Related Applications This application claims priority to United States application serial no. 60/032,273, filed December 2, 1996, the entire contents of which are incorporated herein by reference.

Field of the Invention This invention relates to methods for the treatment of pain and, in particular, to the alleviation of chronic pain and its varieties, e.g., neuropathic pain, and acute persistent pain by administration of an N-butyl tetracaine and/or an N-P-phenylethyl lidocaine.

Background of the Invention The use of long-acting local anesthetics that elicit complete neural blockage for more than several hours frequently is desirable in the management of chronic and acute pain. In general, chronic pain is associated with a known tissue pathology (e.g., cancer pain, arthritic pain) and may be associated with inflammation of an injured body tissue (e.g., surgery).

Neuropathic pain is thought to be a consequence of damage to peripheral nerves or to regions of the central nervous system. Neuropathic pain frequently occurs as a form of chronic pain, although neuropathic pain also can present as an acute pain.

Pain relief research during the last two decades has focused on the identification of new local anesthetics to produce analgesia of long duration with minimal impairment of autonomic function and low toxicity. Two of the best known "long-acting" local anesthetics developed to date, bupivacaine and etidocaine, reportedly block major nerve block for three to twelve hours.

Unfortunately, each of these local anesthetics also is highly cardiotoxic. The development of alternative "long-acting" local anesthetics has met with limited success.

Pain relief research also has focussed on the identification of new neurolytic agents for the treatment of chronic pain and intractable cancer pain. Historically, spinal opiate administration, surgical intervention, or both have been used to alleviate chronic and intractable cancer pain. When these methods fail or provide insufficient pain relief phenol or absolute alcohol reportedly have been used as neurolytic agents to destroy the pathogenic nerve regions that are responsible for pain manifestation. However, these agents exert only weak local

anesthetic effects and, accordingly, have been difficult to administer to alert patients without inducing additional pain. To date, a long-acting local anesthetic with a neurolytic activity has not been available for the treatment of chronic and intractable cancer pain.

In view of the foregoing limitations of the existing local anesthetics to prolong the duration of anesthesia, a need still exists to develop new and useful long-acting local anesthetics and neurolytic agents for pain management. Preferably such long-acting anesthetics also will exhibit neurolytic properties. Such novel drugs would be useful and desirable, for example, for treating postoperative analgesia, intractable cancer pain and chronic pain. Preferably, such agents would have sufficient potency to permit administration of a single, relatively low dosage of the agent, thereby minimizing the likelihood of side reactions that have been attributed to the existing local anesthetic agents.

Summarv of the Invention The invention involves in one respect the discovery of two novel long-acting local anesthetics that are useful for pain management: an N-butyl tetracaine and a N-P-phenyl ethyl lidocaine. The methods for preparing the preferred compounds of the invention are provided in the Examples and are summarized below. These anesthetics exhibit unexpectedly improved analgesic properties compared to related compounds that previously have been used for pain management. Although not intending to be bound to any particular theory or mechanism, it is believed that the anesthetics disclosed herein act by blocking voltage-gated sodium channels and inhibiting the propagation of action potentials in excitable membranes. The availability of neurolytic compounds with strong local anesthetic properties, such as N-butyl tetracaine described herein, are advantageous over the existing compounds for pain management because conventional neurolytic agents typically exert only weak local anesthetic effects or none at all.

Thus, as used herein, a "neurolytic agent" refers to a chemical that is capable of irreversibly damaging the axonal membranes, i.e., these agents are locally neurotoxic; A "local anesthetic" refers to a chemical that is capable of inhibiting the propagation of action potentials in excitable membranes. N-butyl tetracaine is a long-acting anesthetic that also displays neurolytic properties at concentrations 2 1%.

According to one aspect of the invention, a composition containing an N-butyl tetracaine is provided. The preferred N-butyl tetracaine derivative is illustrated in Figure 1. Preferably, the composition further includes a pharmaceutically acceptable carrier and the N-butyl tetracaine is present in the composition in a therapeutically effective amount. In general, a therapeutically

effective amount is that amount necessary to alleviate pain in a subject. More preferably the pharmaceutically acceptable carrier is a pharmaceutically acceptable topical or intrathecal carrier.

In some embodiments, the pharmaceutically acceptable carrier is a topical or intrathecal carrier that is not suitable for oral delivery to the subject.

According to yet another aspect of the invention, a composition containing N- - phenylethyl lidocaine (also referred to herein as a "tonicaine") is provided. The preferred tonicaine is illustrated in Figure 2. Preferably, the composition further includes a pharmaceutically acceptable carrier and the tonicaine is present in the composition in a therapeutically effective amount. More preferably, the pharmaceutically acceptable carrier is a pharmaceutically acceptable topical or intrathecal carrier. In some embodiments, the pharmaceutically acceptable carrier is a topical or intrathecal carrier that is not suitable for oral delivery to the subject.

According to yet another aspect of the invention, a method for alleviating pain (e.g., a localized pain) in a subject is provided. The method involves administering to the subject a therapeutically effective amount of the above-described N-butyl tetracaine and/or the above- described tonicaine, alone or together with another therapeutically useful ingredient. The subject is a mammal that is either exhibiting pain or is about to be subjected to a pain-causing event. In general, the subject to whom the compounds of the invention are administered is diagnosed as having a localized pain that is attributable to a condition selected from the group consisting of phantom pain, rheumatoid arthritis or osteoarthritis, post-operative analgesia, intractable cancer pain, chronic pain, shingles, and painful diabetic neuropathy. In particular, the method for alleviating pain is used to treat a subject who is diagnosed as having localized pain that is a nociceptor-mediated pain. Thus, the method of this invention can be applied to alleviate chronic pain (e.g., chronic neuropathic) as well as to alleviate acute pain (e.g., inflammatory pain resulting from surgery, injuries, bronchial asthma, and so forth). In addition, the method of the invention can be applied to the treatment of pain that is associated with a skin condition such as psoriasis, seborrheic dermatitis and eczema, and shingles (Herpes zoster).

According to still another aspect of the invention, the novel intermediate compounds used in the preparation of the above-described compounds of the invention and the methods for making and using these intermediate compounds to formulate the above-described compounds of the invention are provided. The intermediates and related methods for making and using such compounds are provided in the Examples.

According to yet another aspect of the invention, a method for making a medicament for alleviating pain is provided. The method involves placing at least one of the above-identified compounds of the invention is a pharmaceutically acceptable carrier.

