WO2005044233A1 - Formulations of n-oxide prodrugs of local anesthetics for the treatment of pulmonary inflammation associated with asthma, brochitis, and copd - Google Patents

Abstract

A prodrug of lidocaine and related local anesthetic composition or formulation for delivery by aerosolization is described. The formulation containing an efficacious amount of lidocaine N-oxide prodrug or local anesthetic N-oxide prodrug able to inhibit inflammation in asthmatic lungs. The N-oxide prodrug is formulated in 5 mL solution of a quarter normal saline having pH between 1.0 and 7.0. The method for treatment of respiratory tract inflammation by a formulation delivered as an aerosol having mass medium average diameter predominantly between 1 to 5 µ, produced by nebulization or dry powder inhaler, and as a single N-oxide prodrug therapeutic or in combination with β-agonists.

Description

FORMULATIONS OF N-OXIDE PRODRUGS OF LOCAL ANESTHETICS FOR THE TREATMENT OF PULMONARY INFLAMMATION ASSOCIATED WITH ASTHMA, BRONCHITIS, AND COPD

Field of the Invention The current invention relates to the preparation of novel prodrugs of lidocaine, dibucaine, and related compounds for delivery to the lung and by aerosolization. In particular, the invention concerns the formulation, methods of treatment, and delivery of lidocaine N-oxide, dibucaine N-oxide and related N-oxide compounds such that when delivered to the lung and systemic circulation either by aerosolization or orally, exogenous enzymes and biological processes present in the plasma, lung tissue an d airway reduce the N-oxide prodrug. Lidocaine, dibucaine and related tertiary amine drugs are then released at the site of inflammation. The N-oxide prodrugs are formulated as either liquids or dry powders for aerosolization or tablets for oral administration. The aerosol formulation permits and is suitable for delivery of N-oxide prodrugs to the lung endobronchial space of airways in an aerosol having a mass medium average diameter predominantly between 1 to 5 μ. The formulated and delivered efficacious amount of N-oxide prodrugs is sufficient to deliver therapeutic amounts of lidocaine and dibucaine and related tertiary amine compounds either as a single agent or combination with β-agonists for treatment of acute and chronic respiratory tract inflammation associated with mild to severe asthma, bronchitis, and chronic obstructive pulmonary disease (COPD). Background of the Invention Asthma is a chronic inflammatory disease of the airways resulting from the infiltration of pro-inflammatory cells, mostly eosinophils and activated T lymphocytes (Poston, 1992; Walker, 1991) into the bronchial mucosa and submucosa. The secretion of potent chemical mediators, including cytokines, by these pre-inflammatory cells alters mucosal permeability, mucus production, and causes smooth muscle contraction. All of these factors lead to an increased reactivity of the airways to a wide variety of initant stimuli (Kaliner, et ai, 1988).

Glucocorticoids, which were first introduced as an asthma therapy in 1950 (Carrier, et al, 1950), remain the most potent and consistently effective therapy for this disease, although their mechanism of action is not yet fully understood (Morris, 1985). Available evidence suggests that at least one mechanism by which they exert their potent anti-inflammatory properties is by inhibiting the release and activity of cytokines, which recruit and activate inflammatory cells such as eosinophils (Schleimer, 1990). Ordinarily, eosinophils undergo the phenomenon of apoptosis or programmed cell death, but certain cytokines such as Interleukin 5 (IL-5), Interleukin-3 (IL-3), and granulocyte-macrophage colony stimulating factor (GM-CSF) increase eosinophil survival from 1 or 2 days to 4 days or longer and cause eosinophil activation (Kita, 1992). Wallen, et al. was the first to show that glucocorticoids potently block the cytokine's ability to enhance eosinophil survival in a concentration- dependent manner (Wallen, 1991).

Unfortunately, glucocorticoids are associated with profoundly undesirable side effects such as truncal obesity, hypertension, glaucoma, glucose intolerance, acceleration of cataract formation, bone mineral loss, and psychological effects, all of which limit their use as long-

'term therapeutic agents (Goodman and Gilman). The side effects of glucocorticoid therapy have led to interest in agents, which exhibit similar anti-inflammatory effects. A variety of such agents have been tested. For example, preparations of cyclosporin (Szczeklik, 1991; Mungan, 1995), methotrexate (Dyer, 1991), troleandomycin (TAO) (Wald, 1986; Shivaram,

1991), and gold (Szczeklik, 1991 ; Dykewicz, 2001 ; Bernstein, 1988) have been used in attempts to wean patients off of orally-administered steroids. Similarly, leukotriene receptor antagonists (e.g., montelukast [Singulair^®] and zafirlukast [Accolate^®]) (Korenblat, 2001;

Dykewicz, 2001 ; Wechsler, 1999), colchicine (Fish, 1997), salmeterol (Lazarus, 2001; Lemanske, 2001), and anti-immunoglobulin E (IgE) (Dykewicz, 2001) have been used with limited success in efforts to wean patients off inhaled steroids. However, to date, no completely satisfactory substitute for glucocorticoid therapy has been identified.

Serendipitously, Ohnishi, et al. (Ohnishi, 1996) discovered that eosinophil survival is inhibited by lidocaine in a potent and concentration-dependent manner similar to that of corticosteroids. Lidocaine was shown to be effective at low concentrations, which can easily be achieved in the airways by nebulization. The potent activity of lidocaine, combined with its established record of low toxicity when administered to the airways, inspired use of this agent in preliminary clinical trials to determine its effects in patients with severe, glucocorticoid-dependent asthma. Results of these studies demonstrated that treatment with inhaled lidocaine allowed the majority of patients to significantly reduce, or discontinue, their oral glucocorticoid use without any concurrent increase in their asthma symptoms. In consideration of all the problems and disadvantages connected with the local anesthetic properties of lidocaine and related local anesthetics like dibucaine, for example numbing and high first pass metabolism, it would be highly advantageous to provide a prodrug to mask these properties. Such a prodrug would be effectively formulated and delivered directly to the endobronchial space by aerosolization or indirectly by oral delivery and converted to active drug by the action of an enzyme mediated reduction process thereby delivering to the site of inflammation a therapeutic amount of drug. It is therefore a primary object of this invention to provide a formulation and method of treatment of an N-oxide prodrug of lidocaine, dibucaine and related compounds, which are stable as a liquid or solid dosage form for nebulization or dry powder delivery. Such composition contains sufficient but not excessive concentration of the drug which can be efficiently aerosolized by nebulization in jet, ultrasonic, pressurized, or vibrating porous plate nebulizers or by dry powder into aerosol particles predominantly within the 1 to 5 μ size range, and which salinity and pH are adjusted to permit generation of a N-oxide prodrug aerosol well tolerated by patients, and which formulation further has an adequate shelf life. Summary of the Invention The present invention concerns the use of, and formulation for prodrugs of lidocaine, dibucaine and related local anesthetics and their decadeutrated forms delivered by inhalation or orally to treat pulmonary inflammation. The prodrug design is simple and utilizes the N- oxide form of the drug as a polar function (charged water soluble molecule) which blocks the ability of the prodrug to penetrate cells thereby inhibiting the local anesthetic effect

