WO2002062371A1

WO2002062371A1 - Dipeptide inhibiting angiogenesis in ophthalmopathology

Info

Publication number: WO2002062371A1
Application number: PCT/RU2001/000434
Authority: WO
Inventors: Vladimir Khatskelevich Khavinson; Valentina Mikhailovna Khokkanen; Svetlana Vladislavovna Trofimova; Vladimir Viktorovich Malinin
Original assignee: Sankt-Peterburgskaya Obschestvennaya Organizatsia 'sankt-Peterburgskiy Institut Bioregulyatsii I Gerontologii Szo Ramn'
Priority date: 2001-01-25
Filing date: 2001-10-16
Publication date: 2002-08-15
Also published as: RU2177801C1

Abstract

The invention refers to medicine and concerns the ophthalmologic application of dipeptide L-lysyl-L-glutamic acid (L-Lys-L-Glu) as a preparation inhibiting neovascularisation in ocular diseases. There is proposed the application od L-Lys-L-Glu dipeptide or one of its salts as a substance capable of angiogenesis inhibition in ophthalmopathology. There is proposed a pharmaceutical preparation for parenteral administration including the claimed dipeptide or one of its salts in the amount, which is effective for the prevention of and/or treatment for ophthalmopathology requiring angiogenesis inhibition and which does not exceed 10 νg in 1 m1 of a pharmaceutically admissible carrier. There is proposed a method of ophthalmopathology treatment consisting in the preventive and/or therapeutic administration to a patient of an effective amount of the claimed pharmaceutical preparation for a period required to attain a therapeutic effect with regard to the character of the pathology development. The claimed pharmaceutical preparation is active when administered in the does range of 0.1-100 νg/kg of the body weight.

Description

DIPEPTIDE FOR INHIBITING ANGIOGENESIS IN OPHTHALMOPATHOLOGY

Field of the Invention

The invention refers to medicine and concerns the ophthalmologic application of synthetic dipeptide L-lysyl-L-glutamic acid (L-Lys-L-Glu) as a preparation inhibiting neovascularisation in various ocular diseases.

Processes of angiogenesis are known to be the keystone of tissue regeneration and tumour growth. The principal role in the mechanisms of angiogenesis belongs to growth factors stimulating the proliferation of fibroblasts, endotlielial and other cells and promoting the contacts of cells with each other and the matrix.

Background of the Invention

Among the closest analogues in application there are known pharmaceuticals and cytokines, whose inhibitory effect on angiogenesis is studied in oncology (1). Their action is chiefly directed at suppressing the synthesis of the vascular endothelial growth factor (hereinafter in this patent claim referred to as NEGF'). However, in ophthalmology there are known certain preparations applied as angiogenesis inhibitors. There are known the data confirming a positive effect of Danaparoid Sodium in diabetic retinopathy (hereinafter in this patent claim referred to as 'DR') (2, 3). The preparation was administered to patients in the amount of 750 units once a day for 6 weeks. Its therapeutic effect was assessed by the picture of the eye fundus before and after the treatment. Thereby, nearly 80 % of the cases revealed improvement in the eye fundus picture, while the rest 20 % did not have any dynamics (2). The positive clinical effect was attained irrespective of the presence or absence of retinal laser coagulation surgery in the patient's case history. According to these authors, the preparation facilitated NEGF suppression and, consequently, inhibited neovascularisation (2, 3, 4). Yet, Danaparoid Sodium is recommended only as an adjuvant agent and strictly in oedematous-haemorrhagic forms of DR (2, 5).

There is known pharmaceutical Lysinopryl slowing neovascularisation down via suppressed VEGF production (7, 8, 9). NEGF is accumulated in particularly ample amounts in the vitreous body of patients at proliferative stage of DR due to the renin-angiotensin system activation (6, 10, 11). Still - similarly to Danaparoid Sodium - Lysinopiyl is to be applied only at certain stages of DR development.

Besides, the drawbacks of these two preparations consist in a long treatment course and restricted sphere of their application in ophthalmology. There is known L-Lys-L-Glu dipeptide used as a component for peptide synthesis (12).

