DESCRIPTION

FIELD OF THE INVENTION

[0001] Described are dual inhibitors of endothelin converting enzyme- 1 (ECE-l), and neutral endopeptidase (NEP) and/or human soluble endopeptidase (hSEP) alone, and compositions thereof, with the proviso that said pharmaceutical compositions may optionally contain either one or more of carbonic anhydrase inhibitors such as e.g. brinzolamide, a ₂ -adrenergic agonists such as e.g. brimonidine, b-blockers such as e.g. timolol, prostaglandin analogs such as e.g. bimatoprost, rho kinase inhibitors such as e.g. netarsudil, adenosine A _\ receptor agonists such as e.g. trabodenoson, ET _A endothelin receptor antagonists such as e.g. sitaxentan, dual endothelin receptor antagonists such as e.g. bosentan, nitric oxide donors such as e.g. butanediol, parasympathomimetics such as e.g. pilocarpine, acetylcholine, catecholamines such as e.g. adrenaline, muscarinic receptor antagonists such as e.g. atropine, vascular endothelial growth factor inhibitors such as e.g. ranibizumab, corticosteroids such as e.g. dexamethasone, antibiotics such as e.g. vancomycin, tissue regenerating agents such as e.g. poly-carboxymethylglucose, vitamins and provitamins such as e.g. panthenol and retinyl palmitate, chemotherapeutic agents such as e.g. mitomycin, nonsteroidal anti-inflammatory drugs such as e.g. ketorolac, Hi receptor antagonists such as e.g. cetirizine, monoclonal antibodies such as e.g. adalimumab, proteases such as e.g. ocriplasmin, immunosuppressive agents such as e.g. cyclosporine, or none, for prophylaxis and/or treatment of eye diseases.

SCIENTIFIC BACKGROUND OF THE INVENTION

[0002] An increased intraocular pressure and reduced ocular blood flow are the major risk factors for glaucoma in humans. Glaucoma is the most common optic nerve head neuropathy and is associated morphologically with loss of retinal ganglion cells and clinically with visual field deterioration. Treatment of glaucoma is based on lowering of intraocular pressure and preventing the occurrence or progression of optic neuropathy. No therapy is currently available to mitigate ocular blood flow changes during glaucoma, or to limit the death of retinal ganglion cells by means of apoptosis in the course of glaucoma (B.C. Chauhan (2008) Can. J. Ophthalmol. 43, 356-360). [0003] Endothelin is the human body’s most potent vasoactive peptide known to date. As connoted by the term vasoactive peptide, this peptide participates in the regulation of intraocular blood pressure by means of vasoconstrictor activity. Upon release it causes a decrease in ocular blood flow followed by pathology in the retina and the optic nerve head.

[0004] Endothelin, apart from its vasoconstrictor activity, participates in the regulation of intraocular pressure by means of affecting trabecular outflow, which is the main route for fluid outflow from the eye. Endothelin increases contractility of the trabecular meshwork and therefore decreases fluid outflow from the eye elevating intraocular pressure followed by pathology in the retina and the optic nerve head.

[0005] Endothelin, apart from affecting intraocular blood pressure and regulating intraocular fluid pressure, influences apoptosis of retinal ganglion cells acting via ET _B receptors and induces proliferation of human optic nerve head astrocytes acting via both ET _A and ET _B receptors (G. Prasanna, R. Krishnamoorthy, A.F. Clark, R.J. Wordinger & T. Yorio (2002) Invest.

Ophthalmol. Vis. Sci. 43, 2704-2713).

[0006] Endothelin is a peptide composed of 21 amino acids that is synthesized and released by the endothelium. Endothelin is produced by cleavage of a Trp-Val bond in the precursor peptide big endothelin (Big ET-l). Endothelin converting enzyme-l (ECE-l), a membrane-bound metalloprotease, catalyses proteolytic activation of Big endothelin-l to ET-l and constitutes a regulatory site controlling production of the active peptide.

