WO2007027578A2

WO2007027578A2 - Methods and compositions for decreasing cohesivity of pathological respiratory tract secretions

Info

Publication number: WO2007027578A2
Application number: PCT/US2006/033481
Authority: WO
Inventors: Bruce K. Rubin; Allan L. Goldstein
Original assignee: Regenerx Biopharmaceuticals, Inc.
Priority date: 2005-08-31
Filing date: 2006-08-29
Publication date: 2007-03-08
Also published as: WO2007027578A3

Abstract

A combination and method of treatment for decreasing cohesivity of pathological respiratory tract secretions of a subject, includes administering to a subject an effective amount of a combination including dornase alpha and a peptide agent including amino acid sequence LKKTET or LKKTNT, a conservative variant thereof, or an agent that stimulates production of an LKKTET or LKKTNT peptide, or a conservative variant thereof, wherein a ratio of the dornase alpha to the peptide agent is from about 1.7:1 to about 0.1:1 by weight.

Description

METHODS AND COMPOSITIONS FOR DECREASING COHESIVITY OF PATHOLOGICAL RESPIRATORY TRACT SECRETIONS

BACKGROUND OF THE INVENTION

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 60/712,410, filed August 31 , 2005.

Field of the Invention

[0002] The present invention relates to the field of treating pathological respiratory tract secretions.

Description of the Background Art

[0003] U.S. Patent No. 5,656,589 discloses reducing viscosity of pathological mucoid airway contents in patients, such as cystic fibrosis (CF) patients, by administering a combination of DNase I (dornase alpha) and an actin-binding protein such as gelsolin. Synergistic results were reported with a concentration of DNase I twice that of the concentration of gelsolin.

[0004] There remains a need in the art for methods and compositions for decreasing cohesivity of pathological respiratory tract secretions.

SUMMARY OF THE INVENTION

[0005] In accordance with one aspect, a pharmaceutical combination and method of treatment for decreasing cohesivity of pathological respiratory tract secretions of a subject utilize an effective amount of a combination comprising dornase alpha and a peptide agent comprising amino acid sequence LKKTET or LKKTNT, a conservative variant thereof, or a stimulating agent that stimulates production of an LKKTET or LKKTNT peptide, or a conservative variant thereof, in said respiratory tract, wherein a ratio of said dornase alpha to said peptide agent is from about 1.7:1 to about 0.1 :1 by weight.

DETAILED DESCRIPTION OF THE INVENTION

[0006] It has surprisingly been discovered that dornase alpha can be utilized at substantially lower concentrations relative to actin-binding proteins than previously „ repofty in ϋ'!s! pltelitife 5'$56,589 to achieve synergy in decreasing cohesivity of pathological respiratory tract secretions.

[0007] Certain diseases and conditions are caused by or result in airway (respiratory tract) secretions or contents (e.g., mucus or sputum) which contain higher than normal concentrations of actin filaments and DNA polymers which increase the consistency, viscosity and/or cohesivity of the airway contents, thereby preventing normal clearance and leading to pathological consequences.

[0008] The diseases that are amenable to treatment by the methods and combinations of the present invention include, but are not limited to cystic fibrosis, chronic bronchitis, mucopurulent or purulent exacerbation of simple mucoid bronchitis, bronchorrhea, bronchopneumonia, widespread bronchiolitis, purulent pneumonia, pneumonic-alveolar-consideration, asthma, with or without asthmatic bronchitis with mucus plugging, acute and/or chronic purulent sinusitis, empyema, bronchiectasis, bronchocoele, adult respiratory distress syndrome (ARDS), post-transplantation obliterative bronchiolitis, and allergenic bronchiolitis (fibrosing alveolitus).

[0009] A subject being treated in accordance with the present invention preferably is mammalian, most preferably human.

[0010] In accordance with one embodiment, the condition being treated is cystic fibrosis.

[0011] Thymosin.4 was initially identified as a protein that is up-regulated during endothelial cell migration and differentiation in vitro. Thymosin 4 was originally isolated from the thymus and is a 43 amino acid, 4.9 kDa ubiquitous polypeptide identified in a variety of tissues. Several roles have been ascribed to this protein including a role in a endothelial cell differentiation and migration, T cell differentiation, actin sequestration, vascularization and wound healing.

