WO2008137917A1

WO2008137917A1 - Method of treating bacterial infections with antibacterial formulations

Info

Publication number: WO2008137917A1
Application number: PCT/US2008/062868
Authority: WO
Inventors: Frank G. Pilkiewicz; Vladimir Malinin; Xingong Li; Renu Gupta
Original assignee: Transave, Inc.
Priority date: 2007-05-07
Filing date: 2008-05-07
Publication date: 2008-11-13

Abstract

The present invention relates, in part, to a method of treating a bacterial infection in a human comprising administering to a human in need thereof an effective amount of a lipid antibiotic formulation by inhalation once every day or once every greater time interval. In certain embodiments, the formulation is a liposomal antibiotic formulation. In certain embodiments, the antibiotic is an aminoglycoside, such as amikacin.

Description

Method of Treating Bacterial Infections with

Antibacterial Formulations

Background of the Invention Infection with P. aeruginosa is associated with increased morbidity and mortality in

CF subjects. The aggressive use of oral, intravenous, and aerosolized antibiotics has contributed to increased longevity of these subjects over the past three decades. Currently, inhaled antibiotics, specifically inhaled tobramycin and colistin, are indicated for the treatment of established infections of P. aeruginosa in CF subjects. Use of tobramycin 300 mg inhaled twice a day on alternating months has been shown to increase FEVi % predicted, decrease the density of P. aeruginosa in sputum, decrease hospitalizations, and reduce the need for intravenous antibiotics (Ramsey, Pepe et al. 1999). However, patients have to be dosed twice a day for approximately 15-20 minute inhalation periods per dose.

Therefore, there are opportunities for improvement over inhaled tobramycin, including decreasing the frequency of administration, reducing the administration time, improving quality of life, and possibly increasing the potency of antibacterial activity.

Summary of the Invention

In one aspect, the present invention relates to a method of treating a bacterial infection in a human comprising administering to a human in need thereof an effective amount of an antibiotic formulation by inhalation once every day or once every greater time interval. In a further embodiment, the antibiotic formulation is a lipid based antibiotic formulation. In a further embodiment, the antibiotic formulation is a liposomal antibiotic formulation. In a further embodiment, the antibiotic is an aminoglycoside. In a further embodiment, the antibiotic is amikacin. In a further embodiment, the amount of antibiotic formulation is 5 to 2,500 mg. In a further embodiment, the amount of antibiotic formulation is 250 to 1,500 mg. In a further embodiment, the amount of antibiotic formulation is 500 to 1,000 mg.

In a further embodiment, the antibiotic formulation is administered once every day. In a further embodiment, the antibiotic formulation is administered once every two days. In a further embodiment, the antibiotic formulation is administered once every three days. In a further embodiment, the antibiotic formulation is administered once every day for 5 days to 6 months. In a further embodiment, the antibiotic formulation is administered once every day for 5 days to 3 months. In a further embodiment, the antibiotic formulation is administered once every day for 5 days to 2 months. In a further embodiment, the antibiotic formulation is administered once every day for 5 days to 1 month. In a further embodiment, the antibiotic formulation is administered once every day for 5 days to 2 weeks. In a further embodiment, the antibiotic formulation is administered once every day for a week.

In a further embodiment, the antibiotic formulation is administered once every day for a week followed by a week of no administration, wherein this cycle is repeated more than once. In a further embodiment, the antibiotic formulation is administered once every day for 14 days followed by 14 days of no administration, wherein this cycle is repeated more than once. In a further embodiment, the antibiotic formulation is administered once every day for 28 days followed by 28 days of no administration, wherein this cycle is repeated more than once.

In a further embodiment, the antibiotic is amikacin and the amount of antibiotic formulation is 5 to 2,500 mg administered once every day for 5 days to 3 months.

In a further embodiment, the lipid based or liposomal antibiotic formulation comprises a lipid selected from the group consisting of egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), egg phosphatidylinositol (EPI), egg phosphatidylserine (EPS), phosphatidylethanolamine (EPE), phosphatidic acid (EPA), soy phosphatidylcholine (SPC), soy phosphatidylglycerol (SPG), soy phosphatidylserine (SPS), soy phosphatidylinositol (SPI), soy phosphatidylethanolamine (SPE), soy phosphatidic acid (SPA), hydrogenated egg phosphatidylcholine (HEPC), hydrogenated egg phosphatidylglycerol (HEPG), hydrogenated egg phosphatidylinositol (HEPI), hydrogenated egg phosphatidylserine (HEPS), hydrogenated phosphatidylethanolamine (HEPE), hydrogenated phosphatidic acid (HEPA), hydrogenated soy phosphatidylcholine (HSPC), hydrogenated soy phosphatidylglycerol (HSPG), hydrogenated soy phosphatidylserine (HSPS), hydrogenated soy phosphatidylinositol (HSPI), hydrogenated soy phosphatidylethanolamine (HSPE), hydrogenated soy phosphatidic acid (HSPA), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), dioleylphosphatidyl-ethanolamine (DOPE), palmitoylstearoylphosphatidyl-choline (PSPC), palmitoylstearolphosphatidylglycerol (PSPG), mono-oleoyl-phosphatidylethanolamine (MOPE), cholesterol, ergosterol, lanosterol, tocopherol, ammonium salts of fatty acids, ammonium salts of phospholipids, ammonium salts of glycerides, myristylamine, palmitylamine, laurylamine, stearylamine, dilauroyl ethylphosphocholine (DLEP), dimyristoyl ethylphosphocholine (DMEP), dipalmitoyl ethylphosphocholine (DPEP) and distearoyl ethylphosphocholine (DSEP), N-(2, 3- di-(9-(Z)-octadecenyloxy)-prop-l-yl- N,N,N-trimethylammonium chloride (DOTMA), 1, 2-bis(oleoyloxy)-3- (trimethylammonio)propane (DOTAP), phosphatidyl-glycerols (PGs), phosphatidic acids (PAs), phosphatidylinositols (PIs), phosphatidyl serines (PSs), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylacid (DMPA), dipalmitoylphosphatidylacid (DPPA), distearoylphosphatidylacid (DSPA), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphospatidylinositol (DSPI), dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), and mixture thereof. In a further embodiment, the lipid based or liposomal antibiotic formulation comprises a phospholipid and a sterol. In a further embodiment, the lipid based or liposomal antibiotic formulation comprises DPPC and cholesterol. In a further embodiment, the lipid based or liposomal antibiotic formulation comprises DPPC and cholesterol in a 2 to 1 ratio by weight.

In a further embodiment, the antibiotic is amikacin; the amount of antibiotic formulation is 5 to 2,500 mg administered once every other day for a week to 3 months, and the lipid based or liposomal antibiotic formulation comprises DPPC and cholesterol in a 2 to 1 ratio by weight. These embodiments of the present invention, other embodiments, and their features and characteristics, will be apparent from the description, drawings and claims that follow.

Brief Description of the Drawings

Figure 1 depicts mass distribution of Liposomal Amikacin nebulizate collected on impactor stages as a function of cutoff diameter. The three Liposomal Amikacin lots of Table 15 legend (designated as 1, 2, and 3) were used with the eFlow nebulizer and ACI system (solid symbols) or the LC Star nebulizer and NGI system (open symbols). Figure 2 depicts reduction in the LogioCFU/Lungs of Rats after Inhalation of Liposomal Amikacin 75 mg/mL or Tobramycin The symbols represent the LogioCFU/lungs of each rat 18 days after the instillation of PA3064 in agar beads and 3 days after the last inhalation session of saline or one of the above antibiotics. The values at 2.0 Logio CFU represent the lower limit of detection of bacteria in the lung in the method. The bar represents the mean of each group. The means and standard deviations and two-tail t-test results were calculated using Excel software by Microsoft.

Figure 3 depicts reduction in the LogioCFU / lungs of rats after Inhalation of Liposomal Amikacin and Tobramycin for 28 days. Equivalent doses of the above antibiotics were given by inhalation therapy but on different schedules. Tobramycin was given BID daily for a total of 104 min per day for 28 days. Liposomal Amikacin was given once daily for 80 min for 28 days (QlDx28) as was saline. Liposomal Amikacin was also given once daily for 160 min every other day for 28 days (Q2Dxl4) or once daily for 160 min for 14 consecutive days (Q IDX 14) then just observed until the rats were euthanized. The symbols represent the LogioCFU/ lungs of each rat 35 days after the instillation of P. aeruginosa 3064 in agar beads. The means and standard deviations and two-tail t-test were calculated using Excel software by Microsoft).

Detailed Description of the Invention

I. Definitions For convenience, before further description of the present invention, certain terms employed in the specification, examples and appended claims are collected here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included. The term "including" is used to mean "including but not limited to". "Including" and "including but not limited to" are used interchangeably.

The term "pulmonary distress" refers to any disease, ailment, or other unhealthy condition related to the respiratory tract of a human. Generally pulmonary distress results in difficulty of breathing.

It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction.

The term "substituted" is also contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein above. The permissible substituents may be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.

For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Also for purposes of this invention, the term "hydrocarbon" is contemplated to include all permissible compounds having at least one hydrogen and one carbon atom. In a broad aspect, the permissible hydrocarbons include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic organic compounds that may be substituted or unsubstituted.