These and other aspects of the invention, as well as various advantages and utilities will be more apparent with reference to the drawings and the detailed description of the preferred embodiments.

All references, patents, and patent publications identified in this document are incorporated in their entirety herein by reference.

Brief Description of the Drawings Figure 1 shows the chemical formula for the preferred N-butyl tetracaine compound of the invention; and Figure 2 shows the chemical structure for the preferred N- -phenyl ethyl lidocaine compound of the invention.

Figure 3 shows the chemical structure of tetracaine and its derivatives.

Detailed Description of the Invention The present invention in one aspect involves the discovery of two novel long-acting local anesthetics for pain management: an N-butyl tetracaine and an N-P-phenylethyl lidocaine.

Surprisingly, the in vitro and in vivo activities of the compounds of the invention are significantly greater than the activities of the base compounds, tetracaine and lidocaine. (See the Example and Wang, G.K., et al., Anesthesiology 83 (6): 1293-1301 (1995)) In particular, the potency of the compounds of the invention in blocking sodium channels is vastly improved over the local anesthetics that are known in the art. This substantial increase in activity could not have been predicted based upon structural similarity to the base compounds or to known analogs of the base compounds. Thus, one skilled in the art would not have expected the substantially improved activities of the compounds of the invention compared to the base compounds and derivatized base compounds known in the prior art.

Applicants' discovery represents the first identification of compounds which exhibit both local anesthetic and neurolytic properties. In particular, the results presented herein demonstrate that the novel compounds disclosed herein elicit sensory and motor block properties that are vastly improved compared to the properties of the existing local anesthetics. Accordingly, it is believed that the compounds of the invention are particularly useful for alleviating chronic and intractable cancer pain, phantom pain, rheumatoid arthritis, bronchial asthma, shingles, and

various neuropathic pain. In addition, the compounds of the invention are believed to be useful for alleviating topical pain that is, for example, associated with a skin condition such as psoriasis seborrheic dermatitis, eczema, shingles, and post-operative pain.

According to one aspect of the invention, a composition including an N-butyl tetracaine is provided. Preferably, the composition further includes a pharmaceutically acceptable carrier and the N-butyl tetracaine is present in the composition in a therapeutically effective amount. As used herein, an "N-butyl tetracaine" refers to the compound having the chemical structure shown in Figure 1. Thus, the N-butyl tetracaines of the invention include the particularly preferred N- butyl tetracaine (also referred to as an N-butyl tetracaine quaternary ammonium bromide) shown in Figure 1, as well as derivatives of this N-butyl tetracaine chemical structure. Exemplary N- butyl tetracaine derivatives include N-pentyl and N-hexyl tetracaine (i.e., N-pentyl and N-hexyl groups are substituted for the N-butyl group attached to the quaternary ammonium nitrogen), as well as other amphipathic tetracaine derivatives such as N--phenyl ethyl tetracaine (i.e., a phenyl ethyl group is substituted for the N-butyl group attached to the quaternary ammonium nitrogen). Other groups that can be substituted for this N-butyl include: an alkyl, aryl or cyclic functional group containing from four to twelve carbon atoms (e.g., heptyl-, octyl-, nonyl-, decyl-, aryl-, cyclopentyl-, cyclohexyl-, propylphenyl-, butyl-phenyl-, hexylphenyl-), provided that the derivative compound is amphipathic. The foregoing chemical terms have their common meaning known to one of ordinary skill in the art. The above-noted functional groups that can be used in place of N-butyl can be saturated or unsaturated, straight-chained or branched.

Optionally, one or more hydrogen atoms of the functional group can be replaced by a substituent group, such as a chloride, an amino group or a thio group.

The particularly preferred N-butyl tetracaine is a quaternary ammonium salt that has been synthesized from a tetracaine base (Sigma chemical company, St. Louis, MO) and l-bromo butane (Aldrich, Milwaukee, WI) in accordance with the procedure provided in the Example (see, also, Wang, G. K. et al., Biophys. J. 67:1851-1860(1994)). Of course, other salts of this compound and other N-butyl tetracaines alternatively can be used for practicing the invention.

For example, in addition to the bromide salt, chloride, phosphate, sulfate, citrate. and acetate salts can be used. In the preferred embodiments, bromide is the counter ion for this positively charged quaternary ammonium compound.

The ability of the preferred N-butyl tetracaine to elicit sciatic nerve block of sensory and motor functions in vivo was tested in rats by injecting a single dose of 0.1 ml N-butyl tetracaine

at 37 mM into the sciatic notch. Transverse sections of the sciatic nerves subsequently were examined to determine the neurolytic effect of this agent and the local anesthetic properties of N-butyl tetracaine were studied in vitro, both by tonic inhibition and use-dependent inhibition of sodium currents in neuronal GH3 cells under whole-cell voltage-clamp conditions. The results of these experiments are shown in the Example. Briefly, N-butyl tetracaine at 37 mM elicited prolonged sciatic nerve block of the withdrawal response to noxious pinch in rats for more than two weeks with this withdrawal response being fully restored after nine weeks. Motor functions of the hind legs were similarly blocked by injection of this N-butyl tetracaine. Morphological examinations at three and five weeks after a single injection of this drug revealed degeneration of many sciatic nerve fibers and were consistent with the results of the functional tests. The results of the Example demonstrate that N-butyl tetracaine exhibits the requisite local anesthetic and neurolytic properties to render this agent particularly useful as a long-acting local anesthetic for pain management in humans. In particular, the results demonstrate that N-butyl tetracaine is a potent sodium channel blocker in vitro and produced strong tonic and use-dependent inhibition of sodium channels in vitro.

The animal model used in the Example is illustrative of an early stage of neuropathic pain and is, in particular, predictive of neuropathic and chronic pain in humans. The results obtained using this animal model demonstrate that the nociceptive functions of rat sciatic nerves were completely blocked for 14 days following injection of 37 mM N-butyl tetracaine with full functional recovery occurring after 60 days. Since similar symptoms occur in humans months and years after the original injury, these animal model experiments also are predictive of chronic pain conditions and can be used to predict the efficacy of the compounds of the invention in alleviating chronic as well as acute pain in humans. These results suggest a particularly preferred utility of N-butyl tetracaine for pain management in the central nervous system, where myelinated nerve fibers do not regenerate to the extent documented in the peripheral nervous system. In addition, these results suggest that N-butyl tetracaine can relieve pain in the central nervous system for as long as phenol or absolute alcohol and that N-butyl tetracaine appears to display significantly stronger local anesthetic properties in vivo compared to these conventional drugs. As a result, intrathecal delivery of N-butyl tetracaine is not accompanied by the sharp burning sensation associated with intrathecal delivery of conventional agents such as phenol or alcohol. Thus, a composition containing a therapeutically effective amount of N-butyl tetracaine

in a pharmaceutically acceptable intrathecal carrier represents a particularly preferred aspect of the invention.

