Clinical testing of three novel transient receptor potential cation channel subfamily V member 1 antagonists in a pharmacodynamic intradermal capsaicin model.

BACKGROUND: The transient receptor potential cation channel subfamily V 1 (TRPV1) is involved in nociception and has thus been of interest for drug developers, as a target for novel analgesics. However, several oral TRPV1 antagonists have failed in development, and novel approaches to target TRPV1 with innovative chemistry are needed. METHOD: This work describes an intradermal microdosing approach in humans for pharmacodynamic deductions and pharmacological profiling of compounds. First, a human capsaicin model was developed, to generate pharmacodynamic translational data (Study Part A, n = 24). Then, three small molecule TRPV1 antagonists (AZ11760788, AZ12048189 and AZ12099548) were investigated in healthy volunteers (Study Part B, n = 36), applying the established model. Pain and flare were assessed by Visual Analogue Score and laser Doppler, respectively. RESULTS: The developed model proved useful for pharmacologic deductions; all compounds caused a dose-dependent inhibition of capsaicin-induced pain and flare responses, with a rank order potency of AZ11760788 > AZ12048189 â‰« AZ12099548. In addition, the dose-response data showed that the minimal antagonist concentrations needed to inhibit TRPV1 was ≥6-7 times the equilibrium dissociation constant for each compound. CONCLUSION: With careful design of a pharmacodynamic translational human pain model, it was possible to rank order TRPV1 efficacy among three investigational TRPV1 antagonists, and to estimate human efficacious concentrations. SIGNIFICANCE: This fast and cost-effective translational approach allows for generation of human target engagement information early in drug development. This could be of value for other development programmes where pharmacological targets are expressed in peripheral sensory nerves.

Gastrointestinal safety of AZD3582, a cyclooxygenase inhibiting nitric oxide donator: proof of concept study in humans.

BACKGROUND: Cyclooxygenase inhibiting nitric oxide donators (CINODs) are a new class of anti-inflammatory and analgesic drugs that may minimise gastrointestinal toxicity compared with standard non-steroidal anti-inflammatory drugs (NSAIDs) by virtue of nitric oxide donation. METHODS: A proof of concept study of the gastrointestinal safety of AZD3582, the first CINOD available for human testing, was conducted. Thirty one subjects were randomised to receive placebo, naproxen 500 mg twice daily, or its nitroxybutyl derivative AZD3582 in an equimolar dose (750 mg twice daily) for 12 days in a double blind three period crossover volunteer study. At the start and end of each dosing period, gastroduodenal injury was assessed by endoscopy and small bowel permeability by differential urinary excretion of lactulose and L-rhamnose. Pharmacokinetic profiles were assessed at steady state. RESULTS: On naproxen, the mean total number of gastroduodenal erosions was 11.5 (and one subject developed an acute ulcer) versus 4.1 on AZD3582 (p<0.0001). More than half of the subjects had no erosions on AZD3582. Differences were seen for both the stomach and duodenum. Naproxen increased intestinal permeability (lactulose:L-rhamnose ratio 0.030 before v 0.040 after treatment) whereas AZD3582 (0.029 before, 0.029 after; p=0.006 v naproxen) and placebo (0.030 before, 0.028 after; p<0.001 v naproxen) did not. The steady state bioavailability of naproxen metabolised from AZD3582 was 95% (95% confidence interval 87-101%) of that after naproxen administration. CONCLUSIONS: This human study supports animal data showing reduced gastrointestinal toxicity with the CINOD AZD3582. The potential combination of effective pain relief and gastrointestinal protection offered by AZD3582 warrants further evaluation in human clinical studies.

Effects of infusion rate of intravenously administered morphine on physiological, psychomotor, and self-reported measures in humans.

