Note: Descriptions are shown in the official language in which they were submitted.
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INHIBITION OF ANGIOGENESIS USING INTERLEUKIN 12
The present invention relates to the prevention of diseases
mediated by unwanted angiogenesis. More particularly, the
present invention relates to the use of Interleukin-12 (IL-12) for the
manufacture of medicaments for preventing unwanted
angiogenesis, particularly for the treatment of angiogenesis
dependent or associated diseases.
Interleukin 12 (IL-12), formerly called natural killer cell
stimulatory factor (Kobayashi M., et al., J. Exp. Med. 170:827-845,
1989) and cytotoxic lymphocyte maturation factor (Stern A.S., et al.,
Proc. Natl. Acad. Sci. USA 87:6808-6812, 1990), has potent anti-
tumor and antimetastatic activity in several murine tumor models
(Brunda M. J., et al., J. Exp. Med. 178:1223-1230, 1993; Nastala C.
L., et al., J. Immunol. 153:1697-1706, 1994). Although the
mec~h~ni.~m through which IL-12 exerts its anti-tumor effects is
not completely understood, it has been shown that IL-12 induces a
variety of biological effects on natural killer and T cells in vitro
(Manetti R., et al., J. Exp. Med. 179:1273-1283, 1994; Wu C. Y., et
~0 al., J. Immunol. 151:1938-1949, 1993; Tripp C. S., et al.,: Proc. Natl.
Acad. Sci. USA 90:3725-3729, 1993; Seder lR. A., et al., Proc. Natl.
Acad. Sci. USA 90:10188-10192, 1993; Bloom E. T., et al., J.
Immunol. 152:4242-4254, 1994; Cesano A., et al., J. Immunol.
151:2943-2957, 1993; Chan S. H., et al., J. Immunol. 148:92-98, 1992).
Activation of cytotoxic T lymphocytes by IL-12 is considered crucial
in its anti-tumor activity (Brunda M. J., et al., J. E xp. Med.
178:1223-1230, 1993). The IL-12 anti-tumor effect is partially
maintained in severe combined immune deficient (SCID) and nude
~ mice, both of which are T cell-deficient, and in CD8*-depleted
euthymic mice (Brunda M. J., et al., J. Exp. Med. 178:1223-1230,
1993; O'Toole M., et al., J. Immunol. 150:294A, 1993). These results
demonstrate that IL-12 has potent in v*o anittumor and
Wh/Ul 8.12.95
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antimethastatic effects against murine tumors and demonstrate as
well the critical role of CD8+ T cells in me~ t.ing the antitumor
effects against subcutaneous tumors.
The present invention provides the use of Interleukin-12 for
the preparation of medicaments effective in inhibiting unwanted
angiogenesis. IL-12 was observed to inhibit the growth of a wide
spectrum of tumors in vivo, but had no direct effect on tllmor cells
in vitro. In addition, in T cell deficient mice the anti-tumor activity
of IL-12 is not completely abrogated, suggesting that IL-12 has
lo antiangiogenic properties. IL-12 induces a strong inhibition of
neovascularization. This effect is not mediated by a specific cell
type of the immune system. Interferon gamma (IFN-~) appears to
play a critical role as a mediator of the antiangiogenic effects of IL-
12. The surprising recognition of antiangiogenic properties of IL-
12 is central to the proper design of treatment protocols including
its co-~lmini~tration with other inhibitors of neovascularization.
Consequently, the present invention provides the use of
Interleukin-12 for the manufacture of medicaments for the
treatment of diseases mediated by undesired or uncontrolled
ao angiogenesis, especially for the treatment of diseases wherein the
disease mediated by undesired or uncontrolled angiogenesis is
neovascularization, particularly retinal/choroidal
neovascularization. It is another object of the present invention to
provide the above use wherein the retinal/choroidal
neovascularization is associated with diabetic retinopathy or
wherein the retinal/choroidal neovascularization is associated
with macular degeneration.
It is another object of the present invention to provide the use of
Interleukin-12 for the manufacture of medicaments for the
treatment of diseases mediated by uncontrolled angiogenesis,
wherein the disease mediated by undesired or uncontrolled
angiogenesis is corneal neovascularization.
It is yet another object of the present invention to provide the
use of Interleukin-12 for the manufacture of a medicament for the
W 096/25171 CA 02212370 1997-08-0~ PCTAEr9''0C-07
-3-
treatment of diabetic retinopathy and for the treatment of macular
degeneration.
Further, the invention comprises the use of Interleukin-12 for
the manufacture of medic~7nents for the treatment of diseases
mediated by undesired or uncontrolled angiogenesis wherein the
diseases stem from solid tumors or blood-born tllmors and their
metastases .
It is yet another object of the present illvention to provide the
use of Interleukin-12 for the preparation of a medicament for the
lo treatment of all forms of proliferative vitreoretionopathy, whether
or not associated with diabetes.
The above medicaments may contain one or more additional
angiogenesis inhibitors.
Also part of this invention is Interleukin-12 and the use of
Interleukin 12 for the treatment of a disease as mentioned above.
Further, the invention comprises Interlel~kin-12 or the use of
Interleukin-12 in combination with one or more additional
angiogenesis inhibitors, for the treatment of the above diseases.
