Original Research

Delivery and Safety of Inhaled Interferon-cin Idiopathic Pulmonary Fibrosis

Keith T. Diaz, M.D.,1 Shibu Skaria, M.D.,1 Keith Harris, M.D.,1 Mario Solomita, D.O.,1 Stephanie Lau, M.D.,2

Kristy Bauer. M.D.,2 Gerald C. Smaldone, M.D., Ph.D.,1 and Rany Condos, M.D.2

Abstract

Background: Inhaled interferon-c aerosol (aINF-c) may be effective treatment for idiopathic pulmonary fibrosis(IPF). We evaluated safety and delivery of aIFN-c (100 lg 3 times/week) in 10 IPF patients using the I-neb(Philips Respironics, Parsippany, NJ).Methods: IFN-c activity in the aerosol was confirmed by viral inhibition. Ten patients with an average age of 68diagnosed with IPF (American Thoracic Society/European Respiratory Society consensus guidelines) were en-rolled. In vivo deposition was measured via a gamma camera. The nebulizer recorded patient adherence totherapy. Pulmonary function tests [PFTs, forced vital capacity (FVC), total lung capacity (TLC), diffusing ca-pacity for carbon monoxide (DLCO)] and the 6-min walk test were measured at baseline, and every 12–14 weeksfor 80 weeks. Bronchoalveolar lavage (BAL) of the middle lobe was performed at baseline and 28 weeks. BALand plasma samples were analyzed for chemokines and cytokines, including INF-c.Results: All 10 patients tolerated 80 weeks of inhaled IFN-c well, with no systemic side effects. True adherencewith aerosol treatment averaged 96.7 – 4.81% ( – SEM). In vivo lung deposition averaged 65.4 – 4.8lg and oro-pharyngeal deposition 12.6 – 3.0 lg. BAL IFN-c increased 60-fold and profibrotic cytokines (FGP-2, Flt-3 ligand,IL-5) were significantly decreased; IFN-c plasma levels were unchanged. PFTs showed minimal change in FVC.Post hoc analysis indicated that the slope of decline in TLC and DLCO reversed after beginning therapy. The 6-min walk was unchanged.Conclusions: IFN-c is safe in IPF and can be effectively delivered to lung parenchyma. PFTs remained stablethroughout the trial. Reversal of pretherapy PFT decline may define an end-point for future clinical trials.

Key words: clinical trial, nebulizer, scintigraphy, inhaled therapy

Introduction

Idiopathic pulmonary fibrosis (IPF) is a progressive fi-brotic lung disease with no effective treatment that leads to

respiratory failure and death often within 3 years of diagno-sis.(1–7) Our understanding of the pathophysiology of IPF haschanged during the past decade.(8) Lack of clinical response toimmunosuppressive therapies has led to the concept that re-petitive epithelial injury, fibroblast activation, microvascularinjury, and dysregulation of normal wound repair leads tofibrosis.(9) Additionally, lung cells including epithelial cellsand macrophages produce cytokines/chemokines and growth

factors that influence fibroblast proliferation, apoptosis, andmatrix deposition.(10) Although not prominent, the inflam-matory response in IPF is thought to be more consistent with ahelper T-cell (Th2-type) response coupled with a shift to al-ternatively activated (M2) macrophages that impair re-modeling of extracellular matrix.(11) Murine models suggestthat injured lung tissue is more likely to heal with pronouncedfibrosis when Th2 responses predominate in contrast to apredominance of Th1 responses.(12)

Interferon-gamma (IFN-c) is the signature Th1 cytokine thatis endogenously produced by T cells and natural killer cells andexhibits antifibrotic, antiproliferative, and immunomodulatory

1Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, State University of New York, Stony Brook,New York.

2Division of Pulmonary and Critical Care Medicine, Department of Medicine, New York University School of Medicine, New York,New York.

JOURNAL OF AEROSOL MEDICINE AND PULMONARY DRUG DELIVERYVolume 25, Number 2, 2012ª Mary Ann Liebert, Inc.Pp. 79–87DOI: 10.1089/jamp.2011.0919