L-Lys-L-Glu dipeptide is known to possess immunomodulating activity (13) and stimulate repair processes (14).

However, the known properties of L-Lys-L-Glu dipeptide do not expose any evident interrelation with a newly found property of this dipeptide to inhibit angiogenesis in ophthalmopathology, which is objectively confirmed by the below-adduced examples.

Disclosure of the Invention

The proposed patent claim is intended to solve the problem of obtaining a peptide-based preparation capable of inhibiting angiogenesis in ophthalmopathology.

To solve this problem, the dipeptide of the amino acid sequence L-lysyl-L-glutamic acid was employed as a substance capable of inhibiting angiogenesis in ophthalmopathology with the purpose to stabilise a pathologic process and to improve visual functions.

The dipeptide is obtained by a classical method of peptide synthesis in a solution (15). The previously unknown property of L-Lys-L-Glu dipeptide to inhibit angiogenesis in ophthalmopathology was discovered in experimental and clinical studies thereof.

As this patent claim describes, dipeptide L-lysyl-L-glutamic acid of the general formula L-Lys-L-Glu reveals a new biologic activity, which consists in its ability to inhibit angiogenesis in ophthalmopathology. As this patent claim describes, a pharmaceutical preparation capable of inhibiting angiogenesis in ophthalmopathology contains as its active base an effective amount of dipeptide L-lysyl-L-glutamic acid (L-Lys-L-Glu) or one of its salts and a pharmaceutically admissible carrier.

As this patent claim describes, the pharmaceutical preparation inhibiting angiogenesis in ophthalmopathology may contain salts of the amino group (acetate, hydrochloride, oxalate) or of carboxyl groups (salts of metals - sodium, potassium, calcium, lithium, zinc, magnesium, as well as of other organic and inorganic cations - ammonium and triethylammonium). As this patent claim describes, the pharmaceutical preparation is proposed for parenteral administration in ophthalmopathology.

To obtain pharmaceutical preparation meeting the invention, the claimed dipeptide or its pharmaceutically applicable derivatives in the form of salts are blended as an active base and a pharmaceutical carrier in accordance with the methods of compounding accepted in pharmaceutics.

The carrier may have various forms depending on the drug form of the preparation desirable for introduction into a body, for example parenteral administration.

The carrier for parenteral administration usually includes sterile water, though there could be employed other ingredients instrumental for stability or maintaining sterility.

The notion "pharmaceutical preparation" (hereinafter in this patent claim referred to as 'the preparation') under this patent claim implies the use of any drug form appropriate for the parenteral administration to a body, containing the dipeptide or one of its salts and revealing a preventive and/or therapeutic effect in the treatment for the ocular diseases requiring angiogenesis suppression.

The notion "effective amount" under this patent claim implies the use of such an quantity of the active base, which, in compliance with the quantitative indices of its activity and toxicity, as well as with respect to the knowledge available, shall be effective in this drug form and which does not exceed 10 μg in 1 ml of pharmaceutically admissible carrier. As this patent claim describes, the method of treating ophthalmopathology by angiogenesis suppression embraces preventive and/or therapeutic administration to a patient of the proposed preparation in the doses of 0.01-100 μg/kg of the body weight at least once a day during a period required for attaining a therapeutic effect, i.e. 10-40 days depending on the character and severity of the treated pathological process.

