CA2338928A1 - Thymosin .beta.4 promotes wound repair - Google Patents

Abstract

The present invention relates to methods for promoting tissue repair, angiogenesis and cell migration. The method of the invention utilizes thymos in .beta.4 (T.beta.4) peptide to promote tissue repair, angiogenesis and cell migration. The invention further relates to modulating T.beta.4 activity in tissues.

Description

WO 00!06190 PCT/US99/17282 _ THYMOSIN (34 PROMOTES V~VOUND REPAIR
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
This invention was made in part with hands from the National Institutes of Health, Intramural Program. The government may !have certain rights in this invention.

This applicarion claims priority from Provisional Application Serial No.
601094,690, filed 3uly 30, 1998, which is incorporated herein by reference in its entirety and to which application a priority claim is made under 35 U.S.C. ~ I
19{e).
TECHNICAL FIELD OF THIS INVENTION
The present invention relates generally tn tissue repair and more specifically to methods of wound healing using thyrnosin ~i4.
BACKGROUND OF THE INVENTION
Inadequate methods and compositions to effectively heal chronic wounds 1 S is a significant health care problem. Impaired woumd healing increases the chances of mortality and morbidity. This problem is especially prominent in patients with diabetes who develop severe, life threatening wounds on body extremities.
Chronic diabetic foot ulcers often lead to amputations. These wounds are often the result of poor circulation derived from the diabetic patients' :insulin-compromised cells as well as impaired vascularization of the wound bed, reduced infiltration of germ fighting cells and reduced tissue epithelialization. As a resu'.it, most current therapies include attempts to revascularize the wound bed and prevent infection.
Wounds in non-compromised tissues undergo a complex and ordered series of events to repair the tissue. The series of events may include infiltration of immune cells as part of the process to remove and destroy necrotic tissue, increased vascularization by angiogenic factors and increased cell proliferation and extracellular matrix deposition. Although the basic process of tissue repair has been characterized, the individual steps and factors necessary to carry out this complex series of events are not well understood. The identification of individual steps and factors could lead to improved methods for the treatment of diseases resulting from inadequate wound repair processes.
Previous studies have used the "scratch" wound closure assay to assess the potential effects of an agent on in vitro cell migration. Though informative, such a test does not mimic the dynamic in vivo wound healing conditions to the extent that not alI factors involved in wound closure are present in the in vitro assay.
For this reason, in vivo systems have been developed to assess the ability of an agent or factor to modulate wound healing activities.
Using these types of in vitro models, a mxmber of specific growth factors have been recognized for their effect on angiogenesis. One such growth factor is TGF-Vii. This family of dimeric proteins includes TCPF-(31, TGF-~i2, TGF-~i3, TGF-~34, and TGF-~i5 which regulate the growth and differentiation of many cell types.
This family of proteins exhibits a range of biological effects from stimulating the growth of some cell types (Noda et al., (1989) Endocrinology, 124:2991-2995) and inhibiting the growth of other cell types (Goey et al., (1989) J. Immunol., 143:877-880; Pietenpol et al., {1990) Proc. Nat'l. Acad. Sci. LISA, 87:3758-3762). TGF-(3 has also been shown to increase the expression of extracf;llular matrix proteins, including collagen and fibronectin {Ignotz et al., (1986) J. Biol. Chem.; 261:4337-4345) and accelerates the healing of wounds {Mustoe et al., (1987) Science, 237:1333-1335).
Another growth factor recognized for its effect on angiogenesis is Platelet Derived Growth Factor (PDGF). PDGF was originalJly found to be a potent mitogen for mesenchymal derived cells {Ross R. et al. (1974) ,froc Nat'l Acad Sci USA
71(4):1207-1210.; Kohler N. et al. {1974) Exp. Cell R'es. 87:297-301). Further studies have shown that PDGF increases the rate of cellularir~ and granulation in tissue formation. Wounds treated with PDGF have the appearance of an early stage inflammatory response, including an increase in neutnophils and macrophage cell types at the wound site. These wounds also show enhanced fibroblast function (Pierce, GF et al. (1988) J. Exp. Med. 167:974-987). lBoth PDGF and TGF~i have WO 00/06190 PCT/IJS99/17282 .

been shown to increase collagen formation, DNA content, and protein levels in animal studies. (Grotendorst, GR et al. (1985) J. Clin. Invest. 76:2323-2329.;
Sporn, MB et al. (1983) Science 219:1329). The effect of PDGF in wound healing has been shown to be effective in human wounds. In human wounds, PDGF-AA expression is increased within pressure ulcers undergoing healing. The increase of PDGF-AA
corresponds to an increase in activated fibroblasts, c~xtracellular matrix deposition, and active vascularization of the wound. Furthermore, such an increase in PDGF-AA
is not seen in chronic non-healing wounds. A number of other growth factors having the ability to induce angiogenesis and wound healing include, Vascular Endothelial Growth Factor (VEGF), Keratinocyte Growth Factor (KGF) and basic Fibroblast Growth Factor (bFGF).
However, most of these growth and angiogenic factors have side effects.
Accordingly, there is a need for additianal factors useful in promoting wound repair.
SUMMARY OF THE INVENTION
1 S The present invention is based on the discovery that thymasin X34 {T~i4) accelerates wound healing and stimulates wound repair. Based on this finding, it is now possible to develop methods for accelerating vvound healing in subjects having wounds in need of such treatment.
In a first embodiment, the invention provides a method fox promoting wound repair in a subject in need of such treatment by administering to the subject or contacting the site of the wound with a wound-healing effective amount of a composition containing a wound healing polypepti~de comprising the amino acid sequence LKKTET and conservative variants thereof having wound healing activity.
In one aspect of the method, the wound healing pol.ypeptide is T(34 or an isoform of T~i4.
Tn another embodiment, the invention provides a method for promoting tissue repair in a tissue in need of such treatment by contacting the tissue with an effective amount of a composition containing a wound healing polypeptide comprising the amino acid sequence LKKTET and conservative variants thereof WO 00/06190 PCT/US99/17282 .

having wound healing activity, or nucleic acid encoding a wound healing polypeptide.
In one aspect of the method, a wound healing peptide is T[34 or an isoform of T~34.
The tissue may be contacted either in vivo or ex viv.o.
In yet another embodiment, the invention provides a method of modulating wound repair in a subject in need of such treatment by systemic delivery of a wound-healing effective amount of a wound healing polypeptide comprising the amino acid sequence LKKTET and conservative variants thereof having wound healing activity.
In one aspect of the method, a wound healing peptide is T~34 or an isoform of T(34.
In yet another embodiment, the present invention provides a method for stimulating epithelial cell migration at the site of a wound by contacting the wound with an effective amount of a T~i4 polypeptide.
In another embodiment, the invention provides a method of diagnosing a pathological condition in a subject characterized by a wound healing disorder associated with T(34, including obtaining a sample suspected of containing T~i4 from the subject, detecting a level of T~34 in the sample and comparing the level ofT(34 with the level found in a normal sample (i. e., a standard sample).
In another embodiment, the invention provides a method of ameliorating a wound healing disorder associated with T~34, including treating a subject having the disorder with a composition which modulates T~i4 activity or the activity of a T~34 isoform.
In yet another embodiment, the present invention provides pharrnaceuticai compositions comprising a wound healing polypeptide comprising the amino acid sequence LKKTET and conservative variants thereof having wound healing activity and a pharmaceutically acceptable carrier. In one aspect, the wound healing polypeptide is T~i4 or an isoform of T(34.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other featuxes, objects, and advantages of the invention will be apparent from t:he description and drawings, and from the claims.

