EP1940451A1 - Verfahren zur behandlung von lungenerkrankung mit interferonen - Google Patents

Verfahren zur behandlung von lungenerkrankung mit interferonen

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Publication number
EP1940451A1
EP1940451A1 EP05808951A EP05808951A EP1940451A1 EP 1940451 A1 EP1940451 A1 EP 1940451A1 EP 05808951 A EP05808951 A EP 05808951A EP 05808951 A EP05808951 A EP 05808951A EP 1940451 A1 EP1940451 A1 EP 1940451A1
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EP
European Patent Office
Prior art keywords
interferon
pulmonary disease
patients
ifn
treatment
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
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EP05808951A
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English (en)
French (fr)
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EP1940451A4 (de
Inventor
Rany Condos
Gerald Smaldone
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New York University NYU
Research Foundation of the State University of New York
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New York University NYU
Research Foundation of the State University of New York
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Priority to EP12001438.6A priority Critical patent/EP2494982B1/de
Publication of EP1940451A1 publication Critical patent/EP1940451A1/de
Publication of EP1940451A4 publication Critical patent/EP1940451A4/de
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system

Definitions

  • This invention relates to methods of treating pulmonary diseases using aerosol interferons, formulations of one or more interferons for aerosol delivery and methods for determining aerosol deposition.
  • Th2 cytokine IL-4 plays a prominent role in airway inflammation by promoting isotype switching of B cells to IgE synthesis and inducing naive T cell differentiation to Th2 lymphocytes.
  • IL-4 knockout mice challenged with aerosolized antigen failed to produce specific IgE, airway hyperresponsiveness, airway eosinophilia , or Th2 cytokines in the airways (Brusselle et al, Am JRespir Cell MoI Biol , 1995 Mar; 12(3):254-259.)
  • Wild-type mice treated with anti-IL-4 during the initial exposure to antigen but not during challenge inhibited IL-5 production and airways eosinophilia, whereas anti-IL-4 given during antigen challenge did not inhibit airways eosinophilia, indicating that IL-4 is essential for the induction of a local Th2 response (Coyle et al, Am JRespir Cell MoI Biol 1995 JuI; 13(l):54-59
  • IL-10 IL-10-derived neuropeptide
  • eosinophil function has been demonstrated as well.
  • Low concentrations of IL-10 were almost as active as corticosteroids in decreasing CD4 expression on eosinophils and accelerating cell death.
  • GM-CSF is a cytokine directly involved in the homing and activation of eosinophils and neutrophils in inflamed tissues.
  • Kips confirmed this in vivo by administering IL- 12 at the time of immunization and preventing production of specific IgE, airway eosinophilia, and airway hyperreactivity.
  • IL- 12 administration during the aerosol challenge of already sensitized mice prevented airway eosinophilia and airway hyperresponsiveness, it did not decrease specific IgE production, suggesting that IL- 12 stimulates the differentiation of naive Th cells into ThI cells, and can suppress the development of Th2 cells.
  • Increasing IFN- ⁇ levels may drive the immune response to a ThI phenotype and may be beneficial in asthma.
  • Clinical correlation in humans has focused on cytokine levels in serum or stimulated PBMC. Most measurements of cytokines using stimulated PBMC have been performed in children. These studies have demonstrated an increased propensity towards IL-4 and IL-5 production and decreased production of IFN- ⁇ is asthmatic children.
  • IFN- ⁇ is indicated for the treatment of chronic granulomatous disease in which prolonged treatment (average duration 2.5 ,, > , > , i ⁇ ness) (NEngl JMed 324 (8):509-16;
  • interstitial pneumonias have been grouped into seven categories based upon histology. They include usual interstitial pneumonia (UIP), non-specific interstitial pneumonia (NSIP), diffuse alveolar damage (DAD), organizing pneumonia (OP), desquamative interstitial pneumonia (DIP), respiratory bronchiolitis (RB), and lymphocytic interstitial pneumonia (LIP). See, e.g. Nicholson, Histopathology, 2002, 41, 381-391; White, J Pathol 2003, 201, 343-354.
