Involvement of human CYP1A isoenzymes in the metabolism and drug interactions of riluzole in vitro

Cytochrome P450 (CYP) and uridine diphosphate glucuronosyltransferase (UGT) isoenzymes involved in riluzole oxidation and glucuronidation were characterized in (1) kinetic studies with human hepatic microsomes and isoenzyme-selective probes and (2) metabolic studies with genetically expressed human CYP isoenzymes from transfected B-lymphoblastoid and yeast cells. In vitro incubation of [14C]riluzole (15 microM) with human hepatic microsomes and NADPH or UDPGA cofactors resulted in formation of N-hydroxyriluzole (K(m) = 30 microM) or an unidentified glucuroconjugate (K(m) = 118 microM). Human microsomal riluzole N-hydroxylation was most strongly inhibited by the CYP1A2 inhibitor alpha-naphthoflavone (IC50 = 0.42 microM). Human CYP1A2-expressing yeast microsomes generated N-hydroxyriluzole, whereas human CYP1A1-expressing yeast microsomes generated N-hydroxyriluzole, two additional hydroxylated derivatives and an O-dealkylated derivative. CYP1A2 was the only genetically expressed human P450 isoenzyme in B-lymphoblastoid microsomes to metabolize riluzole. Riluzole glucuronidation was inhibited most potently by propofol, a substrate for the human hepatic UGT HP4 (UGT1.8/9) isoenzyme. In vitro, human hepatic microsomal hydroxylation of riluzole (15 microM) was weakly inhibited by amitriptyline, diclofenac, diazepam, nicergoline, clomipramine, imipramine, quinine and enoxacin (IC50 approximately 200-500 microM) and cimetidine (IC50 = 940 microM). Riluzole (1 and 10 microM) produced a weak, concentration-dependent inhibition of CYP1A2 activity and showed competitive inhibition of methoxyresorufin O-demethylase. Thus, riluzole is predominantly metabolized by CYP1A2 in human hepatic microsomes to N-hydroxyriluzole; extrahepatic CYP1A1 can also be responsible for the formation of several other metabolites. Direct glucuronidation is a relatively minor metabolic route. In vivo, riluzole is unlikely to exhibit significant pharmacokinetic drug interaction with coadministered drugs that undergo phase I metabolism.     

A case of multifocal Langerhans cell granulomatosis: a BAL follow up study

UDP-glucuronosyltransferase (UGT) 2B9, isolated from a cynomolgus monkey liver cDNA library, is 89% identical to human UGT2B7 in primary amino acid sequence, and the two expressed enzymes were previously shown to catalyze the glucuronidation of many common endogenous substrates. The purpose of the present study was to characterize the reactivity of expressed UGT2B9 with important therapeutic agents and other xenobiotics. UGT2B9, stably expressed in human embryonic kidney 293 cells, catalyzes the 3-O- and 6-O-glucuronidation of morphine and the 6-O-glucuronidation of codeine. A number of other morphinan (e.g. naloxone, naltrexone, and nalorphine) and oripavine (e.g. buprenorphine) derivatives are substrates for this enzyme. In general, morphinan derivatives are glucuronidated at higher rates, compared with oripavines; however, glucuronidation efficiency values (Vmax/KM) for the compounds are similar. Stably expressed UGT2B9 also catalyzes the glucuronidation of profen nonsteroidal anti-inflammatory drugs, fibrate hypolipidemic agents, and straight-chain fatty acids at the carboxylic acid moiety. Monoterpenoid alcohols and propanolol are glucuronidated at aliphatic hydroxyl positions. Expressed UGT2B9 exhibits enantioselective glucuronidation for (R/S)-ibuprofen, (R/S)-propanolol, and (+)/(-)-menthol. The data suggest that monkey UGT2B9 and human UGT2B7 are functionally similar.     

Cyclin D1 protein is overexpressed in hyperplasia and intraductal carcinoma of the breast

