Human cytochrome P450 3A7 binding four copies of its native substrate dehydroepiandrosterone 3-sulfate.

Human fetal cytochrome P450 3A7 (CYP3A7) is involved in both xenobiotic metabolism and the estriol biosynthetic pathway. Although much is understood about cytochrome P450 3A4 and its role in adult drug metabolism, CYP3A7 is poorly characterized in terms of its interactions with both categories of substrates. Herein, a crystallizable mutated form of CYP3A7 was saturated with its primary endogenous substrate dehydroepiandrosterone 3-sulfate (DHEA-S) to yield a 2.6 Å X-ray structure revealing the unexpected capacity to simultaneously bind four copies of DHEA-S. Two DHEA-S molecules are located in the active site proper, one in a ligand access channel, and one on the hydrophobic F'-G' surface normally embedded in the membrane. While neither DHEA-S binding nor metabolism exhibit cooperative kinetics, the current structure is consistent with cooperativity common to CYP3A enzymes. Overall, this information suggests that mechanism(s) of CYP3A7 interactions with steroidal substrates are complex.

Allele and genotype frequencies of CYP3A4, CYP3A5, CYP3A7, and GSTP1 gene polymorphisms among mainland Tibetan, Mongolian, Uyghur, and Han Chinese populations.

Over 50% prescribed drugs are metabolised by cytochrome P450 3A (CYP3A) and glutathione S-transferase pi (GSTP1) adds a glutathione to the oxidative products by CYP3A, which increases the hydrophilic property of metabolites and facilitates the excretion. Single nucleotide polymorphisms (SNPs) of CYP3A and GSTP1 show a diverse allele and genotype frequencies distribution among the world populations. The present study aimed to investigate the genotype and allele frequency distribution patterns of CYP3A4, CYP3A5, CYP3A7 and GSTP1 polymorphisms among healthy participants in mainland Tibetan, Mongolian, Uyghur, and Han Chinese populations. Blood samples were collected from 842 unrelated healthy subjects (323 Tibetan, 134 Mongolian, 162 Uyghur, and 223 Han) for genotyping analysis. Variant allele frequencies of CYP3A4 rs2242480, CYP3A5 rs776746, CYP3A7 rs2257401, and GSTP1 Ile105Val were observed in Han (0.253, 0.686, 0.312 and 0.188), Tibetan (0.186, 0.819, 0.192 and 0.173), Mongolian (0.198, 0.784, 0.228 and 0.235) and Uyghur (0.179, 0.858, 0.182 and 0.250) respectively. The allele frequency of CYP3A7*1C in Uyghur (0.019) was higher than that in Tibetan (0.002, p < 0.01). There was a strong linkage disequilibrium between CYP3A4 rs2242480, CYP3A5 rs776746, and CYP3A7 rs2257401 among the four ethnic groups. The results might be useful for the precise medication in the Chinese populations.

Variation in Expression of Cytochrome P450 3A Isoforms and Toxicological Effects: Endo- and Exogenous Substances as Regulatory Factors and Substrates.

The CYP3A subfamily, which includes isoforms CYP3A4, CYP3A5, and CYP3A7 in humans, plays important roles in the metabolism of various endogenous and exogenous substances. Gene and protein expression of CYP3A4, CYP3A5, and CYP3A7 show large inter-individual differences, which are caused by many endogenous and exogenous factors. Inter-individual differences can cause negative outcomes, such as adverse drug events and disease development. Therefore, it is important to understand the variations in CYP3A expression caused by endo- and exogenous factors, as well as the variation in the metabolism and kinetics of endo- and exogenous substrates. In this review, we summarize the factors regulating CYP3A expression, such as bile acids, hormones, microRNA, inflammatory cytokines, drugs, environmental chemicals, and dietary factors. In addition, variations in CYP3A expression under pathological conditions, such as coronavirus disease 2019 and liver diseases, are described as examples of the physiological effects of endogenous factors. We also summarize endogenous and exogenous substrates metabolized by CYP3A isoforms, such as cholesterol, bile acids, hormones, arachidonic acid, vitamin D, and drugs. The relationship between the changes in the kinetics of these substrates and the toxicological effects in our bodies are discussed. The usefulness of these substrates and metabolites as endogenous biomarkers for CYP3A activity is also discussed. Notably, we focused on discrimination between CYP3A4, CYP3A5, and CYP3A7 to understand inter-individual differences in CYP3A expression and function.