  • the histological pattern is characterized by heterogeneity that includes patchy chronic inflammation (alveolitis), progressive injury (small aggregates of proliferating myofibroblasts and fibroblasts, termed fibroblastic foci) and fibrosis (dense collagen and honeycomb change). >ee, .e.g. ng ⁇ . , m o esp. an ri ica are e ., _)- . . re men pneumonia is not predictive of successful therapy for idiopathic interstitial fibrosis.
  • Interferons are a family of naturally-occurring proteins that are produced by cells of the immune system. Three classes of interferons have been identified, alpha, beta and gamma. Each class has different effects though their activities overlap. Together, the interferons direct the immune system's attack on viruses, bacteria, tumors and other foreign substances that may invade the body. Once interferons have detected and attacked a foreign substance, they alter it by slowing, blocking, or changing its growth or function.
  • interferon-inducible CXC chemokine I-TAC/CXCL11
  • BAL bronchoalveolar lavage
  • the present invention features a method of treating a pulmonary disease in a subject suffering from a pulmonary disease, comprising administering an aerosolized interferon in a erapeu ica y c ive amoun .
  • n many em o imen s, e pu nary disease is an
  • the aerosolized interferon- ⁇ will be titrated to ensure no undesirable effects are experienced by these patients.
  • the other agents may be delivered by a means in which they are considered to be the most effective. This may include intravenous, intramuscular, subcutaneous, or may be combined with IFN- ⁇ and delivered as an aerosol.
  • FIGURE 1 describes a typical tidal breathing pattern.
  • FIGURE 2 describes a reduction in inspiratory flow and a greatly prolonged inspiratory time characteristic of a method of slow and deep inspiration as compared to tidal breathing.
  • FIGURE 3 represents a deposition pattern in a human subject inhaling 4.5 ⁇ m aerosols using the slow and deep breathing pattern.
  • the images demonstrate minimal deposition of aerosol (less than 10%) in the upper airways illustrated by the small amount of activity in the stomach.
  • the deposition image represents radiolabeled aerosol deposited in the lung periphery of a human subject after 3 breaths using the slow and deep pattern with an inspiratory time of approximately 8 seconds.
  • FIGURE 4 is an illustrative scan in the same subject following 20 breaths of tidal breathing of 1.5 ⁇ m particles which is the present standard mode of inhalation. Analysis of the images indicates that the slow and deep method of breathing which incorporates the use of large particles, slow inspiration and a prolonged inspiratory time is 51 times more efficient per breath in depositing aerosol particles in the lung.
  • FIGURE 5 depicts a deposition scan of a patient suffering with IPF who has been treated three times per week for twelve weeks with 500 ⁇ g of IFN- ⁇ delivered via a nebulizer. Imaging was performed following a treatment. Regions of interest are shown as outlines. sU/L is the distribution of deposited radioactivity in the upper part of the lung to the lower part of the lung normalized for xenon. The horizontal bar in the figure delineates the border between the upper and lower lung quadrants. sC/P means the specific central to peripheral ratio described below. a/Xe means the aerosol to xenon ratio. J - eve s measure via e an ⁇ alter aerosol u ⁇ e py.
  • FIGURE 7 demonstrates the increased percent predicted total lung capacity after treatment in the five patients treated in a study of aerosol rIFN- ⁇ for five patients with IPF. All patients reported subjective improvements in their shortness of breath. By the end of three months of treatment, patients in the study had a statistically significant increase in total lung capacity. There was also an improvement of greater than 200 cc's (200 and 500 cc, respectively) in the Forced Vital Capacity in two of the five study patients.
  • FIGURE 9 A and 9B demonstrate the reduced portion of TGF- ⁇ of total protein in the five patients treated with aerosol rIFN- ⁇ for IPF.
