Title: Improved Method for Inhibition of Neovascularization

FIELD OF THE INVENTION

This application is directed to the use of the aminosterol compound squalamine for the inhibition of neovascularization as an improved method for the treatment of macular degeneration, cancer and other diseases associated with inappropriate neovascularization.

BACKGROUND OF THE INVENTION

Each of the body's cells must maintain its acid-base balance or, more specifically, its hydrogen ion or proton concentration. Only slight changes in hydrogen ion concentration cause marked alterations in the rates of chemical reactions in the cells-some being depressed and others accelerated. In very broad and general terms, when a person has a high concentration of hydrogen ions (acidosis), that person is likely to die in a coma, and when a person has a low concentration of hydrogen ions (alkalosis), he or she may die of tetany or convulsions. In between these extremes is a tremendous range of diseases and conditions that depend on the cells involved and level of hydrogen ion concentration experienced. Thus, the regulation of hydrogen ion concentration is one of the most important aspects of homeostasis.

A shorthand method of expressing hydrogen ion concentration is pH: ρH=log 1/(H ⁺ concentration) ³ -log (H ⁺ concentration). The normal cell pH is 7.4, but a person can only live a few hours with a pH of less than 7.0 or more than 7.7. Thus, the maintenance of pH is critical for survival.

There are several mechanisms of maintaining pH balance. For example, during quiescence and constitutive growth, cells appear to utilize the chloride/bicarbonate exchanger, a well- studied device which provides for proton exchange across cells such as the red cell.

In addition, during accelerated periods of growth, which are induced by mitogens, growth factors, sperm, etc., cells engage another piece of cellular equipment to handle the impending metabolic burst. This is the sodium/proton (Na ⁺ / H ⁺ ) exchanger-the "NHE," which is also called an "antiporter." Because the NHE iunctions in a number of roles and in a number of tissues, the body has developed a family of NHEs, and recent work has elucidated a family of NHE "isoforms" that are localized in certain tissues and associated with various functions. The NHE isoforms listed below are most likely to be significant.

NHEl is a housekeeping exchanger and is believed to be unregulated in hypertension. It is thought to play a role in intracellular pH conduct. Also, it is believed that control of this exchanger will protect a patient from ischemic injury.

NHEl is associated genetically with diabetes and, thus, inhibition might alter evolution of diabetes through effects on beta cells in the pancreas. In addition, vascular smooth muscle proliferation, responsive to glucose, is associated with increased expression of NHEIa.

NHE lβ is present on nucleated erythrocytes. It is inhibited by high concentrations of amiloride. This NHE isoform is regulated by adrenergic agents in a cAMP-dependent fashion.

NHE2 is associated with numerous cells of the GI tract and skeletal muscle. Inhibition could alter growth of hyperplastic states or hypertrophic states, such as vascular smooth muscle hypertrophy or cardiac hypertrophy. Cancers of muscle origin such as rhabdomyosarcoma and leiomyoma are reasonable therapeutic targets.

NHE3 is associated with the colon. The work described below shows it to be associated with endothelial cells. Inhibition would affect functions such as water exchange in the colon (increase bowel fluid flux, which is the basis of, e.g., constipation), colonic cancer, etc. On endothelial cells, normal growth would be inhibited through inhibition of the exchanger.

NHE4 is associated with certain cells of the kidney. It appears to play a role in cellular volume regulation. Specific inhibitors might affect kidney function, and hence provide therapeutic benefit in hypertension.

NHE5 is associated with lymphoid tissue and cells of the brain. Inhibition of NHE5 should cause inhibition of proliferative disorders involving these cells. NHE5 is a likely candidate for the proliferation of glial cells in response to HIV and other viral infections.

As indicated by the above, although the NHE functions to assist the body, the inhibition of NHE function should provide tremendous therapeutic advantages. For example, although the NHE normally operates only when intracellular pH drops below a certain level of acidity, upon growth factor stimulation the cell's NHEs are turned on even though the cell is poised at

a "normal" resting pH. As a consequence, the NHEs begin to pump protons from the cell at a pH at which they would normally be inactive. The cell undergoes a progressive loss of protons, increasing its net buffering capacity or, in some cases, actually alkalinizing. In settings where the pump is prevented from operating, the growth stimulus does not result in a cellular effect. Thus, inhibitors of the NHE family are likely to exert growth-inhibitory effects.

