Wild-type CYP3A5 Research Articles

A descriptive study of the single-nucleotide polymorphisms known to affect the Tacrolimus trough concentration per dose, among a population of kidney failure patients in a tertiary hospital in Ghana

BackgroundThe burden of chronic kidney disease (CKD) and kidney failure in Ghana is on the ascendency, with the prevalence of CKD estimated at 13.3%. Patients with CKD who progress to kidney failure require life sustaining kidney replacement therapy (KRT) which is almost exclusively available in Ghana as haemodialysis. Kidney transplantation is considered the best KRT option for patients with irreversible kidney failure due to its relative cost efficiency as well as its superiority in terms of survival and quality of life. However, because transplants may trigger an immune response with potential organ rejection, immunosuppressants such as tacrolimus dosing are required.ObjectiveThis study sought to determine single nucleotide polymorphisms in CYP3A5, CYP3A4 and MDR1 genes that affect the pharmacokinetics of Tacrolimus in a population of Ghanaian patients with kidney failure.MethodThis cross-sectional study comprised of 82 kidney failure patients undergoing maintenance haemodialysis at the Renal and Dialysis unit of Korle-Bu Teaching Hospital (KBTH). Clinical and demographic data were collected and genomic DNA isolated. Samples were genotyped for specific SNPs using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP).ResultsParticipants, 58/82 (70.73%) harbored the wildtype CYP3A5*1/*1 AA genotype, 20/82 (24.39%) carried the heterozygous CYP3A5*1/*3 AG genotype, and 4/82 (4.88%) had the homozygous mutant CYP3A5*3/*3 GG genotype. Also, 6/82 (7.32%) carried the wildtype AA genotype, 11/82 (13.41%) had the heterozygous AG genotype, and 65/82 (79.27%) harbored the homozygous mutant GG genotype of CYP3A4*1B (-290 A>G). For MDR1_Ex21 (2677 G>T), 81/82 (98.78%) carried the wildtype GG genotype, while 1/82 (1.22%) had the heterozygous GT genotype. For MDR1_Ex26 (3435 C>T), 63/82 (76.83%) had the wildtype CC genotype, while 18/82 (21.95%) carried the heterozygous CT genotype, and 1/82 (1.22%) harbored the mutant TT genotype.ConclusionSNPs in CYP3A4, CYP3A5, and MDR1 genes in a population of Ghanaian kidney failure patients were described. The varying SNPs of the featured genes suggest the need to consider the genetic status of Ghanaians kidney failure patients prior to transplantation and tacrolimus therapy.

The impact of CYP3A5*3 on oral quetiapine: A population pharmacokinetic model in Chinese bipolar disorder patients

BackgroundThere is great interindividual difference in the plasma concentration of quetiapine, and optimizing quetiapine therapy to achieve a balance between efficacy and safety is still a challenge. In our study, a population pharmacokinetic (PPK) model considering genetic information was developed with the expectation of comprehensively explaining this observation in Chinese patients with bipolar disorder. MethodsPatients who were dispensed quetiapine and underwent the therapeutic drug monitoring (TDM) were included. The genotypes of CYP3A5*3, CYP2D6*10, and ABCB1 C3435T/G2677T were analyzed. Finally, a multivariable linear regression model was applied to describe the PPK of quetiapine considering the covariates weight, height and genotype information. ResultsA total of 175 TDM points from 107 patients were adopted for PPK model development. Resultantly, the CL/F of quetiapine in CYP3A5 expressers was 81.1 CL/h, whereas it was 43.6 CL/h in CYP3A5 nonexpressers. The interindividual variability in CL/F was 47.7 %. However, neither the ABCB1 nor CYP2D6 genotype was significantly associated with the predictor of quetiapine clearance in our study. LimitationsOnly trough concentrations were collected, and the span between different points was relatively wide, impeding the application of the typical nonlinear compartment model for PPK analysis. In addition, this was a single-center study which limited the sample of wild-type CYP3A5 carriers. ConclusionsThe currently established PPK model of quetiapine considering the contribution of the CYP3A5 genotype could efficiently predict the population and individual pharmacokinetic parameters of Chinese bipolar disorder patients, which could better guide the personalized therapy with quetiapine, thus to achieve the best clinical response.

Abstract 1409: RNA sequencing reveals that African Americans carrying wild-type CYP3A5 differentially express genes known to promote aggressive prostate cancer

