The activation of supraspinal GPR40/FFA1 receptor signalling regulates the descending pain control system.

BACKGROUND AND PURPOSE: The ω-3 polyunsaturated fatty acids exert antinociceptive effects in inflammatory and neuropathic pain; however, the underlying mechanisms remain unclear. Docosahexaenoic acid-induced antinociception may be mediated by the orphan GPR40, now identified as the free fatty acid receptor 1 (FFA1 receptor). Here, we examined the involvement of supraspinal FFA1 receptor signalling in the regulation of inhibitory pain control systems consisting of serotonergic and noradrenergic neurons. EXPERIMENTAL APPROACH: Formalin-induced pain behaviours were measured in mice. Antinociception induced by FFA1 receptor agonists was examined by intrathecal injections of a catecholaminergic toxin, 5-HT lowering drug or these antagonists. The expression of FFA1 receptor protein and c-Fos was estimated by immunohistochemistry, and the levels of noradrenaline and 5-HT in the spinal cord were measured by LC-MS/MS. KEY RESULTS: FFA1 receptors colocalized with NeuN (a neuron marker) in the medulla oblongata and with tryptophan hydroxylase (TPH; a serotonergic neuron marker) and dopamine ß-hydroxylase (DBH; a noradrenergic neuron marker). A single i.c.v. injection of GW9508, a FFA1 receptor agonist, increased the number of c-Fos-positive cells and the number of neurons double-labelled for c-Fos and TPH and/or DBH. It decreased formalin-induced pain behaviour. This effect was inhibited by pretreatment with 6-hydroxydopamine, DL-p-chlorophenylalanine, yohimbine or WAY100635. Furthermore, GW9508 facilitated the release of noradrenaline and 5-HT in the spinal cord. In addition, GW1100, a FFA1 receptor antagonist, significantly increased formalin-induced pain-related behaviour. CONCLUSION AND IMPLICATIONS: Activation of the FFA1 receptor signalling pathway may play an important role in the regulation of the descending pain control system.

Inhibition of a medium chain acyl-CoA synthetase involved in glycine conjugation by carboxylic acids.

Molecular characteristics of carboxylic acids were investigated for the ability to inhibit a purified medium chain acyl-CoA synthetase, using hexanoic acid as a substrate. Salicylic acid, 4-methylsalicylic acid, 2-hydroxynaphtoic acid, and 2-hydroxyoctanoic acid, which do not act as substrates for the medium chain acyl-CoA synthetase, were potent as inhibitors. Valproic acid was not an inhibitor. Salicylic acid, 2-hydroxynaphthoic acid, and 2-hydroxyoctanoic acid inhibited the medium chain acyl-CoA synthetase with Ki values of 37, 5.2, and 500 microM, respectively. 4-Methylsalicylic acid was more potent than salicylic acid. The inhibitory carboxylic acids were competitive with respect to hexanoic acid. The distance of the hydroxyl group from the carboxylic acid group of the benzene ring influenced the inhibitory activity. The hydroxyl group on the carbon adjacent to the carboxylic acid group was required for inhibitory activity. In addition, there was a good correlation between the lipophilicity of the carboxylic acids and the Ki values, suggesting that the lipophilicity of the carboxylic acids is a major determinant for inhibition of the medium chain acyl-CoA synthetase.

Participation of a medium chain acyl-CoA synthetase in glycine conjugation of the benzoic acid derivatives with the electron-donating groups.

Glycine conjugation of a series of benzoic acid derivatives was investigated in bovine liver mitochondria. Benzoic acids with chlorine, methyl, methoxy or ethoxy substituents in the para-or meta-positions of the benzene ring showed a high degree of glycine conjugation. In contrast, the acids with cyano, nitro, amino, or acetylamino groups were conjugated to a small extent with glycine. A medium chain acyl-CoA synthetase that activates carboxylic acids was purified from bovine liver mitochondria. The purified medium chain acyl-CoA synthetase accepted not only medium chain fatty acids but also aromatic and arylacetic acids as substrates. There was a good correlation between the activity of the purified medium chain acyl-CoA synthetase and glycine conjugation of ten benzoic acids with electron-donating substituents. These findings indicate that the purified medium chain acyl-CoA synthetase is a major enzyme for glycine conjugation of benzoic acids with electron-donating groups in bovine live mitochondria.

