Adelman, Ron A. (52 Quarry Lane, Hamden, CT, 06518, US)
Zhiwei HU. (470 Prospect Street, Apartment#52 New Haven, CT, 06511, US)
Garen, Alan (278 Canner Street, New Haven, CT, 06511, US)
Adelman, Ron A. (52 Quarry Lane, Hamden, CT, 06518, US)
Zhiwei HU. (470 Prospect Street, Apartment#52 New Haven, CT, 06511, US)
Verteporfin has an absorption spectrum with several peaks, including a strong absorption in the 680 - 695 nm region. Verteporfin absorbs light efficiently at a wavelength of 689 nm (red light), which can penetrate a thin layer of blood, melanin, or fibrotic tissue. Verteporfin can be conjugated to the effector domain of fVII via amide bonds or ester bonds. For example, lysine residues in the effector domain can react with an ester group on verteporfin to form an amide. Another patent that describes or identifies suitable photodynamic dyes is U.S. Patent No. 5,910,510. Several photodynamic dyes are currently approved by the FDA, or are in clinical trials, for use in treating cancer. For example, Photofrin.™ (porfimer sodium, manufactured by QLT PhotoTherapeutics and distributed by Sanofi Pharmaceuticals, Inc. and Beaufour Ipsen) is currently FDA-approved for early and late stage lung cancer and esophageal cancer. The following other photodynamic dyes are presently undergoing clinical trials and/or animal studies: Bopp™ (boronated porphyrin, Pacific Pharmaceuticals) for brain cancer, FoscanTM for head and neck cancer (Scotia QuantaNova), Lutrin.TM. for breast cancer (lutetium texaphyrin (Lu-Tex), Pharmacyclic) and PH-IO for cancerous tumors (Photogen Technologies, Inc.). Although described above with reference specific to compounds, one can also utilize enantiomers, stereoisomers, metabolites, derivates and salts of the active compounds. Methods for synthesis of these compounds are known to those skilled in the art. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, and alkali or organic salts of acidic residues such as carboxylic acids. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. Conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric and nitric acid ; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2- acetoxybenzoic, fumaric, tolunesulfonic, methanesulfonic, ethane disulfonic, oxalic and isethionic acids. The pharmaceutically acceptable salts can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington 's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, PA, 1985, p. 1418). A prodrug is a covalently bonded substance which releases the active parent drug in vivo. Prodrugs are prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to yield the parent compound. Prodrugs include compounds wherein the hydroxy or amino group is bonded to any group that, when the prodrug is administered to a mammalian subject, cleaves to form a free hydroxyl or free amino, respectively. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups. Metabolites result from biochemical processes by which living cells interact with the active parent drug or other formulas or compounds of the present invention in vivo. Metabolites include products or intermediates from any metabolic pathway. c. Linkers Linkers The term "linker" as used herein refers a molecule capable of covalently connecting a cytotoxic compound to an amino acid side chain of a protein. The term "linker" may be a non-peptidyl linker or a peptidyl linker. The linker may have covalently bonded thereto a tether, as defined below, for covalently linking a cytotoxic compound to the linker. The term "peptidyl linker" as used herein refers to a peptide comprising at least two amino acids and which can be coupled to an amino acid side-chain of a protein. The linker may have a reactive group at the carboxyl terminus such as, but not limited to, a chloromethylketone. The peptide of the peptidyl linker may be cleavable by proteolytic enzymes found within a cell. One suitable linker is a peptidyl methylketone linker covalently bonded to the polypeptide, most preferably to the side chain of an amino acid within the catalytic triad of the serine protease domain of fVIIa. In the human and bovine fVII proteins, the amino acids which form a catalytic "triad" are Ser344, Asp242, and His 193. The catalytic sites in fVII from other mammalian species may be determined using presently available techniques including, among others, protein isolation and amino acid sequence analysis. Catalytic sites may also be determined by aligning a sequence with the sequence of other serine proteases, particularly chymotrypsin, whose active site has been previously determined by Sigler et al., J. MoI. Biol., 35:143-164 (1968), and determining from the alignment the analogous active site residues. Attachment of the peptidyl linker to this domain will