The term "treating" is art-recognized and refers to curing as well as ameliorating at least one symptom of any condition or disease. The term "prophylactic" or "therapeutic" treatment is art-recognized and refers to administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate or maintain the existing unwanted condition or side effects therefrom). A "patient," "subject" or "host" to be treated by the subject method may mean either a human or non-human animal.

The term "mammal" is known in the art, and exemplary mammals include humans, primates, bovines, porcines, canines, felines, and rodents (e.g., mice and rats).

The term "bioavailable" is art-recognized and refers to a form of the subject invention that allows for it, or a portion of the amount administered, to be absorbed by, incorporated to, or otherwise physiologically available to a subject or patient to whom it is administered.

The term "pharmaceutically-acceptable salts" is art-recognized and refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds, including, for example, those contained in compositions of the present invention.

The term "pharmaceutically acceptable carrier" is art-recognized and refers to a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the subject composition and its components and not injurious to the patient. Some examples of materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

Contemplated equivalents of the compositions described herein include compositions which otherwise correspond thereto, and which have the same general properties thereof, wherein one or more simple variations of substituents or components are made which do not adversely affect the characteristics of the compositions of interest. In general, the components of the compositions of the present invention may be prepared by the methods illustrated in the general reaction schema as, for example, described below, or by modifications thereof, using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants which are in themselves known, but are not mentioned here.

II. Introduction

Lipid based or liposomal aminoglycoside, such as amikacin, formulations for inhalation are sustained-release formulations of aminoglycosides encapsulated inside nanoscale liposomal carriers designed for administration via inhalation. It is hypothesized that the sustained-release targeting of high concentrations of amikacin in the lungs and bio film penetration properties of this formulation will have several advantages over inhaled tobramycin in treating CF subjects with chronic infection caused by P. aeruginosa. These advantages include: 1. The ability to attain a prolonged antibiotic effect of amikacin in the lung by achieving high concentrations and a prolonged half life due to sustained release.

2. The ability to target and increase the effective concentration of amikacin in the lung with low systemic levels of the aminoglycoside.

3. The potential to better target bacteria growing in a bio film as a result of unique properties of lipid based or liposomal aminoglycosides.

4. Additional release of the drug at the site of infection in the lungs of CF patients, due to targeted action of secreted phospho lipase C and rhamno lipids from bacteria and/or phospholipase A2 or defensins from activated polymorphonuclear leukocytes. 5. Amikacin is a semisynthetic aminoglycoside with a unique resistance to aminoglycoside inactivating enzymes. Consequently, some P. aeruginosa strains which are resistant to tobramycin will remain susceptible to amikacin.

6. Amikacin has less binding affinity than other aminoglycosides for megalin, the transporter responsible for renal cortical aminoglycoside accumulation, and thus inherently has a lower potential for nephrotoxicity.

7. The increase in both the half life, and the area under the concentration curve (AUC) of lipid based or liposomal amikacin, along with bio film penetration should allow for less frequent administration, enhanced bactericidal activity and reduced potential for selection of resistant organisms .

Peclinical pharmacokinetics have demonstrated that the AUC (0-48 hr) of amikacin in the lungs of rats that received a 60 mg/kg dose aerosol of Liposomal Amikacin was five-fold higher than the AUC of tobramycin in the lungs of rats that received an equal dose of tobramycin by inhalation. Generally, 10% of the administered antibiotic is deposited in the lungs for rats. Conversely, the AUC of drug in the kidneys of rats that received an equal dose of tobramycin was significantly higher than the kidney AUC of rats that received aerosols of Liposomal Amikacin. Additionally, data from 30-day inhalation toxicology studies in rats and dogs suggest that there will be no safety pharmacology issues with inhaled Liposomal Amikacin. In 14 days rat model studies of pseudomonas infection, it was noted that 60 mg/kg of Liposomal Amikacin (75 mg/mL) administered every other day for 14 days (Q2D x 7), which effectively delivered half the cumulative dose of aminoglycoside than the other groups, was as effective as 60 mg/kg of Liposomal Amikacin given once per day, and Tobramycin given twice per day daily for 14 days. With 28 day dosing in this model, there were equivalent reductions in CFUs in animals receiving Liposomal Amikacin dosed daily at ~60 mg/kg or dosed every other day at -120 mg/kg. Liposomal Amikacin administered at 120 mg/kg once a day for 14 days was as effective as Tobramycin 60 mg/kg/day (administered twice a day) for 28 days, which suggests a higher AUC and possibly a prolonged post-antibiotic effect with Liposomal Amikacin at 120 mg/kg dosed once per day (see Example 3).

The administration of Liposomal Amikacin via inhalation in the animal model resulted in increased lung (AUC) above the MIC of the bacteria, and demonstrated sustained therapeutic effect, with a reduced frequency, and duration of dosing as compared to Tobramycin. Importantly, the preclinical data for Liposomal Amikacin appear supportive of the hypothesis that this specific formulation may be advantageous over other inhalation products that are hindered by a rapid clearance from lung tissue, necessitating frequent dosing (Geller, Pitlick et al. 2002), which poses a burden for patients and might limit patient compliance. This, along with the safety pharmacology profile of the molecule is supportive of further development of this formulation in the clinic.

Additionally, clinical experience demonstrated that nebulized Liposomal Amikacin 50 mg/mL administered as 500 mg once per day for 14 days is well tolerated, and elicits a clinically relevant effect on pulmonary function and decrease in P. aeruginosa density in CF patients. Also, evaluation of the PK data indicates the systemic exposure to Liposomal Amikacin, even at the 500 mg dose, is very low. By either Cmax or AUC or mg of aminoglycoside which is recovered in the urine, the observed systemic exposure to amikacin, associated with Liposomal Amikacin, given by inhalation is approximately 1/5 to 1/4 the exposure seen with 600 mg/d of TOBI and is less than 1/200 compared to normal parenteral doses of Amikacin. Data indicate high levels of Amikacin are achieved in the sputum. Median AUC values for sputum were 290 and 980 fold greater than the median AUC values for serum on day 1 and day 14 respectively.

III. Overview of Clinical Pharmacology Sustained release of inhalation antimicrobials via a lipid/liposome delivery system could provide the opportunity to maintain prolonged targeted lung exposures and enhance the uptake of drug to the site of infection. Transave, Inc. has developed a sustained release targeting formulation of amikacin encapsulated inside nanoscale liposomal carriers designed for administration via inhalation (Liposomal Amikacin). Using data from a human clinical Phase lb/2a study in which CF patients who were chronically infected with P. aeruginosa received multiple doses of Liposomal Amikacin 50 mg/ml, the objectives of the analyses described herein were three-fold: (1) to use population pharmacokinetic (PK) modeling to characterize amikacin systemic exposure, including approximate systemic bioavailability; (2) to characterize the disposition of liposomal amikacin in sputum; and 3) to characterize the pharmacokinetic-pharmacodynamic (PK- PD) relationship between change in forced expiratory volume in one second (FEVi), change in percent predicted forced expiratory volume in one second (FEVi % predicted), forced expired flow between 25-75% of forced vital capacity (FEF₂5-75%), and forced vital capacity (FVC), in P. aeruginosa colony forming units (CFU) relative to baseline at Days 7 and 14, and amikacin exposure.

IV. Methods Study Design

Data used for this population PK analysis were obtained from two human clinical Phase lb/2a studies (103 and 104) in which CF patients, chronically infected with P. aeruginosa, were administered a total of 500 mg of Liposomal Amikacin daily (as two 20 minute sessions with a 5 minute rest period in between) for 14 days. Amikacin serum samples were obtained pre-dose, and 1, 2, 4, 6, 8, 12 and 24 hours post-dose on Days 1 and 14, while urine samples were collected over 6 hour intervals on Day 1 and Day 14 for a period of 24 hours. Sputum samples were also collected on Day 1 and Day 14, soon after the dose was administered, between 4 and 6 hours after dosing and prior to dose administration on the following day, as well as on Days 14, 21, and 28. Serum, sputum and urine samples were assayed for amikacin using Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS).

Pulmonary function tests (PFT) were carried out during screening from Day -14 to 0) and at baseline (i.e., prior to dose administration on Day 1) and on Day 1, 7, 14, 21, 28, 35, and 42. Sputum samples for microbiology were also collected at baseline and on each of these days. Additional PFTs were carried out 1.5 hours and 3 hours post-dose on Day 1 and Day 14.

V. Pharmacokinetic Analysis

The data were fit by candidate PK models, using Monte Carlo Parametric Expectation Maximization (MC-PEM), as is implemented in S-ADAPT 1.53, initially fitting the plasma concentrations, then co-modeling the serum and urine data. Model discrimination was based on the fit of the data and change in objective function. The 24 hour area under the curve (AUC) at steady state for serum amikacin values were calculated using the post-hoc parameter estimates from the final population PK model. Covariate relationships between patient demographics and individual post-hoc parameters were assessed first graphically, then by means of statistical models created using SYSTAT^® 11 (SYSTAT Software, Inc., Richmond, CA). Sputum AUC values from 0 to 24 hours on Day 1 and Day 14 were obtained using the linear trapezoidal rule.

