NZ523693A

NZ523693A - Macrolide formulations for inhalation and methods of treatment of endobronchial infections

Info

Publication number: NZ523693A
Application number: NZ523693A
Authority: NZ
Inventors: William R Baker
Original assignee: Chiron Corp
Priority date: 2000-07-10
Filing date: 2001-07-01
Publication date: 2004-08-27
Also published as: KR20030020924A; MXPA03000321A; CN1471401A; AU2001283491B2; BR0112330A; WO2002003998A2; AU8349101A; WO2002003998A3; IL153875A0; CN1202831C; JP2004502736A; EP1309332A2; CA2415498A1

Abstract

Macrolide formulations, such as an erythromycylamine formulation, for delivery by aerosolization are disclosed, wherein the concentrated erythromycylamine formulations contain an amount of erythromycylamine effective to treat pulmonary infections caused by susceptible bacteria. Unit dose devices having a container containing a formulation of the macrolide antibiotic in a physiologically acceptable carrier are also described. Methods for treatment of pulmonary infections by a formulation (liquid solution, suspension, or dry powder) delivered as an aerosol having mass median aerodynamic diameter predominantly between 1 to 5 mm are also described.

Description

<div class="application article clearfix" id="description"> WO 02/03998 -1- 523693 PCT/US01/41328 MACROLIDE FORMULATIONS FOR INHALATION AND METHODS OF TREATMENT OF ENDOBRONCHIAL INFECTIONS Field of the Invention This invention concerns novel and improved macrolide formulations, such as 5 erythromycylamine formulations, for delivery by inhalation and to improved methods of-treatment of susceptible acute or chronic endobronchial infections. In particular, the invention relates to formulations comprising at least one concentrated macrolide antibiotic in a physiologically acceptable liquid solution or dry powder form. The formulations are suitable for delivery of a macrolide antibiotic drug, such as 10 erythromycylamine, to the lung endobronchial airway space of a liquid aerosol or dry powder aerosol form, wherein a substantial portion of the aerosolized droplets or particles of the formulation have a mass median aerodynamic diameter between 1 to 5 |im. Formulated and aerosol delivered efficacious amounts of the macrolides are effective for the treatment and/or prophylaxis of acute and chronic endobronchial 15 infections, and pneumonia, particularly those caused by Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae. The novel formulations have small volume yet deliver an effective dose of the macrolide antibiotic to the site of infection. In yet other aspects, this invention relates to new 20 and improved unit dose formulations of macrolide antibiotics for delivery by aerosol inhalation. Background of the Invention Streptococcus pneumonia and other typical and atypical pathogens infect the endobronchial space in the lung of individuals who suffer from chronic obstructive WO 02/03998 PCT/US01/41328 -2- pulmonary disease (COPD) [S. Chodosh et al. Clinical Infectious Diseases 1998; 27: 730-738]. COPD is most commonly manifested as chronic bronchitis (CB) and emphysema. Chronic. bronchitis is a pulmonary disease that is characterized by the 5 inflammation and progressive destruction of lung tissue. The debilitation of the lungs in CB patients is associated with chronic cough, increased daily sputum production, and accumulation of purulent sputum produced as a result of chronic endobronchial infections caused by compromised pulmonary function. Acute exacerbation of chronic bronchitis (AECB) is often characterized by increasing cough, purulent 10 sputum production, and clinical deterioration caused by Streptococcus pneumonia, H. influenzae, and Moraxella catarrhalis. Pneumonia may also result from infection by these organisms either de novo or as a complication of COPD. Despite the controversy over the appropriateness of antimicrobacterial therapy for the treatment of CB and in particular acute exacerbations of CB, Saint et al. (JAMA 1995; 273: 957-15 960) demonstrated that oral antimicrobial therapy provided some clinical benefit when compared to no therapy. Furthermore, the dose of antimicrobial agent was important with respect to time to relapse. Thus, higher doses of oral antimicrobial agents were associated with a higher median infection free-interval (S. Chodosh et al., Clinical Infectious Diseases 1998; 21'. 730-738). 20 Presently, oral administration of macrolides and fluoroquinolones active against typical and atypical pathogens are treatments of choicte for CB. However, oral administration of macrolide antibiotics has adverse side effects. The most common side effects associated with the treatment of oral/parental macrolide antibiotics are diarrhea/loose stools, nausea, abdominal pain and vomiting (R. N. Brogden D. Peters, 25 Drugs, 1994; 48: 599-616 and H. D. Langtry, R. N. Brogden Drugs 1997; 53: 973-1004 and references cited therein). In addition, pseudomembranous colitis is a serious side effect associated with oral antibiotic therapy including oral macrolide therapy (S. H. Ahmad et al. Indian J. Pediatr. 1993, 60: 591-594). Penetration of macrolides into lung tissue after oral administration varies according to dose and composition (R. N. 30 Brogden D. Peters, Drugs, 1994; 48: 599-616 and H. D. Langtry, R. N. Brogden Drugs 1997; 53: 973-1004 and references cited therein). Furthermore, macrolides are associated with alterations in the systemic concentrations of unrelated drugs, such as theophylline, due to interactions with the cytochrome-based metabolic system of the liver. Such drug-drug interactions often require dosage adjustment or elimination of 35 one component from treatment regimes. WO 02/03998 PCT/US01/41328 -3- Erythromycylamine is a 14-membered ring macrolide belonging to the erythromycin family of antibiotics and possesses a similar in vitro antibiotic spectrum to erythromycin A, and like erythromycin A, is an effective treatment of typical and atypical pneumonias. Erythromycylamine has a C-9 amino function having the S-5 configuration in place of the C-9 carbonyl group found in erythromycin A. One significant limitation of erythromycylamine is its lack of oral absorption, thus, in order to achieve useful therapeutic concentrations a prodrug, dirithromycin, was developed. The prodrug of erythromycylamine is dirithromycin, which features a bridged acetal function between the C-9 amino and C-l 1 hydroxy groups (see Fig. 1). 10 The cyclic acetal is rapidly hydrolysized in plasma by a nonenzymatic process (half-life of approximately 30 minutes). Dirithromycin has been shown to successfully treat exacerbations that occur in patients with CB (M. Cazzola et al., Respiratory Medicine-, 1998; 92: 895-901). A major advantage of erythromycylamine is its long half-life (30-44 hours) (R. N. Brogden D. Peters, Dmgs, 1994; 48: 599-616). 15 Unfortunately, oral bioavailability of dirithromycin is only 10-14% in humans with high elimination (62-81%) into the feces mostly as erythromycylamine. Because erythromycylamine is not absorbed and its prodrug, dirithromycin, is poorly absorbed, limited amounts of active drug substance are available systemically to treat lung infections caused by typical and atypical bacteria. While enough erythromycylamine 20 concentrates at the site of infection to provide a therapeutic effect, the concentration of drug is limited. Higher oral doses or more frequent dosing of dirithromycin increase , drug concentration at the site of action; however, increased adverse events are likely to occur and may increase patient hardship and compliance. One of the first studies using aerosolized antibiotics for the treatment of lung 25 infections was reported in Lancet, 22:1377-9 (1981). A controlled, double-blind study on twenty CF patients demonstrated that aerosol administration of carbenicillin and the aminoglycoside gentamicin can improve the health of CF patients. Since that time, scattered reports in the literature have examined aerosol delivery of aminoglycosides in general and tobramycin in particular (see, for example, U.S. 30 Patent No. 5,580,269). However, evaluation and comparison of these studies is often difficult because of the differences in antibiotic formulations, breathing techniques, nebulizers and compressors. Moreover, aerosol delivery is often difficult to evaluate because of differences in the formulations, aerosol delivery devices, dosages, particle sizes, regimens, and the like. When, for example, the mass median aerodynamic 35 diameter (MMAD) is greater than 5 jj-itl, the particles are typically deposited in the WO 02/03998 -4- PCT/USO1/41328 upper airways, decreasing the amount of antibiotic delivered to the site of infection in the lower respiratory tract. An article published in Arch. Dis. Child., 68:788 (1993) emphasized the need for standardized procedures and for improvement in aerosol administration of drugs to CF patients. 5 Effective aerosol administration is currently compromised by the lack of additive-free and physiologically compatible formulations and particularly by the inability of certain nebulizers to generate small and uniform particle sizes. The size range of aerosolized particles needed to deliver the drug to the endobronchial space and peripheral lung, the sites of the infection is preferably between about 1 and 5 pm. 10 Many nebulizers that aerosolize therapeutics, including aminoglycosides, produce a large number of aerosol particles having sizes less than 1 (im or greater than 5 (am. In order to be therapeutically effective, the majority of aerosolized antibiotic particles should not have a MMAD larger than 5 jj.m. When the aerosol contains a large number of particles with a MMAD larger than 5 nm, the larger-sized particles are 15 deposited in the upper airways, decreasing the amount of antibiotic delivered to the site of infection in the lower respiratory tract. Currently, three types of available nebulizers, jet nebulizers, vibrating porous plate nebulizers and ultrasonic nebulizers, can produce and deliver aerosol particles with diameter sizes between 1 and 5 um, a particle size that is preferable for 20 treatment of bacterial infections of the lung. Therefore, it would be highly advantageous to provide a macrolide formulation that could be efficiently aerosolized in a jet, vibrating porous plate, and ultrasonic nebulizer. In addition, newer aerosol generating technologies are now available, including mechanical extrusion and both passive and energized diy powder inhalers that are useful for the delivery of 25 therapeutic agents in dry powder form. Another requirement for an acceptable formulation is adequate shelf life. Generally, antibiotics, and particularly antibiotic solutions for intravenous administration, contain phenol or other preservatives to maintain potency and to minimize the production of degradation products. However, phenol and other 30 preservatives, when aerosolized, may induce