WO1996011016A1

WO1996011016A1 - TREATMENT OF OBSTRUCTIVE AIRWAY DISEASE BY ADMINISTERING THYMOSIN β4, OR COADMINISTRATION OF THYMOSIN β4 AND DNase I

Info

Publication number: WO1996011016A1
Application number: PCT/US1995/012543
Authority: WO
Inventors: David R. Crockford; Bruce K. Rubin; Michael L. Berman; Allan L. Goldstein; Christian Bauman; Arnon Kater
Original assignee: Alpha 1 Biomedicals, Inc.; The George Washington University Medical Center
Priority date: 1994-10-06
Filing date: 1995-10-06
Publication date: 1996-04-18
Also published as: AU3733295A

Abstract

A method of treating obstructive airway disease (OAD) such as cystic fibrosis involves contacting OAD sputum with a viscoelasticity-reducing amount of Thymosin β4, or a combination of Thymosin β4 and DNase I.

Description

TREATMENT OF OBSTRUCTIVE AIRWAY DISEASE BY ADMINISTERING THYMOSIN B4, OR COADMINISTRATION OF THYMOSIN B4 AND DNase I

The present invention relates to methods and compositions for treating obstructive airway disease in mammals.

Description of Background Art

Obstructive airway disease (OAD) encompasses a number of respiratory disorders and is associated with viscoelastic secretions or exudate (sputum) in

the patient's airways which contribute significantly to respiratory distress and

may also contribute to progressive lung destruction.

OAD sputum is a complex material known to contain DNA and other materials, including proteins such as actin. OAD sputum is produced in

patients with cystic fibrosis (CF), and may also be produced in patients with

various forms of bronchitis, bronchiolitis, pneumonia, asthma, sinusitis,

bronchorrhea, adult respiratory distress syndrome (ARDS), empyema,

bronchiectasis, bronchocoele and emphysema.

Recombinant human DNase I (rhDNase I) has been reported to diminish viscosity of CF sputum in vitro (Shak et al. , PNAS USA, 57:9188-9192 [1990]).

Human DNase I has been approved in the United States for treating certain CF patients.

Thymosin β₄ (T β₄) is a peptide which has been reported as containing 43 amino acids. Amino acid sequence information on Tβ₄ is disclosed in U.S.

Patent No. 4,297,276, herein incorporated by reference.

Tβ₄ has been found to be present in numerous tissue types in mammals and has also been implicated in a wide variety of cellular and physiological

processes including actin sequestration within cells, inducing terminal

deoxynucleotidyl transferase activity of bone marrow cells, stimulating secretion of hypothalamic luteinizing hormone releasing hormone and luteinizing hormone, inhibiting migration and enhancing antigen presentation of

macrophages, and inducing phenotypic changes in T-cell lines in vitro.

There remains a need in the art for new methods of treating OAD.

Summary of the Invention

In accordance with the present invention, methods and compositions for treating OAD in a mammal utilize Tβ₄ or co-administerion of Tβ₄ and DNase I to the mammal.

Brief Description of the Drawings

Fig. 1 shows amino acid sequences ID Nos. 1-16, respectively, of Tβ₄ compounds useful in the invention.

Fig. 2 A is a bar graph showing effect of Tβ4 on OAD sputum viscosity. Fig. 2B is a bar graph showing the effect of Tβ₄ and DNase I on storage modulus of OAD sputum at 1 radian/sec.

Fig. 3A is a graph showing Tβ₄ and actin depolymerization.

Fig. 3B is a bar graph showing the effect of Tβ₄ and DNase I on storage modulus of OAD sputum at 100 radian/sec.

Fig. 4 is a bar graph showing the effect of Tβ₄ and DNase I on the vectorial sum of storage modulus and loss modulus of OAD sputum at 100 radian/sec.

Fig. 5 is a bar graph showing the effect of Tβ₄ and DNase I on OAD sputum loss modulus at 1 radian/sec.

Fig. 6 is a bar graph showing the effect of Tβ₄ and DNase I on the ratio of loss modulus to storage modulus of OAD sputum at 100 radian/sec.

Fig. 7 is a bar graph showing the effect of Tβ₄ and DNase I of the ratio of loss modulus to storage modulus of OAD sputum at 1 radian/sec.

