MXPA99005399A - Use of green porphyrins for the manufacture of a medicament for the treatment of secondary cataracts - Google Patents

Abstract

Photodynamic therapy to prevent secondary cataracts is effected using photosensitizers such as green porphyrins as photoactive agents to destroy remnant lens epithelial cells.

Description

USE OF GREEN PORFIRINES FOR THE MANUFACTURE OF A DRUG FOR THE TREATMENT OF CATARATAS HIGH SCHOOLS FIELD OF THE INVENTION The present invention relates to the use of treatment by photodynamic therapy (PDT) to prevent secondary cataracts, more particularly to the use of green porphyrins for such treatment of PDT.

DESCRIPTION OF THE PREVIOUS TECHNIQUE The elimination of cataracts is one of the most common surgical procedures in the United States. Secondary cataracts, more specifically opacification of the posterior capsule, are the most common complication of cataract extraction procedures, with or without intraocular lens implants of the posterior chamber. Depending on their age, this condition affects 15% to 50% of all patients, and is generally "secondary" to a proliferation and migration of residual epithelial cells of the REF .: 30578 crystalline. While ophthalmic surgeons are aware of the incidences of secondary cataracts and are careful to remove as many residual epithelial cells from the lens as possible, for example, before implanting an artificial intraocular lens, it is difficult to identify all such cells and frequently difficult to reach them on the inner surface of the lens capsule. Secondary cataracts, as a post-surgical effect, are also referred to as "late cataracts". One commentator has observed that the term "secondary cataract" is ambiguous because it is often also used to refer to a cataract that appears secondary to various eye diseases. See, for example, Kappelhoff, J.P. and collaborators, "The Pathology of After-Cataract, A Mini Revie", Ac t a Opthamol. Suppl. 205: 13 (1992). For purposes of the present patent application, however, the term secondary cataract means the proliferation, based on histological observations, of the lenticular epithelial cells, fibroblasts, macrophages and even pigment cells derived from the iris on the posterior capsule after the cataract removal, but not the result of unrelated changes in the remaining posterior capsule itself. Although implanted infra-ocular lenses are thought to inhibit capsule opacification by themselves, the mechanisms by which this results are poorly understood. It has been suggested that intraocular lenses influence the formation of secondary cataracts by limiting the space available for the lentoide formation and by maintaining a linear scaffold for epithelial fibrous metaplasia of the lens. Nasisse, M.P. and collaborators, "Lens _ Capsule Opacification in Apha ic and Pseudophakic Eyes", Graefes Arch. Clin. Exp. Opthalmol. 233 (2): 63 (1995). Other people have suggested that intraocular lenses stimulate the development of secondary cataracts. Nishi, O. and collaborators, "Intercapsular Cataract Surgery with Lens Epithelial Cell Removal", J. Ca tara ct Refra ct. Surg. 17: 471-477 (1991). However, secondary cataracts occur frequently and require medical intervention. The various techniques for reducing the opacification of secondary cataracts include, for example, atraumatic surgery and cortical cleaning. A review of these and other techniques is presented in Apple, D. J. et al., "Posterior Capsule Opacification", Survey of Oph th almol ogy, 37 (2): 73 (1992). These "conventional" treatments for secondary cataracts have several serious side effects, including retinal detachment and damage to the implanted intraocular lens. See, for example, Lundgren, B. et al., "Secondary Cataract: An In vi vi Model for Studies on Secondary Cataract in Rabbits" Acta Oph thalmol. Suppl. 205: 25 (1992). In this way, the technique selected for the prevention of the formation of secondary cataracts is of particular importance with respect to a successful result of the original cataract surgery. A variety of experimental techniques for the prevention of secondary cataracts have been proposed or evaluated in this way. These include the use of heparin to inhibit the migration and proliferation of fibroblasts on the posterior capsular surface. Xia, X.P. and collaborators, "A Cytological Study of Inhibition of Secondary Cataract with Heparin," Ch ung Hua Yen Ko Tsa Chih. , 30 (5): 363 (1994); and Xia, X.P. and collaborators "A Clinical Study of Inhibition of Secondary Cataract with Heparin", Ch ung Hua Yen Ko Tsa Chih. , 30 (6): 405 (1994). Other methods for the prevention of secondary cataracts include chemical modification of the posterior surface of the lens capsule through the covalent binding of certain compounds and their subsequent polymerization. Lindquist, B. and collaborators, "Method for Preventing Secondary Cataract" North American Patent No. 5,375,611 (1994). An alternative method refers to the injection of a substance that kills cells, between the anterior capsule and the natural lens prior to the removal of the natural crystalline lens from the eye. Such a substance that kills cells is preferably a relatively strong acid or base adjusted in aqueous solution and may include a viscoelastic material or a dye. Dubroff, S., "Composition for Preventing Clouding of Posterior Capsule After Extracapsular Cataract Eye Surgery and Method of Performing Cataract Surgery", US Patent No. 5,273,751 (1993). Somewhat related are chemical methods to prevent or reverse the formation of cataracts involving the administration of chemical compositions that lower the phase separation temperature of a lens or crystalline lens and prevent or inhibit the formation of opacities, high molecular weight aggregates and other physical characteristics of cataracts. See, for example, Clark, J.I. and collaborators, "Chemical Prevention or Reversal of Cataract by Phase Separation Inhibitors" US Patent No. 5, 401, 880 (1995). The use of monoclonal antibodies in the prevention of secondary cataracts has also been reported. For example, monoclonal antibodies that fix complement, specific for lens epithelial cells can be introduced into the anterior chamber of the eye, after extracapsular extraction. After binding of such monoclonal antibodies to any lens epithelial cells present, the complement is introduced into the anterior chamber, whereby the lysis of the remaining epithelial cells of the lens is effected. Emery, J.M. and collaborators, "Monoclonal Antibodies Against Lens Epithelial Cells and Methods for Preventing Proliferation of Remnant Lens Epithelial Cells After Extracapsular Extraction", US Patent No. 5, 202, 222 (1993). The application of electrical or thermal energy by means of a probe inserted between the iris and the lens capsule has also been used to destroy the residual epithelial cells of the lens within the lens capsule. Bretton, R.H., "Method and Apparatus for Preventing Posterior Capsular Opaci fication," U.S. Patent No. 5,455,637 (1995). Photodynamic therapy for the control of lens epithelial proliferation has also been described. In photodynamic therapy, the photosensitizers used are capable of being located in the target cells, either by the natural tendency or because they have been intentionally directed to a specific type of tissue, or both. When they are irradiated, they are able to fluoresce and, thus, can be useful in diagnostic methods related to the detection of target tissue. However, more importantly, the photosensitizer has the capacity, when irradiated with light at a wavelength that the compound absorbs, to cause a cytotoxic effect against the cells in which the photosensitizer has been located. Although it has not been definitively established, it is thought that this cytotoxic effect is due to the formation of singlet oxygen after irradiation. With respect to therapy with PDT for secondary cataracts, experimental studies using Photofrin II (PII) have been reported by Parel, J.M. and collaborators, "Endocapsular Lavage with Photofrin II as a Photodynamic Therapy by Lens Epithelial Proliferation", Lasers and Medi cal Sci en 5: 25 (1990). These authors noted several technical difficulties with their technique, including a constant leak of PII from the capsular bag and a minimum capture time of less than 15 minutes after rinsing. The fluorescence from PII was still discernible after 30 hours, but it provided insufficient specificity to ensure safe treatment with photodynamic therapy. A related study by Lingua, R. et al., "Preclinical Evaluation of Photodynamic Therapy to Inhibit Lens Epithelial Proliferation," Lasers and Ligh t in Ophthalmol ogy 2 (2): 103 (1988) reported the early testing of photodynamic therapy using Photofrin II. These authors reported that their techniques resulted in local death of the epithelial cells, but also death of the fibrous cells, and that the efflux of these agents from the capsule resulted in local ocular toxicities such as uveitis and corneal edema. They suggested that effective inhibition of lens cell proliferation might require capsular containment of the photoactive agent and a method for even distribution of light within the capsule during photoirradiation. One application of the compound included its mixing with Healon® in order to increase the viscosity of the preparation. In another reported study, aluminum phthalocyanine was applied to preconfluent cultures of crystalline porcine epithelial cells. After the uptake for one hour, the cultures were exposed to red light for five minutes, and a toxic effect was found. Wunderlich, K. et al., "Photodynamic Activity of Phthalocyanines in Cultivated Lens Epithelial Cells of the Pig", Oph thalmol ogy 92 (3): 346 (1995). As a related practical constraint, surgeons have expressed interest that even a ten minute delay for the uptake of photosensitizers is too long in cataract procedures. In this way, the rate of uptake and the clearly localized administration of the photosensitizing compositions are of particular importance. The compositions of the prior art do not exhibit such optimal uptake parameters. According to the present invention, an alternative PDT technique has been developed, which utilizes a preferred group of photosensitizers that are easily picked up by the remaining epithelial cells of the lens, and which can be contained within the capsule and distributed to the cells target during relatively short incubation periods. The containment of the photosensitizer in the capsule prevents photosensitization of non-target parts of the eye. Because these photosensitizers appear green instead of red, they have been dubbed "green porphyrins". These compounds have made it possible to conduct photodynamic therapy with light having a wavelength range outside that normally strongly absorbed by blood or other normal tissues, specifically around 670-780 nm. In addition to providing effective treatment in vivo, contained in the target, and thereby reducing the hypersensitivity of the non-target tissues, an appropriate depth of penetration by the light that is irradiated can also be easily achieved. It is known that green porphyrins can be used to detect and treat atherosclerotic plaques in a photodynamic therapy protocol. See, for example, Levy et al., US Patent No. 5,399,583 issued March 21, 1995 (column 2, lines 14-15); Levy et al., U.S. Patent No. 4,920,143 issued April 24, 1990 (column 10, lines 58-59); Levy et al. U.S. Patent No. 5,095,030 issued March 10, 1992 (column 2, lines 8-9 and column 15, lines 29-30); and Levy et al., U.S. Patent No. 5,171,749 issued December 15, 1992 (column 2, lines 12-13 and column 18, lines 1-4 and 35-47). A particular class of green porphyrins of some clinical interest is the class of compounds called benzoporphyrin (BPD). These photosensitizers have been tested to some degree in relation to other ocular conditions. For example, Schmidt, U. and coworkers described experiments using BPD for the treatment of Greene melanoma (a non-pigmented tumor) implanted in rabbit eyes, and achieved necrosis in this context (IOVS 33: 1253 Excerpt 2802 (1992)) . Lin, C.P. and collaborators describe the measurement of kinetics and distribution in the retinal and choroidal vessels by fluorescent imaging using a 458 n line from an argon ion laser to excite BPD (TOVAS 34: 1168 Extract 2293 (1993)) . In addition, Lin, S.C. and collaborators described the photodynamic closure of the choroidal vessels using BPD in TOVAS 34: Extract 2953 (1993). Researchers associated with the assignee of this application have described the treatment of choroidal neovascularization using BPD in various extracts published on March 15, 1993 and in their patent application (S. N. 08 / 390,591) incorporated by reference herein. These extracts include Schmidt-Erfurth, U. and collaborators "Photothrombosis of Ocular Neovascularization Using BPD"; Haimovici, R. et al. "Localization of Benzoporphyrin Derivative Monoacid in the Rabbit Eye"; and Walsh, A.W. and collaborators "Photodynamic Therapy of Experimental Choroidal Neovascularization Using BPD-MA". All of the above were published in IOVS 34: 1303 as Excerpts 256, 255 and 254 (1993) and Moulton, R.S. and collaborators "Response of Retinal and Choroidal Vessels to Photodynamic Therapy Using Benzoporphyrin Derivative Monoacid", IOVS 34: 1169 Extract 58 (1993). PDT formulations and methods involving green porphyrins according to the present invention offer advantages in their selectivity for cells that give rise to secondary cataracts and in their ability to effect the photodynamically mediated destruction of such cells. Green porphyrins also show a relatively faster uptake by the target cells, thereby decreasing the delay during the operative procedures associated with current PDT techniques. Since they are applied to the prevention of secondary cataracts, these types of green porphyrins also have therefore particularly advantageous properties in terms of selectivity and rapidity of uptake.

