MXPA01008291A - Compositions and methods for use in targeting vascular destruction - Google Patents

Abstract

Treatment of warm-blooded animals having a tumor or non-malignant hypervascularization, by administering a sufficient amount of a cytotoxic agent formulated into a phosphate prodrug form having substrate specificity for microvessel phosphatases, so that microvessels are destroyed preferentially over other normal tissues, because the less cytotoxic prodrug form is converted to the highly cytotoxic dephosphorylated form.

Description

COMPOSITIONS AND METHODS FOR USING THE VASCULAR DESTRUCTION APPROACH CROSS REFERENCE TO RELATED REQUEST This application claims the benefit of the priority of the co-pending provisional patent application of the United States of America Series No. 60 / 120,478 filed on February 18, 1999.

BACKGROUND OF THE INVENTION This invention relates to methods of compositions for effecting targeted vascular destruction of warm-blooded animals, including humans, and methods for identifying drugs capable of such use.

The importance of the vasculature to the growth of tumors is an unquestioned scientific reality. Because a blood vessel feeds thousands of tumor cells, focusing the vasculature of tumors as a molecular approach to cancer chemotherapies is becoming one of the highest scientific priorities. Two drug models are emerging, for example, one that prevents the formation of new blood vessels in the tumor (antiangiogenesis) and one that marks vascular destruction as a means to limit the tumor's feeding and / or the impermeability of the luminal surface. from vessels of endothelial cells to cancer drugs such as immunotherapies (New England Journal of Medicine 339: 473-474, 1998). The antianginal model is basically a cytostatic approach where the angiotensin factors generally produced by tumors such as vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor are blocked by anti-angiogenic compounds such as polypeptide angiostatin. natural endostatin to prevent new growth of blood vessels (The Cancer Journal Scientific American 4 (4): 209-216, 1998; Cell 88: 277-285, 1997) On the other hand, the model of vascular destruction is a cytotoxic approach where the tumor vessels are focused for cytotoxicity in order to increase the cytotoxicity of the tumor cell by means of hypoxia or direct action chemotherapy.

One of the most potent classes of cancer therapeutic drugs are the antimitotic d tubulin polymerization inhibitors (Biochem Molecular Biology Int (6): 1153-1159, 1995; Br. Journal Cancer 71 (4): 705-711, 1995 Journal Med. Chem. 34 (8): 2579-2588, 1991; Biochemistry 28 (17) 6904-6991, 1989). These characteristically have IC cell cytotoxicities in vitro in the range of nM-μM, but often show poor specificity to kill the tumor on tissues in vivo, examples of such drugs include the co bretastatins, taxol (and other taxanes) , vinblastine (other vinca alkaloids), colchicines, dolastatins, podophyllotoxins, steganacines, amphetamines, flavanoids, rhizoxins, curacin A, epothilones AB, welwistatins, fentastins, 2-strylquinazoline-4 (3H ) onas, stilbenes, 2-aryl-1, 8-naphthyridine-4 (1H) -ones, 5,6-dihydroindolo (2, 1-a) isoquinolines, 2,3-benzo (b) thiophenes , the 2, 3-substituted benzo (b) furans and the 2,3 substituted indoles (Journal of Med. Chem. 41 (16): 3022-3032 1998; Journal Med. Chem. 34 (8): 2579-2588, 1991, Anticancer Drug 4 (1): 19-25, 1993; Pharm. Res. 8 (6): 776-781, 1991 Experimentia 45 (2): 209-211, 1989; Med. Res. Rev. 16: 2067 , 1996 Tetrahedron Lett.34: 1035, 1993; Mol. Pharmacol. 49: 288, 1996 J. Med. Chem. 41: 1688-1695, 1998; J. Med. Chem. 33: 1721, 1990 J. Med. Chem. 34: 2579, 1991; J. Md. Chem. 40: 3049; 1997; J Med. Chem. 40: 3525, 1997; Bioorg. Med. Chem. Lett. 9: 1081-1086 1999; International Application (PCT) No. US 98/04380; provisional patent application of the United States of America No. 60 / 154,639. Although tubulin-binding agents in general can mediate effects on the flow of blood tumors, the effective doses are often also toxic to other normal tissues and not particularly toxic to tumors (Br. J. Cancer 74 (Suppl. ): 586-88, 1996).

