TITLE OF THE INVENTION

USE OF SWINHOLIDE A FOR SEVERING AND DEPOLYMERIZING ACTIN

FIELD OF THE INVENTION This application is a continuation-in-part 5 application of United States Serial No. 08/272,188 filed July 8, 1994.

This invention relates to methods for severing and depolymerizing filamentous actin and to therapeutic methods for conditions or diseases involving pathological

10 accumulations of extracellular actin. In particular, to therapeutic methods to alleviate physiological defects in lung airways associated with some respiratory conditions or diseases. More specifically, this invention provides a method for severing and depolymerizing filamentous actin

15 in viscous sputum which accumulates in the lungs of individuals afflicted with the condition or disease.

BACKGROUND OF THE INVENTION Cystic fibrosis is one of the most common, fatal, genetic diseases in the world today. The life

20 expectancies of those effected with this disease is approximately 28 years. Cystic fibrosis effects some 30,000 children and young adults in the United States and approximately 24,000 children and young adults in Europe. Approximately one new case occurs in every 2,000 births

25 with 1,000 new cases of cystic fibrosis being diagnosed each year (Collins, F.S. (1992) Science, 256:774-779; Boat TF, Welsh MJ and Beaudet AL (1989) , in The Metabolic Basis of Inherited Disease (McGraw-Hill, New York) pp. 2649- 2680) .

3 Cystic fibrosis is an autosomal recessive disease that causes abnormalities in fluid and electrolyte transport in exocrine epithelia in the lung. In cystic fibrosis the luminal border of the airway mucosal cell is unresponsive to cyclic-AMP dependent protein kinase

,._ activation of the membrane chloride ion channels (Rich,

D.P. et al. (1990) Nature 347:358-363; Kartner Ν et al. (1991) Cell 64:681-691; Anderson, M.P. et al. (1991) Science 253:202-205; Tabcharani, J.A. et al. (1991) Nature 352:628-631; Bear, C.E. et al. (1991) J. Biol . Chem. 266:19142-19145; Bear, C.E. et al. (1992) Cell 68:809-818; Denning, G.M. et al. (1992) J ^" . Clin . Invest . 89:339-349) . The cells are impermeable to chloride ions and as a result, sodium absorption across the cell membrane is accelerated. The subsequent electrolyte imbalances tend to reduce the level of hydration of the airway mucous, thus contributing to the viscosity of the lung secretions characteristic of cystic fibrosis. Bacteria and microplasma frequently establish colonies within the mucous. As a result of the viscosity of the mucous, mucosal clearance is reduced in cystic fibrosis patients, and therefore bacterial clearance is also reduced and lung congestion and infection are thus common. The prolonged presence of these pathogenic agents invariably initiates inflammatory reactions that further compromise lung function and result in the accumulation of inflammatory cells and necrotic cellular debris in respiratory passages thereby adding to the viscous lung secretions or mucous. Accumulation of these thick mucosal secretions within the airways obstructs the airways and causes progressive pulmonary destruction. The presence of the viscous mucous or sputum in cystic fibrosis patients presents problems from maintenance therapy. Cystic fibrosis mucous sputum is a complex material but a major cause of its thick consistency is puss derived from masses of degenerating leukocytes (Rosenbluth and Chernick (1974) Arch. Dis .

Child. 49:606; Picot et al. 1978 Thorax 33 :235; Puchelle, E. et al. (1985) Europ. J. of Clin . Invest . , 15:389; Lethern, M.I. et al. (1990) Am. Rev. Respir. Dis. 142:1053) . Leukocyte derived DΝA is thought to contribute to the viscosity of cystic fibrosis sputum. (Libermann et

al. (1968), J ^" . Am. Med. Assoc . 205-312; Shak et al. , (1990) Proc. Natl . Acad. Sci . (USA) 87:9188; Altken et al.

(1992) J. Am. Med. Assoc. 267:1947) . Recently, it has been determined that another major contributing factor to the viscosity of cystic fibrosis sputum is filamentous actin (Vasconcellos, CA. et al. (1994) Science 263:968- 970) . Classical modalities of treating individuals affected with cystic fibrosis include antibiotic therapy, and bronchial dilators (β2 agonists) . Suggested newer therapies include the administration of DNAse to target the DNA rich mucous or sputum (Shak, et al . (1990) Proc. Natl . Acad. Sciences (USA), 87:9188-9192; Hubbard, R.C. et al. (1991) N. Engl . J. Med. 326:812) and gene therapy

(Rosenfeld, MA wt al. (1992) Cell 68:143; Zabner et al. ,

(1993) Cell 75:207) . In vi tro data suggests that gelsolin, a plasma protein, reduces mucous viscosity by severing actin filaments (Vasconellos et al. , (1994) Nature, 263:968-969) . Other respiratory conditions such as chronic and acute bronchitis or pneumonia also exhibit pathologically high viscosities of mucous or sputum in the lungs of those affected. Characterization of additional or more efficacious or alternative therapies to target the mucosal secretions or sputum are needed.