(numbing). Water solubility at neutral pH offers additional benefit such that any prodrug deposited in the oropharyngeal cavity will be swallowed quickly. This process is depicted below.

The present invention relates to liquid and dry powder formulations of a N-oxide derivative of a local anesthetic selected from the group consisting of lidocaine, dibucaine, procaine, procainamide, tetracaine, bupivacaine, the decadeutrated forms thereof and pharmaceutically acceptable salts thereof for the treatment of a disorder selected from severe to mild asthma, bronchitis, and COPD which comprise a therapeutically effective amount of the anesthetic and a pharmaceutically acceptable carrier. More specific embodiments of this invention include liquid formulations of lidocaine

N-oxide A, decadeutrolidocacine N-oxide B, dibucaine N-oxide D, decadeutrodibucaine N- oxide D, as individual substances or in combination with β-agonists such as salbutamol E or salmeterol F:

and the pharmaceutically acceptable salts of the foregoing compounds. The invention also relates to a method of treatment and a pharmaceutically acceptable composition for the treatment of a disorder selected from severe to mild asthma, bronchitis, and COPD which comprises a therapeutically effective amount of a compound of the invention or in combination with a β-agonist, a pharmaceutically accepted salt thereof, and a pharmaceutically accepted carrier. Brief Description of the Drawings Figure 1 is a graph that shows the average plasma concentration of lidocaine versus time following intravenous or intratracheal administration in rats. Figure 2 is a graph that shows the average plasma concentration of lidocaine and lidocaine N-oxide versus time following intratracheal administration in rats. Figure 3 is a graph that shows the average lung homogenate concentration of lidocaine versus time following intravenous or intratracheal administration in rats. Figure 4 is a graph that shows the average lung homogenate concentration of lidocaine and lidocaine N-oxide versus time following intratracheal administration in rats. Figure 5 is a graph that shows the average plasma contration of 2,6-dimethylanaline versus time following intratracheal administration of lidocaine and lidocaine N-oxide in rats. Figure 6 is a graph that shows the average plasma concentration of 2-amino-N-(2,6- dimethylphenyl) acetamide versus time following intratracheal administration of lidocaine and lidocaine N-xoide in rats. Figure 7 is a graph that shows the average plasma concentration of N-(2,6- dimethylphenyl) -2-ethylaminoacetamide versus time following intratracheal administration of lidocaine and lodocaine N-oxide in rats.

Detailed Description of the Invention As used herein, the term "pharmaceutically acceptable salts" refers to the nontoxic acid or alkaline earth metal salts of the compounds of the invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting the base or acid functions with a suitable organic or inorganic acid or base, respectively. Representative acid salts include the hydrochloride, hydrobromide, bisulfate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, citrate, maleate, tartrate and the like. Representative alkali metals of alkaline earth metal salts include sodium, potassium, calcium, and magnesium salts. The term "treating", as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term "treatment", as used herein, refers to the act of treating, as "treating" is defined immediately above. The term "normal saline" means water solution containing 0.9% (w/v) NaCl. The term "diluted saline" means normal saline containing 0.9% (w/v) NaCl diluted into its lesser strength. The term "quarter normal saline" or "V* NS" means normal saline diluted to its quarter strength containing 0.225% (w/v) NaCl. The compounds of the invention may comprise asymmetrically substituted carbon atoms. Such asymmetrically substituted carbon atoms can result in the compounds of the invention comprising mixtures of stereoisomers at a particular asymmetrically substituted carbon atom or a single stereoisomer. As a result, racemic mixtures, mixtures of diastereomers, as well as single diastereomers of the compounds of the invention are included in the present invention. The terms "S" and "R" configuration, as used herein, are as defined by the IUPAC 1974 RECOMMENDATIONS FOR SECTION E, FUNDAMENTAL STEREOCHEMISTRY, Pure Appl. Chem. 45: 13-30 (1976). The term configuration, as used herein, is defined by the CHEMICAL ABSTRACTS INDEX GUIDE- APPENDIX IV (1987) paragraph 203. I. PREPARATION OF THE COMPOUNDS OF THE INVENTION Referring to reaction Scheme I, specific compounds useful in the present invention are prepared. In general, tertiary amines serve as the starting materials for the preparation of N-oxide prodrugs and are prepared by reaction of the amine with an oxidizing agent selected from but not limited to 30-35%o hydrogen peroxide (Cope, 1957) meta-chloroperbenzoic acid (Chaudhuri, 1984) perfluoro cis-2,3-dialkyloxaziridines (Amone, 1998) and the like in an appropriate solvent such as methanol, ethanol, dichloromethane with or without the presence of acid. Dibucaine N-oxide was prepared according to literature procedures (Chaudhuri., 1984). In the case of decadeutrolidocaine and decadeutrodibucaine N-oxides 7 and 11, their preparation in volves the reductive alkylation of amino amides 5 and 9 with NaBD₄ / d₄- acetaldehyde and oxidation as indicated in Scheme I. Alternatively, chloro amide 8 is alkylated with decadeutroethylamine in THF to give dio-Hdocaine 6.