Fig. 8 is a bar graph showing the effect of Tβ₄ and DNase I on the vectorial sum of storage modulus and loss modulus of surface OAD sputum at 1 radian/sec. Fig. 9 is a bar graph showing the effect of Tβ₄ and DNase I on the vectorial sum of storage modulus and loss modulus of internal OAD sputum at 1 radian/sec.

Fig. 10 is a bar graph showing the effect of Tβ₄ and DNase I on OAD sputum loss modulus at 100 radian/sec.

Description of the Preferred Embodiments

One embodiment of the present invention involves administration of Tβ₄ to mammals to treat OAD including respiratory disorders such as acute and chronic respiratory distress syndromes, chronic bronchitis, asthma, emphysema and cystic fibrosis. Without being bound to any particular theory, it is believed that these respiratory disorders may be associated with excess actin

polymerization, i.e., polymerization of G-actin (monomeric form) into F-actin.

The terms "Thymosin β₄" and "Tβ₄" refer to peptides having the amino acid sequence disclosed in U.S. Patent No. 4,297,276, supra.

According to one aspect of the present invention, effective amounts of Tβ₄ are administered to a mammal, such as a human patient having a respiratory disorder, so as to depolymerize F-actin, or alternatively prevent G-actin polymerization. Such effective amounts can be referred to as actin-antipolymerizing amounts.

Thus, Tβ₄ can be utilized in accordance with the present invention to treat respiratory disorders mediated by excess actin polymerization.

Accordingly, Tβ₄ can be utilized to treat patients having a respiratory disorder selected from the group consisting of acute and chronic respiratory distress syndromes, and advantageously can be utilized to treat chronic bronchitis, asthma, emphysema and cystic fibrosis.

A preferred embodiment of the present invention involves treating cystic fibrosis with Tβ₄. Patients with cystic fibrosis accumulate thick secretions (sputum) in their airways that cause progressive pulmonary destruction. Cystic fibrosis sputum is a complex material, but a major cause of its thick consistency is pus, derived from masses of degenerating leukocytes. Treatment of cystic fibrosis in accordance with this embodiment involves administering to a CF patient a sputum viscosity-reducing amount of Tβ₄.

Since filamentous actin may be responsible for at least some of the viscosity of cystic fibrosis sputum, the amount of Tβ₄ administered to a CF patient may be characterized as an actin-antipolymerizing amount thereof.

Effective dosage amounts of Tβ₄ for treatment of respiratory disorders including acute respiratory distress syndrome, chronic bronchitis, asthma, emphysema and cystic fibrosis, are generally less than about 10 mg/kg of body weight of the recipient, and are preferably within the range of from about 100 μg/kg to about 1 mg/kg. A dose can be administered to the patient daily, one or more times per day of administration, e.g., one to six times or more per day, and doses can be administered one or more days per week, e.g., two, three, four, five, six or seven days per week. The precise dose administered will depend on the age, condition and other factors of the recipient.

According to preferred embodiments of the present invention, compositions containing Tβ₄ may be formulated in a conventional manner for administration by any suitable route. Preferred methods of administration include inhalation of a composition containing Tβ₄ into the patients' lungs through the mouth and/or nose. In this embodiment, the Tβ₄ composition can be an aerosol.

Other preferred routes of administration may include injection/infusion (including parenteral, subcutaneous, intramuscular, intravenous and

intradermal). It will be appreciated that the preferred route may vary with the condition and age of the recipient.

Alternatively, oral or other routes of administration may be utilized.

In preferred embodiments, Tβ₄ is administered as part of a

pharmaceutical formulation. The formulations of the present invention comprise Tβ₄ together with one or more pharmaceutically acceptable carriers. The carrier(s) are "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The formulations include those suitable for inhalation, injection/infusion (including parenteral, subcutaneous, intramuscular, intravenous and intradermal) or other routes of administration. The formulations may conveniently be presented in unit dosage form, including aerosol, liquid, solid, or powered unit dosage form, and may be prepared by any suitable pharmaceutical method.

Such methods include, but are not limited to, the step of bringing into association Tβ₄ with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association Tβ₄ with liquid carriers or finely divided solid carriers or both.

Formulations of the present invention suitable for oral administration may be presented as discrete units each containing a predetermined amount of Tj3₄; as an aerosol; as a powder; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion, etc.

