CYP8B1 Catalyzes 12alpha-Hydroxylation of C27 Bile Acid: In Vitro Conversion of Dihydroxycoprostanic Acid into Trihydroxycoprostanic Acid.

Sterol 12α-hydroxylase (CYP8B1) is the unique P450 enzyme with sterol 12-oxidation activity, playing an exclusive role in 12α-hydroxylating intermediates along the bile acid (BA) synthesis pathway. Despite the long history of BA metabolism studies, it is unclear whether CYP8B1 catalyzes 12α-hydroxylation of C27 BAs, the key intermediates shuttling between mitochondria and peroxisomes. This work provides robust in vitro evidence that both microsomal and recombinant CYP8B1 enzymes catalyze the 12α-hydroxylation of dihydroxycoprostanic acid (DHCA) into trihydroxycoprostanic acid (THCA). On the one hand, DHCA 12α-hydroxylation reactivity is conservatively detected in liver microsomes of both human and preclinical animals. The reactivity of human tissue fractions conforms well with the selectivity of CYP8B1 mRNA expression, while the contribution of P450 enzymes other than CYP8B1 is excluded by reaction phenotyping in commercial recombinant enzymes. On the other hand, we prepared functional recombinant human CYP8B1 proteins according to a recently published protocol. Titration of the purified CYP8B1 proteins with either C4 (7α-hydroxy-4-cholesten-3-one) or DHCA yields expected blue shifts of the heme Soret peak (type I binding). The recombinant CYP8B1 proteins efficiently catalyze 12α-hydroxylation of both DHCA and C4, with substrate concentration occupying half of the binding sites of 3.0 and 1.9 µM and kcat of 3.2 and 2.6 minutes-1, respectively. In summary, the confirmed role of CYP8B1 in 12α-hydroxylation of C27 BAs has furnished the forgotten passageway in the BA synthesis pathway. The present finding might have opened a new window to consider the biology of CYP8B1 in glucolipid metabolism and to evaluate CYP8B1 inhibition as a therapeutic approach of crucial interest for metabolic diseases. SIGNIFICANCE STATEMENT: The academic community has spent approximately 90 years interpreting the synthesis of bile acids. However, the 12α-hydroxylation of intermediates catalyzed by CYP8B1 is not completely mapped on the classic pathway, particularly for the C27 bile acids, the pivotal intermediates shuttling between mitochondria and peroxisomes. This work discloses the forgotten 12α-hydroxylation pathway from dihydroxycoprostanic acid into trihydroxycoprostanic acid. The present finding may facilitate evaluating CYP8B1 inhibition as a therapeutic approach of crucial interest for metabolic diseases.

Reevaluate In Vitro CYP3A Index Reactions of Benzodiazepines and Steroids between Humans and Dogs.

Cytochrome P450 3A (CYP3A), the most important class of drug-metabolizing enzymes, participates in the metabolism of half of clinically used drugs. The CYP3A index reactions of dogs, one of the most widely used preclinical nonrodent species, are still poorly understood. This work evaluated the activity and selectivity of 10 CYP3A index reactions, including midazolam (MDZ) 1'- and 4-hydroxylation, alprazolam (APZ) and triazolam (TRZ) α- and 4-hydroxylation, testosterone (T) 6ß-hydroxylation, lithocholate (LCA) 6α-hydroxylation, deoxycholate (DCA) 1ß- and 5ß-hydroxylation, with quantitative reaction phenotyping and kinetic analysis in human and canine recombinant CYP enzymes (rCYPs). In human studies, all reactions are reconfirmed as mixed index reactions of CYP3A with minor contributions from non-CYP3A isoforms. In canine studies, all reactions are also primarily catalyzed by CYP3A12 with lower contributions from CYP3A26. However, the canine CYP2B11 appreciably contributes to the hydroxylation of benzodiazepines except for APZ 4-hydroxylation. The canine CYP3A isoforms have lower activity than human isoforms toward T 6ß-hydroxylation and LCA 6α-hydroxylation and both substrates undergo non-CYP3A catalyzed side reactions. DCA 1ß- and 5ß-hydroxylation are validated as the CYP3A index reactions in both humans and dogs with limited non-CYP3A contributions and side reactions. In conclusion, this work provides a comprehensive overview for the selectivity and activity of in vitro CYP3A index reactions in humans and dogs. The validated CYP3A index reactions between humans and dogs may benefit future practices in drug metabolism and drug interaction studies. SIGNIFICANCE STATEMENT: Dogs are one of the most important nonrodent animals with limited studies of cytochrome P450 enzymes than humans. This work provides the most comprehensive quantitative data to date for the selectivity and activity of CYP3A index reactions in humans and dogs. The canine CYP2B11 was found to appreciably contribute to hydroxylation of midazolam, alprazolam and triazolam, the well-known probes for human CYP3A. Deoxycholate 1ß- and 5ß-hydroxylation are validated as the CYP3A index reactions in both humans and dogs.

Tertiary Oxidation of Deoxycholate Is Predictive of CYP3A Activity in Dogs.