BRIEF DESCRIPTION OF THE INVENTION The invention is directed to the use of photodynamic therapy (PDT) to prevent secondary cataracts using photodynamic methods, mainly using green porphyrins as the photoactive compounds. These materials offer advantages of rapid uptake by lens epithelial cells, selectivity and effectiveness when used in protocols aimed at the destruction of the remaining epithelial cells of the lens. Accordingly, in one aspect, the invention is directed to a method for preventing secondary cataracts, which method comprises administering to a subject in need of such treatment, an amount of a green porphyrin that will be located in the epithelial cells of the lens. that give rise to a secondary cataract; and irradiating the cells with light absorbed by the green porphyrin. In a particular aspect, the invention is directed to a method for preventing secondary cataract in the eye of a subject, which involves the steps of administering to the lens capsule of a subject, an amount of a green porphyrin, sufficient to allow that an effective amount is located in the lens epithelial cells; allowing enough time to allow an effective amount of the green porphyrin to be located in the lens epithelial cells; and irradiating said lens epithelial cells with light absorbed by the green porphyrin, at a level of energy sufficient to substantially destroy all the epithelial cells of the lens. Specifically, the method involves the destruction of lens epithelial cells that remain after lens removal during cataract surgery. In still another aspect, the invention includes a step in which a viscous solution is applied to protect the cornea before administration of the green porphyrin. Also, green porphyrin can be combined with an agent that increases viscosity. Such viscosity increasing agents may optionally be selected from the group consisting of hyaluronic acid and its derivatives, starches and cellulose and their derivatives. In a specific formulation, the methods of the invention include the administration of an effective amount of a green porphyrin in the range of about 0.2 to about 2 mg / ml and about 150 to about 250 μl. More specifically, the dose administered is in the range of from about 0.3 to about 0.5 g, and even more specifically, about 0.3 mg. These formulations can also be administered in a liposomal formulation, in which the green porphyrin can be formulated in liposomes before the liposomes are mixed with a viscous vehicle such as Ophthalin, Hymacel or AMVISC. The methods of the present invention are contemplated specifically for the administration of green porphyrin which is selected from the group consisting of BPD-DA, BPD-DB, BPD-MA and BPD-MB as well as the derivatives of these compounds. Particularly preferred BPDs include BPD-MA and BPD-DA. In one aspect of the composition, the present invention contemplates pharmaceutical compositions for preventing or inhibiting the development of secondary cataracts, such compositions containing an amount of a green porphyrin, effective to prevent or inhibit the development of secondary cataracts, when administered to a subject suffering from cataract surgery; and a pharmaceutically acceptable carrier or excipient.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be more clearly understood by reference to the following drawings, in which: Figure 1 shows the typical green porphyrin formulas, useful in the method and composition of the invention.

Figure 2 shows the formulas of four particularly preferred embodiments of the green porphyrins of the invention, BPD-DA, BPD-DB, BPD-MA and BPD-MB.

Figure 3 shows the survival of human lens epithelial cells after incubation with BPD in the absence of light. The cells were incubated in an incubator for 10 minutes in BPD at a concentration range of 0 to 800 ng / ml in the absence of light. Cell viability was determined 5 days after incubation by means of a colorimetric assay (MTT).

Figure 4 shows the cytotoxic effect of BPD and light on human lens epithelial cells. Cells obtained from two different donors were incubated for 10 minutes with BPD at a range of concentrations, and after removal of the excess drug they were exposed to 10 J / cm2 of LED light (690 nm Cell viability was determined by the assay of MTT and the percentage of cell death was calculated with reference to the cell exposed to light only.