[0012] In accordance with one embodiment, the invention is a method of treatment for decreasing cohesivity of pathological respiratory tract secretions a subject, comprising administering to a subject an effective amount of a pharmaceutical composition or combination comprising dornase alpha and a peptide agent, which may be a polypeptide comprising amino acid sequence LKKTET or LKKTNT, or a conservative variant thereof, wherein a ratio of said dornase alpha to said peptide agent is from about 1.7:1 to about 0.1:1 by weight. The peptide agent preferably is Thymosin β4, and/or Tβ4 isoforms, analogues or derivatives, including KLKKTET, LKKTETQ, N- terminal variants of Tβ4, C-terminal variants of Tβ4 and antagonists of Tβ4. The invention also may utilize oxidized Tβ4. In accordance with other embodiments, the antimicrobial agent is other than thymosin beta 4 or oxidized Tβ4. _ι

[omif ¹¹As hbtiδMBό^'vs;tne ratio of the dornase alpha to the peptide agent utilized in compositions, combinations and methods in accordance with the present invention is from about 0.7:1 to about 0.1:1 by weight. In preferred embodiments, the compositions, combinations and methods of the invention utilize dornase alpha and a peptide agent as herein described in a respective ratio of from about 1.5:1 to 0.5:1 , 1.4:1 to about 0.6:1 , 1.3:1 to about 0.7:1 , 1.2:1 to about 0.8:1 , or from about 1.1 :1 to about 0.9:1 , with a ratio of about 1 :1 by weight being most preferred.

[0014] Compositions or combinations which may be used in accordance with the present invention include peptide agents such as Thymosin β4 (Tβ4), and/or Tβ4 isoforms, analogues or derivatives, oxidized Tβ4, N-terminal variants of Tβ4, C-terminal variants of Tβ4 and antagonists of Tβ4, polypeptides or peptide fragments comprising or consisting essentially of the amino acid sequence LKKTET or conservative variants thereof having actin-binding activity. International Application Serial No. PCT/US99/17282, incorporated herein by reference, discloses isoforms of Tβ4 which may be useful in accordance with the present invention as well as amino acid sequence LKKTET and conservative variants thereof, which may be utilized with the present invention. International Application Serial No. PCT/GB99/00833 (WO 99/49883), incorporated herein by reference, discloses oxidized Thymosin β4 which may be utilized in accordance with the present invention. Although the present invention is described primarily hereinafter with respect to Tβ4 and Tβ4 isoforms, it is to be understood that the following description is intended to be equally applicable to amino acid sequence LKKTET or LKKTNT, peptides and fragments comprising or consisting essentially of LKKTET or LKKTNT, conservative variants thereof having actin-binding activity, and/or Tβ4 isoforms, analogues or derivatives, including N-terminal variants of Tβ4, C-terminal variants of Tβ4 and antagonists of Tβ4, and other actin-binding peptides as described herein. The invention also may utilize oxidized Tβ4 in certain embodiments.

[0015] In one embodiment, the invention provides a method of treatment for decreasing cohesivity of pathological respiratory tract secretions of respiratory tissue of a subject, by contacting respiratory tract tissue secretions with an effective amount of a composition or combination which contains dornase alpha and a peptide agent as described herein. As non-limiting examples, the tissue may be selected from respiratory tract or airway tissue of said subject. The contacting may be directly or systemically. Examples of direct administration include, for example, contacting the respiratory tract tissue secretions, by direct application or inhalation, with an aerosol, _, solutlli^'nf fόa'fnrϊotibή;^' salve, get cfeam, paste, spray, suspension, dispersion, hydrogel, ointment, or oil comprising dornase alpha and a peptide agent as described herein. Systemic administration includes, for example, intravenous, intraperitoneal, intramuscular injections or infusions of a composition containing dornase alpha and a peptide agent as described herein, in a pharmaceutically acceptable carrier such as water for injection. If desired, the dornase alpha and peptide agent as described herein may be administered in separate pharmaceutical formulations. Thus, the dornase alpha and peptide agent as described herein may be administered together in a single pharmaceutical composition or as a separately administered combination. [0016] The dornase alpha and peptide agent(s) for use in the invention, as described herein, may be administered in any suitable cohesivity-decreasing or reducing amount. For example, dornase alpha and a peptide agent as described herein may each be administered in dosages within the range of about 0.0001-1 ,000,000 micrograms, or in amounts within the range of about 0.1-5,000 micrograms, or within the range of about 1-30 micrograms.

[0017] A composition or combination in accordance with the present invention can be administered daily, every other day, every other week, every other month, etc., with a single application or multiple applications per day of administration, such as applications 2, 3, 4 or more times per day of administration.

[0018] Many Tβ4 isoforms have been identified and have about 70%, or about 75%, or about 80% or more homology to the known amino acid sequence of Tβ4. Such isoforms include, for example, Tβ4^ala, Tβ9, Tβ10, Tβ11 , Tβ12, Tβ13, Tβ14 and Tβ15. Similar to Tβ4, the Tβ10 and Tβ15 isoforms have been shown to sequester actin. Tβ4, Tβ10 and Tβ15, as well as these other isoforms share an amino acid sequence, LKKTET or LKKTNT, that appears to be involved in mediating actin sequestration or binding. For example, Tβ4 can modulate actin polymerization (e.g. β-thymosins appear to depolymerize F-actin by sequestering free G-actin). Tβ4's ability to modulate actin polymerization may therefore be due to, all or in part, its ability to bind to or sequester actin via the LKKTET sequence. Thus, as with Tβ4, other proteins which bind or sequester actin, or modulate actin polymerization, including Tβ4 isoforms having the amino acid sequence LKKTET, are likely to be effective, alone or in a combination with Tβ4, as set forth herein.

