WO2003007953A1 - Methods and compositions for modulating t-type calcium channels - Google Patents

Abstract

The present invention provides methods and compositions for the modulation of activity of T-type calcium 2+ channels. Included is a method for treatment, inhibition or prevention of pain perception in a subject in need thereof comprising administering to the subject an amount of an inhibitor of T-type Ca2+ channel activity or a pharmaceutically acceptable salt or prodrug thereof effective to treat, inhibit or prevent pain perception in the subject.

Description

METHODS AND COMPOSITIONS FOR

MODULATING T-TYPE CALCIUM CHANNELS

This application claims priority to provisional application 60/306,298 filed on July 18, 2001 , the contents of which are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention provides methods and compositions for the modulation of activity of T-type calcium 2+ channels. These methods and compositions are useful for producing analgesia and sensory enhancement, particularly applicable to the treatment, inhibition or other modulation of chronic, persistent and/or neuropathic pain perception. In particular, oxidizing agents, such as 5,5'-dithio-bis-(2~ nitrobenzoic acid) (DTNB) alone or in combination with T-type Ca 2+ channel bloc ers, such as mibefradil, are effective to decrease pain perception by reducing transmission along nociceptive pathways of the T-type channels. Also, reducing agents, such as dithiothreitol (DTT) and L-cysteine, are effective to enhance pain perception by increasing transmission along nociceptive pathways of the T-type Ca 2+ channels.

In a related aspect, methods are provided to screen potential therapeutic compositions using T-type Ca 2+ channels, active fragments, derivatives, analogs or mimics to identify pain perception modifying agents.

BACKGROUND

Chronic or intractable pain, such as may occur in conditions such as bone degenerative diseases and cancer, is a debilitating condition which is treated with a variety of analgesic agents, and often with opioid compounds, such as morphine. Although brain pathways governing the perception of pain are still incompletely understood, sensory afferent synaptic connections to the spinal cord, termed "nociceptive pathways," have been documented in some detail. Analgesia, or the reduction of pain perception, can be effected by decreasing transmission along such nociceptive pathways.

Calcium is an essential signaling molecule for many normal physiological functions in the human body. These include all electrical signaling in the nervous system, as well as controlling heart and smooth muscle contraction, and hormone release. The entry of calcium into cells is regulated by a diverse set of proteins called calcium channels. Several types of calcium channels have been identified: L, N, P/Q, R and T-types. L-types are the targets of certain drugs given to treat cardiovascular disorders including hypertension, angina and certain cardiac arrhythmias. Two of the other channels, N - and P/Q-types are mainly found in the nervous system where they are implicated in stroke, chronic pain, migraine headache and epilepsy. Proposed roles for neuronal T-type channels include promotion of calcium- dependent burst firing, generation of low-amplitude intrinsic neuronal oscillations, elevation of calcium entry and boosting of dendritic signals, possibly contributing to pacemaker activity, wakefulness, and seizure susceptibility. These calcium channels provide important clinical targets for the development of agents useful for the treatment of epilepsy, cardiac arrhythmias and diabetes.

Voltage-gated calcium channels are present in neurons, and in cardiac, smooth, and skeletal muscle and other excitable cells. These channels are known to be involved in membrane excitability, muscle contraction, and cellular secretion, such as in exocytotic synaptic transmission. In neuronal cells, voltage-gated calcium channels have been classified by their electrophysiological as well as by their biochemical and pharmacological properties. More recently, further classification has been made based on the molecular biology of the channels.

Calcium channels are generally classified according to their electrophysiological properties as Low-voltage activated (LVA) or High-voltage activated (HVA) channels. HVA channels are currently known to comprise at least three groups of channels, known as L-, N- and P/Q-type channels. These channels have been distinguished from one another electrophysiologically as well as biochemically on the basis of their pharmacology and ligand binding properties. Thus, dihydropyridines, diphenylalkylamines and piperidines bind to the alphai subunit of the L-type calcium channel and block a proportion of HVA calcium currents in neuronal tissue, which are termed L-type calcium currents. N-type calcium channels are sensitive to omega conopeptides, but are relatively insensitive to dihydropyridine compounds, such as nimodipine and nifedipine. P/Q-type channels, on the other hand, are insensitive to dihydropyridines, but are sensitive to the funnel web spider toxin Aga IIIA. R-type channels are insensitive to dihydropyridines and omega conopeptides, but, like P/Q, L and N channels, are sensitive to the funnel web spider toxin Aga IVA. There are no known specific or selective ligands for the Class E or R-type neuronal calcium channel. Although the spider peptide omega-Aga IIIA antagonizes this channel, it also potently blocks N, P/Q- and L-type calcium currents (Cohen et al., 1993; Ertel, S. I. and Clozel, J-P. (1997). Mibefradil (Ro 40-5967): the first selective T-type Ca²⁺ channel blocker. Exp. Opin. Invest. Drugs 6, 569-582 vlibefradil (Ro 40-5967): the first selective T-type Ca²⁺ channel blocker. Exp. Opin. Invest. Drugs 6, 569-582; Mibefradil (Ro 40-5967): the first selective T-type Ca²⁺ channel blocker. Exp. Opin. Invest. Drugs 6, 569-582) and therefore lacks specificity. Unlike the family of HVA Ca²⁺ channels, no natural toxins or venom components have been identified that alter the LVA, or T-type channel's selectivity. The lack of specific ligands for this channel has heretofore impeded elucidation of its role(s) in neuronal function.

Accordingly, a need exists for the inhibition, and conversely, in certain conditions, the enhancement, of pain perception. Moreover, in view of the importance of specific calcium channels in neuronal function, it would be useful to identify pharmacological agents that specifically inhibit or potentiate the class T calcium channel. The present invention meets these needs.

SUMMARY OF THE INVENTION

Among the several aspects of the invention, therefore, is provided a method for the treatment, inhibition, or prevention of pain perception in a subject in need thereof. The method comprises administering to the subject an amount of an inhibitor of T-type Ca2+ channel activity or a pharmaceutically acceptable salt or prodrug thereof effective to treat, inhibit or prevent unwanted pain perception in the subject. In another aspect, a method is provided for the treatment, inhibition or prevention of insufficient pain perception in a subject in need thereof. The method includes administering to the subject a pain sensitizing amount of an enhancer of T- type Ca2+ channel activity or a pharmaceutically acceptable salt or prodrug thereof to treat, inhibit or prevent insufficient pain perception in the subject.

In yet another embodiment, methods are provided for the potentiation and/or inhibition of a T-type Ca2+ channel in a subject in need of such potentiation/inhibition or in a tissue comprising a T-type Ca2+ channel. The method comprises administering to the subject or tissue either an amount of a reducing agent or of an oxidizing agent or a pharmaceutically acceptable salt or prodrug thereof effective either to potentiate or inhibit the T-type Ca2+ channel's activity as desired.

In a further aspect of the invention a method of screening candidate pain perception modifying agents is provided. Such method comprises administering a candidate to an expression system comprising a T-type Ca2+ channel or its active fragment, derivative, analog or mimic and determining whether the channel, fragment, derivative, analog or mimic's activity is thereby modified.

The invention further comprises pharmaceutical compositions to carry out the above described methods.

Other objects and features of the invention will be in part apparent and in part pointed out hereinafter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 A-E are depictions of the effect of DTT on T-Type Ca²⁺ and other voltage and ligand-gated currents in acutely dissociated DRG neurons.

FIG. 2A-F are depictions demonstrating redox modulation of T-Type Ca²⁺ currents in rat sensory neurons and Ca_v3.2 currents in HEK cells.

FIG. 3A-D are depictions of the induction of thermal hyperalgesia in adult rats by tested reducing agents.

FIG. 4A-D are depictions of the effect of DTNB in inducing analgesia in thermal PWL testing and in blocking hyperalgesia induced by reducing agents. FIG. 5A-C are depictions of the effect of mibefradil in blocking the effects of reducing agents in vivo.

FIG. 6A-D are depictions o fthe effect of redox agents in augmenting the effects of mibefradil in vitro and in vivo.

FIG. 7A-D are depictions of the role of reducing agents as modulators of peripheral mechanical nociception.

More detailed descriptions of the Figures are contained in the Figure Legends below, as well as in relevant portions of the following specification.

ABBREVIATIONS AND DEFINITIONS

To facilitate understanding of the invention, a number of terms and abbreviations as used herein are defined below:

The term "prevention" includes preventing the onset of clinically evident unwanted pain perception or insufficient pain perception. This definition includes prophylactic treatment.

The term "inhibition" as used herein in connection with pain therapy means to decrease the severity of pain perception or insufficient pain perception as compared to that which would occur in the absence of the application of the method of the present invention.