  • TGF- ⁇ is one of the key mediators of fibrosis in the lung. Its activation leads to collagen production. Decreases in its levels should lead to less collagen deposition and less fibrosis in the lung.
  • FIGURE 10 demonstrates the amount of interferon- ⁇ measured in the lungs of tuberculosis patients and patients with idiopathic pulmonary fibrosis both before and after aerosol treatment with interferon- ⁇ .
  • FIGURE 11 represents the percentage change in peak flow hi asthma patients after treatment with aerosol IFN- ⁇ . All patients receiving aerosol interferon- ⁇ were studied with spirometry to assess reversible airways disease. At each aerosol treatment, patients had monitoring of peak flows before and after treatment.
  • FIGURE 12 provides a summary of the percent change in peak flow measurements referred to in Figure 2.
  • the average peak flow increased after aerosol interferon ⁇ , with significant increases in a few patients.
  • peak flow measurements decreased after interferon ⁇ , none developed cough or other complaints.
  • the phrase "unresponsive to treatment with one or more of corticosteroid, cyclophosphamide, and azathioprine” means a patient population that is unresponsive to conventional prior art treatments. '. air that can be moved in and out of the lungs.
  • Fevl means the forced expiratory volume of air in one second.
  • Fevl/FVC ratio means the ratio of forced expiratory volume in one second and forced vital capacity.
  • pulmonary disease refers to any pathology affecting at least in part the lungs or respiratory system.
  • the term is meant to encompass both obstructive and non-obstructive conditions such as, for instance, asthma, emphysema, chronic obstructive pulmonary disease, pneumonia, tuberculosis, and fibrosis in all its forms including but not limited to idiopathic pulmonary fibrosis.
  • obstructive pulmonary disease refers to any pulmonary disease that results in reduction of airflow in or out of the respiratory system.
  • the reduction in airflow relative to normal may be measured in total or over a finite time, for example, by FVC or FEVl .
  • IDF idiopathic pulmonary fibrosis
  • CFA cryptogenic fibrosing alveolitis
  • the histological pattern is characterized by heterogeneity that includes patchy chronic inflammation (alveolitis), progressive injury (small aggregates of proliferating myofibroblasts and fibroblasts, termed fibroblastic foci) and fibrosis (dense collagen and honeycomb change).
  • the term "asthma” refers to a common disease that involves inflammation (cellular injury) and narrowing of the airways leading to the lungs. Asthma occurs in children and adults. Childhood asthma may continue into adolescence and adulthood, but some adults who develop asthma did not have asthma when they were younger. Millions of people worldwide are affected by asthma, which has becomemore common in recent years. g pa rii ierein e time of expiration. Such a pattern features a duty cycle (time of inspiration/total time of breath) of greater than 0.5. During normal tidal breathing the duty cycle is always less than or near 0.5. That is, the time of inspiration is always less than the time for expiration.
  • STAT-I Upon translocating to the nucleus and binding to its cognate regulatory element in the promoters of many genes, STAT-I activates transcription. STAT-I can work with other preexisting transcription factors that are constitutively active, and thus transcription of some genes is maximally induced without a need for new protein synthesis. Other genes are regulated by STAT-I together with transcription factors that are newly synthesized in response to IFN- ⁇ .
  • the IRF-I gene which also encodes a transcription factor, is also regulated by STAT-I in response to IFN- ⁇ (Pine, R.
  • the promoter of the IRF-I gene also contains binding sites for nuclear factor kappa B (NF-kB), which mediates tumor necrosis factor alpha (TNF- ⁇ )-activated transcription of the IRF-I gene (Harada et al., (1994) MoI Cell Biol 14(2): 1500-9; R. Pine, unpublished).
  • NF-kB nuclear factor kappa B
  • TNF- ⁇ tumor necrosis factor alpha
  • IRF-I has been synthesized, it activates transcription of a temporally downstream set of genes.
