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Easily accessible normal tissues expressing the same molecular site(s) of drug action as malignant tissue offer an enhanced potential for early proof of anticancer drug mechanism and estimation of the biologically effective dose. Studies were undertaken in healthy male volunteers to assess the tolerability of single and multiple (four in 24 h) 3 mm punch biopsies of the buccal mucosa, and to determine the feasibility of detecting and quantifying a range of proliferation, cell-cycle arrest and apoptosis markers by immunohistochemistry (IHC) for use as potential pharmacodynamic (PD) end points. The biopsy procedure was well tolerated with 100% of volunteers stating that they would undergo single (n = 10) and multiple (n = 12) biopsies again. Total retinoblastoma protein (pRb), phosphorylated pRb (phospho-pRb), total p27, phosphorylated p27 (phospho-p27), phosphorylated-histone H3 (phospho-HH3), p21, p53, Cyclin A, Cyclin E, Ki67 all produced good signal detection, but M30, cleaved caspase 3 and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling did not. Total pRb, phospho-pRb, total p27 and phospho-p27 were quantified further in a multiple biopsy study to allow components of variability to be addressed to inform future sizing decisions on intervention studies. Neither site of biopsy within the oral cavity, nor the nominal time of biopsy had any significant impact on any of the four markers expression levels. Inter- and intrasubject coefficients of variation (CVs) that could be used to size future intervention studies for pRb, phospho-pRb, total p27 and phospho-p27 were 14, 19, 18 and 16%; and 18, 29, 25 and 19%, respectively. In conclusion, quantitation of such markers in 3 mm buccal punch biopsies would be suitable to explore as PD end points within intervention studies of drugs acting on these pathways.

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AIMS: The aim of this study was to assess the efficacy of dextromethorphan and ketamine relative to placebo on the acute nociceptive threshold and wind-up of second pain response in healthy male volunteers. METHODS: The trial was a randomized, double-blind, placebo-controlled, three period crossover, double dummy design in 12 healthy male volunteers. During each of the three periods (which were separated by a 1 week washout period) each volunteer received either a single oral dose of 0.7 mg kg(-1) dextromethorphan and placebo to ketamine, or placebo to dextromethorphan followed by a single intravenous injection of 0.375 mg kg(-1) ketamine, or placebo to both dextromethorphan and ketamine. The trial did not schedule administration of both ketamine and dextromethorphan together. Acute nociceptive thresholds and wind-up of second pain were measured in the skin of the thenar eminence of the ventral surfaces of the right and left hands, using a SOMEDIC thermotest apparatus, before and at the estimated tmax for dextromethorphan (i.e. 2.15 h). Blood pressure and heart rate were also monitored before dosing and after the dosing regimen. RESULTS: Neither dextromethorphan nor ketamine had any significant effect on acute nociceptive thresholds on either hand (P>0.05). Moreover, dextromethorphan was without any significant effect (P>0.05) on the wind-up of the second pain response on either hand. The lsmean number of stimuli tolerated vs placebo (95% confidence intervals of the difference in number of stimuli in parentheses) were 15.84 vs 16.48 (-5.52, 4.24) and 11.75 vs 15.25 (-11.89, 4.90) for left- and right-hand, respectively, following dextromethorphan administration. In contrast ketamine produced significant reductions in wind-up to second pain in both the left and right hands (P=0.0002 and 0.0386, respectively). The lsmean numbers of stimuli tolerated vs placebo (95% confidence intervals of the difference in number of stimuli in parentheses) were 28.41 vs 16.48 (6.60, 17.25) and 25.00 vs 15.25 (0.58, 18.93) for left- and right-hand, respectively. CONCLUSIONS: Wind-up of second pain induced by noxious heat is sensitive to intervention by ketamine, which is known to block the NMDA receptor. These data infer that the wind-up phenomenon evoked by noxious heat involves the activation of NMDA receptors. This volunteer model of pain may have utility in the evaluation of agents that modulate their antinociceptive actions via NMDA mechanisms.