During severe acid stress-the condition that a tissue might find itself in when deprived of oxygen (or a blood supply)-the NHE family is believed to contribute to subsequent irreversible damage. For example, when blood flow to the heart is impaired, local acidosis occurs. Heart muscle cells develop a profound internal acidity. The acidity, in turn, activates otherwise dormant NHEs. These exchangers readily eliminate protons from the cell, but in exchange for sodium. As a consequence, intracellular sodium concentrations rise. Subsequently, the sodium-calcium exchanger is activated, exchanging internal sodium for external calcium. The rise in internal Ca ²⁺ concentrations leads to cell death, decreased contractility, and arrhythmias. Thus, post ischemic myocardial damage and associated arrhythmias are believed to arise from an NHE-dependent mechanism, and inhibition of this NHE should therefore prevent such occurrences. If the NHE inhibited the internalization of Na ⁺ and slowed down metabolic activity as a consequence of the depressed pH, damage of the cell could be avoided. Hence, there is an interest in the development of NHE inhibitors for use in cardiac ischemia.

Other members of the NHE family appear to play a more classical role in water and sodium transport across epithelial surfaces. Specifically, the NHE3 isoform found in the colon is believed to play a role in regulating the fluid content of the colonic lumen. This pump is inhibited in cases of diarrhea. The NHE3 isoform present on the proximal tubules of the kidney is believed to play a similar role with respect to renal salt and acid exchange. Accordingly, inhibitors of the NHE family have been regarded as therapeutic modalities for the treatment of hypertension.

In view of the expected value of the inhibition of NHE action, scientists have sought out NHE inhibitors. The most widely studied inhibitor of NHE is amiloride, a guanidine- modified pyrazine used clinically as a diuretic. A number of derivatives have been generated, incorporating various alkyl substitutions. These derivatives have been studied with the

several isoforms of NHE that are known and described above, except for NHE5, for which there is no known inhibitor.

The NHE inhibitors described by Counillon et al. exhibit specificity for NHEl . They therefore serve a therapeutic value in the treatment of conditions where inhibition of this isoform is beneficial. However, these inhibitors do not target the other known NHE isoforms- e.g., NHE3 is unaffected.

NHE3, as is demonstrated below, is expressed on endothelial cells, and its inhibition results in anti-angiogenic effects. The spectrum of NHE isoforms inhibited by squalamine in accordance with the invention are different from those inhibited by the amiloride or the Counillon et al. compounds, and have different, distinct pharmacological effects.

Those in the art have therefore continued to search for NHE inhibitors that exhibit selective action against a single, specific NHE. Such inhibitors would permit more precise inhibition of a tissue by perturbing the effect of the NHE on its growth.

Thus, artisans have recognized that the development of various NHE-specific inhibitors would allow for the development of new therapies for a whole host of diseases or conditions, including: treating arrhythmias; treating and preventing cardiac infarction; treating and preventing angina pectoris and ischemic disorders of the heart; treating and preventing ischemic disorders of the peripheral and central nervous system; treating and preventing ischemic disorders of peripheral organs and limbs; treating shock; providing anti- arteriosclerotic agents; treating diabetic complications; treating cancers; treating fibrotic diseases, including fibroses of lung, liver and kidney; and treating prostatic hyperplasia. Other therapeutic targets include: treatment of viral disease, such as HIV, HPV and HSV; prevention of malignancies; prevention of diabetes (i.e., islet cell injury); prevention of vascular complications of diabetes; treatment of disorders of abnormal neovascularization, e.g., macular degeneration (such as age-related, both wet and dry forms), rheumatoid arthritis, psoriasis, cancer, malignant hemangiomas; prevention of vascular retenosis; prevention of hypertension-associated vascular damage; immunosuppression; and treatment of collagen vascular disorders.

Inhibitors of NHEs of bacteria fungi and protozoa would also be valuable as specific

antimicrobials. It is known that all living cells use an NHE of one form or another to maintain intracellular Na ⁺ and pH homeostasis. NHEs have been cloned from numerous bacteria and fungi, and bear some sequence homology to the mammalian isoforms. Using a highly specific bacterial or fungal NHE as a target, it should be possible to develop a highly specific inhibitor of such an exchanger, one that is particularly advantageous or that lacks activity against the mammalian isoforms. Such compounds would be useful as antibiotics of a different mechanism.

