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Effect of a novel TRPV1 antagonist on capsaicin nociceptors

Laura B. García1*, Isabel D. Giner2, Asia F. Carvajal1, Armando G. -3, Tracey P. -3 and Antonio F. Montiel1
  • 1 Universidad Miguel Hernández de Elche, Biología Molecular y Celular, Spain
  • 2 AntalGenics, SL., Spain
  • 3 Università degli Studi del Piemonte Orientale, Italy

TRPV1 is a polymodal ion channel that is activated by noxious stimuli including high temperature (>42ºC), acid pH (~6), basic pH (~7,8), membrane voltage changes and vanilloids (1). This channel is widely expressed in several cell types from sensory neurons to non-neuronal skin cells (3). The activation of TRPV1 generates a cationic flow that in neurons triggers the firing of action potentials transducing the noxious information from the peripheral nervous system to the central nervous system for a proper nocifensive response. Additionally, TRPV1 has been implicated in pathological conditions underlying inflammation and/or chronic pain. The pathophysiological implications of TRPV1 validate this channel as a therapeutic target for drug development. The most well-known agonist of TRPV1 is capsaicin, which directly activates and desensitizes the channel inducing both inducing neuronal excitability and inactivation upon continuous or repeated exposure. Capsaicin-induced desensitization of TRPV1 has been used therapeutically to alleviate painful symptoms. However, patients do not adhere to the treatment due to the burning effect of capsaicin application. Here, we used the capsaicin pharmacophore to design antagonists that act as soft drugs and display an improved therapeutic index (4). The most promising candidate attenuates capsaicin-induced TRPV1 action potential triggering in primary cultures of nociceptors. 1.Background Capsaicin is a vanilloid that activates the TRPV1 channel in sensory neurons, increasing their excitability. Continuous or repeated exposure to the vanilloid produces TRPV1 desensitization, a property that underlies the therapeutic activity of capsaicin (1). However, the clinical use of capsaicin is limited because of its burning side effect that reduces patient adherence to the treatment. Thus, there is a need to design capsaicin analogues that negatively modulate TRPV1 but not have the burning side effect of the vanilloid. By using the capsaicin pharmacophore and the Passerini reaction, we have obtained a library of TRPV1 agonists and antagonists. We have investigated the best candidate (compound 36) using the MEA technology. 2.Methods -Cell Culture Neonatal dorsal root ganglia from Wistar rats (3–5 days-old) were isolated and digested in order to obtain a pure neuronal culture. Then, DRGs neurons were seeded on MEA chambers in a drop. After 2h, medium was added, supplemented with 2.5S NGF and cytosine arabinoside (37 °C, 5% CO2). All experiments were performed after 48h. -MEA experiments Extracellular Extracellular recordings were carried out using MEA of 60-electrode (30μm diameter / 200μm inter-electrode) and integrated reference electrode. The electrical activity of primary sensory neurons was recorded by the MEA1060 System (Multi Channel Systems GmbH), and MC_Rack 4.3.0 software version at 25 kHz sampling rate. TRPV1-mediated neuronal firing activity was evoked by two repetitive 15s-applications of 500nM capsaicin. Upon desensitization by first capsaicin application (P1), compound 36 at 10, 1 and 0.1µM, capsazepine at 10µM or vehicle (DMSO 0.01% v/v) were applied together with capsaicin in P2. Finally, 40mM KCl pulse was applied to ensure cell viability of the culture. P2/P1 mean spike frequency was calculated and normalized to vehicle-treated value. All data are expressed as mean±SEM. Statistical analysis was performed using One-way ANOVA followed by Bonferroni´s post hoc test to compare treatments versus vehicle group. ****p<0.0001. 3.Results The effect of compound 36, a TRPV1 competitive antagonist, on primary cultures of DRG sensory neurons excited by capsaicin has been characterized using MEA technology. Capsaicin application promoted bursts of action potentials. Compound 36 significantly suppressed TRPV1-mediated neuronal excitability at 0.1, 1 and 10 µM (Figure 1). The inhibitory activity of compound 36 was similar to that exhibited by capsazepine at 10 µM. Notably, the vanilloid-derived antagonist appears to be an excellent candidate for modulating TRPV1 overactivation in pathological conditions. -- Figure 1. Effect of compound 36 on TRPV1-mediated neuronal excitability. (A) Representative MEA recording showing TRPV1-mediated neuronal spiking upon two repetitive 15-s applications of 500 nM capsaicin. Compound 36 at 10, 1 or 0.1 µM, vehicle or capsazepine at 10 µM, applied together with second pulse of capsaicin. Finally, 40 mM KCl to ensure cell viability and excitably. (B) Dose response effect of compound 36 on TRPV1-mediated neuronal excitability. Mean Spike Frequency registered upon first (P1) and second (P2) capsaicin application were normalized (%) to that of vehicle-treated cells. Statistical analysis was one-way ANOVA followed by Bonferroni´s post-test; ****p<0.0001. Data are expressed as mean ± SEM, N=3 independent cultures, n > 100 total registered electrodes. -- 4.Conclusion Compound 36 displays an inhibitory effect on TRPV1-mediated neuronal excitability with a micromolar potency. This compound has been selected for further development for the treatment of pain and other sensory modalities.

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Acknowledgements

This work has been funded by the AEI-MINECO (SAF2015-66275-C2-1) the GVA (PROMETEO 2014/011) and Compagnia di San Paolo (Grant No. C61J12000280007).

References

1. Planells-Cases R, Valente P, Ferrer-Montiel A, Qin F and Szallasi A (2011) Complex Regulation of TRPV1 and Reelated Thermo TRPS: Implications for Therapeutic Intervention. Transient Receptor Potential Channels, Advances in Experimental Medicine and Biology, vol: 704,491-515.
2. Fattori, V.; Hohmann, M. S. N.; Rossaneis, A. C.; Pinho-Ribeiro, F. A.; Verri, W. A. Capsaicin: current understanding of its mechanisms and therapy of pain and other pre-clinical and clinical uses. Molecules 2016, 21, 844-876.
3. Caterina, M. J.; Pang, Z. TRP channels in skin biology and pathophysiology. Pharmaceuticals 2016, 9, 77-104.
4. Devesa, G. I.; Genazzani, A. A.; Pirali, T.; Fernandez-Carvajal, A.; Ferrer-Montiel, A. V. TRPV1 Modulator Compounds. May 11, 2017. EP17382266.9.

Keywords: TRPV1 channels, Microelectrode Array (MEA), Nociceptors, Capsaicin, antagonists

Conference: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018.

Presentation Type: Poster Presentation

Topic: Assay development

Citation: García LB, Giner ID, Carvajal AF, - AG, - TP and Montiel AF (2019). Effect of a novel TRPV1 antagonist on capsaicin nociceptors. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00096

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Received: 18 Mar 2018; Published Online: 17 Jan 2019.

* Correspondence: PhD. Laura B García, Universidad Miguel Hernández de Elche, Biología Molecular y Celular, Elche, Alicante, 03202, Spain, laurabutrongarcia@gmail.com