Ulla G. Sidelmann

Small-molecule agonists for the glucagon-like peptide 1 receptor.

The peptide hormone glucagon-like peptide (GLP)-1 has important actions resulting in glucose lowering along with weight loss in patients with type 2 diabetes. As a peptide hormone, GLP-1 has to be administered by injection. Only a few small-molecule agonists to peptide hormone receptors have been described and none in the B family of the G protein coupled receptors to which the GLP-1 receptor belongs. We have discovered a series of small molecules known as ago-allosteric modulators selective for the human GLP-1 receptor. These compounds act as both allosteric activators of the receptor and independent agonists. Potency of GLP-1 was not changed by the allosteric agonists, but affinity of GLP-1 for the receptor was increased. The most potent compound identified stimulates glucose-dependent insulin release from normal mouse islets but, importantly, not from GLP-1 receptor knockout mice. Also, the compound stimulates insulin release from perfused rat pancreas in a manner additive with GLP-1 itself. These compounds may lead to the identification or design of orally active GLP-1 agonists.

Contemporary issues in toxicology - The role of metabonomics in toxicology and its evaluation by the COMET project

The role that metabonomics has in the evaluation of xenobiotic toxicity studies is presented here together with a brief summary of published studies. To provide a comprehensive assessment of this approach, the Consortium for Metabonomic Toxicology (COMET) has been formed between six pharmaceutical companies and Imperial College of Science, Technology and Medicine (IC), London, UK. The objective of this group is to define methodologies and to apply metabonomic data generated using (1)H NMR spectroscopy of urine and blood serum for preclinical toxicological screening of candidate drugs. This is being achieved by generating databases of results for a wide range of model toxins which serve as the raw material for computer-based expert systems for toxicity prediction. The project progress on the generation of comprehensive metabonomic databases and multivariate statistical models for prediction of toxicity, initially for liver and kidney toxicity in the rat and mouse, is reported. Additionally, both the analytical and biological variation which might arise through the use of metabonomics has been evaluated. An evaluation of intersite NMR analytical reproducibility has revealed a high degree of robustness. Second, a detailed comparison has been made of the ability of the six companies to provide consistent urine and serum samples using a study of the toxicity of hydrazine at two doses in the male rat, this study showing a high degree of consistency between samples from the various companies in terms of spectral patterns and biochemical composition. Differences between samples from the various companies were small compared to the biochemical effects of the toxin. A metabonomic model has been constructed for urine from control rats, enabling identification of outlier samples and the metabolic reasons for the deviation. Building on this success, and with the completion of studies on approximately 80 model toxins, first expert systems for prediction of liver and kidney toxicity have been generated.

Direct quantitative trait locus mapping of mammalian metabolic phenotypes in diabetic and normoglycemic rat models

Characterizing the relationships between genomic and phenotypic variation is essential to understanding disease etiology. Information-dense data sets derived from pathophysiological, proteomic and transcriptomic profiling have been applied to map quantitative trait loci (QTLs). Metabolic traits, already used in QTL studies in plants, are essential phenotypes in mammalian genetics to define disease biomarkers. Using a complex mammalian system, here we show chromosomal mapping of untargeted plasma metabolic fingerprints derived from NMR spectroscopic analysis in a cross between diabetic and control rats. We propose candidate metabolites for the most significant QTLs. Metabolite profiling in congenic strains provided evidence of QTL replication. Linkage to a gut microbial metabolite (benzoate) can be explained by deletion of a uridine diphosphate glucuronosyltransferase. Mapping metabotypic QTLs provides a practical approach to understanding genome-phenotype relationships in mammals and may uncover deeper biological complexity, as extended genome (microbiome) perturbations that affect disease processes through transgenomic effects may influence QTL detection.

Directly coupled HPLC-NMR and HPLC-MS approaches for the rapid characterisation of drug metabolites in urine: application to the human metabolism of naproxen

High resolution nuclear magnetic resonance (NMR) spectroscopy is a very powerful tool for the structural identification of xenobiotic metabolites in complex biological matrices such as plasma, urine and bile. However, these fluids are dominated by thousands of signals resulting from endogenous metabolites and it is advantageous when investigating drug metabolites in such matrices to simplify the spectra by including a separation step in the experiment by directly-coupling HPLC and NMR. Naproxen (6-methoxy-alpha-methyl-2-naphthyl acetic acid) is administered as the S-enantiomer and is metabolised in vivo to form its demethylated metabolite which is subsequently conjugated with beta-D-glucuronic acid as well as with sulfate. Naproxen is also metabolised by phase II metabolism directly to form a glycine conjugate as well as a glucuronic acid conjugate at the carboxyl group. In the present investigation, the metabolism of naproxen was investigated in urine samples with a very simple sample preparation using a combination of directly-coupled HPLC-1H NMR spectroscopy and HPLC-mass spectrometry (MS). A buffer system was developed which allows the same chromatographic method to be used for the HPLC-NMR as well as the HPLC-MS analysis. The combination of these methods is complementary in information content since the NMR spectra provide evidence to distinguish isomers such as the type of glucuronides formed, and the HPLC-MS data allow identification of molecules containing NMR-silent fragments such as occur in the sulfate ester.