VI. Pharmacokinetic-Pharmacodynamic Analysis

Dependent variables for the PK-PD analysis included the change in PFT values for FEVi, FEVi % predicted, FEF25_75<>_/0 and FVC, on Day 7 and 14 relative to baseline (i.e., prior to dose administration on Day 1) and the change in logio CFU on each of these days relative to baseline. Independent variables evaluated included the ratio of the average 24 hour AUC for serum and sputum to the baseline minimum inhibitory concentration (MIC), AUC:MIC ratio for P. aeruginosa. The average 24 hour serum and sputum AUC was computed by taking the average of the Day 1 and Day 14 AUC values.

Using a one-sample t-test, the statistical significance of mean changes from baseline for each of the above-described dependent variables were assessed. Using Spearman's rank correlation (r_s), the direction and strength of the relationship between each of the dependent variables and AUC:MIC ratio for serum and sputum were assessed. The direction and strength of the relationship between change in each of the PFT values from baseline and change in log₁₀ CFU from baseline were also assessed.

VII. Results Patients

A total of 24 patients completed the two studies with 13 patients from Study 103 and 11 patients from Study 104. The median (min, max) age of all the patients was 23.7 (14, 38) years with a median (range) creatinine clearance (CrCL) at baseline of 126 (76.8, 173) mL/min/1.73m².

Pharmacokinetic Analysis

The most robust fit to the serum concentration data was obtained using a two- compartment model (one absorption site, the lung, and the central compartment) with zero- order drug input into the lungs, a first-order process from lungs to the central compartment and linear elimination. Allowing inter-occasional variation on apparent total clearance (CLt/F) and apparent central volume of distribution (Vc/F) between Day 1 and Day 14 improved the objective function statistically. Urine data was modeled by fitting the amounts of amikacin recovered in the collection intervals, as a function of serum concentrations and renal clearance (CLr). Table 1 is a summary of the fitted PK parameter values. Table 1. Structural population pharmacokinetic model for liposomal amikacin for inhalation with inter-occasional variability - Parameter estimates and standard errors.

Parameter Population mean Inter-mdividual variability (%CV)

Final %SE Final %SE estimate estimate

CLt/F Day 1 (L/hr) 68.4 10.3 48.7 29.9 Vc/F Day 1 (L) 286 12.3 59.0 29.7 ka (hr^"1) 3.34 32.5 99.8 50.5 CLr (L/hr) 3.40 15.4 63.9 36.7 CLt/F Day 14 (L/hr) 45.2 8.01 37.1 30.7 Vc/F Day 14 (L) 250 8.51 27.0 30.8

SDintserum 0.05 6.02

SDslpuπne 0.70 9.16

SDintuπne 0.03

Minimum value of the objective function = -258.6

The goodness of fit for observed versus Bayesian post-hoc individual fitted serum concentration data was excellent, with an overall r² of 0.98. As evidenced by an overall r² of 0.38 for observed versus individual fitted urine data, the goodness of fit for urine data was poor. The post-hoc covariate analysis, using generalized linear modeling (GLM) revealed a significant relationship between height and gender and Day 14 estimates of CLt/F and Vc/F. The post-hoc renal clearances obtained appeared to be falsely low, which was likely due to incomplete urine collection and poor documentation.

The AUC values for the serum and sputum data are shown in Tables 2 and 3, respectively. Median AUC values for sputum were 286 and 978 fold greater than the median AUC values for serum on Day 1 and Day 14, respectively. As evidenced by the higher CV% values, greater variability was evident in sputum (117% on Day 1 and 91.2% on Day 14) compared to serum AUC (51.9% on Day 1 and 42.4% on Day 14) values. Table 2. Summary of serum AUC values¹ - All patients

Study Day N Mean SD Min Median Max

Day 1 24 8.27 4.29 3.67 6.88 20.1 Day 14 24 12.0 5.08 5.65 10.8 30.1

AUC values in mcg/mL»hr

Table 3. Summary of sputum AUC values¹ - All patients

Study Day N Mean SD Min Median Max

Day 1 20 3830 4500 78.70 1970 17200 Day 14 19 12500 11400 1740 10578 50000

¹AUC values are in mcg/mL'hr

A 2-compartment model with zero-order input into the lungs, a first-order process from the lungs to the central compartment, and linear elimination was fit to the PK data. Serum (r² = 0.98) and urine (r² = 0.38) concentrations were well and modestly fit by model, respectively. On Day 7, 14 and 21, the observed change for FEF25_75<>_/0 was 0.49 (p < 0.001), 0.42 (p = 0.02) and 0.34 L/sec (p = 0.04), respectively. On Day 7 and 14, the observed change for FEVi was 0.24 (p = 0.002) and 0.13 L (p = 0.10), respectively, and was 7.49 (p <0.001) and 4.38 L/sec (p = 0.03) for FEVi% predicted. Significant relationships (p < 0.05) between logio CFU and serum AUC:MIC ratio, and between changes in logio CFU and FEVi, FEVi% predicted and FVC were identified.

Pharmacokinetic-Pharmacodynamic Analysis

Baseline and Day 14 PFT data were available for all 24 patients and for PFTs carried out on Day 7 and 21, such data were available for 23 patients. Microbiology data were available for all 24 patients. Since MIC values collected prior to dosing on Day 1 for Study 104 were not reported, the screening MIC values as well as CFU counts were used as baseline values. Using a one-sample t-test, the statistical significance of mean changes from baseline for each of the above-described dependent variables was assessed. Using Spearman's rank correlation (r_s), the direction and strength of the relationship between each of the dependent variables and AUC:MIC ratio for serum and sputum was assessed. Mean changes in PFT values on Day 7 relative to baseline were statistically significant for all PFT endpoints. Mean changes in FEVi % predicted and FEF25-750_/0 on Day 14 relative to baseline were also statistically significant (p = 0.029 and p = 0.016, respectively). By Day 21, mean change in FEF25-750_/0 relative to baseline was the single PFT that remained statistically significant (p = 0.036). Regardless of the study day considered, mean change in log 10 CFU from baseline was not statistically significant.

As shown in Table 4, correlations between change in PFT values from baseline and either sputum or serum AUC:MIC ratio were not statistically significant, regardless of whether changes on Day 7 or 14 were evaluated. As shown in Table 5, the correlation between change in logio CFU from baseline and serum AUC:MIC ratio was statistically significant for both Day 7 or 14. Increasing serum AUC:MIC ratios were associated with larger decreases in log₁₀ CFU on Day 7 (r_s= -0.46, p = 0.048 ) and 14 (r_s= -0.45, p = 0.048) relative to baseline.

Correlations between change in both PFT value and logio CFU on Day 7 and 14 relative to baseline were statistically significant for FEVi, FEVi % predicted, and FVC (p < 0.05).

Table 4. Relationship between change in pulmonary function test values from baseline and AUC:MIC ratio for serum and sputum - All patients

Change in PFT values from baseline

Spearman's FEVi FEVi % FEF25-75% FVC

Study Day AUC :MIC rank predicted correlation

Day 7 serum 0.072 0.0066 < 0.0001 0.021 p value 0.21 0.71 0.97 0.51

Day 14 serum r_s ² 0.046 0.0073 0.00018 0.0012 p value 0.31 0.69 0.95 0.87

Day 7 sputum r_s ² 0.033 0.040 0.0085 0.19 p value 0.46 0.41 0.71 0.06

Day 14 sputum r_s ² 0.025 0.052 0.0053 0.06 p value 0.51 0.35 0.77 0.31

Table 5. Relationship between change in logio CFU and AUC:MIC ratio for serum and sputum - All patients

Study Day

Spearman's rank correlation logio CFU AUC:MIC

Day 7 serum 0.21 p value 0.048

Day 14 serum r_s ² 0.20 p value 0.048

Day 7 sputum 0.017 p value 0.64

Day 14 sputum r_s ² 0.0031 p value 0.84

Conclusion Liposomal amikacin for inhalation pharmacokinetics was best described using a two-compartment model (one absorption site, the lung, and one central compartment) with zero-order input into the lung, a first-order process from the lungs to the central compartment and linear elimination.

It is not possible to adequately assess CLr, fraction absorbed or systemic bioavailability, due to, we believe, incomplete urine data collection and poor documentation. As a result, lower than expected estimates of renal clearances were obtained making bioavailability, the exposure of the kidneys to amikacin per unit time, and actual renal clearance, difficult to estimate.

The median AUC values for sputum were 286 and 978 fold greater than the median AUC values for serum on Day 1 and Day 14, respectively. The high degree of variability associated with sputum AUC values precluded further insight into the pulmonary bioavailability of liposomal amikacin for inhalation. Mean changes in PFT values on Day 7 relative to baseline were statistically significant for all PFT endpoints. Similar such results were evident for FEVi % predicted and FEF25-750_/0 on Day 14 for relative to baseline and for FEVi, FEVi % predicted and FEF₂₅_₇₅O_/O on Day 21 relative to baseline. However, correlations between change in PFT values from baseline and sputum or serum AUC:MIC ratio were not statistically significant when either changes on Day 7 or 14 relative to baseline were evaluated.