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properties. Its potential therapeutic role in fibrosis, althoughsupported by in vitro and animal models, was not proven to beeffective in two randomized, placebo-controlled clinical trialswhere it failed to show any benefit.(6,13) Although subcutaneousIFN-c did lead to some antiangiogenic and immunomodulatorychanges in the immune environment of the lung, levels of in-terferon gamma recovered from the lung were low.(14) Despitethe high profile failure of IFN-c as therapy in IPF, its use remainsattractive in view of data supporting the role of a Th2, alter-natively activated macrophage lung milieu that may drive fi-brosis. As such, we studied the use of aerosol IFN-c in IPF. Withan aerosol, the concentration of drug delivered to the lungmay be considerably higher than that achievable by systemicadministration. In support of this concept, data from normalvolunteers has shown that subcutaneous delivery of IFN-c doesnot increase levels of IFN-c in epithelial lung lining fluid oractivate alveolar macrophages, whereas aerosol deliveryachieves both.(15) Furthermore, in patients with active pulmo-nary tuberculosis, inhaled IFN-c has been shown to induce in-tracellular signaling of INF-c-specific transcription factors andto improve clinical response to antituberculosis therapy.(16–18)

Currently there are no data on effective aerosol depositionin IPF patients. In IPF, delivery of a therapeutic aerosol to theperipheral lung may be suboptimal because of airspace en-largement and increased minute ventilation.(19,20) Combinedwith the inherent inefficiency of conventional nebulizers,delivery and deposition may be inadequate.(16) Controllingventilatory parameters and using an efficient breath actuatednebulizer may overcome impediments to lung delivery.(21)

The present study was undertaken with several goals:first, to establish that IFN-c can be aerosolized without af-fecting activity of the protein; second, to assess the efficiencyof a novel breath actuated nebulizer using feedback controlto regulate breathing; and finally, to determine the safety oflong-term IFN-c aerosol therapy in patients with IPF as de-fined according to American Thoracic Society/EuropeanRespiratory Society consensus guidelines.

Materials and Methods

In vitro studies

The nebulizer. The I-neb Adaptive Aerosol Delivery(AAD) System (Philips Respironics, Parsippany, NJ) a vi-brating membrane aerosol generator is breath actuated,minimizing aerosol losses during expiration.(21,22) In addition,the I-neb AAD system, utilizes ‘‘adaptive aerosol delivery,’’by measuring the patient’s breathing pattern and via feed-back, training the patient to perform slow, deep inspirations,thereby facilitating parenchymal aerosol deposition. The I-neb records the treatment date, treatment time, and if thetreatment was completed.(21,23) Daily cleaning and monthlyreplacement maintained membrane performance.

Formulations

Drug activity studies were performed with single vials ofIFN-c (Actimmune, InterMune, Brisbane, CA; 2 million U or100 lg/0.5 mL). Radioactivity labeling studies and nebulizerfunction studies were performed with 0.5 mL of albuterolsulfate (2.5 mg/3 mL, Nephron Pharmaceuticals Corp., Or-lando, FL) or 0.6 mL 0.9 NaCl (normal saline, NS). Radi-olabels consisted of 99m-technetium pertechnetate (99mTc)

and 99mTc sulfur colloid (Pharmalucence, Inc., Bedford, MA;99mTc SC).

Effect of vibrating mesh nebulization on IFN-c activity

Vials of IFN-c were nebulized with a continuous mode I-neb vibrating mesh nebulizer (Philips Respironics). Theaerosol was propelled by the energy of the vibrating mem-brane and simply allowed to pass into a glass jar cooled tozero degrees F. In the jar particles circulated and settled outdepositing on the walls. The collected aerosols were QS to5.0 mL with PBS buffer and serially diluted to a final dilutionof 1:160,000 of the initial 5.0 mL sample. IFN-c activity wasmeasured by viral inhibition bioassay (PBL Biomedical La-boratories, Piscataway, NJ).

Aerosol distribution using saline

The I-neb AAD system was connected to a breathingsimulator (Harvard pump, Harvard Respiratory Apparatus,model# 618;Millis, MA). To simulate a patient breathingslowly and deeply, the pump was set to a tidal volume of1.5 L, respiratory rate 5 per min, and duty cycle 0.7. A filter(Pari, Starnberg, Germany) was placed between the nebu-lizer and the pump to prevent aerosol from returning to thenebulizer during expiration. The breathing mode adoptedwas targeted inhalation mode (TIM) and nebulizer treat-ments were run to ‘‘dryness.’’ To measure particle distribu-tion, a cascade impactor (Marple 8-stage impactor, ThermoFischer Scientific, Waltham, MA, 2 liter per minute flow) wasplaced in-line between the pump and the nebulizer.(22,24)

There was a fixed starting dose of 100 lg and treatment timestook approximately 4–6 min to complete. Data were reportedas mass median aerodynamic diameter (MMAD).