inactivate the serine protease activity, thereby reducing the potential of the composition, when administered to an animal, to induce blood coagulation. The terms "methylketone" and "chloromethylketone" as used herein refer to the carboxy terminus reactive moiety that may form the covalent bond between a peptide linker and an amino acid side chain of a recipient polypeptide. During the linkage reaction, the chloro group is removed. Thus, the unlinked peptidyl linker will have a chloromethylketone moiety and the covalently attached peptide will have a methylketone moiety without a halogen atom thereon. Peptidyl linkers suitable for use, before being bonded to the polypeptide, have a carboxy-terminus chloromethylketone group that may react with a suitable amino acid side chain of the polypeptide. Preferably, but not necessarily, the carboxy terminal amino acid having the chloromethylketone group thereon is an arginine. Although any peptidyl chain sequence or length may be used, a suitable peptide is a tripeptide. Preferred peptidyl linkers include, but are not limited to, tyrosine-glycine- arginine-chloromethylketone (YGR-ck); phenylalanine-phenylalanine- arginine-chloromethylketone (FFR-ck), glutamine-glycine-arginine- chloromethylketone (QGR-ck), and glutamate-glcine-arginine chloromethylketone (EGR-ck). A most preferred linker is FFR-ck. It will be understood by those of skill in the art that upon covalently attaching the chloromethylketone to the recipient polypeptide, the chloro- moiety is displaced. Accordingly, the term "FFR-ck-VIIa", for example, refers to FFR-methylketone tripeptidyl linker bonded to fVIIa and not having a chloro- atom attached thereto. It is believed that a complex, formed from photosensitizer- phenylalanyl-phenylalanyl-arginyl-ck-VIIa (FFR-ck-VIIa) and tissue factor (TF) expressed on the plasma membrane of cells, may be internalized in a FFR-ck-VIIa concentration-dependent manner by ligand-receptor mediated endocytosis. The ligand-receptor complex is endocytosed into early and late endosomes and is delivered to lysosomal vesicles and degraded by lysosomal enzymes. Accordingly, the peptide selected for use as a linker peptide is also suitable for cleavage by an intracellular hydrolytic activity of the target cell enzyme. When so cleaved, after endocytotic internalization, the photosensitizer attached to the linker may be released from a polypeptide such as fVIIa. The released photosensitizer may then modulate a physiological function of the target cell. A number of different linkers can be used. For example, linkers can be an arginyl methylketone such as phenylalanine-phenylalanine-arginine methylketone, tyrosine-glycine-arginine methylketone, glutamine-glycine- arginine methylketone, glutamate-glycine-arginine methylketone or phenylalanine-proline-arginine methylketone. In a preferred embodiment, the linker is phenylalanine-phenylalanine-arginine methylketone. In another preferred embodiment, the linker is tyrosine-glycine-arginine methylketone. In another preferred embodiment, a linker is covalently bonded to an amino acid side chain within a serine protease active site of fVIIa, thereby inactivating the serine protease active site. Tethers The term "tether" as used herein refers to a molecule that can form a hydrolysable bond such as, but not limited to, a carbamate, an amide, an ester, a carbonate or a sulfonate, bond with a cytotoxic compound such as, but not limited to, a curcuminoid, and which can also be covalently bonded to a linker such as, but not limited to, the N-terminus of a linker, including a peptidyl linker, thereby connecting the cytotoxic compound to the linker via the tether. Suitable tethers include a dicarboxylic acid, a disulfonic acid, an omega-amino carboxylic acid, an omega-amino sulfonic acid, an omega- amino carboxysulfonic acid, or a derivative thereof, wherein the tether may comprise 2-6 carbons in any arrangement such as a linear, branched or cyclic carbon arrangement, and wherein the tether is capable of forming a hydrolysable bond. The photosensitizer may be covalently bonded to a tether, which preferably is a dicarboxylic acid or caproyl moiety. Another exemplary tether is succinate that may be bonded to a curcuminoid by the addition of succinic anhydride. The hydrolysable bond can be a carbamate, an amide, an ester, a carbonate and a sulfonate. In one embodiment, the photodynamic compound is linked to the peptide via a tether that functions as a linker and as a tether. In a preferred embodiment, the photodynamic compound is connected to the peptide via both a tether and a linker. The tether forms a hydrolysable bond with the photosensitizer and is covalently bonded to a linker, which is covalently bonded to an amino acid side