Figure 5 shows that the cytotoxic effect of PDT is long lasting. The survival of the HLE cells was determined 24 hours (A) and 20 days (B) after PDT (BPD at 0-800 ng / ml, incubation of 10 minutes, 10 J / cm2) by means of the MTT assay. The percentage of cell death was calculated with reference to cells exposed to light only.

Figure 6 shows the effect of a shortened incubation time, with BPD. The HLE cells from two different donors were incubated for 1-1.5 minutes with BPD (0-30 μg / ml), after which they were exposed to 10 J / cm2 of LED light. Cell survival was determined by means of the MTT assay and the percentage of cell death was calculated with reference to cells exposed to light only.

Figure 7 shows the photosensitization of the human epithelial cells of the lens, with ZnPc. Cells were incubated for 1-15 minutes with ZnPc (CGP 55 847; 0-200 μg / ml) and exposed to 10 J / cm2 of LED light (672 nm) Cell survival was determined by means of the MTT assay and the Percentage of cell death was calculated with reference to cells exposed to light only. Two separate experiments were carried out at different times after the trypsinization.

Figure 8 shows the effect of the distribution of BPD in a viscous solution. The HLE cells were incubated with BPD, at a range of concentrations distributed in either 5% FCS (A) or 25% Amvisc (B) and were exposed to 10 J / cm2 of LED light. Cell viability was determined 24 hours after the use of the MTT assay. The percentage of cell death was calculated with reference to cells exposed to light only.

Figure 9 shows the diffusion of BPD from either Amvisc or Hymecel in the balanced salt solution. The diffusion was evaluated spectrophotometrically after "0", 1 and 5 minutes of contact.

Figure 10 shows rabbit epithelial cells treated with BPD and red light. Figure 10A is the dark control and Figure 10B shows the cells exposed to light. The epithelial cells of the lens were treated with 30 μg / ml of BPD in 100% Hymecel (1 minute) and red LED light (1 J / cm2) in itself (over the capsule). The cells were stained with a cytosonde assay kit where the live cells are stained green and the dead cells are stained red.

Figure 11 shows the treatment of the cells. Vero treated with BPD and red light. Vero cells were treated with BPD (1 minute incubation) and red light (1 J / cm2). The cells were stained with the cytosonde assay kit. In this system, living cells stain green, and dead cells stain red. (A) Untreated cells, (B) BPD at 17 μg / ml in 100% Oftalin, (C) BPD at 30 μg / ml in 100% Hymecel, (D) BPD at 30 μg / ml in 100% of Amvisc.

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to a method in which photodynamic therapy (PDT) is directed to certain cells, for example, lens epithelial cells, which give rise to secondary cataracts at a certain point in time (perhaps months to years) after cataract surgery. For the prevention of secondary cataracts, this treatment is considered as an additional procedure during conventional cataract surgery. Thus, after removal of the lens and prior to insertion, for example, of a replacement infra-ocular lens, the normal cataract surgical procedure will be interrupted. An appropriate porphyrin compound, preferably a green porphyrin, will be introduced into the capsule for about one minute and then removed. During this period of time, the porphyrin compound will be located in the epithelial cells of the lens. Light of appropriate frequency and intensity will be applied and using an appropriate light source, which activates the porphyrin and more to the epithelial cells. After this PDT procedure, the normal surgical procedure will be resumed and completed. The formulations and methods of the claimed invention generally relate to the administration of a green porphyrin to a subject, which is in the class of compounds referred to as benzoporphyrin derivatives (BPD). A BPD is a synthetic porphyrin similar to chlorine, with various structural analogues, as shown in Figure 1. Preferably, the green porphyrin is the diacid or monoacid A ring derived from the benzoporphyrin (BPD-DA or BPD-MA), which absorbs light at approximately a wavelength of 692 nm with improved tissue penetration properties. BPD-MA, for example, is a powerful, lipophilic photosensitizer, and it also appears to be phototoxic for neovascular tissues, tumors and epithelial cells remaining from the lens. After approximately a period of less than one minute for the uptake of an appropriate formulation, for example of a BPD-MMA composition by the lens epithelial cells, the light of the appropriate wavelength is distributed substantially uniformly to all the epithelial cells of the lens located on the anterior and equatorial internal surfaces of the capsule. Due to its pharmacokinetics, BPD seems to be the best candidate for use in this indication, but other green porphyrins such as BPD-DA or other derivatives may also be used (see Formulas 1-6 in Figure 1). Other photosensitizers, such as phthalocyanines, could be used in high concentrations, sufficient to displace their relatively slower uptake. However, with such compounds, one skilled in the art would need to formulate any selected sensitizer to control the possibility of accidental contamination of other parts of the eye.

A particularly preferred formulation according to the present invention will satisfy the following general criteria. First, a photosensitizer capable of rapidly entering the epithelial cells of the lens must be used. Second, the viscosity of the product must ensure that it is substantially contained within the capsule during the incubation period. Third, the components that facilitate the uptake of this photosensitizer by the target cells can be included. As an example of a specific formulation, 0.1 ml of stock solution of BPD-MA is added to 1.9 ml of a viscoelastic material such as Ophthalin and mixed according to the following examples. During cataract surgery, the anterior wall of the lens is either cut to facilitate removal of the extracapsular lens, or partially removed (capsuloresis) to facilitate the introduction of a phacoemulsifier to perform intercapsular lens extraction. Accordingly, the successful containment of a photosensitizer in the capsule for the indicated incubation period will depend on the viscosity of the formulation in which the photosensitizer is introduced into the capsule. The time of such incubation is also significant in the containment, and preferably it will be for less than about one minute, ideally less than about 45 seconds and more preferably less than about 30 seconds. The cut on the inner surface of the capsule and the administration of the PDT compound can result in effusion of the photosensitizer administered to prevent secondary cataracts, and may limit its retention at the intended intracapsular site. Thus, a further important aspect of the present invention is the coating of the corneal endothelium with the drug-free viscoelastic material, prior to the application of the photosensitizer, in order to protect this sensitive part of the eye from contamination with the photosensitizer. Other considerations should also be taken into account when administering PDT to prevent secondary cataracts. For example, the shape of the capsule, for example, an elongated sphere, with cells that have high mitotic potential in the equatorial position, and its small size make it difficult to evenly distribute light when considering photodynamic therapy. As an additional problem, the schedules or busy operating schedules of the eye surgeons, do not allow time for a prolonged procedure to be added to the cataract procedure itself. Accordingly, an optimal photosensitizer for the prevention of PDT from secondary cataracts must be rapidly picked up by the lens epithelial cells and must be physically contained, to a significant degree, within the capsule. After the green photosensitizing porphyrin has been administered, preferably, the compound is allowed to be taken up by the epithelial cells of the lens for less than about one minute. The lens epithelial cells are then irradiated at the absorbance wavelength of the green porphyrin, usually between about 550 and 695 nm, as discussed above. In particular, red light is advantageous, due to its relatively lower energy and the resultant lack of toxicity it imposes on ocular tissue, while lens epithelial cells are destroyed.

The compositions and methods of the present invention provide a useful treatment of PDT to prevent secondary cataracts. The following describes the compositions and formulations of the present invention and their clinical application. The experimental data are also presented and described.