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AIMS: The aim of the current study was to assess the viability of the interdigital web pinch model as a test for analgesic activity in volunteer-based early phase drug development. METHODS: Pain thresholds and sensitization to a series of four sessions of interdigital web pinching (12 Newtons force) were measured in 26 male volunteers before and 1 and 3 h after oral dosing with ibuprofen (800 mg) or placebo to ibuprofen. Within each time point, the pain thresholds were measured by calculating the average visual analogue scores (VAS) for the first session of pinching (VAS-1). Sensitization to pinching was assessed by calculating the average changes in these scores for the three subsequent sessions of pinching (VAS-2). Moreover, the difference between the VAS score after the first session of pinching and that obtained at the end of the fourth session of pinching was calculated as a secondary endpoint (VAS-3). RESULTS: Treatment with ibuprofen had no significant effect on VAS-1 at either 1 or 3 h after dosing. However, the mean values of VAS-2 and VAS-3, were significantly reduced (P < 0.05) following treatment with ibuprofen. CONCLUSIONS: This model has been able to detect an antinociceptive effect with ibuprofen. However, large numbers of subjects were required in order to demonstrate this effect and this feature would restrict the model's utility in early phase clinical trials where small numbers of subjects are normally employed.

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AIMS: The aim of this study was to compare the effects of aspirin on platelet function as measured by the 'classical' template bleeding time with a new ex vivo method measuring closure times using the PFA-100 machine. Platelet aggregation in response to arachidonic acid was also measured ex vivo. METHODS: The trial was a randomized, double-blind, placebo-controlled crossover design, with each volunteer taking 750 mg aspirin (BP) or placebo, three times a day for 5 days, with an 18 day wash-out period between treatments. Bleeding times and closure times were measured before the first dose on the first day and 0.5 h after the last dose on the fifth day of each treatment period. They were also measured 2 weeks after the last day of the trial. RESULTS: Baseline bleeding times (pre-placebo) were 415 s using the Simplate, whilst baseline closure times were 115 s using the PFA-100. Aspirin treatment caused an increase of both the template bleeding time (61%) and the closure time of the PFA-100 (79%) when compared with the effects of placebo. The platelet aggregatory response to arachidonic acid was completely inhibited following aspirin treatment and was unaffected following placebo. Two weeks after the end of the trial, all values had returned to pre-treatment levels. The template bleeding time was unaltered in 1 of the 12 volunteers during aspirin treatment and was significantly prolonged in 3 of the 12 volunteers during placebo treatment. The PFA-100 closure time was unaltered in 1 of the 12 volunteers during aspirin treatment and was prolonged in 1 subject during placebo treatment. CONCLUSIONS: The change in closure time using the PFA-100 is as sensitive and reproducible to the effects of aspirin on platelet function as is the template bleeding time test. However, the PFA-100 produced less variable effects with fewer false positive results.

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1. The relaxant effects of Zeneca ZD7114, BRL37344 (putative beta 3-adrenoceptor agonists) and various phenylethylamine-based agonists were studied in isolated ileum of the rat where tone was increased with carbachol (0.5 microM). Agonist-induced relaxation.was measured under equilibrium conditions with alpha-, beta 1- and beta 2-adrenoceptors inhibited. 2. Relaxant responses were obtained to isoprenaline, noradrenaline, and BRL37344, although, the efficacy of this latter agent was significantly.lower than that of isoprenaline. Salbutamol caused weak relaxation (< 20%) at high concentrations (10 microM) and ZD7114 was without significant relaxant effect even at high concentrations (10 microM). 3. Relaxant responses to isoprenaline and BRL37344 were weakly antagonized by high concentrations of (+/-)-propranolol (10 and 100 microM) yielding pKB values of 5.7 with isoprenaline as the agonist and 5.5 with BRL37344 as the agonist. 4. The non-selective beta-adrenoceptor antagonist, (+/-)-alprenolol (1-100 microM) caused competitive antagonism of the relaxant responses to isoprenaline (pA2 value = 6.5). A similar pKB value was obtained when BRL37344 was used as the agonist (6.4). 5. Relaxant effects of isoprenaline and BRL37344 were also antagonized by ZD7114 (1-100 microM) yielding pA2 and pKB values of 6.3 and 6.7 respectively. 6. The low potencies of (+/-)-propranolol and (+/-)-alprenolol as antagonists of the relaxant responses to isoprenaline and BRL37344 indicate that both the agonists and antagonists employed in the current study may interact with beta 3-adrenoceptors in the rat isolated ileum. Contrary to the previous findings in guinea-pig ileum, where BRL37344 and ZD7114 were full agonists, in the current study, BRL37344 was a partial agonist and ZD7114 an antagonist at the beta 3-adrenoceptor in rat ileum.

