WO2003101390A2 - Endobronchial delivery of antibiotic in individuals with impaired lung tissue or lung function - Google Patents

Abstract

Methods for producing a significant anti-P. aeruginosa effect in a cystic fibrosis patient 6 years of age or younger that presents with early P. aeruginosa infection are disclosed. Such methods involve endobronchial administration of an aerosolized tobramycin formulation, wherein said formulation is administered as a monotherapy for a period of about one month. In addition, methods for selecting a cystic fibrosis patient 6 years of age or younger that my benefit from the claimed methods, as well as methods for monitoring anti- P. aeruginosa aerosol antibiotic treatment outcome in such patients, are disclosed.

Description

ENDOBRONCHIAL DELIVERY OF ANTIBIOTIC IN INDIVIDUALS WITH IMPAIRED

LUNG TISSUE OR LUNG FUNCTION

This invention was made with government support under Grant No. DK57755- 02, awarded by the National Institute of Diabetes and Digestive and Kidney Diseases. The United States Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

In individuals with impaired lung tissue or function, certain bacteria grow and often persist in the pulmonary spaces of these individuals. A prototypical example is Pseudomonas aeruginosa infection of patients with cystic fibrosis (CF) or bronchiectasis. The hallmark of CF disease is chronic bacterial endobronchial infection with alveolar sparing (until late in the disease) associated with an intense neutrophilic airway inflammatory response. The earliest bacterial pathogens include Staphylococcus aureus, Hemophilus influenzae, and P. aeruginosa. By the end of the first decade, P. aeruginosa emerges as the predominant pathogen and remains as such until the CF patient's death. P. aeruginosa, chronically infecting the lower airways of CF patients, establishes a consistent mucoid phenotype, which lacks both O-antigens on its lipopolysaccharide and flagella. These changes appear to be an adaptation to the unique CF lung environment. These mucoid strains frequently have modified porins and are highly resistant to multiple antibiotics, such as aminoglycosides and quinolones. This bacterial ecology of CF patients is consistent worldwide, and reflects a host-pathogen interaction unique to this genetic disorder. Individuals that suffer from chronic infection (and progressive tissue damage due to chronic inflammation) generally experience acute exacerbations of the infection. These chronically infected patients are generally treated with intravenous, oral and/or inhaled antibiotics, especially during these acute exacerbations of infection. Patients with chronic P. aeruginosa infections require many courses of antibiotic treatment, which may be associated with an increased risk of development of resistant microorganisms.

There is substantial evidence demonstrating that P. aeruginosa is pathogenic in the CF lung, i.e., that P. aeruginosa contributes to more rapid disease progression, rather than being a benign epiphenomenon. CF patients colonized with P. aeruginosa have a shortened life span of 30 years, as compared to a life span of 40 years for CF patients not colonized with P. aeruginosa; exhibit a more rapid decline in pulmonary function; and experience more frequent hospitalizations from pulmonary exacerbations than non-colonized CF patients (CFF 1997 National Data Registry). Appropriate treatment with anti-pseudomonal antibiotics, which decrease sputum P. aeruginosa burden, is associated with improved lung function, decreased white blood count, and improvement in clinical scores. Patients with CF develop vigorous antibody responses to P. aeruginosa virulence factors, consistent with a host response to invasive disease. However, these vigorous immune responses are ineffective in clearing P. aeruginosa infection. Thus, development of more effective therapies for treating P. aeruginosa endobronchial infections is critical for decreasing morbidity and mortality in this disease.

Chronic lower airway infection with mucoid P. aeruginosa has been shown to form a biofilm, an accumulation or aggregation of mucoid P. aeruginosa embedded in an extracellular polysaccharide matrix and adherent to the airway surface. P. aeruginosa biofilms can result in bacterial-density dependent signaling that results in the expression of specific virulence factors (for example, proteases) that promote biofilm differentiation and may contribute to lung inflammation and injury. Biofilms are difficult to treat with anti-microbials, due to poor antibiotic penetration, antibiotic inactivation, and/or increased microbial resistance. These properties of P. aeruginosa biofilms may in part explain the suboptimal microbiologic efficacy (lack of eradication of bacteria) following treatment with intravenous or inhaled anti-pseudomonal antibiotics in established P. aeruginosa infection in CF subjects greater than six years of age.

The primary cause of morbidity and mortality in patients with CF is progressive obstructive pulmonary disease associated with chronic P. aeruginosa infection and an intense neutrophilic inflammatory response. Currently available intravenous anti- pseudomonal antibiotics transiently improve lung function and clinical status, but they do not eradicate P. aeruginosa. Despite such symptom-guided treatment of pulmonary exacerbations, there is an absolute decline in pulmonary function of about 2% per annum, leading to eventual respiratory failure. Until specific therapies to correct or replace the dysfunctional gene are available, there is a critical need for improved antibiotic regimens to treat P. aeruginosa infections in CF patients. There is also a significant need for improved antibiotic regimens to treat patients with impaired lung function or impaired host defense mechanisms that present with bacteria (for example, P. aeruginosa) in their pulmonary spaces. SUMMARY OF THE INVENTION

Within one aspect, the present invention provides methods for producing a significant anti-P aeruginosa effect in a cystic fibrosis patient 6 years of age or younger that presents with early P. aeruginosa infection, comprising administering an aerosolized tobramycin formulation.

Within another aspect, the invention provides methods producing a significant anti-P aeruginosa effect in a cystic fibrosis patient 6 years of age or younger that presents with early P. aeruginosa infection, comprising administering an aerosolized tobramycin formulation, wherein the patient has been diagnosed with cystic fibrosis based on the following criteria: (a) sweat chloride > 60 mEq/L, or a genotype with two mutations consistent with cystic fibrosis; (b) two clinical features consistent with cystic fibrosis, and (c) one historical oropharyngeal culture positive for P. aeruginosa within 2 weeks to 12 months prior to the screening step.

In yet another aspect, the invention provides methods for selecting cystic fibrosis patients 6 years of age or younger for aerosol tobramycin treatment, comprising detecting P. aeruginosa in at least one positive oropharyngeal culture, at a time following at least one negative oropharyngeal culture.

In a further aspect, the invention provides methods for monitoring anti-P. aeruginosa aerosol tombramycin treatment outcome, comprising obtaining an oropharyngeal sample from a treated recipient after completion of the tombramycin treatment; and determining the presence or absence of P. aeruginosa in the sample, wherein the presence of P. aeruginosa indicates a need for additional treatment and the absence of P. aeruginosa indicates effective treatment.

These and other aspects of the invention will become evident upon reference to the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to the understanding thereof to define the following terms:

The term "normal saline" is used herein to denote a water solution containing 0.9% NaCl.

The term "diluted saline" is used herein to denote normal saline that has been diluted to a lesser concentration.

The term "quarter normal saline" or "1/4 NS" is used herein to denote normal saline diluted to quarter strength (i.e., about 0.225% NaCl). The term "TOBI®" is used herein to denote the FDA approved product (tobramycin for inhalation) that is provided as a single use 5 mL ampule containing 300 mg tobramycin.

The term "TSI" is used herein to denote a tobramycin solution for inhalation; TSI includes TOBI®.

The term "Pa" is used herein to denote Pseudomonas aeruginosa.

The term "CF" is used herein to denote cystic fibrosis.

The term "FEV-T is used herein to denote forced expiratory volume in 1 second.

The term "FVC" is used herein to denote forced vital capacity. The term "BAL" is used herein to denote bronchoalveolar lavage.

The term "aerosol" is used herein to denote a gaseous suspension of fine solid or liquid particles.

The term "solution" is used herein to denote a homogeneous mixture of two or more substances, which may be solids, liquids, gases, or a combination of these. All references cited herein are incorporated by reference in their entirety.

The present invention provides, in part, a method for producing a significant antimicrobial effect in children with cystic fibrosis that are 6 years of age or younger using endobronchial delivery of tobramycin, wherein such children present with early. Pa infection. In one embodiment, the present invention provides methods for producing a significant anti-P. aeruginosa effect in a cystic fibrosis patient 6 years of age or younger that presents with early P. aeruginosa infection, comprising administering an aerosolized tobramycin formulation. In another embodiment, an aerosolized tobramycin formulation is administered as a monotherapy. In another embodiment, the aerosolized tobramycin is a liquid formulation or a dry powder formulation. In yet another embodiment, the aerosolized tobramycin is administered for a period of about a month. In another embodiment, the administering step occurs twice daily. In another aspect, the aerosolized tobramycin administration is repeated for at least 28 days. In another embodiment, the aerosolized tobramycin administration is repeated for at least 28 days, but for less than 180 days. In another embodiment, the aerosolized tobramycin is administered for at least 14 days. In another embodiment, the aerosolized tobramycin is administered for at least 14 days, but for less than 30 days.

In another embodiment, the effective amount of tobramycin administered is about 150 mg to about 400 mg. In yet another embodiment, the effect amount of tobramycin administered is about 180 mg to about 350 mg. In another embodiment, the effective amount of tobramycin administered is about 300 mg.

In one embodiment, the significant anti- P. aeruginosa effect is a reduction of P. aeruginosa density greater than 1.6 logio CFU/mL in a post-treatment endobronchial sample compared to a pre-treatment endobronchial sample. In another embodiment, the reduction of P. aeruginosa density is greater than 3.0 log₁₀ CFU/mL. In another embodiment, the reduction of P. aeruginosa density is greater than 5.0 logio CFU/mL.