DESCRIPTION OF DF:AWINGS
FiG. 1 is a schematic drawing of a wound..
FIG. 2 is a bar graph which shows the effect of topical and systemic delivery of T(34 on the width of a punch wound as compared to control. (A) Topical delivery of 5 ~.g/50 wI was performed on three of the six wounds in each animal on the day of wounding and at 48 hours after wounding. (B) Intraperitoneal injections of 60 ~gI300~.1 were done on the day of the wounding and thereafter every other day.
Control animals were treated similarly with saline. Measurements are expressed as the mean percent decrease ~ SEM.
FIG. 3 is a bar graph which shows the effect of topical and systemic delivery of T~i4 on the gap of a punch wound as compared to control. (A) Topical delivery of 5 ~gISO wI was performed on the day of wounding and at 48 hours after wounding. (B) Intraperitoneal injections of 60 ~.~;I300~,1 were done on the day of the wounding and thereafter every other day. Measurements are expressed as the mean percent decrease ~ SEM.
FIG. 4 is a histological section, stained with H&E, demonstrating the appearance of control and thymosin ~i4 treated wounds at low magnification and higher magnification. Wounds are from day 7 as described in the legend to figure 2.
Arrows indicate the edges of the original wound. (A) Control wound treated with 20 saline. Migration of the epithelium is visible at the wound edges and debris are visible over the unhealed wound. {B) Increased :re-epithelialization of the wound occurred when T(34 was injected intraperitoneally (60 ~g1300~,1 on alternate days).
(C) Topical treatment {S~g/50~.1 of T~i4) resulted in complete reepithelialization of the wound epidermis. Boxed areas are the locatiion of the .higher magnification fields 25 (D-F). (D-F) Dermis near dermal and epidermal. junction. (D) Control showing few cells near the dermis and little neovascularizatio~n. (E) and (F) Dermis showing granulation tissue infiltrated with fibroblasts and extensive neovascularization (arrowheads). (E) Intaraperitoneal treatment and (F) topical application both resulted in significant new capillaries. (Scale bar =1 mm).

FIG. 5 shows histological sections of 7 ~day wounds showing collagen deposition/accumulation. Masson's trichrome stainng shows collagen and endothelial cells. {A) Low magnification view of a control wound treated with saline.
(B) and (C). Low magnification views of wounds 'where Tj34 was injected 5 intraperitoneally (B) or applied topically (A). Boxed areas are the lacation of the higher magnification fields (D-F). An'ows indicate the edges of the original wound.
(D) Control wound at higher magnification showing baseline collagen accumulation.
Treatment intraperitoneally (E) or (F) topically resulted in enhanced collagen production/accumuiation compared to wounds treated with saline. (Scale bar = 1 mm).
FIG. 6 shows T~4 stimulated keratinocyte migration in Boyden chamber assays. (A) T(34 in the lower wells of the chamber resulted in a 2-3 fold increase in migration on filters coated with collagen IV. The positive control, conditioned media, also showed increased migration over media alone.
FIG. 7 shows a graph demonstrating the migration of corneal epithelial cells at various concentrations of T~i4.
FIG. 8 shows a graph representing coxneal re-epithelialization in rat corneas in the presence and absence of T(34.
FIG. 9 shows a graph representing corneal re-epithelialization in the 20 presence and absence of various concentrations of T~i4.
FIG. lU shows an amino acid sequence of T~4.
FIG. 11 shows the amino acid sequence of several known isoforms of T~34, and their phylogenetic distribution. N-terminal .~cetylation is indicated by "ac."
Residues between 13 and 24 are thought to be innportant for actin binding.
2~ DETAILED DESCRIPTION OF THE INVENTION
Thymosin ~i4 was initially identified as a protein that is up regulated during endothelial cell migration and differentiation in vitro. Thymosin ~i4 was originally isolated from the thymus and is a 43 ~unino acid, 4.9 kDa ubiquitous polypeptide identified in a variety of tissues. Se;verai roles have been ascribed to this WO 00/06190 PCT/US99/17282 .
- '7 protein including a role in endothelial cell differenttiation and migration, T
cell differentiation, actin sequestration and vascularization. One biological activity of thymosin ~i4 (T~i4), as shown herein, effects tissue repair and wound healing.
Another activity of T(34 is anti-inflammatory activity.
The present invention resulted from investigation of the effects of T~34 on wound healing. In vivo results have demonstrated that topical and systemic delivery of T~34 promotes wound healing. Additional experiments demonstrated that T(34-treated wounds have increased extracellular matrix deposition in the wound bed.
The present invention identifies T~i4 as an active factor in promoting 10 wound closure and tissue repair in vivo as well as :increasing epithelial cell migration.
In vivo administration of T(34 indicates that cell migration, angiogenesis and extracellular matrix deposition are stimulated at or above the levels observed for migration, angiogenesis and matrix deposition in control animals. T(34 promotes wound closure when administered systemically (e.g.; infra-peritoneally) and topically 15 in wounded animal models. Increased levels of collagen were also observed in treated wounds showing that T(34 treatment can also accelerate wound contraction and stimulate the healing process.
The methods of the invention result from the identification of the effect of T~i4 on wound healing. In vivo, T(34 stimulates vvound healing in a full thickness 20 punch wound (see Example 1) and in repair of eye-related wounds (Example 4).
When given either topically or systemically (e.g., infra-peritoneally) T(34 accelerated closure and healing of wounds (see Example 1; 4, and 5).
Promoting Tissue Regeneration In one embodiment, the invention provides a method for accelerating 25 wound healing in a subject by contacting a wound with a wound-healing effective amount of a composition which contains T~i4 or a T~i4 isoform. 'The contacting may be topically or systemically. Examples of topical administration include, for example, contacting the wound with a lotion, salve, gel, cream, paste, spray, suspension, dispersion, hydrogel, ointment, or oil comprisini; T(34. Systemic administration WO OO/Ob190 _g_ includes; for example, intravenous, intraperitoneal, intramuscular injections of a composition containing T~i4 or a T(34 isoform. A :>ubject may be any rnamrnal, preferably human.
In addition, T~34 or a T~4 isoform is thc,rapeutically valuable in cases where there is an impaired wound healing process.,, such as in wound healing compromised subjects. By "wound healing compromised" is meant subjects which have a reduced, decreased, or inability to recover fiom a wounding or trauma, due to recurrent wounding, trauma or inability of the subyect's natural system to properly effectuate wound healing. For example, steroids reduce the ability of a subject to heal 10 as compared to a subject which is not on steroids. Other such wounds present in compromised subjects include, but are not limitedl to, skin wounds such as diabetic ulcers, venus ulcers or pressure ulcers. Additionally, T~i4 or a T~34 isoform is therapeutically valuable to augment the normal healing process.
As used herein, a "wound-healing effective amount" of a composition 15 containing T~i4 or a T~i4 isoform for use in wound healing is defined as that amount that is effective in promoting tissue regeneration and repair. The "wound-healing effective amount" may be the therapeutically effective amount. Diseases, disorders or ailments possibly modulated by T~34 or a T~i4 isoform include tissue repair subsequent to traumatic injuries or conditions invluding arthritis, osteoporosis and 20 other musculo-skeletal disorders, burns, ulcers a~ld other skin lesions, neurological and nerve disease and cardiovascular diseases including ischemia and atherosclerosis:
Other potential tissues which can be treated by the methods and compositions of the invention include epidermal, eye, uro-genital, gastro-intestinal, cardiovascular, muscle, connective, and neural tissues. The term "induce", "induction" or "effect" as 25 used herein, refers to the activation, stimulation;, enhancement, initiation and/or maintenance of cellular mechanisms or processE;s necessary for the formation of a tissue or a portion thereof, repair process or tissue development as described herein.