Abstract Background: African American men (AAs) have the highest incidence of prostate cancer (PC) and often develop therapeutic resistance leading to high mortality. The androgen receptor (AR) is paramount in the growth and progression of PC, and insufficient blockade of AR leads to therapeutic resistance. Our previous work shows that CYP3A5 facilitates the activation of AR, promoting the transcription of genes supporting PC growth. CYP3A5 is polymorphic, and the wild-type (wt; *1) variant encoding the full-length active protein is expressed in 73% of AAs and only in 5% of non-Hispanic White Americans (NHWA). The race-linked expression of wt-CYP3A5*1 in AAs can lead to hyperactive AR contributing to therapeutic resistance. We performed an RNA sequencing study utilizing samples from AAs and NWHA patients to understand the allelic influence of CYP3A5 on PC severity. Methods: Illumina RNA sequencing was performed using RNA from 13 AA and 12 NHWA prostatic adenocarcinomas. CYP3A5 genotyping separated them into two groups: a) carrying wt-CYP3A5 (*1/*1 and *1/*3; N=14); and b) mutant CYP3A5 (*3/*3; N=11) variants. The samples’ ancestry was confirmed using markers previously described. Differentially expressed genes (DEGs) between wt-*1 and mutant-*3 CYP3A5 carrying groups were identified using DESeq2, at a false discovery rate of adjusted p < 0.05. Alternately we also used the Kallisto method for quantifying the abundances of the transcript from RNA seq data. Results: Aligned RNA reads with the human genome using DESeq2 resulted in the identification of 30 DEGs between the two groups, of these, 14 genes are known to be dysregulated in PC and promote aggressive disease (SEMG2, ZNRF3, CCL18, U3, MT-ND6, SCARNA9, NRCAM, LINC00853, TCF4, SLC38A6, HHLA3, DECR1, THTPA, and NEFH). Aberrant TCF4 along with ZNRF3 is known to promote PC growth via Wnt-beta catenin signaling. SLC38A6 is a glutamine transporter and is often upregulated in PC to promote glutamine uptake in an AR-dependent manner. NEFH is an intermediate filament protein that is downregulated in PC metastasis. Kallisto method identified 84 DEGs, 11 of those genes overlap with the previous DFGs identified using the transcriptome alignment method. The additional DFGs identified with the Kallisto method include aberrant expression of multiple genes (MT-CO1/2, MT-ND1/6) involved in mitochondrial dysfunction known to contribute towards cell growth and tumorigenesis beyond the Warburg effect. The wt-*1 CYP3A5 expressing group also shows increased expression of several pseudogenes, dysregulation of pseudogenes has been observed in several cancers and can act as a master regulator for gene expression, promoting tumorigenesis. Conclusion: The presence of wt-*1 CYP3A5 may be one of the factors leading to aggressive PC in AAs as it shows differential expression of several genes known to accelerate PC growth. Citation Format: Jeetesh Sharma, Richard Tillett, Shirley Shen, Jabril Johnson, Rick A. Kittles, Mohammad Saleem Bhat, Oscar Goodman Jr, Edwin C. Oh, Ranjana Mitra. RNA sequencing reveals that African Americans carrying wild-type CYP3A5 differentially express genes known to promote aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1409.

Cytochrome P450 oxidoreductase variant A503V contributes to the increased CYP3A5 activity with tacrolimus in vitro

ABSTRACT Background Tacrolimus is a immunosuppressant drug in transplantation with a narrow therapeutic range and high pharmacokinetic variability. Clinical studies have revealed that POR*28 contributes enhanced tacrolimus clearance in CYP3A5 expressers. However, it remains unknown that how exactly the POR polymorphism could influence the metabolism of tacrolimus via CYP3A5 in vitro. Research design & methods A503V is an amino acid sequence variant encoded by POR*28. Wild-type (WT) and A503V POR, with WT CYP3A5 were expressed in recombinant HepG2 cells and reconstituted proteins. Michaelis constant (K m) and maximum velocity (V max) of CYP3A5 with tacrolimus as substrates were determined, and catalytic efficiency is expressed as V max/K m. Results Both WT and A503V POR down-regulated the CYP3A5 mRNA expression, and WT POR rather than A503V down-regulated the protein expression of CYP3A5 in recombinant HepG2 cells. Compared with WT POR, A503V increased metabolism of tacrolimus by CYP3A5 in both cellular and protein levels. Conclusion A503V can affect CYP3A5-catalyzed tacrolimus metabolism in vitro, which suggests that A503V has the potential to serve as a biomarker for tacrolimus treatment in transplantation recipients.

Abstract 5414: RNA sequencing reveals that CYP3A5 siRNA treatment induces cell cycle blockade and senescence in MDAPCa2b human prostate cancer cells

Abstract Background: Previously we have demonstrated that CYP3A5 inhibition blocks prostate cancer (PC) cell growth partly by attenuating androgen receptor (AR) activation and downstream signaling. We further investigated whether CYP3A5 inhibition blocks PC cell growth independently of AR signaling. Initially, most PCs respond to androgen deprivation therapy (ADT), but eventually PC becomes castration resistant and does not respond to ADT. With very limited options available for patients with castration resistant prostate cancer (CRPC), there is an urgent need for the development of novel targeted therapies for this subtype of PC. Additionally, understanding the role of CYP3A5 as promoter of PC growth in African Americans (AA) is very relevant as AAs preferentially carry wild type CYP3A5 gene, often present with aggressive disease, and have higher mortality rate versus Caucasian (CA) patients, who typically express the mutant CYP3A5. Method: We used MDAPCa2b PC cell line of AA origin carrying one copy of wild type CYP3A5 for our study. After inhibiting the CYP3A5 using siRNA pool of 3 siRNAs, we performed Illumina RNA sequencing. Differentially expressed genes (DEGs) between non-target and CYP3A5 siRNA triplicate samples were identified using DESeq2, at a false discovery rate of adjusted p < 0.05. Furthermore, KEGG pathway analysis was performed to identify the roles of the differentially expressed genes in regulating prostate cancer growth. Results: We identified 1814 DEGs after inhibiting CYP3A5 using siRNA pool in MDAPCa2b cells, 825 of which increased or decreased expression greater than two-fold. The differentially down-regulated genes associate with several growth regulatory pathways: cell cycle progression (39), cellular senescence (16) DNA replication (18) and base excision repair (9). Additionally, we identified that CYP3A5 siRNA downregulates several genes which are dysregulated (including oncogenes and tumor suppressors) in different cancer type including RAS, c-Met, ERBB2, FGFR2, cyclin E, p53, Myc, PTEN, RARβ and CDK2. The upregulated genes correspond to two pathways, retinol metabolism pathway (arrests proliferation) and steroid hormone biosynthesis. We also identified several unique genes which are down regulated with CYP3A5 siRNA treatment like Glo1, FoxM1 (often upregulated in several cancers) and Tk1 (marker for chemotherapy sensitivity). Conclusion: CYP3A5 siRNA inhibits several genes involved in cell growth and proliferation. Targeting CYP3A5 represents a potential novel therapeutic approach, especially for AAs expressing wild type CYP3A5. Citation Format: Richard L. Tillett, Shirley Shen, Edwin C. Oh, Oscar B. Goodman Jr., Ranjana Mitra. RNA sequencing reveals that CYP3A5 siRNA treatment induces cell cycle blockade and senescence in MDAPCa2b human prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5414.