In yet another embodiment, the invention provides methods for producing a significant anti-P. aeruginosa effect in a cystic fibrosis patient 6 years of age or younger that presents with early P. aeruginosa infection, and optionally comprises a step of detecting P. aeruginosa before the step of administering an aerosolized tobramycin formulation to the patient. In one embodiment, P. aeruginosa is detected in two consecutive oropharyngeal cultures after a negative oropharyngeal culture.

In another embodiment, after the administration of the aerosolized tobramycin formulation, colony forming units of P. aeruginosa in an endobronchial sample are decreased to less than 100 CFU/mL. In yet another embodiment, the colony forming units of P. aeruginosa in an endobronchial sample are decreased to less than 20 CFU/mL.

In one embodiment, a cystic fibrosis patient 6 years of age or younger for aerosol tobramycin treatment is a non-expectorating patient. In another embodiment, the patient exhibits one or more of the following characteristics: (a) minimal symptoms of bacterial infection; (b) relatively normal lung function; (c) minimal inflammatory response; (d) a first P. aeruginosa infection after a period of non-infection with P. aeruginosa, or a first infection after eradication of a prior P. aeruginosa infection; (e) a serum antibody titer against P. aeruginosa Exotoxin A that is <1 :200; and (f) infection with a P. aeruginosa population that is >60% non-mucoid.

In another embodiment, the invention provides methods producing a significant anti-P. aeruginosa effect in a cystic fibrosis patient 6 years of age or younger that presents with early P. aeruginosa infection, comprising administering an aerosolized tobramycin formulation, wherein the patient has been diagnosed with cystic fibrosis based on the following criteria: (a) sweat chloride > 60 mEq/L, or a genotype with two mutations consistent with cystic fibrosis; (b) two clinical features consistent with cystic fibrosis, and (c) one historical oropharyngeal culture positive for P. aeruginosa within 2 weeks to 12 months prior to the screening step. At present, only one inhaled aminoglycoside, tobramycin for inhalation (TOBI®), is approved by the Food and Drug Administration (FDA). The results of two Phase III clinical trials using TOBI® demonstrated a 99% reduction (a 1.6 logι₀-fold reduction) in sputum Pa density and improved FEVi (12% treatment effect compared to placebo) after 28 days of 300 mg tobramycin twice daily. These trials did not include children less than 6 years of age. In CF patients chronically infected with Pa, intravenous or inhaled antibiotic therapy generally results in significant clinical improvement, but the reduction in lower airway Pa density is only 1-2 logio-fold on average.

Previously, infants and young children with CF have not been included in clinical trials because of a lack of validated noninvasive measures of both safety and efficacy. The primary outcome measures for studies of antimicrobial therapy in older children and adults are sputum microbiology and pulmonary function testing. Young children are unable to reliably expectorate sputum or perform lung function tests, necessitating the use of oro- or naso-pharyngeal samples for bacterial culture. Studies comparing simultaneous upper and lower airway cultures show both poor positive and negative predictive values, limiting the utility of upper airway cultures in predicting lower airway colonization status. Thus, the gold standard for identifying a lower airway infection in patients less than 6 years of age remains direct sampling of lower airway secretions via flexible bronchoscopy. Bronchoalveolar lavage (BAL) allows simultaneous measurement of airway inflammatory cells and soluble inflammatory mediators.

However, BAL is an invasive procedure requiring technical proficiency and stringent patient monitoring to assure safety. Thus, it is more applicable to small, short-term Phase l/ll studies than to large, multicenter Phase III trials.

The second major obstacle to studying this young population is a lack of standardized pulmonary function testing, particularly measures of airway obstruction. Forced expiratory volume at one second (FEV-i), the primary outcome measure for most CF clinical trials, is an effort-dependent measure of expiratory flow. Measurements of expiratory flow in patients less than 3 years old require passive chest wall compression in a sedated child to achieve a flow-volume loop. These techniques are best accomplished in infants who have a compliant chest wall and an intact Hering- Breuer reflex. Toddlers (ages 18 months-3 years) remain the most challenging age group, as they are not readily amenable to either infant or "adult" pulmonary function techniques. A recent multicenter trial in young children with human immunodeficiency virus infection suggested that acceptable flow-volume loops can be obtained via standard spirometry in the majority of children 3-5 years of age.

Until recently, there have been no standardized techniques to initiate passive chest wall compression from full inspiratory volume, so that only partial flow volume loops were measured in infants. These measures are difficult to use in multicenter trials, due to large intra- and inter-subject variability. These measures cannot be compared to FEVi values in older patients. Although these partial flowvolume loops have been used in a multicenter trial in young children with HIV infection, they have not been used in a multicenter trial involving infants with CF, who have much more significant small airway disease at a young age. In spite of these limitations, partial flow volume loops have been used in single center studies to show that airflow obstruction, as measured by Vmax FRC, occurs at a very young age in CF patients. In addition, airflow obstruction can be improved by anti-inflammatory therapy and intravenous anti-pseudomonal antibiotics in hospitalized infant CF patients. In recent years, investigators in the U.S. and Australia have developed new techniques in sedated infants to raise lung volume to total lung capacity (TLC) prior to the passive compression maneuver, so that a full "adult-like", raised-volume flowvolume loop is achieved. These newer techniques may provide a more reliable and reproducible measure of airway obstruction for future therapeutic studies. However, there are currently no published normative data or baseline data in CF patients, no commercially available equipment, and no published standardized procedures, precluding use of this improved technique in multicenter clinical trials at this time.

Early Pa infection is associated with increased morbidity and mortality among young children with CF. The prevalence of Pa infection increases with age, with positive respiratory tract cultures reported for up to 30% of infants, 30-40% of children 2-10 years of age, and 60-80% of adolescents and adults with CF. Appropriate intravenous (IV) or inhaled antibiotic therapy for established Pa endobronchial infection results in significant clinical improvement, but the reduction in lower airway Pa density is only 2-log fold on average, and eradication of Pa is rare. Most previous studies of inhaled anti-pseudomonal antibiotics excluded very young patients, under the general belief that very young patients would not execute the inhalation procedure correctly. In addition, safety and the possible emergence of resistant pathogens that may be associated with aggressive early intervention directed against Pa are a concern for young children with CF, and avoidance of oto- and nephro-toxicity are especially important in this age group. Inhaled anti-pseudomonal antibiotics, in particular aminoglycosides, can deliver high antibiotic concentrations directly to the lower airways with limited systemic absorption. A recent publication reported that a single 300 mg TOBI^® dose administered to children with CF that were younger than age 6 was safe and produced therapeutic lower airway concentrations of tobramycin (Rosenfeld et al., J. Pediatr. 2001 ;139:572-577).

Some investigators have reported that aggressive antimicrobial treatment of initial Pa infection in CF children achieved reduction or eradication of Pa, or delayed chronic Pa infection of the upper respiratory tract (Wiesemann et al., Pediatr. Pulmonol. 1998;25:88-92; Frederickson et al., Pediatr. Pulmonol. 1997;23:330-335; Ratjen et al, Lancet 2001 ;358(9286):983-984; Munck et al., Pediatr. Pulmonol. 2001 ;32:288-292; Nixon et al., J. Pediatr. 2001 ;138:699-704). However, limitations of these prior studies include a lack of controls or use of historical controls; a lack of focus on safety; small sample sizes; and the absence of evaluation of lower airway inflammation or lower airway microbiology. The need for controlled studies is highlighted by reports of transient Pa infection in young children with CF.

Prior intervention trials directed to the treatment of initial Pa infection in CF patients have varied widely in the age of the patient population, the site of respiratory tract cultures (OP, endolaryngeal, sputum, and BAL), the presence of serum antibodies against Pa at enrollment, the duration of Pa infection prior to treatment, treatment regimen, and study endpoints (Wiesemann et al., Frederickson et al., Ratjen et al., Munck et al., and Nixon et al., all supra). Previous intervention trials for early Pa infection, though limited by lack of controls, lack of safety data, and focus on upper airway cultures, have demonstrated a microbiologic effect. Observed results are likely dependent upon patient selection criteria, treatment regimen and outcome measures. The Danish CF Center first reported that a step-wise approach to early Pa infection with inhaled colistin and oral ciprofloxacin prevented or delayed chronic Pa infection; three months of inhaled colistin and oral ciprofloxacin resulted in the best outcomes (Frederickson et al., supra). Two prior studies treated school-aged children presenting with early, seronegative Pa infection (endolaryngeal/sputum cultures) with inhaled tobramycin (80 mg twice daily) for 12 months, and reported high rates of Pa reduction or eradication (Wiesemann et al., supra; Ratjen et al., supra); Ratjen et al. observed that one year after this aggressive treatment, 14 of 15 patients were both seronegative and culture negative for Pa. Munck et al. treated initial Pa colonization (non-mucoid strains, < 2 precipitating antibodies) in young children with IV antibiotics for 21 days and inhaled colistin for 2 months and reported transient eradication of Pa from endolaryngeal/sputum cultures (mean duration of 8 months). Five of 19 patients were re-colonized with the same Pa genotype, while 14 of 19 were colonized with a new Pa genotype, suggesting true eradication in more than 75% of patients.

