CA2138644A1 - Method of treating ophthalmic disorders using tgf-.beta. - Google Patents

Abstract

This invention is a method for the treatment of ophthalmic disorders. The method is suitable for treatment of a variety of disorders including macular holes, macular degeneration, and retinal detachment and tears, cataracts, and corneal and scleral injuries.
The method entails application of an effective amount of Transforming Growth Factor-.beta. (TGF-.beta.) to the affected region.

Description

WO 94~01124 2 ~. 3 ~ i PCr/US93/06420 ' ' ;.

METHOD QF I~EATING OPHTHAI~IIC DISORDERS U5ING TGF~
~ ,"
Field of the_Invention ~
This invention is a method for treating a ~:-~ariety of ophthalmic disorders in which wound healing is 10 impaired or requires modulation, includinq macular holes, ~
macular degeneration, retinal detachment and tears, retinal edema, retinal vascular disorders, retinal i~`;
neovascularization, wound healing disorders, i -~
prollferative disorders, anti degenerative disorders, lS anti-angiogenesis disorders, dry eye syndromes, uveitis, ~`
secondary cataracts, corneal epithelial wounds, corneal ...
neo~ascularization, Sjogren's syndrome, and surgical ~;.
wounds. The method entails application of Transforming .
Growth Factor-~ (TGF-~) to the affected region. TGF-~2 `.`~
is the preferred form of TGF-~. Other growth factors which have wound healing and neurotrophic effects may .
also be applied.

Back~round of_the Invention -~ :
- Major ophthalmic disorders affect the retina, ¦ :`
lens and cornea. Among the most important retinal disor~ers-are-macular holes and degeneration, retinal ~:
tears-, diabetlc retinopathy, and miscellaneous disorders.
The most important disorders of the lens are cataracts ¦
39 and refracti~e errors. The most important disorders of . :~-the-cornea are those related to corneal defects, including corneal ulcers and wounds and the consequences ~ ~.:
of dry--eye-/ Sjogren's syndrome. These are discussed I ~.
briefly below. .
35 _ ~

WO94/U1124 ~ l3 ~ 2- PC~/US93/0~20 Retinal PhYsioloav The retina is the light-sensitive portion of the eye. Supported by the choroid and retinal pigment cells and found at the posterior of the eye, the retina contains the cones and rods which detect colors. When the rods and cones are excited, they transmit signals which pass through successive neurons in the retina to the optic nerve and finally to the cerebral center, where a "visual picture" is integrated.
In the center of the retina is the macula lutea, which is about l/3 to ll2 cm in diameter. The macula provides detailed vision, particularly in the -~
center (the fovea), because the cones are higher in ;;
density. Blood vessels, ganglion cells, inner nuclear layer and cells, and thë plexiform layers are all displaced to one side (rather than resting above the ¦
cones), thereby allowing light a more direct path to the ; ~ ~ cones.
; Under the retina are the choroid, a collection of blood vessels embedded within a fibrous tissue, and the deeply piqmented epithelium, which overlays the choroid layer. The choroidal blood vessels provide nutrition to the retina (particularly its visual cells).

~- 25 ~ Retinal_Pisorders There are a variety of retinal disorders, whose current treatment is~not optimal. The retina may tear, form holes and separate from the underlying choroid. of particular concern are macular holes which produce ` 30 blurred central vision or metamorphopsia. The cause of most macular holes is unknown. However, trauma, cystic degeneration, and vitreoretinal traction have all been associated with hole formation. Full thickness macular holes also appear following myopic degeneration, laser photocoagulation, lightning strike and pilocarpine administration. There also is a higher frequency of macular holes after cataract extraction.

WO94/~1124 213 ~ PCT/US93/~20 -3~
-',` ~' '.

The idiopathic senile macular hole is a disorder occurring generally in healthy women who are in ~;
their sixth decade of life or beyond. The more severe -~
holes involve the full thickness of the macula and are surrounded by a halo of retinal detachment. In the early stage, there may be a sudden decrease or distortion in `~
vision. But early changes are difficult for physicians -~
to spot. Patients may experience sudden vision changes ~:
or may not notice symptoms if the condition slowly l0 evolves. Some experts believe that macular holes begin -with central or foveolar detachment, which eventually develops into a full-depth macular hole. See, Gass, i "Idiopathic Senile Macular Hole--Its Early Stages and Pathogenesis", Arch. Ophthalmol. (1988~ 108:629-639.
15 Partial holes, that is, holes which are partial in depth ~-or shape, having a new moon or horseshoe shape, are worth 1 `
diagnosing early, particularly if an effective way to ¦ ~-stop progression to macular holes were available. `:
For macular holes of unknown origin, certain operations, such as trans-pars plana vitrectomy, may interrupt the progress of macular degeneration toward full-thickness hole formation. However, the surgery may permanently damage central vision. Current metbods for treating-macular holes improve vision in only 40% of -eyes. ~
Other retinal vascular and macular diseases which may xhibit voids, tears, or separations in the retina resu-lting from lack of fibrous or supporting ~ ~' tissue. -- !j , Macular Deqeneration ~
- --Age-related macular degeneration (AMD) is the :` major cause of severe visual loss in United States citizens-aver the age of 55. Most AMD patients have a build up of deposits within and under the retinal pigment epithe-lium-in the macular region resulting in atrophy of the retina and the retinal pigment epithelium. The W094/01124 PCT/US93/0~20 21~ ~4~
retinal pigment cells are long-lived. They scavenge for photoreceptor discs from the rods and cones for years and accumulate intracellular wastes. The incompletely digested residues reduce cytoplasmic space (Feeny-Burns, L. et al., Invest. Ophthal. Mol._ Vis. Sci. (1984) 25:195-200) and interfere with metabolism. As the cell volume available to the organelles diminishes, the capacity to digest photoreceptors decreases, and this may be the basis for macular degeneration.
Some patients also experience exudative AMD
with choroidal neovascularization, detachment and tears of the retinal pigment epithelium, fibrovascular scarring, and vitreous hemorrhage. This process is responsible for more than 80% of cases of significant visual loss in patients with AMD.
Age-related macular degeneration (AMD) is a 11 , sight-threatening disorder which occurs in either an atrophic or (less commonly) an exudative form. In exudative AMD, blood vessels grow from the choriocapillaris through defects in Bruch's membrane, and in some cases the underlying retinal pigment epithelium (RPE). Organization of serous or hemorrhagic exudates escaping from these vessels results in fibrous scarring -~ of the macular region with attendant degeneration of the neuroretlna and permanent loss of central vision.
- _ Several studies have recently described the use of laser photocoagulation in the treatment of initial or recurrent neovascular lesions associated with AMD
(Macular Photocoagulation Study Group tl99l) Arch.
- 30 Oshtha1. iO9:l220; Macular Photocoagulation Study Group (l99l) Arch. O~hthal. 109:1232; Macular Photocoagulation Study Group (l99l) Arch. O~hthal. lO9:1242.
Unfortunately, AMD patients with subfoveal lesions subjected to laser treatment experienced a rather precipitous reduction in visual acuity (mean 3 lines) at 3 months follow-up. Moreover, at two years post-treatment treated eyes had only marginally better ., ., .... .. .. . .. . .. . .... ,.. ,., .. .. .. _.... , ". , .. .~ ,, ... . .. , ... . ,. .,.. ., . ,.. . ., . . . . ..
; ., . . . . ~

W094tO1124 213 ~ 5 ~ 4 PCT/US93/06420 -5~

visual acui~y than their unkreated counterparts (means of ~`
20/320 and 20/400, respectively). Another drawback of the procedure is that vision after surgery is immediately worse.
Retinal Tears The retina may tear or separate from the choroid, and the c~oroid may rupture, for a wide variety of reasons. ~
Other situations in which tissue separation is ~ -observed include such widely disparate conditions as detachment of retina and pigment epithelium, degenera~ive myopia, as may be evidenced by visible breaks in Bruch's membrane (lacquer crac~s), acute retinal necrosis 15 syndrome (ARN), and traumatic chorioretinopathies or `
contusion (Purtscher's Retinopathy).

Other Retinal Disorders -~
Other retinal disorders include edema and ischemic conditions. Macular and retinal edema are often associated with metabolic illnesses such as diabetes mellitus. Retinal edema is found in a large percentage of individuals who have undergone cataract extraction and othPr surgicaL procedures upon the eye. Edema is also ~`
found with acceIerated or malignant hypertension.
Macular edema is a common complication of prolonged inflammation ~ia ~Yei~is, Eales disease, or other diseases. Local edema is associated with multiple cytoid bodies ("cotton bodies") as a result of AIDS.
Ret1nal-ischemia can occur from either choroidal or retinal vascular diseases, such as central or branch retinal vein occlusion, collagen vascular diseases and~--tfirbmbocytopenic purpura. Retinal vasculitis and occlusion is seen with Eales disease and systemic lupus erythematosus.

6 ~ 6-= Proliferative Diabetic_Retino~athY (PDR) Sebag and McMeel reviewed the pathogenesis of PD~ (Survev of O~hthalmol. (1986) 30:377~84. The initiating event may be inadequate tissue oxygenation which causes vasodilation. Inadequate oxygenation may occur after the arterial basement membrane has thickened with diabetes-related deposits and because of endothelial cell proliferation, which is associated with pericyte degeneration. Basement membrane thickening and loss of pericytes are believed to result from low insulin and hyperglycemia, two important metabolic abnormalities of diabetes.
The neovascularization of PDR has been attributed to the subtle vascular abnormalities described above. Even this slight disruption may permit normally absent chemicals to enter the eye across the blood-retinal barrier.
Several growth factors besides TGF-~ appear to be involved in diabetic retinopathy, including fibroblast ~ -20 growth factors (FGF), an interplay of FGF and TGF-~, ¦
tumor necrosis factor (TNF-~ and ~), which are known to have angiogenic properties. (Wiedemann, Survey of - - Qphthalmol. (1992) 36:373-84). Others have proposed that because retinal blood vessels appear to have a unique response to diabetic ischemia, there may be specific _retina-derived ~rowth factors. Berritault et al.
-- - Differentiation (1981) 18:29-42; Chen and Chen Invest.
Oph~halmol. Vis. Sci. (1980) 19: 596-02; D'Amore and Klagsburn J. Cell. Biol. (1984) 99: 1545-49; Elstow et , , ; , , , - 3~ al. Invest. O~hthalmol Vis. Sci. (1985) 26:74-79; Glaser -: et al. Ophthalmoloov (1980) 87:440-46; and Ruelius---_ Altemose et al. Invest. O~hthalmol. Vis. Sci. (1985) 26 Y
(ARVO Suppl):25.
Potential inhibitors of retinal angiogenesis - 35 have been sought. Tumor-induced angiogenesis was prevented with an extract of cartilage, which weighed about 16,000 daltons and inhibited protease activity.

WO94/011~4 ~ PCT/US93/0~20 Langer- et al. Sclence (lg76) lg3:70-71. Later studies indicated that normal vitreous humor contained such an inhibitor. For example, a vitreous protein with a molecular weight of 6200 was found to inhibit RDGF-induced proliferation and thymidine incorporation by vascular endothelial cells in vitro. Raymond and ;
Jacobson, Ex. Eve Res. (1982) 34:267-86.
Clinicallyt the appearance of cotton wool spots in the retina signifies the ons~t of retinal ischemia.
lO Sebag and McMeel, ibid. These spots are irregular -~`
patches of fibrous tissue. ;
.
Uveitis Uveitis refers to inflammation of the uveal tract. It includes iritis, cyclitis and iridocyclitis and choroiditis and usually occurs with inflammatlon of I I
additional structures of the eye. These disorder has a variety of causes but is typically treated with systemic steroids, topical steroids or cyclosporin. The disease 2V frequently presents with a chronic inflammation occurring !
either in the anterior segment (70%) or in the posterior segment ~30%) which is complicated by episodes of severe exacerbation that may not be controllable with conventional-medlcations. Reports in the literature suggest that 30,000 individuals become legally blind each year in the United States from uveitis. In addition, an estimated-~2~,000 individuals suffer significant loss of visual acuity from this disorder. Additional means to control this condition, without suppressing infection fighting abi-lities with steroids, would be highly beneficial~. ;~
Cataracts _ - - Cataracts are opacities in what should be perfectly clear lenses. Cataracts interfere with the vision by causing blurred vision, glare, altered color perception and monocular diplopia. They are related to a variety of factors, including x-ray exposure and WO94/01124 PCT~US9310~0 ~13~ 8-metabolic diseases such as diabetes, Wilson's disease (copper accumulation) and galactosemia. Cataracts are also a side effPct of cortisone, methotrexate and nitrogen mustard therapy.
S ::
Corneal E~ithelial Wounds The cornea and conjunctiva are vulnerable to damage from pathogenic agents or direct trauma, drying associated with disorders of tearing, exposure to radiant energy (ultraviolet light, sun and welding guns), allergens such as pollen and mold, and infectious agents. I -Keratoconjunctivitis can also occur in patients with Stevens-~ohnson syndrome, Wegener's granulomatosis, rheumatoid arthritis, atopic dermatitis and cicatricial pemphigoid. Corneal ulcers may occur.
After corneal surgery, the cornea must heal.
Popular types of corneal surgery include cataract extraction, with or without lens replacement; corneal transplants, to treat viral infection or penetrating keratoplasty (PKP); glaucoma filtration surgery; and ~ radial keratotomy and other types of surgery to correct -~ ~ refraction. ~ -- Cataract incisions are fu}l thickness wounds in ~--~ the cornea which are as large as 8 mm in length with conventional intraocular lenses (IOLs) and as small as 3 _ mm or less with foldable silicone IOLs. These wounds ~~ typically heal without difficulty, although they take `~- several months to stabilize and are associated with -warpage of the corneal tissues leading to permanent -~ 30 astigmatism. Treatment which could speed stabilization of vision and avoid astigmatism would be highly desirable.
Penetrating keratoplasty (PXP) and corneal transplant are characterized by full-thickness wounds 35 around the entire circumference of the cornea. These --wounds tend to remain weak for one or more years.
Patients experience drift in visual acuity and increasing ;
~, :~

