Gunnar Grönberg

Novel Metabolites of Amodiaquine Formed by CYP1A1 and CYP1B1: Structure Elucidation Using Electrochemistry, Mass Spectrometry, and NMR

An aldehyde metabolite of amodiaquine and desethylamodiaquine has been identified. The aldehyde was the major metabolite formed in incubations with two recombinantly expressed human cytochromes P450 (rP450s), namely, CYP1A1 and CYP1B1. The aldehyde metabolite was also formed, to a lesser extent, in both human and rat liver microsomes. When comparing results from incubations with liver microsomes from 3-methylcholanthrene-treated rats (inducing CYP1A1 and CYP1B1) with those from noninduced rats, a 6-fold increase of the aldehyde metabolite was observed in the rat liver microsomes after 3-methylcholanthrene treatment. The metabolic oxidation was mimicked by the electrochemical system, and the electrochemical oxidation product was matched with the metabolite from the in vitro incubations. The electrochemical generation of the aldehyde metabolite was repeated on a preparative scale, and the proposed structure was confirmed by NMR. Trapping of the aldehyde metabolite was done with methoxyl amine. Trapping experiments with N-acetyl cysteine revealed that the aldehyde was further oxidized to an aldehyde quinoneimine species, both in the rP450 incubations and in the electrochemical system. Three additional new metabolites of amodiaquine and desethylamodiaquine were formed via rCYP1A1 and rCYP1B1. Trace amounts of these metabolites were also observed in incubations with liver microsomes from 3-methylcholanthrene-treated rats. Tentative structures of the metabolites and adducts were assigned based on liquid chromatography/tandem mass spectrometry in combination with accurate mass measurements.

Electrochemical generation of electrophilic drug metabolites: characterization of amodiaquine quinoneimine and cysteinyl conjugates by MS, IR, and NMR.

The chemical reactivity of electrophilic metabolites usually prevents their detection in vivo since, by definition, they are relatively short-lived and are likely to undergo one or more structural modifications to form more stable final products. Electrochemical oxidation provides a means to generate reactive metabolites in an environment without the presence of such nucleophiles. This paper describes the results of our MS, MS/MS, NMR, IR, and computational studies on oxidation products (and conjugates) that have been generated electrochemically from the antimalarial agent amodiaquine. The electrophilic quinoneimine metabolite of amodiaquine was the major oxidation product following electrochemical oxidation at +600 mV. The absence of biological nucleophiles in the electrochemical experiment facilitated (i) the acquisition of a clean IR spectrum of the amodiaquine quinoneimine and (ii) the addition of biologically relevant nucleophiles under controlled conditions. The addition of cysteine gave four cysteinyl conjugates, while the addition of glutathione gave four glutathionyl conjugates. The product ion spectra of the conjugates formed in the electrochemical experiment were used to identify suitable fragments for selected reaction monitoring (SRM) to selectively search for these conjugates in human liver microsomal (HLM) incubations. The four cysteinyl conjugates, as well as the four glutathionyl conjugates, were also detected as metabolites in HLM. The experiment with cysteine was repeated on a preparative scale that allowed characterization of the major conjugates by (1)H NMR. Desethylamodiaquine, the major metabolite formed in human liver microsomes, was also generated electrochemically by oxidation of amodiaquine at +1200 mV followed by reduction at -800 mV. In conclusion, the EC-ESI/MS technique provides the unique opportunity to generate reactive metabolites in the absence of biological nucleophiles, which enables studies that can give insight into the nature of these reactive intermediates. Such knowledge is valuable for risk assessment of new compound classes and can be complementary to computer-based structure-activity relationships of carcinogenicity, mutagenicity, and teratogenicity.

Structural analysis of five new monosialylated oligosaccharides from human milk

The total monosialylated oligosaccharide fraction from pooled human milk was isolated by gel filtration and ion-exchange chromatography. Further separation by HPLC using a mobile phase containing an ion-pairing reagent of triethylamine gave five new monosialylated oligosaccharides. Structural analysis was carried out by chemical analyses, fast atom bombardment mass spectrometry, and 500-MHz NMR spectroscopy. Combined structural data revealed the following new structures: [formula: see text]

Electrochemical oxidation of troglitazone: identification and characterization of the major reactive metabolite in liver microsomes.