chain of the protein. In yet another preferred embodiment, the photosensitizer compound is bonded to a tether, which is covalently linked to an N-terminal amino acid of a peptidyl linker such as phenylalanine-phenylalanine-arginine, the C- terminal amino acid of which comprises a methylketone. The methylketone group forms a covalent bond with an amino acid side group of fVIIa that does not prevent the conjugated construct from selectively binding to tissue factor expressed on a cell membrane. In yet other embodiments, the tether can be a dicarboxylic acid, a disulfonic acid, an omega-amino carboxylic acid, an omega-amino sulfonic acid, an omega-amino carboxysulfonic acid, or a derivative thereof, wherein the tether comprises 2-6 carbons, and wherein the tether is capable of forming a hydrolysable bond. The tether can also be succinate. II. Method of Making Targeting Molecule-Photosensitizer Conjugates A. Conjugation The compositions comprise one or more photosensitizers bound to a targeting molecule. The photosensitizer can be bound directly to the targeting molecule, for example, through the reaction of reactive functional groups on the photosensitizer and the targeting molecule. Alternatively, the photodynamic compound can be conjugated to the targeting molecule via a linker and/or tether molecule. Porphyrins can be conjugated to targeting molecules, such as proteins, by the formation of ester or amide bonds. For example, verteporfin is reacted with dichloroethane ("EDC") in the presence of N- hydroxysulfosuccinimide ("Sulfo-NHS") and fVII to form the verteporfin- fVII conjugate as shown below. Sulfo-NHS can be used to aid in binding primary amines to carboxylic acid groups.
Dye +EDC, with or without Sulfo-NHS + fVIIP ->
Dye-(H-N): : : :C=O-(fVIIP)
Porphyrins can also be conjugated to proteins using carbodiimide or N- hydroxysuccinimide ester (HNS)-based protocols as described in Hamblin et al, J. Photochem. Photobiol, B 26, 45-56 (1994); Mew et al, J. Innumol, 130, 1473-1477 (1983); Donald et al, Otolaryngol. -Head Neck Surg., 6, 781-787 (1991); Hamblin et al., Photochem. Photobiol., 72, 533-540 (2000); Del Govematore et al. ; Br. J. Cancer, 82, 56-64 (2000); Schmidt et al, Br. J. Cancer, 75, 54-61 (1997). Sutton et al describe a method of conjugating porphyrins, chlorins, and bacteriochlorins to protein by synthesizing porphyrins, chlorins, and bacteriochlorins bearing a single amine-reactive isothiocyanate group. Sutton et al, Bioconjugate Chemistry, 13, 249-263 (2002). III. Methods of Treatment A. Pharmaceutically Acceptable Formulations Pharmaceutically acceptable compositions are provided that comprise a therapeutically-effective amount of conjugate, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents for use as a therapeutic agent for the treatment of a pathological condition of an animal or human such as a macular degeneration, diabetic retinopathy, cancer or other neovascular based disease. The phrase "therapeutically- effective amount" as used herein means that amount of a compound, material, or composition which is effective for producing a desired therapeutic effect against cancer or other pathological comprising neovascularization. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or an encapsulating material such as liposomes, polyethylene glycol (PEG), PEGylated liposomes, or particles, which is compatible with the other ingredients of the formulation and not injurious to the patient. The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intravitreal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's vascular system. The compositions may be delivered to an animal or human by any of these routes, depending on the disorder to be treated. The preferred route of administration is intravenous injection so that the effective dose of the compound can be delivered via the vascular system. The dose may be delivered by subcutaneous injection, intraperitoneal injection, direct injection into the tissue to be treated or a proximal blood vessel feeding the tissue to be treated for reducing dilution of the effective therapeutic composition, and to achieve more rapid application of the composition to the vascular cells. The affinity of the targeting molecule (i.e. fVII) for the cell surface marker (i.e. tissue factor) will localize the effective dose of the therapeutic composition for selectively targeting abnormal vasculature. As described in detail below, the pharmaceutical compositions may be specially formulated for administration in solid or liquid form, for example, a sterile solution or suspension. Conventional techniques for preparing pharmaceutical compositions which can be used are described in Remington's Pharmaceutical Sciences, 1985. Suitable pharmaceutical