The diflavin NADPH-cytochrome P450 reductase (CYPOR) plays a critical role in human cytochrome P450 (CYP) activity by sequentially delivering two electrons from NADPH to CYP enzymes during catalysis. Although electron transfer to forty-eight human CYP enzymes by the FMN hydroquinone of CYPOR is well-known, the role of the linker between the NH2-terminus membrane-binding domain (MBD) and FMN domain in supporting the activity of P450 enzymes remains poorly understood. Here we demonstrate that a linker with at least eight residues is required to form a functional CYPOR-CYP2B4 complex. The linker has been shortened in two amino-acid increments from Phe44 to Ile57 using site directed mutagenesis. The ability of the deletion mutants to support cytochrome P450 2B4 (CYP2B4) catalysis and reduce ferric CYP2B4 was determined using an in vitro assay and stopped-flow spectrophotometry. Steady-state enzyme kinetics showed that shortening the linker by 8-14 amino acids inhibited (63-99%) the ability of CYPOR to support CYP2B4 activity and significantly increased the Km of CYPOR for CYP2B4. In addition, the reductase mutants decreased the rate of reduction of ferric CYP2B4 (46-95%) compared to wildtype when the linker was shortened by 8-14 residues. These results indicate that a linker with a minimum length of eight residues is necessary to enable the FMN domain of reductase to interact with CYP2B4 to form a catalytically competent complex. Our study provides evidence that the length of the MBD-FMN domain linker is a major determinant of the ability of CYPOR to support CYP catalysis and drug metabolism by P450 enzymes. PREAMBLE: This manuscript is dedicated in memory of Dr. James R. Kincaid who was the doctoral advisor to Dr. Freeborn Rwere and a longtime collaborator and friend of Dr. Lucy Waskell. Dr. James R. Kincaid was a distinguished professor of chemistry specializing in resonance Raman (rR) studies of heme proteins. He inspired Dr. Rwere (a Zimbabwean native) and three other Zimbabweans (Dr. Remigio Usai, Dr. Daniel Kaluka and Ms. Munyaradzi E. Manyumwa) to use lasers to document subtle changes occurring at heme active site of globin proteins (myoglobin and hemoglobin) and cytochrome P450 enzymes. Dr. Rwere appreciate his contributions to the development of talented Black scientists from Africa.

Human pregnane X receptor (PXR) is critical for regulating the expression of key drug-metabolizing enzymes such as CYP3A and CYP2C. Our recent study revealed that treatment with rodent-specific PXR agonist pregnenolone-16α-carbonitrile (PCN) significantly induced hepatomegaly and promoted liver regeneration after two-thirds partial hepatectomy (PHx) in mice. However, it remains unclear whether PXR activation induces hepatomegaly and liver regeneration and simultaneously promotes metabolic function of the liver. Here, we investigated the metabolism activity of CYP1A2, CYP3A1/2 and CYP2C6/11 during PXR activation-induced liver enlargement and regeneration in rats after cocktail dosing of CYP probe drugs. For PCN-induced hepatomegaly, a notable increase in the metabolic activity of CYP3A1/2 and CYP2C6/11, as evidenced by the plasma exposure of probe substrates and the AUC ratios of the characteristic metabolites to its corresponding probe substrates. The metabolic activity of CYP1A2, CYP3A1/2 and CYP2C6/11 decreased significantly after PHx. However, PCN treatment obviously enhanced the metabolic activity of CYP2C6/11 and CYP3A1/2 in PHx rats. Furthermore, the protein expression levels of CYP3A1/2 and CYP2C6/11 in liver were up-regulated. Taken together, this study demonstrates that PXR activation not only induces hepatomegaly and liver regeneration in rats, but also promotes the protein expression and metabolic activity of the PXR downstream metabolizing enzymes such as CYP3A1/2 and CYP2C6/11 in the body.

CYP2A7 is one of the most understudied human cytochrome P450 enzymes and its contributions to either drug metabolism or endogenous biosynthesis pathways are not understood, as its only known enzymatic activities are the conversions of two proluciferin probe substrates. In addition, the CYP2A7 gene contains four single-nucleotide polymorphisms (SNPs) that cause missense mutations and have minor allele frequencies (MAFs) above 0.5. This means that the resulting amino acid changes occur in the majority of humans. In a previous study, we employed the reference standard sequence (called CYP2A7*1 in P450 nomenclature). For the present study, we created another CYP2A7 sequence that contains all four amino acid changes (Cys311, Glu169, Gly479, and Arg274) and labeled it CYP2A7-WT. Thus, it was the aim of this study to identify new substrates and inhibitors of CYP2A7 and to compare the properties of CYP2A7-WT with CYP2A7*1. We found several new proluciferin probe substrates for both enzyme variants (we also performed in silico studies to understand the activity difference between CYP2A7-WT and CYP2A7*1 on specific substrates), and we show that while they do not act on the standard CYP2A6 substrates nicotine, coumarin, or 7-ethoxycoumarin, both can hydroxylate diclofenac (as can CYP2A6). Moreover, we found ketoconazole, 1-benzylimidazole, and letrozole to be CYP2A7 inhibitors.