Molecular specificity of a medium chain acyl-CoA synthetase for substrates and inhibitors: conformational analysis.

Amino acid conjugation is an important route of detoxification of xenobiotic and endogenous carboxylic acids. The specificity of the purified medium chain acyl-CoA synthetase catalyzing the first reaction of amino acid conjugation was investigated further for substrates and inhibitors. Molecular modeling techniques were applied to derive the molecular characteristics of substrates and inhibitors for the medium chain acyl-CoA synthetase. The purified enzyme accepted not only straight medium chain fatty acids but also aromatic acids. Of the arylacetic acids, activity was obtained with naphthylacetic acids, whereas introduction of a methyl group at the alpha-position caused loss of activity. High activity was also observed with cyclohexanoic acid. Diflunisal, 2-hydroxydodecanoic acid, and nalidixic acid inhibited the medium chain acyl-CoA synthetase activity for hexanoic acid, with Ki values of 0.8, 4.4, and 12.3 microM, respectively. The inhibitory carboxylic acids were competitive with respect to hexanoic acid. The hydroxyl or ketone (oxo) groups at the beta-position of carboxylic acids were an important determinant for inhibitory activity. All substrates and inhibitors contained a flat hydrophobic region coplanar to the carboxylate group. In addition, the substrates had negative values for charge on the carbon in the beta-position of carboxylic acids.

Inhibitory effect of quinolone antimicrobial and nonsteroidal anti-inflammatory drugs on a medium chain acyl-CoA synthetase.

The inhibitory effects of quinolone antimicrobial agents and nonsteroidal anti-inflammatory drugs on purified mouse liver mitochondrial medium chain acyl-CoA synthetase catalyzing the first reaction of glycine conjugation were examined, using hexanoic acid as a substrate. Enoxacin, ofloxacin, nalidixic acid, diflunisal, salicylic acid, 2-hydroxynaphthoic acid, and 2-hydroxydodecanoic acid, which do not act as substrates, were potent inhibitors. Diflunisal, nalidixic acid, salicylic acid, 2-hydroxynaphthoic acid, and 2-hydroxydodecanoic acid inhibited competitively this medium chain acyl-CoA synthetase with K(i) values of 0.6, 12.4, 19.6, 13.4, and 15.0 microM, respectively. Enoxacin and ofloxacin inhibited this medium chain acyl-CoA synthetase in a mixed-type manner with K(i) values of 23.7 and 38.2 microM, respectively. Felbinac, which is a substrate, inhibited the activity of this medium chain acyl-CoA synthetase for hexanoic acid (IC50 = 25 microM). The concomitant presence of enoxacin and felbinac strongly inhibited this medium chain acyl-CoA synthetase. These findings indicate that medium chain acyl-CoA synthetases may be influenced by quinolone antimicrobial and nonsteroidal anti-inflammatory drugs.

Effect of a pyridinium metabolite derived from haloperidol on the activities of striatal tyrosine hydroxylase in freely moving rats.

The effects of a pyridinium metabolite (HPP+) derived from haloperidol (HP) on in vivo tyrosine hydroxylation was evaluated in freely moving rats. As an index of the in vivo activity of tyrosine hydroxylase (TH), the rat striatum was perfused with NSD-1015, and extracellular 3,4-dihydroxyphenylalanine (DOPA) levels were measured. HPP+ (1 mM) gradually reduced tyrosine hydroxylation to 30% of the basal level, although the effect was less potent than 1-methyl-4-phenylpyridinium ion (MPP+). On the contrary, HPP+ at a 0.1 mM dose decreased in 5-hydroxyindoleacetic acid (5-HIAA) level, but did not affect dopamine metabolites. The present study revealed that HPP+ irreversible inhibited in vivo tyrosine hydroxylation by the same manner of MPP+. However, the neurotoxic effects of HPP+ in vivo would be selective for serotonergic over dopaminergic neurons, which distinguishes the toxic profile of this compound compared to that of MPP+.