Correction for sulfur colloid radiolabel using albuterol

To prevent free 99mTc from diffusing into the blood duringimaging, it must be bound to a large molecule. The onlysuitable agent available in the United States is sulfur colloid(SC). Using SC as a radiolabel may lead to an underestima-tion of drug delivery because the colloid can stick to plasticin the nebulizer.(25) The degree of SC adhesion to plastic canbe measured by simultaneous assay of nebulized drug andradioactivity in captured aerosol. To define this process for I-neb, we used 0.5 mL of albuterol sulfate mixed with eitherfree 99mTc or 0.1 mL of 99mTc SC. Using the I-neb AAD sys-tem and the Harvard respirator serial filters capturingaerosol particles over different time periods of nebulizationprovided a range of values. After radioactivity captured onthe filter was measured, the same filter was analyzed fordrug activity. Drug was extracted using sodium hydroxidesolution, vortexed, and centrifuged for 10 min at 2000 rpm.Absorption was measured using a Microplate Spectro-photometer (Spectra Max plus, Sunnyvale, CA) at a wave-length of 243 nm, which quantified the amount of drug.(26)

Drug concentration and amount of radioactivity present onthe filter were calculated as a percent of that initially placedin the nebulizer and the relationship between radioactivityand albuterol compared using linear regression analysis.Linear equations for formulations with free 99mTc and 99mTc-SC were calculated and utilized to correct human scintigra-phy studies to define actual drug deposition.

80 DIAZ ET AL.

In vivo studies

Inclusion and exclusion criteria are listed in Table 1. Pa-tients chosen for the study were between the ages of 40 and75 years old and had a diagnosis of IPF made within 1 yearof enrollment defined according to American Thoracic So-ciety/European Respiratory Society consensus guidelines.(27)

To facilitate repeated bronchoalveolar lavage (BAL) we ex-cluded patients with severe restriction (FVC < 55% pre-dicted).

IFN-c deposition

To assess nebulizer function and measure lung dose ofaerosol IFN-c, two deposition studies (baseline and after 28weeks of therapy) were performed using techniques pre-viously reported in detail. In brief, with the patient sittingin front of a computer controlled gamma camera (MaxiCamera 400, General Electric, Horsholm, Denmark, PowerComputing, Model 604/150/D, Austin, TX, Nuclear MAC,Version 4.2.2, Scientific Imaging, Inc., CA) a 15 min 99mTcroom background was obtained. The lung outline, regionalvolume, and regional ventilation were all measured byxenon (133Xe) equilibrium and washout.(16) After xenonscanning, the patient swallowed a measured amount(*500 lCi) of 99mTc-macroaggregated albumin absorbed ona small cracker with water. An image of the stomach wasacquired, which allowed the calculation of stomach atten-uation. Then, after a repeat background image, the patientinhaled a treatment of radiolabeled IFN-c, using the I-nebAAD system. Immediately following the treatment the pa-tient drank a glass of water to wash oropharyngeal contentsinto the stomach. A repeat scan encompassing both thelungs and stomach defined lung and upper airway (stom-ach activity) deposition.

In order to measure the attenuation correction for chestgeometry, a calibrated injection of a known quantity of99mTc-macroaggregated albumin (5–10 mCi) was giventhrough a peripheral intravenous line followed by an imageof lung perfusion. Counts from the previous deposition im-age were subtracted and net counts from the perfusion imagewere divided by the activity injected to yield a correctionfactor for the thorax (units = counts per min per lCi). Thechest attenuation measurement was performed once at thetime of the first deposition study.

Regional deposition calculations

The 133Xe equilibrium images defined lung volume. Usingthe computer, an outline was drawn around both lungs re-presenting the whole lung zone. Then a second region cen-tered over the central airways was outlined, which representedthe central zone; this zone accounted for about 33% of theentire lung. We subtracted the central zone from the entirelung zone and the remaining area was labeled as the periph-eral zone. These images were superimposed on the depositionimages and allowed a calculation of regional deposition perunit volume. The ratio between the central and peripheral lungzones per unit volume (sC/P) allowed comparison betweenpatients and a quantification of regional airway deposition. Aratio of 1.0 indicates deposition in small airways and alveoli.As the ratio increases more aerosol is deposited in the centralairways.(16)

In addition to the standard central and peripheral regionsof interest, we created a region approximating the middlelobe. The RML shape and size was drawn using an ap-proximation of a two-dimensional anatomical outline of thecorresponding lobe. Activity in this region was used to cor-relate deposition with levels of IFN-c measured in bronch-oalveolar lavage (BAL) fluid.