Aerosols suitable for inhalation generally contain liquid or solid particles of less than about 100 microns in size, preferably less than about 50 microns in size, and more preferably less than about 25 microns in size. In particularly preferred embodiments, the aerosol particle size is in the range of about 0.1-10 microns, more preferable less than about 4 microns, and most preferable about 0.1-3 microns.

Formulations suitable for injection/infusion, or parenteral administration, include aqueous and non-aqueous sterile injection solutions which may optionally contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with body fluids of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, aad may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous aerosol or injection solutions or suspensions may be prepared from solid or liquid formulations of the kind previously described.

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other suitable agents having regard to the type of formulation in question, for example, those suitable for oral administration may include flavoring agents.

According to one embodiment of the present invention, the

viscoelasticity of mammalian OAD sputum is reduced by contacting OAD sputum with a viscoelasticity-reducing amount of a combination of Tβ₄ and DNase I.

The term "DNase I" refers to peptides having the amino acid sequence disclosed in Shak et al., supra, and elsewhere.

Without being bound to any particular theory, the high viscoelasticity of OAD sputum may be due to interaction between DNA and excess polymerized actin (F-actin) formed by polymerization of G-actin (monomeric actin).

While DNase I is known to reduce viscosity of sputum from CF patients, and Tβ₄ is a known actin sequestration protein, it has surprisingly been found that combinations of Tβ₄ and DNase I reduce OAD sputum viscoelasticity to a significantly greater extent than would be expected from corresponding individual amounts of Tβ₄ and DNase I.

A preferred embodiment of the present invention involves treating OAD by contacting OAD sputum with a viscoelasticity-reducing amount of a combination of Tβ₄ and DNase I. Patients with obstructive airway disease such as CF accumulate thick secretions of sputum in their airways that cause progressive pulmonary destruction. As noted above, OAD sputum is a complex material, but a major cause of its thick consistency in CF is pus, derived from masses of degenerating leukocytes. Treatment of OAD in accordance with this embodiment involves administering to a human OAD patient a pharmaceutical formulation including a viscoelasticity-reducing amount of a combination of Tβ₄ and DNase I in a pharmaceutically-acceptable liquid carrier. In preferred embodiments, the liquid carrier is an aqueous carrier, e.g. , water for injection, and may contain anti-oxidants, buffers, bacteriostats, antibiotics, solutes, and/or other ingredients.

In particularly preferred embodiments, the inventive pharmaceutical formulation including Tβ₄ and DNase I is administered to an OAD patient by introducing the formulation into one or more airways of the patient so as to contact the formulation with OAD sputum present in the patient's airways. Preferred methods of administration including inhalation of the inventive pharmaceutical formulation into the patient's lungs through the patient's mouth and/or nose. In this embodiment, the inventive formulation can be an aerosol. Injectable or infusible compositions may also be administered, either

concurrently, separately or alone.

Effective amounts of Tβ₄ are amounts sufficient to depolymerize F-actin in OAD sputum or, alternatively, prevent G-actin polymerization in OAD sputum. Such effective amounts can be referred to as actin-antipolymerizing amounts.

Effective amounts of DNase I are capable of further reducing the viscoelasticity of OAD sputum in conjunction with Tβ₄ by cleaving elongate strands of DNA present in OAD sputum and/or further preventing

polymerization of actin. Tβ₄ may also enhance DNase I activity in cleaving DNA by preventing DNase I from binding to actin.

In preferred embodiments, the Tβ₄ and DNase I are each present in the inventive pharmaceutical formulation in a respective ratio of from about 1:2 to about 2:1, more preferably at a ratio of about 1:1.

In accordance with one embodiment, the concentration of Tβ₄ in the inventive formulation is within a range of about 0.1-200 mcg/ml, preferably about 0.3-150 mcg/ml, more preferably about 0.5-30 mcg/ml, even more preferably about 1-10 mcg/ml, still more preferably about 2-5 mcg/ml, and most preferably about 3 mcg/ml.

The concentration of DNase I in the inventive formulation can be within a range of about 0.1-200 mcg/ml, preferably about 0.3-150 mcg/ml, more preferably about 0.5-30 mcg/ml, even more preferably about 1-10 mcg/ml, still more preferably about 2-5 mcg/ml, and most preferably about 3 mcg/ml.