Early Colonization - Infant Bronchoscopy Study

A longitudinal, multicenter observational study to define the early natural history of lower airway Pa colonization in CF infants ages 0 to 3 years has been conducted. CF infants were enrolled at three centers between 1993 and 1996 and followed prospectively to age three. Evaluations included bronchoalveolar lavage (BAL) performed annually at the subject's first, second and third birthdays to obtain fluid for bacterial cultures and inflammatory markers. Quarterly evaluations were conducted to measure clinical status and to obtain oropharyngeal (OP) cultures and serum for anti- Pa exotoxin A serology. A total of 42 infants were enrolled in the study at an average of 11 months of age, and 33 subjects completed the 3 year study.

Prevalence of Lower Airway Pa Infection by Age

The primary aim of this study was to determine the prevalence of lower airway Pa colonization in infants and young children with CF. Prevalences of 18% (7/40) at age one year, 34% (12/35) at age two years, and 33% (11/33) at age three years were observed, with colonization defined as any growth of Pa from BAL fluid. All isolates were sensitive to tobramycin (defined as tobramycin MIC < 8 μg/ml), with the exception of one isolate from a patient at age three years. Pa densities ranged from 1.8 to 8.4 logio CFU/mL (colony forming units per mL), with median densities of 3.9, 4.9, and 5.2 logio CFU/mL at ages one, two, and three years, respectively. The Pa densities from BAL samples in these CF children 0-3 years of age were 2-4 logio-fold lower than the sputum Pa densities observed in subjects > 5 years of age in the Phase III inhaled tobramycin trials, and only a small percentage of this difference could be attributed to the dilutional effect of the BAL procedure.

Lower airway infection with mucoid Pa is associated with greater mortality than non-mucoid Pa in CF patients. In the phase III studies of inhaled tobramycin, the incidence of the mucoid Pa phenotype at baseline in CF subjects > 5 years old was 57%. In contrast, only one subject in the longitudinal infant bronchoscopy study (1-3 years of age) had a mucoid Pa isolate (<1% of cultures). Evidence of Transient Lower Airway Pa Colonization in Young Children with CF

Of the forty CF subjects enrolled in the longitudinal infant bronchoscopy study,

37 subjects completed two or more BAL cultures at annual visits at ages 1 , 2, or 3 years of age. Of these thirty-seven subjects, 20 CF patients (54%) were never colonized with Pa in the lower airway, while 17 (46%) were colonized with Pa in the lower airway at some time during the study. Interestingly, 5 CF patients (14%) demonstrated transient Pa colonization with a negative culture occurring after one or two positive cultures. Assuming that the observed culture results represent true negatives, Pa colonization may occur transiently in CF patients in this young age group. Possible explanations may be related to antibiotic treatment and/or host inflammatory responses that reduce or clear the organism in CF subjects less than 3 years of age. Also, the reduced incidence of mucoid Pa in this young age group may contribute to the occurrence of transient Pa colonization.

The possibility of transient lower airway Pa infection in young CF patients may explain the observation of lower airway inflammation in the absence of concurrent infection in prior studies. Evidence of transient lower airway Pa infection also reinforces the importance of a placebo-control group for any trial of anti-microbial therapy in this age group. The placebo control group permits accounting for the

"natural" incidence of Pa eradication that might otherwise be erroneously attributed to the antibiotic therapy.

Treatment of Very Young Patients with TSI

Twenty eight (28) days of TSI treatment (300 mg BID), as compared to placebo, resulted in both an increased frequency of Pa eradication and a markedly greater reduction in Pa density from the lower airways. The observed antimicrobial treatment effect was much more profound than that observed in older, chronically infected patients (Ramsey et al, NEJM, supra), and represents a new treatment paradigm for younger, newly colonized patients. This method of treatment is the first to demonstrate eradication of Pa from the lower airways of CF patients in a placebo-controlled trial.

This dramatic effect may be the result of several factors, including the characteristics of Pa colonizing the younger CF patients (i.e., more susceptible to tobramycin, less mucoid), lower Pa density, or possibly different clearance of tobramycin from the lower airways with a more prolonged dwell time. Unexpectedly, therapeutic concentrations of tobramycin were found in the BAL fluid at the time of follow-up bronchoscopy. However, several facts corroborate the validity of data relating to the primary efficacy endpoint. First, in vitro studies demonstrated that the concentration of tobramycin in the Day 28 BAL fluid did not eradicate any patients' isolates, with 6 of 8 patients' Pa isolates reduced less than one log-fold in density under the conditions of specimen shipping. Second, the Day 28 lower airway cultures performed at the local sites, with timely dilution and plating, yielded nearly identical results for frequency of eradication in the two treatment groups as did the specimens shipped to the core laboratory. Third, sustained eradication of Pa from OP cultures through Day 56 in 6 of 8 patients in the TSI group was observed. In this study, at baseline many CF patients had well-established lower airway Pa infection, with a mean BAL Pa density greater then 10⁵ CFU/ml, a mean time interval from first positive Pa culture to enrollment of 1.4 years, mucoid isolates in the majority of TSI group patients, and elevated exotoxin A titers in the majority of patients. Potential sites of undetected, persistent Pa infection in our patients include lower airway micro-environments not sampled by lobar BAL, and/or the paranasal sinuses. Treatment of seronegative patients with first Pa isolation from the upper airway may result in a longer period of Pa eradication. In addition, treatment with inhaled anti- pseudomonal antibiotics for greater than 1 month, optionally combined with systemic treatment (IV or oral), may result in more prolonged eradication of Pa infection in CF. Extensive safety data on inhaled tobramycin in young children with CF were generated. No significant safety concerns were observed for TSI in these young children with CF, based on adverse events (i.e., cough, bronchospasm), serum tobramycin concentrations, renal function, audiology testing, stable tobramycin MICs, and lack of emergence of new pathogens.

Due to the profound microbiologic treatment effect, the clinical trial was stopped prematurely. However, the resultant small numbers in the treatment groups made interpretation of any secondary endpoints difficult. The young children enrolled in this study had a marked degree of lower airway inflammation at baseline consistent with prior studies of young, infected CF patients (Rosenfeld et al., Pediatr. Pulmonol. 2001 ;32:356-366; Armstrong et al., Am. J. Respir. Crit. Care Med. 1997; 156: 1197- 1204; Noah et al., J. Infect. Dis. 1997;175:638-647; Muhlebach et al., Am. J. Respir. Crit. Care Med. 1999;160:186-191), and stable adult patients with mild lung disease (Konstan et al., Am. J. Respir. Crit. Care Med. 1994;150:448-454). The degree and duration of Pa infection at baseline, the short duration of treatment and follow-up, the small sample size and high measurement variability may explain the lack of an observed anti-inflammatory effect of TSI after 28 days of treatment. Alternatively, this robust inflammatory response in young patients infected with Pa may be, at least in part, intrinsic to the defect in CF (Khan et al., Am. J. Respir. Crit. Care Med. 1995;151 :1075-1082).

In CF patients greater than 6 years of age, peak sputum tobramycin concentrations occur in 10-60 minutes after tobramycin inhalation (Eisenberg et al., CΛesf 1997;111 :966-962; Mukhopadhyay et al., Respir. Med. 1994;88:203-211), with an estimated half-life in sputum of < 2 hours (Eisenberg et al., supra; Geiler et al., Chest 2002; 122:219-226). The sputum tobramycin concentration decays to the limit of quantitation by 8 hours after administration of 300 mg of TOBI^® (Geiler et al., supra). In contrast, we observed that ELF (epithelial lining fluid) tobramycin concentrations 4- 24 hours after the last dose of TSI, 300 mg twice daily for 28 days, had no relationship to the interval since last TSI dose, and were comparable to ELF concentrations observed 45 minutes after inhalation of a single dose in a similar population of young CF patients (Rosenfeld et al., supra). There are no studies directly comparing sputum and ELF tobramycin concentrations; all ELF data are from young, nonexpectorating children with CF. Possible explanations for the apparent delayed tobramycin clearance from ELF in young children with CF include: 1) reduced cough clearance; 2) serum elimination time being inversely proportional to age (Horrevorts et al., Chest 1985;88:260-264); 3) increased distal deposition and binding of tobramycin to macromolecules (Mendelman et al., Am. Rev. Respir. Dis. 1995;132:761-765; Ramphal et al., J. Antimicrob. Chemother. 1988;22:483-490), possibly due to reduced amounts of central airway purulent secretions; 4) drug accumulation in the lower airways after chronic exposure compared to single dose exposure (Eisenberg et al.; Mukhopadhyay et al; Geiler et al.); and 5) sampling of lower airway secretions rather than sputum. Further studies are needed to confirm this observation and to elucidate the mechanism for delayed clearance of tobramycin from ELF in young CF patients. These data may be important for determining dosing interval for future early intervention trials.