Modulation of Wound Healing Wound healing, tissue regeneration and tissue repair result from a complex process that includes the proliferation and migration of inflammatory cells, endothelial cells, stromal cells and parenchyma) cell, the deposition of extxacellular matrix materials and the growth of new blood vessels, particularly capillaries. This complex process plays a crucial role in such beneficial functions as ernbryogenesis, the female reproductive cycle, as well as such abnormal functions as arthritis, chronic ulcerations and neuro-degenerative diseases.
In another embodiment, the invention provides a method for modulating wound healing in a subject or a tissue including contacting the subject or tissue with an effective wound-healing amount of a composition containing T~i4 or a T~34 isoform. It is envisioned that T(34 or a T(34 isoform can be administered topically ar systemically to prevent or treat a damaged tissue including, for example, tissues damaged due to ischemia, including ischemic brain, bone and heart disease, damage to corneal or retinal tissue of the eye, and damage to epithelial tissue, including skin.
In addition, the method of the invention is useful in promoting wound healing in tissues by promoting angiogenesis in tissue deprived of adequate blood flow. For example, a composition containing T~i4 can promote the healing of chronic ulcers by increasing blood supply to the tissue site as well as increasing keratinocyte migration to close a wound.
T~i4 isoforms have been identified and have about 70%, or about 75%, or about 80% or more homology to the amino acid sequence of Tø4 set forth in Fig.
10.
Such isoforms include, for example, T~i4a~a, T~39, T(310, T~ 11, T(312, Tai 13, Tai I4 and T(315 (Fig. 11; see also, Mihelic et al., (1994) Amino Acids, &:I-13, which describes the amino acid sequence of other T~i4 isoforms, and is incorporated herein by reference}. Similar to T(34, the Tai 10 and T[315 isoforms have been shown to sequester actin. T~i4, T~ilO and T(315, as well as these other isoforms share an amino acid sequence, LKKTET, that appears to be involved in mediating actin sequestration or binding. Although not wishing to be bound ~to any particular theory, the wound healing activity of T(34 and T~i4 isoforms may be due, in part, to the ability to WO 00/06190 PCTIUS99/17282 _ polymerize actin. For example,T(34 can modulate actin polymerization in wounds to promote healing (e.g.; (3-thymosins appear to depolymerize F-actin by sequestering free G-actin}. T~i4'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.
5 Thus, as with T~i4, other proteins which bind or sequester actin, or modulate actin polymerization, including T(34 isoforms having the; amino acid sequence LKKTET, are likely to promote wound healing alone, or in a combination with T(34, as set forth herein.
Thus, it is specifically contemplated that known T(34 isoforms, such as T(34~'~, T~i9, T(310, T(311, Tai 12, Tai 13, Tai 14, and 'T~i 15, as well as T(34 isoform;s not yet identified, will be useful in the methods of the invention. As such T~i4 isoforms are useful in the methods of the invention, including the methods practiced in a subject, the invention therefore further provides plharmaceutical compositions comprising T~i4 isoforms T~34a'a, T(39, T~iIO; T~31:l, T(312, T(313, T~il4, and T(315 and a pharmaceutically acceptable carrier.
In addition, other proteins having actin sequestering or binding capability, or that can mobilize actin or modulate actin polymerization, as demonstrated in an appropriate sequestering, binding, mobilization o:r polymerization assay, or identified by the presence of an amino acid sequence that mediates actin binding, such as 20 LKKTET, for example, can similarly be employed in the methods of the invention.
Such proteins include gelsolin, vitamin D binding; protein (DBP}, profilin, cofilin, depactin, DNaseI, vilin, fragmin, severin, capping protein, (3-actinin and acumentin, for example. As such methods include those practiced in a subject, the invention further provides pharmaceutical compositions comprising gelsolin, vitamin D
binding 25 protein (DBP), profilin, cofilin, depactin, DNase:I, vilin, fragmin, severin, capping protein, ~i-actinin and acumentin as set forth herE;in. Thus, the invention includes the use of waund healing polypeptide comprising the amino acid sequence LICKTET
and conservative variants thereof.

WO 00106190 PCTIUS99/17282 .

As used herein, the term "conservative variant" or grammatical variations thereof denotes the replacement of an amino acid rcaidue 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 5 replacement of a polar residue for another, such as the substitution of arginine fox lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like.
T~i4 has been localized to a number of fiissue and cell types and thus, agents which stimulate the production of T~34 can lbe added to a composition to effect T~i4 production from a tissue and/or a cell. Agents that effect wound repair can also 10 be included in such a composition to augment the 'wound healing process.
Such agents 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-Vii), basic fibroblast growth factor (bFGF), thymosin a I (Ta I ) and vascular endothelial growth factor (VEGF).
More 15 preferably, the agent is transforming growth factor beta (TGF-(3) or other members of the TGF-(3 superfamily. T(34 compositions of the invention aid in wound healing by effectuating growth of the connective tissue through extracellular matrix deposition, cellular migration and vascularization of the wound bed.
Additionally, agents that assist or stimulate the wound healing process 20 may be added to a composition along with T(34 or a T[34 isoform to further modulate the wound healing process. Such agents include <~ngiogenic agents, growth factors, agents that direct differentiation of cells, agents that promote migration of cells and agents that stimulate the provision of extracelluiar matrix materials in the wound bed.
For example, and not by way of limitation, T(34 or a T(34 isoform alone or in 25 combination can be added in combination with any one or more of the following agents: VEGF, KGF, FGF, PDGF, TGF~i, IGF-I, IGF-2; IL-I; prothymosin a and thymosin al in a wound-healing effective amount.
In another aspect, the invention is useful for repair of tissue resulting from injuries due to surgical procedures, irradiation, laiceration, toxic chemicals, vixal 30 infections, bacterial infections or burns. Additionally, the invention is useful for WO 00/06190 PCT/US99117282 _ revitalizing scar tissue resulting from any number of procedures, accidents or trauma.
The term "scar tissue" means fibrotic or collagenous tissue formed during the healing of a wound or other morbid process. For example, T~34 can be included in a controlled release matrix which can be positioned i.n proximity to damaged tissue 5 thereby promoting regeneration, repair andlor reva.scularization of such tissue. The term "controlled release matrix" means any composition that allows for the release of a bioactive substance which is mixed or admixed therein. The matrix can be a solid composition, a porous material (such as a scaffold, mesh, or sponge), or a semi-solid, gel or liquid suspension containing bioactive substances. The term "bioactive 10 material" means any composition that modulates tiissue repair when used in accordance with the method of the present invention. The bioactive materials or matrix can be introduced by means of injection, surgery, catheters or any other means suitable for modulating tissue repair.
It is envisioned that the methods and compositions of the invention can be 15 used to aid wound healing and repair in guided tissue regeneration (GTR) procedures.
Such procedures are currently used by those skilled in the medical arts to accelerate wound healing. Typically, nonresorbable or bioabsorbable materials are used to accelerate wound healing by promoting the repopulation of the wound area with cells which form the architectural and structural matrix of the tissue. For example, the 20 methods and compositions of the invention can bc~ used in aiding tissue repair or regeneration at au ulcer site in a human or other subject by placing a composition containing a bioreasorbable polymer and T~4 at tlhe site in need of tissue repair or regeneration such that the composition is effective for aiding tissue regeneration by releasing a wound-healing effective amount of T~f4 at the site.
25 In another aspect, the invention is usejCul for the purposes of promoting tissue growth during the process of tissue engineering. As used herein, "tissue engineering" is defined as the creation, design, arid fabrication of biological prosthetic devices; in combination with synthetic or natural materials, for the creation, augmentation or replacement of body tissues and organs. Thus, the present method 30 can be used to augment the design and growth of human tissues outside the body, for WO 00/06190 PCT/US99I17282 _ later implantation inside the body, or augment the design and growth of a tissue inside the body to repair or replace diseased or damaged tissue. For example, T(34 may be useful in promoting the growth of skin graft replacements which are used as a therapy in the treatment of burns and ulcers.
In another aspect of tissue engineering, T~34 can be included in external or internal devices containing human tissue designed to replace the function of a diseased internal tissue. This approach involves isolating cells from the body, placing them on or within a three-dimensional matrices and implanting the new system inside the body or using the system outside the body. The methods and compositions of the 10 invention can be used and included in such matricf;s to promote the growth of tissues contained in the matrices. For example, T(34 can be included in a tissue engineered construct to promote the growth of the cells contained in the construct. It is envisioned that the method of the invention can be used to augment tissue repair, regeneration and engineering in endothelial cell-related products which may contain 15 cartilage, cartilage-bone composites, bone, central nervous system tissues, muscle, liver, pancreatic islet (insulin-producing) cells, uro~genital tissues, breast and tissues for gene therapy applications.
The present invention further provides methods and compositions for modulating female reproductive tract function. Growth factors have been shown to 20 play a role in cyclic mitosis and differentiation of c:ndometrial cellular components, recruitment of macrophages in decidualizing the e:ndometrium, endometrial-trophoblast interactions, early pregnancy maintenance, and endometrial functional regeneration. The term "modulate" as used herein, denotes a modification of an existing condition or biologic state. Modulation of a condition as defined herein, 25 encompasses both an increase or a decrease in the determinants affecting the existing condition. For example, administration of T~i4 could be used to augment uterine functions in a condition where the promotion of endothelial cell growth is desired.
For example, the uterus may be treated with T~i4 to promote the growth and development of placental membranes or endomet;~ial growth or the repair of these 30 tissue following tissue injury. Furthermore, treatment with T(34 may be used to WO 00/06190 PCT/US99/17282 .