The phrase "therapeutically-effective amount" or "effective amount" is intended to qualify the amount of each agent which will achieve the goal of improvement in disorder or condition severity and the frequency of incidence over no treatment.

The term "subject" for purposes of treatment includes any mammalian subject who is susceptible to unwanted pain perception or who is in need of sensory enhancement. The subject can be a domestic livestock species, a laboratory animal species, a zoo animal or a companion animal. In one embodiment, the subject is a human being.

The term "T-type Ca2+ channel selective inhibitor" or "T-type Ca2+ channel selective enhancer" denotes a compound able to inhibit or enhance T-type Ca2+ channel activity without significant inhibition or enhancement of other Ca2+ channels.

DTNB = 5,5'-dithio-bis-(2-nitrobenzoic acid).

DTT = dithiothreitol.

DRG =dorsal root ganglion.

LVA = low-voltage activated.

HVA = high-voltage activated.

PWL = pain withdrawal latencies.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Applicants have discovered that peripheral pain perception can be altered by the modulation of T-type Ca2+ channels. Further, applicants have discovered that T- type Ca2+ channels can be modulated by the administration of redox agents. Hence, the treatment, inhibition or prevention of unwanted pain perception is provided by administration of oxidizing agents or T-type channel blockers. Relatedly, sensory enhancement can be provided by the administration of reducing agents or T-type Ca2+ channel potentiators. Applicants research containing their experimental results and findings is set forth in the Addendum entitled "Redox Modulation of T- Type Calcium channels in Rat Peripheral Nociceptors" attached hereto and fully incorporated herein and included as part of this application.

Applicants have discovered that redox agents modulate T-currents but not other voltage- and ligand-gated channels thought to mediate pain sensitivity. Similarly, applicants have shown that redox agents modulate currents through Cav3.2 recombinant channels. Thus, applicants have demonstrated that T-type calcium channels play a role in peripheral pain transmission. Additionally, redox agents may be utilized to potentiate or inhibit T-type channel activity in various tissues including at least central, peripheral and enteric neural tissue, muscle, including cardiac and vascular smooth muscle, kidney cells, liver cells, and cells of the immune system.

Applicants have demonstrated that reducing agents including DTT and the endogenous amino acid, L-cysteine, promote cutaneous thermal and mechanical hyperalgesia via effects on T-channels. Conversely, the oxidizing agent, 5,5'- dithio- bis-(2-nitrobenzoic acid)(DNTB), and the T-type calcium channel antagonist, mibefradil, produce analgesia to cutaneous thermal stimuli. These results demonstrate a previously unknown role for peripheral T-type Ca²⁺ channels in boosting nociceptive signals.

Direct injection of L-cysteine into peripheral receptive fields induced hyperalgesia to thermal and mechanical stimuli. Other preferred reducing agents include thiol-containing compounds like L-homocysteine and glutathione.

Applicants' results indicate that modulation of peripheral T-type Ca²⁺ channels influences thermal and mechanical nociceptive inputs. Since many pathological pain states are associated with exaggerated thermal and mechanical nociception, peripherally applied T-channel antagonists and/or oxidizing agents are believed to represent new classes of drugs to treat these forms of chronic pain.

The invention includes, therefore, in one aspect, a method of treating, inhibiting or preventing pain perception, including chronic, persistent and pathological pain states. Chronic persistent, pathological pain conditions often are accompanied by heightened sensitivity to painful thermal and mechanical stimuli. This state of increased sensitivity to thermal (heat) stimuli is referred to as thermal hyperalgesia. Similarly, increased sensitivity to mechanical stimuli is referred to as mechanical hyperalgesia. Both conditions are often found in conditions causing direct mechanical nerve injury such as compression syndromes (e.g. sciatica), constrictive injury (compartment syndrome) or metabolic diseases (e.g. diabetes mellitus). All of these conditions can cause chronic disfunction of nerves with mechanical and/or thermal hyperalgesia: often called neuropathic pain.

The other most common cause of hyperalgesia is inflammation of tissue involving peripheral nerve endings. Examples of tissue inflammation are arthritis (joint inflammation), sun burns, (epidermal skin inflammation) and fibromyalgia (inflammation of muscles and tendons).

The T-type Ca2+ channel selective modulators useful in the practice of the present methods can be formulated into pharmaceutical compositions and administered by any means that will deliver a therapeutically effective dose. These compositions, for example, can be, if appropriate, administered orally, parenterally, by inhalation spray, rectally, intradermally, transdermally, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired.

Topical formulations may be especially useful by providing direct local delivery in the vicinity of sensory nerve endings that contain the target channels. Pharmaceutical compositions suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol or oil. Carrier materials that can be used include vaseline, lanolin, polyethylene glycols, alcohols and combinations thereof. The active compound is generally present at a concentration of from 0.1 to 15% w/w of the composition, for example, from 0.5 to 2%.

Topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoresis devices. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, or intrasternal injection, or infusion techniques. Formulation of drugs is discussed in, for example, Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania (1975), and Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y. (1980).

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are useful in the preparation of injectables. Dimethyl acetamide, surfactants including ionic and non- ionic detergents, and polyethylene glycols can be used. Mixtures of solvents and wetting agents such as those discussed above are also useful.

Suppositories for rectal administration of the compounds discussed herein can be prepared by mixing the active agent with a suitable non-irritating excipient such as cocoa butter, synthetic mono-, di-, or triglycerides, fatty acids, or polyethylene glycols which are solid at ordinary temperatures but liquid at the rectal temperature, and which will therefore melt in the rectum and release the drug.

Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compounds are ordinarily combined with one or more adjuvants appropriate to the indicated route of administration. If administered peros, the compounds can be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted or encapsulated for convenient administration. Such capsules or tablets can contain a controlled-release formulation as can be provided in a dispersion of active compound in hydroxypropylmethyl cellulose. In the case of capsules, tablets, and pills, the dosage forms can also comprise buffering agents such as sodium citrate, or magnesium or calcium carbonate or bicarbonate. Tablets and pills can additionally be prepared with enteric coatings.

For therapeutic purposes, formulations for parenteral administration can be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions can be prepared from sterile powders or granules having one or more of the carriers or diluents mentioned for use in the formulations for oral administration. The compounds can be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and/or various buffers. Other adjuvants and modes of administration are well and widely known in the pharmaceutical art.

Liquid dosage forms for oral administration can include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions can also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.

The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the patient and the particular mode of administration. In general, the pharmaceutical compositions may contain a T-type Ca2+ channel selective inhibitor in the range of about 0.1 to 2000 mg, preferably in the range of about 0.5 to 500 mg and most preferably between about 1 and 200 mg. A daily dose of about 0.01 to 100 mg/kg body weight, preferably between about 0.1 and about 50 mg/kg body weight and most preferably from about 1 to 20 mg/kg body weight, may be appropriate. The daily dose of the oxidizing agent and T-type Ca2+ channel antagonist can be administered in a sequential manner or a substantially simultaneous manner in an appropriate number of doses per day if a combination of an oxidizing agent and channel antagonist is employed.

Dosage unit compositions may contain such amounts of sub-multiples thereof to make up the daily dose. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the subject treated and the particular mode of administration. For instance, systems such as transdermal administration or oral administration, which are substantially less efficient delivery systems, may require dosages at least an order of magnitude above those required for parenteral administration. The dosage regimen for treating a disease condition with the compounds and/or compositions of this invention is selected in accordance with a variety of factors, including the type, age, weight, sex, diet and medical condition of the patient, the severity of the disease, the route of administration, pharmacological considerations such as the activity, efficacy, pharmokinetic and toxicology profiles of the particular compound employed, whether a drug delivery system is utilized and whether the compound is administered as part of a drug combination. Thus, the dosage regimen actually employed may vary widely and therefore may deviate from the preferred dosage regimen set forth above. Those of ordinary skill in the art can readily determine appropriate dosages for any particular subject based on the teachings in this specification and routine analysis of the subject.

While the compounds of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more compounds which are known to be effective against the specific disease state or condition that one is targeting for treatment.

The biological activity of candidates for use as pain perception modifying agents can be tested using the in vitro and in vivo assays developed by the applicants as described herein. In summary, these screening methods employ T- type Ca2+ channels or their active fragments, derivatives, analogs or mimics. Such methods involve administering the pain perception modifying candidate(s) to an expression system comprising a T-type Ca2+ channel, active fragment, derivative, analog or mimic and determining whether the T-type Ca2+ channel's nociceptive transmission is thereby modified. The expression system can be a biological or chemical expression system, including a cell, tissue or animal subject, as exemplified in the experimental description set forth in the attached Addendum. While native T- type calcium channel proteins may be used in this screening process, those skilled in the art may substitute active protein fragments, derivatives, analogs or mimics through procedures well known in the art. For example, derivatives of T-type Ca2+ channels can include various structural forms of the primary proteins which retain. biological activity. Due to the presence of ionizable amino and carboxyl groups, the channel may be in the form of acidic or basic salts, or may be in neutral form. Individual amino acid residues may also be modified by oxidation or reduction. The primary amino acid structure may be modified by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like, or by creating amino acid sequence mutants.

