Preclinical characterization of three transient receptor potential vanilloid receptor 1 antagonists for early use in human intradermal microdose analgesic studies.

BACKGROUND: The transient receptor potential vanilloid receptor 1 (TRPV1) is a nonselective cation channel involved in the mediation of peripheral pain to the central nervous system. As such, the TRPV1 is an accessible molecular target that lends itself well to the understanding of nociceptive signalling. This study encompasses preclinical investigations of three molecules with the prospect to establish them as suitable analgesic model compounds in human intradermal pain relief studies. METHODS: The inhibitory effectiveness was evaluated by means of in vitro assays, TRPV1 expressing Chinese hamster ovary cells (CHO-K1) and rat dorsal root ganglion cultures in fluorescent imaging plate reader and whole cell patch clamp systems, as well as in vivo by capsaicin-evoked pain-related behavioural response studies in rat. Secondary pharmacology, pharmacokinetics and preclinical safety were also assessed. RESULTS: In vitro, all three compounds were effective at inhibiting capsaicin-activated TRPV1. The concentration producing 50% inhibition (IC50 ) determined was in the range of 3-32 nmol/L and 10-501 nmol/L using CHO-K1 and dorsal root ganglion cultures, respectively. In vivo, all compounds showed dose-dependent reduction in capsaicin-evoked pain-related behavioural responses in rat. None of the three compounds displayed any significant activity on any of the secondary targets tested. The compounds were also shown to be safe from a toxicological, drug metabolism and pharmacokinetic perspective, for usage in microgram doses in the human skin. CONCLUSION: The investigated model compounds displayed ideal compound characteristics as pharmacological and translational tools to address efficacy on the human native TRPV1 target in human skin in situ. SIGNIFICANCE: This work details the pharmaceutical work-up of three TRPV1-active investigational compounds, to obtain regulatory approval, for subsequent use in humans. This fast and cost-effective preclinical development path may impact research beyond the pain management area, as it allows human target engagement information gathering early in drug development.

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.

Calcium spikes and calcium currents in neurons from the medial preoptic nucleus of rat.

Ca2+ spikes, their contribution to firing patterns, and the underlying Ca2+ currents in neurons of the medial preoptic nucleus of rat were investigated by tight-seal whole-cell recordings in a slice preparation. Two different types of spikes were recorded: Low-threshold spikes were generated from membrane potentials <-75 mV. High-threshold spikes were recorded when K+ currents were reduced, and were readily evoked from membrane potentials near -40 mV. Both types of spikes were blocked by substitution of Co2+ for Ca2+ in the external medium, but were insensitive to 2.0 microM TTX. Under voltage-clamp conditions, two main types of Ca2+ currents were characterized: low-threshold currents that activated at membrane potentials >-60 mV, and high-threshold currents that activated at potentials >-30 mV. The low-threshold current and the low-threshold spike were more sensitive to block by external Ni2+ than to block by Cd2+, whereas the high-threshold current and the high-threshold spike were more sensitive to block by external Cd2+ than to block by Ni2+. Significant fractions of the high-threshold currents were blocked by 10 microM nifedipine, 1.0 microM omega-conotoxin GVIA, 50 nM omega-agatoxin IVA and 1.0 microM omega-conotoxin MVIIC, suggesting the presence of L-, N-, P- and Q-type Ca2+ channels. There were also a high-threshold current component insensitive to the above mentioned toxins. It is proposed that the low-threshold current serves as a trigger for short bursts of fast spikes from hyperpolarized levels, whereas the high-threshold current is involved in the Cd2+-sensitive burst firing seen in relatively depolarized neurons.

IL-6 is essential in TNF-alpha-induced fever.

Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine that orchestrates an array of local and systemic effects. For instance, acute exposure to a high dose of TNF-alpha results in septic shock and fever. We have used interleukin-1beta (IL-1beta)- and interleukin-6 (IL-6)-deficient mice, along with their wild-type equivalents, to define a role for TNF-alpha in fever. Briefly, the mice produced prostaglandin E2-dependent fevers in response to recombinant murine TNF-alpha (rmTNF-alpha). Furthermore, rmTNF-alpha (12 microgram/mouse ip) triggered a febrile response in IL-1beta-deficient mice as well as in their corresponding wild-type controls. In contrast, the IL-6-deficient mice were resistant to rmTNF-alpha (4.5 microgram/mouse ip), although their wild-type counterparts readily mounted a fever. In the IL-6-deficient mice, moreover, the febrile response to rmTNF-alpha could be restored by a central administration of rat recombinant IL-6 (500 ng/mouse icv). We thus conclude that TNF-alpha can trigger fever independent of IL-1beta but dependent on IL-6. We also suggest that central, rather than peripheral, IL-6 (plasma IL-6 was measured 2 h after pyrogenic challenge) is essential in TNF-alpha-induced fever.

Simultaneous measurement of brain and core temperature in the rat during fever, hyperthermia, hypothermia and sleep.

The neuropathological outcome of metabolic, vascular or mechanical insults to the CNS depends on brain temperature; mild hypothermia is neuroprotective, whereas elevated brain temperature can cause additional neural damage. Studies in both animals and humans have shown that the core and the brain temperature do not always concur with one another. It is therefore important to develop methods for monitoring brain temperature. This paper describes an animal model (the rat) in which we have developed a method to measure, at thermoneutral ambient temperature, the brain and core temperature concomitantly, during different drug treatments. We have used this animal model to study body temperature during fever (induced by human recombinant IL-1 beta, 5 microgram/kg, i.p.), stress-induced hyperthermia (handling of the animal), hypothermia (induced by (+/-)-8-hydroxy-2-dipropylaminotetralin hydrobromide, 0.5 mg/kg, i.p. ) and sleep (non-induced, other than by light and diurnal variation). We show that the thermal curves are similar in the brain and the peritoneum, independent of the thermal state.

Acute-phase responses in transgenic mice with CNS overexpression of IL-1 receptor antagonist.

The interleukin-1 (IL-1) receptor antagonist (IL-1ra) is an endogenous antagonist that blocks the effects of the proinflammatory cytokines IL-1alpha and IL-1beta by occupying the type I IL-1 receptor. Here we describe transgenic mice with astrocyte-directed overexpression of the human secreted IL-1ra (hsIL-1ra) under the control of the murine glial fibrillary acidic protein (GFAP) promoter. Two GFAP-hsIL-1ra strains have been generated and characterized further: GILRA2 and GILRA4. These strains show a brain-specific expression of the hsIL-1ra at the mRNA and protein levels. The hsIL-1ra protein was approximated to approximately 50 ng/brain in cytosolic fractions of whole brain homogenates, with no differences between male and female mice or between the two strains. Furthermore, the protein is secreted, inasmuch as the concentration of hsIL-1ra in the cerebrospinal fluid was 13 (GILRA2) to 28 (GILRA4) times higher in the transgenic mice than in the control animals. To characterize the transgenic phenotype, GILRA mice and nontransgenic controls were injected with recombinant human IL-1beta (central injection) or lipopolysaccharide (LPS, peripheral injection). The febrile response elicited by IL-1beta (50 ng/mouse icv) was abolished in hsIL-1ra-overexpressing animals, suggesting that the central IL-1 receptors were occupied by antagonist. The peripheral LPS injection (25 micrograms/kg ip) triggered a fever in overexpressing and control animals. Moreover, no differences were found in LPS-induced (100 and 1,000 micrograms/kg ip; 1 and 6 h after injection) IL-1beta and IL-6 serum levels between GILRA and wild-type mice. On the basis of these results, we suggest that binding of central IL-1 to central IL-1 receptors is not important in LPS-induced fever or LPS-induced IL-1beta and IL-6 plasma levels.