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R J Barnaby

Publications and source records attributed to R J Barnaby.

12 recordsLinked to original sources
PubMed2003

Pharmacokinetics, metabolism and excretion of the glycine antagonist GV150526A in rat and dog.

1. The pharmacokinetics, metabolic fate and excretion of 3-[-2(phenylcarbamoyl) ethenyl-4,6-dichloroindole-2-carboxylic acid (GV150526), a novel glycine antagonist for stroke, in rat and dog following intravenous administration of [C14]-GV150526A were investigated. 2. Studies were also performed in bile duct-cannulated animals to confirm the route of elimination and to obtain more information on metabolite identity. 3. Metabolites in plasma, urine and bile were identified by HPLC-MS/MS and NMR spectroscopy. 4. GV150526A was predominantly excreted in the faeces via the bile, with only trace metabolites of radioactivity in urine (< 5%). Radioactivity in rat bile was predominantly due to metabolites, whereas approximately 50% of the radioactivity in dog bile was due to parent GV150526. 5. The principal metabolites in bile were identified as glucuronide conjugates of the carboxylic acid, whereas in rat urine the main metabolite was a sulphate conjugate of an aromatic oxidation metabolite. Multiple glucuronide peaks were observed and identified as isomeric glucuronides and their anomers arising from acyl migration and muta-rotation.

Animals↗
PubMed2000

Human hepatic metabolism of a novel 2-carboxyindole glycine antagonist for stroke: in vitro-in vivo correlations.

1. The hepatic metabolism of 3-[-2(phenylcarbamoyl) ethenyl]-4,6-dichloroindole-2-carboxylic acid (GV150526), a novel glycine antagonist for stroke, was investigated. 2. After a single intravenous administration of 800 mg GV150526 to healthy volunteers, six metabolites were observed. The major metabolites detected in human plasma have been shown by mass spectrometry to be glucoronides and one sulphate conjugate. 3. After incubation of GV150526 for 6 and 24 h with human liver slices, three glucuronide metabolites were observed. After incubation of GV150526 with pooled human liver microsomes, only one metabolite was observed, with the same molecular weight and HPLC retention time as the synthetic standard GV217053 (GV150526 hydroxylated on the para-position of the phenyl ring). 4. GV150526 hydroxylase enzyme kinetics--a step before sulphation--was determined using pooled human microsomes and was shown to be catalysed by cytochrome P4502C9. Glucuronidation kinetics towards GV150526 using microsomal preparations were also determined. Glucuronidation of GV150526 was observed with UGT1A1 cDNA-expressed protein, but not with UGT1A6. 5. The above enzyme kinetic data were used to calculate intrinsic clearance after scaling-up and hepatic clearance were calculated. Since GV150526 has a high plasma protein binding capacity, the effect of GV150526 binding to microsomal protein was determined. Thus, enzyme kinetic data were corrected, plotting the free (unbound) concentration of GV150526 versus enzymatic velocities: apparent Vmax did not alter significantly but apparent Km was approximately 10-fold lower. Correlation of these corrected enzyme kinetic data to predict clearance with in vivo clearance of GV150526 was good when both fu(plasma) and fu(microsomes) were included in the clearance calculations.

Adult↗
PubMed2000

Identification of UDP-glucuronosyltransferases involved in the human hepatic metabolism of GV150526, a novel glycine antagonist.

The major metabolic pathway for elimination of GV150526 is by glucuronidation exerted by glucuronosyl transferases (UGTs). Potential exists for the modification of GV150526 pharmacokinetics by drugs capable of inhibiting the glucuronidation of GV150526. Using human liver microsomes, 44 compounds were screened for inhibition of GV150526 glucuronidation. These compounds were selected because they are extensively glucuronidated themselves or are used as concomitant medication in the treatment of acute stroke. For 11 compounds out of the 44, full inhibition kinetics were performed to determine their Ki-value and mechanism of inhibition. To predict possible in vivo drug-drug interactions, the theoretical percentage of inhibition (i) was determined, based on in vitro determined Ki-values, and the expected Cmax plasma levels of GV150526 and the inhibitor. Of the 11 compounds examined, only propofol had an i-value of 6.6; for all other compounds i-values were lower than 2.1. These results indicate that although in vitro inhibition is observed, the likelihood of in vivo drug-drug metabolic interactions occurring is low. The inhibition results suggest that in addition to UGT1A1, also UGT1A3, UGT1A8/9, and UGT2B4 are involved in the glucuronidation of GV150526. The involvement of UGT1A1 and UGT1A8/9 was confirmed from studies using cDNA expressed human UGT cell lines.

DNA, Complementary↗
PubMed1999

Substituted analogues of GV150526 as potent glycine binding site antagonists in animal models of cerebral ischemia.

