Polymorphic debrisoquin metabolism in a Turkish population.

Debrisoquin hydroxylation polymorphism was studied in 326 unrelated healthy Turkish volunteers. Debrisoquin sulfate (10 mg) was administered to subjects, and debrisoquin and 4-hydroxydebrisoquin were determined in the 0- to 8-hour urine samples. Debrisoquin oxidation was polymorphic, with 11 subjects (3.37%; 95% confidence interval, 1.69% to 6.07%) phenotyped as poor metabolizers. The metabolic ratio between debrisoquin and 4-hydroxydebrisoquin in 8-hour urine samples ranged from 0.02 in extensive metabolizers to 263.8 in poor metabolizers. The proportion of poor metabolizers was found to be in the range observed in the other white populations studied.

Low frequency of defective alleles of cytochrome P450 enzymes 2C19 and 2D6 in the Turkish population.

BACKGROUND AND OBJECTIVES: The genetically polymorphic cytochrome P450 enzymes 2Cl9 (CYP2Cl9) and 2D6 (CYP2D6) contribute to the metabolism of about 30% of all drugs. For analysis of the ethnic-related differences in drug disposition and as a preparation for routine genotyping, we examined CYP2C19 and CYP2D6 mutations in a large Turkish population. METHODS: CYP2C19 and CYP2D6 alleles were determined with use of genomic deoxyribonucleic acid from 404 unrelated Turkish individuals. CYP2C19 alleles *1 to *5 and CYP2D6 alleles *1 to *12, and *14, *15, and *17 were measured by polymerase chain reaction-restriction fragment length polymorphism assays. RESULTS: From 404 subjects genotyped for CYP2C19, allele frequencies of CYP2C19*1 (wt), CYP2C19*2 (ml), and CYP2C19*3 (m2) were 0.88, 0.12, and 0.004, respectively; mutations m3 and m4 were not found. Four individuals (1.0%) were predicted to be poor metabolizers (CYP2C19*2/*2), a significantly lower frequency compared to Middle European populations. Among 404 subjects genotyped for CYP2D6, most frequent alleles were CYP2D6*1 (allele frequency 0.37), *2 (0.35), *4 (0.11), *10 (0.06), duplications *1x2, *2x2, or *4x2 (0.06), *5 (0.01), and *17(0.01). Overall, six subjects (1.49%) were predicted to be CYP2D6 poor metabolizers, and 35 subjects (8.66%) were predicted to be ultrarapid metabolizers as a result of CYP2D6 gene duplications. CONCLUSION: Obviously, within Europe there is a north-south gradient, with decreasing frequency of poor metabolizers of CYP2C19 and CYP2D6 to the south and a corresponding increase of ultrarapid metabolizers of CYP2D6. As in other white groups, only CYP2C19*2 plays a relevant role for the CYP2C19 poor metabolizer phenotype. The mutational spectrum of CYP2D6 indicated partial ethnic relationships to Asian and African populations.

The effects of yohimbine and neuroleptics on apomorphine-induced pecking behaviour in the pigeon.

The effects of yohimbine and some neuroleptics were investigated on pecking behaviour, induced by apomorphine. The total pecking counts, recorded for 18 min, elicited by a standard dose of apomorphine (1 mg/kg) were inhibited dose-dependently by yohimbine and the neuroleptics. Haloperidol was found to be the most potent, trifluoperazine, yohimbine, chlorpromazine and the atypical neuroleptics were less effective in decreasing order. Chronic treatment with yohimbine (2.5 mg/kg per day for 20 days) caused an increase in pecking behaviour. These results suggest that apomorphine-induced pecking behaviour in the pigeon could be used as a reliable method for screening neuroleptic drugs and the antidopaminergic activity of yohimbine is verified in this model.

Muscarinic component of splanchnic-adrenal transmission in the dog.

1. The effect of atropine on catecholamine release evoked by stimulation of the splanchnic nerve was studied in the adrenal medulla of the dog. The magnitude of the catecholamine release was estimated biologically on the basis of the pressor response occurring in the perfused and acutely sympathectomized hindquarters of the same dog by comparing it with responses elicited by intravenous injection of adrenaline.2. Atropine reduced the responses to nerve stimulation, and appeared to have a more prominent effect on the responses elicited by stimulation at moderate frequency (10 pulses/sec).3. Hexamethonium or nicotine caused a more powerful, but not complete, blockade of transmission; the subsequent injection of atropine caused a further inhibition of the residual responses, leading to a complete, or near complete, abolition of the release by nerve stimulation.4. The data were taken as evidence that transmission of impulses through muscarinic receptors occurs in normal conditions in the adrenal medulla of the dog, though this type of transmission is less prominent than that through nicotinic receptors.

