Drug Metabolism Letters - Volume 14, Issue 1, 2021

Background: CYP450 enzymes in the liver have a significant role in the metabolism of xenobiotics. Probe drug strategy is broadly used to evaluate the pharmacodynamic and pharmacokinetic drug/ herb-drug interactions/ food-drug interactions. Probe drugs reveal the exact pathway of drug metabolism in the liver by their targeted tractability property. The CYP3A4 isoenzyme metabolizes the majority of the drugs (65%). Methods: The characteristics of targeted probe drugs were observed from the admetSAR (version2) online database. Results: Midazolam is widely used as a probe drug because of its peculiar character. Midazolam affirms the accurate and consistent prediction of pharmacokinetic mediated drug interactions even in nanogram concentrations with or without a potent CYP3A inhibitor. Remarkably, midazolam is used as a CYP3A4 substrate in the majority of in vivo studies. Conclusion: It is concluded that midazolam shows a good response in all clinical studies because of its lesser half-life and bioavailability when compared with other probe drugs.

Background: In healthy volunteers, the probe drug method is widely practised to assess the pharmacokinetic mediated herb-drug interactions (HDI). We analyzed the clinical evidence of CYP3 A4 probe drug, Midazolam. Methods: Literatures, where Midazolam was used as a probe drug for prediction of herb-drug interaction, were surveyed through an online database such as google scholar, Scopus, Cochrane, PubMed and clinicaltrials.gov. Results: Midazolam was considered a sensitive probe for CYP3A4 substrates due to its bioavailability. We observed that not all the herbs are causing drug interaction. However, significant changes of the Midazolam pharmacokinetics were found after high-dose and long-term intake of some herbs and food supplements, suggesting the induction and/or inhibition of CYP activities. Conclusion: Probe drug technique is one of the easiest ways for predicting CYP enzyme-mediated herb-drug interactions. Midazolam shows a good response in clinical studies because of short halflife and low harmfulness compared with other probe drugs.

Background: Saini et al. recently investigated the pharmacokinetics of darolutamide and its diastereomers in vitro and in vivo in Balb/c mice, reporting higher levels of (S,S)-darolutamide than (S,R)-darolutamide following intravenous or oral dosing, and interconversion of (S,R)-darolutamide to (S,S)-darolutamide. Objective: To present our in vitro and in vivo studies of darolutamide pharmacokinetics in mice, which contrast with the findings of Saini et al. Methods: Nude male Balb/c mice were orally dosed for 7 days with 25, 50, or 100 mg/kg of darolutamide twice daily. Pharmacokinetic parameters in plasma and tissue samples were assessed by liquid chromatography-tandem mass spectrometry. Metabolism and interconversion of darolutamide and its diastereomers were investigated in cryopreserved Balb/c mouse hepatocytes. Protein binding was determined in plasma samples by equilibrium dialysis. Results: On day 7, Cmax was reached 30 min after the last dose. Rapid formation and greater exposure of keto-darolutamide versus darolutamide were observed. Plasma exposure of (S,R)-darolutamide was 3-5-fold higher than that of (S,S)-darolutamide. The fraction of unbound keto-darolutamide was almost 6-fold lower than for darolutamide. In mouse hepatocytes, the conversion of (S,S)- to (S,R)-darolutamide was observed, but the conversion of (S,R)- to (S,S)-darolutamide was not detectable. Back-formation of keto-darolutamide to both diastereomers occurred at low levels. Conclusion: The darolutamide diastereomer ratio changes upon administration in mice and other species due to interconversion through keto-darolutamide. This is not considered clinically relevant since both diastereomers and keto- darolutamide are pharmacologically similar in vitro. Based on the high protein binding of keto-darolutamide, its contribution in vivo in humans is considered low.

Background: Cytochrome P450 (CYP) contributes to a huge collection of medicinal products' Phase I metabolization. We aimed to summarize and investigate the current evidence regarding the frequency of CYP2D6, CYP2C9, CYP2C19, and MDR1 in Saudi Arabia. Methods: A computerized search in four databases was done using the relevant keywords. The screening process was done in two steps; title and abstract screening and full-text screening. Data of demographic and characteristics of included studies and patients were extracted and tabulated. Results: Ten studies were eligible for our criteria and were included in this systematic review. The age of participants ranged between 17-65 years. Only two subjects showed PM phenotype of CYP2C19 in the Saudi population. The most frequent alleles were CYP2C19*1 (62.9%), CYP2C19*2 (11.2%-32%), and CYP2C19*17 (25.7%). The CYP2C19m1 was observed in 97 cases of extensive metabolizing (EM) phenotype CYP2C19. Concerning the CYP2C9, the most frequent alleles were CYP2C9*1 and CYP2C9*2, and the most frequent genotype was CYP2C9*1*1. The CYP2D6*41 allele and C1236T MDR1 were the most frequent allele in this population. Conclusion: The current evidence suggests that Saudi resembled European in the frequency of CYP2C19, Caucasians in both the incidence of CYP2C9 and CYP2C19m1, and the absence of CYP2C19m2. The CYP2D6*41 allele frequency in Saudi is relatively high. We recommend further research to evaluate the basic and clinical relevance of gene polymorphism in such ethnicity.