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Contractile responses to 5-hydroxytryptamine (5-HT) and to a number of 5-HT-receptor agonists have been compared on the rat isolated caudal artery and stomach fundic strip. On the caudal artery, 5-HT was the most potent agonist tested. The 5-HT 1-like agonist, 5-carboxamidotryptamine (5-CT), was less potent than 5-HT and produced a lower maximum response. 8-Hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) and RU24969 (5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)1Hindole) were inactive as agonists and 8-OH-DPAT was not an antagonist. Ketanserin, ICI 169,369 (2-(2-dimethylaminoethylthio)-3-phenylquinoline hydrochloride) and ICI 170,809 (2-(2-dimethylamino-2-methylpropylthio)-3- phenylquinoline hydrochloride) were competitive antagonists of 5-HT on this preparation, indicating that 5-HT is acting via 5-HT2 receptors. In contrast, all the agonists produced contractions of the fundic strip (rank order of potency, 5-HT = 5-CT > RU24969 > 8-OH-DPAT). The maximum response to RU24969 was significantly lower than the maximum responses to the other agonists. Ketanserin was only a weak antagonist of 5-HT in the fundic strip, demonstrating that 5-HT2 receptors were not involved, but ICI 169,369 and ICI 170,809 were non-surmountable antagonists of 5-HT responses, as were methysergide and methiothepin. Since ICI 169,369 and ICI 170,809 are devoid of activity at 5-HT3 and 5-HT4 receptors, then these two subtypes would not appear to be implicated, a view that was confirmed in the case of 5-HT3 receptors by experiments using ondansetron.(ABSTRACT TRUNCATED AT 250 WORDS)

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1. Experiments were performed to characterize the effects of the novel brown adipocyte stimulant, ICI D7114, in the guinea-pig isolated ileum, right atrium and tracheal chain. In the ileum, agonist-induced inhibition of the contractile response to either histamine or prostaglandin E2 (PGE2) was assessed, along with effects on resting rate in the atrium and resting tone in the tracheal chain. In the latter two preparations, antagonism of isoprenaline-induced responses by ICI D7114 was also assessed. 2. Inhibitory responses were obtained in the ileum to ICI D7114, isoprenaline, BRL37344, and noradrenaline. The responses to ICI D7114, isoprenaline and BRL37344 were resistant to blockade with propranolol (5 microM), naloxone (1 microM), methysergide (0.1 microM), cimetidine, indomethacin and 8-phenyltheophylline (all 10 microM). These responses to isoprenaline, in the presence of propranolol (5 microM), were competitively antagonized by alprenolol (1-100 microM) with a pA2 value of 6.44. The responses to ICI D7114 and BRL37344 were antagonized by single concentrations of alprenolol (1 microM) with apparent pKB values of 6.53 and 6.57 respectively. These data indicate an effect of ICI D7114 at the atypical beta-adrenoceptor in the guinea-pig ileum. 3. The order and relative potency of agonists at the atypical beta-adrenoceptor was BRL37344 (4) < isoprenaline (1) = ICI D7114 (1.1) > noradrenaline (0.5). 4. ICI D7114 (1 nM - 10 microM) caused no significant change in the rate of beating or the resting tone of the guinea-pig right atrium or tracheal chain respectively. It did, however, cause selective blockade of the responses to isoprenaline in these tissues (apparent pKB values 7.63 and 5.85 in atrium and tracheal chain respectively). Responses to histamine (atrium) and aminophylline (tracheal chain) were not significantly affected by 10 microM ICI D7114.5. These results demonstrate that ICI D7114 possesses selective agonist activity at atypical beta-adrenoceptors in the guinea-pig ileum and its use as a tool may help to establish a role for the atypical beta-adrenoceptor in the control of gastrointestinal motility.

