Further structural analysis of urinary metabolites of suprofen in the rat

The urinary metabolites of 2-(4-(2-thienylcarbonyl)phenyl)propionic acid (suprofen, S) in rats were analyzed by radio-GC, GC/MS, or 1H NMR technique. Radio-GC analysis of trimethylsilylated materials after TLC separation of intact urine showed the presence of three radioactive peaks with the retention times corresponding to the authentic S, 2-(4-(2-thienylhydroxymethyl)phenyl)propionic acid, and 2-(4-carboxyphenyl)propionic acid. About 40% of the total radioactivity appearing in the 0-24-hr urine was accounted for by the three metabolites and their conjugates. The identification of these metabolites was confirmed by comparison of the MS spectra of urine, in which rats were administered an equimolar mixture of S and S[phenyl-d4], with those of synthetic standards. The labile metabolites of S, corresponding to about 32% of the total radioactivity appearing in the 0-24-hr urine, were isolated and purified by ether extraction from the fresh urine and GC/MS or HPLC. GC/MS of the methylated metabolite revealed the consistent presence of the ion peaks at m/z 304, 245, 217, and 141, indicative of a dimethylated product with monohydroxy group on the thiophene ring. Analysis of the 1H NMR spectrum demonstrated the metabolite to be 2-(4-(5-hydroxy-2-thienylcarbonyl)phenyl)propionic acid.     

The metabolic fate and effects of 2-Bromophenol in male Sprague–Dawley rats

Abstract

1. The metabolic fate and urinary excretion of 2-bromophenol, a phenolic metabolite of bromobenzene, was investigated in male Sprague–Dawley rats following single intraperitoneal doses at either 0, 100, or 200?mg/kg.

2. Urine was collected for seven days and samples analysed using ^1?H NMR spectroscopy, inductively coupled plasma (ICP)MS, and UPLC-MS.

3. ^1?H NMR spectroscopy of the urine samples showed that, at these doses, 2-bromophenol had little effect on endogenous metabolite profiles, supporting histopathology and clinical chemistry data, which showed no changes associated with the administration of 2-bromophenol in this study.

4. The use of ICP-MS provided a means for the selective detection and quantification of bromine-containing species and showed that between 15 and 30% of the dose was excreted via the urine over 7?days of the study for both the 100 and 200?mg doses, respectively.

5. The bulk of the excretion of Br-containing material had occurred by 8?h post administration. UPLC-MS of urine revealed a number of metabolites of 2-bromophenol, with 2-bromophenol glucuronide and 2-bromophenol sulphate identified as the major species. A number of minor hydroxylated metabolites were also detected as their glucuronide, sulphate, or O-methyl conjugates. There was no evidence for the production of reactive metabolites.     

NMR analysis of male fathead minnow urinary metabolites: a potential approach for studying impacts of chemical exposures

The potential for profiling metabolites in urine from male fathead minnows (Pimephales promelas) to assess chemical exposures was explored using nuclear magnetic resonance (NMR) spectroscopy. Both one-dimensional (1D) and two-dimensional (2D) NMR spectroscopy was used for the assignment of metabolites in urine from unexposed fish. Because fathead minnow urine is dilute, we lyophilized these samples prior to analysis. Furthermore, 1D 1H NMR spectra of unlyophilized urine from unexposed male fathead minnow and Sprague-Dawley rat were acquired to qualitatively compare rat and fish metabolite profiles and to provide an estimate of the total urinary metabolite pool concentration difference. As a small proof-of-concept study, lyophilized urine samples from male fathead minnows exposed to three different concentrations of the antiandrogen vinclozolin were analyzed by 1D 1H NMR to assess exposure-induced changes. Through a combination of principal components analysis (PCA) and measurements of 1H NMR peak intensities, several metabolites were identified as changing with statistical significance in response to exposure. Among those changes occurring in response to exposure to the highest concentration (450 microg/L) of vinclozolin were large increases in taurine, lactate, acetate, and formate. These increases coincided with a marked decrease in hippurate, a combination potentially indicative of hepatotoxicity. The results of these investigations clearly demonstrate the potential utility of an NMR-based approach for assessing chemical exposures in male fathead minnow, using urine collected from individual fish.     