bronchospasm, an unwanted occurrence in patients with lung diseases such as chronic bronchitis. Administration of macrolide antibiotics, such as erythromycylamine,, for inhalation in the form of a liquid or dry powder aerosol has the advantage of overcoming poor oral bioavailability associated with the prodrug, while providing 35 efficacious concentrations of the antibiotic to the lung that can not be achieved by INTELLECTUAL property office of n.z. 1 6 JUN 2004 received either the oral or intravenous route. An additional advantage of aerosol delivery of erythromycylamine is its inherent high affinity for lung tissue and persistence in the plasma department (long plasma/tissue half-life). The combination of a high-concentration aerosol delivery, long plasma/tissue half-life and high lung affinity would 5 allow for safer macrolide therapy, which is capable of eradicating or substantially reducing endobronchial infections after a single aerosol dose. It would be highly advantageous, therefore, to provide macrolide antibiotic formulations, such as erythromycylamine formulations, containing no preservatives, at a pH adjusted to levels that slow or prevent degradation, and are tolerable for a patient, 10 and that provide adequate shelf life suitable for commercial distribution, storage and use. It is therefore an object of this invention to provide concentrated formulations of macrolide antibiotics, such as erythromycylamine, erythromycin A, roxithromycin, azithromycin and clarithromycin, that contain effective concentrations of the macrolide 15 antibiotic in a form that can be efficiently aerosolized by nebulization, such as by the use of jet, vibrating porous plate, or ultrasonic nebulizers, or dry powder inhalers, into aerosol particle sizes predominately within a range from 1 and 5 jam. Summary of the Invention 20 In accordance with the present invention, it has now been discovered that human and non-human animal subjects suffering from or at risk for endobronchial infection, such as an infection by bacterial Streptococcus pneumoniae, Haemophilius influenzae, Staphylococcus aureus, Moraxella catarrhalis and/or the atypical pathogens Legionella pneumophila, Chlamydia pneumoniae, and/or Mycoplasma pneumoniae, can be 25 effectively and efficiently treated by administering to the subject by inhalation an antibacterially effective amount of a macrolide antibiotic, such as erythromycylamine, erythromycin A, roxithromycin, azithromycin or clarithromycin, in a liquid solution or dry powder form suitable for aerosol generation. Thus, one aspect of the current invention relates to concentrated formulations 30 suitable for efficacious delivery by inhalation of a macrolide antibiotic drug, such as erythromycylamine, erythromycin A, roxithromycin, azithromycin or clarithromycin, into the endobronchial space of a subject suffering from or at risk for a bacterial pulmonary infection. Another aspect of the invention provides formulations suitable for efficacious 35 delivery of a macrolide antibiotic drug, such as erythromycylamine, erythromycin A, roxithromycin, azithromycin or clarithromycin, into the endobronchial space of a intellectual property office of n.7. 1 6 JUN 2004 received subject suffering from the bacterial Streptococcus pneumoniae, Haemophilius influenzae, Staphylococcus aureus, Moraxella catarrhalis, and or the atypical pathogens Legionella pneumophila, Chlamydia pneumoniae, and/or Mycoplasma pneumoniae pulmonary infection. 5 Another aspect of the current invention provides formulations suitable for efficacious delivery of a macrolide antibiotic drug, such as erythromycylamine, erythromycin A, roxithromycin, azithromycin or clarithromycin, into the endobronchial space of a subject to prevent or substantially reduce the risk of pulmonary infection in at-risk patients caused by Streptococcus pneumoniae, Haemophilius influenzae, 10 Staphylococcus aureus, Moraxella catarrhalis and/or the atypical pathogens Legionella pneumophila, Chlamydia pneumoniae, and/or Mycoplasma pneumoniae. Still another aspect of the current invention provides liquid formulations comprising the equivalent of 50 to 750 mg of a macrolide antibiotic drug, such as erythromycylamine, erythromycin A, roxithromycin, azithromycin or clarithromycin, in 15 0.5 to 5 ml of a physiologically acceptable carrier, such as saline diluted into a quarter normal saline strength wherein said formulation has a physiologically tolerated osmolarity, salinity, and pH and is suitable for delivery to a subject in concentrated form by aerosol inhalation. Still another aspect of the current invention provides dry powder formulations 20 comprising the equivalent of 25 to 250 mg of a macrolide antibiotic drug, such as erythromycylamine, erythromycin A, roxithromycin, azithromycin or clarithromycin, in a physiologically acceptable dry powder carrier for delivery to a subject in concentrated form by aerosol inhalation, wherein the dry powder formulations comprise about 50 to 90% by weight of the macrolide antibiotic drug. 25 A related aspect of the current invention provides methods for the treatment of pulmonary infections caused by susceptible bacteria by administering to a subject requiring such treatment by inhalation an aerosol formulation comprising an antibacterially effective amount of a macrolide antibiotic drug, such as erythromycylamine, erythromycin A, roxithromycin, azithromycin or clarithromycin, 30 formulated in a physiologically compatible liquid solution or dry powder form, wherein the mass medican aerodynamic diameter (MMAD) of particles in aerosol formulation is predominately between 1 and 5 jam. In other aspects, the present invention provides unit dose formulations and devices adapted for use in connection with a high efficiency inhalation system, the unit 35 dose device comprising a container designed to hold and store the relatively small intellectual property office of n.2. 1 6 JUN im received volume of the macrolide antibiotic formulations of the invention, and to deliver the formulations to an inhalation device for delivery to a subject in aerosol form. In one aspect, a unit dose device of the invention comprises a sealed container, such as an ampoule, containing less than about 2.0 ml of a liquid macrolide antibiotic formulation 5 comprising from about 50 to 150 mg/ml of a macrolide antibiotic in a physiological acceptable liquid carrier. Alternatively, the container of the unit dose device may contain less than about 1.5 ml, or lass than about 1.0 ml, of the liquid macrolide antibiotic formulation, and the macrolide antibiotic formulation may comprise from about 80 to about 180 mg/ml, or from about 90 to about 120 mg/ml, of macrolide 10 antibiotic. In another aspect, a unit dose device of the invention comprises a sealed container, such as an ampoule, containing a dry powder macrolide antibiotic formulation comprising from about 20 to 250 mg of a macrolide antibiotic in a physiologically acceptable dry powder carrier. The sealed unit dose containers of the invention are preferably adapted to deliver the macrolide antibiotic formulation to a 15 high efficiency inhalation device for aerosolization and inhalation by a subject. According to an aspect of the invention there is provided an aerosol formulation for inhibition of susceptible bacteria in the endobronchial space of a subject suffering from an endobronchial infection, said formulation comprising from 50 mg to 750 mg of a macrolide antibiotic and a pharmaceutically acceptable carrier, in the form of 20 particles having a mass median aerodynamic diameter between 1 and 5 |im in size when administered using a jet nebulizer, an ultrasonic nebulizer, a vibrating porous plate nebulizer or a dry powder inhaler. According to another aspect of the invention there is provided a use of an aerosol formulation comprising 50 mg to 750 mg of a macrolide antibiotic and a 25 pharmaceutically acceptable carrier in the manufacture of a medicament for the treatment of susceptible bacterial infections by inhalation from a jet nebulizer, an ultrasonic nebulizer, a vibrating porous plate nebulizer or a dry powder inhaler of particles having a mass median aerodynamic diameter between 1 and 5 jxm in size. According to another aspect of the invention there is provided a unit dose device 30 for inhalation administration, comprising a container containing less than 2.0 ml of a macrolide antibiotic formulation comprising from 50 to 150 mg/ml of a macrolide antibiotic in a liquid physiologically acceptable carrier. According to another aspect of the invention there is provided a unit dose device for inhalation administration, comprising a container containing a macrolide antibiotic 35 formulation comprising from 25 to 250 mg of a macrolide antibiotic in a dry powder physiologically acceptable carrier. INTELLECTUAL property , office of n 7 16 JUN 2004 7a received Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. Brief Description of the Drawings The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same becomes better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein; FIGURE 1 illustrates the chemical structure of erythromycylamine and dirithromycin; FIGURE 2 is a geographical representation of the stability of erythromycylamine hydrochloride in aqueous solution at 60,100, and 150 mg/mL and pH 5.0, 6.0, and 7.0, at 4 degrees centigrade, as described in Example 4; FIGURE 3 is a graphical representation of the stability of erythromycylamine hydrochloride in aqueous solution at 60, 100, and 150 mg/mL and pH 5.0, 6.0, and 7.0, at 25 degrees centigrade, as described in Example 4; FIGURE 4 is a graphical representation of the stability of erythromycylamine hydrochloride in aqueous solution at 60, 100, and 150 mg/mL and pH 5.0, 6.0, and 7.0, at 40 degrees centigrade, as described in Example 4; FIGURE 5 is a graphical representation of the stability of erythromycylamine hydrochloride in aqueous solution at 60, 100, and 150 mg/mL and pH 5.0, 6.0, and 7.0, at 60 degrees centigrade, as described in Example 4; WO 02/03998 PCT/US01/41328 -8- FIGURE 6 is a graphical representation of the stability of erythromycylamine sulfate in aqueous solution at 60, 100, and 150 mg/mL and pH 5.0, 6.0, and 7.0, at 60 degrees centigrade, as described in Example 4; FIGURE 7 is a graphical representation of the stability of erythromycylamine 5 acetate in aqueous solution at 60, 100, and 150 mg/mL and pH 5.0, 6.0, and 7.0, at 60 degrees centigrade, as described in Example 4; FIGURE 8 illustrates mean plasma concentrations of erythromycylamine following a single 25 mg/kg intravenous dose, or single inhalation dose of 30 or 60 mg/ml solution for 30 minutes (0.7 or 1.77 mg/kg pulmonary dose) in rats (n=3), as 10 described in Example 6; FIGURE 9 illustrates mean lung concentrations of erythromycylamine following a single 25 mg/kg intravenous dose, or single inhalation dose of 30 or 60 mg/ml solution for 30 minutes (0.7 or 1.77 mg/kg pulmonary dose) in rats (n=3), as described in Example 6. 