Comparison of the Stimulatory and Inhibitory Effects of Steroid Hormones and α-Naphthoflavone on Steroid Hormone Hydroxylation Catalyzed by Human Cytochrome P450 3A Subfamilies.

The inhibitory and stimulatory effects of steroid hormones and related compounds on the hydroxylation activity at the 6ß-position of two steroid hormones, progesterone and testosterone, by CYP3A4, polymorphically expressed CYP3A5, and fetal CYP3A7 were compared to clarify the catalytic properties of the predominant forms of the human CYP3A subfamily. Hydroxylation activities of progesterone and testosterone by CYP3A4, CYP3A5, and CYP3A7 were estimated using HPLC. The Michaelis constants (Km) for progesterone 6ß-hydroxylation by CYP3A5 were markedly decreased in the presence of dehydroepiandrosterone (DHEA) and α-naphthoflavone (ANF), whereas progesterone and DHEA competitively inhibited testosterone 6ß-hydroxylation mediated by CYP3A4, and progesterone competitively inhibited CYP3A5-mediated activity, which was weaker than that for CYP3A4. ANF noncompetitively inhibited testosterone 6ß-hydroxylation mediated by both CYP3A4 and CYP3A5. Progesterone and testosterone 6ß-hydroxylation mediated by CYP3A7 was inhibited or unaffected by DHEA, pregnenolone, and ANF. These results suggested that DHEA and ANF stimulated progesterone 6ß-hydroxylation by CYP3A5 but not by CYP3A4 and CYP3A7; however, progesterone, DHEA, and ANF inhibited testosterone 6ß-hydroxylation mediated by all CYP3A subfamily members. The inhibitory/stimulatory pattern of steroid-steroid interactions is different among CYP3A subfamily members and CYP3A5 is the most sensitive in terms of activation among the CYP3A subfamily members investigated.

Structural Basis for the Diminished Ligand Binding and Catalytic Ability of Human Fetal-Specific CYP3A7.

Cytochrome P450 3A7 (CYP3A7) is a fetal/neonatal liver enzyme that participates in estriol synthesis, clearance of all-trans retinoic acid, and xenobiotic metabolism. Compared to the closely related major drug-metabolizing enzyme in adult liver, CYP3A4, the ligand binding and catalytic capacity of CYP3A7 are substantially reduced. To better understand the structural basis for these functional differences, the 2.15 Å crystal structure of CYP3A7 has been solved. Comparative analysis of CYP3A enzymes shows that decreased structural plasticity rather than the active site microenvironment defines the ligand binding ability of CYP3A7. In particular, a rotameric switch in the gatekeeping amino acid F304 triggers local and long-range rearrangements that transmit to the F-G fragment and alter its interactions with the I-E-D-helical core, resulting in a more rigid structure. Elongation of the ß3-ß4 strands, H-bond linkage in the substrate channel, and steric constraints in the C-terminal loop further increase the active site rigidity and limit conformational ensemble. Collectively, these structural distinctions lower protein plasticity and change the heme environment, which, in turn, could impede the spin-state transition essential for optimal reactivity and oxidation of substrates.

Comparison of steroid hormone hydroxylation mediated by cytochrome P450 3A subfamilies.

