Repaglinide-gemfibrozil drug interaction: inhibition of repaglinide glucuronidation as a potential additional contributing mechanism

AIM

To further explore the mechanism underlying the interaction between repaglinide and gemfibrozil, alone or in combination with itraconazole.

METHODS

Repaglinide metabolism was assessed in vitro (human liver subcellular fractions, fresh human hepatocytes, and recombinant enzymes) and the resulting incubates were analyzed, by liquid chromatography-mass spectrometry (LC-MS) and radioactivity counting, to identify and quantify the different metabolites therein. Chemical inhibitors, in addition to a trapping agent, were also employed to elucidate the importance of each metabolic pathway. Finally, a panel of human liver microsomes (genotyped for UGT1A1*28 allele status) was used to determine the importance of UGT1A1 in the direct glucuronidation of repaglinide.

RESULTS

The results of the present study demonstrate that repaglinide can undergo direct glucuronidation, a pathway that can possibly contribute to the interaction with gemfibrozil. For example, [³H]-repaglinide formed glucuronide and oxidative metabolites (M2 and M4) when incubated with primary human hepatocytes. Gemfibrozil effectively inhibited (∼78%) both glucuronide and M4 formation, but had a minor effect on M2 formation. Concomitantly, the overall turnover of repaglinide was also inhibited (∼80%), and was completely abolished when gemfibrozil was co-incubated with itraconazole. These observations are in qualitative agreement with the in vivo findings. UGT1A1 plays a significant role in the glucuronidation of repaglinide. In addition, gemfibrozil and its glucuronide inhibit repaglinide glucuronidation and the inhibition by gemfibrozil glucuronide is time-dependent.

CONCLUSIONS

Inhibition of UGT enzymes, especially UGT1A1, by gemfibrozil and its glucuronide is an additional mechanism to consider when rationalizing the interaction between repaglinide and gemfibrozil.     

Genotoxicity studies of organically grown broccoli (Brassica oleracea var. italica) and its interactions with urethane,methyl methanesulfonate and 4-nitroquinoline-1-oxide genotoxicity in the wing spot test of Drosophila melanogaster

Broccoli (Brassica oleracea var. italica) has been defined as a cancer preventive food. Nevertheless, broccoli contains potentially genotoxic compounds as well. We performed the wing spot test of Drosophila melanogaster in treatments with organically grown broccoli (OGB) and co-treatments with the promutagen urethane (URE), the direct alkylating agent methyl methanesulfonate (MMS) and the carcinogen 4-nitroquinoline-1-oxide (4-NQO) in the standard (ST) and high bioactivation (HB) crosses with inducible and high levels of cytochrome P450s (CYPs), respectively. Larvae of both crosses were chronically fed with OGB or fresh market broccoli (FMB) as a non-organically grown control, added with solvents or mutagens solutions. In both crosses, the OGB added with Tween–ethanol yielded the expected reduction in the genotoxicity spontaneous rate. OGB co-treatments did not affect the URE effect, MMS showed synergy and 4-NQO damage was modulated in both crosses. In contrast, FMB controls produced damage increase; co-treatments modulated URE genotoxicity, diminished MMS damage, and did not change the 4-NQO damage. The high dietary consumption of both types of broccoli and its protective effects in D. melanogaster are discussed.     

Permissive and suppressive effects of dexamethasone on enzyme induction in hepatocyte co-cultures

