The effect of AMG9810 in various pain tests and on capsaicin-induced neuropeptide release T. Kark1, A. Tóth2, J. Szilvási1, T. Kobezda1, A. Szalai1, J. Németh1*, R. Pórszász1*

Department of Pharmacology and Pharmacotherapy1, Division of Clinical Physiology, Institute of Cardiology2 University of Debrecen, Hungary

Santorini Island, 19-21th of April *Equal contribution.

Abstract

Background and Aims. The transient receptor potential vanilloid-1 (TRPV1) receptor of primary sensory afferent neurons plays an important role in pain mechanisms. Physiological activators are the heat (>42 °C) and the acidic pH (pH<=5). Capsaicin and other vanilloids cause TRPV1 excitation. Antiinflammatory and analgesic effects of TRPV1 antagonists are described. The formerly developed TRPV1 antagonist molecules show limited selectivity and the moderate inhibitory potency requires new compounds to be synthesized. The effects of AMG9810 as TRPV1 receptor antagonist were tested on various animal pain models and we measured the inhibitory effect on capsaicin-induced neuropeptide release in vitro. Methods. Writhing test, eye-wiping test, plasmaextravasation measurement, capsaicin-induced hypothermia, pulmonary chemoreflex and neuropeptide release measurement. Results. After 30 min of AMG9810 administration the number of writhes decreased in dose-dependent manner (1 mg/kg: 22.4±2.9, 3 mg/kg: 16.0±6.6 vs. control 26.6±3.8, x mean ± S.E.M.). One mg/kg AMG9810 evoked maximal inhibitory effect in eye-wiping test (1 mg/kg: 17.4±1.8, 3 mg/kg: 18.8±2.5 vs. control 24.8±2.3). The lower dose (1 mg/kg i.p.) AMG9810 has no effect on capsaicininduced plasmaextravasation, but we found an increased plasmaextravasation in response to 3 mg/kg administration (1 mg/kg: 36.3±5.5, 3 mg/kg: 90.2±11.4 vs. control: 40.9±7.1). The capsaicin-induced body core temperature decrease was not affected by AMG9810. The capsaicin-induced pulmonary chemoreflex was dose-dependently inhibited by AMG9810 and time dependence was verified in the antagonist effect. AMG9810 evoked a significant, dose-dependent decrease in the capsaicin-induced substance P release in isolated rat tracheae (control: 4.28±0.12 fmol/mg, 500 nM AMG9810 3.84±0.15, 2 μM AMG9810: 3.36±0.15). The release of CGRP was not reduced significantly. Conclusion. AMG9810 significantly decreased the capsaicin and acetic acid induced pain. The capsaicin-induced plasmaextravasation was enhanced in the dose of 3 mg/kg. AMG9810 reduced the substance P release from isolated rat trachea. State funding sources. OTKA T046244, ETT 493/2006, ETT 430/2006, AT is supported by Bolyai fellowship

Introduction

Transient receptor potential (TRP) proteins comprise a large family of non-selective cation channels responsible for diverse cellular functions, including the transduction of thermal, chemical and mechanical stimuli, as well as the regulation of cellular calcium levels. TRP vanilloid (TRPV1–4) family members are directly activated by either heat or cold and by compounds that modulate sensations of warmth, cooling, and burning pain. Capsaicin, the active ingredient of hot chili peppers, and its ultrapotent vanilloid analog, resiniferatoxin (RTX), from Euphorbia resinifera, elicit burning pain by activating TRPV1 channels on sensory nerve endings (Caterina et al., 1997; Szallasi and Blumberg, 1999). TRPV1 emerged as a promising therapeutic target for pain because TRPV1 knock-out mice demonstrate reduced thermal hypersensitivity after inflammatory tissue injury, agonists of TRPV1 cause desensitization of the channel, which activated and upregulated during inflammatory conditions. There is a sore need of potent and selective TRPV1 receptor antagonists. By this time capsazepine is the only TRPV1 antagonist that has been studied profoundly, but unfortunately it shows only moderate inhibitory potency and very limited selectivity. Several TRPV1 antagonists were discovered by high throughput screening, but none of them brought breakthrough in the development of TRPV1-mediated antiinflammatory and analgesic drugs. In our study, the effects of AMG9810 as TRPV1 receptor antagonist were tested on various animal pain models and we measured the inhibitory effect on capsaicin-induced neuropeptide release in vitro.