Evidence for muscarinic receptors in the adrenal medulla of the dog.

1. The release of catecholamines from the adrenal medulla by the cholinergic drugs was monitored by the perfusion pressure rise in autoperfused and sympathetically denervated hindlimbs of the dog.2. Acetylcholine, dimethylphenylpiperazinium (DMPP) and methacholine, given in relatively small doses into the aorta proximal to the blood supply to the adrenal glands, caused a marked rise in perfusion pressure which was due to the release of catecholamines from the adrenal medulla.3. Atropine (1 mg/kg intravenously) blocked the pressor response to methacholine only. The ganglion-blocking agents (mecamylamine 4-5 mg/kg or hexamethonium 10 mg/kg) given subsequently blocked the pressor responses to the other two cholinergic drugs. The ganglion-blocking agents, when given before atropine, blocked the pressor action only of DMPP. These agents partly depressed, rather than potentiated, the pressor response to methacholine.4. The results suggest that muscarinic as well as nicotinic receptors are present in the adrenal medulla of the dog.5. Neither atropine nor ganglion-blocking drugs alone reduced the pressor response to acetylcholine. It is postulated that the blockade of one set of receptors makes more acetylcholine available for the other set of receptors and so inactivation of the former receptors are compensated by the increased release through the activation of the latter's.

Regional distribution of cholinergic muscarinic receptors in spinal cord.

Quinuclidinylbenzilate ([3H]QNB) binding sites are present in the rat spinal cord. The binding site are muscarinic in character based on displacement of [3H]QNB by cholinoceptive drugs. They are distributed rather uniformly along the cord, although the receptor density is greater in gray matters than in white matter. Binding to white matter may be associated with glial cells. Within the gray matter, the receptor density is higher in the ventral horn than in the dorsal horn. In the thoracic region receptor density is about equal in the intermediate zone and ventral horn. Mid-thoracic transection of the cord does not change the receptor density or the dissociation constant of [3H]QNB in the lumbar cord. In contrast, treatment with the neurotoxin, 6-aminonicotinamide, which produces lesions of the cord, loss of motor control and paralysis, reduces the receptor density and affinity of [3H]QNB for lumbar gray matter but not white matter. The presence of [3H]QNB binding sites thrughout the spinal cord as well as the documented presence of acetylcholine-containing neurons, suggest that muscarinic receptors play a role in all phases of spinal cord physiology.

Muscarinic receptor binding in the rat adrenal medulla.

Muscarinic receptor binding sites are present in rat adrenal gland andare associated primarily with the medulla. A Scatchard analysis using the ligand [3H]-quinuclidinyl benzylate (eH-QNB) revealed a KD of 0.064 nM and a Bmax of 66 fmol/mg prot. Denervation of the adrenal gland had no significant effect on 3H-QNG binding. The presence of muscarinic receptor binding sites in the adrenal medulla is consistent with reports that muscarinic receptors play a role in the release of adrenal catecholamines and modulation of cycle 3',5'-guanosine monophosphate.

The rat anococcygeus muscle is a convenient bioassay organ for the airway epithelium-derived relaxant factor.

The response to epithelium-derived relaxant factor (EpDRF) released from guinea-pig trachea by acetylcholine was studied in the precontracted rat anococcygeus muscle in a coaxial bioassay system. Acetylcholine caused no relaxation of rat anococcygeus muscle which was precontracted with phenylephrine. When the same muscle was placed into a guinea-pig trachea with intact epithelium, acetylcholine produced a slowly developing relaxation which was potentiated by neostigmine and antagonized by atropine but was not altered by indomethacin, propranolol, hydroquinone nor methylene blue. The rat anococcygeus muscle was not relaxed by acetylcholine when it was placed in an epithelium-free trachea. The results indicate that acetylcholine releases EpDRF from guinea-pig trachea and that rat anococcygeus muscle is a convenient organ for the bioassay of EpDRF.

High frequency discharges in sensory nerves following ischemia.

The effects of ischemia and recovery from ischemia on afferent discharges recorded from dorsal root filaments were assessed in anesthesized cats. During ischemia produced by cross-clamping the lumbar aorta, some of the hindlimb afferents originating in muscle spindles exhibited a sustained high frequency discharge of 120--160 Hz, the so-called "explosion." During recovery from ischemia, nerve discharges appeared in previously inactive dorsal root afferent nerve fibers not of muscle spindle origin. The post-ischemic high frequency discharges appeared in bursts of action potentials at frequencies of 200--500 per second, with 6--32 spikes per burst; the bursts recurred at intervals of 120--600 msec. The nerve fibers exhibiting post-ischemic bursts could not be activated by muscle stretch, muscle tension, palpation, or tactile skin stimulation prior to or during the ischemia. No post-ischemic burst discharges could be detected in analogous experiments with the sural nerve. Intermediate size fibers from normal cutaneous and muscle mechanoreceptors were eliminated as possible origins of the post-ischemia activity. Possible sources include dorsal root ganglion cells or fibers whose sensory endings lie in deep structures such as the walls of the larger blood vessels.