  • IRF-I has been shown to regulate the IFN- ⁇ -induced expression of key _ genes invo ve in an n processing an presen a ion, inc u ing p , LJVLP-2, ana J ⁇ LA- antigens (Johnson et al., (1994) MoI Cell Biol 14(2):
  • IRF-I is phosphorylated, and manipulating the extent of phosphorylation affects its DNA- binding activity (Pine et al, (1990) MoI Cell Biol 10(6): 2448-57; Nunokawa et al, (1994J Biochem Biophys Res Commun 200(2): 802-7).
  • phosphorylation of IRF-I is regulated in vivo.
  • STAT-I activity is dependent on tyrosine phosphorylation and is affected by the extent of serine phosphorylation.
  • the abundance of latent STAT-I is also regulated.
  • a method of treating pulmonary diseases including asthma and idiopathic pulmonary fibrosis (IPF) in a subject suffering from the pulmonary disease comprising administering an aerosolized interferon such as interferon- ⁇ in a therapeutically effective amount wherein the symptoms of the pulmonary disease are improved or ameliorated.
  • the improved symptoms may be an increase of at least 10% of predicted FVC relative to values prior to treatment.
  • aerosolized IFN- ⁇ may be used for treating subjects suffering from asthma or IPF wherein the subjects are unresponsive to treatment with one or more corticosteroid, cyclophosphamide, and azathioprine.
  • the administration of an aerosolized interferon such as IFN- ⁇ is calculated and optimized in patients with pulmonary fibrosis. Such administration may result in improvement in pulmonary function tests in patients.
  • Interferons such as IFN- ⁇ may be administered by several different routes, including intravenous, intramuscular, subcutaneous, intranasally and via aerosol. However, when treating a pulmonary process alone, delivery of medication directly to the lung avoids exposure to other organ systems. Effective administration of 500 ⁇ g IFN- ⁇ via aerosol three times per week for two weeks has been shown by bronchoalveolar lavage (BAL) analysis in normal patients to result in increased levels of IFN- ⁇ post-administration. Likewise, about 500 micrograms of interferon- ⁇ three times per week and about 0.25 mg of interferon- ⁇ three times per week is thought to be effective.
  • BAL bronchoalveolar lavage
  • interferon- ⁇ it is an object of the present invention to deliver the interferon such as interferon- ⁇ via the pulmonary route of administration.
  • Interferons like IFN- ⁇ are delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream.
  • Adjei et al PHARMACEUTICAL RESEARCH, VOL. 7, No. 6, pp. 565- 569 (1990); Adjei et al, International Journal of Pharmaceutics, 63: 135-144 (1990); Braquet et al., Journal of Cardiovascular Pharmacology, Vol. 13, suppl. 5, s.
  • Ultravent nebulizer manufactured by Mallinckrodt, Inc., St. Louis, Missouri
  • Acorn II nebulizer manufactured by Marquest Medical Products, Englewood, Colorado
  • the Ventolin metered dose inhaler manufactured by Glaxo Inc., Research Triangle Park, North Carolina
  • the Spinhaler powder inhaler manufactured by Fisons Corp., Bedford, Massachusetts, MistyNeb, manufactured by Allegiance, McGraw Park, IL
  • AeroEclipse manufactured by Trudell Medical International, Canada.
  • each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants and/or carriers useful in therapy. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.
  • Chemically modified protein may also be prepared in different formulations depending on the type of chemical modification or the type of device employed.
  • Formulations suitable for use with a nebulizer may typically comprise protein dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per inL of solution.
  • the formulation may also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure).
  • the nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol.
  • Formulations for use with a metered-dose inhaler device may generally comprise a finely divided powder containing the protein suspended in a propellant with the aid of a surfactant.
  • the propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and , , , -tetra uoroe e, or com nat ons t ereo .
  • u ta e sur acta p nc u e sor r an rio ea e also be useful as a surfactant.
  • Formulations for dispensing from a powder inhaler device may comprise a finely divided dry powder containing protein and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.