[0019] Thus, it is specifically contemplated that known Tβ4 isoforms, such as Tβ4^ala, Tβ9, Tβ10, Tβ11, Tβ12, Tβ13, Tβ14 and Tβ15, as well as Tβ4 isoforms not yet identified, will be useful in the methods of the invention. As such Tβ4 isoforms are useful in the methods of the invention, including the methods practiced in a subject. The invention therefore further provides pharmaceutical compositions and combinations comprising Tβ4, as well as Tβ4 isoforms Tβ4^ala, Tβ9, Tβ10, Tβ11 , Tβ12, Tβ13, Tβ14 and Tβ15, and one or more pharmaceutically acceptable carriers. [0020] In addition, other antimicrobial agents or proteins having actin sequestering or binding capability, or that can mobilize actin or modulate actin polymerization, as demonstrated in an appropriate sequestering, binding, mobilization or polymerization assay, or identified by the presence of an amino acid sequence that mediates actin binding, such as LKKTET or LKKTNT, for example, can similarly be employed in the methods, compositions and combinations of the invention. Such proteins may include gelsolin, vitamin D binding protein (DBP), profilin, cofilin, depactin, vilin, fragmin, severin, capping protein,, β-actinin and acumentin, for example. Thus, the invention further provides pharmaceutical compositions and combinations comprising gelsolin, vitamin D binding protein (DBP), profilin, cofilin, depactin, vilin, fragmin, severin, capping protein, β-actinin and acumentin as set forth herein. [0021] As used herein, the term "conservative variant" or grammatical variations thereof denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the replacement of a hydrophobic residue such as isoleucine, valine, leucine or methionine for another, the replacement of a polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. [0022] Tβ4 has been localized to a number of tissue and cell types and thus, agents which stimulate the production of an LKKTET or LKKTNT peptide such as Tβ4 or another actin-binding agent as described herein, can be added to or comprise a composition or combination to effect production an actin-binding agent from a tissue and/or a cell. Such stimulating agents may include members of the family of growth factors, such as insulin-like growth factor (IGF-1), platelet derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor beta (TGF-β), basic fibroblast growth factor (bFGF), thymosin α1 (Tα1) and vascular endothelial growth factor (VEGF). More preferably, the stimulating agent is transforming growth factor beta (TGF.-β) or other members of the TGF.-β superfamily.

[0023] In accordance with one embodiment, subjects are treated with domase alpha and a stimulating agent that stimulates production in the subject of a peptide agent as defined herein. [0024] Additionally, other agents that assist in decreasing cohesivity of pathological respiratory tract secretions or reduction of respiratory microbial infection of respiratory tissue or secretions may be added to a composition along with an agent as described herein. For example, and not by way of limitation, a peptide agent as described herein alone or in combination can be added in combination with any one or more of the following agents: antibiotics, VEGF, KGF, FGF, PDGF, TGFβ, IGF-1 , IGF-2, IL-I₁ prothymosin a and/or thymosin α1 in an effective amount. [0025] The invention also includes a pharmaceutical composition comprising a therapeutically effective amount of dornase alpha and a peptide agent as described herein in a pharmaceutically acceptable carrier such as water for injection. [0026] The actual dosage or reagent, formulation or composition that provides treatment may depend on many factors, including the size and health of a subject. However, persons of ordinary skill in the art can use teachings describing the methods and techniques for determining clinical dosages as disclosed in PCT/US99/17282, supra, and the references cited therein, to determine the appropriate dosage to use. [0027] Suitable formulations may include dornase alpha and a peptide agent as described herein, each at a concentration within the range of about 0.001 - 45% by weight, more preferably within the range of about 0.01 - 0.1% by weight, most preferably about 0.05% by weight.

[0028] In preferred embodiments, the dornase alpha and peptide agent are administered together as an aerosol. The amount of aerosolized actin-binding peptide agent may be determined according to those procedures used to determine the doses of dornase alpha inhalation effective for treatment of patients with dornase alpha. The dose delivered in vivo may be related to the effective amount determined in vitro. For example, if sputum is successfully solubilized in vitro by amounts within a range of 0.01-1,000 mcg/ml, a thousand-fold increase over the in vitro dose may be appropriate to be administered to a patient. Of course, it would be understood that the amounts and regimens for the administration of actin-binding peptide agent can be determined readily by those of ordinary skill in the art of treating respiratory disorders. Generally, the dosage of actin-binding peptide agent treatment will vary depending upon considerations such as the type of the actin-binding peptide agent employed, the age of the subject, the health of the subject, the severity of the disease, the kind of concurrent treatment, the extent of tissue damage, the gender of the patient, the duration of the symptoms, counterindications (if any), and other variables understood as needing adjustment by the individual physician. The correct effective dose could routinely be determined by monitoring the airway contents by aspiration or as sputum. [0029] Thus, cohesivity-decreasing amounts of each of dornase alpha and peptide agent as described herein may be within a range of about 0.01 mcg/ml to about 1,000 mόg ffil, dr wff n ranges , about 0.05 - 500 mcg/ml, 0.1-300 mcg/ml or 0.3 - 150mcg/ml. According to the above formula, suitable concentrations of each of dornase alpha and peptide agent as described herein may be within a range of from about 0.01 mg/ml to about 1 ,000 mg/ml, or within concentration ranges of about 0.1- 500 mg/ml, 0.01-300 mg/ml or 0.3-150 mg/ml.