Application of diffusion-edited NMR spectroscopy for the structural characterization of drug metabolites in mixtures

Diffusion-edited NMR spectroscopy is used to enable the structural characterization of low level metabolites in the presence of endogenous compounds, and organic solvents. We compared data from standard one-dimensional (1D) ¹H, 1D NOESY-presaturation, and 1D diffusion-edited experiments run on 20 μg and 100 μg samples of ethacrynic acid glutathione thioether (EASG) and a previously unreported metabolite of mefenamic acid, mefenamic acid glutathione thioester (MSG). The 1D NOESY-presaturation technique gave spectra with the best signal-to-noise (S/N) ratio, approximately three times that observed with the standard ¹H experiment, with respect to the metabolite signals. However, it was not selective for solvent signals as overlapping metabolite signals were also suppressed by this technique. In some cases, these signals were key to determining the site of glutathione attachment on the parent molecule. 1D NOESY-presaturation spectra also produced baseline distortions and inconsistent integration values. By comparison, 1D diffusion-edited experiments were found to selectively and simultaneously remove multiple solvent signals, resolve overlapping metabolite signals, and provide more uniform integration for metabolite signals overlapping with or proximal to solvent peaks, without producing baseline distortions. However, the diffusion-edited experiments caused significant signal attenuation of the metabolite signals when compared with a standard ¹H spectrum. Partially purified metabolites isolated from biological matrices were also characterized by using two-dimensional diffusion-ordered spectroscopy (DOSY). DOSY spectra acquired on a sample of EASG purified from rat bile proved useful in ‘separating’ the signals of EASG, from those of a co-eluting bile acid and parent drug ethacrynic acid (EA) in the diffusion-dimension in regions where there was no spectral overlap. In the low-field regions of high overlap, the DOSY experiment did not effectively separate the signals from the individual components. Diffusion based experiments provide a way to determine the total number of components that are present in a metabolite sample as well as an ability to identify them based on the chemical shift information, without the need for laborious chromatography on small samples.     

19F and 1H magnetic resonance strategies for metabolic studies on fluorinated xenobiotics: application to flurbiprofen [2-(2-fluoro-4-biphenylyl)propionic acid

Strategies for the use of 1H and 19F nuclear magnetic resonance (NMR) spectroscopy as an aid to the study of the metabolic fate of fluorinated drugs are discussed with reference to the application of these methods to flurbiprofen metabolism in man. 1H and 19F NMR analysis of untreated urine enabled the detection of two major and eight minor metabolites of the drug. The two major metabolites were identified using a combination of NMR spectroscopy, solid-phase extraction chromatography with 19F and 1H NMR detection and chemical hydrolysis to a flurbiprofen glucuronide and the glucuronide of the 4-hydroxy metabolite. 1H-19F 2D shift correlated spectroscopy and spin-echo difference experiments are discussed in relation to their use in the structural identification of drug metabolites.     

High Resolution 1H NMR Spectroscopic Studies of the Metabolism and Excretion of Ampicillin in Rats and Amoxycillin in Rats and Man

Abstract— High resolution proton nuclear magnetic resonance (¹H NMR) spectroscopy has been used to investigate the metabolism and urinary excretion of the aminopenicillins, ampicillin and amoxycillin, in rats and of amoxycillin in man. ¹H NMR resonances of the aminopenicillins, together with those for their 5R, 6R and 5S, 6R penicilloic acids and diketopiperazine metabolites were detected, assigned and quantified in urine samples with the aid of spin-echo NMR techniques. The dimer of amoxycillin was detected in rat urine for the first time together with novel drug-related resonances assigned to amoxycillin carbamate. Quantitative ¹H NMR spectroscopic results were consistent with HPLC and microbiological data considering that only single measurements were recorded. Due to the short analysis time and simple sample preparation, NMR was particularly useful for studying the metabolism of the aminopenicillins for which sample degradation poses analytical problems. The non-invasive character of ¹H NMR spectroscopic analysis of urine also provided unique information on a reversible reaction between amoxycillin and bicarbonate, an endogenous urinary metabolite.     

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.     