The Green Porphyrins The green porphyrins useful in the method of the invention are described in detail in Levy et al., US Patent No. 5,171.7490 issued December 15, 1992, which is incorporated by reference herein. "Green porphyrins" refers to the porphyrin derivatives obtained by the reaction of a porphyrin nucleus with an alkyne in a Diels-Alder type reaction, to obtain a monohydrobenzoporphyrin. Typically, green porphyrins are selected from a group of porphyrin derivatives obtained by Diels-Alder reactions of acetylene derivatives, with photoporphyrin under conditions that promote the reaction in only one of the two available, present, conjugated, non-aromatic diene structures. in the protoporphyrin IX ring system (rings A and B). Various structures of the typical green porphyrins are shown in Figure 1. The Diels-Alder reaction initially results in the formation of a cyclohexadiene - herein referred to as "idrobenzo" - fused to the pyrrole ring A or B, as shown in Figures 1 and 2. The rearrangement or rearrangement of the system p in the hexadiene ring results in the formation of compounds of formulas 3 and 4, and the reduction provides compounds of formulas 5 and 6. These compounds are shown in formulas 1-6 with hydrogen occupying the nitrogens of the inner rings. However, it should be understood that methylated forms, in which a cation replaces one or both of these hydrogens, can also be used. The preparation of the green porphyrin compounds, useful in this invention, are described in detail in U.S. Patent No. 5,095,030, which is incorporated by reference herein. • For convenience, an abbreviation of the term hydromonobenzoporphyrin derivative ("BPD") is generally used to refer to the compounds of formulas 3 and 4 of Figure 1. The compounds of formulas 3 and 4 and mixtures thereof are particularly preferred. As shown in Figure 1, R1, R2, R3 and R4 are non-interfering substituents, which do not appreciably affect the activity of the compound in the method and composition of the invention. More specifically, the term "non-interfering substituents" is used to imply substituents that do not interfere with the pharmacological functions of BPD. For the compounds of Figures 1 and 2, in general, R1 and R2 are each, independently, substituents, moderately electron withdrawers or any other activating substituents that do not remove enough electrons to result in the Diels-Alder reaction proceeding with rings A and b, instead of just one. Examples of suitable R1 and R2 groups include carbalkoxy of 2 to 6 carbon atoms, (alkyl of 1 to 6 carbon atoms) sulfonyl or (aryl of 6 to 10 carbon atoms) sulfonyl, aryl of 6 to 10 carbon atoms , cyano, and -CONR5CO- wherein R5 is aryl of 6 to 10 carbon atoms or alkyl of 1 to 6 carbon atoms. One of R1 and R2 can also be hydrogen, as long as the other is a substituent that extracts electrons, of sufficient strength to facilitate the Diels-Alder reaction. Most commonly, R1 and R2 are carbalkoxy groups, preferably methyl or ethyl carboxylic esters. Preferred compounds are those in which R1 and R2 are the same and are carbalkoxy, particularly carboethoxy. As used herein, the term "carboxyl" is, as is conventionally defined, -COOH, while "carbalkoxy" represents -COOR where R is alkyl. "Carboxyalkyl" refers to the substituent -R'-COOH where R 'is alkylene. "Carbalkoxyalkyl" refers to -R'-COOR where R 'and r are alkylene and alkyl respectively. "Alkyl" generally represents a straight or branched chain, saturated, hydrocarbyl portion of 1 to 6 carbon atoms, such as methyl, n-hexyl, 2-methylpentyl, t-butyl, n-propyl, and so on. "Alkylene" is the same as "alkyl" except that the group is divalent instead of monovalent. "Aryl" represents a phenyl group, optionally substituted with 1 to 3 substituents, which may be selected from the group consisting of halo, such as fluoro, chloro, bromo or iodo; lower alkyl of 1 to 4 carbon atoms; and lower alkoxy of 1 to 4 carbon atoms. The "aryl" or "alkylsulfonyl" groups have the formula -S02R where R is alkyl or aryl as defined above. R3 is independently a? -carboxyalkyl group of 2 to 6 carbon atoms, or a salt, amide, ester or acylhydrazone thereof, or is alkyl of 1 to 6 carbon atoms. Preferably, R3 is 2-carboxyethyl or the alkyl ester thereof, and R4 is vinyl. These modalities, however, are preferred because of the availability of native porphyrins, rather than dictated by considerations of biological efficacy. As shown in Figure 1, adducts or addition complexes formed by the reaction of R1-C = C-R2 with a protoporphyrin IX ring system (where R3 is an appropriate form of 2-carboxyethyl, such as 2- carbomethoxyethyl or 2-carboethoxyethyl, and R 4 is -CH = CH 2) are compounds of formulas 1 and 2. The compounds of formula 1 result from the addition to ring A, and the compounds of formula 2 result from addition to the ring B. Suitable starting materials for the green porphyrin compounds of the present invention include naturally occurring porphyrins where R3 is either -CH2CH2COOH or -CH2CHRC00R where R is alkyl of 1 to 6 carbon atoms. However, the exact nature of R3, unless it contains a p-linked conjugate to the p-bond of the ring, is ordinarily not relevant to the progress of the Diels-Alder reaction or to the effectiveness of the resulting product. R3 may thus be any of a wide variety of groups such as, for example, lower alkyl of 1 to 4 carbon atoms; and? -carboxyalkyl of 2 to 6 carbon atoms and the esters and amides thereof The R- "substituent may also be substituted with halogen, such as fluoro, chloro, bromo or iodo, or with other non-reactive substituents. is -CH2CHRCOOR, it has been found advantageous to hydrolyze, or partially hydrolyze, the esterified carboxyl group Typically, hydrolysis at the R3 position conveniently occurs at a much faster rate than that of the ester groups of R1 or R2. The solubility and biodistribution characteristics of the resulting compounds are more desirable than those of the non-hydrolyzed form The hydrolysis results in diacidic or monoacid products (or their salts).

In the compounds of formulas 1 and 2, R4 is usually -CH = CH2, at least initially, but this vinyl group is rapidly derivatized to other embodiments of R4 by addition to, or oxidation of, the vinyl substituent of ring B or A in formula 1 or 2 respectively. Thus, R4 can be any of a wide variety of substituents that are consistent with that formed with an easy reaction reaction. For example, an exemplary addition reagent may be of the form HX where H is added to the carbon adjacent to the ring, to provide a position R4 having the formula: -CHCH3 I X Thus, in one embodiment, one of the added substituents is a hydrogen, and the other is selected from the group consisting of hydrogen, halo such as fluoro, chloro, bromo or iodo; hydroxyl; lower alkoxy; Not me; amide; sulfhydryl; or an organosulfide. By. example, the addition of Markovnikov water provides a substituent structure analogous to a ring system of hetophorphyrin in the relevant ring. The vinyl group can also be oxidized to obtain, as a substituent at the position R4, -CH20H, -CHO, or COOH or its salts or esters. The addition or oxidation products can themselves be substituted if the added substituents are functional leaving groups. For example, when Br is a substituent, it can be replaced by portions such as -OH, -OR where R is alkyl of 1 to 6 carbon atoms as described above, halo, -NH2, -NHR, -NR2 and the like . Thus, in general, R4 represents any substituents to which the vinyl group -CH = CH2 is easily converted by cleavage or addition, and additional substituents formed by the reaction of the good leaving groups with additional portions. Preferably, however, R4 is vinyl (-CH = CH2); -CHOR4 'wherein R4' is hydrogen or alkyl of 1 to 6 carbon atoms, optionally substituted with a hydrophilic substituent such as -CH2OH; -CHO; -COOR4 'such as COOH or -COOCH3; -CH (OR 4 ') CH 3 such as -CH (OH) CH 3 or -CH (OCH 3) CH 3; -CH (OR4 ') CH2OR4'; -CH (OH) CH2OH; -CH (SR4 ') CH3 such: o or -CH (SCH3) CH3 and the disulfide thereof; -CH (NR4 ') CH3; -CH (CN) CH3; -CH (pyridinium bromide) CH3; -CH (COOR4 ') CH3; -CH (COOCR4) CH3; -CH2 (halo) CH3 such as -CHBrCH3; or -CH (halo) CH2 (halo). Alternatively, R4 may be an organic group of less than 12 carbon atoms that results from the direct or indirect derivatization of the vinyl. Or R4 may provide additional porphyrin ring systems or porphyrin-related systems, such as a group containing from 1 to 3 tetrapyrrole-type cores of the formula -L-P, as defined below. Those compounds in which R4 is -CH = CH2, -CH (OH) CH3, -CH (halo) CH3, or a group containing 1 to 3 tetrapyrrole-type nuclei of the formula -LP, as defined below, are preferred. As used herein, the term "tetrapyrrole type core" represents a system of four skeletal rings: or a salt, ester, amide, or acylhydrazone thereof, which is highly conjugated. This includes the porphyrin system, which is in effect a fully conjugated system; the chlorine system, which is in effect a dihydro form of the porphyrin; and the reduced chlorine system, which is a tetrahydro form of the conjugated porphyrin system. When the "porphyrin" is specified, the fully conjugated system is indicated. The green porphyrins are effectively a dihydro form of the porphyrin system. In one embodiment, the substituent R4 includes at least one additional core of the tetrap-iron type. The resulting compounds of the invention are dimers or oligomers in which at least one of the tetrapyrrole ring systems is a green porphyrin. The link between the green porphyrin portion at the R4 position to an additional tetrapyrrole ring system can be via an ether, amine or vinyl bond. The porphyrin ring systems having two available substituent positions (in both rings, A and B) corresponding to R4 can be further derivatized, as explained below.