promote and maintain a pregnancy by facilitating endometrial-trophoblast interaction.
Alternatively, antagonist to T(34 could be administered to modulate conditions of excessive endometrial growth in which the level of'T~i4 is excessive in comparison to a normal biological condition. In addition, T~34 in combination with other agents, such as thymosin al, may be desirable for treating dlisorders of the reproductive tract.
The therapeutic approaches described herein involve various routes of administration or delivery of reagents or compositions comprising the T(34 of the invention including any conventional administration techniques {for example, but not limited to, topical administration, local injection, inhalation, or systemic administration), to a subject with a wound or tissue in need of healing or repair.
Administration of T~34, as described above, can accelerate wound healing, increase cell migration into a wound site, induce the formation of tissue repair or regeneration, or promote the growth and development of the enda~metrium. The reagent, formulation or composition may also be targeted to specific cells or receptors by any method described herein ar by any method known in the art of delivering, targeting T~i4 polypeptides and expressing genes encoding T~34. For example, the methods and compositions using or containing T(34 of the invention may be formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable non-toxic excipients or corners. Such compositions may be prepared for parenteral administration, particularly in the form of liquid solutions or suspensions in aqueous physiological buffer solutions; for oral administration, particularly in the form of tablets or capsules; or for intranasal administration, particularly in the form of powders, nasal drops, or aerosols. Sustained release: compositions are also encompassed by the present invention. Compositions for other routes of administration may be prepared as desired using standard methods.
A composition of the invention containing T~i4 may be conveniently administered in unit dosage form, and may be prepared by any of the methods well known in the pharmaceutical art, for example, as described in Remin~-ton's Pharmaceutical Sciences (Mack Pub. Co., Easton, PA, 1990). Formulations for parenterai administration may contain as common excipients sterile water or saline, WO 00/06I90 PCTlUS99/1?282 .

polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphtalenes, and the like. In particular, biocompatible, biodegradable lactide polymer, lactidelglycolide copolymer, or polyoxethylene-polyoxypropylene copolymers are examples of excipients for controlling the release of a compound of the invention in vivo. Other suitable parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation administration may contain excipients such as lactose, if desired. Inhalation formulations may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate a~:zd deoxycholate, or they may be oily solutions for administration in the form of nasal drops. If desired, the compounds can be formulated as a gel to be applied intranasally. Formulations for parenteral administration may also include giycocholate for buccal administration.
The composition ofthe liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phaspholipids or other lipids may also be used. The physical characteristics of l.iposomes depend on pH, ionic strength, and the presence of divalent cations.
Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine;
phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, .and gangliosides.
Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated.
Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidyl-choline.
The targeting of liposomes has been classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinus~~idal capillaries.
Active WO 00106190 PCT/US991I7282 .

targeting, on the other hand, involves alteration of the iil?osome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturalhr occurring sites of localization.
The surface of the targeted delivery system may be modified in a variety of ways. In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in orf,er to maintain the targeting ligand in stable association with the liposomal bilayer. 'Various linking groups can be used for joining the lipid chains to the targeting ligand. In general, the compounds bound to the surface of the targeted delivery system will be ligands and receptors which will allow the targeted delivery system to find and "home in" on the desired cells. A ligand may be any compound of interest which will bind to another compound, such as a receptor.
The therapeutic agents useful in the method ofthe invention can be administered parenterally by injection or by gradual perfusion over time.
Administration may be intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
Preparations far parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholiclaqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents and inert gases and the like.

WO 00/06190 PCT/US99117282 .

The invention also includes a pharmaceutical composition comprising a therapeutically effective amount of T~i4 or a T~34 isofornri in a pharmaceutically acceptable carrier. Such Garners include those listed above with reference to parenteral administration.
The actual dosage or reagent, formulation or composition that modulates a tissue repair process, fibrotic disorder, a sclerotic disorder, a cell proliferative disorder, or wound healing depends on many factors, including the size and health of a subject. However, one of ordinary skill in the art can use the following teachings describing the methods and techniques for determining clinical dosages (Spilker B., Guide to Clinical Studies and Developing Protocols, Raven Press Books, Ltd., New York, 1984, pp. 7-13, 54-60; Spilker B., Guide to Clinical Trials, Raven Press, Ltd., New York,1991, pp. 93-101; Craig C., and R. Stitzel, edls. , Modern Pharmacology, 2d ed., Little, Brown and Co., Boston, 1986, pp. 127-33; T. Speight, ed., Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3d ed., Wiiliams and Wilkins, Baltimore,1987, pp. 50-56; R. Tallarida, R. Raffa and P.
McGonigle, Principles in General Pharmacology, Springer-Verlag, New York, 19$$, pp. 18-20) or to determine the appropriate dosage to use.
Antibodies that Bind to Antibodies to T(34 peptide or fragments could be valuable as diagnostic tools to aid in the detection of diseases in which T~i4 is a pathological factor. Further, use of antibodies which bind to T(34 and inhibit or prevent the actions of T(34 are included in the present invention. Therapeutically, antibodies or fragments of the antibody molecule could also be used to neutralize the biological activity of T(i4 in diseases where T~i4 is over expressed. Such antibodies can recognize an epitope of T(34 or fragments thereof suitable for antibady recognition and neutralization of T~34 activity. As used in this invention, the term "epitope" refers to an antigenic determinant on an antigen, such as a T~34 peptide, to which the paratope of an antibody, such as an T(34-specific antibody, binds. Antigenic determinants usually consist of chemically active surface groupings of molecules, such as amino acids or w0 00/06190 PCT/iJS99/17282 sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics.
Preparation of an antibody requires a substantially purified moiety that can provide an antigenic determinant. 'The term "substantially pure" as used herein refers to T~34, or variants thereof, which is substantially fi°ee of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
Substantially purified or "isolated" refers to molecules, either nu<;leic or amino acid sequences, that are removed from their natural envirox>nnent, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated. One skilled in the art can isolate T(34 or a T[34 isoform using standard techniques for protein liurif cation. The substantially pure peptide will yield a single major band on a non-reducing polyacrylamide gel.
The purity of the T~i4 peptide can also be determined by amino-terminal amino acid sequence analysis. T~i4 or a T(34 isoform peptide includes functional fragments of the peptide, as long as the activity of T~i4 or a T(34 isoforxn remains. Smaller peptides containing the biological activity of T~34 or a T(34 isoform are included in the invention. As used in the present invention, the term "antibody" includes, in addition to conventional antibodies, such protein fragments that have the ability to recognize specifically and bind the T~34 protein or variants thereof. Regions of the gene that differ at the protein level are well defined. A proteiin can be raised by expression of the wild type (wt) gene or of the variants, or, preferably, fractions therefore. For example, the nucleic acid sequence can be cloned into expression vectors.
According to this embodiment, the sequence of interest can first be obtained by employing PCR, as described above, or from a synthetic gene construction with overlapping and ligated synthetic oligonucleotides. Another alternative would involve synthesis of a short peptide. All those methodologies are well known to one skilled in the art. See, for example, Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR
B10LOGY, Volumes 1 and 2 (1987), with supplexr~ents, and Maniatis et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Laboratory, all of which are incorporated herein by reference.

WO 00106I90 PCTIUS99117282 .

The invention provides a method for detecting T(34, or variants thereof, which includes contacting an anti-T~i4 antibody with a sample suspected of containing T~i4, (e.g., cell or protein) and detecting binding to the antibody. An antibody which binds to T~i4 peptide is labeled with a compound which allows 5 detection of binding to T(i4. There are many different labels and methods of labeling known to those of ordinary skill in the art. ExamplE;s of the types of labels which can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, and bioluminescent compounds. Those of ordinary skill in the art will know of other suitable labels for binding to the antibody, or will be able to ascertain such, using routine experimentation. For purposes of the invention, an antibody specific for T(34 peptide may be used to detect the level of T~i4 in biological fluids and tissues. Any specimen containing a detectable ,amount of antigen can be used. The level of T(34 in the suspect cell can be compared with the level in a normal cell to 15 determine whether the subject is predisposed to a T(34 associated increase in angiogenesis or wound healing.
Use of antibodies for the diagnostic methods of the invention includes, fox example, immunoassays in which the antibodies can be utilized in liquid phase or bound to a solid phase carrier. in addition, the antibodies in these immunoassays can be detectably labeled in various ways. Examples of types of immunoassays which can utilize antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay.
Detection of the antigens using the antibodies of the invention can be done utilizing 25 immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.

WO 00/06190 PCT/US99/172$2 .