Human glucagon receptor antagonists based on alkylidene hydrazides.

A series of alkylidene hydrazide derivatives containing an alkoxyaryl moiety was optimized. The resulting hydrazide-ethers were competitive antagonists at the human glucagon receptor. Pharmacokinetic experiments showed fast clearance of most of the compounds tested. A representative compound [4-hydroxy-3-cyanobenzoic acid (4-isopropylbenzyloxy-3,5-dimethoxymethylene)hydrazide] with an IC50 value of 20 nM was shown to reduce blood glucose levels in fasted rats.

Novel glucagon receptor antagonists with improved selectivity over the glucose-dependent insulinotropic polypeptide receptor.

Optimization of a new series of small molecule human glucagon receptor (hGluR) antagonists is described. In the process of optimizing glucagon receptor antagonists, we counter-screened against the closely related human gastric inhibitory polypeptide receptor (hGIPR), and through structure activity analysis, we obtained compounds with low nanomolar affinities toward the hGluR, which were selective against the hGIPR and the human glucagon-like peptide-1 receptor (hGLP-1R). In the best cases, we obtained a >50 fold selectivity for the hGluR over the hGIPR and a >1000 fold selectivity over the hGLP-1R. A potent and selective glucagon receptor antagonist was demonstrated to inhibit glucagon-induced glycogenolysis in primary rat hepatocytes as well as to lower glucagon-induced hyperglycemia in Sprague-Dawley rats. Furthermore, the compound was shown to lower blood glucose in the ob/ob mouse after oral dosing.

Measurement of Internal Acyl Migration Reaction Kinetics Using Directly Coupled HPLC-NMR: Application for the Positional Isomers of Synthetic (2-Fluorobenzoyl)-d-glucopyranuronic Acid.

Ester glucuronides (1-O-acyl-β-d-glucopyranuronates) of many drugs may undergo internal acyl migration reactions, resulting in the formation of new positional isomers with both α- and β-anomers. We illustrate here a novel approach for the direct investigation of the acyl migration kinetics of ester glucuronides and show the application with respect to the isomers of synthetic (2-fluorobenzoyl)-d-glucopyranuronic acid. Individual isomers were separated from an equilibrium mixture containing the β-1-O-acyl, α- and β-2-O-acyl, α- and β-3-O-acyl, and α- and β-4-O-acyl isomers at pH 7.4 in 20 mM phosphate buffer. The interconverting isomers were separated using reversed-phase HPLC and pumped directly into a dedicated on-line NMR flow probe in a 600 MHz NMR spectrometer. The flow was stopped with each isomer in the NMR flow probe, and sequential NMR spectra were collected at 25 °C, allowing direct measurement of the production of positional isomers from each selectively isolated glucuronide isomer. All of the positional isomers and anomers were characterized, and relative quantities determined, and a kinetic model describing the rearrangement reactions was constructed. The acyl migration reaction kinetics were simulated using a theoretical approach using nine first-order rate constants determined for the acyl migration reactions and six first-order rate constants describing the mutarotation each of the 2-, 3-, and 4-positional isomers. The rate constants (in h(-)(1)) for the rearrangement reactions of the 2-fluorobenzoyl glucuronide isomers were as follows:  β-1-O-acyl, 0.29 ± 0.01; α-2-O-acyl, 0.11 ± 0.01; β-2-O-acyl, 0.07 ± 0.01; α-3-O-acyl, 0.10 ± 0.01; β-3-O-acyl, 0.09 ± 0.01; α-4-O-acyl, 0.09 ± 0.01; and β-4-O-acyl, 0.06 ± 0.01. The α- and β-anomerization rates were estimated on the basis of the kinetics model; the anomerization rates of the 4-O-acyl isomers were additionally determined experimentally using directly coupled HPLC-NMR. The fitted anomerization rates for the 4-O-acyl isomer were 0.80 (α → β) and 0.50 h(-)(1) (β → α), whereas the experimentally estimated anomerization rates were 0.89 ± 0.1 and 0.52 ± 0.1 h(-)(1), respectively. The dynamic stop-flow HPLC-NMR approach allows unique kinetic information to be obtained relating to glucuronide reactivity, and this approach will be useful in future structure-reactivity studies on drug ester glucuronides and their properties.