BAL

BAL was performed at baseline and after 28 weeks oftreatment with inhaled IFN-c. The second BAL at 1 h afterthe treatment near the 28th week.(15) Briefly, after localanesthesia with lidocaine, the bronchoscope was insertedvia the nasal passage to the lower respiratory tract,wedged into the middle lobe bronchus, and a lavage wasperformed using five 60-mL aliquots of normal saline. TheBAL fluid was processed on the same day as the bron-choscopy in the following manner. BAL fluid was filteredthrough two layers of sterile cotton gauze to removemucus. Alveolar cell populations were counted in a he-mocytometer. Cell viability was determined by trypanblue exclusion and all recovered cells showed > 90% via-bility. Cell pellets and supernatants were separated bycentrifugation at 1000 rpm for 10 min. BAL fluid superna-tant was divided into aliquots and stored at minus 70�Cuntil assayed. The cell pellet was washed twice in serumfree RPMI (Cellgro, Manassas, VA) and resuspended in

Table 1. Inclusion and Exclusion Criteria

Inclusion Exclusion

Between 40 and 75 yearsof age

Presence of interstitial lung disease due to conditions other than IPFPulmonary function indicating severe restriction [e.g., forced vital capacity (FVC) < 55%

predicted]Diagnosis of IPF made within

1 year of enrollment asdefined according toAmerican Thoracic Society/European RespiratorySociety consensusguidelines(27)

A diffusing capacity of carbon monoxide (DLCO) corrected for hematocrit < 30%Obstructive lung disease (e.g., forced expiratory volume in 1 sec/FVC (FEV1/FVC)

< 65%]Echocardiographic evidence of severe pulmonary hypertension (systolic pulmonary

pressure > 40 mmHg)A 6-minute walk test < 200 mHome oxygen therapyKnown hypersensitivity to the study medication or its excipientsSevere cardiac disease, peripheral vascular disease, or seizure disorderPregnancy and inability to follow instructions regarding use of the nebulizer and drug

INHALED INTERFERON-c IN IPF 81

RPMI at 106 cells per mL and cultured in suspensionfor 24 h at 37�C. Supernatants from the cell culture werecollected and frozen at - 70�C until assayed. In a singlebatch, IFN-c and a panel of potentially important cyto-kines and chemokines were measured in aliquots from theBAL and the 24-h cell culture supernatant by LuminexBeadlyte ELISA assay. Protein concentrations (albumin)for BAL fluid measurements were corrected with the BCAProtein Assay (Pierce, Rockford, IL).

Treatment protocol

Baseline assessment included physical examination, vitalsigns, ECG, oxygen saturation by pulse oximetry, and a 6-min walk test. Full pulmonary function testing (PFTs) andblood sampling were performed at baseline and every 12–14weeks until the conclusion of the study. Retrospective PFTdata were obtained by reviewing patient records for 5months prior to study entry. The same laboratories measuredthe study and retrospective PFTS. Complete blood count,liver, and renal function were monitored. Plasma and BALIFN-c levels were measured at baseline and after the secondbronchoscopy. After the initial assessment and signing ofconsents, the baseline bronchoscopy was performed fol-lowed by nebulizer education and the first deposition study.Subjects were treated with 100 lg of IFN-c via the I-neb AADsystem three times a week for 80 weeks. The dose was cho-sen based on the efficiency of the nebulizer and the measuredlung dose from previous studies in tuberculosis patients withless efficient devices.(16,18)

The treatment protocol was performed at New YorkUniversity Medical Center. The in vitro aerosol studies andthe in vivo deposition studies were performed at Stony BrookUniversity Medical Center. All patients gave written, in-formed consent to participate in the study, which wasapproved by the appropriate independent institutional re-view boards of both institutions. The study is registered atClinicalTrials.gov (NCT00563212).

Data analysis

Deposition, pulmonary function testing, and cytokinelevels were analyzed using descriptive statistics includingmean and standard error. Linear regression analysis wasused to evaluate the aerosol formulations to compareamount of radioactivity and drug present. Wilcoxon mat-ched-pairs rank test was used to compare plasma and BALcytokine levels pre- and posttreatment. Cytokine data wasconsidered exploratory. In exploratory studies in which dataare collected with an objective but not a prespecified keyhypothesis, multiple test adjustments are not required. Themultiple tests used in this study were for descriptive pur-poses and not for decision making. Further studies withdefined hypotheses will be needed to confirm these possibleassociations.(28) Patient adherence was quantified by down-loading I-neb AAD system usage data at the end of the trial.True adherence was defined as the product of adherence andcompliance. Adherence was defined as the number of treat-ments started by each patient expressed as a percentage ofthe number of treatments expected in a given time periodbased on the prescribed regimen for that patient; complianceas the number of treatments started by each patient that werecompleted (i.e., ‘‘Full’’ dose recorded by I-neb AAD system)

expressed as a percentage of the total number of treatmentsstarted by the patient.(23)

Results

Effects of vibrating mesh nebulization on IFN-c

Figure 1 summarizes the viral inhibition assay results fromcaptured aerosols. IFN-c activity in control aerosols of nor-mal saline (NS) and samples of assay buffer (PBS) was un-detectable. Viral inhibition at serial dilutions was consistentwith the nominal concentration of interferon in the originalvial (e.g., 2 million units/0.5 mL diluted 160,000 times, ex-pected activity 5 IU) confirming that nebulization with the I-neb AAD system did not diminish IFN-c bioavailability.