Methods Animals and anaesthesia The experiments were performed on Wistar rats weighing 250-450 g and ‘CD-1 IGS Swiss mouse’ (2555g) raised on a standard laboratory food and water ad libitum. Anaesthesia was performed with 50 mg/kg i.p. thiopental (Trapanal). All procedures used in this study are in agreement with the rules of the Ethics Committee on Animal Research of the University Medical School of Debrecen. Drugs and chemicals Capsaicin, acetic acid, AMG9810, and the thiopental were purchased from Sigma. Core body temperature was measured with a rectal thermometer manufactured by Experimetria ltd. Ewans Blue concentration was measured by using a Beckmann photometer. Animals were anesthetized with thiopental (50 mg/kg i.p ,Trapanal) Writhing test Mice weighing 25–55g were used. The study was carried out as described by Collier et al. (1968) and modified by Nakamura et al. (1986). Each mouse was given an injection of 0.9 % acetic acid aqueous solution (5 ml/kg) into the peritoneal cavity and the animals were then placed in a transparent plastic box. The number of writhes was counted for 10 min beginning from 5 min after the acetic acid injection. AMG9810 were administered i.p. 30 min before the acetic acid injection. Eye-wiping test One drop (10 μl) of capsaicin solution (50 μg/ml) was put into the right or left conjunctiva of the animal, in a random order. When the capsaicin concentration was irritant, the animals immediately began to wipe the eye with ipsilateral forepaw, and the number of eye wipes was counted during 60 s. Pretreatment with AMG9810 (1 or 3 mg/kg) was performed i.p. 20 min before capsaicin administration. In the control group the solvent was injected i.p. in the same volume.

Plasmaextravasation measurement Anesthesia was induced by the i.p. administration of thiopental (50 mg/kg) A tail vein was cannulated for the injection of Evans blue dye (30 mg/kg). One min after Evans blue administration 1mg/kg of capsaicin injection (i.v.) was performed. Each animal was sacrificed by transcardiac perfusion with 50 ml of 0.9% w/v saline, at 37°C, into the left cardiac ventricle. 10 min after intravenous injection of Evans blue. After this, the urinary bladder was removed and weighed, and the content of dye was determined by spectrophotometry (at 620 nm wavelength), after extraction in 1 ml of formamide for 24 h. Plasma protein extravasation was expressed as the content of Evans blue dye in micrograms per gram of wet tissue. AMG9810 (1 or 3 mg/kg) treatment was performed i.p. 1 min before capsaicin administration. Capsaicin-induced hypothermia Capsaicin injection (300 μg/kg, s.c.) produced a hypothalamus-mediated rapid decrease of core body temperature. This reduction was measured with a rectal thermometer in every 10 min for 1 h. Pretreatment with AMG9810 (1 or 3 mg/kg) was performed i.p. 20 min before capsaicin administration. Examination of the pulmonary chemoreflex Bolus intravenous capsaicin injection evokes a triple response consisting of hypotension, bradycardia and apnoea known as Bezold–Jarisch reflex or pulmonary chemoreflex (Porszasz J. et al, 1955) Rats were anaesthetized with thiopental (50 mg/kg i.p.). A tracheal cannula was inserted after tracheotomy to facilitate spontaneous respiration and to register the respiratory movements. Blood pressure was continuously recorded through a cannula inserted into the left carotid artery via a pressure transducer connected to the Haemosys hemodynamic setup (Experimetria Ltd., Budapest, Hungary). The body core temperature was maintained at 37±0.5 °C. For administration of capsaicin and AMG9810 the left jugular vein was cannulated and a catheter was advanced until its tip was positioned just above the right atrium. The pulmonary chemoreflex was evoked by increasing doses of capsaicin. Area under the curve (AUC) of the capsaicin-induced hypotension was determined for quantitative analysis. The experiment was carried out after 5, 30 or 60 min after intravenous injection of AMG9810 (1 and 3 mg/kg).

Neuropeptide release measurement 2–2 tracheae were removed after exsanguination and perfused (1 ml/min) in an organ bath (1.8 ml) at 37 °C for 60 min with oxygenated (95% O2 and 5% CO2) Krebs solution containing 119mM NaCl; 25 mM NaHCO3; 1.2mM KH2PO4; 1.5mM MgSO4; 4.7mM KCl; 2.5mM CaCl2; 11mM glucose. The solution was changed 3 times for 8 min (prestimulated–stimulated–poststimulated) after stopping the flow. Chemical stimulation with capsaicin (10−6 M) or electrical field stimulation (40 V, 0.1 ms, 10 Hz, 2 min) was performed to induce peptide release in the second fraction. AMG9810 (500 and 2000 μM) was added into the incubation medium at the beginning of each 8 min period. In control experiments stimuli were applied in the absence of the antagonists. The fractions were collected in ice-cold tubes and the wet weight of each trachea was measured. Concentrations of substance P, CGRP and somatostatin were determined by specific and sensitive radioimmunoassay (RIA), and peptide output was expressed as the released amount per tissue weight. Detection limits of the assays were 2 fmol/tube (substance P), 0.2 fmol/tube (CGRP), 2 fmol/tube (somatostatin) (Nemeth et al., 1996). Statistical analysis The results are expressed as mean ±S.E.M. Mean values was considered significantly different when P < 0.05.