Studies on the metabolism of haloperidol (HP): the role of CYP3A in the production of the neurotoxic pyridinium metabolite HPP+ found in rat brain following ip administration of HP.

The levels of haloperidol (HP) and its pyridinium metabolite HPP+ were estimated in plasma and brain tissues of rats treated i.p. with HP (10 mg/kg). HP and HPP+ levels in plasma decreased linearly during the 0-3 hour period following drug administration. On the other hand, HPP+ levels in brain tissues increased gradually during the same period. HPP+ levels in brain tissues increased further when HP (10 mg/kg) was injected for three consecutive days. The formation of HPP+ also was studied in rat brain mitochondrial and liver microsomal preparations. Enzyme activity responsible for the conversion of HP to HPP+ was not found in brain mitochondria. Liver microsomal enzymes catalyzed the oxidation of HP and its tetrahydropyridine dehydration product HPTP to HPP+ with about the same efficiency. Studies employing several cytochrome P450 inhibitors and anti-cytochrome P450 antibodies were carried out in an effort to identify the forms of cytochrome P450 that are responsible for catalyzing the oxidation of HP and HPTP to HPP+. The formation of HPP+ in liver microsomes was strongly inhibited by ketoconazole and nifedipine and by an anti-CYP3A antibody. These results suggest that formation of HPP+ from HP and HPTP in rat liver microsomes is catalyzed mainly by CYP3A although the participation of other P450 forms cannot be ruled out.

Characterization of a renal medium chain acyl-CoA synthetase responsible for glycine conjugation in mouse kidney mitochondria.

Glycine conjugation of a series of benzoic acid derivatives was investigated in mouse kidney mitochondria. The chlorine and methyl substitutions in the para- and meta-positions of the benzene ring yielded an increase in glycine conjugation. The acids with a methoxy group showed a low degree of glycine conjugation. In addition, the acids with nitro or amino groups were conjugated to a slight extent with glycine. The in vitro conjugation of salicylic acid with glycine occurred not in liver but in kidney. The specificity of the renal medium chain acyl-CoA synthetase catalyzing the first reaction of glycine conjugation was also examined. The enzyme accepted not only medium chain fatty acids but also aromatic and arylacetic acids. The highest activity was shown with hexanoic acid. High activities were observed for benzoic acid derivatives with alkyl and alkoxyl groups in the para- and meta-positions of the benzene ring. An ortho-substituted acid exhibited no activity. In addition, the enzyme was less active with valproic acid, tranexamic acid, indomethacin and ketoprofen. The enzyme was inhibited by diflunisal, 2-hydroxydodecanoic acid and salicylic acid, which did not act as substrates. There was a poor correlation between the activity of the medium chain acyl-CoA synthetase and glycine conjugation of eleven substituted benzoic acids. These findings suggest that the present medium chain acyl-CoA synthetase is involved in glycine conjugation of the substituted acids in mouse kidney mitochondria, but there may be a larger contribution of another isoenzyme.

Difference of the liver and kidney in glycine conjugation of ortho-substituted benzoic acids.

The relative importance of the liver and kidney for glycine conjugation of ortho-substituted benzoic acids was investigated. Glycine conjugation of ortho-substituted benzoic acids was investigated in mouse liver and kidney mitochondria. The extent of glycine conjugation of benzoic acids with the halogen group decreased in the order F > Cl > Br > I. The conjugation of salicylic acid with glycine took place in only the kidney. 2-Methoxybenzoic acid exhibited no activity in the liver and kidney. The difference in glycine conjugation of ortho-substituted benzoic acids was observed between liver and kidney. The kidney was more active in glycine conjugation of ortho-substituted acids than the liver. In addition, the relationship between glycine conjugation and the chemical structure of ortho-substituted acids was examined in the liver and kidney. The size of the substituent had a far greater influence over glycine conjugation in the liver and kidney. Glycine conjugation was also dependent on the substituent electronegativity. It may be important that the substrates undergoing glycine conjugation contain a flat region coplanar to the carboxylate group.