15 FIGURE 10 illustrates efficacy of erythromycylamine in the S. pneumoniae pulmonary infection model after 30 minute inhalation administration daily for three (3) days to rats (n = 3) and comparing 5 mg/mL (0.13 mg/kg), 25 mg/mL (0.27 mg/kg), and 50 mg/mL (1.3 mg/kg) inhalation dose as described in Example 7; and FIGURE 11 illustrates efficacy of erythromycylamine in the S. pneumoniae 20 pulmonary infection model after 30 minute inhalation administration as a single dose to rats (n = 3) and comprising 1 mg/mL (0.03 mg/kg), 5 5 mg/mL (0.13 mg/kg), 25 mg/mL (0.27 mg/kg), and 50 mg/mL (1.3 mg/kg) inhalation dose as described in Example 8. FIGURE 12 illustrates the mean plasma and whole lung concentrations of 25 erythromycylamine following a single dose, 30 minute inhalation administration of a 60 mg/mL sulfate solution in dogs, as described in Example 9. FIGURE 13 illustrates the mean lung concentrations of erythromycylamine in individual lung lobes following a single dose, 30 minute inhalation administration of a 60 mg/mL sulfate solution in dogs as described in Example 9. 30 Detailed Description of the Preferred Embodiment Erythromycylamine and dirithromycin are macrolides having a chemical structure depicted in FIG. 1. Dirithromycin, a prodrug of erythromycylamine, is a broad-spectrum macrolide antibiotic used for treatment of AECB and pneumonia. Macrolide antibiotics useful in the present invention include, for example, 35 erythromycylamine, dirithromycin (a prodrug of erythromycylamine), erythromycin WO 02/03998 -9- PCT/US01/41328 A, clarithromycin (6-O-methyl erythromycin), azithromycin, and roxithromycin. Other newer macrolides such as the ketolides (for example, ABT-773 (39th ICAAC (1999), September 26-29, abstracts F-2133-2141, and HMR-3647 (Drugs of the Future, 23, 591 (1998), 38th ICAAC (1998), September 24-27, abstract A-49), and 5 ahhydrolides (see, J. Med. Chem., 1998, 41, 1651-1659 and 1660-1670) may also be used in the practice of the invention. In one aspect of the present invention, the macrolide antibiotic used in the aerosol formulations described herein is erythromycylamine or dirithromycin. Erythromycylamine and dirithromycin have the chemical structures depicted in FIG. 1. 10 In accordance with the present invention, methods are provided for the treatment of a subject in need of treatment, such as a subject suffering from an endobronchial infection, comprising administering to the subject by inhalation an antibacterially effective amount of a macrolide antibiotic formulation. This aspect of the invention is particularly suitable for formulation of concentrated macrolides, such 15 as erythromycylamine, for aerosolization by small volume, breath actuated, high output rate and high efficiency inhalers to produce a macrolide aerosol particle size between 1 and 5 nm desirable for efficacious delivery of the macrolide into the endobronchial space to treat susceptible microbial infections. . The formulations preferably contain minimal yet efficacious amounts of the macrolide formulated in 20 small volumes of a physiologically acceptable solution. For example, an aqueous solution having a salinity adjusted to permit generation of macrolide aerosol particles that are well-tolerated by patients but prevent the development of secondary undesirable side effects such as bronchospasm and cough. By way of example, a quarter normal saline solution is useful for this purpose. By the more efficient 25 administration of the macrolide formulation provided by the present invention, substantially smaller volumes of macrolide than the conventional administration regime are administered in substantially shorter periods of time, thereby reducing the costs of administration and drug waste, and significantly enhancing the likelihood of patient compliance. 30 Thus, in accordance with one aspect of the present invention, methods are provided for the treatment of a subject in need of treatment, such as a subject WO 02/03998 PCT/US01/41328 -10- suffering from a susceptible endobronchial infection, comprising administering to the subject for inhalation a dose of a nebulized aerosol formulation comprising from about 50 to about 750 mg of a macrolide and a pharmaceutically acceptable carrier. In other aspects of the invention, the aerosol formulations administered in the practice 5 of the invention may be liquid formulations comprising from about 50 to about 150 mg/ml of a macrolide antibiotic, preferably from about 70 to about 130 mg/ml of a macrolide antibiotic, and more preferably from about 90 to about 110 mg/ml of a macrolide antibiotic. Preferably, small volumes of aerosol formulation are administered to the subject. Thus, in this aspect a dose of less than about 2.0 ml of a 10 nebulized liquid aerosol formulation is administered to the subject. In another aspect, a dose of less than about 1.5 ml of a nebulized aerosol formulation is administered to the subject. In yet another aspect, a dose of less than about 1.0 ml of a nebulized aerosol formulation is administered to the subject In other aspects, the macrolide compounds of the invention may be formulated 15 for aerosol delivery as a dry powder. As used herein, the term "powder" means a composition that consists of finely dispersed solid particles that are free flowing and capable of being readily dispersed in an inhalation device and subsequently inhaled by a subject so that the particles reach the lungs to permit penetration and deposition in the peripheral airways. Thus, powder formulations of the invention are said to be 20 "respirable." Preferably the average powder particle size is less than about 10 p.m in diameter with a relatively uniform spheroidal shape. More preferably the diameter is less than about 7.5 nm and most preferably less than about 5.0 jim. Usually the particle size distribution is between about 0.1 prn and about 5 |xm in diameter, particularly about 1 fim to about 5 jim. Dry powder formulations of the invention 25 have a moisture content such that the particles are readily dispersible in an inhalation device to form an aerosol. This moisture content will generally be below about 10% by weight (% w) water, usually below about 5% w water and preferably less than about 3% w water. Dry powder formulations of the invention generally comprise a therapeutically 30 effective amount of a macrolide compound of the invention together with a WO 02/03998 PCT/US01/41328 -11- pharmaceutically acceptable carrier. The dry powder formulations of the invention may comprise from about 25 to about 250 mg of a macrolide antibiotic, preferably from about 50 to about 200 mg of a macrolide antibiotic, and more preferably from about 75 to about 150 mg of a macrolide antibiotic. In this aspect of Ihe invention, 5 the dry powder formulations may comprise from about 50% to about 90% by weight of the macrolide antibiotic, preferably from about 60% to about 88% by weight of the macrolide antibiotic, and more preferably from about 75% to about 85% by weight of the macrolide antibiotic. Suitable pharmaceutically acceptable carriers include carriers that can be taken 10 into the lungs of a patient with no significant adverse toxicological effects on the lungs, including, for example, stabilizers, bulking agents, buffers, salts and the like. A sufficient amount of the pharmaceutically acceptable carrier is employed to obtain desired stability, dispersibility, consistency and bulking characteristics to ensure a uniform pulmonary delivery of the composition to a subject in need thereof. The 15 actual amount of pharmaceutically acceptable carrier employed may be from about 0.05% w to about 99.95% w. More preferably, from about 5% w to about 95% w of the pharmaceutically acceptable carrier will be used. Most preferably, from about 10% w to about 90% w of the pharmaceutically acceptable carrier will be used. Pharmaceutical excipients useful as carriers in this invention include 20 stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two. Preferred bulking agents include compatible carbohydrates, polypeptides, amino acids or combinations thereof. Suitable 25 carbohydrates include monosaccharides such as galactose, D-mannose, sorbose, and the like; disaccharides, such as lactose, trehalose, and the like; cyclodextrias, such as 2-hydroxypropyl-p-cyclodextrin; and polysaccharides, such as raffinose, maltodextrins, dextrans, and the like; alditols, such as mannitol, xylitol, and the like. A preferred group of carbohydrates includes lactose, threhalose, raffinose 30 maltodextrins, and mannitol. Suitable polypeptides include aspartame. Amino acids INTELLECTUAL property i office of n7. 1 6 jun 2004 received 12 include alanine and glycine, with glycine being preferred. Additives, which may be included as minor components of the dry powder formulations of the invention, may be included for conformational stability during spray drying and for improving dispersibility of the powder. These additives include hydrophobic amino acids such as 5 tryptophan, tyrosine, leucine, phenylalanine, and the like. Suitable pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred. In other aspects, the present invention relates to concentrated macrolide formulations, such as a concentrated erythromycylamine formulation, suitable for 10 efficacious delivery of the macrolide by aerosolization into endobronchial space. The invention is suitable for formulation of concentrated erythromycylamine for aerosolization by jet, vibrating porous plate, ultrasonic or dry powder nebulizers to produce erythromycylamine aerosol particle size between 1 and 5 jam preferable for efficacious delivery of erythromycylamine into the endobronchial space to treat 15 Streptococcus pneumoniae, Haemophilius influenzae, Staphylococcus aureus, Moraxella catarrhalis and Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae infections. The formulations preferably contain minimal, yet efficacious amounts of erythromycylamine formulated on a relatively small volume of physiologically acceptable solution having a salinity, or a dry powder, adjusted to 20 permit generation of an erythromycylamine aerosol that is well-tolerated by patients but preventing the development of secondary undesirable side effects such as bronchospasm and cough. Primary requirements for any aerosolized formulation are its safety and efficacy. Additional advantages are lower treatment cost, practicality of use, long-shelf life, 25 storage and optimization of nebulizer. The aerosol formulation is nebulized predominately into particle sizes which can be delivered to the terminal and respiratory bronchioles where the Streptococcus pneumoniae, Haemophilius influenzae, Staphylococcus aureus, and Moraxella catarrhalis and the atypical bacteria Legionella pneumophila, Chlamydia pneumoniae, 30 and Mycoplasma pneumoniae or other susceptible bacteria reside in patients with chronic bronchitis and pneumonia. Streptococcus pneumoniae, Haemophilius influenzae, Staphylococcus aureus, Moraxella catarrhalis, Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae are present throughout the airways including the bronchi, bronchioli and lung parenchema. However, they are intellectual property office of n 7. 