Many tubulin binding agents and such combretastatins and analogous taxolls are insoluble in water and require formulation prior to clinical evaluation. An approach that has been successfully used to overcome this problem of clinical development is the formulation of water-soluble biolabilag prodrugs, such as the salt derivatives of combretastain A4 phosphate and taxol, which allows metabolic conversion back into the form insoluble to water (Anticancer Drug Des. 13 (3): 183-191, 1998; United States Patent No. 5,561,122; Bioorganic Med. Chem Lett. 3: 1766, 1993; Bioorganic Med. Chem. Lett. : 1357, 1993) A prodrug is a precursor which may undergo l metabolic activation in vivo to the active drug. Disclosed further reference to the aforesaid phosphate salt derivatives, the concept here is that specific phosphatases such as alkaline phosphatases in mammals are capable of dephosphorylating the phosphate prodrugs in the biologically original active forms. This prior art teaches how to administer an insoluble drug to water to warm-blooded animals for therapeutic purposes under conditions of maximum ma absorption and bioavailability by formulating in water-soluble biolabile form (Krogsgaard-Larsen, P. Bundegaard, H., eds., a textbook on the Drug Development Drug Design, Harvard Academic Publishers, page 148, 1991).

When the combretastatin A4 phosphate prodrug was used in vitro and in vivo, the animal and cell models exhibited an exceptional specificity for vascular toxicity (Int. J. Radiat, Oncol. Biol. Phys. 42 (4): 895-903 1998; Cancer Res. 57 (10): 1829-1834, 1997). It was not obvious by this one with a skill in the art that phosphat prodrugs in general, which serve as substrates of alkaline phosphates, had nothing whatsoever to do with the vascular approach. However, the data reported in the combretastatin A4 phosphate prodrug did describe the principle of preferential vascular toxicity, even though there was no understanding or appreciation of the fact that it produced it by itself was responsible for the vascular approach. In other words, prior art teaches that A4 and not the A4 prodrug was responsible for vascular toxicity by assuming that there was no difference in vascular toxicity between the two non-obvious forms described above is simplified by the fact that although the A4 phosphate prodrug and other taxol phosphate prodrugs were promoted as susceptible to the conversion of phosphatase to the cytotoxic tubulin binding forms, it has never been recognized that this enzyme was elevated in microvessels that focused on cytotoxicity.

SYNTHESIS OF THE INVENTION An object of the invention is to provide compositions and methods useful in approaching the destruction model of microvessels for treatment, in warm-blooded animals including (but not limited to) humans, e cancer, Kaposi's sarcoma, and others. , non-malignant proliferative vascular disorders such as macular degeneration, psoriasis and restenosis, and in general, inflammatory diseases characterized by vascular proliferation.

Another object is to provide methods for identifying drugs that are capable of being used in the production of such compositions and effecting such methods.

For these and other purposes, the present invention in a first aspect broadly contemplates the provision of such a method to treat a warm-blooded animal having a vascular proliferative disorder, which comprises administering to the animal an amount of a prodrug instead of a phosphate of disodium d combretastatin A4 effective to achieve vascular destruction focused on a locality of proliferating vasculature, where the prodrug is substantially non-cytotoxic but is converted to a substantially cytotoxic drug by the action of a selectively induced enzyme in increasing levels in sites of vascular proliferation.

In a second aspect, the invention contemplates the provision of a method to treat a warm blood animal having a non-malignant vascular proliferative disorder, which comprises administering to the animal, an amount of an effective prodrog to achieve targeted vascular destruction in a proliferating locality of the vasculature, where the prodrug is substantially non-cytotoxic but is convertible to a substantially cytotoxic drug by the action of a selectively induced endothelial enzyme at increased levels and sites of vascular proliferation.

In a further aspect, the invention contemplates the delivery of compositions for treating a warm blooded animal that has a vascular proliferative disorder to achieve targeted vascular destruction in a locality of proliferating vasculatur, comprising a prodrug, instead of the phosphate prodrugs. of taxol, pancrastistatin and combretastatin A4, which is substantially non-cytotoxic but is convertible to a substantially cytotoxic drug by the action of a selectively induced endothelial enzyme at increased levels and sites of vascular proliferation.

