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.

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.

Adenosine injection into the brachial artery produces ischaemia like pain or discomfort in the forearm.

To determine whether pain or discomfort could be provoked by adenosine in skeletal muscle and, if so, whether it was dependent on the vasodilatation produced by adenosine, eight male volunteers were given intra-arterial bolus injections of adenosine and glyceryl trinitrate into the forearm. Local pain was assessed on a scale rate, forearm blood flow was measured by venous occlusion plethysmography, and blood was sampled simultaneously from the deep vein of the same arm. Five different doses of adenosine, ranging between the maximum tolerable and the lowest producing pain or discomfort, were given intra-arterially in random order and repeated in reverse order. Glyceryl trinitrate was given intra-arterially in increasing doses from 1 to 20 micrograms. Pain or discomfort began 12(1)(SEM) s after administration reached its maximum after 17(1) s, and disappeared after 40(2) s. Pain or discomfort appeared 8(1) s (p less than 0.001) after the first recorded increase in forearm blood flow, whereas maximum pain or discomfort preceded maximal forearm blood flow by 5(1) s (p less than 0.001). The flow remained increased after the disappearance of pain or discomfort. The effects of adenosine on pain or discomfort and vasodilatation were dose dependent. Glyceryl trinitrate provoked a similar increase in flow to that with adenosine without producing pain or discomfort. Arterial occlusion for 5 min at rest or forearm exercise with arterial occlusion increased forearm blood flow to the same extent as the maximum dose of adenosine. In addition, ischaemic work slightly increased the plasma concentration of adenosine. The pain or discomfort after ischaemic work was not considered different from the adenosine provoked pain or discomfort by four of the subjects. It is concluded that the symptoms did not appear to be dependent on vasodilatation and therefore adenosine may contribute to ischaemic pain or discomfort.

The calcium antagonist nifedipine inhibits arterial smooth muscle cell proliferation.

Migration of smooth muscle cells from the media to the intima of the arterial wall and proliferation of intimal smooth muscle are major early events in the formation of an atherosclerotic lesion. The start of proliferation requires that the cells have passed through a modulation from contractile to synthetic phenotype and that they are stimulated with growth factors. Here, we have examined the effects of the calcium antagonist nifedipine on phenotypic modulation and growth of isolated rat arterial smooth muscle cells cultivated in vitro. The results indicate that micromolar concentrations of nifedipine slow down the rate of transformation of the cells from a contractile to a synthetic phenotype and inhibit initiation of DNA synthesis as well as cellular proliferation. The inhibitory effect on DNA synthesis was seen both in cells stimulated with whole blood serum and with purified platelet-derived growth factor. The results raise the possibility that nifedipine may be used to prevent atherogenesis and to inhibit progression of fibromuscular lesions by interfering with the proliferation of arterial smooth muscle cells.

The adenosine receptor activity of EMD 28422, a purine derivative with reported actions on benzodiazepine receptors.

The effects of a novel purine derivative, N6-[2-(4-chlorophenyl)-bicyclo-2.2.2.octyl-(3)]-adenosine (EMD 28422), that has been found to influence central benzodiazepine receptors, has been compared to those of other adenosine analogues such as L-phenylisopropyladenosine (L-PIA), cyclohexyladenosine (CHA) and adenosine-5'-N-ethyl-carboxamide (NECA). EMD 28422 was about 30 times less potent than CHA and 4 times less potent than NECA in displacing bound [3H]-L-PIA from specific binding sites in the rat brain, presumably reflecting adenosine A1-receptors. A similar relative potency was found using depression of field e.p.s.p. in the hippocampal slice in vitro. In isolated fat cells EMD 28422 was antilipolytic, but some 1000 times less potent than L-PIA. In rat isolated hippocampal slices, which have adenosine A2-receptors, EMD 28422 was more than 300 times less potent than NECA and in guinea-pig thymocytes, which similarly have A2-receptors, EMD 28422 was about 60 times less potent. The results are compatible with the opinion that EMD 28422 is a rather weak agonist at adenosine receptors, with limited selectivity for A1- or A2-receptors. The compound is highly lipophilic, which plays a role in determining its potency in a given biological system. The results are discussed in relation to reported adenosine modulation of benzodiazepine receptors.