Scheme I

II. FORMULATION OF LIDOCAINE N-OXIDE IN WATER The solubility of lidocaine N-oxide in water and eighteen other solvent systems was investigated. The solubility of lidocaine N-oxide was greater than or equal to -25 mg/ml in the following solutions: ethanol (A); 80% ethanol/20% glycerol (B), 80% ethanol/20% propylene glycol (C); 80%> ethanol/20%) polyethylene glycol 300 (D); water (E); aqueous solutions of 0.1 M zinc chloride (F), 0.1 M magnesium chloride (G), dilute hydrochloric acid (pH 1.1) (H), 0.1 M citric acid (I), and 0.2 M maleic acid (J) (Table 2). Lidocaine N-oxide was not completely soluble at 25 mg/ml in several dilute aqueous acids, including phosphoric acid (pH ~ 2) acetic acid (pH 3.6), sulfiiric acid (pH 2.1), and 0.2 M citric, lactic, succinic, fumaric, malic, or tartaric acids (Table 1).

Table 1 Solubility of Lidocaine N-oxide at 25 mg/ml in Various Solvents

Solution Solvent Soluble at 25 mg/ml A Ethanol Yes B 80:20 ethanol/glycerol Yes C 80:20 ethanol/propylene glycol Yes D 80:20 ethanol/polyethylene glycol 300 Yes E Water Yes F ZnCl₂, 0.1 M Yes G MgCl₂, 0.1 M Yes H Hydrochloric acid (pH 1.1) Yes I Citric acid, 0.1 M Yes J Maleic acid, 0.2 M Yes Phosphoric acid (pH ~ 2) No Acetic acid (pH 3.6) No Sulfiiric acid (pH 2.1) No Tartaric acid, 0.2 M No Citric acid, 0.2 M No Malic acid, 0.2 M No Succinic acid, 0.2 M No Fumaric acid, <0.2 M (saturated) No Lactic acid, 0.2 M No

The stability of lidocaine N-oxide in solutions A-J was determined as a function of temperature (55° C) and time (1, 2, and 5 days, Table 2). After 24 hours, it was noted that the concentration of lidocaine N-oxide in solutions (A-H) significantly decreased (2.7% to 47.2%>) when submitted to these conditions. However, lidocaine N-oxide was stable in 0.1 M citric acid (solution I) and 0.2 M maleic acid (solution J). Heating solutions I and J for an additional 24 hours (2 days total) caused further degradation of only solution I by 2.5 %. The concentration of the remaining lidocaine N-oxide solution J (0.2 M maleic acid ) remained unchanged after 5 days at 55° C (Table 2). Thus it was shown that lidocaine N- oxide was most stable in 0.2 M aqueous maleic acid solution J at a concentration of 25 mg/mL. Two degradation products with molecular mass of 410 and 413 appeared in all examples where the concentration of lidocaine N-oxide decreased as the result of time and temperature effects. The increase in the two degradation products coπelated with the decrease in concentration of lidocaine N-oxide (Table 3).

Table 2

Percent Recovery of Lidocaine N-oxide in Solvents After Storage at 55°C

Solution Solvent 1 Day 2 Days 5 Days

A Ethanol 64.7% 49.7%

B 80:20 ethanol/glycerol 52.8% 40.9%

C 80:20 ethanol/propylene glycol 61.1% 49.4%

D 80:20 ethanol/polyethylene glycol 300 61.3% 49.1%

E Water 88.7% 89.1%

F ZnCl₂/water, 0.1 M 92.5% 88.4%

G MgCl₂/water, 0.1 M 91.5% 88.2%

H Hydrochloric acid, pH 1.1 97.5% -

I Citric acid water, 0.1 M 99.5% 94.1%

J Maleic acid, 0.2 M - - 99.9%>

Table 3

Total Percent Area of Degradation Products in Lidocaine N-oxide Solutions After Storage at 55°C

III. PHARMACOKINETICS AND IN VIVO ACTIVATION OF LIDOCAINE N-OXIDE AFTER INTRATRACHEAL DELIVERY TO THE LUNG Bioavailability is a measure of the extent (amount) of a therapeutically active drug, which reaches the systemic circulation after dosing by various routes. Thus bioavailability is an important biological determinant of therapeutic efficacy. Various prodrug strategies have been developed to enhance oral bioavailability of poorly absorbed drugs. In particular, N- oxide prodrugs of analgesic morphinans have shown improved oral bioavailability (Boswell, 1988). However, N-oxide prodrugs of tertiary amine compounds and formulations thereof are unknown as prodrugs for aerosol or intratracheal (lung) delivery, and thus represent a new and unprecedented way to safely deliver directly to the lung tertiary amine drugs like lidocaine, dibucaine and the like. In the following pharmacokinetic study, the bioavailability of lidocaine and lidocaine N-oxide was determined in rats after dosing intratracheally. In addition to bioavailability, a number of pharmacokinetic parameters such as AUC o-_t, volume of distribution (Vss), half- life (T ₂), Cmax, T_max and clearance (CLs) were measured. The lung-to-plasma concentration ratios for lidocaine and lidocaine N-oxide for each rat were also determined. Lidocaine had a total clearance (as denoted by CLs, in this report) of 93.0 mL/min kg after intravenous dosing, and a half-life of 28.1 minutes. Intratracheal administration of lidocaine at 20 mg/kg resulted in systemic bioavailability of 30%. Lung concentrations were much greater after intratracheal lidocaine dosing than after intravenous dosing, with 58-fold greater AUC while the intratracheal dose was only 10-fold greater than the intravenous dose. Intratracheal dosing of lidocaine N-oxide resulted in the appearance of lidocaine in plasma with an AUC similar to that after intratracheal lidocaine dosing. The resulting systemic bioavailability of lidocaine was 36.5%. Plasma concentrations of lidocaine N-oxide were greater than lidocaine concentrations with AUC being 3.3-fold greater. Lidocaine N-oxide conversion to lidocaine was also apparent in lung tissue samples, and the lidocaine lung concentration versus time AUC was similar to the lidocaine N-oxide AUC. Lung/plasma lidocaine concentration ratios after intravenous lidocaine dosing were generally within a range of 3-10, and increased somewhat above this at later times (Figure 1). After intratracheal lidocaine dosing, lidocaine had very high lung/plasma lidocaine concentration ratios at 5 minutes, but reached similar ratios as after intravenous dosing at later times (Figure 3). After intratracheal lidocaine N-oxide dosing, lung/plasma lidocaine concentration ratios were usually greater than lung/plasma lidocaine N-oxide concentration ratios (Figures 2 and 4). This suggests rapid conversion of lidocaine N-oxide by the lungs in vivo. While bioanalytical analysis showed the presence of lidocaine metabolites DMA, MEGX, and GX in plasma after It. administration of a 20 mg/kg dose of both lidocaine and lidocaine N-oxide treated rats, unexpectedly it was discovered that the relative proportions of the metabolites were significantly reduced in the lidocaine N-oxide treated group (Table 4). In particular, the production of toxic and carcinogenic metabolite, 2,6-dimethyl aniline (DMA, Figure 5) was markedly reduced when compared to plasma levels of DMA from the lidocaine treated group. Thus, not only was lidocaine N-oxide found to be non-numbing but it greatly minimized the production of unwanted lidocaine metabolites, DMA, GX and MEGX (Table 4, Figures 5-7).