The treatment features and host/environmental factors that determine the interval until reinfection with Pa are not well understood. Often these young patients with early Pa infection have minimal symptoms with normal lung function. Thus, there remains a need for new clinical outcome measures for young children, such as raised- volume chest tomography to assess early bronchiectasis, or raised volume infant lung function tests to detect air trapping and reduced small airway flows. Any early intervention should focus on safety (i.e., adverse events, microbial resistance, emergence of new pathogens), seek to determine the minimum amount of anti- pseudomonal therapy required to eradicate the original Pa isolate, and/or determine the host and environmental factors that result in re-infection with a new environmental Pa isolate. Such studies will aid in formulating informed and effective clinical strategies for prolonged eradication of early Pa colonization in young children with CF. This "eradication" trial presented several unique features as compared to other reported "eradication" trials: (1) The mean duration of Pa colonization in the treated group was about 1 year; (2) one of the inclusion criteria was that the patient demonstrate two consecutive Pa-positive OP cultures; (3) another inclusion criteria was that the patient have a subsequent Pa-positive BAL sample; (4) the majority (5/8) of treated patients presented with mucoid Pa isolates; (5) several (4/7) of the treated patients presented with high Pa Exotoxin A titers; and (6) this trial featured the least aggressive antibiotic regimen of any studies reported to date (28 days bid treatment; monotherapy using inhaled tobramycin only).

Early anti-pseudomonal therapy in children 6 years of age and younger can eradicate both upper and lower airway Pa colonization.

Treatment of Other Patients Susceptible to Chronic Endobronchial Infection

Patient populations other than very young CF patients may benefit from endobronchial delivery of antibiotic upon first or early infection with bacteria that, if not significantly reduced in number or eradicated, can lead to chronic infection (with its accompanying pulmonary damage). Patients susceptible to bronciectasis, either through genetic predisposition, environmental insults or other causes, or to chronic bronchitis may also benefit from early intervention, such as that detailed herein for very young CF patients experiencing a first or early infection with Pa. More generally, any patient that presents with impaired airway clearance and/or host defense mechanisms, or with a ciliary disorder or mechanical insult, such that the patient faces a likelihood of chronic endobronchial infection, may benefit from the methods of the present invention. Preferably, the patient that is treated according to the claimed methods has minimal symptoms associated with the infection and relatively normal lung function. Also, recipients that exhibit little or no inflammatory response, as well as those that are seronegative for the infectious agent's virulence factors, may also be preferred. Antibiotics

Suitable antibiotics for use within the claimed invention include polymyxins (such as colistin), beta-lactams (such as ticarcillin and ceftazidime), macrolides (such as azithromycin), carbapenems (such as meropenem), fluoroquinolones (such as ciprofloxacin) and aminoglycosides. Prototypical aminoglycosides include tobramycin, gentamicin, amikacin, kanamycin, streptomycin, neomycin, and netilmicin. A preferred aminoglycoside antibiotic within the present invention is tobramycin. Aminoglycoside formulations according to the invention typically contain from about 100 to about 500 mg aminoglycoside, more preferably from about 200 to about 400 mg aminoglycoside, and most preferably about 300 mg of aminoglycoside.

When delivered in nebulized aerosol form, a preferred amount of aminoglycoside for delivery is about 20 to about 100 mg/ml, more preferably about 40 to about 80 mg/ml, and most preferably about 60 mg/ml of aminoglycoside. Typically, about 300 mg of aminoglycoside antibiotic is dissolved in 5 ml of about 0.225% NaCl. Depending on the nebulizer/aerosolizer device or other delivery device that performs the same function, other volumes of diluent or vehicle may be appropriate (optinally, with an adjustment of aminoglycoside amount). Also, other diluents or vehicles may be used within the present invention, so long as such diluent or vehicle provides an appropriate balanced osmolarity, ionic strength and chloride concentration, and such diluent or vehicle does not negatively affect the functionality of a recipient's airways or cause undesirable side effects (such as bronchospasm and cough). For example, the commercially available TOBI® product is formulated in 1/4 NS, has an osmolarity in the range of 165-190 Mosm/L, and a pH in the range of 5.5 to 7.0. If a decreased volume of diluent or vehicle is compatible with the aerosol delivery device selected for administration, the desired amount of antibiotic may be delivered to a recipient's endobronchial spaces in a shorter period of time, which may lead to improved recipient comfort and better compliance with the treatment regimen. However, the present invention is not limited to the use of TOBI^® as a liquid formulation of tobramycin for aerosolization. Any TSI (tobramycin solution for inhalation) or other antibiotic solution for inhalation may be suitable for use within the claimed methods, so long as the TSI or other antibiotic solution for inhalation is compatible with aerosolization and can be used with a nebulizer or other aerosol delivery device, and so long as the tobramycin solution or other antibiotic solution does not elicit negative responses in patients (for instance, bronchospasm or cough). With respect to other antibiotic solutions for inhalation that are suitable for use within the present invention, the concentration of antibiotic and the diluent or vehicle solution will be selected to achieve a delivered dose of antibiotic that is effective in reducing or eradicating bacteria in a recipient's endobronchial space. Aerosolization

Aerosolized formulations of concentrated antibiotic may be nebulized by a jet, ultrasonic, electronic or functionally comparable nebulizer capable of producing an antibiotic aerosol having a particle size predominately between 1 and 5 microns.

Particles of these sizes facilitate delivery of the drug into the terminal and respiratory bronchioles, and efficaciously deliver concentrated antibiotic into the endobronchial space. To achieve high concentrations of antibiotic solution in both the upper and lower airways and in sputum, the antibiotic is preferentially nebulized in jet nebulizers, particularly those modified with the addition of one-way flow valves, such as, for example, a Pah LC Plus® nebulizer, commercially available from Pari Respiratory Equipment, Inc., Richmond, Va., which delivers up to 20% more drug than unmodified nebulizers. In addition, for use with very young patients, a nebulizing or aerosolization device equipped with a face mask, rather than a mouthpiece, may be beneficial. The AeroDose 5. RP inhaler (Aerogen; Mountain View, CA) may also be suitable for use herein. Additional nebulizers suitable for use within the present invention are described in US Patent Nos. 5,508,269 and 6,387,886 (both incorporated herein by reference in their entirety).

Alternatively, methods of the present invention may employ endobronchial administration of a dry powder formulation of an antibiotic, using dry powder or metered dose inhalers (for example). Depending on the efficiency of the dry powder delivery device, which is typically about 70%, prototypical effective dry powder dosage levels are within the range of about 20 to about 60 mg. As with nebulized aerosol antibiotic, in dry powder or metered dose form the drug particle sizes should be within a range of 1 to 5 microns. Dry powder or metered dose inhalers offer convenience, portability and potentially longer shelf life than nebulizer-compatible, liquid-based formulations. However, these dry powder delivery devices have not been tested and successfully used with patients to the extent that nebulizers have been (for instance, TOBI® is administered via nebulizer). Additional features of dry powder formulations and delivery devices that are preferred for use within the present invention are described in US Patent No. 6,387,886 (incorporated herein by reference in its entirety).

Related Methods

In one embodiment, the present invention also provides a method for selecting patients, particularly those 6 years of age or younger with early Pa infection, for endobronchial TSI treatment, comprising the steps of: detecting Pa in at least two consecutive positive oropharyngeal cultures, at a time following at least one Pa- negative oropharyngeal culture; and, optionally, testing for the presence of Pa in a subsequent bronchioalveolar lavage sample. In another embodiment, the present invention further provides a method for monitoring anti-P. aeruginosa endobronchial TSI treatment outcome, comprising the steps of: obtaining an oropharyngeal sample from a treated recipient after completion of said TSI treatment; and determining the presence or absence of P. aeruginosa in said sample, wherein the presence of P. aeruginosa indicates a need for additional TSI treatment and the absence of P. aeruginosa indicates effective TSI treatment.

The invention is further illustrated by the following non-limiting examples.

EXAMPLES Example 1. Patient Inclusion

A double-blind, randomized, placebo-controlled multicenter trial that featured administration of TSI (300 mg twice daily for 28 days) to children with CF less than 6 years of age was undertaken to determine whether such treatment would result in a beneficial antimicrobial effect and would be safe.

Inclusion criteria for the screening visit were: 1) age > 6 months and < 6 years;

2) diagnosis of CF based upon the following criteria: i) sweat chloride > 60 mEq/L by quantitative pilocarpine iontophoresis; OR ii) genotype with two identifiable mutations consistent with CF; AND iii) two clinical features consistent with CF; 3) informed consent by parent or legal guardian; and 4) one historical oropharyngeal (OP) culture positive for Pa within 2 weeks to 12 months prior to screening. Exclusion criteria included: history of adverse reaction to anesthesia or sedation; aminoglycoside hypersensitivity; unresolved anemia or thrombocytopenia; hemoptysis within 30 days prior to screening; abnormal renal function; chronic hearing loss; or administration of an investigational drug within 30 days prior to screening.

If the OP culture obtained at screening was Pa positive, patients were eligible for baseline bronchoscopy and BAL. Patients were excluded from baseline BAL for the following conditions: acute respiratory infection, pulmonary exacerbation or receiving intravenous or inhaled antibiotics within 14 days prior to bronchoscopy, or oxygen saturation less than 90% on room air. Evaluation on the day of baseline BAL included physical examination, modified Shwachman score, chest radiograph, OP culture, and a blood draw for complete blood count (CBC) with differential, creatinine, urea, Pa exotoxin A serology, cytokines, and neutrophil elastase activity.