Thus, there is a need in the art for specific inhibitors of NHEs. There is further a need to develop NHE inhibitors for various therapeutic uses. Selected aminosterol compounds have been found to inhibit various NHEs.

This invention relates to various methods for using squalamine. Squalamine, the structure of which is illustrated in FIG. 5, is an aminosterol which has been isolated from the liver of the dogfish shark, Squalus acanthias. Methods for synthesizing squalamine have been devised, such as the methods described in U.S. Patents 6,262,283, 6,933,383 and 6,610,866. U.S. Patent 5,721,226 describes the use of squalamine as an antiangiogenic agent and U.S. Patents 6,147,060, 6,596,712 and 6,962,909 describe its use to treat cancer and neovascularization in the eye. Additional uses of squalamine (e.g., as a sodium/proton exchanger (isoform 3), or NHE3, inhibiting agent and as an agent for inhibiting the growth of endothelial cells) and squalamine synthesis techniques are disclosed in U.S. Patent No. 5,792,635.

Vascular endothelial growth factor (VEGF) stimulates the production or activity of endothelial nitric oxide synthetase (eNOS) which results in increased nitric oxide (NO) production. NO is a known vasodilator which results in hypotension. And- VEGF (Avastin™) is used as a systemic anti-angiogenesis therapy and causes hypertension, presumably through the reduction in the production or stimulation of eNOS, with the resulting reduction of NO causing an increase in vascular tone.

Squalamine, an anti-angiogenic agent, does not inhibit VEGF-induced eNOS and the linked NO production, as monitored by cGMP production, and therefore should not induce hypertension.

SUMMARY OF THE INVENTION

According to the present invention, an inhibitor of angiogenesis that does not inhibit the induction of eNOS is provided.

An aspect of the invention is a method for treating an individual with a disorder associated with neovascularization, comprising administering to the individual an effective amount of squalamine, or a pharmaceutically acceptable salt thereof, to inhibit the neovascularization, wherein the individual is diagnosed with hypertension.

BRIEF DESCRIPTION OF DRAWING FIGURES

FIGS. IA and IB show the inhibition of rabbit sodium/proton exchanger isoform 3 (NHE3) by squalamine. FIG. IA is a plot of the rate of pH recovery (y-axis) as a function of restored extracellular sodium ion concentration (x-axis) for cells acid-preloaded by exposure to 40 mM NH ₄ CI, with the curve marked by "+" being for control (no drug) and the curve marked by "δ" being for squalamine. FIG. IB shows the actual internal pH (y-axis) as a function of time (x-axis) following addition of 5 .mu.g/ml of squalamine for cells not acid-preloaded.

FIG. 2 A shows the lack of inhibition of rabbit sodium/proton exchanger isoform 1 (NHEl) by squalamine. FIG. 2B shows the lack of inhibition of human NHEl by squalamine. In these plots of internal pH vs. time, the curve marked by "0" is for squalamine and that marked by "+" is the control (cells incubated in the absence of squalamine).

FIGS. 3 A, 3B and 3C show the suppression of the growth of murine melanoma, respectively through the subcutaneous, intraperitoneal and oral administration of squalamine.

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

FIG. 5 shows the structure of squalamine.

FIG. 6 indicates that squalamine treatment does not affect VEGF-induced eNOS activity.

DETAILED DESCRIPTION OF THE INVENTION Synthesis of Squalamine

The steroid known as squalamine is the subject of U.S. Pat. No. 5,192,756 to Zasloff et al, 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, compound 1256, is shown in FIG. 5. The total chemical synthesis of squalamine was reported in 1994.