When R4 is "-L-P", -L- is selected from the group consisting of: (a) -CH-O-CH-, CH CH-. (b) -CHNHCH-, CH3 CH3 (c) -CH = CH-CH- CH; (d) -CH ~ CH = CH- CH; e.) = CH-C-CH-, and O CH. (f) -CH-C-CH =; CH3 O and P is a porphyrin structure or a second green porphyrin of the formulas 1-6 shown in Figure 1, except that any second group R4 is replaced by L above. (It is also understood that, when -L- is of the formula (e) or (f) shown above, the ring system to which the double bond is coupled, will have a resonance system corresponding to in the ring to which the double bond is attached, as shown). Hydromonobenzoporphyrins resulting directly from the Diels-Alder reaction described above can be isomerized to the BPD compounds of formulas 3 and 4 of Figure 1. The descriptions of compounds 3 and 4 in Figure 1 do not show the relative position of the group exocyclic methyl (ring A of formula 3 and ring B of formula 4) with respect to the substituent R2. Any isomer is available. The compounds of formulas 3 and 4 are particularly preferred in the methods and compositions of the invention.

In addition, the Diels-Alder products can be selectively reduced by treatment with hydrogen in the presence of a catalyst, such as palladium on mineral coal, to give the saturated ring analogues, shown as formulas 5 and 6 in Figure 1, which correspond to the respective Diels-Alder products of rings A and b. The description given above with respect to the compounds of formulas 1 and 2 with respect to the conversion of the remaining vinyl substituent (R4) and with respect to the variability of R3, also applies to the compounds of formulas 3, 4, 5 and 6 The preferred embodiments of the green porphyrins of the invention are those in which the Diels-Alder product is rearranged and partially hydrolyzed. Even more preferred are the compounds of formulas 3 and 4 (BPDs) in which the carbalkoxy groups in the R3 positions have also been hydrolyzed or partially hydrolyzed. The compounds of the invention containing -COOH can be prepared either as the free acid or in the form of salts with organic or inorganic bases. Figure 2 shows four particularly preferred compounds of the invention covered by formulas 3 and 4, which are collectively designated as benzoporphyrin derivatives, for example, BPD-DA, BPD-DB, BPD-MA and BPD-MB. These are idolized or partially hydrolysed forms of the rearranged products of formulas 3 and 4, wherein one or both of the protected carboxyl groups of R3 have been hydrolyzed. The ester groups in R1 and R2 are relatively slowly hydrolyzed, so that conversion to the forms shown in Figure 2 is easily effected. The most preferred of these green porphyrin compounds is BPD-MA. In Figure 2, R3 is -CH2CH2COOR3 'where R3' varies by individual compound. Specifically, in BPD-DA, R1 and R2 are carbalkoxy, R3 'is hydrogen, and derivatization is in ring A. BPD-DB is the corresponding compound with derivatization in ring B. BPD-MA represents partially hydrolyzed form of BPD-DA and BPD-MB represents the partially hydrolyzed form of BPD-DB. Thus, in these latter compounds, R1 and R2 are carbalkoxy, one of R3 'is hydrogen, and the other R3' is alkyl of 1 to 6 carbon atoms. The compounds of the formulas BPD-MA and BPD-MB can be homogeneous, in which only the carbalkoxyethyl of the C ring or only the carbalkoxyethyl of the D ring could be hydrolyzed, or they can be mixtures of the hydrolysates of ring substituents C and D In addition, mixtures of any two or more of BPD-MA, -MB, -DA and -DB can be used in the methods and compositions of the invention. It should be noted that many of the compounds of Figure 1 contain at least one chiral center, and, in this way, they can exist as optical isomers. The method of the invention can utilize compounds having both chiral carbons configurations, whether the compounds are supplied as single stereoisomer isolates or are mixtures of enantiomers and / or diastereoisomers. The separation of diastereomeric mixtures can be effected by any conventional means. Mixtures of enantiomers can be separated by any of the usual techniques, such as by reacting them with optically active preparations and separating the resulting diastereoisomers. It should also be noted that the reaction products can be mixtures not separated from additions of ring A and b, for example, mixtures of formulas I and 2 or 3 and 4 or 5 and 6. Either of the separate forms, for example, Formula 3 alone or 4 alone, or mixtures in any ratio, can be used in the methods and compositions of the invention. Still further, the dimeric forms of the green porphyrin and the dimeric or multimeric forms of the porphyrin / green porphyrin combinations can be used. The dimeric and oligomeric compounds of the invention can be prepared using analogous reactions to those for the dimerization and oligomerization of the porphyrins. The green porphyrins or green porphyrin / porphyrin bonds can be made directly, or the porphyrins can be coupled, followed by a Diels-Alder reaction of either or both terminal porphyrins, to convert them to the corresponding green porphyrins. The green porphyrin compounds of the invention can be administered as a simple compound, preferably BPD-DA or BPD-MA, or as a mixture of various green porphyrins. Suitable formulations include those suitable for the administration of therapeutic compounds to the eye. In addition, other components can be incorporated into such formulations. These include, for example, visible dyes or various enzymes to facilitate access of a compound photosensitization to the target cells, through the remnants of the lens material.

Green Porphyrin Formulations: The compositions of the present invention may also comprise additional components, such as conventional administration vehicles and excipients including isotonization agents, pH regulators, solvents, solubilizers, colorants, gelling agents and thickeners and buffers, and combinations thereof. Typically, the photosensitizing agent is formulated by mixing it, at an appropriate temperature, for example, at ambient temperatures and at appropriate pHs, and the desired degree of purity, with one or more physiologically acceptable carriers, eg, carriers, which are non-toxic at the doses and concentrations used. In general, the pH of the formulation depends mainly on the particular use, and the concentration of the photosensitizer, but it is in the range preferably anywhere from about 3 to about 8.

Preferably, the photosensitizer is maintained at a pH in the physiological range (for example about 6.5 to 7.5). The presence of salts is not necessary, and therefore the formulation is preferably not an electrolyte solution. Suitable non-antigenic ingredients, such as human serum albumin, can optionally be added in amounts that do not interfere with the photosensitizing agent that is captured by the lens epithelial cells. Because the formulation of the photosensitizing agent is to be applied to the lens capsule during a cataract extraction procedure where leakage is undesirable, it is preferable to use a viscous solution such as a gel, instead of a non-solution. viscose. Preferably, the formulation is prepared such that its viscosity is sufficient to contain the photosensitizing drug substantially within a capsule that has been cut to open widely.

A preferred formulation uses approximately 5% (v / v) green porphyrin formulated in liposomes and approximately 95% (v / v) sodium hyaluronate such as Ophthalin.

