# Patent

## Title
Antisense oligonucleotides capable of inhibiting the formation of capillary tubes by endothelial cells and methods of treating ophthalmic and dermatological diseases

## Abstract

A pharmaceutical composition blocks angiogenesis and contains as an active agent at least one nucleotide sequence from nucleic acid molecule SEQ ID NO. 3, fragments thereof containing at least twelve contiguous nucleotides and derivatives thereof; and nucleic acid sequences containing at least twelve contiguous nucleotides of the nucleic acid molecule SEQ ID NO 30 and derivatives thereof.

## Background
RELATED APPLICATION
This is continuation-in-part of U.S. patent application Ser. No. 10/735,512 filed Dec. 12, 2003, which is a continuation of International Application No. PCT/FR02/02067, with an international filing date of Jun. 14, 2002, which is based on French Patent Application No. 01/07805, filed Jun. 14, 2001.


TECHNICAL FIELD
This disclosure relates to antisense oligonucleotides capable of inhibiting the expression of the protein IRS-1 and inhibiting the formation of capillary tubes by endothelial cells. Thus, the disclosure relates to antiangiogenic agents and anti-cell-multiplication agents, particularly, antitumor agents. The disclosure also pertains to pharmaceutical compositions containing the oligonucleotides, methods of treating diseases such as ophthalmic and skin diseases and the use of the oligonucleotides as analysis reagents.
BACKGROUND
Angiogenesis is a fundamental process by means of which new blood vessels are formed. This process is essential in multiple normal physiological phenomena such as reproduction, development and even cicatrization. In these normal biological phenomena, angiogenesis is under strict control, i.e., it is triggered during a short period (several days) and then completely inhibited. However, many pathologies are linked to uncontrolled, invasive angiogenesis: arthritis, a pathology due to the damaging of cartilage by invasive neovessels; diabetic retinopathy or the invasion of the retina by neovessels leading to blindness of patients; neovascularization of the ocular apparatus which is a major cause of blindness. This neovascularization is involved in about twenty different eye diseases. Moreover, the growth and metastasis of tumors which are linked directly to neovascularization are dependent on angiogenesis. The tumor stimulates the growth of neovessels by its own growth. Moreover, these neovessels are escape routes for tumors which thereby join up with the blood circulation and induce metastases in sites remote from the initial tumor focus, such as the liver, lungs or bones.
Angiogenesis, the formation of neovessels by endothelial cells, involves the migration, growth and differentiation of endothelial cells. Regulation of these biological phenomena is directly linked to genetic expression.
SUMMARY
We provide pharmaceutical compositions comprising as an active agent at least one nucleotide sequence selected from the group consisting of nucleic acid molecule SEQ ID NO. 3, fragments thereof comprising at least twelve contiguous nucleotides and derivatives thereof; and nucleic acid sequences comprising at least twelve contiguous nucleotides of the nucleic acid molecule SEQ ID NO 30 and derivatives thereof.
In a preferred aspect, the nucleic acid sequences comprises at least twelve contiguous nucleotides of the nucleic acid molecule SEQ ID NO 30 are selected from the group consisting of SEQ ID NO 31 to SEQ ID NO 44 and fragments thereof comprising at least twelve contiguous nucleotides.
We also provide methods of inhibiting angiogenesis including administering a pharmaceutically effective amount of the pharmaceutical composition to a mammal.
We further provide methods of treating ophthalmic disease including administering a pharmaceutically effective amount of the pharmaceutical composition to a mammal.
We still further provide methods of treating corneal graft rejection, neovascular glaucoma or retinopathy of prematurity comprising administering a pharmaceutically effective amount of the pharmaceutical composition to a mammal.
We yet further provide methods of treating age related macular degeneration or diabetic retinopathy comprising administering a pharmaceutically effective amount of the pharmaceutical composition to a mammal.
We also provide methods of treating dermatological diseases including administering a pharmaceutically effective amount of the pharmaceutical composition to a mammal.
We further provide methods of treating psoriasis or rosacea, comprising administering a pharmaceutically effective amount of the pharmaceutical composition to a mammal.



BRIEF DESCRIPTION OF THE DRAWINGS
Other advantages and characteristics will become clear from the representative examples below in which the term “oligonucleotide” is used to designate the oligonucleotide of SEQ ID NO. 3 and which refer to the attached figures in which:
FIG. 1A is a Western Blot of images obtained from supernatant samples stemming from unstimulated cells (track NS) and cells stimulated with bFGF (track S) developed with an anti-IRS-1 antibody,
FIG. 1B is a Western Blot of images obtained after staining with silver nitrate obtained from the same supernatant samples stemming from unstimulated cells (track NS) and cells stimulated with bFGF (track S),
FIG. 2 is a Western Blot of images obtained from supernatant cells stemming from unstimulated cells (track NS) and cells stimulated with bFGF (track B) when the membrane is incubated with an anti-phosphotyrosine monoclonal antibody and developed with an anti-isotope antibody tagged at the peroxidase as indicated in Example 3,
FIGS. 3A to 3D show the images of the cultures on a type I collagen surface of the different lots of endothelial cells:
FIG. 3A shows the culture of untreated endothelial cells,
FIG. 3B shows the culture of endothelial cells stimulated with 3 ng/ml of bFGF,
FIG. 3C shows the culture of endothelial cells incubated with 100 μg/ml of oligonucleotide of SEQ ID NO. 3 for 4 hours and then stimulated with 3 ng/ml of bFGF,
FIG. 3D shows the culture of endothelial cells incubated with 100 μg/ml of oligonucleotide of SEQ ID NO. 3 for 4 hours,
FIGS. 4A to 4F illustrate the results of tests of the inhibition of corncal neovascularization in rats:
FIG. 4A shows the results obtained by subconjunctival injection of an antisense oligonucleotide at a concentration of 60 μm,
FIG. 4B shows the results obtained after subconjunctival injection of a sense oligonucleotide at a concentration of 60 μm,
FIG. 4C shows the results obtained after topical application of an antisense oligonucleotide at a concentration of 200 μm,
FIG. 4D shows the results obtained after topical application of a sense oligonucleotide at a concentration of 200 μm,
FIG. 4E illustrates the state of the cornea in the absence of any treatment,
FIG. 4F illustrates the state of the cornea when treated with subconjunctival injections of PBS,
FIGS. 5A to 5J illustrate the results of the inhibition of corneal neovascularization obtained in different groups of rats after de-epithelialization and limbic resection of the corneas of the rats on day 4 (FIGS. 5A to 5E) and on day 9 (FIGS. 5F to 5J). These are slit lamp photographs showing the comparison of the growth of the vessels in the various groups of rats. Enlargement ×10.