EXAMPLE 1

Preparation of Shark Liver Isolates

Squalamine has been recovered from extracts of dogfish shark liver. To prepare the squalamine, shark liver was extracted in methanokacetic acid. The aqueous extract was adsorbed to Cl 8 silica and eluted with 70% acetonitrile, and the eluate was adsorbed to SP- sephadex and eluted with 1.5 M NaCl. The eluate was adjusted to 5M NaCl, and the steroids salted out. The precipitate was filtered over Celite and eluted with hot water, followed by methanol. The eluate was reduced in volume and applied to a 1-inch Cl 8 column, and subjected to chromatography utilizing an increasing gradient in acetonitrile. Fractions were collected, concentrated by evaporation, and analyzed separately by thin layer chromatography (TLC). Squalamine elutes beginning at about fraction 62 and continues until fraction 80. The squalamine was isolated, purified, characterized, and the structure determined by NMR as described in U.S. Patent 6,962,909, incorporated by reference herein.

Therapeutic Activities and Utilities

Aminosterol compounds such as squalamine have been discovered to be effective inhibitors of NHE. In seeking to elucidate the antimicrobial mechanism of action for squalamine, squalamine has been found to advantageously inhibit a specific NHE isoform-the compound inhibits NHE3, but not NHEl. In addition, squalamine has been determined to inhibit the exchanger through a special mechanism. The special and advantageous effects and utilities of squalamine and other aminosterols are further evident from the results of the experimental tests discussed below.

Specific Inhibition of NHE3:

To determine the specificity of squalamine's inhibition of NHEs, squalamine was assayed against a cell line expressing either human NHEl or human NHE3 following procedures

outlined in Tse et al., J. Biol. Chem. 268, 1993, 11917-11924. Internal pH was measured either following acid loading or in the absence of an acid-loading ^' challenge, with the results shown in FIGS. IA and IB.

Specifically, PS 120 fibroblasts transfected with rabbit NHE3 were grown in supplemented Dulbecco's-modified Eagle's medium as described by Levine et al., J. Biol. Chem. 268, 1993, 25527-25535. Transfected cells grown on glass coverslips were then assayed for internal pH changes following treatment with 5 μg/ml squalamine using the fluorescent dye BCECF-AM (2 ^l ,7'-bis(carboxyethyl)-5(6)-carboxyfiuorescein-acetoxymethyl ester) as a pH indicator as described by Levine et al. For cells acid-preloaded by exposure to 40 mM NH4CI the rate of pH recovery as a function of restored extracellular sodium ion concentration was monitored, with the results being shown in FIG. IA. For cells not acid-preloaded, the actual internal pH value was monitored as a function of time following addition of squalamine, with the results depicted in FIG. IB.

As seen in FIGS. IA and IB, squalamine inhibited NHE3 with respect to proton concentration at both K _m and V _max levels. In contrast, existing agents such as amiloride affected only V _max .

Thus, the aminosterol squalamine not only reduces the absolute number of protons that can be secreted by the cell (the V _max effect), but also forces the cell to fall to a lower pH in the presence of this inhibitor (the K _n , effect). As a consequence, the sodium/proton exchanger is more profoundly inactivated by squalamine than by amiloride.

In contrast to its effects on NHE3 shown in FIGS. IA and IB, squalamine exhibited no inhibitory activity against human NHEl or rabbit NHEl as shown in FIGS. 2A and 2B. PS 120 fibroblasts transfected with rabbit or human NHEl were grown as described above. Transfected cells expressing rabbit NHEl (FIG. 2A) or human NHEl (FIG. 2B) grown on glass coverslips were then assayed for internal pH changes following treatment with 5 .mu.g/ml squalamine using the fluorescent dye BCECF-AM with cells acid-preloaded by exposure to 40 mM NH4CI. The rate of pH recovery as a function of restored extracellular sodium ion concentration was monitored.

In addition, as demonstrated by FIG. IB, the resting pH of these cells was also inhibited.

Thus, squalamine's effect on proton exchange causes the cell to drop to a lower pH in its presence before activation of the pump occurs.

Through these studies, squalamine has been discovered to be a distinct inhibitor with specificity for NHE3 over NHE 1. Moreover, squalamine has been identified as an inhibitor that causes a cell to drop to a lower pH before the pump is activated. The results shown in FIGS. IA, IB, 2A and 2B demonstrate that squalamine exhibits a unique NHE specificity.

A variety of endothelial cell growth/shape related events are inhibited by squalamine and functionally related compounds. The experimental tests discussed below were conducted to assess this aminosterol's effects.