The above proportion of the photosensitizing agent to the viscous carrier is preferable, and may be varied depending on the particular viscosity agent and any optional ingredients that are also used in the formulation to be administered. In addition, the same viscosity can be achieved with a variety of different ingredients, such as polysaccharides, preferably a water-soluble polysaccharide, for example, hyaluronic acid, starches and cellulose derivatives (such as methylcellulose, hydroxyethylcellulose, and carboxymethylcellulose). When a polysaccharide is present in a gel formulation, the amount usually present is in the range of about 1-90% by weight of the gel, more preferably, about 1-20%. Examples of other polysaccharides suitable for this purpose and a determination of the solubility of polysaccharides are found in European Patent EP 267,015 published on May 11, 1988. Other thickeners can be added to the formulations of the invention in standard amounts, typically organic cellulose ethers such as hydroxypropylmethylcellulose, or salts of hyaluronic acid such as the sodium salt of hyaluronic acid. Customary shock absorbers can be added in standard quantities. The particular concentration of a given green porphyrin should be adjusted according to its photosensitizing power. For example, BPD-DA can be used but approximately at a concentration five times greater than that of BPD-MA. In addition, BPD can be solubilized in a different way than by formulation in liposomes. For example, reserves of BPD-MA or any other green porphyrin can be diluted in DMSO (dimethyl sulfoxide), polyethylene glycol or any other solvent acceptable for use in the eye.

Still, the ratio between the volume of the drug and the volume of the viscous material should be kept relatively constant, in order to preserve the viscosity and thus the capacity of drug containment in the capsule. Normally, pH adjustment is not required when liposomal BPD-MA is used, since both components (eg, BPD and viscous material) have a neutral pH. However, when solvents other than liposomes are used, the pH must be adjusted before mixing the drug with the viscous material. Bacteriostatic agents approved for use in the eye may be optionally added to the formulation, but antioxidants may interfere with the treatment and thus should be generally avoided. The preparation of formulations that are reconstituted immediately before use, are also contemplated. The preparation of anhydrous or lyophilized formulations of the compositions of the present invention can also be carried out in a known manner, conveniently from the solutions of the invention. The anhydrous formulations of this invention are also storable. By conventional techniques, a solution can be evaporated to dryness under mild conditions, especially after the addition of the solvents for the azeotropic removal of water, typically a mixture of toluene and ethanol. The residue is thereafter conveniently dried, for example for a few hours in a drying oven. Suitable isotonicity agents are preferably nonionic isotonic agents such as urea, glycerol, sorbitol, mannitol, aminoethanol or propylene glycol. In contrast, ionic isotonic agents such as sodium chloride are generally not suitable in the context of this invention. The solutions of this invention will contain the isotonic agent, if present, in an amount sufficient to give rise to the formation of an approximately isotonic solution. The expression "an approximately isotonic solution" will mean in this context, a solution having an osmolarity of approximately 300 milliosmol (mOsm), conveniently 300 + 10% mOsm. It must be taken into account that all the components of the solution contribute to osmolarity. The non-ionic isotonicity agent, if present, is added in customary amounts, for example, preferably in amounts of about 1 to about 3.5 weight percent, preferably in amounts of about 1.5 to 3 weight percent. Solubilizers such as the Cremophor types, preferably Cremophor RH 40, or the Tween types or other customary solubilizers, can be added to the solutions of the invention in standard amounts. A further preferred embodiment of the invention relates to a solution comprising a green porphyrin compound, and a partially etherified cyclodextrin, the ether substituents of which are the hydroxyethyl, hydroxypropyl or dihydroxypropyl groups, a non-ionic isotonicity agent, a buffer and an optional solvent. However, the appropriate cyclodextrins should be of appropriate size and conformation for use with the photosensitizing agents described herein.

Administration of the Green Porphyrin Compound: As noted above, the treatment of the present invention is carried out between the removal of the lens and the introduction of an infra-ocular lens. This represents a relatively short interlude in typical cataract surgery procedures. In this way, the surgery on the eye was performed according to the standard procedures hast? that the crystalline lens has been extracted. The lens cavity is preferably washed with a balanced physiological saline solution, and the green porphyrin composition according to this invention is applied, preferably in a viscous formulation as described above, in order to increase retention within the capsule of the lens and reduce the leak. The preparations of the present invention are preferably administered through a cannula attached to a reservoir such as a syringe. The solution is allowed to act for about 1/2 minute to about 10 minutes, preferably for about 1 minute to about 3 minutes, more preferably for less than about 1 minute, and more preferably for 15 to 30 seconds. After such incubation, the lens cavity is preferably washed once more with the balanced salt solution to remove excess green porphyrin. Appropriate light is applied immediately after washing. Using a suitable light source, preferably a laser or laser diode, in the range of about 550 to about 695 nm, the lens epithelial cells are exposed to light for a period of time sufficient to destroy the residual epithelial cells of the lens. An appropriate and preferred wavelength for such a laser could be 690 ± 12.5 nm at maximum mean. In general, the destruction of epithelial cells occurs within 60 seconds, and is probably sufficiently complete for about 15 to about 30 seconds. Finally, optionally, the lens capsule is washed once more with an appropriately buffered saline solution, and the cataract operation is completed according to standard procedures by insertion, for example, of an artificial lens. Alternatively, the cells within the epithelium of the lens capsule can also be killed by injection of a green porphyrin solution subcapsularly directly into the lens still intact. (hydrodissection). The excess compound is washed together with the fragments and thereafter the irradiation of the target cells with an appropriate light source is carried out. The complete surgical procedure could be modified appropriately. In an alternative and preferred embodiment, the green porphyrin is prepared as a liposomal preparation or is coupled to a ligand, such as a monoclonal antibody, which binds to a specific surface component of the lens epithelial cells to further improve its effectiveness as a photosensitizer. Preferably, the ligand comprises an antibody or an immunologically reactive fragment thereof. As noted above, the ability for the selective localization of a green porphyrin can be further improved during eye surgery by being provided in a composition with a higher viscosity than aqueous preparations, thereby reducing leakage or spillage from the capsule during the surgical procedure. This procedure distributes higher concentrations of the green porphyrin to the target tissue. The dose of the green porphyrin can be optimized by the person skilled in the art depending on the physical distribution system in which it is carried, such as in the form of liposomes, or if this is coupled to a specific ligand of the target, such as an antibody or an immunologically active fragment. It should be noted that the various parameters used for selective photodynamic therapy, effective, in the invention, are interrelated. Therefore, the dose should also be adjusted with respect to other parameters, for example, fluence, irradiation, duration of light used in photodynamic therapy, and the time interval between dose administration and irradiation. therapy. All these parameters must be adjusted to produce significant damage to the remaining epithelial cells of the lens, without causing significant damage to surrounding tissue. Typically, the dose of the green porphyrin will be administered by applying less than about 200 microliters by volume of the formulation to the interior of the lens capsule. More or less of the formulation can be applied depending on the size of the incision made to the lens capsule, the condition of the lens, and the presence or absence of other materials such as blood or wash solutions. In general, one skilled in the art should attempt to provide local exposure to a sufficient amount of a green porphyrin compound, and any excess compound must be washed and in any case no substantial risk of toxicity is present. The concentration of the green porphyrin used is generally in the range of from about 0.2 to about 2.0 mg / ml, preferably from about 0.5 to about 1.5 mg / ml, and even more preferably about 1.0 mg / ml. The above concentrations can be varied by the person skilled in the art so that the parameters of cellular uptake and destruction are consistent with the therapeutic objectives described above. As a result of being irradiated, the green porphyrin in its triplet state is thought to interact with oxygen and other compounds to form reactive intermediates, such as singlet oxygen, which can cause disorganization of cellular structures. Possible cellular targets include the cell membrane, the mitochondria, the lyosomal membranes. The dose of light administered during the PDT treatment contemplated herein may vary, but preferably is in the range of about 10 to about 150 J / cm2. The interval between about 50-100 J / cm2 is preferred. The increase in irradiation can reduce exposure times. The time of light irradiation after administration of the green porphyrin may be important as a way to maximize the selectivity of the treatment, and thereby minimizing damage to the different structures of the target cells and facilitating the conclusion of the surgical procedure. The treatment immediately after the application of the photosensitizer should be generally attempted.

