Effect of polyethylene glycol 400 on the pharmacokinetics and tissue distribution of baicalin by intravenous injection based on the enzyme activity of UGT1A8/1A9.

Baicalin (BG) is a bioactive flavonoid extracted from the dried root of the medicinal plant, Scutellaria radix (SR) (dicotyledonous family, Labiatae), and has several biological activities. Polyethylene glycol 400 (PEG400) has been used as a suitable solvent for several traditional Chinese medicines (TCM) and is often used as an excipient for the compound preparation of SR. However, the drug-excipient interactions between BG and PEG400 are still unknown. Herein, we evaluated the effect of a single intravenous PEG400 administration on the BG levels of rats using pharmacokinetic and tissue distribution studies. A liver microsome and recombinant enzyme incubation system were used to further confirm the interaction mechanism between PEG400 and UDP-glucuronosyltransferases (UGTs) (UGT1A8 and UGT1A9). The pharmacokinetic study demonstrated that following the co-intravenous administration of PEG400 and BG, the total clearance (CLz) of BG in the rat plasma decreased by 101.60% (p < 0.05), whereas the area under the plasma concentration-time curve (AUC)0-t and AUC0-inf increased by 144.59% (p < 0.05) and 140.05% (p < 0.05), respectively. Additionally, the tissue distribution study showed that the concentration of BG and baicalein-6-O-ß-D-glucuronide (B6G) in the tissues increased, whereas baicalein (B) in the tissues decreased, and the total amount of BG and its metabolites in tissues altered following the intravenous administration of PEG400. We further found that PEG400 induced the UGT1A8 and UGT1A9 enzyme activities by affecting the maximum enzymatic velocity (Vmax) and Michaelis-Menten constant (Km) values of UGT1A8 and UGT1A9. In conclusion, our results demonstrated that PEG400 interaction with UGTs altered the pharmacokinetic behaviors and tissue distribution characteristics of BG and its metabolites in rats.

UGT1A1 and UGT1A9 Are Responsible for Phase II Metabolism of Tectorigenin and Irigenin In Vitro.

Tectorigenin and irigenin are biologically active isoflavones of Belamcanda chinensis (L.) DC. Previous studies indicated that both compounds could be metabolized in vivo; however, the kinetic parameters of enzymes involved in the metabolization of tectorigenin and irigenin have not been identified. The aim of this study was to investigate UGTs involved in the glucuronidation of tectorigenin and irigenin and determine enzyme kinetic parameters using pooled human liver microsomes (HLMs) and recombinant UGTs. Glucuronides of tectorigenin and irigenin were identified using high-performance liquid chromatography (HPLC) coupled with mass spectrometry and quantified by HPLC using a response factor method. The results showed that tectorigenin and irigenin were modified by glucuronidation in HLMs. One metabolite of tectorigenin (M) and two metabolites of irigenin (M1 and M2) were detected. Chemical inhibition and recombinant enzyme experiments revealed that several enzymes could catalyze tectorigenin and irigenin glucuronidation. Among them, UGT1A1 and UGT1A9 were the primary enzymes for both tectorigenin and irigenin; however, the former mostly produced irigenin glucuronide M1, while the latter mostly produced irigenin glucuronide M2. These findings suggest that UGT1A1 and UGT1A9 were the primary isoforms metabolizing tectorigenin and irigenin in HLMs, which could be involved in drug-drug interactions and, therefore, should be monitored in clinical practice.

The pharmaceutical excipient PEG400 affect the absorption of baicalein in Caco-2 monolayer model by interacting with UDP-glucuronosyltransferases and efflux transport proteins.

The bioavailability of drugs is often related to intestinal metabolism and transport mechanisms. In previous studies, pharmaceutical excipients were recognized as inert substances in clinical safety evaluations. However, a large number of studies have shown that pharmaceutical excipients regulate the metabolism and transport of drugs in the body and improve the bioavailability. The pharmaceutical excipient polyethylene glycol 400 (PEG400) as a good solubilizer and surfactant has the potential to improve the bioavailability of drugs. The combined action of UDP-glucuronosyltransferases (UGTs) and efflux transport proteins is responsible for the intestinal disposition and poor bioavailability of baicalein. Our aim is to study the effect of PEG400 on the absorption of baicalein on the Caco-2 monolayer, and confirm the interaction of PEG400 with UGTs (UGT1A8 and UGT1A9) and efflux transports. We initially found that baicalein in the Caco-2 monolayer would be metabolized into glucuronide conjugates BG and B6G under the action of UGT1A8 and UGT1A9 on the endoplasmic reticulum membrane, and then mainly excreted to different sides by acting of MRP and BCRP. The addition of PEG400 significantly accelerated the metabolism of B in Caco-2 cells and increased the penetration of BG and B6G. Furthermore, PEG400 also significantly decreased the efflux ratio of BG and B6G, which was the evidence of the interaction with the efflux transporters. In the in vitro intestinal microsome regeneration system, low concentration PEG400 decreased the Km value of UGT1A8 and UGT1A9 (key enzymes that mediate the production of BG and B6G); high concentration PEG400 enhanced the Vmax value of UGT1A8 and UGT1A9. In conclusion, our results determined that PEG400 interacted with some UGTs and efflux transporters, which were the main factors affecting the absorption of baicalein.

Overexpression of MRP3 in HeLa-UGT1A9 Cells Enhances Glucuronidation Capability of the Cells.

