WO2006119211A2 - Methods and compositions for treating ocular disorders - Google Patents

Abstract

Aminosterol compounds are described that are useful in combination with photodynamic therapy with verteporfin, rostaporfin or other photo-activated compounds for treating ocular disorders. Compositions comprising such aminosterols and photo-activated compounds and methods of using such aminosterols compounds in combination with photodynamic therapy with photo-activated compounds are also employed for treating ocular disorders.

Description

Title: METHODS AND COMPOSITIONS FOR TREATING OCULAR DISORDERS

Inventors: Michael McLane, Roger Vogel, Avinash Desai, Kenneth Holroyd and Roy C. Levitt

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application 60/676,284 (filed May 2, 2005). U.S. Provisional Application 60/676,284 is related to U.S. Patent Application 09/985,417 (filed November 2, 2001, now U.S. Patent 6,962,909), which is a continuation of U.S. Patent Application 09/198,486 (filed on November 24, 1998, now abandoned), which is a continuation of U.S. Patent Application 08/487,443 (filed June 7, 1995, now U.S. Patent 5,847,172). Each of these documents is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical compositions useful in treating ocular disorders. The invention is also directed to pharmaceutical compositions comprising a combination therapy and their use for inhibiting neovascularization, especially as it relates to the mammalian eye. The invention is further directed to pharmaceutical combinations useful in treating age related macular degeneration.

BACKGROUND OF THE INVENTION

The aminosterol squalamine is the subject of U.S. Patent 5,192,756 to Zasloff et ah, the disclosure of which is herein incorporated by reference. This compound is a broad-spectrum antibiotic, killing bacteria, fungi and protozoa. The absolute stereochemistry for squalamine (represented as compound 1256) is shown in U.S. Patent 5,192,756. The total chemical synthesis of squalamine is described in U.S. Patents 6,262,283 and 6,610,866, which are incorporated herein by reference in their entirety.

Squalamine was originally isolated from the liver of the dogfish shark, Squalus acanthias, and was produced synthetically as squalamine lactate. Squalamine was originally identified as a broad-spectrum antibiotic in vitro, with activity against gram negative and gram-positive organisms as well as fungi and protozoa. Squalamine was also found to be an inhibitor of new blood vessel formation (anti-angiogenic) as judged by a set of in vifro and in vivo assays described in U.S. Patents 5,792,635 and 5,721,226 which are incorporated herein by reference in their entirety.

Following discovery of its anti-angiogenic activity, squalamine lactate (i.e., the salt formed by combining squalamine with lactic acid) became a development candidate for treatment of advanced malignancy, and most recently for the treatment of age-related macular degeneration (AMD). An extensive battery of nonclinical pharmacodynamic and toxicology studies have been conducted to support clinical development of squalamine lactate for the treatment of solid tumors described in U.S. Patents 6,147,060 and 6,596,712, which are incorporated herein by reference in their entirety. Multiple Phase 1 and 2 human clinical trials evaluating squalamine lactate for the treatment of non-small cell lung cancer, ovarian cancer, and other adult solid tumors have been performed.

AMD is the leading cause of irreversible central vision loss among people in the United States aged 52 or older and is the most common overall cause of blindness in the United States, Canada, Great Britain and Australia. AMD encompasses several types of abnormalities that develop in the macula of affected individuals. Two forms of macular degeneration exist: dry (also known as atrophic) and wet (also known as disciform, exudative, subretinal neovascular or choroidal neovascular). The dry form, which may be a precursor to the wet form, results from an inability of the pigment epithelium of the macula to remove waste materials generated by the retina. The wet form occurs when new blood vessels begin to grow under the retina, particularly the macula. Since angiogenesis has been implicated in the development of AMD, inhibition of choroidal neovascularization should preserve and/or improve vision. Squalamine lactate, with its anti- angiogenic properties, has been identified for injection as a development candidate for the treatment of patients with subfoveal choroidal neovascularization associated with AMD.

Another treatment of wet macular degeneration involves the technique known as photodynamic therapy (PDT). In order to understand this treatment approach, it is important to remember that the difficulty with the wet form of macular degeneration is the growth of abnormal blood vessels beneath the retina, which leak fluid and bleed. The fluid and blood cause scar formation that damages the vision in patients with this disease. The concept of PDT is to selectively close the abnormal blood vessels, eliminating the leakage and bleeding, and thus stabilizing or improving the vision. This is accomplished without the damaging effect of a conventional laser on the normal structures of the retina and back of the eye. Photodynamic therapy is typically a two-step process. In an exemplary embodiment, a patient receives in a first step an injection of a photosensitive drug containing a photo-activated agent. In one such treatment, the photo-activated agent comprises a dye. In an exemplary embodiment, the dye is verteporfin, which forms part of the pharmaceutical formulation known as Visudyne^® (i.e., liposomal BPD-MA, verteporfin). Visudyne^® may be introduced through a vein in the hand or arm of the subject. In another treatment example, the dye is rostaporfin, which foπns part of the pharmaceutical formulation known as Photrex™. These dyes have unique properties, which allow them to be effectively used for this treatment. Specifically, the dye circulates through the body and sticks to the walls of the blood vessels beneath the macula. At this point in the procedure, a laser is used to shine a light into the back of the eye. The energy produced by this laser is of a very low power and is not damaging like that of regular laser treatment. Instead, the light from the laser simply activates the dye that is bound to the abnormal blood vessel wall. When the light beam activates the dye such as verteporfin, there is closure of the blood vessel. The result of this action is that the fluid and blood that had been leaking beneath the retina is stopped. Over time, the body is able to absorb the blood and fluid, which results in stabilization or improvement in visual function. The blood vessel itself has not been completely destroyed, but rather is no longer leaking or actively growing.

A major draw back of photodynamic therapy for the treatment of choroidal neovascularization is the induction of the angiogenic agent VEGF, leading to a post-treatment angiogenic response with malperfusion that is maximal approximately one week after treatment. (Schmidt-Erfurth et al. (2003) Invest Ophthalmol Vis Sci. 44, 4473-4480). Squalamine has been shown to inhibit VEGF induced neovascularization and therefore is proposed for use in combination with photodynamic therapy for the treatment of AMD.

SUMMARY OF THE INVENTION

In one embodiment of the invention, the aminosterols described herein may be used in combination therapy with at least one other therapeutic agent that is useful in PDT. In other embodiments, the invention encompasses pharmaceutical compositions comprising a combination of a photo-activated agent and an aminosterol described herein. In an exemplary embodiment, the photo-activated agent is a benzoporphyrin derivative. In a particular embodiment, the photo- activated agent is verteporfin. In another particular embodiment, the photo-activated agent is rostaporfin. In an exemplary embodiment, the invention encompasses a pharmaceutical composition comprising a combination of a photo-activated agent and squalamine. Such a composition is suitable for use in a human undergoing PDT and in need of such therapy (e.g. , is suffering from one or more ocular disorders as described herein). In a particular embodiment, the photo-activated agent is verteporfm or rostaporfm.