While mean change in logio CFU of P. aeruginosa from baseline on both Day 7 and 14 was not statistically significant, the correlation between change in logio CFU from baseline at both of these time points and serum AUC:MIC ratio was statistically significant; increases in serum AUC:MIC ratio were associated with decreases in logio CFU. In contrast, this relationship did not hold with sputum AUC:MIC and confirms the large variability in sputum kinetics of Liposomal Amikacin, that is also shown with TOBI (Geller, Pitlick et al. 2002).

The significant relationships between changes in logio CFU and serum AUC:MIC ratio, and between changes in PFT values and logio CFU, and the lack of significant decrease in logio CFU of P. aeruginosa during the two weeks of treatment with liposomal amikacin for inhalation suggests that higher doses may be required to be more reliably effective in a large patient population.

VIII. Overview of Efficacy Two Phase lb/2a (studies 103 and 104), using the Liposomal Amikacin 50 mg/mL have been completed. The two studies were similar in design. A total of 24 CF patients (with FEVi >40% of predicted) received 500 mg Liposomal Amikacin daily for 14 days. The drug was administered using a PARI LC Star nebulizer, over a period of two 20-minute inhalation sessions with a 5 minute rest period between sessions. There were 13 patients enrolled in Study 103 and 11 patients in Study 104. Patient demographics were similar, with the exception of Pseudomonas MICs at baseline. In Study 103, the mean MIC (μg/mL) was 8 (range 1.5-16) and in Study 104, the mean MIC was 41 μg/mL (range 8- 192). The patients enrolled in Study 104 had prior experience with inhalation antibiotics, and per protocol, were permitted to resume treatment with TOBI^®/Colistin after Day 28 of the study. The patients in Study 103 were naϊve to inhalation antibiotics, and did not receive additional inhalation antibiotics during the follow-up period. The 500 mg dose of Liposomal Amikacin (50 mg/mL) was well tolerated, and in select patients improved pulmonary function and decreased the density of P. aeruginosa in sputum. The details of patient demographics for Studies 103 and 104 (combined) are shown in Table 6.

Table 6. Patient demographics in studies 103 and 104.

All efficacy analyses in these human clinical Phase lb/2a studies were exploratory in nature. The efficacy endpoints included:

Change from Baseline in density of P. aeruginosa (logio CFU/g) in sputum;

Change from Baseline in pulmonary function tests (FEVi, FEVi % predicted, FVC, and

FEF(25-75%). Changes in P. aeruginosa sputum density, FEVi, and FEVi % predicted at Day 14 were identified as the primary efficacy endpoints.

Quantitative culture of sputum samples and subsequent amikacin susceptibility testing of each morphologically distinct P. aeruginosa were performed. The MIC of amikacin for the isolates with the highest MIC cultured from each subject at screening and Day 14 was documented. The density (CFU per gram of sputum) of P. aeruginosa in sputum was calculated as the log₁₀ value for the sum of all morphotypes.

A summary of the baseline characteristics for the combined population (n=24) are shown in Table 7.

Table 7. Baseline measurements for patients in Studies 103 and 104.

Study 103: In this study CF patients infected with P. aeruginosa isolates sensitive to amikacin (amikacin MIC <64μg/mL), and those subjects naϊve to inhaled antibiotics were enrolled. Administration of Liposomal Amikacin 500 mg once daily for 2 weeks showed a mean change in log sum of counts of P. aeruginosa from baseline to Day 14 of 1.09

(n=13; 95% confidence interval, 2.09 to 0.09). The reductions in counts were observed in 9 of the 13 subjects. Treatment with Liposomal Amikacin did not result in selection of resistant strains of P. aeruginosa. The mean P. aeruginosa amikacin MIC was 8.04μg/mL at Day 0 and 30.79μg/mL at Day 14. On Day 14, a single isolate in one subject had a non sensitive MIC (>256μg/mL); all other Day 14 isolates were sensitive to amikacin. No human was hospitalized or received intravenous anti-pseudomonas antibiotics. Additionally, there was improvement in lung function as measured by an increase in FEVi from baseline to Day 14 of +260 mL (n=13; 95% confidence interval, +30 mL to +500 mL). The corresponding change in FEVl % predicted from baseline to Day 14 was +7.32%. Increases in FEVl were observed in 9 of the 13 subjects. Also noted were increases in FEF₍₂₅_₇₅O_/O) (mean: 570 mL) and FVC (mean: 180 mL).

Study 104: Study 104 was conducted in a population of CF patients who were infected with P. aeruginosa, and were inhalation antibiotic treatment experienced. In these patients, the administration of Liposomal Amikacin 500 mg q.d. for 2 weeks did not show any significant change in P. aeruginosa density during the study (p-values >0.297 for change from Day 1). The proportion of patients with mucoid P. aeruginosa remained constant throughout the study. No statistically significant changes in FEVi, FEVi % predicted, FVC, and FEF (25-75%) were observed after administration of Liposomal Amikacin 500 mg. However, trends suggesting improvement in FEVi % predicted, FVC, and FEF(25_75%) were observed at Day 7, Day 14 (end of treatment), and Day 15. Integrated Efficacy Summary: Studies 103 and 104

Data from the combined population of 24 patients in studies 103 and 104 are summarized below in Tables 8, 9, 10, and 11. The microbiologic end-point of change in log CFU of P. aeruginosa, demonstrated a reduction in bacterial density in the combined population, but this did not achieve statistical significance. However, when data were analyzed from the inhalation antibiotic naϊve patients (study 103), a statistically significant reduction in CFU was observed at end of treatment. Factors that might explain this effect are the inherent variability in sputum samples, the inter-laboratory variability in methodology, and reporting of quantitative microbiology, and the enrollment of patients with higher MICs (including resistant isolates) in study 104. All of the above are further compounded by the small sample size of each study.

Assessment of clinical benefit by measurement of pulmonary function tests showed a statistically significant improvement in lung function as measured by an increase in FEVi from baseline to Day 7 of +240 mL (n=23; p- value 0.0024). The effect at day 14 was a 126 mL increase from baseline in FEVl, which was not statistically significant. A corresponding statistically significant increase in FEVl % predicted from baseline to Day 7 was +7.49% (n=24; p-value 0.0002), and at Day 14 was +4.37% (n=24; p-value 0.0285). The improvement in lung function was also noted with the assessment of small airways as measured by FEF(₂5-75%) at day 7, an increase in +494 mL (n=23; p-value 0.001), and at Day 14, +423 mL (n=24; p-value 0.0162). These data support a clinically meaningful improvement in lung function in CF patients with chronic pseudomonas infection who have received a 14 day course of treatment with Liposomal Amikacin.

Table 8. Change in FEV from baseline at various times in all patients.

Table 9. Change in % predicted FEV from baseline at various times in all patients.

Table 10. Change in FEF25-75 from baseline at various times in all patients.

Table 11. Change in CFU from baseline at various times in all patients.

IX. Overview of Safety

Two Phase 1 single dose clinical studies have been completed with the 20 and 50 mg/mL formulations of Liposomal Amikacin in healthy volunteers and in CF patients, respectively. Six healthy volunteers received a single dose of 120 mg of Liposomal Amikacin and tolerated it well, and exhibited prolonged retention of the radiolabeled liposomes in the lungs, with a measured half-life of 46 hours.

Liposomal Amikacin was administered to CF subjects with chronic P. aeruginosa infections in a human clinical Phase I study (Study 101). Single doses of 90 mg (n=6), 270 mg (n=6), or 500 mg (n=4) were administered to CF subjects to evaluate the safety, tolerability and pharmacokinetics of liposomal amikacin for inhalation. A total of 24 patient dosing sessions of a single dose administration of Liposomal Amikacin or placebo by inhalation via the Pari LC Star nebulizer were evaluated. Two serious adverse events were reported (both occurring in placebo group). Both events recovered without sequelae. A total of 41 adverse events (AEs) were experienced by 17 of the 24-patient sessions dosed (71%) during the trial. Of the AEs reported, 10 of the 16 patients (62.5%) who reported adverse events were in the active group and 7 of the 8 patients (87.5%) were in the placebo group. Headache was the most common AE reported in the active group and no patients were discontinued from the study due to AEs. Liposomal Amikacin was well tolerated and safe up to a single dose of 500 mg administered via inhalation.

Additionally, the PK data confirm minimal systemic drug levels, and high sputum levels of drug, and pharmacodynamic modeling estimates long elimination half life presumably due to slow release from liposomes.

