Interplay of Ritonavir-Boosted Oral Cabazitaxel with the Organic Anion-Transporting Polypeptide (OATP) Uptake Transporters and Carboxylesterase 1 in Mice.

Intravenously administered chemotherapeutic cabazitaxel is used for palliative treatment of prostate cancer. An oral formulation would be more patient-friendly and reduce the need for hospitalization. We therefore study determinants of the oral pharmacokinetics of cabazitaxel in a ritonavir-boosted setting, which reduces the CYP3A-mediated first-pass metabolism of cabazitaxel. We here assessed the role of organic anion-transporting polypeptides (OATPs) in the disposition of orally boosted cabazitaxel and its active metabolites, using the Oatp1a/b-knockout and the OATP1B1/1B3-transgenic mice. These transporters may substantially affect plasma clearance and hepatic and intestinal drug disposition. The pharmacokinetics of cabazitaxel and DM2 were not significantly affected by Oatp1a/b and OATP1B1/1B3 activity. In contrast, the plasma AUC0-120 min of DM1 in Oatp1a/b-/- was 1.9-fold (p < 0.05) higher than that in wild-type mice, and that of docetaxel was 2.4-fold (p < 0.05) higher. We further observed impaired hepatic uptake and intestinal disposition for DM1 and docetaxel in the Oatp-ablated strains. None of these parameters showed rescue by the OATP1B1 or -1B3 transporters in the humanized mouse strains, suggesting a minimal role of OATP1B1/1B3. Ritonavir itself was also a potent substrate for mOatp1a/b, showing a 2.9-fold (p < 0.0001) increased plasma AUC0-120 min and 3.5-fold (p < 0.0001) decreased liver-to-plasma ratio in Oatp1a/b-/- compared to those in wild-type mice. Furthermore, we observed the tight binding of cabazitaxel and its active metabolites, including docetaxel, to plasma carboxylesterase (Ces1c) in mice, which may complicate the interpretation of pharmacokinetic and pharmacodynamic mouse studies. Collectively, these results will help to further optimize (pre)clinical research into the safety and efficacy of orally applied cabazitaxel.

In vitro and in vivo metabolic activation and hepatotoxicity of chlorzoxazone mediated by CYP3A.

Chlorzoxazone (CZX), a benzoxazolone derivative, has been approved for the treatment of musculoskeletal disorders to relieve localized muscle spasm. However, its idiosyncratic toxicity reported in patients brought attention, particularly for hepatotoxicity. The present study for the first time aimed at the relationship between CZX-induced hepatotoxicity and identification of oxirane intermediate resulting from metabolic activation of CZX. Two N-acetylcysteine (NAC) conjugates (namely M1 and M2) and two glutathione (GSH) conjugates (namely M3 and M4) were detected in rat & human microsomal incubations with CZX (200 µM) fortified with NAC or GSH, respectively. The formation of M1-M4 was NADPH-dependent and these metabolites were also observed in urine or bile of SD rats given CZX intragastrically at 10 mg/kg or 25 mg/kg. NAC was found to attach at C-6' of the benzo group of M1 by sufficient NMR data. CYPs3A4 and 3A5 dominated the metabolic activation of CZX. The two GSH conjugates were also observed in cultured rat primary hepatocytes after exposure to CZX. Inhibition of CYP3A attenuated the susceptibility of hepatocytes to the cytotoxicity of CZX (10-400 µM). The in vitro and in vivo studies provided solid evidence for the formation of oxirane intermediate of CZX. This would facilitate the understanding of the underlying mechanisms of toxic action of CZX.

Physiologically based pharmacokinetic modeling for confirming the role of CYP3A1/2 and P-glycoprotein in detoxification mechanism between glycyrrhizic acid and aconitine in rats.