In another exemplary embodiment, the invention encompasses treating or preventing disorders of a mammalian eye including, but not limited to, subfoveal choroidal neovascularization, age related macular degeneration, pathologic myopia, and presumed ocular histoplasmosis, comprising administering a benzoporphyrin derivative, preferably verteporfm, and an aminosterol described herein for use in a human undergoing photodynamic therapy. In a particular embodiment, the invention encompasses treating or preventing disorders of the eye comprising administering a composition comprising squalamine and verteporfϊn or rostaporfin for use in photodynamic therapy.

In various exemplary embodiments the photo-activated agent and/or squalamine is administered intravenously, intraoccularly and/or orally. In an exemplary embodiment, the photo-activated agent is administered in an amount of from about 0.01 mg to about 20 mg. In another exemplary embodiment, the squalamine is administered in an amount of from about 0.001 mg to about 100 mg.

In an exemplary embodiment, squalamine is administered simultaneously with the photo- activated agent. In another exemplary embodiment, squalamine is administered subsequent to the administration of the photo-activated agent or subsequent to exposing the eye of the subject to an energy source. In another embodiment the squalamine is administered prior to the administration of the photo-activated agent. In any of these embodiments, the subject may treated more than one time. In a particular embodiment, the photodynamic therapy utilizes a laser that is a non-theπnal laser.

In an exemplary embodiment, the anti-angiogenic aminosterol is used in combination with another anti-angiogenic compound. In a particular embodiment, the aminosterol is squalamine, including, but not limited to, squalamine lactate and other pharmaceutically acceptable salts.

BRIEF DESCRIPTION OF THE FIGURES

The following figures are simply illustrative of exemplary embodiments of the invention and are not intended to define the scope of the invention. FIG. IA, IB and 1C illustrate that endothelial cells exhibit greater sensitivity to squalamine (bar above 3 on the x-axis) than to other membrane-active agents, and that endothelial cells are more sensitive to squalamine than are epithelial cells and fibroblasts. FIG. IA is for the administration of 1 μg/ml of the agent against bovine pulmonary endothelial cells, whereas FIGS. IB and 1C are for administration of 10 μg/ml of the membrane-active agents to human epithelial cells and to human foreskin fibroblasts, respectively.

FIG. 2 A, 2B and 2C illustrate the suppression of the growth of murine melanoma, through the subcutaneous, intraperitoneal or oral administration of squalamine respectively.

FIG. 3 illustrates the suppression of murine melanoma in mice by intraperitoneal administration of compound 319.

FIG. 4 demonstrates the suppression of the growth of human melanoma 1205 Lu in immunocompromised (RAG-I) mice by administration of squalamine at various dosages ("o" = 10 mg/kg/d, "+" = 20 mg/kg/d, "•" = 40 mg kg/d; d=day).

FIG. 5 illustrates that squalamine and compound 1436 exhibit synergy in suppressing growth of murine melanoma in mice.

FIG. 6A illustrates that squalamine significantly (pO.OOl) inhibits hypoxia-induced retinopathy compared to no drug treatment in an oxygen-induced retinopathy mouse model.

FIG. 6B illustrates that squalamine significantly (p<0.001) inhibits neovascular nuclei compared to no drug treatment in the oxygen-induced retinopathy mouse model.

FIG. 7 illustrates inhibition of the growth of VEGF-activated bovine retina endothelial cells (BREC) by squalamine.

FIG. 8 illustrates human visual acuity data including improved vision and OCT data including improved central retinal thickness in one patient.

FIG. 9 illustrates human visual acuity data including improved vision and OCT data including improved central retinal thickness in one patient. DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term "therapeutically effective amount" means an amount of an aminosterol described herein and a photo-activated agent in combination that is sufficient to provide the desired local or systemic effect and performance at a reasonable benefit/risk ratio attending the medical treatment. Such an effect may include the inhibition, totally or partially, of the progression of the condition or the alleviation, at least partially, of one or more symptoms of the condition. A therapeutically effective amount may also be an amount that is prophylactically effective. In an exemplary embodiment, a "therapeutically effective amount" is an amount that will suppress or inhibit VEGF-induced neovascularization commonly seen following photodynamic therapy. In another exemplary embodiment, a "therapeutically effective amount" is an amount effective to inhibit angiogenesis in the eye of a mammal that has undergone PDT. The amount that is therapeutically effective will depend upon the patient's size and gender, the condition to be treated, the severity of the condition and the result sought. For a given patient, a therapeutically effective amount can be determined by methods known to those of skill in the art.

As used herein, "squalamine lactate" refers to the compound formed from the combination of lactic acid with squalamine. Squalamine lactate typically contains about 15 to about 25 mole percent of lactic acid. In an exemplary embodiment, the squalamine lactate exists primarily as squalamine dilactate (i.e., two molecules of lactic acid per molecule of squalamine).

As used herein, "photodynamic therapy" or "PDT" refers to the use of the combination of light and light sensitive (i.e., photo-activated) agents (such as porphyrins) in an oxygen-rich environment to selectively destroy or disable certain cells that are typically associated with a disease state.

Aminosterol Compounds and Photo-activated Compounds

In an exemplary embodiment, the aminosterol is squalamine or a pharmaceutically acceptable salt thereof.

In an exemplary embodiment, the photo-activated agent is verteporfin, which belongs to a class of compounds known as benzoporphyrins. Verteporfin is a 1:1 mixture of two regioisomers (I and II). The chemical names for the verteporfin regioisomers are: 9-methyl (I) and 13-methyl (II) trans-(+/-)- 18-ethenyl-4,4a-dihydro-3,4-bis-(methoxycarbonyl)-4a,8, 14, 19-tetramethyl-23H,25H- benzo[b]porphine-9, 13-dipropanoate. In another exemplary embodiment, the photo-activated agent includes, but is not limited to, MV9411, MV6401 or rostaporfin (also known as SnET2). Rostaporfin belongs to a class of compounds known as purpurins. U.S. Patent 6,376,483 describes photo-activated agents and is incorporated by reference in its entirety.

Photodvnamic Therapy

The invention also encompasses a photodynamic therapy. U.S. Patents 6,849,058; 6,800,086; 6,609,014; 6,548,542; 6,248,734; 6,107,325; 6,100,290; 6,096,776; 6,013,053; 6,008,241; 5,910,510 and 5,756,541, each of which are incorporated by reference in their entirety, disclose various aspects of PDT. In particular, the invention described herein encompasses compositions comprising a therapeutically effective amount of an aminosterol and a photo-activated agent, such as a benzoporphyrin derivative, for use in a PDT. The invention also encompasses methods to improve visual acuity using photodynamic treatment methods. The photodynamic therapeutic compositions and methods of use thereof encompassed by the invention are effective in decreasing unwanted neovasculature, especially neovasculature of the choroid. Accordingly, in an exemplary embodiment, the invention encompasses methods to enhance visual acuity, which comprise administering to a subject in need of such treatment a therapeutically effective amount of an aminosterol described herein and a photo-activated agent sufficient to permit an effective amount of these compounds to localize in the eye of the subject, and irradiating the eye with light of a frequency typically absorbed by the photo-activated agent.