DETAILED DESCRIPTION
Our work performed made it possible to identify and prepare nucleic acid sequences involved in the regulation of angiogenesis.
Other studies pertaining to angiogenesis have shown a noteworthy expression and phosphorylation at the level of a tyrosine residue of an intracellular 180-kDa protein by endothelial cells cultured on a surface of type I collagen and stimulated by an angiogenic factor such as bFGF. The noteworthy expression and phosphorylation at the level of the tyrosine residue of the intracellular 180-kDa protein accompanies the formation of capillary tubes by the endothelial cells.
That protein is already known as a substrate of the insulin receptor (called IRS-1). It has been partially identified and investigated by certain diabetes researchers (Quon et al., J. Biol. Chem. (1994), 269 (45), 27920-27924). Those authors studied the role of IRS-1 in (i) the translocation of GLUT 4 stimulated by insulin and (ii) the transport of glucose in rat adipose cells. They constructed a plasmid containing:

    
    
        a double chain oligonucleotide obtained from the sense oligonucleotide of the following sequence SEQ ID NO. ID No. 1: 5′-TCGATGTGAC GCTACTGATG AGTCCGTGAG GACGAAACTC TGGCCTAG-3′ and
        cDNA coding for human IRS-1,
    
    


and transfected rat adipose cells with said plasmid to do this.
Our work revealed that the expression of the protein IRS-1 is also induced in endothelial cells when those cells are stimulated by the angiogenic factor bFGF.
We thus provide pharmaceutical compositions active on angiogenesis phenomena comprising as an active agent at least one nucleotide sequence selected from the group consisting of nucleic acid molecule SEQ ID NO.3, fragments thereof comprising at least twelve contiguous nucleotides and derivatives thereof; and nucleic acid sequences comprising at least twelve contiguous nucleotides of the nucleic acid molecule SEQ ID NO 30 and derivatives thereof.
In a preferred aspect, the nucleic acid sequences comprising at least twelve contiguous nucleotides of the nucleic acid molecule SEQ ID NO 30 are selected from the group consisting of SEQ ID NO 31 to SEQ ID NO 44 and fragments thereof comprising at least twelve contiguous nucleotides.
These oligonucleotides have remarkable antiangiogenic and antitumor activities. They are therefore particularly useful in the treatment of diseases linked to invasive angiogenesis not controlled by gene therapy methods including administering to an individual a composition containing at least one of these oligonucleotides.
The disclosure pertains most particularly to the oligonucleotide of formula SEQ ID NO. 3










5′-TATCCGGAGGGCTCGCCATGCTGCT-3′,







a fragment of this sequence comprising at least 12 contiguous nucleotides or derivatives of this sequence.
The term “derivative” is understood to mean a sequence capable of hybridizing under stringent conditions with one of the above mentioned sequences or with a fragment of these of at least 12 contiguous nucleotides. Derivatives may be derived from insertion, deletion or substitution of nucleic acids. Stringent conditions may be selected, as appropriate, by a person skilled in the art. A low stringent condition is, for example, 42° C., 2*SSC, and 0.1% SDS, and preferably 50° C., 2*SSC, and 0.1% SDS. A high stringent condition is for example 65° C., 2*SSC, and 0.1% SDS.
The disclosure pertains also most particularly to nucleic acid sequences of at least twelve contiguous nucleotides of SEQ ID NO 30 and derivatives thereof, SEQ ID NO 30 having the following formula:








5′-



 


TAGTACTCGAGGCGCGCCGGGCCCCCAGCCTCGCTGGCCGCGCGCAGTAC


 


GAAGAAGCGTTTGTGCATGCTCTTGGGTTTGCGCAGGTAGCCCACCTTGC


 


GCACGTCCGAGAAGCCATCGCTCTCCGGAGGGCTCGCCATGCTGCCACC


 


G -3′.






The disclosure pertains also most particularly to the oligonucleotide of formula SEQ ID NO. 31










5′- TCTCCGGAGGGCTCGCCATGCTGC -3′,







a fragment of this sequence comprising at least 12 contiguous nucleotides or derivatives of this sequence.
The disclosure also pertains to the oligonucleotide of formula SEQ ID NO 32










5′- CGGAGGGCTCGCCATGCTGCCACCG -3′,







a fragment of this sequence comprising at least 12 contiguous nucleotides or derivatives of this sequence.
The disclosure also pertains to the oligonucleotide of formula SEQ ID NO 33










5′- CGGAGGGCTCGCCATGCTGCCACC -3′,







a fragment of this sequence comprising at least 12 contiguous nucleotides or derivatives of this sequence.
The disclosure also pertains to the oligonucleotide of formula SEQ ID NO 34










5′- CGGAGGGCTCGCCATGCTGCCAC -3′,







a fragment of this sequence comprising at least 12 contiguous nucleotides or derivatives of this sequence.
The disclosure also pertains to the oligonucleotide of formula SEQ ID NO 35










5′- CGGAGGGCTCGCCATGCTGCCA -3′,







a fragment of this sequence comprising at least 12 contiguous nucleotides or derivatives of this sequence.
The disclosure also pertains to the oligonucleotide of formula SEQ ID NO 36










5′- CGGAGGGCTCGCCATGCTGCC -3′,







a fragment of this sequence comprising at least 12 contiguous nucleotides or derivatives of this sequence.
The disclosure also pertains to the oligonucleotide of formula SEQ ID NO 37