In vitro correction for sulfur colloid

Figure 2 depicts radioactivity captured on serial filtersplotted against albuterol activity both as a percent of nebu-lizer charge. For aerosols labeled with free 99mTc the datawell approximated the line of identity (y = 0.946x + 0.272,R = 0.983, p < 0.001) indicating that free 99mTc accurately re-presented albuterol activity. For 99mTc-SC there was a shift ofthe line to the right (y = 0.637x + 0.276, R = 0.924, p < 0.001),indicating that more drug was nebulized per unit of radio-activity. This observation was consistent with retention of99mTc-SC in the nebulizer.

Patient deposition and particle diameter

Individual results of deposition studies and particle sizeexpressed as MMAD are listed in Table 2. All deposition datawere corrected for SC adherence to plastic using the equationfrom Figure 2.

drug deposited (lg)¼ [scintigraphic activity (lCi)=0:637]� 2:76

Percentages refer to the initial amount of drug placed in thenebulizer (100 lg = nebulizer charge).

FIG. 1. Viral inhibition activity of interferon gamma (INF-c) aerosol [in international units (IU/mL)] for serial dilutionsof aerosol condensate (filled bars), Predicted levels from di-lution of nominal dose in vial (2 million IU) (open bars);captured normal saline aerosol and sample buffer controlshad undetectable activity.

82 DIAZ ET AL.

In vivo lung deposition averaged 65.4 – 4.8% ( – SEM) of thenebulizer charge with 16.5 – 1.4% depositing in the middlelobe. Variability in deposition was attributed primarily todifferences in oropharyngeal deposition. Oropharyngeal de-position averaged 12.6 – 3.0%. Regional lung deposition,quantified by the ratio of central to peripheral distribution ofdeposited particles (sC/P ratio), averaged 1.20 – 0.6 indicat-ing relatively peripheral distribution (1.0 represents unde-

tectable deposition in central airways). Inspection of Table 2indicates some negative values of deposition as well as valuestotaling (lung depo plus stomach depo) above 100%. Thesereflect experimental error.

Particle diameters ranged from 1.2 to 2.8 lm with a meanMMAD of 1.7 – 0.11 lm.

BAL fluid results

An average of 40 million cells/mL were recovered fromBAL both before and after treatment with a macrophagepredominance (pre: 76.7% macrophages vs. post: 81.7%macrophages). Pretreatment versus posttreatment changes incell count and differential were not significant. Cytokine andchemokine analyses were performed on BAL fluid, 24-h cellculture supernatants, and plasma (Table 3). Albumin correc-tion of BAL fluid allowed calculation of cytokine or chemo-kine per milligram of protein. The mean baseline BAL fluidlevel of IFN-c corrected for protein increased from 5.23 – 3.16to 320 – 79.5 pg/mg, p = 0.002. Mean 24-h cell culture super-natant levels of IFN-c also increased from 8.25 – 5.40 to36.9 – 11.0 pg/mL, p = 0.027. Mean 24-h cell culture superna-tant levels of FGP-2, Flt-3 ligand, and IL-5 were significantlydecreased.

Correlation of deposition studies with levelsof interferon in BAL fluid

Figure 3 depicts changes in the level of IFN-c (pg/mgprotein/mL) removed by BAL from the middle lobe, plottedagainst lung deposition (highest of two studies) in the mid-dle lobe region of the gamma camera image (R = 0.651,p = 0.035, Spearman correlation).

Clinical testing; Safety and tolerability

Ten patients were sequentially enrolled in the clinical trialstarting in June 2008. Three patients had a diagnosis con-firmed by open lung biopsy reviewed by a pulmonary pa-thologist while the rest met clinical criteria and had consistentchest high-resolution computed tomography scans. Patientswere enrolled within 4 weeks of screening. The last patient

FIG. 2. Radioactivity captured on filters for albuterol aerosols versus drug activity (measured by assay) as percent ofnebulizer charge from the same filter: aerosols labeled with free 99mTc lie close to the line of identity (y = 0.946x + 0.272,R = 0.983, p < 0.001) left panel; aerosols labeled with 99mTc-sulphur colloid (SC) are shifted to the right, indicating some 99mTc-SC is systematically retained in the nebulizer (y = 0.637x + 2.76, R = 0.924, p < 0.001) right panel.