The most recent multidose human clinical Phase lb/2a studies (Study 103 and Study 104) enrolled a total of 24 patients with Cystic Fibrosis. Patients received 500 mg daily dose of Liposomal Amikacin by inhalation for 14 days. Inhaled Liposomal Amikacin 50 mg/mL was well tolerated, and in select patients improved pulmonary function. In summary, the two studies were similar in design. A total of 24 CF patients (with

FEVi > 40% of predicted) received 500 mg Liposomal Amikacin daily for 14 days. Drug was administered using a PARI LC Star nebulizer, over a period of two 20-minute inhalation sessions with a 5 minute rest period between sessions. There were 13 patients enrolled in Study 103 and 11 patients in Study 104. Patient demographics were similar, with the exception of Pseudomonas MICs at baseline, and history of prior exposure to inhalation antibiotics. In Study 103, the mean MIC (μg/mL) was 8 (range 1.5-16) and in Study 104 the mean MIC was 41 μg/mL (range 8-192). The patients enrolled in study 104 had prior experience with inhalation antibiotics, and per protocol, were permitted to resume treatment with TOBI^®/Colistin after Day 28 of the study. The patients in Study 103 were naϊve to inhalation antibiotics, and did not receive additional inhalation antibiotics during the follow-up period. Overall, the treatment was safe and well tolerated, with the most frequent AEs being dyspnea and headache of mild to moderate severity. There were 12/49 (24.4%) AEs reported as possibly or probably related to Liposomal Amikacin. No AEs required discontinuation of the study drug. Tables 12 and 13 below, summarize the adverse effects and the possible or probable relatedness to study drug.

Evaluation of the PK data indicate that in CF patients, systemic exposure to Liposomal Amikacin, even at the 500 mg dose is very low. Analyses of serum Cmax, AUC or mg of aminoglycoside which is recovered in the urine, the observed systemic exposure to Amikacin associated with Liposomal Amikacin, administration by inhalation is approximately 1/5 to 1/4 the exposure seen with 600 mg/d of TOBI and is less than 1/200 compared to normal parenteral doses of Amikacin. To date, the multiple-dose studies of Liposomal Amikacin in CF patients have shown no nephro- or ototoxicity. Study 103: Liposomal Amikacin 50 mg/mL formulation was well tolerated at a daily dose of 500 mg for 14 days in a group of 13 CF patients with chronic P. aeruginosa infection. Twenty-one AEs were experienced by 9 of the 13 patients. AEs reported by more than a single subject included: productive cough, dysgeusia, myalgia, and hemoptysis. All AEs were judged moderate or mild in intensity. No subjects were discontinued from the study due to an AE.

Study 104: Thirty-two AEs were experienced by 10 of the 11 patients. Adverse events experienced by more than one subject included headaches, dyspnea, rales, musculoskeletal chest pain, and cough. Abnormalities in safety parameters were mostly related to the underlying CF condition and were present at baseline. Treatment-emergent abnormalities were infrequent and transient. One subject experienced viral bronchopneumonia that started 9 days after the end of treatment with the study drug, and was reported as an SAE. This resolved within 5 months. The SAE was deemed to be doubtfully related to the study drug by the investigator. All adverse events were judged moderate or mild in intensity, except for AEs associated with the episodes of viral bronchopneumonia. A summary of AE's from the human clinical phase l/2a studies is found in Table 12.

Table 12. Summary of AEs in Studies 103 and 104.

TR02-103 TR02-104

Adverse Events No. of Patients (%) No. of Patients

N = 13 (%)

N = 11

Total patients with AEs

9 (69%) 10 (91%)

Dyspnea 0 (0%) 4 (36%)

Haemoptysis 2 (15%) 1 (9%)

Headache 1 (8%) 3 (27%)

Myalgia 2 (15%) 1 (9%)

Thoracic pain 0 (0%) 2 (18%)

The relationship between the AEs and Liposomal Amikacin are noted in Table 13. Table 13. AE Relationship to Study Drug.

In summary, Liposomal Amikacin appeared to be safe and well tolerated up to the highest inhaled dose level (500 mg) in this group of CF patients with chronic P. aeruginosa infection.

X. Benefits and Risks Conclusions

Infection with P. aeruginosa is associated with increased morbidity and mortality in cystic fibrosis subjects. The aggressive use of oral, intravenous, and aerosolized antibiotics has contributed to increased longevity of these subjects over the past 3 decades. Currently, inhaled antibiotics, specifically inhaled tobramycin, are indicated for the treatment of established infections of P. aeruginosa in CF subjects. Use of tobramycin 300 mg inhaled twice a day on alternating months has been shown to increase FEVi % predicted, decrease the density of P. aeruginosa in sputum, decrease hospitalizations, and reduce the need for intravenous antibiotics (Ramsey, Pepe et al. 1999). Liposomal Amikacin technology offers two advantages over inhaled tobramycin: the first being decreased frequency of dosing (i.e., 1 time per day or less versus 2 times per day), and the second being sustained, high local drug concentrations with concomitant low systemic levels of drug. The lipids used in the Liposomal Amikacin formulation are the same as the endogenous surfactant layer of human lung and are not expected to elicit any adverse reaction.

The single most prevalent compound in pulmonary surfactant is the disaturated phospholipid, dipalmitoylphosphatidylcholine (DPPC), which is crucial for surface tension lowering. The ratio of DPPC: Amikacin in Liposomal Amikacin, 50 mg/ml is 1.0 w:w. Hence, for the nominal 500 mg dose of Liposomal Amikacin, 50 mg/ml approximately 75.0 mg amikacin, and 75.0 mg of DPPC was delivered to the lungs. For a 50 kg adult, this corresponds to a delivered dose of DPPC of 1.5 mg/kg. This is 1-2 orders of magnitude less than the 50-100 mg/kg doses of exogenous lung surfactants that have been instilled into neonate and adult lungs in the acute treatment of respiratory distress syndrome (Gunther, Ruppert et al. 2001). For the 70 mg/ml formulation, the DPPC:Amikacin ratio is 0.4 w:w, and thus the estimated amount of lipid delivered to the lungs is further reduced. Liposomes deposited in the conducting airways will be cleared by the mucociliary escalator, and are not expected to contribute to the endogenous phospholipid pool. Martini and coworkers (Martini, Chinkes et al. 1999) have shown that DPPC is recycled into lamellar bodies of alveolar type II cells at a rate of 216 nmol/hr/g tissue for ventilated pigs. For a 400 g human lung this corresponds to a basal absorption rate for DPPC of about 70 mg/hr. Hence, the phospholipid dose delivered from Liposomal Amikacin can be easily cleared and recycled using existing metabolic pathways.

The use of liposomes for the inhalational administration of drugs has been studied in both rodents and humans and has demonstrated few adverse effects (Taylor, Taylor et al. 1989; Thomas, Myers et al. 1991; Myers, Thomas et al. 1993; Vidgren, Waldrep et al. 1994; Hung, Whynot et al. 1995; Gilbert, Knight et al. 1997; Skubitz and Anderson 2000; Landyshev Iu, Grigorenko et al. 2002; Ten, Anderson et al. 2002). Further, liposomal formulations have been prepared for a number of drugs in these studies, and in many cases it is has been shown that the inhaled liposomal drug has a longer half life in the lung than its free drug (non-liposomal formulation).

XI. Nonclinical Studies with Liposomal Amikacin Several preclinical studies were conducted with the 20 and 50 mg/mL formulations.

Preclinical testing of the lead 70 mg/ml formulation is summarized below. It has been demonstrated the penetration of liposomes through CF sputum and bio film, shown anti-pseudomonas activity of Liposomal Amikacin in in vitro and in vivo models, confirmed that virulence factors secreted by Pseudomonas facilitate the further release of amikacin from Liposomal Amikacin, and characterized the deposition and sustained release of amikacin in the lungs of rats, and dogs. Most importantly, the safety of 30 day administration of Liposomal Amikacin in two species was established.

In 30-day inhalation GLP toxicology studies in rats and dogs with Liposomal Amikacin at doses up to approximately 100 and 31 mg/kg/day, respectively, alveolar foamy macrophage accumulation in the lung was the principal finding. The macrophage accumulation was considered a normal clearance response of the lung to aerosolised materials and not a direct toxic effect of Liposomal Amikacin. Following 1 -month recovery periods in both species, the macrophage accumulation showed evidence of reversibility. In a separate investigative study, it was documented that alveolar macrophages retained their normal function following daily inhalation of Liposomal

Amikacin at approximately 50 mg/kg for 14 days in rats. The lack of any kidney toxicity (a known target organ of Amikacin) in the 30-day studies in rats and dogs is consistent with the low Amikacin plasma levels following inhalation of Liposomal Amikacin in these studies. In a battery of genetic toxicology studies, Liposomal Amikacin showed no evidence of genotoxicity . Liposomal Amikacin was tested up to cytotoxic levels (Ames assay), at concentrations limited by precipitation (chromosome aberration assay in Chinese hamster ovary cells), or up to the testing limit of 5,000 μg/mL (mouse lymphoma assay).