2 1 ~ 5 ~ ~ ~ PCT/U~93/0~20 _g_ :, risk of wound dehiscence and/or endophthalmitis. It would be highly desira~le to st bilize visual acuity and accelerate wound maturation as early as possible, to -, avoid sight-threatening adverse effects.
Radial keratotomy (RK) is the most widespread "
technique for altering the shape of the cornea. The most '' commonly used form of RK is based on the placement of 4-8 surgical incisions in a radial pattern across the cornea.
These incisions are typically 70-80% of the depth of the lO cornea, and are therefore non-penetrating wounds. New ' laser and mechanical methods of altering the corneal '~, curvature are emerging, with the wound healing issues being a major hurdle that has limited the development and clinical application of these techniques.
Normally, the cornea heals rapidly. FGF is known to be involved in the proliferation of corneal epithelial cells and scleral fibroblasts. TGF-~ is ' believed to encourage fetal scleral development but effects later in life have not been reported. ~
A method of enhancing healing of corneal '-epithelial wounds without scarring would help maintain vision after the cornea is wounded. Such predictable healing would be highly beneficial in contributing to a , more pred-ietable-surgical outcome in RK. , Stoaren' 5 Syndrome 5jagren's syndrome is an immune system disorder which manifest~ itself in the eyes as conjunctival and corneal dryness (keratoconjunctivitis sicca syndrome) and 30 a gritty sensation in the eyes. This is due to lack of ,, tear resulting from dèstruction of the lacrimal (or tear) glands by pr,,ogressive mononuclear cell infiltrate and scarring-o~ t,,he gland. If the cornea is too dry, corneal ulcerations can develop.
- "There is currently no effective treatment for the ongoing exocrine gland destruction. Treatment is geared toward symptomatic relief of mucosal dryness . . .

2,l'~6 4~ -lo-and includes artificial tears [and] ophthalmologic lubricating ointments." Harrison's Principles of Internal Mediclne, 12th ed., McGraw-~ill, pages 144~-50, 1991 .
Neovascularizat on Neovascularization is a serious complication of a large variety of ocular disorders affecting the various tissues of the eye because it can lead to blindness.
Corneal neovascularization occurs in many conditions and diseases, including trauma, chemical burns and corneal transplantation. ' -Corneal transplantation is successful in many patients because of the absence of blood vessels in the corneal tissue. Because there are no blood vessels in the cornea, the circulating components of the immune system are not exposed to the new cornea and there is normally no problem of host-graft rejection. Induction of neovascularization in the cornea would expose the cornea to the immune system and lead to graft rejection.
In addition, a subsequent graft is less likely to be successful, too. Treatments of these various causes of neovascularization may include the administration of ~ immunosuppressives to modulate the inflammatory process, including neovascularization. However, - _ immunosuppressives may inhibit appropriate wound healing in the cornea and interfere with the a~ility to fight - infections. Delayed wound healing leaves the cornea vulnerable to infections for longer periods. Hence, `~
1, ~ ' : ' i ' , , I ` ` -- --~ 30 vision-threatening infections can result from current ;~
`~ ~ treatments. j`~
~-- Neovascularization of the iris, and its-- attendant scarring can result in glaucoma and blindness. l~
Neovascularization of this portion of the eye can arise :`
as a consequence of diabetic retinopathy, venous occlusion, ocular tumors and retinal detachment. Most -commonly, laser treatment to cauterize the blood vessels .

~ "~;~;" "~ "~; ~ ,, WOg4/01124 21~i6l~ ~1 PCT/US93/0~20 is tried;= however, that has the attendant risk of causing additional scarring. -Retinal and intravitreal neovascularization `
occurs in a wide range of disorders including diabetic ~ -retinopathy, vein occlusions, sickle cell retinopathy, retinopathy of prematurity, retinal detachment, ocular ~;
ischemia and trauma. ~-Subretinal pigment epithelial (RPE) and sub- ~-retinal neovascularization are common, yet very severe, ~`
disorders of the eye. The growth of new bload vessels interferes with the normal anatomy of the visual and pigmentary cells in the eye, leading to severe visual loss. The new blood vessels leak fluid and blood under the macula causing marked distortion and loss of vision. ~;~
When these blood vessels develop in the avascular foveal region of the eye, the result is central visual loss and legal blindness.
The~specific causes of this type of neovascularization are unknown; however, this disease `~;
20 most often affects patients over the age of 50 years old, ~
who may or may not have a family history of subfoveal -neovascularization. The visual loss is usually ~
sufficient~to result in leqal blindness. These is no `~`
;proven~trea~ment-once the blood~vessels invade the foveal ~5~ ~region. In fact, there are few warning signs that a patient ~LS~ developing this disorder and there are no preventative~measu~res. ~Even under close monitoring by an ophthalmoloqist~ patients with subfoveal neovascularization have a poor prognosis. In eyes in which a natura~l history course is followed with no treatment, vi-sual acuity tends to decrease gradually to a mean of 20/4Q~ (Macular Photocoagulation Study Group, - ~ 1991). Alternatively, in eyes treated with macular ~- ~ photocoagulation, visual acuity measurements after one 35 year of laser treatment yielded a mean visual acuity of `~
~-~ only 20/320 (Nacular Photocoagulation Study Group, 1991).

~: :
.
.

WO94/01124 PCT/US93/0~20 2 13 ~ 6~ 12-Effective therapy of sub-retinal neovascularization is needed to save vision.
Choroidal neovascularization is caused by such retinal disorders as age-related macular degeneration, S presumed ocular histoplasmosis syndrome, myopic degeneration, angioid streaks and ocular trauma. Macular degeneration was discussed above. Choroidal neovascularization has proven recalcitrant to treatment v in most cases. In only 10% of cases can laser 10 photocoagulation be attempted. There is no other treatment available. Even with successful laser photocoagulation, neovascularization recurs in about 60 70% of eyes.
'` '~
15 Growth Facto~s The family of peptides known as TGF-~ can both regulate cell growth and differentiation. These polypeptides can both stimulate and inhibit cell proliferation depending largely on the cell type and 20 environment. TGFs of some type have been found in almost all tissues from all species of animals which have been `~
examined so far.
~ TGF-~2 is a well-rharacterized material. As ~ noted above, it is a polypeptide and has a molecular 2S weight of about 25,000 D and is a dimer composed of two _ - 12,500 D subunits which are linked by a disulfide ~: ~-- -- (Chiefetz et al., 5~1L (1987) 48:408-415; Ikeda et al., Biochemistry (1987) 26:2406-2410) and has been iso~ated from bo~ine demineralized bone (Seyedin et al., J. Biol.
- - 30 Çhg~ (1987) 262:1946-1949), porcine platelets (Cheifetz - et al., Cell (1987) 48:409-415), human prostatic _=- adenocarcinoma cell line, PC-3 (Ikeda et al., 1987, `
Biochemistry 26:2406-2410), etc. Methods for separating and purifying TGF-~2 are given in U.S. Patent 4,774,322 35 to Seyedin et al.
TGF-~l and TGF-~2 are found in many of the same cells. However, their mature sequences have only about ~ ;

WO94/01124 2 ~ 3 & ~ A 4 PcT/usg3/o~2n -13~

75-80% homology (Derynck et al., EMBO J. (1987) 7: 3737-3743). It has been established that the several species of T~F-~ are coded for by different genes. (Madisen et al., DNA (1988) 7: 1-8) It has been observed that TGF-~ ~only TGF-~1 is mentioned in the article's materials section) appeared to seal the edge of surgical retinotomy in rabbits (See, Smidd~ et al., "Transforming Growth Factor-~--A Biologic Chorioretinal Glue", Arch. O~hthal. Mol. (1989) 107:577- `
580). Smiddy et al. showed the formation of fibrotic tissue around the retinotomy which sealed the retina to the choroid layer.
TGF-~2 has been found to stimulate collagen ~;
glycoprotein synthesis as well as cellular proliferation 15 and migration involved in the wound healing process. ;~
See, ~gnotz, "Transforming Growth Factor-~ Stimulates the Expression of Fi~ronectin in Collagen and their Incorporation into the Extracellular Matrix", J. Biol.
- Chem. (1986) 261:4337-45.
TGF-~ has been found to inhibit [3H~thymidine ;~
incorporatiorl by retinal pigment epithelial cells which is stimulated by platelet-derived growth factor, a-FGF, b-FGF and EG~. According to Leschey, this could be due to TGF-B be~ng linked to a strong inhibitory pathway capable of oyerriding stimulatory signals from other growth factors. Leschey et a}., Invest. Ophthalmol. Vis.
Sci. (19g0j 31:g39-46.
In contrast, TGF-~ positively modulates the bioactivity of FGF in corneal endothelial cells. Plouet et al., J. Ce~I-.- Phvsiol. (1989) 1~:392-99.
None of these documents discloses the application-o~TGF-~ to retinal disorders with the result of healing and the improvement of sight. `~

Summary of the Invention This invention is a method of significantly improving the ocul~r vision in retinal disorders of the WO94/01l~ PCT/US93/0~20 ~ 3~ 6~ 14-mammalian eye, in which the retinal disorders are characterized by a loss or impending loss of fibrous tissue, and in which the method comprises administering ~-to the mammal about 1 to 10 ~g of T~F-~. Preferably, the type of TGF-~ is TGF-~2.
A further aspect of the invention is that TGF-~is administered by intraocular, subretinal, subscleral, intrascleral, intrachoroidal and subconjunctival injection or by topical, oral or parenteral modes of administration.
In another embodiment of the above invention, ~
the method comprises two additional preceding steps: -;
removing the vitreous humor from the eye; and peeling the -:
epiretinal membrane, if present, from the retina. In this -~
method, TGF-~ is administered in an effective amount as a concentrated solution by cannula to the portion of the -retina requiring treatment. l`
In a more preferred embodiment, the retinal disorder to be treated is a macular hole.
20~ In another e~bodiment, there is provided a ; method of maintaining or improving the ocular vision in m~acular degeneration. The method calls for administering ~-to the~mammal an amount of TGF-~ effective to stabilize ~`
or improve vision. ~
- In yet another embodiment, there is provided a method of maintaining or improving the ocular vision in cystoid~ma~cular edema.~ The method calls for ~ administering to the mammal an amount of TGF-~ effective to stabilize or improve vision.
30 i ~ ~ In yet another embodiment, the method of ~
treating retinal disorders, which are characterized by `
decreased connective or fibrous tissue, comprises the -- steps of removing the vitreous humor from the eye;
removing the epiretinal membrane, if present, from the eye; and administering a concentrated solution of TGF-~by cannula to place the TGF-~ solution immediately over the portion of the retina requiring treatment.