Growth Inhibition of Endothelial Cells, Fibroblasts and Epithelial Cells in Vitro: 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 1 OA, and human foreskin fibroblasts were incubated in the presence of 12 different membτane-active agents, including peptides and squalamine.

LPS-Induced Neutrophil Adherence to Human Umbilical Venous Endothelial Cells: When endothelial cells are exposed to certain stimuli, including lipopolysaccharide (LPS) and certain cytokines, specific adhesion molecules are induced on the plasma membrane that enhance the binding of leukocytes. These leukocyte-endothelial cell interactions are believed to be necessary to localize leukocytes to sites of bacterial invasion and to facilitate extravasation of the leukocytes from the capillary into the surrounding tissue space. Leukocyte-adhesion molecules include the Selectins and ICAM-I .

To determine if squalamine inhibited this particular endothelial cell function, standard adhesion assays were performed as outlined in Gamble et al., J. Imm. Methods 109, 1988, 175-184. The expression of cell surface ligands in an endothelial-based system has been shown to effect adherence to granulocytes with a system using human umbilical venous endothelial cells, purified neutrophils, and inducers of cell surface ligands such as LPS (100 ng/ml) and TNF-.alpha. (40 ng/ml). In these experiments, approximately 2xlO ⁵ human umbilical venous cells (passage 2-6) were plated per well. The cells were grown in serum-

free media overnight. For induction, either TNF-α (40 ng/ml) was added to endothelial cells for 6 hours prior to adding neutrophils or LPS (100 ng/ml) was added for 4-6 hours. It was found that the LPS response was increased by adding 1% FBS to the wells to provide a source of LPS-binding protein. After activation of the endothelial cells, approximately 5OxIO ⁶ neutrophils were added per well. The plates were gently rocked for 30 minutes at room temperature, followed by removal of the media and washing in serum-free media three times and then photographing of each well. Experiments to test the effects of squalamine were performed by adding squalamine at 10 μg, 1.0 μg, or 0.1 μg at the time of adding LPS or TNF-α. A second repeat dose of squalamine was added at the time of adding neutrophils. A monoclonal Ab to ICAM-I was a positive control.

Using three different subjects, there was no inhibition of squalamine on neutrophil adherence using activated human endothelial cells. There was approximately 50% inhibition of adherence when adding 40 μg/ml of a monoclonal Ab to ICAM-I prior to adding neutrophils.

These results indicate that inhibition of the endothelial NHE by squalamine affects both growth and capillary formation in vitro, but does not inhibit all signal transduction pathways in this cell. Thus, certain "housekeeping" functions, such as the capacity of the endothelial cell to attract leukocytes to the site of an infection, should not be impaired by squalamine. This demonstrates that squalamine can be used to inhibit angiogenesis but will not otherwise disrupt certain important endothelial cell functions, such as leukocyte irecruitment to sites of infection or inflammation.

Anti-Proliferative Activity:

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 novo" 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., Pharm. Ther. 51, 1991, 1-11. Results are tabulated below.

INHIBITION OF CAPILLARY GROWTH IN CAM MODEL

3-Day Squalamine Percentage positive

Embryo: Applied (wg) 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 vascular irritation noted

As seen from the above table, 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.

The Vitelline Capillaries of 3-5 Day Chick Embryo 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 (0.1 ml in 15% Ficol in PBS) 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. In 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," squalamine was observed to inhibit vascular reactivity as concentrations as low as 0.01 μg in 0.1 ml medium. This experiment demonstrates that squalamine can dramatically restructure capillaries over a time interval amounting to several minutes. The results reflect that squalamine exerts this effect through inhibition of NHE.

Tadpole Assay:

A newly developed assay employing tadpoles, preferably Xenopus laevis Stages 59-60, were employed to study the effect of a compound by monitoring capillary occlusion in the tadpole's tail. Animals at these stages were used because they represent the period of transition through metamorphosis at which time the animals possess both embryonic and adult stage tissues. The compounds of the invention affect the shape, viability and integrity of the embryonic tissues while not affecting the adult tissues, providing a powerful, highly specific screen. For example, substances that destroy all of the animal's epithelium, both adult

and embryonic, could be regarded as toxic. Substances that destroy only the embryonic tissues exhibit a very unique specificity.