Hydroxylation activity at the 6ß-position of steroid hormones (testosterone, progesterone, and cortisol) by human cytochromes P450 (CYP) 3A4, polymorphic CYP3A5, and fetal CYP3A7 were compared to understand the catalytic properties of the major forms of human CYP3A subfamily. Testosterone, progesterone, and cortisol 6ß-hydroxylation activities of recombinant CYP3A4, CYP3A5, and CYP3A7 were determined by liquid chromatography. Michaelis constants (Km) for CYP3A7-mediated 6ß-hydroxylation of testosterone, progesterone, and cortisol were similar to those of CYP3A4 and CYP3A5. The maximal velocity (kcat) and kcat/Km values for CYP3A4 were the highest, followed by CYP3A5 and those for CYP3A7 were the lowest among three CYP3A subfamily members. A decrease in Km values for progesterone 6ß-hydroxylation by CYP3A4, CYP3A5, and CYP3A7 in the presence of testosterone was observed, and the kcat values for CYP3A5 gradually increased with increasing testosterone. This indicated that testosterone stimulated progesterone 6ß-hydroxylation by all three CYP3A subfamily members. However, progesterone inhibited testosterone 6ß-hydroxylation mediated by CYP3A4, CYP3A5, and CYP3A7. In conclusion, the kcat values, rather than Km values, for 6ß-hydroxylation of three steroid hormones mediated by CYP3A7 were different from those for CYP3A4 and CYP3A5. In addition, the inhibitory/stimulatory pattern of steroid-steroid interactions would be different among CYP3A subfamily members.

CYP3A5 and CYP3A7 genetic polymorphisms affect tacrolimus concentration in pediatric patients with nephrotic range proteinuria.

PURPOSE: The purpose of this study was to investigate the potential impact of CYP3A4, CYP3A5, and CYP3A7 polymorphisms on the concentration and efficacy of tacrolimus in a cohort of pediatric patients with nephrotic range proteinuria. METHODS: Genetic variants including CYP3A5*3 (rs776746), CYP3A4*1G (rs2242480), rs4646437, and CYP3A7 rs2257401 and rs10211 were detected in 70 pediatric patients with nephrotic range proteinuria. The relationships of dose-adjusted trough concentration (C0) of tacrolimus with corresponding genotypes were investigated. RESULTS: The tacrolimus concentration in patients without CYP3A5*3 A allele was 94% higher than those with A allele (90.7 vs 54.2, P = 0.00006). The CYP3A7 rs2257401 was also associated with the concentration of tacrolimus. The C allele carriers had an obviously lower C0 than the non-carriers (62.4 vs 90.7, P = 0.001). In addition, there were significant differences in tacrolimus concentration among CYP3A7 rs10211 G carriers and non-carriers; the latter had an almost twofold C0 of the former (101.8 vs 59.6, P = 0.0004). CONCLUSIONS: Our study demonstrated the associations between CYP3A5*3, CYP3A7 rs2257401 and rs10211, and tacrolimus concentration in pediatric patients with nephrotic range proteinuria. Children with CYP3A5*3 A, CYP3A7 rs2257401 C, and rs10211 G alleles might need a higher dose of tacrolimus.

The use of a unique co-culture model of fetoplacental steroidogenesis as a screening tool for endocrine disruptors: The effects of neonicotinoids on aromatase activity and hormone production.