Although the rate of onset of a drug effect is commonly believed to contribute to a drug's abuse liability, only a few systematic experimental studies have been conducted examining this notion. The present study determined the profile of physiological, psychomotor, and self-reported effects of infusion rate (a key means of manipulating onset of drug action) of intravenously administered morphine, the prototypical analgesic with a known abuse liability in human participants. Two doses of morphine sulfate (5 and 10 mg/70 kg, i.v.) and a placebo dose (0 mg/70 kg, i.v.) were administered to healthy volunteers under three infusion rates (2 min bolus, 15 min, and 60 min). Faster infusions of morphine produced greater positive subjective effects than slower infusions on visual analog scale measures of good drug effect, drug liking, and high. Faster infusions also resulted in greater self-reported drug effects and opioid agonist effects, without producing significant physiological or psychomotor impairment. Importantly, faster rates of drug infusion produced significantly higher morphine plasma levels than slower rates, and morphine plasma levels followed a similar pattern and timing of peak effect as the self-reported effects of the drug. Moreover, morphine produced dose-dependent increases in self-reported drug effects, opioid agonist effects, and morphine plasma levels in the study. Results suggest that the pharmacokinetic properties of a drug, including the dosage administered and the rate of at which it is administered may function to jointly affect the abuse liability of the drug.

Lack of polymorphism of the conversion of losartan to its active metabolite E-3174 in extensive and poor metabolizers of debrisoquine (cytochrome P450 2D6) and mephenytoin (cytochrome P450 2C19).

OBJECTIVE: Losartan was given to subjects with known phenotypes of the polymorphic enzymes CYP2D6 and CYP2C19 to study any possible influence on the metabolism of the drug. METHODS: Plasma concentrations of losartan and E-3174 were studied after oral intake of 50 mg losartan in 24 healthy, male, Swedish Caucasian subjects who were extensive or poor metabolizers (EM/PM) of debrisoquine [cytochrome P450 2D6 (CYP2D6)] or mephenytoin [cytochrome P450 2C19 (CYP2C19)]. RESULTS: The areas under the curve (AUCinfinity) of losartan and E-3174 did not differ between poor and extensive metabolizers of debrisoquine or mephenytoin, respectively. CONCLUSION: About 14% of the antihypertensive drug losartan is metabolized to the active carboxylic acid metabolite E-3174, which contributes to the effect of losartan. The present study suggests that CYP2D6 and CYP2C19 are not involved to any major extent in the in vivo conversion of losartan to E-3174.

Characterization and partial purification of a keratinocyte-derived growth factor with wound-healing properties.

We have previously described the mitogenic and wound-healing properties of keratinocyte-conditioned medium (KCM). In this study we investigated the effect of KCM on the activation of second messenger systems and the expression of proto-oncogene in cultured human skin fibroblasts. We also present a partial purification of the factor responsible for the mitogenic and wound-healing effects of KCM. KCM was shown to increase the expression of the proto-oncogenes c-fos, c-myc and c-jun. The effect of KCM on three second messenger systems was investigated. The extracellular release of choline metabolites was increased by 40 per cent when cells were stimulated with KCM whereas the formation of cAMP and hydrolysis of phosphatidyl inositol (PI) was unaffected. KCM was purified by ion exchange chromatography and filtration. The biologically active fraction was eluted from an SP column and retained its activity after filtration through a 3-kDa filter. The fraction was inactivated by heat and acid, indicative of a peptide origin. Furthermore, the active fraction was shown to increase the extracellular release of choline metabolites and to stimulate re-epithelialization in wounds in human skin in vitro comparable to KCM. The study indicates that human keratinocytes produce a < 3 kDa peptide which may be partly responsible for the growth stimulatory and wound-healing properties of KCM.

Effects of adenosine on ex vivo filtragometry platelet aggregation in relation to plasma levels.