preparations are made by mixing the pharmaceutical composition, preferably in purified form, with suitable adjuvants and a suitable carrier or diluent. Suitable physiological acceptable carriers or diluents include sterile water and saline. Suitable adjuvants, in this regard, include calcium, proteins (e.g. albumins), or other inert peptides (e.g. glycylglycine) or amino acids (e.g. glycine, or histidine) to stabilise the purified fVIIa. Other physiological acceptable adjuvants are non-reducing sugars, cyclodextrins (cyclic carbohydrates derived from starch), polyalcohols (e.g. sorbitol, mannitol or glycerol), polysaccharides such as low molecular weight dextrins, detergents (e.g. polysorbate) and antioxidants (e.g. bisulfite and ascorbate). The adjuvants are generally present in a concentration of, but not limited to, from 0.001 to 4% w/v. The pharmaceutical preparation may also contain protease inhibitors, e.g. aprotinin, and preserving agents. The preparations may be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions. They can also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile medium suitable for injection prior to or immediately before use. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, preservatives and antioxidants can also be present in the compositions. Pharmaceutical compositions suitable for parenteral administration may comprise one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers which may be employed include water, saline, balanced salt solution, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and other antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid. It may also be desirable to include isotonic agents, such as sugars or sodium chloride into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as polyethylene glycol (PEG), aluminum monostearate and gelatin. In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by coupling to PEG, the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon size, form and amount of PEG, crystal size and crystalline form. Alternatively, delayed absorption of a parenterally- administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsuled matrices of the subject peptides or peptidomimetics in biodegradable polymers such as the polyhydroxy acids such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The pharmaceutical compositions are intended for parenteral, topical or local administration for prophylactic and/or therapeutic treatment. Most preferably, the pharmaceutical compositions are administered parenterally, i.e., intravenously, so that the compositions may be rapidly transported to a selected target cell such as a cancer cell or neovascular endothelial cell. Thus, compositions are provided for parenteral administration which comprise a solution of the fVIIPD dissolved in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine . The fVIIPD can also be formulated into liposome preparations for delivery or targeting to sites of injury. The compositions may be sterilized by conventional, well known sterilization techniques. The resulting solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. B. Disorders to Be Treated Disorders to be treated include any disorder characterized by uncontrolled or pathological angiogenesis, and preferably those characterized by overexpression of tissue factor. A switch from a quiescent state to an angiogenic state in the pathologic vasculature is usually activated by vascular endothelial growth factor (VEGF). One response activated by the binding of VEGF to receptors on vascular endothelial cells is the expression of tissue factor, a transmembrane receptor that binds plasma factor VII/VIIa to initiate blood coagulation. Because only the vascular endothelial cells that have bound VEGF express tissue factor, a putative target for the tumor vasculature is tissue factor expressed on endothelial cells which should bind factor VIWIIa circulating in the blood. In addition to macular degeneration, this method should be useful for other neovascular disorders such as macular degeneration, diabetic retinopathy, retinopathy of prematurity, pathologic myopia, neovascular glaucoma, sickle cells disease, corneal neovascularization, angiomas, melanomas, carcinomas, sarcomas, and other solid tumors 1. Corneal Neovascularization Neovascularization (NV) of the cornea represents a state of disease secondary to a variety of corneal insults, including contact lens (CL) wear. Diseases associated with corneal neovascularization that can be treated include but are not limited to, corneal graft rejection, , contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, Sjogren's, acne rosacea, phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration, chemical bums, bacterial ulcers, fungal ulcers, herpes simplex infections, herpes zoster infections, protozoan infections, trauma, rheumatoid arthritis, systemic lupus, polyarteritis, Wegener's sarcoidosis, scleritis, Stevens-Johnson disease, pemphigoid, radial keratotomy, and corneal graft rejection. Conjunctival neovascularization that can be treated include but are not limited to Pinguecula, pterygium, squamous cell carcinoma, pre- malignant lesions, scarring. 