Nonclinical pharmacokinetics have demonstrated that the AUC (0-48 hr) of amikacin in the lungs of rats that received a 60 mg/kg dose of Liposomal Amikacin via nebulization, was five-fold higher than the AUC of tobramycin in the lungs of rats that received an equal dose of tobramycin by inhalation. High levels of amikacin were sustained in the lung (>250 μg/mL through 150 hr), suggesting a depot effect. In contrast, lung levels of tobramycin were undetectable within 6 hours of cessation of administration. Conversely, the AUC of drug in the kidneys of rats that received an equal dose of tobramycin was significantly higher than the AUC of rats that received aerosols of Liposomal Amikacin. There were no significant differences in the AUC of aminoglycosides in the serum and urine of the animals; serum levels were undetectable after 24 hr. This profile supports the intended sustained release of amikacin in the lung following administration of nebulized Liposomal Amikacin, potentially representing an enhanced efficacy profile. These data for Liposomal Amikacin appear supportive of the hypothesis that this specific formulation may be advantageous over other inhalation products that are hindered by a rapid clearance from lung tissue, necessitating frequent dosing (Geller, Pitlick et al. 2002), and placing a burden on patients. Additionally, toxicokinetic data from 30-day inhalation GLP toxicology studies in rats and dogs show that there is a 15 fold increase in lung deposition of amikacin in Liposomal Amikacin high dose treated dogs as compared to the free amikacin treated group, with comparable plasma and urine levels, indicating high lung concentrations with low systemic exposure. This suggests that there will be no safety pharmacology issues with inhaled Liposomal Amikacin, and the possibility of enhanced targeted antibacterial effect.

The safety pharmacology profile of Liposomal Amikacin is currently derived from GLP general toxicology studies in rats and dogs. In a 30-day inhalation toxicology study in dogs at Liposomal Amikacin doses up to approximately 30 mg/kg, there were no indications of adverse effects on respiratory or electrocardiographic parameters. In this study and a companion 30-day inhalation toxicology study in rats, there were no substantive in-life changes and no drug-related histopathologic changes in any organ except the lung (both species) and upper respiratory tract (rats); this is likely related to the very low plasma levels of Amikacin in both species following inhalation dosing with Liposomal Amikacin.

The pharmacodynamic effect of Liposomal Amikacin was evaluated in vivo in a rat model of chronic pulmonary infection with Pseudomonas (Cash, Woods et al. 1979). In the 14 days pseudomonas infection model, it was noted that 60 mg/kg of Liposomal Amikacin (75 mg/mL) administered every other day for 14 days (Q2D x 7), which effectively delivered half the cumulative dose of aminoglycoside than the other groups, was as effective as 60 mg/kg of Liposomal Amikacin (given once per day), and tobramycin (given twice per day) daily for 14 days. When dosing was extended in this model to 28 days, there were equivalent reductions in CFUs for animals receiving Liposomal Amikacin dosed daily at ~60 mg/kg or dosed every other day at -120 mg/kg. Also, in this experiment, Liposomal Amikacin administered at 120 mg/kg once a day for 14 days was as effective as tobramycin 60 mg/kg/day (administered twice a day) for 28 days. This indicated a higher AUC and possibly a prolonged post-antibiotic effect with Liposomal Amikacin at 120 mg/kg dosed once per day. The preclinical pharmacodynamic data are consistent with a sustained antimicrobial benefit enhanced by the site-specific delivery of drug to the lungs via inhalation, and the drug's unique pharmacologic properties.

Thus, administration of Liposomal Amikacin via inhalation resulted in increased lung concentrations (AUC) several fold above the MIC of the bacteria, with the potential to provide a sustained therapeutic effect with a reduced frequency and duration of dosing as compared to Tobramycin. This, along with the property of bio film penetration, and the safety pharmacology profile of the molecule is supportive of further development in the clinic.

XII. Clinical Studies with Liposomal Amikacin Treatment with Liposomal Amikacin up to 500 mg, qd was safe and well tolerated.

The most recent multidose human clinical Phase lb/2a studies (Study 103 and Study 104) demonstrate that nebulized Liposomal Amikacin 50 mg/mL administered as 500 mg once per day for 14 days is well tolerated, and in select CF patients improved pulmonary function and decreased the density of P. aeruginosa in sputum. In summary, the two studies were similar in design. A total of 24 CF patients (with

FEVi > 40% of predicted) received 500 mg Liposomal Amikacin daily for 14 days. Drug was administered using a PARI LC Star nebulizer, over a period of two 20-minute inhalation sessions with a 5 minute rest between periods. There were 13 patients enrolled in Study 103 and 11 patients in Study 104. Patient demographics were similar, with the exception of Pseudomonas MICs at baseline, and history of prior exposure to inhalation antibiotics. In

Study 103, the mean MIC (μg/mL) was 8 (range 1.5-16) and in Study 104 the mean MIC was 41 μg/mL (range 8-192). The patients enrolled in study 104 had prior experience with inhalation antibiotics, and per protocol, were permitted to resume treatment with TOBI^®/Colistin after Day 28 of the study. The patients in Study 103 were naϊve to inhalation antibiotics, and did not receive additional inhalation antibiotics during the follow-up period. Overall, the treatment was safe and well tolerated, with the most frequent AEs being dyspnea and headache of mild to moderate severity. There were 12/49 (24.4%) AEs reported as possibly or probably related to Liposomal Amikacin. No AEs required discontinuation of the study drug. Toxicities associated with Liposomal Amikacin administration are expected to be less frequent and less severe than with intravenous amikacin due to the low systemic levels achieved with inhalation compared to what would be expected with intravenously administered amikacin. The adverse effects of amikacin given by the intravenous route are primarily nephrotoxicity and ototoxicity. Evaluation of the PK data from studies 103 and 104 indicate that in CF patients, systemic exposure to Liposomal Amikacin, even at the 500mg dose is very low. Analyses of serum Cmax, AUC or mg of aminoglycoside which is recovered in the urine, the observed systemic exposure to Amikacin associated with Liposomal Amikacin administration by inhalation is approximately 1/5 to 1/4 the exposure seen with 600 mg/d of TOBI. This is less than 1/200 compared to normal parenteral doses of Amikacin. To date, the multiple-dose studies of Liposomal Amikacin in CF patients have shown no nephro- or ototoxicity.

Measurement of pulmonary function tests showed a statistically significant improvement in lung function as reported by an increase in FEVi from baseline to Day 7 of +240 mL (n=23; p- value 0.0024). The effect at day 14 was a 126 mL increase from baseline in FEVl, which was not statistically significant. A corresponding statistically significant increase in FEVl % predicted from Day 14 was +4.37% (n=24; p-value 0.0285). The improvement in lung function was also noted with the assessment of small airways as measured by FEF25-750_/0 at day 7, with an increase in +494 mL (n=23; p-value 0.001), and at Day 14, +423 mL (n=24; p-value 0.0162).

These data support a clinically meaningful improvement in lung function in CF patients with chronic pseudomonas infection who have received a 14 day course of treatment with Liposomal Amikacin. The pre-clinical, and clinical data to date serve as the basis for further development of Liposomal Amikacin 70 mg/ml in patients with cystic fibrosis. Liposomal Amikacin is formulated for the treatment of pulmonary gram-negative bacterial infections, and pulmonary mycobacterial infections. The current focus of the development program is for the treatment of CF subjects with P. aeruginosa infections. Conclusion

CF occurs primarily in individuals of central and western European origin. In the United States, the median age at death has increased from 8.4 years of age in 1969 to 14.3 years of age in 1998. The mean age of death has increased from 14 years in 1969 to 32.4 years of age in 2003 (Cystic Fibrosis Foundation). A major contributor to the significant increase in life expectancy is improved antibiotic treatment of chronic respiratory tract infections in CF subjects (Goss and Rosenfeld 2004) as well as improved nutrition and earlier diagnosis.

A major factor in the respiratory health of CF subjects is acquisition of chronic Pseudomonas aeruginosa infections. The infection rate with P. aeruginosa increases with age and by age 18 years, 80% of CF subjects in the U.S. are infected. The difficulties treating this infection are multifactorial, including poor penetration of antibiotics into sites of infection including mucus plugs, inactivation of antibiotics by CF sputum, growth of bacteria in a bio film, changes in phenotype including conversion to a mucoid form of P. aeruginosa, and emergence of multi-drug resistance (Chmiel and Davis 2003; Gibson, Burns et al. 2003). The cornerstone of pulmonary therapy is optimizing treatment of P. aeruginosa as infection with this pathogen is associated with a poor clinical outcome (Doring, Conway et al. 2000; Chmiel and Davis 2003; Gibson, Burns et al. 2003; Gibson, Emerson et al. 2003).

One of the current approaches to management of chronic P. aeruginosa infection in humans with CF includes the use of suppressive therapy with inhaled tobramycin (TOBI^®). Inhaled tobramycin, 300 mg, administered twice a day for cycles of 28 days followed by 28 days off drug has been shown to reduce P. aeruginosa colony counts, increase FEVi % predicted, reduce hospitalizations, and decrease antibiotic use (Ramsey, Pepe et al. 1999). However, patients have to be dosed twice a day for approximately 15-20 minute inhalation periods per dose.

Possible improvements over inhaled tobramycin would be to decrease the frequency of administration, reduce the administration time, improve quality of life, and to possibly increase the potency of antibacterial activity. In addition, the current challenges with treatment with TOBI, include the declining clinical benefit, emerging resistance and the treatment burden. This highlights the need for new therapies for treatment of lung infections due to P. aeruginosa in CF patients. Liposomal Amikacin technology provides high levels of sustained release of antibiotic in the lung, with potential for drug concentrations well above the MICs for Pseudomonas aeruginosa during the dosing interval, and biofilm penetration. These features may contribute to improved clinical efficacy with reduced dosing frequency, and improvement in patient compliance.