Table 4

Average Plasma Concentration of 2,6-Dimethylanaline (DMA), 2-Amino-N-(2,6- dimethyl-phenyl)-acetamide (GX), and N-(2,6-Dimethyl-phenyl)-2-ethylamino- acetamide (MEGX) Versus Time Following Intratracheal Administration of Lidocaine and Lidocaine N-oxide in Rats

MEGX (ng/ml) GX (ng/ ml) DMA (ng/ml) Time Lidocaine N-oxide Lidocaine N-oxide Lidocaine N-oxide (minutes) 10 393.1 51.9 181.7 9.2 277.7 1.4 30 165.1 87.3 145.9 33.0 23.7 4.9 60 103.9 40.1 216.3 48.1 35.3 4.1 240 7.2 45.7 45.6 0.9

In conclusion, lidocaine was delivered to male rats by three different routes, intravenous lidocaine, intratracheal lidocaine, and intratracheal lidocaine-N-oxide. When delivered intravenously, lidocaine exhibited rapid clearance, with low to moderate volume of distribution and Ty₂ ( 28 minutes, consistent with what has previously been reported in the literature. A small percentage, approximately 0.3% of theoretical, was absorbed into the lung from the intravenous route. When delivered intratracheally, lidocaine was systemically cleared from the lung at an initial rapid rate, reaching similar plasma levels to intravenous delivery after 2 hours. However, the systemic absorption did not appear to be complete, as evidenced by the lung/plasma ratios, where significant levels of lidocaine remained in the lungs for at least four hours. The bioavailability of lidocaine was about 30%>, with a biphasic clearance pattern, suggesting that the systemic levels of lidocaine were limited not only by its absorption from the lung, but also by its high extraction rate by the liver. When lidocaine was delivered via the prodrug, lidocaine-N-oxide, the prodrug was rapidly, but incompletely, reduced to lidocaine in the lung. Both lidocaine and lidocaine-N- oxide were systemically absorbed from the lung, where the overall bioavailability of lidocaine was approximately 36%>. The lung/plasma distribution ratios of lidocaine-N-oxide and lidocaine reached equilibrium between 10-30 minutes. This data suggest that lidocaine- N-oxide is more easily absorbed from the lung than lidocaine. It also appears that lidocaine- N-oxide continues to be reduced to lidocaine in the blood, possibly by a mechanism similar to what has been reported for imipramine-N-oxide (Bickel, 1968) III. AEROSOL DELIVERY DEVICES The use of N-oxide prodrugs of lidocaine and local anesthetics with a suitable formulation for liquid nebulization, or as a dry powder provides sufficient prodrug to the lungs for a local therapeutic effect. Prodrugs are suitable for aerosolization using jet, electronic, or ultrasonic nebulizers as well as for delivery by dry powder or metered dose inhalers. The pure powder form has long-term stability permitting the drug to be stored at room temperature. The aerosol formulation comprises a concentrated solution of 10 to 500 mg/mL of pure lidocaine N-oxide prodrug or its pharmaceutically acceptable salt as a single agent or in combination with a β-agonist or its pharmaceutically acceptable salt, dissolved in aqueous solution having a pH between 4.0 and 7.5. Preferred pharmaceutically acceptable salts are inorganic acid salts including hydrochloric acid, hydrobromic acid, sulfiiric acid, and phosphoric acid as they may cause less pulmonary irritation. The therapeutic amount of the pure lidocaine prodrug is delivered to the lung endobronchial space by nebulization of a liquid aerosol or dry powder having an average mass medium diameter between 1- 5 μ. An indivisible part of this invention is a device able to generate aerosol from the formulation of the invention into aerosol particles predominantly in the 1-5 μ size range. Predominantly in this application means that at least 70%> but preferably more than 90% of all generated aerosol particles are within the 1-5 μ size range. Typical devices include jet nebulizers, ultrasonic nebulizers, vibrating porous plate nebulizers, and energized dry powder inhalers. A jet nebulizer utilizes air pressure to break a liquid solution into aerosol droplets. An ultrasonic nebulizer works by a piezoelectric crystal that shears a liquid into small aerosol droplets. A pressurized nebulization system forces solution under pressure through small pores to generate aerosol droplets. A vibrating porous plate device utilizes rapid vibration to shear a stream of liquid into appropriate droplet sizes. However, only some formulations of lidocaine prodrugs can be efficiently nebulized, as the devices are sensitive to the physical and chemical properties of the formulation. The formulations, which can be nebulized typically, must contain large amounts of lidocaine N-oxide prodrugs, which are delivered in large volumes (up to 5 ml) of aerosol. IV. UTILITY The compositions of the invention are useful (in humans) for treating pulmonary inflammation. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. This small volume, high concentration formulation of lidocaine N-oxide prodmg can be delivered as an aerosol and be delivered at efficacious concentrations to the respiratory tract in patients suffering from mild to severe asthma. The solid dosage formulation is stable, readily manufactured, and very cost effective. Furthermore, the formulation provides adequate shelf life for commercial distribution. The prodrug masks the anesthetic properties of lidocaine thus numbing in the oropharyngeal cavity is completely eliminated. The drug is released by enzymes or reductive processes in the lung and plasma compartment, thereby releasing the therapeutic amount of lidocaine or local anesthetic at the site of inflammation. The foregoing may be better understood from the following examples, which are presented for the purposes of illustration and are not intended to limit the scope of the inventive concepts. Example 1 Lidocaine N-oxide