Brief description of the drawings
Figure 1. Effect of recombinant murine IL-12 on bFGF-induced
mouse corneal neovascularization. These photos represent
corneas of either vehicle (control) or IL-12-treated C57BL/6 and
SCID mice, 5 days after implantation of the basic fibroblast growth
factor pellet (P). There are prominer-t new vessels in the control
corneas, whereas almost no vascular response is seen after
treatment with IL-12. (Note that SCID mice have preexistent iris
vessels which are visible through the cornea since their iris is
hypopigmented. Thus, the vessels seen in the IL-12 treated panel
are in the plane of the iris and are not corneal vessels induced by
the basic fibroblast growth factor pellet.)
Figure 2. Angiogenic response 5 days after implantation of the
basic fibroblast growth factor pellets in C57BL/6 mice. Treatment
WO96/2~171 CA 02212370 1997-08-0~ PCTAEP9"~0~07
consisted of either vehicle (21 corneas), IL-12 (30 corneas) or a
monomeric mixture of IL-12 (10 corneas) as described below.
Vessel length in mm and number of clock hours are presented as
mean + SEM.
Figure 3. Effects of IFN-~-antibodies on IL-12-induced
inhibition of mouse corneal neovascularization. Male C57BL/6
mice were treated with either single intraperitoneal injections of
rat XMG1.2 IFN-~ antibodies or rat IgG as described below. Vessel
length and clock hours of neovascularization were measured on
day 5. This experiment was repeated on two separate occasions
with ~imil~r results. Data are presented as the mean + SEM of at
least 13 corneas.
Figure 4. The effect of treatment with IFN-Ay on basic fibroblast
growth factor-induced mouse corneal neovascularization. Male
C57BL/6 mice were either treated with intraperitoneal bolus
injections of IFN-~ starting on the day of pellet impl~nt~t.ion or by
continuous infusion of IFN starting 3 days before implantation of
the pellet. Vessel length and clock hours were measured on day 5
after impl~nt~t.ion of the basic fibroblast growth factor pellet and
are presented as mean + SEM of 10 corneas in each group.
Figure 5. Effect of IL-12 and AGM-1470 on growth of Lewis
lung carcinoma. Male C57BL/6 mice were inoculated with Lewis
lung carcinoma on day 0 and treatment with either saline, IL-12 or
AGM-1470 was started after the tumor became measurable.
Treatment protocol and measurement procedures are described
below. Results are representative of a single experiment of 4
~nirn~l.c in each group.
Figure 6. Effect of IL-12 and AGM-1470 on spontaneous lung
surface metastases of Lewis lung carcinoma. Treatment protocol
and counting procedure are described below. Results are
representative of a single experiment of 4 ~nim~lc in each group.
Angiogenesis is fundamental for tumors and metastases to
enlarge beyond a few millimeters in diameter (Folkman J., N.
Engl. J. Med. 285:182-1186, 1971). Strategies to prevent the
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development of new blood vessels in tumors and metastases have
been effective in suppressing growth of these tumors (Millauer B.,
et al., Nature 367:576-579, 1994; ~;m K. J., et al., Nature 362:841-
844, 1993). To determine whether IL-12 has antiangiogenic
properties, IL-12 was evaluated in a model of basic fibroblast
growth factor-induced mouse corneal neovascularization. The
results show that IL-12 is a potent inhibitor of angiogenesis in vivo
and that this effect is mediated by IFN-~.
Angiogenesis is the generation of new blood vessels into a
lo tissue or organ. Under normal physiological conditions, hllm~n.~:
or ~3nim~1~ only undergo angiogenesis in very specific and
restricted situations. For example, angiogenesis is normally
observed in wound healing, fetal and embryonal development and
formation of the corpus luteum, endometrium and placenta. The
control of angiogenesis is a highly regulated system of angiogenic
stimulators and inhibitors. The control of angiogenesis has been
found to be altered in certain disease states and, in many cases, the
pathological damage associated with the disease is related to the
uncontrolled angiogenesis.
Both controlled and uncontrolled angiogenesis are thought to
proceed in a .cimil:~r manner. Endothelial cells and pericytes,
surrounded by a basement membrane, form capillary blood
vessels. Angiogenesis begins with the erosion of the basement
membrane by enzymes released by endothelial cells and leukocytes.
The endothelial cells, which line the ll~men of blood vessels, then
protrude through the basement membrane. Angiogenic
stimulants induce the endothelial cells to migrate through the
eroded basement membrane. The migrating cells form a "sprout"
off the parent blood vessel, where the endothelial cells undergo
mitosis and proliferate. The endothelial sprouts merge with each
other to form c~pill~ry loops, creating the new blood vessel. In the
disease state, prevention of angiogenesis could avert the ~l~m~ge
caused by the invasion of the new microvascular system.
Persistent angiogenesis occurs in a multiplicity of disease
states, tllmor ~ow~h (both as primary tumor and metastasis) and
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abnormal growth by endothelial cells, and supports the
pathological damage seen in these conditions. The diverse
pathological states created due to unregulated angiogenesis have
been grouped together as angiogenesis dependent or angiogenesis
associated diseases. Therapies directed at control of the angiogenic
processes could lead to the abrogation or mitigation of these
diseases.