According to yet another aspect of the invention, an alternative long-acting local anesthetic, namely, N- -phenyl ethyl lidocaine, is provided. Preferably, the composition further includes a pharmaceutically acceptable carrier and the tonicaine is present in the composition in a therapeutically effective amount. As used herein, a "tonicaine" refers to the compound having the chemical structure shown in Figure 2. Thus, the tonicaines of the invention include the particularly preferred tonicaine (also referred to as N- P-phenyl ethyl lidocaine quaternary ammonium bromide) shown in Figure 2, as well as derivatives of this tonicaine chemical structure. Exemplary tonicaine derivatives include N-P-aminophenyl ethyl lidocaine and N-p- chlorophenyl ethyl lidocaine and other amphipathic derivatives such as N-hexyl lidocaine.

Other groups that can be substituted for the phenylethyl group include: an alkyl, aryl or cyclic functional group containing from four to twelve carbon atoms (e.g., heptyl-, octyl-, nonyl-, decyl-, aryl-, cyclopentyl-, cyclohexyl-, propylphenyl-, butyl-phenyl-, hexylphenyl-), provided that the derivative compound is amphipathic. The above-noted functional groups that can be used in place of the N- -phenyl ethyl group can be saturated or unsaturated, straight-chained or branched. Optionally, one or more hydrogen atoms of the functional group can be replaced by a substituent group, such as a chloride, an amino group or a thio group.

The preferred tonicaine has the chemical structure shown in Figure 2. The method for synthesizing this tonicaine is provided in the Example. (See, also, Wang, G. I(., et al., <BR> <BR> <BR> <BR> Anesthesiology 83(6):1293-1301 (1995).) Briefly, this preferred tonicaine was synthesized from lidocaine (base) and (2-bromo ethyl) benzene. As noted above in reference to the N-butyl tetracaine derivative, various counter ions (e.g., phosphate, sulfate, citrate, acetate and so forth) can be used in place of bromide for synthesizing this compound.

The ability of the preferred tonicaine to inhibit sodium currents in cultured rat neuronal GH3 cells was tested in vitro under whole-cell voltage clamp conditions. Neurologic evaluations of sciatic nerve block of sensory and motor functions in vivo for tonicaine also were determined in rats. The results demonstrate that tonicaine is a potent sodium channel blocker in vitro and that tonicaine produced both tonic and use-dependent blocks of sodium currents that were significantly greater than the corresponding effects induced by the underivatized lidocaine base compound. In vivo, tonicaine elicited a prolonged and complete sciatic nerve block of motor function with a withdrawal response to noxious pinch that was significantly greater than that of

the corresponding lidocaine base compound. Administration of tonicaine achieved complete sciatic neural blockade for more than three hours with sensory blockage being prolonged to a greater extent than the motor blockade. These results demonstrate the particular advantages of tonicaine over existing local anesthetics in a clinical setting in which differential block of sensory and of motor function is desirable during regional anesthesia.

The compositions of the invention are useful for alleviating pain in a subject. As used herein, "alleviating pain" refers to treating a subject so as to remove existing pain as well as to suppress or inhibit pain which would otherwise ensue from a pain-causing event. When used therapeutically, the above-described compounds of the invention are administered in therapeutically effective amounts. In general, a therapeutically effective amount means that amount necessary to delay the onset of, inhibit the progression of, or halt altogether the particular condition being treated. The method of this invention can be applied to the treatment of chronic (e.g., neuropathic) pain as well as the acute pain (e.g., inflammatory pain) that can occur following trauma to body tissues, e.g., surgery, injury and so forth. Preferably, the condition being treated is a localized pain that is associated with, for example, postoperative analgesia, intractable cancer pain, chronic pain, bronchial asthma, shingles, phantom pain, rheumatoid arthritis, and painful diabetic neuropathy. As used herein, "localized pain" or "local pain" refers to sensory processes signaling tissue injury (nociceptor). In the preferred embodiments, the compounds of the invention are administered to a subject who is diagnosed as having a localized pain that is a nociceptor-mediated pain. Exemplary conditions that are manifested by a nociceptor-mediated pain include cutaneous, deep somatic or visceral pain.

In general, a therapeutically amount of the compounds of the invention will vary with the subject's age, condition, and sex, as well as the nature and extent of the disease in the subject, all of which can be determined by one of ordinary skill in the art. The dosage may be adjusted by the individual physician or veterinarian, particularly in the event of any complication. A therapeutically effective amount typically varies from 0.01 mg/kg to about 1000 mg/kg, preferably from about 0.1 mg/kg to about 200 mg/kg and most preferably from about 0.2 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or more days. The therapeutics of the invention are "nontoxic", i.e., the therapeutics have been approved by the United States Food and Drug Administration ("FDA") for administration to humans or, in keeping with established criteria, are susceptible to approval by the FDA for administration to humans.

The therapeutics of the invention can be administered to a patient immediately before the patient is subjected or exposed to a pain-causing event (i.e., as preemptive analgesia), or while the patient is experiencing pain. The therapeutics of the invention can be administered by any conventional route, including injection, gradual infusion over time, infiltration anesthesia, regional anesthesia, or spinal anesthesia. The administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intra cavity, subcutaneous, or transdermal. In the preferred embodiments, the compounds of the invention are administered in a pharmaceutically acceptable topical carrier or a pharmaceutically acceptable intrathecal carrier. Such pharmaceutically acceptable carriers include components which will not significantly impair the biological properties of the compounds of the invention. Those of skill in the art can readily determine the various parameters and conditions for producing such pharmaceutical preparations without resort to undue experimentation.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions. emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. As would be apparent to those of ordinary skill in the art, the compounds of the invention alternatively can be delivered using controlled release drug delivery systems. Preferably such systems are biodegradable and bioerodible. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. The compounds of the invention may also be used in combination with other therapeutic agents, for example, bronchodilators or anti-inflammatory agents. In general, the therapeutics of the invention are sterile preparations and can be sterilized, for example, by gamma irradiation.