Troglitazone (TGZ) was developed for the treatment of type 2 diabetes but was withdrawn from the market due to hepatotoxicity. The formation of reactive metabolites has been associated with the observed hepatotoxicity. Such reactive metabolites have been proposed to be formed via three different mechanisms. One of the proposed mechanisms involves the oxidation of the chromane moiety of TGZ to a reactive o-quinone methide. The two other mechanisms involve metabolic activation of the thiazolidinedione moiety of TGZ. In the present study, it is shown that electrochemical oxidations can be used to generate a reactive metabolite of TGZ, which can be trapped by GSH or N-acetylcysteine. From incubations of TGZ with rat and human liver microsomes in the presence of either GSH or N-acetylcysteine, it was shown that similar conjugates were formed in vitro as formed from electrochemical oxidations of TGZ. One- and two-dimensional NMR studies of the troglitazone- S-( N-acetyl)cysteine conjugate revealed that N-acetylcysteine was attached to a benzylic carbon in the chromane moiety, showing that the conjugate was formed via a reaction between the o-quinone methide of TGZ and N-acetylcysteine. From electrochemical oxidations of rosiglitazone, pioglitazone, and ciglitazone in the presence of GSH, no GSH conjugates could be identified. These three compounds all contain a thiazolidinedione moiety. In conclusion, it has been shown that the primary reactive metabolite of TGZ formed from electrochemical oxidation was the o-quinone methide, and this metabolite was similar to what was observed to be the primary reaction product in human and rat liver microsomes.

An improved approach to the analysis of the structure of small oligosaccharides of glycoproteins: application to the O-linked oligosaccharides from human glycophorin A

Treatment of purified human glycophorin A with alkaline borohydride cleaved the oligosaccharide side chains to yield alditol derivatives that were separated by gel filtration into three mixtures of low molecular weight compounds. Each mixture was oxidised with periodate, and the products were reduced with borohydride and analysed after acetylation or methylation by GLC-MS and FABMS. The resulting data allowed the monosaccharide sequence and linkage positions to be assigned to each component of the mixtures. The anomeric configuration was determined by 1H NMR spectroscopy of the intact fractions. The structures of a desialylated tetrasaccharide, two monosialylated trisaccharides, and five other minor products were defined.

A P450 fusion library of heme domains from Rhodococcus jostii RHA1 and its evaluation for the biotransformation of drug molecules.

The actinomycete Rhodococcus jostii RHA1 contains a multitude of oxygenase enzymes, consonant with its remarkable activities in the catabolism of hydrophobic xenobiotic compounds. In the interests of identifying activities for the transformation of drug molecules, we have cloned genes encoding 23 cytochrome P450 heme domains from R. jostii and expressed them as fusions with the P450 reductase domain (RhfRED) of cytochrome P450Rhf from Rhodococcus sp. NCIMB 9784. Fifteen of the fusions were expressed in the soluble fraction of Escherichia coli Rosetta (DE3) cells. Strains expressing the fusions of RhfRED with genes ro02604, ro04667, ro11069, ro11320, ro11277, ro08984 and ro04671 were challenged with 48 commercially available drugs revealing many different activities commensurate with P450-catalyzed hydroxylation and demethylation reactions. One recombinant strain, expressing the fusion of P450 gene ro11069 (CYP257A1) with RhfRED, and named Ro07-RhfRED, catalyzed the N-demethylation of diltiazem and imipramine. This observation was in accord with previous reports of this enzymes activity as a demethylase of alkaloid substrates. Ro07-RhfRED was purified and characterised, and applied in cell-free biotransformations of imipramine (7 μM) giving a 63% conversion to the N-desmethyl product.

Isolation of two novel sialyl-Lewis X-active oligosaccharides by high-performance liquid affinity chromatography using monoclonal antibody Onc-M26

A monoclonal antibody, Onc-M26, that recognizes a cancer-associated antigen expressed by most human adenocarcinomas of the breast was shown previously to recognize a carbohydrate epitope carried on a hexaglycosyl ganglioside carrying the sialyl-Lewis X (SLex) antigen (P.S. Linsley et al., 1988, Cancer Res. 48, 2138-2148). Evidence that the antibody binds even more avidly to minor gangliosides containing more complex carbohydrate chains prompted us to search for a higher affinity epitope among sialylated oligosaccharides from pooled human milk. Affinity chromatography of a partially purified fraction of monosialylated milk oligosaccharides on a column containing monoclonal antibody Onc-M26 bound to a macroporous silica matrix gave a peak with a retention volume significantly greater than that of a standard SLex-active hexasaccharide. The retained material consisted of two nonasaccharides, each containing the SLex tetrasaccharide sequence, Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3) GlcNAc, linked beta 1-6 to a 3,6-disubstituted galactosyl residue.