In accordance with another embodiment, the concentration of Tβ₄ in the inventive formulation is within a range of about 0.1-10 mg/ml, preferably about 0.3-7 mg/ml, more preferably about 0.5-5 mg/ml.

The concentration of DNase I in the inventive formulation can be within a range of about 0.1-10 mg/ml, preferably about 0.3-7 mg/ml, more preferably about 0.5-5 mg/ml.

In accordance with one aspect, the inventive formulation is administered to an OAD patient so that about 0.5-10 mg per day of each of Tβ₄ and DNase I is administered to the patient, and preferably about 2.5-5 mg per day of each is administered to the patient. The daily dose can be administered to the patient all at once, or portions of the daily dose can be administered in a regimen spread over the day, e.g. , in increments of one to six times or more per day. Furthermore, doses can be administered one or more days per week, e.g. , 2, 3, 4, 5, 6 or 7 days per week. The precise dose administered will depend on the age, condition and other factors of the recipient.

The present invention is also directed to pharmaceutical formulations comprising an OAD sputum viscoelasticity-reducing amount of a combination of Tβ₄ and DNase I. As indicated above, the preferred inventive formulations are aerosols suitable for inhalation. Such aerosols generally contain particles (preferably liquid) of less than about 100 μ in size, preferably less than about 50 μ in size, and more preferably less than about 25 μ in size. In particularly preferred embodiments, the aerosol particle size is in the range of about 0.1-10 μ, more preferably less than about 4 μ, and most preferably about 0.1-3 μ.

The invention is applicable to native (i.e., naturally occurring) Tβ₄ as well as synthetic Tβ₄ and recombinant Tβ₄ having the amino acid sequence of native Tβ₄, biologically active amino acid sequences substantially similar thereto, or a biologically active abbreviated sequence form thereof, and their biologically active analogs (including muteins) having substituted, deleted, elongated, replaced, or otherwise modified sequences which possess bioactivity substantially similar to that of native Tβ₄. Representative sequences are shown in Fig. 1.

The invention also is applicable to native (i.e. , naturally-occurring) human DNase I, as well as other DNase I peptides which are compatible with human patients, along with synthetic DNase I and recombinant DNase I having an amino acid sequence of native DNase I, biologically-active amino acid sequences substantially similar thereto, or a biologically-active abbreviated sequence form thereof, and their biologically-active analogs (including muteins) having substituted, deleted, elongated, replaced or other modified sequences which posses bioactivity substantially similar to that of human DNase I.

The following examples are for illustrative purposes only, and are not to be construed in a limiting sense.

Example 1

Synthetic Tβ₄ was provided by Alpha 1 Biomedicals, Inc. (Two

Democracy Center, 6903 Rockledge Drive, Ste. 1200, Bethesda, Maryland 20817). Tβ₄ was prepared by solid phase peptide synthesis.

Methods

CF Sputum Viscosity Assay

For measuring the difference in viscosity between the samples incubated with Tβ₄ and water an apparatus was utilized that was used in a sliding assay which measured a rate of migration of sputum samples that were treated with varying amounts of Tβ₄ and corresponding water controls. The apparatus was a grooved plastic surface that could lie in a flat position and upon addition of samples be turned upright at a right angle and the sliding of the sample was measured (a modified tube gel casting stand). The surface was coated with silicon-spray to compensate for any variations of the surface of the apparatus. The migration distance of the apparatus was 6.9 cm.

Each sample contained 100 ug of sputum. The sputum was spread on a plate and the 10 ug samples were cut and weighed and placed in a siliconized eppendorf tube. For each sample varying amounts of Tβ₄, as indicated below, was added to the 100 ug of sputum. For each Tβ₄ sample a corresponding control was done that contains an equal volume of water that was added to the Tβ₄ sample. Samples were incubated for 1 hour at 37°C. Samples were then placed on the apparatus and the migration distance was measured for 3 min. and a migration rate then calculated in mm/ sec.

DNasel Assay with Tβ₄

In defining the relationship between Tβ₄ and actin, an assay was used that utilizes the ability G-actin to bind to and inhibit DNasel, in a one to one stoichiometric fashion. This relationship was used to indirectly measure the presence of G-actin in solution. The assay was a simple spectrophotometric assay that measured a relative change in absorbance at 260 nm. DNasel will digest DNA in solution causing an increase in absorbance at 260 nm. G-actin inhibits this digestion, therefore inhibiting the change in absorbance at 260 nm. Buffers

DNA Buffer - 0.1 M Tris/4 mM MgSO₄/1.8 mM CaCl₂.pH 7.5.