A series of analogues of the indole-2-carboxylate GV150526, currently in clinical trials as a potential neuroprotective agent for the control of the cerebral damage after stroke onset, was designed based on previous studies dealing with the electronic features of the north-east region of the glycine binding site associated with the NMDA receptor. In particular, the substitution of the para position of the terminal phenyl ring of GV150526 with suitable hydrophilic groups resulted in the identification of a new class of glycine antagonists. These compounds exhibited nanomolar in vitro affinity to the glycine binding site, high receptor selectivity, and outstanding in vivo potency. In particular, 3-[(E)-2-[(4-ureidomethylphenyl)aminocarbonyl]ethenyl]-4, 6-dichloroindole-2-carboxylic acid was found to be highly effective in the middle cerebral artery occlusion (MCAo) model in the rat, an animal model of focal ischemia, when given both prior to and after the occlusion of the middle cerebral artery. Notably, a significant neuroprotective effect was seen in this model postischaemia, when the administration of this compound was delayed up to 6 h from the occlusion of the middle cerebral artery, further confirming the wide therapeutic window seen for GV150526A.

Animals↗
PubMed1994

Automated assay for GV104326, a novel tribactam antibiotic, in human plasma by high-performance liquid chromatography and solid-phase extraction.

A highly automated, rapid, robust and specific plasma assay for GV104326, a novel tribactam antibiotic, has been developed to monitor human volunteer trials. The method involves automated solid-phase extraction with a strong anion-exchange phase and HPLC on a reversed-phase column with ultraviolet detection. The calibration range for the assay is 0.05-2 micrograms/ml. The assay is linear over this range and is specific with respect to endogenous interference and likely metabolites of GV104326. Both intra- and inter-assay variability were < 8% and intra- and inter-day bias < 10%.

Anion Exchange Resins↗
PubMed1991

Mass assay for inositol 1-phosphate in rat brain by high-performance liquid chromatography and pulsed amperometric detection.

A high-performance liquid chromatographic method for direct mass measurement of inositol 1-phosphate (I(1)P) in rat brain is described. Separation of I(1)P from its isomers and from endogenous components is achieved by polymeric anion-exchange chromatography with a sodium hydroxide/sodium acetate mobile phase. Detection is performed at high pH by pulsed amperometric detection at a gold electrode. Sample preparation involves liquid-liquid extraction and ion-exchange solid-phase extraction, prior to HPLC. The method is sufficiently sensitive and selective to enable facile determination of basal levels of I(1)P in small amounts of brain tissue. The applicability of the method is demonstrated by the in vivo monitoring of I(1)P levels in rat brain after administration of the inositol monophosphatase inhibitor lithium and the cholinergic agonist pilocarpine. The method is a significant improvement over existing published mass assays for I(1)P by virtue of its simplicity, speed, sensitivity, and ruggedness.

Animals↗
PubMed1989

Evidence for at least four different inositol bisphosphatases in bovine brain.

Bovine brain supernatant contains at least four enzymes capable of hydrolysing inositol bisphosphates. These activities may be distinguished on the basis of their metal, salt and pH dependence, sensitivity to Li+ ions and thiol-modification reagents, and on their molecular sizes. In addition to Li+-sensitive Ins(1,4)P2/Ins(1,3,4)P3 1-phosphatase [Gee et al. (1988) Biochem. J. 253, 777-782] which has an absolute requirement for Mg2+, two (Li+-insensitive and Mg2+-independent) phosphatases, capable of hydrolysing Ins(3,4)P2 and Ins(1,3)P2, respectively, have been identified. Both enzymes were inhibited by only moderate concentrations of salt, although for the former there was no obvious correlation between inhibitory potency and either the nature of the anion/cation or the ionic strength of the buffer. Ins(3,4)P2 phosphatase had a pH optimum of 7.6 and this activity could be resolved on gel-filtration columns into a two overlapping peaks of molecular mass 170 kDa and 450 kDa. Mg2+-independent Ins(1,3)P2 phosphatase had a pH optimum of 7.1 and displayed a single broad activity peak on gel-filtration columns. However, if assays were performed in the presence of Mg2+, a second Ins(1,3)P2 phosphatase was revealed (35 kDa), which had a pH optimum of 8.8 Ins(1,4)P2/Ins(1,3,4)P3 1-phosphatase, Ins(3,4)P2 phosphatase, Mg2+-independent Ins(1,3)P2 phosphatase and inositol monophosphatase were all inhibited by 5,5'-dithiobis(2-nitrobenzoic acid) with IC50 values of 34 microM, 65 microM and 560 microM and 1100 microM, respectively. The metabolism of Ins(1,3,4)P3 by brain supernatant was also examined. Product specificity was shown to be entirely dependent on the buffer conditions employed. In Mg2+-containing buffers, Ins(1,3,4)P3 was hydrolysed predominantly to Ins(3,4)P2, consistent with hydrolysis by Ins(1,4)P2/Ins(1,3,4)P3 1-phosphatase. In the presence of EDTA, Ins(1,3,4)P3 was degraded exclusively by a 4-phosphatase enzyme generating Ins(1,3)P2. Under these conditions, high concentrations of Ins(3,4)P2 blocked the hydrolysis of Ins(1,3,4)P3.