Therapeutic agents that target the cytoskeleton are limited mostly to compounds that affect microtubules. Recently, however, several drugs that directly or indirectly affect the organization of the actin cytoskeleton have been investigated as potential therapies for neoplastic, immunologic and cardiovascular diseases (Senderowicz A.M.J. et al. (1994), Proc. Am. Assoc. Cane. Res . , 35:409; Bubb M.R. et al. (1994), J. Biol . Chem. , 269:4869; Van Leenen, D. et al. (1993), J. Immunol . , 151:2318; Rao, K.M.K. et al. (1988), J ^" . Cell . Physiol . , 137:577; Korthuis, et al. R.J. (1991), J. Appl . Physiol . , 71:1261) . Monomeric macrolides, such as latrunculin and tolytoxin have been shown to disrupt microfilament

organization by sequestering monomeric actin, but they do not sever actin filaments. (Spector, I., et al. (1989) Cell . Mot . and Cytoskel . 13:127-144; Patterson et al. (1993) Cell Mot . and Cytoskel . 24:39-48; Coue, M. , et al. (1987) FEB . Lett, 213 (2) .316-318) . Swinholide A, is a cytotoxic 44-carbon ring dimeric macrolide isolated from the marine sponge, Theonella swinhoei (F. Carmeli and Y. Kashman, (1985), Tetrahedron Lett . , 26:511; Kobayashi M. et al. (1990), Chem Pharm. Bull . 38:2409-2418; Kobayashi M. et al. (1990) , Chem Pharm. Bull . 38:2960; Kitagawa, I. et al. (1990) J. Am. Chem. Soc . 112:3710-3712); Kobayashi, M., et al. (1994) Chem. Pharm. Bull . 42 (1) :19-26) . The present invention demonstrates that Swinholide A is capable of both severing and depolymerizing actin. Among the known drugs that disrupt actin organization only Swinholide A has the ability to both sever and depolymerize actin filaments.

SUMMARY OF THE INVENTION

This invention relates in general to a method of severing and/or depolymerizing filamentous actin by contacting the filamentous actin or a locus containing filamentous actin with an effective amount of Swinholide A or modification thereof.

This invention relates to the use of Swinholide A and modifications thereof in the treatment of mammals afflicted with diseases or conditions in which extracellular actin is pathological.

This invention also relates to the use of Swinholide A and modifications thereof in treatment of mammals afflicted with respiratory conditions or diseases involving thick mucous secretions or sputum accumulations in the respiratory passages.

This invention further relates to the use of Swinholide A and modifications thereof in the treatment of mammals afflicted with cystic fibrosis.

This invention also relates to methods of use of Swinholide A and modifications thereof in isolation and purification of cellular components.

This method also relates to a treatment method comprising administering therapeutically effective amounts of Swinholide A to patients afflicted with diseases characterized by accumulation of inflammatory cells and necrotic cellular debris in respiratory passages resulting in the accumulation of pathological thick mucous or sputum containing filamentous actin.

This invention relates to compositions suitable for pulmonary administration comprising effective amounts of Swinholide A and modifications thereof to sever and depolymerize the filamentous actin present in cystic fibrosis mucous or sputum.

DESCRIPTION OF THE FIGURES

Figures 1A-E show the effect of Swinholide A on mouse fibroblasts. Fluorescence micrographs of Balb/C 3T3 (Fig.lA and Fig.IB) and Swiss 3T3 (Fig.lC and Fig.ID) cells labeled with TRITC-phalloidin. The decrease in fluorescence intensity in (Fig.IB) and (Fig.ID) was much greater than it appears to be because the exposure times were controlled automatically to optimize visualization. (Fig.lA) Control cells grown to high density. (Fig.IB) Cells grown to high density and then treated for 24 h with 50 nM Swinholide A. (Fig.lC) Exponentially growing control cells. (Fig.ID) Exponentially growing cells treated with 10 nM Swinholide A for 24 h. (Fig.IE) The cells shown in (Fig.ID) stained with DAPI; both are binuclear. Bar = 20 μm.

Figures 2A-C shows the sequestration of dimeric actin by Swinholide A. (Fig.2A) . Effect of Swinholide A on the apparent actin critical concentration. Pyrenyl- labeled, gel-filtered skeletal muscle F-actin (J.A. Spudich and S. Watt (1971), J ^" . Biol . Chem. , 246:4865; T. Kouyama and K. Mihashi (1981), Eur. J. Biochem. , 114:33)