The activation of pregnane X receptor (PXR) or peroxisome proliferator-activated receptor α (PPARα) can induce liver enlargement. Recently, we reported that PXR or PPARα activation-induced hepatomegaly depends on yes-associated protein (YAP) signaling and is characterized by hepatocyte hypertrophy around the central vein area and hepatocyte proliferation around the portal vein area. However, it remains unclear whether PXR or PPARα activation-induced hepatomegaly can be reversed after the withdrawal of their agonists. In this study, we investigated the regression of enlarged liver to normal size following the withdrawal of PCN or WY-14643 (typical agonists of mouse PXR or PPARα) in C57BL/6 mice. The immunohistochemistry analysis of CTNNB1 and KI67 showed a reversal of hepatocyte size and a decrease in hepatocyte proliferation after the withdrawal of agonists. In details, the expression of PXR or PPARα downstream proteins (CYP3A11, CYP2B10, ACOX1, and CYP4A) and the expression of proliferation-related proteins (CCNA1, CCND1, and PCNA) returned to the normal levels. Furthermore, YAP and its downstream proteins (CTGF, CYR61, and ANKRD1) also restored to the normal states, which was consistent with the change in liver size. These findings demonstrate the reversibility of PXR or PPARα activation-induced hepatomegaly and provide new data for the safety of PXR and PPARα as drug targets.

OBJECTIVE: Develop a cytochrome P450 (CYP) phenotyping cocktail for dogs using specific substrates for hepatic P450 enzymes CYP2B11, CYP2D15, and CYP3A12 and determine whether alternative sampling methods (saliva and urine) or single time point samples could be used instead of multiple blood sampling.
METHODS: 12 healthy client-owned dogs (8 females and 4 males) from February 2019 to May 2019.
METHODS: In a randomized crossover study, dogs received oral administration of the probe drug bupropion (75 mg), dextromethorphan (30 mg), or omeprazole (40 mg) alone or as a 3-drug combination (Program in Individualized Medicine [PrIMe] cocktail) to evaluate simultaneous phenotyping of CYP2B11, CYP2D15, and CYP3A12. Pharmacokinetic profiles for the probe drugs and metabolites were determined using plasma, saliva, and urine. Dogs received probe drugs alone or combined. Pharmacokinetic profiles up to 6 hours postdose for the probe drugs and metabolites were determined using plasma, saliva, and urine.
RESULTS: The PrIMe cocktail was well tolerated. There was no statistically significant interaction between the probe drugs when administered together. Single time point plasma metabolic ratios at 4 hours postdose for all probe drugs strongly correlated with the corresponding area under the plasma concentration-versus-time curve (AUC) ratios. Saliva AUC metabolic ratios for CYP3A12 and CYP2D15 and 6-hour urine for CYP2B11 and CYP2D15 were correlated with plasma AUC ratios.
CONCLUSIONS: The PrIMe cocktail can be used for simultaneous CYP phenotyping using plasma 4-hour single time point sample metabolic ratios. Saliva and urine sampling are suitable for specific CYPs.
CONCLUSIONS: The PrIMe cocktail has potential as a useful tool in dogs to detect clinically important CYP-mediated drug-drug interactions, identify novel pharmacogenes, determine the drug-metabolizing phenotype of individual dogs, aid in individualized dose selection, and evaluate the effects of various physiological states on drug metabolism.

In this brief report, we provide an analysis of the influence of a novel CYP2C haplotype (CYP2C:TG) on proton pump inhibitor (PPI) pharmacokinetics (PK) in children. The CYP2C:TG haplotype has been proposed to be associated with increased CYP2C19 activity. We sought to determine if this CYP2C:TG haplotype resulted in similar alterations in metabolism for proton pump inhibitors, which are primarily metabolized by CYP2C19. In a cohort of 41 children aged 6-21 participating in a PPI pharmacokinetic study, effects of the CYP2C:TG allele were assessed by fitting two linear regression models for each of the six PK outcomes assessed, the second of which accounted for the presence of the CYP2C:TG allele. The difference in R2 values between the two models was computed to quantify the variability in the outcome that could be accounted for by the CYP2C:TG allele after adjustment for the CYP2C19 genotype. We found the CYP2C:TG haplotype to have no measurable additive impact on CYP2C19-mediated metabolism of PPIs in vivo in older children and adolescents. The findings of this study do not support the clinical utility of routine testing for the CYP2C:TG haplotype to guide PPI dose adjustments in children.