Results

Effect of AMG9810 on acetic acid-evoked abdominal writhing motion AMG9810 (1 and 3 mg/kg) administered i.p. 30 min before acetic acid exposure dose-dependently decreased the number of writhes compared to the solvent-treated control group (Figure 1.), but it proved not significant. (AMG9810 1 mg/kg: 22,4±2,9; 3 mg/kg: 16,0±6,6 vs. control 26,6±3,8)

Effect of AMG9810 on capsaicin-evoked wiping movements Instillation of 10 μl capsaicin solution into the eye of the rat evoked 24,8±2,3 wiping movements (Figure 2.). Pretreatment with AMG9810 i.p. significantly decreased the number of eye wipes. (1 mg/kg 17,4±1,8; 3 mg/kg 18,8±2,5)

Effect of AMG9810 on capsaicin-induced plasmaextravasation Pretreatment with 1 mg/kg AMG9810 has no effect on capsaicin evoked systemic inflammation (Figure 3.). Unexpectedly in higher dose of AMG9810 attained agonistic effect and significantly increased the extent of plasmaextravasation. (AMG9810 1 mg/kg: 36,3±5,5; 3 mg/kg: 90,2±11,4 vs. control: 40,9±7,1)

Effect of AMG9810 on capsaicin-induced hypothermia None of the tested doses of AMG9810 influenced the hypothermic effect of capsaicin (Figure 4.). (AMG9810 1 mg/kg: 4,9±0,2; 3 mg/kg: 4,3±0,3 vs. control: 4,6±0,3)

Effect of AMG9810 on capsaicin-evoked pulmonary chemoreflex The capsaicin-induced hypotension was dose-dependently inhibited by AMG9810. This inhibition showed time dependence. Increasing doses of capsaicin were administered after 5, 30 and 60 min of AMG9810 (1 and 3 mg/kg) administration. The dose-response curves were shifted to the right by the applied doses of AMG9810. A decreased inhibitory effect of AMG9810 was observed as time progressed (Figure 5., 6.).

Effect of AMG9810 on capsaicin-evoked neuropeptide release Capsaicin (10−6 M) evoked increase of CGRP, substance P and somatostatin release (Figure 7., 8., 9.). The release of the three peptides was diminished in the presence of 0.5 and 2 μM AMG9810. AMG9810 evoked a significant, dose-dependent decrease in the capsaicin-induced substance P release in isolated rat tracheae (control: 4.28±0.12 fmol/mg, 500 nM AMG9810 3.84±0.15, 2 μM AMG9810: 3.36±0.15). The release of CGRP and SS was not reduced significantly.

Conclusion As shown in Figure. 1., none of the tested doses of AMG9810 influenced the writhing response induced by an intraperitoneal injection of acetic acid in mice. For measuring nociception evoked by capsaicin, the number of wiping movements was reduced significantly. Pretreatment with 1 mg/kg AMG9810 was able to significantly reduce capsaicin evoked systemic inflammation, contrary to all expectations in higher dose AMG9810 acted as an agonist, significantly increasing the extent of plasmaextravasation. None of the tested doses of AMG9810 altered the capsaicin-induced drop of body core temperature. AMG9810 dose- and time-dependently inhibited the capsaicin-induced hypotension. The level of SP was diminished dose-dependently, but the release of CGRP and SOM was not influenced in the presence of AMG9810. Discussion This study aimed at assessing effects of AMG9810 as TRPV1 receptor antagonist. The results well correlate with previous experiments in which AMG9810 was found potent antagonist of TRPV1 in vitro, and blocks capsaicin-induced eye wiping behavior (Gavva et al., 2005). Lower doses of AMG 9810 (1, 3 mg/kg) alone failed to significantly inhibit the acetic acid-evoked writhing movements, capsaicininduced hypothermia and causes hyperthermia by itself, and were ineffective in blocking the capsaicininduced release of CGRP and SOM from rat trachea. Only the SP release was decreased significantly. In the light of previous results, higher doses of AMG9810 have significant antagonistic effect on TRPV1 receptor. (Gavva et al., 2007) In our experiments we used much lower dose to test the effectiveness of the substance. Further studies on AMG9810 will help to define the role of TRPV1 as target for the next generation of analgesics.