Irinotecan (CPT-11) is a promising antitumor agent, recently approved for use in patients with metastatic colorectal cancer. Its active metabolite, SN-38, is glucuronidated by hepatic uridine diphosphate glucuronosyltransferases (UGTs). The major dose-limiting toxicity of irinotecan therapy is diarrhea, which is believed to be secondary to the biliary excretion of SN-38, the extent of which is determined by SN-38 glucuronidation. The purpose of this study was to identify the specific isoform of UGT involved in SN-38 glucuronidation. In vitro glucuronidation of SN-38 was screened in hepatic microsomes from normal rats (n = 4), normal humans (n = 25), Gunn rats (n = 3), and patients (n = 4) with Crigler-Najjar type I (CN-I) syndrome. A wide intersubject variability in in vitro SN-38 glucuronide formation rates was found in humans. Gunn rats and CN-I patients lacked SN-38 glucuronidating activity, indicating the role of UGT1 isoform in SN-38 glucuronidation. A significant correlation was observed between SN-38 and bilirubin glucuronidation (r = 0.89; P = 0.001), whereas there was a poor relationship between para-nitrophenol and SN-38 glucuronidation (r = 0.08; P = 0.703). Intact SN-38 glucuronidation was observed only in HK293 cells transfected with the UGT1A1 isozyme. These results demonstrate that UGT1A1 is the isoform responsible for SN-38 glucuronidation. These findings indicate a genetic predisposition to the metabolism of irinotecan, suggesting that patients with low UGT1A1 activity, such as those with Gilbert's syndrome, may be at an increased risk for irinotecan toxicity.     

UDP-glucuronosyltransferases in human intestinal mucosa

While UDP-glucuronosyltransferases (UGTs) are known to be expressed at high levels in human liver, relatively little is known about extrahepatic expression. In the present study, UGT2B family isoforms involved in the glucuronidation of steroid hormones and bile acids have been characterized in microsomes prepared from jejunum, ileum and colon from six human subjects. Glucuronidation of androsterone and testosterone was highly significant and increased from proximal to distal intestine. In contrast, hyodeoxycholic acid was glucuronidated at a low level in jejunum and ileum and activity was barely detectable in colon. No significant glucuronidation of lithocholic acid was found. Small phenols were glucuronidated with much lower activity than found in liver. High levels of UGT protein were detected with polyclonal anti-rat androsterone- and testosterone-UGT antibodies, whereas UGT2B4, a major hepatic hyodeoxycholic acid-specific UGT, was undetectable using a highly specific anti-human UGT2B4 antibody. Screening for RNA expression by RT-PCR confirmed the absence of UGT2B4 and UGT1A6 and showed expression of UGT2B7, a hepatic isoform shown to glucuronidate androsterone, in all intestinal segments. To our knowledge, the presence of functional androsterone and testosterone directed isoforms in human intestine is a novel finding which supports the idea that the intestinal tract functions as a steroid-metabolizing organ and plays a significant role in steroid hormone biotransformation.     

Glucuronidation of retinoids by rat recombinant UDP: glucuronosyltransferase 1.1 (bilirubin UGT)

Rat liver recombinant BR1UGT1.1 was found to have significant activity toward retinoid substrates. UGT1.1 glucuronidation activity was 91 +/- 18 pmol/mg x min for atRA and 113 +/- 19 pmol/mg x min for 5,6-epoxy-atRA. The apparent K(M) and V(max) of atRA acid glucuronidation by UGT1.1 were 59.1 +/- 5.4 microM and 158 +/- 43 pmol/mg x min, respectively. SDS-PAGE and Western blot analysis of UGT1.1-transfected HK293 membrane proteins photolabeled with [11,12-3H]atRA revealed a protein of approximately 56 kDa that was labeled by [3H]atRA, detected by anti-pNP UGT antibody and not present in membranes from nontransfected HK293 cells. Liver microsomes from Gunn rats, which lack UGT1.1, had significant activity toward atRA (111 +/- 28 pmol/mg x min).     

Appendiceal inflammation in ulcerative colitis

The epoxyalkanoyl derivatives were designed and synthesized as ACE inhibitors. Coupling of unsaturated carboxylic acids with amino acids and following epoxidation with dimethyldioxirane gave the epoxyalkanoyls with high yield. The inhibitory activity of synthesized compounds on angiotensin converting enzyme was IC50 values of 0.06-5.5 microM.     

Drug glucuronidation by human renal UDP-glucuronosyltransferases

The UDP-glucuronosyltransferases catalyse the conjugation of glucuronic acid to a wide variety of endobiotics and xenobiotics, representing one of the major conjugation reactions in the conversion of both exogenous (e.g. drugs and pesticides) and endogenous compounds (e.g. bilirubin and steroid hormones). The liver is the major site of glucuronidation, however a number of extrahepatic tissues exhibit particular UDP-glucuronosyltransferase activities. The present study was undertaken to assess the human renal UDP-glucuronosyltransferase system. Enzymatic analysis of human kidney showed that a limited number of UDP-glucuronosyltransferase isoforms were expressed in this tissue. However the level of renal UGT activity towards the anaesthetic propofol was higher compared with human liver. The glucuronidation of propofol is catalysed by UGT1A8/9 suggesting higher levels of this isoform in the kidney. Immunoblot analysis revealed two major UDP-glucuronosyltransferase immunopositive bands to be present in human kidney as compared to four major bands in human liver. The human kidney was capable of conjugating various structurally diverse drugs and xenobiotics.     