Table 2. Deposition Summary: Each Patient

Had Two Studies; Stomach Depo Represents

Upper Airway Activity Swallowed Before Scanning

Patient #/study

Lungdepo%a

MLdepo%a

Stomdepo%a

MeansC/P depo MMAD

1.1 67.0 22.1 35.3 1.311.2 91.8 20.0 27.6 1.00 1.92.1 75.2 19.5 10.4 1.10 2.82.2 49.7 15.5 8.1 2.213.1 91.8 26.3 - 1.3 0.88 1.83.2 75.3 1.4 - 2.3 1.074.1 95.1 26.0 - 0.4 1.16 1.84.2 49.5 15.2 8.9 1.10 1.45.1 65.1 15.0 28.0 1.17 2.05.2 77.4 16.7 4.0 1.21 1.76.1 74.2 21.5 - 2.8 1.22 2.16.2 52.2 12.4 5.7 1.14 2.07.1 43.6 9.5 29.9 1.13 1.37.2 21.6 5.7 34.9 1.26 1.38.1 65.9 17.3 4.3 1.21 1.38.2 92.9 21.4 0.4 1.05 1.39.1 86.8 21.9 4.9 1.42 1.29.2 32.6 15.9 29.9 1.01 2.210.1 44.8 13.0 7.8 1.35 1.310.2 55.4 12.9 18.0 1.07 1.4

Mean 65.4 16.5 12.6 1.20 1.70SEM 4.8 1.4 3.0 0.06 0.11

aData are presented as percent of initial nebulizer charge.ML, middle lobe; sC/P, central to peripheral ratio of deposition

image divided by 133Xe image; MMAD, mass median aerodynamicdiameter.

INHALED INTERFERON-c IN IPF 83

finished treatment in December 2009. All 10 patients werefollowed for a minimum of 80 weeks with a range of 80 to 130weeks. The majority of patients were male and White, with amean age of 68 years. Clinical and pulmonary function pa-rameters were similar across the group at the time of entryand details of the demographics are shown in Table 4. Allpatients completed > 90% of scheduled visits.

True adherence with aerosol treatment averaged 96.7 –4.81%. Treatment time ranged from 3–20 min.

No significant systemic side effects were reported. Themost common side effect was cough, which affected fivepatients during the initial few breaths of each treatment. Twopatients coughed throughout a typical treatment session.There were no significant changes in 6-minute walk test over

the 80 weeks of treatment (pre- vs. posttreatment, 386 – 41.35vs. 382 – 49.38 meters, p = 1.000).

Pulmonary function tests [forced vital capacity (FVC),total lung capacity (TLC), and diffusion capacity (correctedfor Hb, DLCO)] are shown in Figure 4. Values are plotted aspercent predicted change from baseline. For most of thepatients PFT data obtained from 20 weeks prior to baselineand enrollment are included. TLC and DLCO were declin-ing at the initiation of aerosol therapy. FVC appeared sta-ble, which is consistent with a decreasing residual volume.With therapy the decline in TLC and DLCO reversedand all parameters including FVC improved. After ap-proximately 1 year of therapy values peaked and began todecline.

Discussion

This study illustrates the potential use of inhaled IFN-c asa novel therapy for IPF, bringing pharmacologic doses of thedrug directly to the site of disease. We attained levels in BALfluid that are not possible with parenteral therapy and thesystemic effects of subcutaneous IFN-c were not seen in ourstudy. The most common side effect was cough, possiblysecondary to upper airway irritation. It is possible that somepatients with airways disease may not tolerate inhaled IFN-cbut the present study in IPF, combined with our previouswork with inhaled IFN-c in tuberculosis, indicates that thedrug is safe in most humans with serious lung diseases.(18)

Biologic proteins made via recombinant techniques mayhave to overcome higher safety standards than chemicallysynthesized aerosolized agents.(29,30) Because of this concern,our study has increased value. All of our patients weretreated for 80 weeks and some for up to 2 years, far beyondthe usual phase 1 safety study for a new drug. Future studiesshould include a dose-escalation component to fully assessthe range of tolerability. As with all reported studies ofproposed treatments for IPF there are no clear indicators of

Table 3. Cytokine Data

BALF a (pg/mg) 24 h Supernatant (pg/mL)