In a preferred embodiment, the aminosterol is squalamine and the photo-activated agent is verteporfm or rostaporfin. The invention also encompasses the use of aminosterols and a photo- activated agent in PDT. In a particular embodiment, the invention encompasses use of verteporfin or rostaporfin and squalamine in photodynamic therapy for disorders of the eye including, but not limited to, subfoveal choroidal neovascularization, age related macular degeneration, pathologic myopia, and presumed ocular histoplasmosis.

The invention also encompasses a three-step procedure that can be performed on an outpatient basis. First, the light-sensitive drug is injected intravenously into the subject's arm. As the drug flows naturally through the body's vascular system, it will pass into the abnormal blood vessels in the macula at the back of the eye. A number of energy sources can be used to activate the drug in the patient's eyes by focusing the energy source on the lesion or site to be treated. These energy sources include: a broad spectrum energy source or intense light, or a monochromatic energy source including, for example, a laser, such as a non-thermal or "cold" laser, or thermal laser. Prior to or subsequent to exposure of the eye of the subject to an energy source, the subject can receive administration of squalamine. Administration can be a one-time administration or can be prescribed as a longer-term treatment. In yet another embodiment, the invention encompasses a multi-course therapy, wherein the subject can receive more than one PDT treatment, squalamine, or laser treatment if the practitioner believes it will be beneficial to the subject.

The PDT of the invention can be used to treat various disorders of the eye including, but not limited to AMD and preferably for treatment of wet AMD, a disease that involves abnormal blood vessel growth in the macula. In another embodiment, PDT with a photo-activated agent and squalamine can stabilize vision and significantly reduce the risk of vision loss in certain subjects with wet AMD. Subjects are preferably mammals and more preferably humans. Additionally, the invention encompasses PDT with a photo-activated agent and an aminosterol to help slow abnormal vessel growth in subjects with wet AMD.

In an exemplary embodiment, PDT is a multistep process, wherein a subject is administered a photo-activated agent and an aminosterol such as squalamine. Administration of the aminosterol can be simultaneous, prior to, immediately after, or subsequent to administration of the photo- activated agent. In a particular embodiment, the photo-activated agent is administered intravenously, intravitreally or intraoccularly followed by squalamine. The subject's eye is then subjected to a non-thermal or "cold" laser. Optionally, administration of the aminosterol may be continued for a necessary period as prescribed by a medical or veterinary practitioner. In an exemplary embodiment, the amount of photo-activated agent administered is from about 0.01 mg to about 50 mg. In another embodiment, the amount administered is from about 0.1 mg to about 30 mg. In another embodiment, the amount administered is from about 1 mg to about 15 mg.

In an exemplary embodiment, the amount of squalamine administered is from about 0.01 mg to about 100 mg. In another embodiment, the amount of squalamine administered is from about 0.1 mg to about 50 mg. In another embodiment, the amount of squalamine administered is from about 1 mg to about 40 mg. However, different photo-activated agents require different dosage ranges. For example, if green porphyrins are used, a typical dosage is in the range of about 0.1 to about 50 mg/m² (of body surface area). In a particular embodiment, the dosage is from about 1 to about 10 mg/m². In another particular embodiment, the amount of squalamine administered is from about 2 to about 8 mg/m². Thus, the dose of photoactive compound can vary widely depending on the mode of administration and the formulation in which it is carried, such as in the form of liposomes. These parameters can readily be determined by the treating physician. For example, without being limited by theory, it is generally recognized that there is a nexus between the type of aminosterol and photo-activated agent, the formulation, the mode of administration, the relative time duration between administrations, and the dosage level. Adjustment of these parameters to fit a particular combination is possible and will depend on the physical and physiological parameters of subject to be treated and the extent of treatment required, which can readily be determined by the physician treating the subject.

The aminosterols and photo-activated agents or combinations thereof can be administered in any of a wide variety of ways, for example, orally, parenterally, or rectally, or the compound may be placed directly in the eye. In an exemplary embodiment, the route of administration is parenteral, such as intravenous, intramuscular, or subcutaneous. In a particular embodiment, the route of administration is intravenous injection.

The various parameters used for effective, selective photodynamic therapy in the invention are interrelated. Therefore, the dose should also be adjusted with respect to other parameters, for example, fluence, irradiance, duration of the light used in photodynamic therapy, and time interval between administration of the dose and the therapeutic irradiation. All of these parameters should be adjusted to produce significant enhancement of visual acuity without significant damage to the eye tissue and the treating physician can readily determine these parameters. After the aminosterols and photo-activated agents or combinations thereof have been administered, the target ocular tissue is optionally irradiated at the wavelength absorbed by the agent selected. The absorption spectra for benzoporphyrin derivatives are known in the art. For example, for green porphyrins the desired wavelength range is generally between about 550 and 695 nm. In an exemplary embodiment, a wavelength in this range is utilized for enhanced penetration into bodily tissues.

As a result of being irradiated and without being limited by theory, it is believed that the photoactive compound in its excited state is thought to interact with other compounds to form reactive intermediates, such as singlet oxygen, which can cause disruption of cellular structures. Possible cellular targets include the cell membrane, mitochondria, lysosomal membranes, and the nucleus. Evidence from tumor and neovascular models indicates that occlusion of the vasculature is a major mechanism of photodynamic therapy, which occurs by damage to endothelial cells, with subsequent platelet adhesion, degranulation, and thrombus formation. The fluence during the irradiating treatment can vary widely, depending on type of tissue, depth of target tissue, and the amount of overlying fluid or blood. In an exemplary embodiment, the fluence varies from about 50 to about 200 Joules/cm², hi an exemplary embodiment, the irradiance varies from about 150 to about 900 mW/ cm². In another embodiment, the range varies from between about 150 to about 600 mW/cm². The use of higher irradiances may be selected as effective and having the advantage of shortening treatment times. The optimum time following administration of the photoactive agent until the treatment by light can also vary widely depending on the mode of administration, the foπn of administration and the specific ocular tissue being targeted. In an exemplary embodiment, the times after administration of the photoactive agent range from about 1 minute to about 2 hours. In another embodiment, the times range from about 5 to about 30 minutes. In yet another embodiment, the time ranges from about 10 to about 25 minutes.

The duration of light irradiation depends on the fluence desired. For example, for an irradiance of 600 mW/cm², a fluence of 50 J/cm² requires 90 seconds of irradiation, while 150 J/cm² requires 270 seconds of irradiation.