Growth-inhibitory properties of vasoactive intestinal polypeptide.

It has recently been demonstrated that several neuropeptides can affect cell growth. The mammalian tachykinins substance P and neurokinin A, which are present in peripheral sensory neurons, stimulate growth of cultured connective tissue cells. Substance P-like immunoreactivity has been demonstrated in neuroblastoma cell lines. Neuroblastoma cells also produce other neuropeptides, among them vasoactive intestinal polypeptide (VIP). We report here that VIP is a potent inhibitor of serum-induced DNA synthesis in cultured smooth muscle cells (SMC), whereas no growth-inhibition was seen in SMC exposed to neurokinin A, calcitonin-gene related peptide, neuropeptide Y, somatostatin, or cholecystokinin. The growth-inhibitory effect of VIP was closely related to its ability to induce formation of cyclic AMP. Our results raise the possibility that peptides released by neurons, endocrine cells, as well as by transformed cells, may not only function as mitogens but also as inhibitory modulators of cell growth.

Purine levels in the intact rat brain. Studies with an implanted perfused hollow fibre.

A thin dialysis tube was implanted stereotaxically under halothane anesthesia in the caudate nucleus of Sprague-Dawley rats and perfused with Ringer solution at a rate of 2 microliters/min. Initially there was a high rate of purine outflow but after 1-2 h of perfusion the rate was essentially constant (anesthetized - adenosine 0.4 +/- 0.04 microM, inosine 0.8 +/- 0.2 microM; non-anesthetized - adenosine 0.33 +/- 0.03 microM, inosine 0.21 +/- 0.07 microM). Hypoxia (9% O2) increased the levels more than 3-fold. The adenosine deaminase inhibitor erythro-2-(2-hydroxy-3-nonyl)adenine (EHNA) increased the adenosine level and decreased the inosine level. In vitro recovery of adenosine was about 30%. Therefore, we conclude that the free exchangable concentration of adenosine in the rat brain is likely to be 102 micro M. This level is high enough to potentially affect central nervous function.

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.

Effect of adenosine receptor agonists and other compounds on cyclic AMP accumulation in forskolin-treated hippocampal slices.

The effect of adenosine analogues and some putative neurotransmitters have been studied on cyclic AMP accumulation in rat hippocampal slices treated with the adenylate cyclase activator forskolin. The effects of PGE2 and histamine were potentiated by forskolin (0.1 microM). Isoprenaline and NECA had essentially additive effects with 0.1 microM forskolin and serotonin (above 10(-4) M) inhibited forskolin-stimulated cyclic AMP accumulation. The A1-adenosine receptor selective adenosine analogue R-PIA inhibited forskolin stimulated cyclic AMP accumulation in low doses and stimulated in high. NECA, adenosine and 2-chloroadenosine uniformly stimulated cyclic AMP accumulation. 2',5'-dideoxyadenosine inhibited, but only at high concentrations. Both the stimulatory and the inhibitory effects of R-PIA were antagonized by 8-phenyltheophylline (10 microM). Enprofylline (100 microM) selectively inhibited the stimulatory effect. In the presence of enprofylline both 2-chloroadenosine showed an inhibitory effect on cyclic AMP accumulation. It is concluded that the forskolin-treated rat hippocampal slice is a useful preparation to study both stimulatory and inhibitory effects of transmitters and modulators on adenylate cyclase. The results also show that the rat hippocampus has both A1-receptors that are linked to inhibition of cyclic AMP accumulation and A2-receptors that are linked to stimulation. Furthermore, enprofylline is shown to selectively antagonize the stimulatory response, revealing inhibitory effects of compounds such as 2-chloroadenosine and adenosine.

Adenosine receptor mediated inhibition of noradrenaline release from slices of the rat hippocampus.