In this assay, tadpoles are introduced into Petri dishes containing a solution of the test compound in distilled water, preferably about 100 ml. The preferred concentration of the test compound is from about 1 μg/ml to about 10 μg/ml. The volume of liquid is sufficient for the animal to swim freely and drink from the solution. Thus, the effect observed results from oral absorption and subsequent systemic distribution of the agent. If the volume of liquid is not sufficient to permit oral intake, the effects that are observed would result from absorption through the surface epithelium. Thus, this simple assay can identify if an compound has characteristics of oral availability.

In another embodiment of this assay, a solution of a compound in water can be injected directly into the abdomen of the animal using standard techniques. Concentrations of the compound from about 0.05 mg/ml to about 0.5 mg/ml in about 0.05 ml of water are preferred.

After an amount of time, typically about 60 minutes, the occlusion of blood flow through capillaries in the tadpole's tail are observed under an inverted microscope at a magnification of roughly 10Ox.

When the tadpoles were introduced into distilled water containing squalamine at 10 μg/ml, it was observed that blood flow through the capillaries of the tail shut down. The process occurred from the caudal to cranial direction. Blood flow within the most distal vessels stopped initially, followed by the larger vessels. During this period, it was observed that the cardiovascular system was otherwise robust, as evidenced by a continued heartbeat, pulsatile expansion of the great vessels, and, most curiously, unaltered blood flow through the fine capillaries of the hands and feet. Thus, selective cessation of blood flow was seen in localized regions. If the animals are maintained in squalamine for several days, enhanced regression of the most distal aspects of the tail, as well as the peripheral aspects of the tail fin are observed, corresponding to regions of the animal perfused by the occluded vasculature. This effect apparently results from selective change in the resting diameter of the capillaries of the tail. Inhibition of the endothelial cell NHE evidently leads to a change in shape of the cell making up the capillary, resulting in diminished flow. The continued functioning of capillary beds in the "adult" portions of the tadpole (the limbs) indicates that squalamine is

selective for certain capillaries. From the results of the tadpole tail capillary occlusion assay, squalamine was found to induce a vascular occlusive effect.

Suppression of Melanoma Growth:

Suppression of Growth of Melanomas in Mice by Oral and Parenteral Routes of

Administration:

The growth of B 16 melanoma in C57B mice is dependent upon neovascularization. Hence, this is a recognized model for evaluating the impact of inhibitors of angiogenesis on the growth of cancer.

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. 3A-3C.

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. 3A), intraperitoneally (FIG. 3B) or orally (FIG. 3C), a dose-dependent suppression of tumor volume was observed. Measurement of both body weight and hematologic parameters demonstrated no significant depression. 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.

Suppression of Growth of Human Melanoma in Izmunocompromised Mice: As apparent from FIG. 4, 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.

Suppression of Tumor-Induced Corneal Neovascularization in Rabbits: The implantation of VX2 carcinoma into the rabbit comea results in the induction of new blood vessels within several days (Tamargo et al., Cancer Research 51, 1991, 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 releases 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, Wilmington, Del.) inhibited new blood vessel formation by about 60% at days 7 and 14, and by about 25% at day 21.

As demonstrated by the experiments described above, squalamine provides a potent inhibitor of NHE3. Squalamine therefore should provide invaluable therapeutic intervention wherever new blood vessel formation in vivo is implicated.

Indeed, any pathological processes dependent on new blood vessel formation can be treated through inhibition of NHE3. As an agent that interferes with the process of neovascularization, squalamine has therapeutic utility in the treatment of diseases or disorders dependent on continued neovascularization where interruption of neovascularization diminishes the intensity of the pathological process. Thus, squalamine has utility for treating disorders including solid tumor growth and metastasis, rheumatoid arthritis, psoriasis, diabetic retinopathy, macular degeneration, neovascular glaucoma, papilloma, retrolental fibroplasia, and organ rejection.