Medications as a potential source of exposure to phthalates among women of childbearing age

ObjectiveTo evaluate the association between the use of medications potentially containing phthalates and urinary concentrations of specific phthalate metabolites around conception.MethodsWomen enrolled in the Environment and Reproductive Health project from 2006 to 2009 completed questionnaires about the use of medications and provided multiple urine samples before and after conception. We compared the mean urinary concentration of phthalate metabolites between users of phthalate containing medications and a matched unexposed control group.ResultsOne woman used Asacol^® (mesalamine), which utilizes dibutyl phthalate (DBP) as a delayed release coating material, and had a mean urinary concentration of the main DBP metabolite 200 times higher than the controls (8176 μg/L vs. 37.5 μg/L). The three users of stool softeners had a higher concentration of the main diethyl phthalate (DEP) metabolite (8636 μg/L vs. 714.2 μg/L). Neither the three additional Prilosec^® (omeprazole) users nor one cyclobenzaprine user had higher urinary concentration than controls.ConclusionSelected medications may be important sources of DBP and DEP exposures around conception.     

Detection and in vitro metabolism of the confiscated peptides BPC 157 and MGF R23H

A new peptide, body protecting compound (BPC), BPC 157, and a variant of mechano‐growth factor (MGF), MGF R23H, were identified in confiscated vials. BPC 157 has the amino acid sequence, GEPPPGKPADDAGLV, and is currently under investigation for the promotion of healing and recovery in a variety of tissues. In vitro metabolism experiments in plasma demonstrate that MGF R23H has good stability and should be detectable in urine, while BPC 157 forms a stable metabolite that should be detectable in urine. A weak cation exchange solid phase extraction method was validated for detection of BPC 157 in urine. The method has a limit of detection of 0.1 ng/mL, precision of less than 20%, and good linearity, r² 0.998. BPC 157 was stable in urine for at least 4 days. The specificity of the method is improved by measurement of a potential BPC metabolite along with the parent peptide. Copyright © 2016 John Wiley & Sons, Ltd.     

NMR study of whole rat bile: the biliary excretion of 4-cyano-N,N-dimethyl aniline by an isolated perfused rat liver and a liver in situ

The structure of two biliary metabolites of 4-cyano-N,N,-dimethyl aniline (CDA) contained in whole rat bile have been studied in detail by NMR at 400 MHz. A 4-cyano-N-methyl glutathione-N-aniline conjugate was identified as a biliary metabolite of CDA using relatively simple ¹H NMR techniques. Isotopically ¹³C labelled CDA was used to generate ¹³C labelled xenobiotic conjugates. Our use of ¹H/¹³C heteronuclear NMR techniques, in particular a ¹³C-selective HMQC-TOCSY experiment, allowed a N-β-glucuronide conjugate, a previously unknown biliary metabolite of CDA, to be identified. Bile samples obtained from both the isolated perfused rat liver and the rat liver in situ were analysed.     

Mass spectrometric and NMR characterization of metabolites of roxifiban,a potent and selective antagonist of the platelet glycoprotein IIb/IIIa receptor

1. The methylester prodrug roxifiban is an orally active, potent and selective antagonist of the platelet glycoprotein GPIIb/IIIa receptor and is being developed for the prevention and treatment of arterial thrombosis. 2. Roxifiban was rapidly hydrolyzed to the zwitterion XV459 in vivo and by liver slices from the rat, mouse and human and by intestinal cores from dog. XV459 was metabolized to only a small extent in vitro and in vivo. 3. Studies with rat and dog givenradiolabelled roxifibanshowedlimited oralabsorption with the majority of the radiolabel being excreted in faeces. After i.v. doses of ¹⁴C-roxifiban, most of the radioactivity was recovered in the urine of rat whereas the dog excreted significant amounts of radioactivity in bile and urine. 4. XV459 could be metabolized extrahepatically by dog gut flora to produce an isoxazoline ring-opened metabolite. In vitro hepatic metabolism of XV459 was mainly by hydroxylation at the prochiraland chiralcentres of the isoxazolinering. These hydroxylated metabolites were not detected in the urine and plasma of human volunteers administered roxifiban. 5. Initial LC/MS identification of metabolites was achieved by dosing the rat with an equimolar mixture of d₀:d₄ roxifiban and detecting isotopic clusters of pseudomolecular ions. Unequivocal characterization of these metabolites was achieved by LC/MS, LC/NMR and high-field NMR techniques using synthetic standards of the metabolites. 6. The synthesis of one hydroxylated metabolite enabled the assignment of the correct stereochemistry of the substituted hydroxyl group on the isoxazoline ring.     

Pharmacokinetics and metabolism of mespirenone,a new aldosterone antagonist,in rat and cynomolgus monkey

Abstract

1. The pharmacokinetics and metabolism of mespirenone were examined in rat and cynomolgus monkey using the tritiated drug.