Metabolism of benoxinate in humans.

The metabolism of benoxinate hydrochloride [2-(diethylamino)ethyl 4-amino-3-butoxybenzoate monohydrochloride; oxybuprocaine] was examined in humans after administration of a single oral dose. The drug was almost completely absorbed and was rapidly excreted in the urine (92.1% of dose in 9 h). Nine metabolites and unchanged drug were isolated from the urine and identified by comparison of TLC, GC, and GC-MS with authentic compounds. Any metabolites reflecting initial loss of the butyl side chain of benoxinate could not be detected. This suggests that the ester portion is metabolized more rapidly than the O-butyl side chain. 3-Butoxy-4-aminobenzoic acid, the hydrolyzed product of benoxinate, was primarily excreted (70-90% of dose) as the glucuronide together with a trace of the glycine conjugate (0.35% of dose). In addition, 3-butoxy-4-acetylaminobenzoic acid, 3-hydroxy-4-aminobenzoic acid, and 3-hydroxy-4-acetylaminobenzoic acid were identified, the latter two being detected partly as the glucuronides (1.20 and 1.43% of dose, respectively).

The analysis of cocaine and its metabolites by liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS).

The method for simultaneous determination of cocaine and its four metabolites (benzoylecgonine, ecgonine methyl ester, ecgonine and norcocaine) in urine by liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS) was studied. The mass spectra showed the quasi-molecular ions, [M+H]+ as the base peak. LC/APCI-MS analysis was performed by focusing the characteristic ions at m/ = 186, 290, 200, 304 and 290 for ecgonine, benzoylecgonine, ecgonine methyl ester, cocaine and norcocaine, respectively. Cocaine and its four metabolites were well separated by high performance liquid chromatography (HPLC). The recoveries of cocaine and its metabolites from the spiked urine were 40.3-94.7% by solid-phase extraction with two type cartridges (Bond Elut Certify and Bond Elut SCX).

Determination of bromvalerylurea and its metabolites in biological samples by frit-fast atom bombardment liquid chromatography-mass spectrometry.

A simple, rapid, and sensitive method which allows us to simultaneously determine bromvalerylurea (BVU) and its three metabolites (3-methylbutyrylurea [MVU], alpha-(cystein-S-yl)isovalerylurea [CVU], and alpha-(N-acetylcystein-S-yl)isovalerylurea [AcCVU]) was investigated by frit-fast atom bombardment liquid chromatography-mass spectrometry (frit-FAB LC-MS). The LC-MS analysis was performed after the solid-phase extraction from tissue and urine samples with a Sep-Pak C18 cartridge. Tissue homogenates and urine were adjusted to pH 4.0 and applied to the cartridges. The retained BVU and its metabolites were eluted from the cartridge with 2 mL of acetonitrile/10 mM ammonium acetate buffer (pH 3.5, 50:50, v/v). The eluate was analyzed by LC-MS, which employs a semimicro type L-column ODS column. The proposed conditions are as follows: mobile phase A, 0.4% glycerol in acetonitrile/10 mM ammonium acetate buffer (pH 3.5) (5:95, v/v); mobile phase B, 0.4% glycerol in acetonitrile; elution mode, linear gradient, 100% A (5 min) to 100% B in 15 min; flow rate, 0.2 mL/min; split ratio, 1:40. Extraction recoveries of BVU and its metabolites were 91.90-97.79% from the spiked liver homogenate and 89.68-96.13% from the spiked urine. The detection limits ranged from 10 to 25 ng/g in selected ion monitoring mode.

Metabolism of chlorpheniramine in rat and human by use of stable isotopes.