Following the procedure described by Cope et al. (Organic Synthesis Collective

Volume IV, 612-615) a solution of 41.0g (0.17 mole) of lidocaine, 22 mL of methanol and 20g of aqueous 35% hydrogen peroxide was allowed to stand at room temperature for 18 hours. An additional 20g of 35% hydrogen peroxide was then added to the reaction solution and the solution kept at room temperature for 6 hours. Finally, a third portion of 35%> hydrogen peroxide (20g) was added and the reaction was allowed to stand at room temperature for 18 hours. The reaction solution was cooled in an ice-water bath and a suspension of 75 mg of platinum black in 3 mL of water was added portion wise to the cooled reaction mixture to destroy excess H₂O₂. After gas evolution (oxygen) ceased, the reaction solution was filtered through a pad of celite and the solvents (methanol/water) were removed by rotoevaporation under reduced pressure then high vacuum. The crude product was

recrystallized from hot ethyl acetate to give a white crystalline solid. Yield: 30 g (71%>) *H NMR (400 MHz, CDC1₃) 7.26-7.18 (m, 3H), 4.23 (s, 2H), 3.65 - 3.55 (m, 4H), 2.21 (s, 6H),

1.37 (t, 6H); LC/MS (M+H)⁺ 251. Anal. Calcd for Ci₄H₂₂N₂O₂ C, 67.17; H, 8.86; N, 11.19.

Found: C, 67.20; H, 9.02; N, 1 1.39. Example 2 2-Butoxy-quinoline-4-carboxylic acid (2-diethylamino-ethyl)-amide N-oxide

Title compound was prepared according to the procedure of Chaudhuri, N. K., (1984). + (J. of Labeled Compounds and Radiopharmaceuticals, 22: 117-125). LC/MS (M+H) 360.

Anal. Calcd for C₂oH₂₉N₃O₃ C, 66.83; H, 8.13; N, 11.69. Found: C, 66.60; H, 8.11 ; N, 11.59.

Example 3 Decadeutrolidocaine

Method A: A solution of 2,6-dimethylchloroacetanilide 1 (10 mmol), diethyl-dio- amine (15 mmol) in 25 mL of dry tetrahydrofuran (THF) are stir at room temperature for 3 days. TLC [30:70:1, ethyl acetate:hexane:triethylamine (TEA)] shows complete reaction. Solvents are removed by rotoevaporation and the residue redissolved in chloroform, washed with dilute KOH, water, and dried (MgSO₄). Evaporation of solvents affords crude amine in good yield as a white solid. Method B: A solution of 10 mmol of amine prepared in Example 6 and 40 mmol of d₄-acetaldehyde in 35 mL of dry methanol are stirred at ice-water bath temperature and 15 mmol of NaBD was added. The reaction was warmed to room temperature and IN HCl was added. Solvents were concentrated under vaccum and CHC is added. The CHC|₃ layer was separated and dried (MgSO₄). Concentration gives the title compound. Example 4 Decadeutrolidocaine N-oxide

Using the procedure in Example 1 and replacing lidocaine with dio-lidocaine prepared in Example 3 gives the title compound. Example 5 2-Azido-N-(2.6-dimethylphenyl)-acetamide

A solution of sodium azide (5.5 g, 85 mmol) and 2,6-dimethylchloroacetanilide 1

(12.9 g, 65 mmol) in 40 mL of anhydrous DMSO was heated to 40-45 °C for 24 hours. The reaction was cooled to room temperature, chloroform (350 L) was added followed by 400 mL of water. The organic layer was separated, dried (MgSO₄) and concentrated under reduced pressure to give a white solid which was recrystallized from ethyl acetate/hexane.

Yield 1 1.1 g: ^XH NMR (400 MHz, CDC1₃) δ 7.63 (brs IH) 7.14-7.06 (m 3H) 4.15 (s 2H) 2.21 (s 6H); LC/MS (M+H)+ 205. Anal. Calcd for Cι₀Hι₂N₄O: C, 58.81; H, 5.92; N, 27.43. Found: C, 58.81; H, 5.63; N, 27.51.

Example 6 2-Amino-N-(2.6-dimethylphenyl)-acetamide

The azide prepared in Example 5 (3.0 g, 14.6 mmol) was dissolved in ethanol (40 mL) and palladium catalyst (10%o Pd/C, 300 mg) suspended in ethanol (10 mL) was added with stirring. The flask was evacuated and rinsed 3 times with hydrogen from the balloon. The reaction mixture was stined under hydrogen atmosphere overnight and then filtered through a pad of celite. The filtration cake was washed several times with ethanol and combined filtrates were evaporated to yield the crude product which crystallized under diethyl ether. Yield: 1.422 g (54%); 'HNMR (400 MHz, CDC1₃): 8.80 (bs, IH), 7.06 - 7.10 (m, 3H), 3.56 (s, 2H), 2.26 (s, 6H); LC/MS (M+H)⁺ 179. Anal. Calcd. for Cι₀H₁₄N₂O C, 67.39; H, 7.92; N,