in buffer F was diluted to the concentrations shown in the presence of 0 (D) , 0.3 (Δ) , 0.9 (o) or 1.8 (O) μM Swinholide A and the fluorescence determined at steady state. The solid lines show the expected result if Swinholide A binds two actin subunits with K _eq =9.2-10 ¹³ M ^"2 and infinite cooperativity. The deviation of the data for 0.9 μM Swinholide A to the left of the theoretical line implies the probable existence of a small amount of Swinholide A with a single bound actin subunit and finite cooperativity. (Fig.2B) . Sedimentation velocity analysis of the Swinholide A-induced actin dimer. Actin (15 μM) in buffer G was sedimented in a Beckman XLA analytical ultracentrifuge at 53,000 RPM at 18 °C alone (Δ) and with 10 μM Swinholide A (D) . Optical absorbance scans at 290 nm were obtained at 7-min intervals. (Fig.2C) . SDS-PAGE analysis (U.K. Laemlli (1970), Nature, 227:680) of PDM- crosslinked (phenylenebis maleimide) actin. Actin (20 μM) was crosslinked with an equal volume of PDM in 20 mM sodium borate for 5 min at room temperature. Lane 1-3: actin crosslinked in buffer G (actin:PDM=2 :1) in the presence of 0, 5 and 15 μM Swinholide A. Lane 4: actin crosslinked in the absence of Swinholide A by addition of PDM (actin:PDM=2 :1) simultaneously with 2 mM MgCl ₂ . The dimer observed in lanes 2-4 had the same electrophoretic mobility (apparent mass = 86 kDa) as the dimer obtained by crosslinking the gelsolin-actin dimer complex (data not shown) in which the two actin subunits are anti-parallel (Hesterkamp, T. et al. (1993), Eur. J. Biochem. , 218:507) . Lane 5: filamentous actin crosslinked in buffer F (actin:PDM=l:2) in the absence of Swinholide A produced a ladder of actin oligomers including a dimer (apparent molecular mass = 116 kDa) resulting from crosslinking two adjacent parallel subunits across the genetic helix (M. Elzinga and J.J. Phelan (1984), Proc. Natl . Acad. Sci . , 81: 6599) .

Figures 3A-C. Destabilization of F-actin by Swinholide A. (Fig.3A) . Time course of depolymerization of Mg ²⁺ -pyrenyl actin monitored by decrement in fluorescence intensity. Ca ²⁺ -pyrenyl actin in buffer G was converted to Mg ²⁺ -pyrenyl actin with 125 μM EGTA and 50 μM MgCl ₂ , polymerized to give a stock 10 μM F-actin solution in 2.0 mM MgCl ₂ and diluted at time t=0 to a final concentration of 190 nM actin and either 2.0 ( ^{• • •} ) or 0.1 (—) mM MgCl ₂ with either 0 (D) or 158 nM (O) Swinholide A. The samples were mixed by inversion of the cuvettes for 10 seconds (s) and fluorescence intensity was first measured 25 s after dilution. (Fig.3B) . Depolymerization rates of Ca ²⁺ -pyrenyl F-actin. F-actin was diluted, as in (Fig.3A), in varying concentrations of Swinholide A in 0.1 mM MgCl ₂ in the absence (O) or presence (D) of gelsolin-actin dimer added to the stock F- actin at a concentration of 1 complex:20 F-actin subunits immediately prior to dilution. (Fig.3C) . Initial depolymerization rates of Mg ²⁺ -pyrenyl F-actin. F-actin prepared as in Fig. 2A was diluted in a 3-syringe stopped flow apparatus to a final concentration of 400 nM in 2.0 mM MgCl ₂ containing 0 (—), 152 (+++), 280 (•••), 480 (- - - ) , and 1000 (ooo) nM Swinholide A and the decrease in fluorescence measured over time. The injection was complete at t=0.5 s.

Figures 4A-4B show examples of isomers in which the size or the conformation of the macrocyclic dilactone ring has been altered.

DETAILED DESCRIPTION OF THE INVENTION

For the purpose of a more complete understanding of the invention, the following definitions are described herein. Mammal includes, but is not limited to, humans, monkeys, dogs, cats, mice, rats, hamsters, cows, pigs, horses, sheep and goats.

By the term filamentous actin we mean F-actin or

- i/i - polymers of actin or polymeric actin.

- 8 -

By the term effective amount is meant amounts of Swinholide A and modifications thereof sufficient to sever and depolymerize filamentous actin. Preferred Swinholide A effective amounts are in the range of about 5 nM to about 100 μm.

By the term therapeutically effective amount is meant dosages of Swinholide A and modifications thereof administered within pharmaceutical compositions as described herein and resulting in the severing and depolymerization of filamentous actin. The therapeutically effective amount of Swinholide A and modifications thereof will depend, for example, upon the therapeutic objective, the rate of administration and the emulation of the patient. Accordingly it may be necessary for the therapist to titer the dosage and modify the rate of administration and dosage as required to obtain the optimal therapeutic effect.

This invention is meant to encompass the use of Swinholide A (Formula I R ^X =R ² =R ³ = CH ₃ ; Kobayashi, et al. (1994) Chem. Pharm. Bui1. 42(1) 19-26, herein incorporated by reference) and modifications thereof. The term "modification" includes isomers, stereo-isomers, derivatives, homologs, congners, chemical derivatives, and minor modifications, such as esters, ethers and amides, or any other chemical modifications resulting in compounds which are the functional equivalents of Swinholide A as described herein. Swinholide A and modifications thereof may be natural or synthetic in origin.

- 9

In a preferred embodiment this invention may encompass Swinholdes of the Formula I:

OR*

FORMULA I

- 10 -

Wherein, R ¹ =H or optionally substituted lower straight chain or branched chain alkyl, said alkyl having 1 or more carbons, preferably having 1 to 6 carbon atoms and said optional substituents preferably are, for example, hydroxy; OR ⁴ wherein R ⁴ is lower alkyl or halo- alkyl; lower alkyl; haloalkyl; amino; monoalkylamino; dialkylamino; halogen; cycloalkyl; aryl or heteroaryl; R ² , R ³ are each independently H, optionally substituted straight chain or branch chain alkyl, cycloalkyl, aryl, or heteroaryl; said optional substituents being the same as those mentioned hereinabove.

