Note: Descriptions are shown in the official language in which they were submitted.
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.
WO94/01124 PCT/US93/06420
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 ;
~,
:~
WO94/01124 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 . . .
WO94/01124 PCT/US93/06420
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
-17-
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 ~
-21-
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-
- EXAMPLE 1
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 ~
-25-
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 ~-
- 1
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.
WO94/01124 PCT/US93/0~20
-27-
-= 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
-28-
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.
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WO94/01124 213 ~ 5 -1 'I PCT/US93/0~20
-37
- - EXAMPLE 2
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~
-38-
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
-39-
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
-42-
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
- - - =. ,
- ~ .
,:
~
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.- ,,.
~,
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WO 94/01 124 PCI /US93/06420
.~ 3~6 ~ 44-
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~ ~ I ~ ~
WO94~01124 ~ ~ PCT/US93/0~20 ';~
-45-
- 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
-47-
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 -;
-49-
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
-51-
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 -
WO94/01124 PCT/US93/06420
~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
-53-
doses, effects on cellular function which could inhibit `;
neovascularization. :::
EXAMPLE 6
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 ~;
-55-
.
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
-59-
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
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