Bergamottin a CYP3A inhibitor found in grapefruit juice inhibits prostate cancer cell growth by downregulating androgen receptor signaling and promoting G0/G1 cell cycle block and apoptosis.

Prostate cancer is the second leading cause of cancer related death in American men. Several therapies have been developed to treat advanced prostate cancer, but these therapies often have severe side effects. To improve the outcome with fewer side effects we focused on the furanocoumarin bergamottin, a natural product found in grapefruit juice and a potent CYP3A inhibitor. Our recent studies have shown that CYP3A5 inhibition can block androgen receptor (AR) signaling, critical for prostate cancer growth. We observed that bergamottin reduces prostate cancer (PC) cell growth by decreasing both total and nuclear AR (AR activation) reducing downstream AR signaling. Bergamottin’s role in reducing AR activation was confirmed by confocal microscopy studies and reduction in prostate specific antigen (PSA) levels, which is a marker for prostate cancer. Further studies revealed that bergamottin promotes cell cycle block and accumulates G0/G1 cells. The cell cycle block was accompanied with reduction in cyclin D, cyclin B, CDK4, P-cdc2 (Y15) and P-wee1 (S642). We also observed that bergamottin triggers apoptosis in prostate cancer cell lines as evident by TUNEL staining and PARP cleavage. Our data suggests that bergamottin may suppress prostate cancer growth, especially in African American (AA) patients carrying wild type CYP3A5 often presenting aggressive disease.

P546 The safety and efficacy of the new-generation budesonide-MMX in the aspect of the cytochrome P-450 enzyme genotype

Abstract Background Unlike previous forms of budesonide absorbing from the ileal and ascending colon region, the new-generation budesonide-MMX contains a formula, that allows absorption throughout the whole colon. Budesonide is degraded in the liver by cytochrome P450 3A enzyme, but so far, there is no study examining the relationship between the budesonide’s effect and the enzyme activity. CYP3A5 is absent in 90% of the European/caucasian population due to a functional loss mutation (CYP3A5*3), whereas patients with the wild-type CYP3A5*1 allele may be expected to have increased metabolism. The most common genetic polymorphisms in CYP3A4 (CYP3A4*1B and CYP3A4*22) result in increased and decreased expression, respectively. Methods We enrolled 33 patients with UC in this prospective study until January of 2021. Patients received 9 mg oral budesonide-MMX once daily until 8 weeks. Laboratory parameters (cholesterol, triglyceride, CRP) and serum hormone levels (parathormone, dehydroepiandrosterone and cortisol) were monitored before and after the 8-week therapy to follow metabolic and hormonal changes. During these visits, body composition analysis was also performed with InBody 770 machine to observe the adverse effects of budesonide-MMX in respect of body fat mass, body mass index, protein content of the body and bone mineral content. We examined the CYP450 3A (CYP3A5 and CYP3A4) enzyme genotype of the patients, to see, whether the different alleles of this drug-degrading enzyme affect the efficacy and safety. Results 33 patients had received the 2-month therapy. By the end of follow-up, based on partial Mayo score, 26 (78.8%) patients experienced remission and 6 patients (18.2%) were primary non-responders. Mean pMayo score decreased from 4.18 to 1.63 (p<0.001). No significant changes were observable regarding body composition. Serum cholesterol level showed significant increase (p<0.001), while triglyceride and CRP did not show significant changes. Serum cortisol levels were decreased (p<0.001), while PTH and DHEA showed no significant decrease. Only two patients experienced side effects: one of them hypertonia, headache and acnes, while the other mild diarrhoea. 3 patients have CYP3A5*1/*3 genotype, and 16 have CYP3A5*3/*3. There was no significant difference between the two groups, regarding safety and efficacy. Only 1 patient have CYP3A4*1B genotype, while the rest have CYP3A4*1, hence, no statistics could be performed. Conclusion In our study, budesonide-MMX proved to be safe and effective in the therapy of UC, however, cholesterol was elevated in the serum. Based on our cohort, different genotypes of CYP3A don’t have an impact on the effect of the drug, however, CYP3A allele variants are rare, therefore, further examinations should be performed.

Abstract 2467: Interaction of CYP3A5 with AR-HSP90 complex regulating AR activation in prostate cancer cells

Abstract Background: Previously we have demonstrated that CYP3A5 stimulates prostate cancer cell growth by promoting AR activation and downstream signaling. We further investigated the mechanism of CYP3A5's interaction with the AR-HSP90 complex known to regulate AR activation process. AR activation is tightly regulated and it involves several chaperons (HSP90, HSP70, HSP40, and HSP27), multiple cofactors and protein kinases. We hypothesize that stoichiometric CYP3A5 binding to the HSP90-AR complex can induce conformational changes in the chaperone complex, promoting AR nuclear migration and activation. Method: We used co-immunoprecipitation to identify the interaction of CYP3A5 with the components of the AR-HSP90 complex. Immunofluorescence studies were performed to identify the components of the AR-HSP90 complex that co-localize with CYP3A5. FRET analysis was used to confirm the interactions of CYP3A5 with the AR-HSP90 complex. Results: We performed co-immunoprecipitation (co-IP) experiments with extracts from flag-tagged CYP3A5 transfected LNCaP cells. Our data demonstrates that CYP3A5 co-immunoprecipitates with HSP90, AR, HSP70 and HSP40, all known components of the HSP90-AR complex. We further confirmed using reverse immunoprecipitation with AR/ HSP90 antibodies and agarose A/G beads that CYP3A5 is a part of the HSP90-AR mature complex. Empty vector transfected cell extract and IgG were used as controls for pull down and reverse IP respectively. Immunofluorescence studies demonstrated that HSP70 and HSP40 co-localized with CYP3A5 in the cytoplasm. Interestingly, the MDAPCa2b and LNCaP cells show co-localization of CYP3A5 and AR in the nucleus, in contrast to non-transformed RWPE1 cells where CYP3A5 remains in the cytoplasm. The nuclear presence of CYP3A5 is more pronounced in MDAPCa2b cells carrying the wild type CYP3A5. The FRET analysis further confirmed direct interaction between CYP3A5 and HSP40 a chaperon in the AR-HSP90 complex. Conclusions: Our data confirms that CYP3A5 directly interacts with the AR-HSP90 complex regulating AR activation process. Further understanding of this interaction can help establish CYP3A5-AR axis as a novel therapeutic target. Citation Format: Priyatham Gorjala, Oscar Goodman Jr., Ranjana Mitra. Interaction of CYP3A5 with AR-HSP90 complex regulating AR activation in prostate cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2467.