16 JUN 2004 received most predominant in terminal and respiratory bronchioles. During exacerbation of infection, bacteria can also be present in alveoli. Therefore, in one aspect, the present invention provides a formulation that is delivered throughout the endobronchial tree to the terminal bronchioles and eventually to the parenchymal tissue. 5 Aerosolized erythromycylamine formulation is formulated for efficacious deliveiy of erythromycylamine to the lung endobronchial space. A specific jet, vibrating porous plate or ultrasonic nebulizer is selected to allow the formulation of an erythromycylamine aerosol particles with a mass median aerodynamic diameter predominately between 1 to 5 |a,m. The formulated and delivered amount of 10 erythromycylamine is efficacious for treatment and/or prophylaxis of endobronchial infections, particularly those caused by the bacteria Streptococcus pneumoniae, Haemophilius influenzae, Staphylococcus aureus, and Moraxella catarrhalis and the atypical pneumonias Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae. The formulation has salinity adjusted to permit generation 15 of erythromycylamine aerosol well tolerated by patients. Further, the formulation has suitable osmolarity. The formulation has a small aerosolizable volume and is able to deliver an effective dose of erythromycylamine to the site of the infection. Additionally, the aerosolized formulation does not impair negatively the function of the airways by causing undesirable side effects. 20 The antibiotic formulation may be administered with the use of an inhalation device having a relatively high rate of aerosol output, high emitted dose efficiency, and emission limited to periods of actual inhalation by the patient. Thus, while conventional air-jet nebulizers exhibit a rate of aerosol output on the order of 3 |j,l/sec, inhalation devices useful for use in the practice of the present invention will typically 25 exhibit a rate of aerosol output of not less than about 5 |il/sec, more preferably not less than about 6.5 (al/sec, and most preferably not less than about 8 p.l/sec. In addition, while conventional air-jet nebulizers have a relatively low emitted dose efficiency and typically release about 55% (or less) of the nominal dose as aerosol, inhalation devices useful for use in the practice of the present invention will typically release at least about 30 75%, more preferably at least about 80% and most preferably at least about 85% of the loaded dose as aerosol for inhalation by the subject. In other aspects, conventional air-jet nebulizers typically continually release aerosolised drug throughout the delivery period, without regard to whether the subject is inhaling, exhaling or in a static portion of the breathing cycle, thereby wasting a substantial portion of the loaded drug dose. In 35 contrast, preferred inhalation devices for use in WO 02/03998 PCT/US01/41328 -14- the present invention will be breath actuated, and restricted to delivery of aerosolized particles of the macrolide formulation to the period of actual inhalation by the subject. A representative inhalation device meeting the above criteria and suitable for use in the practice of the invention is the Aerodose™ inhaler, available from Aerogen, Inc., 5 Sunnyvale, California. The Aerodose™ inhaler generates an aerosol using a porous membrane driven by a piezoelectric oscillator. Aerosol delivery is breath actuated, and restricted to the inhalation phase of the breath cycle, i.e., aerosolization does not occur during the exhalation phase of the breath cycle. The airflow path design allows normal inhale-exhale breathing, compared to breath-hold inhalers. Additionally, the 10 Aerodose™ inhaler is a hand-held, self-contained, and easily transported inhaler. Although piezoelectric oscillator aerosol generators, such as the Aerodose™ inhaler, are presently preferred for use in the practice of the invention, other inhaler or nebulizer devices may be employed that meet the above performance criteria and are capable of delivering the small dosage volumes of the invention with a relative high 15 effective deposition rate in a comparatively short period of time. In other aspects of the present invention, unit dose formulations and devices are provided for administration of a macrolide antibiotic formulation to a subject with an inhaler, in accordance with the methods of the invention as described supra. Preferred unit dose devices comprise a container designed to hold and store the 20 relatively small volumes of the macrolide antibiotic formulations of the invention, and to deliver the formulations to an inhalation device for delivery to a patient in aerosol form. In one aspect, unit dose containers of the invention comprise a plastic ampoule filled with a macrolide antibiotic formulation of the invention, and sealed under sterile conditions. Preferably, the unit dose ampoule is provided with a twist-off tab or other 25 easy opening device for opening of the ampoule and delivery of the macrolide antibiotic formulation to the inhalation device. Ampoules for containing drug formulations are well known to those skilled in the art (see, for example, U.S. Patent Nos. 5,409,125, 5,379,898, 5,213,860, 5,046,627, 4,995,519, 4,979,630, 4,951,822, 4,502,616 and 3,993,223, the disclosures of which are incorporated herein by this 30 reference). The unit dose containers of the invention may be designed to be inserted directly into an inhalation device of the invention for delivery of the contained macrolide antibiotic formulation to the inhalation device and ultimately to the subject. WO 02/03998 PCT/US01/41328 -15- In accordance with this aspect of the invention, a unit dose device is provided comprising a sealed container containing less than about 5.0 ml, preferably less than about 3.0 ml and most preferably less than about 2.0 ml of a liquid macrolide antibiotic formulation comprising from about 50 to about 150 mg/ml of a macrolide 5 antibiotic in a physiologically acceptable carrier, the sealed container being adapted to deliver the macrolide antibiotic formulation to an inhalation device for aerosolization. Suitable macrolide antibiotics for use in connection with this aspect of the invention include those macrolide antibiotics described in detail, supra. In a presently preferred embodiment, the macrolide antibiotic employed in the unit dose devices of the 10 invention is erythromycylamine. In other aspects of the invention, the unit dose devices of the invention may contain a liquid macrolide antibiotic formulation comprising from about 70 to about 130 mg/ml of macrolide antibiotic. In yet other aspects of the invention, the unit dose devices of the invention may contain a liquid macrolide antibiotic formulation comprising from about 90 to about 110 mg/ml of 15 macrolide antibiotic. In preferred liquid unit dose formulations of the invention, the physiologically acceptable carrier may comprise a physiological saline solution such as a solution of one quarter strength of normal saline, having a salinity adjusted to permit generation of erythromycylamine aerosol well-tolerated by patients but to prevent substantially 20 the development of secondary undesirable side effects such as bronchospasm and cough. In yet other aspects of the invention, dry powder formulations of the invention are placed within a suitable unit dose receptacle in an amount sufficient to provide a subject with a macrolide antibiotic compound of the invention for a unit dosage 25 treatment by dry powder inhalation. Preferred dry powder unit dosage receptacles fit within a suitable inhalation device to allow for the aerosolization of the macrolide-based dry powder composition by dispersion into a gas stream to form an aerosol and then capturing the aerosol so produced in a chamber having a mouthpiece attached for subsequent inhalation by a subject in need of treatment. Such a dosage receptacle 30 includes any container enclosing the formulations known in the art such as gelatin or plastic capsules with a removable portion that allows a stream of gas (e.g., air) to be directed into the container to disperse the dry powder formulation. Such containers are exemplified by those shown in U.S. Patent Nos. 4,227,522, 4,192,309, and WO 02/03998 PCT/US01/41328 -16- 4,105,027. Suitable containers also include those used in conjunction with Glaxo's Ventolin Rotohaler brand powder inhaler or Fison's Spirihaler brand powder inhaler. Another suitable unit-dose container which provides a superior moisture barrier is formed from an aluminum foil plastic laminate. The macrolide powder is filled by 5 weight or by volume into the depression in the formable foil and hermetically sealed with a covering foil-plastic laminate. Such a container for use with a powder inhalation device is described in U.S. Pat. No. 4,778,054 and is used with Glaxo's Diskhaler.RTM. (U.S. Patent Nos. 4,627,432, 4,811,731; and 5,035,237). All of these references are incorporated herein by reference. 