Target engagement biomarkers for pain receptors

The clinical development of novel analgesics is facing extreme challenges. The highest attrition is caused in the proof-of-concept phase (phase II) due to lack of efficacy and/or safety in the target population. Lack of efficacy is most prominent in CNS related diseases, including chronic pain conditions, in which animal models of efficacy often are insufficient to predict target engagement and efficacy in patients. A potential approach for developing novel analgesics is to target the very beginning of the pain pathway as such at these receptors and ion channels.

The aim of this thesis is to develop models that can provide both insight in the physiopathology of these channels and can be used in early development to give a first indication about the target engagement of drugs targeting these channels.

This thesis consisted of 9 chapters and 3 parts. In chapter 1, a general overview of the current knowledge regarding neurogenic inflammation and the associated receptors, second messengers and diseases was given. As well as a review of the capsaicin model as example of a target engagement model in practice. Chapter 2 describes the objectives of this thesis in more detail.

Part I contains all clinical studies targeting the TRPV1 receptor.

Chapter 3 describes for the first time a dysfunction of TRPV1 in cystinosis patients, homozygous for the 57-kb deletion. Cystinosis is a rare disease caused by a deletion of the cystinosin (CTNS) gene which also affects the non-coding region of the TRPV1 gene. As TRPV1 is activated by capsaicin and high temperatures, the capsaicin response and temperature detection and pain thresholds were tested in these patients. The homozygous patients clearly showed a reduced sensitivity for capsaicin (both vasodilatory and pain response) and a disturbed detection of high temperatures, compared to heterozygous patients and healthy volunteers.

In chapter 4, the feasibility of the capsaicin model with topical treatments is investigated. In this study, brimonidine, an alpha-adrenergic agonist and the standard treatment of rosacea, is investigated in its ability to block the capsaicin- induced dermal blood flow response (CIDBF). The rationale behind this study was that in the pathophysiology of the skin disease rosacea, which is characterized by flushing of the facial skin, neurogenic inflammation and thus the TRPV1-CGRP pathway is involved. Pre-treatment with brimonidine for 3 hours was able to reduce the CIDBF in a modest way, probably because brimonidine is not directly interfering with the TRPV1-CGRP pathway and only has an indirect effect on the vasodilatation.

Part II contains all clinical studies regarding the TRPA1 receptor.

To investigate the TRPA1 receptor and future antagonists, a reproducible and robust target-engagement biomarker was developed as described in chapter 5. Based on preclinical literature, cinnamaldehyde (CA) was chosen as a selective agonist to develop this model. First, a dose-finding study was performed to find a safe and robust dose of CA. From 3 different doses, 10% CA was chosen as the most appropriate dose. With this dose, the reproducibility of the CA-induced DBF response between arms and periods was tested. Both inter-arm and inter-period reproducibility was found sufficient and sample size calculations showed that this model can be used to detect a 30-50% change between 2 groups with a maximum of 15 subjects.

In chapter 6, we tried to get a better understanding of the second messengers involved in the CA-induced activation of TRPA1 and the related vasodilatory response. Surprisingly, we showed that the CA-induced DBF response can be reduced using non-selective COX inhibitors aspirin and indomethacin and not with selective COX-2 inhibitor Celecoxib or NK1-receptor antagonist aprepitant. Additionally, aspirin showed a stronger inhibitory effect compared to indomethacin, which needs further investigation. With this chapter, we showed that COX-1 dependent PG play an important role in CA-induced vasodilatation.   

As TRPA1 is thought to play an important role in neurogenic inflammation and migraine, the CA-induced DBF response between migraine patients, with and without aura, and healthy volunteers was investigated in chapter 7. CA-induced DBF was not different between migraine patients, with and without aura, and healthy volunteers, probably because TRPA1 was activated peripherally (on the skin of the forearm) and not on the trigeminal nerves where migraine most possibly originates. Immediately after CA application, the CA-induced pain response was lower in migraine patients compared to healthy volunteers. However, this needs further investigation to draw any conclusions.

In the last part, part III, the PAC1 receptor is investigated via the development of a selective and reproducible target-engagement model, the maxadilan model, as described in chapter 8. Maxadilan, a peptide derived from the sand fly, is a selective exogenous agonist of the PAC1 receptor. In accordance with the cinnamaldehyde model, also first a safe and robust dose was searched. As there was no dose-dependent effect between our 3 doses tested, the lowest dose of 0.9ng maxadilan was chosen to continue. Dermal blood flow response to intradermal injection of 0.9ng maxadilan was shown to be nicely reproducible between arms and periods. Sample size calculations showed that this biomarker is feasible to detect a 30-50% change between 2 groups with a maximum of 15 subjects. An unexpected peak in DBF response was observed 24 hours after injection which need further investigation.

In chapter 9, the results of the thesis are discussed in a broader context and recommendations for future research are given. In general, we can conclude that we successfully developed 2 new in vivo in human models for the TRPA1 (the cinnamaldehyde model) and PAC1 (the maxadilan model) receptors and contributed to the fine-tuning of the already existing capsaicin model for the TRPV1 receptor. We showed that these models can not only be used to support GO/NO GO decisions and characterization of patient stratification, but also to gain more insight in the pathophysiology of these receptors and associated diseases.