2. Skin and eyelids Skin and eyelid lesions associated with angiogenesis that can be treated include but are not limited to squamous cell carcinoma, basal cell carcinoma, angioma, hamangioma, scar, granuloma, other tumors of skin, eyelids, and orbit. 3. Retinal/Choroidal Neovascularization With choroidal neovasculatization, abnormal blood vessels stemming from the choroid grow up through the retinal layers. Diseases associated with retinal/choroidal neovascularization that can be treated include, but are not limited to, diabetic retinopathy, macular degeneration, retinopathy of prematurity, sickle cell retinopathy, myopic degeneration, histoplasmosis, sarcoidosis, angioid streaks, syphilis, pseudoxanthoma elasticum, Paget's disease, vein occlusion, artery occlusion, carotid obstructive disease, chronic uveitis/vitritis, mycobacterial infections, Lyme disease, systemic lupus erythematosus, Eales' disease, choroidal angioma, retinal angioma, ocular melanoma, Behcet's disease, retinitis or choroiditis, presumed ocular histoplasmosis, Best's disease, optic pits, Stargardt's disease, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma and post-laser complications. Other diseases include, but are not limited to, diseases associated with rubeosis (neovascularization of the iris ), neovascularization of angle, neovascular galucoma and diseases caused by the abnormal proliferation of fibrovascular or fibrous tissue including all forms of proliferative vitreoretinopathy, whether or not associated with diabetes. 4. Hemangiomas One of the most frequent angiogenic diseases of childhood is hemangioma. In most cases, the tumors are benign and regress without intervention. In more severe cases, the tumors progress to large cavernous and infiltrative forms and create clinical complications. Systemic forms of hemangiomas, the hemangiomatoses, have a high mortality rate. Therapy- resistant hemangiomas exist that cannot be treated with therapeutics currently in use. 5. Cancer and Benign Tumors Angiogenesis is prominent in solid tumor formation and metastasis. Angiogeneic factors have been found associated with several solid tumors such as rhabdomyosarcomas, retinoblastoma, Ewing sarcoma, neuroblastoma, and osteosarcoma. A tumor cannot expand without a blood supply to provide nutrients and remove cellular wastes. Tumors in which angiogenesis is important include solid tumors, and benign tumors such as carcinmoas, sarcomas, angiomas, and pyogenic granulomas. Angiogenesis is important in two stages of tumor metastasis. The first stage where angiogenesis stimulation is important is in the vascularization of the tumor, which allows tumor cells to enter the blood stream and to circulate throughout the body. After the tumor cells have left the primary site, and have settled into the secondary, metastasis site, angiogenesis must occur before the new tumor can grow and expand. Therefore, prevention or control of angiogenesis could lead to the prevention of metastasis of tumors and possibly contain the neoplastic growth at the primary site. C. Dosages The regimen for any patient to be treated with a pharmaceutical composition should be determined by those skilled in the art. The daily dose to be administered in therapy can be determined by a physician and will depend on the treated organ, cause of neovascularization, particular compound employed, on the route of administration and on the weight and the condition of the patient. The amount of fVIIPD that should be administered is that sufficient to provide an amount of photosensitizer which is capable, upon excitation, to damage the targeted tissue. This amount can vary widely depending upon the mode of administration and the formulation in which it is carried. As it is generally recognized that there is a relationship between the type of photosensitizer, the formulation, mode of administration, and dosage level, adjustment of these parameters to fit the particular combination to ensure delivery of an effective amount of the fVIIPD to the targeted tissue is possible. For retinal and choroidal disorders like macular degeneration and diabetic retinopathy, the preferred route of treatment is by intravitreal injection or intravenous injection with subsequent activation with non¬ thermal laser. The route of administration is the same for