Fuzi, an effective common herb, is often combined with Gancao to treat disease in clinical practice with enhancing its efficacy and alleviating its toxicity. The major toxic and bioactive compounds in Fuzi and Gancao are aconitine (AC) and glycyrrhizic acid (GL), respectively. This study aims to elucidate detoxification mechanism between AC and GL from pharmacokinetic perspective using physiologically based pharmacokinetic (PBPK) model. In vitro experiments exhibited that AC was mainly metabolized by CYP3A1/2 in rat liver microsomes and transported by P-glycoprotein (P-gp) in Caco-2 cells. Kinetics assays showed that the Km and Vmax of AC towards CYP3A1/2 were 2.38 µM and 57.3 pmol/min/mg, respectively, whereas that of AC towards P-gp was 11.26 µM and 147.1 pmol/min/mg, respectively. GL markedly induced the mRNA expressions of CYP3A1/2 and MDR1a/b in rat primary hepatocytes. In vivo studies suggested that the intragastric and intravenous administration of GL significantly reduced systemic exposure of AC by 27% and 33%, respectively. Drug-drug interaction (DDI) model of PBPK predicted that co-administration of GL would decrease the exposure of AC by 39% and 45% in intragastric and intravenous dosing group, respectively. The consistency between predicted data and observed data confirmed that the upregulation of CYP3A1/2 and P-gp was the crucial detoxification mechanism between AC and GL. Thus, this study provides a demonstration for elucidating the compatibility mechanisms of herbal formula using PBPK modeling and gives support for the clinical co-medication of Fuzi and Gancao.

Docetaxel, cyclophosphamide, and epirubicin: application of PBPK modeling to gain new insights for drug-drug interactions.

The new adjuvant chemotherapy of docetaxel, epirubicin, and cyclophosphamide has been recommended for treating breast cancer. It is necessary to investigate the potential drug-drug Interactions (DDIs) since they have a narrow therapeutic window in which slight differences in exposure might result in significant differences in treatment efficacy and tolerability. To guide clinical rational drug use, this study aimed to evaluate the DDI potentials of docetaxel, cyclophosphamide, and epirubicin in cancer patients using physiologically based pharmacokinetic (PBPK) models. The GastroPlus™ was used to develop the PBPK models, which were refined and validated with observed data. The established PBPK models accurately described the pharmacokinetics (PKs) of three drugs in cancer patients, and the predicted-to-observed ratios of all the PK parameters met the acceptance criterion. The PBPK model predicted no significant changes in plasma concentrations of these drugs during co-administration, which was consistent with the observed clinical phenomenon. Besides, the verified PBPK models were then used to predict the effect of other Cytochrome P450 3A4 (CYP3A4) inhibitors/inducers on these drug exposures. In the DDI simulation, strong CYP3A4 modulators changed the exposure of three drugs by 0.71-1.61 fold. Therefore, patients receiving these drugs in combination with strong CYP3A4 inhibitors should be monitored regularly to prevent adverse reactions. Furthermore, co-administration of docetaxel, cyclophosphamide, or epirubicin with strong CYP3A4 inducers should be avoided. In conclusion, the PBPK models can be used to further investigate the DDI potential of each drug and to develop dosage recommendations for concurrent usage by additional perpetrators or victims.

Induction of human hepatic cytochrome P-450 3A4 expression by antifungal succinate dehydrogenase inhibitors.

Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these agrochemicals, the interactions of 15 SDHIs with expression and activity of human cytochrome P-450 3A4 (CYP3A4), a major hepatic drug metabolizing enzyme, were investigated in vitro. 12/15 SDHIs, i.e., bixafen, boscalid, fluopyram, flutolanil, fluxapyroxad, furametpyr, isofetamid, isopyrazam, penflufen, penthiopyrad, pydiflumetofen and sedaxane, were found to enhance CYP3A4 mRNA expression in human hepatic HepaRG cells and primary human hepatocytes exposed for 48 h to 10 µM SDHIs, whereas 3/15 SDHIs, i.e., benzovindiflupyr, carboxin and thifluzamide, were without effect. The inducing effects were concentrations-dependent for boscalid (EC50=22.5 µM), fluopyram (EC50=4.8 µM) and flutolanil (EC50=53.6 µM). They were fully prevented by SPA70, an antagonist of the Pregnane X Receptor, thus underlining the implication of this xenobiotic-sensing receptor. Increase in CYP3A4 mRNA in response to SDHIs paralleled enhanced CYP3A4 protein expression for most of SDHIs. With respect to CYP3A4 activity, it was directly inhibited by some SDHIs, including bixafen, fluopyram, fluxapyroxad, isofetamid, isopyrazam, penthiopyrad and sedaxane, which therefore appears as dual regulators of CYP3A4, being both inducer of its expression and inhibitor of its activity. The inducing effect nevertheless predominates for these SDHIs, except for isopyrazam and sedaxane, whereas boscalid and flutolanil were pure inducers of CYP3A4 expression and activity. Most of SDHIs appear therefore as in vitro inducers of CYP3A4 expression in cultured hepatic cells, when, however, used at concentrations rather higher than those expected in humans in response to environmental or dietary exposure to these agrochemicals.