Based on the preliminary profile of Liposomal Amikacin seen in the phase l/2a studies, there is a possible opportunity for liposomal amikacin via inhalation to improve and sustain clinical benefit, have potential for increased anti-bacterial activity, with once a day or less frequent administration.

Exemplification Example 1

Amikacin was encapsulated in liposomes composed of dipalmitoylphoshatidylcholine (DPPC) and cholesterol, at a targeted lipid-to-drug ratio of 0.6-0.7:1 (w/w). The quantitative formula for liposomal amikacin, 70 mg/mL is presented in Table 14.

Table 14. Composition of Liposomal Amikacin, 70 mg/mL.

Added to the formulation as Amikacin sulfate, USP

Liposomal amikacin was made using an aseptic process that involves the preparation of three solutions, sterile filtration of the solutions into a sterilized reactor utilizing in-line mixing, followed by diafϊltration and concentration of the resulting liposomal suspension to form the final product as described below.

1. Solution Preparation Sufficient quantities of the following three solutions are prepared.

(a) Amikacin Solution: Amikacin Sulfate, USP, in Water for Injection, pH adjusted with NaOH to 6.6-6.8.

(b) Lipid Solution: DPPC/Cholesterol (2:1 w/w) in Ethanol.

(c) 1.5% Sodium Chloride Solution: 1.5% Sodium Chloride, USP, in Water for Injection, pH adjusted to 6.6-6.8. 2. Infusion/Initial Concentration The Amikacin Solution (a) and Lipid Solution (b) are warmed (40⁰C) and passed through separate sterilizing filters into an in-line infusion module at controlled rates of addition. The streams mix as they pass through the infusion module and are then collected in a pre-sterilized reactor vessel. Simultaneously, 1.5% Sodium Chloride Solution is passed through a sterilizing filter and introduced into the infusion stream. Once a sufficient volume of material is in the reactor, mixing is initiated and the product is circulated through the 500 kDa diafiltration cartridge to begin the removal of ethanol and free amikacin. Upon conclusion of the infusion, the product is concentrated. 3. Diafiltration Diafiltration is initiated upon the completion of the infusion and initial concentration. Diafiltration occurs at approximately 30⁰C via the same diafiltration cartridge used for the Infusion/Initial Concentration (2). The bulk solution is maintained at a constant mass while 1.5% Sodium Chloride Solution is added to the reactor. A total volume of 1.5% Sodium Chloride Solution, equal to six 6 washes added and removed from the reactor at equal rates. This step functions to remove the ethanol from the bulk solution and to wash away any "un-associated" or free amikacin.

Example 2

Nebulization of Liposomal Amikacin

The aerosol properties of Liposomal Amikacin produced from the eFlow 4OL are shown in Table 15. When compared to nebulizate generated from the LC Star, the mass median aerodynamic diameter (MMAD) values for the eFlow are ~0.5 μm larger. The actual size dependent mass distributions from both ACI (with eFlow) and NGI (with LC Star) cascade impactors for nebulized Liposomal Amikacin are shown in Figure 1. Aerosol from the eFlow/ ACI measurements was slightly narrower in size distribution than that from the LC Star/NGI. This difference is reflected in the lower mean geometric standard deviation (GSD) (1.66 versus 1.99) which is a measure of the width of the distribution around the MMAD, see values in Table 15. This narrower distribution offsets any potential effect of a larger MMAD and therefore, the amount of nebulized drug in the respirable range (< 5 μm droplet size) is comparable for both eFlow and LC Star. Table 15. Properties of Liposomal Amikacin Nebulized with the eFlow and LC Star Nebulizers.

The Andersen cascade impactor was used at a flow rate of 28.3 L/min, 18⁰C, and 50% humidity. The NGI impactor was used at a flow rate of 15L/min and 5⁰C to achieve >60% humidity. ^Percent mass of the nominal drug dose that is less than 5 μm in diameter.

Example 3

Effect of Liposomal Amikacin on P. aeruginosa Lung Infections in Rat

The efficacy of Liposomal Amikacin for Inhalation, Liposomal Amikacin was studied using a model for chronic pulmonary infection (Cash, Woods et al. 1979) where P. aeruginosa, embedded in an agarose bead matrix, was instilled in the trachea of rats. This mucoid Pseudomonas animal model was developed to resemble the chronic Pseudomonas infections seen in CF patients (Cantin and Woods 1999). Rat lungs were inoculated with 10⁴ CFUs of a mucoid P. aeruginosa strain (mucoid strain 3064) originally isolated from a CF patient. Three days later, 60 mg/kg Liposomal Amikacin (75 mg/mL) was administered by inhalation once daily for 14 doses (QlD x 14) or every other day for 7 doses (Q2D x 7) (6 mg/kg per dose). For comparison, tobramycin was administered by inhalation BID for 14 days (30 mg/kg per dose for a total of 60 mg/kg daily). There was a significant reduction in bacterial density in all three treatment groups as compared to the saline control (see Figure 2). There were no significant differences in the reduction of logioCFU/lung between the three treatment groups of rats. It should be noted that Liposomal Amikacin (75 mg/mL) administered every other day for 14 days (Q2D x 7), which effectively delivered half the cumulative dose of aminoglycoside, was as effective as the daily dosing regimen in this model.

As shown in Figure 3 when dosing was extended in this model to 28 days, there were equivalent reductions in CFUs for animals receiving Liposomal Amikacin dosed daily at -60 mg/kg or dosed every other day at -120 mg/kg. However, this was only seen as statistically significant for the latter group when compared to animals that received 1.5% saline on the same schedules (p= 0.24 and 0.03, respectively). In both cases, there was a significant number of animals in the saline control groups that also experienced 2 log reductions in the CFUs. The longer duration (post 14 days) of saline inhalation treatment seemed to enhance the spontaneous ability of rats to clear their lungs of infection and presumably the agar beads which maintain the chronic infection condition. Rats that received Liposomal Amikacin -120 mg/kg daily for 14 days, were observed for another 14 days, and then euthanized on day 35. Lungs of these animals had bacteria below the limit of detection, as was the case in the group that received tobramycin 60 mg/kg (given twice per day) daily for 28 days, and then euthanized. Data indicate that in this experiment,

Liposomal Amikacin administered at 120 mg/kg once a day for 14 days was as effective as tobramycin 60 mg/kg/day (administered twice a day) for 28 days. This result suggests a higher AUC and possibly a prolonged post-antibiotic effect with Liposomal Amikacin at 120 mg/kg.

REFERENCES

1. Cash, H. A., D. E. Woods, et al. (1979). "A rat model of chronic respiratory infection with Pseudomonas aeruginosa." Am Rev Respir Pis 119(3): 453-9. 2. Challoner, P. B., M. G. Flora, et al. (2001). Gamma Scintigraphy Lung Deposition Comparison of TOBI in the PARI LC PLUS Nebulizer and the Aerodose Inhaler. American Thoracic Society 97th International Conference, San Francisco, California, Aerogen, Inc.

3. Chmiel, J. F. and P. B. Davis (2003). "State of the art: why do the lungs of patients with cystic fibrosis become infected and why can't they clear the infection?" Respir

Res 4: 8. 4. Cow, G. D. (2007a). Exploratory 28 Day Inhalation Toxicity Study of SLIT™ Amikacin in Rats, Charles River Laboratories: 259.

5. Cow, G. D. and A. Morgan (2007c). 30 Day Inhalation Toxicity Study of SLIT™ Amikacin in Rats with a 30 day Recovery Period, Charles River Laboratories: 870. 6. Cow, G. D. and A. Morgan (2007d). 30 Day Inhalation Toxicity Study of SLIT™

Amikacin in Dogs with a 30 Day Recovery Period, Charles River Laboratories: 777.

7. Doring, G., S. P. Conway, et al. (2000). "Antibiotic therapy against Pseudomonas aeruginosa in cystic fibrosis: a European consensus." Eur Respir J 16(4): 749-67.

8. Geller, D. E., W. H. Pitlick, et al. (2002). "Pharmacokinetics and bioavailability of aerosolized tobramycin in cystic fibrosis." Chest 122(1): 219-26.

9. Gibson, R. L., J. L. Burns, et al. (2003). "Pathophysiology and management of pulmonary infections in cystic fibrosis." Am J Respir Crit Care Med 168(8): 918-51.

10. Gibson, R. L., J. Emerson, et al. (2003). "Significant microbiological effect of inhaled tobramycin in young children with cystic fibrosis." Am J Respir Crit Care Med 167(6): 841-9.

11. Gilbert, B. E., C. Knight, et al. (1997). "Tolerance of volunteers to cyclosporine A- dilauroylphosphatidylcholine liposome aerosol." Am J Respir Crit Care Med 156(6): 1789-93.

12. Goss, C. H. and M. Rosenfeld (2004). "Update on cystic fibrosis epidemiology." Curr Opin PuIm Med 10(6): 510-4.

13. Gunther, A., C. Ruppert, et al. (2001). "Surfactant alteration and replacement in acute respiratory distress syndrome." Respir Res 2(6): 353-64.

14. Hug, M. (2007a). Characterization of the PARI eFlow® (4OL to 50L) and Liposomal Amikacin™ (48 to 79 mg/ml⁽¹⁾) PARI GmbH, Aerosol Research Institute: 10.