iris disorders. Topical (preferred), intracular or intravenous injection can be used for administration to the cornea. Either topical or systemic administration can be used to treat eyelids and skin. The dosage is adjusted based on the route of administration and the disease. The treatment may be repeated as needed. The pharmaceutical composition can be administered in a single dose, but it can also be given in multiple doses with intervals between successive doses depending on the dose given and the condition of the patient. The pharmaceutical composition may be administered intravenously or it may be administered by other routes. The dosage of the compound will depend on the condition being treated, the particular compound, and other clinical factors such as weight and condition of the patient and the route of administration of the compound. For the treatment of retinal and choroidal neovascularization the compositions may be administered by intravitreal injection or intravenous injection. A slow release device may be used for slow release in the eye or other organs. For the treatment of skin disorders, the compositions can be administered systemically or topically. For treatment of certain disorders, the fVIIPD may be applied locally to treat cancer, angiomas, pre-malignant conditions and other diseases and conditions in which angiogenesis occurs. The fVIIPD is administered as required to alleviate the symptoms of the disorder. Assays can be performed to determine an effective amount of the agent, either in vitro and in vivo. Methods are known to those skilled in the art, and can be used to determine an effective dose of these and other agents for the treatment and prevention of diseases or other disorders as described herein. After fVIIPD is administered, the application of dye excitation radiation, preferably via laser light, should be withheld until the targeted tissues selectively retain the dye, i.e, until the dye concentration in the target tissue is optimal. This will minimize damage to the non-targeted tissue. The optimum time following administration until the application of radiation to the treatment site will vary, and depends upon the mode of administration, the form of administration, and the nature of the targeted tissue should any radiation inadvertently be administered to non-targeted tissue. By way of example, if a choroidal neovascularization is the targeted tissue, the time between administration and application of radiation ranges from about 1 minute to about 2 hours, advantageously between about 15 to about 60 minutes, and preferably about 30 minutes. The fluence and irradiation during the treatment with the radiation source can also vary, depending upon the type of tissue undergoing treatment, the depth of treatment desired in the targeted tissue, and the amount of overlying fluid or blood. Generally, however, the fluence will vary between about 50-200 joules/cm2. The irradiance typically varies from about 150-900 mW/cm2, with a range between about 150-600 mW/cm2 being preferred. However, the use of a higher irradiance level may be preferable because higher levels shorten the treatment time. C. Photodynamic Therapy Preferably, electromagnetic radiation, such as from ultraviolet to visible and infra red light, is delivered after administration of the compositions and formulations. "Low-dose PDT", refers to a total photodynamic therapy experience at substantially lower levels of intensity than that ordinarily employed. Generally, there are three significant variables: the concentration of the photosensitizing drug, the intensity of the radiation employed and the time of exposure to light, which determines the total amount of energy ultimately delivered to the target tissue. Generally, an increase in one of these factors permits a decrease in the others. For example, if it is desired to irradiate only for a short period of time the energy of irradiation or the concentration of the drug may be increased. Conversely, if longer time periods of irradiation are permitted, lower irradiation intensities and lower drug concentrations are desirable. The use of low dose PDT offers an additional advantage in the form of reducing the likelihood of PDT side effects such as damage to unintended tissues or vessels. Additionally the targeting will increase the efficacy of the treatment by aiming at the abnormal blood vessels and preserving normal vessels. It is understood that the manipulation of these parameters will vary according to the nature of the tissue being treated and the nature of the photosensitizer (PS) employed. However, in general, low-dose PDT employs combinations of the drug concentration, radiation intensity, and total energy values which are several fold