In one particular embodiment, the preferred pharmaceutical composition is contained in an implant that is suitable for implantation into the mammalian recipient. Exemplary bioerodible implants that are useful in accordance with this method are described in PCT International application no. PCT/US/03307 (Publication No. WO 95/24929, entitled "Polymeric Gene Delivery System", claiming priority to U.S. patent application serial no. 213,668, filed March 15,

1994). PCT/US/03307 describes a biocompatible, preferably biodegradable polymeric matrix for sustained release of an exogenous agent in the patient. In accordance with the instant invention, the compositions described herein are encapsulated or dispersed within the biocompatable, preferably biodegradable polymeric matrix disclosed in PCT/US/03307. The polymeric matrix preferably is in the form of a micro particle such as a micro sphere (wherein the composition is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the composition is stored in the core of a polymeric shell). Other forms of the polymeric matrix for containing the composition include films, coatings, gels, implants, and stents. The size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix device is implanted. The size of the polymeric matrix devise further is selected according to the method of delivery which is to be used, typically injection into a tissue or administration of a suspension by aerosol into the nasal and/or pulmonary areas. The polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when the devise is administered to a mucosal or other surface. The matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time.

Both non-biodegradable and biodegradable polymeric matrices can be used to deliver the compositions of the invention to the subject. Biodegradable matrices are preferred. Such polymers may be natural or synthetic polymers. Synthetic polymers are preferred. The polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable. The polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross- linked with multi-valent ions or other polymers.

In general, the compositions of the invention are delivered using the bioerodible implant by way of diffusion, or more preferably, by degradation of the polymeric matrix. Exemplary synthetic polymers which can be used to form the biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose,

hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinyl chloride, polystyrene and polyvinylpyrrolidone.

Examples of non-biodegradable polymers include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

Examples of biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.

Bioadhesive polymers of particular interest include bioerodible hydrogels described by H.S. Sawhney, C.P. Pathak and J.A. Hubell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate). Thus, the invention provides a composition of the above-described therapeutic agents for use as a medicament, methods for preparing the medicament and methods for the sustained release of the medicament in vivo.

When the compositions of the invention are to be used to alleviate epidural or intrathecal pain, injection or administration of the compositions through a catheter is the most simple mode

of administration. The compounds of the invention can be administered for example in an aqueous medium, such as an isotonic solution, or in a non-aqueous medium. such as glycerol or an oil. Alternatively, the compounds of the invention can be administered as an injectable suspension to achieve a more prolonged effect. The preferred medium for intrathecal injection is isotonic dextrose. Other exemplary pharmaceutically acceptable intrathecal carriers include hypobaric (e.g., 50% normal saline) and hyperbaric (e.g., 5-8% glucose) solutions. (See, e.g., Neural Blockade, ed., Cousins & Bridenbaugh, pp. 213-251 (1988).) In general, the compounds of the invention are present in an intrathecal formulation in an amount ranging from about 0. 1% to about 10% by weight, based upon the total weight of the composition. Preferably, the compounds of the invention are present in an amount ranging from about 0.25% to about 2.5% by weight and, most preferably, the compounds are present in an amount ranging from about 0.5% to about 1% by weight. In some embodiments, it is preferred that the compounds of the invention be formulated in a pharmaceutically acceptable intrathecal or topical carrier that is not suitable for oral administration.

When the compositions of the invention are to be used to alleviate a topical pain, the compounds can be administered as a pure dry chemical (e.g., by inhalation of a fine powder via an insufflator) or, more preferably, as a pharmaceutical composition further including a pharmaceutically acceptable topical carrier. Thus, the pharmaceutical compositions of the invention include those suitable for administration by inhalation or insufflation or for nasal, intraocular or other topical (including buccal and sub-lingual) administration.

For administration to the upper (nasal) or lower respiratory tract by inhalation, the compounds of the invention can be delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray. Alternatively, the compounds of the invention can be delivered as a dry powder composition containing, for example, the pure compound together with a suitable powder base (e.g., lactose, starch).

For intra-nasal administration, the compounds of the invention can be administered via nose drops, a liquid spray, such as via a plastic bottle atomizer or metered-dose inhaler.

Exemplary atomizers are known to those of ordinary skill in the art. Drops, such as eye drops or nose drops, can be formulated with an aqueous or non-aqueous base which optionally further includes one or more dispersing agents, solubilizing agents or suspending agents. Apparatus and methods for delivering liquid sprays and/or drops are well known to those of ordinary skill in the ai.

For topical administration to the eye, nasal membranes or to the skin, the compounds according to the invention may be formulated as ointments, creams or lotions, or as a transdermal patch or intraocular insert or iontophoresis. For example, ointments and creams can be formulated with an aqueous or oily base alone or together with suitable thickening and/or gelling agents. Lotions can be formulated with an aqueous or oily base and, typically, further include one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. (See, e.g., U.S. 5,563,153, entitled "Sterile Topical Anesthetic Gel", issued to Mueller, D., et al., for a description of a pharmaceutically acceptable gel-based topical carrier.) In general, the compounds of the invention are present in a topical formulation in an amount ranging from about 0.01% to about 30.0% by weight, based upon the total weight of the composition. Preferably, the compounds of the invention are present in an amount ranging from about 0.5 to about 30% by weight and, most preferably, the compounds are present in an amount ranging from about 0.5 to about 10% by weight. In a preferred embodiment, the compositions of the invention comprise a gel mixture to maximize contact with the surface of the localized pain and minimize the volume and dosage necessary to alleviate the localized pain. GELFOAM (g) (a methylcellulose-based gel manufactured by Upjohn Corporation) is a preferred pharmaceutically acceptable topical carrier. Other pharmaceutically acceptable carriers include iontophoresis for transdermal drug delivery.

In a particularly preferred aspect of the invention, the compounds of the invention are formulated in a composition to alleviate pain in the oral cavity. An exemplary pharmaceutically acceptable topical carrier for the sustained release of a pain relieving substance in the oral cavity is a polyvinyl alcohol matrix such as that described in U.S. 5,520,924, entitled "Methods and articles for administering drug to the oral cavity", issued to Chapman, R., et al. Alternative formulations suitable for topical administration in the mouth or throat include lozenges comprising the compound(s) of the invention in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the compound(s) in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

Other suitable carriers for delivery to the oral cavity or other topical surface and/or for the sustained release of a pain alleviating compound are known to one of ordinary skill in the art.