Industrial Application

The invention is illustrated by an example of synthesis of dipeptide L-lysyl-L- glutamic acid (L-Lys-L-Glu) (Example 1), examples of testing the dipeptide for toxicity and biological activity (Examples 2, 3) and examples of the clinical application of the preparation demonstrating its pharmacological properties and confirming the possibility of attaining a preventive and/or therapeutic effect (Examples 4, 5). Example 1. Synthesis of L-Lys-L-Glu dipeptide and its salts

1. N_fl,N_ε-dibenzyloxycarbonyl-lyzyI-γ-benzyl-glutamic acid [I].

0.154 g (0.65 mmole) of L-glutamic acid-γ-benzyl ester is suspended in 3 ml of dimethylformamide. The suspension is stirred with the consecutive introduction of 0.091 ml

(0.65 mmole) of triethylamine and 0.300 g (0.59 mmole) of N-oxysuccinimide ester of N_α,N_ε- dibenzyloxycarbonyl-lyzine. The reacting mixture is blended for 12 hours at room temperature. Afterwards, the solvent is removed by evaporation in vacuo at 40°C and 10 ml of

IN H₂S0₄ are added to the residue. The product is twice extracted by ethyl acetate (30 x 2). The organic layer is twice washed with IN H₂S0₄ and water until neutralisation and then dried over Na₂S0₄. The solution is filtered and evaporated in vacuo at 40°C. The residue is dissolved in 1-2 ml of ethyl acetate, precipitated with hexane and recrystallised in ethyl acetate/hexane system. The product is filtered out and dried in vacuo over P₂0₅. The yield is

0.330 g (88 %). Coefficient of retention (R_f) makes 0.81 (benzene-acetone, 1:1, Silufol).

2. L-lysyl-L-glutamic acid.

0.330 g of the defended dipeptide [I] is dissolved in 10 mg of methanol, added 3 ml of water and hydrated by palladium-on-charcoal catalyst. The reaction is monitored by thin layer chromatograms (TLC). At the completion of hydrogenolysis the catalyst is filtered out, the residue is dissolved in the minimal amount of water and precipitated with methanol. The product is filtered out, washed with ethanol and dried in vacuo over P₂0₅. The yield is 0.110 g (85 %). Melting point (T_m]) - 194-196°C. [α]_D ²⁰= +20.0° (c=3.0; H₂0). Rp0.54 (acetonitrile- water, 1:3; Merk). Electrophoresis: E_Gι_y=1.96; E_his=0.98, (1400 V, 45 min, 2 % acetic acid, Watmann 3MM).

In order to obtain corresponding salts of carboxyl groups, the free dipeptide is added a calculated amount of the aqueous solution of a corresponding metal hydroxide (NaOH, KOH, Zn(OH)₂, LiOH, Ca(OH)₂, Mg(OH)₂, NH₄OH).

To obtain the salt of triethylammonium, the processing is carried out similarly, triethylamine being used as the base.

The preparation in the form of solution is obtained the following way: the dipeptide or one of its salts obtained by the above-described method is dissolved in 0.9 % isotonic natural saline solution. The concentration of the dipeptide equals 10 μg per 1 ml of natural saline solution. Example 2. Study of L-Lys-L-Glu dipeptide for toxicity

L-Lys-L-Glu dipeptide was studied for general toxicity in compliance with "The Rules of Pre-Clinical Assessment of Safety of Pharmacological Substances (GLP)". This study was designed to define the tolerable toxic doses of L-Lys-L-Glu dipeptide, assess the degree and character of pathologic alterations in various organs and systems of the organism and reveal the dependence of toxic effects on the dosage and duration of the dipeptide administration.

Acute toxicity of L-Lys-L-Glu dipeptide was defined in a murine model according to Kerber. Examined were 66 white mongrel male mice weighing 20-23 g. The mice were kept in vivarium under standard regimen and fed upon standard rations. They were randomised to six equal groups, 11 mice in each. The animals were intramuscularly injected with the dipeptide once in the doses of 1 mg/kg, 2 mg/kg, 3 mg/ kg, 4 mg/kg and 5 mg/kg 0.25 ml of natural saline solution (several thousand times exceeding the therapeutic dose recommended for clinical trials). The control animals received the same amount of natural saline solution.

During 72 hours and later in 14 days none of the animals died in either of the groups. No alterations in their general state, behaviour, motor activity, hair and skin integument, physiological discharge were registered.

Consequently, L-Lys-L-Glu dipeptide administered in doses, which several thousand times exceed the therapeutic one recommended for clinical trials, does not entail any acute toxic reactions, thus revealing a wide therapeutic applicability of the preparation.