Combination Therapies

In an exemplary embodiment of the invention, the aminosterols described herein can be used in combination therapy with at least one other therapeutic agent

In a particular embodiment of PDT, the therapeutic agent that is used in combination with an aminosterol of the invention is verteporfin. In another embodiment, the therapeutic agent is another anti-angiogenic agent such as another aminosterol. The aminosterol and the therapeutic agent can act additively or synergistically. In one embodiment, a composition comprising an aminosterol described herein is administered concurrently with the administration of another therapeutic agent, which can be part of the same composition as an aminosterol described herein or a different composition. In a preferred embodiment, a composition comprising the aminosterol is administered prior or subsequent to administration of another therapeutic agent. As many of the disorders for which the aminosterols are useful in treating are chronic disorders, in one embodiment combination therapy involves alternating between administering a composition comprising an aminosterol described herein and a composition comprising another therapeutic agent (e.g., to minimize the toxicity associated with a particular drug). The duration of administration of each drug or therapeutic agent can be, for example, one week, one month, three months, six months, or a year, hi certain embodiments, when an aminosterol described herein is administered concurrently with another therapeutic agent that potentially produces adverse side effects including, but not limited to, toxicity, the therapeutic agent can advantageously be administered at a dose that falls below the threshold at which the adverse side effects are elicited.

In still another embodiment, the invention encompasses a combination therapy comprising a photodynamic therapy.

Administration of the Aminosterols

The patient to be treated can be any animal, and is preferably a mammal. More preferably, the patient is a human, including a human suffering from an ocular disease.

Pharmaceutical compositions for use in vitro or in vivo in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Examples of carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, phospholipids, liposomal carriers, gelatin and polymers such as polyethylene glycols.

One example of a pharmaceutical carrier for the aminosterols of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied. For example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and sugars or polysaccharides, such as dextrose.

In addition to carriers, the pharmaceutical compositions of the invention may also include stabilizers and preservatives. For an exemplary listing of typical carriers, stabilizers and adjuvants known to those of skill in the art, see Gennaro (2005) Remington: The Science and Practice of Pharmacy. Mack Publishing.

Pharmaceutically acceptable salts of the aminosterols of the invention include the conventional non-toxic salts or the quaternary ammonium salts which are formed from inorganic or organic acids or bases. Examples of such acid addition salts include acetate, adipate, benzoate, benzenesulfonate, citrate, camphorate, dodecylsulfate, hydrochloride, hydrobromide, lactate, maleate, methanesulfonate, nitrate, oxalate, pivalate, propionate, succinate, sulfate and tartrate. Base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts and salts with amino acids such as arginine. Also, the basic nitrogen- containing groups may be quaternized with, for example, alkyl halides. In addition to carriers, the pharmaceutical compositions of the invention may also include stabilizers and preservatives.

The active aminosterol may be administered alone or preferably as a pharmaceutical formulation comprising the aminosterol together with at least one pharmaceutically acceptable carrier. Optionally, other therapies known to those of skill in the art may be combined with the administration of the aminosterols of the invention. More than one aminosterol may be present in a single composition.

In vivo administration of the aminosterols of the invention can be effected in one dose, multiple doses, continuously or intermittently throughout the course of treatment. In an exemplary embodiment, the dose ranges from about 0.05 mg/kg to about 5 mg/kg in single or divided daily doses. In another embodiment, the dose ranges between about 0.5 mg/kg to about 1 mg/kg in single or divided daily doses. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated and the subject being treated. Single or multiple administrations may be carried out with the dose level and pattern being selected by the treating physician.

Pharmaceutical compositions containing the aminosterols of the invention can be administered by any suitable route, including oral, rectal, intranasal, topical (including transdermal, aerosol, buccal and sublingual), parenteral (including subcutaneous, intramuscular, intravenous, intraocular), intraperitoneal and pulmonary. It will be appreciated that the preferred route will vary with the condition and age of the subject, and the disease being treated. For treatment of age-related macular degeneration, for example, the preferred routes of administration are oral, topical, subcutaneous, intramuscular and/or intravenous.

For oral administration, the aminosterols can be formulated readily by combining them with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by combining the active compound with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross- linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Pharmaceutical compositions for topical administration of the aminosterols of the invention may be formulated in conventional ophthalmologically compatible vehicles, such as, for example, an ointment, cream, suspension, lotion, powder, solution, paste, gel, spray, aerosol or oil. These vehicles may contain compatible preservatives such as benzalkonium chloride, surfactants such as polysorbate 80, liposomes or polymers such as methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone and hyaluronic acid, which may be used for increasing viscosity. For diseases of the eye, preferred formulations are ointments, gels, creams or eye drops containing at least one of the aminosterols of the invention which can be administered to the eye.

For administration by inhalation, the aminosterols for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The aminosterols can be formulated for parenteral administration by injection, e.g., bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as buffers, bacteriostats, suspending agents, stabilizing agents, thickening agents, dispersing agents or mixtures thereof.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. In a preferred embodiment, the aminosterols are dissolved in a 5% sugar solution, such as dextrose, before being administered parenterally.

For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks 's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

EXAMPLES

A. Anti-angiogenic Effects of Aminosterols-J« Vitro Studies

1. Growth Inhibition of Endothelial Cells. Fibroblasts and Epithelial Cells When non-transformed human cells are grown in the presence of increasing concentrations of squalamine, endothelial cells exhibit a particular sensitivity to squalamine, as shown by the following experiment. Bovine pulmonary endothelial cells, human epithelial cell line MCF 1OA, and human foreskin fibroblasts were incubated in the presence of 12 different membrane-active agents, including peptides and squalamine.

Specifically, cells were incubated in the presence of the following twelve membrane-active agents: (1) RGD[KIAGKIA]3-NH2; (2) d-[KKLLKKL]2-NH2; (3) squalamine; (4)

SWLSKTAKKLENSAKKRISEGIAIAIQGGPR; (5) FLGGLIKIVPAMICAVTKKC; (6) Magainin 2; (7) PGLA; (8) GFASFLGKALKAALKIGANLLGGTPQQ; (9) PR-39; (10) 1- [KKLLKKL]2-NH2 (11) Cecropin B; and (12) [KIAGKIA]3-NH2. Cell growth was measured by absorbance at 600 nm. Results are shown in FIGS. 1A-1C. As evident from FIG. IA, squalamine inhibited the growth of bovine pulmonary artery endothelial cells (BPE) at 1 μg/ml. In contrast, at 10 μg/ml it exerted no effect on the growth of either epithelial (FIG. IB) or fibroblast (FIG. 1C) lines. However, peptides that inhibited the growth of epithelial cells exhibited no effect on BPE. Thus, endothelial cells, which form the lining of blood vessels, are more sensitive to squalamine than are either fibroblasts or epithelial cells.

2. Inhibition of Endothelial Cell Cord Formation in vitro

Endothelial cells have the capacity in vitro to form tubular aggregates resembling capillaries in various early stages of formation. This conversion occurs under relatively specific conditions, in which essential growth factors along with an effective substratum are provided. It has been shown that both the interaction of growth factors with the endothelial cell and its attachment to a substratum activate the NHE. The activation of this exchanger is believed to be required for subsequent morphologic transformation of the endothelial cell into a multicellular tubular structure.