In accordance with the present invention, aryl is preferably phenyl or napthyl, heteroaryl includes, for example, aromatic hetero groups containing 1 or more oxygen, sulfur, or nitrogen atoms. Preferred examples of groups include pyridyl, quinolinyl and indolinyl. Halogen is preferably chlorine, fluorine or bromine.

Since the unusual dimeric ring structure of the Swinholide A molecule probably accounts for its interaction with an actin dimer, and may explain its severing activity, included within the scope of this invention are monomeric lactone macrolides. Examples of such monomeric macrolides include, but are not limited to, a 21-carbon macrolide and 23-carbon macrolides (Kobayashi, et al. (1994) Chem Pharm Bull 42(1) 19-26, herein

- 11 incorporated by reference) . Such monomeric macrolides may have the general Formulas II and III :

FORMULA II

FORMULA III

- 12

Wherein R ² being the same as those mentioned hereinabove.

Also included within the scope of this invention are a group of isomers on which the size or conformation of the macrocyclic dilactone ring is altered. Such derivatives can be obtained by acidic treatment of Swinholide A (Kobayashi, et al. (1994) Chem. Pharm Bull 42(1) 19-26, herein incorporated by reference) which may yield a mixture of compounds. By way of example, such mixtures may include those compounds shown in Figures 4A and 4B and Formula IV below. Such derivatives may have the general Formula IV:

FORMULA IV

Wherein R ² and R ³ are the same as described hereinabove.

Further included within the scope of the invention are several monomeric "preswinholide" molecules

- 13 with open chains in which the spatial structure of the original molecule is lost. These compounds can be synthesized using the procedures described in (Kobayashi, et al. (1994) Chem Pharm Bull 42(1) 19-26, herein incorporated by reference) . Such compounds may have the the general Formula V:

FORMULA V

Wherein R , is the same as described herein and abov .

Wherein R = CH ₂ 0H or COOR ⁵ , and

R ⁵ = H or optionally substituted lower straight chain or branch chain alkyl and the optional substituents are mentioned herein above.

A still further embodiment of this invention is the octaformat which may have the structure shown in formula VI.- By way of example, such compounds may be prepared by reacting Swinholide A with acyl chlorides at about a 2:1 equivalent ratio in pyridene for about 24

- 14 hours at room temperature, or may be prepared by reacting Swinholide A with dibasic acyl chlorides of the general type Z0 ₂ C- ( ) _n -COCl or monobasic acyl chlorides of the ZNH- ( ) _n -C0Cl type where Z is a protecting group which will be removed after the completion of the reaction which may be separated based on their cellular and in vitro effects. Formula VI, as shown below:

Wherein R ⁶ = hydrogen or alkanoyl, preferably Cj - C ₅ alkonoyl, typical formyl, acetyl or propionyl, preferably R ⁷ , R ⁸ are either jointly or independently alkanoyl or alkyl straight chain or branch chain lower alkyl which can be optionally substituted as described hereinabove.

Also intended to be encompassed are epoxidized derivatives of any of the compounds shown as formulae I - VI, in which some or all of the double bonds have been epoxidized as to form either c_, β or γ,δ- epoxides. Furthermore, the epoxy group(s) may be ring-opened to provide hydroxy derivatives.

- 15 -

Epoxidation products of Swinholide A with OH in the presence of H ₂ 0 ₂ will yield a, /3-epoxides, while epoxidation with known epoxidation reagents will yield γ, δ - expoxides.

In a preferred embodiment the Swinholide A modifications are modified so as to be more polar or bulky thus less able to pass through the cell membrane.

By respiratory condition or disease is meant an affliction in a mammal wherein there are accumulations of viscous mucous or sputum or pathological accumulations of mucous or sputum blocking the air passages of the afflicted mammal.

Examples of respiratory conditions or diseases that may be treated by the therapeutic methods described herein include, but are not limited to, cystic fibrosis, pneumonia, acute bronchitis, chronic bronchitis and any respiratory condition characterized by pathological accumulation of sputum resulting in obstruction of the afflicted mammal's airways.

The disclosure demonstrates that Swinholide A, a natural marine product, disrupts the actin cytoskeleton of cells grown in culture, severs F-actin with high cooperativity, depolymerizes actin filaments rapidly when the molar ratio of Swinholide A to actin subunits is approximately one and sequesters actin dimers in both polymerizing and non-polymerizing buffers. The binding stoichiometry is one Swinholide A molecule per actin dimer. One skill in the art will appreciate these characteristics can be varied but still be within the scope of this invention. This invention provides- a method of severing and depolymerizing filamentous actin by exposing or contacting the filamentous actin with an effective amount of Swinholide A or modifications thereof. Examples of filamentous actin-containing biological materials include but are not limited to, a cell, continuous cell lines,

- 16 - primary cultures of cells, cells, detergent-insoluble cell fractions and cell membrane preparations, cellular homogenates, nuclear homogenates, tissues, organs, extracellular actin, mucous or sputum samples isolated from mammals affiliated with respiratory conditions or diseases, and respiratory secretions. Examples of cells include but is not limited to, fibroblasts, neutrophils endothelial, epithelial, leukocytes and platelets. A suitable effective amount of Swinholide A to be used may be in the range of about 5 nM to about 100 μM, the most preferred range is about 30 nM to about 80 μm and the most preferable range is about 50 nM to about 100 nM.