Subacute toxicity of L-Lys-L-Glu dipeptide was investigated on 60 white mongrel rats weighing 150-250 g. The animals were injected with the dipeptide intramuscularly in the doses of 1 μg/kg, 0.3 mg/kg, 3 mg/kg in 0.5 ml of natural saline solution, once a day for 90 days. The control animals were administered with natural saline solution in the same amount.

The animals were observed daily for the whole period of investigation. Their behaviour, food and water consumption, state of hair integument and mucous membranes were noted.

The rats were weighed weekly. Before the dipeptide administration and on the 30^th, 60^th and

90^th days thereof, the morphological composition and properties of the animals' peripheral blood were examined. Biochemical and coagulological properties of the blood were studied at the experiment completion.

The dipeptide obtained by the claimed method was investigated for chronic toxicity in a long-term experiment on 64 white mongrel rats weighing 150-250 g. The animals were intramuscularly injected with the dipeptide in the doses of 1 μg/kg, 0.1 mg/kg, 1 mg/kg in 0.5 ml of natural saline solution, once a day for 6 months. Their behaviour, food and water consumption, state of hair integument and mucous membranes were examined. The rats were weighed daily during the first three months of the experiment and then once a month.

In three months after the administration onset and at the experiment completion, haematological and biochemical investigations were conducted. The functions of the animals' cardiovascular system, liver, pancreas, kidneys and adrenal glands were assessed. At the end of administering the tetrapeptide, part of the animals were subjected to a post mortem examination to assess the state of various segments of their brain and spinal marrow, heart, aorta, lungs, liver, kidneys, endocrine and immune organs. Assessment of the animals' general state, morphological and biochemical indices of their peripheral blood, morphological state of their intrinsic organs, cardiovascular and respiratory systems, liver and kidney functions revealed no pathologic alterations.

Study of L-Lys-L-Glu dipeptide for subacute and chronic toxicity demonstrates the lack of any side effects in case of long-term application of the tetrapeptide in doses, which exceed the therapeutic one 100- 1000 times.

Example 3. Effect of L-Lys-L-Glu dipeptide on the mechanisms of angiogenesis in toxic retinal degeneration in rats

The action of L-Lys-L-Glu dipeptide in case of experimental retinal degeneration was studied in the eyes of 17 puberal Chinchilla rabbits weighing 2-3 kg. Experimental toxic degeneration of the retina was induced by an intravenous administration of 3 % potassium iodide solution in the dose of 30 mg/kg. The experimental animals (9 rabbits) were intramuscularly injected with the dipeptide in the dose of 1 μg/kg once a day for 7 days starting since the 10^th day after the introduction of the toxic agent. The animals of the control group were injected with sterile natural saline solution by the same scheme.

All the animals were subjected to daily ophthalmoscopic examination. Besides, their eye tissues were histologically examined at different time intervals after administering potassium iodide.

In an hour after the toxic agent administration, we observed oedemas of the animals' optic disc and retina. The oedemas were more expressed along the retinal periphery.

The histological examination revealed severe oedema of the retina, loosening of its layers, significant accumulation of fluid in the region of the 1^st and 3^rd neurones and drenching of the iris blood vessels on the 2^nd day after the introduction of potassium iodide. On the 7^th day ophthalmoscopy detected the appearance of amorphous greyish-black foci along the eye fundus periphery.

Henceforth, the retinal oedema remained and the pigment cells located closer to the eye corner were found to accumulate along the retinal periphery. In the ciliary body inter-adhesion of the processes and formation of cavernous lacunae were observed.

Five days later, the histological examination still revealed a remaining moderate oedema of the retina and amorphous foci at the retinal periphery in the control animals. Ophthalmoscopy of the dipeptide-injected rabbits demonstrated a decrease in the retinal oedema and diminished size of the amorphous foci. Results of the histological investigation conducted in 5 days after the end of the dipeptide administration confirmed these observations. By that time, the experimental animals did not have any retinal oedema and the remaining foci were less in size that those in control rabbits.