Potent inhibitors of acyl-CoA:cholesterol acyltransferase. 2. Structure-activity relationships of novel N-(2,2-dimethyl-2,3-dihydrobenzofuran-7-yl)amides

Novel N-(2,2-dimethyl-2,3-dihydrobenzofuran-7-yl)amide derivatives 1 were synthesized and tested for their ability to inhibit rabbit small intestinal ACAT (acyl-CoA:cholesterol acyltransferase) and lower serum total cholesterol in cholesterol-fed rats. Among the synthesized compounds, N-(2,2,4,6-tetramethyl-2,3-dihydrobenzofuran-7-yl)amide derivatives showed potent ACAT inhibitory activity. The synthesis and structure-activity relationships of these compounds are described. A methyl group at position 6 of the 2,3-dihydrobenzofuran moiety was important for potent ACAT inhibitory activity. In the series of N-(2,2,4,6-tetramethyl-2,3-dihydrobenzofuran-7-yl) amides, lipophilicity of the acyl moiety was necessary for the potent ACAT inhibitory activity. The highly lipophilic acid amides N-(2,2,4,6-tetramethyl-2,3-dihydrobenzofuran-7-yl)-2,2- dimethyldodecanamide (10) and 6-(4-chlorophenoxy)-N-(2,2,4,6-tetramethyl-2,3-dihydrobenzofuran-7-y l)-2,2-dimethyloctanamide (50) showed potent activity. Introduction of a dimethylamino group at position 5 of the 2,3-dihydrobenzofuran moiety resulted in highly potent activity. The most potent compound, N-[5-(dimethylamino)-2,2,4,6-tetramethyl-2,3-dihydrobenzofuran-7-yl ]-2,2-dimethyldodecanamide (13, TEI-6620), showed highly potent ACAT inhibitory activity (rabbit small intestine IC50 = 0.020 microM, rabbit liver IC50 = 0.009 microM), foam cell formation inhibitory activity (rat peritoneal macrophage IC50 = 0.030 microM), extremely potent serum cholesterol-lowering activity in cholesterol-fed rats (71% at a dose of 0.3 mg/kg/day po), and good bioavailability in fed dogs (Cmax = 2.68 microg/mL at 1 h, 10 mg/kg po).     

Autotransplantation of the spleen in the rat: donor leukocytes of the splenic fragment survive implantation to migrate and proliferate in the host

A rabbit liver UDP-glucuronosyltransferase cDNA that is related to human and rat UGT1A7 has been identified. The predicted amino acid sequence of the UGT1A71 displays 80% similarity to that encoded by human HP4 (UGT1A9), but 81% to that predicted for human UGT1A7 and 77% to the rat UGT1A7 (UGTA2). The exons encoding human UGT1A7 and rat UGTA2 are the seventh of the series of cassette exons that flank the 3' common exon series of the UGT1A locus. Southern blot analysis demonstrates that the exon sequence encoding UGT1A71 is part of a larger cluster of highly related genes. The UGT1A71 RNA is expressed in both neonatal and adult liver, and unlike rat UGT1A2 which is inducible with Ah receptor ligands such as polycyclic aromatic hydrocarbons, rabbit UGT1A7 is not regulated when animals are exposed to these inducers. Following expression of UGT171 in COS-1 cells, glucuronidation activity was identified for small phenolic molecules like 4-nitrophenyl, bulky phenols as represented by 4-hydroxybiphenol and octylgallate, as well as 4-hydroxyestrone. In addition, UGT1A71 possesses catalytic activity toward tertiary amines like the tricyclic antidepressant imipramine. The pattern of UGT1A71 glucuronidation is similar to that observed for human UGT1A9, except tertiary amines are not subject to glucuronidation by human UGT1A9. Glucuronidation of tertiary amines is catalyzed principally by human UGT1A4 as well as rabbit UGT1A4. Although rabbit UGT1A7 catalyzes the formation of quarternary ammonium glucuronides, the Vmax is considerably less than that observed for rabbit UGT1A4. Overall, the characterization of rabbit UGT1A7 suggests that this protein represents the ortholog of the human UGT1A7, which to date has not been identified.     