10 In accordance with this aspect of the invention, a unit dose device is provided comprising a sealed container containing a dry powder formulation comprising from about 25 to about 250 mg of a macrolide antibiotic, preferably from about 50 to about 200 mg of a macrolide antibiotic, and more preferably from about 75 to about 150 mg of a macrolide antibiotic in a physiologically acceptable dry powder carrier, the sealed 15 container being adapted to deliver the macrolide antibiotic formulation to an inhalation device for aerosolization. In this aspect of the invention, the dry powder formulations may comprise from about 50% to about 90% by weight of the macrolide antibiotic, preferably from about 60% to about 88% by weight of the macrolide antibiotic, and more preferably from about 75% to about 85% by weight of the 20 macrolide antibiotic. Aerosol Erythromycylamine Formulation In order to assess the stability of erythromycylamine in aqueous solutions three salt forms of the antibiotic were prepared and submitted to varying conditions of temperature, time, concentration, and pH. Erythromycylamine concentrations were 25 determined by HPLC methodology. The data from these stability studies are shown in Figures 2-7 and several important findings are revealed. First, the stability of erythromycylamine hydrochloride as expected, was directly proportional to temperature of the solution (see Figures 2-5). Second, erythromycylamine solutions were more stable at neutral pH 7 than acidic pH 5 and 6 (Figure 5). This result is 30 consistent with the known effects of pH on the degradation of macrolide antibiotics. One of the main degradation pathways is loss of the neutral sugar, cladinose (see J Chrom. A, 812m 1998, 255-286). Third, solutions of erythromycylamine acetate were more stable at pH 6 and 7 than the corresponding hydrochloride and sulfate salts at the same pH (compare Figure 7 to Figure 5 and 6). intellectual property . office of n1 16 JUN 2004 17 received Liquid and dry powder formulations according to the invention contain from about 50 to about 750 mg, preferably from about 75 to about 600 mg, and most preferably from about 100 to about 500 mg of a macrolide antibiotic drug, such as erythromycylamine acetate, per dose. This corresponds to minimal yet efficacious amounts of 5 erythromycylamine to suppress Streptococcus pneumoniae, Haemophilius influenzae, Staphylococcus aureus, Moraxella catarrhalis, Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae infections in the endobronchial space. Presently preferred liquid aerosol erythromycylamine formulations according to the invention comprise from about 90 to about 110 mg of erythromycylamine sulfate 10 per 1 mL of quarter normal saline. This corresponds to a representative efficacious amount of erythromycylamine to suppress bacterial infections of AECB. Both patients and aerosol generating devices are sensitive to the osmolarity, pH, and ionic strength of the formulation. It has now been discovered that this problem is conveniently solved by formulating erythromycylamine solutions in quarter normal 15 saline, that is saline containing 0.225% of sodium chloride, and that normal quarter saline is a suitable vehicle for delivery of erythromycylamine into the endobronchial space. Chronic bronchetic patients and other patients with chronic endobronchial infections have a high incidence of bronchospastic or asthmatic airways. These 20 airways are sensitive to hypotonic and hypertonic aerosols, to the concentration of a permeant ion, particularly a halide such as chloride, as well as to aerosols that are acidic or basic. The effects of irritating the airways can be clinically manifested by cough or bronchospasm. Both of these conditions can prevent efficient delivery of aerosolized erythromycylamine into the endobronchial space. 25 The erythromycylamine acetate, hydrochloride, and sulfate formulation containing 60-100 mg of erythromycylamine per ml of quarter saline has an osmolarity in the range of 130-400 mOsm/kg. This is within the safe range of aerosols administered to a chronic bronchitis patient (Table 1). WO 02/03998 PCT/USO1/41328 -18- TABLE1 Osmolality of Erythromycylarnine Solutions as a Function of Salt Form, pH, and Concentration: Experimental and Theoretical Results Cone. Experimental Theoretical Salt pH (mg/ml) (mOsm/kg) (mOsm/kg) Acetate 5.0 60 300 245 100 518 408 150 840 613 Acetate 6.0 60 365 100 639 150 701 Acetate 7.0 60 501 100 891 150 746 Hydrochloride 5.0 60 227 245 100 382 408 150 601 613 Hydrochloride 6.0 60 224 100 382 150 598 Hydrochloride 7.0 60 226 100 386 150 594 Sulfate 5.0 60 130 163 100 239 272 150 396 409 Sulfate 6.0 60 132 100 238 150 395 Sulfate 7.0 60 132 100 241 150 391 5 The pH of the formulation is equally important for aerosol delivery. As noted previously, when the aerosol is either acidic or basic, it can cause bronchospasm and cough. The safe range of pH is relative; some patients will tolerate a mildly acidic aerosol that in others will cause bronchospasm. Any aerosol with a pH of less than 10 4.5 usually will induce bronchospasm in a susceptible individual; aerosols with a pH between 4.5 and 5.0 will occasionally cause this problem. An aerosol with a pH between 5.0 and 7.0 is considered to be safe. Any aerosol having pH greater than intellectual PROPERTY office of n.z. 1 6 JUN 2004 19 received 10.0 is to be avoided since irritation resulting in bronchospasm may occur. The optimum pH for the aerosol formulation was determined to be between pH 5.0 and 7.0. In one aspect, liquid formulations of the invention are preferably nebulized predominately into particle sizes allowing a delivery of the drug into the terminal and 5 respiratory bronchioles and lower airways where the bacteria reside. For efficacious delivery of erythromycylamine to the lung endobronchial airway space by aerosol, the formulation of aerosol particles having a mass median aerodynamic diameter predominately between 1 to 5 p.m is necessary. The formulated and delivered amount of erythromycylamine for treatment and prophylaxis of endobronchial infections, 10 particularly those caused by the bacteria Streptococcus pneumoniae, Haemophilius influenzae, Staphylococcus aureus, Moraxella catarrhalis, Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae, must effectively target the endobronchial surface. Delivered doses of the formulations preferably have the smallest practical aerosolizable volume able to deliver an effective dose of 15 erythromycylamine to the site of the infection. Preferred formulations additionally provide conditions that do not adversely affect the functionality of the airways. Consequently, preferred formulations contain a sufficient amount of the drug formulated under conditions that allow its efficacious delivery, while avoiding undesirable reactions. The new formulations according to the invention meet all these 20 requirements. According to the invention, erythromycylamine is formulated in a dosage form intended for inhalation therapy by patients with chronic bronchitis and pneumonia. Since the patients reside throughout the world, it is desirable that the formulation has reasonably long shelf life. Storage conditions and formulation stability thus become 25 important. As discussed above, the pH of the solution is important. A pH between 5.0 and 7.0, preferably about 6.0, is optimal from the storage and longer shelf-life point of view. The formulation is typically stored in a one- to two- millimeter low-density 30 polyethylene (LDPE) vials. The vials are aseptically filled using a blow-fill-seal process. The vials are sealed in foil overpouches. Stability of the formulation with respect to the oxidation is another very important issue. If the drug is degraded from aerosolization, a smaller amount of the drug is delivered to the lung, thus impairing the treatment as well as provoking 35 conditions WO 02/03998 PCT/US01/41328 -20- that could lead to the development of resistance to erythromycylamine, because the delivered dose would be too small. Moreover, erythromycylamine degradation products may provoke bronchospasm and cough. To prevent oxidative degradation of erythromycylamine and in order to provide acceptable stability, a product with low 5 oxygen content is produced by packaging the LDPE vials in oxygen-protective packaging comprising foil overpouches, six vials per overpouch. Prior to vial filling, the solution in the mixing tank is nitrogen sparged and the annular overpouch headspace is nitrogen purged. In this way, both hydrolysis and oxidation of erythromycylamine is prevented. 10 II. Aerosolization Devices Aerosolization devices, such as a jet, vibrating porous plate or ultrasonic nebulizers, useful in the practice of the invention are generally able to nebulize the formulation of the invention into aerosol particles predominantly in the range from 1-5 fj.m. Predominantly in this application means that at least 70% but preferably more 15 than 90% of all generated aerosol particles are within 1-5 pp. range. Nebulizers such as jet, ultrasonic, vibrating porous plate, and energized dry powder inhalers, that can produce and deliver particles between the 1 and 5 p.m particle size that is optimal for treatment of Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis Legionella pneumonia, 20 Chlamydia pneumoniae, and Mycoplasma pneumoniae infections, are currently available or can be produced using known methods and materials. A jet nebulizer works by air pressure to break a liquid solution into aerosol droplets. Vibrating porous plate nebulizers work by using a sonic vacuum produced by a rapidly vibrating porous plate to extrude a solvent droplet through a porous plate. An ultrasonic 25 nebulizer works by a piezoelectric crystal that shears a liquid into small aerosol droplets. However, only some formulations of erythromycylamine can be efficiently nebulized by these three nebulizers, as these devices are sensitive to the pH and ionic strength of the formulation. While a variety of devices are available, only a limited number of these 30 nebulizers are suitable for the purposes of this invention. Preferred nebulizers useful rr-rm r rrri r in the present invention include, for example, AeroNeb and AeroDose vibrating porous plate nebulizers (AeroGen, Inc., Sunnyvale, California), Sidestream (r) nebulizers (Medic-Aid Ltd., West Sussex, England), Pari LC Plus and Pari LC Star jet nebulizers (Pari Respiratory Equipment, Inc., Richmond, Virginia), and 35 Aerosonic™ (DeVilbiss Medizinische Produkte (Deutschland) GmbH, Heiden, WO 02/03998 PCT/US01/41328 -21- Germany) and UltraAire (Omron Healthcare, Inc., Vernon Hills, Illinois) ultrasonic nebulizers. HI. Aerosol Pharmacokinetics Solutions of erythromycylamine were administered to rats by the IV and 5 inhalation routes and drag Concentrations in plasma and lung were measured. Data from these studies is shown in Figures 8 and 9. Two dose levels were selected for the inhalation deliveiy route, 1.7 and 0.7 mg/kg, and were compared to a single intravenous dose (25 mg/kg). Dose normalized AUC of erythromycylamine in the lung for IV (25 mg/kg), 10 inhalation (1.7 mg/kg), and (0.7 mg/kg) was 24.21, 1067.84 and 848.34 j_ig*h/gram, respectively. Therefore, by administering erythromycylamine directly to the lung via inhalation route, lung drug levels achieved were approximately 40 times higher on a milligram basis than by the intravenous route of delivery. Thus, antibiotic therapy by inhalation should be more efficacious than treatment by oral or IV routes. 15 IV. Aerosol Efficacy Erythromycylamine was very effective by both intravenous and aerosol administration. At the lowest dose tested (10 mg/kg per day) intravenous erythromycylamine reduced the lung burden of S. pneumonia to below the limits of detection (10 CFU/gram of lung) as shown in Example 7. Aerosol was also very 20 effective (see Figure 10) with only detectable recovery of S. pneumonia at 5 mg/ml aerosol solution (calculated dose 0.13 mg/kg per day). In addition, erythromyclamine was very effective when administered as a single aerosol dose at concentrations greater than those required for single daily doses for 3 days. A single dose of 0.13 mg/kg was less effective (less than 2 orders of magnitude reduction in CFU/gram) 25 compared with 0.13 mg/kg for three consecutive days (5 orders of magnitude reduction). However, a single dose of 0.67 mg/kg achieved almost complete clearance of the organism from lung tissue, an effect similar to the multiple dose efficacy, indicating that at this concentration, the second and third doses added little value (see Figure 11). 