Preparation of fragments can be achieved using techniques known to isolate a desired portion of an active region. Also, unique restriction sites or PCR techniques that are known in the art can be used to prepare numerous truncated forms which can be expressed and analyzed for activity. Analogs or mimics of the calcium channels may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. Generally, substitutions should be made conservatively. The most preferred substitute amino acids are those which do not adversely affect the biological activity of the calcium channel. Examples of possible substitutions include substituting one aliphatic residue for another, such as lie, Val, Leu, or Ala for one another.

The following examples are intended to provide illustrations of the application of the present invention. The examples are not intended to completely define or otherwise limit the scope of the invention. Examples

Experimental procedures

Electrophysiological methods.

Acutely dissociated DRG neurons. Dissociated DRG cells were prepared from adult rats (100-420 gm) and used within 4 hours for whole-cell recordings as described elsewhere (Todorovic, S.M. and Lingle, C.J. (1998). Pharmacological properties of T-type Ca²⁺ current in adult rat sensory neurons: effects of anticonvulsants and anesthetic agents. J. Neurophysiol. 79, 240-252; Todorovic, S.M., Perez-Reyes, E. and Lingle, C.J. (2000). Anticonvulsants but not general anesthetics have differential blocking effects on different T-type current variants. Mol. Pharmacol. 58, 98-108. Briefly, 8-10 DRG's from thoracic and lumbar regions were dissected and incubated at 36° C for 60-100 min in Tyrode solution containing (in mM): 140 NaCI, 4 KCI, 2 MgCfe, 10 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), adjusted to pH 7.4 with NaOH. This solution was supplemented with 5 mg/ml collagenase (Sigma type I) and 5 mg/ml dispase II (Boehringer-Manheim). The duration of enzymatic treatment did not alter the effects of redox agents on T- type calcium currents. Single neuronal somata were obtained by trituration in Tyrode solution at room temperature. For recordings, cells were plated onto glass coverslip and placed in a culture dish that was perfused with external solution. All data were obtained from small diameter neurons (21-27 μm) without visible processes.

Transfected cells. HEK cells were stably transfected with human Cav3.2 constructs (cell lines AH-13, or Q31) as described previously (Lee JH, Daud AN, Cribbs LL, Lacerda AE, Pereverzev A, Klockner U, Schneider T, Perez-Reyes E (1999). Cloning and expression of a novel member of the low voltage-activated T- type calcium channel family. J Neurosci 19,1912-1921 ; Todorovic, S.M., Perez- Reyes, E. and Lingle, C.J. (2000). Anticonvulsants but not general anesthetics have differential blocking effects on different T-type current variants. Mol. Pharmacol. 58, 98-108). Cells were typically used 1-3 days after plating. Because these cells are routinely grown with 10% FBS (fetal bovine serum) which may contain endogenous redox agents, we routinely incubated these cells in our external solution for at least 30 minutes at room temperature before experiments. Electrophysiological recordings. Recordings were made with standard whole cell voltage-clamp techniques (Hamill, O.P., Marty, E., Neher, E., Sakmann, B., and Sigworth, F.J. (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfluegers Arch. 381 , 85-100). Electrodes were fabricated from microcapillary tubes and fire-polished to resistances of 1-4 MΩ. Voltage commands and digitization of membrane currents were done with Clampex 6.0 of the pClamp software package (Axon Instruments, Foster City, CA) running on a IBM-compatible computer. Neurons were typically held at -90 mV and depolarized to -35 mV every 20 seconds to evoke inward T-currents. Membrane currents were recorded with an EPC 7 patch-clamp amplifier (List Instruments). All T- currents are measured as peak response relative to the end of the depolarizing pulse to avoid small contaminating HVA currents that could be present in some cells. Data were analyzed using Clampfit (Axon Instruments) and Origin 4.0. Currents were filtered at 5 kHz. Reported series resistance values were taken directly from the reading of the amplifier. Average uncompensated series resistance for DRG cells was 7.9 ± 4.5 MΩ (mean ± SD) and the average capacitance was 14 + 5 pF (mean ± SD, N=107 neurons). For HEK cells, average capacitance (Cm) was 19.5 + 6 pF and average series resistance was (R_s) 4.5 ± 2 MΩ (N=14). Series resistance typically was compensated 50-70% during experiments. In most experiments, a P/5 protocol was used for on-line leakage subtractions.

Solutions. A glass syringe served as a reservoir for a gravity-driven local perfusion system that consisted of multiple, independently controlled glass capillary tubes. Switching between solutions was accomplished by manually controlled valves except for applications of capsaicin, heat, acid and ATP when computer- driven electronic valves designed for fast drug applications were used. Most experiments were done at room temperature (20-24°C) except when the effect of higher temperature on redox modulation of T-currents and heat gated-currents was studied. In this case, extracellular solution was heated in the syringe and perfusion tube by a thermoresistive device. The temperature in the bath was measured with a microprobe positioned close to the recorded cell. All drugs were prepared as stock solutions (ATP, MK-801 and ketamine as 10 mM, L-cysteine and DTT and D-APV as 100 mM stocks in water) and freshly diluted to appropriate concentrations at the time of experiment. Final L-cysteine dilutions were done at the time of experiment and used within 1 hour because of instability resulting from spontaneous oxidation in the presence of trace metal ions. Stock solutions of DTNB (600 mM), capsaicin (10 mM) and mibefradil (100 mM) were prepared in DMSO. The maximal concentration of DMSO was 0.5 % for in vitro experiments. At these concentrations, DMSO had no effect on either T-currents or holding potential (data not shown). Mibefradil (Ro 40- 5967) was a kind gift of F. Hoffman-La Roche, Basel to Dr. Christopher Lingle. All other chemicals were obtained from Sigma (St. Louis, MO) and/or Aldrich Chemicals (Milwaukee, Wl).

The standard external solution used to isolate Ca²⁺ currents contained (in mM): 5-10 BaCI₂, 160 tetraethylammonium (TEA) chloride, 10 N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), with pH adjusted to 7.4 with TEAOH. To isolate T- currents, we blocked most HVA currents in these cells with intracellular F^" and with application of 5 μM nifedipine (L-type HVA channel blocker) and 1 μM ώCgTx-GVIA (ω-conotoxin-GVIA, N-type HVA blocker) in the external solution (Todorovic, S.M. and Lingle, C.J. (1998). Pharmacological properties of T-type Ca²⁺ current in adult rat sensory neurons: effects of anticonvulsants and anesthetic agents. J. Neurophysiol. 79, 240-252). The standard external solution for recording voltage-gated K⁺ and Na⁺ currents, and capsaicin-, heat-, proton- and ATP- gated currents contained (in mM): 140 NaCI, 5 KCI, 2 MgCfe, 0.5 CaCI₂, 10 glucose and 10 HEPES, pH 7.4 with NaOH. The standard pipette solution used to record T and HVA Ca²⁺ currents, capsaicin-, heat-, proton- and ATP- gated currents and voltage-gated Na⁺ currents contained (in mM): 110 Cs-methane sulfonate, 14 phosphocreatine, 10 HEPES, 9 EGTA, 5 Mg-ATP and 0.3 Tris-GTP, pH adjusted to 7.2 with CsOH. For recording voltage-gated potassium currents, KCI replaced Cs-methane sulfonate and 2 mM QX 314 was added to pipette solution to block voltage-gated Na⁺ currents. For recording T-type Ca²⁺ currents in isolation, we used the following F^" based intracellular solution (in mM): 135-140 TEAOH, 10 EGTA, 40 HEPES, and 2 MgCI₂, titrated to pH 7.15-7.25 with HF.

Current analysis. Concentration-response data were fit to the function:

PB([MIBEFRADIL]) = PB_max/ (1+(IC₅₀/[MIBEFRADIL])ⁿ) where PB_maχ is the maximal percent block by mibefradil of peak T-currents, the IC₅o is the concentration that produces half-maximal block and n is the apparent Hill coefficient. Fitted values are reported with 95% linear confidence limits. The time course of T-current inactivation was examined using a single exponential fit to current decay. Fitting was done either with Origin 4.0 (Microcal Software, Northhampton, MA) or Clampfit 6.0 (Axon Instruments).