The chemotherapeutic agent tegafur, a prodrug that prolongs the half-life of fluorouracil (5-FU), exerts antitumor effects against various cancers. Since tegafur is metabolized to 5-FU by CYP2A6 in the liver, the expression of CYP2A6 determines the effect of tegafur. Here, we report that the expression rhythm of Cyp2a5, a homolog of human CYP2A6, in female mice causes dosing time-dependent differences in tegafur metabolism. In the livers of female mice, CYP2A5 expression showed a circadian rhythm, peaking during the dark period. This rhythm is regulated by RORA, a core clock component, and abrogation of the CYP2A5 activity abolished the time-dependent difference in the rate of tegafur metabolism in female mice. Furthermore, administration of tegafur to mice transplanted with 4T1 breast cancer cells during the dark period suppressed increases in tumor size compared to female mice treated during the light period. Our findings reveal a novel relationship between 5-FU prodrugs and circadian clock machinery, potentially influencing antitumor effects, and contributing to the development of time-aware chemotherapy regimens for breast cancer.

BACKGROUND: Warfarin is widely used for the prevention and treatment of thrombotic events. This study aimed to examine the influence of gene polymorphisms on the early stage of warfarin therapy in patients following heart valve surgery.
METHODS: Nine single nucleotide polymorphisms were genotyped using microarray chips, categorizing patients into three groups: normal responders (Group I), sensitive responders (Group II), and highly sensitive responders (Group III). The primary clinical outcomes examined were time in therapeutic range (TTR) and international normalized ratio (INR) variability. To investigate potential influencing factors, a generalized linear regression model was employed.
RESULTS: Among 734 patients, the prevalence of CYP2C9*3-1075A > C, CYP2C19*3-636G > A, and CYP2C19*17-806C > T variants were 11.2%, 9.9%, and 1.9% of patients, respectively. VKORC1-1639G > A or the linked -1173C > T variant was observed in 99.0% of the patients. Generalized linear model analysis revealed an impact of sensitivity grouping on INR variability. Compared to Group I, Group II showed higher TTR values (p = 0.023), while INR variability was poorer in Group II (p < 0.001) and Group III (p < 0.001). Individual gene analysis identified significant associations between CYP2C9*3-1075A > C (p < 0.001), VKORC1-1639G > A or the linked -1173 C > T (p = 0.009) and GGCX-3261G > A (p = 0.019) with INR variability.
CONCLUSIONS: The genotypes of CYP2C9, VKORC1, and GGCX were found to have a significant impact on INR variability during the initial phase of warfarin therapy. However, no significant association was observed between TTR and gene polymorphisms. These findings suggest that focusing on INR variability is crucial in clinical practice, and preoperative detection of gene polymorphisms should be considered to assist in the initiation of warfarin therapy.

Nivasorexant was the first orexin-1 selective receptor antagonist entering clinical development. Despite encouraging preclinical evidence in animal models, a proof-of-concept trial in binge-eating patients recently failed to demonstrate its clinical utility in this population.Across species, nivasorexant clearance was driven by metabolism along seven distinct pathways, five of which were hydroxylation reactions in various locations of the molecule. The exact sites of metabolism were identified by means of mass spectrometry, the use of deuterated analogues, and finally confirmed by chemical references.CYP3A4 was the main cytochrome P450 enzyme involved in nivasorexant metabolism in vitro and accounting for about 90% of turnover in liver microsomes. Minor roles were taken by CYP2C9 and CYP2C19 but individually did not exceed 3-7%.In the rat, nivasorexant was mostly excreted via the bile after extensive metabolism, while urinary excretion was negligible. Only traces of the parent drug were detected in urine, bile, or faeces.

Aflatoxin B1 (AFB1), a naturally-occurring mycotoxin, can cause severe toxicological and carcinogenic effects in livestock and humans. Given that the chicken is one of the most important food-producing animals, knowledge regarding AFB1 metabolism and enzymes responsible for AFB1 transformation in the chicken has important implications for chicken production and food safety. Previously, we have successfully expressed chicken CYP1A5 and CYP3A37 monooxygenases in E. coli, and reconstituted them into a functional CYP system consisting of CYP1A5 or CYP3A37, CPR and cytochrome b5. In this study, we aimed to investigate the roles of CYP1A5 and CYP3A37 in the bioconversion of AFB1 to AFM1. Our results showed that chicken CYP1A5 was able to hydroxylate AFB1 to AFM1. The formation of AFM1 followed the typical Michaelis-Menten kinetics. The kinetics parameters of Vmax and Km were determined as 0.83 ± 0.039 nmol/min/nmol P450 and 26.9 ± 4.52 μM respectively. Docking simulations further revealed that AFB1 adopts a \"side-on\" conformation in chicken CYP1A5, facilitating the hydroxylation of the C9a atom and the production of AFM1. On the other hand, AFB1 assumes a \"face-on\" conformation in chicken CYP3A37, leading to the displacement of the C9a atom from the heme iron and explaining the lack of AFM1 hydroxylation activity. The results demonstrate that chicken CYP1A5 possesses efficient hydroxylase activity towards AFB1 to form AFM1.