In general, the N-butyl tetracaine and tonicaine compounds of the invention can be administered in accordance with known methods for administering the base compounds

tetracaine and lidocaine, respectively. In addition to the above-described carriers, exemplary pharmaceutically acceptable carriers and modes of administration for administering the tetracaine and/or the lidocaine base compounds to a subject are described in at least the following United States patents: U.S. 5,502,058 (entitled "Method for the treatment of pain", issued to Mayer, D., et al.); U.S. 5,346,903 (entitled "Aqueous suspension preparation for injection, method for producing the same and use thereof for producing pain relief", issued to Ackerman, E., et al.); U.S. 5,563,153 (entitled "Sterile topical anesthetic gel", issued to Mueller, D., et al.); U.S.

5,520,924 (entitled "Methods and articles for administering drug to the oral cavity", issued to Chapman, R., et al.); U.S. 5,510,339 (entitled "Method for the treatment of bronchial asthma by administration of topical anesthetics", issued to Gleich, G., et al.); and U.S. 5,433,954 (entitled "Method and composition for treating psoriasis, seborrheic dermatitis and eczema, issued to Smith, S., et al.). In general, the concentration ranges of the N-butyl tetracaine and tonicaine compounds which define the therapeutically effective amounts of these active agents are approximately the same or slightly less than the concentration ranges of the tetracaine and lidocaine base compounds that define the therapeutically effective amounts of these base compounds, respectively. Because the N-butyl tetracaine and tonicaine compounds described herein are significantly more potent than the base compounds from which they are derived, a lower dosage can be administered to a subject to induce a therapeutically effective result.

It should be understood that the preceding is merely a detailed description of the preferred embodiments. It should therefore be apparent to those of ordinary skill in the art that various modifications equivalent can be made without departing from the spirit and scope of the invention.

All references, patents and patent publications that are identified in this application are incorporated in their entirety herein by reference. The specific examples presented below are illustrative only and are not intended to limit the scope of the invention described herein.

EXAMPLES MATERIALS AND METHODS Chemicals Tetracaine (pKa = 8.5) was obtained from Sigma Chemical Co. (St. Louis, MO). A tetracaine homolog, 2-(dimethylamino) ethyl benzoate, was purchased from Pfaltz and Bauer, Inc. (Waterbury, CT). Tetracaine and its homolog were dissolved in dimethylsulfoxide (DMSO) at 100-500 mM and maintained as stock solutions at 4 0C. Batrachotoxin (BTX) was provided by

Dr. John Daly (National Institutes of Health, Bethesda, MD) and was dissolved in DMSO at 0.5 mM.

Lidocaine base was purchased from Sigma Chemical Co. (St. Louis, MO); 1- bromododecane and (2-bromoethyl) benzene were from Aldrich, Chemical Company, Inc.

(Milwaukee, WI). QX-314 chloride was donated by Astra Pharmaceutical Products (Worcester, MA). Silica gel G was obtained from Brinkmann Instruments, Inc. (Westbury, NY). All chemicals were reagent grade from commercial sources.

Synthesis of N-butyl tetracaine quaternary ammonium salt (QA) A 1:2 molar ratio of tetracaine base and l-bromobutane (Aldrich, Milwaukee, WI) was mixed in 15 ml of absolute ethanol and refluxed at 85 "C for 30 h. The reaction was stopped and excess ethanol was evaporated. The jelly-like residue was washed several times with warm hexane to remove the remaining starting reactants, and the product was dried under a vacuum.

The yield for N-butyl tetracaine QA was -90%, and the product was >97% pure was judged with a thin layer chromatography system. N-Butyl tetracaine QA was dissolved in DMSO at 100 mM as a stock solution. The chemical structures of tetracaine and its derivatives are shown in Figure 3.

Analysis of N-butyl tetracaine QA by mass spectrometry revealed a molecular mass of 321.4 daltons. Proton NMR spectrum (500 MHZ) of N-butyl tetracaine was undertaken in CDC13. Peak assignments were made by inspecting chemical shifts, peak intensities, and spin couplings. The following peaks were identified (5 = parts per million from TMS): 0.94 (t, 6H); 1.43 (m,4H); 1.73 (m,4H); 3.18 (t,2H); 3.49 (s,6H); 3.65 (t,2H); 4.15 (br,s,2H); 4.72 (br.s,2H); 6.92 (br,s,2H); and 7.86 (d, 2H). The peak at 7.26 was from CDC13. The results of NMR spectrum and mass spectrometry were consistent with the proposed structure of N-butyl tetracaine QA. Bromide was the counter ion for this positively charged QA compound.

Synthesis of Tonicaine The conventional method for QA synthesis was used to modify the lidocaine structure.

Tonicaine was synthesized from lidocaine (base) and (2-bromoethyl) benzene. A 2:1 molar ratio of lidocaine and (2-bromoethyl) benzene were refluxed at 80-900C in absolute ethanol for 5 days.

Excess ethanol was evaporated. The product was washed several times each with 25 ml warm hexane (600C). Residual hexane was removed under vacuum. The product, N-P-phenylethyl lidocaine bromide [diethyl-(2,6-dimethylanilinocarbonyl) methyl-P-phenylethyl ammonium bromide, was purified by silica gel column chromatography. The mobile phases were

chloroform/ethyl acetate (13/1, vol/vol) followed by chloroform/ethanol (80/20, vol/vol).

Tonicaine was eluted in the chloroform/ethanol. The eluted fractions containing the product were pooled and the solvents were removed under vacuum. The product was >98% pure as judged by thin layer chromatography systems. Thin layer chromatography systems employed in the synthesis were normal phase thin layer chromatography plates (Fisher Scientific, PA) developed with ethanol, 96% ethanol/0.8 M NH4C (80/20, vol/vol), or chloroform/ethyl acetate (13/1, vol/vol). The practical yield was 39%. Structural analysis of tonicaine by mass spectrometry yielded a molecular mass of 339.2, which is consistent with the structure of tonicaine cation.

Synthesis of N-dodecyl Lidocaine Quaternary Ammonium N-dodecyl lidocaine was synthesized from lidocaine (base) and 1-bromododecane by the method similar to that for tonicaine synthesis (see above). The product was >98% pure as judged by thin layer chromatography systems. Structural analysis of N-dodecyl lidocaine QA by mass spectrometry yielded a molecular mass of 403.2, which agreed with the structure of N-dodecyl lidocaine.

Sensory and Motor Block of Rat Sciatic Nerve A neurologic evaluation of sciatic nerve block in the rat was conducted according to the method described by Thalhammer and colleagues (Thalhammer JG et al., ANESTHESIOLOGY 1995; 82:1013-25). These protocols were approved by Harvard Medical Area Standing Committee on Animals. All sciatic nerve functions were observed in rats handled under free- behavior conditions. Handling of rats, which took -3 weeks before drug testing, reduced stress during neurologic examination without adversive behavior of biting, escaping, and vocalization.