Behavioral studies. All experimental protocols were approved by the Washington University Animal Studies Committee. The nociceptive response to thermal (heat) stimulation was tested in a commercially available paw thermal stimulation system as described elsewhere (Jevtovic-Todorovic, V., Wozniak, D. F., Powell, S., Nardi, A. and Olney, J.W. (1998). Clonidine potentiates the neuropathic pain-relieving action of MK-801 while preventing its neurotoxic and hyperactivity side effects. Brain. Res. 781 , 202-211). Briefly, the device consists of a clear plastic chamber (10x20x24 cm) that sits on a clear elevated glass floor and is temperature regulated at 30°C. Adult female Sprague-Dawley rats were placed in the plastic chamber and given 10-15 minutes to accommodate. A radiant heat source mounted on a portable holder beneath the glass floor was positioned to deliver a thermal stimulus to the plantar side of the hind paw. When an animal withdraws its paw, a photocell detects interruption of a light reflection and an automatic timer shuts off. This provides an accurate record of paw withdrawal latency (PWL). The PWL can be measured with this apparatus with a precision of 0.1 sec. To prevent thermal injury, the thermal source is automatically discontinued after 20 sec if the rat fails to withdraw its paw.

For measurements of mechanical nociception rats were placed in a clear plastic, wire mesh-bottomed cage, divided into 4 individual compartments permitting free movement of animals while allowing access to the paws. Von Frey Filaments (Stoelting, Wood Dale, IL) were used to assess the mechanical threshold for paw withdrawal. These filaments are designated by manufacturer as the log- (milligram weight required to cause bending X10). We identified the filament (4.93) that applied to the plantar surface of the foot causes a noxious response resulting in an average of 3-4 paw withdrawals in 10 trials. Baseline withdrawal scores were determined first in both paws 1-2 days prior to actual testing and compared to time (just prior to injection, marked as "0" time on our graphs). If these two values differed by more than 2 withdrawals, animals were not used for further experiments.

To test the effects of drugs in peripheral receptive fields, we injected 100 μL of test compounds intradermally in the ventral side of the left hind paw. The non- injected side (right hind paw) was used as a control in each animal. All solutions were pH balanced to 7.4 to avoid skin irritation. No signs of skin inflammation, discoloration or irritation were noted at the sites of injection with test compounds. All doses are expressed in μg per 100 μL. For animals included in this study, baseline values were compared to thermal PWLs or withdrawal scores on non-injected and injected paws at various time points during the testing as indicated in the figures (post-treatment values). In the data displayed, every point is an average of at least 8 animals and values represent mean ± SE. Statistical analysis was performed using an ANOVA comparing within-subject variables: paw condition (injected vs. non- injected) and test session (prior to drug administration or 10, 20 or 60 minutes post- treatment).

Results

In vitro studies. The DRG contains cell bodies of primary afferent (sensory) fibers that originate as pain endings in the periphery and terminate in the dorsal horn of the spinal cord. Whole-cell recordings from dissociated DRG neurons of adult rats are used to study peripheral nociceptive mechanisms because the small size of peripheral nerve endings precludes direct measurement of currents from sensory endings. We limited our experiments to small diameter (< 27 μm) acutely dissociated neurons because the majority of these cells are involved in nociceptive processing (Gardens, C.G., Del Mar, L.P. and Scroggs, R.S. (1995). Variation in serotonergic inhibition of calcium channel currents in four types of rat sensory neurons differentiated by membrane properties. J. Neurophysiol. 74, 1870-1879 ; Coderre, T.J., Katz, J., Vaccarino, A.L. and Melzack, R. (1993). Contribution of central neuroplasticity to pathological pain: review of clinical and experimental evidence. Pain 52, 259-285; Levine, J. D., Fields, H. L and Basbaum, A. I. (1993). Peptides and the primary afferent nociceptors. J. Neurosci. 13, 2273-2286 ; Snider, W. D. and McMahon, S. B. (1998). Tackling pain at the source: new ideas about nociceptors. Neuron 20, 629-632).

In an effort to identify endogenous modulators of excitability in sensory neurons, we initially examined whether redox agents alter voltage-gated currents that regulate the excitability of nociceptive cells. We found that the reducing agent DTT (0.1 and 1 mM), at a test potential of -35 mV, selectively augmented T-type Ca²⁺ currents by 128 ±16 % (N=14) and 142 ± 18 % (n=11), respectively, while leaving HVA Ca²⁺ currents in the same cells unaffected (N=5 for 0.1 mM and N=7 for 1 mM, Figures 1A and 1B). In all, DTT enhanced T-currents in 34 of 39 cells (86%), and this effect was completely reversible within 2 minutes after the removal of DTT from the bath. An apparent steady state effect was observed with up to 1 minute application of DTT (0.1-1 mM) and no change in maximal effect was observed with longer applications up to 5 minutes (N=3, data not shown). The effect of DTT on T- currents was accompanied by a change in current kinetics. Activation times, measured as the 10-90% rise times of peak currents, were accelerated in the presence of DTT. At -35 mV, the control 10-90% rise time was 17.2 ± 2.5 ms and in the presence of DTT it was 10.1 ± 1.4 ms (mean ± SE, N=5). At -35 mV, the inactivation time constant (t) was 50 ± 3.7 ms in control and 25.5 ± 1.6 ms in the presence of DTT (N=5). We also examined whether increasing the temperature of in vitro experiments to 37°C (the normal in vivo temperature) altered responses to reducing agents since higher temperatures are likely to increase the amplitude of T- currents and accelerate activation and inactivation kinetics in the absence of redox modulation. At 37°C, 0.1 mM DTT increased peak T-currents about 2-fold, similar to the effect observed at 22° C. However, changes in current kinetics with DTT were less apparent at higher temperatures (N=3, data not shown).

We also examined the effects of DTT upon voltage-gated K⁺ and Na⁺ currents in small size sensory neurons. Figure 1C illustrates the lack of effect of 4 mM DTT on inward Na⁺ currents in these cells (N=7) and Figure 1 D shows the lack of effect of 3 mM DTT on a family of outward potassium currents (N=5). T-channels are heterogeneously expressed in DRG cells of different sizes but are uniformly present in small diameter acutely dissociated cells of adult rat (Scroggs, R.S. and Fox, A.P. (1992). Calcium current variation between acutely isolated rat dorsal root ganglion neurons of different size. J. Physiol. (Lond), 445, 639-658). Small DRG cells have electrical properties of nociceptors and sensitivity to capsaicin (Todorovic, S.M and Anderson, E. G. (1992). Serotonin preferentially hyperpolarizes capsaicin-sensitive C type sensory neurons by activating 5-HT1A receptors. Brain Research, 585, 212-218; Gardens, C.G., Del Mar, L.P. and Scroggs, R.S. (1995). Variation in serotonergic inhibition of calcium channel currents in four types of rat sensory neurons differentiated by membrane properties. J. Neurophysiol. 74, 1870- 1879), features that identify these cells as chemical and thermal nociceptive sensory neurons (Snider, W. D. and McMahon, S. B. (1998). Tackling pain at the source: new ideas about nociceptors. Neuron 20, 629-632; Reichling, D.B. and Levine, J.D. (2000). In hot pursuit of the elusive heat transducers. Neuron 26, 555-558; Caterina, M.J. and Julius, D. (1999). Sense and specificity: a molecular identity for nociceptors. Current Opinion in Neurobiology 9, 525-530). Therefore, we tested the sensitivity of these cells to a variety of noxious stimuli including capsaicin, heat, acid pH and ATP. Capsaicin (1 μM) evoked inward currents in 9 of 12 small DRG cells (75%) that had T-currents sensitive to DTT, indicating that our in vitro experiments include cells that participate in thermal and chemical nociception. However, up to 3 mM DTT had no effect on capsaicin-induced currents (1 ± 4% change, N=6, Figure 1 E). To test the sensitivity of heat channels in these cells to DTT, we applied heated external solution (45 ± 2° C) to these cells. Nineteen small DRG cells were studied and 10 of them (47%) responded with inward currents as depicted in Figure 1E. All DRG neurons responded to low pH (pH 5.3, N=9) but only 3 of 15 cells (20%) responded to application of 100 μM ATP. Currents elicited by heat, low pH and ATP were not significantly altered by 1-3 mM DTT (Figure 1 E). These data strongly suggest that DTT selectively modulates T-type Ca²⁺ currents in capsaicin-sensitive sensory neurons, but not other voltage-gated currents or channels thought to mediate heat or chemical sensitivity.