1. Steroids are known to act as permissive factors in hepatocytes. This study shows that dexamethasone (DEX) is a permissive factor for induction of CYP2B1/2, CYP3A1, CYP2A1 and probably also CYP2C11 in cultures with primary rat hepatocytes. 2. The induction factor of phenobarbital (PB)-induced formation of 16 β -hydroxytestosterone (OHT), a testosterone biotransformation product predominantly formed by CYP2B1, is increased 18-fold by the addition of 32 nM DEX to the culture medium. Interestingly, higher concentrations of DEX up to 1000 nM led to a concentration-dependent maximally 5-fold decrease (p = 0.002) of phenobarbital-induced 16 β -OHT formation compared with the effect observed with 32 nM DEX. Thus, DEX shows permissive and suppressive effects on enzyme induction depending on the concentration of the glucocorticoid. 3. Qualitatively similar but smaller permissive and suppressive effects of DEX were observed for PB-induced CYP3A1 activity as evidenced by formation of 2 β -, 6 β - and 15 β -OHT. 4. DEX is a permissive factor for induction of CYP2A1 activity by 3-methylcholanthrene (3MC), as evidenced by the formation of 7 α -OHT. Without addition of DEX, 3MC did not induce formation of 7 α -OHT, whereas an almost 3-fold induction occurred in the presence of DEX. In contrast to CYP2B and CYP3A, concentrations up to 1000 nM DEX were not suppressive for the induction of CYP2A1. 5. We described recently a technique that allows preparation of cultures from cryopreserved hepatocytes. An almost identical influence of dexamethasone on enzyme induction was observed here in cultures from cryopreserved compared with freshly isolated hepatocytes. 6. Cultures with primary hepatocyte cultures represent a well-established technique for the study of drug-drug interactions. However, a large interlaboratory variation is known. Our study provides evidence that differences in glucocorticoid concentration in the culture medium contribute to this variation.     

Assessment of UDP-glucuronosyltransferase catalyzed formation of Picroside II glucuronide in microsomes of different species and recombinant UGTs

1. This study compared the hepatic glucuronidation of Picroside II in different species and characterized the glucuronidation activities of human intestinal microsomes (HIMs) and recombinant human UDP-glucuronosyltransferases (UGTs) for Picroside II.

2. The rank order of hepatic microsomal glucuronidation activity of Picroside II was rat > mouse > human > dog. The intrinsic clearance of Picroside II hepatic glucuronidation in rat, mouse and dog was about 10.6-, 6.0- and 2.3-fold of that in human, respectively.

3. Among the 12 recombinant human UGTs, UGT1A7, UGT1A8, UGT1A9 and UGT1A10 catalyzed the glucuronidation. UGT1A10, which are expressed in extrahepatic tissues, showed the highest activity of Picroside II glucuronidation (K_m?=?45.1 μM, V_max?=?831.9 pmol/min/mg protein). UGT1A9 played a primary role in glucuronidation in human liver microsomes (HLM; K_m?=?81.3 μM, V_max?=?242.2 pmol/min/mg protein). In addition, both mycophenolic acid (substrate of UGT1A9) and emodin (substrate of UGT1A8 and UGT1A10) could inhibit the glucuronidation of Picroside II with the half maximal inhibitory concentration (IC₅₀) values of 173.6 and 76.2 μM, respectively.

4. Enzyme kinetics was also performed in HIMs. The K_m value of Picroside II glucuronidation was close to that in recombinant human UGT1A10 (K_m?=?58.6 μM, V_max?=?721.4 pmol/min/mg protein). The intrinsic clearance was 5.4-fold of HLMs. Intestinal UGT enzymes play an important role in Picroside II glucuronidation in human.

     

Comparison of Inhibition Capability of Scutellarein and Scutellarin Towards Important Liver UDP‐Glucuronosyltransferase (UGT) Isoforms

Scutellarin is an important bioactive flavonoid extracted from Erigeron breviscapus (Vant.) Hand‐Mazz, and scutellarein is the corresponding aglycone of scutellarin. The present study aims to compare the inhibition potential of scutellarin and scutellarein towards several important UDP‐glucuronosyltransferase (UGT) isoforms, including UGT1A1, UGT1A6, UGT1A9 and UGT2B7. It was demonstrated that scutellarein exerted stronger inhibition towards the tested UGT isoforms than scutellarin. Furthermore, the inhibition kinetic type and parameters (K_i) were determined for the scutellarein's inhibition towards these UGT isoforms. Competitive inhibition of scutellarein towards all these UGT isoforms was demonstrated, and the K_i values were calculated to be 0.02, 5.0, 5.8 and 35.9 μM for UGT1A1, 1A6, 1A9 and 2B7, respectively. Using in vivo maximum plasma concentration of scutellarein in rat, the in vitro–in vivo extrapolation was performed to predict in vivo situation, indicating the most possible in vivo adverse effects due to the inhibition of scutellarein towards UGT1A1. All these results remind us to monitor the utilization of scutellarin and scutellarein, and the herbs containing these two components. Copyright © 2013 John Wiley & Sons, Ltd.     