Penetration of topical, oral, and combined administered ofloxacin into the subretinal fluid.

AIMS: To assess the subretinal fluid (SRF) levels of ofloxacin following topical, oral or combined administration. METHODS: 31 patients undergoing conventional retinal reattachment surgery were randomly assigned to three groups. Nine patients received topical ofloxacin, 11 patients received oral ofloxacin, and the other 11 patients received combined administration. Collected SRF samples were analysed for drug level by using high performance liquid chromatography. RESULTS: SRF drug levels after oral and combined administration were significantly higher than that after topical administration (p=0.0002 and p=0.0002, respectively) while there was no significant difference between oral and combined administration (p=0.0844). CONCLUSIONS: Ocular bioavailability of ofloxacin in SRF after oral and combined administration is equivalent. The addition of oral ofloxacin to topical therapy increased drug SRF penetration sixfold.

Nitric oxide synthase inhibition attenuates signs of ethanol withdrawal in rats.

The effects of N(G)-nitro arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI), nitric oxide synthase inhibitors, and L-arginine, a nitric oxide precursor, on ethanol withdrawal signs were investigated in rats. Ethanol (7.2% v/v) was given to rats by a liquid diet for 16 days. L-NAME (30 and 60 mg/kg), 7-NI (40 and 80 mg/kg), L-arginine (100 mg/kg), a combination of L-arginine (100 mg/kg) and 7-NI (40 mg/kg), and saline or vehicle were injected to rats intraperitoneally 30 min before ethanol withdrawal. A second series of injections was given at 6 hour after the first one, and subjects were then tested for audiogenic seizures. 7-NI (40 mg/kg), vehicle and saline were also administered to naive rats. 7-NI (40 mg/kg) did not produce any significant change in locomotor activity in naive rats. Both L-NAME and 7-NI significantly inhibited locomotor hyperactivity from the 2nd to the 6th hour of the withdrawal period. They also reduced the total ethanol withdrawal score from the 30th min to the 6th hour, and they significantly decreased audiogenic seizures. Neither drug increased locomotor activity nor total ethanol withdrawal score, which were increased significantly by L-arginine (100 mg/kg); however, L-arginine (100 mg/kg) prevented the inhibitory effects of 7-NI (40 mg/kg) on increased locomotor activity, total ethanol withdrawal score, and audiogenic seizure. Our results suggest that nitric oxide synthase inhibition by L-NAME and 7-NI alleviates the signs of ethanol withdrawal. The data also support the hypothesis that nitric oxide may take part in the neuroadaptation that develops during chronic ethanol ingestion in rats.

Effect of changes in swimming area on results of "behavioral despair test".

Our previous observations have revealed that the total time spent in immobility and time to reach complete immobility (latency) vary with the diameter of the cylindric chamber where mice are forced to swim in the behavioral despair test. Therefore, we investigated the effect of changing the test conditions of the original Porsolt test. Mice were forced to swim for 15 min in chambers with 10 cm (original diameter of the Porsolt's forced swimming chamber), 20, 30, and 50 cm diameter in 20 cm deep water. Total time spent in spells of immobility during the observation period from third to sixth (inclusive) minutes and time to reach complete immobility were measured. In addition, a possible correlation between the rotatory locomotor activity of mice during swimming as assessed by the number of tours per minute and effect of antidepressant drugs on it was investigated. Total duration of spells of immobility was shorter and the latency was longer in tests carried out in chambers with 10 cm diameter. Increasing the diameter of the cylinders made it possible to distinguish the antidepressant drugs from caffeine, anticholinergics, and antihistaminics, which gave a false positive response in 10 cm diameter cylinders, but not in cylinders with larger diameters. Increasing the diameter of the chambers to 20 and 30 cm also allowed to study the selective effect of the antidepressants, namely, the rotatory locomotor activity during swimming. The extension of the test period to 15 min increased the reliability of the measurement of the time to reach complete immobility.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of bromocriptine and haloperidol on ethanol withdrawal syndrome in rats.