Estrogen biosynthesis during pregnancy is dependent on the collaboration between the fetus producing the androgen precursors, and the placenta expressing the enzyme aromatase (CYP19). Disruption of estrogen production by contaminants may result in serious pregnancy outcomes. We used our recently developed in vitro co-culture model of fetoplacental steroidogenesis to screen the effects of three neonicotinoid insecticides on the catalytic activity of aromatase and the production of steroid hormones. A co-culture of H295R human adrenocortical carcinoma cells with fetal characteristics and BeWo human choriocarcinoma cells which display characteristics of the villous cytotrophoblast was exposed for 24h to various concentrations of three neonicotinoids: thiacloprid, thiamethoxam and imidacloprid. Aromatase catalytic activity was determined in both cell lines using the tritiated water-release assay. Hormone production was measured by ELISA. The three neonicotinoids induced aromatase activity in our fetoplacental co-culture and concordingly, estradiol and estrone production were increased. In contrast, estriol production was strongly inhibited by the neonicotinoids. All three pesticides induced the expression of CYP3A7 in H295R cells, and this induction was reversed by co-treatment of H295R cells with exogenous estriol. CYP3A7 is normally expressed in fetal liver and is a key enzyme involved in estriol synthesis. We suggest that neonicotinoids are metabolized by CYP3A7, thus impeding the 16α-hydroxylation of fetal DHEA(-sulfate), which is normally converted to estriol by placental aromatase. We successfully used the fetoplacental co-culture as a physiologically relevant tool to highlight the potential effects of neonicotinoids on estrogen production, aromatase activity and CYP3A7 expression during pregnancy.

The effect of fetal androgen metabolism-related gene variants on external genitalia virilization in congenital adrenal hyperplasia.

The 21-hydroxylase deficiency (21OHD) is caused by CYP21A2 mutations resulting in severe or moderate enzymatic impairments. 21OHD females carrying similar genotypes present different degrees of external genitalia virilization, suggesting the influence of other genetic factors. Single nucleotide variants (SNVs) in the CYP3A7 gene and in its transcription factors, related to fetal 19-carbon steroid metabolism, could modulate the genital phenotype. To evaluate the influence of the 21OHD genotypes and the CYP3A7, PXR and CAR SNVs on the genital phenotype in 21OHD females. Prader scores were evaluated in 183 patients. The CYP3A7, PXR and CAR SNVs were screened and the 21OHD genotypes were classified according to their severity: severe and moderate groups. Patients with severe genotype showed higher degree of genital virilization (Prader median III, IQR III-IV) than those with moderate genotype (III, IQR II-III) (p < 0.001). However, a great overlap was observed between genotype groups. Among all the SNVs tested, only the CAR rs2307424 variant correlated with Prader scores (r(2) = 0.253; p = 0.023). The CYP21A2 genotypes influence the severity of genital virilization in 21OHD females. We also suggest that the CAR variant, which results in a poor metabolizer phenotype, could account for a higher degree of external genitalia virilization.

Augmentation of Pectoral Fin Teratogenicity by Thalidomide in Human Cytochrome P450 3A-Expressing Zebrafish.

The pharmacological and toxicological effects of active metabolites of enzymes including cytochrome P450 (CYP) are important. While it has been believed for a long time that thalidomide causes characteristic limb malformation only in rabbits and primates including humans, the involvement of their CYP3A subtypes (CYP3As) has been suggested. Recently, however, it was reported that zebrafish were sensitive to thalidomide, showing defects of pectoral fins, homologous organs of forelimbs in mammals, as well as other deformities. In this study, we prepared human CYP3A7 (hCYP3A7)-expressing zebrafish (F0) using a transposon system. Thalidomide caused pectoral fin defects and other malformations including pericardial edema in hCYP3A7-expressing embryos/larvae but not in wild-type and hCYP1A1-expressing embryos/larvae. Thalidomide also reduced the expression of fibroblast growth factor 8 in pectoral fin buds in only hCYP3A7-expressing embryos/larvae. The results suggest the involvement of human-type CYP3A in thalidomide teratogenicity.

Per- and polyfluoroalkyl substances (PFAS) inhibit cytochrome P450 CYP3A7 through direct coordination to the heme iron and water displacement.