  • the protein should most advantageously be prepared in particulate form with an average particle size of less than 10 ⁇ m (or microns), most preferably 0.5 to 5 ⁇ m, for most effective delivery to the distal lung.
  • a particularly preferred approach to breathing slow and deep inspiration may, when compared with standard (tidal breathing), increase deposition efficiency in the lung periphery by a factor of up to about
  • This process can be further enhanced by using particles that are relatively large (e.g., about 4.5 ⁇ m) that ordinarily would deposit in the oropharynx.
  • the prolonged inspiration of slow and deep breathing is particularly suited for delivery of drugs to the lungs of patients whose peripheral airway pathology results in reduced deposition of conventional smaller aerosols as well as promoting avoidance of deposition in the oropharynx.
  • Diseases of the lung periphery that may be treated by this method include, for example, idiopathic pulmonary fibrosis and emphysema. Both these entities result in enlarged airspaces that result in minimal deposition during tidal breathing.
  • Figure 3 represents a deposition pattern in a human subject inhaling 4.5 ⁇ m aerosols using the slow and deep breathing pattern.
  • the images demonstrate minimal deposition of aerosol (less than 10%) in the upper airways illustrated by the small amount of activity in the stomach.
  • the deposition image represents radiolabeled aerosol deposited in the lung periphery of a human subject after 3 breaths using the slow and deep pattern with an inspiratory time of approximately 8 seconds.
  • Figure 4 is an illustrative scan in the same subject following 20 breaths of tidal breathing of 1.5 ⁇ m particles which is the present standard mode of inhalation. Analysis of the images indicates that the slow and deep method of breathing which incorporates the use of large particles, slow inspiration and a prolonged inspiratory time is 51 times more efficient per breath in depositing aerosol particles in the lung.
  • Nasal delivery of the protein is also contemplated.
  • Nasal delivery allows the passage of the protein to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung.
  • Formulations for nasal delivery include those with dextran or cyclodextran.
  • interferon- ⁇ dosage will be between 250 ⁇ g of biologically active protein (calculating the mass of the protein alone, without chemical modification) to 750 ⁇ g (based on the same) given three times per week. More preferably, the dosage may be about 500 ⁇ g given three times per week.
  • interferon- ⁇ dosage is generally 250 to 750 micrograms administered one to five times per week, preferably about 500 micrograms administered three times per week. In the instance of interferon- ⁇ , dosage is generally 0.10 to 1 mg one to three times per week, preferably about 0.25 mg three times per week.
  • the dosing schedule may vary, depending on the circulation half-life of the protein, and the formulation used.
  • Each patient is evaluated for bronchoscopy as per Bellevue Hospital Protocol.
  • Each evaluation includes Hgb, platelets, BUN/CR, coagulation panel, ABG with PO2 > 75 mm Hg, EKG, CXR.
  • Contradictions to bronchoscopy include lack of patient cooperation, recent myocardial infarction, malignant arrythmias, uncorrectable hypoxemia, unstable bronchial asthma, pulmonary hypertension, partial tracheal obstruction or vocal cord paralysis, bleeding diathesis, and uremia.
  • the patient must be NPO at least 8 hours prior to bronchoscopy.
  • An intravenous line will be placed, supplemental oxygen will be administered, and continuous pulse oximetry and blood pressure monitoring will be performed.
  • BAL fluid After BAL fluid is obtained from the patient, it is processed in the GCRC core laboratory under standardized protocol used for processing all BAL. BAL fluid is filtered through sterile gauze. A total cell count with differential is performed in a hemocytometer. Cell viability is e . o cen i re prepareu jx ⁇ m ca lobe of BAL fluid and frozen at -7O 0 C. 24 hour superaatants are collected at a concentration of
  • cytokine ELISA assays 10 cells/ml for cytokine ELISA assays.
  • the volume of epithelial lining fluid is determined according to the protein method. Following cent ⁇ fugation, BAL fluid supernatant is concentrated 10X-50X using the AMICON filter method.