The effects of bromocriptine and haloperidol, either alone or in combination, on ethanol withdrawal syndrome (EWS) have been investigated in rats. Bromocriptine (5 mg/kg 1P) inhibited wet dog shakes behavior and catatonia but potentiated the intensity of abnormal gait. The latency of the audiogenic seizures was prolonged by bromocriptine treatment. Haloperidol (0.5 mg/kg SC) decreased the intensity of stereotyped behavior but potentiated catatonia and agitation. It did not antagonize the behaviors induced by bromocriptine when injected in combination except the increased latency of the audiogenic seizures. The total intensity score of the EWS was not significantly different from that in untreated control. The results suggest that brain dopaminergic system may be involved to a limited extent in mediating the EWS in rats.

Determination of debrisoquine and 4-hydroxydebrisoquine in urine by high-performance liquid chromatography with fluorescence detection after solid-phase extraction.

A simple, selective and sensitive method has been developed to determine debrisoquine and 4-hydroxydebrisoquine in human urine. Separation of the analytes was obtained using a mobile phase of 0.1 M sodium dihydrogen phosphate-acetonitrile (87:13, v/v) and a muBondapak C18 column. The column effluent was monitored with fluorescence detection at 210 nm (ex) and 290 nm (em). Rapid sample preparation was achieved by solid-phase extraction columns (Bond Elut CBA, 3 ml capacity) which provided excellent recovery values for both compounds. The cost per sample using this approach could be minimized by column regeneration and re-use. The within-day and the day-to-day reproducibilities were less than 7% for both components. The method was shown to be suitable for the study of the debrisoquine-sparteine type genetic polymorphism in man.

Rapid liquid chromatographic assay of ciprofloxacin in human aqueous humor.

A simple, selective and sensitive method has been developed to determine ciprofloxacin in human aqueous humor. Separation of ciprofloxacin was carried out with pipemidic acid as internal standard using a Novapak C18 reversed-phase cartridge column (100 x 8 mm i.d., particle size 4 microns) and a mobile phase consisting of methanol-acetonitrile-citric acid (0.4 M) (3:1:10, v/v/v) at a flow rate of 1 ml min-1. The column effluent was monitored with fluorescence detection at 278 nm (excitation) and 450 nm (emission) after direct injection. The retention times were 4.88 min for pipemidic acid and 7.52 min for ciprofloxacin. The within-day and day-to-day reproducibilities were less than 7% for ciprofloxacin at 0.1 and 1 microgram ml-1 (n = 6). The mean recovery from aqueous humor was found to be 101.37 +/- 6.7% for ciprofloxacin at 0.1 micrograms ml-1 (n = 6 and the detection limit corresponding to a signal-to-noise ratio of 2.5:1 was 250 pg ml-1. The method was shown to be suitable for determining ciprofloxacin levels in human aqueous humor samples.

Determination of ofloxacin in human aqueous humour by high-performance liquid chromatography with fluorescence detection.

A reversed-phase high-performance liquid chromatographic method is described for the determination of ofloxacin in human aqueous humour; the method involves fluorescence detection (excitation at 290 nm; emission at 500 nm) after direct injection of samples. The method utilized a 100 mm x 8 mm i.d. cartridge column packed with 4 microns Novapak C18 with a mobile phase methanol-acetonitrile-0.4 M citric acid (3:1:10, v/v/v) and a flow rate of 1 ml min-1 at ambient temperature. The retention times for the internal standard pipemidic acid and for ofloxacin were 4.82 and 7.32 min respectively. The mean recovery (+/- ISD) from human aqueous humour was 103.24 +/- 4.45% for ofloxacin at 1 microgram ml-1 (n = 6). The within-day and day-to-day RSDs at 0.1 microgram ml-1 and 1 microgram ml-1 were less than 6.71% (n = 6) and the lower limit of reliable determination corresponding to a signal-to-noise ratio of 2.5:1 was 20 ng ml-1. The assay was shown to be suitable for measuring ofloxacin levels in human aqueous humour samples after topical, oral and intravenous administration.

Heterogeneity of tolerance developed to effects of ethanol on rotarod and accelerod performances in rats.

The ethanol-induced impairment of rotarod and accelerod performances has been investigated in rats. Ethanol (7.2%, v/v) was given orally ad lib to rats in a modified liquid diet for 15 days. Rotarod and accelerod performances were recorded before and at the 2nd, 4th, 7th, and 15th days of ethanol intake. The daily ethanol consumption of the rats ranged from 12.03 to 16.4 g/kg. Mean blood ethanol level was estimated as 282.3 and 242.5 mg/dl on the 7th and 15th days of ethanol consumption, respectively. Ethanol significantly decreased (p < 0.01 rotarod performance on the 2nd and 4th days. But tolerance developed to the ethanol-induced impairment of rotarod performance from the 7th day. It also significantly decreased (p < 0.01) accelerod performance of the rats throughout the 15 days. So no tolerance was seen to this action of ethanol. Our results suggest that rotarod and accelerod performance tests seem to have differential characteristics in the context of tolerance development to ethanol in rats.