References: Almási R., Pethő G., Bölcskei K. and Szolcsányi J., Effect of resiniferatoxin on the noxious heat threshold temperature in the rat: a novel heat allodynia model sensitive to analgesics, Br. J. Pharmacol. 139 (2003), pp. 49–58. Balázs Jakab et al., Pharmacological characterization of the TRPV1 receptor antagonist JYL1421 (SC0030) in vitro and in vivo in the rat, European Journal of Pharmacology Volume 517, Issues 1-2 , 4 July (2005), Pages 35-44 Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D., The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature. (1997) Oct 23;389(6653):816-24. Coleridge G. and Coleridge H.M., Afferent vagal C fibre innervation of the lungs and airways and its functional significance, Rev. Physiol., Biochem. Pharmacol. 99 (1984), pp. 1–110. Collier H.O., Dinneen L.C., Johnson C.A. and Schneider C., The abdominal constriction response and its suppression by analgesic drugs in the mouse, British Journal of Pharmacology 32 (1968), pp. 295–310. Gavva NR et al., AMG9810, a novel vanilloid receptor 1 (TRPV1) antagonist with antihyperalgesic properties. J PharmacolExp Ther (2005) 313:474–484. Gavva NR et al., The Vanilloid Receptor TRPV1 Is Tonically Activated In Vivo and Involved in Body Temperature Regulation, The Journal of Neuroscience, March 28, 2007, 27(13):3366-3374 Jancso-Gabor, J. Szolcsanyi and N. Jancso, Irreversible impairment of thermoregulation induced by capsaicin and similar pungent substances in rats and guinea-pigs, J. Physiol. 206 (1970), pp. 495–507. Nakamura H., Shimoda A., Ishii K. and Kadokawa T., Central and peripheral analgesic action of non-acidic non-steroidal anti-inflammatory drugs in mice and rats, Archives Internationales de Pharmacodynamic et de Therapie 282 (1986), pp. 16–25. Nemeth J., Oroszi G., Than M., Helyes Zs., Pinter E., Farkas B. and Szolcsanyi J., Substance P radioimmunoassay for quantitative characterization of sensory neurotransmitter release, Neurobiology 7 (1999), pp. 437–444. Porszasz J, GYorgy L, Porszasz-Gibeszer K., Cardiovascular and respiratory effects of capsaicin, Acta Physiol Hung. (1955);8(1):61-76. Szallasi A, Blumberg PM., Vanilloid (Capsaicin) Receptors and Mechanisms, Pharmacol Rev. (1999) Jun;51(2):159-212. Szolcsányi J et al., Analgesic effect of TT-232, a heptapeptide somatostatin analogue, in acute pain models of the rat and the mouse and in streptozotocininduced diabetic mechanical allodynia, European Journal of Pharmacology Volume 498, Issues 1-3 , 13 September (2004), Pages 103-109

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Figure. 1. Effect of AMG9810 (i.p.) on acetic acid-induced writhing movements. Results are means ± S.E.M., n=10, NS=non-significant

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Figure. 2. Effect of i.p. pretreatment with AMG9810 on the number of wiping movements evoked by instillation of 10 μl capsaicin solution (50 μg/ml) into the eye. Results are means ± S.E.M. (*p<0.05) n = 10

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Figure. 3. Effect of AMG9810 (i.p.) on plasmaextravasation after injection of Evans blue (30 mg/kg) expressed in μg/g wet tissue. Results are means ± S.E.M. (**p<0.01) n=10

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Figure. 4. Effect of i.p. pretreatment with AMG9810 on maximal decrease in body core temperature. Results are means ± S.E.M. n = 10

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Figure 5. Effect of 1 mg/kg of AMG9810 (i.v.) on capsaicin-evoked hypotension

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Figure 6. Effect of 3 mg/kg of AMG9810 (i.v.) on capsaicin-evoked hypotension.

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Figure 7. Effect of AMG9810 on capsaicin-evoked CGRP release (fmol/mg). Results are means ± S.E.M.

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Figure 9. Effect of AMG9810 on capsaicin-evoked SS release (fmol/mg). Results are means ± S.E.M.