oral and stable-labeled intravenous administration in young and elderly adult epilepsy patients: Effect of age.

OBJECTIVE: The objectives of this study were to investigate the effect of age on pharmacokinetic parameters of lamotrigine (LTG) and estimate parameter variability. METHODS: Patients (>18 years old) who were already on a steady-state dose of LTG therapy with no interacting comedications were enrolled. Patients with significant cardiac disease, severe kidney dysfunction, or moderate-to-severe liver dysfunction were excluded. Fifty milligrams of a stable-labeled intravenous LTG formulation (SL-LTG) replaced 50 mg of a patient's normal daily oral LTG dose. Thirteen blood samples were collected in each person over 96 hours. SL-LTG and unlabeled LTG concentrations were measured simultaneously by gas chromatography-mass spectrometry. Concentration-time data were analyzed by nonlinear mixed-effects modeling (NONMEM version 7.3). RESULTS: Twenty-eight patients representing 16 young (18-48 years old) and 12 elderly (63-87 years old) patients were included, yielding 382 unlabeled and 351 SL-LTG concentrations. A two-compartment model with first-order absorption and elimination adequately described the plasma concentration-time data. Bioavailability of oral LTG was approximately 74% and did not differ by age. LTG clearance was 27.2% lower in elderly than in young patients (1.80 L/h for a 70-kg patient). SIGNIFICANCE: Although LTG bioavailability was not affected by age, LTG clearance was 27.2% lower in elderly versus young patients of comparable body weight, possibly indicating lower dosages being needed in this population.

Attempted validation of 44 reported SNPs associated with tacrolimus troughs in a cohort of kidney allograft recipients.

AIM: Multiple genetic variants have been associated with variation in tacrolimus (TAC) trough concentrations. Unfortunately, additional studies do not confirm these associations, leading one to question if a reported association is accurate and reliable. We attempted to validate 44 published variants associated with TAC trough concentrations. MATERIALS & METHODS: Genotypes of the variants in our cohort of 1923 kidney allograft recipients were associated with TAC trough concentrations. RESULTS: Only variants in CYP3A4 and CYP3A5 were significantly associated with variation in TAC trough concentrations in our validation. CONCLUSION: There is no evidence that common variants outside the CYP3A4 and CYP3A5 loci are associated with variation in TAC trough concentrations. In the future rare variants may be important and identified using DNA sequencing.

Concepts of Genomics in Kidney Transplantation.

PURPOSE OF REVIEW: Identification of genetic variants to aid in individualized treatment of solid organ allograft recipients would improve graft survival. We will review the current state of knowledge for associations of variants with transplant outcomes. RECENT FINDINGS: Many studies have yet to exhibit robust and reproducible results, however, pharmacogenomic studies focusing on cytochrome P450 (CYP) enzymes, transporters and HLA variants have shown strong associations with outcomes and have relevance towards drugs used in transplant. Genome wide association study data for the immunosuppressant tacrolimus have identified multiple variants in the CYP3A5 gene associated with trough concentrations. Additionally, APOL1 variants had been shown to confer risk to the development of end stage renal disease in African Americans. SUMMARY: The field is rapidly evolving and new technology such as next generation sequencing, along with larger cohorts, will soon be commonly applied in transplantation to understand genetic association with outcomes and personalized medicine.

CRISPR/Cas9 Genetic Modification of CYP3A5 *3 in HuH-7 Human Hepatocyte Cell Line Leads to Cell Lines with Increased Midazolam and Tacrolimus Metabolism.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 engineering of the CYP3A5 *3 locus (rs776746) in human liver cell line HuH-7 (CYP3A5 *3/*3) has led to three CYP3A5 *1 cell lines by deletion of the exon 3B splice junction or point mutation. Cell lines CYP3A5 *1/*3 sd (single deletion), CYP3A5 *1/*1 dd (double deletion), or CYP3A5 *1/*3 pm (point mutation) expressed the CYP3A5 *1 mRNA and had elevated CYP3A5 mRNA (P < 0.0005 for all engineered cell lines) and protein expression compared with HuH-7. In metabolism assays, HuH-7 had less tacrolimus (all P < 0.05) or midazolam (MDZ) (all P < 0.005) disappearance than all engineered cell lines. HuH-7 had less 1-OH MDZ (all P < 0.0005) or 4-OH (all P < 0.005) production in metabolism assays than all bioengineered cell lines. We confirmed CYP3A5 metabolic activity with the CYP3A4 selective inhibitor CYP3CIDE. This is the first report of genomic CYP3A5 bioengineering in human cell lines with drug metabolism analysis.

Multigene predictors of tacrolimus exposure in kidney transplant recipients.

AIM: Determine the effect of the genetic variants beyond CYP3A5*3 on tacrolimus disposition. PATIENTS & METHODS: We studied genetic correlates of tacrolimus trough concentrations with POR*28, CYP3A4*22 and ABCC2 haplotypes in a large, ethnically diverse kidney transplant cohort (n = 2008). RESULTS: Subjects carrying one or more CYP3A5*1 alleles had lower tacrolimus trough concentrations (p = 9.2 × 10(-75)). The presence of one or two POR*28 alleles was associated with a 4.63% reduction in tacrolimus trough concentrations after adjusting for CYP3A5*1 and clinical factors (p = 0.037). In subset analyses, POR*28 was significant only in CYP3A5*3/*3 carriers (p = 0.03). The CYP3A4*22 variant and the ABBC2 haplotypes were not associated. CONCLUSION: This study confirmed that CYP3A5*1 was associated with lower tacrolimus trough concentrations. POR*28 was associated with decreased tacrolimus trough concentrations although the effect was small possibly through enhanced CYP3A4 enzyme activity. CYP3A4*22 and ABCC2 haplotypes did not influence tacrolimus trough concentrations. Original submitted 19 December 2014; Revision submitted 2 April 2015.