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

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

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Publication number
WO2005044233A1
WO2005044233A1 PCT/US2004/036926 US2004036926W WO2005044233A1 WO 2005044233 A1 WO2005044233 A1 WO 2005044233A1 US 2004036926 W US2004036926 W US 2004036926W WO 2005044233 A1 WO2005044233 A1 WO 2005044233A1
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lidocaine
oxide
formulation
treatment
aerosol
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PCT/US2004/036926
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English (en)
French (fr)
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William R. Baker
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Corus Pharma
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Priority to AU2005310312A priority Critical patent/AU2005310312A1/en
Priority to PCT/US2005/003532 priority patent/WO2006060027A2/en
Priority to EP05790821A priority patent/EP1807123A4/en
Priority to CA002581053A priority patent/CA2581053A1/en
Priority to JP2007532308A priority patent/JP2008513445A/ja
Publication of WO2005044233A1 publication Critical patent/WO2005044233A1/en
Priority to NO20071654A priority patent/NO20071654L/no

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/04—Nitro compounds

Definitions

  • the current invention relates to the preparation of novel prodrugs of lidocaine, dibucaine, and related compounds for delivery to the lung and by aerosolization.
  • 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 ⁇ .
  • 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 ⁇ -agonists for treatment of acute and chronic respiratory tract inflammation associated with mild to severe asthma, bronchitis, and chronic obstructive pulmonary disease (COPD).
  • COPD chronic obstructive pulmonary disease
  • 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.
  • 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).
  • 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).
  • IL-5 Interleukin 5
  • IL-3 Interleukin-3
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • 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-
  • lidocaine 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.
  • lidocaine 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.
  • 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 ⁇ 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.
  • 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
  • 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
  • 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 ⁇ -agonist, a pharmaceutically accepted salt thereof, and a pharmaceutically accepted carrier.
  • 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.
  • 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.
  • treating 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.
  • treatment refers to the act of treating, as “treating” is defined immediately above.
  • normal saline means water solution containing 0.9% (w/v) NaCl.
  • diluted saline means normal saline containing 0.9% (w/v) NaCl diluted into its lesser strength.
  • 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.
  • racemic mixtures, mixtures of diastereomers, as well as single diastereomers of the compounds of the invention are included in the present invention.
  • S and R are as defined by the IUPAC 1974 RECOMMENDATIONS FOR SECTION E, FUNDAMENTAL STEREOCHEMISTRY, Pure Appl. Chem. 45: 13-30 (1976).
  • 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).
  • 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).
  • lidocaine N-oxide 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).
  • 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 co ⁇ elated with the decrease in concentration of lidocaine N-oxide (Table 3).
  • 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.
  • N- oxide prodrugs of analgesic morphinans have shown improved oral bioavailability (Boswell, 1988).
  • 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.
  • the bioavailability of lidocaine and lidocaine N-oxide was determined in rats after dosing intratracheally.
  • 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).
  • 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).
  • 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.
  • lidocaine metabolites DMA, MEGX, and GX 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).
  • lidocaine was delivered to male rats by three different routes, intravenous lidocaine, intratracheal lidocaine, and intratracheal lidocaine-N-oxide.
  • lidocaine When delivered intravenously, lidocaine exhibited rapid clearance, with low to moderate volume of distribution and Ty 2 ( 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.
  • lidocaine was systemically cleared from the lung at an initial rapid rate, reaching similar plasma levels to intravenous delivery after 2 hours.
  • lidocaine 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.
  • 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 ⁇ -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 ⁇ .
  • 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 ⁇ 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 ⁇ 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.
  • 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.
  • 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 (MgSO 4 ). 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 d 4 -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
  • Example 4 Decadeutrolidocaine N-oxide
  • Example 5 2-Azido-N-(2.6-dimethylphenyl)-acetamide
  • Example 5 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.
  • 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 Ca 2+ free OR-2 solution (96 mM NaCl, 2 mM KC1, 1 mM MgCl 2 , 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).
  • oocytes were injected with cRNA (50 nL) and maintained at 18°C in frog saline solution (96 M NaCl, 1 mM KC1, 1 mM CaCl 2 , mM MgCl 2 , 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.
  • 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.
  • 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.
  • 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 ⁇ g/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 ⁇ L of blank plasma, 25 ⁇ L of internal standard spiking solution, and 25 ⁇ L of one of the calibration curve spiking solutions.
  • Sample solutions were prepared by combining 50 ⁇ L of sample plasma, 25 ⁇ L of internal standard spiking solution, and 25 ⁇ L of 50:50 methanol/water. Each calibration curve standard solution or sample solution was analyzed according to the following procedure: 200 ⁇ l 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 ⁇ l of the supernantant was combined with 400 ⁇ l of 5 mM ammonium acetate solution, and mixed. A 50 ⁇ L aliquot of each standard or sample solution was injected and analyzed by LC -API/MS/MS.
  • a 2 x 100 mm, 5 ⁇ m Betasil C ⁇ 8 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.
  • CD ® (SD)IGS BR] rats 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.
  • 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
  • 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.
  • 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.
  • test solutions 250 ⁇ L 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.
  • Solution A was prepared by mixing 200 ⁇ L of the stock solution with 50 ⁇ L ethanol.
  • Solution B was prepared by mixing 200 ⁇ L of the stock solution with 50 ⁇ L glycerol.
  • Solution C was prepared by mixing 200 ⁇ l of the stock solution with 50 ⁇ L propylene glycol.
  • Solution D was prepared by mixing 200 ⁇ L of the stock solution with 50 ⁇ L polyethylene glycol 300.
  • Solution E was prepared by mixing 50 ⁇ L of stock solution with 200 ⁇ L water.
  • Solution F was prepared by mixing 50 ⁇ L of stock solution with 200 ⁇ L of a solution consisting of 5.0 mg ZnC dissolved with 200 ⁇ L water.
  • Solution G was prepared by mixing 50 ⁇ L of stock solution with 200 ⁇ L of a solution consisting of 7.2 mg MgCi 2 (6H 2 0) dissolved in 200 ⁇ L water.
  • Solution H was prepared by mixing 50 ⁇ L of stock solution with 200 ⁇ L of a solution consisting of 7.2 mg citric acid dissolved in 200 ⁇ L 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.
  • 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.
  • 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.