1. The metabolism of chlorpheniramine (I) was examined in vivo in rats and a human volunteer; in the rats a stable isotope was used. 2. In addition to the unchanged drug (I) and the N-demethylated metabolites (II and III), nine further metabolites were identified in rat urine, four of which were also found in human urine. Chlorpheniramine N-oxide (IV), 3-(p-chlorophenyl)-3-(2-pyridyl) propanol (V), 3-(p-chlorophenyl)-3-(2-pyridyl)-N-acetylaminopropane (VII) and 3-(p-chlorophenyl)-3-(2-pyridyl)-propionic acid (XIII) were identified in rat and human urine. 3. The hydroxylated metabolites of the pyridyl ring of the unchanged drug, II, V and VII, and the glucuronide of XIII were identified only in rat urine. XIII was found in rat urine as long as 6 days after the last dose.

Oxybuprocaine and five metabolites simultaneously determined in urine by gas chromatography and gas chromatography-mass spectrometry after extraction with Extrelut.

We describe a gas-liquid chromatographic (GC) method for determination of oxybuprocaine, and a gas chromatographic-mass spectrometric (GC-MS) method for simultaneous determination of four of its nine metabolites in urine. We used an Extrelut column to simply and rapidly extract oxybuprocaine and its metabolites from urine. For the GC-MS analyses, we monitored the characteristic fragment ions at m/z 353, 395, 369, 411, and 235 for 3-butoxy-4-aminobenzoic acid (metabolite 2, M-2), 3-butoxy-4-acetylaminobenzoic acid (M-3), 3-hydroxy-4-aminobenzoic acid (M-4), 3-hydroxy-4-acetylaminobenzoic acid (M-5), and methaqualone (internal standard), respectively. We quantified the glucuronide of M-2 after enzymic treatment. The assay's selectivity and reproducibility (within-day and between-day CVs less than 8% for all metabolites) make it applicable to determine oxybuprocaine and its metabolites in human urine. Mean 9-h urinary excretion of oxybuprocaine and its five metabolites from four healthy volunteers was 89.2% after a 100-mg oral dose.

Metabolism of dibucaine. II. Disposition and metabolism of dibucaine in rats.

The disposition and metabolism of dibucaine were studied in rats. After intraperitoneal administration of 3H-labelled dibucaine, the blood concentration of total radioactivity reached a maximum at 10 min and declined thereafter, with a biphasic curve having half-lives of 37.7 min and 11.2 h. Radioactivity in the tissues after administration was high in small intestine, lung, spleen, liver, kidney and stomach. Urinary and fecal excretion of radioactivity were 39.4 and 49.0% of the dose, respectively, in 3 d after administration. Biliary excretion of radioactivity in bile duct-cannulated rats was 53% of the dose within 48 h. About 40% of urinary, fecal and biliary excretion was found in the basic metabolites, and about 12% of urinary excretion was in the acidic metabolites. In addition, 9.5% of urinary excretion and 39.5% of biliary excretion were conjugated metabolites which could be extracted following enzymatic hydrolysis. Most of the conjugates were glucuronides. Remaining metabolites were highly polar and water-soluble ones which were not hydrolyzed by enzyme treatment and could not be extracted into organic solvents.

Metabolism of dibucaine: isolation and identification of urinary basic metabolites in the rat, rabbit and man.

The metabolism of dibucaine was studied in the rat, rabbit and man. A total of ten basic metabolites other than dibucaine were detected in the urine samples of three species by thin-layer chromatography (TLC) and gas chromatography (GC), and structures of these metabolites were identified by comparison of the properties given by TLC, GC and gas chromatography-mass spectrometry (GC-MS) with those of authentic compounds. Four of these metabolites were new metabolites which were found in the rabbit or human urine; two were identified as the 2', 3'-dihydroxybutoxy product (M-6, diol) and its N-deethyl product (M-2), and others were identified as the 2'-hydroxyethoxy product (M-8, alcohol) and its N-deethyl product (M-3). One of the two hydroxyl groups on M-5 was at 6-position on the quinoline ring, while another was assumed to be at 3'-position on the O-alkyl side chain. There were apparent species differences with regard to the major metabolites found in each species; i.e. M-10 and M-5 in rat, M-6 and M-4 in rabbit, and M-8 and M-4 in man. Small amounts of the conjugated basic metabolites were observed in the urine of all three species. The new metabolic pathways to the diols (M-2 and M-6) or the alcohols (M-3 and M-8) were also discussed.