WO94/01124 ~ ~ 3 g ~ PCT/US93/06420 -- In accordance with another embodiment of the present invention, there is provided a method for treating an individual with an ophthalmic disorder or poor pealing in the eye which can benefit from the '' administration of TGF-~ orally, topically or systemically to an individual in need of such treatment. TGF-~ is administered in an amount suffîcient to improve healing.
In accordance with a further embodiment of the present invention, the ocular disorder is selected from ' lO the group consisting of retinal and corneal wounds, `' macular degeneration, secondary cataracts, corneal disease and dry eye / Sjogren's syndrome. In a further embodiment, TGF-~ is administered by intraocular injection or by application to the cornea. TGF-~ can be applied to the cornea by means of eyedrops or a timed release capsule~placed in the cul de sac.
In yet another embodiment, the method provides 1 '~
for admin~istration of TGF-~ in an amount sufficient to promote~healing and reduce symptoms associated with poor -20 healing. In a further embodiment, the amount of TGF-~ !
administered is at least about O.5 to 50 ~g of TGF-~ per treated eye.
In another embodiment, there is provided a method f-~ treating a mammal who has undergone or is about to undergo or is undergoing ophthalmic surgery to promote healing without excessive scasring, said method comprising adm-i~istering to said mammal TGF-~ in an ~'~amount sufficiênt to promote healing without excessive scarring.
The`'-ophthalmic surgery can be, but is not limited tQ, cataract extraction, with or without lens replacement,_corneal transplants, to treat viral infection'or-penetrating keratoplasty (PXP); glaucoma filtration surgery; and radial keratotomy and other types of surgery to correct refraction.
In another embodiment, there is provided a - method for treating a mammal for ocular WO94~01124 PCT/US93/06420 . , 2~Çi~ -16-neovascularization, said method comprising administering to a mammal an effective amount of TGF-~2.
In another embodiment, there is provided a method for treating a mammal for uveitis, in which the method comprises injecting an effective amount of TGF-~intraocularly.
While not wishing to be bound by any particular theory, the Inventors propose that the administered TGF-~
aids healing by modulating a fibrotic response in the -- 10 ocular tissues. We have also found that application of an effective dose of TGF-~ to the retina appears to ~ `
provide positive neural regenerative effects as evidenced by the fact that such TGF-~ application significantly -~
improves the eyesight of the individuals having the `
15 macular hol-s. ~ i `

Detailed Descri~tion of the Invention The method of this invention is suitable for the treatment of ophthalmic disorders, particularly 20 ~retinal~disorders involving macu}ar degeneration, nèovascularization, holes, separations, tears, and the like~in the retina or between the retina and its `;
underlying ch~oroidal tissue, or involving_choroidal tissue, as~described above.

Definitions~
"Ophthalmic~disorder" refers to physiologic abnormalities of~ the eye. They may involve the retina,; ~`~
he ~itreousihumor, lens, cornea, sclera or ot~er ~ 30 portions~of the eye, or physiologic abnormalities which adver9ely affect the eye, such as inadequate tear production.
"Retinal wounds" include, but are not limited ~ to, tears and holes in the retina and separation from the ~ 35~; underlying choroid. Retinal wounds appear after trauma, cystic degeneration, vitreoretinal traction, myopic degeneration, laser photocoagulation, lightning strike, - ~, .-~

WO94~01124 2 ~ 3 8 w ~ I PCT/US93/06420 pilocar~-ine administration and cataract extraction. To :
help the retina heal in a modulated process, TGF-~ can be ;
administered.
"Macular degeneration" is characterized by the excessive buildup of fibrous deposits in the macula and retina and the atrophy of the retina} pigment epithelium.
The administration of TGF-~ can help promote healing of the atrophied retinal pigment epithelium in a controlled fashion, which is designed to limit excessive fibroproliferation that may occur without such treatment.
"Secondary cataracts" are opacities in the ocular lens which interfere with vision. Secondary cataracts occur after x-ray exposure, in diabetes, -Wilson's disease and galactosemia, and as side effects in drug therapy. TGF-~ can be used to promote healing of the lens after damage in a modulated fashion which is designed to limit hyperproliferation which can occur ;~-natura}ly. `~
The term "diseased corneal tissue" includes damage to the cornea by a variety of causes including, but not limited to, trauma, dry eyes (in which the conjunctiva on the inside of the eyelid may abrade the cornea),-excessive light, allergens and infectious agents. ~TGF-~`ca~-be used to promote gradual healing of ~ 25 diseased corneal tissues and avoid excessive scarring ;~ which can inter~ere with vision.
'rSjogren's syndrome" is an autoimmune disorder which frequent}y is characterized by dry eyes, due to destruction of the tear glands by the autoimmune process. -TGF-~ can be-used to control at least the ocular manifestations of Sjogren's syndrome. While not wishing to be boun~-by any particular theory, it appears that , first TGF-~-can promote gradual healing without scarring of the tear gland and that se~ond, TGF-~ also promotes healin~ of corneal epithelial wounds which arise from the dry eye syndrome caused by lack of tear glands.

wos4/o~l24 PCT/US93/0~20 ~;3~ 18~

~ocular neovascularization" is herein defined as the unwanted new growth of blood vessels into the ocular tissues. Unchecked, such growth can result in `~
blindness. The ocular tissues which can be invaded by 5 neovascularization include the cornea, iris, retina, -vitreous, and choroid. Many diseases and conditions -cause or contribute to ocular neovascularization. Causes of corneal neovascularization include ~ut are not limited to trauma, chemical burns or corneal transplantation.
lo Causes of neovascularization of the iris include but are not limited to diabetic retinopathy, vein occlusion, ocular tumor and retinal detachment. Causes of retinal and intravitreal neovascularization include but are not !~
limited to diabetic retinopathy, vein occlusion, sickle 15 cell retinopathy, retinopathy of prematurity, rëtinal detachment, ocular ischemia and trauma. Causes of choroidal neovascularization include but are not limited ¦
- to retinal disorders of age-related macular degeneration, presumed ocular histoplasmosis syndrome, myopic r 20 degeneration, angioid streaks and ocular trauma.
"Treating a mammal for ocular neovascularization" is herein defined as treating ocular ~ neovascularization which has already become detectable. ;~
~ "Mammals" are defined as humans and mammalian farm and sport animals and pets. Farm animals include, but are n~t limited to, cows, hogs, and eheep- Sport !~
animals include, but are not limited to, dogs and horses.~;
~ The category pets includes, but is not limited to, cats, ~ogs, and hamsters.
~ ~~ 30 The method can involve the placement of at least an effective amount of a growth factor such as TGF-1 `
, preferably TGF-~2, on the ophthalmic abnormality.
-- - Specifically, for treatment of macular holes, a concentrated solution of TGF-~ or TGF-~2 is placed on the 35 macular hole itself and/or the edges of the macular hole. ~`~
Such treatments provide improvement of vision and healing by decreasing the thickness of the edge of the hole. The I ~.

WO 94/0l 124 2 1 3 ~ .1 PCT/US93/0~20 --ls--edges of the hole appear to adhere to choroid or reconnect with the posterior hyaloid membrane.
Similarly, use of the growth factor on other retinal abnormalities is effective.
In one aspect of the invention, TGF-~2 is applied using known surgical techniques, such as those described in the example which follows. It is desirable that the TGF-~2 stay in place for a substantial period of - time after application. For instance, a day is typically considered adequate for this purpose. To help retain TGF-~2 in place, known pharmaceutic combinations may be used. Hyaluronic acid is typically used in the eye for this purpose; however, as indicated by the data below, hyaluronic acid does not appear to increase treatment ~5 effectiveness.
A TGF-~ dose of at least 1000 ng is preferred for-at least partial alleviation of macular hole detachment. More preferred is a TGF-~ dose of at least about 1300 ng. These doses appear to be the approximate dosage for improvement of vision (at least two lines on the~Snellen Yision Chart). Although it is permissible to --dissolve or suspend TGF-~2 in suitable ophthalmic carriers suc~ as normal saline solution, we prefer to apply the matéria~ in a relatively concentrated form.
The concentration may be measured by known light ~- transmittance (21Q Qr 280 nm wavelength) techniques and -- comparison with~a~standard curve.
This inventive treatment is applicable to retinal disorders, particularly to macular degeneration and holes, where-it promotes healing and significantly improvès vision. The treatment also may be used on peripheral_ret-~-nal holes and tears.
The-formulation, method of administration and dosage will depend upon the disorder to be treated, the point at which the disorder is being treated, and perhaps other aspects of the medical history of the patient.
These factors are readily determinable in the course of .

,.. ,~, .. .... ........ . ... .

WO94/01124 PCT/US93/06420 ~
~3~fi 1~ -20- ~

therapy. Suitable patients with an ophthalmic disorder can be identified by medical history, physical findings and laboratory tests. The medical history reveals such facts as time of onset of symptoms such as red sclera, 5 pain, photophobia, dry or gritty eyes, and vision ~
changes, such as blurred vision not correctable with ~-eyeglasses and double vision in an eye. Patients -`
sometimes complain of inability to engage in their usual activities, such as watching television or driving a car lO at night. ;
Patients with ophthalmic disorders associated with impaired healing may have physical findings such as -~
injected sclera, cotton-wool spots on the retina, a macular hole, bleeding behind the retina. Indicative 15 laboratory results include low levels of TGF-~ in the `~
serum or in eye tissues, such as the vitreous.
TGF-~ may be administered by any of a variety ; of routes known in the art, including but nct limited to, intraocular, subretinal, subscleral, intrascleral, intrachoroidal, and subconjunctiva} injectian, depending on the nature and location of the pathology being treated. Also contemplated in the present invention are `
~ administration by intravenous injection, subcutaneous ~ injection, or oral administration, provided that sufficient TGF-~ reaches the condition being treated. In one preferred embodiment, a concentrated solution of TGF-~ is injected into the eye and placed immediately over - - the lesion, for example, on the retina.
- TGF-~ may be a~ministered in any ~ ~ 30 pharmaceutically acceptable formulation, including, but -- not limited to, solutions, suspensions, and timed-release . . .
preparations, such as microcapsular particles and - implantable articles. i ~
To achieve wound healing and improve visual ~;
acuity after treatment of retinal disordersj the preferred dose is greater than about lO00 ng (measured at an absorbance of 210 or 280 nm wavelength). Unless WO94/01124 PCTtUS93/0~20 21~ 4 ~

otherwise specified, all weights of TGF-~ are based on measurements performed at 210 or 280 nm wavelenqth. More preferably, the dose is about 1300 ng. For some conditions, the preferred dose is about 2500 ng.
Alternatively, TGF-~2 may be administered in a slow-release device embedded in the tissue stroma or in a compartment adjacent to the affected tissue. For example, 1,000 ug of TGF-~ in a pellet of ethylene vinyl copolymer 2 mm in diameter could be surgically implanted in the vitreous cavity or suprachoroidal space to release ~GF-~ over time. This modality is believed to be particularly beneficial for neovascularization of the iris or choroid. -~
Patients at risk for ophthalmic healing lS problems include those who have undergone or about to undergo surgery. Examples of such surgery include, but are not limited to, Cataract extraction, with or without lens replacement;
20 ~ Corneal transplant for treating viral infections or penetrating keratoplasty (PKP);
Glaucoma filtration surgery; and Radial Xeratotomy and other types of surgery to correct- refraction.
~ In these conditions, the administration of TGF-~ promotes ~prompt, gradual h-aling_without excessive fibrous tissue -- formation. ` ~ ~~
Other growth factors which have both wound healing and neurotrophic effects can be applied in certain of these inventive treatments. These factors ..
- include, bùt are not limited to, acidic and basic ;~ fibroblast growth ~actor, insulin, insulin-like growth _ _ .
factor, platelet-derived growth factor, nerve growth ~- factor, epidermal growth factor, transforming growth 35 factor ~, colony-stimulating factor, keratinocyte growth ~`
~actor, and tissue plasminogen activator.

I .
- !

WO94/01124 pcT/us93/o~2n 2 ~'3~,6~ 22-In this example, the effectiveness of TGF-~2 in alleviating macular holes is shown. Clinical data are provided in a table which follows this example~
Materials and Methods Sixty eyes (60 patients~ with Stage 2, 3, or 4 -macular holes were treated. Thirty-two of the 60 ~`
patients had macular holes in both eyes. Patients ranged `~
in age from 11-76 years, with a mean age of 63. All treated eyes had biomicroscopic evidence of a Stage 2, 3, or 4 macular hole confirmed by at least two independent I ~;
observers. All but S eyes had the macular holes present ~}~
for one year or less; all patients had subjective `
decreases in visual acuity as well as subjective distortions of vision. None of the patients had previous histories of cystoid macula edema, diabetic retinopathy, ¦
or exudative age-related macular degeneration. ~ ~
fiefore treatment, technicians who were not told `
the planned treatment obtained best corrected Snellen visual acuity and performed intraocular pressure measurements, fundus photographs, and fluorescein - angiography. Each macular hole was graded as Stage 2, 3, or 4 according to the criteria described by Gass (Arch.
O~hthalmol. (1988) 106:629-39). Briefly, eyes with Stage - - ~ holes have a retinal dehiscence along the margin of the ~., - --area of deep retinal cyst formation. In Stage 3, typically there is a full-thickness hole with overlying operculum. Macular holes are classified as Stage 4 when ~ 3Q a posterior vitreous detachment is present. Treatment ~"
- was scheduled within 2 weeks of the baseline examination.
Under the criteria, patients were excluded if they had - greater than 2+ nuclear sclerotic or posterior subcapsular lens changes. Patients were followed for 6-lO months, with mean follow-up of 8 months.