15. Hug, M. (2007b). Aerosol Characterization of the PARI eFlow® 4OL an Transave Liposomal Amikacin™ for Inhalation (70 mg/ml⁽¹⁾), PARI GmbH, Aerosol Research Institute: 12.

16. Hung, O. R., S. C. Whynot, et al. (1995). "Pharmacokinetics of inhaled liposome- encapsulated fentanyl." Anesthesiology 83(2): 277-84. 17. Landyshev Iu, S., A. A. Grigorenko, et al. (2002). "[Clinico-experimental aspects of liposomal therapy of bronchial asthma patients with hydrocortisone therapy]." Ter Arkh 74(8): 45-8.

18. Lass, J. S., A. Sant, et al. (2006). "New advances in aerosolised drug delivery: vibrating membrane nebuliser technology." Expert Opin Drug Deliv 3(5): 693-702.

19. Li, Z. (2007). Droplet Size of Liposomal Amikacin™: Comparison of Nebulizate for the eflow Electronic Nebulizer and the PARI LC STAR Jet Nebulizer. Monmouth Junction, Transave Inc.: 20.

20. Martini, W. Z., D. L. Chinkes, et al. (1999). "Lung surfactant kinetics in conscious pigs." Am J Physiol 277(1 Pt 1): E 187-95.

21. Myers, M. A., D. A. Thomas, et al. (1993). "Pulmonary effects of chronic exposure to liposome aerosols in mice." Exp Lung Res 19(1): 1-19.

22. Niven, R. W., T. M. Carvajal, et al. (1992). "Nebulization of liposomes. III. The effects of operating conditions and local environment." Pharm Res 9(4): 515-20. 23. Niven, R. W. and H. Schreier (1990). "Nebulization of liposomes. I. Effects of lipid composition." Pharm Res 7(11): 1127-33.

24. Niven, R. W., M. Speer, et al. (1991). "Nebulization of liposomes. II. The effects of size and modeling of solute release profiles." Pharm Res 8(2): 217-21.

25. Ramsey, B. W., M. S. Pepe, et al. (1999). "Intermittent administration of inhaled tobramycin in patients with cystic fibrosis. Cystic Fibrosis Inhaled Tobramycin

Study Group." N Engl J Med 340(1): 23-30.

26. Skubitz, K. M. and P. M. Anderson (2000). "Inhalational interleukin-2 liposomes for pulmonary metastases: a phase I clinical trial." Anticancer Drugs 11(7): 555-63.

27. Taylor, K. M., G. Taylor, et al. (1989). "The influence of liposomal encapsulation on sodium cromoglycate pharmacokinetics in man." Pharm Res 6(7): 633-6.

28. Ten, R. M., P. M. Anderson, et al. (2002). "Interleukin-2 liposomes for primary immune deficiency using the aerosol route." Int Immunopharmacol 2(2-3): 333-44.

29. Thomas, D. A., M. A. Myers, et al. (1991). "Acute effects of liposome aerosol inhalation on pulmonary function in healthy human volunteers." Chest 99(5): 1268- 70. 30. Vecellio, L. (2006). "The Mesh Nebuliser: A Recent Technical Innovation for Aerosol Delivery." Breath 2(3): 253-260.

31. Vidgren, M. T., J. C. Waldrep, et al. (1994). "A study of ^99mtechnetium-labelled beclomethasone dipropionate dilauroylphosphatidylcholine liposome aerosol in normal volunteers." Int J Pharm: 8.

32. Wenker, A. (2006). In vitro characterization of nebulized Amikacin, Activaero GmbH: 28.

33. Wolff, R. K. and M. A. Dorato (1993). "Toxicologic testing of inhaled pharmaceutical aerosols." Crit Rev Toxicol 23(4): 343-69.

Claims

We claim:

I . A method of treating a bacterial infection in a human comprising administering to a human in need thereof an effective amount of an antibiotic formulation by inhalation once every day or once every greater time interval.

2. The method of claim 1 , wherein the antibiotic formulation is a lipid based antibiotic formulation.

3. The method of claim 1, wherein the antibiotic formulation is a liposomal antibiotic formulation.

4. The method of claim 1, wherein the antibiotic is an aminoglycoside.

5. The method of claim 1, wherein the antibiotic is amikacin.

6. The method of claim 1, wherein the amount of antibiotic formulation is 5 to 2,500 mg.

7. The method of claim 1, wherein the amount of antibiotic formulation is 250 to 1,500 mg.

8. The method of claim 1, wherein the amount of antibiotic formulation is 500 to 1,000 mg.

9. The method of claim 1, wherein the antibiotic formulation is administered once every day.

10. The method of claim 1, wherein the antibiotic formulation is administered once every two days.

I 1. The method of claim 1 , wherein the antibiotic formulation is administered once every three days.

12. The method of claim 1, wherein the antibiotic formulation is administered once every day for 5 days to 6 months

13. The method of claim 1, wherein the antibiotic formulation is administered once every day for 5 days to 3 months.

14. The method of claim 1, wherein the antibiotic formulation is administered once every day for 5 days to 2 months.

15. The method of claim 1, wherein the antibiotic formulation is administered once every day for 5 days to 1 month.

16. The method of claim 1, wherein the antibiotic formulation is administered once every day for 5 days to 2 weeks.

17. The method of claim 1, wherein the antibiotic formulation is administered once every day for a week.

18. The method of claim 1 , wherein the antibiotic formulation is administered once every day for a week followed by a week of no administration, wherein this cycle is repeated more than once.

19. The method of claim 1, wherein the antibiotic formulation is administered once every day for 14 days followed by 14 days of no administration, wherein this cycle is repeated more than once.

20. The method of claim 1, wherein the antibiotic formulation is administered once every day for 28 days followed by 28 days of no administration, wherein this cycle is repeated more than once.

21. The method of claim 1 , wherein the antibiotic is amikacin and the amount of antibiotic formulation is 5 to 2,500 mg administered once every day for 5 days to 3 months.

22. The method of claim 2 or 3, wherein the lipid based or liposomal antibiotic formulation comprises a lipid selected from the group consisting of egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), egg phosphatidylinositol (EPI), egg phosphatidylserine (EPS), phosphatidylethanolamine (EPE), phosphatidic acid (EPA), soy phosphatidylcholine (SPC), soy phosphatidylglycerol (SPG), soy phosphatidylserine (SPS), soy phosphatidylinositol (SPI), soy phosphatidylethanolamine (SPE), soy phosphatidic acid (SPA), hydrogenated egg phosphatidylcholine (HEPC), hydrogenated egg phosphatidylglycerol (HEPG), hydrogenated egg phosphatidylinositol (HEPI), hydrogenated egg phosphatidylserine (HEPS), hydrogenated phosphatidylethanolamine (HEPE), hydrogenated phosphatidic acid (HEPA), hydrogenated soy phosphatidylcholine

(HSPC), hydrogenated soy phosphatidylglycerol (HSPG), hydrogenated soy phosphatidylserine (HSPS), hydrogenated soy phosphatidylinositol (HSPI), hydrogenated soy phosphatidylethanolamine (HSPE), hydrogenated soy phosphatidic acid (HSPA), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylcholine

(DSPC), distearoylphosphatidylglycerol (DSPG), dioleylphosphatidyl-ethanolamine (DOPE), palmitoylstearoylphosphatidyl-choline (PSPC), palmitoylstearolphosphatidylglycerol (PSPG), mono-oleoyl- phosphatidylethanolamine (MOPE), cholesterol, ergosterol, lanosterol, tocopherol, ammonium salts of fatty acids, ammonium salts of phospholipids, ammonium salts of glycerides, myristylamine, palmitylamine, laurylamine, stearylamine, dilauroyl ethylphosphocholine (DLEP), dimyristoyl ethylphosphocholine (DMEP), dipalmitoyl ethylphosphocholine (DPEP) and distearoyl ethylphosphocholine (DSEP), N-(2, 3- di-(9-(Z)-octadecenyloxy)-prop-l-yl-N,N,N-trimethylammonium chloride (DOTMA), 1, 2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP), phosphatidyl-glycerols (PGs), phosphatidic acids (PAs), phosphatidylinositols (PIs), phosphatidyl serines (PSs), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylacid (DMPA), dipalmitoylphosphatidylacid (DPPA), distearoylphosphatidylacid (DSPA), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphospatidylinositol (DSPI), dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), and mixture thereof.

23. The method of claim 2 or 3, wherein the lipid based or liposomal antibiotic formulation comprises a phospholipid and a sterol.

24. The method of claim 2 or 3, wherein the lipid based or liposomal antibiotic formulation comprises DPPC and cholesterol.

25. The method of claim 2 or 3, wherein the lipid based or liposomal antibiotic formulation comprises DPPC and cholesterol in a 2 to 1 ratio by weight.

26. The method of claim 2 or 3, wherein the antibiotic is amikacin; the amount of antibiotic formulation is 5 to 2,500 mg administered once every other day for a week to 3 months, and the lipid based or liposomal antibiotic formulation comprises DPPC and cholesterol in a 2 to 1 ratio by weight.