One example of a disease mediated by angiogenesis is ocular
neovascular disease. This disease is characterized by invasion of
0 new blood vessels into the structures of the eye such as the retina or
cornea. It is the most common cause of blindness and is involved
in appr-xim~tely twenty eye diseases. In age-related macular
degeneration, the associated visual problems are caused by an in
growth of choroidal capillaries through defects in Bruch's
membrane with proliferation of fibrovascular tissue beneath the
retinal pigment epithelium. Angiogenic d~rnage is also associated
with diabetic retinopathy, retinopathy of prematurity, corneal graft
rejection, neovascular glaucoma and retrolental fibroplasia. Other
diseases associated with corneal neovascularization include, but
are not limited to, epidemic keratoconjunctivitis, Vitamin A
deficiency, contact lens overwear, atopic keratitis, superior limbic
keratitis, pterygium keratitis sicca, sjogrens, acne rosacea,
phylectenulosis, syphilis, Mycobacteria infections, lipid
degeneration, chemical burns, bacterial ulcers, fungal ulcers,
Herpes simplex infections, Herpes zoster infections, protozoan
infections, Kaposi sarcoma, Mooren ulcer, Terrien's mariginal
degeneration, marginal keratolysis, rheumatoid arthritis,
systemic lupus, polyarteritis, trauma, Wegeners sarcoidosis,
Scleritis, Steven's Johnson disease, periphigoid radial keratotomy,
and corneal graph rejection.
Diseases associated with retinal/choroidal neovascularization
include, but are not limited to, diabetic retinopathy, macular
degeneration, sickle cell anemia, sarcoid, syphilis,
pseudoxanthoma elasticum, Pagets disease, vein occlusion, artery
occlusion, carotid obstructive disease, chronic uveitis/vitritis,
mycobacterial infections, Lyme's disease, systemic lupus
WO 96/25171 CA O 2 2 l 2 3 7 0 l 9 9 7 - O 8 - O ~ PCT/EP9G/0 ~ -07
eryth~m~tosi.q, retinopathy of prematurity, Eales disease, Bechets
disease, infections causing a retinitis or choroiditis, presumed
ocular histoplasmosis, Bests disease, myopia, optic pits, Stargarts
disease, pars planitis, chronic retinal det~hment, hyperviscosity
syndromes, toxoplasmosis, trauma and post-laser complications.
Other diseases include, but are not limited to, diseases associated
with rubeosis (neovascularization of the angle) and diseases
caused by the abnormal proliferation of fibrovascular or fibrous
tissue including all forms of proliferative vitreoretinopathy.
Another disease in which angiogenesis is believed to be involved is
rheumatoid arthritis. The blood vessels in the synovial lining of
the joints undergo angiogenesis. In addition to forming new
vascular n~wolk~, the endothelial cells release factors and
reactive o~y~ell species that lead to pannus growth and cartilage
destruction. The factors involved in angiogenesis may actively
contribute to, and help maintain, the chronically infl~med state of
rheumatoid arthritis.
Factors associated with angiogenesis may also have a role in
osteoarthritis. The act*ation of the chondrocytes by angiogenic-
related factors contributes to the destruction of the joint. At a later
stage, the angiogenic factors would promote new bone formation.
Therapeutic intervention that prevents the bone destruction could
halt the progress of the disease and provide relief for persons
suffering with arthritis. Chronic infl~mm~tion may also involve
pathological angiogenesis. Such disease states as ulcerative colitis
and Crohn's disease show histological changes with the ingrowth
of new blood vessels into the infl~med tissues. Bartonellosis, a
bacterial infection found in South America, can result in a chronic
stage that is characterized by proliferation of vascular endothelial
cells. Another pathological role associated with angiogenesis is
found in atherosclerosis. The plaques formed within the lumen of
blood vessels have been shown to have angiogenic stimulatory
activity.
One of the most frequent angiogenic diseases of childhood is
the hem~ngioma. In most cases, the tumors are benign and
regress without intervention. In more severe cases, the tl~mors
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progress to large cavernous and infiltrative forms and create
clinical complications. Systemic forms of ~em~ngiomas, the
h~m~ngiomatoses, have a high mortality rate. Therapy-resistant
h~m~ngiomas exist that cannot be treated with therapeutics
currently in use. Angiogenesis is also responsible for ~m~e
found in hereditary diseases such as Osler-Weber-Rendu disease,
or hereditary hemorrhagic telangiectasia. This is an inherited
disease characterized by multiple small angiomas, tumors of blood
or lymph vessels. The angiomas are found in the skin and mucous
membranes, often accompanied by epistaxis (nosebleeds) or
gastrointestinal bleeding and sometimes with pulmonary or
hepatic arteriovenous fistula.
Angiogenesis is prominent in solid tumor formation and
metastasis. Angiogenic factors have been found associated with
several solid tumors such as rhabdomyosarcomas, retinoblastoma,
Ewing sarcoma, neuroblastoma, and osteosarcoma. A tumor
cannot expand without a blood supply to provide nutrients and
remove cellular wastes. Tumors in which angiogenesis is
important include solid tumors, and benign tumors such as
acoustic neuroma, neurofibroma, trachoma and pyogenic
granulomas. Prevention of angiogenesis could halt the growth of
these tumors and the resultant damage to the ~nim~l due to the
presence of the tumor.