If reducing agents enhance currents through T-type Ca²⁺ channels, we would expect that oxidizing agents should decrease current flow through these channels. Indeed, application of 1 mM DTNB blocked peak T-currents by about 50% (46 ± 2.7%, N=9) in all tested cells (Figure 2B) and when applied immediately after DTT, reversed the effects of DTT on current kinetics (Figure 2A). In contrast to DTT, DTNB had little effect on the time course of activation or inactivation (at -35 mV the 10-90% rise time was 17 ± 4.5 ms, and the inactivation 150.7 ± 5.3 ms, N=4, p>0.05). Furthermore, this effect was selective for T-type Ca²⁺ channels because applications of up to 3 mM DTNB, which completely blocked T-currents (Figure 2B), did not alter HVA Ca²⁺ (N=6), voltage-gated Na⁺ (N=3) or K⁺ (N=3) currents (data not shown).

The effects of DTT on T-currents were mimicked by the endogenous reducing agent, L-cysteine (Figure 2C). L-cysteine augmented T-currents with a threshold of 10 μM and maximal effects at about 100 μM (130 ± 11 % increase, N=7). Similar to DTT, L-cysteine also increased rates of current activation and inactivation (Figure 2C). At maximal concentrations, L-cysteine and DTT consistently increased T- currents by about 2-fold. A low concentration of DTNB (0.1 mM) had little effect on T-currents when administered alone (3.3 ± 3.3% block), but completely reversed the effects of both L-cysteine and DTT when applied immediately after a reducing agent (Figure 2D). The half time of spontaneous oxidation in 4 DRG cells after exposure to reducing agent was 76.6 ±12 seconds while in the presence of 0.1 mM DTNB the recovery half-time was only 30 ± 3 seconds.

Effects of redox agents on recombinant T-type Ca²* channels. Molecular studies have shown that Cav3.2 mRNA is the most abundant isoform present in small DRG cells (Talley, E.M., Cribbs, L.L., Lee, J.H., Daud, A., Perez- Reyes, E., Bayliss, D.A.(1999). Differential distribution of three members of a gene family encoding low voltage-activated (T-type) calcium channels. J Neurosci 19:1895-1911). This sub-type of T-channels also has the most similar pharmacological profile to the native DRG currents (Todorovic, S.M., Perez-Reyes, E. and Lingle, C.J. (2000). Anticonvulsants but not general anesthetics have differential blocking effects on different T-type current variants. Mol. Pharmacol. 58, 98-108). Therefore, we examined the redox sensitivity of Cav3.2 channels expressed in HEK cells as a representative nociceptor T-type current. DTT (0.1 and 1 mM) increased peak T-currents by an average of 2.5 fold in all cells tested (157 ± 50% increase for 0.1 mM DTT, N=5, Figure 2E). Similar to DRG cells, the increased current was accompanied by acceleration of activation and inactivation kinetics (Figure 2E). The effects of DTT were mimicked by L-cysteine (N=2). Also, similar to DRG cells, 1 mM DTNB blocked Ca_v3.2 currents by 50 ± 3.4% (N=7, Figure 2E) while 3 mM DTNB completely blocked these currents in HEK cells (N=6) with little effect on current kinetics. On average, the control 10-90% rise time at a V_t of -30 mV was 8 ± 0.9 ms and the control inactivation D was 24.7 ± 2 ms. In the presence of DTT, rise times were 6.2 ± 0.9 ms and inactivation D's were 15.8 ± 2 ms N=5). In the presence of DTNB these times were 7.7 ± 0.9 ms and 28 ± 5 ms (N=6), respectively. At lower concentrations, DTNB fully reversed effects of DTT on current kinetics (N=3, data not shown), peak current amplitude and time course of recovery (Figure 2F). The average half-time for the fully reduced Cav3.2 channel to spontaneously return to baseline levels was 83 ±12 seconds in control conditions and 43 ± 3 seconds in the presence of 0.1 mM DTNB (N=3, p<0.05). These results suggest that Ca 3.2 T- type channels, the predominant isoform in sensory neurons (Talley, E.M., Cribbs, L.L., Lee, J.H., Daud, A., Perez-Reyes, E., Bayliss, D.A.(1999). Differential distribution of three members of a gene family encoding low voltage-activated (T- type) calcium channels. J Neurosci 19:1895-1911), have sensitivity to redox agents similar to native DRG cells.

In vivo studies. Because redox agents modulate T-currents in isolated neurons that participate in thermal nociception, we examined whether these agents modify in vivo responses to radiant thermal (heat) stimulation (Jevtovic-Todorovic et al., 1998; Hargreaves, K., Dubner, R., Brown, F., Flores, C. and Joris, J. (1988). A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32, 77-88). In these studies, 100 μl of test compounds were injected directly into peripheral receptive fields of sensory neurons in the hind paw of adult rats, and the latency to paw withdrawal in the presence of a radiant heat stimulus was measured. Injection of the reducing agent, L-cysteine, produced an exaggerated thermal response in a dose-dependent fashion (Figure 3B). L-cysteine (12 μg/100μl) reversibly decreased paw withdrawal latencies (PWLs) by about 40% ten to twenty minutes (p<0.005) following injection. Injection of the same volume of vehicle (saline) had no effect on PWLs (Figure 3A). Similar but less potent hyperalgesic responses were observed following injection of DTT. Compared with contralateral, non-injected paws in the same animals, 150 μg/100 μL DTT decreased PWLs by >25% ten to twenty minutes after injection (p<0.05, Figure 3C). These changes in thermal PWLs are of similar magnitude to effects observed in animals with neuropathic pain following injury to sensory nerves (Jevtovic-Todorovic, V., Wozniak, D. F., Powell, S., Nardi, A. and Olney, J.W. (1998). Clonidine potentiates the neuropathic pain-relieving action of MK-801 while preventing its neurotoxic and hyperactivity side effects. Brain. Res. 781, 202-211). L-cysteine and DTT injections did not cause animals to lick or flinch their paws suggesting that the reducing agents do not directly produce pain but rather augment transmission of thermal nociceptive signals from peripheral nerve endings.

It has been shown previously that intrathecal administration of redox agents can influence pain perception, putatively by interaction with spinal NMDA receptors (Laughlin, T. M., Kitto, K. F. and Wilcox, G. L. (1998). Redox manipulation of NMDA receptors in vivo: alteration of acute pain transmission and dynorphin-induced allodynia. Pain 80, 37-43). To explore the potential role of NMDA receptors in cutaneous thermal hyperalgesia, we injected 25 μg/100 μl of ketamine, a non- competitive NMDA antagonist, into hind paws and found that ketamine neither altered thermal PWLs (N=8) nor affected the thermal hyperalgesia induced by DTT (N=8, data not shown). In prior studies, injected locally (2.5 μg/100μl) attenuated the development of thermal hyperalgesia and allodynia associated with chemically induced joint inflammation (Lawand, N.B., Willis, W.D. and Westlund, K.N. (1997). Excitatory amino acid receptor involvement in peripheral nociceptive transmission in rats. Eur. J. Pharmacol., 324, 169-177). Similarly, another even more potent and selective non-competitive NMDA antagonist, MK-801 (0.35 μg/100 μl), did not affect baseline thermal PWL's (N=8) nor the hyperalgesic response to L-cysteine when injected into hind paws (Figure 3D, N=8). At this dose, MK-801 has been reported to block hyperalgesia to peripherally applied glutamate (Zhou, S., Bonasera L. and Carlton, S.M. (1996). Peripheral administartion of NMDA, AMPA or KA results in pain behaviors in rats. NeuroRepoήl , 895-900).

If the cutaneous thermal hyperalgesia observed following DTT and L-cysteine results from the reducing properties of these agents, then oxidizing agents should antagonize the hyperalgesia. Indeed, co-injection of 4 μg/100 μl DTNB completely reversed the effects of L-cysteine (Figure 4C) and DTT (Figure 4D), while having no effect on thermal PWLs alone (Figure 4B). Moreover, a higher dose of DTNB (40μg/100μl) prolonged PWLs indicating an analgesic effect (Figure 4B). At 10 minutes following injection, DTNB increased the latency time by 4.3 s (P<0.0005, N=12). This analgesic effect was completely reversed by 1.5 μg/100μl DTT which had no effect on its own (N=6, data not shown). DMSO (1%), the vehicle used to dissolve DTNB, had no effect on PWLs (Figure 4A) and did not interfere with the effects of the reducing agents (N=6, data not shown).