Comparison of aqueous humour and vitreous humour levels of two 0.3% ciprofloxacin eyedrops.

BACKGROUND: Two ophthalmic solutions of 0.3% ciprofloxacin eyedrops are available in Turkey: Ciloxan and Siprogut. The objective of this study was to compare the concentrations of drug produced by the two products in the aqueous humour and vitreous humour after local administration. METHODS: Twenty-one patients undergoing primary vitreoretinal surgery received either Ciloxan (10 patients) or Siprogut (11 patients). Six hours before surgery, two drops of solution were instilled onto the operative eye. Drops were then instilled every 30 minutes for the first 3 hours and then hourly for the next 3 hours. Aqueous and vitreous samples were collected 30 minutes after administration of the last dose and were assayed for ciprofloxacin concentration by means of high-performance liquid chromatography with fluorometric detection. RESULTS: The mean aqueous humour concentrations of Ciloxan and Siprogut were 0.36 (standard error of the mean [SEM] 0.09) microgram/mL and 0.44 (SEM 0.17) microgram/mL respectively. The corresponding vitreous humour concentrations were 0.21 (SEM 0.05) microgram/mL and 0.22 (SEM 0.06) microgram/mL. Neither of these differences was statistically significant. The aqueous and vitreous levels of both products exceeded the minimum inhibitory concentrations for certain bacterial species that frequently cause intraocular infections. INTERPRETATION: Our results show that the ocular bioavailability of Ciloxan and Siprogut after local administration is equivalent. Penetration of ciprofloxacin into the vitreous humour seems to be poorer than that into the aqueous humour.

A study on the mediators of antidromic vasodilatation elicited by sciatic nerve stimulation in cats.

This study was undertaken to ascertain whether a purinergic substance, histamine, dopamine or prostaglandins are mediators involved in antidromic vasodilatation. This type of vasodilatation was elicited by stimulation of the distal end of sectioned sciatic nerve at 40 V with 2 msec pulses for 30 sec in autoperfused hindlimb of reserpine-pretreated and anesthetized cats. Artificial respiration was applied because of curarization that was done to abolish the vascular responses due to contractions of striated muscle. Stimulation of sciatic nerve caused a sustained vasodilatation which was more marked at a frequency of 30 pps. The responsiveness of the perfused vessels to vasodilator stimuli tended to increase by increasing the duration of perfusion as controlled by papaverine administration at certain intervals. Both mepyramine and subsequent administration of metiamide did not produce any change in the vasodilator response to nerve stimulation though they blocked the vasodilator response to histamine. Dopamine injected intra-arterially elicited a pressor response which was reversed into a vasodilator one by phentolamine. This vasodilator response to dopamine tended to be reduced by haloperidol which produced either no change or a slight increase in antidromic vasodilatation. Pretreatment with theophylline to antagonize the likely purinergic mediators or with indomethacin to inhibit the synthesis of prostaglandins had no effect on antidromic vasodilatation elicited by sciatic nerve stimulation. These results suggest that the above-mentioned compounds might not be involved in antidromic vasodilatation.

Metabolic ratios of four probes of CYP2D6 in Turkish subjects: a cross-over study.

The relationships among the metabolic ratios for the standard probe drugs of CYP2D6 activity, such as debrisoquine, sparteine, metoprolol and dextromethorphan, were studied in 32 Turkish subjects. All subjects were randomly selected according to their phenotypes from a group of 111 Turkish subjects whose oxidation status had been tested for debrisoquine previously. All subjects were given a 10 mg debrisoquine tablet, a 100 mg sparteine tablet, a 100 mg. metoprolol tablet and a 20 mg dextromethorphan capsule orally with a wash-out period of at least 1 week between each probe administration. Metabolic ratios were calculated as percentage of dose excreted as parent drug/percentage of dose excreted as its hydroxymetabolite of parent drug in 0-8 h urine. Three poor metabolisers (PM) of debrisoquine were identified. They were also PMs of the other test probes and no misclassification by the 4 phenotyping methods was observed. All six correlations among the metabolic ratios of the 4 probe drugs assessed by Spearman's rank test were highly significant (P < 0.001). The present findings indicate that the oxidative metabolism of debrisoquine, sparteine, metoprolol and dextromethorphan is catalysed by the same cytochrome P450 in the Turkish subjects.