Angiogenesis has been associated with blood-born tumors
such as leukemias, any of various acute or chronic neoplastic
diseases of the bone marrow in which unrestrained proliferation of
white blood cells occurs, usually accompanied by anemia,
impaired blood clotting, and enlargement of the lymph nodes, liver,
and spleen. It is believed that angiogenesis plays a role in the
abnormalities in the bone marrow that give rise to lellkemi~-like
tumors.
Angiogenesis is important in two stages of tumor metastasis.
The first stage where angiogenesis stimulation is important is in
the vascularization of the tumor which allows tumor cells to enter
the blood stream and to circulate throughout the body. After the
CA 022l2370 l997-08-0~
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tumor cells have left the primary site, and have settled into the
secondary, metastatic site, angiogenesis must occur before the new
tumor can grow and expand. Therefore, prevention of angiogenesis
could lead to the ~Le:ve~ltion of metastasis of tumors and possibly
contain the neoplastic growth at the primary site. Knowledge of
the role of angiogenesis in the maintenance and metastasis of
tumors has led to a prognostic indicator for breast cancer. The
Pmount of neovascularization found in the primary tumor was
determined by counting the microvessel density in the area of the
0 most intense neovascularization in invasive breast carcinoma. A
high level of microvessel density was found to correlate with tumor
recurrence. Control of angiogenesis by therapeutic means could
possibly lead to cessation of the recurrence of the tumors.
Angiogenesis is also involved in normal physiological
processes such as reproduction and wound healing. Angiogenesis
is an important step in ovulation and also in implantation of the
blastula after fertilization. Prevention of angiogenesis could be
used to induce amenorrhea, to block ovulation or to prevent
implantation by the blastula. In wound healing, excessive repair
or fibroplasia can be a detrimental side effect of surgical
procedures and may be caused or exacerbated by angiogenesis.
Adhesions are a frequent complication of surgery and lead to
problems such as small bowel obstruction.
Several kinds of compounds have been used to prevent
25 angiogenesis. Taylor et al.. have used prolPmine to inhibit
angiogenesis, see Taylor et al., Nature 297:307 (1982). The toxicity
of prolPmine limits its practical use as a therapeutic. Folkman et
al.. have disclosed the use of heparin and steroids to control
angiogenesis. See Folkman et al., Science 221:719 (1983) and U.S.
Patent Nos. 5,001,116 and 4,994,443. Steroids, such as
tetrahydrocortisol, which lack gluco and mineral corticoid activity,
have been found to inhibit angiogenesis.
Other factors found endogenously in :~nim~l.c7 such as a 4 kDa
glycoprotein from bovine vitreous h7lmor and a cartilage derived
factor, have been used to inhibit angiogenesis. Cellular factors
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- 10 -
such as interferon inhibit angiogenesis. For example, interre~oll a
or human int~- ~lOll ,~ has been shown to inhibit tumor-induced
angiogenesis in mouse dermis stimulated by human neoplastic
cells. Inte. re. ~ll ,B is also a potent inhibitor of angiogenesis
induced by allogeneic spleen cells. See Sidky et al., Cancer
Research 47:5155-5161 (1987). Human recnmhin~nt oc inte. re. Oll
(alpha/A) was reported to be successfully used in the treatment of
pulmonary hem~ngiomatosis, an angiogenesis-induced disease.
See, White et al., N. Engl. J. Med. 320:1197-1200, 1971.
In accordance with the present invention, compositions and
methods are provided that are effective in inhibiting unwanted
angiogenesis in an ~nim~l, both human and non-human. These
compositions are easily ~tlmini.ctered by different routes including
parenteral and can be given in dosages that are safe and provide
angiogenic inhibition at internal sites. The present invention
provides a method of treating m~mm~ n diseases mediated by
undesired and uncontrolled angiogenesis by ~lmini~tering a
composition comprising Interleukin-12 in a dosage sufficient to
inhibit angiogenesis.
The present invention is especially useful for treating certain
ocular neovascular diseases such as macular degeneration. The
compositions which are contemplated as part of the present
invention preferably can be given parenterally to the patient and
thereby halt the progression of the disease. Other diseases that can
be treated using the present invention are diabetic retinopathy,
neovascular glaucoma and retrolental fibroplasia.
Interleukin-12 may be prepared by methods known in the art,
e. g. described in European Patent Application No. 433827, in
International Patent Applications WO 9005147 and WO 9205256, in
Kobayashi M., et al., J. Exp. Med. 170:827-845, 1989 and Stern A. S.,
et al., Proc. Natl. Acad. Sci. USA 87:6808-6812, 1990. Interleukin-12
may be produced by known conventional chemical synthesis,
recombinant methods or may be purified form natural sources.
The term "Interleukin-12" also comprises polypeptides .~imil~r to
-
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those of the purified and/or recomhin~nt protein but which
modifications are naturally provided or deliberately engineered.
This invention provides evidence that inhibition of
angiogenesis is a new biological activity of IL-12. This inhibition of
neovascularization was profound and occurred at concentrations of
IL-12 which also result in an optimum anti-tllmor effect (Brunda
M. J., et al., J. Exp. Med. 178:1223-1230, 1993). IL-12 is species
specific which is in agreement with the lack of inhibition of
angiogenesis when IL-12 was used in the chick chorioallantoic
lo membrane assay. The mouse corneal neovascularization model
was therefore the assay of choice to evaluate the antiangiogenic
properties of IL-12. Using this model in strains of mice with
different immunological backgrounds, no individual cell type of the
immune system (natural killer or T cells) could be recognized as
1~ the mediator of the anti-angiogenic effects of IL-12.