Impact of CYP3A5 Polymorphisms on Pediatric Asthma Outcomes.

Genetic variation among inhaled corticosteroid (ICS)-metabolizing enzymes may affect asthma control, but evidence is limited. This study tested the hypothesis that single-nucleotide polymorphisms (SNPs) in Cytochrome P450 3A5 (CYP3A5) would affect asthma outcomes. Patients aged 2-18 years with persistent asthma were recruited to use the electronic AsthmaTracker (e-AT), a self-monitoring tool that records weekly asthma control, medication use, and asthma outcomes. A subset of patients provided saliva samples for SNP analysis and participated in a pharmacokinetic study. Multivariable regression analysis adjusted for age, sex, race, and ethnicity was used to evaluate the impact of CYP3A5 SNPs on asthma outcomes, including asthma control (measured using the asthma symptom tracker, a modified version of the asthma control test or ACT), exacerbations, and hospital admissions. Plasma corticosteroid and cortisol concentrations post-ICS dosing were also assayed using liquid chromatography-tandem mass spectrometry. Of the 751 patients using the e-AT, 166 (22.1%) provided saliva samples and 16 completed the PK study. The e-AT cohort was 65.1% male, and 89.6% White, 6.0% Native Hawaiian, 1.2% Black, 1.2% Native American, 1.8% of unknown race, and 15.7% Hispanic/Latino; the median age was 8.35 (IQR: 5.51-11.3) years. CYP3A5*3/*3 frequency was 75.8% in White subjects, 50% in Native Hawaiians and 76.9% in Hispanic/Latino subjects. Compared with CYP3A5*3/*3, the CYP3A5*1/*x genotype was associated with reduced weekly asthma control (OR: 0.98; 95% CI: 0.97-0.98; p < 0.001), increased exacerbations (OR: 6.43; 95% CI: 4.56-9.07; p < 0.001), and increased asthma hospitalizations (OR: 1.66; 95% CI: 1.43-1.93; p < 0.001); analysis of 3/*3, *1/*1 and *1/*3 separately showed an allelic copy effect. Finally, PK analysis post-ICS dosing suggested muted changes in cortisol concentrations for patients with the CYP3A5*3/*3 genotype, as opposed to an effect on ICS PK. Detection of CYP3A5*3/3, CYPA35*1/*3, and CYP3A5*1/*1 could impact inhaled steroid treatment strategies for asthma in the future.

Bone marrow niche chemoprotection of metastatic solid tumors mediated by CYP3A4.

BACKGROUND: The bone/bone marrow is one of the most common sites for metastatic solid tumors. Moreover, the tumor microenvironment is an essential part of cancer homeostasis. Previously, it was shown that cytochrome P450 enzymes (CYPs) are present in the bone marrow (BM) microenvironment, particularly in the mesenchymal stroma cells, at levels comparable to those of hepatocytes. It was found that the CYPs play important roles in nurturing and maintaining normal hematopoietic stem cells as well as multiple myeloma and leukemia cells, including protecting them from toxic insults. It was hypothesized that the CYPs in the BM microenvironment might play a similar role in solid tumors metastatic to bone. METHODS: The interaction between the BM microenvironment and malignant cells that routinely metastasize to the bone (lung, breast, and prostate cancer) was modeled. Via genetic engineering and pharmacological approaches, the role of stromal cytochrome P450 3A4 (CYP3A4) in drug resistance promoted by the BM microenvironment in niche-cancer models in vitro and in vivo was interrogated. RESULTS: BM stroma protected prostate, breast, and lung cancer cells from cytotoxic chemotherapy. Stromal CYP3A4 was at least partially responsible for this protection in vitro and in vivo. Moreover, inhibiting CYP3A4 with clarithromycin overcame the stroma-mediated chemoresistance toward prostate, breast, and lung cancer cells. CONCLUSIONS: These results suggest that, similar to observations from hematologic malignancies, the BM microenvironment, through expression of CYPs, creates a sanctuary site from chemotherapy for metastatic solid tumors. Targeting these sanctuaries holds promise for eradicating bone metastasis in solid tumors.

Enhancement of the Oral Availability of Cabazitaxel Using the Cytochrome P450 3A (CYP3A) Inhibitor Ritonavir in Mice.