15.72. Found: C, 67.09; H, 8.13; N, 15.60.

Example 7 Percent Inhibition of Lidocaine. Lidocaine N-oxide. Dibucaine. and Dibucaine N-oxide at 1 mM concentration in the Sodium Channel Blockade Assay Local anesthetics cause numbing by blocking sodium channel activity. Xenopus oocytes were used as an expression system to study the effect of test articles on the alpha subunit of the NAV 1.4 sodium channel derived from human skeletal muscle. Oocytes were harvested from female Xenopus laevis (Xenopus I, Dexter,MI), previously injected with human chorionic gonadotropin. Frogs were anesthetized by immersion in 0.2%> 3-aminobenzoic acid ethyl ester and the ovarian tubes surgically removed. Oocytes were dissociated by gentle agitation for 1 hour in 1 mg/ml collagenase D (Boehringer-Mannheim), and then washed extensively in Ca²⁺ free OR-2 solution (96 mM NaCl, 2 mM KC1, 1 mM MgCl₂, 5 mM HEPES, pH 7.4). Stage V and VI oocytes were collected with the aid of a dissecting microscope. Plasmid containing cDNA for the NAV 1.4 alpha subunit of the human skeletal muscle Na channel was linearized, and capped cRNAs synthesized in vitro (Message Machine RNA polymerase kit; Ambio, Austin TX). RNA was purified with an RNAid kit (BiolOl, Vista, CA). Individual oocytes were injected with cRNA (50 nL) and maintained at 18°C in frog saline solution (96 M NaCl, 1 mM KC1, 1 mM CaCl₂, mM MgCl₂, 10 mM Hepes, ImM theophylline, 2 mM Na pyruvate, pH 7.4, 50U/ml penicillin G, and 50 ug/mL streptomycin. Electrophysiological recordings were performed at 2 days post- cRNA injection. Sodium channel currents were recorded from oocytes with a two-electrode voltage clamp using a Geneclamp 500B amplifier (Axon Instruments, Foster City, CA). Voltage-measuring and cunent passing electrodes were filled with 3 M KC1 and adjusted to a resistance of 0.3 to 1 M. Currents were sampled at 5 kHz and filtered at 1-2 kHz. Oocytes were perfused continuously with an external solution containing 96 mM NaCl, 2 mM KC1, 2 mM CaCi2, 1 mM MgC , 10 mM HEPES, pH 7.4. Oocytes were clamped at -70 mV and step depolarized to -20mV to activated the channels. Compounds were tested with five (5) replicates, and each experiment was repeated in triplicate. Example ICsn (uM) % Inhibition (1 mM) SEM(%) 1 >1,000 0 0 Dibucaine 88 93 3 Lidocaine 290 98 9 Example 8 Pharmacokinetics of Lidocaine and Lidocaine N-oxide in Rats

Preparation of Dosing Formulations

To prepare the vehicle, 0.9% sodium chloride for Injection, USP, the required amount of vehicle was aseptically dispensed into an amber glass serum bottle while under a laminar flow hood. The vehicle was dispensed prior to handling the test article. To prepare the lidocaine-N-oxide and higher dose lidocaine hydrochloride solutions, the required amount of test article was weighed directly into a beaker. An appropriate amount of vehicle was added to the beaker and the contents were stirred using a magnetic stir bar and stir plate until dissolved. The pH was measured and adjusted to between 6.0 and 6.5 using IN hydochloric acid and/or 2N sodium hydroxide, as appropriate. The solution was transferred to a graduated cylinder and additional vehicle was added to yield the required volume of prepared test article. The cylinder was shaken thoroughly and the contents were filtered under a laminar flow hood through a 0.2 (m syringe filter into a sterile amber glass serum bottle. To prepare the lower dose lidocaine hydrochloride solution, the required volume of higher dose lidocaine hydrochloride solution was measured using a sterile needle and syringe and transfened into a graduated cylinder. Vehicle was added to the cylinder to yield the required volume of prepared test article. The cylinder was shaken thoroughly and the contents were filtered under a laminar flow hood through a 0.2 (m syringe filter into a sterile amber glass serum bottle. Analysis of Dosing Formulations

A. Homogeneity On Days 1 and 2, prior to initiation of test article administration, duplicate samples (1 mL each) of the dose formulation were collected from the top and bottom of the container, using a syringe, and were placed in a brown bottle. The samples were stored refrigerated until analyzed to determine homogeneity. No samples from the control formulation were analyzed.

B. Stability Because the dose formulations were administered as a single one-time dose, and because the dose formulations were analyzed predose and postdose, no separate stability analysis was required.

C. Concentration On Days 1 and 2, duplicate predose and postdose samples (1 mL each) of each formulation were collected from the container using a syringe, and were placed in a brown bottle. The samples were stored refrigerated until analyzed to determine the concentration of test article. The predose and postdose samples were analyzed the day of dosing. No samples from the control formulation were analyzed.

D. Analysis Procedure 1. Lidocaine and Lidocaine N-oxide Standard curves for lidocaine and lidocaine-N-oxide were analyzed by HPLC to determine the retention time and UV response of the test materials. A stock solution of lidocaine was prepared by adding 14.7 mg of lidocaine to a vial and then diluting with 14.7 mL of HPLC grade water to give a final concentration of 1.0 mg/mL. A stock solution of lidocaine-N-oxide was prepared by adding 21.3 mg of lidocaine-N-oxide to a vial and then diluting with 21.3 mL of HPLC grade water to give a final concentration of 1.0 mg/mL. Serial dilutions of each stock solution were prepared at 0.5, 0.2, and 0.05 mg/mL. A 10 μL aliquot of each of the diluted stock solutions was analyzed by HPLC to generate a standard curve for analysis of the samples. Aliquots of the 2 mg/mL lidocaine dose solutions (0.5 mL) were diluted with 4.5 mL of HPLC grade water. Aliquots of the 20 mg/mL lidocaine and lidocaine-N-oxide dose solutions (0.5 mL) were diluted with 49.5 mL of water. A 10 μL aliquot of each dilution was then analyzed by HPLC to determine the concentration of the dose solution aliquots. Analysis of rat plasma Standard curves for DMA, GX, and MEGX were analyzed by LC-API/MS/MS to determine the retention time and responses of the test materials. Stock solutions of DMA, GX, and MEGX were prepared in HPLC grade methanol at known concentrations of approximately 100 μg/mL. A calibration curve stock solution having known concentrations of 1000 ng/mL DMA, 10,000 ng/mL MEGX and 10,000 ng/mL GX, was prepared by combining aliquots of the individual stock solutions and diluting with 50:50 methanol/water. The calibration curve spiking solutions were prepared by serially diluting the calibration curve stock solution with 50:50 methanol water to known concentrations ranging from 50 to 0.5 ng/mL for DMA and ranging from 500 to 5 ng/mL for MEGX and GX. An internal standard stock solution of lidocaine-dio was prepared in HPLC grade methanol at a known concentration of approximately 100 μg/mL. The internal standard spiking solution was prepared by diluting the stock solution with 50:50 methanol/water to a known concentration of approximately 200 ng/mL. Calibration curve standard solutions were prepared by combining 50 μL of blank plasma, 25 μL of internal standard spiking solution, and 25 μL of one of the calibration curve spiking solutions. Sample solutions were prepared by combining 50 μL of sample plasma, 25 μL of internal standard spiking solution, and 25 μL of 50:50 methanol/water. Each calibration curve standard solution or sample solution was analyzed according to the following procedure: 200 μl of 3:1 acetonitrile/methanol was added to the solution. The solution was vortexed for 1 minute, allowed to stand at room temperature for at least 5 minutes, and then centrifuged at 3400 RPM for 5 minutes. 100 μl of the supernantant was combined with 400 μl of 5 mM ammonium acetate solution, and mixed. A 50 μL aliquot of each standard or sample solution was injected and analyzed by LC -API/MS/MS. A 2 x 100 mm, 5 μm Betasil Cι₈ column was installed in the HPLC; the mobile phase was 0.1 %_> formic acid/18%) acetonitrile/water mobile phase at a flow rate of 0.6 ml/minute. The effluent was nebulized in the atmospheric pressure ionization interface of the MS, and the resulting ions were analyzed by tandem mass spectrometry. For each analyte, a specific ion was isolated and fragmented, and the signal from a specific fragment ion was used to quantify the concentration of the analyte or internal standard. The m/z's of the parent/child ions selected were: DMA, 122/105; GX, 179/122; MEGX, 207/58; lidocaine-dio, 245/96. The ratios of the peak area of each analyte to the peak area of the internal standard were calculated for each injection. The known concentrations and peak area ratios of each analyte in the standard solutions were used to calculate a calibration curve by least squares regression analysis. The concentration of each analyte in each sample was calculated from its peak area ratio using the calibration curve. E. Reserve Sample and Test Article Disposition