T(34 antibodies can be bound to many different carriers and used to detect the presence of an antigen comprising the peptide o:Pthe invention. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable Garners for binding antibodies, ar will be able to ascertain such, using routine experimentation.
Another technique which may also result in greater sensitivity consists of coupling the antibodies to low molecular weight havptens. These haptens can then be specifically detected by means of a second reaction. For example, it is common to use such haptens as biotin, which reacts with avidin, or dinitrophenyl, puridoxal, and fluorescein, which can react with specific antihaptem antibodies.
The invention includes use of antibodies immunoreactive with T~i4 peptide or functional fragments thereof. Antibody which consists essentially ofpooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided. M~onaclonal antibodies are made from antigen containing fragments of the protein by methods well known to those skilled in the art (Kohler, et al., Nature, 256:495, 1 ~~75). The team antibody as used in this invention is meant to include intact molecules as well as fragments thereof, such as Fab and F(ab')2, Fv and SCA fragments which arcs capable of binding an epitapic determinant on T(34.
(1) An Fab fragment consists of a monowalent antigen-binding fragment of an antibody molecule, and can be produced by digestion of a whole antibody molecule with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain.
(2) An Fab' fragment of an antibody molecule can be obtained b~ treating a whole antibody molecule with pepsin, followed b;y reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab' fragments are obtained per antibody molecule treated in this manner.

WO 00/06190 P~T/US99/17282 .

(3) An (Fab')Z fragment of an antibody can be obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. A
(Fab')z fragment is a dimer of two Fab' fragments, held together by two disulfide bonds.
(4) An Fv fragment is defined as a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains.
(5) A single chain antibody ("SCA") is a genetically engineered single chain molecule containing the variable region of a Eight chain arid the variable region of a heavy chain, linked by a suitable, flexible polyp>eptide linker.
Alternatively, a therapeutically or diagnostically useful anti-T(34 antibody may be derived from a "humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementary determining regions from heavy and light variable chains of the mouse i~:nmunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the marine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of marine constant regions. General techniques for cloning marine immunoglobulin variable domains are described, for example, by Orlandi et al., Proc.
Natl. Acad. Sci. USA 86: 3833 (1989), which is hereby incorporated in its entixety by reference. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321: 522 (1986); Riechmann et al., Nature 332:
323 (1988); Verhoeyen et al., Science 239: 1534 (1!x88); Carter et al., Proc.
Nat'l Acad. Sci. USA 89: 4285 (1992); Sandhu, Crit. Rev. Biotech. I2: 437 (1992);
and Singer et al., J. Immunol. 150: 2844 (1993), which ~~re hereby incorporated by reference.
Antibodies of the invention also may be derived from human antibody fragments isolated from a combinatorial immunoglobulin library. See, for example;
Barbas et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 119 (1991); Winter et al., Anna. Rev. Immunol. 12: 433 (1994), which are hereby incorporated by reference. Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from STRA.TAGENE Cloning Systems (La Jolla, CA).
Methods and Compositions for Treating or Diagnosing T,134 Associated Disorders In another embodiment of the invention, a method of diagnosing a pathological state in a subject suspected of having a. pathology characterized by a disorder associated with T~34 is provided. The metriod includes obtaining a sample suspected of containing T(34 from the subject, determining the level of T~34 in the sample and comparing the level of T~i4 in the sample to the level of T(34 in a normal standard sample. Such conditions include, but are not limited to subjects having cell proliferative disorders, recurrent wounds, tissue repair disorders, f brotic tissue disorders, chronic ulcers and other disorders described herein. Such disorders further include those associated with the various T(34 isofo:rms; known or not yet identified.
The term "cell-proliferative disorder" denotes malignant as well as non-malignant cell populations which often appear to differ from the surrounding tissue 1 S both morphologically and genotypically. Malignant cells (i. e. cancer) develop as a result of a muitistep process. Such disorders may b~e detected using the methods of the current invention. For example, a sample suspected of containing T~i4 is obtained from a subject, the level of T(34 peptide is determined and 'compared with the level of T~i4 peptide in a normal tissue sample. The level of T(34 can be determined by any number of methods including, for example, immunoassay using anti-T(34 peptide antibodies. Other variations of such assays include radioimmunoassay (RIA), ELISA
and immunofluorescence. Alternatively, nucleic acid probes can be used to detect and quantify T(34 peptide mRNA for the same purpose. Such detection methods are standard in the art.
In another embodiment, the invention provides a method for ameliorating a wound healing disorder associated with T~i4 or a 'T~i4 isoform, including treating a subject having the disorder with a composition that regulates T~i4 activity.
The term "ameliorate" denotes a lessening of the detrimental effect of the disease-inducing response in the subject receiving therapy. Where tree disease is due to an abnormally WO 00/06190 PCT/tJS99117282 _ high level of T[34, the administration of an agent, such as an antagonist of T(~4 activity, may be effective in decreasing the amount of T(34 activity.
Alternatively, where the disease is due to an abnormally low level of T~i4, the administration of T(34 or an agent that increases T~i4 activity, such as an a~;onist, may be effective in increasing the amount of T~34 activity.
Jn yet another embodiment, the invention provides a method of treating a subject having a wound healing disorder characteri2:ed by recurrent or slow to heal wounds or wounds that are chronic non-healing woo~xnds associated with altered T(34 or T(34 isoform gene expression in a subject. The method includes administering to a I O subject having the disorder a wound-healing effective amount of an agent which modulates T~34 gene expression, thereby treating th<; disorder. The term "modulate"
refers to inhibition or suppression of T~i4 expression when T(34 is over expressed, and induction of expression when T~i4 is under expressf;d. The term "wound-healing effective amount" means that amount of T(34 agent which is effective in modulating T(34 gene expression resulting in reducing the symptoms of the T~34 associated wound healing disorder.
An agent which modulates T(34 or T~i4 i.soform gene expression may be a polynucleotide for example. The polynucleotide m;ay be an antisense, a triplex agent, or a ribozyme. For example, an antisense may be directed to the structural gene region or to the promoter region of T~i4 may be utilized.
When a wound healing disorder is associated with the expression of Tj34, a therapeutic approach which directly interferes with the translation of T~i4 mRNA into protein is possible. For example, an antisense nucleic acid or a ribozyme can be used to bind to the T(34 RNA or to cleave it. Antisense ItNA or DNA molecules bind specifically with a targeted gene's RNA message, i~iterrupting the expression of that gene's protein product. The antisense binds to the mRNA forming a double stranded molecule which cannot be translated by the cell. Antisense oligonucleotides of about I S-25 nucleotides are preferred since they are easily synthesized and have an inhibitory effect just like antisense RNA molecules. In addition, chemically reactive group, such as iron-linked ethylenediaminetetraacetic acid (EDTA-Fe) can be WO 00/06190 PCTIUS99i17282 attached to an antisense oligonucleotide, causing elE;avage of the RNA at the site of hybridization. These and other uses of antisense methods to inhibit the in vitro translation of genes are well known in the art (Marcus-Sakura, Anal., Biochem., 172:289, 1988).
Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mFNA molecule (Weintraub, Scientific American, 262:40, 1990). In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule. 'The antisense nucleic acids interfere with the translation of the mIZNA, since the cell will not translate a mRNA that is double-stranded. Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced intro the target T(34 producing cell.
The use of antisense methods to inhibit the in vitro translation of genes is well known in the art (Marcus-Sakura, Anal.Biochem.; 172:289, 1988).
Use of an oligonucleotide to stall transcription is known as the triplex strategy since the oligomer winds around double-helical DNA, forming a three-strand helix. Therefore, these triplex compounds can be designed to recognize a unique site on a chosen gene (Maher, et al., Antisense Res. and Dev., .1~3 :227, 1991;
Helene, C., Anticancer Drug Design, 6~6,~:Sd9, 1991).
Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences which encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech,. J.Amer.Med. Assn., 260:3030, 1988). A major advantage ofthis approach is that, because they are sequence-specific, only mRNAs with particular sequences are inactivated.
There are two basic types of ribozymes namely, tetrahymena-type (HasselhofF, Nature, 334:585,1988) and "hammerhead°'-type. Tetrahymena-type ribozymes recognize sequences which are four bases in length; while "hammerhead"-type ribozymes recognize base sequences 11-i8 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species.
These and other uses of antisense methods to inhibit the ih vivo translation of genes are well known in the art (e.g., De Mesmae'ker, et al., 1995.
Backbone modif cations in oligonucleotides and peptide nucleiic acid systems. Cu~-r.
Opin.
Struct. Biol. 5:343-355; Gewirtz, A.M., et al., 1996b. Facilitating delivery of antisense oligodeoxynucleotides: Helping antisense deliver on its promise;
Proc. Natl.
Acad. Sci. U.S.A. 93:3161-3163; Stein, C.A. A discussion of G-tetrads 1996.
Exploiting the potential of antisense: beyond phosplxorothioate oligodeoxynucleotides. Chem. and Biol. 3:319-323).
Delivery of antisense, triplex agents, ribozymes, competitive inhibitors and the like can be achieved using a recombinant ex;pressian vector such as a chimeric virus or a colloidal dispersion system. Various viral vectors which can be utilized for gene therapy as taught herein include adenovirus, herpes virus, vaccinia, or, preferably, an RNA virus such as a retrovirus. Preferably, the retroviral vector is a derivative of a marine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney marine leukemia virus (MoMuLV), Harvey marine sarcom;~ virus (HaMuSV), marine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. By inserting a polynucleotide sequence of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target specific.
Retroviral vectors can be made target specific by inserting, for example, a polynucleotide encoding a sugar, a glycolipid, or a protein. Preferred targeting; is accomplished by using an antibody to target the retroviral vector. Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome to allow target specific delivery of the retroviral vector containing the antisense polyn~acleotide.