The aim of the present study was to investigate the concentration effect of adenosine on unstimulated platelet aggregation in humans. Adenosine infusion was given intravenously to 12 volunteers in the antecubital vein with infusion rates increasing from 20 to 100 micrograms kg-1 min-1. Filtragometry measurements were obtained from the contralateral antecubital vein before and during 100 micrograms kg-1 min-1 or during maximal tolerable infusion rate. In another set of experiments with 10 volunteers, basal filtragometry measurements were obtained before and after infusion of various concentrations of adenosine into the filtragometer test unit. With intravenous infusion aggregation time tended to increase from 333 +/- 42 to 418 +/- 8 s (mean +/- SEM) and increased the venous plasma adenosine concentration from 0.42 +/- 0.09 microM to 1.52 +/- 0.38 microM. Adenosine infusion into the filtragometer tubing system dose-dependently inhibited aggregation (P < 0.05). Adenosine was rapidly eliminated with a half-life of adenosine in the filtragometry tubing system calculated to be about 6 s. These data extend our knowledge from an in vitro to an ex vivo situation that adenosine dose-dependently has a platelet antiaggregatory effect.

Effects of sera, basic fibroblast growth factor, heparin and cyclic AMP-stimulation on proliferation of human vascular endothelial cells.

The aim of this study was to find culture conditions that optimize the proliferation of human endothelial cells (ECs). The effects of different sera, growth factors and other additives on ECs derived from the adult human great saphenous or the umbilical vein were studied. Human serum (HS) at 10 and 40% was significantly more effective than fetal calf serum at 10 and 30%, respectively. The addition of basic fibroblast growth factor (bFGF) increased proliferation alone and in combination with heparin. Heparin alone increased EC growth only in medium containing 40% HS. The addition of cholera toxin (CT) and a phosphodiesterase inhibitor (IBMX) to raise cAMP-levels, stimulated proliferation of both cell types but was more pronounced on the ECs from the saphenous vein. The cAMP-levels were elevated equally in both cell types. However, db-cAMP stimulated proliferation only of the ECs from the saphenous vein. An additive stimulatory effect was observed when bFGF and CT/IBMX were combined. For saphenous vein ECs, a medium containing HS (40%), bFGF, heparin, CT and IBMX was found optimal for proliferation. We conclude that these compounds may be used to increase EC growth and that, even a limited number of donor ECs may be sufficient starting material for in vitro studies on the human endothelium.

Cyclic AMP, early response gene expression, and DNA synthesis in rat smooth muscle cells.

Smooth muscle cells (SMC) isolated from neonatal and adult rats differ markedly in their growth characteristics. The growth of neonatal cells is mainly due to autocrine stimulation, whereas the growth of adult SMC is dependent upon addition of exogenous mitogens. Increasing intracellular cyclic AMP (cAMP) levels effectively inhibits DNA synthesis in adult cells, but is essentially without effect on the rate of DNA synthesis in neonatal cells. In the present study we investigated whether this difference in cAMP sensitivity is due to an effect of cAMP on early response genes. The results show that increasing intracellular levels of cAMP by exposing the cells to the synthetic adenosine analogue N-ethyl-carboxamido adenosine (NECA) results in an accumulation of c-jun and c-fos mRNA in both cell types. NECA also lowered c-myc mRNA levels in neonatal cells, whereas it marginally increased the presence of c-myc mRNA in adult cells. Exposure to NECA also resulted in a limited increase in alpha-actin mRNA levels. NECA did not inhibit DNA synthesis or growth of adult SMC actively proliferating in the presence of 10% serum, suggesting that cAMP interferes with processes taking place during the early G1 phase or in the entry of growth-arrested cells into the G1 phase of the SMC cell cycle. It is concluded that the growth-inhibitory effect of NECA is unlikely to be due to actions of cAMP on early response genes. However, it cannot be completely excluded that an increased synthesis of jun/fos transcription factors may induce the transcription of other, growth-suppressing genes in the cells.

DMSA administration to patients with alleged mercury poisoning from dental amalgams: a placebo-controlled study.