Per- and polyfluoroalkyl substances (PFAS) are a chemical class of highly stable, fluorinated compounds popular for use in a variety of consumer products. PFAS environmental persistence in drinking water contributes to acute exposure in humans and subsequent bioaccumulation of the compounds in the liver and lung tissue. Prenatal PFAS exposure has been associated with lowered birth weight, premature birth, and developmental defects including cranio-facial abnormalities. The cytochrome P450 enzyme CYP3A7 is responsible for facilitating a variety of reactions essential for proper fetal development in humans. In addition to drug metabolism, CYP3A7 is responsible for metabolizing endogenous ligands in the developing human liver, including the steroid precursor dehydroepiandrosterone 3-sulfate (DHEA-S), essential for estriol synthesis during pregnancy, along with the morphogen all-trans-retinoic acid (atRA). Interference with estriol synthesis during pregnancy, as well as atRA clearance, is known to result in similar effects associated with prenatal PFAS exposure including lowered birth weight, premature birth, and developmental defects. We hypothesized that PFAS compounds bind to the CYP3A7 enzyme resulting in its inhibition. We implemented a series of binding studies using spectral characterization of six PFAS compounds (PFOA, PFOS, GenX, PFNA, PFNS, and PFHxS), and evaluated their interactions with recombinant CYP3A7. In addition, we screened PFAS for their ability to inhibit CYP3A7 oxidative activity using dibenzylfluorescein, a fluorescent probe, and DHEA-S, an endogenous substrate of CYP3A7. Our data demonstrate that of the six PFAS tested, PFOA, PFOS, PFNA, and PFHxS bind to and inhibit CYP3A7.

Stimulatory and Inhibitory Effects of Steroid Hormones and Human Cytochrome P450 (CYP) 3A Inhibitors on Cortisol 6ß-Hydroxylation Catalyzed by CYP3A Subfamilies.

OBJECTIVE: The inhibitory and stimulatory effects of several compounds, including steroid hormones and azole antifungal agents, on cortisol 6ß-hydroxylation activity by cytochrome P450 (CYP) 3A4, polymorphically expressed CYP3A5, and fetal CYP3A7 were compared with those on testosterone 6ß-hydroxylation to clarify the catalytic properties of the predominant forms of the human CYP3A subfamily. METHODS: 6ß-Hydroxylation activities of cortisol and testosterone by CYP3A4, CYP3A5, and CYP3A7 in the absence or presence of dehydroepiandrosterone (DHEA), α-naphthoflavone (ANF), ketoconazole, itraconazole, and voriconazole were measured using high-performance liquid chromatography. RESULTS: Lower concentrations of DHEA and ANF increased cortisol 6ß-hydroxylation activities catalyzed by CYP3A4 but not those catalyzed by CYP3A5 and CYP3A7. The inhibition strength of azole antifungal agents against cortisol 6ß-hydroxylation catalyzed by all CYP3A subfamilies was similar to that of testosterone 6ß-hydroxylation. Although the Michaelis constant (Km) increased 2-fold in the presence of 20 µM DHEA compared to that of the control, the maximal velocity (Vmax) values gradually increased with increasing DHEA. For ANF, both Km and Vmax values increased, although the Km value decreased at 2.5 µM concentrations. Ketoconazole and itraconazole competitively inhibited cortisol 6ß-hydroxylation mediated by CYP3A4 with similar inhibition constants. CONCLUSION: The inhibitory/stimulatory pattern among CYP3A subfamily members differed between cortisol and testosterone, and CYP3A4 was found to be the most sensitive in terms of inhibition by azole antifungals among the CYP3A subfamily members investigated.

Mechanism-based inhibition of CYP3A subfamilies by macrolide antibiotics and piperine.