WO94/01124 PCT/US93/0~20 -23- 2~3~

Treatment--Groups Doses of 70 ng, 330 ng, and 1330 ng of TGF-~2 were administered. The 70 ng dose was chosen to provide a negative control for the higher doses. The 330 ng dose was believed to be at the low end of the e~fective range, and the 1330 ng dose was believed to be well within the effective range.
Eyes were randomly chosen f or the indicated doses of intravitreal TGF-~2. In addition, some eyes separately received 100 ~1 of intravitreal hyaluronic acid at the time of instillation of TGF-~2 in an attempt to delay clearance of TGF-~2 from the area of the macular hole. Although effective in conjunction with TGF-~2, co-administration of hyaluronic acid appeared to lessen benefits fsom TGF-~2.
Surq~cal Procedure j All surgery was done under local anesthesia with sedation. After the eye was prepped and draped, a standard three-port vitrectomy was performed. In eyes i~
with Stage 2 and Stage 3 macular holes r a core ~itrectomy was performed~ In many of these cases, a large central lacuna was found in the posterior one-third of the vitreous cavity, initially giving the impression that the .
posterior h~aloid--was detached. However, in all of these eyes, further investigation revealed that the cortical vitreous remalned on the retinal surface. In Stage 4 macular holes, a-complete pars plana vitrectomy was -performed at this point.
In some cases, an epiretinal membrane was ! 30 found; however~,-no definite edges of this membrane could be found. Where encountered, tha epiretinal membrane was peeled from the surface of the retina and removed from the eye. In-Qther cases, no definite epiretinal membrane could be found; however, there appeared to be some gelatinous condensation on the inner surface of the retina surrounding the macular hole for approximately 200-400~, with a firm adhesion along the margin of the WOg4/01124 PCT/US93/0~20 ~

3 ~ 24-macular hole. This was carefully dissected where possible; however, great care was exercised in order to limit traction on the edgee of the macular hole or damage to the nerves. -After ~ short period of time to allow peripheral fluid to drain posteriorly, the fluid that had migrated posteriorly wa~ aspirated. Typically, -~
approximately 0.1-0.5 cc of fluid had reaccumulated on `~
the retinal surface during this time. The center of the macular hole was gently aspirated to remove the last remaining amounts of fluid in the region of the macular hole.
A tapered, bent-tipped cannula was then connected to a 1 cc syringe containing a solution of 15 TGF-~2 . ~n each case, TGF-~2 was thawed and mixed within . 2 hours of use. The TGF-~2 was supplied by Celtrix Pharmaceuticals, Inc., Santa Clara, CA, and kept at -70C
until ready for use. It was always used within 2 hours of thawing and kept on ice until used. The TGF-~2 was `;
20 highly purified (greater than 95% purity), and derived l`
from bo~ine bone. The reconstituted formulation contained either 70, 330, or 1330 ng/O.1 cc of TGF-~2 ~-- after dilution with a diluent solution. Eyes were `
~ -- - randomly assigned a dose of TGF-~2. About 0.1 cc of TGF-25 ~2 solution was gently infused into the macular hole. In ;`
- _ about 50% of eyes, a comparable volume of hyaluronic acid ~ ~-- -- was also introduced in order to determine if this might maintain the presence of the T~F-~2 solution, thereby improving efficacy.
- - 30 After surgery, the patient was instructed to - lie in a supine position for the first 24 hours following _ - surgery; thereafter, the patient was instructed to remain in a face-down position as much as possible over the ensuing two weeks.
After surgery, patients were examined at 1 day, 2 weeks, 4-6 weeks, and monthly thereafter. Fluorescein angiography was performed at 4 to 6 weeks, 3 months, and WO94tO1124 PCT/~'S93/0~20 ~ 1 3 ~

6 mont~s. Best corrected Snellen visual acuity, intraocular pressure, lens status, bubble size, status of macular hole, and occurrence of adverse effects were determined at each examination.
Statistical Analysis Treatment effects were assessed using logistic regression. The dependent variable was improvement in visual acuity of two or more lines on the Snellen Chart, and the independent variables were TGF-~2 dose and hyaluronic acid use.
, Results ~ At the time of surgery the status of the posterior hyaloid surface as well as the stage of the macular hole could be readily confirmed. As is shown in :~: the table, of the 60 treated eyes, four had Stage 2 ¦
macular holes, 34 had Stage 3 macular holes, and 22 eyes had Stage 4 macular holes associated with pasterior .
vitreous detachment.
Using the techniques described above, the posterior hyaloid surface was completely separated from the ret na-in all eyes with Stage 2 and Stage 3 macular holes. ~Howevér, in all eyes with Stage 2 macular holes, . .
this separation of the posterior hyaloid surface from the retina extended.~arginal dehiscence and formed an ~ : operculum that ~as ele~ated from the posterior hyaloid -::~ surface.- In one-eye, the retina was torn in the inferotemporal quadrant between the ora and the equator and accompanied by an adjacent intraretinal hemorrhage.
- This retinal tear was treated without consequence with . ~: transscleral cryopexy.
A~ter. ~itrectomy, all eyes had a 200-400 ~ band .
of gelatinous material on the inner retinal surface along : 35 the margin of the macular hole. Small amounts of this material could be dissected from the retina, but it could not be removed in a continuous sheet, as is typical for I ~-W094/01124 PCT/US93/0642n ,~
~3~ 26- ~

idiopathic fibrocellular epiretinal membranes.
Aggressive dissection was avoided to minimize trauma to the mac~la.
On the first and second post-operative days, the anterior chamber had only trace amounts of flare and cell in all eyes except six. In these six eyes, a fine, red-~rown precipitate was found on the endothelial surface of the cornea along with minimal striae. In all cases, the precipitate and striae resolved within two weeks without sequelae. All eyes with intact lenses had mild to moderate posterior lens feathering which resolved within two weeks.
All eyes had bubbles filling at least 75% of the vitreous cavity on the first and second post- --operative day. The intraocular pressure was not higher than 30 mm Hg. None of the eyes had a significant ¦ `
inflammatory response two weeks post-operatively. All ! ::
eyes had a bubble filling at least 60% of the vitreous at that time. After four to six weeks, the bubble filled 30% to 40~ of the vitreous cavity.
At four to six weeks, the macular region could be adequately examined using a biomicroscope with a contact lens or a 78 diopter lens. Microscopic retinal - detachment and retinal thickening surrounding the macular hole could be readily assessed at this time. As is shown _ in the table, flattening of the detachment and thinning , = , , -- - of the adjacent retina to a normal-appearing thickness occurred in 12/12 eyes treated with 330 ng of TGF-~2 without hyaluronic acid and ll/ll eyes treated with~1330 ng of TGF-~2 without hyaluronic acid. In contrast, only 6/ll eyes treated with 70 ng TGF-~2 without hyaluronic acid had the edges of the macular hole flatten after 4-6 weeks. The addition of hyaluronic acid to the TGF-~2 gave unexpected results. As the data in the table indicate, hyaluronic acid significantly suppressed the rate of flattening of the retina arount the ~acular hole.

4 PCT/US93/0~20 -= Visual acuity did not improve in eyes with no improvement in retinal flattening. Final visual acuity improved two lines or more in lO/ll eyes treated with 1330 ng TGF-~2 without hyaluronic acid, 4/12 eyes treated with 330 ng TGF-~2 without hyaluronic acid, and 5/ll eyes treated with 70 ng TGF-~2 without hyaluronic acid. -In contrast, the addition of hyaluronic acid appeared to suppress visual improvement. Final visual acuity improved two lines or more in 4/9 eyes treated with 1330 ng TGF-~2 and hyaluronic acid, 2/8 eyes treated with 330 ng TGF-~2 and hyaluronic acid, and 0/9 eyes treated with 70 ng TGF-~2 and hyaluronic acid.
Logistic regression analysis was performed using two-line improvement in visual acuity as the lS outcome variable and use of TGF-~2 and hyaluronic acid as independent-variables. ~he analysis demonstrated a statistically significant beneficial effect of TGF-~2 on visual improvement (ps.003). In contrast, the analysis demonstrated that the use of hyaluronic acid reduced the ~ ' chance for visual improvement (ps.002).
Most eyes developed a subtle, localized layer of fibrous tissue along the edge of the macular hole.
This~'-fibrous tissue could sometimes be seen to span the ~ ~ .
~ macular hole. In spite of this fibrous tissue formation, ~, .
no eyes developed a traction retinal detachment or significant_macular traction.
~Additionally, angiographic findings improved.
-~ Preoperatively, fluorescein angiography revealed a central hyperfluorescent window defect corresponding to the'bas'e of the macular hole in most eyes.
Postoperatively, angiography showed a decrease of the centr~ hyperfluorescence in most eyes in which the edges of~-the macular hole had flattened. Howaver, the '~
hyperfluorescence persisted in all eyes with persistent ;~ 35 ~ubretinal fluid and retinal thickening surrounding the macular hole.

:
.

WO94/01124 PCT/US93/0~20 3~ .4 Discussion We consider the control of wound healing to be important in the treatment of numerous retinal disorders.
This example describes the first use of TGF-~ in the

5 treatment of a retinal disorder with improvement of -vision.
The rationale for treatment in this example was to induce flattening of the edges of the macular hole in order to resolve retinal detachment and thickening surrounding the hole. Knowledge of the behavior of peripheral retinal holes suggests that reducing the ;
traction force which elevates the retina around the hole coupled with the inducing chorioretinal adhesion a~ong -~
the edge of the hole may be required. Unlike peripheral retinal holes where surgical techniques can be used to reattach the retina and a small area of destxuction i5 ¦ `.
not noticeable, macular holes require gentle induction of - chorioretina} adhesion to avoid the destruction of adjacent neurosensory tissue and per~anent destruction of centra} vision.
Visual improvement was achievable when significant degeneration of the neurosensory retina had not occurred secondary to the localized foveal - detachment, and significant destruction of tissue does ¦
- 25 not result from surgical intervention. Again, experience -~ _ with peri~heral retinal holes and tears suggests that not all retinal traction need be removed, provided that the method induces a chorioretinal adhesion of sufficient strength to counteract the existing traction.
In this technique, the posterior hyaloid was separated from the retina in eyes having Stage 2 or Stage 3 macular holes. Next, a gelatinous, friable material accumulated along the margins of the holes, but only limited attempts were made to remove the material for fear of damaging the adjacent neurosensory tissue.
To avoid significant tissue damage but relieve traction along the margin of the hole, this method limits WO 94/01124 PCr/US93/06420 -2g~

manipulation of the retina and induces chorioretinal adhesion using TGF-~.
AS is shown in the Table, the edges of the macular holes were flattened in 23/23 (100%) eyes treated with 330 or 1330 ng TGF-~2 without hyaluronic acid. In many of these eyes, a fine bead of fibrous tissue could be observed along the margin of the macular hole after it flattened. ThiS f ibrous tissue was accompanied with good visual recovery (vision improvement of two or more Snellen lines).
In this example, with limited manipulation of the macular hole, there was no enlargement of the macular hole or evidence of mottling of the retinal pigment epithelium surrounding the macular hole. r~"`
In this example, the fluorescein angiographical}y demonstrable hyperfluorescence over the base of the macular hole disappeared after the edges of the hole had flattened. The reduced fluorescence could be due to formation of an overlying fibrous membrane.
However, judging by the thin, relatively clear-appearance of the membrane, we believe this is not the cause. More likely is a redistribution of pigment within the retinal pigment epithelium (RPE) cells. Regardless of the exp-lanatlon, the higher level of treatment improved the visual acuity of the treated eyes.
A major concern in this study was that TGF-~2 might-~cause excessive fibrosis which can increase macular contraction and result in proliferative vitreoretinopathy ~PVR) . TGF-~2 is known to be present in significant concentr-ation in eyes with PVR, and has been implicated . . .
in its~formation (See, Connor, J. Clin. Invest. (198~) ~:16~L-66). However, the concentration of TGF-~2 used _ in these cases was significantly less than that seen in ~ eyes with PVR. Notably, neither of these potential - 35 complications was seen in any of the 60 eyes treated; and all eyes were observed for at least six months after treatment.
I

WO94/01124 PCT/US93/0~20 :
~3S6i ~ -30-Visual acuity improved in eyes with flattened edges of the macular hole, resorption of subretinal fluid, and thinning of the adjacent retina in response to ;
TGF-~2.
As shown in the table, although the edges of the macular holes flattened successfully in all eyes in both the 330 ng and the 1330 ng non-hyaluronic acid groups, visual improvement surprisingly occurred more ~ommonly in eyes receiving the higher dose. More fibrous 10 tissue formation is unlikely to account for this finding. !
Whil~ not wishing to be bound by a theory, we may postulate that TGF-~2 also enhanced recovery of the -photoreceptor outer segment function, possibly by neural regeneration or stimulation of accessory tissues which in turn help stabilize and align neural retinal cells.
In a follow-up study, to ascertain the I ;
best dose, there were 30 eyes treated with placebo, 29 ¦
treated with 660 ng of TGF-~2 and 29 eyes treated with 1330 ng of TGF-~2. The 1330 ng dose waC surprisingly more effective than the 660 ng dose, particularly in improving visual acuity by three or more lines ETDRS by 12 months post-operatively. 30~ of placebo eyes had such ;
an improvement, 62% of 660 ng-treated eyes had such an improvement (p=0.019, not significantly different from placebo), but 75.9~ of 1330 ng-treated eyes had such an improvement (p~0.001, highly significant). As for two- :
line improvement at 12 months, 69.0S of 660 ng-treated eyes improved (p=0.004) but 79% of 1330 ng-treated eyes improved (p<0.001). Mean line changes were calculated :
for each group and TGF-~2 groups were compared with placebo. At 12 months, placebo eyes improved 0.6 lines, 660 ng-treated eye improved 2.5 lines (p=0.037), and 1330 `~
ng-treated eyes improved 3.8 lines (p~0.001). Thus, even .
compared to a dose of about 700 ng, there was marked improvement with the 13~0 ng dose.

