Ligand binding assays for vanilloid receptors

Details Ligand binding assays for vanilloid receptors Ligand binding assays for vanilloid receptors

2001-10-11

2004-08-24

Ulm, John D. (Department: 1646)

Chemistry: analytical and immunological testing

Biospecific ligand binding assay

C435S007210

Reexamination Certificate

active

06780650

ABSTRACT:

BACKGROUND OF THE INVENTION
Noxious chemical, thermal and mechanical stimuli excite peripheral nerve endings of small diameter sensory neurons (nociceptors) in sensory ganglia (eg., dorsal root, nodose and trigeminal ganglia) and initiate signals that are perceived as pain. These neurons are crucial for the detection of harmful or potentially harmful stimuli (for example heat), tissue damage caused by local tissue acidosis, and physical movement (for example tissue stretch) that arise from changes in the extracellular space during inflammatory or ischaemic conditions (Wall and Melzack, 1994).
Capsaicin (8-methyl-N-vanillyl-6-nonenamide), the main pungent ingredient in “hot” capsicum peppers, and its analogs interact at specific membrane recognition sites called vanilloid receptors. These receptors are expressed almost exclusively by primary sensory neurons involved in nociception and neurogenic inflammation (Bevan and Szolcsanyi, 1990). Capsaicin is a very selective activator of thinly or unmyelinated nociceptive afferents (Szolcsanyi, 1993; Szolcsanyi, 1996). Capsaicin can be blocked by a selective antagonist, capsazepine. Another ligand is the potent tricyclic diterpene resiniferatoxin (RTX), (Szolcsanyi et al., 1991), a molecule that binds with nanomolar affinity at the capsaicin-binding site.
Recently, one receptor for capsaicin (VR1) was cloned from rat (Caterina et al., 1997) and shown to be a coincidence detector for H+ (low pH) and heat (Tominaga et al., 1998). VR1 is expressed in small nociceptive neurons of the dorsal root ganglion, consistent with its role in modulating peripheral pain (Tominaga et al., 1998). VR1 is a ligand-gated non-selective cation channel that shows pronounced outward rectification (Caterina et al., 1997). The vanilloid (“capsaicin”) receptor VR1 is activated by capsaicin and RTX, and activation of VR1 is blocked by the antagonists capsazepine (CPZ); (Bevan et al., 1992) and ruthenium red (RR; (Wood et al., 1988)). Recently, rat VR1 and VR2 and a partial cDNA sequence of human sequences were disclosed in the WIPO publication WO 99/09140.
The densities of VR1 receptors can be tested using a [
3
H]RTX binding assay (Szallasi and Blumberg, 1990; Szallasi and Blumberg, 1993). Indeed, high expression of VR1 receptors was observed in rat and human spinal cord and dorsal root ganglia (Szallasi et al, 1993; Szallasi and Goso, 1994; Acs et al., 1994). Protons inhibited [
3
H]RTX binding to VR1 receptors (Szallasi et al. 1995).
Prior ligand binding assays using the VR-1 receptor teach that the pH must be near physiological conditions. In these assays, ligand binding was reduced by 50% and 70% at pH 8.0 and pH 9.0, respectively (Szallasi and Blumberg, 1993).
SUMMARY OF THE INVENTION
In contrast to what is suggested in the art, the present invention provides the surprising discovery that the binding capacity of certain ligands of the Vanilloid receptor increases at pH values that are greater than pH 7.4. The present invention provides improved assays to measure competitive vanilloid receptor binding of a known radiolabeled ligand and a test compound binding in aqueous buffers at a pH in the range of about 7.5-10.0. The present invention also provides the discovery that divalent cations also increase the binding capacity of certain ligands for the Vanilloid receptor. Therefore the aqueous solutions used for the methods of the present invention advantageously may include, as one component, a divalent cation.
The methods of the present invention are useful to find compounds that bind to Vanilloid receptors.


REFERENCES:
patent: WO 99/09140 (1999-02-01), None
patent: WO 00/29577 (2000-05-01), None
Acs, G., et al, “{3H} Resiniferatoxin binding by the human vanilloid (capsaicin) recepor”, Molecular Brain Research 1994 23:185-190.
Bevan, S., et al, “Sensory neuron-specific actions of capsaicin: mechanisms and applications,” Trends Pharmacolo. Sci. 1990 11:330-333.
Bevan, S., et al., “Capsazepine: a competitive antagonist of the sensory neuron excitant capsaicin,” British Journal Pharmacolo. 1992 107:544-552.
Caterina, M. J., et al. “The capsaicin receptor: a heat-activated ion channel in the pain pathway,” Nature 1997 389:816-824.
Szallasi, A., et al., “Specific binding of resiniferatoxin, an ultrapotent capsaicin analog, by dorsat root ganglion membranes,” Brain Research 1990 524:106-111.
Szallasi, A., et al., “{3H} resiniferatoxin binding by the vanilloid receptor: species-related differences, effects of temperature and sulfhydryl reagents,” Naunyn-Schmiedeberg's Archives of Phamacology, 1993 347:84-91.
Szallasi, A., et al., “Proton inhibition of {3H} resiniferatoxin binding to vanilloid (capsaicin) receptors in rat spinal cord,” Molecular Pharmacology 1995 289:181-187.
Szallasi, A., et al., “Characterization by {3H} resiniferatoxin binding of a human vanilloid (capsaicin) receptor in post-mortem spinal cord,” Neuroscience Letters 1994 165:101-104.
Szallasi, A., et al., “Competitive inhibition by capsazepine of {3H} resiniferatoxin binding to central (spinal cord and dorsal root ganglia) and Peripheral (urinary bladder and Airways) vanilloid (capsaicin) receptors in the Rat,” Journal Pharmacol. Exp. Ther. 1993 267:728-33.
Szallasi, A., et al., “Vanilloid (capsaicin) receptors in the rat: distribution in the brain, regional differences in the spinal cord, axonal transport to the periphery, and depletion by systemic vanilloid treatment,” Brain Research 1995 703:175-183.
Szolcsanyi, J., “Capsaicin-sensitive sensory nerve terminals with local and systemic efferent functions: facts and scopes of an unorthodox neuroregulatory mechanism,” Progress in Brain Research 1996 113:343-359.
Szolcsanyi, J., “Actions of capsaicin on sensory receptors,” Capsaicin in the study of pain 1993 J.N. Wood, ed.: Academic, London, UK pp. 1-26.
Szolcsanyi, J., et al., “Resiniferatoxin: an ultrapotent neurotoxin of capsaicin-sensitive primary afferent neurons,” Annual N.Y. Academy of Sciences, 1991 632:473-475.
Tominaga, M., et al., “The cloned capsaicin receptor integrates multiple pain-producing stimuli,” Neuron 1998 21:531-543.
Wood, J.N., et al., “Capsaicin-induced ion fluxes in dorsal root ganglion cells in culture,” Journal of Neuroscience 1988 8:3208-3220.
Genbank Accession#NM_018727, Homo Sapiens Vanilloid receptor subtype 1 (VRI), MRNA.
PCT Search Report of PCT/US 01/42602 dated Mar. 11, 2002.

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