The mouse corneal neovascularization assay used in this
study is a basic fibroblast growth factor-driven model of
angiogenesis. It may the.e~o~e be argued that IL-12 specifically
inhibits basic fibroblast growth factor-induced angiogenesis.
ao However, IL-12 was equally inhibitory when the basic fibroblast
growth factor pellet was replaced by a pellet cont~ining vascular
endothelial growth factor (160 ng/pellet).
Treatment with IL-12 induces a sustained elevation of IFN-yin
the bloodstre~m of mice (Gately M. K., et al., Int. Immunol. 6:157-
167, 1994). The ~mini.~tration IFN-~ antibodies prevented the IL-
12-induced inhibition of neovascularization. In addition, treatment
with either bolus injections or continuous infusion of IFN-y
resulted in inhibition of neovascularization. These findings
suggest that IFN-~ is a necessary and sufficient mediator of the
antiangiogenic act*ity of IL-12. In support of an important role of
IFN-~ in the anti-tumor activity of IL-12 is the observation that
treatment of euthymic mice with IFN-~-antibodies resulted in loss
of anti-tumor efficacy of IL-12 (Nastala C. L., et al., J. lmmunol.
153:1697-1706, 199). IFN- y has been used in murine tumor models
~5 (Brunda M. J., et al., Int. J. C~ancer 40:807-810, 1987) but the
-
W 096/25171 CA 02212370 1997-08-0~ PCT~EP9~'OC'07
clinical use of IFN-y as an anti-cancer agent has not been ve~
successful. The pharmacokinetics of IFN-~ may have contributed
to the disappointing results with this drug in clinical trials. After
intravenous bolus ~rlminictration, IFN-~y has a relatively short half
life (hours) (Rutenfranz I., et al., J. Interferon Res. 8:573-580, 1988)
and subcutaneous injections do not result in detectable levels of the
drug in serum (Cross S. E., et al., J Interferon Res 45:606-609,
1993). The observation that, in comparison with bolus injections,
continuous intraperitoneal infusion of IFN-~ achieved enhanced
lo inhibition of angiogenesis, suggests a pharmacokinetic difference
between the two methods of ~lmini~tration. However, it cannot be
excluded that the additional 3 days of continuous IFN-r treatment
before implantation of the basic fibroblast growth factor pellet may
have had a beneficial effect on the outcome of the experiment.
Since either IFN-~y or serum obtained from IL-12-treated
s~nim~ had a significant effect on endothelial cell proliferation in
vitro, it is presently unclear how IFN-~ exerts its effect on blood
vessels. The literature on IFN-~ as an antiangiogenic agent is
controversial and mainly based on observations in vitro (Sato N., et
al., J. Invest. Dermatol. 95:85S-89S, 1990; Saegusa Y., et al., J. Cell.
Physiol. 142:488-495, 1990; Friesel R., et al., J. Cell. Biol. 104:689-
696, 1987; Saiki I., et al., lnt. J. Cancer 51:641-645, 1992; Kobayashi
S., et al., Immunopharmacol. 27:23-30, 1994). Since IFN-~ is
involved in the regulation of numerous genes (Sen G. C., et al., J.
Biol. Chem. 267:5017-5020, 1992) it seems reasonable to assume that
actions downstream of IFN- y may be involved in the antiangiogenic
effects.
The experiments with Lewis lung carcinoma bearing mice
confirm the potent anti-tumor activities of both IL-12 and the
angiogenesis inhibitor AGM-1470 as single agents (Brunda M. J.,
et al., J. Exp. Med. 178:1223-1230, 1993; Ingber D., et al., Nature
348:555-557,1990). The observation that simultaneous treatment
with IL-12 and AGM-1470 has additive effects in the Lewis lung
carcinoma model suggests that these agents act on endothelial
cells through different pathways. Combinations of antiangiogenic
agents may enhance this strategy to treat m~lign~ncies. The
.
CA 02212370 1997-08-0~
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invention clearly demonstrates that IL-12 is a potent inhibitor of
angiogenesis in vivo, an effect which appears to be mediated by
inducing a sustained release of IFN-~.
Pharmaceutically acceptable formulations of IL-12 in
connection with this invention can be made using formulation
methods known to those of ordinary skill in the art. These
formulations can be Allmini.ctered by standard routes. In general,
the formulations may be atlmini~tered parenterally (e.g.,
intravenous, subcutaneous or intramuscular) with topical,
0 transdermal, oral, or rectal routes also being contemplated. In
addition, the formulations may be incorporated into biodegradable
polymers allowing for sustained release of IL-12, the polymers
being implanted in the vicinity of where drug delivery is desired,
for example, at the site of a tumor. The biodegradable polymers
and their use are described, for exAmple, in detail in Brem et al., J.
Neurosurg. 74:441-446 (1991). The dosage of IL-12 will depend on
the condition being treated, the particular compound, and other
clinical factors such as weight and condition of the human or
AnimAl and the route of A~mini.ctration of IL-12. It is to be
understood that the present invention has application for both
human and veterinary use. For parenteral A~miniqtration to
humans, a dosage of between approximately 0.1 to 20 mg/kg 1 to 5
times a week, ~lefe~ably between ap~.~ x;..-Ately 0.5 and 10 mg/kg 1
to 3 times a week, and most preferably between approximately 1 to
10 mg/kg 1 to 3 times a week, is generally sufflcient.