5′- CGGAGGGCTCGCCATGCTGC -3′,







a fragment of this sequence comprising at least 12 contiguous nucleotides or derivatives of this sequence.
The disclosure also pertains to the oligonucleotide of formula SEQ ID NO 38










5′- CGTCCGAGAAGCCATCGCTCTCCGGAG -3′,







a fragment of this sequence comprising at least 12 contiguous nucleotides or derivatives of this sequence.
The disclosure also pertains to the oligonucleotide of formula SEQ ID NO 39










5′- GCGCAGGTAGCCCACCTTGCGCACGTC -3′,







a fragment of this sequence comprising at least 12 contiguous nucleotides or derivatives of this sequence.
The disclosure also pertains to the oligonucleotide of formula SEQ ID NO 40










5′- CCCACCTTGCGCACGT -3′,







a fragment of this sequence comprising at least 12 Contiguous nucleotides or derivatives of this sequence.
The disclosure also pertains to the oligonucleotide of formula SEQ ID NO 41










5′- GAAGAAGCGTTTGTGCATGCTCTTGGGTTT -3′,







a fragment of this sequence comprising at least 12 Contiguous nucleotides or derivatives of this sequence.
The disclosure also pertains to the oligonucleotide of formula SEQ ID NO 42










5′- GCCCCCAGCCTCGCTGGCCGCGCGCAGTACGAA -3′,







a fragment of this sequence comprising at least 12 contiguous nucleotides or derivatives of this sequence.
The disclosure also pertains to the oligonucleotide of formula SEQ ID NO 43










5′- TAGTACTCGAGGCGCGCCGGGCCCCC -3′,







a fragment of this sequence comprising at least 12 contiguous nucleotides or derivatives of this sequence.
The disclosure also pertains to the oligonucleotide of formula SEQ ID NO 44