Patients with no Pa isolated from their baseline BAL were withdrawn from the study, and the CF pathogens identified from the BAL were reported to clinicians caring for the patient. Patients with Pa isolated from their baseline BAL sample were randomized in a 1 :1 ratio to receive TSI (300 mg) or placebo (study drug vehicle=1/4 NS, pH 6.0) twice daily for 28 days at home. The first dose of study drug was administered on Day 0 (< 10 days after BAL). Randomization was stratified by study center and patient age (< 36 months or > 36 months). Patients who received IV or inhaled antibiotics between baseline BAL and Day 0 were ineligible for randomization. Subsequent study visits occurred on Days 14, 28, 42, and 56, with interim history, clinical evaluation, and OP culture at each visit. At Day 14, blood was obtained for trough and peak tobramycin levels, serum creatinine, CBC with differential, and Pa exotoxin A serology. At Day 28, a second bronchoscopy and BAL were performed, and baseline tests and procedures except the chest radiograph were repeated. Other inhaled antibiotics beyond study drug were not to be administered until study completion at Day 56.

Example 2. Study Endpoints

The primary efficacy endpoint was change in Pa density in BAL cultures from baseline to Day 28. The frequency of patients in whom Pa was eradicated from BAL cultures was also examined. Eradication of lower airway Pa was defined as having no Pa isolated from the Day 28 BAL culture (limit of detection is 20 colony forming units (CFU) per mL). The primary safety endpoints were adverse events, changes in renal function and hearing acuity, peak and trough serum tobramycin concentrations, incidence of bronchospasm or acute respiratory distress, and rate of isolation of lower airway Pa isolates resistant to tobramycin. Secondary endpoints included eradication of Pa from OP cultures, Pa exotoxin A serology titers, concentrations of inflammatory markers in BAL fluid and blood, and clinical parameters (oxygen saturation, weight, and modified Shwachman scores). Example 3. Techniques and Methodology A. Bronchoscopy and BAL Processing

The BAL was performed, under anesthetic or sedation per institutional guidelines, at each center according to a standard operating procedure. The Day 28 BAL was to occur 12 to 48 hours after the last dose of study drug. BAL specimens were placed immediately on ice and processed for qualitative (site laboratory) and quantitative culture (core laboratory), cell count with differential, urea, cytokines, and elastase activity.

More specifically, patients underwent anesthesia or sedation per institutional protocol for the bronchoscopy and bronchoalveolar lavage (BAL). The BAL was performed at each site according to a standard operating procedure developed by the Cystic Fibrosis Therapeutics Development Network (CFTDN). Topical anesthesia (lidocaine) was applied to the airways as necessary. The flexible bronchoscope was introduced into the lower airway with suctioning only used to maintain visibility in order to avoid contamination with upper airway secretions. The bronchoscope tip was wedged in a segmental bronchus of the lingula or right middle lobe. An aliquot of sterile non-bacteriostatic physiologic saline, 1 mL/kg body weight (maximum 30 mL, minimum 10 mL), was introduced via the scope and aspirated by suction into a Lukey trap to collect BAL fluid. The procedure was repeated two additional times (maximum lavage, 3 mlJkg). The BAL fluid was placed immediately on ice and processed for quantitative culture, cell count with differential, urea, cytokines, and elastase activity. Two placebo patients and 4 TSI patients had Day 28 BAL performed less than 12 hours after their last dose of study drug. All patients had baseline and Day 28 BAL specimens collected from a single segmental bronchus at the same site for both procedures (right middle lobe for one patient; lingula for all others).

BAL fluid was kept on wet ice until transfer to sterile polypropylene plasticware. Two aliquots (1 mL each) were removed for quantitative bacterial culture, with one aliquot sent to the site lab for immediate dilution and plating on selective agar and the other aliquot sent to the core laboratory. Total BAL cell count was determined using a hemocytometer after dilution of an aliquot in Turk's solution, with an average determined from counts on each side of the chamber. Cytospin slides for differential cell counts were prepared from an aliquot of diluted BAL fluid at cell density of 1-2x10⁵, and two unstained and stained (Hema 3 Kit) slides were used for determination of differential cell counts. The remaining BAL fluid was centrifuged at 250xg for 10 minutes at 4°C and the pellet resuspended in 0.5 mL 0.9% saline then stored at -70°C. The supernatant was recentrifuged at 4,000xg for 20 minutes at 4°C and the subsequent supernatant filtered through a 0.22 micron filter unit (Corning). One-third of the filtrate was stored in aliquots at -70°C, and the remaining filtrate was treated with protease inhibitors (PMSF1 μl/ml of 100 mM stock; EDTA 25 μl/mL of 200 mM stock). Aliquots of the untreated filtrate were used for measurement of BAL fluid urea, tobramycin, and free elastase activity; aliquots treated with protease inhibitors were used to measure IL-6, IL-8, and elastase-α1-AT complexes.

B. Bacterial Cultures

OP specimens were obtained from the posterior oropharyngeal wall and tonsillar pillars using a commercially available collection and transport system. OP specimens obtained at baseline and Day 28 visits were collected after the patient was sedated, and prior to introduction of any medications or instruments into the airway. Screening OP cultures were performed at the clinical lab at each center for identification of Pa. All subsequent OP cultures, quantitative BAL cultures, and minimal inhibitory concentrations (MICs) for Pa isolates were performed by the core laboratory.

More specifically, all specimens were sent on wet ice by overnight express shipment to the core laboratory, based on previous demonstration by the laboratory that bacterial density of Pseudomonas aeruginosa (Pa) is not significantly affected by 48-72 hours on wet ice.

OP swabs were processed by vortexing thoroughly in 0.5mL Sputolysin (Calbiochem) followed by addition of 0.5 mL of phosphate buffered saline containing 0.1% gelatin (PBSG). BAL fluid was homogenized by adding 1 :1 (volume:volume) Sputolysin and allowing the sample to sit for 5 minutes at room temperature with vigorous mixing by vortex. OP and BAL specimens were diluted 1 :10, 1 :1000, and 1 :10,000 with sterile PBSG. From each dilution, 0.1 ml was spread on the following six solid media using a sterile glass rod (final dilutions 10^"1, 10^"3, 10^"5, 10^"6). The media included: 1) MacConkey agar for identification of non-lactose fermenters, 2) OFPBL agar (oxidation-fermentation base supplemented with agar, polymyxin, bacitracin, and lactose) for the identification of Burkholderia cepacia, 3) DNAse agar for the identification of Stenotrophomonas maltophilia, 4) streptococcal selective agar, 5) Haemophilus selective agar, 6) mannitol salt, 7) chocolate age for detection of fastidious organisms, and 8) mycosel agar for identification of fungi. The plates were incubated at 37°C (Streptococcal and Haemophilus selective agar were incubated in an anaerobic chamber) and evaluated at 48 hours (all plates) and 72 hours (OFPBL). All organisms were identified using standard techniques. OP culture results were reported qualitatively, while BAL culture results were reported quantitatively with bacterial density expressed as colony forming units per mL (CFU/mL). Growth of P. aeruginosa at any density was considered a positive lower airway culture for the purposes of this protocol.

Minimal inhibitory concentrations (MIC) of antibiotics against P. aeruginosa were determined using a semi-automated microbroth dilution method (Sensititre, AccuMed, Westlake, OH). For BAL isolates, the antibiotics tested were amikacin, aztreonam, ceftazidime, ciprofloxacin , gentamicin, imipenem, pipericillin, ticarcillin, tobramycin, and trimethoprim. For OP isolates, only tobramycin was tested.

C. BAL Tobramycin Concentrations

Tobramycin concentrations in Day 28 BAL fluid samples were measured using a high pressure liquid chromatography (HPLC) procedure. The lower limit of quantitation was 0.2 μg/mL. A subset of samples from the baseline BAL served as controls. Concentrations of bioactive tobramycin in Day 28 BAL fluid samples were measured using a bioassay procedure. Briefly, agar plates were prepared by addition of a suspension of highly-tobramycin sensitive Bacillus subtilis to Mueller Hinton agar. Replicates of each BAL sample were plated and incubated for 18 hours at 35°C.

Zones of growth inhibition were measured for each replicate, and mean zone diameter was calculated by averaging the zone sizes from individual replicates. The tobramycin concentration (ug/mL) for each sample was determined by comparing the mean zone diameter to a standard curve.

D. Viability Assay of Pa Isolates Exposed to Tobramycin

An in vitro Pa viability assay mimicking BAL shipping conditions was performed to determine if negative cultures at Day 28 were due to residual tobramycin in the BAL fluid (on-line data supplement). Briefly, Pa isolates from baseline BAL samples of TSI group patients (17 isolates from 8 patients) were grown overnight in Mueller Hinton agar and then inoculated into phosphate buffered saline (PBS) at a density of approximately 1 x 10⁹ CFU/mL. The sample was diluted with PBS to a final concentration that approximated the density in the baseline BAL sample. For each patients' isolate(s), tobramycin was added at a concentration corresponding to that measured in their Day 28 BAL fluid. Aliquots were incubated at 4°C with colony counts performed after 24, 36, and 48 hours of incubation.