2. Following i.v. administration, mespirenone exhibited a short half-life and a high plasma clearance; after oral administration the unchanged compound was not detectable. Thus, mespirenone has to be considered a pro-drug.

3. From rat urine three metabolites were isolated by?h.p.l.c. and identified by mass spectra and ¹H n.m.r., all having retained the 7α-sulphur substituent as a thiomethyl or a sulphinylmethyl group.

4. The 7α-thiomethyl metabolite had a half-life of six hours in rat plasma and its AUC value was 35% of that for total ³H.

5. As this metabolite has marked anti-aldosterone activity, it is an active metabolite that contributes to the pharmacological effect of mespirenone.     

Urinary metabolites of a novel quinoxaline non-nucleoside reverse transcriptase inhibitor in rabbit,mouse and human: identification of fluorine NIH shift metabolites using NMR and tandem MS

1. The urinary metabolites of (S)-2-ethyl-7-fluoro-3-oxo-3,4-dihydro-2H-quinoxaline-carboxylic acid isopropylester (GW420867X) have been investigated in samples obtained following oral administration to rabbit, mouse and human. GW420867X underwent extensive biotransformation to form hydroxylated metabolites and glucuronide conjugates on the aromatic ring, and on the ethyl and isopropyl side-chains in all species. In rabbit urine, a minor metabolite was detected and characterized as a cysteine adduct that was not observed in mouse or man. 2. The hydroxylated metabolites and corresponding glucuronide conjugates were isolated by semi-preparative HPLC and characterized using NMR, LC-NMR and LCMS/MS. The relative proportions of fluorine-containing metabolites were determined in animal species by ¹⁹F-NMR signal integration. 3. The fluorine atom of the aromatic ring underwent NIH shift rearrangement in the metabolites isolated and characterized in rabbit, mouse and human urine. 4. The characterization of the NIH shift metabolites in urine enabled the detection and confirmation of the presence of these metabolites in human plasma.     

Study of acute biochemical effects of thallium toxicity in mouse urine by NMR spectroscopy

Thallium (Tl) is a toxic heavy metal and its exposure to the human body causes physiological and biochemical changes due to its interference with potassium‐dependent biological reactions. A high‐resolution ¹H NMR spectroscopy based metabonomic approach has been applied for investigating acute biochemical effects caused by thallium sulfate (Tl₂SO₄). Male strain A mice were divided in three groups and received three doses of Tl₂SO₄ (5, 10 and 20 mg kg^?1 b.w., i.p.). Urine samples collected at 3, 24, 72 and 96 h post‐dose time points were analyzed by ¹H NMR spectroscopy. NMR spectral data were processed and analyzed using principal components analysis to represent biochemical variations induced by Tl₂SO₄. Results showed Tl‐exposed mice urine to have distinct metabonomic phenotypes and revealed dose‐ and time‐dependent clustering of treated groups. The metabolic signature of urine analysis from Tl₂SO₄‐treated animals exhibited an increase in the levels of creatinine, taurine, hippurate and β‐hydroxybutyrate along with a decrease in energy metabolites trimethylamine and choline. These findings revealed Tl‐induced disturbed gut flora, membrane metabolite, energy and protein metabolism, representing physiological dysfunction of vital organs. The present study indicates the great potential of NMR‐based metabonomics in mapping metabolic response for toxicology, which could ultimately lead to identification of potential markers for Tl toxicity. Copyright © 2011 John Wiley & Sons, Ltd.     

Biotransformation of trans-1,1,1,3-tetrafluoropropene (HFO-1234ze)