30 The pharmacokinetic evaluation of aerosolized erythromycylamine suggests, and the efficacy data indicates, that equivalent lung concentrations to multiple daily IV, oral, or aerosol doses can be achieved by a single aerosol dose and that the single dose would be about 3-5 fold greater than required for similar effectiveness as three daily doses. WO 02/03998 PCT/US01/41328 -22- Utility One aspect of the utility of this invention is that small volume, high concentration formulations of macrolide antibiotics, such as erythromycylamine, can be used with suitable nebulizers to deliver an efficacious dose of erythromycylamine 5 to the endobronchial space in people with chronic bronchitis, bronchiectasis, and pneumonia caused by macrolide susceptible bacteria or other infections. The formulation is safe and very cost effective. Furthermore, the formulations may be kept in a nitrogen environment, with pH controlled for tolerance, to provide adequate shelf life for commercial distribution. 10 EXAMPLE 1 General Procedure For The Preparation Of Erythromycylamine Salts: Synthesis Of Erythromycyclamine Acetate To a solution of 10.0 g (13.6 mmol) of erythromyclamine in lOOmL of MeOH cooled in an ice bath was added dropwise 1.56 mL (27.2 mmol, 2.0 eq) of glacial 15 acetic acid. The solution was wanned to ambient over a period of 30 min, then the solvent removed under reduced pressure. E^O (50 mL) was added and the slurry concentrated. This was repeated to provide 11.52 g (96.9%) of erythromyclamine acetate monohydrate as a white powder; IR (KBr, cm"1) 1718, 1560, 1406, 1168, 1080, 1055, 1012; *H NMR (400 MHz, CD3OD) 5 0.89 (t, 3H, J = 7.2 Hz), 1.06-1.32 20 (m, 27H), 1.35-1.47 (m, 4H), 1.52-1.66 (m, 3H), 1.85-2.02 (m, 8H), 2.03-2.26 (m, 2H), 2.45-2.49 (m, 1H), 2.66-2.77 (m, 5H), 2.91-3.09 (m, 3H), 3.21-3.40 (m, 6H), 3.58 (d, 1H, J = 7.0 Hz), 3.67 (s, 1H)„ 3.78-3.83 (m, 2H), 4.10-4.13 (m, 1H), 4.59 (d, 1H, J = 7.0 Hz), 4.88-5.01 (m, 12H); MS m/z 735.6 (M+-2Ac0H-2H20); KF 2.33 % h2o. 25 Anal. Calcd for C41H80N2OI7: C, 56.40; H, 9.24; N, 3.21. Found: C, 56.38; H, 9.21; N, 3.16. EXAMPLE 2 Synthesis Of Erythromvcylamine Sulfate To a solution of 10.0 g (13.6 mmol) of erythromyclamine in lOOmL of MeOH 30 cooled in an ice bath was added dropwise 0.73 mL (13.6 mmol, 1.0 eq) of concentrated sulfuric acid. The solution was warmed to ambient over a period of 30 min, then the solvent removedUnder reduced pressure. Et20 (50 mL) was added and the slurry concentrated. This was repeated to provide 11.13 g (96.1%) of erythromyclamine sulfate monohydrate as a white powder; IR (KBr, cm"1) 1718, 35 1384, 1168, 1122, 1078, 1012; *H NMR (400 MHz, CD3OD) 5 0.89 (t, 3H, J = 7.2 WO 02/03998 PCT/US01/41328 -23- Hz), 1.08-1.32 (m, 27H), 1.45-1.63 (m, 7H), 1.89-2.04 (m, 2H), 2.23-2.31 (m, 2H), 2.44-2.47 (m, 1H), 2.84-2.89 (5H), 2.99-3.07 (m, 3H), 3.30-3.49 (m, 6H), 3.58 (d, 1H, J = 7.0 Hz), 3.69 (s, 1H), 3.78-3.86 (m, 2H), 4.09-4.11 (m, 1H), 4.60 (d, 1H, J = 6.8 Hz), 4.87-4.99 (m, 12H); MS m/z 735.7 (]Vr-H2S04-2H20); KF 2.93 % H20. 5 Anal. Calcd for C37H74N2016S: C, 52.22; H, 8.76; N, 3.29. Found: C, 52.55; H, 8.91; N, 3.27. EXAMPLE 3 Synthesis Of Erythromycylamine Hydrochloride To a solution of 10.0 g (13.6 mmol) of erythromyclamine in lOOmL of MeOH 10 cooled in an ice bath was added dropwise 2.34 mL (27.2 mmol, 2.0 eq) of 37% hydrochloric acid. The solution was warmed to ambient over a period of 30 min, then the solvent removed under reduced pressure. Et,0 (50 mL) was added and the slurry concentrated. This was repeated to provide 11.24 g (97.9%) of erythromyclamine hydrochloride dihydrate as a white powder; IR (KBr, cm"1) 1718, 1466, 1383, 1170, 15 1078, 1055, 1011; NMR (400 MHz, CD3OD) 5 0.87-0.91 (m, 3H), 1.10-1.31 (m, 27H), 1.43-1.65 (m, 7H), 1.89-2.01 (m, 2H), 2.25-2.27 (m, 2H), 2.45-2.48 (m, 1H), 2.82-3.10 (m, 8H), 3.34-3.42 (m, 8H), 3.57-3.58 (m, 1H), 3.67 (s, 1H), 3.80-3.82 (m, 2H), 4.08-4.11 (m, 1H), 4.61-5.00 (m, 13H); MS m/z 735.6 (M+-2HC1-2H20); KF 4.38 % HzO. 20 Anal. Calcd for C37H76C12N2012: C, 52.65; H, 9.08; N, 3.32. Found: C, 52.21; H, 9.18; N, 3.20. EXAMPLE 4 Aqueous Formulation And Stability Of Erythromycylamine Salts Preparation of Solutions Erythromyclamine (9.0g, 12.2 mM) free base was 25 added to a tared 100 mL Erlenmeyer flask . De-ionized water (25 mL) was added to the flask with agitation by magnetic stirrer. IN sulfuric acid (24.5 mL, 2 equivalents) was gradually added while stirring. When the solution was clear, it was removed from the stir plate and re-weighed. Deionized water was added dropwise to obtain a final solution weight of 62.9g. The solution was divided into three 20 mL portions, 30 and the pH was adjusted to the desired value (5.0, 6.0, or 7.0) by dropwise addition of IN sodium hydroxide or sulfuric acid while monitoring with a pH meter. The above procedure was used to prepare solutions at 100 mg/mL and 60 mg/mL by adjusting the weight of erythromyclamine (6.0g and 3.6g) and the volume of IN sulfuric acid (16.3 and 9.8 mL). WO 02/03998 PCT/US01/41328 -24- Solutions of the acetate and hydrochloride salts of erythromyclamine at 150, 100 and 60 mg/mL were prepared as described above, except that IN Acetic acid and IN Hydrochloric acid (2 equivalents) were added to prepare the salts and adjust the pH. Aliquots of each salt form at each concentration and each pH were stored at 4, 40, 5 and 60 °C, and at ambient temperature. Stability Determination All solutions were analyzed immediately after preparation (t — 0) and at 24 hours, 48 hours, eight days, 15 days and 22 days following preparation, excepting that samples that appeared substantially degraded at eight days were omitted from subsequent analyses. 10 Refrigerated and heated samples were equilibrated to ambient temperature for at least one hour prior to sample preparation. Final dilution volume for all samples was 10 mL. The diluent for all samples consists of an. 80:20 (v/v) mixture of 50 mM phosphate buffer at pH 6.5 and acetonitrile. . An appropriate amount of sample (40 microliters for a 150 mg/mL solution, 15 50 microliters for a 100 mg/mL, or 100 microliters for a 60 mg/mL solution) was transferred to a 20 mL scintillation vial. 10 mL of the diluent were added to the vial and mixed thoroughly. Standard Preparation Standards were prepared in duplicate and used for a maximum of three days. Ery-amine free base (30 mg) was transferred to a tared 50 20 mL volumetric flask and exact weight was recorded. Sample diluent (45 mL) is added and sonicated briefly to dissolve. The standard was cooled and diluted to volume with diluent. Sample and Standard Analysis Samples and standards were analyzed by reversed-phase high performance liquid chromatography. A 250 x 4.6 mm 25 Phenomenex Luna CN column with 5 micron particle size was used to perform the separation. All analyses were performed on an Agilent Technologies HP 1100 chromatography system, and the data were acquired and stored using an Agilent Technologies ChemStation data system. Analytical parameters were as shown below in Table 2. WO 02/03998 PCT/USO1/41328 -25- 10 Flow Rate Column Temperature Injection Volume Detector Run Time Mobile Phase A Mobile Phase B Composition Table 2 l.OmL/min 30 °C 20 |j.L UV absorbance at 200 rvm 10 min 50 mM Phosphate pH2.1 Acetonitrile 80/20 A/B EXAMPLE 5 Osmolality Of Erythromyclamine Salt Solutions Three portions of erythromyclamine HC1 salt (0.6g, l.Og and 1.5g) were weighed into separate 10 mL volumetric flasks. Easypure UV water (8 mL) was added to each flask and sonicated until completely dissolved, then diluted to volume. This procedure was repeated for the Erythromyclamine sulfate and acetate. The pH and osmolality of each solution were measured, and the measured osmolality compared to the theoretical values. The salts prepared in Example 4 (4°C) were allowed to equilibrate to room temperature, and the osmolality was measured. The results are shown in Table 3: Table 3 Erythromyclamine Salt Osmolality Study Erythromyclamine HC1 Salt Lot# TEM-702-171 dihydrate mw=843.91 g/m Target Weight (g) 0.600 1.00 1.50 Actual Weight (g) 0.59957 1.00443 1.50258 Theoretical Osmolarity (mOsm) 279 474 733 Actual Osmolarity (mOsm) 213 357 534 pH 7.57 7.59 7.60 Comments wh. cloudy wh. cloudy wh. cloudy Erythromyclamine Sulfate Salt Lot # TEM-702-169 monohydrate mw=883.14 g/m 0.600 0.60368 146 137 77.62 wh.cloudy 1.00 1.00430 258 227 77.63 wh.cloudy 1.50 1.49941 . 408 340 77.61 wh.cloudy Erythromyclamine Acetate Salt Lot # TEM-702-167 monohydrate mw= 873.08 g/m 0.600 0.60189 g 195 207 6.62 si. cloudy 1.00 1.00713 g 345 346 6.67 si. cloudy 1.50 1.50178 g 552 516 6.62 si. cloudy (((wt. in grams/molecular wt )*number of species)/0.01L)*(1000 m0sm/10sm)= X mOsm WO 02/03998 PCT/US01/41328 -26- EXAMPLE 6 Aerosol Delivery Of Erythromycylamine To Rats: Characterization Of Aerosol Pharmacokinetics IV Pharmacokinetics: Erythromycylamine (250 mg) was dissolved in a 5 mL 5 of DI water, and 12 mL of concentrated sulfuric acid was added. A solution of dilute sulfuric acid (1:10 v/v) was added gradually to the solution to dissolve the drug completely. The solution of dilute sulfuric acid was added gradually to bring the pH of solution to 6.8-7.2. By adding DI water, the total volume of solution was brought up to 8 mL. A 200 |nL solution of erythromycylamine sulfate (25 mg/kg) was 10 delivered to male Sprague-Dawley rats (Simonsen Laboratories, 1180 C Day Road, Gilroy, CA 95020) by intravenous administration via the lateral tail vein. Animals were anesthetized with 1-4% isoflurane and lung and blood samples were collected from 3 rats at 0.083, 0.25, 0.5 1, 2, 4, 6, 8 and 24 hours post dosing. The blood samples were collected via cardiac puncture using heparin as an anticoagulant. Lungs 15 were removed surgically following blood sampling, and the bronchi and trachea were removed and discarded. The remaining lung tissue was processed as described below. Both the lung and blood samples were immediately placed on ice, and the blood samples were centrifuged immediately following collection to harvest plasma samples. Both lung and plasma samples were stored at -80°C until assayed. 