A reserve sample from each batch of test article used for this study was taken and archived. A reserve sample of the prepared dosing formulations was not taken. Experimental Design

A. Animal Acquisition and Acclimation

Eighty-five male CD^®[Crl: CD^®(SD)IGS BR] rats, approximately six weeks of ®age, were received from Charles River Laboratories, Portage, Michigan. The rats were weighed the day after arrival. During the one week acclimation period, the rats were observed daily for any clinical signs of disease and given a detailed clinical examination prior to selection for study. Randomization, Assignment to Study, and Maintenance

Rats considered suitable for study were weighed prior to selection. All rats placed on study had body weights that fell within (20%) of the mean body weight. A standard, by weight, simple randomization procedure was used to select the rats for study. Seventy-six male rats, weighing 149 to 172 grams at randomization, were assigned to the groups as identified in the following table.

Group Assignment Group Number Dose Level Dose Volume Number of Males (mg kg) Dose Route (mL/kg) 1 24 2 Lidocaine Intravenous 1 2 24 20 Lidocaine Intratracheal 1 3 24 20 Lidocaine-N- Intratracheal 1 Oxide 4 4 0 (Control) Intratracheal 1

Two males at 20 mg/kg lidocaine were replaced on study on day 2. Animal number 941 died during the predose blood collection and animal number 943 died immediately following dosing. These males were replaced on study with male animals numbered 10941 and 10943, respectively. Each animal was individually identified by an ear tag. Each cage was identified by the study number, animal number, group number, and sex. The individual animal number plus the study number comprised a unique identification number for each animal. The rats were individually housed in suspended, stainless steel cages with wire mesh bottoms. Fluorescent lighting was provided for approximately 12 hours per day via an automatic timer. Temperature and humidity were monitored and recorded daily, and maintained between 68 to 77 °F and 30 to 56%, respectively. Block Lab Diet^® (Certified Rodent Diet #5002, PMI Nutrition International) and water were available ad libitum to all rats. The lot number from each diet lot used was recorded. Certification analysis of each diet lot was performed by the manufacturer. The water supply is monitored for the presence of specific contaminants at periodic intervals according to SOPs. The results of food and water analyses applicable to the study are maintained in the archives. Test Article Administration

A. Justification for Route of Administration The intratracheal route is the intended route of administration of this test article in man. The intravenous route was used to determine lung/plasma bioavailability. B. Justification for Dose Levels The dose levels were selected based on available data from previous studies. C. Dose Administration

The test article was administered as a single dose to half of the rats in each group on day 1 and the other half in each group on day 2. The first group received the test article via intravenous injection into the tail vein; the other three received the test article via intratracheal injection. The first two groups received lidocaine at dose levels of 2 and 20 mg/kg and the third group received lidocaine-N-oxide at the dose level of 20 mg/kg. The dose volume for all treated groups was at 1 mL/kg. A fourth group served as a control and received the vehicle of saline via intratracheal injection at the same dose volume as the treated groups. In-life Examinations

A. Mortality and Cageside Observations

All rats were observed for morbidity, mortality, injury, and the availability of food and water twice a day throughout the duration of the study.

B. Body Weights Individual body weights were measured and recorded for all rats the day after receipt

(randomization) and prior to administration of the test article. The body weights recorded the day after receipt are not reported but are maintained in the study file. Sample Collection

A. Blood Collection Whole blood (approximately 1 to 2 mL) was collected from all rats predose, from all treated rats at approximately 5, 10, and 30 minutes, and 1, 2, 4, 8, and 24 hours postdose (three rats per group per time point), and from all control rats at approximately 24 hours postdose. The blood was collected from the jugular vein into mbes containing EDTA. Each blood sample was stored on ice until being centrifuged for 10 minutes, at approximately 4°C and at 3000 rpm. The samples were stored on wet ice until centrifuged. The centrifugation was completed within one hour of collection.

B. Tissue Collection and Animal Disposition Following euthanasia by intraperitoneal injection of sodium pentobarbital, the lungs were removed, blotted dry, weighed, and stored frozen until shipment for analysis. C. Sample Identification, Storage, and Shipment All samples were identified with the study number, test article, animal number, group number, sample matrix (plasma or lung), sample weight or volume, and collection interval. All samples were stored at approximately -20° C, protected from light, before and after analysis. The samples were shipped on dry ice to Absoφtion Systems LP, Exton, Pennsylvania, for analysis of test article concentration.

D. Sample Analysis by Absoφtion Systems LP

Absoφtion Systems LP analyzed all plasma and lung samples: Pharmacokinetic analysis was performed on the plasma concentration of lidocaine or lidocaine N-oxide using the average plasma concentration for all three rats in the group for each time point. The data were subjected to non-compartmental analysis using the pharmacokinetic program

WINNONLIN.

E. Computer Systems

The computer systems used during the conduct of this study are presented in the following table.