Inhibitory effects of uridine diphosphate on UDP-glucuronosyltransferase

Inhibitory effects of uridine diphosphate on the enzymatic activity of UDP-glucuronosyltransferase (UGT) were investigated. Pyrimidine nucleotides such as UDP, UTP and cytidine diphosphate reduced the activity of rat purified UGT (phenol UGT) to about 10%, 48% and 46% of the control, respectively, at the same concentration as a donor substrate, UDP-glucuronic acid. Purine nucleotides, uridine monophosphate, glucuronic acid and some UDP-sugars were only slightly inhibitory toward the transferase. Similar effects were observed in the expressed UGT (UGT1A6; corresponding to phenol UGT) in yeast cells and rat liver microsomal membrane-binding UGT, indicating that uracil and diphosphate residues are essential for the UDP inhibition. Interestingly, 2'-deoxy UDP was found to be a less effective inhibitor (about 50% inhibition) than UDP on the purified, the expressed (UGT1A6 and UGT2B1) and microsomal membrane-binding UGTs. These results indicate that not only uracil and diphosphate residues but also 2'-hydroxyl residue of UDP ribose participates in the interactions between UDP and UDP-glucuronosyltransferase.     

DNA vaccines for bacterial infections

The uptake and transportation of purine and pyrimidine based nucleosides by trophozoites of axenically grown Entamoeba histolytica (HMI-IMSS) were studied. The trophozoites transported adenosine and its analog tubercidin (1 microM) at a significant rate but poor transportation was observed in case of uridine (about 10% relative rate), inosine (3%), thymidine (2%) and formycin B (1%). The Km for adenosine was 160 +/- 42 microM. Unlabeled nucleosides (100 microM) inhibited adenosine and tubercidin transport. Adenosine related compounds 5'-deoxyadenosine and nebularin inhibited adenosine and tubercidine transport by 50% or more. However, inosine related compounds guanosine, 3'-deoxyinosine and formycin B were less inhibitory. The pyrimidine nucleosides uridine, thymidine and cytidine were poorly inhibitory. 6-[(4 nitrobenzyl)-mercapto] purine ribonucleoside, an inhibitor of mammalian nucleoside transporter, inhibited adenosine or tubercidin transport in E. histolytica variably between 0-30% at 10 microM, but dilazep, a known inhibitor, was inactive upto 10 microM. Increase in temperature from 22 degrees C to 33 degrees C enhanced the rate of transport of adenosine 4.5 fold, tubercidin 7.3 fold and of inosine 4 fold. These findings along with the structure activity figures suggested that transport was mediated and not passive.     

Novel disaccharide inhibitors of human glioma cell division

Several alpha-L-Fuc-(1-->3)-alpha-D-GlcNAcOC8H17 disaccharide derivatives bearing different hydroxylated alkyl chains, with or without sulfate groups at C-4 and/or C-6 positions of the GlcNAc unit, have been synthesized and tested as inhibitors of human astrocytoma lines U-373 and U-118. The antimitotic activity was dependent on the structure and position of the hydroxylated chain linked to the disaccharide. The compounds with a pentaerythritol or L-glyceryl chain at the C-6 position showed the best inhibitory properties, with an ID50 value of ca. 200 microM. On the contrary, sulfated disaccharide derivatives were inactive. The antimitotic activities of the compounds tested were essentially independent of the mitogen used to stimulate cell division.     

Theophylline derivatives as potential histamine H3-receptor antagonists

Previous results of histamine H3-receptors investigations allowed to formulate a general structure of H3-receptor antagonists. According to this model a series of compounds were obtained. As heterocycles they contained a theophylline moiety connected with a polar group (amine, ester, amide, and thiourea function) via an alkyl chain linked by a spacer to a lipophilic residue. The common distance between xanthine moiety and lipophilic rest was a six-link-chain. Selected compounds did not show significant H3-receptor antagonist activity and were weak antagonists at histamine H1-receptors.     

Isolation of novel and known genes from a human fetal cochlear cDNA library using subtractive hybridization and differential screening