20 Erythromycylamine concentrations in plasma and the lung (per gram of lung tissue) were determined using a validated LC-MS method. Plasma samples (100 fig) were spiked with oleandomycin (internal standard, 1 ng/mL) before extraction. Plasma samples (100 p,L) were deproteinated with 3.3% trichloroacetic acid (TCA). Samples were centrifuged (10,000 rpm, 10 min.) and the supernatant was transferred 25 to HPLC centrifilter for centrifiltration (10,000 rpm, 10 min). The mobile phase consisted of 0.1% acetic acid-acetonitrile (70:30, v/v, pH=3.2) solution at a flow rate of 0.5 ml/min for 3 minutes, followed by 0.1% acetic acid-acetonitrile (60:40, v/v) at a flow rate of 0.8 ml/min for 3 minutes. A stainless steel analytical column (Zorbax SB-C18, 2.1 mm ID x 150.0 mm, 5 (im with a Phenomenex cartridge guard column) 30 was used as the stationary phase. The column temperature was 50° C. Quantification of the erythromycylamine was performed using a HP 1100 LC/MSD API-Electrospray System. Data acquisition was set in the selective ion monitoring mode. The method was linear (r>0.9990) in the concentration range of 0.01 to 50 jj.g/ml. The absolute recovery was 95.0 ± 2.19%. WO 02/03998 PCT/US01/41328 -27- Lung samples were homogenized with DI water. Oleandomycin was added to the samples as an internal standard. The homogenate was deproteinated with 0.9 M TCA. Samples were centrifuged at 10,000 rpm for 10 minutes and the supernatant was transferred to HPLC centrifilters for centrifiltration. The mobile phase consisted 5 of 0.1% acetic acid-acetonitrile (70:30, v/v, pH=3.2) at a flow rate of 0.5 ml/min for 3 minutes, followed by 0.1% acetic acid-acetonitrile (60:40, v/v) at a flow rate of 0.8 ml/min for 3 minutes. A stainless steel analytical column (Zorbax SB-C18, 2.1 mm ID x 150.0 mm, 5 p.m with a Phenomenex cartridge guard column) was used as the stationary phase. The column temperature was 50° C. Quantification of the 10 erythromycylamine was performed using a HP 1100 LC/MSD API-Electrospray System. Data acquisition was set in the selective ion monitoring mode. The linearity (r>0.9990) of the assay ranged from 0.1 to 200 fig/g. The extraction efficiency was 93.8 + 2.54%. Pharmacokinetic parameters, area under the curve (AUC) and mean residence-15 time (MRT), were estimated based on the statistical moment theory using WinNonlin™ Professional Version 2.0 software (Pharsight Corporation). The peak concentration (Cm,v) was not estimated but observed. Inhalation Pharmacokinetics: For a 60 mg/mL solution, 3.191 g (4-08 mmol) of erythromycylamine (94% purity) was added to 43 mL of DI water and 4.27 mL 20 (4.27 mmol) of 1 M sulfuric acid in a 50 ml volumetric flask. The solution was then adjusted to pH 6.5 with the addition of another 53 pL (0.053 mmol) of 1 M sulfuric acid. The volume was brought up to 50 mL with additional DI water. The 30 mg/mL solution was made by diluting the 60 mg/mL solution in 1/2 normal saline. The osmolality of the resulting solutions were 148 mOsm as determined using The 25 Advanced™ Micro-Osmometer Model 3300 (Advanced Instruments, Inc., Norwood, Mass.) Rats were exposed once to either 30 or 60 mg/mL solution of erythromycylamine sulfate via inhalation in a 32-port nose-only rodent exposure system (Battelle, Richland, WA) for 30 minutes. The Battelle system nose-only 30 rodent exposure system is based on the Cannon Flow-Past Nose only system (Am. Ind Hyg Assoc J1983 Dec;44(12)923-8) and is made up of four stackable stain-less steel tiers with a total of 32 ports. The system includes inlet and exhaust flow monitoring and control, aerosol data was collected using the NORES version 1.1.4 software provided by Battelle. Erythromycylamine solutions were aerosolized using the PARI 35 LC STAR™ nebulizer. Mean aerosol concentrations were determined by gravimetric WO 02/03998 -28- PCT/US01/41328 analysis of filter samples taken at 10 and 20 minutes following the start of exposure. The mean aerosol concentrations were 0.54±0.06 and 1.36±0.30 mg/L, respectively, for 30 and 60 mg/mL solutions. Lung and blood samples were collected from 3 rats at 0.083, 0.25, 0.5 1, 2, 4, 5 8 and 24 hours post dosing as described above. The sample collection and handling procedures for the inhalation study were same as for the intravenous study. Bioanalytical assay procedures for the inhalation study were the same as for the intravenous study. The calculated deposited dose in the lung (pulmonary dose) was approximately 0.70 or 1.77 mg/kg following an inhalation dose of 30 or 60 10 mg/mL erythromycylamine solution for 30 minutes, respectively. The pulmonary dose in the lung was calculated as follows: LDD = MAC x MV x DE x FLD + MBW Where, LDD = Lung Deposited Dose 15 MAC = Mean Aerosol Concentration = 0.54 and 1.36 mg/L for 30 and 60 mg/mL solutions, respectively. MV = Minute Volume = 0.1 L/min. DE = Duration of Exposure = 30 minutes FLD = Fraction of Lung Deposit = 0.1 20 MBW = Mean Body Weight = 0.23 kg Pharmacokinetic parameters in the lung following intravenous and inhalation administration of erythromycylamine are summarized below in Table 4: WO 02/03998 -29- PCT/US01/41328 Table 4 Phannacokinetic parameters of erythromycylamine in the lung following an intravenous or two inhalation doses in the rat (N=3). Drug Administration Route and Dose Inhalation Inhalation 30 mg/mL, 60 mg/mL, Pharmacokinetic Intravenous (Pulmonary (Pulmonary Dose: Parameter 25 mg/kg Dose: 0.7 mg/kg) 1.77 mg/kg) C^Gng/gram) 68.99 89.33 155.24 AUC (ng^h/gram)1 605.19 854.27 1357.35 AUC (ng#h/gram)2 24.21 1220.39 766.86 MRT(h)3 10.8 10.5 11.2 5 1. Area under the curve estimated 0-24 hours postdose. 2. Area under the curve dose-normalized to 1 mg/kg. 3. Mean residence time estimated 0-24 hours postdose. n.e.: Not estimated. EXAMPLE 7 10 Aerosol And IV Efficacy Of Erythromycylamine In The S. Pneumonia Rat Lung Model Of Infection Methods Male Sprague-Dawley rats were infected by intratracheal administration with 50-100 microliters of S. pneumoniae A66 (Strain # PGO 4716) prepared in agar beads. The inoculum was prepared by suspending a broth culture of 15 PGO 4716 in molten agar, suspending the agar suspension in sterile mineral oil with mixing to generate small beads of agar containing the bacteria. The beads are recovered by centrifugation, resuspended in sterile saline, and administered to each animal through a tracheal incision by injection directly into the lung. Erythromycylamine solutions are prepared in sterile saline. Antibiotic was 20 administered either by intravenous injection into the tail vein or by aerosol exposure. The aerosol exposure was accomplished by nose-only exposure using the In-Tox Aerosol Exposure System (model No. 04-1100). This system is a closed aerosol delivery system designed to expose rodents that are confined in plastic tubes, open to the system at one end (nose port) and sealed at the other to maintain system integrity. 25 The aerosol is generated by a Pari LC Star ™ air-jet nebulizer at a flow of approximately 6.5 liters per minute. Vacuum is set at 9 liters per minute such that the total flow through the system with diluter air is 7.5 liters per minute. Treatment is initiated 24 hours after infection and continued once per day, for 3 days. Aerosol was administered for 30 minutes each day. On day four after WO 02/03998 PCT/US01/41328 . -30- infection and 12 hours after the last dose, animals are sacrificed and lungs surgically removed. After removal, lungs are homogenized, diluted and quantitatively plated onto blood agar. Plates are incubated for 24 hours and colonies of S. pneumoniae counted to determine bacterial load. The results are shown in Table 5: 5 Table5 Efficacy of Brythromycylamine vs. S. pneumoniae in the Rat Pneumonia Model Dose CFU/gram Route (mg/kg per day) Recovered IV 0 8.5 x 107 10 BQL* 20 BQL* 40 BQL* Aerosol 0 4.1 x 107 0.13 3.5 x 102 0.67 BQL* 1.33 BQL* *BQL=Below Quantitation Limit EXAMPLE 8 Aerosol Efficacy Of Erythromycylamine 10 In The S. Pneumonia Rat Pulmonary Model Of Infection After a Single Dose Treatment of Erythromycylamine Male Sprague-Dawley rats were infected and exposed to aerosol treatment as described in Example 7. The single treatment was initiated 24 hours after infection with aerosol administered at the doses indicated for 30 minutes. No further treatment 15 was undertaken and animals were observed until surgery. On day four after infection (day 3 after dosing), the animals were sacrificed and their lungs were surgically removed. After removal, the lungs are homogenized, diluted and quantitatively plated onto blood agar. The plates are incubated for 24 hours and colonies of S. pneumoniae are counted to determine bacteria load. The results after a single dose administration 20 are shown in Figure 11. Further results are shown in Table 6: WO 02/03998 PCT/US01/41328 -31- Table 6 Efficacy of Erythromycylamine vs. S. pneumoniae in the Rat Pneumonia Model Number Dose CFU/gram of Doses (mg/kg/day) Recovered 3 0 4.1 x 107 3 0.13 3.5 x 107 3 0.67 BQL 3 1.33 BQL BQL = below quantitation limit EXAMPLE 9 5 Aerosol Delivery Of Erythromycylamine To Dogs Characterization Of Aerosol Pharmacokinetics Inhalation Pharmacokinetics: For a 60 mg/mL solution, 3.191 g (4.08 mmol) of erythromycylamine (94% purity) was added to 43 mL of DI water and 4.27 mL (4.27 mmol) of 1 M sulfuric acid in a 50 ml volumetric flask. The solution was then 10 adjusted to pH 6.5 with the addition of another 53 |iL (0.053 mmol) Of 1 M sulfuric acid. The volume was brought up to 50 mL with additional DI water. Dogs were exposed once to either 60 mg/mL solution of erythromycylamine sulfate via an inhalation mask exposure system (Inveresk Research, Scotland, UK) for 30 minutes. The dogs were removed from their pen in the dog holding area and transferred 15 to the dosing laboratory. During dosing the animals were either restrained by an animal attendant or in a sling/harness system. Inhalation dosing was undertaken using a closed facemask connected to a nebulizer that was suitably characterized prior to commencement of dosing. The dosing apparatus incorporates a facemask and mouthpiece attached to flexible tubing, which was connected to the nebulizer device. 