Thus, the investigation results show that the application of L-Lys-L-Glu dipeptide in case of progressing toxically induced retinal degeneration reduces the degree of manifested destructive changes, prevents the subsequent development of the pathologic process and facilitates normalisation of the retinal structures. L-Lys-L-Glu dipeptide exerts an anti- inflammatory effect due to normalisation of the regional immunity indices, microcirculation and rheological characteristics of the blood.

Example 4. Efficacy of applying the pharmaceutical preparation in the lesions of the anterior eye section

Constituting over one third of all the ophthalmopathologies, diseases of the cornea are of higher incidence than pathologies of any other ocular layers. Frequent post-inflammatory corneal complications consist in opacifications of different intensity pierced through with newly formed vessels. Neovascularisation spreads from the conjunctiva vessels or from deeper episcleral and scleral vasculature. There are distinguished magisterial, dispersed and branch types of newly formed corneal vessels. The type of a new vessel depends upon its location and square of the affected corneal focus. A centrally located pathologic process can reduce visual functions considerably - up to mere photoperception. It is known that newly formed vessels - even after complete disappearance of the corneal infiltration foci - can remain in the cornea for a long time thus decreasing the acuity of vision.

The claimed preparation was applied in the patients with damaged cornea accompanied by neovascularisation. The main group consisted of 39 patients (71 eyes) with corneal lesion of various aetiology and concomitant neovascularisation. The preparation was parabulbarly injected to the patients in the dose of 10.0 μg, once a day for 10 days.

The control group embraced 16 patients (30 eyes) with a similar clinical picture, treated by conventional therapeutic methods. The clinical picture in all the patients was marked by photophobia, excessive lacrimation, blepharospasm, pericorneal injection of the eye pupil, corneal infiltration with disturbed transparency, spherical form of the cornea and - in particular - formation of blood- drenched vasculature. In some patients keratitis was accompanied by the signs of uveitis.

The patients of both groups were subjected to fluorescein keratography for diagnostic purposes. Temporal parameters ("hand-conjunctiva" time, time of complete limbus contrast), state of the limbic and corneal tissue angio-architectonics and permeation type of limbic and corneal vessels were chosen as the criteria of microcirculation assessment.

All the patients exposed disturbed angio-architectonics of the limbic vasculature due to changes in their radial location and growing of the new vessels both into the superficial and deep layers of the cornea. Ampoule-formed dilatations with increased fluorescein emission at the late experimental phase were observed at the terminal branches of the newly formed vessels. The "hand-conjunctiva" time decreased twofold, while the time of complete limbus contrast - 1.5-fold as compared to the norm.

All the preparation-treated patients revealed a much faster reduction or disappearance of photophobia, lacrimation and inflammatory signs as compared to the control patients. The infiltration sites decreased in size significantly, while the corneal transparency in the opacity segments enhanced.

Moreover, no blood flow in the new vessels was registered in any of the examined cases - only solitary semitransparent and utterly bloodless empty vessels were found. The improved clinical picture resulted in a considerable visual acuity rise as compared to the relevant control parameter, which is exhibited in Table 1. Table 1

Parameters of the visual acuity in control and dipeptide-treated patients with the lesions of the anterior eye section

*p<0.05, as compared to the index before treatment; #p<0.05, as compared to the control index.

The data of fluorescein keratography confirmed the significant improvement in the clinical picture of the main group. After cupping off the inflammation signs the temporal parameters of microcirculation extended. By the end of the treatment course, they attained the normal values in 81.6 % of the cases. Permeation of the dye from the newly formed vessels lowered in all the cases. In 70.4 % of the cases, the central vascular segments became empty, fluorescein permeation from them and microcirculation in them stopped. The temporal parameters of microcirculation in the control group normalised only in

36.6 % of the cases. Emptiness of the newly formed corneal vasculature was registered in none of the control eyes.

Patient K. Female, born in 1978, underwent an in-patient treatment from August 24 to

November 22, 2000.