[0030] The therapeutic approaches described herein involve various routes of administration or delivery of dornase alpha and a peptide agent as described herein, including any conventional administration techniques (for example, but not limited to, direct administration, local injection, inhalation, or systemic administration), to a subject. The methods and compositions using or containing dornase alpha and a peptide agent as described herein may be formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable non-toxic excipients or carriers.

Example 1

[0031] Background: Filamentous actin (F-actin) forms polymers that contribute to the abnormal biophysical properties of sputum. Thymosin β4 (Tβ4) is the major G-actin sequestering peptide in cells and can depolymerize F-actin. Tβ4 could potentially decrease sputum viscoelasticity and adhesivity and improve sputum clearance. The objective was to evaluate the effects of concentration- and time-dependent effects of TB4 on the properties of cystic fibrosis (CF) sputum.

[0032] Methods: Sputum was collected during pulmonary function testing from 17 subjects during a routine CF center visit. Sputum rheology, cough and ciliary transportability, and interfacial tension was measured before and after the addition of dornase alfa at 30 μg/ml, Tβ4 at 0.3, 3, 30, and 150 μg/ml, and Tβ4 with dornase alfa at 1.5 μg/ml each. Sputum was separately incubated with Tβ4 30 μg/ml for 0, 10, 20, or 60 minutes.

[0033] Results: There was a direct relationship between actin filament length and sputum cohesivity. There was a dose-dependent threshold decrease in cohesivity with Tβ4 and a time-dependent decrease in cohesivity over 60 minutes at 30 μg/ml. [0034] Conclusions: Actin polymer filament length is correlated with sputum cohesivity. Tβ4 depolymerizes CF sputum actin in both a dose-dependent and a time- dependent manner. Synergy of TB4 on actin and dornase on DNA may be explained by the combined effect of actin depolymerization and DNA filament severing or by virtue of actin depolymerization increasing the effectiveness of dornase alfa. Abbreviations

CF: cystic fibrosis

CTR: cough transportability of sputum in vitro

LSM: laser scanning confocal microscopy

MCTR: mucociliary transportability of sputum in vitro

TB4: thymosin beta 4 rhDNase: recombinant human deoxyribonuclease

Introduction

[0035] There has long been speculation that persistent pulmonary infections in the cystic fibrosis (CF) airway are caused, in part, by airway secretions that are abnormally viscous or sticky. This purulent sputum is poorly transported by cough or by cilia when compared to sputum from patients with other hypersecretory conditions such as chronic bronchitis. Several biopolymers in CF sputum have been identified that contribute to abnormal sputum properties. These include neutrophil derived DNA and filamentous actin (F-actin) derived from the cytoskeleton of damaged epithelial cells and neutrophils. The effects of dornase alfa (Pulmozyme, Genentech, S San Francisco, CA) on CF sputum have been well studied. This agent reduces sputum viscosity and adhesivity and improves sputum transport in vitro. Dornase alfa also improves pulmonary function, and decreases the likelihood of hospitalization in patients with CF while on treatment. A number of other agents including gelsolin acetylcysteine and NaCI have also been reported to affect CF sputum by lowering viscoelastic properties. These agents act directly to either fragment actin polymers or by reducing anionic surface charges in mucin.

[0036] The presence of large amounts of polymerized F-actin in CF sputum indicates the possibility that thymosin β4 (Tβ4), because of its unique properties related to actin, may be useful in lowering the abnormal viscoelectric properties in CF patients. Tβ4 is the major actin-sequestering peptide in eukaryotic cells. Tβ4 plays an important role in remodeling and healing of tissues and wounds by virtue of its ability to regulate the actin-dynamics of the cell and by sequestering the bulk of monomeric actin, its ability to depolymerize F-actin, and by its anti-microbial and anti-inflammatory properties. [0037] This study tested the hypotheses that sputum viscoelasticity and cohesivity was related to actin filament length, and that TB4 would reduce the viscoelasticity and cohesivity of CF sputum thus improving mucociliary and cough clearability in vitro. Materials and methods

[0038] Studies were conducted using spontaneously expectorated sputum from 17 subjects with CF. Subjects age ranged from 14 to 38 years (mean age 24.6) and 11 were male. All had chronic sputum expectoration and forced expired volume in 1 second (FEV1) percent predicted was 28-68% (mean 60% predicted). All subjects were chronically infected with Pseudomonas aeruginosa and all but 1 were taking dornase alfa by inhalation but this was held for 24 hours before the clinic visit used for sputum collection. Subjects were excluded if they had been admitted to hospital within the preceding month for an exacerbation of pulmonary disease. [0039] Subjects were asked to swallow their saliva and then to expectorate all secretions mobilized during routine pulmonary function testing. Eight subjects gave 2 specimens at different visits. Six of the expectorated sputa were greater than 5 ml in volume and these were used for the more extensive dose response studies. Sputum was visually separated from saliva in clinic and immediately stored in separate aliquots of 150 μl_ in dehydration-protected O-ring containers. Collection of sputum for analysis was approved by the Institutional Review Board for Human Research.