20 The mouthpiece was located inside the animal's mouth, on top of the tongue, and the facemask sealed around the dog's snout by means of a rubber sleeve. An exhaust valve from the mask was connected to an extract system. When the dosing apparatus is fully assembled and fitted to the dog, inspiration is shown by movement of the aerosol through the flexible tubing to the dog. 25 Lung samples were collected from 2 dogs at 2, 24, 48, 72, 96 and 120 hours post-dosing. Lungs were removed surgically from the dogs, and each lobe (right caudal, left caudal, right cranial, left cranial, right middle and accessory), was separated for assay. Plasma samples were collected from all surviving animals at 2, 24, 48, 72,96 and 120 hours post. 30 Erythromycylamine concentrations in plasma and the lung (per gram of lung tissue) were determined using a LC-MS method. Plasma samples (100 fig) were WO 02/03998 -32- PCT/USO1/41328 spiked with oleandomycin (internal standard, 1 |i,g/mL) before extraction. Plasma samples (100 (xL) were deproteinated with 3.3% trichloroacetic acid (TCA). Samples were centrifuged (10,000 rpm, 10 min.) and the supernatant was transferred to HPLC centrifilter for centrifiltration (10,000 rpm, 10 min). The mobile phase consisted of 5 0.1% acetic acid-acetonitrile (70:30, v/v, pH=3.2) solution at a flow rate of 0.5 ml/min for 3 minutes, followed by 0.1% acetic acid-acetonitrile (60:40, v/v) at a flow rate of 0.8 ml/min for 3 minutes. A stainless steel analytical column (Zorbax SB-C18, 2.1 mm ID x 150.0 mm, 5 jxm with a Phenomenex cartridge guard column) was used as the stationary phase. The column temperature was 50°C. Quantification of 10 erythromycylamine was performed using a HP 1100 LC/MSD API-Electrospray System. Data acquisition was set in the selective ion monitoring mode. The method was linear (r>0.9990) in the concentration range of 0.01 to 50 p.g/ml. The absolute recovery was greater than 90%. Lung samples were homogenized with DI water. Oleandomycin was added to 15 the samples as an internal standard. The homogenate was deproteinated with 0.9 M TCA. Samples were centrifuged at 10,000 rpm for 10 minutes and the supernatant was transferred to HPLC centrifilters for centrifiltration. The mobile phase consisted of 0.1% acetic acid-acetonitrile (70:30, v/v, pH=3.2) at a flow rate of 0.5 ml/min for 3 minutes, followed by 0.1% acetic acid-acetonitrile (60:40, v/v) at a flow rate of 0.8 20 ml/min for 3 minutes. A stainless steel analytical column (Zorbax SB-C18, 2.1 mm ID x 150.0 mm, 5 jxm with a Phenomenex cartridge guard column) was used as the stationary phase. The column temperature was 50° C. Quantification of the erythromycylamine was performed using a HP 1100 LC/MSD API-Electrospray System. Data acquisition was set in the selective ion monitoring mode. The linearity 25 (r>0.99) of the assay ranged from 2 to 100 jig/g for lung. The extraction efficiency was greater than 90%. Pharmacokinetic parameters, area under the curve (AUC) and mean residence time (MRT) and half-life (T1/2) were estimated baised on the statistical moment theory using WinNonlin™ Professional Version 3.1 software (Pharsight Corporation). The 30 peak concentration (Cmax) was not estimated but observed. Pharmacokinetic parameters in the lung and plasma inhalation following administration of erythromycylamine in the dog are summarized in Table 7 below, and in Figures 13 and 14: WO 02/03998 PCT/US01/41328 -33- Table 7 Pharmacokinetic Parameters of Erythromycylamine in the Lung and Plasma following 30-minutes Inhalation Administration of 60 mg/mL Solution in the Dog (N=2' Phannacokinetic Parameter (unit) Matrix c ^max (Hg/gram) AUC(0-120h) (Hg-h/gram)1 ti/2 (h) MRT (h)1 Whole Lung 69 2085 27 29 Plasma 1.0 29 n.e. 37 Right Caudal 77 2078 26 26 Left Caudal 68 2000 26 28 Right Cranial 87 2517 24 27 Left Cranial 54 1857 29 31 Right Middle 48 1979 30 34 Accessory 68 2256 31 31 Mean residence time Not estimated. EXAMPLE 10 Liquid Aerosol Delivery Of Erythromycylamine A solution of erythromycylamine sulfate (100 mg/mL) in quarter normal 10 saline at pH 7.0 is prepared in accordance with the general procedure of the foregoing examples. A 1.0 mL dose of the solution is administered by aerosol inhalation in less than 10 minutes to a human subject suffering from acute exacerbation of chronic bronchitis (AECB) using an AeroGen Aerodose™ inhaler. A reduction in the bacteria associated with AECB and symptoms of AECB is observed. 15 EXAMPLE 10 Dry Powder Aerosol Delivery Of Erythromycylamine A dry powder formulation of erythromycylamine sulfate (100 mg) and a dry powder carrier (equal parts of lactose, 2-hydroxypropyl-p-cyclodextrin, mannitol and aspartame; - total weight 25mg) is prepared. The formulation is administered by 20 aerosol inhalation in less than 2 minutes to a human subject suffering from acute exacerbation of chronic bronchitis (AECB) using a Glaxo Ventolin Rotohale™ inhaler. A reduction in the bacteria associated with AECB and symptoms of AECB is observed. WO 02/03998 PCT/US01/41328 -34- While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention. </div>

Claims

<div class="application article clearfix printTableText" id="claims"> intellectual property office of n.z. 16 JUN 2004 35 received THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1.
An aerosol formulation for inhibition of susceptible bacteria in the 5 endobronchial space of a subject suffering from an endobronchial infection, said formulation comprising from 50 mg to 750 mg of a macrolide antibiotic and a pharmaceutically acceptable carrier, in the form of particles having a mass median aerodynamic diameter between 1 and 5 |xm in size when administered using a jet nebulizer, a ultrasonic nebulizer, a vibrating porous plate nebulizer or a dry powder 10 inhaler. 2.
The aerosol formulation of Claim 1 wherein the macrolide antibiotic is selected from the group consisting of erythromycylamine, dirithromycin, erythromycin A, clarithromycin, azithromycin, and roxithromycin. 3.
The aerosol formulation of Claim 1 or Claim 2 wherein the macrolide antibiotic is erythromycylamine. 4.
The aerosol formulation of any one of Claims 1 to 3 having a pH which is in the 20 range of 5.0 to 7.0. 5.
The aerosol formulation of any one of Claims 1 to 4 wherein the nebulizer is a jet nebulizer. 6.
The aerosol formulation of any one of Claims 1 to 4 wherein the nebulizer is an ultrasonic nebulizer. 7.
The aerosol formulation of any one of Claims 1 to 4 wherein the nebulizer is a vibrating porous plate nebulizer. 30 8.
The aerosol formulation of any one of Claims 1 to 7 wherein the susceptible bacteria are selected from the group consisting of Streptococcus pneumoniae, Hemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae. 35 25 intellectual property office of n.z. 16 JUN 2004 36 received 9.
The aerosol of any one of Claims 1 to 8 wherein the pH is 6.0. 10.
Use of an aerosol formulation comprising 50 mg to 750 mg of a macrolide antibiotic and a pharmaceutically acceptable carrier in the manufacture of a 5 medicament for the treatment of susceptible bacterial infections by inhalation from a jet nebulizer, an ultrasonic nebulizer, a vibrating porous plate nebulizer or a dry powder inhaler of particles having a mass median aerodynamic diameter between 1 and 5 \xm in size. 10 11.
The use of Claim 10 wherein the macrolide antibiotic is selected from the group consisting of erythromycylamine, dirithromycin, erythromycin A, clarithromycin, azithromycin, and roxithromycin. 12.
The use of either Claim 10 or Claim 11 wherein the macrolide antibiotic is 15 erythromycylamine. 13.
The use of any one of Claims 10 to 12 wherein the pH of the aerosol formulation is in the range of 5.0 to 7.0. 20 14.
The use of any one of Claims 10 to 13 wherein the nebulizer used for administration of the aerosol formulation is a jet nebulizer. 15.
The use of any one of Claims 10 to 13 wherein the nebulizer used for administration of the aerosol formulation is a ultrasonic nebulizer. 16.
The use of any one of Claims 10 to 13 wherein the nebulizer used for administration of the aerosol formulation is a vibrating porous plate nebulizer. 17.
The use of any one of Claims 10 to 16 wherein the susceptible bacteria are 30 selected from the group consisting of Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae. 18.
The use of any one of Claims 10 to 17 wherein a dose of less than about 2.0 ml 35 of a nebulized liquid aerosol formulation comprising from 50 to 150 mg/ml of the macrolide antibiotic is administered to the subject in less than about 10 minutes. 'HTELLECtual property OFFICE OF n.7. 16 JUN 2004 RECEIVED^ 19.
The use of Claim 18 wherein the dose comprises less than 1.5 ml of the nebulized aerosol formulation. 5 20.
The use of either Claim 18 or Claim 19 wherein the dose comprises less than 1.0 ml of the nebulized aerosol formulation. 21.
The use of any one of Claims 18 to 20 wherein the aerosol formulation comprises from 70 to 130 mg/ml of the macrolide antibiotic. 22.
A unit dose device for inhalation administration, comprising a container containing less than 2.0 ml of a macrolide antibiotic formulation comprising from 50 to 150 mg/ml of a macrolide antibiotic in a liquid physiologically acceptable carrier. 15 23.
The unit dose device of Claim 22 which contains less than 1.5 ml of the macrolide antibiotic formulation. 24.
The unit dose device of either Claim 22 or Claim 23 which contains less than 1.0 ml of the macrolide antibiotic formulation. 20 25.
The unit dose device of any one of Claims 22 to 24 wherein the macrolide antibiotic formulation comprises from 70 to 130 mg/ml of the macrolide antibiotic. 26.
The unit dose device of any one of Claims 22 to 25 wherein the macrolide 25 antibiotic formulation comprises from 90 to 110 mg/ml of the macrolide antibiotic. 27.
The unit dose formulation of any one of Claims 22 to 26 wherein the macrolide antibiotic is selected from the group consisting of erythromycylamine, dirithromycin, erythromycin A, clarithromycin, azithromycin, and roxithromycin. 30 28.
The unit dose device of any one of Claims 22 to 27 wherein the macrolide antibiotic is erythromycylamine. 35 29.
A unit dose device for inhalation administration, comprising a container which contains less than 2.0 ml of a macrolide antibiotic formulation comprising from 20 to 200 mg/ml of erythromycylamine. 38 intellectual property office of n.z. 1 6 JUN 2m received 30.
A unit dose device for inhalation administration, comprising a container containing a macrolide antibiotic formulation comprising from 25 to 250 mg of a 5 macrolide antibiotic in a dry powder physiologically acceptable carrier. 31.
The unit dose device of Claim 30 wherein the macrolide antibiotic formulation comprises from 50 to 200 mg of the macrolide antibiotic. 10 32.
The unit dose device of either Claim 30 or Claim 31 wherein the macrolide antibiotic formulation comprises from 75 to 150 mg of the macrolide antibiotic.
34. The unit dose device of any one of Claims 30 to 33 wherein the macrolide antibiotic is selected from the group consisting of erythromycylamine, dirithromycin, 15 erythromycin A, clarithromycin, azithromycin, and roxithromycin.
35. The unit dose device of any one of Claims 30 to 34 wherein the macrolide antibiotic is erythromycylamine. 20
36. The unit dose device of any one of Claims 30 to 35 wherein the macrolide antibiotic formulation comprises from about 50% to about 90% by weight of the macrolide antibiotic.
37. An aerosol formulation for inhibition of susceptible bacteria in the 25 endobronchial space of a subject suffering from an endobronchial infection, substantially as herein described with reference to any one of the Examples.
38. Use of an aerosol formulation comprising 50 mg to 750 mg of a macrolide antibiotic and a pharmaceutically acceptable carrier in the manufacture of a 30 medicament for the treatment of susceptible bacterial infections by inhalation, substantially as herein described with reference to any one of the Examples. 35 intellectual property office of n 7. 1 6 JUN 2004 received
39. A unit dose device, substantially as herein described with reference to any one of the Examples. Dated this eleventh day of June 2004 Chiron Corporation Patent Attorneys for the Applicant: F B RICE & CO END OF CLAIMS </div>