E. Genotyping of Pa Isolates

DNA was isolated from an overnight culture of a single bacterial colony, using the DNeasy kit (Qiagen, Valencia, CA). Three primers (208, 270, and 272) were used to prime random amplified polymorphic DNA polymerase chain reactions (RAPD-PCR) using a previously published technique. RAPD products were separated by electrophoresis in agarose and visualized following staining in ethidium bromide. Polymorphisms that differed by two or more bands were considered distinct genotypes.

F. Exotoxin A Serology

Anti-exotoxin A titers were tested by indirect microtiter ELISA assay. All serum samples from each subject were run simultaneously to assure that titers were compared under identical conditions. An exotoxin A IgG titer of ≥ 1 :200 was defined as a positive result. Briefly, each microtiter plate well was coated overnight with 300 ng of exotoxin A in carbonate buffer, pH 9.6. Plates were washed 6 times with phosphate- buffered saline with 5% Tween (PBST) between each step. Bovine serum albumin (5%) solution was added to block nonspecific binding sites. Human sera diluted serially in PBST, range 1 :100 to 1 :12,800, were incubated for 1 hour at 37°C. Horseradish peroxidase goat/antihuman IgG and IgM were added and incubated for 1 hour at 37°C. Peroxidase substrate, phenylene diamine, and hydrogen peroxide were then added and the reaction was allowed to continue for 10 minutes at room temperature in the dark. The reaction was stopped with 2.5M sulfuric acid. Optical densities of wells were read at 492 nm using a Dynatech MR5000 plate reader (Dynex Technologies, Chantilly, VA). Each sample was run in triplicate and a mean value determined. Samples from age-matched healthy children with no known history of Pa infection served as negative controls.

G. BAL Cell Counts and Differential

The total white cell count was performed on fresh BAL fluid at each center. Differential counts were performed on stained slides. Briefly, total cell counts were performed on fresh BAL fluid according to a CFTDN standard operating procedure. Cells were stained by gentle mixing of 100 μL specimen with 100 μL Turk's solution, and then both sides of a hemacytometer chamber were loaded with the cell suspension. The four corners in each side of the hemacytometer were counted and used in the calculation of the total number of cells per side. The calculation included a dilution factor for the Turk's solution and a factor to correct the calculation to cells per milliliter (cells/mL). The totals from each side were averaged to determine the total cell count.

H. Serum and BAL Urea and Inflammatory Mediators

All assays were performed by the CFTDN Inflammatory Mediator Core, University of Colorado, Denver, CO. The concentrations of inflammatory mediators reported from BAL samples were corrected for dilution using urea as a marker. Urea was measured on BAL fluid using a standard assay. Levels of IL-8 (BAL), IL-6 (BAL and serum), and elastase-α1-anti-trypsin complexes (BAL and serum) were assayed by quantitative indirect ELISA assays (Quantikine R&D Systems, Minneapolis, MN), and free elastase activity (BAL) was measured by spectrophotometric kinetic assay (Sigma).

All serum and BAL samples were simultaneously analyzed in triplicate and a mean value reported. The concentrations of inflammatory mediators reported from BAL samples were corrected for urea dilution. Urea-adjusted concentrations were calculated by multiplying the BAL concentration times the plasma to BAL urea ratio.

I. Safety Monitoring

Determinations of CBC with differential, serum creatinine, and blood urea nitrogen were performed by clinical laboratories at each center. Serum tobramycin concentrations were measured using the Abbott/TdxFLx method (Abbott, Abbott Park, IL; limit of quantitation was 0.2 μg/mL)). Audiology testing was performed by an audiologist at Day 0 and Day 28 using standard techniques. Briefly, audiology evaluations included visual reinforcement audiometry for patients 6 to 36 months of age, and play or conventional audiometry for patients >36 months of age. Audiometric responses were recorded from 500 to 8000 Hz, with tympanometry performed to detect the presence of fluid in the middle ear. Abnormal hearing was defined as an auditory threshold >25 dB at any frequency (500-8000 Hz) in either ear. Any abnormal tests at Day 28 were to be followed-up at Day 42.

J. Statistical Considerations

Randomization of 98 patients was planned to detect a difference of 1.6 logio CFU/mL between treatment groups in mean change in lower airway Pa density. One interim analysis with early stopping for futility or efficacy was planned. Slow enrollment, due to lower than expected rates of culture positivity at screening and baseline BAL, prompted an additional early interim analysis to allow the Data Monitoring Committee (DMC) to evaluate the primary endpoint with respect to futility. The group sequential design was modified to include this additional review, and stopping boundaries for futility at both the first and second interim reviews were revised using a less conservative stopping boundary than the original design, to give a better probability of early stopping if there were truly no difference between treatment groups. O'Brien-Fleming type boundaries were planned for both stopping rules.

At the first interim analysis, statistical review of the data suggested that the DMC should also receive unblinded efficacy data, however, there was no efficacy stopping rule in effect for this early look. The DMC thus implemented a stopping rule for efficacy based on the O'Brien-Fleming efficacy boundary that would have been in effect if such a boundary had been specified in advance. Additional analyses were performed to evaluate the sensitivity of the efficacy inference to a myriad of stopping rules that the DMC could have considered; results were highly consistent with the putative stopping rule used by the DMC. Thus, appropriate steps were taken to address statistical ramifications of this unplanned efficacy analysis.

For the primary efficacy analysis, change in Pa density between baseline and Day 28 was calculated as the baseline BAL Pa density minus the Day 28 Pa density, with density expressed as logio CFU/mL). Mean change in Pa density was compared between treatment groups under an intent-to-treat analysis. Treatment effect, 95% confidence interval, and p-value estimates were adjusted for the efficacy stopping rule. The proportion of patients with lower airway eradication of Pa was also compared between treatment groups. Pa density was calculated as the sum of all strain-specific (mucoid and non-mucoid) densities isolated from a particular sample. Mean reduction in log o Pβ density was compared between treatment groups under an intent-to-treat analysis, and 95% confidence intervals and p-values were calculated. All estimates were adjusted for the efficacy stopping rule. The proportion of patients in whom eradication of Pa was achieved at Day 28 was also compared between treatment groups, and binomial 95% confidence intervals were calculated for the proportion with eradication in each treatment group. Other study endpoints were summarized using descriptive statistics, with a restricted number of exploratory analyses performed to compare differences in secondary endpoints between treatment groups. The exploratory analyses used two-sample t-tests and were not adjusted for multiple comparisons. All analyses were performed using SAS 8.0 (SAS Institute, Inc., Cary, NC) and/or SPLUS 2000 (Insightful Corporation, Seattle, WA) and were independently verified. Group sequential designs and monitoring were implemented using S+SeqTrial (Insightful Corporation, Seattle, WA).

Example 4. Patient Overview and Demographics

Though the planned sample size was 98 patients, the trial was stopped early by the DMC after interim analysis of data from the first 21 randomized patients showed a statistically significant microbiologic treatment effect. At the time the trial was stopped, 113 patients had been screened at 9 participating centers. Among screened patients, 37 qualified for baseline bronchoscopy with BAL, of whom 21 were positive for lower airway Pa and were randomized into the study (13 to placebo and 8 to TSI). All randomized patients completed the study.

Of the 21 randomized patients, 6 were younger than 36 months and 15 were older than 36 months of age. Treatment groups were fairly similar with regard to patient demographic and clinical characteristics at baseline, as shown in Table 1. Adherence to study drug was good for both groups, based on comparing counts of returned drug ampules as a surrogate for doses administered; mean number of doses was 53.2 (standard deviation (SD) 6.7) for the placebo group and 54.5 (SD 4.1) for the TSI group.

Table 1. Demographics and Prognostic Variables at Baseline by Treatment Group

Placebo Group TSI Group

(N=13) (N=8)

N (%) N (%)

Gender

Female 7 (53.8%) 3 (37.5%)

CF Genotype

ΔF508 Homozygous 7 (53.8%) 4 (50.0%)

ΔF508 Heterozygous 4 (30.8%) 3 (37.5%)

Race

Caucasian 9 (69.2%) 6 (75.0%)

Hispanic 2 (15.4%) 0 (00.0%)

African American 1 (07.7%) 0 (00.0%)

Native American 0 (00.0%) 1 (12.5%)

Other 1 (07.7%) 1 (12.5%)

Mean (SD) Mean (SD)

Age (years) 3.7 (1.6) 4.0 (1.5)

Height (centimeters) 98.6 (13.3) 99.0 (9.7)

Weight (kilograms) 14.9 (3.6) 16.0 (3.0)

Oxygen Saturation (%) 98.2 (1.7) 98.0 (1.9)

Modified Shwachman Score 67.5 (9.0) 67.4 (3.9)

Sweat Chloride^* (mEq/L) 106.9 (15.6) 116.8 (14.2)

^* Sweat chloride results available for 10 placebo and 5 TSI group patients. If genotype results were consistent with CF, no sweat chloride test was required.