Adenosine and adenosine analogues inhibited electrically evoked 3H-noradrenaline (3H-NA) release from slices of the rat hippocampus in vitro in a dose -dependent manner in the concentration range 0.01-100 M. L-phenylisopropyladenosine (L-PIA) was more potent than 5'-N-carboxamidoadenosine (NECA), which was more potent than adenosine. The adenosine uptake blocker dipyridamole (3 M) enhanced the effect of exogenous adenosine, and had a slight inhibitory effect per se. The effect of L-PIA on NA release was competitively antagonized by 8-phenyltheopylline; pA2 = 7.1. Enprophylline (300 M), theophylline (300 M) and 8-phenyltheophylline (1-10 M) enhanced the evoked 3H-NA release per se, while no such enhancement was seen with the non-xanthine phosphodiesterase inhibitor ZK 62.711 (Rolipram) (30 M). It is concluded that adenosine, at physiologically relevant concentrations, inhibits electrically evoked NA release from terminals in the central nervous system. Alkylxanthines increase evoked NA release from hippocampal terminals, which probably not related to cyclic AMP but may partly involve inhibition of endogenous adenosine acting as a modulator of transmitter release in the hippocampal slice preparation.

On the mechanism by which methylxanthines enhance apomorphine-induced rotation behaviour in the rat.

Methylxanthines, such as caffeine and theophylline, potentiate the rotation behaviour induced by dopamine receptor agonists in rats with unilateral lesions of the nigro-striatal pathway. In the present study we have examined the possibility that interaction with central adenosine mechanisms could influence rotation behaviour. Under in vitro conditions adenosine and N6-phenylisopropyl-adenosine (PIA) stimulate cyclic AMP accumulation. This effect was enhanced by the phosphodiesterase inhibitor rolipram, but blocked by alkylxanthines such as caffeine, theophylline and, particularly, 8-phenyl-theophylline. Rotation behaviour induced by apomorphine (0.05 mg/kg), was inhibited by PIA and rolipram and by a low dose of the adenosine deaminase inhibitor EHNA (2 mg/kg). By contrast, theophylline and 8-phenyl-theophylline caused a potentiation. The former drug stimulated rotation behaviour per se, while the latter did not. 8-Phenyl-theophylline entered the brain poorly and its concentration in brain it was less than 1/10 of theophylline. It is concluded that theophylline does not potentiate rotation behaviour secondarily to inhibition of phosphodiesterase. Antagonism of endogenous adenosine may partly explain the effect of methylxanthines. Possibly, some as yet unknown mechanism may also contribute to the effects of xanthine-derivatives on rotation behaviour.

Adenosine mechanisms are not affected by antidepressant concentrations of desipramine.

We have evaluated the proposal that adenosine may mediate some of the effects of tricyclic antidepressant therapy. In-vitro desipramine (DMI) (1-10 microM) did not affect adenosine or 2-chloroadenosine-induced inhibition of lipolysis or the adenosine stimulated formation of cyclic (c) AMP in the hippocampal slice. However, very high concentrations of desipramine (0.2-0.5 mM) as well as some detergents potentiated the stimulatory effect of adenosine on cAMP formation. The ATP, ADP and AMP contents in slices were unaffected as was the electrically evoked release of purines. Long-term treatment in-vivo with antidepressants in clinically relevant doses did not alter the sensitivity of adenosine receptor mediated cAMP formation in-vitro while the beta-adrenoceptor-mediated formation was depressed by desipramine or imipramine treatment but not by zimelidine or fluoxetine treatment. It is concluded that actions on central adenosine mechanisms are unlikely to play any important role in the therapeutic effects of tricyclic antidepressants.

Quinacrine and release of purines from the rat hypothalamus.

The acridine derivative quinacrine accumulates in certain endocrine cells and nerve endings. It has been suggested that it binds to ATP stored in granules in such cells. For this reason the uptake and release of quinacrine and the ATP-precursor adenine was studied in slices and in crude synaptosomes from the rat hypothalamus. Both quinacrine and purines were taken up and after uptake there was a spontaneous release. The rate of purine release could be stimulated by nerve stimulation, the depolarizing agent veratridine, and by omitting glucose from the medium. Veratridine-induced purine release was inhibited by dipyridamole, an inhibitor of carrier-mediated nucleoside transport, whereas release induced by glucose deprivation was not. None of the procedures caused release of labelled quinacrine. The results are not compatible with the opinion that the bulk of the purines are released from specific storage vesicles that have the capacity specifically to bind quinacrine.