Mibefradil overcomes the effects of reducing agents both in vivo and in vitro. The results described above suggest that redox agents modulate peripheral thermal nociception via effects on T-type Ca²⁺ channels in primary sensory neurons. To test this further, we used mibefradil, a peripherally acting antihypertensive drug that has been shown to block T-type Ca²⁺ currents preferentially over HVA currents in vascular smooth muscle (Ertel, S. I. and Clozel, J-P. (1997). Mibefradil (Ro 40- 5967): the first selective T-type Ca²⁺ channel blocker. Exp. Opin. Invest. Drugs 6, 569-582;_Mibefradil (Ro 40-5967): the first selective T-type Ca²⁺ channel blocker. Exp. Opin. Invest. Drugs 6, 569-582, Ertel, E.A. and Clozel, J. (1997). T-type Ca²⁺ channels and pharmacological blockade: potential pathophysiological relevance. Cardiovascular Drugs and Therapy 11, 723-739) and cerebellar Purkinje cells (Magee, J. C. and Johnston, D. (1995). Synaptic activation of voltage-gated channels in the dendrites of hippocampal pyramidal neurons. Science 268, 301-304). In DRG neurons, we previously found that mibefradil is one of the most potent blockers of T-currents yet described with an IC₅₀ of 3 μM (Todorovic, S.M. and Lingle, C.J. (1998). Pharmacological properties of T-type Ca²⁺ current in adult rat sensory neurons: effects of anticonvulsants and anesthetic agents. J. Neurophysiol. 79, 240-252). In vivo, injection of 6 Dg/100Dl mibefradil into rat hind paws had no significant effect on thermal PWLs (N=12 animals, Figure 5A). We were unable to examine doses of mibefradil above 6 Dg/100 Dl because mibefradil injections caused hyperemia and local increases in skin temperature presumably due to local vasodilating effects at higher doses. However, co-injection of 6 Dg/100Dl mibefradil into hind paws with either L-cysteine or DTT completely blocked the thermal hyperalgesia induced by the reducing agents (Figure 5B and 5C, N=8 for L-cysteine and N=14 animals for DTT).

These results indicate that the idea that the hyperalgesic responses to reducing agents result from modulation of T-channels in nociceptors. However, we were surprised that mibefradil alone had no effect on PWLs yet blocked the effects of L-cysteine and DTT, given that DTNB had analgesic effects at higher doses and also inhibited DRG T-currents. This led us to examine interactions between mibefradil and redox agents in vitro. Figure 6A shows data from an experiment in which 100 μM L-cysteine enhanced DRG T-currents about 2-fold and 1 μM mibefradil abolished the cysteine-enhanced portion of the current (55 % block of the total T-current). Also 0.3 μM mibefradil alone did not block baseline T-currents, but, when coapplied with L- cysteine, it blocked about 66% of L-cysteine enhanced and 28% of total T-current. This suggests that reduced T-channels may have increased affinity for mibefradil. Indeed, we found that L-cysteine shifted mibefradil's IC₅₀ for block of T-currents from 3 μM (Todorovic, S.M. and Lingle, C.J. (1998). Pharmacological properties of T-type Ca²⁺ current in adult rat sensory neurons: effects of anticonvulsants and anesthetic agents. J. Neurophysiol. 79, 240-252). to 0.7 μM (Figure 6C).

We also examined the effect of DTNB on the T-channel blocking actions of mibefradil. Figure 6B shows an experiment in which a subthreshold concentration of DTNB (0.2 mM) enhanced the block of T-currents by 1 μM mibefradil by about 2-fold. On average, 1 μM mibefradil alone blocked 23.7 ± 1.5 %, while the combination of 1 μM mibefradil and 0.2 mM DTNB blocked 49.2 ± 1.1 % of the current (p<0.001, N=4). Consistent with this, 0.2 mM DTNB shifted the mibefradil IC₅o to 1.1 μM (Figure 6C).

In vivo, we found that a combination of ineffective doses of DTNB and mibefradil induced analgesia as indicated by an increase in thermal PWLs (Figure 6D) prolonging PWLs by 2.5 sec ten minutes after injection (N=12, p<0.001). This analgesic effect of DTNB plus mibefradil in vivo mirrors the synergistic blocking effect of the two agents on T-currents in vitro.

Reducing agents modulate mechanical nociception. The majority of small size sensory neurons are polymodal nociceptors that respond to a variety of mechanical, thermal and chemical stimuli. If T-type channels serve as a general amplifier of peripheral nociception, they might be involved in pain sensation generated through other modalities. We therefore tested the possibility that mechanical sensation could also be modulated by redox agents injected in peripheral receptive fields of sensory neurons. We first determined the size of von Frey filament (4.93) which when applied to the ventral side of the hind paw causes nociceptive behavior (measured as the number of paw withdrawals per 10 trials). These withdrawal scores were compared in injected and non-injected paws before (baseline and 0 minutes), 10 and 60 minutes following injections of 100 μl of redox agents in hind paws. Figure 7A indicates that injections of saline or 1% DMSO did not cause any change in paw withdrawal scores. However, injection of 12 μg/100 μl L-cysteine in saline caused a reversible hyperalgesic response (Figure 7B) increasing the withdrawal scores by 2.6 fold after 10 minutes (p<0.00001 , N=8 animals). When 40 μg/100 μl of DTNB was injected (Figure 7C) withdrawal scores after 10 minutes indicated an analgesic effect (60 % less than control, p<0.005, N=12 animals). A lower dose of DTNB (4 μg/100 μl) had little effect on its own but completely reversed the hyperalgesic effect of L-cysteine (Figure 7D, N=8). Similarly, a lower dose of L-cysteine completely reversed the analgesic effects of DTNB (N=8, data not shown). These data indicate that redox agents modulate peripheral mechanical nociception as well as thermal nociception.

Discussion

Mechanisms of T-type Ca²⁺ current modulation by redox agents.

We provide data indicating that the extracellular reducing agents, DTT and L- cysteine, augment T-currents in rat sensory neurons. The oxidizing agent, DTNB, which is also relatively membrane impermeant (Aizenman, E., Lipton, S.A. and Loring, R.H. (1989). Selective modulation of NMDA responses by reduction and oxidation. Neuron 2, 1257-1263; Tang, L. and Aizenman, E. (1993). The modulation of N-methyl-d-aspartate receptors by redox and alkylating reagents in rat cortical neurons in vitro. J. Physiol. (London), 465, 303-323) blocks T-currents on its own at higher concentrations, and, at lower concentrations, reverses the effects and speeds recovery from the effects of the reducing agents. Reducing agents produce prominent changes in T-current kinetics as evidenced by acceleration of both activation and inactivation. In contrast, DTNB had little effect on current kinetics. Furthermore, spontaneous recovery from the effect of DTNB was faster in both in vitro and in vivo experiments than recovery from the effects of the reducing agents.

Putative endogenous redox modulators in rat sensory neurons. The examples demonstrate that the endogenous amino acid, L-cysteine, acts as a potent redox modulator of T-channels in DRG neurons. In the plasma of healthy human subjects, free L-cysteine concentrations are reported to be as high as 140 μM (Suliman, M. E., Anderstam, B., Lindholm, B. and Bergstrom, J. (1997). Total, free, and protein-bound sulfur amino acids in uremic patients. Nephrol. Dial. Transplant, 12, 2332-2338). Because significant enhancement of T-currents in sensory neurons occurs with L-cysteine concentrations in the range 30-100 μM, it appears that L- cysteine could be an important endogenous modulator of T-currents. Although the concentrations of amino acids in the extracellular milieu of nociceptive endings is unknown, it is possible that inflammation, burns, tissue hematoma or trauma result in plasma extravasation and local accumulation of L-cysteine and other thiol-containing amino acids in proximity to putative nociceptive endings. We show here that direct injection of L-cysteine into peripheral receptive fields induces hyperalgesia to thermal and mechanical stimuli.

The effects of mibefradil on redox modulators of T-type Ca²⁺ channels.

The examples show that concentrations of mibefradil thought to be selective for T- currents completely block L-cysteine and DTT-enhanced T-currents. These results also demonstrate that among the calcium channels in sensory neurons, T-channels are selectively and reversibly augmented by reducing agents and that reducing agents produce hyperalgesia in response to thermal stimulation.

FIGURE LEGENDS

Figure 1. DTT selectively modulates T-type Ca²⁺ currents but not other voltage- and ligand-gated currents in acutely dissociated DRG neurons.

A: The traces depict results from an experiment in which a double-pulse protocol was used to record T-type (LVA) and HVA Ca²⁺ currents in a DRG cell. Note that

0.1 mM DTT selectively increased the T-current about 2-fold while the HVA current was not affected. The amplitude of T-currents was measured as the difference from the peak of the inward current to the current remaining at the end of a 200 ms test step to avoid contamination by a small residual HVA component. Note acceleration of current activation and inactivation kinetics during application of DTT.

B: The trace shows the lack of effect of 1 mM DTT on isolated HVA Ca²⁺ currents.

Bars indicate calibration.

C: The raw traces show the lack of effect of 4 mM DTT upon voltage-gated Na⁺ currents in another DRG cell. These currents were obtained by holding the neuron at -60 mV and stepping to 0 mV. Traces in the absence and presence of DTT are overlaid.