5′- AGGCGCGCCGGGCCCCC -3′,







a fragment of this sequence comprising at least 12 contiguous nucleotides or derivatives of this sequence.
All or part of the phosphodiester bonds are advantageously protected. This protection is generally implemented via the chemical route using methods that are known by art. The phosphodiester bonds can be protected, for example, by a thiol or amine functional group or by a phenyl group.
The 5′- and/or 3′-ends of the oligonucleotides are also advantageously protected, for example, using the technique described above for protecting the phosphodiester bonds.
The oligonucleotides can be synthesized using conventional techniques that are known art, for example, using one of the DNA synthesizers marketed by various companies.
Although their mechanism of action has not been entirely elucidated, the oligonucleotides inhibit the expression of the protein IRS-1 within endothelial cells. These oligonucleotides block the formation of neovessels by endothelial cells (i.e., they inhibit angiogenesis) and thus they inhibit the multiplication of tumor cells in mice.
The composition of the invention advantageously comprises as an active agent at least one oligonucleotide as defined above advantageously combined in the composition with an acceptable vehicle.
It was found that the protein IRS-1 represents a cellular constituent which is essential in the angiogenesis process. In fact, inhibition of the expression of the protein IRS-1 by the antisense oligonucleotides leads to the inhibition of the formation of capillary tubes by endothelial cells.
The oligonucleotides and the compositions containing them are thus antiangiogenic agents. They are also anti-cell-multiplication agents, particularly as antitumor agents, and consequently are particularly useful for the treatment of tumors. Thus, the invention includes the use of the oligonucleotides for the preparation of a composition intended for the treatment or prevention of pathologies linked to invasive, uncontrolled angiogenesis such as, as a nonlimitative example: the treatment of tumor vascularization, eye diseases such as those linked to the neovascularization of the ocular apparatus such as retinopathies such as retinopathy of prematurities and diabetic retinopathy, corneal graft rejection, neovascular glaucoma, AMD (age-related macular disease), rheumatoid arthritis, Crohn's disease, atherosclerosis, hyperstimulation of the ovary, skin disease such as rosacea and psoriasis, endometritis associated with neovascularization, restenosis due to balloon angioplasty, tissue superproduction due to cicatrization, peripheral vascular disease, hypertension, vascular inflammation, Raynaud's disease and Raynaud's phenomena, aneurysm, arterial restenosis, thrombophlebitis, lymphangitis, lymphedema, tissue cicatrization and repair, ischemia, angina, myocardial infarction, chronic heart disease, cardiac insufficiencies such as congestive heart failure, age-related macular degeneration and osteoporosis.
The pharmaceutical composition is in a form capable of subcutaneous, intramuscular, intravenous, transdermal or topical administration.
Preferably, the pharmaceutical composition is in a form capable of topical administration.
More preferably, the pharmaceutical composition is in the form of eye drops.
According to one aspect, the pharmaceutical composition of the invention is in the form of a hydrogel. In one embodiment, said hygrogel comprises the active agent, a water-soluble polymer such as carboxymethylcellulose, and water. Preferably, the amount of water-soluble polymer is about 0.001 to about 5 g per 100 g and the amount of water is about 95 to about 99.9 g per 100 g. In another aspect, the hydrogel comprises the active agent, paraffine and vaseline. Preferably, the amount of paraffine is about 40 to about 85 g per 100 g and the amount of vaseline is about 15 g to about 60 g per 100 g.
According to one aspect, the pharmaceutical composition is supplied in bi-compartmental vials, one compartment comprising dry powder of the pharmaceutical composition and the other compartment comprising a solvent such as NaCl, 0.9% solution.
The above pharmaceutical compositions are more particularly implemented in a manner such that they can be administered via the subcutaneous, intramuscular, intravenous, transdermal or topical route, for example. For such administration, use is made of aqueous suspensions, isotonic saline solutions or sterile, injectable solutions containing pharmacologically compatible dispersion agents and/or wetting agents such as, for example, propylene glycol or butylene glycol.
The usual unit dose to be administered contains from about 0.001 mg to about 50 mg of active principle.
According to another aspect, the pharmaceutical composition contains about 0.001 mg/ml to about 10 mg/ml of the active agent, preferably about 0.1 mg/ml to about 5 mg/ml, more preferably about 0.2 to about 4 mg/ml and most preferably about 0.4 mg/ml to 2 mg/ml.
According to another aspect, the total dose per day per eye of the active principle is comprised from about 1 μg to about 500 μg, preferably from about 10 μg to about 250 μg and more preferably from about 40 μg to about 200 μg.
The oligonucleotides are also useful as research reagents, notably for the in vitro study of signalization routes involving the 180-kDa protein, for example, on tumor cells or non-tumor cells transfected by the oligonucleotides. They are also useful for the in vivo study of signalization routes involving the 180-kDa protein in a large number of physiological and pathological phenomena such as angiogenesis or carcinogenesis essentially from the kinase/phosphatase ratio.
The disclosure also relates to methods for inhibiting angiogenesis, comprising administering a pharmaceutically effective amount of the pharmaceutical composition of the invention to a mammal.
The disclosure also relates to methods for treating ophthalmic disease in a subject in need thereof, comprising administering a pharmaceutically effective amount of the pharmaceutical composition to the subject.
According to an aspect, the methods comprise administering at least one drop, preferably two drops per day and per affected eye of a pharmaceutically effective amount of the pharmaceutical composition to the subject.
According to one aspect, the duration of the administration is at least three months, preferably six months.
The disclosure also relates to methods for restoring visual acuity and/or corneal sensitivity in a subject in need thereof, comprising administering a pharmaceutically effective amount of the pharmaceutical composition to the subject.
The disclosure also relates to methods for treating corneal graft rejection in a subject in need thereof, comprising administering a pharmaceutically effective amount of the pharmaceutical composition to the subject.
The disclosure also relates to methods for treating corneal graft rejection in a subject having experienced corneal lesions leading to neovascularization, comprising administering a pharmaceutically effective amount of the pharmaceutical composition to the subject.
According to one aspect, we provide methods for treating corneal graft rejection in a subject suffering from keratitis or keratouveitis, comprising administering a pharmaceutically effective amount of the pharmaceutical composition to the subject, wherein the keratitis or keratouveitis is due to contact lens, bacteria, microorganisms such as Chlamydia, Staphylococcus, Streptococcus, Pseudomonas; viruses, such as herpes simples, herpes zoster; protozoa, such as Onchocerca volvulus, Leishmaniabrasiliensis, Acanthamoeba keratitis; immunologic diseases, such as Stevens-Johnson syndrome, rosacea keratitis; or keratitis or keratouveitis due to trauma and prior surgery, alkali burns, graft rejection and degenerative disorders.
According to an aspect, the method for treating corneal graft rejection comprises the administration of one to two drops per day and per affected eye of the pharmaceutical composition, the composition comprising about 0.1 mg/ml to about 5 mg/ml of the active agent, preferably about 0.2 to about 4 mg/ml and most preferably about 0.4 mg/ml to 2 mg/ml of the active agent.
In a preferred aspect, the pharmaceutical composition comprises about 0.8 mg/ml to about 1 mg/ml of the active agent. In this embodiment, each drop contains about 40 μg to about 50 μg of the active agent. Administration of two drops per day corresponds thus to the administration of about 80 μg to about 100 μg of the active agent per day.
The disclosure also relates to methods for treating neovascular glaucoma or retinopathy of prematurity in a subject in need thereof, comprising administering a pharmaceutically effective amount of the pharmaceutical composition to the subject.
According to an aspect, the methods comprise administration of one to two drops per day and per affected eye of the pharmaceutical composition the composition comprising about 0.1 mg/ml to about 5 mg/ml of the active agent, preferably about 0.2 to about 4 mg/ml and most preferably about 0.4 mg/ml to 2 mg/ml of the active agent.
The disclosure also relates to methods for treating age related macular degeneration in a subject in need thereof, comprising administering a pharmaceutically effective amount of the pharmaceutical composition to the subject.
The disclosure also relates to methods for treating diabetic retinopathy in a subject in need thereof, comprising administering a pharmaceutically effective amount of the pharmaceutical composition to the subject.
The disclosure also relates to methods for treating dermatological diseases in a subject in need thereof, comprising administering a pharmaceutically effective amount of the pharmaceutical composition to the subject.
The disclosure also relates to methods for treating psoriasis in a subject in need thereof, comprising administering a pharmaceutically effective amount of the pharmaceutical composition to the subject.
The disclosure also relates to methods for treating rosacea in a subject in need thereof comprising administering a pharmaceutically effective amount of the pharmaceutical composition to the subject.
In a preferred aspect, the pharmaceutical composition is in the form capable of transdermal or topical administration.
According to one aspect, the pharmaceutical composition comprises about 0.1 mg/ml to about 5 mg/ml of the active agent, preferably about 0.2 to about 4 mg/ml and most preferably about 0.4 mg/ml to 2 mg/ml of the active agent.
EXAMPLE 1
Demonstration of the Induction of the Expression of IRS-1 (the 180-kDa Protein) in Endothelial Cells Resulting from the Stimulation of these Cells with bFGF.
The 180-kDa protein was demonstrated in the following manner:
The endothelial cells were cultured in a 6-well microtitration plate previously covered with type I collagen as described in (Montesano et al., J. Cell. Biol., 1983, 83, 1648-1652). The culture medium was DMEM (Sigma) enriched with 10% of fetal calf serum, 4 mM glutamine, 500 U/ml penicillin and 100 μg/ml streptomycin. After 3 to 4 days of culture, there resulted a semi-confluent layer of endothelial cells. The culture medium of six wells was aspirated and replaced by fresh culture medium. Three wells were enriched with 3 ng/ml of bFGF. After incubation for 48 hours, the wells were washed three times with a phosphate buffer and the cells were used to extract the messenger RNA (mRNA) according to protocols known in the art. The mRNAs were reverse transcribed by a polymerization chain reaction (PCR) using each of four degenerated groups of oligo (dT) (T12MN) (SEQ ID NO: 45) primers, M can be G, A or C; and N is G, A, T and C. Each group of primers is imposed by the base in position 3′(N) with a degeneration in the (M) position. Example: the set of primers in which N=G is constituted by:











SEQ ID NO. 24:
5′-TTTTTTTTTTTTGG-3′




 



SEQ ID NO. 25:
5′-TTTTTTTTTTTTAG-3′



 



SEQ ID NO. 26:
5′-TTTTTTTTTTTTCG-3′.