[0007] Neutral endopeptidase (NEP), a zink metallopeptidase, degrades atrial natriuretic peptide (ANP) and constitutes a regulatory site controlling concentration of the active peptide. In serum- deprived pheochromocytoma cells, ANP inhibits apoptosis by causing cGMP elevation.

Spreading depression in the cortex is followed by long lasting elevation of the ANP expression and intracellular cGMP concentration. Natriuretic peptide receptor NPrA regulates intracellular cGMP concentration by stimulating particulate guanylyl cyclase (pGC), which activates cGMP- dependent protein kinase G pathway.

[0008] Since up-regulation of ET-l receptors and down-regulation of ANP receptors has been reported to occur in cells subjected to metabolic or oxidative insults, it prompted us to explore the effects of dual ECE/NEP inhibition on rodent cells undergoing apoptosis similar to those reported in humans during ophthalmic diseases such as during e.g. glaucoma.

INDUSTRIAL BACKGROUND OF THE INVENTION

[0009] The invention relates to a novel use of benzazepine, benzoxazepine, benzothiazepine-N- acetic acid and phosphono-substituted benzazepinone derivatives having neutral endopeptidase (NEP) and/or human soluble endopeptidase (hSEP) inhibitory activity. The compounds of the invention are useful for the preparation of pharmaceutical compositions for prophylaxis and treatment of ophthalmic diseases.

[0010] The invention relates to the use of compounds disclosed in the patent EP1706121 Bl for the manufacture of medicaments giving a beneficial effect. A beneficial effect is disclosed herein or apparent to a person skilled in the art from the specification and general knowledge in the art. The invention also relates to the use of compounds of the invention for the manufacture of medicaments for treating or preventing a disease or condition. More particularly, the invention relates to a new use for the treatment of a disease or condition disclosed herein or apparent to a person skilled in the art from the specification and general knowledge in the art. In embodiments of the invention specific compounds disclosed herein are used for the manufacture of a medication.

[0011] EP 1706121 Bl refers to the use of certain compounds with a combined inhibitory activity on both neutral endopeptidase, and/or human soluble endopeptidase (hSEP), and endothelin converting enzyme (ECE) for the treatment and/or prophylaxis of neurodegenerative diseases such as traumatic brain injury, acute disseminated encephalomyelitis, epilepsy related brain damage, spinal cord injury, bacterial or viral meningitis and meningoencephalitis, prion diseases, poisonings with neurotoxic compounds, and radiation-induced brain damage, and for prophylaxis of ischemic stroke, with the proviso that said pharmaceutical compositions do not contain an aldosterone receptor antagonist.

[0012] WO 2004/082637, filed on March 18, 2004, and published on September 30, 2004, discloses a method for the prophylaxis or treatment of a very large number of pathological conditions, comprising administering an aldosterone receptor antagonist and an ECE inhibitor. Among the pathological conditions listed are glaucoma, hypertensive or diabetic retinopathy, and elevated intraocular pressure. However, no disclosure regarding the benefit of preferred compounds in these indications was made.

105 [0013] The goal of the present invention was to identify specific metalloprotease inhibitors

which are of therapeutic value when administered in a pharmaceutical composition not containing aldosterone receptor antagonist.

[0014] Surprisingly, it now has been found that benzazepine, benzoxazepine, benzothiazepine-N-

110 acetic acid and phosphono-substituted benzazepinone derivatives having neutral endopeptidase (NEP) and/or human soluble endopeptidase (hSEP) inhibitory activity prevent apoptosis in rodent models similar to that observed in the course of eye diseases in humans. This property makes them useful for treatment and/or prophylaxis and/or preparation of pharmaceutical compositions for treatment and/or prophylaxis of optic and/or eye diseases selected from the

115 group consisting of such diseases as e.g. (i) all forms of primary and secondary glaucoma,

preferably such as e.g. primary open-angle glaucoma, normal-tension glaucoma, primary angle- closure glaucoma, pseudoexfoliation syndrome and glaucoma, pigment dispersion syndrome and glaucoma, neovascular glaucoma, inflammatory glaucoma, lens-related glaucoma, traumatic glaucoma, primary congenital glaucoma, iatrogenic induced glaucoma, and malignant glaucoma;

