Pharmacokinetics and brain distribution studies of 6-hydroxykynurenic acid and its structural modified compounds.

OBJECTIVES: 6-Hydroxykynurenic acid (6-HKA) is an organic acid component in extracts of Ginkgo biloba leaves and acts as a major contributor to neurorestorative effects, while its oral bioavailability was low. Therefore, using prodrug method to improve the bioavailability and brain content of 6-HKA is significant. METHODS: Three structural modified compounds of 6-HKA were synthesized, and ultra performance liquid chromatography-tandem mass spectrometry methods for quantification of these structural modified compounds in rat plasma and rat brain homogenate were established and comprehensively validated. The methods were effectively applied to investigate the effects of structural modification on apparent permeability coefficients in cells, the pharmacokinetics and the brain distribution in rats. KEY FINDINGS: The results illustrated that esterification can greatly improve the apparent permeability coefficient and bioavailability of 6-HKA. Comparing with direct oral administration of 6-HKA, the bioavailability of isopropyl ester was greatly improved (from 3.96 ± 1.45% to 41.8 ± 15.3%), and the contents of 6-HKA in rat brains (49.7 ± 9.2 ng/g brain) were significantly higher after oral administration. CONCLUSIONS: The bioavailability and the brain content of 6-HKA can be improved by the prodrug method. Among three structural modified compounds, isopropyl-esterified 6-HKA was the most promising treatment.

The characterisation of the in vitro metabolism and transport of 6-hydroxykynurenic acid, an important constituent of Ginkgo biloba extracts.

6-Hydroxykynurenic acid (6-HKA) is a nitrogen-containing phenolic acid compound in Ginkgo biloba leaves. The pharmacological activities of 6-HKA have been reported and shown that 6-HKA has the potential to become a therapeutic drug and may play an important role in the treatment of nervous system diseases. However, there are few studies on the drug metabolism and transport of 6-HKA. The aim of this study is to investigate the in vitro metabolism of 6-HKA and its interaction with multiple important drug transporters.The in vitro metabolism experiments in the present study demonstrate that 6-HKA might not undergo phase-I or phase-II metabolism in hepatic microsomes/S9 of rats. In addition, some drug transporters, including OAT1/3, OCT2, MDR1, OATP1B1, MATE1/2K and OCTN2, were investigated. The cellular uptake assays indicate that 6-HKA exhibits inhibition to the transport of classical substrates mediated by OAT3, OCT2, MATE2K and OCTN2 but has no significant effect on the transport of substrates mediated by MDR1, OAT1, OATP1B1 or MATE1. Further investigation of cellular accumulation assays shows that 6-HKA might be the substrate of OAT3, but not OCT2 or OCTN2. The bidirectional transport study suggests that 6-HKA is not a substrate of MDR1.The information about the in vitro metabolism of 6-HKA and the interaction between 6-HKA and some transporters will help us to better understand the pharmacokinetic properties of 6-HKA and provide reference for its pharmacodynamics, DDIs and drug-food interactions studies.

Influence of organic anion transporter 1/3 on the pharmacokinetics and renal excretion of ginkgolides and bilobalide.

ETHNOPHARMACOLOGICAL RELEVANCE: The major terpene lactones of ginkgo biloba extract (GBE) include ginkgolide A, B, C and bilobalide are used for the protection of cardiovascular, cerebrovascular and neurodegenerative diseases. Terpene lactones are orally bioavailable and predominantly eliminated via the renal pathway. However, information on the transporters involved in the pharmacokinetics (PK) and renal excretion of terpene lactones is limited. AIM OF THE STUDY: The objective of this study is to assess the role of OAT1/3 which are important transporters in the human kidney in the PK and renal excretion ginkgolide A, B, C and bilobalide. MATERIALS AND METHODS: Uptake of ginkgolide A, B, C and bilobalide in Madin-Darby Canine Kidney (MDCK) and human embryonic kidney 293 (HEK293) cells overexpressing OAT1 or OAT3, respectively were studied. To verify the result from in vitro cell models, the studies on PK, kidney accumulation and urinary excretion of ginkgolide A, B, C and bilobalide were carried out in rats. RESULTS: The result showed that ginkgolide A, B, C and bilobalide are low-affinity substrates of OAT1/3. Following co-administration with probenecid, a typical inhibitor of OAT1/3, the rat plasma concentrations of ginkgolide A, B, C and bilobalide increased significantly. AUC showed a significant increase in the probenecid-treated rats compared to control rats (893.48 vs. 1123.85, 314.91 vs. 505.74, and 2724.97 vs. 3096.40 µg/L*h for ginkgolide A, B and bilobalide, respectively), while the clearance of these compounds significantly decreased. The accumulation of ginkgolide A, B and bilobalide in the kidney of the probenecid-treated rats was reduced by 1.8, 2.4, and 1.5-fold, respectively; further reducing the cumulative urinary recovery of these compounds. CONCLUSION: The findings indicated that ginkgolide A, B and bilobalide are excreted via OAT1/3-mediated transport in the kidney and OAT1/3 inhibitor significantly influence the PK ginkgolides and bilobalide.

The metabolism and hepatotoxicity of ginkgolic acid (17 : 1) in vitro.

Pharmacological activities and adverse side effects of ginkgolic acids (GAs), major components in extracts from the leaves and seed coats of Ginkgo biloba L, have been intensively studied. However, there are few reports on their hepatotoxicity. In the present study, the metabolism and hepatotoxicity of GA (17 : 1), one of the most abundant components of GAs, were investigated. Kinetic analysis indicated that human and rat liver microsomes shared similar metabolic characteristics of GA (17 : 1) in phase I and II metabolisms. The drug-metabolizing enzymes involved in GA (17 : 1) metabolism were human CYP1A2, CYP3A4, UGT1A6, UGT1A9, and UGT2B15, which were confirmed with an inhibition study of human liver microsomes and recombinant enzymes. The MTT assays indicated that the cytotoxicity of GA (17 : 1) in HepG2 cells occurred in a time- and dose-dependent manner. Further investigation showed that GA (17 : 1) had less cytotoxicity in primary rat hepatocytes than in HepG2 cells and that the toxicity was enhanced through CYP1A- and CYP3A-mediated metabolism.

Mechanism for ginkgolic acid (15 : 1)-induced MDCK cell necrosis: Mitochondria and lysosomes damages and cell cycle arrest.

Ginkgolic acids (GAs), primarily found in the leaves, nuts, and testa of ginkgo biloba, have been identified with suspected allergenic, genotoxic and cytotoxic properties. However, little information is available about GAs toxicity in kidneys and the underlying mechanism has not been thoroughly elucidated so far. Instead of GAs extract, the renal cytotoxicity of GA (15 : 1), which was isolated from the testa of Ginkgo biloba, was assessed in vitro by using MDCK cells. The action of GA (15 : 1) on cell viability was evaluated by the MTT and neutral red uptake assays. Compared with the control, the cytotoxicity of GA (15 : 1) on MDCK cells displayed a time- and dose-dependent manner, suggesting the cells mitochondria and lysosomes were damaged. It was confirmed that GA (15 : 1) resulted in the loss of cells mitochondrial trans-membrane potential (ΔΨm). In propidium iodide (PI) staining analysis, GA (15 : 1) induced cell cycle arrest at the G0/G1 and G2/M phases, influencing on the DNA synthesis and cell mitosis. Characteristics of necrotic cell death were observed in MDCK cells at the experimental conditions, as a result of DNA agarose gel electrophoresis and morphological observation of MDCK cells. In conclusion, these findings might provide useful information for a better understanding of the GA (15 : 1) induced renal toxicity.