Example 1 Evaluation of BPD Toxicity in Cultured HLE Cells In order to evaluate its complete toxicity, a technique was established to cultivate human lens epithelial cells (HLE), obtained either from donor eyes or from cataract surgeries. The majority of human lens epithelial cells (HLEs) were developed from donor eye lenses 12 to 48 hours post-mortem delivered as posterior poles (corneas previously excised) by the British Bank of Eyes (Eye Bank of British Columbia). Occasionally, complete eyes were supplied ::. Also, excised lenses stored for 1 to 5 hours in cold PBS were occasionally supplied. The lens capsules were harvested from male and female donors in the range of 20 to 72 years of age. In all cases, a circumferential incision was made at the equator (and / or up to 0.5 mm anterior to the equator) in the anterior capsule of the lens. This explanted tissue was placed on the cell side down on a plastic tissue culture box (35 mm) with DME supplemented with 15% FCS. The capsules were then cut into a series of parallel strips of 1 mm width by use of an oscillating movement of a rounded scalpel blade which welded the edges of the capsule strips on the surface of the box. The explants were fed fresh medium every two to three days. Cell development was allowed to continue until the monolayer was confluent or nearly confluent and then 0.25% trypsin in EDTA was passed. The HLE cells were used in any two or three pass. Mono Verde VERO kidney cells (supplied from QLT) were developed under the same media conditions as HLE, and were used before reaching pass 20 of the frozen reserves. In this system, it was determined that a 10 minute incubation with BPD (in 5% fetal calf serum, FCS) at a range of concentrations (0-800 μg / ml) does not affect cell survival (Figure 3).

Example 2 Treatment of HLE Cells with Light After Incubation with BPD The cells of Example 1 were exposed to J / cm2 of red light (690 ± 12.5 nm) distributed with light-emitting diodes (LED), immediately after a 10-minute incubation with BPD, but subsequent to the withdrawal of the excess drug. As anticipated, cell survival was greatly reduced. In the presence of 5% FCS, 400-800 ng of BPD / l followed by red light resulted in 80 to 100% cell death. In related experiments conducted in vi tro, the sensitivity of HLE cells to BPD and light differed, depending on the donors and the number of passages in culture (Figure 4). Cell survival was determined by means of a colorimetric assay, MTT, which measures the metabolic status of the cells and the proliferation thereof, and thus reflects the cytotoxic and cytostatic effects. See, Mossman, "Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays," Journal of Immunol ogi cal Me th ods 65: 55-63 (1983). Therefore, it was of interest to verify if the effects of PDT were of long duration. The results of the experiments, in which the HLE cells were tested for survival either 1 or 20 days (the same culture was divided) after incubation with BPD (in 5% FCS or 25% Amvisc) and exposure at 10 J / cm2 of red light, it showed a comparable cell death (Figure 5). This indicated that the effects of PDT are long lasting. According to the opinions of surgeons in cataracts, even an incubation time of 10 minutes, considered appropriate at the beginning of the study, was unacceptably prolonged. Thus, the effects of PDT were also tested after 1 minute of incubation with BPD. The results of these experiments showed thatAlthough BPD concentrations had to be increased from nanograms to micrograms per milliliter, satisfactory brain death was obtained (Figure 6). One minute of contact of the cells with BPD at 3-5 μg / ml and 10 J / cm2 of red light, resulted in a very substantial cell death. Some individual differences in the sensitivity of the cells were observed (Figure 6). However, these concentrations are relatively low in pharmacological terms and could be easily optimized at a higher level resulting in total cell death. Rapid uptake by cells is very characteristic of BPD. Another photosensitizer, ZnPc, was tested in this system (1 minute of incubation, 10 J / cm2 of light). The highest concentration tested (200 μg / ml), which was limited by the concentration of the ZnPc buffer solution (200 μg / ml in water) resulted in a maximum of 62-78% cell death (Figure 7 ).

Example 3 Treatment of HLE Cells with BPD in a Viscose Solution As noted above, optimally, in a clinical situation BPD will be distributed in a viscous solution. To date, hydroxypropylmethylcellulose (HPMC, Hymecel®) or sodium hyaluronate (SH, Amvisc, Ophthalin, Biolon) are suitable reagents for this purpose and are known to those of relevant skill in the art. Therefore, the photosensitization of the HLE cells with BPD distributed in a viscous solution was evaluated in other vehicles. Due to its high viscosity, only 25% of the Amvisc sodium hyaluronate in a balanced salt solution (BSS, as used in surgery) was tested in an HLE culture system and compared to 5% FCS in the culture medium. of Example 2. The data showed that 10 minutes of incubation of the HLE cells with BPD in any of the solutions followed- by 10 J / cm2 of red light, caused a similar cell death (Figure 8). This indicates that BPD can be effectively distributed to the cells when it is in a viscous solution. The BPD containment in the lens capsule and the prevention of BPD contact with other ocular cells / tissues depends to a certain extent on its diffusion from the viscous solution to any other drug-free viscous solution or balanced saline solution (BBS). which are used by surgeons to maintain eye pressure during surgery. Preliminary in vi tro experiments showed that BPD does not diffuse into BSS quickly either from Hymecel or Amvisc, much less from Amvisc than from Hymecel (Figure 9). Thus, Amvisc or any other sodium hyaluronate solution such as Ophthalin may be preferred vehicles for BPD. The capsules of the rabbit lens with epithelial cells still intact were also treated with BPD at 30 μg / ml in 100% Hymecel. These were incubated for 1 minute and exposed to 10 J / cm2 of red light, resulting in very high cell death (Figure 9).

Example 4 Treatment of Vero cells with BPD in a Viscose Solution Due to the difficulties in obtaining a sufficient number of HLE cells for the test, some experiments were carried out using Vero cells as a model. The cells were developed on coverslips, and treated with BPD in 100% viscous solution (Hymecel, Amvisc and Ophthalin). It was used throughout one minute of incubation, but the doses of light and BPD concentrations were varied. BPD was tested at 5-30 μg / ml using light doses of 1, 5 and 10 J / cm2. Cell survival was determined by fluorescence, using a Cytoprobe Assay Kit (cytosonde) (PerSeptive Bio Science, ethidium homodimer, and calcein-AM). The results indicate death of the HLE cells and are presented in photographs (Figure 10).

Example 5 Preparation of Ophthalmic Formulation A Under aseptic conditions and low light, a bottle of liposomal BPD-MA (concentration of the stock solution of 2 mg / ml) is reconstituted with sterile water for injection. Take 0.1 ml of BPD-MA solution and add 1.9 ml of Ophthalin in a 3 ml glass syringe. In this step, other components may optionally be added, such as agents to facilitate the penetration of the cells or dyes to visualize the formulation during application in the eye. The excess air is removed, and the syringe is connected to another 3 ml glass syringe by means of a stainless steel luer connector. The BPD is mixed with the viscous material by pushing alternately from one syringe to the other, and a minimum of about 30 thrusts may be necessary to obtain a homogeneous formulation. The color of the final formulation is green and contains BPD-MA at a final concentration of approximately 0.1 mg / ml. A 20 gauge needle is attached to the syringe for administration, and the formulation is applied within the lens capsule, after removal of the lens, taking care to cover the equatorial region of the capsule.