Abstract B017: Role of CYP3A5 in modulating androgen receptor signaling and its relevance in African American prostate cancer patients

Abstract Introduction: African-American men (AA) often present with aggressive castration-resistant prostate cancer (CRPC), due to highly active androgen receptor (AR). AR is a ligand-activated transcription factor that promotes expression of genes responsible for cell proliferation and growth. Previously, we have shown that CYP3A5 promotes AR nuclear translocation and activation leading to increased prostate cancer (PC) growth. 73% of AAs carrying wild-type CYP3A5 (*1/*1) express full-length functional CYP3A5, whereas 95% of Caucasians carry mutant (*3/*3) variant producing truncated nonfunctional protein. Difference in CYP3A5 expression leads to highly active AR and aggressive disease in AAs. Additionally, most of the PC patients are prescribed concomitant medications to manage age-related comorbidities. Many of these drugs are known modulators of CYP3A5; modulation of CYP3A5 may alter efficacy of ADT in these patients. Our work is focused on characterizing the effect of CYP3A5 modulating drugs on AR signaling in AAs. Methods: q-RT-PCR based profiler assay was used to study effect of CYP3A5 modulation on AR-regulated genes using cDNA from nontarget (NT) and CYP3A5 siRNA treated cells. Confocal microscopy and cell fractionation assays were performed to evaluate and confirm the effect of CYP3A5 modulating drugs on AR nuclear translocation. Genes shortlisted from q-PCR based profiler assays were analyzed for change of expression in response to CYP3A5 modulating drugs. MDAPCA2b (AA origin, *1/*3) and LNCaP (Caucasian, *3/*3) cell lines were used for above experiments. Results: CYP3A5 siRNA pool treatment downregulates AR regulated genes identified using q-PCR based profiler assay, performed with cDNA from CYP3A5 siRNA pool and NT treated MDAPCA2b cells. These downregulated genes include SCL45A3, FKBP5, NCAPD3, MYC, MME, ELL2, PIK3R3, HPRT1 and SPDEF with p-value of ≤0.005. These genes are known to regulate AR nuclear translocation, cell cycle progression, and cell growth. CYP3A5 siRNA treated MDACPA2b/ LNCaP cells showed decreased AR nuclear translocation and PSA production. Commonly prescribed drugs that are either CYP inhibitors (amiodarone, ritonavir) or inducers (phenytoin, rifampicin) were tested for their ability to alter AR signaling. The results show that the CYP inducers promoted AR nuclear migration and downstream signaling whereas CYP3A5 inhibitors blocked it. Further, CYP3A5 siRNA treated MDAPCa2b cells do not show any increase in AR nuclear migration with phenytoin treatment (CYP3A inducer), confirming that the activation of AR activity is specific to changes in CYP3A5 activity. Conclusions: Concomitantly prescribed CYP3A5 modulating drugs can alter downstream AR signaling, cell growth, and ADT efficacy in men, more so in AAs expressing wild-type CYP3A5. We propose to further characterize drug-induced CYP3A5 modulation of AR signaling to develop a guideline for physicians coprescribing CYP3A5 modulating drugs to treat comorbidities in elderly patients undergoing ADT. Citation Format: Priyatham Gorjala, Oscar B. Goodman Jr, Ranjana Mitra. Role of CYP3A5 in modulating androgen receptor signaling and its relevance in African American prostate cancer patients [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B017.

Role of CYP3A5 in Modulating Androgen Receptor Signaling and Its Relevance to African American Men with Prostate Cancer.

Androgen receptor signaling is crucial for prostate cancer growth and is positively regulated in part by intratumoral CYP3A5. As African American (AA) men often carry the wild type CYP3A5 and express high levels of CYP3A5 protein, we blocked the wild type CYP3A5 in AA origin prostate cancer cells and tested its effect on androgen receptor signaling. q-PCR based profiler assay identified several AR regulated genes known to regulate AR nuclear translocation, cell cycle progression, and cell growth. CYP3A5 processes several commonly prescribed drugs and many of these are CYP3A5 inducers or inhibitors. In this study, we test the effect of these commonly prescribed CYP3A5 inducers/inhibitors on AR signaling. The results show that the CYP3A5 inducers promoted AR nuclear translocation, downstream signaling, and cell growth, whereas CYP3A5 inhibitors abrogated them. The observed changes in AR activity is specific to alterations in CYP3A5 activity as the effects are reduced in the CYP3A5 knockout background. Both the inducers tested demonstrated increased cell growth of prostate cancer cells, whereas the inhibitors showed reduced cell growth. Further, characterization and utilization of the observation that CYP3A5 inducers and inhibitors alter AR signaling may provide guidance to physicians prescribing CYP3A5 modulating drugs to treat comorbidities in elderly patients undergoing ADT, particularly AA.