Biomarker Baseline Post IFN-c p-value Baseline Post IFN-c p-value

PDGF-AA 187 – 36.5 204 – 25.0 0.441 21.6 – 4.55 13.1 – 3.51 0.065RANTES 263.1 – 55.4 416 – 106.6 0.065 182 – 49.7 230 – 75.9 0.846Eotaxin 15.1 – 6.20b 8.09 – 2.06b 0.375 8.01 – 1.87b 5.54 – 2.17b 0.375FGP-2 18.2 – 2.84 21.0 – 3.08 0.275 12.00 – 1.80b 9.60 – 1.66b 0.031Flt-3 ligand 6.49 – 1.29 7.67 – 1.49 0.375 5.48 – 1.01 3.61 – 0.83b 0.039Fractalkine 28.3 – 6.99 26.9 – 4.53 0.922 21.7 – 5.32 19.0 – 10.0 0.275IFN-c 5.23 – 3.16 320 – 79.5 0.002 8.25 – 5.40 36.9 – 11.0 0.027IL-10 2.34 – 0.57b 2.67 – 0.58b 0.286 34.6 – 15.4b 55.1 – 51.4b 0.875IL-5 1.08 – 0.18 1.05 – 0.16 0.625 0.81 – 0.04 0.74 – 0.03 0.049IL-6 48.4 – 26.0 73.4 – 53.0 0.922 445 – 195 690 – 633 0.492IL-8 637.7 – 181 642 – 176 0.557 2729 – 185 2196 – 284 0.092IP-10 4365 – 1149 6581 – 1460 0.084 1383 – 608 2508 – 967 0.275MIP-1b 53.4 – 19.7 64.9 – 21.7 0.695 708 – 226 1390 – 1192c 0.375TGF-b1 66.7 – 15.0 77.1 – 22.1 1.000 15.3 – 3.78b 15.0 – 3.78b 1.000TNF-a 2.07 – 0.48 3.22 – 1.00 0.139 598 – 271 384 – 325 0.432IFN-c (plasma levels) 4.18 – 0.68 4.41 – 0.83 0.557

aBALF, bronchoalveolar lavage fluid, corrected for albumin.bMean contains value(s) below assay sensitivity; these values were replaced with value of lowest standard in assay.cMean contains value(s) above assay sensitivity; these values were replaced with value of highest standard in assay.Data expressed as MEAN – SEM; p-value calculated using Wilcoxon signed-rank test.

FIG. 3. Change in BAL levels of interferon gamma (INF-cpg/mg protein) following aerosol therapy versus middlelobe deposition (highest of two studies) (y = 29.4x - 251,R = 0.651, p = 0.035, Spearman correlation).

84 DIAZ ET AL.

efficacy. Therefore, the optimal level of drug in the lung isunknown. However, we know that the range of lung dosethat we have reported in tuberculosis is sufficient to treatthat disease, evidence for macrophage activation at the levelsreached by our aerosols.

In previous studies we measured deep lung deposition intuberculosis patients averaging only 7% of the nebulizercharge with more aerosol deposited in upper airways (10%of the nebulizer charge) than in the lungs.(16) In the presentstudy, we achieved an order of magnitude increase in lungdeposition per treatment utilizing a novel delivery system(65% vs. the previous 7%). Upper airway deposition remainsa problem (13%) likely secondary to the fraction of aerosolparticles above 3 lm.(24,31) In the current study gammacamera data confirmed peripheral lung deposition with ansC/P ratio of 1.20. BAL data demonstrated significantly in-creased levels of IFN-c in the epithelial lining fluid followingaerosol therapy in all IPF patients. These levels appeared tobe dose dependent illustrating the importance of choice ofthe delivery device and control of deposition. The absence ofa concomitant increase in plasma levels likely explains thefavorable side effect profile of aerosolized interferon gammawhen compared to subcutaneous administration.

The effects of pharmacologically administered IFN-c on thecytokine/chemokine milieu of the alveolar space are un-known. In our exploratory studies in tuberculosis and IPF wehave measured a range of cytokines thought to be important.In tuberculosis, we have shown that aerosol IFN-c treatmentleads to significant decreases in pro-inflammatory cytokinesin BAL fluid (IL-1b, IL-6, IL-8, and IL-10).(18) In IPF, 24 h cellsupernatant levels of IFN-c were increased while FGP-2, Flt-3ligand, and IL-5 were significantly decreased. In models ofpulmonary fibrosis, cytokine deficient-mice have worseningfibrotic disease with a shift in cytokine balance to a TH2 CD4+