To assess the effect of compounds on the cord-like structures formed by human microvascular cells when plated in the presence of VEGF (Vascular Endothelial Growth Factor) and basic fibroblast growth factor on a collagen matrix, a standard cord formation assay was used. The results are shown in Table 1 below.

TABLE 1

Effect of Various Aminosterols on Endothelial Cord Formation

0.01 μg/ml 0.1 μg/ml 1.0 μg/ml 10.0 μg/ml

Fumagillin — +/- + squalamine - + + +

Compound 319 - + + +

Compound 353 + + + Compound 410 - + +* Compound 411 - - + Compound 412 - - + Compound 413 - - + Compound 415 - - +/T Compound 371 T T

Compound 432 - -

Compound 449 - +/-

Compound 467 __.₌ _₌_₌______--₌ - -

Notes:

+ = Inhibition of angiogenesis;

- = No inhibition of angiogenesis;

T = Toxic

* = cell rounding @ 10 μg/ml.

As shown in Table 1, squalamine inhibits cord formation at about 0.1 μg/ml, compared with fumagillin, which exhibits comparable activity at 10 μg/ml. At these concentrations, squalamine does not appear to profoundly affect cell viability or proliferation. This property in vitro roughly correlates with anti-angiogenic activity in more complex in vivo models (see Goto et al. (1993) Lab Investigation 69, 508-518).

3. Specific inhibition of VEGF stimulated endothelial cells by squalamine The specificity of squalamine for activated endothelial cells was evaluated in culture using bovine retina endothelial cells (BREC). The endothelial cell line was grown in DMEM containing ten percent fetal bovine serum, 1% L-glutamine, 25 mM HEPES and genticine with or without 20 ng/mL vascular endothelial growth factor (VEGF) in the presence of squalamine. The growth factor was added to the cells prior to the addition of squalamine. Squalamine exposure was observed to specifically inhibit VEGF-stimulated endothelial cell proliferation for BREC, but not unstimulated cells. The degree of inhibition of VEGF-stimulated BREC, increased with increasing squalamine concentration. For example, the net growth of BREC was inhibited by more than 30% with 7 pg/mL squalamine, and at 21 pg/mL, inhibition was more than 70% (FIG. 7). By contrast, squalamine had no effect on the survival or growth of unstimulated BREC (FIG. 7).

B. Anti-angiogenic Effects of Aminosterols-/« Vivo Studies

3. The Chorioallantoic Membrane Model

Using the classical chorioallantoic membrane model, it has been found that squalamine is an inhibitor of capillary growth. The growing capillaries within the chorioallantoic membrane model (CAM model) have been used as a system in which to evaluate the effect of agents on their potential to inhibit new vessel growth. Neovascularization occurs most aggressively over the first week of embryonic development. Thereafter capillary growth is characterized by principally "elongation" rather than "de novό" formation.

In the standard assay, agents are applied locally to a region of the embryo over which neovascularization will occur. Agents are assessed by their ability to inhibit this process, as evaluated by visual examination about 7 days after application. Agents which disrupt vascular growth during the period of de novo capillary formation, but do not interfere with subsequent capillary growth, are generally regarded as "specific" inhibitors of neovascularization, as distinguished from less specific toxic substances. The assay utilized is described in detail in Auerbach et al. (1991) Pharm. Ther. 51, 1-11. Results are tabulated below in Table 2.

As seen from Table 2, applying as little as 0.65 μg squalamine to a 3-day CAM resulted in inhibition of CAM vessel neovascularization. In contrast, applying ten times that amount of squalamine onto a 13 -day old chick exerted no inhibitory effect.

Thus, in a classical angiogenesis assay, squalamine exhibited potent but specific inhibitory activity, equal in potency to the most active compounds described to date in the literature. The effect is compatible with suppression of neovascularization rather than toxic inhibition of capillary growth. TABLE 2

Inhibition of Capillary Growth In CAM Model

3-Day squalamine Percentage positive

Embryo: Applied (μg) Assay 1 Assay 2 Mean

0.65 28

1.25 18 18 18

2.5 35 18 27

5.0 91 57 74

20 52* 58* 55

40 50* 13* 32

13-Day squalamine

Embryo: Applied (μg) Percentage positive

5.0 0/26

Note: * = Some bascular irritation noted.

4. The Vitelline Capillaries of Chick Embrvo Model

In the course of evaluating squalamine in the "classical" chick chorioallantoic membrane model, it was noted that this steroid exerted a dramatic and rapid effect on capillary vessel integrity in the three- to five-day old chick embryo. Using the chick embryo vitelline capillaries assay, compounds were tested for their ability to induce capillary regression. Each compound was applied in 0.1 ml of 15% Ficol 400 and PBS onto the embryo, and vascular regression was assessed after 60 minutes. Squalamine was found to disrupt vitelline capillaries in 3-to 5-day chick embryos. The 3-day chick embryo consists of an embryonic disc from which numerous vessels emerge and return, forming a "figure 8"-shaped structure - the embryo in the center with vascular loops extending outward over both poles. Application of squalamine onto the embryonic structure resulted in progressive "beading up" of the vitelline vessels, with the finest capillaries being the first to exhibit these changes. Following a lag period of around 15 minutes, the constriction of continuity between capillary and secondary vessels, generally on the "venous" side, was observed. Continued pulsatile blood flow progressed, resulting in a "swelling" of the blind tube, followed by a pinching off of the remaining connection and formation of an enclosed vascular sac resembling a "blood island." This process progressed until only the largest vessels remained intact. The embryonic heart continued to beat vigorously. No hemorrhage was seen, reflecting the integrity of the capillary structure, hi addition, no obvious disruption of circulating red cells was observed microscopically, demonstrating the absence of hemolysis. Utilizing this assay, which appears to demonstrate what is commonly called capillary "regression," a minimum concentration of squalamine required to observe an effect in 60 minutes can be determined. Results are summarized in Table 3 below.

TABLE 3

Effects of Various Aminosterols in Chick Embryo Vitelline Capillary Regression Assay

Amount of Compound Applied (μg) Compound 10 1 OJ O₁Ol 0.001

Compound 1436 + + + + +/-

Compound 319 + + + + +/- squalamine + + + + 0

Compound 415 + + + 0

Compound 410 + +/- +/- 0

Compound 412 + 0 0 0

Compound 411 +/- 0 0 0

As apparent from Table 3, 0.1-0.01 μg of squalamine in 0.1 ml medium can induce changes. Compounds having various ranges of activities were found, with squalamine, compound 319 and compound 415 being especially active. This experiment demonstrates that the steroids tested can dramatically restructure capillaries over a time interval amounting to several minutes.

5. Suppression of Melanoma Growth Using the growth of B16 melanoma cells in C57B mice, a recognized model for the evaluation of inhibitors of angiogenesis on the growth of cancers, the effects of subcutaneous, intraperitoneal and oral administration of squalamine were evaluated. An inoculum of B16 melanoma cells was implanted subcutaneously on the dorsum of the C57B mouse, which resulted in the progressive growth of melanoma lesions over 30-40 days as shown in FIGS. 2A-C.