The present investigation was performed to determine the effect of 14-day oral administration of meso-2.3-dimercaptosuccinic acid (DMSA) on the urinary mercury excretion and the potential reduction of blood and plasma mercury concentrations, and also to relate these effects to possible decrease of symptoms, allegedly associated with amalgam fillings. Twenty subjects, relating their symptoms to mercury from amalgam fillings, received 20 mg/kg DMSA or placebo for 14 days. Their symptoms and mood states were recorded during the study and at a check-up 3 months later. Interpretation was based on intra-individual differences. DMSA-treatment resulted in an average increase in urinary mercury excretion by 65% and a decrease in blood mercury levels of 0.04 microgram/L/day. At the check-up after 3 months, urinary mercury excretion had returned to the pre-treatment level. No treatment effect of DMSA was apparent on subjective symptoms and mood state. One statistically significant treatment effect was noted-a decrease in fatigue-inertia in the DMSA-group-but there was no demonstrable correlation with increased urinary excretion or decreased blood concentration of mercury. Three subjects showed hypersensitive reactions, probably DMSA-specific, at the end of the treatment period. This placebo-controlled study provides no scientific support for diagnostic or therapeutic administration of DMSA for symptoms allegedly associated with chronic mercury exposition from dental amalgam fillings.

Levels of cyclosporin-A (CsA) in saliva in children after oral administration of the drug in mixture or in capsule form.

The cyclosporin-A (CsA) level in human unstimulated whole saliva was studied in 5 children, aged 9-16 yr, receiving the immunosuppressive drug CsA following renal allograft transplantation. The time-concentration relationship of CsA in saliva was determined in the children who were taking the drug orally in mixture form (n = 3) as well as in capsule form (n = 3). For the mixture, the median maximal level of CsA in whole saliva was 2867 ng/ml compared to 5.4 ng/ml for the capsule. The oral mucosal exposure of CsA during the dosage interval was approximately 130 times higher when the drug was administered in mixture form than in capsule form. The study demonstrates that gingival tissue is exposed to a considerable concentration of CsA throughout the dosage interval in patients taking CsA in mixture form. Therefore the vehicle in which the drug is administered should be considered in the pathogenesis of CsA-induced gingival overgrowth.

Adenosine receptors, cyclic AMP accumulation, and DNA-synthesis in aortic smooth muscle cell cultures of adult and neonatal rats.

The effects of two adenosine analogs on cyclic AMP (cAMP) accumulation and DNA synthesis were studied in cultured smooth muscle cells (SMCs) isolated from adult and neonatal rat arteries. N-ethylcarboxamido adenosine (NECA) dose-dependently increased intracellular cAMP levels and appeared to be more potent in adult than in neonatal SMCs. R-phenylisopropyl adenosine (R-PIA), in nanomolar concentrations, counteracted the increase in cAMP evoked by 10 microM forskolin in adult but not in neonatal SMCs, indicating that the enhanced "A2" response seen in adult SMCs was not due to a lack of "A1" receptors in these cultures. Binding experiments performed using the adenosine antagonist XAC did not reveal any differences in the number or affinity of the adenosine receptors between neonatal and adult SMCs. This indicates effects presumably on the G-protein level. A high capacity to spontaneously synthesize DNA and a weak response to platelet-derived growth factor (PDGF) were seen in the neonatal SMCs. Furthermore, NECA had no effect on PDGF-induced DNA synthesis in these cells. In contrast, adult SMCs presented a low rate of spontaneous DNA synthesis and a marked proliferative response to PDGF, which was inhibited by NECA. This inhibition paralleled the increase in cAMP elicited by NECA. Our findings suggest that neonatal and adult SMCs differ both in their response to growth factors and growth inhibitors.

Nicotine enhances angina pectoris-like chest pain and atrioventricular blockade provoked by intravenous bolus of adenosine in healthy volunteers.

An attempt was made to study possible interaction between neuromodulation by adenosine and nicotine stimulatory effects. Dose-effect curves were made double blind in 7 nonsmoking, nonsnuffing healthy volunteers (25-49 years) before and during exposition to nicotine roughly corresponding to the nicotine of one cigarette, 2 mg ingested from a chewing gum (800 chews during 20 min). Chest pain was estimated by the Borg CR-10 scale. ECG was followed, and respiration was recorded continuously by spirometry. Maximal tolerable dose of adenosine was 12.7 +/- 3.0 mg. Chest pain increased dose dependently to 5.7 +/- 1.7 units. Nicotine increased the pain response by 20 +/- 15%, (p less than 0.02). The total time with atrioventricular (AV) block provoked by adenosine increased with nicotine (7 +/- 12%, p less than 0.03) while increased ventilation provoked by adenosine was unaffected by nicotine. In conclusion, interaction between adenosine and nicotine was demonstrated. Nicotine enhanced both stimulatory (chest pain) and inhibitory actions (AV-block) of adenosine.