Glycine conjugation of the substituted benzoic acids in vitro: structure-metabolism relationship study.

The relationships between the chemical structure and glycine conjugation of 10 para- and 8 meta-monosubstituted, and 6 disubstituted benzoic acids were examined in the rat liver and kidney mitochondria. For the simultaneous determination of the acid and its glycine conjugate, a simple and specific high performance liquid chromatographic method was developed. The extent of glycine conjugation of a series of substituted benzoic acids in liver mitochondria was similar to that in kidney mitochondria. Glycine conjugation increased with greater lipid solubility. On the other hand, more bulky substituents at the p- or m-position on the benzene ring reduced the glycine conjugation. The dependence on van der Waals volume (Vw) values for substituents reflects the importance of steric effects in the glycine conjugation. However the steric effect of the substituent was slightly less pronounced at the m-position than at the p-position. These results indicate that an active site of the enzyme possess limited steric bulk tolerance.

Glycine conjugation of the substituted benzoic acids in mice: structure-metabolism relationship study II.

Glycine conjugation of a series of substituted benzoic acids was investigated in the mouse liver and kidney mitochondria. Correlations between the structure of 24 substituted benzoic acids and glycine conjugation were obtained. The extent of glycine conjugation of a series of substituted benzoic acids in liver mitochondria was different from that in kidney mitochondria. Glycine conjugation increased with greater lipid solubility in the mouse liver and kidney. The steric effect of the substituent had a far greater influence over the glycine conjugation in kidney, while the size of the substituent played a small role in the pattern of conjugation in liver. The formation of the glycine conjugate in liver was also dependent on the substituent electronegativity.

Determination of ornithine conjugates of some carboxylic acids in birds by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method for the determination of ornithine conjugation of some carboxylic acids in vitro has been developed. The ornithine conjugates of benzoic acid, p-nitrobenzoic acid, furancarboxylic acid and phenylacetic acid in an incubation mixture with kidney mitochondria were well separated on a reversed-phase C18 column using a mixture of 10 mM ammonium acetate buffer and methanol as the mobile phase. In addition, by varying the pH of the mobile phase and utilizing the absorption wavelengths (nm) of the conjugates it was possible to resolve and specifically detect each conjugate. The calibration curves were linear in the range of 0.2-16 micrograms/ml for all compounds and the detection limits were about 50 ng/ml except for the ornithine conjugate of phenyl acetic acid (S/N = 2). The ornithine conjugation of some carboxylic acids with chicken kidney mitochondria were determined by this assay method. The activity of ornithine conjugation of benzoic acid, furancarboxylic acid, p-nitrobenzoic acid and phenylacetic acid were 14.5, 5.5, 0.5 and 6.9 nmol/mg of protein, respectively. Moreover, the ornithine conjugation and the glycine conjugation of benzoic acid were examined in birds and rodents. The ornithine conjugation was observed only in chicken (14.5 nmol/mg of protein) and mallard (0.99 nmol/mg of protein).

Purification and characterization of a medium chain acyl-coenzyme A synthetase.

Glycine conjugation is an important route of detoxification of many xenobiotic and endogenous carboxylic acids. A medium chain acyl-coenzyme A synthetase that catalyzes the first reaction of glycine conjugation was purified from bovine liver mitochondria by chromatographies on anion exchange, hydroxylapatite, affinity, and finally by gel filtration. The purified enzyme not only conjugates medium chain fatty acids, but also aromatic and arylacetic acids. The highest activity was shown with hexanoic acid. High activities were observed for benzoic acid derivatives with large alkyl and alkoxyl groups in the para- or meta-positions of the benzene ring. Ortho-substituted derivatives exhibited no activity. The enzyme was inhibited by iodoacetamide and salicylic acid, and activated by albumin. Salicylic acid was a competitive inhibitor of the enzyme, with an apparent Ki value of 37 microM. Enzyme activity increased 74% when the pH was raised from 7 to 10. Molecular weight of the purified medium chain acyl-coenzyme A synthetase was 65.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.