V79 (Chinese hamster lung fibroblast) cell lines expressing a functional recombinant phenobarbital-inducible rat liver UDP-glucuronosyltransferase (UGT), i.e., UGT2B1, were established. Western blot analysis of positive colonies, using anti-rat liver UGT antibodies, revealed the presence of an immunoreactive polypeptide of the expected molecular mass of 52 kDa. The substrate specificity of the recombinant enzyme toward > 100 compounds was determined. Phenolic and alcoholic substrates included 4-methylumbelliferone, 4-hydroxybiphenyl, chloramphenicol, and testosterone, but a range of carboxylic acids of both endogenous (medium-chain saturated fatty acids, long-chain polyunsaturated fatty acids, and bile acids) and exogenous (profen nonsteroidal anti-inflammatory drugs, fibrate hypolipidemic agents, and sodium valproate) origin were also accepted, indicating that the enzyme was capable of forming both ether- and ester-type glucuronides from various structurally unrelated compounds. Determination of apparent kinetic constants for the glucuronidation by UGT2B1 of selected aglycones revealed a high maximal velocity toward the 3-position of morphine (49.3 +/- 2.2 nmol/min/mg of protein), compared with other known substrates such as 4-methylumbelliferone (2.67 +/- 0.11 nmol/min/mg of protein) or clofibric acid (0.06 +/- 0.02 nmol/min/mg of protein). To gain a better insight into the mechanisms underlying the apparently wide substrate specificity of UGT2B1, series of structurally related compounds were tested as potential substrates. The rate of glucuronidation of unbranched saturated fatty acids and omega,omega,omega-triphenylalkanoic acids increased progressively with increasing alkyl chain length and then declined, with the best substrates in these two homologous series being decanoic acid and 4,4,4-triphenylbutanoic acid, respectively. Glucuronidation of para-substituted phenols always proceeded at a higher rate than that of the corresponding para-substituted benzoic acids. This could mean that the aglycon hydroxyl group was better positioned in the enzyme active site in the case of phenols. Alternatively, if the initial interaction with the enzyme required the aglycon to be in the protonated uncharged form, then the observation could be explained by the difference in ionization between phenols and benzoic acids at the incubation pH used. The introduction of a bulky alkyl group into the para-position led to increases of up to 300-fold in the rate of glucuronidation, probably as a result of the increased aglycon lipophilicity. Finally, the enzyme showed a degree of stereo- and regiospecificity, preferring (S)-ibuprofen to the R-enantiomer (Vmax/Km, 3.06 and 1.10 microliters/min/mg of protein, respectively) and glucuronidating lithocholic acid but not hyodeoxycholic acid, which differs by only a single hydroxyl group.(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies on 5-lipoxygenase inhibitors. Synthesis of and structure--activity relationship in p-hydroxyarylalkenylbenzoazoles

In a previous paper we reported that 2-(p-hydroxyarylbutadienyl)benzoxazoles are highly potent 5-lipoxygenase inhibitors. We synthesized their ethenyl homologues and benzothiazole derivatives, and evaluated their 5-lipoxygenase inhibitory activity in vitro with cell-free rat basophilic leukemia (RBL-1). In most cases the replacement of benzoxazolyl with benzothiazolyl resulted in an enhancement of the activity. All compounds with butadienyl spacers tested herein exhibited strong inhibitory activities. While most of the ethenyl homologues showed weaker activities than their corresponding butadienyl homologues, some ethenyl compounds in the benzothiazole derivatives were found to be as potent as their corresponding butadienyl homologues. The inhibitory activity was also affected by the variation in the p-hydroxyaryl functionality.     

Pneumocystis carinii infections in patients with neoplasms and AIDS--clinical-pathologic changes in the lungs

Administration of 0.5 or 1% lyophilized green tea (5 or 10 mg tea solids per ml, respectively) as the sole source of drinking fluid to female Long-Evans rats for 18 days stimulated liver microsomal glucuronidation of estrone, estradiol and 4-nitrophenol by 30-37%, 15-27% and 26-60%, respectively. Oral administration of 0.5% lyophilized green tea to female CD-1 mice for 18 days stimulated liver microsomal glucuronidation of estrone, estradiol and 4-nitrophenol by 33-37%, 12-22% and 172-191%, respectively. The in vitro addition of a green tea polyphenol mixture, a black tea polyphenol mixture or (-)-epigallocatechin gallate inhibited rat liver microsomal glucuronidation of estrone and estradiol in a concentration-dependent manner and their IC50 values for inhibition of estrogen metabolism were approximately 12.5, 50 and 10 microg/ml, respectively. Enzyme kinetic analysis indicates that the inhibition of estrone glucuronidation by 10 microM (-)-epigallocatechin gallate was competitive while inhibition by 50 microM (-)-epigallocatechin gallate was noncompetitive. Similarly, several flavonoids (naringenin, hesperetin, kaempferol, quercetin, rutin, flavone, alpha-naphthoflavone and beta-naphthoflavone) also inhibited rat liver microsomal glucuronidation of estrone and estradiol to varying degrees. Naringenin and hesperetin displayed the strongest inhibitory effects (IC50 value of approximately 25 microM). These two hydroxylated flavonoids had a competitive mechanism of enzyme inhibition for estrone glucuronidation at a 10 microM inhibitor concentration and a predominantly noncompetitive mechanism of inhibition at a 50 microM inhibitor concentration.