WO 94J01124 2 1 3 S 8 ~
-~ ~ Z; Z Z; :~ ~ Z. Z Z.
, ~ ,~ + ~ ~ _ o o o o I ~t ~ C o I +
_I ~ o o o o o o o o o o . t ~ r ~ 0 ~n ~: o o o o o o o o o --. _ ~`1 ~ ~ ~ ~ ~ ~ ~ ~ ~ N , Ll C IOO'~ + OOOO~
I _ ~
I o o o o Q o o o o o o o ¦ ~ ~ ~ ~ ~ ~ ~ ~
15~
. ~ ~ I
~ Z Z ~ ~ Z Z ~ ~ ~ .
O ~ .:
~, ~. .
~ ~1 J~ ~ C
U~ J~

_ 2 5 ~ 3 ~ ~ ~ O
- .

~ Z ~ ~ ~ ~ ~ ~ , - ~t~
, ~ ~ O ~
5 ~ ~ :1 _ ;

C~ o 3 ..

WO 94/01124 PCr/US93/06420 2,~3~6 -~4 -32- :

_ æ ~ ~ z; ~ z x æ æ æ z ~ æ
_ ~, o ~ o o o o o o + ~ ~ :
_I ooooooooooo ~ O U~ ~1 r ~ ~ ~ ~ ~ ~ u~
~ ~________ ~_O~
.~ _ooooooooooo~ ,-~
~ ~ ~ ~ N ~ ~ ~ ~ ~ ~1 ~ N

O O O O O + O O I I +
1 0 o O O O O O O O O O O O O _ ~I) ~
~1 O O O C~ ~ o O o o o o o o I .:
t~ ~ ~ N ~ ~ ~ ~ ~ ~ ~ ~ N

I

C
.

~ ~ o ~ ~ g .
.--` - , O ~ a~ ~ ~o ~ ~ ~ r~ o ~ ~ a~

u ~ ~ O
o J~ ~ ~
~o ~ ~ ~ ~ ..
O ~ 3 :~ U
C~

P~/US93/06420 WO 94/01124 ~ 3 ~ 6 ,, ~ + ~ o 3 ~ ~ N t~l t~ ~ ~I t~ N

N N N ~ N 1` N N N N N

a ,:

,, _ ' `' - ~ 0 0 ~ _ - . , ~:
:~: a~ o ~

,, i , U ;~
- - - c~ o ~ `.
~ o . ~ _ I
- O ~1 3 ~ u - ~:

WO 94~0~124 P~/US93/06420 23~3~ 34--,.
~ z a~ z; æ z z ~ z ~ :

, ~
o o o o ~ o o o o o o ~ o ~ o o o o o 1~ ~ el~ ~ el' ~ t~ P N ~1 C ~ ~ '~
O O O O O O O C~ O ~ ' ' ~4 ~ ~ ~ ~ t~ t~l N t~ ~
O
~1 ~
S ~ o o o o + o o o O OOOO~OOOo_I I
O ~
O O O O O O O O

~ .
' ~ ~ Z ~ ~ Z Z Z ~ Z Z

O ~ .
U . ~1 2 0 31 ~ c r 14 ' .
,~ O O ~ 30 - I

,. _ . -=. .
- ' .
,, . O ~ ~ o ~ N N

~ _ 5: U ,~
C ~ C _ , ~ ~ o _ ~o o ~ ~
- --, - o ~ U
~ ¢

WO94/011~4 2I3~,`6~ I Pcr/US93/06420 z :z z ~ x Z j.
S
,~ 0 o o o o o o , o o o o o o o o o o ~ -~

. h ~ ~ _ ~ ~ ~ o o o O

v O I , . , ~ ~ r ~o .=.

Z~ o u~ a~ o ~ ~ .,~
3 0 ~ .

e e 1~.3 0.

_ .

WO g4/01124 Pcr/~lss3/o642n 3~i6 ~ -36-_ ~ ~ z z z :æ ~ z _ + I o o I ~
,~ o o ~ o o o o o o ~, ~ ~ u~ f~ ~ o ~ o C _ _ ~ _ _ ~ _ ..
.,, o o ~ o _ o o _ o ~
O : .;
,i ,1 ~ ~ ..
1 0 ,, , ~ , + ~ o o o o I o o o o o o o o o CO ~ O ~
I ~
o o _ o o o o o o O

~ ,, z z z; z ~ z ~ ' oO .~ 1 ~ I ~
_I .

. :
.
o : ~ ~ ~ :
_ ~

., ~ r~ ~ O ~
-. . a -l 3 :~ ~ .

WO94/01124 213 ~ 5 -1 'I PCT/US93/0~20 In this example, treatment of AMD is described.
The therapeutic efficacy and safety of locally administered bovine TGF-~2 (bTGF-B2) was comparison to a placebo or no treatment in patients with exudative age-related macular degeneration and visual acuity of 20/160 or better. The primary efficacy variable was change in visual acuity from baseline in the respective ~reatment groups. The secondary efficacy variable was -stabilization (within 2 lines on ETDRS) or improvement (>2 lines on ETDRS~ of visual acuity compared to baseline measurements. Further efficacy measurements were quantitative changes on fluorescein angiograms and biomicroscopy photographs, but determining the size of the neovascular net proved to be very difficult and did not seem to be a reliable measure~ent.
Patients with exudative AMD, who met the other entry requirements of the study were assigned to treatment groups. Patients were chosen according to the 20 following criteria: ~;
l. Biomicroscopic and fluorescein angiographic documentation of subfoveal -neovascularization tclassic/occult) ---2. --Patient has age-related macular degeneration 3. At least one high risk characteristic:
~ a. subretinal serous exudate - b. subretinal hemorrhagic exudate c. presence of lipid 4.- Best corrected vision 20/160 or bstter as measured by ET~RS (Early Treatment Diabetic --~etinopathy Study) !
- S. Visual symptoms of six months duration or less in the treated eye

- 6. Ability to comply with all aspects of the treatment and evaluation schedule (at least l year postoperatively) WO94/011~4 PCT/US93/0~20 3~6~

7. Ability to provide voluntary informed consent However, patients meeting the following criteria were excluded from the study:
l. Previous treatment with bTGF-~2 in the operative eye 2. Previous laser treatment in the study eye 3. Patients eligible for laser photocoagulation (reference 3), unless the patient refused treatment with this modality 4. Photographic documentation of fibrosis exceeding 25% of the lesion area 5. Blood present in greater than a twelve disc area 6. Inability to complete entire follow-up schedule with the investigator 7. Presence of other vision threatening diseases such as proliferative diabetic retinopathy, macular hole, retinal detachment, uncontrolled glaucoma, or advanced visual field loss

8. Use of concomitant medications which would interfere with the evaluation of the study ~ (determined by the investigator)

9. Nursing or pregnant patient Each patient was randomly assigned to one of three groups. Stratification of pati`ents at baseline was performed to ensure that all groups were represented with minimum bias. The stratification parameters included ~ 30 visual acuity (20/lOO mid point), age t70 years), and visual loss duration (3 months). Following vitrectomy, the patients in Group I received 50 ~l (665 ng) of - bTGF-B2 subretinally in the area of the neovascular net, with another 150 ~l (1995 ng) of bTGF-B2 applied directly onto the fovea at its interface with the vitreous cavity.
Subjects assigned to Group II were treated in the same manner as those in Group ~ except that they received a WO94/01124 2~ ~ ~ 6 ,1 q PCT/US93/06420 pl?cebo (vehicle) solution identical to the drug product ~
but without bTGF-B2, while those in Group III were '' untreated. ;
Following treatment (or admission to the study if a subject is in Group III), all patients were examined at 2 weeks, 4 weeks, 6 weeks, 3 months, 6 months and 12 ~' months after baseline. The study parameters assessed included best corrected visual acuity for both distance and near vision, intraocular pressure, lens status, and refraction. The amount of serous and hemorrhagic exudate, size of the neurosensory detachment, presence of epiretinal membrane, presence and size of ~ ~-hyperfluorescence from classic/ occult ~ neovascularization, total lesion size and foveal jl 15 involvement were also measured on fluorescein angiography ` ' and ICG (indocyanine green) angiography.
Patients were treated with bTGF-B2 extracted I -' -~ ~ from bovine bone in a highly purified form (>95S purity) -'~ and~supplied as a concentrated acidic solution (Vial l, MS 2004) which was mixed with diluent containing human serum albumin (Vial 2, MS 2005) prior to subretinal or intravitreal injection. The final concentration of ~.~ '!,,.
bTGF-B~ following mixing with the diluent was 2660 ng per 200~ volume of solution. ~he placebo preparation was an acidic solution without bTGF-B2 (Yial l, MS 2009) which~was mixed with diluent (Vial 2, MS 2005) prior to subret-inai'-~or lntravitreal injection.
- The total dose administered was 200 ~l (2660 ng) of bTGF-~2 (Group I), or 200 ~l of placebo (Group '' 30 II). As for the route of administration, S0 ~l was ''in~ected into the subretinal space through a site at least one disc diameter from the center of the fovea, and l50~l appiied directly onto the retinal surface with underlying neovascularization. One treatment was given fQll~wing vitrectomy, except for Group III patients who received neither a vitrectomy nor the experimental or placebo solutions (~.e. will remain untreated).

WO94/0l124 PCT/~IS93/06420 p~3 36 ;~5~3~ ~

Baseline findings not consistent with the requirements of the study were cause for discontinuation of the patient from the study. Baseline studies included the following. A complete medical and ophthalmologic 5 history were obtained. Using the ETDRS eye chart, best -corrected visual acuity measurements were obtained. Near visual acuities were also measured using the Bailey-Lovie Word Chart. Subfoveal neovascularization were visualized by biomicroscopy, fundus photography, and fluorescein angiography. A subset of 5 patients per group was further studied using ICG angiography. Standard 30 stereo fundus photographs of the disc and macula (photographic fields I and II) of the involved eye were obtained. In addition, a stereo angiogram was obtained at this visit. Stereo black and white red-free photographs were taken of the disc and macula of the study~and fellow eye. The transit frames of the angiogram were centered on field II of the eye. Stereo angiographic views were taken during the transit phase, and at 30, 40, 60, 90 seconds, 2, 3, 5, and 10 minutes centered on field II. At 10 minutes there were also a stereo view of field I of the eye. Additional views of the opposite eye and of other fields were obtained at the discretion of the treating ophthalmologist. In addition, the ICG angiogram were taken using a similar protocol, but ICG views were taken at 40 minutes as well. All study photographs and angiograms were labeled with the patient's code at the clinic. Photographs and angiograms w!ere read by an observer masked with respect to~patient information and randomization code. Patients eligible ~- ~ for the study were randomly assigned to treatment groups, using a computer program to stratify on the basis of baseline visual acuity t20/100), age (70 years) and visual loss duration (3 months). The treatment of Group I and II patients was scheduled within one week (se~en days) of the date the baseline tests are completed.

' .