OBJECTIVE: The mechanism-based inhibition of macrolide antibiotics, such as erythromycin and clarithromycin, and piperine on testosterone 6ß-hydroxylation activities by cytochrome P450 (CYP) 3A4, polymorphically expressed CYP3A5, and fetal CYP3A7 were compared. METHODS: 6ß-Hydroxy testosterone was determined by high-performance liquid chromatography. RESULTS: Although preincubation with erythromycin and clarithromycin decreased CYP3A4-meditaed testosterone 6ß- hydroxylation in a time-dependent manner, and the estimated maximum inactivation rate constant (k inact ) and the inactivation rate constant reaching half of k inact (K i ) for erythromycin were approximately 1/2 and 1/5, respectively, of those for clarithromycin. Obvious preincubation time-dependent inhibition of erythromycin against CYP3A5 and CYP3A7 was not observed. Piperine exhibited preincubation time- dependent inhibition, and the calculated K i and k inact values for CYP3A4 were approximately 1/7 and 1/2, respectively, of those for CYP3A5. CONCLUSION: It is speculated that the preincubation-dependent inhibition by piperine would be more potent in CYP3A5 non-expressors than CYP3A5-expressors.

CYP3A7, CYP3A4, and CYP3A5 genetic polymorphisms in recipients rather than donors influence tacrolimus concentrations in the early stages after liver transplantation.

AIM: The purpose of this study was to investigate the effect of CYP3A7, CYP3A4, and CYP3A5 genetic polymorphisms in liver transplant recipients and donors on tacrolimus concentrations in the early stages after liver transplantation. METHODS: One hundred and thirty-eight liver transplant recipients and matched donors were genotyped for CYP3A7 (rs10211 and rs2257401), CYP3A4 (rs4646437 and rs2242480), and CYP3A5*3 (rs776746) polymorphisms. The relationships between dose-adjusted trough concentrations (C0/D) of tacrolimus and corresponding genotypes were investigated. RESULTS: Recipient CYP3A polymorphisms were associated with tacrolimus concentrations. The CYP3A7 rs10211 AA carriers (186.2 vs 90.5, p < 0.001), CYP3A4 rs4646437 CC carriers (184.0 vs 88.8, p < 0.001), CYP3A4*1G rs2242480 CC carriers (189.8 vs 99.7, p < 0.001), and CYP3A5*3 rs776746 GG carriers (197.3 vs 86.0, p < 0.001) had an almost twofold increase in the tacrolimus C0/D compared to that of the non-carriers. We further investigated the effect of the combination of recipient (intestinal) and donor (hepatic) genotypes on tacrolimus concentrations. Regardless of the genotype of the matched donor, CYP3A7 rs10211, CYP3A4*1G (rs2242480), and CYP3A5*3 (rs776746) polymorphisms of recipients could affect tacrolimus concentrations. For the CYP3A4 rs4646437 polymorphisms, when the donor carried CYP3A4 rs4646437 CC, the recipient CYP3A4 rs4646437 polymorphism was associated with the C0/D of tacrolimus, and when the donor carried CYP3A4 rs4646437 CT/TT genotype, the recipient CYP3A4 rs4646437 polymorphism also affected on tacrolimus C0/D, although the effect was not significant. CONCLUSION: The large inter-individual variation in tacrolimus concentrations in the early stages after liver transplantation is influenced by genetic polymorphisms of CYP3A7, CYP3A4, and CYP3A5. Recipient (intestinal) CYP3A7, CYP3A4, and CYP3A5 polymorphisms seem to contribute more to such variation than donors. Therefore, the detection of CYP3A polymorphisms in recipients could help to predict the tacrolimus starting dose in the early stages after liver transplantation.

Identification of Selective CYP3A7 and CYP3A4 Substrates and Inhibitors Using a High-Throughput Screening Platform.