Laser-scanning confocal microscopy (LSM) analysis of actin polymers in sputum

[0040] Sputum samples, 30 μL in volume, were placed onto glass microscope slides partitioned into two lanes using double-stick tape to a total thickness of approximately 70 μm. After this, 7.5 μL of buffered saline was layered onto the sample with 1 μL of rhodamine-phalloidin added for visualization of F-actin. A glass cover slip was placed over the sample and onto the double-stick tape. This was incubated for 30 minutes at room temperature. The slide was then inverted onto the stage of the LSM. [0041] The samples were scanned using the argon/krypton laser setting of the Zeiss 410 LSM (Carl Zeiss LSM Version 3.92 Beta, Oberkochen, Germany). Fluorescence images were transformed by pseudo color. Images were analyzed for the intensity of fluorescence, distribution of the F-actin filaments, filament number, and filament length using the KS400 image analysis software. For the purpose of geometric analysis of filament length and number, the red fluorescence was converted into a gray scale with pixel intensity scaled from 0 to 255 for Marr analysis. This is an edge analysis by region defining the contours and delineating boundaries of shapes at discrete intensity changes. On the transformed images, filament length and size distribution were calculated. The shortest filaments were less than 10 μmin length and the longest were more than 20 μm with the maximal calculated length limited by the diagonal of the viewi g ieic ^';S^;!atsticarana!ysιs was completed using the LSM computer software and Fiberscore analysis, optimized for sputum biopolymer detection and analysis.

Biophysical properties

[0042] Analysis was performed using 150 μl_ of freshly obtained sputum layered with

30 μl_ of each test agent and incubated at 37° C. All test agents were prepared using modified amphibian Ringer's solution containing NaCI = 98.3, KCI = 2.7, and CaCb =

1.5.

[0043] To evaluate the time course of the action of Tβ4 on expectorated secretions, 6 specimens with volume greater than 5 ml each, were treated with Tβ4 at 30 μg/ml for 0,

10, 20 and 60 minutes of incubation at 37 degrees C. Evaluation of cohesivity, mucociliary and cough transportability, and surface and bulk viscoelasticity was performed on these specimens as described below. For time course experiments, domase alfa at 30 μg/ml was chosen to match that reported to be achieved in the airway after aerosolization.

[0044] These analysis were made using 150 μl_ of sputum layered with 30 μl_ of test substance and incubated for 30 minutes at 37° C. Each specimen was completely analyzed in a single day. The order of treatment and analyses was randomly determined.

[0045] The test substances used were:

1) amphibian Ringer's solution (negative control),

2) dornase alfa at a concentration of 30 μg/ml (positive control)

3) TB4 at a concentration of 0.3 μg/ml

4) TB4 at a concentration of 3 μg/ml

5) Tβ4 at a concentration of 30 μg/ml

6) TB4 at a concentration of 150 μg/ml

7) dornase alfa 15 μL at a concentration of 3 mcg/ml and Tβ4 15 μL at 3 μg/ml

The physical properties of sputum

Dynamic viscoelasticity (rheology)

[0046] Viscosity is the loss of energy from an object moving through a substance and thus the resistance to flow. Elasticity is the stored energy transmitted back to a rheologic probe. Viscoelasticity is a property of non-Newtonian fluids such as gels. In the magnetic microrheometer a dissecting microscope was used to position a small steel ball, 100 μm in diameter in a 3 μL sample of mucus and this was then placed in the field of an electromagnet where was it was oscillated at driving frequencies of 1 and 100 rkύlsfτheirf]a^' ge of me ban was magnified and projected onto photocells where the magnitude of displacement of the ball and its phase lag with respect to the driving force were used to calculate the dynamic loss modulus (viscosity) G" and the storage modulus (elasticity) G' of the specimen, each measured in dyne/cm-cm.

Sputum surface mechanical impedance G *s by the rolling ball technique

[0047] The rolling ball technique is used in industry to measure the adhesiveness of glue strips. A steel microprobe is placed on the surface of the sputum in the magnetic microrheometer. The magnetic force needed to roll the sphere across the surface of the sample is used to calculate the surface mechanical impedance (G*s) at 1 rad/s in dyne/cm-cm.