Abstract 1311A: CYP3A5 regulation of androgen receptor signaling: Relevance in African American prostate cancer patients and its development as novel target

Abstract Background: Previously we demonstrated that CYP3A5 positively regulates androgen receptor (AR) signaling and growth of prostate cancer (PC) cells. CYP3A5 inhibition decreases nuclear AR localization and downregulates AR downstream signaling (e.g. PSA and TMPRSS2), resulting in decreased PC cell growth both with and without dihydrotestosterone (DHT) induction. Our current work focusses on the effect of CYP3A5 modulation on AR signaling and therapeutic resistance, mostly in African Americans (AA) widely carrying (73%) wild type CYP3A5 (*1/*1). AAs and have higher incidence of PC and double the mortality rate compared to Non-Hispanic White Americans (NHWA) carrying mutated CY3A5 gene (*3/3, 95%) and expressing truncated non-functional CYP3A5 protein. Methods: Immunoprecipitation was used to identify binding partners of AR. q-RT-PCR based profiler assays was used to detect changes in AR downstream signaling and effect on genes responsible for therapeutic resistance after CYP3A5 inhibition. Effect of CYP3A5 on AR nuclear translocation and induction of synthetic lethality in combination with PARP inhibitors were evaluated using cell fractionation and immunocytochemistry studies. Results: Co-immunoprecipitation of flag-tagged CYP3A5 transfected LNCaP cell extracts showed that CYP3A5 was in a complex with HSP90, AR, HSP70 and HSP40, also confirmed with reverse immunoprecipitation using AR/ HSP90 antibodies and agarose A/G beads. AR regulated genes (SCL45A3, FKBP5, MYC, ELL2, and MT2A) were downregulated with CYP3A5 siRNA treatment in MDA PCa 2b cells (*1/*3, AA origin) in a qPCR-based profiler assay with high fold difference and a P value of ≤0.005. PC patients are often prescribed medications for comorbidities, many of which are CYP3A modulators. Our results indicate that CYP inhibitors (amiodarone, ritonavir, fluoxetine etc.) decrease AR nuclear translocation and inducers (phenytoin, rifampicin, pioglitazone and hyperforin) increase AR nuclear translocation and activation. CYP3A5 siRNA treatment downregulates genes involved in cancer drug therapeutic resistance (TOP2A, BRCA1/2, CCNE1, CDK2/4, DHFR, MVP, MYC, RARB and HPRT1, P ≤ 0.005 ). Loss of TOP2A, BRCA2 and BRCA1is known to impair homologous recombination (HR). In combination with topoisomerase inhibitor etoposide, CYP3A5 siRNA in PC cells increases number of gamma-H2AX foci indicating synthetic lethality. Conclusion: A-Developing CYP3A5-AR-HSP90 interface as a target may provide a novel strategy to block PC growth. B-CYP3A5 modulation of AR signaling can affect the efficacy of androgen deprivation therapy specifically AA patients carrying wild type CYP3A5. C-Since CYP3A5 inhibition, downregulates DNA damage repair genes and impairs HR, CYP3A5 inhibition in combination with PARP inhibitors can be a novel strategy to induce synthetic lethality in PC patients. Citation Format: Priyatham Gorjala, Oscar B. Goodman, Ranjana Mitra. CYP3A5 regulation of androgen receptor signaling: Relevance in African American prostate cancer patients and its development as novel target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1311A.

Abstract B001: CYP3A5 promotes AR downstream signaling and contributes to therapeutic resistance

Abstract Purpose: Androgen receptor (AR) plays a major role in promoting growth and progression of prostate cancer and thus has been successfully targeted therapeutically. Therapeutic resistance to androgen deprivation therapy (ADT) accounts for much of the morbidity and mortality attributable to prostate cancer. Therapeutic resistance is typically accompanied by insufficient blockade of AR activation, whose initial step involves translocation of AR to the nucleus. In recent studies we demonstrated that CYP3A5 facilitates the nuclear translocation of AR, promoting prostate cancer growth. This work focuses on understanding the role of CYP3A5 in regulating AR nuclear translocation and therapeutic resistance. Method: To understand how CYP3A5 effects AR downstream signaling, we used RT2 profiler PCR arrays to help identify set of genes in AR signaling pathway that are affected by CYP3A5 inhibition. Confocal microscopy and cell fractionation experiments were used to further confirm the effect of CYP3A5 modulation of AR signaling. As concomitant CYP3A5 inducer/inhibitor medications can affect intratumoral CYP3A5 activity and thus may modify AR downstream signaling, we utilized cell fractionation and confocal microscopy to analyze the effect of these medications on AR nuclear translocation. Further, to decipher the role of CYP3A5 in the AR translocation process we performed a series of co-immunoprecipitation (co-IP) experiments with CYP3A5 and components of HSP90-AR complex. We used extracts of LNCaP transfected with a CYP3A5-Flag-tagged construct for IP, with either Flag-agarose or HSP90 and AR antibodies with A/G beads. Finally, to decipher CYP3A5’s role in therapeutic resistance we analyzed cDNA after CYP3A5 siRNA treatment using drug resistance RT2 profiler PCR array. Results: The q-PCR based profiler assay revealed a set of nine AR regulated genes (SCL45A3, FKBP5, NCAPD3, MYC, MME, ELL2, PIK3R3, HPRT1, and SPDEF) downregulated with CYP3A5 siRNA treatment with a p value of ≤0.005. MYC and PIK3R3 are known to be involved in regulating cell growth by either affecting cell cycle progression and/or apoptosis. PSA, known to aid in the cell growth, is regulated by SPDEF, a transcription factor. Downregulation of these genes with CYP3A5 siRNA treatment indicates that CYP3A5 modifier medications can alter AR downstream signaling regulating growth. Concomitantly prescribed CYP3A inhibitors amiodarone and ritonavir blocked AR nuclear migration and decreased PSA expression whereas inducers phenytoin and rifampicin promoted increased AR nuclear migration and increased PSA production, supporting our hypothesis. Co-immunoprecipitation experiments revealed that CYP3A5 is a part of the HSP90-AR complex. Both Flag-immunoprecipitation and indirect immunoprecipitation revealed that CYP3A5 associates with the HSP90-AR complex. The CYP3A5 siRNA also downregulated 11 genes (TOP2A, BRCA1/2, CCNE1, CDK2/4, DHFR, MVP, MYC, RARB, and HPRT1) known to confer drug resistance, with a p value of ≤0.005. The role of TOP2A, MYC, and BRCA is well known in prostate cancer drug resistance; thus, their downregulation with CYP3A5 siRNA treatment may predict better outcome. Conclusion: Our results indicate that CYP3A5 is a part of the HSP90-AR complex and positively regulates AR nuclear translocation promoting AR downstream signaling and therapeutic resistance. Therefore, targeting CYP3A5 to block AR nuclear translocation may be a therapeutic adjunctive approach in advanced prostate cancer. As CYP3A5 siRNA also downregulates drug-resistance genes, targeting CYP3A5 may also help overcome therapeutic resistance. As many concomitant medications can alter CYP3A5 activity, thus they can modify AR signaling. We hypothesize that AR directed therapeutic efficacy can be optimized by proper drug selection through minimizing the use of AR inducers while maximizing the use of AR inhibitors. These observations may play a significant role in African Americans who express high level of wild-type CYP3A5 and often have aggressive prostate cancer. Citation Format: Priyatham Gorjala, Oscar B. Goodman Jr., Ranjana Mitra. CYP3A5 promotes AR downstream signaling and contributes to therapeutic resistance [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B001.