  • Cytokine assays are carried out with commercially available kits (R&D Systems, Minneapolis, MN) All samples are assayed in triplicate and the amount of cytokine is quantified at the end of the assay by a microliter plater reader Transbronchiall bipsy specimens are processed for isolation of fibroblasts as previously described (Raghu et al (1989) Am Rev Resp Dis 140 95-100) and analyzed for collagen production using H proline incorporation into collagenous proteins.
  • bronchoscopy including but not limited to fever, shortness of breath, hemoptysis, and pneumothorax for 4 hours post procedure in the GCRC by the clinical nursing staff.
  • Concomitant medications will be recorded in the patient's medical record.
  • culture supernatants are .assa ⁇ .ed for cytokines IL-l ⁇ , IL-4, IL-6, IL-8 and TNF- ⁇ by ELISA (R&D, Minneapolis).
  • Cell-free BAL supernatant is used to measure TGF- ⁇ activity by ELISA and luciferase reporter assay.
  • Transbronchial biopsy (TBBX) specimens are used to measure TGF- ⁇ gene transcription by semi-quantitative RT-PCR. Fibroblasts are obtained from TBBX specimens, and the quantities of collagen I, III, and fibronectin RNA measured by RT-PCR.
  • RNA (10 ⁇ g) is obtained from TBBX or cell culture of TBBX, and Northern Blot analysis is performed.
  • BAL was guided by lung deposition images, so that the areas of highest drug deposition was analyzed and compared to areas of lowest delivered drug and pre-aerosol IFN- ⁇ samples. In this way, total dose to each area of the lung can be calculated and determined. Depending on clinical response and BAL data, dose may be adjusted to reflect optimal clinical and deposition parameters. Attempts will be made to sample similar segments pre- and post- treatment, when possible. Each patient has a follow up evaluation at one month post therapy. The results of all procedures, laboratory evaluations, radiological studies, and pulmonary physiology evaluations are documented in the patient's medical record. All study evaluations are conducted at the GCRC of NYU Medical Center. - m i ,
  • a ⁇ roEclrose, wtosephale generation is dependent on patient breathing through the nebulizer. i . ⁇ • ' ' IiJ ⁇ 3 l,,li ib ,/ ,,;;lêt:;!' / "+ / It produces aerosol only during inspiration.
  • the nebulizers were connected to the circuit in the manner of their clinical use.
  • a ten stage, low flow (1.0 L/m) cascade impactor (California measurements, Sierra Madre, CA) was connected using a T connector (T connector cascade , Hudson Respiratory Care, Temecula, CA).
  • An inspiratory filter that prevented particles from entering the cascade impactor during expiration, was placed between the piston pump and cascade impactor.
  • a second filter was placed in the system to capture the excess particles directed neither to the inspiratory filter nor to the impactor.
  • a piston pump Harmonic Apparatus, Millis, MA
  • Aerosol deposition studies After 133 Xe imaging, the camera was switched to 99m Tc. Then, the patient inhaled radiolabeled aerosolized IFN- ⁇ from the nebulizer. For each device an expiratory filter was present to capture exhaled particles. The nebulizers were run until dry. After final inhalation, the patient drank a glass of water to wash material from the oropharynx to the stomach. Measuring stomach activity assessed upper airway deposition.
  • stomach deposition was measured using the gamma camera by quantifying activity in the lung regions and applying the appropriate attenuation correction. Oropharyngeal depostion was determined by subtracting the lung activity from the total activity on the deposition image Appropriate corrections were made for stomach attenuation.
  • Figure 7 emons ra es e mc e percen pre ic e o a ung capaci y a ?eatment m tnree oi me five patients tre ⁇ tad ⁇ ,,,There,was also an improvement of greater than 200 cc's (200 and 500 cc, ⁇
  • Figure 8 demonstrates the increased percent predicted forced vital capacity after treatment in three of the five patients treated.