The formulations include those suitable for parenteral
(including subcutaneous, intramuscular, intravenous,
intradermal, intratracheal, and epidural) A~lminictration. The
formulations may conveniently be presented in unit dosage form
and may be prepared by conventional pharmaceutical techniques.
Such techniques include the step of bringing into association the
IL-12 and the pharmaceutical carrier(s) or excipient(s). In
general, the formulations are prepared by uniformly and
intimately bringing into association the IL-12 with liquid carriers.
Formulations suitable for parenteral A~lmini.~tration include
aqueous and non-aqueous sterile injection solutions which may
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contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents. The
formulations may be presented in llnit-dose or multi-dose
containers, for ex~mple, seated ~n~poules and vials, and may be
stored in a freeze-dried (lyophilized) conditions requiring only the
addition of the sterile liquid carrier, for example, water for
injections, immediately prior to use.
lo Preferred unit dosage formulations are those cont~ining a
daily dose or unit, daily sub-dose, as herein above recited, or an
appropriate fraction thereof, of the ~mini~tered ingredient.
Diseases associated with corneal neovascularization that can
be treated according to the present invention include but are not
limited to, diabetic retinopathy, retinopathy of prematurity, corneal
graft rejection, neovascular glaucoma and retrolental fibroplasia,
epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens
overwear, atopic keratitis, superior limbic keratitis, pterygi~
keratitis sicca, sjogrens, acne rosacea, phylectenulosis, syphilis,
Mycobacteria infections, lipid degeneration, chemical bums,
bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes
zoster infections, protozoan infections, Kaposi sarcoma, Mooren
ulcer, Terrien's mariginal degeneration, mariginal keratolysis,
trauma, rheumatoid arthritis, systemic lupus, polyarteritis,
Wegeners sarcoidosis, Scleritis, Steven's Johnson disease,
periphigoid radial keratotomy, and corneal graph rejection.
Diseases associated with retinal/choroidal neovascularization
that can be treated according to the present invention include, but
are not limited to, diabetic retinopathy, macular degeneration,
sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum,
Pagets disease, vein occlusion, artery occlusion, carotid obstructive
disease, chronic uveitis/vitritis, mycobacterial infections, Lyme's
disease, systemic lupus erythematosis, retinopathy of prematurity,
Eales disease, Bechets disease, infections causing a retinitis or
choroiditis, presumed ocular histoplasmosis, Bests disease,
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myopia, optic pits, Stargarts disease, pars pl~nitis, chronic retinal
detachment, hyperviscosity syndromes, toxoplasmosis, trauma
and post-laser complications.
~.
Other diseases include, but are not limited to, diseases
associated with rubeosis (neovascularization of the angle) and
diseases caused by the abnormal proliferation of fibrovascular or
fibrous tissue including all forms of proliferative vitreoretinopathy,
whether or not associated with diabetes.
Another disease which can be treated according to the present
0 invention is rhellm~toid arthritis. It is believed that the blood
vessels in the synovial lining of the joints undergo angiogenesis.
In addition to forming new vascular networks, the endothelial cells
release factors and reactive oxygen species that lead to pannus
growth and cartilage destruction. The factors involved in
angiogenesis may actively contribute to, and help maintain, the
chronically inflamed state of rheumatoid arthritis.
Another disease that can be treated according to the present
invention are h~ ngiomas, Osier-Weber-Rendu disease, or
hereditary hemorrhagic telangiectasia, solid or blood borne tumors
and acquired immune deficiency syndrome.
A model of basic fibroblast growth factor-induced corneal
neovascularization in mice was used to evaluate the effects of IL-12
on angiogenesis in vivo. Different strains of mice were treated with
1 mg IL-12 per day intraperitoneally for 5 consecutive days. Extent
of neovascularization was measured using vessel length and
number of corneal clock hours of new blood vessel formation in
response to a basic fibroblast growth factor cont~ining pellet. The
anti-tumor act*ities of IL-12 and the angiogenesis inhibitor AGM-
1470 were evaluated in Lewis lung carcinoma-bearing mice. In
3~ vitro proliferation studies were performed on bovine capillary
endothelial cells, mouse pancreas endothelial cells, and a mouse
h~ n~ioendothelioma cell line. Corneal neovascularization in
immune competent C57BL/6 mice, severe combined immune
deficient (SCID) mice and natural killer cell deficient, beige mice
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W 096/25171 PCT/~~
was almost completely inhihited as a result of treatment with IL-
12. This potent ~,u~. ession of angiogenesis was prevented by the
:~llmini.ctration of IFN-~ neutr~ ing antibodies. In addition, IFN-
~ given either as intraperitoneal bolus injections or as a continuous
infusion from an implanted osmotic pump intraperitoneally
reproduced the antiangiogenic effects observed during treatment
with IL-12. Treatment with IL-12 and AGM-1470 had an additive
anti-tumor effect in Lewis lung carcinoma-bearing mice
suggesting a different antiangiogenic me~h~ni.~m of action.