The Effect of Squalamine on VEGF-Induced eNOS Activity

Human Umbilical Vein Endothelial Cells (HUVECs) were plated at a density of 300,000 cells per 10cm2 plastic tissue culture dish and cultured for 2 days in Endothelial Growth Media-2 (EGM-2) supplemented with manufacturer supplied serum, cytokines, and growth factors (Cambrex). Squalamine was added at a final concentration of 3 μM for 23 hrs as

indicated, after which cells were washed in unsupplemented media to remove serum and growth factors. The cells were incubated in HBSS at 37 ⁰ C for 30 minutes, and then recombinant VEGF-A (165) was added at a final concentration of 12.5 ng/mL as indicated. IBMX was added to all dishes at a final concentration of 0.5 mM to inhibit the non-specific phosphodiesterases from lowering guanosine 3',5' cyclic monophosphate (cGMP) levels after cell lysis. After 15 minutes of VEGF and IBMX incubation, the supernatant was discarded and cells scraped off the plates in 500 μL of 0.1 N HCl. Cell lysates were assayed for cGMP levels using a cGMP competitive enzyme-linked immunoassay (Cayman Chemical). Protein levels were measured with the colormetric microBCA Protein Assay (BioRad) to insure normalization and plot pmol cGMP per mg of protein in the cell lysates.

The increased production of cGMP is a surrogate marker for the activity of eNOS and therefore also an indicator of an increase in NO production. The results of this assay, shown in FIG. 6, indicate that squalamine does not affect the VEGF-induced induction of eNOS. Therefore, unlike anti-VEGF agents, squalamine should not cause a hypertensive side effect when used for the inhibition of neovascularization.

Administration of the Squalamine

The patient to be treated can be any animal, and is preferably a mammal. More preferably, the patient is a human.

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 squalamine 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 Remington: The Science and Practice of Pharmacy, 21 ^st ed. (2005).

Pharmaceutically acceptable salts of the squalamine 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 quatemized with, for example, alkyl halides.

In addition to carriers, the pharmaceutical compositions of the invention may also include stabilizers and preservatives. For examples of typical carriers, stabilizers and adjuvants known to those of skill in the art, see Remington: The Science and Practice of Pharmacy, 21 ^st ed. (2005).

The squalamine may be administered alone or preferably as a pharmaceutical formulation comprising the squalamine 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 squalamine.

A "therapeutically effective amount" is an amount of squalamine which inhibits, totally or partially, the progression of the condition or alleviates, at least partially, one or more symptoms of the condition. A therapeutically effective amount can also be an amount that is

prophylactically effective. 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.

In vivo administration of the squalamine can be effected in one dose, multiple doses, continuously or intermittently throughout the course of treatment. Doses range from about 0.05 mg/kg to about 5 mg/kg, preferably 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 can be carried out with the dose level and pattern being selected by the treating physician.

Pharmaceutical compositions containing the squalamine can be administered by any suitable route, including oral, rectal, intranasal, topical (including transdermal, aerosol, buccal an sublingual), parenteral (including subcutaneous, intramuscular, intravenous), intraperitoneal and pulmonary. It will be appreciated that the preferred route will vary with the condition and age of the recipient, 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 squalamine can be formulated readily by combining it 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 are, in particular, 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 squalamine maybe 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 squalamine.

For administration by inhalation, the squalamine for use according to the present invention is 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 squalamine 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 squalamine is 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.

The squalamine may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

All cited patents and publications are incorporated by reference in their entireties.

REFERENCES

1. Yang R, Thomas GR, Bunting S. Ko A, Ferrara N, Keyt B, Ross J, and Jin H: "Effects for vascular endothelial growth factor on hemodynamics and cardiac performance", L Cardtovasc. Pharmacol. 27:838-44, 1996.

2. Ku DD, Zaleski JK, Liu S, Brock TA. "Vascular endothelial growth factor induces EDRF- dependent relaxation in coronary arteries", Am J Physiol. 1993;265:H586-H592.

3. van der Zee R, Murohara T, Luo Z, Zollmann F, Passeri J, Lekutat C, Isner JM. "Vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF) augments nitric oxide release from quiescent rabbit and human vascular endothelium", Circulation 1997;95: 1030- 1037.

4. Papapetropoulos A. et ai, "Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells," J. Clin Invest. 1997; 100:3131-3139.

5. Gelinas DS et ai, "Immediate and delayed VEGF-mediated NO synthesis in endothelial cells: role of PDK, PKC and PLC pathways." Br. J, Pharmacol. 2002:137: 1021-1030.