Abstract B48: Effect of concomitant CYP3A5-modulating drugs on androgen-receptor signaling in African American men undergoing androgen deprivation therapy

Abstract Introduction: In a recent study we have established that CYP3A5 augments nuclear translocation of androgen receptor (AR) and promotes prostate cancer (PC) growth. CYP3A isoforms are responsible for metabolizing almost 50% of the commonly prescribed drugs, many of which are CYP3A5 inhibitors and inducers. PC patients are elderly with comorbidities and are often prescribed concomitant medications while undergoing androgen-deprivation therapy (ADT), the most preferred treatment for PC. CYP3A5 is polymorphic, and African Americans (AA, 73%) express higher levels of CYP3A5 due to presence of wild-type CYP3A5 (*1/*1) compared to Caucasians (95%) expressing the mutant (*3/*3) producing only 13% of the active full-length CYP3A5 protein. Due to the expression of wild-type CYP3A5 in AA, concomitant medications modulating CYP3A5 activity can drastically alter AR activity and ADT efficacy in these patients. Our current work is focused on deciphering the mechanism of how commonly used CYP3A5-modulating medications can alter AR signaling in AA carrying the wild-type CYP3A5. Method: We genotyped the androgen-responsive prostate cancer cell lines to determine their CYP3A5 status using TaqMan® Drug Metabolism assay. We used cDNA from cells treated with either nontarget or CYP3A5 siRNA in RT2 profiler PCR arrays to identify set of genes in AR signaling pathway that are affected by CYP3A5 inhibition. Confocal microscopy was used to study AR nuclear localization after exposure to commonly used CYP3A5 inducer/inhibitor drugs. Cell fractionation experiments were used to further confirm the effect of these drugs on AR signaling. Luciferase-based reporter assay was used to test effect of CYP3A5-modulating drugs on AR activity. Results: Our genotyping assay revealed that MDA PCa 2b (African American) carries *1/*3 CYP3A5 gene compared to LNCaP (Caucasian) carrying *3/*3 genotype. To compare the differences due to CYP3A5 genotype on AR signaling, we selected MDAPCa2b and LNCaP cells for further studies. Consistent with earlier observations with LNCaP, MDA PCa 2b cell lines also showed decreased nuclear translocation of AR with CYP3A5 siRNA in both confocal and cell fractionation experiments. We also observed blocking of AR nuclear localization with azamulin, a CYP3A-specific inhibitor in both cell lines. The q-PCR-based profiler assay revealed a set of nine AR-regulated genes (SCL45A3, FKBP5, NCAPD3, MYC, MME, ELL2, PIK3R3, HPRT1, and SPDEF) downregulated with CYP3A5 siRNA treatment with a p value of ≤0.005 in MDA PCa 2b cells. MYC is known to be involved in cell cycle progression and apoptosis whereas PIK3R3 codes for the regulatory subunit of PI3 kinase regulating cell growth. SPDEF codes for a transcription factor promoting PSA known to aid in the cell growth. Downregulation of these genes with CYP3A5 siRNA treatment indicates that CYP3A5 modifier medications can alter AR downstream signaling regulating growth. We tested some commonly prescribed drugs that are either CYP inhibitors (amiodarone, ritonavir) or inducers (phenytoin, rifampicin) and evaluated their ability to alter AR signaling. The results revealed that the CYP inducers promoted increased AR nuclear migration and increased PSA production whereas CYP3A5 inhibitors blocked AR nuclear migration, decreasing PSA expression in both cell lines. A luminescence-based reporter assay using androgen response elements also supported that these CYP3A inducer and inhibitor drugs alter AR signaling. Conclusion: Our results indicate that concomitant medications that can alter CYP3A5 activity can modify AR signaling and PC growth. These results suggest that these CYP-modulating drugs can alter efficacy of ADT severely in AA men predominantly expressing wild-type CYP3A5 (*1/*1). We hypothesize that AR-directed therapeutic efficacy can be optimized by proper drug selection through minimizing the use of AR inducers while maximizing the use of AR inhibitors in patients undergoing ADT. Citation Format: Priyatham Gorjala, Oscar B. Goodman Jr., Ranjana Mitra. Effect of concomitant CYP3A5-modulating drugs on androgen-receptor signaling in African American men undergoing androgen deprivation therapy [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr B48.