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  • Health & Medical Sciences (AREA)
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  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Otolaryngology (AREA)
  • Pulmonology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
PCT/US2004/036926 2003-11-04 2004-11-04 Formulations of n-oxide prodrugs of local anesthetics for the treatment of pulmonary inflammation associated with asthma, brochitis, and copd WO2005044233A1 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
AU2005310312A AU2005310312A1 (en) 2004-09-20 2005-01-28 A method for improvement of tolerance for therapeutically effective agents delivered by inhalation
PCT/US2005/003532 WO2006060027A2 (en) 2004-09-20 2005-01-28 A method for improvement of tolerance for therapeutically effective agents delivered by inhalation
EP05790821A EP1807123A4 (en) 2004-09-20 2005-01-28 METHOD FOR IMPROVING TOLERANCE ON THERAPEUTICALLY EFFECTIVE MEANS THROUGH INHALATION
CA002581053A CA2581053A1 (en) 2004-09-20 2005-01-28 A method for improvement of tolerance for therapeutically effective agents delivered by inhalation
JP2007532308A JP2008513445A (ja) 2004-09-20 2005-01-28 吸入による送達で治療効果のある薬剤の許容性の改善方法
NO20071654A NO20071654L (no) 2004-09-20 2007-03-29 Fremgangsmate for a forbedre toleransen for terapeutiske effektive midler levert ved inhalering

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US60/517,284 2003-11-04

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103601650A (zh) * 2013-01-16 2014-02-26 四川大学华西医院 N-二乙氨基乙酰-2,6-二甲基苯胺衍生物、制备方法及用途
WO2019077356A1 (en) * 2017-10-18 2019-04-25 Biotherics Limited LIDOCAINE N-OXIDE FOR USE IN THE PROPHYLAXIS OF CARDIAC SUDDEN DEATH
CN112521315A (zh) * 2019-09-17 2021-03-19 鲁南制药集团股份有限公司 一种利多卡因降解杂质的制备方法
IT202100031637A1 (it) 2021-12-17 2023-06-17 Universita’ Degli Studi Di Parma Polveri per inalazione e procedimento di produzione

Citations (2)

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Publication number Priority date Publication date Assignee Title
US4722928A (en) * 1985-12-02 1988-02-02 E. I. Du Pont De Nemours And Company N-oxide prodrug derivatives of 3-hydroxy morphinans and partial morphinans having improved oral bioavailability, pharmaceutical compositions, and processes
US5837713A (en) * 1997-02-26 1998-11-17 Mayo Foundation For Medical Education And Research Treatment of eosinophil-associated pathologies by administration of topical anesthetics and glucocorticoids

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4722928A (en) * 1985-12-02 1988-02-02 E. I. Du Pont De Nemours And Company N-oxide prodrug derivatives of 3-hydroxy morphinans and partial morphinans having improved oral bioavailability, pharmaceutical compositions, and processes
US5837713A (en) * 1997-02-26 1998-11-17 Mayo Foundation For Medical Education And Research Treatment of eosinophil-associated pathologies by administration of topical anesthetics and glucocorticoids

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103601650A (zh) * 2013-01-16 2014-02-26 四川大学华西医院 N-二乙氨基乙酰-2,6-二甲基苯胺衍生物、制备方法及用途
CN103601650B (zh) * 2013-01-16 2014-08-06 四川大学华西医院 N-二乙氨基乙酰-2,6-二甲基苯胺衍生物、制备方法及用途
WO2019077356A1 (en) * 2017-10-18 2019-04-25 Biotherics Limited LIDOCAINE N-OXIDE FOR USE IN THE PROPHYLAXIS OF CARDIAC SUDDEN DEATH
GB2569225A (en) * 2017-10-18 2019-06-12 Biotherics Ltd Prophylactic treatment
GB2569225B (en) * 2017-10-18 2020-05-13 Biotherics Ltd Prophylactic treatment
JP2021500361A (ja) * 2017-10-18 2021-01-07 バイオセリックス リミテッド 心臓突然死の予防に用いるリドカインｎ−オキシド
US11266613B2 (en) 2017-10-18 2022-03-08 Biotherics Limited Lidocaine n-oxide for use in the prophylaxis of sudden cardiac death
JP7201261B2 (ja) 2017-10-18 2023-01-10 バイオセリックス リミテッド 心臓突然死の予防に用いるリドカインｎ－オキシド
AU2018351893B2 (en) * 2017-10-18 2023-12-07 Biotherics Limited Lidocaine N-oxide for use in the prophylaxis of sudden cardiac death
CN112521315A (zh) * 2019-09-17 2021-03-19 鲁南制药集团股份有限公司 一种利多卡因降解杂质的制备方法
IT202100031637A1 (it) 2021-12-17 2023-06-17 Universita’ Degli Studi Di Parma Polveri per inalazione e procedimento di produzione

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