These procedures were taken to measure baseline behavior and to determine the influence of regional block on behavior in a reproducible manner. Drugs at a concentration of 37 mM in isotonic saline (equivalent to 1.11% tetracaine-hydrochloric acid concentration were injected in a volume of 0.1 ml at the sciatic notch, as described previously (Wang GK et al., ANESTHESIOLOGY 1995; 83:1293-1301). Functional impairment was measured by comparing values obtained before and at various intervals after injection. Changes of function were estimated and normalized as percentages of maximal possible effect (% MPE) (Thalhammer JG et al., ANESTHESIOLOGY 1995; 82:1013-25; Wang GK et al., ANESTHESIOLOGY 1995; 83:1293-1301). Complete block of function was defined as 100% MPE and unchanged function as 0% MPE. This definition did not imply that our functional

assays used a continuous variable but was applied to normalize the data for comparison.

Throughout the experiment all animals were observed for abnormalities in behavior, such as alertness, responsiveness of environment, weight loss, motor activity, gait, and resting posture.

This was to verify that rats were not under extreme stress induced by drug injection or by the researcher during behavior tests. Well-handled rats permitted a more precise neurologic examination and yielded reliable reproducible results. All behavior tests were not blinded and were performed by one person. Detailed descriptions of behavior tests can be found in Thalhammer and colleagues (Thalhammer JG et al., ANESTHESIOLOGY 1995; 82:1013-25) and are summarized briefly here.

Proprioception was evaluated based on the combined postural reaction (such as "hopping" and "tactile placing"; see below) and was scored from 3 (normal postural reactions) to 0 (complete lack of postural reactions); a score of 0 was considered to represent 100% MPE.

To evaluate tactile placing, the toes of one foot were ventroflexed and their dorsi were placed onto the supporting surface while the animal was kept in normal resting posture. The ability to reposition the toes was evaluated (3 = normal reposition ability, 2 = slightly impaired, 1 = severely impaired, 0 = no attempt).

To assess hopping, the front half of the animal was lifted so that the body's weight was supported by the hind limbs. One hind limb at a time was then lifted and the animal's body was moved laterally. The ability of the animal to follow the lateral movement of the body by hopping with the weight-supporting limb was evaluated (3 = normal hopping ability, 2 = slightly impaired. 1 = severely impaired, 0 = no hoping ability).

Motor function of the hind limbs was evaluated by the "extensor postural thrust." The rat was held upright so that the hind limbs were extended and the body's weight was supported by the foot. The force (measured in grams) necessary to bring the heel into contact with the platform of a balance was measured. The reduction in force resulting from reduced extensor muscle tone was considered the motor deficit. A force of <15 g was considered to represent an absence of extensor postural thrust or 100% motor block.

Nociception was evaluated by measurement of the different degree of the withdrawal response to noxious pinch. Care was taken to avoid tissue injury resulting in hyperalgesia by properly spacing the stimulations. The fifth toe was pinched (to 300 g) with a force-calibrated serrated forceps for 2 s, and the withdrawal response was graded as 4 (normal, brisk, generalized motor reaction; withdrawal of the stimulated hind limb; attempts to bite the forceps; and

vocalization); 3 (the same as 4, but slower than on the control side); 2 (the same as 3, but with one of the responses lacking); 1 (only a weak attempt to withdraw); or 0 (no response).

Morphologic Changes in Treated Sciatic Nerves At 21 and 35 days after N-Butyl tetracaine injection. rats were killed by pentobarbital overdose. Sciatic nerves near the distal end of the injected site and the uninjected contralateral site were carefully dissected. Excess adipose tissue was trimmed, and the nerves were rinsed with saline solution. Each sample was placed in a separate vial containing 4% glutaraldehyde in 0.1 M cacodylate buffer (Poly Scientific Research & Development Corp., Bay Shore, NY).

Histologic cross-sectioning and staining were performed as described by Anthony and coworkers (Anthony DC et al., J Neuropathol Exp Neurol 1983; 42:548-60).

Voltage Clamp Experiments in GH3 Cells Cell Culture. Rat clonal pituitary GH3 cells were purchased from the American Type Culture Collection (Rockville, MD) and maintained as described by Cota and Armstrong (Cota G. Et al., J Gen Physiol 1989; 94:213-32) in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclone Labs, Logan, UT). For Na+ current recording, cells were grown in a 35-mm culture dish, which was then used as a recording chamber.

Whole-cell Voltage Clamp. The whole-cell variant of the patch-clamp method was used to measure Na+ currents in rat clonal GH3 cells (Cota G. Et al., J Gen Physiol 1989; 94:213-32).

The external solution contained 150 mM choline Cl, 0.2 mM CdCl2, 2 mM CaC12, and 10 mM hydroxyethylpiperazine-ethane sulfonic acid adjusted to pH 7.4 with tetramethylammonium hydroxide. Micropipettes were fabricated and had a tip resistance of ~ 1 MQ when filled with an Na+ solution containing 100 mM NaF, 300 mM NaC1, 10 mM EGTA, and 10 mM hydroxyethylpiperazine-ethane sulfonic acid adjusted to pH 7.2 with CsOH. The junction potential of electrodes was nulled before seal formation. After the patch membrane ruptured, the cell was allowed to equilibrate with the pipette solution for at least 15 min at a holding potential of-100 mV. Tetracaine and N-butyl tetracaine QA at appropriate concentrations were applied to cells with a flow rate of about 0.12 ml/min through a series of narrow-bored capillary tubes positioned within 200 pm ofthe cell (Wang GK et al., ANESTHESIOLOGY 1995; 83:1293- 1301). Washout of drugs was performed using a tube containing the external solution without drug. Voltage clamp protocols were created with pClamp software (Axon Instruments, Foster City, CA). Leak and capacitance were subtracted by a leak and capacitance compensator, as described by Hille and Campbell (Hille B et al., J Gen Physiol 1976; 67:265-93). Additional

compensation was achieved by the patch-clamp device (EPC7: List-Electronic, Dramstadt/Ebertstadt, Germany). All experiments were conducted at room temperature (23 +/- 2°C). At the end of the experiments, the drift in the junction potential was generally less than 2mV.