Sputum cohesivity

[0048] Cohesivity is defined as interfacial tension multiplied by the new area created after a test substance is pulled apart. This = 2 x y for ideal or Newtonian fluids where y is the interfacial tension. For a non-Newtonian gel such as mucus, a distraction device (Filancemeter) is used to stretch the mucus until breaking. The measurement is performed with a 25 μl_ mucus sample at a distraction velocity of 10 mm/sec. An electric signal conducted through the sample is interrupted at the point where the stretched mucus thread is broken. Assuming a cone with a mean diameter of 1 mm (circumference of π) to the point of breaking this would mean that cohesivity = y x π x length in mm/100.

Sputum interfacial tension y by distraction (de Nouy ring method)

[0049] A specially instrumented platinum-iridium ring, a metal that is completely wettable, is pulled from the mucus sample at a distraction velocity of 10 mm/sec until separation is achieved. The force of separation is measured by a strain gauge connected to the ring. This gives the work of adhesion, Wad, in dyne/cm. A semi- automated tensiometer was used for these measurements (Fischer Tensiomat Model #21, Fischer Scientific, Pittsburgh, PA). A platinum-iridium ring of circumference 1.7145 ± 0.0381 cm so that with a 0.5 cm was calibrated depth chamber we need only a volume of 0.117 ml to accurately measure sputum interfacial tension.

The transport properties of sputum

In vitro cough clearability (CTR)

[0050] A simulated cough machine was used to measure the airflow-dependent clearability of sputum. A model plexiglas trachea, rectangular in cross section (1.2 x 2 cm) wlsVoiWeicfecrtό a o.^'4 L tank^' containing air pressurized to 12 psi giving a flow rate of 11 L/s. A solenoid valve controlled air release through a flow constrictive element used to mimic the airflow pattern of a natural cough. A sinusoidal constriction (length 7.7 cm and height 8 mm) was used to decrease the airway diameter while minimizing the Reynolds number and thus the turbulence of the system. Peak linear air velocity was about 120 m/s. A sample, 40 μl_ in volume and 0.5 mm in depth, was placed in a thin line across the base of the plexiglas trachea. The bulk transport of the sample was measured in mm after each cough maneuver.

In vitro mucociliary transportability (MCTR)

[0051] A mature leopard frog was pithed and the jaw was disarticulated and the palate removed by cutting through from the junction of the posterior pharynx and esophagus out to the skin of the back. The excised palate was placed on a piece of gauze saturated with amphibian Ringer's solution and allowed to rest in a refrigerator at 4 degrees C for 12-18 hours to deplete of mucus. The palate was then placed in a box with a fitted glass top where humidity was maintained at 95-100% and temperature kept at 24 degrees C. The palate was focused under a microscope so that a 5 mm micrometer scale ran between the optic bulges to the opening of the esophagus. The movement of a 5 μl_ sputum specimen was timed as the trailing edge moves across a 3 mm segment. Three to six measurements of mucus transport rate were taken to minimize variability and the mean transport rate was normalized to the transport rate for collected endogenous frog mucus. Frog palate measurement of mucociliary transportability was approved by the Institutional Animal Care and Use Committee.

Statistical analysis

[0052] Data were analyzed using the StatView™ 5 statistics package (SAS Institute, Cary, NC). All raw data were visually confirmed to be normally distributed about the mean. ANOVA was used to compare results of treating sputum either with modified amphibian Ringer's solution, with dornase alfa, or with different concentrations of Tβ4. Fisher's protected least significant difference (PLSD) test was done to determine significance with multiple comparisons. All data are presented as group means ± 1 standard deviation. By convention p < 0.05 was considered statistically significant.

Results

Laser scanning confocal microscopy (LSM)

[0053] All sputa demonstrated staining for filamentous actin (F-actin). There was a log linear correlation between sputum cohesivity and filament length calculated from ac n p i l (T θ ; = 0.0028).

Biophysical properties

Cohesivity

[0054] There was a significant decrease in sputum cohesivity compared with amphibian Ringer's treated specimens (28.4 mm) after either dornase alfa 30 μg/ml (23.4 mm, p = 0.003) or after 3, 30 or 150 μg/ml of Tβ4 (23.7, 23.9., and 22.1 mm respectively - p < 0.03 for each). There was a significant (p = 0.0007) dose dependent reduction in cohesivity, with a depolymerization threshold between 0.3 and 3 μg/ml. There was also a time dependent decrease in cohesivity with Tβ4 treatment (30 μg/ml) when compared with amphibian Ringer's solution (p < 0.01). Consistent with depolymerization kinetics, the greatest decrease occurred within the first 10 minutes of exposure.

Bulk and surface rheology

[0055] Both bulk or viscosity (G^" at 1 rad/sec, below), and surface rheology (G*s at 1 rad/sec, below) decreased by about half after exposure to amphibian Ringer's solution. This decrease took place within the first 10 minutes after exposure and remained unchanged after this for a one hour observation period and is consistent with rapid hydration of the specimen. TE4 treatment (30 μg/ml, N = 6 at each time) produced a greater decrease in G^', G^", and G*s which was significantly different from Ringer's control only at 1 hour.