trans-1,1,1,3-Tetrafluoropropene (HFO-1234ze) is a non-ozone-depleting fluorocarbon replacement with a low global warming potential and is developed as foam blowing agent. The biotransformation of HFO-1234ze was investigated after inhalation exposure. Male Sprague-Dawley rats were exposed to air containing 2000; 10,000; or 50,000 ppm (n = 5/concentration) HFO-1234ze. Male B6C3F1 mice were only exposed to 50,000 ppm HFO-1234ze. All inhalation exposures were conducted for 6 h in a dynamic exposure chamber. After the end of the exposures, animals were individually housed in metabolic cages and urines were collected at 6 or 12 h intervals for 48 h. For metabolite identification, urine samples were analyzed by ¹H-coupled and ¹H-decoupled ¹⁹F-NMR and by LC/MS-MS or GC/MS. Metabolites were identified by ¹⁹F-NMR chemical shifts, signal multiplicity, ¹H-¹⁹F coupling constants and by comparison with synthetic reference compounds. In urine samples of rats exposed to 50,000 ppm HFO-1234ze, the predominant metabolite was S-(3,3,3-trifluoro-trans-propenyl)-mercaptolactic acid and accounted for 66% of all integrated ¹⁹F-NMR signals in urines. No ¹⁹F-NMR signals were found in spectra of rat urine samples collected after inhalation exposure to 2000 or 10,000 ppm HFO-1234ze likely due to insufficient sensitivity. S-(3,3,3-Trifluoro-trans-propenyl)-l-cysteine, N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-l-cysteine and 3,3,3-trifluoropropionic acid were also present as metabolites in urine samples of rats and mice. A presumed amino acid conjugate of 3,3,3-trifluoropropionic acid was the major metabolite of HFO-1234ze in urine samples of mice exposed to 50,000 ppm and related to 18% of total integrated ¹⁹F-NMR signals. Quantification of three metabolites in urines of rats and mice was performed, using LC/MS-MS and GC/MS. The quantified amounts of the metabolites excreted with urine in both mice and rats, suggest only a low extent (< 1% of dose received) of biotransformation of HFO-1234ze and 95% of all metabolites were excreted within 18 h after the end of the exposures (t_1/2 app. 6 h). The obtained results suggest that HFO-1234ze is likely subjected to an addition-elimination reaction with glutathione and to a CYP 450 mediated epoxidation at low rates.     

More acidic metabolites of Δ1-tetrahydrocannabinol isolated from rabbit urine

The in vivo metabolism of Δ¹-trahydrocannabinol (Δ¹-THC) was further investigated in the rabbit after i.v. administration. Nine acidic metabolites were isolated from a previously not investigated fraction of the urine and identified by gas chromatography-mass spectrometry and by proton magnetic resonance spectroscopy. The major metabolites were side-chain hydroxylated monocarboxylic acids. Three side-chain monocarboxylic acids hydroxylated in allylic positions in the isoprene moiety were also characterized. The metabolites 4″-hydroxy-Δ¹-THC-7-oic acid and 7-hydroxy-4”,5″-bisnor-Δ¹-THC-3”-oic acid were hitherto not identified. An earlier described dicarboxylic metabolite was present in high concentration. Further, the identity of an O-glucuronide as an in vivo urinary metabolite of Δ¹-THC was here for the first time unambiguously established by m.s. and p.m.r.     

NMR-based metabolic profiling for serum of mouse exposed to source water

¹H nuclear magnetic resonance (NMR) based metabonomic method was used to characterize the profile of low-molecular-weight endogenous metabolites in mouse (Mus musculus) serum following exposure to Taihu Lake source water for 90 days. The ¹H NMR spectra of mice sera were recoded and a total of 21 metabolites were identified. Data reduction and latent biomarkers identification were processed by pattern recognition (PR) analysis. The principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) identified differences in metabolic profiles between control and treatment groups. A number of serum metabolic perturbations were observed in sera of source water treatment mice compared to control mice, including decreased levels of acetone, pyruvate, glutamine, lysine and citrate. These results indicated that Taihu Lake source water could induce energy metabolism changes in mouse related to fatty acid β-oxidation, tricarboxylic acid (TCA) cycle, citric acid cycle, and metabolism of some amino acids. ¹H NMR-based metabolic profiling provides new insight into the toxic effect of Taihu Lake source water, and suggests potential biomarkers for noninvasive monitoring of health risk.     

Metabolism of the hydrochlorofluorocarbon 1,2-dichloro-1,1-difluoroethane.