WO 00/06190 PCTIUS99/17282 .
- 2~ _ Since recombinant retroviruses are defective, they require assistance in order to produce infectious vector particles. This assistance can be provided, for example, by using helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR.
These plasrnids are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encahsidation. Helper cell lines which have deletions of the packaging signal include but are not limited to '~'2, PAS 17 and PA12, for example. These cell lines produce empty.virions, since no genome is packaged. If a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced lby other genes of interest, the vector can be packaged and vector virion produced.
Alternatively, NIH 3T3 or other tissue culture cells can be directly transfected with plasmids encoding the retroviral stnxctural genes gag, pal and env, by conventional caIciurn phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. 'The resulting cells release the retroviral vector into the culture medium.
A targeted delivery system for delivery of nucleic acids as described herein includes a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, mierosphe;res, beads, gene activated matrices and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome. Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 ~m can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci., f :77, 1981 ). In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells. In order for a liposome to b~e an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells;
(3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al., Biotechniques, 6_:682,1988).
Pathologically, T~i4 may be involved in diseases in which there is an overgrowth of blood vessels, such as cancer, tumor formation and growth, diabetic retinopathy, neovascular glaucoma, rheumatoid arthritis and psoriasis.
The ingrowth of capillaries and ancillary blood vessels is essential for growth of solid tumors and is thus an unwanted physiological response which facilitates the spread of malignant tissue and metast~~ses. Inhibition of angiogenesis and the resultant growth of capillaries and blood vessels is therefore a component of effective treatment of malignancy in use of treatment of cancer patients.
Thus, in another embodiment, the invention provides a method of inhibiting angiogenesis in a subject, including administering to the subject a composition containing an agent which regulates T(34 activity. The composition may include agents that regulate angiogenesis, for example agents that affect thymosin al, PDGF, VEGF, IGF, FGF and TGF(3. For example, the inhibition of angiogenesis and endothelial cell migration can be beneficial in controlling the growth of solid tumors.
Most, if not all solid tumors, like normal tissue, require a steady and sufficient blood supply for optimal growth. Tumors are known to make use of angiogenic growth factors to attract new blood vessels and ascertain supply with sufficient amounts of nutrients to sustain their growth. Many tumors are well vascularized and the inhibition of the formation of an adequate blood supply to the tumor by inhibition of tumor vascularization, as a result of inhibition of ar~giogenesis, is beneficial in tumor growth control. Without a strong blood supply, rapid and prolonged growth of tumor tissue cannot be sustained. Thus, agents that inhibit T(34 activity may be used to prevent neoplastic growth. The T~i4 inhibiting agent may be administered orally, parenterally, topically, intravenously, or systemically. In addition, far inhibiting tumor cell proliferation and tumor growth, the agent may be administered locally WO 00/06190 PCT/US99/17282 .

directly to the tumor or as a part of a deposited slov~r release formulation.
Administration may be on a daily basis for as long ~~s needed to inhibit angiogenesis, endothelial cell proliferation, tumor cell proliferation or tumor growth.
Alternatively, a slow release formulation may continue for as long as needed to control tumor S growth. This dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may ibe administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
In this regard, the compositions of this invention that are useful as inhibitors of angiogenesis, endothelial cell proliferation, tumor cell proliferation and tumor growth contain a pharmaceutically acceptable corner and an amount of T[34 modulating agent effective to inhibit tumor or endothelial cell proliferation.
Such compositions may also include preservatives, antio:cidants, immunosuppressants and other biologically and pharmaceutically effective agents which do have effects on 1 S tumor growth but which do not exert a detrimental .effect on the T(34 modulating agent. For treatment of tumor cells the composition may include a chemotherapeutic agent, for example an anti-cancer agent which selectively kills the faster replicating tumor cells, many of which are known and clinically used. Exemplary anticancer agents include mephalan, cyclophosphamide, meth.otrexate, adriamycin and bleomycin.
Screen or comwounds which modulate T,a4 activity In another embodiment, the invention provides a method for identifying a compound that modulates T[34 activity, angiogenes:is activity or wound healing activity. The method includes incubating components including the compound and 2S T~34 under conditions sufficient to allow the components to interact and determining the effect of the compound on T(34 activity before a.nd after incubating in the presence of the compound. Compounds that affect T(34 activity (e.g., antagonists and agonists}
include peptides, peptidornimetics, polypeptides, chemical compounds, minerals such WO 00/06190 PCT/US991I7282 .

as zincs, and biological agents. T~i4 activity can be assayed using the methodology as described in the present Examples.
The present Examples are meant to illusi:rate, but not limit the scope of the appended claims. ,Accordingly, one skilled in the art will recognize a number of equivalent materials and methods, which are intend to be covered by the present invention and disclosure.

In vivo wound healingis accelerated bv'T~i4 T(34, whether administered topically or intraperitoneal, significantly accelerated wound healing as compared to untreated wounds (Fig. 2 and 3). Full thickness 8 mm punch biopsy wounds were made on the dorsal surface of rats as previously reported (Bhartiya et al., J. Cell. Physiol. 150:312, 1992; Sihhu et al., J.
Cell. Physiol. 169:108,1996) and T~4 was given topically at the time of wounding (5 ~cg in 50 ~l) and again after 48 hours. Controls fox 'the topical treatment received identical amounts of saline at the time of wounding; and at 48 hours.
Additional rats received intraperitoneal injections at the time of wounding (60 ~.g in 300 pl) and again every other day (e.g., days 0, 2, 4, and 6). Controls for these animals received identical amounts of saline infra-peritoneally on thf; same injection schedule. On days 4 and 7 post-wounding, measurements were made on the wound size. At days 8 and post-wounding, tissue was collected and fixed in 10% buffered formalin. The samples were sectioned and stained with H&E and Masson's Trichrome (American Histolabs, Gaithersburg, MD).
Histological sections were used to measure the re-epithelialization and the contraction of the wound using an ocular micrometer. Epidermal migration was determined by measuring the lengths of the tongues of epithelium migrating form either side of the wound over the wound bed from the zone of proliferation at the margin of the uninjured and wounded skin. Epidemnal thickness was also measured beginning at the junction of the uninjured and proliferating epidermis. The thickness was measured vertically from the basement membrane to the most superficial layer of WO 00/06190 PCT/US99/17282 .

the migrating epidermis at every 200 microns. The; mean epidermal thickness of each migrating tongue of epidermis was then computed from each wound. Vessel counts were performed by first identifying vascular space:. by their endothelial lining. All such vessels in the wound bed were counted including those at the junction of the 5 dermis and the subcutis, since angiogenesis into the wounds occurs to a great extent from these vessels. The numbers were averaged into vessel counts per 10 high powered fields (40x).
The effect of T~34 on wound healing w~us studied in a full thickness cutaneous rat wound model. FIG. 1 shows a diagr<~tn of the wound site that extends form the epidermis to the fatlmuscle layer. This model allowed measurement of two parameters: the re-epitheliaiization (gap) and the contraction (width) of the wound.
Wounds treated topically with T~34 showed about a 15% decrease in width and about 15% decrease in gap in the treated versus controls (FIG. 2 and 3, respectively).
Figure 2 shows a 15% decrease in wound width as compared to the saline 15 controls as early as 4 days after wounding and conitinued until day 7.
Intraperitoneal injection of T~i4 resulted in a 18% decrease in wound width relative to saline treated controls at day 4 and 11 % decrease at day 7. This trend was observed on the 4th day post wounding and continued through day 7 (*P~CL0001, **P<_0.08, significant difference from media alone, student's t-test). These data demonstrate that:
T~i4, when given either topically or systemically, increases wound re-epithelialization and contraction. Both topical and systemic treatment ~~re equally effective. Lower doses of T~i4 were tested including 2.5 pg and 0.5 ~.g in 50 ~,l for topical and 30 g,g and 6 p,g in 300 ~,1 for intraperitoneal injection but reduced or no effect, respectively, was observed on wound healing.
25 Figure 3 shows an 18% decease in gap length as compared to saline controls when T(34 is administered topically, as early as 4 days after wounding. This trend continued to termination at day 7 (*P<_0.04, student's t-test).
Intraperitoneal injections resulted in a 42% decrease in gap size rE;lative to saline treated controls.
This decrease was observed on the 4th day post wounding and continued through day 7 (**P<_0.0007, student's t-test). The increase in re-epithelialization was observed in WO 00/06190 PCTIUS99117282 .