[0056] There was no dose dependent change in bulk or surface rheology with TB4 when compared with control. However with the combination of dornase alfa and Tβ4 3 μg/ml each at one half volume, there was a 70% in G*s at 1 rad/sec (p = 0.017) and a 65% decrease in G^' at 1 rad/sec (p = 0.013).

Mucociliary and cough transportability (MCTR and CTR) and interfacial tension (Table 1, below)

[0057] There was a 44% increase in CTR (p = 0.037) relative to Ringer's control with the combination of dornase alfa and TB4 each at 3 μg/ml but no significant change with dornase alfa or Tβ4 alone at any dose. Similarly there was a 71% increase in MCTR (p = 0.013) with the combination of dornase alfa and Tβ4 each at 3 μg/ml but no significant change from amphibian Ringer's control with dornase alfa or Tβ4 alone. There was no significant change in MCTR or CTR with any duration of exposure to Tβ4 at 30 μg/ml when compared with amphibian Ringer's control. There was no significant dose- or time-dependent change in interfacial tension with exposure to Tβ4 in vitro. [0058] Thymosin β4 is a regulator of actin assembly whose major function is the sequestration of G-actin monomers. Isolated from mammalian thymus in a search for immunoregulatory peptides, it was originally proposed that Tβ4 might function as a thymic hormone based on its ability to induce the expression of terminal deoxynucleotidyl transferase in T-lymphocytes, and to inhibit macrophage migration independent of antigen. Related studies indicated that Tβ4 is expressed ubiquetously in other mammalian cells such as neutrophils and macrophages at concentrations up to 200 μM and at concentrations as high as 600 μM in platelets. TB4 is the most abundant G-actin-sequestering molecule in eukaryotic cells, and sequesters over 85% of the G- actin in the cytoplasm and can depolymerize F-actin.

[0059] In the lungs of patients with CF significant quantities of actin sputum and DNA are released into the extracellular lung fluids as cells die during episotic bacterial, fungal, and viral infections and inflammation of the lungs. It is generally accepted that the "rigidity" of the spectrum of CF patients is due in part to the accumulation of these molecules.

[0060] Cohesivity is the internal force of attraction between similar molecules in a fluid or gel. Because polymerization is one of the strongest attractive forces between like molecules, depolymerization of macromolecules that contribute to cohesive forces should have a profound effect on sputum cohesivity. In concentrated polymer solutions consisting of a single species, cohesivity is proportional to the 3.4-power of the molecular weight of the monomer forming monomolecular linear polymers. In the study reported here, it was determined that there is a log-linear relationship between actin filament length and cohesivity. Actin depolymerization was most probably responsible for the threshold decrease in cohesivity with Tβ4 at concentrations greater than 3 μg/ml and for the predominant decrease in cohesivity occurring within the first 10 minutes of exposure.

[0061] Synergy was indicated with the combination of Tβ4 and dornase alfa at a concentration ratio of about 1:1 by weight, e.g., at 1.5 μg/ml each. F-actin and DNA copolymerize in sputum stabilizing the polymer structure. The additive effect of Tβ4 and DNase at lower concentrations might be explained by enhanced depolymerization of F- actin in combination with severing DNA. As well, because dornase alfa also binds G- actin, which in turn inactivates the DNase activity, this synergy might also be due to enhancing DNase activity by blocking the formation of actin-DNase complexes. [0062] Mucociliary clearability on the frog palate (MCTR) is a bioassay that yields information on the interaction between mucus and cilia. MCTR decreases as cohesivity / _U or viscosity increases. The MCTR assay also provides us with information about the interaction of sputum with beating cilia. The usual range of MCTR for CF sputum, normalized to endogenous frog mucus, is 0.6 to 0.7. The MCTR of 0.77 observed after amphibian Ringer's solution is at the upper end of this range. The MCTR of uninfected respiratory mucus from man or dog is 0.85 to 1.0. This was achieved after both dornase alfa 30 μg/ml (0.95) and after 30 μg/ml and 150 μg/ml treatment with Tβ4 (0.93 and 0.87 respectively).

[0063] Cough transportability (CTR) measures how clearable secretions are on a plexiglas "trachea" when exposed to air flow. The typical CTR of CF sputum is 15 to 25 mm increasing slightly after treatment with saline. All CTR values observed in these experiments were low, with means less than 19 mm regardless of treatment. CTR is critically dependent on sputum tenacity; the product of adhesivity and cohesivity. Although there was a significant decrease in cohesivity with either Tβ4 or dornase alfa, adhesivity was probably unchanged as reflected in the lack of change in interfacial tension.

[0064] Tβ4 appears to be capable of favorably altering the cohesivity of expectorated CF sputum at dosages that are achievable by aerosol administration. These changes are similar in direction and magnitude to those noted with dornase alfa treatment of these secretions.

Legend for table

[0065] Table 1 : Mucociliary transportability (MCTR) and in vitro cough transportabiliry (CTR) of CF sputum after exposure to thymosin beta 4 (TB4) or dornase alfa (Pulmozyme).

Each sputum specoment (N=6) was divided into 150 meg aliquots Data are presented as mean.