The cDNAs obtained in this manner were amplified and tagged by means of an arbitrary decamer in the presence of isotopically tagged ATP. The electrophoresis analysis of the cDNAs revealed the presence of an amplified 326-bp cDNA fragment in the sample stemming from the endothelial cells stimulated with bFGF, identified in the attached sequence listing as number SEQ ID NO. 27. However, this same fragment is weakly present or present in the trace state in the sample stemming from the endothelial cells that were not stimulated with bFGF. The sequencing of this fragment and the subsequent interrogation of the databases revealed that this fragment corresponds to a part of an already known gene, coding for the substrate of the insulin receptor (an intracellular 180-kDa protein).
EXAMPLE 2
Demonstration of the Induction of the Expression of IRS-1 (the 180-kDa Protein)
Endothelial cells cultured on a layer of type I collagen stimulated or not stimulated with bFGF (cf. example 1) were lysed in a cellular lyse buffer containing sodium orthovanadate. These solutions were then clarified by centrifugation at 14,000 g for 15 minutes. Supernatant samples stemming from unstimulated cells and cells stimulated with bFGF containing equivalent amounts of proteins were then taken up with an electrophoresis solution containing 2% SDS and 15 mM of dithiothreitol, heated at 100° C. for 5 minutes then deposited on polyacrylamide gel (gradient from 4 to 15% of acrylamide) under denatured conditions (in the presence of 2% SDS). After migration, the proteins were transferred onto a nitrocellulose membrane. The membrane was blocked by incubation at ambient temperature in a 5% milk solution in a PBS buffer. The membrane was then washed three times with a PBS buffer, incubated in a PBS buffer containing 1 μg/ml of anti-IRS-1 monoclonal antibody for 2 hours at ambient temperature and washed three times with a PBS buffer. The proteins were then developed with a secondary anti-isotope antibody coupled to peroxidase. The presence was noted of a protein of molecular weight 180 kDa recognized by the monoclonal anti-IRS-1 antibody in the preparations stemming from the endothelial cells stimulated with bFGF; this protein was weakly present in the preparation stemming from the endothelial cells not simulated with bFGF (FIG. 1).
EXAMPLE 3
Demonstration of the Induction of Phosphorylation at the Level of IRS-1 Tyrosine (the 180-kDa Protein)
Human endothelial cells cultured on a layer of type I collagen stimulated or not stimulated with bFGF were lysed in a cellular lyse buffer containing sodium orthovanadate. These solutions were then clarified by centrifugation at 14,000 g for 15 minutes (cf example 2). The IRS-1 protein was extracted by means of an anti-IRS-1 monoclonal antibody. This extraction was performed after immunoprecipitation by means of an anti-IRS-1 monoclonal antibody (Sigma). After addition of the anti-IRS-1 antibody coupled to agarose, the suspension was incubated for 2 hours at ambient temperature then centrifuged at 4000 g for 15 minutes. The resultant precipitate was taken up with an electrophoresis solution containing 2% SDS and 15 mM of dithiothreitol, heated at 100° C. for 5 minutes, then deposited on polyacrylamide gel (acrylamide gradient of 4 to 15%) under denaturing conditions (in the presence of 2% SDS). After migration, the proteins were transferred onto a nitrocellulose membrane. The membrane was blocked by incubation at ambient temperature in a 5% milk solution in a PBS buffer. The membrane was then washed three times with a PBS buffer, incubated in a PBS buffer containing 1 μg/1 ml of anti-phosphotyrosine monoclonal antibody for 2 hours at ambient temperature, and then washed three times with a PBS buffer. The proteins were then developed by means of a secondary anti-isotope antibody coupled to peroxidase. It was found that the IRS-1 protein of molecular weight 180 kDa was phosphorylated at the level of the tyrosine residue in the preparations stemming from the endothelial cells stimulated with bFGF; this protein was very weakly phosphorylated at the level of the tyrosine residue in the preparation stemming from the endothelial cells not stimulated with bFGF (FIG. 2).
EXAMPLE 4
Evaluation of the In Vitro Antiangiogenic Activity of the Oligonucleotide
Human endothelial cells were cultured on a layer of type I collagen. The culture wells were divided into four lots on the seventh day of culture:

    
    
        Lot 1: Wells corresponding to the culture of untreated endothelial cells (FIG. 3A).
        Lot 2: Wells corresponding to the culture of endothelial cells stimulated with 3 ng/ml of bFGF (FIG. 3B).
        Lot 3: Wells corresponding to the culture of endothelial cells incubated with 100 μg/ml of oligonucleotide of SEQ ID NO. 3 for 4 hours then stimulated with 3 ng/ml of bFGF (FIG. 3C).
        Lot 4: Wells corresponding to the culture of endothelial cells incubated with 100 μg/ml of oligonucleotide of sequence SEQ ID NO. 3 for 4 hours (FIG. 3D).
    