There is currently great interest in developing oral taxanes due to their lower costs and greater patient friendliness. We here wanted to test whether oral ritonavir, a cytochrome P450 3A (CYP3A) inhibitor, could boost the pharmacokinetics and tissue distribution of orally administered cabazitaxel (10 mg/kg) in male wild-type, Cyp3a-/-, and Cyp3aXAV (transgenic overexpression of human CYP3A4 in liver and intestine) mice. Ritonavir was initially administered at a dose of 25 mg/kg, but lower dosages of 10 and 1 mg/kg were also studied to assess the remaining amount of boosting, aiming to minimize possible side effects. Compared to the respective vehicle groups, plasma exposure of cabazitaxel (AUC0-24h) was enhanced 2.9-, 10.9-, and 13.9-fold in wild-type mice and 1.4-, 10.1-, and 34.3-fold in Cyp3aXAV mice by treatment with 1, 10, and 25 mg/kg ritonavir, respectively. Upon treatment with 1, 10, and 25 mg/kg of ritonavir, the peak plasma concentration (Cmax) was increased by 1.4-, 2.3-, and 2.8-fold in wild-type mice, while it increased by 1.7-, 4.2-, and 8.0-fold in Cyp3aXAV mice, respectively. AUC0-24h and Cmax remained unchanged in Cyp3a-/-. Biotransformation of cabazitaxel to its active metabolites still took place when coadministered with ritonavir, but this process was delayed due to the Cyp3a/CYP3A4 inhibition. These data indicate that CYP3A is the primary limiting factor in the plasma exposure to cabazitaxel and that cabazitaxel oral bioavailability could be dramatically enhanced by coadministration of an effective CYP3A inhibitor such as ritonavir. These findings could be a starting point for the setup of a clinical study, which would be needed to verify the boosting of cabazitaxel by ritonavir in humans.

Cluster headache polygenetic risk and known functional variants of CYP3A4 are not associated with treatment response.

BACKGROUND AND PURPOSE: The response to cluster headache treatments has a high interindividual variation. To date, treatment response has only been assessed by a candidate gene approach and no investigations into metabolic pathways have been performed. Our aim was to investigate the association between the polygenetic risk of cluster headache and treatment response to first-line cluster headache treatments as well as known functional variants of CYP3A4 and the response to verapamil. Further, it was aimed to replicate previous single nucleotide polymorphisms found to be associated with treatment response in cluster headache and/or migraine. METHODS: In, 508 cluster headache patients diagnosed according to the International Classification of Headache Disorders were genotyped and participated in a semi-structured interview to evaluate treatment response. Polygenetic risk scores were calculated by the effect retrieved from a meta-analysis of the latest two genome-wide association studies on cluster headache. RESULTS: Inferior treatment response to oxygen, triptans and verapamil is associated with chronicity of cluster headache were confirmed but no evidence was found that a response could be predicted by a high genetic risk of cluster headache. Likewise, verapamil response was not associated with functional variants of CYP3A4. No support of the genetic variants previously reported to be associated with treatment response to triptans or verapamil was found. CONCLUSION: The clinically relevant variation in treatment response for cluster headache was not influenced by genetic factors in the present study.

Evaluation of effects of indoxyl sulfate and parathyroid hormone on CYP3A activity considering the influence of CYP3A5 gene polymorphisms.

AIMS: Indoxyl sulfate and parathyroid hormone (PTH), which accumulate in chronic kidney disease (CKD), have been reported to reduce cytochrome P450(CYP)3A activity. Homozygotes of the CYP3A5*3 allele have reduced CYP3A5 activity compared to carriers of at least one CYP3A5*1 allele. 4ß-Hydroxycholesterol (4ß-OHC) has been established as an endogenous substrate reflecting CYP3A activity. 4ß-OHC is produced through hydroxylation by CYP3A4 and CYP3A5 and by autoxidation of cholesterol, whereas 4α-hydroxycholesterol (4α-OHC) is produced solely by autoxidation of cholesterol. This study focused on CKD patients and evaluated the effects of plasma indoxyl sulfate and intact-PTH concentrations on plasma 4ß-OHC concentration, 4ß-OHC/total cholesterol ratio and 4ß-OHC-4α-OHC, with consideration of the influence of CYP3A5 polymorphism. METHODS: Sixty-three CKD patients were analysed and divided into CYP3A5 carrier group (n = 26) and non-carrier group (n = 37). RESULTS: Plasma indoxyl sulfate significantly correlated inversely with 4ß-OHC concentration and with 4ß-OHC-4α-OHC in both the CYP3A5*1 carrier group (r = -0.42, P = .034; r = -0.39, P = .050, respectively) and the non-carrier group (r = -0.45, P = .0054; r = -0.39, P = .019, respectively). However, multiple regression analysis did not identify plasma indoxyl sulfate concentration as a significant independent factor associated with any of the CYP3A activity indices. There was no significant correlation between plasma intact-PTH concentration and any of the CYP3A activity indices. CONCLUSIONS: The present results suggest that plasma indoxyl sulfate and intact-PTH concentrations do not have clinically significant effects on CYP3A activity in patients with CKD.