Diagnosis: tuberculous allergic keratitis of the right eye; tuberculosis of the internal thoracic lymph nodes (minor form).

According to her case history, the patient got ill in May 2000. Non-specific therapy with corticosteroids, wide-spectrum antibiotics and other preparations brought no effect.

Ophthalmologic state at admission. Visual acuity in the right eye made 1.0. Two rounded infiltration foci (2.0 and 2.7 mm in diameter), towards which several newly formed vessels shot from the limbus, were detected in the superficial layer and stroma. Ophthalmoscopy exposed blood flow in the newly formed vasculature, signs of epithelial oedema and descemiitis around the opacifications. Other structures were preserved unchanged.

The "hand-conjunctiva" time at fluorescein keratography constituted 9.1±0.4 sec, while the time of complete limbus contrast was below the normal limit as well and equalled 10.8+1.1 sec.

The patient underwent a course of parabulbar injections with the preparation into both eyes in the dose of 10.0 μg, once a day for 10 days.

During this therapy course, we registered resorption of the infiltrates accompanied by emptying of the newly formed vessels. We observed only semitransparent, linear and hardly detectable bloodless opacifications in the superficial layers of the cornea in place of these vessels. By the end of the treatment course, one of the opacity foci resorped completely and the second became transparent in its centre.

The results of fluorescein keratotomy showed that the pathology was blocked: the time of "hand-conjunctiva" reaction and complete limbus contrast approached the norm (10.9±1.1 and 14.8+0.9, respectively). No permeation of fluorescein from the new vessels was registered.

Patient Z. Female, born in 1946, underwent an in-patient treatment from September 18 to November 04, 2000.

Diagnosis: tuberculous allergic keratitis of both eyes.

According to the patient's case history, her disease had a sinuous remittent character, was hardly amenable to any treatment for the span of 3 years. The last acute phase of keratitis occurred in March 2000. Specific therapy with tuberculostatics, desensitisers and physiotherapy resulted only in a short-term effect.

Ophthalmologic state at admission. The patient complained of expressed photophobia, excessive lacrimation and blepharospasm. The visual acuity in the right eye made 0.2 with the sphere of +1.5 = 0.6, incorrigible. The left eye visual acuity - 0.01, incorrigible. Ophthalmoscopy of the right eye exposed two small grey infiltrates in the central layers of the corneal paraoptic zone. A network of deep and superficial corneal neovasculature spreading from the limbus to the infiltrates was observed in the "three o'clock" position. Ophthalmoscopy of the left eye revealed three small rounded grey infiltrates in the central layers of the corneal optic zone. A bundle of newly formed corneal vessels protruding from the limbus towards the infiltrates was detected in the "three o'clock" position. The patient underwent a course of parabulbar injections with the preparation into both eyes in the dose of 10.0 μg, once a day for 10 days.

Photophobia and excessive lacrimation disappeared after a course of the therapy with the preparation. The visual acuity grew considerably, especially in the left eye. Visual acuity in the right eye equalled 0.5, with the sphere of +1.5 = 0.7, incorrigible; in the left eye - 0.5, incorrigible either.

Ophthalmoscopy of both eyes detected a significant increase in the transparency of the cornea in the opacification zone. One infiltrate in the right eye resorped completely and the other became significantly diminished in its square and much more condensed. Infiltrates in the left eye almost resorped.

All the newly formed vessels emptied. Only separate semitransparent and utterly empty bloodless vessels remained.

Consequently, the claimed preparation is an effective inhibitor of angiogenesis in the lesions of the anterior eye section.