D: These panels depict a family of outward potassium currents elicited from a holding potential of -60 mV to test potentials of 0 to +60 mV under control conditions

(left) and in the presence of 3 mM DTT (right). Note the lack of effects on current kinetics and amplitudes.

E: Small sensory neurons are sensitive to 1μM capsaicin, heat, protons and 100 μM

ATP which induce inward currents at -60 mV. Representative traces are shown for each current on the left, horizontal bars indicate times of application. Baseline pH was 7.4 in experiments with proton-gated currents. The bar graph on the right summarizes that DTT had little effect on any of these currents. For capsaicin-, proton- and ATP-gated currents 1-3 mM DTT was coapplied with second application of ligand. In the case of ATP, we waited for 1 minute between applications to allow full recovery of responses (Cook, S.P. and McCleskey, E.W. (1997). Desenzitization, recovery and Ca²⁺-dependent modulation of ATP-gated P2X receptors in nociceptors. Neuropharmacology 36, 1303-1308). For heat-gated currents we measured control responses first and then incubated approximately 1-2 mM DTT from 3-15 minutes in the bath before subsequent application of heat. Both direct application of DTT and bath incubation with DTT result in augmentation of T- currents in our experiments.

Figure 2. Characterization of redox modulation of T-type Ca²⁺ currents in rat sensory neurons and Ca_v3.2 currents in HEK cells.

A: Raw traces from an experiment showing that DTNB blocks T-currents in rat DRG cells without affecting current kinetics but reverses DTT-induced kinetic changes of T-currents. Currents at -55 and -40 mV are depicted here. The 10-90% rise time at -40 mVfor control was 12.8 ms, 9.6 ms in the presence of 0.1 mM DTT and 13.6 ms in the presence of DTNB which was applied immediately after DTT. Inactivation t at - 40 mV for control was 53.5 ms, for DTT 35.3 ms and for DTNB 50 ms.

B: DTNB (1 mM and 3 mM) induced a fully reversible blockade of T-currents currents in DRG cells. Drug applications are denoted by the horizontal bars. C: L-cysteine mimicked the effects of DTT in sensory neurons. The traces show results from an experiment in which T-currents were evoked from a V_h of -90 mV to V_t -35 mV. One hundred μM L-cysteine increased the peak T-current about 2-fold and increased the speed of current activation and inactivation as evidenced by crossover of traces prior, during and after application of L-cysteine. Bars indicate calibration.

D: This graph displays results from experiments in which 0.1 mM DTNB increased the rate of recovery from the effects of reducing agent L-cysteine on T-currents. Bars indicate times of drug applications. E: One mM DTNB blocked about 60% of the peak current in HEK cells expressing Cav3.2 without obvious changes in current kinetics. DTT (0.1 mM) in another HEK cell increased the peak Cav3.2 current 2.5 fold and accelerated both activation and inactivation of current. 10-90% time rise was 9.25 and 6 sec, inactivation t was 20.5 and 11.7 sec before and during application of 0.1 mM DTT, respectively. F: The time course from another experiment in Cav3.2 -transfected HEK cells which illustrates that DTNB accelerates recovery from the fully reduced state 80 vs. 180 sec with and without DTNB, respectively).

Figure 3. Reducing agents induce thermal hyperalgesia in adult rats.

A: Injections of saline, the vehicle for DTT and L-cysteine, had no effect on PWLs. B indicates baseline PWL obtained 2 days before study and pretreatment indicates value just prior to test injection.

B: L-cysteine induced a dose-dependent decrease of thermal PWLs. Four (open triangles), 12 (filled triangles) and 120 μg/100 μl (open squares) L-cysteine significantly decreased PWLs ((*) p<0.005) 10 and 20 minutes post injection when compared with non-injected paws. PWLs return to control values by 60 minutes following injection.

C: DTT, at 15 and 150 μg/100 μl, also produced a dose-dependent decrease of

PWLs; (^*) p< 0.05, injected vs. non-injected paw. Note that the effects of L-cysteine and DTT are fully reversible.

D: MK-801 (0.35 μg/100μll) failed to block the hyperalgesia induced by 12 μg/100 μl of L-cysteine. (*) p< 0.05 injected vs. non-injected paw.

Figure 4. DTNB induces analgesia in thermal PWL testing and blocks hyperalgesia induced by reducing agents

A: One percent DMSO, the vehicle used for DTNB, has no effect on PWLs when administered alone. All points are averages of at least 8 animals and vertical bars indicate ± SE. B: The oxidizing agent, DTNB, induced a brief dose-dependent analgesia. (*) p<

0.0005, injected vs. non-injected paws with full recovery after 20 minutes from injection.

C: At 4 μg/100 μl, DTNB had no effect on PWL alone. However, this dose completely reversed L-cysteine-induced thermal hyperalgesia.

D: Similarly, 4 μg/100 μl DTNB also blocked the effects of DTT on PWLs. There was no statistical difference between injected and non-injected paws in panels C and D.

Dotted lines in panels C and D are taken from Figure 3 B and C, respectively.

Figure 5. Mibefradil blocks the effects of reducing agents in vivo.

A: Mibefradil, at a dose of 6 μg/100 μl (filled circles), had a small but non-significant effect on PWLs under control conditions (open circles) (N=12 animals). B: Hyperalgesic responses to 12 μg/100 μl L-cysteine (dashed trace, see Figure 3B) were completely blocked when 6 μg/μDl mibefradil was co-injected with L-cysteine. C: In another experiment in vivo, 6 μg/100 μl mibefradil abolished the hyperalgesic response to 150 μg/100 μl DTT (dashed trace, see Figure 3C). No statistically significant differences were observed between injected paws (solid circles) and non- injected paws (N=14 animals).

Figure 6. Redox agents augment the effects of mibefradil in vitro and in vivo.

A: The graph shows the time course of mibefradil's blocking effect on L-cysteine modulated-current from a DRG cell. The horizontal solid bar indicates the time of L- cysteine application, dotted bars indicate the times of mibefradil applications. One hundred μM L-cysteine increased the peak T-current by about 2-fold in this cell. One μM mibefradil completely blocked the L-cysteine potentiated-current and 3 μM mibefradil blocked the total T-current by > 80%. Note that 0.3 μM mibefradil applied before and after L-cysteine did not produce any inhibition of peak T-current. However, when coapplied with L-cysteine, it blocked about one third of the total current.

B: DTNB potentiates the blocking effect of mibefradil. The graph shows the time course of an experiment in which DTNB alone (dotted horizontal bar) had little effect on the baseline T-current. μDM mibefradil (solid horizontal bar) blocked about 23% of the current. When DTNB (dotted bar) was coapplied with mibefradil the magnitude of block increased to about 50%.

C: DTNB induces a 3-fold shift and L-cysteine induces a 4-fold shift to the left of the mibefradil concentration-response curve in acutely dissociated DRG neurons. The dotted line is a concentration-response curve to mibefradil obtained with identical recording conditions in these cells with an IC₅₀ of 3 μM and Hill n of 1.28 (Todorovic, S.M. and Lingle, C.J. (1998). Pharmacological properties of T-type Ca²⁺ current in adult rat sensory neurons: effects of anticonvulsants and anesthetic agents. J. Neurophysiol. 79, 240-252). All points are averages of at least 4 μM and n of 1.04 ± 0.2 in the presence of 200 μM DTNB. Mibefradil had an IC₅₀ of 0.7 ± 0.07 μM and n of 1.24 ± 0.14 for T-current blockade in the presence of 100 μM L-cysteine D: Mibefradil (6 μg/100 μl), in combination with an ineffective dose of DTNB (4 μg/100 μl, filled circles), produced an analgesic effect. Thermal PWL's were significantly prolonged 10 minutes after injection from 12.1 ± 0.5 s to 14.7 ± 0.8 s (N=12 animals, p<0.005).

Figure 7. Redox agents modulate peripheral mechanical nociception.

A. Injections of saline or 1% DMSO did not change paw withdrawal scores in animals tested with a 4.93 von Frey filament (N=8 for each condition, control (non- injected) sides are grouped together.

B. L-cysteine (12 μg/100 μL) induced an increase in the withdrawal score (injected vs. non-injected side) 10 minutes after injection. Note a full return of the response to baseline at 60 minute μg/100 μl) was injected in peripheral receptive fields, fewer paw withdrawals were measured to filament stimulation 10 minutes after injection indicating an analgesic response. * indicates p<0.005, N=12 animals.