Animals↗
PubMed1988

Purification and properties of inositol-1,4-bisphosphatase from bovine brain.

Inositol-1,4-bisphosphatase has been purified 13,000-fold from bovine brain supernatant. The enzyme is monomeric, with an apparent subunit Mr of 40,000. Maximal hydrolytic rates were observed in Tris buffer, pH 7.8, in the presence of 9 mM-Mg2+. The enzyme acted as a 1-phosphatase, hydrolysing both inositol 1,4-bisphosphate [Ins(1,4)P2] (Km 0.04 mM) and inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] (Km 0.5 mM) to inositol 4-phosphate and inositol 3,4-bisphosphate respectively. Li+ inhibited the hydrolysis of both substrates in an uncompetitive manner, with apparent Ki values of 9.63 mM and 0.46 mM for Ins(1,4)P2 and Ins(1,3,4)P3 respectively.

Animals↗
PubMed1988

The dephosphorylation of inositol 1,4-bisphosphate to inositol in liver and brain involves two distinct Li+-sensitive enzymes and proceeds via inositol 4-phosphate.

1. Hydrolysis of both enantiomers of inositol 1-phosphate and both enantiomers of inositol 4-phosphate to inositol is inhibited by LiCl in liver and brain. 2. The phosphatase activity is predominantly soluble. 3. Inositol 1,4-bisphosphate is also hydrolysed by the soluble fraction of liver and brain. 4. Bisphosphatase activity is inhibited by LiCl, but is less sensitive than monophosphatase activity. 5. The product of bisphosphatase in liver and brain is inositol 4-phosphate.

Animals↗
PubMed1979

Incorporation of N-fluoroacetyl-D-glucosamine into hyaluronate by rabbit tracheal explants in organ culture.

1. Incubation of rabbit tracheal explants with N-[(3)H]acetyl-d-glucosamine and N-acetyl-d-[1-(14)C]glucosamine led to labelling of a number of soluble macromolecular products separable from the medium, after papain digestion, by ion-exchange chromatography. 2. With N-acetyl-d-[1-(14)C]glucosamine in the incubation medium, a neutral glycoprotein, two acidic glycoprotein fractions, hyaluronic acid and a glycosaminoglycan fraction were obtained and all were radioactively labelled. Similar labelling occurred with N-fluoroacetyl-d-[1-(14)C]glucosamine or N-fluoro[(3)H]acetylglucosamine as precursor. 3. Maximal labelling was obtained at 96h after incubation of cultures. N-Fluoroacetyl-glucosamine under these conditions was incorporated into hyaluronate less efficiently than N-acetylglucosamine. 4. With N-fluoroacetyl-d-[1-(14)C]glucosamine as precursor, a hyaluronate component was separated that on enzymic degradation by glycosidases (hyaluronidase, beta-glucuronidase and N-acetyl-beta-hexosaminidase) yielded a (14)C-labelled oligosaccharide fraction together with N-acetyl-d-[1-(14)C]glucosamine and N-fluoroacetyl-d-[1-(14)C]glucosamine, consistent with some exchange of N-acetyl groups having occurred. 5. The results on enzymic degradation of labelled macromolecules by glycosidases suggest that the presence of incorporated N-fluoroacetyl side chains may render the hyaluronate analogue more resistant to hyaluronidase.

Acetylglucosamine↗
PubMedDate not supplied

Pharmacokinetics of a glycine site antagonist (gavestinel) following multiple dosing in patients with acute stroke.

OBJECTIVE: The objective of this study was to characterize the pharmacokinetics of gavestinel in patients with acute stroke. METHODS: Gavestinel was administered as an 800-mg loading dose and followed by either 100-, 200-, or 400-mg maintenance doses given every 12 h for five doses. Blood and urine samples were collected for pharmacokinetic evaluation. The pharmacokinetics of gavestinel were determined using compartmental analysis. RESULTS: The mean clearance (CL) and central (Vc) and steady-state (Vss) volumes of distribution across the dose groups were 0.31-0.40 l x h(-1), 3.3-3.9 l, and 9.8-17 l, respectively. The mean terminal half-life ranged from 29 h to 56 h. Gavestinel was extensively bound to plasma protein (median percentage free <0.01). During gavestinel administration, some patients exhibited elevated levels of bilirubin, which may be the result of shared mechanisms of elimination (glucuronide conjugation and excretion in bile). CONCLUSIONS: This study characterized the pharmacokinetics of gavestinel following multiple doses in acute stroke patients and showed that the pharmacokinetics are similar for increasing maintenance doses. The high protein binding of gavestinel was confirmed in acute stroke patients. A pharmacokinetic interaction between gavestinel and bilirubin may contribute to the increase in bilirubin.

Aged↗