Further information:

[0066] Actin filament length was calculated from spectral intensity in the rhodamine range. Y = -24.101 + 27.358 In (X). R² = 0.34, p = 0.0028.

[0067] Sputum cohesivity was decreased in a dose dependent manner by Tβ4. The log regression is shown (N=17 each; r² = 0.183, p = 0.0007). Analysis were made using 150 μL of sputum layered with 30 μL of test substance and incubated for 30 minutes at 37degrees C. Kinetics suggest a depolymerization threshold between 0.3 and 3 μg/ml concentrations of Tβ4 with a small dose dependent effect at higher concentrations. The cohesivity of sputum treated with amphibian Ringer's solution (TB4 = 0 μg/ml),

and Tβ4 and dornase at 1.5 μg/ml each

(synergy evaluation) were compared.

[0068] There was a significant decrease in cohesivity with Tβ4 (30 μg/ml) treatment when compared with amphibian Ringer's solution at each time tested (N = 6 each) with the greatest decrease occurring within the first 10 minutes of exposure.

[0069] Viscosity (G^" 1 rad/sec) decreased by about half after exposure to amphibian

Ringer's solution and remained unchanged for a one hour observation period. Tβ4 treatment (30 μg/ml, N = 6 at each time point) produced a greater decrease in G^" which was significant by 1 hour (p = 0.01).

[0070] Surface mechanical impedance (G*s 1 rad/sec) was significantly decreased by

Tβ4 treatment (30 μg/ml, N = 6 at each time point) by 20 minutes of exposure (p = 0.03 at 20 minutes and 1 hour when compared with amphibian Ringer's solution).

Table 1

Claims

1. A pharmaceutical combination for decreasing cohesivity of pathological respiratory tract secretions of a subject, comprising dornase alpha and a peptide agent comprising amino acid sequence LKKTET or LKKTNT, a conservative variant thereof, or a stimulating agent that stimulates production of an LKKTET or LKKTNT peptide, or a conservative variant thereof, in said subject, wherein said dornase alpha and said peptide agent are in a respective ratio in said combination of from about 1.7:1 to about 0.1 :1 by weight, wherein said dornase alpha and said peptide agent may be administered to said subject separately or together.

2. The combination of claim 1 wherein said dornase alpha and said peptide are present together in a pharmaceutical composition.

3. The combination of claim 2 wherein said peptide comprises amino acid sequence LKKTET, LKKTNT, KLKKTET, LKKTETQ, and N-terminal variant of Tβ4, a C-terminal variant of Tβ4, and isoform of Tβ4, oxidized Tβ4, Tβ4^ala, Tβ9, Tβ10, Tβ11 , Tβ12, Tβ13, Tβ14, Tβ15, gelsolin, vitamin D binding protein (DBP), profilin, cofilin, depactin, vilin, fragmin, severin, capping protein,, β-actinin or acumentin.

4. The combination of claim 3 wherein said dornase alpha and said peptide agent is present in said pharmaceutical composition in a respective ratio of from about 1.5:1 to about 0.5:1 by weight.

5. The combination of claim 4 wherein said respective ratio is from about 1.4:1 to about 0.6:1 by weight.

6. The combination of claim 5 wherein said respective ratio is from about 1.3:1 to about 0.7:1 by weight.

7. The combination of claim 6 wherein said respective ratio is from about 1.2:1 to about 0.8:1 by weight.

8. The combination of claim 7 wherein said respective ratio is from about 1.1 :1 to about 0.9:1 by weight.

9. The combination of claim 8 wherein said peptide agent is Tβ4.

10. The combination of claim 9 wherein said respective ratio is about 1:1 by weight. " ^{"' "} e co bin^fion of claim 2 wherein said dornase alpha and said peptide agent are each present in said pharmaceutical composition at a concentration within a range of from about 0.01 mcg/ml to about 1 ,000 mcg/ml.

12. The combination of claim 2 wherein said dornase alpha and said peptide agent are each present in said pharmaceutical composition at a concentration within a range of from about 0.01 mg/ml to about 1 ,000 mg/ml.

13. The combination of claim 2 wherein said pharmaceutical composition is an aerosol.

14. A method of treatment for decreasing cohesivity of pathological respiratory tract secretions of a subject, comprising administering to said subject the pharmaceutical combination of claim 1.

15. The method of claim 14 wherein said dornase alpha and said peptide are present together in a pharmaceutical composition.

16. The method of claim 15 wherein said pharmaceutical composition is administered to said subject as an aerosol by inhalation.

17. The method of claim 16 wherein each of said dornase alpha and said peptide agent is present in said composition at a concentration within a range of about 0.001 - 45% by weight.

18. The method of claim 16 wherein said dornase alpha and said peptide agent are present in said composition in a respective ratio from about 1.5:1 to about 0.5:1.

19. The method of claim 18 wherein said ratio is about 1 :1 , and said peptide agent is Tβ4.

20. The method of claim 19 wherein said pathological respiratory tract secretions are a result of cystic fibrosis in said subject.