In this model, there was observed little evidence of metastasis with or without treatment with chemotherapeutic agents. When animals were treated with squalamine either subcutaneously (FIG. 2A), intraperitoneally (FIG. 2B) or orally (FIG. 2C), a dose-dependent suppression of tumor volume was observed. Measurement of both body weight and hematologic parameters demonstrated no significant depression indicative of a toxic effect. Since squalamine itself shows minimal cytostatic activity against B 16 in culture, except at very high concentrations, this response of the tumor was interpreted to be secondary to interference with capillary development.

Compound 319 has been found to exhibit activity against B16 melanoma in vivo. As seen in FIG. 3, which illustrates the results from the murine melanoma assay described above, subcutaneous administration of the compound achieved control of B 16 in C57B mice to an extent almost comparable to squalamine (FIG. 2B).

6. Suppression of Growth of Human Melanoma in Immunocompromised Mice As apparent from FIG. 4, human melanoma 1205Lu develops aggressively in RAG-I mice after implantation. Squalamine has been found to suppress the growth of melanoma 1205Lu in RAG-I mice in a dose-dependent fashion.

Squalamine was administered after tumors had reached about 0.1 ml, and clear suppression of tumor growth in a dose-dependent fashion was found as evidenced by FIG. 4. After cessation of treatment, tumor growth continued at a rate similar to untreated controls, suggesting that the impact of squalamine in this setting is reversible.

7. Synergistic Inhibition of Tumor Growth Based on the idea that tumor growth involves both the clonal expansion of a malignant cell along with the development of a supporting vascular supply, a combination of compound 1436 with squalamine was tested to determine whether it would achieve a synergistic effect on solid tumor growth. This concept was evaluated in the B 16 melanoma model.

Animals were implanted with B16 melanoma followed by treatment with compound 1436 or squalamine administered in a combined schedule or separately. As apparent from FIG. 5, when squalamine was administered at 5 mg/kg/day or compound 1436 was administered at 10 mg/kg/every 3 days, no significant impact on tumor volume was observed. In contrast, when both agents were administered together, a significant reduction in tumor growth was noted. Neither administration of squalamine at 15 mg/kg/day nor compound 1436 alone in a tolerable schedule could achieve this effect. Thus, a combination of these two compounds achieves a therapeutic benefit in tumors dependent on neovascularization that may prevent metastatic spread.

8. Suppression of Tumor-Induced Corneal Neovascularization The implantation of VX2 carcinoma into the rabbit cornea results in the induction of new blood vessels within several days (Tamargo et al. (1991) Cancer Research 51, 672-675). It is believed that this carcinoma secretes growth factors that stimulate new blood-vessel growth. Thus, this model is indicative in vivo evidence of therapeutic utility in the treatment of pathological disorders of vascularization, including the metastatic spread of tumors, diabetic retinopathy, macular degeneration, and rheumatoid arthritis. This experiment followed the published protocol- -tumor was implanted adjacent to a polymer containing a concentration of the agent to be evaluated. The polymer released the agent slowly in the immediate neighborhood of the tumor, providing sustained high local concentrations of the agent. In this experiment, squalamine introduced into a pellet of ELVAX 40 P (DuPont) inhibited new blood vessel formation by about 60% at days 7 and 14, and by about 25% at day 21.

9. Treatment of Iris Neovascularization in Monkeys

Iris neovascularization was induced into one eye each of fourteen cynomolgus monkeys through vein occlusion. The monkeys' eyes were evaluated approximately every three days for iris neovascularization by slit lamp examination and by fluorescein angiography. The observed iris neovascularization was graded using the scale published by Miller et al. (1994) American Journal of Pathology. 145, 574-584.

In one study (test 1), squalamine was administered (1 mg/kg squalamine dissolved in 100 mL of 5% dextrose in water) by continuous intravenous infusion to four of the monkeys. Four other monkeys received a placebo (100 mL of 5% dextrose in water) and were used as a control. The infusions were administered via an infusion pump set for a period of one hour. Infusions began immediately after vein occlusion and were repeated twice weekly for the next two weeks.

In a second study (test 2), squalamine was administered (1 mg/kg squalamine dissolved in 100 mL of 5% dextrose in water) by continuous intravenous infusion to four of the monkeys. Four other monkeys received a placebo (100 mL of 5% dextrose in water) and were used as a control. The infusions were administered via an infusion pump set for a period of one hour. Infusions began on day 7 (after the development of iris neovascularization) and were repeated twice weekly for the next two weeks.

The results of test 1 are reported below in Table 4. Table 4 illustrates the difference between the eyes of monkeys that are treated with squalamine while induced with experimental iris neovascularization and the eyes of monkeys treated with a placebo while induced with experimental iris neovascularization. The control monkeys all developed extensive iris neovascularization within nine days after vein ocjclusion (three developed grade 4 and one developed grade 5 iris neovascularization). In contrast, moneys treated with squalamine all developed only a mild form of iris neovascularization (grade 1 or 2) seven days after vein occlusion. Fourteen days after treatment with squalamine initiated immediately after vein occlusion, two of the four eyes in the squalamine-treated monkey group exhibited no clinical signs of iris neovascularization (grade 0), and two others retained only a mild form of iris neovascularization (grade 2).

___ __ TABLE 4

Grading of Iris Neovascularization in Test 1 Monkey Time after vein occlusion 4 days 7 days 9 days 14 days 17 days 21 days 23 days

1 -control 0 4 4 4 4 4 4

2-control 0 3 4 4 4 4 4

3-control 0 5 5 5 5 5 5

4-control 0 4 4 4 4 4 4

5-drug 0 2 2 2 2 2 2

6-drug 0 1 1 2 2 2 2

7-drug 0 1 1 0 0 0 0

8-drug 0 1 1 0 0 0 0

Grade 0: Grade 0 is an example of a normal, healthy iris. Vessels may or may not be visible, depending on the degree of brown iris pigmentation. On angiography, the vessels fill briefly with fluorescein, are radial, and do not leak any fluorescein.

Grade 1 : The vessels appear more prominent, tortuous and discontinuous than in Grade 0, but still do not leak fluorescein.

Grade 2: The vessels are prominent, nonradial and leak fluorescein late in the angiogram.

Grade 3: The vessels are prominent, nonradial and leak fluorescein early in the angiogram.

Grade 4: Individual vessels cannot be delineated in the early frames of the angiogram and the iris appears as a diffuse, opaque, fluorescent sheet.

Grade 5: Angiographically identical to Grade 4 with the additional association with hyphema and ectropion uveae.