Effect of CYP3A5 Genotypes on the Pharmacokinetics of Carbamazepine when used as Monotherapy or Co-Administered with Phenytoin, Phenobarbital or Valproic Acid in Thai Patients

To determine the effects of CYP3A5 polymorphisms on carbamazepine (CBZ) pharmacokinetic parameters when CBZ is used either as monotherapy or co-administered with phenytoin (PHT), phenobarbital (PB) or valproic acid (VPA). Retrospective data were collected from an electronic database and medical records. Blood samples were obtained and drug concentrations analyzed as a part of routine therapeutic drug monitoring (TDM). Screening for wild-type CYP3A5*1 and CYP3A5*3 single nucleotide polymorphism (rs776746) by allelic discrimination assay using real-time polymerase chain reaction technique (real-time PCR) was performed. Pharmacokinetic parameters of CBZ; clearance and dose-adjusted CBZ levels in patients with different genotypes were calculated and compared. Of the 70 patients assessed, 8 (11%) patients were homozygous CYP3A5*1/*1, 28 (40%) patients were heterozygous CYP3A5*1/*3, and 34 (49%) patients were homozygous CYP3A5*3/*3. The CBZ clearance and dose-adjusted CBZ levels did not significantly differ between patients with CYP3A5*1 and CYP3A5*3 alleles when CBZ was used as monotherapy. For patients who used CBZ in combination with an enzyme-inducing antiepileptic drug (AED: PHT or PB), individuals carrying the CYP3A5*1 allele (CYP3A5 expressers) showed a trend of having higher CBZ clearance and lower dose-adjusted CBZ level as compared to individuals carrying the CYP3A5*3 allele, even though no statistical significance was recorded. Nevertheless, it was observed that AEDs significantly increased CBZ clearance only in patients carrying the active CYP3A5*1 allele. When CBZ was used in combination with enzyme-inducing AED, CYP3A5 expressers yielded a trend toward greater susceptibility to change in CBZ clearance and showed lower dose-adjusted CBZ levels compared to CYP3A5 non-expressers. The dosage regimen should be adjusted accordingly to gain a better clinical outcome.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Cytochrome P450 3A5 Plays a Prominent Role in the Oxidative Metabolism of the Anti-Human Immunodeficiency Virus Drug Maraviroc

Maraviroc is an anti-human immunodeficiency virus drug that acts by blocking viral entry into target cells. With use of ultra-performance liquid chromatography-mass spectrometry several monooxygenated, dioxygenated, and glucuronidated metabolites of maraviroc were identified both in vitro and in vivo. Characterization of the enzymes involved in the production of these metabolites determined that cytochrome P450 3A5 was the principal enzyme responsible for the formation of an abundant metabolite of maraviroc that resulted from oxygenation of the dichlorocyclohexane ring. For the formation of this metabolite, the V(max) values for CYP3A4 and CYP3A5 were 0.04 and 0.93 pmol · min⁻¹ · pmol P450⁻¹, and the K(m) values were 11.1 and 48.9 μM, respectively. Furthermore, human liver microsomes isolated from donors homozygous for the loss-of-function CYP3A5*3 allele exhibited a 79% decrease in formation of this metabolite compared with those homozygous for the wild-type CYP3A5*1 allele. To probe which divergent residues between CYP3A4 and CYP3A5 might play a role in the differential activities of these enzymes toward maraviroc, mutations were introduced into both enzymes and metabolism of maraviroc was measured. A CYP3A5 L57F mutant exhibited a 61% decrease in the formation of this metabolite, whereas formation by a CYP3A4 F57L mutant was increased by 337% compared with that of the wild type. Taken together, these data provide novel insights into the biotransformation of maraviroc as well as the potential role of CYP3A4 and CYP3A5 divergent residues in the enzymatic activities of these two highly homologous enzymes.

Unusual high dose of tacrolimus in liver transplant patient, a case report

We describe the case of a liver transplant patient who had great difficulty in reaching the desired trough blood levels despite the use of high dose tacrolimus. The patient was homozygous for the CYP3A5*3 allele. However, the respective donor carried the wild-type CYP3A5*1/*1 genotype. Regarding ABCB1 SNPs at exon 21 and 26, the patient showed the 2677GT and 3435CC genotypes. For the corresponding donor we observed the 2677GG and 3435CC wild-type genotypes. One, two and three weeks after transplantation the patient received daily 0.219, 0.287 and 0.273 mg/kg of tacrolimus, respectively. However, the corresponding tacrolimus trough blood levels were of 4.6, 5.6 and 6.1 ng/mL. The tacrolimus target level of 10.4 ng/mL was finally reached after 1 month of therapy. During the entire period of observation the kidney showed no sign of damage. No other signs of toxicity were reported except for the occurrence of an isolated systolic hypertension. CYP3A5 genotyping may represent a useful tool to better evaluate the appropriate initial dose of tacrolimus for patients carrying a liver with the CYP3A5*1/*1 genotype.

Vincristine pharmacodynamics and pharmacogenetics in children with cancer: A limited‐sampling, population modelling approach

Vincristine is a key component of many childhood cancer treatment regimens. Pharmacodynamic parameters such as clinical efficacy and toxicity may be influenced by polymorphisms of CYP3A. The aim of this study was to document CYP3A5 genotype, vincristine pharmacokinetics (PK) and neurotoxicity profile for 50 children with cancer and determine whether, in a population of Australian children, the CYP3A5 genotype influenced the pharmacodynamics of vincristine as reflected by peripheral neurotoxicity. Blood for PK analysis was collected after any single dose of vincristine and assayed using high performance liquid chromatography with tandem mass spectrometry detection. CYP3A5*3 and CYP3A5*6 genotype was determined using gel-electrophoresis or automated microfluidic electrophoresis. Neurotoxicity was determined by physical examination. The median age of children sampled was 6.5 years (range 1-16.25). Half the patients received concurrent corticosteroids for acute lymphoblastic leukaemia. Six patients (12%) had experienced grade 3 or 4 neurotoxicity. The median clearance, area under the curve and Cmax of vincristine was 482 mL/min/m(2) (range 132-698), 49.7 mcg/L.h (16.5-143.1) and 3.5 mcg/L (1.0-31.2), respectively. In contrast to prediction, all but three children were homozygous for wild-type CYP3A5*3. No CYP3A5*6 polymorphisms were identified. No correlation was identified between vincristine clearance, vincristine neurotoxicity, age, sex or concomitant steroid therapy. The limited sampling methodology proved acceptable to patients and families and would be suitable for larger scale studies including a wider range of genotypic variants and more detailed prospective evaluation of neurotoxicity.