CYP3A specifically catalyzes 1β-hydroxylation of deoxycholic acid: Characterization and enzymatic synthesis of a potential novel urinary biomarker for CYP3A activity

The endogenous bile acid metabolite 1β-hydroxy-deoxycholic acid (1β-OH-DCA) excreted in human urine may be used as a sensitive CYP3A biomarker in drug development reflecting in vivo CYP3A activity. An efficient and stereospecific enzymatic synthesis of 1β-OH-DCA was developed using a Bacillus megaterium (BM3) cytochrome P450 (P450) mutant, and its structure was confirmed by nuclear magnetic resonance (NMR) spectroscopy. A [2H4]-labeled analog of 1β-OH-DCA was also prepared. The major hydroxylated metabolite of deoxycholic acid (DCA) in human liver microsomal incubations was identified as 1β-OH-DCA by comparison with the synthesized reference analyzed by UPLC-HRMS. Its formation was strongly inhibited by CYP3A inhibitor ketoconazole. Screening of 21 recombinant human cytochrome P450 (P450) enzymes showed that, with the exception of extrahepatic CYP46A1, the most abundant liver P450 subfamily CYP3A, including CYP3A4, 3A5, and 3A7, specifically catalyzed 1β-OH-DCA formation. This indicated that 1β-hydroxylation of DCA may be a useful marker reaction for CYP3A activity in vitro. The metabolic pathways of DCA and 1β-OH-DCA in human hepatocytes were predominantly via glycine and, to a lesser extent, via taurine and sulfate conjugation. The potential utility of 1β-hydroxylation of DCA as a urinary CYP3A biomarker was illustrated by comparing the ratio of 1β-OH-DCA:DCA in a pooled spot urine sample from six healthy control subjects to a sample from one patient treated with carbamazepine, a potent CYP3A inducer; 1β-OH-DCA:DCA was considerably higher in the patient versus controls (ratio 2.8 vs. 0.4). Our results highlight the potential of 1β-OH-DCA as a urinary biomarker in clinical CYP3A DDI studies.

Synthesis of [3H] and [2H6]AZD6642, an inhibitor of 5-lipoxygenase activating protein (FLAP)

An AstraZeneca effort to identify a 5-lipoxygenase activating protein inhibitor with good drug-like properties resulted in the identification of AZD6642. To further understand its drug metabolism and pharmacokinetic properties, it was required labeled with tritium. The tritiation of AZD6642 was effected by Ir-catalyzed exchange chemistry to give an average of one tritium per molecule. Additionally, a stable isotope labeled version of AZD6642 was required to support bioanalytical studies. The synthesis originated from [(2) H6 ]acetone which was converted to the trimethylsilyl cyanide adduct and subsequently reduced to give 2-(aminomethyl)-[1,1,1,3,3,3-(2) H6 ]propan-2-ol in good yield. Carbonylation to give an amide adduct resulted in an intermediate that was converted to the final compound in four steps.

Discovery of a Novel Microsomal Epoxide Hydrolase–Catalyzed Hydration of a Spiro Oxetane

Oxetane moieties are increasingly being used by the pharmaceutical industry as building blocks in drug candidates because of their pronounced ability to improve physicochemical parameters and metabolic stability of drug candidates. The enzymes that catalyze the biotransformation of the oxetane moiety are, however, not well studied. The in vitro metabolism of a spiro oxetane-containing compound AZD1979 [(3-(4-(2-oxa-6-azaspiro[3.3]heptan-6-ylmethyl)phenoxy)azetidin-1-yl)(5-(4-ethoxyphenyl)-1,3,4-oxadiazol-2-yl)methanone] was studied and one of its metabolites, M1, attracted our interest because its formation was NAD(P)H independent. The focus of this work was to elucidate the structure of M1 and to understand the mechanism(s) of its formation. We established that M1 was formed via hydration and ring opening of the oxetanyl moiety of AZD1979. Incubations of AZD1979 using various human liver subcellular fractions revealed that the hydration reaction leading to M1 occurred mainly in the microsomal fraction. The underlying mechanism as a hydration, rather than an oxidation reaction, was supported by the incorporation of 18O from H218O into M1. Enzyme kinetics were performed probing the formation of M1 in human liver microsomes. The formation of M1 was substantially inhibited by progabide, a microsomal epoxide hydrolase inhibitor, but not by trans-4-[4-(1-adamantylcarbamoylamino)cyclohexyloxy]benzoic acid, a soluble epoxide hydrolase inhibitor. On the basis of these results, we propose that microsomal epoxide hydrolase catalyzes the formation of M1. The substrate specificity of microsomal epoxide hydrolase should therefore be expanded to include not only epoxides but also the oxetanyl ring system present in AZD1979.