Example 5. Efficacy of applying the pharmaceutical preparation in the lesions of the posterior eye section

Proliferative chorioretinites develop more frequently as complications after other forms of this pathology (haemorrhagic or exudative chorioretinites). They are usually accompanied by severe circulation disorders and ischemisation of the tissue spawning trophicity exacerbation. An intensive venous congestion entails a stasis in the capillary segment of the vessel bed, thus provoking VEGF production. This process expedites the growth of new vessels. Deficient wall of a newly formed vessel results in so-called preretinal haemorrhagic and exudative alterations, as well as in vitreous haemorrhages. Furthermore, the newly formed vasculature can spread along the posterior hyaliod membrane of the vitreous body and subsequently, having overcome this hurdle, grow into the vitreous body using vitreous fibres as the substructure. Appearance of the fibrovascular tissue penetrating into the posterior hyaloid membrane and contraction of this tissue provokes the formation of vitroretinal traction, which can result in retinal detachments. Hence, the search for neoangiogenesis inhibiting preparations belongs to the most important targets in ophthalmology. The preparation was applied in 34 patients (67 eyes) with focal pathology of various geneses (chorioretinites, central serous chorioretinopathy, macular degeneration, etc.) accompanied by neovascularisation.

The preparation was parabulbarly injected to the patients in the dose of 10.0 μg once a day for 10 days.

The control group included 12 patients (24 eyes) with similar disorders. The patients of this group underwent a relevant conventional treatment by a worked-up scheme.

Subjective symptoms of focal ocular lesions in the patients consisted in visual acuity decrease (especially significant when the lesion focus occupied the central region of the eye fundus) in 89.5 %, scotomas in the field of vision in 43.2 %, appearance of photopsias, metamorphopsias, macro- or micropsias in 44.7 % of the cases.

The clinical picture was characterised by cellular infiltration of the vitreous body. Thereby, a diffuse cellular exudative opacity of the vitreous body, which impeded ophthalmoscopy, was observed at biomicroscopy in case of medium and highly intensive inflammation.

Ophthalmoscopy of the eye fundus exposed solitary or multiple foci in the central or peripheral retinal segments in all the patients. Inflammatory foci in the choroid were registered as bounded yellowish-grey rounded formations with indistinct borders. Grey inflammatory foci looked as white grains of fibrous tissue with pigment and clear borders. Fluorescein angiography of the eye fundus (hereinafter in this patent claim referred to as 'FAEF') revealed signs of neovascularisation in all the cases.

Angiography conducted at earlier phases registered a radial distribution of contrasted capillaries. Vascular branches could be isolated, but - in the majority of cases - they formed large vascular fields with adducting vessels approaching to them. The neovascularisation zone had advancing brims, which consisted of compactly located capillaries. The margin of the choroid neovascularisation zone had expressed hyperfluorescence resultant from profuse permeation of the dye from the deformed vessels.

During later phases, the entire zone of choroid neovascularisation was diffusely contrasted with fluorescein at angiography. All the preparation-treated patients subjectively noted an increase in their visual functions - images became clearer and brighter. Photopsias, metamorphopsias, macro- or micropsias disappeared (in 44.3 % of the cases) or became less manifested (43.6 % of the cases). In 87.3 % of the cases scotomas in the field of vision diminished in size and quantity. Table 2 displays the visual acuity dynamics. Table 2 Parameters of the visual acuity in control and dipeptide-treated patients with the lesions of the posterior eye section

*p<0.05, as compared to the index before treatment;

#ρ<0.05, as compared to the control index.

Bioophthalmoscopy revealed the signs of decreased chorioretinitis activity in all the patients. Cellular infiltration of the vitreous body reduced considerably, thus heightening its transparency. Fresh chorioretinal foci in the eye fundus acquired distinct contours, perifocal oedema disappeared in 52.4 % and diminished essentially in 43.2 % of the cases.

The preparation promoted an improvement in the FAEF results in all the patients due to the decreased activity of chorioretinitis. The square of choroid neovascularisation reduced and hyperfluorescence diminished substantially. Moreover, the square of pigmented epithelium detachment decreased considerably, which indirectly indicated a lower probability of VEGF emission, as well as of growing of the newly formed vessels from the choroid into the retina.

Patient P. Female, born in 1953.