The results of test 2 are reported below in Table 5. Table 5 illustrates the difference between the eyes of monkeys that are treated with squalamine after experimental iris neovascularization has been induced and the eyes of monkeys treated with a placebo after experimental iris neovascularization has been induced. After vein occlusion, all of the test monkeys and two of the control monkeys developed neovascularization. The two control monkeys that didn't develop grade 4 iris neovascularization were removed from the study and the remaining control monkeys, were treated with placebo, all retained the extensive levels of iris neovascularization (grade 4) throughout the placebo-treatment period. In contrast, moneys treated with squalamine all showed significant improvement upon the implementation of the squalamine treatment. Seven days after treatment with squalamine was initiated, two of the eyes in squalamine-treated monkey group completely recovered, showing no clinical signs of iris neovascularization (grade 0). The other two eyes improved from a severe case of iris neovascularization (grade 4) to only a mild form of iris neovascularization (grade 2).

TABLE 5

Grading of Iris Neovascularization in Test 2

Monkey Grade of iris Grade of iris neovascularization during the time after the neovascularization 7 days development of iris neovascularization after vein occlusion 4 days 7 days 11 days 15 days

1 -control 4 4 4 4 4

2-control 4 4 4 4 4

3-drug 4 4 2 2 2

4-drug 4 3 2 2 2

5-drug 4 3 0 0 0

6-drug 4 2 0 0 0

10. Oxvεen-induced Retinopathy in Mice

Systemic squalamine was effective as an antiangiogenic compound in oxygen-induced retinopathy (OIR) in mice, a model of retinopathy of prematurity. C57BL/6J mice were exposed to 75% oxygen for post-natal days (P) 7 through 12 and subsequently returned to normoxic conditions, which induced neovascularization of the retina. Control mice were raised in room air only. Retinopathy was assessed by quantification of neovascular nuclei on retinal sections and by a retinopathy scoring system evaluation of retinal whole mounts. Single dose squalamine lactate improved retinal neovascularization. The degree of suppression of neovascular nuclei in hypoxia exposed mice after treatment with a single dose (25 mg/kg, subcutaneously) of squalamine lactate (64%) was comparable with that seen after 5 days (25 mg/kg/day, subcutaneously) of squalamine lactate treatment (78%). Animals exposed to oxygen had an average of 46.2 ± 24.1 neovascular nuclei/retinal sections whereas animals that received squalamine lactate on P12 had 16.3 ± 6.8 neovascular nuclei/retinal sections (p<0.001) (Fig 6B). Control animals had nuclei counts (4.9 ± 3.1) which were similar to squalamine lactate treated controls (6.6 ± 4.4). Improvement was seen in total retinopathy scores: animals that received a single administration of squalamine lactate after oxygen exposure had a median retinopathy score of 4 whereas only oxygen exposed animals had a median retinopathy score of 9 (p<0.001) (Fig. 6A). Untreated control animals had retinopathy scores of 0 similar to those control animals receiving squalamine lactate which had a median score of 1. Vehicle treatment did not affect retinopathy scores. There were no differences in neonatal growth in any of the treatment groups. Squalamine lactate did not appear to adversely affect the general health or retinal vascular development of neonatal mice.

In another set of experiments using the oxygen-induced retinopathy model in mice, arrest and possible regression of retinal neovascularization with late squalamine lactate treatment was demonstrated. The study was performed to investigate the effects of late squalamine lactate treatment on oxygen-induced retinopathy in the mouse once retinal neovascularization had ensued. Animals were given squalamine lactate (25 mg/kg subcutaneously) in a single dose on day 15, 16, or 17. These timepoints coincided with a point in time in the mouse model when maximal retinal neovascularization occurred. Retinopathy was assessed at day 19-21 by a retinal scoring system and by quantification of extraretinal neovascularization on retinal sections. Squalamine lactate, as a single administration on Day 15 or 16, significantly inhibited retinal neovascularization while single administration on Day 17 had no effect. Animals reared in oxygen (n=12) had total retinopathy median scores of 8.5 [25th, 75th quartile] compared to oxygen reared animals with squalamine lactate treatment (n=l 1) at day 15 with retinopathy score of 1.5 with p<0.05. Room air reared animals (n=8) had retinopathy scores of 0 and room air reared and squalamine lactate treated (n=4) also had scores of 0. The number of neovascular nuclei extending beyond the inner limiting membrane in the oxygen and squalamine lactate treated animals (n=7) was 6.4 ± 4.1 compared to the oxygen only treated mice (n=10) with 34.8 ± 25.1 with p<0.01. Room air reared animals (n=7) had 4.3 ± 3.4 and room air with squalamine lactate treatment (n=4) had 4.8 ± 1.7 neovascular nuclei per retinal section. Similar effects were seen when animals were treated with squalamine lactate on Day 16. Animal growth was not affected by single dose squalamine lactate treatment at any treatment timepoint (Days 12, 15, 16, 17). Squalamine lactate reduced retinal neovascularization when utilized at day 15 or 16 in the mouse model of oxygen-induced retinopathy. Since abnormal angiogenesis was already occurring at the time of treatment in this study, squalamine lactate arrested abnormal vessel growth. It was speculated that squalamine lactate caused vessel regression in the mouse model. Another arm of the study consisted of mice being treated with squalamine lactate as a single administration on Day 12 at doses from 1.00 to 25 mg/kg (3 to 75mg/m²). Squalamine lactate, at all doses, inhibited retinal neovascularization in this model of retinopathy. 11. Choroidal Neovascularization in Rats

The efficacy of squalamine as an antiangiogenic agent was evaluated in experimental choroidal neovascularization induced in rats. Choroidal neovascularization was induced in the eyes of anesthetized male Brown Norway rats irradiated with eight krypton red laser lesions per eye. Half of the animals were subsequently given intraperitoneal injections of 5 mg/kg/injection of squalamine lactate, dosed twice per day on days 0-4, 7-8, 14 and 21; the other animals were dosed under the same regimen with vehicle only (5% dextrose in water). Eyes were examined by color fundus photography and fluorescein angiography on days 14 and 28, and all animals were sacrificed on day 28. Histological specimens were prepared for sacrificed animals and the presence or absence of experimental choroidal neovascularization was scored. By fundus photography, squalamine treated laser-induced lesions often, but not always, appeared less distinct in contrast to surrounding tissues. Lesion sites in eyes of animals treated with squalamine appeared to have less vascular density and tangential spread, and vascular leakage assessed by fluorescein angiography seemed less intense for squalamine-treated animals when compared to that seen in the eyes of control animals. Histological analyses were more quantifiable and revealed that choroidal neovascular sites in squalamine-treated eyes had a mean thickness of 47 +/- 11 microns, while control eyes had a mean thickness of 63 +/- 14 microns (p < 0.001). It was concluded that systemic squalamine treatment partially inhibited the development of choroidal neovascularization at the lesion sites. It was also noted that squalamine reduced the frequency of tangential anastomosizing spread of neovascularization from one laser damage site to another: 38% of control eye sites recovered experienced this problem, while only 21% of the sites in the eyes of squalamine-treated rats demonstrated this effect.

12. Treatment of Macular Degeneration in Humans Forty patients affected with wet AMD (choroidal neovascularization) were treated intravenously weekly over a four-week period with squalamine (25 mg/m² or 50 mg/m² dosage levels). Visual acuity examinations were conducted on the patients at four weeks, two months and four months. Changes in visual acuity were measured using standard methods employing the Early Treatment Diabetic Retinopathy Study (ETDRS) charts and scoring system (see Dong et al. (2003), Ophthalmic Epidemiology 10, 149-165).