  • Low dose inhaled corticosteriod use includes 168-500 meg/day of beclomethasone dipropionate, 200-400 meg/day of budesonide DPI, 500-1000 meg of fiunisolide, or 400-1000 meg/day of triamcinolone acetonide.
  • Subjects will characterize symptoms in a daily diary before, during, and after aerosolized IFN- ⁇ treatment. They will rate symptoms of cough, wheeze, and shortness of breath on a scale. They will also record daily peak flow measurements.
  • Fiberoptic bronchoscopy with BAL will be performed the day after the last IFN- ⁇ treatment or no treatment.
  • We will analyze BAL cell count/differential and levels of IFN- ⁇ , IL-4, IL-5, GM-CSF, IL-IO, IL- 12, and IL- 13 by ELISA of 24-hour culture supernatants.
  • [0127JBAL specimens will be obtained from the 30 asthma patients. We will administer aerosol IFN- ⁇ to 15 of these patients for 8 weeks in order to assess whether EPN- ⁇ modulates cytokine production. These patients will have pre- and post-treatment BAL and blood draws.
  • Fiberoptic bronchoscopy Subjects will be pre-screened with medical history and physical examination, spirometry, oximetry, assessment of bronchial hyperresponsiveness, coagulation tests (PT, PTT, platelets), and CBC and screening chemistries.
  • patients with have continuous monitoring of heart rate and 02 saturation, recording of subject symptoms and medication doses, intravenous catheter m place, premedication with inhaled b- agonist, subcutaneous atropine (0.4mg), and sedation (midazolam, iv), and supplemental oxygen.
  • the fiberoptic bronchoscope is introduced after light premedication and topical anesthesia of the nose and upper airway.
  • the tip of the bronchoscope is wedged into a segmental, or subsegmental, bronchus of the right middle lobe or lingula.
  • One hundred milliliters of 37 ° C normal saline are instilled into the bronchus in aliquots of 20 ml.
  • the warmed saline should avoid thermally induced bronchospasm in asthma subjects.
  • Gentle intermittent suction is used to recover the effluent. Fluid recovery of 60 to 80% is expected in mild asthmatics.
  • ,.wi,ll include, foUow ⁇ n, appointment within the week, and contact telephone number.
  • A Alveolar macrophages
  • BAL bronchoalveolar lavage
  • the fluid is filtered through one layer of sterile gauze to remove clumps of mucus.
  • a total cell count is done in a hemocytometer and cell differentials performed on cytocentrifuge slides stained with modified Wright-Giemsa stain with a total of 500 cells counted.
  • Cell viability is determined by Trypan Blue exclusion, and in all cases recovered cells to be used for experiments will be greater than 90% viable.
  • Twenty cytocentrifuge slides will be prepared from each lobe of BAL and once fixed in 10% formalin, frozen at -70 0 C.
  • BAL cells will be washed and cultured (37 0 C) in RPMI (GIBCO) supplemented with 10% heat-inactivated fetal calf serum (FCS) and lOOu/mL penicillin and lOOmcg/ml streptomycin at a concentration of I ⁇ 6 cells/ml for 24 hours.
  • RPMI RPMI
  • FCS heat-inactivated fetal calf serum
  • PBMCs will be isolated from heparinized venous blood by Ficoll-Hypaque density gradient centrifugation. Heparinized venous blood is layered on Ficoll-Hypaque and centrifuged at 2500rpm for 20 minutes.
  • the low density layer of PBMCs will be aspirated and washed with phosphate buffered saline (PBS) and resuspended at a concentration of 10 6 cells/ml of RPMI-1640 (GIBCO) with 10% heat-inactivated FCS, 100 U/ml penicillin, and 100 mcg/ml streptomycin.
  • PBS phosphate buffered saline
  • FCS fetal bovine serum
  • the clinical treatment protocol will have clear-cut effects on the abundance and activity of transcription factors that regulate gene expression in response to IFN- ⁇ and that correlation of these data with the cytokine profile will extend the criteria by which the immune response in asthma can be evaluated. Furthermore, the data obtained will allow mechanistic interpretation of the results from analysis of cytokine production and expression of cytokine and other genes.