This invention is further illustrated by the following ex~mples,
which are not to be construed in any way as imposing limitations
upon the scope thereof On the contrary, it is to be clearly
understood that resort may be had to various other embodiments,
modifications, and equivalents thereof which, after reading the
description herein, may suggest themselves to those skilled in the
art without departing from the spirit of the present invention
and/or the scope of the appended claims.
Examples
1. Materials
Recombinant murine IL-12 (IL-12), recombinant murine
Interferon g~mma (IFN-y), and rat IgGl XMG1.2 IFN-~ blocking
antibodies were of H~-fr...~nn-La Roche, Nutley, NJ. AGM-1470
(TNP-470) and basic fibroblast ~ . ~.w Lh factor were obtained from
Takeda Industries, Osaka, Japan. All other materials were
purchased from Sigma, St Louis, Mo.
A monomeric mixture of IL-12 was made by reducing IL-12
with dithiothreitol (10 mM) and iodoacetamide (50 mM). Hereafter
the mixture was dialyzed for 3 hours (molecular weight cut off
point 6-8000 D, Spectra/Por dialysis membrane, Houston, Tx) to
elimin~te the reducing compounds. The presence of monomers
and absence of dimers in the mixture was confirmed by SDS-
PAGE.
2. Cells and Culture Conditions
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Bovine c~pill~ry endothelial celis=, a primary culture of mouse
pancreatic islet endothelial cells and a mouse
h~m~n~ioendothelioma cell line were used in this study.
Monolayer culturing was performed in Dulbecco's modified
6 Eagle's minim~l essential medium (DMEM) supplemented with
100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glut~mine
("full medium"), and 10 % bovine serum (GIBCO BRL, Grand
Island, NY.) in an atmosphere of 10% C02. Bovine c~rill~ry
endothelial cells were maintained in culture in the presence of 4
lo ng/ml basic fibroblast growth factor (bFGF) whereas mouse
pancreatic islet endothelial cells were grown in the presence of 6
ng/ml basic fibroblast growth factor and 10% NUSERUM IV
culture supplement (Becton Dickinson Labware, Bedford, MA).
Experiments involving bovine and mouse endothelial cells were
1~ performed between passage 10 and 15.
3. Mice
Male C57BL/6, SCID (C57BL/6/SCID/s:~j), and Beige
(C57BL/6/bgj) mice were purchased from the Jackson Laboratories,
Bar Harbor, ME. Nude mice (NCR Nu/sed, Swiss white
background) were obtained from the M~.s~husetts General
Hospital, Boston, MA). All ~nim~l studies were conducted on
male, 6-8 weeks old mice.
4. In Vitro Assays
To evaluate effects on endothelial cell proliferation, bovine
capillary endothelial cells, mouse pancreatic islet endothelial cells
and h~m~n~ioendothelioma cells were plated at a density of 10,000 -
12,500 cells/well in a 24 well plate. Twenty-four hours later, cells
were incubated in full medium supplemented with 1 ng/ml basic
fibroblast growth factor and 5% bovine serum and challenged with
the compound to be tested. After 72 hours, cells were harvested by
trypsini~tion and counted with a Coulter counter.
.
5. In Vivo Assays
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To study the effect of IL-12 and IFN-y on angiogenesis in vivo, a
modification of a previously described mouse corneal angiogenesis
assay was used (Polakowski I. J., et al., Am. J. Pathol. 143:507-517,
1993; Muthukkaruppan V., et al., Science 205:1416-1418, 1979). In
brief, corneal micropockets were made in both eyes re~ ing
within 1 mm of the limbus and a pellet cont~ining basic fibroblast
growth factor (~80 ng), sucralfate and hydron was implanted in
both eyes. The vascular response measured as the m~im~l vessel
length and nl~mber of clock hours of neovascularization was
lo assessed daily. Data presented in this study were obtained on the
fifth day after implantation of the basic fibroblast growth factor
pellet, which was found to be the day of m~im~l angiogenic
response .
An osmotic pump (Alzet 2002, Alza Corporation, Palo Alto,
CA) was implanted intraperitoneally in experiments designed to
ensure continuous infusion of either saline or IFN-~. Mice were
allowed to recover from the laparotomy for 3 days before
implantation of the basic fibroblast growth factor pellets. After
termination of the experiment the r~m~ining pump volumes were
checked to ensure adequate function and delivery.
Serum of IL-12-treated mice was obtained by cardiac puncture
24 hours after the fifth daily injection of IL-12.
6. Tumor experiments
Male C57BL/6 mice were inoculated with 106 Lewis lung
carcinoma cells. Treatment with either saline, IL-12, AGM-1470
or IL-12 plus AGM-1470 was initiated after the tumor volume
reached 75 mm3. IL-12 was given at a dose of 1 mg/day
intraperitoneally for 5 consecut*e days. After 2 days of rest this
cycle was repeated again. AGM-1470 was a~mini.~tered
subcutaneously every other day at a dose of 30 mg/kg. Serial
caliper measurements of perpendicular di~meters were used to
calculate tumor volumes in mm3 using the formula: longest
diameter x shortest diameter2 x 0.52. Three weeks after
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inoculation, tumors and lungs were ~esected and weighed. Lung
surface metastases were counted under a dissecting microscope.
7. Statistical analysis
The statistical significance of dirre.ellces between groups was
calculated by applying Student's 2-tailed t-test. Results are
presented as the mean + standard error of the mean.