A New CYP3A5 Variant,CYP3A5*11, Is Shown to Be Defective in Nifedipine Metabolism in a Recombinant cDNA Expression System

A new CYP3A5 variant, CYP3A5*11, was found in a white European subject by DNA sequencing. The CYP3A5*11 allele contains a single nucleotide polymorphism (SNP) (g.3775A>G) in exon 2, which results in a Tyr53Cys substitution, and a g.6986A>G splice change, the latter SNP previously reported in the defective CYP3A5*3 allele. However, the CYP3A5*3 is not a null allele because this variant is associated with leaky splicing, resulting in small amounts of functional protein still being produced. Therefore, we constructed a cDNA coding for the newly identified CYP3A5.11 protein by site-directed mutagenesis, expressed it in Escherichia coli, and partially purified it. Whereas bacteria transformed with wild-type CYP3A5*1 cDNA expressed predominantly cytochrome P450 (P450), those transfected with CYP3A5*11 expressed a significant amount of denatured cytochrome P420 in addition to P450, suggesting the protein to be unstable. CYP3A5.11 exhibited a 38% decrease in the V(max) for nifedipine metabolism, a 2.7-fold increase in the K(m), and a 4.4-fold decrease in the CL(int) of nifedipine compared with CYP3A5.1. A polymerase chain reaction-restriction fragment length polymorphism genotyping procedure was developed and used to genotype DNA of 500 white individuals for CYP3A5*11. No additional examples of this allele were identified. In summary, individuals carrying the rare CYP3A5*11 allele are predicted to have lower metabolism of CYP3A5 substrates than individuals expressing CYP3A5*3.

Significance of the minor cytochrome P450 3A isoforms.

Cytochrome P450 (CYP) 3A4 is responsible for most CYP3A-mediated drug metabolism but the minor isoforms CYP3A5, CYP3A7 and CYP3A43 also contribute. CYP3A5 is the best studied of the minor CYP3A isoforms. It is well established that only approximately 20% of livers express CYP3A5. The most common reason for the absence of expression is a splice site mutation. The frequency of variant alleles shows interethnic differences, with the wild-type CYP3A5*1 allele more common in Africans than Caucasians and Asians. In individuals who express CYP3A5, the percentage contributed to total hepatic CYP3A by this isoform is still unclear, with estimates ranging from 17% to 50%. CYP3A5 is also expressed in a range of extrahepatic tissues. Only limited information is available on the regulation of CYP3A5 expression but it appears to be inducible via the glucocorticoid receptor, pregnane X receptor and constitutive androstane receptor-beta, as for CYP3A4. Although information on the substrate specificity of CYP3A5 is limited compared with CYP3A4, there have been a number of recent pharmacokinetic studies on a small range of substrates in individuals of known genotype to investigate the contribution of CYP3A5. In the case of midazolam, ciclosporin, nifedipine and docetaxel, clearance by individuals with a CYP3A5-expressing genotype did not differ from that for nonexpressors, but in the case of tacrolimus, eight independent studies have demonstrated faster clearance by those carrying one or two CYP3A5*1 alleles. This may reflect faster turnover of tacrolimus by CYP3A5 than the other substrates. CYP3A5 genotype may affect cancer susceptibility. Certain combined CYP3A4/CYP3A5 haplotypes show differential susceptibility to prostate cancer and there is a nonsignificant increase in the risk of small-cell lung cancer for a CYP3A5*1/*1 genotype. Females positive for CYP3A5*1 appear to reach puberty earlier, which may affect breast cancer risk. CYP3A5*1 homozygotes may have higher systolic blood pressure.CYP3A7 is predominantly expressed in fetal liver but is also found in some adult livers and extrahepatically. The molecular basis for expression in adult liver relates to upstream polymorphisms, which appear to increase homology to CYP3A4 and make regulation of expression more similar. CYP3A7 has a specific role in hydroxylation of retinoic acid and 16alpha-hydroxylation of steroids, and is therefore of relevance both to normal development and carcinogenesis.CYP3A43 is the most recently discovered CYP3A isoform. In addition to a low level of expression in liver, it is expressed in prostate and testis. Its substrate specificity is currently unclear. Polymorphisms predicting absence of active enzyme have been identified.

Differences in CYP3A5*3 Genotype Distribution and Combinations With Other Polymorphisms Between Spaniards and Other Caucasian Populations

The goal of this study was to detect genotypic differences between Spaniards and other related populations regarding CYP3A4*1B, CYP3A5*3, and ABCB1 (MDR1) C3435T polymorphisms. DNA from 177 Spanish patients were analyzed for the presence of these mutations using PCR-restriction fragment length polymorphism or direct sequencing. The observed frequencies for CYP3A4*1B, CYP3A5*3, and C3435T alleles were within normal values in Caucasians (0.04, 0.91, and 0.5, respectively). However, 2.8% of the patients were homozygous for the wild-type CYP3A5*1 allele, an extremely uncommon genotype in other Caucasians. In addition, analysis of CYP3A4-3A5 haplotypes revealed the existence of 2 unusual subgroups: patients who were homozygous wild-type for both polymorphisms, and patients showing a CYP3A4*1A/*1B-CYP3A5*3/*3 genotype combination. The incidence of CYP3A5*1/*1 carriers and the occurrence of subjects combining the 2 above-mentioned unusual genotype combinations were more frequent in Spanish-Caucasians compared with American- or European-Caucasians. ABCB1 C3435T genotype frequencies were equally distributed between both single and combined CYP3A4 and 3A5 genotypes. These findings suggest that dose requirements for drugs metabolized by CYP3A and certain allele-disease association studies in white populations could show discrepancies in Spaniards.