  • the design of the project incorporates several controls to help establish the effect of the aerosol IFN- ⁇ treatment, distinct from any other variable. These include obtaining BAL and blood samples before and after the course of treatment, and collection of BAL samples from uninvolved as well as involved lobes. All experiments for this aim will be done with protein extracts prepared from BAL or PBM cells. Cytoplasmic and nuclear proteins will be obtained and analyzed separately. To gain more definitive results, BAL cells will be separated into adherent and nonadherent populations. The former will include predominantly alveolar macrophages. The latter will be comprised predominantly of lymphocytes and granulocytes. PBMC will be extracted without further separation.
  • Immunoblot detection will be the primary technique used. Cytoplasmic or nuclear extract from up to 5 x 10 cells will be used for each analysis. Obtaining cells as described above will yield 10 samples for each patient. All the extracts of PBMC and BAL cells from one patient will be included in a single experiment, which will facilitate relative quantitation within a set of samples. Control cytoplasmic and nuclear extracts prepared from cultured cell lines will also be included in each experiment. On the basis of previous studies, these samples will be known to contain the target proteins, and can thus provide positive controls for the immunoblot detection. Additionally, they can be used to validate that the data obtained are quantitative or reveal the limits of the quantitative detection.
  • the proteins will be separated by SDS-PAGE, then transferred to a membrane.
  • the membrane will be developed with reagents to detect STAT-I, IRP-I, and CIITA, one after the other.
  • the membrane will be probed finally to detect b-tubulin, which will be present in both cytoplasmic and nuclear protein extracts and can thus serve as an internal standard for quantitative comparison of cytoplasmic or nuclear extracts within and between experiments. All the antibodies needed are available in the laboratory or can be commercially obtained and are known to work for immunoblot protocols.
  • the membrane will be treated to disrupt antibody binding without releasing the target proteins. , i - i jse ⁇ uenpe, and comparing the signals obtained in the first and second round. Negative controls / U S O b ,.• ⁇ ' ' ,,:;f .37' " ⁇ T " / for specificity of detection are provided for each protein by the antibodies against the other two.
  • the membrane will be developed a final time without inclusion of a primary antibody.

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EP05808951A 2005-09-20 2005-09-20 Verfahren zur behandlung von lungenerkrankung mit interferonen Withdrawn EP1940451A4 (de)

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EP2343081A1 (de) * 2009-12-31 2011-07-13 Rijksuniversiteit Groningen Interferon-Analoga
EP3793545A4 (de) * 2018-05-17 2022-04-06 Actuate Therapeutics Inc. Behandlung von idiopathischer lungenfibrose mit beta-inhibitoren in form von glykogensynthasekinase 3
US12257093B2 (en) * 2019-10-15 2025-03-25 Indian Institute Of Technology Madras Walk-in lab test for lung morphometry characterization

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US6030609A (en) * 1995-05-19 2000-02-29 Case Western Reserve University Method and composition for treating paramyxovirus
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US6964761B1 (en) * 2001-12-12 2005-11-15 New York University Method of treating idiopathic pulmonary fibrosis with aerosolized IFN-γ
AU2003262811A1 (en) * 2002-08-27 2004-03-19 Intermune, Inc. Methods of treating idiopathic pulmonary fibrosis
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EP2494982A1 (de) 2012-09-05
CA2622987A1 (en) 2007-03-29
JP5175198B2 (ja) 2013-04-03
AU2005336519B2 (en) 2012-05-10
CA2622987C (en) 2018-02-27
JP2009508947A (ja) 2009-03-05
WO2007035180A1 (en) 2007-03-29
EP1940451A4 (de) 2010-03-17
KR20130110225A (ko) 2013-10-08
CN101378774A (zh) 2009-03-04
EP2494982B1 (de) 2016-10-26
BRPI0520564A2 (pt) 2009-05-19

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