Identification of UGTs and BCRP as potential pharmacokinetic determinants of the natural flavonoid alpinetin

1. Alpinetin is a natural flavonoid showing a variety of pharmacological effects such as anti-inflammatory, anti-tumor and hypolipidemic activities. Here, we aim to determine the roles of UDP-glucuronosyltransferases (UGTs) and breast cancer resistance protein (BCRP) in disposition of alpinetin.

2. Glucuronidation potential of alpinetin was evaluated using pooled human liver microsomes (pHLM), pooled human intestine microsomes (pHIM) and expressed UGT enzymes supplemented with the cofactor UDPGA. Activity correlation analyses with a bank of individual HLMs were performed to identify the main contributing UGT isozymes in hepatic glucuronidation of alpinetin. The effect of BCRP on alpinetin disposition was assessed using HeLa cells overexpressing UGT1A1 (HeLa1A1) cells.

3. Alpinetin underwent extensive glucuronidation in pHLM and pHIM, generating one glucuronide metabolite. Of 12 test UGT enzymes, UGT1A3 was the most active one toward alpinetin with an intrinsic clearance (CL_int?=?V_max/K_m) value of 66.5?μl/min/nmol, followed by UGT1A1 (CL_int?=?48.6?μl/min/nmol), UGT1A9 (CL_int?=?21.0?μl/min/nmol), UGT2B15 (CL_int?=?16.7?μl/min/nmol) and UGT1A10 (CL_int?=?1.60?μl/min/nmol). Glucuronidation of alpinetin was significantly correlated with glucuronidation of estradiol (an activity marker of UGT1A1), chenodeoxycholic acid (an activity marker of UGT1A3), propofol (an activity marker of UGT1A9) and 5-hydroxyrofecoxib (an activity marker of UGT2B15), confirming the important roles of UGT1A1, UGT1A3, UGT1A9 and UGT2B15 in alpinetin glucuronidation. Inhibition of BCRP by its specific inhibitor Ko143 significantly reduced excretion of alpinetin glucuronide, leading to a significant decrease in cellular glucuronidation of alpinetin.

4. Our data suggest UGTs and BCRP as two important determinants of alpinetin pharmacokinetics.

     

Spectrum of UGT1A1 mutations in Crigler-Najjar (CN) syndrome patients: identification of twelve novel alleles and genotype-phenotype correlation

Crigler-Najjar syndrome types I and II (CN1 and CN2) are usually inherited as autosomal recessive conditions and are characterized by non-hemolytic unconjugated hyperbilirubinaemia. CN1 is the most severe form, associated with the absence of hepatic bilirubin-uridinediphosphoglucuronate glucuronosyltransferase (UGT1A1) activity. CN2 presents intermediate levels of hyperbilirubinaemia as a result of an incomplete deficiency of hepatic UGT1A1 activity. Here, we present the analysis of UGT1A1 gene in 31 unrelated Crigler-Najjar (CN) syndrome patients. This analysis allowed us to identify 22 mutations, 12 of which were not previously described, expanding the spectrum of known UGT1 mutations to 77. Novel mutations, considered pathogenic, including one nonsense mutation, two altered splice sites, one single base deletion and nine missense mutations were identified in coding exons of the UGT1A1gene and flanking introns. Several novel missense mutations localize in critical domain of UGT1A1 enzyme. In addition, the evaluation of Gilbert-type promoter of UGT1A1in Crigler-Najjar (CN) syndrome patients was performed. The polymorphisms of the promoter region can modify the UGT1A1 mutation phenotype. This study represents the molecular characterization of the largest cohort of Italian Crigler-Najjar Gilbert syndrome patients studied so far; increase the mutational spectrum of UGT1A1 allelic variants worldwide and provide a new insight useful for clinical diagnosis and genetic counseling.