Statistical Analysis An unpaired Student's test (Sigmastat, Jandel Scientific Software, San Rafael, CA) was used to evaluated the significance of drug-induced changes in the rate and the steady state of tonic and use-dependent changes in the rate and the steady state of tonic and use-dependent block. A Mann-Whitney rank sum test or a Kruskal-Wallis one-way analysis of variance on ranks (Sigmastat) as used to assess the significance of differences in the magnitude and duration of functional changes detected by neurologic evaluation after tetracaine and N-butyl tetracaine injection. A probability value less than 0.05 was considered statistically significant.

RESULTS Onset of Sciatic Nerve Block by N-butyl Tetracaine Within 5 min after its injection into the sciatic notch of rats, 0. l ml of 37 mM N-butyl tetracaine elicited complete functional impairment of proprioception, motor function, and response to noxious pinch. This onset of the blocking of proprioception and motor function by N-butyl tetracaine was as fast as that by tertiary-amine tetracaine. Tetracaine did not completely block the response to noxious pinch in all rats tested. Its effect was maximal (~95% MPE) 10 min after tetracaine injection. Thus, despite its permanent positive changes. N-butyl tetracaine elicited complete sciatic nerve block with a rapid onset comparable to or faster than that measured for tetracaine.

Duration of Complete Sciatic Nerve Block by N-butyl Tetracaine Complete functional impairment of the response to noxious pinch by N-butyl tetracaine in rats lasted longer than 2 weeks (336 h). This prolonged block of nociceptive function in the sciatic nerve has not been documented for any reversible LAs but resembles the long sensory analgesia produced by dodecyl triethyl QA ions on trigeminal nerve (Scurlock JE et al., ANESTHESIOLOGY 1981; 54:265-9). Proprioception and motor function were also completely blocked for at least 24 h after N-butyl tetracaine injection; the block remained operative up to 85% to 90% MPE for 2 weeks (336 h). In contrast, complete block of proprioception and motor function by tetracaine lasted less than 60 min. For each functional test, the duration of complete block by tetracaine was significantly shorter than that of complete block by N-butyl tetracaine (P

< 0.005). Thus N-butyl tetracaine elicited prolonged block of rat sciatic nerve functions after a single injection into the sciatic notch, whereas tetracaine at the same concentration failed to do so.

Recovery of Sciatic Nerve Functions With a single injection of 0.1 ml of 37 mM B-butyl tetracaine, impaired sciatic nerve functions recovered completely only after 8 weeks (1.344 h), at which time the %MPE decreased to near 0 (with six of eight rats recovering completely). For the response to noxious pinch, partial recovery began between 3(504 h) and 8(1.344 h) weeks after injection of N-butyl tetracaine. For proprioceptive and motor functions, partial recovery began earlier. For example, 8 of 14 rats started to recover their motor function at 1 week, although 6 rats remained completely blocked. Similarly, 4 of 14 rats had partially recovered their proprioceptive function as 1 week. Some of the early recovery in proprioceptive and motor functions might be due to adaption of the rats. Nevertheless, this result showed that rats began to recover from proprioceptive and motor block at about 1 week after injection, but the time course could vary considerably in humans. The recovery of functions continued slowly until it was complete after 8 week.

Neurolytic Effects of N-butyl Tetracaine on Sciatic Nerve Fibers Extensive degeneration of myelinated nerve fibers that was attributable to N-butyl tetracaine was evident with light microscopic examination of the transverse sections of rat sciatic nerve from the injected region. We observed axonal loss and abundant axonal degeneration 3 weeks (504 h) after a single injection of N-butyl tetracaine. In sections of contralateral sciatic nerve, a normal density of axons and an absence of axonal degeneration were evident. In addition, proliferation of Schwann cells and macrophages was apparent in the drug-treated nerve.

Some myelinated nerve fibers remained intact in the section, although their numbers varied widely in individual rats. Similar morphologic changes were found in sciatic nerve 5 weeks (840 h) after injection. Morphological changes were not evident by light microscopy in sciatic nerve 3 days after injection despite complete functional block. These results show that N-butyl tetracaine can elicit local acute axonal neuropathy and that its neurolytic activity causes ultralong block of sciatic nerve functions.

Tonic Block of Na+ Channel by N-butyl Tetracaine Typical tertiary amine LAs, including tetracaine, block voltage-gated Na+ channels in a complicated manner. Generally they elicit tonic inhibition of Na+ currents when the nerve is

stimulated infrequently (Butterworth JF et al., ANESTHESIOLOGY 1990; 72:711-34). We observed show the tonic inhibition of Na+ currents by N-butyl tetracaine and tetracaine, respectively. The rapid wash-in tetracaine block at 100 pM was characteristic of tertiary amine LAs. Within 1 to 2 min, the block reached its steady-state level of about 80% of the control current. The wash-off of tetracaine was equally rapid, reaching its completion within 2 min. N- butyl tetracaine at 100 I1M had a significantly slower wash-in effect than did tetracaine (P <0.0001); an interval of more than 20 min was required to reach the steady-state block of up to 80% to 90% of Na+ current. The drug's wash-off effect was also very slow. After 30 min of continuous perfusion of drug-free solution, about 40% of Na+ current remained blocked. This wash-out time course of N-butyl tetracaine was significantly longer than that for tetracaine (P < 0.0001).

Use-dependent Block of Na+ Channels by N-butyl Tetracaine When a nerve was stimulated repetitively, additional use-dependent block occurred in the presence of tetracaine. This use-dependent block phenomenon was commonly noted when tertiary amine LAs were used (Wang GK et al., Biophys J 1994; 67:1851-60). 20 pM tetracaine produced 40% to 50% tonic block of peak Na+ current. Repetitive depolarization of the cell at 2 Hz causes an additional 30% inhibition. At the end of 60 pulses, only about 30% of peak current remained. This use-dependent block by tetracaine is significantly greater than that measured for the control (s 10%) in the absence of drug (P <0.0001). For N-butyl tetracaine, a concentration of 100 I1M is used because of its slow wash-in time course. Use-dependent block by this drug significantly exceeded the control value as early as 5 min after application (P<0.0001). Between 20 and 30 min, only about 20% of the peak Na+ current remains. However, approximately 50% of this remaining current is blocked after repetitive pulses. The rate of use-dependent block for N-butyl tetracaine is significantly slower than that for tetracaine (P< 0.0001). A similar rate difference has been found for tonicaine (a QA derivative of lidocaine) and for lidocaine (Wang GK et al., ANESTHESIOLOGY 1995; 83:1293-1301). Clearly, N-butyl tetracaine QA retains the ability of its parent compound, tetracaine, to elicit tonic and use-dependent block.