In one embodiment Balb/c 3T3 cells and Swiss Wesbster 3T3 fibroblast cells are cultured under standard condition and exposed to Swinholide. A preferred range of Swinholide A is about 5 nM to about 100 μM. The cells are exposed to tetramethyl rhodaminyl-phalloidin (TRITC- phalloidin, Cano, Manuale et al., Cell Mot . and Cytoskel . (1992) 21:147-158) fluorescent derivative of the F-actin binding toxin, and cytological stains. Severing and depolymerization of actin is assessed by fluorescent microscopy.

The severing and depolymerizing effect of Swinholide A or modifications thereof on filamentous actin may be assessed by conventional methods. Examples of such methods include but are not limited to, electron microscopy (Small, J.V. and Celis, J.E. (1978) Cytobiologie 16:308; Small, J.V. and Celis, J.E. (1978) J ^" . Cell Sci . 31:393); fluorescence microscopy with rhodamine- labeled phalloidin (Wang, K. et al. (1982) Methods in Enzymology 85:514) and immunoflourenee using actin antibodies (Ausebel, et al., (eds) (1987) in "Current Protocols In Molecular Biology") John Wiley and Sons, New York, New York) .

In another embodiment mucous or sputum samples isolated from mammals afflicted with a respiratory

- 17 - condition or disease such as cystic fibrosis are exposed to Swinholide A or modifications thereof. Severing and depolymization of filamentous actin is assessed by the viscolacistity of the samples. Assessment of severing and depolymerization of filamentous actin can also be determined by viscosity measurements of the filamentous actin contacted with Swinholide A or modifications thereof. Viscosity assessment of biological samples can be performed by standard methods. Examples of such methods includes, but is not limited to, falling ball viscometry (Pollard, T.D. (1982) Methods in Cell Biology 24: 301); torsion pendulum assay (Janmey, P.A. (1991) J. Biochem. Biophys . Methods 22:41); elastic shear modulus measurements (Janmey, P.A., et al. (1988) Biochemistry 27:8218), pourability assay (Keal, E.E. (1971) Postgard Med. J. , 47:171-177) Brookfield cone-plate viscometer (Lieberman, J. (1968) Am. Rev. Respir. Dis . , 97:654-661; Lieberman, J. (1968) Am. Rev. Respir. Dis. , 97:662-672).

In yet another embodiment of this invention the actin severing and/or depolymerizing effect of Swinholide A or modifications thereof is used in purification of cellular components or cellular fractions in biological preparations. For example, Swinholide A can be added to a biological preparation thereby allowing for the separation of plasma membranes from associated filamentous actin. Examples of biological preparations include, but is not limited to, cellular homogenates, and membrane fractions prepared from them by differential centrifugation (Miyata H. et al. (1989) J ^" . Cell Biol . 109: 1519; Clarke, BJ et al. (1988) J. Protozool . 35:408) and detergent-insoluble membrane skeletons (Apgar J. (1990) J. Immunol. 45:3814). Suitable concentrations of Swinholide may be in the range of about 5 nM to about 100 μM. Examples of cellular components include, but are not limited to, plasma membranes, nuclear membranes, and cytoskeletons.

- 18 -

Another embodiment of this invention relates to the use of Swinholide A or modifications thereof in the treatment of mammals afflicted with a disease or condition involving pathological extracellular actin. By way of example, Swinholide A or modifications thereof may be used to therapeutically treat mammals afflicted with respiratory conditions or disease. Examples of conditions that can be treated by the therapeutic methods disclosed herein include but are not limited to, pneumonia, acute bronchitis, chronic bronchitis and cystic fibrosis.

In yet a further embodiment of this invention mammals preferably humans afflicted with cystic fibrosis are administered therapeutically effective amounts of Swinholide A which functions to sever and depolymerize the filamentous actin present in the pathological mucous or sputum in their lungs. An individual having cystic fibrosis is initially administered amounts of Swinholide A in the range of about 0.10 mg to about 5 mg per dosage, twice daily. Preferred dosages are in the range of about 0.25 mg to 1.0 mg, twice daily or as determined by the minimum effective concentration from sputum samples. A pharmaceutical composition of the Swinholide A is administered as fluid containing Swinholide A deliverable as a spray from a nebulizer. The Swinholide A composition is preferably to be delivered alone until mucous viscosity is decreased or together with other therapeutic agents such as antibiotics. One skilled in the art will understand that the exact dosage regime may be varied both in frequency and in quantity to a given patient. It is anticipated that this regime will be optimized for an individual in accordance with the conventional evaluation techniques. Efficacy will be determined by assaying for improved lung function in afflicted mammals. This assessment can include viscoelastic measurements of sputum, improvements in pulmonary function including

- 19 - improvements in forced exploratory volume of sputum and maximal midexpirator flow rate. The aforementioned therapeutic regime can be given in conjunction with adjunct therapies such as antibiotics, DNAse I or other current therapies for the treatment of cystic fibrosis. If antibiotics are coadministered as part of the patients therapy, bacterial quantitation following therapy can be included to assess the efficacy of the treatment by decreased bacterial growth indicating decreased viscosity of mucous or sputum and increase of the mucous or sputum lung clearance. Pulmonary function tests as well as diagnostic tests for the clinical progression of cystic fibrosis are well known to those individuals with skill in this art. The therapeutic amount of Swinholide A is effective to reduce the viscosity of pulmonary mucous in a cystic fibrosis patient thereby facilitating lung clearance of the patients. In an alternative embodiment, Swinholide A or modifications thereof can be coupled to hydrophilic elements or other molecules by standard methods so as to prevent entry of the Swinholide A or modifications thereof into the cells of the respiratory tract.