    


The various wells were examined by means of an inverted phase optical microscope after 3 to 4 days of culture. Upon reading the results, it was found that the human endothelial cells in lot 2 formed capillary tubes following stimulation with bFGF. It was also found that the oligonucleotide inhibits the formation of neovessels by these same cells stimulated with bFGF in lot 3. Finally, it was found that that the oligonucleotide does not modify in a pronounced manner the growth of the endothelial cells. In fact, the numbers of endothelial cells in the lot 1 wells and in the lot 4 wells were comparable.
EXAMPLE 5
Evaluation of the In vivo Activity of the Oligonucleotide
Three lots of naked mice were used. Each lot was constituted by 5 mice.
Lot no. 1: This lot was used as control. Each mouse was inoculated on day 0 with 200 μl of a suspension of B16 melanoma cells (provided by Institut Gustave Roussy, Villejuif) dispersed in PBS at the level of 106 cells/ml. These mice did not receive subsequent treatment.
Lot no. 2: Each mouse was inoculated subcutaneously on day 0 with 200 μl of a suspension of B16 melanoma cells dispersed in PBS at the level of 106 cells/ml. On day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9 and day 10 each mouse received a subcutaneous injection of 200 μl of an oligonucleotide solution diluted in PBS at a concentration of 500 μg/ml. The oligonucleotide injection was performed close to the cell injection site.
Lot no. 3: The mice of this lot were not inoculated with the B16 melanoma cells. However, each of the mice received an injection of 200 μl of an oligonucleotide solution in PBS at a concentration of 500 μg/ml; the injections were performed on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9 and day 10.
The following results were obtained:
In the mice of lot no. 1, the tumor mass developed very rapidly after inoculation. In fact, the tumor mass reached a size of 1.6 to 2.5 cm in diameter after ten days in the mice of said lot no. 1 (untreated mice). The evolution of the tumor mass in the mice of lot no. 2 (mice treated after inoculation by injection of oligonucleotide on day 1, day 2 and day 3), exhibited a clearly lower increase in the volume of the tumor mass. The tumor mass in the mice of lot 2 did not exceed 0.8 cm in diameter on the tenth day. On the fourteenth day, the difference between the tumor mass of the mice of lot no. 2 and those of lot no. 1 was remarkable.
In the mice of lot no. 3 (mice not having received B16 melanoma cells but treated by injection of oligonucleotide for three days), an unexpected general effect was observed on the skin. It was identical to that observed on all of the mice treated with the oligonucleotide (lot 2). The skin had an aged, crumpled appearance. The emergence of hairs was also observed on all of the treated mice. There was a parallelism during the evolution between the regression of the cutaneous signs and the resumption of tumor growth.
Thus, it was found that the oligonucleotide inhibits the development and formation of neovessels by endothelial cells in vitro. The oligonucleotide also has a remarkable in vivo antitumor activity in the naked mouse.
EXAMPLE 6
Evaluation of the Antiangiogenic Oligonucleotide on a Corneal Neovascularization Model in the Rat
I employed, modified and analyzed a model of the formation of corneal neovessels in the rat after de-epithelialization and limbectomy (FIGS. 5A to 5J). It is reproducible, allows direct slit-lamp examination and quantification of the neovessels. The details are described below. The model was then used for testing the efficacy of the antiangiogenic agents of the invention. Animals and corneal neovascularization model
Male Wister rats (Rattus norvegicus), aged five weeks (Charles River France, St-Aubin les Elbeufs, France), free of specific pathogens, were fed and allowed to drink water freely, and maintained in the laboratory animal facility under fixed temperature and humidity conditions, with cycles of 12 hours of light/12 hours of darkness.
The rats were anesthetized with a mixture of ketamine (Kétamine 1000, UVA, Ivry-sur-Seine, France; 128 mg/kg) and chlorpromazine (Largactil 25 mg/ml; Specia Rhône Poulenc, Paris, France; 5 mg/kg), injected via the intramuscular route. A drop of oxybuprocaine (Novésine, Chibret, Clermont-Ferrand, France) was instilled in the right eye. Using an enlargement system (macroscope Wild MPS 51 S, LEICA, Heerbrugg, Switzerland), the corneal epithelium was removed by a microsponge impregnated with 70% ethanol. A 1.5-mm band of conjunctiva, at the limbus, was excised with microsurgical scissors, and the eyelids were closed by a temporary blepharorraphy with a Vicryl 5.0 thread (Dacron, Alcon, Rueil-Malmaison, France). The eye was then rinsed abundantly with 1×PBS, an oxytetracycline cream was applied (Posicycline, Alcon, France) and the blepharorraphy was opened on the fourth day [8, 9].
Treatment by Subconjunctival Injections and Topical Applications of Antiangiogenic Oligonucleotide
The rats were divided into 6 groups:

    
    
        Group A: model+subconjunctival injection of a 60-μM antisense oligonucleotide solution in 1×PBS,
        Group B: model+topical application of a 200-μM antisense oligonucleotide solution in 1×PBS,
        Group C: model+subconjunctival injection of a 60-μM sense oligonucleotide solution in 1×PBS,
        Group D: model+topical application of a 200-μM sense oligonucleotide solution in 1×PBS,
        Group E: model+subcutaneous injection of 1×PBS,
        Group F: model without treatment.
    
    


All of the rats were subjected to de-epithelialization as described above; the treatment was performed every 24 hours starting on the fourth day and continuing until the ninth day. Neovascularization was examined at the beginning, in the middle and at the end of the protocol by slit-lamp examination; photographs were taken on day 0 and day 9.
Visualization and Quantification of the Neovascularization
The animals were euthanized 10 days after the de-epithelialization by lethal injection of pentobarbital (intraperitoneal injection). In order to fill the microvessels and quantify the corneal neovascularization, the upper part of the animals' bodies were perfused with fluorescein-dextran 2×1,000,000. The eyes were enucleated and immersed in paraformaldehyde/1×PBS 4% for 3 hours, then overnight in 1×PBS. The cornea was then isolated with 1 mm of limbus under surgical microscope and inserted in the flat state between plate and cover by means of 3 to 5 radial incisions. The flat corneas were then examined and photographed using fluorescence microscopy. After the whole corneas were reconstituted, they were scanned and the surfaces were measured by image analysis; a software program (NIH image) was used for the quantification of the neovascularization. For each photo, the total corneal surface was measured three times as was the neovascularized surface; the ratio of the means—neovascularized surface/total corneal surface—was used to obtain the percentage of neovascularization and to measure the inhibition obtained.
Statistical Analysis
The results were expressed as means±SD. The percentages of neovascularized surface/total surface were compared with the nonparametric test of Mann-Whitney. Values of P<0.05 were considered to be significant.
Dilution of the Oligonucleotide
The oligonucleotide was diluted in 1×PBS at pH 7.2. Based on the data in the literature and the experiments performed with other oligonucleotides, it was decided to use a concentration of 60 μM for the subconjunctival injections and a concentration of 200 μM for the topical applications.
Results
Using the model of corneal neovessels, treatment was performed with the 5′-TATCCGGAGGGCTCGCCATGCTGCT-3′ oligonucleotides identified under SEQ ID NO. 3 in the attached sequence listing modified in phosphorothioate form, daily, from day 4 to day 9, according to the following protocol:

    
    
        Group A: subconjunctival injection of the antisense oligonucleotide at 60 μM (AS 60),
        Group B: topical application of the antisense oligonucleotide at 200 μM (AS 200),
        Group C: subconjunctival injection of the sense oligonucleotide at 60 μM (S 60),
        Group D: topical application of the sense oligonucleotide at 200 μM (S 200),
        Group E: subconjunctival injection of 1×PBS (PBS),
        Group F: no treatment (0 Tt).
    
    


On the tenth day of the protocol, the rats were perfused with a solution of FITC/dextran and then euthanized. The corneas were collected and fixed in a 4% PAF solution. The corneas were then inserted in the flat state between plate and cover in a glycerol solution. The fluorescent neovessels were observed and photographed using the fluorescence microscope. The photographs were scanned and the neovascularization percentages were measured for each animal.
The results observed are presented in Table 1 below:














TABLE 1







Group A
Group B
Group C
Group D
Group E
Group F



AS 60
AS 200
S 60
S 200
PBS
0 Tt






















Mean
0.6157
0.5058
0.9431
0.9392
0.9552
9.9170


SD
0.2194
0.1172
0.0964
0.0308
0.0481
0.0751


Number of
15
15
15
12
9
9


measurements


SEM
0.0566
0.0303
0.0249
0.0089
0.0160
0.0250









The statistical analysis of the results using a nonparametric Mann-Whitney test yielded the following results:

    
    
        The subconjunctival injections of 60-μM of the antisense oligonucleotide (A) reduced neovascularization in relation to the control groups E and F (very significant results, P<0.0001 and P=0.0011); topical application of the antisense oligonucleotide at a concentration of 200 μM (B) reduced neovascularization in relation to the control groups E and F (extremely significant results, P<0.0001).
    
    


Compared to the subconjunctival administration of the sense oligonucleotide at 60 μM (C) or the topical application of the sense oligonucleotide at 200 μM (D), injection of the antisense oligonucleotide at 60 μM (A) and topical application of the antisense oligonucleotide at 200 μM (B) reduced neovascularization. These results were extremely significant (P<0.0001) (FIGS. 4A to 4F).
The inhibition of neovascularization was not significantly different depending on whether the antisense oligonucleotide was administered via the subconjunctival route (60 μM) or applied topically (200 μM). It was approximately 35% in relation to the controls (E and F).
The subconjunctival injection of the sense oligonucleotide at 60 μM (C) and the topical application of the sense oligonucleotide at a concentration of 200 μM (D) did not modify the neovascularization in relation to the control groups (E and F). In contrast, there was a small effect of the sense oligonucleotide in topical application (D) compared to the sense oligonucleotide in subconjunctival injections (C) (P=0.0117).
Moreover, there was seen in the groups treated with the antisense oligonucleotide (A and B), a smaller diameter and density of the neovessels. Their distribution did not differ in relation to the control groups nor was any difference observed in relation to the level of inflammation (FIG. 4).
Secondary Effects
No noteworthy secondary effects were seen in any of the groups during the two experimental series: after 6 days of treatment at the doses specified above, the skin of the rats was not crumpled, the fur was unchanged and the general condition of the animals was good; they fed normally until the last day and no suspicious mortality was observed. Although neither autopsies nor blood tests were performed, the general status of the animals at the end of the experiments did not suggest hepatic disorders. The only symptom observed was a transitory whitish deposit at the site of the conjunctival injections in 60% of the rats of group A, 60% of the rats of group C and 10% of the rats of group E. This deposit had been resorbed by the end of the experiments in all cases.
This example shows that—contrary to expectations—the subcutaneous injections of antisense oligonucleotide at a concentration of 60 μM did not inhibit neovascularization to a greater extent than the topical application of the antisense oligonucleotide at a concentration of 200 μM.
This can perhaps be explained by the difference in the concentrations employed; but this results suggests also a penetration of the oligonucleotide via the topical route rather than via the limbus. It also suggests the absence of prolonged release of the product from the injection site.
CONCLUSION
The application of the antisense oligonucleotide via the topical route or in subconjunctival injections reduces neovascularization in our model of corneal neovessels in the rat.
The purpose of this study was to test the efficacy of the antisense oligonucleotides stemming from the sequence of the gene IRS-1 on a previously developed model of corneal neovascularization in the rat.
The subject matter of the publications below is incorporated by reference:
1. Aiello L P. Keeping in touch with angiogenesis. Nat Med 2000; 6: 379-381.
2. D'Amore P A. Mechanisms of Retinal and Choroidal Neovascularization. Invest Ophthalmol Vis Sci 1994; 35(12): 3974-3979.
3. Hélène C. Rational design of sequence-specific oncogene inhibitors based on antisense and antigens oligonucleotides. Eur. J Cancer 1991; 27: 1466-1471.
4. Agrawal S, Bunnel B A, Crooke S T, Davidkova G, Gyurko R, Iyer K et al. Antisense oligonucleotides and antisense RNA. Benjamin Weiss edition (Philadelphia, USA) 1997; 1-11, 19-40.
5. Pierga J Y, Cammilleri S, Benyahia B, Magdelénat H. Applications of antisense oligonucleotides in cancer research. Bull Cancer 1994; 81: 1023-1042.
6. Robinson G S, Pierce E A, Rook S L, Foley E, Webb R, Smith L E H. Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative retinopathy. Proc Natl Acad Sci USA 1996; 93: 4851-4856.
7. Aiello L P. Vascular endothelial growth factor. Invest Ophthalmol Vis Sci 1997; 38: 1647-1652.
8. Amano S, Rohan R, Kuroki M, Tolentino M, Adamis A P. Requirement for vascular endothelial growth factor in wound and inflammation-related corneal neovascularization. Invest Opthalmol Vis Sci 1998; 39: 18-22.
9. Hoang-Xuan T, Prisant O. Restoration of corneal epithelium from limbic stem cells. Med Sci 1998; 14: 1375-1377.
10. Parry T J, Cushman C, Gallegos A M, Agrawal A B, Richardson M, Andres L E et al. Bioactivity of antiangiogenic ribozymes targeting Flt-1 and KDR in RNA. Nucleic Acids Research 1999; 27: 2569-2577.
11. Ozaki H, Seo M S, Ozaki K, Yamada H, Yamada E, Okamoto N et al. Blockade of vascular endothelial cell growth factor receptor signaling is sufficient to completely prevent retinal neovascularization. Am J Pathol 2000; 156: 697-707.
12. Berdugo Polak M. Iontophoresis administration of antisense oligonucleotides in the anterior segment of the eye: application to a corneal neovascularization model in the rat. DEA “Biology and Pathology of the Epithelia”; University of Paris VII, Feldmann G; Inserm U450, Director Courtois Y, under the direction of Bellar Cohen F. 2000.