DISCUSSION In Vivo Ultralong Block of Sensory and Motor Functions in Rat Sciatic Nerve This example shows that N-butyl tetracaine QA at 37 mM (equivalent to 1.11% tetracaine-hydrochloride) elicits neurolytic sensory block of rat sciatic nerve in vivo that generally lasts for more than 2 weeks. Scurlock and Curtis (Scurlock JE et al.,

ANESTHESIOLOGY 1981: 54:265-9) reported that dodecyl triethyl ammonium ions at 14 mM elicit ultralong sensory block of the rat trigeminal nerve for 17 to 20 days. This interval is comparable to that of neurolytic sensory block elicited by N-butyl tetracaine QA ions.

Partial recovery of nociceptive function occurs as early as 21 to 60 days, with apparently full recovery after 60 days, with apparently full recovery after 60 days. Despite the paralysis of the drug-injected leg, all treated animals behaved normally. There was no apparent differential block between sensory and motor functions during the initial onset phase. The onset of block in vivo for N-butyl tetracaine is as fast as that for tetracaine. Because N-butyl tetracaine with a permanent charge is not as rapidly membrane permeable as tetracaine, we surmise that N-butyl tetracaine has a high binding affinity toward Na+ channels, which could explain the rapid onset of block in vivo. The recovery time course of motor and proprioception function after tetracaine injection is slower than that of the response to noxious pinch. This difference in partial-block duration is statistically significant (P<0.05; Kruskal-Wallis analysis of variance on ranks). Such a result is consistent with the clinical observation that pain sensation returns sooner than motor function in patients given tetracaine (Concepcion M et al., Anesth Analg 1984; 63:134-8).

In Vivo Degeneration of Myelinated Nerve Fibers in Drug-treated Sciatic Nerve Histologic studies have revealed degeneration of myelinated nerve fibers after a single injection of N-butyl tetracaine QA. At 3 and 5 weeks after injection, the proliferation of Schwann cells is evident in treated sciatic nerves but not in their untreated counterparts. The degeneration of most myelinated nerve fibers at week 3 after drug injection demonstrates severe neurolytic effect of N-butyl tetracaine. If the axonal degeneration caused by N-butyl tetracaine parallels that documented in nerve-crushing experiments, regenerative nerve fibers may be present 4 weeks after drug treatment (Myers RR et al., Neural Blockade. Edited by MJ Cousins.

PO Bridenbaugh. Philadelphia. JB Lippincott. 1988. pp 1031-51). The partial recovery of functions of the hind legs during the 2-month period may be due in part to sprouting of residual intramuscular axons and to the longitudinal regeneration of axons. Proof of the regeneration of nerve fibers will require more extensive morphologic studies, such as tease fiber analysis and electron microscopy. Clearly, however, N-butyl tetracaine possesses strong neurolytic properties that elicit prolonged block of the sciatic nerve functions.

In Vivo block of Voltage-gated Na+ Channels by N-Butyl Tetracaine Quaternary Ammonium

N-butyl tetracaine QA exhibits not only neurolytic but also LA characteristics. It blocks voltage-gated Na+ channels effectively when applied externally to GH3 cells in vivo. At 100 1M, this drug produces profound tonic block of Na+ current by more than 80%. Only 0.27% of the concentration is used in experiments conducted in vitro (37 mM versus 100 1M). The wash-in of this drug is relatively slow; it takes more than 30 min to reach steady state. The wash-out is equally slow; again, at least 30 min is required for partial removal of the block. These slow kinetics are to be expected because this compound has a permanent positive charge. A comparable slowness of the wash-in and wash-out of tonicaine, a permanently charged lidocaine derivative, was recently reported (Wang GK ANESTHESIOLOGY 1995; 83:1293-1301). In contrast, tetracaine, as a tertiary amine drug, can penetrate the membrane barrier rapidly and complete is blocking effect within 2 min. Similarly, tetracaine can be washed out of the cell within 2 to 5 min.

Like most tertiary amine LAs, N-butyl tetracaine elicits profound use-dependent block of Na+ current at 2Hz. This is not surprising because QA compounds produce use-dependent block in the same manner as their tertiary amine LA counterparts (Strichartz GR, J Gen Physiol 1973; 62:37-57). Without repetitive pulses, closed Na+ channels may not be able to interact with N- butyl tetracaine efficiently. For example, after 5 min of wash-in, the first single pulse elicits about 65% block of Na+ current, whereas the tenth pulse elicits about 50% block (with pulses applied every 30 mins). These results can be explained if the LA receptor is not freely accessible to charged QA compounds when the Na+ channel is in its closed state. Similar results have been found for QX-314 (Strichartz GR, J Gen Physiol 1973; 62:37-57), a quaternary derivative of lidocaine. With as little as 5 min of incubation with external N-butyl tetracaine at 100 pM, more than 80% of Na+ currents can be blocked after 60 repetitive pulses at 2 Hz. This magnitude of use-dependent block is significantly greater than that elicited by tetracaine at 20 I1M (P<0.005).

Because of the slow wash-in of N-butyl tetracaine in GH3 cells, the internal concentration of this drug after incubation for 5 min is probably near 20 1M. Thus, N-butyl tetracaine at <e 100 uM can produce use-dependent block of Na+ current as effective as tetracaine. These results together demonstrate that N-butyl tetracaine QA at low concentrations (100 21M) is an LA that, like typical tertiary amine LAs, elicits both tonic and use-dependent block of Na+ currents.

N-Butyl Tetracaine Quaternary Ammonium as a Dual Local Anesthetic and Neurolytic Agent

Our results show that the nociceptive functions of rat sciatic nerves are completely blocked for 14 days by 37 mM N-butyl tetracaine and that full functional recovery occurs after 60 days. In the central nervous system, where myelinated nerve fibers do not regenerate to the extent documented in the peripheral nervous system, N-butyl tetracaine may be used intrathecally as a neurolytic agent to substitute for currently used drugs such as phenol and absolute alcohol. N-butyl tetracaine may relieve pain in the central nervous system for as long as phenol or absolute alcohol; in addition, it may display much stronger LA properties in vivo than these currently available drugs. Treatment with N-butyl tetracaine may also prove less distressing to patients than intrathecal injection of alcohols, which causes burning sharp pain.

Such burning pain, usually lasting for several hours after alcohol injection, will not be a potential problem for N-butyl tetracaine injection because of its strong and fast LA effect.

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