Swinholide A or modifications thereof can be administered to the afflicted mammal by means of a pharmaceutical delivery system for the inhalation route. Swinholide A or modifications thereof may be formulated for administration as pharmaceutical compositions in physiologically acceptable carriers or excipient, optimally with supplementary therapeutic agents.

The compounds may be formulated in a form suitable for administration by inhalation. The pharmaceutical delivery system is one that is suitable for respiratory therapy by topical administration of Swinholide A and analogs thereof to mucosal linings of the bronchi. This invention can utilize a system that depends on the power of a compressed gas to expel the Swinholide A

- 20 - from a container. An aerosol or pressurized package can be employed for this purpose.

As used herein, the term "aerosol" is used in its conventional sense as referring to very fine liquid or solid particles carried by a propellant gas under pressure to a site of therapeutic application. When a pharmaceutical aerosol is employed in this invention, the aerosol contains the therapeutically active compound, which can be dissolved, suspended, or emulsified in a mixture of a fluid carrier and a propellant. The aerosol can be in the form of a solution, suspension, emulsion, powder, or semi-solid preparation. Aerosols employed in the present invention are intended for administration as fine, solid particles or as liquid mists via the respiratory tract of a patient. Various types of propellants known to one of skill in the art can be utilized. Examples of suitable propellants include, but is not limited to, carbon dioxide or other suitable gas. In the case of the pressurized aerosol the dosage unit may be determined by providing a value to deliver a metered amount.

The present invention can also be carried out with a nebulizer, which is an instrument that generates very fine liquid particles of substantially uniform size in a gas. Preferably, a liquid containing the Swinholide A or analogs thereof is dispersed as droplets. The small droplets can be carried by a current of air or oxygen through an outlet tube of the nebulizer. The resulting mist penetrates into the respiratory tract of the patient.

A powder composition containing Swinholide A or analogs -thereof, with or without a lubricant, carrier, or propellant, can-be administered to a mammal in need—of therapy. This embodiment of the invention can be carried out with a conventional device for administering a powder pharmaceutical composition by inhalation. For example, a powder mix of the compound and a suitable powder base such

- 21 - as lactose or starch may be presented in unit dosage form in for example capsular or cartridges (e.g., - gelatin, or blister packs) from which the powder may be administered with the aid of an inhaler.

The patient to be treated can be a primate, such as a human, or any other animal exhibiting the described symptoms. While the method of the invention is especially adapted for the treatment of a human patient, it will be understood that the invention is also applicable to veterinary practice.

All books, articles, or patents referenced herein are incorporated by reference. The following examples illustrate various aspects of the invention and in no way intended to limit the scope thereof.

Example 1

Severing And Depolymerization Of Filamentous Actin By Swinholide A

Effects of swinholide A on cell morphology and the actin cytoskeleton

Balb/C 3T3 (American Type Culture Tissue Collection, Rockville, MD) and Swiss 3T3 (American Type Culture Tissue Collection, Rockville, MD) cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% calf serum (Gibco) at 37 °C in a humidified atmosphere of 9% C0 ₂ in air. Swinholide A (Carmely, F. and Kashman, Y. (1985) Tetrahedron Lett. 26:511; Kobayashi, M. et al.

(1990) Chem. Pharm. Bull 38:2409) in dimethylsulfoxide was added to the medium at 1:500 to 1:1000 dilution (5 to 100 nM Swinholide A) and cells were examined over a 1 to 24 h ^• period. For fluorescence microscopy, treated and untreated cells grown on coverslips were fixed with 3% paraformaldehyde in PBS, permeabilized with 0.5% Triton-X 100 in buffer containing 50 mM MES, pH 6.1, 2.5 mM EGTA, 2 mM MgCl ₂ and labeled with tetramethylrhodaminyl-phalloidin (TRITC-phalloidin) (Sigma) , to visualize F-actin, and with 4' ,6-diamidino-2-phenylindole (DAPI) , Molecular Probes),