WO~4/011~4 PCT/US93/0~2 -4l--- All surgeries were performed under either local anesthesia with sedation or general anesthesia. After the eye was prepped and draped, a lid speculum was positioned. Two conjunctival flaps were made laterally and medially. A 3 mm infusion cannula was to be placed 4 mm posterior to the limbus, and held in place with a pre-placed 4-0 white silk mattress suture. In each case, the tip of the cannula was seen within the vitreous cavity prior to the onset of infusion. Two additional l0 sclerotomies were made at l0 o'clock and 2 o'clock ~
meridians, 4 mm posterior to the limbus. A light pipe '~!' and vitreous cutter were then introduced. At this point, a core vitrectomy was performed in the involved eye. `-After completing the core vitrectomy, the vitreous cutter lS was removed and replaced with a cannula having a flexible silicone tip. The cannula was then connected to the ;~ aspiration system of the vitrectomy machine and aspiration set at 150 mm Hg. ~he infusion bottle was positioned approximately 50 cm above the level of the 20 patient's head. The tip of the cannula was positioned ~i~
approximately l mm above the surface o the retina just ``
below the superotemporal arcade. ~ull aspiration was `
-applied, and the cannula gently elevated. After the p-osteri-or hyaloid æurface was elevated in the area just ;' ~ 25 inferior to the superotemporal arcade, the cannula was ;~ used to e~tend this posterior hyaloid detachment as far as pos~ible~out to the equator. In some cases, additional manipulation was needed at the disc in order to complete the~detachment of the posterior hyaloid surfaee. Once this was accomplished, the vitreous cutter was introduced and total pars plana vitrectomy performed, `
remQving the vitreous as far out to the periphery as ..
-possible. The flexible-tipped cannula was then reintroduced and again positioned approximately l mm 35 - above the retinal surface, and gently moved side to side.
If the entire posterior hyaloid was removed, there was no bending of the cannula tip.

WO94/01124 PCT/US93/0~20 The instruments were then removed from the eye, and replaced with scleral plugs. The peripheral retina were examined with indirect ophthalmoscopy and scleral depression in order to ensure that no retinal tears occurred. The scleral plugs were then removed, and the light pipe and flexible-tipped cannula were reintroduced.
A fluid-air exchange was performed aspirating all fluid over the optic disc. The instruments were then removed from the eye and replaced with scleral plugs. Fifteen minutes were allowed for peripheral fluid to drain posteriorly. The scleral plugs were once again removed, and the light pipe and flexible-tipped cannula were reintroduced. Additional fluid that migrated posteriorly was aspirated.
The viscodissection cannula and tubing - (Visitec, Inc.) was then connected to a l cc syringe containing freshly diluted bTGF-B2, or placebo. The tip of the cannula was positioned just over the area of neovascùIarization and 200 ~l containing 1330 ng/lO0 ~
bTGF-A2 solution or placebo solution was gently infused.
The instruments were then removed from the eye, and the two superior sclerotomies were closed with 7-0 vicryl. The infusion cannula was removed, and the sclerotomy closed with 7-0 vicryl. The intraocular pressure was checked, and the intra~itreal bubble was adjusted to achieve normal pressure. The conjunctiva was -- closed with interrupted 6-0 collagen. The patient received acetazolamide, 500 mg intravenously (IV) and continued on acetazolamide 250 mg by mouth or IV every six hours, for the next 24 hours. The patient was instructed to lie in a supine position for the first 24 hours following surgery; thereafter to remain in a facedown position as much as possible over the ensuing five days.
- 35 Followup on day l and week l (Group I and II) included intraocular pressure, the size of the remaining gas bubble, the appearance of the lens, adverse events, WO94/01124 2 ~ 3 ~ 6 ~ 4 PCT/US93/06420 ~

-43- ~

- concomitant medications. Followup at weeks 2, 4, and 6 ;-and at months 3, 6, and 12 Post-Treatment included best corrected visual acuity for both distance and near vision~;~
using the EDTRS eye chart and Bailey-Lovie word chart, respectively; refraction; intraocular pressure; presence of epiretinal membrane; results of fluorescein angiography (3, 6 and 12 months only); results of ICG
angiography in selected patients; lens status; occurrence of adverse events; and assessment of concomitant medications.
Twenty patients were treated and assessed as described above. Their data are shown in the following table. Because the patients have not completed the ~
contemplated year-long study, not all anticipated ~-15 changes, including expected visual acuity improvements, , `~
have occurred. Of patients receiving TGF-~2 only over ~ `-th:e area of the macula, 7 of- lO had stable or improved visual acuity, which is considered successful, since ;~
h~istorically 50% of untreated patients become blind within 6 months and all untreated patients become blind within one year. Of the patients receiving ~GF-~2 divided between subretinal and supraretinal injections, ~ a~l pati-nts have been successful. Of the three with : , .
~ 25 - - - =. , - ~ .
,:

~
,,, . .
.- ,,.
~, 35- ~ ~

.~ 3~6 ~ 44-5 ~ ~ ~ o o~

~ ~ I ~ ~

WO94~01124 ~ ~ PCT/US93/0~20 ';~

- unsatisfactory visual responses, two received the low dose ( 665 ng), and a third had a 3~ cataract. Near vision was assessed with the Bailey Lovie system for 18 i.
eyes. Four eyes were unable to read at baseline, but three of these were able to read after treatment, two at the two-week visit and the third at the three-month assessment. Data from other patients indicated that for most patients vision was generally stabilized or ;-improved. Two patients had.a transient elevated IOP
after surgery, but IOP returned to normal by week 4.

EXAMPLE 3 ~
In this example, a process for the treatment of .
subretinal pigment epithelial (sub-RPE) or subretinal .
neovascularization using TGF-~2 is described.

The study involves 50 patients who satisfy the .
study criteria for sub-RPE or sub-retinal ~: : 20 neovascularization. Patients are chosen according to the ¦
following criteria: i ~
l. ~iomicroscopic and fluorescein angiographic .
- - evidence of sub-RPE or sub-retinal neovascularization involving the foveal ! `.`
: 25 avascular zone; ~ :
.2. Subjective visual decrease; and 3. Objective visual loss confirmed by visual acuity measurements. :.

30 - -- However, patients meeting the following ;
. criteria are excluded from the study:
_ l. Patients who have been previously treated .
_, . _ .....
-. - with TGF-~2 in the operative eye;
2. Patients currently pregnant or nursing; -- 35 3. Presence of any clinically significant condition (e.g., active proliferative diabetic `:
;

WO94/01124 PCT/US93/0~20 ~3~ 46-retinopathy) which may be incompatible with participation in this study; and 4. Patients taking medications which would interfere with the evaluation of this study.
One group of patients is randomly assigned to receive a single intraoperative local dose of either 50 ~l or 200 ~l of 1330 ng/ l00 ~l of TGF-~2 applied directly to the foveal ra~ion. Another group of patients will be injected with 50 ~l in the subretinal space and l50 ~l within the vitreous cavity above the area of the sub-RPE or sub-retinal neovascularization. After twenty pati~nts are treated, the data are evaluated to determine whether any safety modifications to the protocol are appropriate. If there are unusual side effects in the twenty treated eyes, such as proliferative vitreoretinopathy (PV~), excessive hemorrhage, or unexplained retinal detachment, the trial is stopped at this point.
8aseline studies include visual acuity measurement (standardized Snellen and ETDRS eye charts) and biomicroscopy, as well as both fluorescein and ICG
angiography to document the presence of the sub-RPE orr .
sub-retinal neovascularization. After treatment, patients are followed for one year. Safety and efficacy assessments include visual acuity measurements, biomicroscopic visualization of the fovea, and fluorescein and ICG angiography.

Surqical Procedures All surgery is performed under either local anesthesia with sedation or genera} anesthesia. Two ;
conjunctival flaps are made laterally and medially. Two additional sclerotomies are made at ~0 o'clock and 2 o'cloc~ meridians, 4 mm posterior to the limbus. A light pipe and vitreous cutter are then introduced. At this point, a core vitrectomy is performed in the involved eye. A~ter the core vitrectomy, the vitreous cutter is W094/01124 ~1 3 ~ PCT/VS93/0~20 removed and replaced with a cannula having a flexible silicone tip. Then the cannula is connected to an aspiration system. The tip of the cannula is inserted and positioned approximately 1 mm above the retinal surface but below the superotemporal arcade. After the posterior hyaloid surface is elevated in the area just inferior to the superotemporal arcade, the posterior hyaloid is detached as far as possible out to the equator. In some cases, additiQnal manipulation is lo neaded at the disc in order to complete the detachment of ~ `
the posterior hyaloid surface. Once this is accomplished, a total pars plana vitrectomy is performed ' `
by removing the vitreous as far out to the periphery as possible. The retina is examined to assure that no retinal tears have occurred.
Freshly thawed TGF-~2 is suspended in buffer containing 2~ human serum albumin for a concentration of 1330 ng/100 ~1 TGF-~2 solution. TGF-~2 solution (either 50 ~1 or 200 ~1) is applied to the area of neovascularization. In patients receiving subretinal TGF-~2, a bent, tapered 33-gauge cannula is used to enter the subretinal space at a site at least one disc diameter from the center of the fovea. Gentle injection of 50 ~1 containing 1330 ng/ 100 ~1 TGF-~2 is performed. The additional 150 ~1 of TGF-~2 is injected within the vitreous cavity just over the area of neovascularization.
-The conjunctival flaps and sclerotomies are closed. The intraocular pressure is checked and the intravitreal bubble adjusted to achieve normal pressure. The patient receives acetazolamide, 500 mg IV, and is continued on acetazolamide, 250 mg PO or IV every six hours, for the next 24 hours. The patient is instructed to lie in a supine position for the first 24 hours f~ll-ow~ng surgery;
thereafter, the patient is instructed to remain in a face-down position as much as possible over the next five days.

WO94/01124 PCT/US93/0642n .~3~5~ ~4~~

Patients are examined at one day, two weeks, - four to six weeks, and at three, six and twelve months ' after surgery. The patients are examined for best corrected visual acuity for both distance and near vision, refraction, intraocular pressure, size of neovascular net, presence of epiretinal membrane, presence of hyperfluorescence on fluorescein angiography, lens status, and results of ICG angiography.

l0 Analysis and Results '~
Data resulting from this trial are analyzed to establish the safety and efficacy of these doses of TGF-~2. A treatment is considered successful if: ' ' - corrected visual acuity improves (about lS two lines) or stabilizes in patients whose vision is 20/200 or better; or corrected visual acuity improves to 20/200 . .
in patients whose vision is worse than 20/200, or - 2~0 - there is a decrease in the size of the 'j neovascular net.

: : - i,.
Other Disorders ~:~~ '~ ~
25 ~ : The inventive treatment is also considered to e beneficial~in:other ocular disorders such as retinal dema,~retinal ~ascular~~disorders, wound healing ~- disorders, proliferative--ocular disorders, anti-degenerative~disorders, anti-angioqenesis disorders, dry;.
eye syndromes, uveitis,''-and various retinal detachments.
~ - EXAMPLE 4 ! ::
: To inves'~i.gate the effects of TGF-~l and TGF-~2 ~ ;
alone~on neovascularization in vivo, various doses of TGF-~ were impla~ted'into the clear cornea of rabbits, ~': and the neovascular response was measured over time.

, , ,~

WO94/01124 21 3 8 5 PCT/US93/0~20 -;

Five to seven pound male and female_New Zealand White rabbits were used. The animals were anesthetized with subcutaneous injections of 20 mg of xylazine and 80 mg of ketamine every other day for a total of 4 anesthesias. While under anesthesia on day 6, the animals were euthanized with an intracardiac injection of '' 325 mg of sodium pentobarbital.
TGF-~l and TGF-~2 and vehicle controls were placed in 2.5 isogel agarose (FMC Corp., Rockland, ME). ~ ' lO Porcine platelet-derived TGF-~l and TGF-~2 lyophilized i ' without bovine serum albumin (BSA) were obtained from Drs. ~nita Robert and Michael Sporn (NIH, Bethesda, MD). 1 '~
Porcine platelet-derived TGF-~l lyophilized with BSA also was obtained from R&D systems, Inc~. (Minneapolis, MN;).
15 The duplication of sources was used to help control for ~ `
the method of procurement, handling and shipment variables. Prior to adding to the agarose, TGF-~l and ¦ `
TGF-~2 were solubilized in 4 mM HCl. The agarose was heated to 60 C, added to the solubilized peptides and then allowed to'gel at room temperature. The gelled agarose was then divided into 2 X l.5 X l mm implants for implantation into the rabbit cornea.
Agarose implants containing l, 5, 25 and lO0 ng of TGF-~ were~placed within pockets in 7, 6, 6 and 4- -` 25 corneas~, respectively, on day 0. Corneas were 'photograph-d on days 2, 4 and 6. The photographs were developed as slides, which in turn were projected-a'nd~the blood vesse~l lengths were measured. ~ ' When TGF-~l was implanted into a nonvascular '~' 30 rabbit cor~ea', there was a dose-dependent stimulation-of blood vessel growth in 82% of corneas implanted--with l, ~
5, 25 and lO0 ng. The majority of corneas implanted with iP~`
~ l ng of TGF-~ showed no neovascular ingrowth. ~-Tfie~ -;~ remaining l-ng-treated corneas had sparse, short blood vessels. As the dose increase from 5 to lO0 ng, the neovascularization became more dense, the blood vessels ~;~ were longer, and the corneas became more edematous. At :::

W094J01124 PCT/US93/0~2 ~ 50-two days post-implantatio~, an intrastromal neovascular response was evident and became more prominent at days 4 and 6.
However, in corneas implanted with loO ng of TGF-~, blood vessel formation appeared to be impeded adjacent to the TGF-~-containing implant. This effect was not observed when neovascularization was stimulated by TGF-alpha or PGE1 (see below), suggesting that the implant did not merely act as a mechanical barrier impeding the growth of new blood vessels.
Thus, administering TGF-~1 or TGF-~2 without any preexisting neovascularization may cause neovascularization to develop.