8. Effect of IL-12 on mouse corneal neovascularization.
Male C57BL/6 mice were treated with either IL-12 (1 mg in 0.1
ml vehicle intraperitoneally/day for 5 consecutive days, starting on
lo the day of pellet implantation) or vehicle (1% syngeneic mouse
serum in phosphate-burrered saline). During treatment, no
obvious toxicity was encountered. C57BL/6 mice treated with IL-12
had almost no corneal neovascularization in response to the basic
fibroblast growth factor pellet whereas mice treated with vehicle
~5 had blood vessels which reached the pellet within 5 days after
implantation of the pellet (p< 0.0001; Figures 1 and 2). Results were
obtained from three independent experiments. When IL-12 was
reduced to a monomeric mixture and the mice were treated daily
with 1 mg of this mixture intraperitoneally for 5 days the in vivo
~30 inhibitory effect on neovascularization was lost (Figure 2).
To investigate which cells of the immune system might
mediate the inhibitory effect of IL-12 on angiogenesis, the mouse
corneal neovascularization assay was used in strains of mice with
an aberrant immune system. We first studied T cell-deficient
SCID mice. IL-12 retained its inhibition of angiogenesis in SCID
mice (vessel length: 0.98+0.06 mm versus 0.22+0.02 mm (p= 0.0002)
and clock hours: 4.6+0.4 h versus 3+0.3 h (p= 0.011) for vehicle and
IL-12 treated mice respectively). The extent of inhibition resembled
that observed in the euthymic C57BL/6 mice. A .c:imil~r pattern of
inhibition was observed when natural killer cell-deficient, beige
mice were treated with IL-12 (vessel length: 0.7_0.05 mm versus no
new vessels (p< 0.0001) and clock hours: 3.6_0.3 h versus no new
vessels (p< 0.0001) for vehicle or IL-12 treated mice respectively).
Nude mice were found to have spontaneous corneal
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neovascularization which masked the development of basic
fibroblast growth factor-induced new blood vessels. A consistent
finding however, suggestive of some degree of inhibition of
angiogenesis, was the lack of capillaries growing into the pellet in
the IL-12-treated ~nim~ls, whereas the vehicle-treated mice had
vessels clearly growing into the pellet.
9. Effects of IL-12 and IFN-~ on endothelial cell proliferation in
vitro.
IL-12 (range 0.001-100 ng/ml) had no effect on the proliferation
lo of either bovine or mouse endothelial cells or
h~m~ngioendothelioma cells. Serum obtained from either
C57BL/6, SCID or nude mice after they were treated with IL-12 for 5
days had no inhibitory effect on proliferation of either type of
endothelial cells.
IFN-y (range 0.0001-200 ng/ml) had only a minim~l effect (16 %
inhibition as compared with control cell numbers) on mouse
pancreatic islet endothelial cell proliferation and no effect on bovine
capillary endothelial cell proliferation.
10. The role of IFN-~ as a mediator of IL-12 activity in vivo.
ao Treatment of C57BL/6 mice with a single injection of IFN-g-
antibodies (1 mg/mouse intraperitoneally on the day of pellet
implantation ~tlmini.stered 2 hours before the first injection with
IL-12) totally abolished the antiangiogenic properties of IL-12
(p<0.0001). Control injections with rat IgG (1 mg/mouse
intraperitoneally on the day of pellet implantation) did not affect the
inhibition of neovascularization by IL-12 (Figure 3).
To investigate whether treatment with IFN-g resulted in
.simil~r inhibition of angiogenesis as seen with IL-12, C57BL/6
mice were treated with daily intraperitoneal injections of IFN-y
(250,000 U/day for 5 consecutive days). In these mice, significant
(p=0.0007) inhibition of vessel length was observed (Figure 4)
whereas no obvious toxicity was encountered. To m~int~in a
constant level of IFN-~, an osmotic mini-pump was implanted
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intraperitoneally which released IFN-~ to a final dose of 130,000
U/day for the duration of the experiment. The basic fibroblast
growth factor-pellets were implanted 72 hours after implantation of
the pump. Implantation of pumps loaded with saline did not affect
the development of new blood vessels in control ~nim~ . The
extent of neovascularization in these ~nim~lc was comparable to
control ~nim~ without a pump. However, ~nim~lc with pumps
cont~ining IFN-g had complete inhibition of new vessel growth in
the cornea (Figure 4; p=0.0002 for vessel length and p=0.0004 for
lo clock hours as compared with controls). Both the control ~nim~
and the IFN-~-treated mice lost weight in the recovery period after
the laparotomy. After implantation of the pellets the control
~nim~l~ gained weight whereas the IFN-y-treated mice had stable
weights and were lethargic.
11. Effect of treatment with IL-12 and AGM-1470 on Lewis lung
carcinoma .
Treatment with either IL-12 or AGM-1470 was effective in
inhibiting primary tumor growth and spontaneous lung
metastases in C57BL/6 mice inoculated with Lewis lung carcinoma
as compared with control ~nim~ treated with saline.
Simultaneous treatment of Lewis lung carcinoma-bearing mice
with IL-12 and AGM-1470 resulted in smaller primary tumor
volumes (Figure 5) and less spontaneous lung metastases (Figure
6) than seen in ~nim~l~ treated with IL-12 or AGM-1470 as single
agents. No obvious toxicity was encountered during treatment in
either of the groups.