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Agonist and antagonist analogues of substance P were synthesized by replacing at least two of the amino acid residues with D-Trp, D-Phe, D-Val, or D-Pro residues. The syntheses of these compounds were achieved by solid-phase methodology using the hydroxymethyl resin. The analogues were tested for agonist and antagonist activity on guinea pig ileum and rat spinal cord preparations. Two types of antagonists were obtained. The first type of compounds, e.g., [N alpha-Z-Arg1,N epsilon-Z-Lys3,D-Trp7,8,D-Met11]-SP-OMe (1), antagonized SP and SP(6-11)-hexapeptide on the ileum but only SP(6-11)-hexapeptide on the spinal cord. The second type of antagonists, e.g., [N alpha-Z-Arg1,N epsilon-Z-Lys3,D-Pro9,10]-SP-OMe (17), were inactive on the ileum but were potent antagonists of the hexapeptide on the spinal cord. Two of the antagonists, [N alpha-Z-Arg1,N epsilon-Z-Lys3,D-Trp7,8,D-Met11]-SP (3) and [D-Trp7,8,9]-SP (43), were also tested in vivo. Both of these depressed hypotensive responses to SP and SP(6-11)-hexapeptide in rabbits.

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Antagonists of SP and the C-terminal (6-11)-hexapeptide have been obtained by multiple D-amino acid substitutions in various positions of SP and by protecting the N alpha-Arg1 and N epsilon Lys3 amino groups with benzyloxycarbonyl groups. On the guinea pig ileum a number of these antagonized both SP and the hexapeptide. Except [N alpha-Z-Arg1,D-Pro2,N epsilon-Z-Lys3,Asn5,Arg6,D-Phe7,D-Trp9]-SP-OMe (4) and the corresponding amide 7, which were more potent antagonists of SP than the hexapeptide, all the others, e.g., [N alpha-Z-Arg1,D-Pro2,4,N epsilon-Z-Lys3,D-Phe7,8,Sar9,D-Met11]-SP-OMe (9), [N alpha-Z-Arg1,D-Pro2,4,N epsilon-Z-Lys3,D-Phe7,8,Sar9,MeLeu10,D-Met11]-SP -OMe (11), were more potent antagonists of the hexapeptide. On the rat spinal cord preparation, most of the antagonists were only active against the hexapeptide. A few antagonized SP, but these also reduced carbachol or both carbachol and glutamate responses. Two of the antagonists, [D-Pro2,Asn5,Lys6,D-Phe7,D-Trp9]-SP-OMe (2) and [Boc-D-Pro4,D-Phe7,8,Sar9,D-Met11]-SP(4-11)-OMe (10), were inactive on the ileum but still antagonized the hexapeptide on the spinal cord. The smallest peptides to antagonize SP and the hexapeptide were two heptapeptides, 6 and 21, [Z-Asn5,Arg6,D-Phe7,8,Gly9 psi (CH2S)D-Leu10,D-Met11]-SP(5-11)-OMe (21) being more potent than 6. None of the antagonists showed significant analgesic activity without side effects. Some of the antagonists were shown to release histamine from isolated rat peritoneal cells.

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Rank order potency data for substance P (SP), physalaemin and eledoisin have been determined in a variety of assays in vitro. On the guinea-pig field stimulated vas deferens, physalaemin was approximately eight and five times more potent than eledoisin and SP respectively whereas in the rat field-stimulated vas deferens, eledoisin was approximately five times more potent than either physalaemin or SP. It would appear that the guinea-pig preparation has a preponderance of 'P'-receptor subtypes, whereas the rat preparation contains mainly 'E'-receptor subtypes. We have attempted to validate the hypothesis that there are multiple tachykinin receptors. Experiments have been performed with the guinea-pig urinary bladder in vitro, using phenoxybenzamine (20 microM, 30 min) to alkylate receptor sites and using excess eledoisin (0.2-0.4 microM, 30 min) to protect receptors selectively against the effects of this agent. Our results showed that after pretreatment with phenoxybenzamine, the responses to eledoisin were significantly attenuated. Moreover, in the presence of excess eledoisin it was found that phenoxybenzamine was unable to produce any significant reduction of the subsequent responses. This difference between the responses to eledoisin and to other agonists suggests the existence of at least two different receptor subtypes for the tachykinins.

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The effects of substance P nonapeptide amide on inactivation of substance P by the guinea-pig ileum and urinary bladder preparations and of substance P hexapeptide amide, bradykinin and acetylcholine by the guinea-pig urinary bladder, have been investigated. Significant inhibition of inactivation of Substance P was found only in the urinary bladder. None of the other agents were significantly affected. These results suggest that the previously reported agonist effects of substance P nonapeptide may be due to inhibition of inactivation of SP in vivo and this effect is possibly specific.

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