Design, synthesis and biological evaluation of chalcone derivatives as potent and orally active hCYP3A4 inhibitors.

Human cytochrome P450 3A4 (hCYP3A4), one of the most important drug-metabolizing enzymes, catalyze the metabolic clearance of ∼50% therapeutic drugs. CYP3A4 inhibitors have been used for improving the in vivo efficacy of hCYP3A4-substrate drugs. However, most of existing hCYP3A4 inhibitors may trigger serious adverse effects or undesirable effects on endogenous metabolism. This study aimed to discover potent and orally active hCYP3A4 inhibitors from chalcone derivatives and to test their anti-hCYP3A4 effects both in vitro and in vivo. Following three rounds of screening and structural optimization, the isoquinoline chalcones were found with excellently anti-hCYP3A4 effects. SAR studies showed that introducing an isoquinoline ring on the A-ring significantly enhanced anti-CYP3A4 effect, generating A10 (IC50 = 102.10 nM) as a promising lead compound. The 2nd round of SAR studies showed that introducing a substituent group at the para position of the carbonyl group on B-ring strongly improved the anti-CYP3A4 effect. As a result, C6 was identified as the most potent hCYP3A4 inhibitor (IC50 = 43.93 nM) in human liver microsomes (HLMs). C6 also displayed potent anti-hCYP3A4 effect in living cells (IC50 = 153.00 nM), which was superior to the positive inhibitor ketoconazole (IC50 = 251.00 nM). Mechanistic studies revealed that C6 could potently inhibit CYP3A4-catalyzed N-ethyl-1,8-naphthalimide (NEN) hydroxylation in a competitive manner (Ki = 30.00 nM). Moreover, C6 exhibited suitable metabolic stability in HLMs and showed good safety profiles in mice. In vivo tests demonstrated that C6 (100 mg/kg, orally administration) significantly increased the AUC(0-inf) of midazolam by 3.63-fold, and strongly prolonged its half-life by 1.66-fold compared with the vehicle group in mice. Collectively, our findings revealed the SARs of chalcone derivatives as hCYP3A4 inhibitors and offered several potent chalcone-type hCYP3A4 inhibitors, while C6 could serve as a good lead compound for developing novel, orally active CYP3A4 inhibitors with improved druglikeness properties.

Mechanism-based inactivation of cytochrome P450 3A by evodol.

Evodol is one of the furanoids isolated from the fruits of Evodia rutaecarpa that has been widely prescribed for the treatment of gastrointestinal diseases in China. The aim of this study was to investigate the inhibitory effect of evodol on CYP3A.A 30-min preincubation of evodol with human liver microsomes raised an obvious left IC50 shift, 3.9-fold for midazolam 1'-hydroxylation and 3.2-fold for testosterone 6ß-hydroxylation. Evodol inactivated CYP3A in a time-, concentration- and NADPH-dependent manner, with KI and kinact of 5.1 µM and 0.028 min-1 for midazolam 1'-hydroxylation and 3.0 µM and 0.022 min-1 for testosterone 6ß-hydroxylation.Co-incubation of ketoconazole attenuated the inactivation while the inclusion of glutathione (GSH) and catalase/superoxide dismutase displayed no such protection.cis-Butene-1, 4-dial (BDA) intermediate derived from evodol were trapped by glutathione and N-acetyl-lysine in microsomes and characterised by HR-MS spectra. The BDA intermediate was believed to play a key role in CYP3A inactivation. CYP3A4 and 2C9 were the primary enzymes contributing to the bioactivation of evodol.To sum up, for the first time evodol was characterised as a mechanism-based inactivator of CYP3A.