Calcitonin gene-related peptide stimulates proliferation of human endothelial cells.

The effects of the vasoactive perivascular neuropeptides calcitonin gene-related peptide (CGRP), neurokinin A (NKA), neuropeptide Y (NPY), and vasoactive intestinal polypeptide (VIP) on proliferation of cultured human umbilical vein endothelial cells (HUVECs) were investigated. CGRP was shown to increase both cell number and DNA synthesis, whereas NKA, NPY, and VIP were ineffective. 125I-labeled CGRP was shown to bind to HUVECs and this binding was displaced by addition of unlabeled CGRP, suggesting the existence of specific CGRP receptors. The effect of CGRP on formation of adenosine 3',5'-cyclic monophosphate (cAMP) and inositol phosphates (InsP), two intracellular messengers known to be involved in regulation of cell proliferation, was investigated. CGRP stimulated cAMP formation but was without effect on the formation of InsP. Proliferation, as well as cAMP formation, was also stimulated by cholera toxin. Basic fibroblast growth factor stimulated growth without affecting cAMP or InsP formation, whereas thrombin, which increased InsP formation, did not stimulate proliferation. We thus suggest that CGRP may act as a local factor stimulating proliferation of endothelial cells; that the mechanism of action is associated with cAMP formation; and that this effect of CGRP may be important for formation of new vessels during physiological and pathophysiological events such as ischemia, inflammation, and wound healing.

Theophylline decreases pain in the ischaemic forearm test.

To study the hypothesis that endogenous adenosine is a mediator of the ischaemic pain sensation, the effect of the adenosine receptor blocker theophylline (5.5 mg of the ethylendiamine salt.kg-1 intravenously) was tested in a placebo controlled double blind cross over study (placebo/theophylline/placebo or placebo/placebo/theophylline) in five healthy volunteers. Ischaemic work was performed with a spring loaded hand ergometer (1 Hz). The pain sensation was continuously reported using the Borg scale. Blood flow was measured by occlusion plethysmography. Pain was reported 18 (SEM 2.4) s after starting the ischaemic work and increased continuously to a maximum after 129(18) s (placebo). Theophylline at a plasma concentration of 75(7) mumol.litre-1 decreased the pain sensation in relation to working time. With theophylline, 12(3)% more work (p less than 0.01) was performed for the same reported pain estimate. Blood flow increased from a basal level of 52(9) to 495(55) ml.min-1.100 ml-1 30 s after work and returned to normal within 30-40 min. Theophylline did not affect blood flow. In conclusion, theophylline has a small but significant inhibitory effect on the ischaemic pain sensation compatible with a hyperalgesic effect of adenosine.

Vasoactive intestinal polypeptide stimulates cell proliferation and adenylate cyclase activity of cultured human keratinocytes.

An increasing body of evidence has suggested trophic effects of peripheral nerves. In this study, the growth stimulatory properties of the sensory neuropeptides vasoactive intestinal polypeptide (VIP), substance P (SP), calcitonin generelated peptide (CGRP), and somatostatin (SOM) on cultured human keratinocytes were investigated. It was shown that VIP, in the presence of lethally treated 3T3 fibroblast feeder cells and epidermal growth factor (EGF), stimulated proliferation of keratinocytes in a dose-dependent manner, whereas SP, CGRP, and SOM were ineffective. VIP stimulated adenylate cyclase activity in membranes obtained from cultured keratinocytes in a dose-dependent manner, indicating an involvement of cAMP as second messenger in this reaction. Furthermore, 125I-labeled VIP was shown to bind to cultured keratinocytes and this binding could be displaced by addition of unlabeled VIP, suggesting the presence of specific receptors. It is therefore possible that VIP, released from sensory nerve endings in the skin, may act as a local mitogenic factor for human keratinocytes by stimulating adenylate cyclase activity via specific VIP receptors.