BACKGROUND: The interplay between phase II enzymes and efflux transporters leads to extensive metabolism and low systemic bioavailability of flavonoids. OBJECTIVE: In this study, the dynamic interplay between multiple UGTs and multiple efflux transporters that occur inside the cells was fully investigated. METHODS: A new HeLa-UGT1A9-MRP3 cell was established to overexpress two dominant efflux transporters MRP3 and BCRP, and two UGT isoforms UGT1A9 and UGT1A3. The metabolism and glucuronides excretion for a model flavonoid genistein were determined in HeLa-UGT1A9-MRP3 cells and HeLa-UGT1A9-Con cells that overexpressed one UGT (1A9) and one efflux transporter (BCRP). RESULTS: The excretion rate grew nearly 6-fold, cellular clearance of glucuronides increased about 3-fold, and fraction of genistein metabolized (fmet) increased (14%, p<0.01) in the new cells. Small interfering (siRNA)-mediated MRP3 functional knockdown resulted in marked decreases in the excretion rates (26%-78%), intracellular amounts (56%-93%), and cellular clearance (54%-96%) in both cells, but the magnitude of the differences in HeLa- UGT1A9-Con cells was relatively small. Reductions in fmet values were similarly moderate (11%-14%). In contrast, UGT1A9 knockdown with siRNA caused large decreases in the excretion rates (46%-88%), intracellular amounts (80%-97%), cellular clearance (80%-98%) as well as fmet value (33%-43%, p<0.01) in both UGT1A9 cells. Comparisons of the kinetic parameters and profiles of genistein glucuronidation as well as UGT mRNA expression suggest that HeLa-UGT1A9-MRP3 has increased expression of both MRP3 and UGT1A3. CONCLUSION: The newly engineered HeLa-UGT1A9-MRP3 cells is an appropriate model to study the kinetic interplay between multiple UGTs and efflux transporters, and a promising biosynthetic tool to obtain flavonoid glucuronides of high purity.

Metabolism and Mass Balance of the Novel Nonsteroidal Androgen Receptor Inhibitor Darolutamide in Humans.

The biotransformation and excretion of darolutamide were investigated in a phase I study. Six healthy male volunteers received a single dose of 300 mg 14C-darolutamide as an oral solution in the fasted state. Plasma, urine, and feces samples were analyzed for mass balance evaluation by liquid scintillation counting (LSC). Metabolite profiling and identification were determined using liquid chromatography mass-spectrometry with off-line radioactivity detection using LSC. Complete mass balance was achieved, with mean radioactivity recovery of 95.9% within 168 hours (63.4% in urine, 32.4% in feces). The administered 1:1 ratio of (S,R)- and (S,S)-darolutamide changed to approximately 1:5, respectively, in plasma. Darolutamide and the oxidation product, keto-darolutamide, were the only components quantifiable by LSC in plasma, accounting for 87.4% of total radioactivity, with a 2.1-fold higher plasma exposure for keto-darolutamide. Aside from darolutamide, the most prominent metabolites in urine were O-glucoronide (M-7a/b) and N-glucuronide (M-15a/b), as well as pyrazole sulfates (M-29, M-24) and glucuronides (M-21, M-22) resulting from oxidative cleavage of the parent. The darolutamide diastereomers were mainly detected in feces. In vitro assays showed that darolutamide metabolism involves a complex interplay between oxidation and reduction, as well as glucuronidation. Interconversion of the diastereomers involves oxidation to keto-darolutamide, primarily mediated by CYP3A4, followed by reduction predominantly catalyzed by cytosolic reductase(s), with aldo-keto reductase 1C3 playing the major role. The latter reaction showed stereoselectivity with preferential formation of (S,S)-darolutamide. SIGNIFICANCE STATEMENT: The metabolism and excretion of darolutamide in humans revealed that oxidation (CYP3A4) and glucuronidation (UGT1A9, UGT1A1) were the main metabolic routes of elimination. Direct excretion also contributed to overall clearance. The two pharmacologically equipotent diastereomers of darolutamide interconvert primarily via oxidation to the active metabolite keto-darolutamide, followed by reduction predominantly by cytosolic reductase(s). The latter reaction showed stereoselectivity with preferential formation of (S,S)-darolutamide. Data indicate a low drug-drug interaction potential of darolutamide with inducers or inhibitors of metabolizing enzymes.

Model-Informed Pediatric Dose Selection for Dapagliflozin by Incorporating Developmental Changes.

This analysis reports a quantitative modeling and simulation approach for oral dapagliflozin, a primarily uridine diphosphate-glucuronosyltransferase (UGT)-metabolized human sodium-glucose cotransporter 2 selective inhibitor. A mechanistic dapagliflozin physiologically based pharmacokinetic (PBPK) model was developed using in vitro metabolism and clinical pharmacokinetic (PK) data and verified for context of use (e.g., exposure predictions in pediatric subjects aged 1 month to 18 years). Dapagliflozin exposure is challenging to predict in pediatric populations owing to differences in UGT1A9 ontogeny maturation and paucity of clinical PK data in younger age groups. Based on the exposure-response relationship of dapagliflozin, twofold acceptance criteria were applied between model-predicted and observed drug exposures and PK parameters (area under the curve and maximum drug concentration) in various scenarios, including monotherapy in healthy adults (single/multiple dose), monotherapy in hepatically or renally impaired patients, and drug-drug interactions with UGT1A9 modulators, such as mefenamic acid and rifampin. The PBPK model captured the observed exposure within twofold of the observed monotherapy data in adults and adolescents and in special population. As a guide to determining dosing regimens in pediatric studies, the verified PBPK model, along with UGT enzyme ontogeny maturation understanding, was used for predictions of dapagliflozin monotherapy exposures in pediatric subjects aged 1 month to 18 years that best matched exposure in adult patients with a 10-mg single dose of dapagliflozin.