DNasel Buffer - 50 mM Tris/10 μM PMSF/100 mM CaCl₂.pH 7.5.

G-Actin Buffer - 2mM Tris/0.5 mM DTT/0.5 mM ATP/0.2 mM CaCl₂/0.01 % NaN₃

Polymerization buffer - 75 mM Immidazole/0.3 m K Cl/6 mM MgCl₂

The assay was standardized before it was used for experiments with samples. Several tests were done using various amounts of DNasel and DNA to define the assay system. The optimal conditions were defined for the DNasel assay using the following method. DNA (Sigma Chemical Co. from bovine) was diluted to an absorbance at 260 nm of approximately 0.9 (0.1 mg/ml). DNasel (Sigma Chemical) was diluted to absorbance of 0.04-0.06 units/min. (4-5 ul of 1 mg/ml). 10 μM of G-actin (in most samples) was incubated with 1/3 volume of polymerization buffer to polymerize the G-actin. 10 ul of polymerized actin (F-actin) was incubated with varying amounts of Tβ₄ protein. Tβ₄ was allowed to incubate with F-actin for 1 hour at room temperature. After each of the sample incubations, 10 ul of DNasel was added to a quartz cuvette and the actin/Tβ₄ sample was added to the cuvette containing the DNasel. This was allowed to incubate at room temperature for 10 minutes. Then 1 ml of DNA was added to the cuvette and the absorbance at 260 nm was measured every 30 seconds for 3 minutes.

Results and Conclusions

Sputum migration assays

Fig. 2 A represents a typical experiment with the CF sputum. From this data it can be seen that Tβ₄ significantly decreased the viscosity at doses 20 ug, 40 ug, and 100 ug. The migration rate measurements at higher volumes (the 150 ug measurement) tended to skew results because of the volume of liquid added to the samples. In samples that had volume increases of over 10% of total volume, the water added decreased the viscosity of the sample. The measurement at the 60 ug sample occurred approximately every 10 samples. In this case the water control slid faster than the treated sample. This was due to the thickness of the sputum before incubation; not every sputum sample was the same density.

Despite a few inconsistencies, the Tβ₄ had a significant effect on the sputum samples. This preliminary data was also supported by the following data from the in vitro DNasel assay with Tβ₄ and F-actin. The results can be seen in Fig. 2 A.

From the Fig. 3 A data it can be seen that with an increase of Tβ₄ there was a decreased percentage of F-actin in the sample. This was seen in a decreased activity of DNasel. This data demonstrates the ability of Tβ₄ to depolymerize actin filaments. Without being bound to any particular theory, this depolymerization activity may be due to Tβ₄ sequestering G-actin monomers, or Tβ₄ may bind directly to the filament and cause its

depolymerization. Actin monomers (i.e., G-actin) are released into the blood in large quantities in certain disease conditions when there is acute tissue injury or chronic infection. The strong tendency of G-actin to polymerize into long strands of F-actin fibrils in the blood quickly overwhelms the blood's "actin sequestering" system, clogging small capillaries in the lungs and elsewhere.

Tβ₄ and Tβ₄ analogs, homologues and fragments are active in the blood where they prevent G-actin from forming capillary-clogging F-actin filaments.

In the lungs, in patients with chronic respiratory distress syndromes, the changes in the microvascular capillaries due to excess F-actin and actin complexes results in severe lung injury and inflammation. In diseases such as acute respiratory distress syndrome (ARDS), this microvascular pathology may be due to activation of the inflammatory cascade, particularly by by-products of bacterial infections such as endotoxin. Inhibitors of the inflammatory cytokines IL-1 or TNF do not appear to significantly improve survival or long-term outcome. Cell death, with the release of actin and the polymerization of G-actin into long fibrils that produce microangiopathy, leads not only to vascular occlusion, but to activation of complement, and elaboration of a large number of inflammatory mediators. Tβ₄ treatment is directed to the role that actin, and particularly actin polymerization, plays in situ in patients with cystic fibrosis, asthma, or other pulmonary diseases, who have increased turnover of airway cells associated with chronic airway inflammation and regeneration.