Simultaneous quantitative determination of the major phase I and II metabolites of ibuprofen in biological fluids by high-performance liquid chromatography on dynamically modified silica

Ibuprofen has previously, after ingestion by man, been demonstrated to yield four major phase I metabolites, which are excreted in the urine partly as glucuronic acid conjugates. However, in previous investigations the quantitative determinations of the conjugates were performed by indirect methods. The purpose of the present investigation was to develop a high-performance liquid chromatographic (HPLC) system for the simultaneous determination of the major phase I and II metabolites of ibuprofen in biological fluids. The separation was performed using bare silica dynamically modified with N-cetyl-N,N,N-trimethylammonium hydroxide ions contained in the mobile phase. The separation of the metabolites of ibuprofen is greatly improved with this system compared to other published reversed-phase HPLC systems intended for the same purpose. The method developed makes it possible to simultaneously determine the intact glucuronic acid conjugates of ibuprofen as well as its phase I metabolites in human urine. In a study involving four healthy volunteers, a total recovery in urine of the dose given was found to be 58-86% within 8 h. This may be compared to an average of 67% earlier reported in the literature.

LC-1H NMR used for determination of the elution order of S-naproxen glucuronide isomers in two isocratic reversed-phase LC-systems.

The reactive metabolite S-naproxen-beta-1-O-acyl glucuronide was purified from human urine using solid phase extraction (SPE) and preparative HPLC. The structure was confirmed by 600 MHz 1H NMR. Directly coupled 600 MHz HPLC-1H NMR was used to assign the peaks in chromatograms obtained when analysing a sample containing S-naproxen aglycone and the 1-, 2-, 3-, and 4-isomers of S-naproxen-beta-1-O-acyl glucuronide in two simple isocratic reversed phase HPLC-systems. Using mobile phase 1 (50 mM formate buffer pH 5.75/acetonitrile 75:25 v/v) the elution order was: 4-O-acyl isomers, beta-1-O-acyl glucuronide, 3-O-acyl isomers, 2-O-acyl isomers, and S-naproxen aglycone. Using mobile phase II (25 mM potassium phosphate pH 7.40/acetonitrile 80:20 v/v) the elution order was: alpha/beta-4-O-acyl isomers, S-naproxen aglycone, beta-1-O-acyl glucuronide, 3-O-acyl isomers, and alpha/beta-2-O-acyl isomers. In both systems the elution order for the 2-, 3- and 4-O-acyl isomers corresponded with previously published results for 2-, 3-, and 4-fluorobenzoic acid glucuronide isomers determined by reversed phase HPLC-1H NMR (U.G. Sidelmann, S.H. Hansen, C. Gavaghan, A.W. Nicholls, H.A.J. Carless, J.C. Lindon, I.D. Wilson, J.K. Nicholson, J. Chromatogr. B Biomed. Appl. 685 (1996) 113-122]. The alpha-1-O-acyl isomer was found to be present at approximately 3% of the initial S-naproxen-beta-1-O-acyl glucuronide concentration in the glucuronide isomer mixture after 6 h of incubation at pH 7.40 and 37 degrees C. In both HPLC systems it eluted just before the beta-1-O-acyl glucuronide well separated from other isomers. Investigators should consider the possible formation of a alpha-1-O-acyl isomer when studying glucuronide reactivity and degradation.

Development of a simple liquid chromatographic method for the separation of mixtures of positional isomers and anomers of synthetic 2-, 3- and 4-fluorobenzoic acid glucuronides formed via acyl migration reactions

Many drugs containing carboxylate groups form beta-1-O-acyl glucuronides as their major phase II metabolites in vivo. These ester glucuronides are potentially reactive due to the susceptibility of the acyl group to nucleophilic reactions resulting in hydrolysis, acyl migration or covalent adduct formation. In the present study, a number of synthetic fluorobenzoic acid glucuronide conjugates were chosen as models for chromatographic studies. A high-performance liquid chromatography method is presented for the simultaneous determination of the 1-, 2-, 3- and 4-positional isomers of the acyl glucuronides, and their alpha- and beta-anomers for the 2-, 3- and 4-fluorobenzoic acids as well as each aglycone formed as a result of hydrolysis. The same elution order was found for the acyl migrated glucuronide isomers of the three fluorobenzoic acids in their equilibrium mixtures. The alpha-4-O-acyl isomer eluted first followed by the beta-4-O-acyl isomer, then the beta-1-O-acyl, the beta-3-O-acyl, the alpha-3-O-acyl, the alpha-2-O-acyl and finally the beta-2-O-acyl isomer eluted. The method was used to determine the overall degradation rates, the acyl migration rates and the hydrolysis rates of 1-O-(2-fluorobenzoyl)-beta-D-glucopyranuronic acid 1-O-(3-fluorobenzoyl)-beta-D-glucopyranuronic acid and 1-O-(4-flurobenzoyl)-beta-D-glucopyranuronic acid in a buffer system pH 7.4 at 25 degrees C. It was found that the order of beta-1-glucuronide acyl migration rates was 2-fluorobenzoyl > 3-fluorobenzoyl > 4-fluorobenzoyl. Both the acyl migration rates and the elution order were interpreted in terms of electronic effect of the fluorine substituent on the carbonyl carbon.