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.

Release of purines, noradrenaline, and GABA from rat hippocampal slices by field stimulation.

Labelled adenine, noradrenaline (NA), and gamma-aminobutyric acid (GABA) were taken up by the transversely cut hippocampal slice. [3H]NA and [14C]GABA were retained as such, [3H]- (or [14C]-) adenine mainly as adenine nucleotides. There was a spontaneous overflow of all three types of compounds ranging from 0.1 (GABA) to 0.21 (NA) %/min. The rate of [3H]NA overflow increased rapidly during electrical field stimulation. The release rate was well maintained over a 15-min period. The rate of [14C]GABA release also increased rapidly but it was not maintained over a 15-min period even if uptake and/or metabolism was inhibited by nipecotic acid (1 mM) and aminooxyacetic acid (AOAA, 0.1 mM). The bulk of the purines was released after the stimulation period. For all compounds the amounts released were frequency- and calcium-dependent. At a frequency of 3 Hz a 10 V stimulation was sufficient to cause a maximal [3H]NA release and 20 V to cause maximal [14C]GABA release, but 14C-purine release was increased further by increasing the voltage to 40 V. The evoked purine release was inhibited by a nucleoside uptake inhibitor (dipyridamole). On stimulation of [3H]NA-labelled slices the released radioactivity was composed of greater than 95% unchanged NA. The specific activities of NA in the slice and in the superfusate were practically identical. In [3H]adenine-labelled slices the released radioactivity was composed of adenosine, inosine, and hypoxanthine, but the activity in the slice of ATP, ADP, and AMP.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

Effect of physical training on different categories of patients with intermittent claudication.

The effect of supervised training was studied in 68 patients with intermittent claudication. Maximal walking distance was measured on a treadmill. Eight of the patients had resting pain in the leg when recumbent (group A), 25 had an initial walking distance of less than 500 m (group B), 11 had an initial walking distance of 500--1 000 m (group C), 24 had coronary insufficiency (group D). The study shows that training should be undertaken for at least three months. In some patients with resting pain, training led to relief of pain and surgical treatment was not necessary. Almost all patients without signs of coronary insufficiency increased their walking distance, compared to only 14 of the 24 patients with coronary insufficiency. Walking distance increased significantly in groups B and C and no significant difference was found between patients and proximal or distal arterial stenosis.

Angina pectoris-like pain provoked by intravenous adenosine in healthy volunteers.

In a study to characterise the chest pain induced by adenosine this agent was given as a bolus into a peripheral vein to six healthy volunteers (five men) aged 30-44. On the first day the maximum tolerable dose was determined in each case. On the second day three doses of adenosine (one third, two thirds, and the full maximum tolerable dose) and three doses of saline were given single blind in randomised order. Thereafter aminophylline 5 mg/kg was given and the procedure repeated in a different randomised order. On the third day between two thirds and the full maximum tolerable dose was given followed by 10 mg dipyridamole intravenously and a second injection of the same dose of adenosine. Heart rate and atrioventricular blocks were recorded by electrocardiography. One minute after each dose of adenosine the chest pain was scored. The maximum tolerable dose of adenosine ranged from 10.6 to 37.1 mg. All subjects experienced uneasy central chest pain provoking anxiety. The pain radiated to the shoulders, ulnar aspect of the arms, epigastric area, back, and into the throat. The pain began about 20 seconds after the injection and lasted 10-15 seconds. Increasing the dose of adenosine increased the intensity of the pain. Administration of aminophylline reduced the pain significantly. Second degree heart block was recorded in five of the six subjects during the time that the pain was experienced. After aminophylline no block was observed. Dipyridamole increased the intensity of pain. The duration of second degree heart block increased in four of the subjects, and in two of these third degree heart block occurred. These findings suggest that adenosine released from the myocardium during ischaemia induces angina pectoris by stimulating theophylline sensitive receptors.

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.