EX~MPLE 5 The triple pocket corneal assay includes first ¦ -~
administra~ion of an aqent to produce neovascularization in one compartment, followed by implantation of TGF~
TGF-~2 or a control on both side~ of the ~ -neovascularization.
; First, neovascularization was induced by implanting a pellet containing either PGEl (Upjohn Co., Xalamazoo, MI) or TGF-alpha (Chemicon International, Inc., El S-gundo, CA). T~e-PGEl---~as- solubilized in absolute alcohol and then added to a casting solution of

10* ethy~ene vinyl coacetate polymer in methylene chloride to form pellets. TGF-~ was solubilized in 1 mM
.
HCl and then added to agarose and divided into implants.
I Two days later (day 0 of the TGF-~ study), TGF-~1, TGF-~2 or control pellets-were implanted next to ... . .
acti~ely growing blood vessels and on either side of the primary implant to test for the effect on the angiogenic !
acti~ity. TGF-~l and TGF-~2 were used at doses of 1, 3, 5, 10, 25, 100 and 200 ng (6 corneas for each dose, ;
except for 8 for the 100-n~ dose). In addition, TGF-~l was implanted at the 50 ng dose. Control agarose implants contained an equivalent volume of vehicle (4 mM

WO94/01124 ,) ~ PCT/US93/0~20 HCl) or lO0 ng of platelet-derived growth factor-(PDGF ;
from R&D Systems, Inc-) solubilized in 4 mM HCl- Ir The pockets for the secondary implants were formed by one-half-thickness incisions which were l.5 mm 5 long and perpendicular to and l.5 mm from the limbus and ~"
3-4 mm from the primary implant. Two pockets were formed on either side of the primary implant by gently inserting :
a cyclodialysis spatula into the incised edge of the cornea and advancing the spatula in a plane parallel to the curvature of the cornea to within l.0 mm of the primary implant such that the pockets lay l.5 mm from and parallel to the limbus. ;
Blood vessel lengths were measured adjacent to the TGF-~-containing implant ~E) and the control implant (C) 2 mm from the center of the primary implant (Figure l). The relative lengths of the blood vessels in these l ~
areas were then expressed as a ratio: E/C (the length of l ~;
the blood vessels in the area of the T~F-~ implant ~;
divided by the length o the blood vessels in the area of ; :
20 the control implant). Percent etimulation or inhibition ;~
was calculat~d by subtracting l.0 from E/C and ~
multiplying by lO0. Measurements of blood vessel length :
adjacent to both the TGF-~ and control implants were made from projected slides taken at days ~, 4 and 6. -- -Serial 5-micron frozen sections were taken from a cornea implanted with lO0 ng of TGF-~ in a triple pocket assay, stained with hematoxylin and examined-by- ~
light microscopy. ;
TGF-~ enhanced neovascularization in 89% of 30 corneas at doses of l, 3 and 5 ng. At l ng, - :
neovascularization was enhanced ~y 47%; at 3 ng, 118%;
and at 5 ng, 67% relative to control on day 4. TGF~
stimulated neovascularization much more than TGF-~2 at~
the l ng dose ~about 55% and 40%, respectively) and 3 ng dose (180% and 56%, respectively).
In contrast, in 100% of corneas receiving 25-lO0 ng of TGF-~l and TGF-~2, neovascularization was -~3~6'` '~ -52-inhibited relative to control-=as shown in the following -table. TGF-~l and TGF-~2 were comparable in effectiveness.
INHIBITION OF NEOVASCULARIZATION
BY DOSE AND LENGTH OF IMPLANTATION TIME
Dose Day 2 Day 4 Day 6 25 ng 52% 42% 33%
l00 ng 68% 53% 56%
200 ng 66% 56% 46%
In addition, the 50 ng dose of TGF-~l inhibited neovascularization by 59% on day 2, 49% on day 4 and 29%
on day 6. ~he dose of l0 ng appeared to be a transitional dose at which two of six corneas showed -`
15 stimulation of neovascuiarization in the area of the TGF- .
implant and four of six had neovascularization relative ~ to the control. ~.
: Thus, both TGF-~l and TGF-~2 can inhibit : neovascularization caused by PGEl or TGF-~. The .
inhibition was found to be dose dependent, with doses greater than l0 ng inhibiting neovascularization. The optimal dose in this experiment appeared to be about l00 ~.
ng. Overall, TGF-~2 is superior-in having less stimulatory effect and greater ~afety~while offering ~:
: 25~ equivalen.t neovascularization inhibition.
.While not~wishing to be-kound by any particular theory, th- inventors propose that--the different effects : ` above:and below l0 ng may be due.to-the interplay of TGF- ..
on multiple functions, including causing chemotaxis in 30 blood monocytes at about 0.l to l-.0 pg/ml, inducing gene -~: expression for interleukin-l (at-least IL~ sp~cific ~ mRNA has been observed in culture~ monocytes) at l.0 to i.
:~ : 25 ng/ml, and inhibiting vascula~ endothelial cell . j .
proliferation at 0.l to l0.0 ng/ml. Thus, at less than 35 l0 ng, the~ effect on vascula-r endothelial cell `-`
: proliferation appears to predominate; whereas, at higher WO~4/01124 ~1 3 '~~ 5 `'1 ~1 PcT/US93/~20 doses, effects on cellular function which could inhibit `;
neovascularization. :::

In this experiment, lO0 ng of PDGF and lO0 ng `~
of TGF-~l and TGF-~2 (four corneas for each peptide) were the secondary implants after the initial l.5 ug PGEl neovascular stimulus. TGF-~ inhibited neovascularization r~latiYe to PDGF in 100% of corneas. TGF-~l and TGF-~2 showed comparable degrees of inhibition. The average blood vessel length in the area of the TGF-~ implant was 19~, 40% and 36S of the average blood vessel length in the area of the PD~F implant on days 2, 4 and 6, respectively (combined data for ~l and ~23.
In this experiment, lO0 ng of PDGF and lO0 ng of TGF-~l and TGF-~2 (six corneas for each peptide) were the secondary implants after the initial 300 ng TGF-~
neovascular stimulus. TGF-~ also inhibited neovascularization stimulated by TGF-~ at a dose of 300 20 ng. TGF-~ inhibited neovascularization in lO0~ of ¦ ~
corneas. TGF-~l and TGF-~2 had compara~le degrees of I `
inhibition. The combined average blood vessel length in the area of the TGF-~ implant was 47%, 51% and 47% of the blood vessel lengths around the control implant on days - -2, 4 and 6, respectively.
In addition, 5-micron frozen sections were taken from a cornea implanted with lO0 ng of TGF-~ in~a triple pocket assay, hematoxylin stained and examined by light microscopy. An increase in cell number was observed in the stroma surrounding the TGF-~ implant when campared to the control implant. No evidence of tissue edema or tissue necrosis was observed in these section._ No strong inflammatory cell infiltrate was observed~ ` -around either of the pellets.
In these experiments, TGF-~l and TGF-~2 were compared with PDGF, which served as a negative protein control. Thus, this experiment indicates that the anti-WO94/01124 ` PCT/US93/0~20 ,~3~,6~ 54-neovascularization effects of TGF-~l and TGF-~2 are specific to these proteins and are not due to administering protein. ' S EXAMPLE 7 ' In this experiment, the effect of TGF-~2 on ''v healing after corneal surgery for correction of myopia ~' and hyperopia is determined by measuring the magnitude of effect that TGF-~2 has on altering the corneal topography in three different types of corneal incisions.
Female cats weighing 7-9 pounds are used - because the eye is similar in size, shape and morphology to that of humans. Similarly, their corneal endothelial ~-~
~ cells have limited mitotic capability. It has also been 15 demonstrated that cat corneas heal very much like human - I ~
corneas. Thus, the cat is an excellent model to study I '' corn-al wound healing.
Radial non-penetrating radial incisions are made. ~On~group of animals receives two radial incisions ¦ '~
and the other group receives four radial in~isions using a knife with micrometer adjusted to cut up to 90% of the c-ntral~corneal thickness. Both` eyes are operated. With the~aid of~an operating microscope, the 3.5 mm central optical zone centered over the pupil is_demarcated with a 25~marker. ~ Radial ~inoiJ~ions start at the central optical zone a~nd~extend peripherally to within about 2-3 mm of j ~"
the li~bu5~. ~Circular inc~ision~ a-re-~madë-with corneal I ~
' tréphines~of different~d'iameters-and penetrate about 90% ! ',`.
of the corneal depth. At the end of surgery, 2.0 ~g or ''~` 30 5.0 ~g TGF-~2 or control solution is applied to each eye.
In some eyes with nasal and t'ëmpora-l incision, TGF-~2 is ~' ~' applied to one incision (after which_a cup is placed over the incision to keep' the medication frôm dispersing) and ~ the~other incision is not treated. This helps assess the !' '~ ~ 35 ~effect of TGF-~2 on the change in topography. Next, ~ antibiotic ointment is topically applied to the eyes.
: :: :

'~

WO94/01124 21386~ PcT/US93/0~20 ~;

.
The eyes are observed under t~e slit lamp-and ~;
corneal topographic measurements are made, both before and at regular intervals after surgery. The slit lamp is used to evaluate corneal vascularization, epithelial healing, depth of incisions (to assess healing) and the amount of scar tissue formed. Corneal topoqraphic measurements will help assess how symmetrically and quickly the eyes stabilize. At the end of the study, the cats are euthanized, and sections of the eyes are mounted lO on slide, stained and compared. The eyes receiving TGF- -~2 treatment have rapid, strong healing and early stabilization of corneal topography.
. .
EXAMP~E 8 In this~example, bovine bone-derived TGF-B2 (bTGF-B2) was applied onto the macula of patients with CME following vitrectomy with the intent to reduce the magnitude of retinal edema and to improve visual acuity. `-; Ten~patiènts with persistent CME, and meeting the othçr entry requirements of the study, receive a vitrectomy ; plus bTGF-B2 applied directly onto the macular lesion at ltS i nterface with the vitreous cavity. Study parti~cipants_are examined at baseline and selected ;interva1s~post-treatment. Parameters to be assessed ~
25 ~ include~visual a~cuity (~DT~S eye chart), macular status (biomicr~scopy and fluorescein angiography), adverse _ ;
events ~lens~status, intraocular pressure, and retinal ~` ~
detachment), and concomitant medications.
Patients are chosen according to the following criteria~
l. Biomicroscopic and fluorescein angiographic evidence of cystoid macular edema_ (CNE) ~- -_ 2. CME present for at least 3 months but not more than 12 months 3. Patient failed to respond to treatment ~ with one or more anti-inflammatory agents :: ;

WO94/01124 PCT/US93/0642~
.~33~ 56-'~"
3. - Previous cataract extraction with or without lens implant 4. Best correct~d visual acuity between 20/60 and 20/800 .:
5. Patient can comply with all aspects of the treatment and evaluation schedule 6. Patient can provide voluntary inf ormed consent ~-However, patients having any of the following are excluded from the study:
l. Prior posterior ~itrectomy with or without ! ~`
TGF-B2 treatment 2. Other ocular disease (e.g. macular degeneration, diabetic retinopathy, macular ,~
- hole, retinal detachment, advanced glaucoma, etc.) which could interfere with mac11lar 1 ' function ¦ '''`' 3. Significant ocular media opacity which `' interferes with determining best corrected ,;
visual acuity 4. Uveitis unrelated to cataract surgery 5. Nursing or pregnant patient ~ ~' ~_ 6. Potentially una~le-to complete entire follow-up schedule '~~` --Patients who appear to meet the subject eligibility criteria undergo basel-ine ev~uation.
Baseline findings that are not consist'ent w-ith the requirements of the study cause discontinuation of the , ~ patient frjom,the study. Baseline studies include a !
complete medical and ophthalmologic-history and an ophthalmic examination with visual'-'acuity measurements.
'~est corrected visual acuity is_me~sured using the ETDRS
eye chart. CME is confirmed by^sl--i-~ lamp biomic~oscopy using a fundus contact lans or 78 diopter lens. Color 35 fundus photographs (30) are taken of the disc and macula , ,, (photographic fields I and II). Fluorescein angiography is used to confirm the diagnosis of CME using ,~
~: ', .~
~';' .