Therapeutic Drug Monitoring of Blood Sirolimus and Tacrolimus Concentrations for Polypharmacy Management in a Lymphangioleiomyomatosis Patient Taking Two Cytochrome P450 3A Inhibitors.

Patients with lymphangioleiomyomatosis (LAM) and lung transplantations are treated with multiple drugs, such as tacrolimus, mycophenolate mofetil, prednisolone, and itraconazole, for long-term suppression of rejection response and prevention of infection. Additional drugs are required when lung transplant recipients develop graft complications. Therefore, managing polypharmacy is critical because of drug-drug interactions caused by various factors, including drug-metabolizing enzymes such as cytochrome P450 3A (CYP3A). The patient was a 48-year-old woman (height 144.9 cm and weight 38.4 kg) who underwent lung transplantation for LAM. Mycophenolate mofetil, tacrolimus (target blood concentration, 4.0-8.0 ng/mL), and prednisolone were administered for immunosuppression, and itraconazole and clarithromycin were administered to manage graft infection. The patient developed unilateral lymphedema, predominantly in the left leg; therefore, sirolimus was initiated with a target blood concentration of 3.0-5.0 ng/mL. In addition to 1.0 mg/day of sirolimus, tacrolimus (0.3 mg/day), itraconazole (100 mg/day), and clarithromycin (800 mg/day) were added. Blood sirolimus concentrations ranged from 18.8 to 36.9 ng/mL on days 6 to 9; thus, treatment with sirolimus was stopped because of over-target blood concentrations. Blood concentrations of sirolimus and tacrolimus were successfully managed without adverse events using therapeutic drug monitoring (TDM) and azole anti-fungal substitution of azithromycin instead of clarithromycin although sirolimus concentration was relatively lower compared to the target range. Thereby, frequent TDM, management of polypharmacy that influences CYP3A activity, and possibly CYP3A genotyping should be appropriately conducted for personalized medicine.

Research progress regarding CYP3A gene family in gastric cancer. / CYP3AåŸºå› å®¶æ—åœ¨èƒƒç™Œä¸­çš„ç ”ç©¶è¿›å±•.

Cytochrome P450 family 3 subfamily A (CYP3A), a major member of cytochrome P450 (CYP) family, is one of the most important drug metabolizing enzymes in human. CYP3A includes 4 gene subtypes (CYP3A4, CYP3A5, CYP3A7, and CYP3A43), which is involved in 60% of drug metabolism in the human. It is not only widely distributed in normal tissues, but also significantly overexpressed in various tumor tissues. Recently, CYP3A has attracted great attention due to its involvement in the progression from chronic atrophic gastritis to gastric cancer, as well as the differential metabolism and resistance of chemotherapeutic drugs. Targeting CYP3A gene mediated-prodrug provides new ideas for the treatment of gastric cancer and is expected to become a new target for the diagnosis and treatment of gastric cancer.

Sensitive UHPLC-MS/MS quantification method for 4ß- and 4α-hydroxycholesterol in plasma for accurate CYP3A phenotyping.

4ß-Hydroxycholesterol (4ß-OHC) is formed by Cytochrome P450 (CYP)3A and has drawn attention as an endogenous phenotyping probe for CYP3A activity. However, 4ß-OHC is also increased by cholesterol autooxidation occurring in vitro due to dysregulated storage and in vivo by oxidative stress or inflammation, independent of CYP3A activity. 4α-hydroxycholesterol (4α-OHC), a stereoisomer of 4ß-OHC, is also formed via autooxidation of cholesterol, not by CYP3A, and thus may have clinical potential in reflecting the state of cholesterol autooxidation. In this study, we establish a sensitive method for simultaneous quantification of 4ß-OHC and 4α-OHC in human plasma using ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Plasma samples were prepared by saponification, two-step liquid-liquid extraction, and derivatization using picolinic acid. Intense [M+H]+ signals for 4ß-OHC and 4α-OHC di-picolinyl esters were monitored using electrospray ionization. The assay fulfilled the requirements of the US Food and Drug Administration guidance for bioanalytical method validation, with a lower limit of quantification of 0.5 ng/ml for both 4ß-OHC and 4α-OHC. Apparent recovery rates from human plasma ranged from 88.2% to 101.5% for 4ß-OHC, and 91.8% to 114.9% for 4α-OHC. Additionally, matrix effects varied between 86.2% and 117.6% for 4ß-OHC and between 89.5% and 116.9% for 4α-OHC. Plasma 4ß-OHC and 4α-OHC concentrations in healthy volunteers, stage 3-5 chronic kidney disease (CKD) patients, and stage 5D CKD patients as measured by the validated assay were within the calibration ranges in all samples. We propose this novel quantification method may contribute to accurate evaluation of in vivo CYP3A activity.