Without being bound to any particular theory, it is believed that the physiological conditions necessary for actin polymerization exist in the airway, and further that the Tβ₄ actin-scavenging and sequestering system also operates across the blood-airway barrier. In the laboratory, Tβ₄ has been shown to significantly reduce the toxicity and severity of septic shock and endotoxin-induced death in rodent experiments, and to down-regulate a number of cytokines associated with inflammatory diseases such as IL-1, IL-6, TNF-α, and PAF. Tβ₄ can also down-regulate a number of other inflammatory molecules such as arachidonic acid metabolites, aldehydes, and free-radicals within the cell, and up-regulate glutathione. According to the invention, Tβ_A is believed to be effective in treating the acute and chronic lung diseases identified above, both by reducing the severity of actin toxicity in the blood (by maintaining actin in its sequestered G-actin form), and by down-regulating a number of cytokines, prostaglandin

intermediates, and free radicals, which in excess are toxic and cause significant inflammation and accumulation of monocytes, neutrophils, and other cells that exacerbate tissue destruction. In preferred embodiments, Tβ₄ is administered by injection or by spraying Tβ₄ directly into the lungs.

Tβ₄ and Tβ₄ analogs, homologues and fragments having Tβ₄ activity appear to have the ability to both sequester actin monomers (G-actin), and down-regulate the major inflammatory cytokines such as IL-1α, IL-6, TNF-α, and PAF; as well as a number of arachidonic acid metabolites such as Txβ₂ and 6-keto-PGF1α; in addition to lipid peroxidation. The cascade of free radicals and inflammatory molecules is deleterious, and contributes to the pathology of the lung diseases described above.

Tβ₄, when sprayed directly into the lungs, reduces inflammation and promotes healing by down-regulating the monocytes, neutrophils, and other white blood cells that exacerbate the inflammatory process. Given

intravenously or by subcutaneous or intramuscular injection, Tβ₄ and Tβ₄ analogs, homologues and fragments reduce the clogging of lung capillaries and thus prevent death and promote healing by down-regulating the inflammatory cytokines and molecules produced during this process.

Example 2

The objective was to determine the effects of Tβ₄ and DNase I on the properties of OAD sputum collected from six patients with stable CF lung disease.

Synthetic Tβ₄ was provided by Alpha 1 Biomedicals, Inc. and recombinant human DNase I (Pulmozyme^®) was obtained from Genentech.

OAD sputum was analyzed untreated, and after the addition of amphibian Ringer's solution (negative control) mixed 1:5 v/v with the sputum, as well as when treated with rhDNase I at 30 mcg/ml, Tβ₄ at 0.3, 3, 30 and 150 mcg/ml, or rhDNase I combined with Tβ₄ at 0.3 mcg/ml each. All specimens were incubated at 37 °C for 30 minutes. Measurements were taken using a magnetic microrheometer. As described in King and Rubin, "Rheology of Airway Mucous" in Airway Secretion, Publisher Marcel Dekker, Inc., New York, Editors Takishima and Shimura, pages 283-314 (1994), the magnetic microrheometer measures viscoelastic properties of very small quantities of sputum. A steel ball was positioned in a 1-5 mcl sample of sputum and oscillated by an electromagnet at two different driving frequencies. The magnitude of displacement of the ball and its phase lag relative to the driving force were used to calculate the viscoelasticity of the sputum. The parameters measured were as follows:

G' is storage modulus (elasticity) measured in dynes/cm²;

G" is loss modulus in dynes/cm²; loss modulus multiplied by rotational velocity corresponds to viscosity;

G* is mechanical impedance within a sputum sample, measured in dynes/cm², and is the vectorial sum of G' and G";

Gs* is surface mechanical impedance, i.e. , mechanical impedance measured on the surface of a sputum sample in dynes/cm², and is the vectorial sum of G' and G"; and

tan δ is a ratio of G'/G' .

For each sample, measurements were taken utilizing the magnetic microrheometer described in King and Rubin, supra, at a driving frequency of 1 radian/sec. (e.g. , G' 1) corresponding to normal ciliary beats, and 100 radian/sec. (e.g. , G' 100) corresponding to clearance by cough.