wounds treated for 7 days and the rate of gap closure was slightly accelerated over that observed at day 4. A 62% decrease in gap size was observed in the T~i4-treated wounds. Quantitadon of epidermal migration showed a statistically significant 1.5 fold increase in migration of epidermal tongues over the wound bed after topical treatment (Table 1). Quantitation of epithelial mil~ation in intraperitoneally treated wounds showed a statistically significant increase in migration of epidermal tongues as compared to controls (Table 1). There was no difference_in the thickness of the migrating epidermis between either of the T(34 treatments and the control (Table 1 ).
Histological sections of the wounds clearly show increased re-epithelialization in the treated wounds as compared to controls in 7 day wounds (FIG 4).
Table 1: Morphometric Measurements of Control and Thymosin ~i4 Treated Samples Parameter Control LP. Topical Epidermal Migration 2403.39.7 3168.3t38.4*3668.7~56.6*
(~.m) Epidermal Thickness 128.2119.3 135.O~I1.7 142.3119.8 (gym) Vessels/10 HPF 1364.015.0 2415:0~24.3*2186.Ot11.8*

HPF: high power field. *P~0.00001 by Welch's t-to;st, significantly different than control.
FIG. 4 shows a comparison of typical control (D) and T~i4-treated (E and F) sections of 7 day wounds. Treatment with T(34 resulted in considerable capillary ingrowth (FIG 4E and F, arrows). Vessel counts showed a significant (about 2 fold) increase in the number of vessels in T(34 treated wounds (Table 1). No increases in the number of macrophages in the wounds were observed. There was no apparent increase in the accumulation/biosynthesis of collagen in treated -T~34 wounds (Fig. SB
and C vs A) supporting a decreased wound width and supporting a role for T(34 in wound contraction. Both the topical and systemically treated wound appeared similar although the wound contraction proceeded slightly more quickly with the topical treatment.

WO 00/06190 PCT/US99/17282 .

Reduction of the wound size was observed in both experimental groups as compared to control groups (Fig. 2 - 4). More and larger blood vessels were noted in the experimental groups as compared to the controls (Fig. 4). Additionally, an increase in the accumulationlbiosynthesis of collagen by T(34 treated wounds as compared to control suggests a role for T~i4 in wound contraction and extracellular matrix deposition. Histological staining of these wounds demonstrated an increase in collagen density and extracellular matrix deposition when compared to controls. (Fig.
5).

Migration Assays of Keratinoc es Primary keratinocytes were prepared from either Balb/c or CD-I newborn mice as described previously (Dlugosz et al.,1995). Cells were plated in calcium-and magnesium-free Eagle's Minimal Essential Medium (EMEM) containing 8%
fetal calf serum treated with 8% Chelex (Bio-Rad Laboratories, Hercules, CA), units/ml penicillin-streptomycin, and the calcium concentration was adjusted to 0.25' mM. The following day, cultures were washed with calcium- and magnesium-free phosphate buffered saline, treated briefly with Trypsin (Life Technologies;
Gaithersburg, MD), washed with culture medium aJnd resuspended in EMEM
containing 0.05 mM calcium. Cells were used immediately in migration assays.
Keratinocyte migration assays were carried out in Boyden chamber using 12 ~.m pore polyester membranes (Poretics, Liverm.ore, CA) coated with a 0.1 mg/ml solution of collagen IV in dH20 (Trevigen, Gaithers.burg, MD). Filters were then dried at least 1 h. Cells were harvested using Versene or Trypsin (Life Technologies, Gaithersburg, MD) and resuspended in Eagle's minimal essential medium with 0.05 2:i mM Caz+. The bottom chamber was loaded with E:MEM containing O.OI, 0.1, 10, 100, and 1000 ng/ml of synthetic T~34. Conditioned medium from primary dermal fibroblasts and/or keratinocyte growth factor was aided to several wells as a positive control. Cells were added to the upper chamber at .a concentration of 50,000 cells per well. Chambers were incubated at 35 C/7% COZ for 4-5 hours and the filters were w0 00106190 PCTlUS99/17282 .

then f xed and stained using Diff Quik (Baxter I Iealthcare Corporation, McGraw Park, IL). The cells that migrated through the filter were quantitated by counting the center of each well at l Ox using an Olympus CK2 microscope. Each condition was assayed in triplicate wells and each experiment was. repeated four times with different preparations of cells.
The results demonstrated that keratinoc;~te migrated in response to T(34 after 4-5 hours of exposure. Migration was enhanced 2-3 fold (P<_0.003} over migration in the presence of media alone (FIG. 6) and at the maximal responding dose exceeded the positive control. The effect of T~i4 on migration, while showing slightly i 0 different dose curves depending on the cell preparation and source, clearly showed a biphasic pattern with 1000 ng/ml and 0.01 ng/ml slowing the most migratian and the middle doses showing less stimulation (but still greater than control media) in all 4 assays.

Mi,~ration Assays of Corneal Epithelial Cells Corneal Epithelial Cell migration assays were earned out in Boyden chamber using 12 ~m pore polyester membranes (1'oretics, Livermore, CA) coated with a 0.1 mg/ml solution of collagen IV in dH20 (Trevigen, Gaithersburg, MD}.
20 Filters were then dried at least 1 h. Cells were cultured and resuspended in Eagle's Minimal Essential Medium with 0.05 mM Ca2+. The bottom chamber was loaded with EMEM containing 0.01, 0.1,10, 100, and lOt?0 ng/ml of synthetic T~34.
Conditioned medium from primary dermal fibroblasts and/or keratinocyte growth factor was added to several wells as a positive control. Cells were added to the upper chamber at a concentration of 50,000 cells per well. Chambers were incubated at 35 C/7% COZ for 4-5 hours and the filters were then fixed and stained using Diff Quik (Baxter Healthcare Corporation, McGraw Park, IL). The cells that migrated through the filter were quantitated by counting the center of each well at l Ox using an Olympus CK2 microscope. Each condition was a;>sayed in triplicate wells and each experiment was repeated four times with different preparations of cells.

WO 00/06190 PCTJUS99/17282 .

The results demonstrated that corneal epithelial cell migrated in response to T~i4 after 4-5 hours of exposure. Migration was enhanced 2-3~ fold over migration in the presence of media alone (FIG. 7) with the highest level of migration seen at nglml of T~i4.

In vivo Corneal Re-Epithelialization To determine the effect of T~i4 on corneal re-epithelialization in vivo, Rat corneas were de-epithelialized and treated with T(34. Filters were soaked in heptanol, applied to the eye for 30 seconds, and then the epithelium was scraped.
Various concentration of T(34 in saline was applied to the eye and at 24 hours the rats were sacrificed. The eyes were fixed, sectioned and the degree of corneal epithelial migration (as measured in pixels) was determined using a microscope with an internal caliper by a masked observer. The results demonstrate that re-epithelialization of the corneawas increased 2-fold over untreated control in the presence of about 1 to 25~.g of T~34 (FIG. 8 and 9}. In addition, it was noted that T~i4 treated eyes had reduced inflammation compared to the non-treated corneas.

Impaired Healing Model Thymosin ~i4 also enhanced wound healing in an impaired model. Steroid treatment reduces the rate of wound repair in marn~ma.ls. Rats treated with steroids such as hydrocortisone serve as a model of impaired wound healing due to the delay observed in wound closure. Animals were injected intramuscularly everyday with hydrocortisone. Steroid treated rats showed a significant increase in the level of healing when T~34 was added topically or injected intraperitoneally. At the initial time point, day 4, topically treated animals showed little response (-~ 7% gap or width closure, N=3) compared to saline treatment. Intraperitoneal injection, however, resulted in a 28% decrease in3 gap size and a 14% decrease in wound width. At day 7, a response was observed with both topical treatment and intraperitoneal injection.

WO 00/06190 PCT/US99117282 .