- 22 - to visualize the nucleus. The stained cells were examined with a Zeiss Axiphot microscope equipped with epifluoresence illumination and photographed using automatic exposure to optimize visualization.). At concentrations as low as 80 nM, Swinholide A caused rounding of mouse embryo 3T3 fibroblast cells within 1 h and massive destruction of the actin cytoskeleton as monitored by TRITC-phalloidin, a fluorescent derivative of the F-actin-binding toxin. Partial cell retraction or arborization and diminution of microfilament bundles (stress fibers) began after exposure of cells to 10-50 nM Swinholide A for 2 to 4 h, with complete loss of stress fibers by 5 to 7 h. The effect of 50 nM Swinholide A on cells grown to high density is shown in Figs. 1A and IB. The intensely fluorescent, well developed arrays of stress fibers (Fig. 1A) completely disappeared with the appearance of weakly fluorescent patches randomly scattered throughout the cytoplasm (Fig. IB) . Exponentially growing cells (Fig. 1C) exposed to 10 nM Swinholide A for 24 h became arborized with diffusely distributed phalloidin-stainable F-actin in the cytoplasm in addition to fluorescent punctate structures (Fig. ID) . Almost all of the cells became binuclear (Fig. IE) indicating that Swinholide A does not interfere with the progression of cells through mitosis but inhibits cytokinesis, presumably by inhibiting formation and function of the contractile ring. Swinholide A did not affect the integrity and organization of the microtubule system (data not shown) .

Sequestration of actin dimers by Swinholide A The. ability of Swinholide A to. sequester unpolymerized actin -subunits was quantified (Fig. 2A) by the increase in the apparent critical concentration of N- pyrenylcarboxyamidoethyl-labeled actin (pyrenyl-actin) in buffer containing 2.0 mM MgCl ₂ , 0.1 mM CaCl ₂ , 0.2 mM dithiothreitol (DTT), 0.1 mM ATP, 0.01% sodium azide, and

- 23 -

5.0 mM Tris, pH 7.8 (buffer F) . The amount of unpolymerized actin sequestered by Swinholide A was consistent with highly cooperative binding of 2 actin monomers to 1 molecule of Swinholide A with an equilibrium association constant K _a = 9.2- 10 ¹³ M ^"2 or (and indistinguishable by this assay) the binding of 1 actin dimer to 1 Swinholide A with K _a = 4-10 ⁷ M ^"1 . Sedimentation velocity data obtained in buffer F were consistent with the formation of a Swinholide A-actin complex with a sedimentation coefficient equivalent to that of an actin dimer and in molar concentration equal to the concentration of total Swinholide A (data not shown) . The same results were obtained when MgCl ₂ was omitted from the buffer (buffer G) (Fig. 2B) demonstrating that Swinholide A can induce the formation of an actin dimer under non- polymerizing as well as polymerizing conditions. Assuming a partial specific volume of 0.73 ml/g (K. Mihashi, (1964) Arch. Biochem. Biophys, 107:441), the sedimentation coefficient (s _20(W =5.1 S) of the actin dimer generated by Swinholide A was exactly as predicted (J.G. Kirkwood, (1954) J. Polymer Science, 12:1; M.R. Bubb, _. J. Knutson, D. Porter, E.D. Korn (submitted).) for a dimer consisting of two contiguous spheres (monomer sedimentation coefficient, s _20/W =3.4 S) . The ability of Swinholide A to bind actin dimers is reasonable given its two-fold axis of symmetry: two identical 22-carbon chains joined end to end to form, a 44-carbon ring (S. Carmeli and Y. Kashman (1985), Tetrahedron Lett . , 26, 511; Kobayashi, M. et al. (1990) Chem Pharm. Bull . 38:2409 (1990); Kobayashi, M. et al. (1990) Chem Pharm. Bull . 38:2960). in the presence of Swinholide A, actin in a non- polymerizing buffer was covalently crosslinked by N,N'- 1,4-phenylenedimaleimide (PDM) to a species (Fig. 2C, lanes 2 and 3) with an electrophoretic mobility identical to that of the actin dimer (apparent molecular mass, 86 kDa) formed when the crosslinking reagent was added

- 24 - immediately after addition of MgCl ₂ (Fig. 2C, lane 4 and Millonig, R. et. al., (1988), J. Cell Biol . , 106:785). This is the same electrophoretic mobility as the dimer obtained by crosslinking the two actin subunits that bind to the F-actin severing protein, gelsolin (Hesterkamp, T. et. al. (1993) J ^" . Biochem. , 218:507), and faster than the electrophoretic mobility of the dimer (apparent molecular mass, 115 kDa) formed when the subunits of F-actin are crosslinked (Fig. 2C, lane 5) . Maximum crosslinking efficiency in the presence of Swinholide A was obtained at a ratio of 1.0 PDM to 2.0 actin subunits (data not shown), as expected if the two subunits in the actin dimer were oriented in anti-parallel fashion so that the Cys-374 residues of the subunits could be crosslinked (Millonig, R. et al. (1988), J ^" . Cell Biol . , 106:785; Hesterkamp, T. et al. (1993), Eur. J. Biochem. , 218:507).

Severing and depolymerization of F-actin by Swinholide A In addition to sequestering non-polymerized actin subunits, stoichiometric concentrations of Swinholide A accelerated the rate of depolymerization of F-actin (Fig. 3A) . The depolymerization of F-actin, both capped and uncapped, was highly cooperative with respect to the concentration of Swinholide A (Fig. 3B) , and, as determined by stopped-flow fluorescence measurements the rate of depolymerization induced by Swinholide A increased during the first several seconds after dilution (Fig. 3C) . The rates of depolymerization were about 15% higher in the absence of Swinholide A than when measured in the steady- state fluorimeter either because the initial rate was determined more accurately or because of fragmentation during injection. Swinholide A appeared to be equally effective with Mg- and Ca-F-actin (compare Fig. 3A and Fig. 3B) .