Adenosine receptor mediated stimulation of ventilation in man.

We wanted to examine how adenosine stimulates ventilation in man. Bolus doses of adenosine were given i.v. in an antebrachial vein in multiples of 2.65 mg. The minute ventilation was increased by adenosine 5.3 to 15.9 mg (median values) from control 12.6 +/- 1.9 l min-1 to 42.5 +/- 4.7 l min-1 in a dose-dependent manner. The adenosine receptor antagonist theophylline, 58.3 +/- 3.3 (mean +/- SEM) mumol l-1 plasma, inhibited the response by approximately 25%. Dipyridamole 10 mg, an adenosine uptake blocker, enhanced the effect of adenosine by approximately 60%. The ventilation was not affected by metoprolol, atropine, naloxone or cromolyn sodium but was attenuated by hyperventilation. The respiratory stimulation started before chest pain and cardiovascular effects such as AV-block were encountered. It is concluded that this respiratory stimulation shows characteristics of adenosine receptor mediated responses but the location of such adenosine receptors is uncertain. The findings are compatible with a stimulatory or facilitating effect of adenosine on afferent pathways.

Angina pectoris-like pain provoked by i.v. bolus of adenosine: relationship to coronary sinus blood flow, heart rate and blood pressure in healthy volunteers.

After finding the maximum tolerated i.v. bolus dose of adenosine, three fractions of this dose were given randomly to five volunteers in a double-blind manner. Pain, estimated by a 10-graded category-ratio scale, ECG and coronary sinus blood flow (CSBF), measured by thermodilution and intra-arterial blood pressure, were continuously recorded. At the highest tolerated dose (10.3 +/- 2.3 mg), the ECG showed short lasting (less than 5 s) AV-block but no ischaemic signs. Following the maximum dose, pain started 15 +/- 2 s after injection, reached a maximum (median 6 of 10 grades) after 25 +/- 4 s and disappeared after 62 +/- 7 s. Basal CSBF was 84 +/- 14 ml/min-1, and increased to 297 +/- 48 ml/min. The rise in CSBF started 2.4 +/- 0.8 s before pain appeared (P less than 0.05), but reached its peak 18 +/- 2 s after maximum pain (P less than 0.005). Although maximum coronary vasodilation was induced at the lowest dose of adenosine given--1/3 of the maximum dose--chest pain increased in a dose-dependent manner. When AV-block did not occur, diastolic pressure did not change from baseline, while systolic blood pressure increased by 5 +/- 2% (ANOVA, P less than 0.0001) and heart rate increased by 40 +/- 7% (ANOVA, P less than 0.0001). Following AV-block, except for a decrease of short duration in heart rate and systolic and diastolic blood pressures, the responses were similar. In conclusion, the vasodilator adenosine given as an i.v. bolus to human volunteers who were awake increased heart rate and systolic blood pressure with unchanged diastolic pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Provocation of chest pain in patients with coronary insufficiency using the vasodilator adenosine.

Chest pain provoked by intravenous injection of adenosine was compared with natural angina pectoris in five patients with ischaemic heart disease. In seven healthy subjects a possible myocardial site for provocation of the chest pain was evaluated by analysis of time delays from injection to symptoms. The healthy volunteers were given the maximum tolerable dose of adenosine intravenously, together with 99Technetium-diethylentriaminpentaacetate (99Tcm-DTPA). Chest pain started after 4.1 +/- 2.4 s and reached its maximum 8.4 +/- 4.1 s after maximum left ventricular radioactivity. The patients with a history of typical angina pectoris were given similar doses of intravenous adenosine and the provoked chest pain did not differ in quality from the patients' habitual angina pectoris. The patients did not develop electrocardiographic signs suggesting myocardial ischaemia. Heart rate and blood pressure did not indicate increased myocardial work. In conclusion, the results concur with the hypothesis that adenosine elicits angina pectoris by stimulation of intracardiac adenosine receptors.