'The gap in topically treated animals decre;ased by 39% compared to saline treatment. The wound width decreased by 23%. Int~~peritoneal injection resulted in a 26% decrease in gap size and a 10% decease in wound width. Taken together, these demonstrate that T~34 is useful to treat chronic, as we;ll as, acute wounds.
A number of embodiments of the present invention have been described.
Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims (67)

WHAT IS CLAIMED IS:

1. A method for promoting wound healing in a subject in need of such treatment comprising administering to the subject a wound-healing effective amount of a composition containing a wound healing polypeptide comprising the amino acid sequence LKKTET and conservative variants thereof having wound healing activity.

2. The method of claim 1, wherein the wound healing polypeptide is thymosin .beta.4 or an isoform of thymosin .beta.4.

3. The method of claim 2, wherein the composition further contains an agent that stimulates the production of thymosin .beta.4 peptide.

4. The method of claim 3, wherein the agent is transforming growth factor beta (TGF-b).

5. The method of claim 1, wherein the wound healing polypeptide is delivered systemically.

6. The method of claim 1, wherein the wound healing polypeptide is delivered topically.

7. The method of claim 6, wherein the wound healing polypeptide is contained in a topical formulation selected from the group consisting of a gel, cream, paste, lotion, spray, suspension, dispersion, salve, hydrogel and ointment.

8. The method of claim 1, wherein the wound healing polypeptide is recombinant or synthetic.

9. The method of claim 2, wherein the isoform of thymosin .beta.4 is at least 70%
homologous to thymosin .beta.34 peptide set forth as SEQ ID NO: 1 in Figure 10.

10. The method of claim 9, wherein the isoform of thymosin .beta.4 is selected from the group consisting of T.beta.4ala, T.beta.9, T.beta.10, T.beta.11, T.beta.12, T.beta.13, T.beta.14 and T.beta.15.

11. The method of claim 1, further comprising contacting the site of the wound with an agent which promotes wound healing.

12. The method of claim 11, wherein the agent is selected from the group consisting of IGF, IGF-1, IGF-2, IL-1, PDGF, FGF, KGF, VEGF, prothymosin .alpha., thymosin .alpha.1 or combinations thereof.

13. A method for promoting wound healing in a subject in need of such treatment comprising administering to the subject a wound-healing effective amount of a composition containing thymosin .beta.4 or an isoform of thymosin .beta.4.

14. The method of claim 13, wherein the composition further contains an agent that stimulates the production of thymosin .beta.4 peptide.

15. The method of claim 14, wherein the agent is transforming growth factor beta (TGF-b).

16. The method of claim 13, wherein the thymosin .beta.4 is delivered systemically.

17. The method of claim 13, wherein the thymosin .beta.4 is delivered topically.

18. The method of claim 17, wherein the thymosin .beta.4 is contained in a topical formulation selected from the group consisting of a gel, cream, paste, lotion, spray, suspension, dispersion, salve, hydrogel and ointment.

19. The method of claim 13, wherein the thymosin .beta.4 is recombinant or synthetic.

20. The method of claim 13, wherein the isoform of thymosin .beta.4 is at least 70%
homologous to thymosin .beta.4 peptide set forth as SEQ ID NO: 1 in Figure 10.

21. The method of claim 13, wherein the isoform of thymosin .beta.4 is selected from the group consisting of T.beta.4ala, T.beta.9, T.beta.10, T.beta.11, T.beta.12, T.beta.13, T.beta.l4 and T.beta.15.

22. The method of claim 13, further comprising contacting the site of the wound with an agent which promotes wound healing.

23. A method for promoting wound healing in a tissue comprising contacting the tissue with a therapeutically effective amount of a composition containing a wound healing polypeptide comprising the amino acid sequence LKKTET and conservative variants thereof having wound healing activity.

24 The method of claim 23, wherein the wound healing polypeptide is thymosin .beta.4 or an isoform of thymosin .beta.4.

25. The method of claim 23, wherein the contacting is in vivo in a subject.

26. The method of claim 23, wherein the contacting is ex vivo.

27. The method of claim 23, wherein the subject is a mammal.

28. The method of claim 27, wherein the mammal is human.

29. The method of claim 24, wherein the composition further contains an agent that stimulates the production of thymosin .beta.4 peptide.

30. The method of claim 29, wherein the agent is transforming growth factor beta (TGF-b).

31. The method of claim 29, wherein the agent is a mineral.

32. The method of claim 29, wherein the mineral is zinc.

33. The method of claim 23, wherein the wound healing polypeptide is delivered topically.

34. The method of claim 23, wherein the wound healing polypeptide is contained in a topical formulation selected from the group consisting of a gel, cream, paste, lotion, spray, suspension, dispersion, salve, hydrogel and ointment.

35. The method of claim 23, wherein the wound healing polypeptide is delivered systemically.

36. The method of claim 23, further comprising contacting the site of the tissue with an agent which promotes wound healing.

37. The method of claim 36, wherein the agent is selected from the group consisting of IGF, IGF-1, IGF-2, PDGF, FGF, KGF, VEGF, prothymosin .alpha., thymosin .alpha.1 or combinations thereof.

38. The method of claim 23, wherein the tissue is selected from the group consisting of epidermal, eye, uro-genital, gastro-intestinal, cardiovascular, muscle, connective, and neural.

39. The method of claim 23, wherein the tissue is skin tissue.

40. The method of claim 23, wherein the tissue is eye tissue.

41. A method of inhibiting wound healing in a subject, comprising administering to the subject a composition containing an agent which regulates thymosin .beta.4 activity.

42. The method of claim 41, wherein the agent is an antibody.

43. The method of claim 42, wherein the antibody is polyclonal.

44. The method of claim 42, wherein the antibody is monoclonal.

45. A method of diagnosing a pathological state in a subject suspected of having pathology characterized by a wound healing disorder associated with thymosin .beta.4, comprising:
obtaining a sample suspected of containing thymosin .beta.4 from the subject;
detecting a level of thymosin .beta.4 in the sample; and comparing the level of thymosin .beta.4 in the sample to the level of thymosin .beta.4 in a normal standard sample.

46. The method of claim 45, wherein the pathology is selected from the group consisting of fibrotic disease, ischemia, atherosclerosis and cell proliferative disorders.

47. A method for ameliorating a wound healing disorder associated with thymosin .beta.4, comprising treating a subject having the disorder, at the site of the disorder, with an agent which regulates thymosin .beta.4 or the activity of a thymosin .beta.4 isoform.

48 The method of claim 47, wherein the thymosin .beta.4 regulating agent is an antagonist of thymosin .beta.4 peptide.

49. The method of claim 48, wherein the antagonist is an antibody which specifically binds to thymosin .beta.4 peptide.

50. A method for identifying a compound which modulates wound healing, angiogenesis or cell migration activity, comprising contacting thymosin .beta.4 or an isoform of thymosin .beta.4 with a compound suspected of having thymosin .beta.4 modulating activity and detecting an effect on thymosin .beta.4 or thymosin .beta.4 isoform activity.

51 The method of claim 50, wherein the compound is an agonist of thymosin .beta.4 activity.

52. The method of claim 50, wherein the compound is an antagonist of thymosin .beta.4 activity.

53 A method of promoting epithelial cell migration, comprising contacting an epithelial cell with a composition comprising thymosin .beta.4 or an isoform of thymosin .beta.4.

54. The method of claim 53, wherein the epithelial cell is a skin cell.

55. The method of claim 54, wherein the skin cell is a keratinocyte.

56. The method of claim 53, wherein the epithelial cell is a corneal epithelial cell.

57. The method of claim 53, wherein the contacting is in vivo.

58. The method of claim 57, wherein the contacting is topical.

59. The method of claim 57, wherein the contacting is systemic.

60. The method of claim 53, wherein the contacting is in vitro or ex vivo.

61. The method of claim 53, wherein the composition is selected from the group consisting of a gel, cream, paste, lotion, spray, suspension, dispersion, salve, hydrogel, ointment, and a biocompatible matrix.

62. A pharmaceutical composition comprising wound healing polypeptide comprising the amino acid sequence LKKTET and conservative variants thereof having wound healing activity, and a pharmaceutically acceptable carrier.

63 The pharmaceutical composition of claim 62, wherein the wound healing polypeptide is thymosin .beta.4 or an isoform of thymosin .beta.4.

64. The pharmaceutical composition of claim 62 in a controlled release formulation.

65. The pharmaceutical composition of claim 62 in a liposomal form.

66. The pharmaceutical composition of claim 62 in a lyophilized form.

67. The pharmaceutical composition of claim 62 in a unit dosage form.