Cytochrome P450 (CYP) 3A7 is one of the major xenobiotic metabolizing enzymes in human embryonic, fetal, and newborn liver. CYP3A7 expression has also been observed in a subset of the adult population, including pregnant women, as well as in various cancer patients. The characterization of CYP3A7 is not as extensive as other CYPs, and health authorities have yet to provide guidance towards DDI assessment. To identify potential CYP3A7-specific molecules, we used a P450-Glo CYP3A7 enzyme assay to screen a library of ∼5,000 compounds, including FDA-approved drugs and drug-like molecules, and compared these screening data with that from a P450-Glo CYP3A4 assay. Additionally, a subset of 1,000 randomly selected compounds were tested in a metabolic stability assay. By combining the data from the qHTS P450-Glo and metabolic stability assays, we identified several chemical features important for CYP3A7 selectivity. Halometasone was chosen for further evaluation as a potential CYP3A7-selective inhibitor using molecular docking. From the metabolic stability assay, we identified twenty-two CYP3A7-selective substrates over CYP3A4 in supersome setting. Our data shows that CYP3A7 has ligand promiscuity, much like CYP3A4. Furthermore, we have established a large, high-quality dataset that can be used in predictive modeling for future drug metabolism and interaction studies.

Human Fetal Liver Metabolism of Oxycodone Is Mediated by CYP3A7.

Oxycodone is an opioid analgesic that is commonly prescribed to pregnant women to treat moderate-to-severe pain. It has been shown to cross the placenta and distribute to the fetus. Oxycodone is mainly metabolized by CYP3A4 in the adult liver. Since CYP3A7 is abundantly expressed in the fetal liver and has overlapping substrate specificity with CYP3A4, we hypothesized that the fetal liver may significantly limit fetal exposure to oxycodone. This study showed that oxycodone is metabolized by CYP3A7 to noroxycodone in fetal liver microsomes (FLMs). The measured CYP3A7 expression was 191-409 pmol/mg protein in 14 FLMs, and an intersystem extrapolation factor (ISEF) for CYP3A7 was 0.016-0.066 in the panel of fetal livers using 6ß-OH-testosterone formation as the probe reaction. Noroxycodone formation in the fetal liver was predicted from formation rate by recombinant CYP3A7, CYP3A7 expression level and the established ISEF value with average fold error of 1.25. Based on the intrinsic clearance of oxycodone measured in FLM, the fetal hepatic clearance (CLh) at term was predicted to be 495 (range: 66.4-936) µL/min, a value that is > 99% lower than the predicted adult liver CLh. The predicted fetal hepatic extraction ratio was 0.0019 (range: 0.00003-0.0036). These results suggest that fetal liver metabolism does not quantitatively contribute to the total systemic clearance of oxycodone in pregnant women nor does it provide a barrier for limiting fetal exposure to oxycodone. Additionally, since CYP3A7 forms noroxycodone, an inactive metabolite, the metabolism in the fetal liver is unlikely to affect fetal opioid activity.

Development of template systems for ligand interactions of CYP3A5 and CYP3A7 and their distinctions from CYP3A4 template.

Starting from established CYP3A4 Template (DMPK. 2019, and 2020), CYP3A5 and CYP3A7 Templates have been constructed to be reliable tools for verification of their distinct catalytic properties. A distinct occupancy was observed on CYP3A4-selective ligands, but not on the non-selective ligands, in simulation experiments. These ligands often invade into Bay-1 region during the migration from Entrance to Site of oxidation in simulation experiments. These results offered an idea of the distinct localization of Bay-1 residue on CYP3A5 Template, in which the Bay-1 residue stayed closely to Template border. The idea also accounted for the higher oxidation rates of CYP3A5, than of CYP3A4, of noscapine and schisantherin E through their enhanced sitting-stabilization. Typical CYP3A7 substrates such as zonisamide and retinoic acids took their placements without occupying a left side region of Template for their metabolisms. In turn, the occupancies of the left-side region were inevitably observed among poor ligands of CYP3A7. Altered extent of IJK-Interaction or localization of a specific residue at the left-side would thus explain distinct catalytic properties of CYP3A7 on Template. These data suggest the alteration of each one of Template region, from CYP3A4 Template, led to the distinct catalytic properties of CYP3A5 and CYP3A7 forms.

Real-time fluorometric evaluation of hepatoblast proliferation in vivo and in vitro using the expression of CYP3A7 coding for human fetus-specific P450.