Table 4. Patient Demographics and Baseline Pulmonary Functions

Pt Age Sex Smoking (pk-yr) Diagnosis Previous Rx FVC% TLC% DLCO%

1 58 M n/a OBLx None 82 70 582 75 M 25 CT NAC 85 78 333 67 M n/a OBLx None 58 59 484 71 M 20 OBLx None 75 79 525 67 F n/a CT None 68 60 476 75 M 35 CT None 69 69 327 63 F 5 CT NAC 72 61 388 64 M n/a OBLx None 93 74 639 75 M 15 CT NAC 104 92 4310 69 M n/a CT NAC 86 75 52

MEAN 68.4 79.2 71.7 46.6SEM 1.82 4.27 3.24 3.24

OBLx, open lung biopsy; CT, CAT scan of the chest; NAC, N-acetylcysteine; FVC, forced vital capacity; TLC, total lung capacity; DLCO,diffusing capacity for carbon monoxide (% predicted).

FIG. 4. Forced vital capacity (FVC), total lung capacity(TLC), and single breath diffusion capacity (DLCO), plottedas change in percent predicated from baseline. Baseline andposttreatment over a period of 80 weeks (10 patients) areshown as well as retrospective data over 20 weeks prior toinitiating treatment (nine patients for TLC and FVC, eight forDLCO). Dotted line represents baseline value and beginningof therapy.

‰

INHALED INTERFERON-c IN IPF 85

T cell response involving IL-4, IL-5, and IL-13.(32) TH2 driveninflammation is involved in the pathogenesis of both hepaticfibrosis(33) and pulmonary fibrosis.(34) IL-5 has been shown toaugment the progression of liver fibrosis by regulating IL-13activity and it is increased in areas of active fibrosis in a mu-rine model of pulmonary fibrosis.(35) Inhaled IFN-c may at-tenuate the fibrotic response in IPF by promoting a TH1cytokine profile. Other pro-fibrotic pathways may be impor-tant. FGF-2 has been associated with increased fibronectinsynthesis in fibroblasts of patients with IPF(36) and fibroblastproliferation.(37) Therefore, decreasing FGF-2 activity may beuseful in IPF. Flt-3 ligand is important as a hematopoieticcytokine and for the development and function of the immunesystem.(38) Its activity in IPF may be a measure of cellularproliferative activity secondary to the underlying diseaseprocess or associated infection, for example, by a virus.

Our study was designed to assess safety of long-termadministration of inhaled IFN-c in IPF patients; therefore, notherapeutic conclusions can be drawn. However, our obser-vations allow some speculation regarding pulmonary func-tion testing that may be useful in design of future studies.Controlled clinical trials of potential therapeutic agents forIPF have focused on pulmonary function. In all reportedstudies, both treatment and placebo arms report a 5–10%decline over 50 weeks (primarily changes in FVC).(5–7,13,39) Inour study, serial measurements of pulmonary function wereobtained with increases in TLC, FVC, and DLCO after initi-ation of aerosol therapy. This change became more apparentwhen prebaseline pulmonary function data obtained retro-spectively was added (the same laboratories performed thepre- and posttreatment testing). Before initiation of aerosoltherapy, the rate of decline in pulmonary function wasconsistent with that expected in IPF.(39) With initiation ofinhaled IFN-c, the decline seen over the initial 20 weeks ofobservation did not continue and, in fact, appeared to re-verse. If this observation is indeed a signal of a treatmenteffect, it may serve as a possible endpoint for future clinicaltrials. It is not known what parameters best signal a drugeffect short of a reduction in mortality. In addition to FVC,our study suggests that data analysis may be enhanced ifpatients are followed for a time before randomization. Thosepatients with declining function may then be randomizedand benefits of therapy more readily detected.

Acknowledgments

The authors thank Lorraine Morra for technical assistancein performing the aerosol studies and preparing the manu-script and Maryann Huie who performed the cytokine anal-ysis. The study was supported in part by Philips Respironics.

Author Disclosure Statement

Dr. Smaldone serves as a consultant to PhilipsRespironics. Stony Brook University and New York Uni-versity jointly hold patents in the treatment of IPF withinhaled IFN-c.

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Received on September 9, 2011in final form, January 20, 2012

Guest Editor:Philip Bromberg

Reviewed by:Steve Newman

Luca Richeldi

Address correspondence to:Gerald C. Smaldone, M.D., Ph.D.

Pulmonary, Critical Care and Sleep MedicineT17-040 Health Sciences Center

Stony Brook University Medical CenterStony Brook, NY 11794-8172

E-mail: gerald.smaldone@stonybrook.edu

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