Computer Systems

In-life System Provantis™ Randomization Provantis™ Reporting Microsoft Office Professional

F. Data and Specimen Retention

All raw data, documentation, records, protocol, reserve samples, tissues, and the final report generated as a result of this study will be retained at MPI Research, Inc., or an approved archive facility, for a period of one year following completion of the study (final report issue date). All data generated by Absoφtion Systems LP will be retained at that laboratory for at least the same time period. Retention of materials after the times stated above will be subject to future contractual agreements between the Sponsor and MPI Research, Inc.

Example 9 Formulation and Solution Stability of Lidocaine N-oxide Two lidocaine stock solutions were prepared in ethanol and water by dissolving 31.8 mg of lidocaine N-oxide in 1 mL of ethanol or by dissolving 24.6 mg lidocaine N-oxide in

250 μL of water. The following test solutions were prepared from the stock solutions and when necessary, the test solutions were shaken, stirred or sonicated to facilitate dissolution of undissolved solids.

From the ethanol stock solution: Solution A was prepared by mixing 200 μL of the stock solution with 50 μL ethanol. Solution B was prepared by mixing 200 μL of the stock solution with 50 μL glycerol. Solution C was prepared by mixing 200 μl of the stock solution with 50 μL propylene glycol. Solution D was prepared by mixing 200 μL of the stock solution with 50 μL polyethylene glycol 300. From the aqueous stock solution: Solution E was prepared by mixing 50 μL of stock solution with 200 μL water. Solution F was prepared by mixing 50 μL of stock solution with 200 μL of a solution consisting of 5.0 mg ZnC dissolved with 200 μL water. Solution G was prepared by mixing 50 μL of stock solution with 200 μL of a solution consisting of 7.2 mg MgCi₂(6H₂0) dissolved in 200 μL water. Solution H was prepared by mixing 50 μL of stock solution with 200 μL of a solution consisting of 7.2 mg citric acid dissolved in 200 μL water Additional solutions were prepared as follows: Solution I was prepared by dissolving 123.7 mg lidocaine N-oxide in 4 mL water, adjusting the pH to 1.1 with 6 N hydrochloric acid, and diluting to 5 mL with water. Solution J was prepared by dissolving 36.6 mg lidocaine N-oxide in 1.5 mL of a solution consisting of 118.2 mg maleic acid dissolved in 5 mL water.

A. Analytical Method for Stability Study 20 μL of each sample solutions was diluted with 20 mL water and analyzed by HPLC with UV and MS detection. A gradient HPLC method was used. Mobile phase A was 0.1% formic acid/5%> acetonitrile/water. Mobile phase B was 0.1% formic acid/acetonitrile. The column was a Zorbax Stablebond SB-C8, 3.5 μm, 2.1 x 50 mm. The gradient program was as follows: 0 min, 2% B; 3 min, 32% B, 6 min, 92% B; 8 min, 92% B; 8.01 min, 2% B; 10 min, 2% B. The flow rate was 0.4 ml/min. 10 μl of each sample was injected. The column was maintained at 30°C by a column oven. The effluent from the column passed through the UV detector and was nebulized in the electrospray interface of the mass spectrometer. The resulting positive ions were analyzed using an ion trap. The lidocaine N-oxide concentration of the samples was compared using the 250 nm UV absorbance signal. The relative amounts of degradation products were determined from the MS signals at m/z 178, 410, or 413.

B. Stability Study Solutions A-J (Table 2) were sealed in vials and stored at 55°C. After 1, 2, or 5 days, the solutions were analyzed by HPLC with UV and MS detection. Portions of the same

solutions, stored at lower temperatures (-20°C or +4°C) were analyzed for comparison. The

percent recovery was calculated by taking the ratio of the lidocaine N-oxide UV peak area for the heated solution to the peak area for the cooled solution.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A pharmaceutical composition comprising an N-oxide of a member selected from the group consisting of lidocaine, dibucaine, procaine, procainamide, tetracaine, bupivacaine the decadeuterated forms thereof and pharmaceutical acceptable salts thereof, and a pharmaceutically acceptable excipient.

2. An aerosol formulation for the prevention and treatment of pulmonary inflammation in asthma patients, said formulation comprising from about 10 mg to about 500 mg of an N-oxide of a composition of claim 1 prepared as liposomes, microscopic particles or other suitable carrier wherein the N-oxide is suspended in about 5 ml of solution containing about 0.225%) (w/v) of sodium chloride; said formulation having a pH between about 5.0 and 7.0; 4.5 to 8.5 said formulation to be administered by aerosolization using a jet, ultrasonic, pressurized, or vibrating porous plate nebulizer able to produce predominantly aerosol particles between 1 and 5 μ .

3. An aerosol formulation for the prevention and treatment of pulmonary inflammation in asthma patients, said formulation comprising from about 10 mg to about 500 mg of a composition of claim 1 prepared as a dry powder for aerosol delivery in a physiologically compatible and tolerable matrix; said formulation to be administered by aerosolization using a dry powder inhaler able to produce predominantly aerosol particles between 1 and 5 μ .

4. An aerosol formulation for the prevention and treatment of pulmonary inflammation in asthma patients, said formulation comprising from about 10 mg to about 500 mg of a composition of claim 1 as a lyophilized powder for reconstitution as a 5 ml solution containing about 0.225%) (w/v) of sodium chloride; said formulation having pH between about 1.0 and 7.0; 4.5 to 8.5 said formulation to be administered by aerosolization using a jet, ultrasonic, or vibrating porous plate nebulizer able to produce predominantly aerosol particles between 1 and 5 μ .

5. A method for the prevention and treatment of pulmonary inflammation comprising administering to a patient in need of such treatment an effective amount of an aerosol formulation comprising about 10-500 mg of a composition as in claim 1.

6. A method for the prevention and treatment of pulmonary inflammation comprising administering to a patient in need of such treatment an effective amount of an aerosol formulation comprising about 10-500 mg of a composition as in claim 1 in combination with a β-agonist such as salbutamol, to be delivered as a mixture or sequentially by aerosol.

7. A method for the prevention and treatment of pulmonary inflammation comprising orally administering to a patent in need of such treatment an effective amount of a composition as in claim 1.

8. A pharmaceutical composition as in claim 1 wherein the composition comprises about 25mg/mL lidocaine N-oxide in 0.2 M aqueous maleic acid.

9. A pharmaceutical composition as in claim 1 wherein the N-oxide is decadeutrolidocaine N-oxide.

10. Decadeutrolidocaine N-oxide and its pharmaceutically acceptable salts.