Deoxycholic acid (DCA, 3α, 12α-dihydroxy-5ß-cholan-24-oic acid) is the major circulating secondary bile acid, which is synthesized by gut flora in the lower gut and selectively oxidized by CYP3A into tertiary metabolites, including 1ß,3α,12α-trihydroxy-5ß-cholan-24-oic acid (DCA-1ß-ol) and 3α,5ß,12α-trihydroxy-5ß-cholan-24-oic acid (DCA-5ß-ol) in humans. Since DCA has the similar exogenous nature and disposition mechanisms as xenobiotics, this work aimed to investigate whether the tertiary oxidations of DCA are predictive of in vivo CYP3A activities in beagle dogs. In vitro metabolism of midazolam (MDZ) and DCA in recombinant canine CYP1A1, 1A2, 2B11, 2C21, 2C41, 2D15, 3A12, and 3A26 enzymes clarified that CYP3A12 was primarily responsible for either the oxidation elimination of MDZ or the regioselective oxidation metabolism of DCA into DCA-1ß-ol and DCA-5ß-ol in dog liver microsomes. Six male dogs completed the CYP3A intervention studies including phases of baseline, inhibition (ketoconazole treatments), recovery, and induction (rifampicin treatments). The oral MDZ clearance after a single dose was determined on the last day of the baseline, inhibition, and induction phases, and subjected to correlation analysis with the tertiary oxidation ratios of DCA detected in serum and urine samples. The results confirmed that the predosing serum ratios of DCA oxidation, DCA-5ß-ol/DCA, and DCA-1ß-ol/DCA were significantly and positively correlated both intraindividually and interindividually with oral MDZ clearance. It was therefore concluded that the tertiary oxidation of DCA is predictive of CYP3A activity in beagle dogs. Clinical transitional studies following the preclinical evidence are promising to provide novel biomarkers of the enterohepatic CYP3A activities. SIGNIFICANCE STATEMENT: Drug development, clinical pharmacology, and therapeutics are under insistent demands of endogenous CYP3A biomarkers that avoid unnecessary drug exposure and invasive sampling. This work has provided the first proof-of-concept preclinical evidence that the CYP3A catalyzed tertiary oxidation of deoxycholate, the major circulating secondary bile acid synthesized in the lower gut by bacteria, may be developed as novel in vivo biomarkers of the enterohepatic CYP3A activities.

Predictive and prognostic biomarkers of bone metastasis in breast cancer: current status and future directions.

The most common site of metastasis in breast cancer is the bone, where the balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation is disrupted. This imbalance causes osteolytic bone metastasis in breast cancer, which leads to bone pain, pathological fractures, spinal cord compression, and other skeletal-related events (SREs). These complications reduce patients' quality of life significantly and have a profound impact on prognosis. In this review, we begin by providing a brief overview of the epidemiology of bone metastasis in breast cancer, including current diagnostic tools, treatment approaches, and existing challenges. Then, we will introduce the pathophysiology of breast cancer bone metastasis (BCBM) and the animal models involved in the study of BCBM. We then come to the focus of this paper: a discussion of several biomarkers that have the potential to provide predictive and prognostic value in the context of BCBM-some of which may be particularly compatible with more comprehensive liquid biopsies. Beyond that, we briefly explore the potential of new technologies such as single-cell sequencing and organoid models, which will improve our understanding of tumor heterogeneity and aid in the development of improved biomarkers. The emerging biomarkers discussed hold promise for future clinical application, aiding in the prevention of BCBM, improving the prognosis of patients, and guiding the implementation of personalized medicine.

Compensatory Transition of Bile Acid Metabolism from Fecal Disposition of Secondary Bile Acids to Urinary Excretion of Primary Bile Acids Underlies Rifampicin-Induced Cholestasis in Beagle Dogs.

Drug induced cholestasis (DIC) is complexly associated with dysbiosis of the host-gut microbial cometabolism of bile acids (BAs). Murine animals are not suitable for transitional studies because the murine BA metabolism is quite different from human metabolism. In this work, the rifampicin (RFP) induced cholestasis was established in beagle dogs that have a humanlike BA profile to disclose how RFP affects the host-gut microbial cometabolism of BAs. The daily excretion of BA metabolites in urine and feces was extensively analyzed during cholestasis by quantitative BA profiling along the primary-secondary-tertiary axis. Oral midazolam clearance was also acquired to monitor the RFP-induced enterohepatic CYP3A activities because CYP3A is exclusively responsible for the tertiary oxidation of hydrophobic secondary BAs. RFP treatments caused a compensatory transition of the BA metabolism from the fecal disposition of secondary BAs to the urinary excretion of primary BAs in dogs, resulting in an infantile BA metabolism pattern recently disclosed in newborns. However, the tertiary BAs consistently constituted limitedly in the daily BA excretion, indicating that the detoxification role of the CYP3A catalyzed tertiary BA metabolism was not as strong as expected in this model. Multiple host-gut microbial factors might have contributed to the transition of the BA metabolism, such as inhibition of BA transporters, induction of liver-kidney interplaying detoxification mechanisms, and elimination of gut bacteria responsible for secondary BA production. Transitional studies involving more cholestatic drugs in preclinical animals with a humanlike BA profile and DIC patients may pave the way for understanding the complex mechanism of DIC in the era of metagenomics.