The fields of drug discovery and regenerative medicine require large numbers of adult human primary hepatocytes. For this purpose, it is desirable to use hepatocyte-like cells (HLCs) differentiated from human pluripotent stem cells (PSCs). Premature hepatoblast-like cells (HB-LCs) differentiated from PSCs provide an intermediate source and steady supply of newly mature HLCs. To develop an efficient HB-LC induction method, we constructed a red fluorescent reporter, CYP3A7R, in which DsRed is placed under the transcriptional control of CYP3A7 coding for a human fetus-type P450 enzyme. Before using this reporter in human cells, we created transgenic mice using mouse embryonic stem cells (ESCs) carrying a CYP3A7R transgene and confirmed that CYP3A7R was specifically expressed in fetal and newborn livers and reactivated in the adult liver in response to hepatic regeneration. Moreover, we optimized the induction procedure of HB-LCs from transgenic mouse ESCs using semi-quantitative fluorometric evaluation. Activation of Wnt signaling together with chromatin modulation prior to Activin A treatment greatly improved the induction efficiency of HB-LCs. BMP2 and 1.7% dimethyl sulfoxide induced selective proliferation of HB-LCs, which matured to HLCs. Therefore, CYP3A7R will provide a fluorometric evaluation system for high content screening of chemicals that induce HB-LC differentiation, hepatocyte regeneration, and hepatotoxicity when it is introduced into human PSCs.

A two-dimensional multiwell cell culture method for the production of CYP3A4-expressing hepatocyte-like cells from HepaRG cells.

Cytochrome P450 enzymes (CYP) function in drug metabolism in the liver. To evaluate numerous drug candidates, a high-content screening (HCS) system with hepatocyte-like cells (HLCs) that can replace adult human hepatocytes is required. Human hepatocellular carcinoma HepaRG is the only cell line capable of providing HLCs with high CYP3A4 expression comparable to that in adult hepatocytes after cell differentiation. The aim of this study was to design an ideal multiwell culture system for HLCs using transgenic HepaRG cells expressing the EGFP coding an enhanced green fluorescent protein under CYP3A4 transcriptional regulation. HLCs were matured on five different types of 96-well black plates. Culturing HLCs on glass-bottom Optical CVG plates significantly promoted cell maturation and increased metabolic activity by twofold under two-dimensional (2D) culture conditions, and these features were enhanced by 2% collagen coating. Three plates for three-dimensional (3D) cell cultures with a gas-exchangeable fabric or dimethylpolysiloxane membrane bottom formed multiple round colonies, whereas they were ineffective for CYP3A4 expression. Under optimized conditions presented here, HLCs lost responsiveness to nuclear receptor-mediated transcriptional induction of CYP3A4, suggesting that CYP3A4 transcription has already been fully upregulated. Therefore, HepaRG-derived HLCs will provide an alternative to human hepatocytes with high levels of CYP3A4 enzyme activity even under 2D culture conditions. This will improve a variety of drug screening methods.

Impact of CYP3A5 phenotype on tacrolimus concentrations after sublingual and oral administration in lung transplant.

Aim: This study evaluated the impact of CYP3A5 genotype and other patient characteristics on sublingual (SL) tacrolimus exposure and compared the relationship with oral administration. Patients & methods: Tacrolimus concentrations were retrospectively collected for adult lung transplant recipients, who were genotyped for CYP3A5*3, CYP3A4*22, CYP3A7*1C, and POR*28. Regression analyses were performed to determine covariates that impacted the SL and oral tacrolimus concentration/dose ratios. Results: An interaction of CYP3A5 genotype and CYP3A inhibitor increased the SL concentration/dose, while cystic fibrosis decreased the SL concentration/dose. The oral concentration/dose was independently associated with these covariates and was increased by serum creatinine and number of tacrolimus doses. Conclusion: This study suggests personalized dosing strategies for tacrolimus likely need to consider characteristics beyond CYP3A5 genotype.