120 (ii) aquired macular disorders, preferably such as e.g. age-related macular degeneration,

idiopathic choroidal neovascularisation, central serous chorioretinopathy, vitreomacular interface disorders, idiopathic macular telangiectasia, cystoid macular oedema, and microcystic macular oedema; (iii) optic neuropathy, preferably such as e.g. anterior or posterior ischemic optic neuropathy; (iv) optic neuritis; (v) uveitis, preferably such as e.g. anterior uveitis, intermediate

125 uveitis, posterior uveitis, and panuveitis; (vi) hereditary fundus dystrophies, preferably such as e.g. retinitis pigmentosa, cone dystrophy, cone-rod dystrophy, rod dystrophy, Stargardt's disease, Bietti's crystalline corneoretinal dystrophy, familial benign fleck retina, Best vitelliform macular dystrophy, adult-onset vitelliform macular dystrophy, North Carolina macular dystrophy, familial dominant drusen, and concentric annular macular dystrophy; (vii) retinal vascular

ISO diseases, preferably such as e.g. diabetic retinopathy, non-diabetic retinopathy, retinal venous occlusive disease, retinal arterial occlusive disease, ocular ischaemic syndrome, hypertensive eye disease, sickle cell retinopathy, thalassemia retinopathy, retinopathy of prematurity, retinal artery macroaneurysm, primary retinal telangiectasia, Eales disease, and radiation retinopathy; (viii) scleritis and episcleritis; (ix) retinal detachments; (x) trauma to the eye globe; (xi) vitreous

135 opacities, preferably such as e.g. vitreous hemorrhage, and asteroid hyalosis; (xii) myopia and degenerative myopia; (xiii) postsurgical trauma, preferably such as e.g. mechanical trauma due to conventional surgery, thermotrauma due to laser surgery, and trauma induced by cryosurgery; (xiv) dry eye disease; (xv) corneal disorders, preferably such as abrasions, lacerations, ulcerations, dystrophies, opacities, endothelial and epithelial decompensation, post-surgical oedema, corneal degenerations, corneal vascularisation; and corneal ectasias, preferably such as keratoconus.

[0015] The compounds of the invention are known from the European patents EP 0 733 642, EP 0 916 679 and EP 1 468 010, containing detailed syntheses, and can be described by the general formula (1):

wherein:

Rl stands for a group with formula (2) or (3):

A represents CE12, O or S,

R2 and R3 independently represent hydrogen or halogen,

R4 and R6 independently represent hydrogen or a biolabile carboxylic ester forming group;

R5 is selected from the group consisting of (Cl-C6)-alkoxy-(Cl-C6)-alkyl which may be substituted by a (Cl-C6)-alkoxy, phenyl-(Cl-C6)-alkyl and phenyloxy-(Cl-C6)-alkyl wherein the phenyl group may be substituted with (Cl-C6)-alkyl, (Cl-C6)-alkoxy or halogen, and naphtyl-(C 1 -C6)-alkyl,

R7 and R8 independently represent hydrogen or a group forming a biolabile phosphonic acid ester. [0016] To the invention belong all compounds having formula (1), racemates, mixtures of diastereomers and the individual stereoisomers, and also include pharmaceutically acceptable salts thereof. Thus compounds in which the substituents on potentially asymmetrical carbon atoms are in either the R-configuration or the S-configuration belong to the invention.

[0017] Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by mixing a compound of the present invention with a suitable metal cation or an organic base, for instance an amine.

[0018] This objective can be achieved by preparing the metal salt of the compounds with the general formula (1) as mentioned above wherein the metal ion is a lithium ion or a bivalent metal ion. Preferred bivalent metal salts are calcium, magnesium and zinc salts. Most preferred is the calcium salt.