When a lower dose of DTNB (4 μg/100 μL) was co-injected with L-cysteine (12 μg/100 μl), the hyperalgesic response to mechanical stimulation typically observed with L-cysteine was completely blocked. CONCLUSION

In light of the detailed description of the invention and the examples presented, it can be appreciated that the several aspects of the invention are achieved.

It is to be understood that the present invention has been described in detail by way of illustration and example in order to acquaint others skilled in the art with the invention, its principles, and its practical application. Particular formulations and processes of the present invention are not limited to the descriptions of the specific embodiments presented, but rather the descriptions and examples should be viewed in terms of the claims that follow and their equivalents, while some of the examples and descriptions above include some conclusions about the way the invention may function, the inventors do not intend to be bound by those conclusions and functions, but puts them forth only as possible explanations.

It is to be further understood that the specific embodiments of the present invention as set forth are not intended as being exhaustive or limiting of the invention, and that many alternatives, modifications, and variations will be apparent to those of ordinary skill in the art in light of the foregoing examples and detailed description. Accordingly, this invention is intended to embrace all such alternatives, modifications, and variations that fall with the spirit and scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. ^"A method for the treatment, inhibition or prevention of pain perception in a subject in need thereof comprising administering to the subject an amount of an inhibitor of T-type Ca2+ channel activity or a pharmaceutically acceptable salt or prodrug thereof effective to treat, inhibit or prevent pain perception in the subject.

2. The method of claim 1 wherein the inhibitor comprises an oxidizing agent.

3. The method of claim 2 wherein the oxidizing agent comprises DTNB.

4. The method of claim 1 wherein the inhibitor comprises a T-type Ca2+ channel blocker.

5. The method of claim 4 wherein the inhibitor comprises mibefradil.

6. The method of claim 2 wherein a T-type Ca2+ channel blocker is administered to the subject in addition to the oxidizing agent.

7. The method of claim 2 wherein the oxidizing agent and channel blocker are administered substantially simultaneously.

8. The method of claim 2 wherein the oxidizing agent and channel blocker are administered sequentially.

9. The method of claim 1 wherein the pain perception which is treated, inhibited or prevented is induced by thermal or mechanical nociceptive inputs.

10. The method of claim 1 wherein the pain perception which is treated, inhibited or prevented is a form of chronic pain.

11. The method of claim 1 wherein the inhibitor is administered systemically.

12. The method of claim 1 wherein the inhibitor is administered locally.

13. The method of claim 1 wherein the inhibitor is administered orally, parenterally, by inhalation, rectally, intradermally, transdermally or topically.

14. The method of claim 1 wherein the inhibitor is an endogenous inhibitor whose concentration is upregulated to treat, inhibit or prevent pain perception.

15. The method of claim 1 wherein the treatment, inhibition or prevention of pain perception is achieved at least in part by substantial inhibition of T-type Ca2+ channel peripheral nociceptive transmission.

16. The method of claim 1 wherein the subject is a zoo mammal, a domestic livestock mammal or a laboratory mammal species.

17. The method of claim 1 wherein the subject is a companion animal.

18. The method of claim 1 wherein the subject is a human.

19. The method of claim 1 wherein the T-type Ca2+ channel inhibitor is in single dose form.

20. The method of claim 15 wherein the single dose form comprises about 0.1 to about 2000 mg of T-type Ca2+ channel inhibitor.

21. The method of claim 15 wherein the single dose form comprises about 1 to about 200 mg of T-type Ca2+ channel inhibitor.

22. A method for the treatment, inhibition or prevention of insufficient pain perception in a subject in need of such treatment comprising administering to the subject a pain sensitizing amount of an enhancer of T-type Ca2+ channel activity or a pharmaceutically acceptable salt or prodrug thereof effective to treat, inhibit or prevent insufficient pain perception in the subject.

23. The method of claim 22 wherein the enhancer comprises a reducing agent.

24. The method of claim 23 wherein the reducing agent is selected from the group consisting of DTT, L-cysteine, L-homocysteine and glutathione.

25. The method of claim 24 wherein the reducing agent comprises DTT.

26. The method of claim 24 wherein the reducing agent comprises L- cysteine.

27. The method of claim 22 wherein the insufficient pain perception which is treated, inhibited or prevented is induced by thermal or mechanical nociceptive inputs.

28. The method of claim 22 wherein the enhancer is administered system ically.

29. The method of claim 22 wherein the enhancer is administered locally.

30. The method of claim 22 wherein the enhancer is administered orally, parenterally, by inhalation, rectally, intradermally, transdermally or topically.

31. The method of claim 22 wherein the enhancer is an endogenous enhancer whose concentration is upregulated to treat, inhibit or prevent insufficient pain perception.

32. The method of claim 22 wherein the treatment, inhibition or prevention of insufficient pain perception is achieved at least in part by substantial potentiation of T-type Ca2+ channel peripheral nociceptive transmission.

33. The method of claim 22 wherein the subject is a zoo mammal, a domestic livestock mammal or a laboratory mammal species.

34. The method of claim 22 wherein the subject is a companion animal.

35. The method of claim 22 wherein the subject is a human.

36. The method of claim 22 wherein the T-type Ca2+ channel enhancer is in single dose form.

37. The method of claim 36 wherein the single dose form comprises about 0.1 to about 2000 mg of T-type Ca2+ channel enhancer.

38. The method of claim 36 wherein the single dose form comprises about 1 to about 200 mg of T-type Ca2+ channel enhancer.

39. A method for the potentiation of a T-type Ca2+ channel in a subject in need of such potentiation or in a tissue comprising a T-type Ca2+ channel, the method comprising administering to the subject or tissue an amount of a reducing agent or a pharmaceutically acceptable salt or prodrug thereof effective to potentiate the T-type Ca2+ channel.

40. The method of claim 39 wherein the tissue comprising the T-type Ca2+ channel is selected from one or more of the group including central neural tissue, peripheral neural tissue, enteric neural tissue, muscle, including cardiac and vascular smooth muscle, kidney, liver and cells of the immune system.

41. A method for the inhibition of a T-type Ca2+ channel in a subject in need of such inhibition or in a tissue comprising a T-type Ca2+ channel, the method comprising administering to the subject or tissue an amount of an oxidizing agent or a pharmaceutically acceptable salt or prodrug thereof effective to inhibit the T-type Ca2+ channel.

42. The method of claim 41 wherein the tissue comprising the T-type Ca2+ channel is selected from one or more of the group including central neural tissue, peripheral neural tissue, enteric neural tissue, muscle, including cardiac and vascular smooth muscle, kidney, liver and cells of the immune system.

43. A method for screening a candidate pain perception modifying agent comprising administering the candidate to an expression system comprising a T- type Ca2+ channel or its active fragment, derivative, analog or mimic and determining whether the channel, fragment, derivative, analog or mimic's activity is thereby modified.

44. A pharmaceutical composition useful for the modulation of T-type Ca2+ channel activity in unit dosage form comprising at least one T-type Ca2+ channel selective inhibitor and a pharmaceutically acceptable carrier.

45. The pharmaceutical composition of claim 44 wherein the at least one inhibitor comprises an oxidizing agent.

46. The pharmaceutical composition of claim 44 wherein the at least one inhibitor comprises a T-type Ca2+ channel blocker.

47. The pharmaceutical composition of claim 44 wherein the at least one inhibitor comprises DNTB and mibefradil.

48. A pharmaceutical composition useful for the modulation of T-type Ca2+ channel activity in unit dosage form comprising at least one T-type Ca2+ channel selective enhancer and a pharmaceutically acceptable carrier.

49. The pharmaceutical composition of claim 48 wherein the at least one T- type Ca2+ channel selective enhancer comprises a reducing agent.

50. The pharmaceutical composition of claim 49 wherein the reducing agent is selected from the group consisting of DTT, L-cysteine, L-homocysteine, and glutathione.

51. The pharmaceutical composition of claim 50 wherein the reducing agent comprises DTT.

52. The pharmaceutical composition of claim 50 wherein the reducing agent comprises L-cysteine.

53. A method of producing an analgesic effect in a subject in need of such treatment comprising administering to the patient an analgesic effective amount of an oxidizing agent.

54. The method of claim 48 wherein the oxidizing agent comprises DTNB.

55. The method of claim 53 wherein the analgesic effect is an effect responsive to thermal hyperalgesia.

56. The method of claim 53 wherein the analgesic effect is an effect responsive to mechanical hyperalgesia.

57. A method of producing a sensory enhancing effect in a subject in need of such treatment comprising administering to the patient a sensory enhancing effective amount of a reducing agent.

58. The method of claim 57 wherein the reducing agent comprises DTT or L- cysteine.

59. The method of claim 57 wherein the sensory enhancing effect is thermal hyperalgesia.

60. The method of claim 57 wherein the sensory enhancing effect is mechanical hyperalgesia.