ABCB1 limits brain exposure of the KRASG12C inhibitor sotorasib, whereas ABCB1, CYP3A, and possibly OATP1a/1b restrict its oral availability.

Sotorasib (Lumakras™) is the first FDA-approved KRASG12C inhibitor for treatment of patients with non-small cell lung cancer (NSCLC) carrying this mutation. Using genetically modified mouse models, we studied the influence of the efflux transporters ABCB1 and ABCG2, the OATP1a/1b uptake transporters, and the CYP3A drug-metabolizing enzyme complex on the plasma pharmacokinetics and tissue distribution of oral sotorasib. In vitro, sotorasib was a potent substrate for human ABCB1 and a modest substrate for mouse Abcg2, but not for human ABCG2. In vivo, the brain-to-plasma ratio of sotorasib (40 mg/kg) was highly increased in Abcb1a/1b-/- (5.9-fold) and Abcb1a/1b;Abcg2-/- (7.6-fold) compared to wild-type mice, but not in single Abcg2-/- mice. Upon coadministering elacridar, an ABCB1/ABCG2 inhibitor, sotorasib brain accumulation increased 7.5-fold, approaching the levels observed in Abcb1a/1b-deficient mice. No acute CNS toxicity emerged upon boosting of the sotorasib exposure. In Oatp1a/1b-deficient mice, we observed a 2-fold reduction in liver disposition compared to wild-type mice, although these uptake transporters had no noticeable impact on sotorasib plasma exposure. However, plasma exposure was limited by mouse Cyp3a and human CYP3A4, as the AUC0-4 h in Cyp3a-/- mice was increased by 2.5-fold compared to wild-type mice, and subsequently strongly decreased (by 3.9-fold) in Cyp3aXAV mice transgenically overexpressing human CYP3A4 in liver and intestine. Collectively, the oral availability of sotorasib was markedly limited by CYP3A and possibly also by ABCB1 and OATP1a/b, whereas its brain accumulation was strongly restricted by ABCB1. The obtained results may help to further optimize the safety and efficacy of sotorasib in clinical use.

The CYP3A5 and ABCB1 Gene Polymorphisms in Kidney Transplant Patients and Establishment of Initial Daily Tacrolimus Dosing Formula.

BACKGROUND: Tacrolimus is an immunosuppressive drug used to prevent organ rejections. Many factors could influence blood concentration of tacrolimus. OBJECTIVE: To detect genotypes of cytochrome P450 3A5 (CYP3A5) and ABCB1 in kidney transplant patients and establish initial daily tacrolimus dosing formula based on genotypes of CYP3A5 and ABCB1 and patients' clinical parameters. METHODS: Sequence specific primer polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism were used to detect genotypes of CYP3A5 and ABCB1. The blood cell, procalcitonin, C-reactive protein, height, weight, age, gender and other clinical parameters were recorded. Multiple linear regression analysis and Pearson correlation analysis were used to conduct date analysis. RESULTS: 102 cases were enrolled in cohort 1, and there were 10 cases of CYP3A5 *1/*1 (9.8%), 28 cases of CYP3A5 *1/*3 (27.5%), and 64 cases of CYP3A5 *3/*3 (62.7%). The distributions of ABCB1 C3435T genotype were CC 36 (35.3%), CT 52 (51.0%), and TT 14 (13.7%). The distributions of ABCB1 G2677T/A genotype were GG 39 (38.2%), GT 40 (39.2%), and TT 23 (22.5%). The formula was 7.499 + (0.053 × Weight) - (0.029 × Hemoglobin concentration) - (1.045 × CYP3A5 genotype) (CYP3A5 genotype: *1/*1 type inputs 0, *1/*3 type inputs 1, *3/*3 type inputs 2). The predicted doses from the established formula had a significant correlation (r = 0.605) with actual clinical doses (P < 0.05). CONCLUSION AND RELEVANCE: Hemoglobin concentration, weight, and CYP3A5 genotype should be considered using tacrolimus. The initial daily tacrolimus dosing formula established can make a good prediction.