[0019] The invention relates to the use of a compound of general formula (1), as defined above, for treatment and/or prophylaxis and/or preparation of pharmaceutical compositions for treatment and/or prophylaxis of optic and/or eye diseases selected from the group consisting of such diseases as e.g. (i) all forms of primary and secondary glaucoma, preferably such as e.g. primary open-angle glaucoma, normal-tension glaucoma, primary angle-closure glaucoma, pseudoexfoliation syndrome and glaucoma, pigment dispersion syndrome and glaucoma, neovascular glaucoma, inflammatory glaucoma, lens-related glaucoma, traumatic glaucoma, primary congenital glaucoma, iatrogenic induced glaucoma, and malignant glaucoma; (ii) aquired macular disorders, preferably such as e.g. age-related macular degeneration, idiopathic choroidal neovascularisation, central serous chorioretinopathy, vitreomacular interface disorders, idiopathic macular telangiectasia, cystoid macular oedema, and microcystic macular oedema;

(iii) optic neuropathy, preferably such as e.g. anterior or posterior ischemic optic neuropathy;

(iv) optic neuritis; (v) uveitis, preferably such as e.g. anterior uveitis, intermediate uveitis, posterior uveitis, and panuveitis; (vi) hereditary fundus dystrophies, preferably such as e.g. retinitis pigmentosa, cone dystrophy, cone-rod dystrophy, rod dystrophy, Stargardt's disease, Bietti's crystalline corneoretinal dystrophy, familial benign fleck retina, Best vitelliform macular dystrophy, adult-onset vitelliform macular dystrophy, North Carolina macular dystrophy, familial dominant drusen, and concentric annular macular dystrophy; (vii) retinal vascular diseases, preferably such as e.g. diabetic retinopathy, non-diabetic retinopathy, retinal venous occlusive disease, retinal arterial occlusive disease, ocular ischaemic syndrome, hypertensive eye disease, sickle cell retinopathy, thalassemia retinopathy, retinopathy of prematurity, retinal artery macroaneurysm, primary retinal telangiectasia, Eales disease, and radiation retinopathy; (viii) scleritis and episcleritis; (ix) retinal detachments; (x) trauma to the eye globe; (xi) vitreous opacities, preferably such as e.g. vitreous hemorrhage, and asteroid hyalosis; (xii) myopia and degenerative myopia; (xiii) postsurgical trauma, preferably such as e.g. mechanical trauma due to conventional surgery, thermotrauma due to laser surgery, and trauma induced by cryosurgery; (xiv) dry eye disease; (xv) corneal disorders, preferably such as abrasions, lacerations, ulcerations, dystrophies, opacities, endothelial and epithelial decompensation, post-surgical oedema, corneal degenerations, corneal vascularisation; and corneal ectasias, preferably such as keratoconus, with the proviso that said pharmaceutical compositions do not contain an aldosterone receptor antagonist.

[0020] Further embodiments of the invention are defined in the dependent claims.

[0021] The invention particularly relates to the use of compounds having general formula (4):

wherein the symbols have the meanings as given above.

[0022] More particular, the invention relates to the use of compounds having general formula (5):

wherein the symbols have the meanings as given above. [0023] The most preferred active substances used according to the present invention are:

• (2R)-2-{ [ 1 -({ [(3 S )- 1 -(carboxymethyl)-2-oxo-2,3,4,5-tetrahydro- \H- 1 -benzazepin-3- yl]amino}carbonyl)cyclopentyl]methyl}-4-phenylbutanoic acid (6):

· (2R)-2- { [ 1 -({ [(3S)- 1 -(carboxymethyl)-2-oxo-2,3,4,5-tetrahydro- \H-\ -benzazepin-3- yl] am ino } carbonyl)cyclopentyl]methyl } -4-( 1 -naphthyl)butanoic acid (7) :

• tert-butyl-((3 S)-3 - { [( 1 - { [(benzyloxy)(ethoxy)phosphoryl]methyl)cyclopentyl)

carbonyl] amino } -2-oxo-2,3 ,4, 5 -tetrahydro- IH-l -benzazepin- 1 -yl)acetate (8) :