The results are shown in Tables 1-10 below and Figures 2B, 3B and 4-9, wherein "Groups" represent tested concentrations of rhDNase and/or Tβ₄, "cell" is a test cell in which a drug "Group" is tested, "cell mean" represents the mean average for a particular test "cell" in the units set forth above, and "count" represents sputum from the number of individuals tested. The Ringer's solution was 98.3 mmols/l NaCl, 2.7 mmols/l KCl, and 1.5 mmols/l CaCl. Data analyses were performed using a StatView™ 4 statistics package (Abacus Concepts, Inc., Berkeley, CA) and a Power PC Macintosh^® computer. The results demonstrate a synergistic effect with the combination of Tβ₄ and DNase I.

Without being bound to any particular theory, the synergistic effect on viscoelasticity brought about with a combination of Tβ₄ and DNase I may be explained by an enhanced effect of depolymerizing F-actin along with severing DNA. As DNase I also binds G-actin, which in turn inactivates DNase I activity, this synergy may also be due to enhanced DNase I activity by blocking the formation of actin-DNase I complexes.

While the invention has been described and illustrated with details and references to certain preferred embodiments, those skilled in the art will appreciate that various modifications, changes, omissions, and substitutes can be made without departing from the spirit of the invention.

Claims

What is claimed is:

1. A method of reducing viscoelasticity of sputum of obstructive airway disease (OAD), comprising contacting OAD sputum with a

viscoelasticity-reducing amount of Thymosin β₄ (Tβ₄) or a combination of Tβ₄ and DNase I.

2. The method of claim 1, wherein said Tβ₄ or said combination of Tβ₄ and DNase I are present in a pharmaceutical formulation including a pharmaceutically-acceptable liquid carrier.

3. The method of claim 2, further including the step of

administering the pharmaceutical formulation to an OAD patient by introducing said formulation into an airway of said patient, so as to contact said formulation with said sputum.

4. The method of claim 3, wherein said pharmaceutical formulation is in aerosol form.

5. The method of claim 4, wherein said Tβ₄ and said DNase I are present in said pharmaceutical formulation in a respective ratio of from about 1:2 to about 2:1.

6. The method of claim 4 or 5, wherein the concentration of said Tβ₄ in said formulation is within the range of from about 0.1 mcg/ml to about 10 mg/ml, or the concentrations of said Tβ₄ and said DNase I in said formulation are each within the range of from about 0.1 mcg/ml to about 10 mg/ml.

7. The method of claim 6, wherein said range is about 0.1-10 mg/ml.

8. The method of claim 7, wherein said range is about 0.3-7 mg/ml.

9. The method of claim 8, wherein said formulation is administered to said patient so that about 0.5-10 mg/day of each of said Tβ₄ and said DNase I is administered to said patient.

10. The method of claim 9, wherein said ratio is about 1:1.

11. The method of claim 10, wherein about 2.5-5 mg/day of each of said Tβ₄ and DNase I is administered to said patient.

12. The method of claim 8 or 11, wherein said patient is a cystic fibrosis (CF) patient.

13. The method of claim 12, wherein each said concentration is within the range of about 0.1-200 mcg/ml.

14. The method of claim 12, wherein each said concentration is within the range of 0.1-10 mg/ml.

15. A pharmaceutical formulation for use in reducing viscoelasticity of sputum of OAD, comprising an OAD sputum viscoelasticity-reducing amount of Tβ₄, or a combination of Tβ₄ and DNase I.

16. The pharmaceutical formulation of claim 15, further including a pharmaceutically-acceptable liquid carrier, wherein Tβ₄ is at a concentration within the range of from about 0.1 mcg/ml to about 10 mg/ml, or wherein said Tβ₄ and said DNase I in said combination each have a concentration within the range of from about 0.1 mcg/ml to about 10 mg/ml, and said Tβ₄ and said DNase I in said combination are present in a respective ratio of about 1:2 to about 2:1.

17. The pharmaceutical formulation of claim 16, wherein said range is about 0.3-7 mg/ml.

18. The pharmaceutical formulation of claim 17, wherein said range is about 0.5-5 mg/ml.

19. The pharmaceutical formulation of claim 16, wherein said range is about 0.1-10 mg/ml, said ratio is about 1: 1 and said pharmaceutical formulation is in aerosol form.

20. The pharmaceutical formulation of claim 19, wherein said range is about 0.5-5 mg/ml.