1,2-Dichloro-1,1-difluoroethane (HCFC-132b) is a potential substitute for some ozone-depleting chlorofluorocarbons and a model for other 1,1,1,2-tetrahaloethanes under consideration as chlorofluorocarbon substitutes. Male Fischer 344 rats were given 10 mmol/kg HCFC-132b dissolved in corn oil by intraperitoneal injection. An NMR assay for covalent binding of HCFC-132b metabolites to liver proteins was negative, whereas binding was observed in halothane-treated rats. Total urinary metabolites excreted by rats given HCFC-132b during the first 24 h amounted to 1.8 +/- 0.1% of the injected dose, as determined by 19F NMR. During the first 6 h, metabolites of HCFC-132b corresponding to 2-chloro-2,2-difluoroethyl glucuronide, unknown metabolite A, chlorodifluoroacetic acid, and chlorodifluoroacetaldehyde hydrate [both free and conjugated (unknown metabolite B)] were excreted in urine in the approximate ratio 100:9:3:7, respectively. Metabolite A is apparently an O-conjugate of 2-chloro-2,2-difluoroethanol; unconjugated 2-chloro-2,2-difluoroethanol was not detected in urine. The 19F NMR spectrum of metabolite B indicates the formation of a hemiacetal of chlorodifluoroacetaldehyde. Repeated exposure of rats to HCFC-132b significantly increased both the rate of chlorodifluoroacetic acid excretion and the relative fraction of the HCFC-132b dose excreted as chlorodifluoroacetic acid in urine. Incubation of HCFC-132b with rat hepatic microsomes yielded chlorodifluoroacetaldehyde hydrate as the only fluorinated product. The in vitro metabolism of HCFC-132b was increased in microsomes from pyridine-treated rats as compared with control rats, and HCFC-132b metabolism was inhibited by p-nitrophenol, indicating that the cytochrome P-450 isoform IIE1 is largely responsible for the initial hydroxylation of HCFC-132b.     

Solid phase extraction chromatography and NMR spectroscopy (SPEC-NMR) for the rapid identification of drug metabolites in urine

The use of solid phase extraction onto disposable columns containing a C18 bonded silica gel provides a rapid and simple procedure for the removal of interfering endogenous components from urine samples containing drug metabolites prior to detection and identification by (1)H NMR spectroscopy. In addition, these columns can be used to retain and concentrate the compounds of interest, thus improving the effective sensitivity of the NMR detection method. Using simple step gradients chromatographic separations can be performed, and metabolites may be rapidly fractionated. This approach (solid phase extraction chromatography with NMR or SPEC-NMR) utilises the multiparametric metabolite detection facility of a Fourier transform NMR spectrometer to monitor a chromatographic separation, as such it has some of the beneficial properties of a directly linked liquid chromatography-NMR system without any of the disadvantages. Applications of the SPEC-NMR method in the investigation of drug metabolism are illustrated here by reference to excretion studies on the drugs ibuprofen, paracetamol, aspirin, oxpentifylline and naproxen.     

Antidiabetic Efficacy of BRL 49653, a Potent Orally Active Insulin Sensitizing Agent,Assessed in the C57BL/KsJ db/db Diabetic Mouse by Non-invasive 1H NMR Studies of Urine

High resolution ¹H nuclear magnetic resonance (NMR) spectroscopic analysis of biofluids is a recently established tool for evaluating inherited and acquired errors in metabolic control. In the present study ¹H NMR analysis of urine was used to monitor efficacy of BRL 49653, a potent and selective antihyperglycaemic agent, following oral administration for up to 36 weeks to the genetically diabetic C57BL/KsJ db/db mouse. The effects of BRL 49653 on carbohydrate and fatty acid metabolism were monitored by determination of changes in concentrations of low molecular weight urinary metabolites. A qualitative comparison of the NMR spectra of urine from untreated diabetic mice with those of lean littermates and literature examples revealed several abnormalities, the majority of which could be explained in terms of the non-insulin dependent diabetes syndrome exhibited by these animals. Quantitatively the most prominent was the extreme glycosuria of both young (8–12 weeks; 0.9 g glucose kg^?1 h^?1) and older (42 weeks; 2 g glucose kg^?1 h^?1) diabetic mice. This was accompanied by the excretion of a number of unassigned sugar derivatives and by ketone bodies. Administration of BRL 49653 (3 μmol kg^?1) to db/db mice for 24 days reduced blood glucose concentrations to values comparable with non-diabetic lean littermates and reduced glycosuria by > 90%. BRL 49653 significantly reduced excretion of unassigned sugars, acetate, lactate, and the ketone bodies, acetoacetate, 3-d-hydroxybutyrate and acetone. The anti-diabetic efficacy of BRL 49653, assessed from the pattern of urinary metabolites, was maintained over a 36-week treatment period. These results demonstrate the value of ¹H NMR to evaluate non-invasively the efficacy of novel therapeutic agents.