PHARMACEUTICAL COMPOSITIONS

[0024] The compounds of the invention can be brought into forms suitable for administration by means of usual processes using auxillary substances such as liquids or carrier materials. The pharmaceutical compositions of the invention may be administered either topically and/or systemically and in particular e.g. topically to the eye, periocularly in such manner as subconjunctival, subtenon, retrobulbar or peribulbar, intraocularly into the eye, but also enterally, orally, parenterally (intramuscularly or intravenously), subcutaneously or rectally. They can be administered in the form of solutions, suspensions, ointments (creams, gels, gel- forming solutions, sprays, ocular inserts/deposits, contact lenses), ocular implants but also tablets, capsules, softgels, powders, suppositories, nano-formulations or via iontophoresis, or by means of pharmaceutical compositions based on nanoparticle carrier systems. Suitable excipients for such formulations are the pharmaceutically customary liquid or solid fillers and extenders, solvents, emulsifiers, lubricants, flavorings, colorings and/or buffer substances. Frequently used auxiliary substances which may be mentioned are magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, lactoprotein, gelatin, starch, cellulose and its derivatives, animal and vegetable oils such as fish liver oil, sunflower, groundnut or sesame oil, polyethylene glycol and solvents such as, for example, sterile water and mono- or polyhydric alcohols such as glycerol.

[0025] Compounds of the present invention are generally administered as pharmaceutical compositions. Types of pharmaceutical compositions that may be used include but are not limited to solutions, suspensions, ointments (creams, gels or sprays), but also tablets, chewable tablets, capsules, softgels, parenteral solutions, suppositories, and other types disclosed herein or apparent to a person skilled in the art from the specification and general knowledge in the art.

The pharmaceutical compositions of the invention do not contain an aldosterone receptor antagonist.

[0026] In embodiments of the invention, a pharmaceutical pack or kit is provided comprising one or more containers filled with one or more of the ingredients of a pharmaceutical composition of the invention. Associated with such container(s) can be various written materials such as instructions for use, or a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals products, which notice reflects approval by the agency of manufacture, use, or sale for human or veterinary administration.

[0027] Very specific formulations suitable for the compounds of the invention have been described in the patent applications WO 03/068266 and WO 04/062692.

[0028] The specific compounds described above are intended to further illustrate the invention in more detail, and therefore are not deemed to restrict the scope of the invention in any way.

EYE DISEASES: DELAYED RETINAL CELL DEATH IN HUMANS AND

EXPERIMENTAL MODELS [0029] Apoptosis is a significant component of cell death in optic and/or eye diseases including (i) all forms of primary and secondary glaucoma, preferably such as e.g. primary open-angle glaucoma, normal-tension glaucoma, primary angle-closure glaucoma, pseudoexfoliation syndrome and glaucoma, pigment dispersion syndrome and glaucoma, neovascular glaucoma, inflammatory glaucoma, lens-related glaucoma, traumatic glaucoma, primary congenital glaucoma, iatrogenic induced glaucoma, and malignant glaucoma (R. Agarwal, S.K. Gupta, P. Agarwal, R. Saxena & S.S. Agrawal (2009) Indian J. Ophthalmol. 57, 257-266); (ii) aquired macular disorders, preferably such as e.g. age-related macular degeneration, idiopathic choroidal neovascularisation, central serous chorioretinopathy, vitreomacular interface disorders, idiopathic macular telangiectasia, cystoid macular oedema, and microcystic macular oedema (J.L. Dunaief, T. Dentchev, G.-S. Ying & A.H. Milam (2002) Arch. Ophthalmol. 120, 1435- 1442); (iii) optic neuropathy, preferably such as e.g. anterior or posterior ischemic optic neuropathy (B.J. Slater, Z. Mehrabian, Y. Guo, A. Hunter & S.L. Bernstein (2008) Invest.