Diagnosis: peripheral tuberculous chorioretinitis of the right eye (put in 1998). According to the patient's case history, she underwent a primary course of anti- tubercular therapy and laser coagulation of the right eye retina in 1998 and has had several conservative treatment courses with tuberculostatics, corticosteroids, hepato- and angioprotectors and vitamins over the last two years. Yet, the specific process kept progressing, although very slowly, and the visual functions decreased greatly. Ophthalmologic state at admission. Visual acuity in the right eye made 0.4, incorrigible, in the left eye - 1.0. Biornicroscopy of the right eye detected fibrosis of the vitreous body and cellular infiltration, which impeded the eye fundus ophthalmoscopy. No pathologic alterations were observed in the left eye. Examination of the left eye fundus exposed no peculiarities in the optic disc. Near the old chorioretinal focus, in the upper inner quadrant at the mid-periphery, a fresh grey focus (two thirds of the optic disc diameter) was found. It had vague borders and an expressed perifocal inflammation and protruded into the vitreous body. At the inner side of the focus, neovascularisation and multiple haemorrhages were detected.

According to the FAEF data, the ophthalmoscopically visible foci before the injections were marked by auto-fluorescence. The old focus was stained only during the circulatory phase and not furthermore. During the choroid phase, the hyperfluorescence area in place of the fresh focus continued for 2 minutes. Simultaneously, spots of light appeared to converge into an amply hyperfluorescent "network" during the arterial phase. Retinal haemorrhages were defined as "spots" of absolute hyperfluorescence. The fresh focus accumulated the dye gradually attaining the maximum by the 10^th minute. The intensity of the focus luminescence persisted for 30 minutes and then weakened. The after-luminescence lasted until the 60^th minute. The patient underwent a course of parabulbar injection with the preparation in the dose of 10.0 μg once a day for 10 days.

After the eighth injection, the patient noted a subjective improvement manifested in clearer and brighter images. By the end of the treatment course her visual acuity in the right eye increased by two decimals and reached 0.6. Biomicroscopy revealed only occasional cellular elements preserved on the vitreous body fibrils. The fresh chorioretinal focus in the eye fundus gained sharp contours. The perifocal oedema disappeared, the haemorrhages resorped and the newly formed vessels emptied completely. The FAEF data verified the improved picture of the eye fundus. The old focus was still stained only during the circulatory phase. The intensity of the fresh focus luminescence persisted just until the 15^th minute to become weaker afterward. There was practically no dye permeation from the newly formed vessels and the signs of retinal haemorrhages at fluorescein angiography vanished.

Consequently, the proposed preparation is an effective inhibitor of angiogenesis in the lesion of the posterior eye segment.

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Claims

1. Application of dipeptide L-lysyl-L-glutamic acid of the general formula L-Lys-L-Glu or one of its salts as a substance capable of inhibiting angiogenesis in ophthalmopathology.

2. Application according to Claim 1 characterised by the fact that the substance is proposed in the form of a pharmaceutical preparation for parenteral administration.

3. Pharmaceutical preparation containing dipeptide L-lysyl-L-glutamic acid of the general formula L-Lys-L-Glu or one of its salts according to Claim 1 in the amount, which is effective for the prevention of and/or treatment for ophthalmopathology requiring angiogenesis inhibition and which does not exceed 10 μg in 1 ml of a pharmaceutically admissible carrier.

4. Preparation according to Claim 3 characterised by the fact that it contains the dipeptide in the form of a salt of the amino group selected from acetate, hydrochloride, oxalate.

5. Preparation according to Claim 3 characterised by the fact that it contains the dipeptide in the form of a salt of carboxyl groups selected from the salts of sodium, potassium, calcium, lithium, zinc, magnesium or of organic and inorganic cations, for example, ammonium, triethylammonium.

6. Method of the treatment for ophthalmopathology including the preventive and/or therapeutic administration to a patient of an effective amount of the pharmaceutical preparation according to Claims 3-5 for a period required to attain a therapeutic effect with regard to the character of the pathology development.

7. Method according to Claim 6 characterised by the fact that the pharmaceutical preparation according to Claims 3-5 is active when administered in the dose range of 0.1-100 μg/kg of the body weight.