Background: In order to avoid drug-induced liver injury (DILI), in vitro assays, which enable the assessment of both metabolic activation and immune reaction processes that ultimately result in DILI, are needed. Objective: In this study, recent progress in the application of in vitro assays using cell culture systems is reviewed for potential DILI-causing drugs/xenobiotics and a mechanistic study on DILI, as well as on the limitations of in vitro cell culture systems for DILI research, was carried out. Methods: Information related to DILI was collected through a literature search of the PubMed database. Results: The initial biological event for the onset of DILI is the formation of cellular protein adducts after drugs have been metabolically activated by drug metabolizing enzymes. The damaged peptides derived from protein adducts lead to the activation of CD4⁺ helper T lymphocytes and recognition by CD8⁺ cytotoxic T lymphocytes, which destroy hepatocytes through immunological reactions. Because DILI is a major cause of drug attrition and drug withdrawal, numerous in vitro systems consisting of hepatocytes and immune/inflammatory cells or spheroids of human primary hepatocytes containing non-parenchymal cells have been developed. These cellular-based systems have identified DILI-inducing drugs, with approximately 50% sensitivity and 90% specificity. Conclusion: Different co-culture systems consisting of human hepatocyte-derived cells and other immune/inflammatory cells have enabled the identification of DILI-causing drugs and of the actual mechanisms of action.

Background: Carbon-carbon bond cleavage of a saturated aliphatic moiety is rarely seen in xenobiotic metabolism. Olanexidine (Olanedine®), containing an n-octyl (C8) side chain, was mainly metabolized to various shortened side chain (C4 to C6) acid-containing metabolites in vivo in preclinical species. In liver microsomes and S9, the major metabolites of olanexidine were from multi-oxidation on its n-octyl (C8) side chain. However, the carbon-carbon bond cleavage mechanism of n-octyl (C8) side chain, and enzyme(s) responsible for its metabolism in human remained unknown. Methods: A pair of regioisomers of α-ketol-containing C8 side chain olanexidine analogs (3,2-ketol olanexidine and 2,3-ketol olanexidine) were synthesized, followed by incubation in human liver microsomes, recombinant human cytochrome P450 enzymes or human hepatocytes, and subsequent metabolite identification using LC/UV/MS. Results: Multiple shortened side chain (C4 to C6) metabolites were identified, including C4, C5 and C6- acid and C6-hydroxyl metabolites. Among 19 cytochrome P450 enzymes tested, CYP2D6, CYP3A4 and CYP3A5 were identified to catalyze carbon-carbon bond cleavage. Conclusion: 3,2-ketol olanexidine and 2,3-ketol olanexidine were confirmed as the key intermediates in carbon-carbon bond cleavage. Its mechanism is proposed that a nucleophilic addition of iron-peroxo species, generated by CYP2D6 and CYP3A4/5, to the carbonyl group caused the carbon-carbon bond cleavage between the adjacent hydroxyl and ketone groups. As results, 2,3-ketol olanexidine formed a C6 side chain acid metabolite. While, 3,2-ketol olanexidine formed a C6 side chain aldehyde intermediate, which was either oxidized to a C6 side chain acid metabolite or reduced to a C6 side chain hydroxyl metabolite.

Background: Darolutamide is recently approved for the treatment of non-metastatic castrate resistance prostate cancer. Hitherto, no stereoselective pharmacokinetic data have been published pertaining to darolutamide and its diastereomers in animals or humans. The key aims of the experiment were to examine darolutamide, S,S-darolutamide and S,R-darolutamide with respect to (a) assessment of in vitro metabolic stability and protein binding and (b) characterization of in vivo oral and intravenous pharmacokinetics in mice. Methods: In vitro (liver microsomes stability and protein binding) and in vivo experiments (oral/intravenous dosing to mice) were carried out using darolutamide, S,S-darolutamide and S,Rdarolutamide. Besides, tissue levels of darolutamide, S,S-darolutamide and S,R-darolutamide were measured following oral and intravenous dosing. Appropriate plasma/tissue samples served to determine the pharmacokinetics of various analytes in mice. Liquid chromatography in tandem with mass spectrometry procedures enabled the delineation of the plasma pharmacokinetics, in vitro and tissue uptake data of the various analytes. Results: Chiral inversion was absent in the metabolic stability study. However, darolutamide showed profound stereoselectivity (S,S-darolutamide greater than S,R-darolutamide) after either intravenous or oral dosing. S,R-darolutamide but not S,S-darolutamide showed conversion to its antipode post oral and intravenous dosing to mice. Regardless of oral or intravenous dosing, active keto darolutamide formation was evident after administration of darolutamide, S,S-darolutamide or S,R- darolutamide. Tissue data supported the observations in plasma; however, tissue exposure of darolutamide, S,Sdarolutamide and S,R-darolutamide was much lower as compared to plasma. Conclusion: In lieu of the human pharmacokinetic data, although the administration of diastereomeric darolutamide was justified, it is proposed to delineate the clinical pharmacokinetics of S,Rdarolutamide and S,S-darolutamide relative to darolutamide in future clinical pharmacology studies.