In still another aspect, the invention provides a method for identifying prodrugs suitable for use in the above methods and compositions, in such a procedure comprising the steps of culturing the endothelial proliferation of cells, and other cells, in the presence of a prodrug, a a disodium phosphate of A4 combretastatin for a limited period; The respective cultures are then compared to determine if the culture of the endothelial cells which proliferate exhibit a significant greater cytotoxic effect than the culture of other cells; and, if so, the cultivation of the other mentioned cells in the presence of the prodrug and of an endothelial enzyme selectively induced in level increased in sites of vascular proliferation, the cytotoxic effect increased with respect to other cells in the presence of the enzyme as compared to the cytotoxic effects in the initial culture of the other cells indicating the convenience of the prodrug for such methods and compositions Conveniently or preferably, the "other cells" may be non-malignant fibroblastomas, eg, fibroblast, to normal humans.

In an important specific sense, to which however the invention is in its broadest and most limited aspects, the prodrug of the methods, compositions, and methods above may be a phosphate within the class of compounds having the following general formula: Z ll R'-X-P-Y R2 and R? where: X is O, NH, or S; And it is O, NH, S, O, NH or S; Z is O or S; each R2 and R3 is an alkyl group, H, is a mono or divalent cationic sa, or a cationic ammonium salt, and and R3 may be the same or different; Y R1 is defined by the formula R! -R qu represents a compound containing at least one group (designated Ra) Which is a group, which contains X, which can form a phosphate or other salt that serves as a substrate for endothelial phosphatases vascular non-specific, and is therefore still converted relatively non-cytotoxic form of phosphate to a cytotoxic form R'-R ".

Currently the preferred prodrugs for the practice of the invention are those having the following formulas: o O II II R? _0_p_0- Na + R? _N_p_0- Na + R1-N-P-OCH2CH3 I I I O O "Na + H O" Na + H 0CH2CH3 More particularly, the compound with the formula R'-Ra can be a tubulin-binding agent. In specific aspects it can be selected from tubulin binding agents and previously listed such as combretastatins, taxanes, vinblastins (vinca alkaloids), colchicines, dolastatins, podophyllotoxins, estganacines, amphetamines, flavanoids, rhizoxins, the curacinas To the epothilones A and B, the welwistatins, the fenstatins, the 2 strilquinazolina-4 (3H) -onas, the stilbenes, the 2-aryl-l, 8 naft ir idina-4 (1H) -ones, the 5,6-dihydroindolo (2, 1 a) isoquinolines, the 2, 3-benzo (b) thiophenes, the 2, 3-substituted benzo (b) furans and the 2, 3-substituted indoles. In still a more specific sense, ttubulin binding agent can be a compound selected from the group consisting of combretastatin (instead of A4 combretastatin), colchicine, and 2-methox estradiol.

Described with reference to phosphate prodrugs, for an understanding of the invention it can be explained that vascular endothelial cells have at high levels of phosphatase activity due to (i) the response to arrest arrest of the microvessels due to the circulation of the blood (J. Invest, Dermatol, 109 (4): 597-603, 1997) and (ii) the induction of phosphatase in vascular endothelial cells by IL-6 produced by inflammatory cells during wound healing or by invasion tumor cells (FEBS Lett.350 (1): 99-103, 1994; Ann. Surg Oncol. 5 (3): 279-286, 1998). The high levels of phosphatases (eg alkaline) only a part of the normal physiology of the microvessels, because together with the blood coagulation mechanism, the calcium deposits generated by the assistance of the alkaline phosphatase activity in the processes of sanitation d the wound. The present invention encompasses the discovery that phosphatase or other appropriate prodrug constructs, which become substrates for phosphatases such as alkaline phosphatases, are useful in focused microvascula toxicity. Examples of phosphatase enzymes suitable for this purpose require an ectoplasmic cellular location due to the poor absorption of phosphorylated molecules through the cytoplasmic membrane. The dephosphorylating enzymes that have an ectoplasmic location are non-specific alkaline phosphatase, ATPase, ADPase, 5 'nucleotidase, and nucleoside purine phosphorylase. Another necessary property for the cytotoxic agents targeted by defoeforilization by means of phosphatase fabrics is that they can utilize a broad spectrum of phosphate prodrugs with substrates. In this regard, alkaline phosphatase is an attractive target to provide selective toxicity to vascular endothelial cells.

Advantages and additional features of the invention may be apparent from the detailed description set forth below, together with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Figures IA, IB and 1C illustrate the structure of various cytotoxic compounds and non-cytotoxic prodrugs thereof as examples of molecular diversity capable of targeting microvascular cell toxicity through the formation of phosphatase prodrugs; Figures 2A and 2B are graphs showing the effect of time exposure on the cytotoxicity of Combretastatin A4 prodrug; Figures 3 and 3B are graphs showing the effect of alkaline phosphatase on an HDF and on a cultured HMVE; Figures 4A and 4B are graphs showing the effect of dose response of alkaline phosphatase added in the cytotoxicity of HMVEC and HDF to prodrug A4; and Figure 5 is a series of graphs showing the effects of time exposure on the clonogenic toxicity induced by a variety of tubulin-binding drugs.