The increase in the rate of F-actin depolymerization was unlikely to have been due simply to sequestration of actin subunits by Swinholide A because:

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(i) under the conditions of the experiments in Figs. 3A- 3C, the initial concentration of actin monomers was very low, (ii) the effect of Swinholide A was greater at higher Mg ²⁺ concentration (Fig. 3A) , even though the actin critical concentration is lower at higher Mg ²⁺ concentrations, and (iii) successive increments in Swinholide A concentration had progressively greater effect for both capped and uncapped filaments (Fig. 3B) , contrary to what would have been expected from simple mass action.

Swinholide A could actively destabilize F-actin by complexing to and increasing the off-rate of terminal subunits and/or by severing actin filaments thus creating more filament ends. The first possibility is inconsistent with the observation that the depolymerization rate was not directly proportional to the concentration of Swinholide A (Fig. 3B) , and the observed increase in depolymerization rate with time (Fig. 3C) is most consistent with an increase in the number of filament ends as a result of severing. The high degree of cooperativity with respect to Swinholide A concentration (Fig. 3B) implies that Swinholide A must bind to several neighboring subunits before the filament breaks.

If Swinholide A, like the protein gelsolin, not only severed actin filaments but also capped the "barbed" ends (the two ends of the polarized filament are designated "barbed" and "pointed" from the arrowhead-like appearance of electron microscopic images of filaments decorated with myosin.) of severed filaments, the rates of depolymerization of uncapped and gelsolin capped filaments would.be expected to converge at high concentrations of Swinholide A. In fact, the opposite behavior-was-observed (Fig. 3B) indicating that the barbed ends of filaments severed in the absence of gelsolin remained uncapped. If Swinholide A capped the "pointed" ends of actin filaments, the rate of depolymerization of gelsolin-capped filaments

- 26 - should have decreased in the presence of Swinholide A, contrary to what was observed (Fig. 3B) , because both ends would then have been blocked. Therefore, it is highly unlikely that Swinholide A caps either end of actin filaments.

Comparison of Swinholide A to other actin disrupting agents

Latrunculin and tolytoxin, two other macrolides that bind to actin, appear to sequester actin monomers (M. Coue, M. et. al. (1993), FEBS Lett . , 213:316; Patterson, G.M.L. et al. (1993), Cell Mot . Cytoskeleton, 24:39) not dimers; neither of these macrolides has the two-fold axis of symmetry that may explain the interaction of Swinholide A with an actin dimer. The data presented for tolytoxin is consistent with the formation of a 1:1 complex with actin monomer. Although cytochalasins have been reported to induce the transient formation of an actin dimer and may have some severing activity, their principal activity is to cap the barbed end of actin filaments and serve as nuclei for filament elongation (J.A. Cooper, (1987) J. Cell Biol . , 105:1473). That neither property is shared by Swinholide A is not surprising because Swinholide A lacks the carbonate or acetate carbonyl groups common to all active cytochalasin derivatives (A. Mozo-Villarias and B.R. Ware, (1988) Arch . Biochem. Biophysics, 264:321) . The effects of Swinholide A on the actin cytoskeleton and cell morphology are more like those of latrunculins (Spector, I. et al. (1989), Cell Mot, Cytoskel . , 13:127) than of cytochalasins which, also, do not usually cause a decrease in the concentration of F-actin (A. Morris and J. Tannenbaum, (1980) Nature, 287:638; Wodnicka, M. et al. (1992), Folia Histochem. Cytobiol . 30:107).

Comparison of Swinholide A To Other Actin Severing Proteins

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The properties of Swinholide A in vi tro are in some ways similar to those of gelsolin in that the severing capacity of gelsolin also increases with increasing Mg ²⁺ concentration (J. Bryan and L.M. Coluccio, (1985) J. Cell Biol . , 101:1236) and the actin dimers complexed to gelsolin and Swinholide A have the same electrophoretic mobility when covalently crosslinked. However, in contrast to Swinholide A, gelsolin caps actin filaments with high affinity and its severing activity is non-cooperative. Actophorin, an actin-severing protein from the soil amoeba, Acanthamoeba castellani , does show cooperativity qualitatively similar to Swinholide A but appears to sever much less effectively and apparently binds to actin monomers (Maciver, S.K. et al. (1991) J. Cell Biol . , 115:1611) .

Example 2

Treatment of Cystic Fibrosis Patient With Swinholide A

Swinholide A may be efficacious in treating mammals afflicted with cystic fibrosis. For example

Swinholide A may be administered to mammals afflicted with cystic fibrosis in the form of an inhalation or nebulizer device. Mammals can be administered ranges or doses of

Swinholide A in the ranges described herein in the ranges of about 0.1 mg to 10 mg per dosage, twice daily.

Preferred dose ranges are 0.25 to 1.0 mg per dosage twice daily. The mammals will be monitored for increased lung capacity, decreased viscosity of mucous or sputum by conventional clinical evaluation methods. Specific parameters to be expressed include the increased production of lung capacity. Such treatments may be administered either alone or in conjunction with other adjuvants therapies, such as lavage, percussion, posteral drainage, antibiotics or DNAse I treatment.

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Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the dependent claims.