Example 5. Determination of Lower Airway (BAL) Pa density

Pa was isolated at similar densities from the placebo and TSI group patients at the baseline BAL (Table 2). At the Day 28 BAL, Pa was eradicated in 8 of 8 patients in the TSI group (100%), with a one-sided 95% confidence interval for the proportion of patients eradicating Pa equal to (0.688,1.000). In contrast, Pa was eradicated in 1 of 13 placebo patients (7.4%) at the Day 28 BAL with 95% confidence interval (0.002,0.360). Table 2. P. aeruginosa Density in BAL Fluid by Treatment Group and Patient

Treatment Group Years Since Baseline Day 28 Log₁₀ 28-Day

& Patient 1^st Pa (+) Log₁₀ CFU/mL* Reduction in

Culture^* CFU/mL⁺ Pa Density*

TSI^® Group

T1 0.15 7.87 0 7.87

T2 0.31 6.38 0 6.38

T3 3.34 6.41 0 6.41

T4 0.11 1.61 0 1.61

T5 0.82 4.66 0 4.66

T6^§ 1.07 8.17 0 8.17

T7 0.06 3.31 0 3.31

T8^§ 0.67 3.53 0 3.53

Group Mean (SD) 0.8 (1.1) 5.25 (2.34) 0.00 (-) 5.25 (2.34)

Placebo Group

P1 0.67 1.32 5.47 -4.15

P2 3.03 7.24 6.92 0.31

P3 0.30 4.00 5.89 -1.89

P4 4.68 4.81 2.68 2.12

P5^§ 0.16 6.34 4.64 1.70

P6^§ 1.20 7.58 6.94 0.64

P7 0.58 7.07 7.23 -0.17

P8 3.94 4.00 6.30 -2.30

P9^§ 1.69 2.60 0 2.60

P10 1.26 NA 6.31 NA

P11^§ 0.34 4.60 3.62 0.99

P12 2.42 2.78 5.99 -3.21

P13 3.62 3.82 4.08 -0.25

Group Mean (SD) 1.8 (1.5) 4.68 (2.01) 5.08 (2.06) -0.30 (2.15)

Time from first positive Pa culture to randomization was determined by medical record review. The log transformation was computed on the total colony count as logιo(Total CFU/mL +1), so that when Total CFU/mL was equal to zero, log₁₀(Total CFU/mL +1) was equal to zero.

* The change in density (28-day reduction) is the Pa density at baseline minus the Pa density at Day 28 for each patient.

^§ These patients were all less than or equal to 36 months of age. The mean reduction in Pa density in the TSI group between baseline and Day 28 was 5.25 log₁₀ CFU/mL, and the mean increase in Pa density in the placebo group was 0.30 logio CFU/mL, for an observed mean difference between treatment groups of 5.55 logio CFU/mL (Table 2). The difference between treatment groups, accounting for the stopping rule, was 5.36 logio with 95% confidence interval (3.52,7.54). These results, combined with the ethical dilemma related to performing invasive bronchoscopy procedures in additional placebo patients, served as the basis for the DMC decision to stop the study early.

The time between the date of first positive culture for Pa (as determined by medical record review) and randomization date was longer on average in the placebo group (Table 2), and for a number of patients the study setting did not represent initial Pa isolation. Four placebo group patients and 5 TSI group patients had mucoid Pa strains isolated from their baseline BAL, and 1 TSI patient had mucoid Pa exclusively.

Example 6. Determination of Tobramycin Concentrations in Day 28 BAL Samples

We had predicted that there would be no detectable lower airway tobramycin at

12 hours after the last dose of TSI, based upon sputum tobramycin pharmacokinetics in patients with CF ages 6 and older. Following early termination of the trial by the

DMC, tobramycin concentrations from Day 28 BAL samples were measured to assist in interpretation of eradication results from the study.

Surprisingly, all Day 28 BAL samples from the TSI group had detectable tobramycin concentrations, as determined by two different methods. The concentrations observed in the TSI group were comparable when measured by bioassay or HPLC. The urea-adjusted HPLC concentrations in the TSI group, which provide an estimate of tobramycin concentrations in the epithelial lining fluid (ELF), were: 8.2, 24.1 , 36.8, 53.6, 98.1 , 104.0, 119.0, and 145.8 μg/mL All Day 28 BAL samples from the placebo group had undetectable tobramycin levels by bioassay and HPLC. Five baseline BAL samples used as controls had no detectable tobramycin by either assay. Four TSI group patients had the Day 28 BAL performed less than the required

12 hours after last dose of study drug. However, there was no clear relationship between measured BAL tobramycin concentrations and the time between the last dose of study drug and the BAL. An in vitro experiment was performed to determine whether exposure of Pa isolates to therapeutic tobramycin concentrations in Day 28 BAL fluid during shipping on ice affected the viability of Pa from the TSI group patients. All baseline isolates from the 8 TSI group patients were studied; for 6 of these patients, there was less than I log-fold reduction in Pa density after 48 hours incubation at 4°C. In the remaining two patients, a maximum of a two log-fold reduction was observed.

Results from unshipped aliquots of Day 28 BAL fluid that were cultured at the site microbiology laboratories were also examined. These samples were exposed much more briefly to tobramycin. Quantitative bacterial cultures were not available at the site laboratories. These samples showed eradication in 2 of 13 in the placebo group patients and in 7 of 8 TSI group patients, which are similar to results from cultures shipped to the core laboratory. These in vitro and site laboratory data support the conclusion from the primary analysis of a significant short-term microbiologic treatment effect of TSI in young children with CF.

Example 7. Results from Oropharyngeal Cultures

Among patients receiving TSI, all eight were positive for Pa at baseline and negative for Pa at Days 14 and 28. For the TSI group, 7 of 8 patients remained negative for Pa at Day 42, and 6 of 8 remained negative at Day 56. No TSI group patients received anti-pseudomonal antibiotics other than study drug. Among patients receiving placebo, there were several patterns of Pa status in the serial OP cultures. Five placebo group patients received anti-pseudomonal antibiotics (oral/inhaled) between Day 29 and Day 56; four patients with available data had Pa negative OP cultures within 3 days of stopping anti-pseudomonal treatment. There were four placebo group patients who had negative OP results at baseline and/or Day 28 that did not agree with their positive BAL culture results. Eight of 13 placebo group patients received no anti-pseudomonal antibiotics between baseline and Day 56; five patients were Pa positive for all OP cultures and three patients were Pa positive for all but one OP culture. These data provide further evidence for a significant microbiologic treatment effect of TSI.

Example 8. Results of Pa Serology

Four of the 7 TSI group patients with available data had positive exotoxin A titers at baseline; all four of these patients' titers were reduced at Day 14 and/or Day 28, including two who had negative titers at Day 28. Three TSI group patients had negative titers throughout the study. Seven of the 11 placebo group patients with available data had positive exotoxin A titers at baseline; four patients had persistent positive titers at Day 14 and Day 28, and three patients had negative titers at Day 14 or Day 28. Four patients in the placebo group had negative titers at baseline; two of these patients developed positive titers at Day 14 or Day 28, and two patients' titers remained negative.

Example 9. Results of Genotyping of Pa Isolates

For the two patients in the TSI group who had recurrence of upper airway Pa at Day 42 or 56, genotyping was performed on each distinct Pa morphotype isolated at baseline (OP or BAL) and at subsequent visits (OP only). At baseline, the first patient had four BAL isolates (two each of 2 genotypes, A and B) and two OP isolates (both genotype A). One OP isolate was identified at the Day 42 visit and two at the Day 56 visit (all genotype B). The second patient had one BAL and one OP isolate at baseline, both the same genotype, and two OP isolates at the Day 56 visit, both the same genotype as at baseline.

Example 10. Results of ELF (Epithelial Lining Fluid) Inflammatory Markers

There was no evidence for a TSI treatment effect at Day 28 for ELF white cell counts, ELF neutrophils, percent neutrophils, and ELF IL-8 concentrations. There was not a significant difference between TSI and placebo groups in the 28-day change in ELF white cell count (Day 28-baseline mean difference between groups: -6.8 x10⁶/mL; 95% CI -24.5, 10.8; p=0.42). There was also no difference between treatment groups in the 28-day change in ELF neutrophil count (Day 28-baseline mean difference between groups: -5.1 x10⁶/mL; 95% CI -19.8, 9.6; p=0.47). Seventy percent of values for free neutrophil elastase activity from BAL samples were below the limit of detection, thus descriptive statistics are not given.

Table 3. Changes in Lower Airway Inflammatory Markers in BAL Fluid Samples by Treatment Group^*

Placebo Group TSI Group

N Mean SD Med* N Mean SD Med^f

White cell count (x10⁶/mL)

Baseline 13 18.1 12.8 15.0 7 29.0 23.2 17.5

Day 28 13 21.5 26.3 15.0 8 24.3 17.0 15.7

Day 28-Baseline 13 3.3 23.2 -1.1 7 -3.5 14.3 -5.1

Neutrophils (x10⁶/mL)

Baseline 13 9.0 10.4 5.0 6 9.8 12.3 5.7

Day 28 13 13.3 21.7 5.5 7 8.2 7.6 4.6

Day 28-Baseline 13 4.3 19.7 0.8 6 -0.8 10.6 -0.3

Neutrophils (%)

Baseline 13 42.7 20.9 44.5 7 40.4 19.3 39.3

Day 28 13 51.8 25.1 63.0 7 35.8 10.8 33.7

Day 28-Baseline 13 9.2 24.5 11.5 7 -4.6 25.2 -6.3

IL-8 (pg/mL)

Baseline 13 29226 23411 26507 8 47082 75583 19367

Day 28 13 61964 95838 30060 8 27258 27330 15978

Day 28-Baseline 13 32738 93813 -5362 8 -19824 54149 -2140

IL-6 (pg/mL)

Baseline 13 483 415 375 8 534 548 341

Day 28 13 699 663 420 8 896 808 619

Day 28-Baseline 13 215 552 -8 8 362 1001 152

White cell count, neutrophil count, and concentrations of IL-8 and IL-6 are corrected for urea. There were missing values for white cell count and differential in the TSI group. For two placebo patients at baseline and one TSI patient at the Day 28 visit, the assay for BAL urea concentration was run on a sample treated with protease inhibitors. For one TSI patient at baseline, a serum urea value was used in place of a missing plasma urea concentration. ^f Med=median

Example 11. Results of Plasma Inflammatory Markers

There was not a significant difference between treatment groups in the 28-day change in peripheral white cell count (Day 28-baseline difference between groups: -2.0 x10⁶/mL; 95% CI -4.19, 0.25; p=0.08), nor was there a significant difference in the 28- day change in neutrophil count between treatment groups (Day 28-baseline difference between groups: -1.9 x10⁶/mL; 95% CI -4.09, 0.30; p=0.09). Sixty-four percent of the values of plasma IL-6 were below the lower limit of detection; therefore no descriptive statistics are given for this marker.