Comparative Muscle Tolerability of Different Types and Intensities of Statins: A Network Meta-Analysis of Double-Blind Randomized Controlled Trials.

PURPOSE: The benefits of statins for ischemic cardio-cerebrovascular diseases are well known. However, concerns around muscle adverse events still exist. We therefore aimed to compare the muscle safety of individual statins in adults. METHODS: PubMed, Embase, Cochrane Central Register of Controlled Trials and Web of Science were searched to include double-blind randomized controlled trials (RCTs) comparing one statin with another or with control treatment. Pairwise meta-analyses and network meta-analyses were undertaken with Stata 14.0 software. Relative risk (RR) with 95% confidence intervals (CIs) was adopted for each outcome. RESULTS: A total of 83 RCTs were included. In the pairwise meta-analysis, statins were significantly associated with only a slight increase in muscle symptoms compared with control (RR=1.05; 95% CI=1.01-1.09). In the drug-level network meta-analyses, no statistically significant difference was found between individual statins in the incidence of muscle symptoms, myalgia, myopathy, rhabdomyolysis, creatine kinase (CK) >10 times the upper limit of normal (ULN) or discontinuation due to muscle adverse events. In the dose-level network meta-analyses, there were no statistically significant dose-dependent effects on any outcomes except that moderate-intensity statins had a higher incidence of muscle symptoms than control (RR=1.13; 95% CI=1.01-1.27). Moderate simvastatin (RR=6.57; 95% CI=1.26-34.41) and moderate pravastatin (RR=5.96; 95% CI=1.00-35.44) had a statistically significantly higher incidence of CK >10×ULN compared with moderate atorvastatin. Lipophilic statins and statins metabolized by liver cytochrome P450 3A4 were not associated with an increased risk of muscle adverse events. CONCLUSION: Statins may be generally safe on muscle. Moderate atorvastatin may be superior to equivalent simvastatin and pravastatin in muscle tolerability.

Highly sensitive simultaneous quantification of indoxyl sulfate and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid in human plasma using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry.

Indoxyl sulfate and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid are uremic toxins that accumulate in renal failure and have been reported to decrease the activities of the drug-metabolizing enzyme cytochrome P450 3A and the drug transporter organic anion transporting polypeptides 1B, respectively. In this study, we established and validated an assay for simultaneous quantification of indoxyl sulfate and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid in human plasma. The samples were pretreated by solid-phase extraction, and measured by ultra-high-performance liquid chromatography-tandem mass spectrometry. The validation results for this assay were within the acceptable limits recommended by the US Food and Drug Administration, with a lower limit of quantitation of 0.05 µg/mL for both indoxyl sulfate and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid. Recovery rates of indoxyl sulfate and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid corrected by internal standard were 100.7-101.9 and 100.2-101.3%, respectively. Matrix effects of indoxyl sulfate and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid corrected by internal standard were 101.1-105.5 and 97.0-103.8%, respectively. The validated assay was used to analyze indoxyl sulfate and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid concentrations in the plasma samples of healthy volunteers and patients with chronic kidney disease. All the measured plasma indoxyl sulfate and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid concentrations were within the calibration ranges. This novel method may contribute to predicting the activities of drug-metabolizing enzymes and drug transporters in individual patients.

TNP and its analogs: Modulation of IP6K and CYP3A4 inhibition.

Inositol hexakisphosphate kinase (IP6K) is an important mammalian enzyme involved in various biological processes such as insulin signalling and blood clotting. Recent analyses on drug metabolism and pharmacokinetic properties on TNP (N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl)purine), a pan-IP6K inhibitor, have suggested that it may inhibit cytochrome P450 (CYP450) enzymes and induce unwanted drug-drug interactions in the liver. In this study, we confirmed that TNP inhibits CYP3A4 in type I binding mode more selectively than the other CYP450 isoforms. In an effort to find novel purine-based IP6K inhibitors with minimal CYP3A4 inhibition, we designed and synthesised 15 TNP analogs. Structure-activity relationship and biochemical studies, including ADP-Glo kinase assay and quantification of cell-based IP7 production, showed that compound 9 dramatically reduced CYP3A4 inhibition while retaining IP6K-inhibitory activity. Compound 9 can be a tool molecule for structural optimisation of purine-based IP6K inhibitors.