lower than those conventionally used for destroying target tissues such as tumors and unwanted neovascularization. One measure may be the product of PS concentration (e.g., in ng/ml) x intensity (e.g., in mW/cm2) x time (e.g., in seconds). However, it is difficult to set absolute numbers for this product since there are constraints on each of the parameters individually. For example, if the intensity is too low, the PS will not be activated consistently; if the intensity is too high, hyperthermic and other damaging effects may occur. Additionally, in some instances, ambient or environmental light available at the target cell or tissue undergoing PDT may be sufficient in the absence of additional deliberate irradiation. Similarly, PS concentrations cannot vary over any arbitrary range. There may also be constraints on the time during which radiation can be administered. Accordingly, the product of the foregoing equation is only a rough measure. However, this approach may provide a convenient index that can be adjusted according to the relative potency of the PS employed, and in general, an increase in intensity would permit a decrease in time of irradiation, and so forth. The present invention will be further understood by reference to the following non-limiting examples. Examples Example 1. Synthesis of fVII-Verteporfin Conjugate Materials and Methods Construction of plasmid containing the nifVIIP cDNAs. The plasmid vector encoding mfVTIP was constructed by amplifying the mouse factor VII cDNA with a K341A mutation from a previously constructed plasmid vector described in U.S. Patent No. 6,924,359. The mouse ("M")fVIIP cDNA contains the coding sequence for mfVII, a BamHI site, ribonuclease S-peptide (wild-type or mutated) and 6 Histidines with a Hind III at the 5 '-end and Not I at the 3 '-end. The PCR amplified cDNAs are sequentially digested with Hind III and Not I and ligated into the Hind III and Not I digested-pcDNA3.1 (+). The sequences of the inserts in the plasmids were confirmed by sequencing. A hfVIIP cDNA also has been constructed with mutated human fVII in place of mouse fVII. Production and purification of the mfVIIP protein. The mfVIIP plasmid was transfected into Chinese Hamster Ovary (CHO) cells using the Superfect reagent (Qiagen), and the cells were selected for tranfected colonies. The colony with the highest expression of mfVIIP protein was grown in CHO serum-free culture medium (Excel 301, JRH Biosciences) supplemented with 1 μg/ml vitamin Kl (Sigma), and the medium was collected every 3-4 days. The media were pooled and the protein was purified using Ni-NTA resin (Qiagen). The purified protein was dialyzed against PBS pH7.4 and concentrated to at least 1 mg/ml. The purified protein was stored at -20C for labeling reaction with the dyes. Extraction of pure verteporfin dye from liposomal Visudyne and conjugation with FVIIP protein. Verteporfin was extracted from liposomal Visudyne (QLT) by acidification with 6 M HCl and separation of organic (verteporfin, lower phase) and aqueous (liposome, upper phase) layers using CH2Cl2. After concentrating, verteporfin was further purified by silica gel chromatography using an eluting solvent CH2Cl2/methanol (3:1) and vacuum dried. For conjugation to the fVIIP protein, verteporfin was dissolved in DMF to a final concentration of 10 mg/ml. The verteporfin was activated by mixing 2 ul of verteporfin solution in DMF with 2 ul of a 25 mg/ml solution of EDC in DMF and adding 6 ul DMF. The mixture was incubated at room temperate for 30 min, followed by addition of 80 ul of fVIIP (1 mg/ml) was added to the activated verteporfin and incubated at RT for 1 hr. The verteporfin- mfVIIP conjugate (mfVTIPD) was separated from unconjugated verteporfin with a Sephadex G50 spin column. The mfVIIPD was scanned on a spectrophotometer from 200nm to 800 run to measure the protein absorbance at 280 run and the verteporfin absorbance at 689nm. For the control PBS was added instead of mfVIIP protein to the activated dye. Results The mfVIIPD molecule absorbed at 280 nm and 689 nm, whereas free verteporfin and mfVIIP protein absorbed at 689 nm or 280 nm, respectively. These data indicate that verteporfin was successfully conjugated to the mfVIIP protein. The same procedure was tested for conjugation of the mfVIIP to another dye, Sn Chlorin e6 (SnCeό). The conjugation to SnCe6 also was successful. Example 2. Binding of mfVIIP protein and mfVIIPD (conjugated to verteporfin) to human tumor cells expressing TF. mfVIIP was conjugated to verteporfin using the procedure described above. The binding activity of the conjugate to human breast cancer cells was tested by flow cytometry The results indicate that the binding activity, presumably to Tissue Factor on the