DETAILED DESCRIPTION This invention encompasses the use of phosphate prodrugs which comprises administering to the warm blood animals having tumor or a non-malignant hypervasculation a sufficient amount of a cytotoxic agent formulated in a prodrug form having substrate specificity for the microvessel phosphatases, so that the microvessels are destroyed preferentially on other normal tissues, because lesser form of cytotoxic prodrug is converted to the highly cytotoxic form of phosphorus. Examples of the preferred cytotoxic agents for the vascular approach are tubulin binding agents, because they can be transformed from insolubility to water to water solubility, tubulin binding agents to non-tubulin binding agents, and cytotoxicity to the non-cytotoxicity by means of the phosphate prodrug formulation (Anti-Cancer Drug Design 13: 183-191, 1998).

Examples of the molecular diversity for cell toxicity of approach microvases by the formation of phosphate prodrugs are presented in Figures IA to 1C and these were selected from the known tubulin binding agents already listed previously such as combretastatins, taxanes, vonblastina (vinca alcaliodes), colchicinoids, dolastatins, podophyllotoxins, steganacines, amphetamines, flavanoids, rhizoxins, curanins A, epothilones A and B, welwistatins, fentastins, 2-strilquinazoline - (3H) -ones, stilbenes, 2-aryl-l, 8-naphthyridine-4 (1H) -ones, 5,6-dihydroindolo (2, 1-a) isoquinolines, 2-3-benzo (b) thiophenes, the 2, 3-substituted benzo (b) furans and the 2, 3-substituted indoles The compounds listed in Figures IA to 1C satisfy the structural requirements of having either aromatic hydroxyl or groups of amines present capable of chemically reacting to produce a phosphate salt, and the further conversion of a cytotoxic agent into a cytotoxic phosphate prodrug construct. Other criteria necessary for focused vascular toxicity are: 1. Tubulin binding agents or other cytotoxic accidents (eg, pancratistatin has not been reported as binding to tubulin polymers) should induce similar levels of toxicity to both human microvessel cells and to other normal human cells such as fibroblastomas when in the cytotoxic form (tubulin binding), or, alternatively, the tubulin binding form must be much less inherently cytotoxic to normal cells than the microvessel cells. If this were not the case the fibroblastomas (for example normal cells) were much more sensitive than the microvessels to the cytotoxic form, then when the form of the cytotoxic prodrug, even though the fibroblastomas had much less phosphatase to activate the cytotoxic form, much Less pruning in turn may be necessary to induce cytotoxicity in fibroblastomas. The net result may be that the prodrugs may still be more toxic to the microvessels instead of the normal cells, due to their increased alkaline phosphatase activity which produces the cytotoxic form. 2. The cytotoxic forms or tubulin binding of the potential phosphate prodrugs should not be cytotoxic in the form of prodrug, which in turn needs to be converted into the cytotoxic form within 1 to 3 hours, preferably within 1 to 2 hours . The tubulin binding agents are cleared from the peripheral circulation within a few hours. So in order to be more effective in vascular destruction focused in vivo, the phosphate prodrug constructions must be converted to the cytotoxic forms within 1 to 3 hours by phosphatase in the microvessels in order to cause preferential toxicity of the cells. Therefore, the kinetics of tubulin binding must necessarily be completed within 1 to 3 hours.

Although higher levels of alkaline phosphates are useful for vascular destruction focused on tubulin binding agents, this invention also encompasses, in a broader sense, than any enzyme or protein specifically amplified in microvessels, and which is metabolically capable. becoming a nontoxic prodrug in a cytotoxic drug, it may be equally useful in focused vascular destruction.

The compositions according to the invention which have use in targeted vascular destruction are actively exemplified, without limitation, by the compounds encompassed within the class of compounds having the general formula: O O II R'-O-P-O R R'-N-P-O R2 wherein R1 is defined by the formula R'-R * which represents a compound containing at least one group (designated Ra) and which is a phenolic hydroxyl group, or an aromatic amine group, or any other amine group or suitable hydroxyl which can form the phosphate metal R2-R3 or amine salts of phosphate esters which serve as substrates for non-specific vascular endothelial phosphatase, and are therefore converted to a relatively non-cytotoxic phosphate form an amine form or cytotoxic hydroxyl.