## Claims

1. A pharmaceutical composition comprising as an active agent at least one nucleotide sequence selected from the group consisting of a nucleic acid sequence comprising at least twelve contiguous nucleotides of the nucleic acid molecule SEQ ID NO 30 and a derivative of said nucleic acid sequence capable of hybridizing with IRS-1 sequence at a temperature from 42° C. to 65° C., 2× SSC and 0. 1% SDS.

2. The pharmaceutical composition according to claim 1, wherein said nucleic acid sequence comprising at least twelve contiguous nucleotides of the nucleic acid molecule SEQ ID NO 30 is selected from the group consisting of SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44 and fragments thereof comprising at least twelve contiguous nucleotides.

3. The pharmaceutical composition according to claim 1, which contains about 0.001 mg to about 50 mg of the active agent and is in a form capable of subcutaneous, intramuscular, intravenous, transdermal or topical administration.

4. The pharmaceutical composition according to claim 1, which is in the form of eye drops.

5. The pharmaceutical composition according to claim 1, which is supplied in bi-compartmental vials, one compartment comprising dry powder of the pharmaceutical composition and the other one comprising solvent.

6. The pharmaceutical composition according to claim 1, which contains about 0.001 mg/ml to 10 mg/ml of the active agent.

7. The pharmaceutical composition according to claim 1, which contains about 0.1 mg/ml to about 5 mg/ml.

8. The pharmaceutical composition according to claim 1, which contains about 0.4 mg/ml to 2 mg/ml.

9. A method of inhibiting angiogenesis comprising administering a pharmaceutically effective amount of the pharmaceutical composition according to claim 1 to a mammal.

10. A method for treating ophthalmic diseases in a subject in need thereof, comprising administering a pharmaceutically effective amount of the pharmaceutical composition according to claim 1 to said subject.

11. A method according to claim 10 for restoring visual acuity and/or corneal sensitivity.

12. The method according to claim 10, comprising administering one to two drops per day and per affected eye of the pharmaceutical composition according to claim 1 to said subject.

13. The method according to claim 10, wherein said pharmaceutical composition comprises about 0.1 mg/ml to about 5 mg/ml of the active agent.

14. A method for treating corneal graft rejection in a subject in need thereof, comprising administering a pharmaceutically effective amount of the pharmaceutical composition according to claim 1 to said subject.

15. The method according to claim 14, comprising administering one to two drops per day and per affected eye of a pharmaceutical composition according to claim 1, wherein said composition comprises about 0.4 mg/ml to about 2 mg/ml of the active agent.

16. A method according to claim 14 for treating corneal graft rejection in a subject having experienced corneal lesions leading to neovascularization.

17. A method according to claim 14 for treating corneal graft rejection in a subject suffering from keratitis or keratouveitis, wherein said keratitis or keratouveitis is due to contact lens, bacteria, microorganisms, viruses, protozoa, immunologic diseases, or due to trauma and prior surgery, alkali burns, graft rejection and degenerative disorders.

18. A method for treating neovascular glaucoma or retinopathy of prematurity in a subject in need thereof, comprising administering a pharmaceutically effective amount of the pharmaceutical composition according to claim 1 to said subject.

19. A method for treating age related macular degeneration in a subject in need thereof, comprising administering a pharmaceutically effective amount of the pharmaceutical composition according to claim 1 to said subject.

20. A method for treating diabetic retinopathy in a subject in need thereof, comprising administering a pharmaceutically effective amount of the pharmaceutical composition according to claim 1 to said subject.

21. A method for treating dermatological diseases in a subject in need thereof, comprising administering a pharmaceutically effective amount of the pharmaceutical composition according to claim 1 to said subject.

22. A method for treating psoriasis or rosacea in a subject in need thereof, comprising administering a pharmaceutically effective amount of the pharmaceutical composition according to claim 1 to said subject.

23. A pharmaceutical composition comprising as an active agent at least one nucleotide sequence selected from the group consisting of a nucleic acid sequence consisting of at least twelve contiguous nucleotides of the nucleic acid molecule SEQ ID NO 30 and a derivative of said nucleic acid sequence capable of hybridizing with IRS-1 sequence at a temperature from 42° C. to 65° C., 2×SSC and 0.1% SDS.

24. The pharmaceutical composition according to claim 23, wherein said nucleic acid sequence consisting of at least twelve contiguous nucleotides of the nucleic acid molecule SEQ ID NO 30 is selected from the group consisting of SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44 and fragments thereof comprising at least twelve contiguous nucleotides.