tumor cells, was identical for the conjugated and non- conjugated molecules. Example 3. Injection of fVII-targeted verteporfin followed by laser activation stops blood vessel leakage in rats. Tissue factor (TF), a transmembrane receptor, forms an exceptionally strong and specific complex with its ligand fVII as the initial step of the blood coagulation pathway. TF is not normally highly expressed on vascular endothelial cells but is expressed in a significantly higher amount on endothelial cells of new vessels such as tumor vasculature, probably induced by angiogenic factors such as Vascular Endothelial Growth Factor (VEGF). A photosensitizer is attached to a targeting molecule called fVII, which selectively binds to endothelial cells of abnormal blood vessels. The model for the targeting molecule is a Camelid IgGl antibody, which is composed of two heavy chains without associated light chains; each heavy chain contains a VH targeting domain conjugated directly to the hinge region of the Fc effector domain. The targeting molecules are composed of one or two chains, each chain containing a fVII targeting domain conjugated to the hinge region of an IgGlFc effector domain. The two fVII domains provide an avidity effect for binding to cells expressing multiple TF molecules, resulting in an affinity for TF that is significantly higher than the strong affinity of the monomelic fVII molecule. The Fc domain can activate a powerful cytolytic attack against cells that bind the targeting molecule , mediated by components of the immune system containing Fc receptors, such as natural killer (NK) cells and the CIq protein that initiates the complement pathway. The efficacy of the fVII/Fc for cancer immunotherapy was demonstrated in a mouse model of human cancer, using an adenoviral vector encoding the fVII as the delivery system (Hu, et al., PNAS, 96:8161-8166 (1999) and Hu and Garen, PNAS 97:9221-9225 (2000)). Clinical trials of the protocol are being arranged for melanoma and prostate cancer. The fVII/Fc shows remarkable specificity for binding to TF on endothelial cells of growing blood vessels induced by exposing the retina of the experimental animals to a laser beam. The experiments have demonstrated the efficacy of FVIIPD-Targeted Photodynamic Therapy ITPT) of choroidal neovascularization (CNV) in a rat model of age-related macular degeneration (ARMD). Methods CNV lesions were induced by laser photocoagulation of the retina in both eyes of Norway-Brown rats. Choroidal neovascularization was induced by photocoagulating the posterior pole of rats with argon green laser using a slit lamp and a fundus contact lens to deliver 200-700 mW power for 0.1 second to a 50μm spot. The power was adjusted to produce a blister or small hemorrhage. If no bleeding was observed, an additional laser-induced lesion was produced next to the first spot. Neovascular membranes usually develop within 2 weeks. After 4 weeks, FVII-verteporfin conjugate (fVIIPD) at doses of 0.5 or 1 mg/m2 was injected intravenously into the tail vein of the rats. Some of the CNV lesions were irradiated with 689 nm laser for 83 seconds at 15, 30 or 60 minutes after intravenous injection, while the other lesions were not irradiated (no light controls). Control rats were intravenously injected with Verteporfϊn (6.0 mg/m2) and laser irradiation was done 15 minutes later. The lesions were examined by fundus photographs and fluorescein angiography 1, 7 and 14 days later to assess the efficacy of ITPT. Choroidal veovascular membranes were treated by intravitreal injection of Verteporfin attached to targeting molecules that bind selectively to the endothelial cells of pathologic vessels. The photosensitizer was activated with an opal laser. A similar experiment was performed by intravitreal injection of fVII-targeted verteporfin. Results Intravitreal injection of fVII-targeted verteporfin followed by laser activation completely stopped blood vessel leakage at 7 days. This method selectively destroys abnormal vessels, to which the photosensitizer is attached, while leaving neighboring tissues intact. The advantage of targeted photodynamic therapy compared to conventional photodynamic therapy is the fact that it is selectively targeted toward abnormal blood vessels, thus it combines the benefits of targeting with photodynamic therapy. Targeted photodynamic therapy increases the efficacy of conventional photodynamic therapy and decreases its side effects, since the normal blood vessels will be relatively preserved. It is understood that the disclosed invention is not limited to the particular methodology, protocols, and reagents described as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
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