Ophthalmol. Vis. Sci. 49, 3671-3676); (iv) optic neuritis (K.S. Shindler, E. Ventura, M. Dutt & A. Rostami (2008) Exp.Eye Res. 87, 208-213); (v) uveitis, preferably such as e.g. anterior uveitis, intermediate uveitis, posterior uveitis, and panuveitis (C.-C. Chan, D.M. Matteson, Q. Li, S.M. Whitcup & R.B. Nussenblatt (1997) Arch. Ophthalmol. 115, 1559-1567); (vi) hereditary fundus dystrophies, preferably such as e.g. retinitis pigmentosa, cone dystrophy, cone-rod dystrophy, rod dystrophy, Stargardt's disease, Bietti's crystalline corneoretinal dystrophy, familial benign fleck retina, Best vitelliform macular dystrophy, adult-onset vitelliform macular dystrophy, North Carolina macular dystrophy, familial dominant drusen, and concentric annular macular dystrophy (H. Zhang, X. Li, X. Dai, J. Han, Y. Zhang, Y. Qi, Y. He, Y. Liu, B. Chang & J.J. Pang (2017) J. Ophthalmol. 2017, 1-13 9721362); (vii) retinal vascular diseases, preferably such as e.g. diabetic retinopathy, non-diabetic retinopathy, retinal venous occlusive disease, retinal arterial occlusive disease, ocular ischemic syndrome, hypertensive eye disease, sickle cell retinopathy, thalassemia retinopathy, retinopathy of prematurity, retinal artery macroaneurysm, primary retinal telangiectasia, Eales disease, and radiation retinopathy (G. Donati, A.

Kapetanios, M. Dubois-Dauphin & C.J. Poumaras (2008) Acta Ophthalmol. 86, 302-306); (viii) scleritis or episcleritis (C. Heinz, N. Bograd, J. Koch & A. Heiligenhaus (2013) Graefes Arch. Clin. Exp. Ophthalmol. 251, 139-142); (ix) retinal detachments (J.G. Arroyo, L. Yang, D. Bula & D.F. Chen (2005) Am. J. Ophthalmol. 139, 605-610); (x) trauma to the eye globe (H.-C.H. Wang, J.-H. Choi, W.A. Greene, M.L. Plamper, H.E. Cortez, M. Chavko, Y. Li, J.J. Dalle Lucca & A.J. Johnson (2014) Military Med. 179, S34-S40); (xi) vitreous opacities such as e.g. vitreous haemorrhage and asteroid hyalosis (A. Alamri, H. Alkatan & I. Aljadaan (2016) Middle East African J. Ophthalmol. 23, 271-273); (xii) myopia and degenerative myopia (G.Z. Xu, W.W. Li & M.O. Tso (1996) Trans. Am. Ophthalmol. Soc. 94, 411-431); (xiii) postsurgical trauma, preferably such as e.g. mechanical trauma due to conventional surgery, thermotrauma due to laser surgery, and trauma induced by cryosurgery (A. Barak, T. Goldkorn & L.S. Morse (2005) Invest. Ophthalmol. Vis. Sci. 46, 2587-2591; X. Liu, S. Ling, X. Gao, C. Xu & F.Wang (2013) JAMA Ophthalmol. 131, 1070-1072; D. Reichstein (2015) Curr. Opin. Ophthalmol. 26, 157- 166); (xiv) dry eye disease (S. Yeh, X.J. Song, D.Q. Li, W. Farley, M.E. Stern & S.C.

Pflugfelder (2003) Invest. Ophthalmol. Vis. Sci. 43, 124-129); (xv) corneal disorders, preferably such as abrasions, lacerations, ulcerations, dystrophies, opacities, endothelial and epithelial decompensation, post-surgical oedema, corneal degenerations, corneal vascularisation, and corneal ectasias, preferably such as keratoconus (R.M. Kaldawy, J. Wagner, S. Ching & G.M. Seigel (2002) Cornea 21, 206-209; N. Szentmary, B. Szende & I. Suveges (2005) Eur. J.