WO94~01124 ~1 3 ~` ~ d '~ PCTtUS93/06420 -57- ;
;( stereoscopic angiography. Stereo ~ed-free photographs are also taken of the macula at the beginning of the angiogram. The transit frames of the angiogram are centered on the macular (field II) of the study eye, wit~ ;
frames taken at 30 seconds, l minute, 2 minutes, 5 minutes, and lO minutes. A stereo photograph of the disc (field I) is also taken at lO minutes. All study photographs are labeled with the patient's study code and the date of the photograph, and subsequently graded by a certified grader who is masked with re~ard to either the patient's study code or the time the photographs were ; -taken relative to treatment. ', Treatment is scheduled within one week (seven days) of the date on which the baseline tests are completed. If for any reason treatment is postponed, all baseline studies must be repeated.
All surgery is performed under local anesthesia with standby anesthesia, unless the investigator or patient wants the procedure to be performed under general ~
20 anesthesia. After the eye is prepped and draped, a lid `
speculum is positioned. Two conjunctival incisions are made in the temporal and nasal conjunctiva, and a 4 mm infusion cannula is sutured 3 mm posterior to the limbus in the inferotemporal guadrant using a preplaced 4-o~
white silk suture. Before the vitreous infusion solution is started, it is established that the infusion cannula tip is indeed in the vitreous cavity. Two additional -- i sclerotomies are made in the superotemporal and - '~
superonasal quadrants, 3 mm posterior to the limbus. A
30 vitreous cutter and fiberoptic light pipe are then - - ~
- introduced into the vitreous cavity. The anterior ànd central vitreous are removed. Any visible ~itreous _ adhesions to the anterior segment, iris, or lens capsu}e_ are severed. The posterior vitreous is then removed with the vitreous cutter. Each eye is examined for a posterior vitreous detachment using a flexible silicone tip cannula attached to the suction line of the vitreous W094/01124 i~6 ~ -58- PCT/US93/~6420 cutter console~ A suction of 150 mm Hg is applied approximately 2-3 mm superior to the fovea, about 0.2 to o.5 mm anterior to the retina, to determine if there is residual posterior cortical vitreous with an attached posterior hyaloid. If the posterior hyaloid is still attached, the silicone tip cannula is used to create a posterior vitreous detachment. The posterior hyaloid is removed with the vitreous cutter to at least the equator.
The silicone tip cannula is used to verify that the posterior hyaloid has been completely removed posterior to the equator.
The ~itreous cutter and fiberoptic light pipe is removed from the eye, and the sclerotomies closed with scleral plugs. The peripheral retina is examined with `
indirect ophthalmoscopy and scleral depression to look for any peripheral retinal breaks. Peripheral breaks, if present, are treated with cryopexy. The scleral plugs ! ~:
are then removed, and the light pipe and silicone tip cannula are reintroduced into the vitreous cavity. A
20 fluid-air exchange is performed, aspirating t~e ! `:
intravitreal fluid over the optic nerve. The instruments are removed, and the scleral plugs replaced. Fifteen minutes is allowed for accumulation of fluid from the peripheral retina and vi~reo~s base-~around the optic nerve. The scleral plugs are again removed, and the remaining fluid anterior to the-optic_nerve aspirated using the silicone tipped cannula~
The viscodissection cannula and tubing -isitec) are then connected to a 1 cc syringe containing 30 TGF-b2 freshly diluted in neutral-buffer supplemented ~
with 2% human serum albumin. The tip of the ^
~iscodissection cannula is posit-loned directly over the i~;
fovea, taking care not to touch--the-fovea with the cannula. A O.l ml aliquot (1330 ng) of TGF-~2 is injected over the macula. -The instruments are removed from the eye, and the two superior sclerotomies closed ~i - with 7-0 vicryl suture. The infusion cannula is removed, W094tO1124 ~1 ~ ~ ~ 4 PCT/US93/06420 and the remaining sclerotomy clQsed with 7-0 vicryl suture. The intraocular pressure is normalized with sterile air to achieve an intraocular pressure of about 10 mm Hg. The conjunctiva is then closed wit~ i ;
interrupted 6-0 collagen suture. The patient is instructed to lie in a supine position for the first 24 hours following surgery; thereafter, the patient is t ;
instructed to remain in a facedown position as much as possible over the ensuing five days.
On Day 1 and Week 1, the following parameters are assessed: Intraocular pressure (if abnormally high, IOP is treated first with topical aqueous suppressants such as ~-blockers or ~-blockers; oral carbonic anhydrase inhibitors are only used if topical therapy has been deemed inadequate to control the intraocular pressure);
the size of the gas bubble remaining; the anterior t-- segment, including the cornea, anterior chamber, and ` ¦ ;
lens, with slit lamp biomicroscopy; adverse events; and concomitant medication.
At weeks 2 and 6, and months 3, 6, and 12 - post-treatment, the following parameters are assessed:
best corrected vision using standard refraction and t~e ETDRS chart; intraocular pressure; lens status including ~- the magnitude of nuclear sclerosis and posterior -~ -~
subcapsular cataract formation, both of which are graied on a scale of 0-4; the severity of cystoid macular edema _ is assessed by slit lamp biomicroscopy on a 0-4 scale;- ~
~- the severity of cystoid macular edema from fluorescein angiographic photographs by a certified grader who is masked with regard to patient identity and visitation--`-date; the presence or absence of an epiretinal membrane, ~ ;
;~ adverse events; and changes in concomitant medications.

The invention has been described by example and by words. It is the Inventors' intent that the examplesnot be used to limit the scope of the invention and , . , WO 94/01 1 24 PCI /lJS93/064~0 ` .
.2 ~ 3 ~6 i' ~ -60- ~ ~

~urther that equivalents to the claims expressed below be considered within the scope of the invention. j S
''.,,''.

` ;'' I

!`~

j:,`
",.

,~.
- ~ _ :~

1~'' ' ' I ; :

Claims (48)

1. A method of significantly improving the ocular vision in retinal disorders of the mammalian eye, said disorders being characterized by an insufficiency, loss or impending loss of fibrous tissue, said method comprising administering to said mammal about 1 to 10 µg of TGF-.beta..

2. The method of claim 1 wherein the TGF-.beta. is TGF-.beta.2.

3. The method of claim 1 wherein TGF-.beta. is administered by a method selected from intraocular, subretinal, subscleral, intrascleral, intrachoroidal, and subconjunctival injection and topical, oral or parenteral modes of administration.

4. The method of claim 3 wherein the intraocular administration comprises placing TGF-.beta.
solution on, under, over, or in the retinal disorder, or any combination thereof.

5. The method of claim 1 further comprising the following steps:
removing the vitreous humor from the eye;
removing the epiretinal membrane, if present; and administering a concentrated solution of TGF-.beta. by cannula to place the solution immediately over the retinal disorder.

6. The method of claim 1 wherein TGF-.beta. is administered once.

7. The method of claim 1 wherein TGF-.beta. is administered repeatedly until the retinal disorder has ameliorated.

8. The method of claim 1 wherein the dosage of TGF-.beta. is greater than about 1300 ng.

9. The method of claim 1 wherein the dosage of TGF-.beta. is greater than 2500 ng.

10. The method of claim 1 wherein the retinal disorder is a macular hole.

11. The method of claim 1 wherein the retinal disorder is macular degeneration.

12. The method of claim 1 wherein the retinal disorder is a retinal tear.

13. The method of claim 1 wherein the retinal disorder is subretinal neovascularization.

14. A method of maintaining or improving the ocular vision in macular degeneration of the mammalian eye, said method comprising administering to said mammal an amount of TGF-.beta. effective to stabilize or improve vision.

15. A method of maintaining or improving the ocular vision in cystoid macular edema, said method comprising administering to said mammal an amount of TGF-.beta. effective to stabilize or improve vision.

16. The method of treating retinal disorders to significantly improve vision, the retinal disorders being characterized by decreased connective or fibrous tissue, said method comprising the following steps:

removing the vitreous humor from the eye;
peeling off the epiretinal membrane, if present; and administering a concentrated solution of TGF-.beta.2 by cannula to place the TGF-.beta.2 solution immediately over the portion of the retina requiring treatment.

17. A method for treating a mammal with an ophthalmic disorder associated with impaired healing, said method comprising administering orally, locally or systemically, to said mammal a pharmaceutical composition of TGF-.beta., wherein said composition is being administered in an amount wherein the dosage range of TGF-.beta. is about 1 µg to about 10 µg.

18. The method of claim 17 wherein the ophthalmic disorder is selected from the group consisting of retinal edema, retinal wounds, macular degeneration, secondary cataracts, corneal epithelial wounds and Sj?gren's syndrome.

19. The method of claim 17 wherein the TGF-.beta. is TGF-.beta.2.

20. The method of claim 19 wherein the TGF-.beta.2 is recombinant human TGF-.beta.2.

21. The method of claim 17 wherein the administration is performed by intraocular, subretinal, subscleral, intrascleral, intrachoroidal injection, or any combination thereof.

22. The method of claim 17 wherein the administration is performed by application to the cornea or sclera.

23. The method of claim 22 wherein the application to the cornea or sclera is placement of a timed release dosage form in the cul de sac.

24. The method of claim 17 wherein the sufficient amount of the pharmaceutical composition is that amount which enhances healing without excessive fibroproliferation.

25. The method of claim 17 wherein the sufficient amount of the pharmaceutical composition is that amount which enhances healing of the ocular condition and improves the mammal's symptoms.

26. The method of claim 17 wherein the amount of amount of the pharmaceutical composition administered to the eye is about 1000 to 5000 nanograms of TGF-.beta..

27. A method for treating a mammal who has undergone or is about to undergo or is undergoing ophthalmic surgery to promote healing without excessive scarring, said method comprising administering to said mammal TGF-.beta., wherein the dosage range of TGF-.beta. is about 1 µg to about 10 µg.

28. The method of claim 27, wherein the TGF-.beta. is administered by direct application in the surgical wound.

29. The method of claim 27, wherein the TGF-.beta. is administered by intraocular, subretinal, subscleral, intrascleral, intrachoroidal injection, or any combination thereof.

30. The method of claim 29, wherein the TGF-.beta. is administered in a solution.

31. The method of claim 29, wherein the TGF-.beta. is administered in a controlled release formulation.

32. The method of claim 27, wherein the TGF-.beta. is TGF-.beta.1 or TGF-.beta.2.

33. The method of claim 27, wherein the TGF-.beta.2 is recombinant human TGF-.beta.1 or TGF-.beta.2.

34. The method of claim 27, wherein the ophthalmic surgery is cataract extraction.

35. The method of claim 27, wherein the ophthalmic surgery is corneal transplantation.

36. The method of claim 27, wherein the ophthalmic surgery is glaucoma filtration surgery.

37. The method of claim 27, wherein the ophthalmic surgery is surgery to correct refraction.

38. The method of claim 37, wherein the surgery to correct refraction is radial keratotomy.

39. The method of claim 27 wherein the amount of amount of the pharmaceutical composition administered per eye is at least about 1000 nanograms of TGF-.beta..

40. A method of treating a mammal for ocular neovascularization, said method comprising administering to a mammal TGF-.beta.2 in a dosage range from about 1 µg to about 10 µg.

41. The method of claim 40 wherein the TGF-.beta.2 is administered intravenously, topically, intraocularly, intramuscularly, locally or in an ocular device.

42. The method of claim 40 wherein the ocular neovascularization is selected from the group including neovascularization of the cornea, iris, retina, vitreous, and choroid.

43. The method of claim 42 wherein the corneal neovascularization is caused by trauma, chemical burns or corneal transplantation.

44. The method of claim 42 wherein the neovascularization of the iris is caused by diabetic retinopathy, vein occlusion, ocular tumor or retinal detachment.

45. The method of claim 42 wherein the retinal neovascularization is caused by diabetic retinopathy, vein occlusion, sickle cell retinopathy, retinopathy of prematurity, retinal detachment, ocular ischemia and trauma.

46. The method of claim 42 wherein the intravitreal neovascularization occurs in diabetic retinopathy, vein occlusion, sickle cell retinopathy, retinopathy of prematurity, retinal detachment, ocular ischemia and trauma.

47. The method of claim 42, wherein the choroidal neovascularization occurs in retinal or subretinal disorders of age-related macular degeneration, presumed ocular histoplasmosis syndrome, myopic degeneration, angioid streaks and ocular trauma.

48. A method of treating uveitis in a mammal, said method comprising injecting intraocularly in said mammal an effective amount of TGF-.beta.2.