Example 12. Clinical Parameters

There were no significant differences between the TSI and placebo groups in change in oxygen saturation, change in weight, or change in modified Shwachman scores between baseline and Day 56.

Example 13. Adverse Events

Study-drug related - There were 107 treatment emergent adverse events (AEs), 72 among the 13 placebo group patients, and 35 among the 8 TSI group patients. Of these treatment emergent AEs, 6 in the placebo group and 4 in the TSI were considered related to study drug. The rate of occurrence of specific AEs was similar between the two groups. The most common AE in both groups was cough, affecting 92% of placebo patients and 88% of TSI patients. There were no episodes of bronchospasm related to study drug. Bronchoscopy related - Two patients had a serious adverse event (SAE) related to bronchoscopy. One patient had transient vomiting and unsteady gait after lorazepam sedation that required hospitalization and overnight observation. A second patient had an acute episode of laryngospasm and hypoxemia as the bronchoscope was introduced into the airway and required intubation; a chest radiograph showed acute bilateral upper lobe atelectasis. The patient stabilized rapidly but was kept on low synchronized intermittent mandatory ventilation support overnight as a precaution. The patient completed a 14-day course of IV anti-pseudomonal antibiotics and was stable on discharge from the hospital.

Example 14. Results of Safety Data Systemic effects- Day 14 peak (1 hour) serum tobramycin concentrations for the

TSI group were 1.0 ± 0.4 μg/mL and trough concentrations were 0.4 ± 0.5 (mean ± SD). There was no detectable serum tobramycin among placebo group patients. Serum creatinine levels were within the normal range for both groups at all evaluations (data not shown). There were no changes in auditory threshold in the TSI group patients.

Microbiology- The distribution of tobramycin MIC concentrations for Pa strains from OP cultures were determined at each study visit. For the placebo patients, the MICs for Pa isolates were fairly stable over time from the baseline visit through Day 56. For the two TSI patients with recurrence of positive OP cultures, the Pa isolates had tobramycin MICs < 0.5 ug/mL.

Tobramycin MICs for Pa isolates from the baseline BAL were comparable for the two treatment groups, and tobramycin MICs for concurrent OP and BAL Pa isolates were similar (data not shown). There were no tobramycin-resistant isolates among TSI group patients. One placebo patient had a tobramycin resistant isolate (MIC=16 μg/mL) at baseline BAL and a Day 28 isolate with MIC < 0.5 μg/mL. There was no significant change in tobramycin MIC values between baseline and Day 28 in BAL isolates for the placebo group (data not shown). Quantitative BAL cultures at Day 28 showed no evidence for emergence of new

CF pathogens in either treatment group. Five of 8 TSI group patients had co-infection with S. aureus at baseline; three patients had eradication of S. aureus at Day 28. No patients in the TSI group had any gram-negative organisms at Day 28. Five of 13 placebo group patients had co-infection with S. aureus or H. influenzae at baseline and Day 28.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

WE CLAIM:

1. A method for producing a significant anti-P. aeruginosa effect in a cystic fibrosis patient 6 years of age or younger, wherein such patient presents with early P. aeruginosa infection, comprising: administering endobronchially to the patient an aerosolized tobramycin formulation, wherein said formulation is administered as a monotherapy, optionally before the step of administering, a step of detecting P. aeruginosa in an oropharyngeal culture.

2. The method of Claim 1 wherein the aerosolized tobramycin formulation is a liquid formulation or a dry powder formulation.

3. The method of Claim 1 wherein the aerosolized tobramycin formulation is administered for a period of about one month.

4. The method of Claim 1 , wherein the administering step is repeated for at least 28 days; preferably for at least 14 days.

5. The method of Claim 1 wherein the effective amount of tobramycin administered is about 150 mg to about 400 mg; preferably about 180 mg to about 350 mg; most preferably about 300 mg.

6. The method of Claim 1 wherein the significant anti-P. aeruginosa effect is a reduction of P. aeruginosa density greater than 1.6 logio CFU/mL in a post-treatment endobronchial sample, as compared to a pre-treatment endobronchial sample; preferably reduction is greater than 3.0 logio CFU/mL in a post-treatment endobronchial sample; most preferably reduction is greater than 5.0 logio CFU/mL in a post-treatment endobronchial sample.

7. The method of Claim 1 , wherein P. aeruginosa is detected in two consecutive oropharyngeal cultures after a negative oropharyngeal culture.

8. The method of Claim 1 wherein the patient is a non-expectorating patient.

1 9. The method of Claim 1 wherein the patient exhibits one or more of the

2 following characteristics: (a) minimal symptoms of bacterial infection; (b) relatively

3 normal lung function; (c) minimal inflammatory response; (d) a first P. aeruginosa

4 infection after a period of non-infection with P. aeruginosa, or a first infection after

5 eradication of a prior P. aeruginosa infection; (e) a serum antibody titer against P.

6 aeruginosa Exotoxin A that is <1:200; and (f) infection with a P. aeruginosa population

7 that is >60% non-mucoid.

1 10. The method of Claim 1 wherein, after the administration step, colony

2 forming units of P. aeruginosa in an endobronchial sample are decreased to less than

3 100 CFU/mL; preferably less than 20 CFU/mL.

1 11. The method of Claim 1 wherein the administering step occurs twice daily.

1 12. A method for producing a significant anti-P. aeruginosa effect in a cystic

2 fibrosis patient 6 years of age or younger, wherein such patient presents with early P.

3 aeruginosa infection, comprising:

4 administering endobronchially to the patient an aerosolized tobramycin

5 formulation, wherein said patient has been diagnosed with cystic fibrosis based upon

6 the following criteria:

7 (a) sweat chloride > 60 mEq/L, or a genotype with two mutations consistent with

8 cystic fibrosis;

9 (b) two clinical features consistent with cystic fibrosis, and

[0 (c) one historical oropharyngeal culture positive for P. aeruginosa within 2

[ 1 weeks to 12 months prior to the screening step.

1 13. The method of Claim 12 wherein the aerosolized tobramycin formulation

2 is a liquid formulation or a dry powder formulation.

1 14. The method of Claim 12 wherein the aerosolized tobramycin formulation

2 is administered as a monotherapy.

1 15. The method of Claim 12 wherein the administering step is repeated for at

2 least 28 days; preferably for at least 14 days.

16. The method of Claim 12 wherein the effective amount of tobramycin administered is about 150 mg to about 400 mg; preferably about 180 mg to about 350 mg; most preferably about 300 mg.

17. The method of Claim 12 wherein the significant anti-P. aeruginosa effect is a reduction of P. aeruginosa density greater than 1.6 logio CFU/mL in an endobronchial sample; preferably reduction is greater than 3.0 logio CFU/mL in an endobronchial sample; most preferably reduction is greater than 5.0 logio CFU/mL in an endobronchial sample.

18. The method of Claim 12 wherein the patient is infected with a P. aeruginosa population that is > 60% non-mucoid.

19. The method of Claim 12 wherein, after the administration step, colony forming units of P. aeruginosa in an endobronchial sample are decreased to less than 100 CFU/mL; preferably less than 20 CFU/mL.

20. The method of Claim 12 wherein the administering step occurs twice daily.

21. A method for selecting a cystic fibrosis patient 6 years of age or younger for aerosol tobramycin treatment, comprising: detecting P. aeruginosa in at least one positive oropharyngeal culture, at a time following at least one negative P. aeruginosa oropharyngeal culture, optionally after the step of detecting P. aeruginosa in at least one positive oropharyngeal culture, a second step of detecting P. aeruginosa in a subsequent endobronchial sample.

22. The method of Claim 21 , wherein at least two consecutive P. aeruginosa- positive oropharyngeal cultures are detected.

23. A method for monitoring anti-P. aeruginosa aerosol tobramycin treatment outcome in a cystic fibrosis patient 6 years of age or younger, comprising: obtaining an oropharyngeal sample from the treated patient after completion of the aerosol tobramycin treatment; and determining the presence or absence of P. aeruginosa in the sample, wherein the presence of P. aeruginosa indicates a need for additional treatment and the absence of P. aeruginosa indicates effective treatment.