The results of the study, summarized in Table 6 below, indicate a measurable visual improvement in patients afflicted with wet AMD at the end of the squalamine therapy period and at two months thereafter. At two months, 96 percent of all patients had preserved or improved vision while 31 percent of patients had significantly improved vision (up to 8 lines of visual acuity improvement was observed). Lesions were noticeably smaller in some patients and stable in others while vessel leakage and sub-retinal blood were diminished.

TABLE 6

Visual Improvement of Eves Affected With Wet AMD After Squalamine Theraov

Visual Acuity Line Changes End of Treatment 2 Months After Treatment

(# of patients/total patients) (# of patients/total patients)

Positive Change (> 3 lines) 17/54 (31%) 15/53 (28%)

Stable (< 2 lines) 36/54 (67%) 36/53 (68%)

Positive Change or Stable 53/54 (98%) 51/53 (96%)

Negative Change (> 3 lines) 1/54 (2%) 2/53 (4%)

Range Lines Change +8 to -4 +8 to -4

Mean Lines Change 1.5 1.2

Median Visual Acuity 20/80 20/100

13. Combination of Squalamine with Photodvnamic Therapv

Squalamine lactate (Evizon™) was infused intravenously at the dose of 40 mg (Group A), 20 mg (Group B) or 10 mg (Group C) once a week for the first 2 weeks. Patents were then treated using PDT with verteporfϊn (Visudyne®) on week three followed by Evizon™ at weeks four and five and then monthly for five months. A vehicle control was used in place of Evizon™ and was infused intravenously on weeks 1, 2, 4 and 5 and then monthly for five months. Changes in visual acuity (VA) were measured using standard methods employing the Early Treatment Diabetic Retinopathy Study (ETDRS) charts and scoring system (see Dong et al. (2003), Ophthalmic Epidemiology 10, 149-165) and central retinal thickness (CRT) was measured by optical coherence tomography. Measurements were made at the start of the study to obtain baseline values and then at 3, 5, 9, 13 and 17 weeks. The results for two patients are shown in Figures 8 and 9 and indicate an improvement in visual acuity and a reduction in CRT compared to baseline with treatment with PDT plus Evizon™.

The present invention is not to be limited in scope by the specific embodiments disclosed in the examples that are intended as illustrations of a few aspects of the invention and any embodiments, which are functionally equivalent, are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art and are intended to fall within the appended claims. A number of references have been cited, the entire disclosures of which are incorporated herein by reference in their entirety.

Claims

We claim:

1. A composition comprising a therapeutically effective amount of an aminosterol and photo- activated agent for use in photodynamic therapy as a combination therapy.

2. The composition of claim 1 , wherein the aminosterol is squalamine or a pharmaceutically acceptable salt thereof.

3. The composition of claim 1, wherein the aminosterol is squalamine lactate.

4. The composition of claim 1, wherein the photo-activated agent is a dye.

5. The composition of claim 1, wherein the photo-activated agent is a benzoporphyrin.

6. The composition of claim 5, wherein the benzoporphyrin is verteporfin.

7. The composition of claim 1, wherein the photo-activated agent is a purpurin.

8. The composition of claim 7, wherein the purpurin is rostaporfin.

9. The composition of claim 1 wherein the composition is suitable for use in a human undergoing photodynamic therapy.

10. A method of treating a subject with an ocular disorder comprising: administering a therapeutically effective amount of photo-activated agent; administering a therapeutically effective amount of an aminosterol, and exposing the eye of the subject to an energy source.

11. The method of claim 10, wherein the aminosterol is squalamine or a pharmaceutically acceptable salt thereof.

12. The method of claim 10, wherein the aminosterol is squalamine lactate.

13. The method of claim 10, wherein the energy source is a laser.

14. The method of claim 10, wherein the photo-activated agent is a dye.

15. The method of claim 10, wherein the photo-activated agent is a benzoporphyrin.

16. The method of claim 15, wherein the benzoporphyrin is verteporfin.

17. The method of claim 10, wherein the photo-activated agent is a purpurin.

18. The method of claim 17, wherein the purpurin is rostaporfin.

19. The method of claim 16, wherein the verteporfin is administered intravenously.

20. The method of claim 16, wherein the verteporfin is administered intraoccularly.

21. The method of claim 18, wherein the rostaporfin is administered intravenously.

22. The method of claim 18, wherein the rostaporfin is administered intraoccularly.

23. The method of claim 10, wherein the photo-activated agent is administered in an amount of from about 0.01 mg to about 20 mg.

24. The method of claim 11, wherein the squalamine or pharmaceutically acceptable salt thereof is administered intravenously.

25. The method of claim 12, wherein the squalamine lactate is administered intravenously.

26. The method of claim 11, wherein the squalamine or pharmaceutically acceptable salt thereof is administered intraoccularly.

27. The method of claim 12, wherein the squalamine lactate is administered intraoccularly.

28. The method of claim 11, wherein the squalamine or pharmaceutically acceptable salt thereof is administered orally.

29. The method of claim 12, wherein the squalamine lactate is administered orally.

30. The method of claim 12, wherein the squalamine lactate is administered in an amount of from about 0.001 mg to about 100 mg.

31. The method of claim 10, wherein the ocular disorder is caused by neovascularization.

32. The method of claim 10, wherein the ocular disorder is age-related macular degeneration.

33. The method of claim 31, wherein the macular degeneration is the wet form.

34. The method of claim 31, wherein the macular degeneration is the dry form.

35. The method of claim 10, wherein the ocular disorder is macular edema.

36. The method of claim 35, wherein the macula edema is due to diabetic disease

37. The method of claim 10, wherein the ocular disorder is central retinal vein occlusion

38. The method of claim 10, wherein the ocular disorder is cancer.

39. The method of claim 11, wherein squalamine or pharmaceutically acceptable salt thereof is administered prior to the photo-activated agent.

40. The method of claim 12, wherein squalamine lactate is administered prior to the photo-activated agent.

41. The method of claim 11, wherein squalamine or pharmaceutically acceptable salt thereof is administered simultaneously with the photo-activated agent.

42. The method of claim 12, wherein squalamine lactate is administered simultaneously with the photo-activated agent.

43. The method of claim 11, wherein squalamine or pharmaceutically acceptable salt thereof is administered subsequent to the photo-activated agent.

44. The method of claim 12, wherein squalamine lactate is administered subsequent to the administration of the photo-activated agent.

45. The method of claim 11, wherein the squalamine or pharmaceutically acceptable salt thereof is administered subsequent to the step of exposing the eye of the subject to the energy source.

46. The method of claim 12, wherein squalamine lactate is administered subsequent to the step of exposing the eye of the subject to the energy source.

47. The method of claim 10, wherein the subject is treated more than one time.

48. The method of claim 10, wherein the energy source is a non-thermal laser.