Ophthalmol. 15, 17-22). In humans, apoptosis has been demonstrated to occur in the course of glaucoma in retinal ganglion cells by means of real-time imaging using annexin 5 labelled with fluorescent dye DY-776 (M.F. Cordeiro, E.M. Normando, M.J. Cardoso, S. Miodragovic, S. Jeylani, B.M. Davis, L. Guo, S. Ourselin, R. A'Hern & P.A. Bloom (2017) Brain 140, 1757- 1767). To model this kind of apoptosis in a non-human experiment, rats are subjected to systemic administration of doxorubicin and apoptosis assessed in large organs such as the liver or heart (R. Gillet, G. Grimber, M. Bennoun, C. Caron de Fromentel, P. Briand & V. Joulin (2000) Oncogene 19, 3498-3507; L.L. Fan, G.P. Sun, W. Wei, Z.G. Wang, L. Ge, W.Z. Fu & H. Wang (2010) World J. Gastroenterol. 16, 1473-1481), or rats are subjected to the action of doxorubicin in the eye and apoptosis assessed in the retinal ganglion cells (I.M. Parhad, J.W. Griffin, A.W. Clark & J.F. Koves (1984) J. Neuropathol. Exp. Neurol. 43, 188-200). We used doxorubicin-induced apoptosis in the rat to assess whether systemic administration of compounds example prevents it.

EXAMPLES

[0030] Methods: Wistar rats, 200-250 g, were anesthetized with chloral hydrate, 400 mg/kg, and osmotic minipumps, primed prior to implantation, were filled with either vehicle or example (6), representative for the compounds of the invention, in the dose of 60 mg/kg/d, and were implanted subcutaneously. Subsequently, rats (n=7) received doxorubicin in the dose of 5 mg/kg i.p. ti.d. on days 1, 2 and 3, or rats (n=8) received doxorubicin in the dose of 5 pg or vehicle in the volume of 5 mΐ into the vitreous body of the eye by means of a Hamilton microsyringe over 5 min on the day 1. Rats were euthanized 5 days after the first i.p. injection of doxorubicin, or 5 days after the administration of doxorubicin into the vitreous body, and were transcardially perfused with a solution containing 4% paraformaldehyde in phosphate buffer. The livers and the eyes were subsequently removed and embedded in paraffin. To visualize apoptosis, a terminal deoxynucleotide transferase-mediated dUTP nick end-label (TUNEL) based staining was performed on 10 pm paraffin sections. The method of stereological disector (L.M. Cruz-Orive & E.R. Weibel (1990) Am. J. Physiol. 258, L148-L156) was used for quantification of apoptosis. An unbiased counting frame (0.05 x 0.05 mm, disector height 0.01 mm) was used for liver and (0.10 x 0.025 mm, disector height 0.01 mm) for the retinal ganglion cell layer for the sampling. The N _v of TUNEL positive cells was determined with 8-10 dissectors and is expressed as number of TUNEL positive cells x l0 ² /mm ³ for liver and as number of TUNEL positive cells x l0 ³ /mm ³ for retinal ganglion cells. Statistical evaluation was performed by means of Student's t- test (Table 1).

[0031] Results: Doxorubicin caused apoptosis either in the liver or in the retina as evidenced by the presence of TUNEL positive cells (Table 1). Example (6) conferred significant protection against doxorubicin induced apoptosis in the liver and in the retina as evidenced by reduction of N _v of TUNEL positive cells vs vehicle-treated subjects (Table 1).

[0032] Conclusions: Example (6) decreased density of apoptotic cells in the rat liver by 23.46 % and in the retina by 8.21 % (Table 1). The apoptosis induced by doxombicin in the liver was characterized by many morphological features similar to those of apoptosis seen in the retinal ganglion cells.

Table 1. Effect on doxorubicin-induced apoptosis.

P<0.05, P<0.0l vs doxombicin + vehicle; Student's t-test; RGC, retinal ganglion cells; N _v , number of TUNEL-positiv cells/mm ³ ± SEM.