Example 6 Preparation of Ophthalmic Formulation B Under aseptic and low light conditions, the pH of a BPD-DA formulation dissolved in PEG 400 (at a concentration of 10 mg / ml) is adjusted to approximately neutrality and then the formulation is sterilized by filtration. Then, 0.1 ml is taken from the sterilized BPD-DA solution and 1.9 ml of Ophthalin is added to it in a 3 ml glass syringe. In this step, other components may optionally be added, such as agents to facilitate the penetration of the cells or dyes to visualize the formulation during application in the eye. The excess air is removed, and the syringe is connected to another 3 ml glass syringe by means of a stainless steel luer connector. The BPD is mixed with the viscous material by pushing alternately from one syringe to the other, and a minimum of about 30 thrusts may be necessary to obtain a homogenous formulation. The color of the final formulation is green and contains BPD-DA at a final concentration of approximately 0.5 mg / ml. A 20 gauge needle is attached to the syringe for administration, and the formulation is applied within the lens capsule, after removal of the lens, taking care to cover the equatorial region of the capsule.

Example 7 In Vivo Application of Formulation A The cytotoxic effect of the BPD-MA irradiation of Example 5 with 690 nm of light is investigated on rabbit lens epithelium. First, the eye of the rabbit to be treated is dilated and an appropriate anesthetic is administered before the operation. The surgical procedure involves a cut at the edge (8 PM) and is performed with a scalpel (anterior chamber stylet blade, 0.9 mm, VISITEO.) Balanced salt solution (BSS) is introduced through a chamber maintainer above, in order to maintain constant pressure in the anterior chamber (based on the height at which the container with BSS is placed on the head of the rabbit). The pressure keeps the camera slightly distended and the iris pressed giving better access to the capsule and more space for manipulation and homeostasis. Next, another entrance is made at the edge (12 AM) using a stylet (0.9 mm in diameter) and a round anterior capsulectomy is performed on the front of the capsule using a blade flexed 25 degrees, on a handle. Using a 3 mm keratome, the opening at 12 AM is enlarged. (This opening is used for the introduction of instruments into the anterior chamber and all manipulations). The phacoemulsifier is then introduced and the lens is systematically removed (this part of the procedure takes approximately 1 to 1.5 minutes). After phacoemulsification the cornea is covered with a viscous drug-free solution for the protection of the spilled BPD. The BPD in viscous solution is applied by systematic coating to the wall of the equatorial capsule, using the Raycroft cannula and a syringe. Special care is taken to cover the entire area, and especially the equatorial region. After the incubation (1 minute, longer times may be used), the BPD is washed using the BSS of the anterior chamber maintainer and the aspiration in the pupil and the bag.

Theoretically, the luminous device must be introduced through the opening at 12 AM and must be such as to allow its introduction into the capsule and irradiation mainly from the equatorial region with very limited illumination of the iris, cornea and the retina (depth expected from the equatorial region from the front of the capsule with the anterior chamber maintainer (ACM) = 1 mm). The irradiation 10 J / cm2 is applied by laser diode for 30 seconds to kill the remaining epithelial cells of the lens. The rest of the surgical procedure is completed by conventional protocols. Based on the description and previous implementation, someone of skill in the art could develop and use a variety of appropriate formulations for the prevention of secondary cataracts. Such formulations may utilize photosensitizing agents other than green porphyrins, as long as the agents are rapidly captured by the remaining epithelial cells of the lens, their formulations may be substantially contained within the interior of the capsule and cause epithelial cell death after the administration of a relatively brief exposure to an appropriate dose of light. Accordingly, the scope of the present invention is defined by the following claims, and not by the foregoing examples. All publications cited above are incorporated by reference.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following claims is claimed as property:

Claims (30)

1. A method for preventing secondary cataracts in the eye of a subject, the method is characterized in that it comprises: the administration, to the lens capsule of a subject, of an amount of a sufficient green porphyrin to allow an effective amount to be located in the lens epithelial cells; allowing a sufficient time to allow an effective amount of the green porphyrin to be located in the lens epithelial cells; and irradiating the lens epithelial cells with light absorbed by the green porphyrin, at a level of energy sufficient to substantially destroy all the epithelial cells of the lens.

2. The method according to claim 1, characterized in that the lens epithelial cells are those that remain after lens removal during cataract surgery.

3. The method according to claim 2, characterized in that a viscous solution is applied to protect the cornea before administration of the green porphyrin.

4. The method according to claim 2, characterized in that the green porphyrin is combined with a viscosity-increasing agent.

5. The method according to claim 4, characterized in that the viscosity enhancing agent is selected from the group consisting of hyaluronic acid and its derivatives, starches and cellulose and their derivatives.

6. The method according to claim 1, characterized in that the effective amount is a green porphyrin formulation which is administered in the range of from about 0.2 to about 10 mg / ml and about 150 to about 250 μl.

7. The method according to claim 1, characterized in that the effective amount is a dose administered in the range of about 0.03 to about 2.5 mg.

8. The method according to claim 7, characterized in that the effective amount is approximately 0.1 mg.

9. The method according to claim 1, characterized in that the green porphyrin is administered in a liposomal formulation.

10. The method according to claim 1, characterized in that the green porphyrin is of the formulas 1-6, as shown in figure 1, wherein R1, R2, R3 and R4 are substituents that do not interfere.

11. The method according to claim 10, characterized in that R1 and R2 are independently carbomethoxy or carboethoxy.

12. The method according to claim 10, characterized in that each R3 is -CH2CH2COOH or a salt, amide, ester or acylhydrazone thereof.

13. The method according to claim 10, characterized in that the green porphyrin has the formula 3 or 4 in Figure 1, wherein R4 is a substituent that does not interfere.

14. The method according to claim 2, characterized in that the green porphyrin is selected from the group consisting of BPD-DA, BPD-DB, BPD-MA, and BPD-MB and its derivatives.

15. The method according to claim 14, characterized in that the green porphyrin is BPD-DA.

16. A pharmaceutical composition for preventing or inhibiting the development of secondary cataracts, characterized in that the composition comprises: an amount of an effective green porphyrin to prevent or inhibit the development of secondary cataract when administered to a subject suffering from cataract surgery; and a pharmaceutically acceptable carrier or excipient.

17. The composition according to claim 16, characterized in that the green porphyrin is BPD.

18. The composition according to claim 16, characterized in that it also comprises a visible dye or enzyme to facilitate the uptake of the formulation by the lens epithelial cells.

19. The composition according to claim 18, characterized in that the green porphyrin is combined with a viscosity-increasing agent.

20. The composition according to claim 19, characterized in that the viscosity enhancing agent is selected from the group consisting of hyaluronic acid and derivatives, starches and cellulose and their derivatives.

21. The composition according to claim 19, characterized in that the effective amount is a green porphyrin formulation with a concentration in the range of about 0. 2 to about 10 mg / ml and administered from about 150 to about 250 μl.

22. The composition according to claim 19, characterized in that the effective amount is a dose administered in the range of from about 0.03 to about 2.5 mg.

23. The method according to claim 22, characterized in that the effective amount is approximately 0.1 mg.

24. The composition according to claim 19, characterized in that the green porphyrin is administered in a liposomal formulation.

25. The composition according to claim 19, characterized in that the green porphyrin is of the formulas 1-6, as shown in figure 1, wherein R1, R2, R3 and R4 are substituents that do not interfere.

26. The composition according to claim 25, characterized in that R1 and R2 are independently carbomethoxy or carboethoxy.

27. The composition according to claim 25, characterized in that each R3 is -CH2CH2COOH or a salt, amide, ester or acylhydrazone thereof.

28. The composition according to claim 25, characterized in that the green porphyrin has the formula 3 or 4 in Figure 1, wherein R4 is a substituent that does not interfere.

29. The composition according to claim 25, characterized in that the green porphyrin is selected from the group consisting of BPD-DA, BPD-DB, BPD-MA and BPD-MB and its derivatives.

30. The composition according to claim 29, characterized in that the green porphyrin is BPD-DA.