Background: Ezetimibe is a cholesterol-lowering agent with an oral bioavailability of 50% by virtue of its poor solubility and extensive hepatic and intestinal metabolism. Objective: The study aimed to overcome low bioavailability issues of ezetimibe by formulating an oral disintegrating film. Methods: The low solubility of ezetimibe was undertaken, preparing solid dispersions using mannitol, β-cyclodextrin, and urea. The mannitol solid dispersion assimilated oral disintegrating film was prepared and optimized using 2³ factorial design, where the concentration of film formers hydroxypropyl methylcellulose (K5& K15) (X1and X2) and super disintegrant, sodium starch glycolate (X3) was used as factors on the response disintegration time (Y). The films were evaluated for physical properties, time of disintegration, and drug release profiles. Results: Mannitol solid dispersion (1:2 ratio) based on the superior drug content, solubility and in vitro release profile was preferred in film formation. The low crystalline nature of the solid dispersion was very evident by the absence of prominent peaks in the X-Ray diffraction pattern and the reduced peak intensity of melting endotherms. The correlation coefficient (R2) and statistical parameter analysis of variance specify the implication of linear factors on responses, which is apparent from confidence intervals (P-values) less than 0.05. The in vitro release profile of all the eight formulations (F1-F8) in a phosphate buffer solution of pH 6.8 revealed a significant increment in comparison to ezetimibe. Conclusion: The study revealed that the formulation approach could overcome the biopharmaceutical challenge of solubility as well as low bioavailability issues of ezetimibe.

Objective: Previous studies have shown that catabolism of adenosine 5’-triphosphate (ATP) in systemic blood is a potential surrogate biomarker for cardiovascular toxicity. We compared the acute toxicity of high doses of doxorubicin (DOX) and isoproterenol (ISO) on hemodynamics and ATP catabolism in the systemic circulation. Methods: sprague Dawley (SD) rats (n = 8 - 11) were each given either a single dose of 30 mg/kg ISO, or a twice-daily dose of 10 mg/kg of DOX or 4 doses of normal saline (control) by subcutaneous injection. Blood samples were collected up to 6 hours for measuring concentrations of ATP and its catabolites. Hemodynamics was recorded continuously. The difference was considered significant at p < 0.05 (ANOVA). Results: Mortality was 1/8, 5/11, and 0/11 for the DOX, ISO, and control groups, respectively. Systolic blood pressure was significantly lower in the DOX and ISO treated rats than in control measured at the last recorded time (76 ± 9 for DOX vs. 42 ± 8 for ISO vs. 103 ± 5 mmHg for control, p < 0.05 for all). Blood pressure fell gradually after the final injection for both DOX and control groups, but abruptly after ISO, followed by a rebound and then gradual decline till the end of the experiment. Heart rate was significantly higher after ISO, but there were no differences between the DOX and control rats (p > 0.05). RBC concentrations of ADP and AMP, and plasma concentrations of adenosine and uric acid were significantly higher in the ISO group. In contrast, hypoxanthine concentrations were significantly higher in the DOX treated group (p < 0.05). Conclusion: Acute cardiovascular toxicity induced by DOX and ISO may be measured by changes in hemodynamics and breakdown of ATP and adenosine in the systemic circulation, albeit a notable qualitative and quantitative difference was observed.

Background: Food-drug interactions may lead to suppression or induction of drug metabolizing enzymes. Pomegranate is a commonly used fruit in folk medicine all over the world. Data concerning the effect of pomegranate on the activity of UDP-glucuronosyltransferases (UGTs) is scarce. Objective: The purpose of this work was to investigate the effect of pomegranate juice ingestion on the transcription of ugt2b1, ugt2a3, and ugt1a9 in the liver and small intestine of male mice. Methods: Pomegranate juice was administered to 10 male mice for 14 days in drinking bottles instead of water. Ten control mice received water in the drinking bottles. On the 15th day, the mice were sacrificed and the liver and the small intestine were removed. The small intestine was divided into 3 parts. Total mRNA was extracted from samples of these specimens, and cDNA was synthesized by quantitative real-time polymerase chain reaction (RT-PCR) using specific primers for each ugt gene. Results: The ugt1a9 mRNA level was reduced by 2.25-fold in the liver and by 6-, 1.5-, and 3-folds in the first, second and third part of the small intestine, respectively. The ugt2b1 mRNA level in the liver and the third part of the small intestine was not affected, while it was reduced by 3.7- and 3-folds in the first and second parts of the small intestine, respectively. The ugt2a3 mRNA level was not affected in the liver and the 3 parts of the small intestine. Conclusion: Some ugt mRNA levels may be reduced by the ingestion of pomegranate juice, which may reduce the metabolism of their drug substrates. The consequences may be an accumulation of such drugs in the body and enhanced toxicity.