Therefore, in the illustrative embodiments, R is defined by the formula R'-R "representing a compound containing at least one group of phenolic hydroxid (designated Ra) which can form a sodium phosphate another suitable room (eg example, R2, R3 can be Li, Na, K, Ca Cs, Mg, Mn, Zn, piperazine, nicotinamide and other examples as found in International Patent Application (PCT) No. 99 / US / 5368, the complete description of which is incorporated herein by this reference) which serves as a substrate for non-specific vascular endothelial phosphatases, and which are thus converted from a relatively cytotoxic n-phosphate form to the cytotoxic phenolic hydroxyl form.

The invention particularly encompasses discovery made in determining henceforth the unknown explanation for the apparent selective approach observed of endothelial cells proliferating by disodium phosphate d of combretastatin A4, and in recognizing the applicability of those discoveries to other drugs instead. of Combretastatin A4 and non-malignant treatment as well as malignant disorders that involve vascular proliferation.The relevant studies regarding the disodium phosphate of combretastatin A4 are now described.

Chemicals The disodium phosphate of combretastatin A was purchased from OXIGENE, Inc. (Boston) dissolved in physiological saline for the addition of cell cultures. The alkaline phosphates were purchased from Sigma as a buffer solution and were added directly to the cell cultures.

Cell culture Four commercially available human cell lines were grown in the medium indicated below in 5% C02 80% humidity and 37 ° C. 1. The human leuchymy cells HL60, a pro-apoptotic cell line - grown in RPMI 1640 fortified with 10% of fetal serum of beef. 2. The human leuchymy cells K562, a resistant d apoptotic cell line - grown in RPMI 164 fortified with 10% serum of fetus of beef. 3. Neonatal microvascular endothelial cells (HMVEC) - cultured in 131 + medium microvascular growth supplement (MVGS) + coupling factor (AF) = 50 milliliters + 25 milliliters (AF is added 2-3 milliliters frasc per T-25; reagents supplied by Casacad Biologics, Inc. Portland, Oregon). 4. Human neonatal dermal fibroblasts (HDF) grown in medium 106 + lower serum growth supplement (LSGS) = 500 milliliters + 10 milliliters (Cascad Biologics, Inc.).

The cells used in all the experiments were first subcultured for 2 to 3 days at an initial density of 2 x 105 cells per milliliter before their use in in vitro tests. This resulted in an exponential growth stage and the cell viability was > 95% trypan blue exclusion.

Cell Survival by Clonocorentric Test. This essay is based on the description reported by Schweitzer and others (Expt Haematol 21: 573-578, 1993) with slight modifications. Briefly, the HMVEC, HDF, K56 and HL60 cells concentrations of 4.2 x 103 milliliters were cultured in 96 wells of flat microculture dishes at the bottom at a volume of 190 μl per well plus different concentrations of disodium phosphate of combretastatin A4 or other tubulin binding agents and their prodrugs or alkaline phosphatase units added in a volume of 10 μl. After 5 days of incubation under the normal culture conditions described above, the colonies (> 40 cells) were counted by a reverse light microscope or estimated by an MTT assay. IC values were obtained from adjusted control percentage curves against drug concentrations.

Metabolism of alkaline phosphatase disodium phosphate of combretastatin A4 to combretastatin A4 cytotoxic superior. There are three types of experiments designed to demonstrate the importance of converting an A4 to A4 prodrug in order to target the toxicity of vascular endothelial cells.

Experiment 1 The HL60, K562, HDF, and HMVE cells were either cultured in well dishes -96 at the indicated concentrations (Figures 2A and 2B) for 5 days in the presence of A4 prodrug, or after 2 hours exposure and medium containing The drug was removed, a fresh medium was added, and the cells were cultured for an additional 5 days. Clonogenic growth was recorded after 5 days d incubation for all treatments.

Experiment 2. HMVEC and HDF were cultured well plates -96 from microtiter initially containing 80 cells per well. The cells were cultured for 1 hour in the presence of the indicated concentrations of alkaline phosphatase ± 1 bound prodrug. The medium was removed, the cells washed, and fresh medium was added, and the cells were incubated for an additional 5 days. The clonogenic growth was then established by the MTT test.

Experiment 3. The HMVEC were cultured in well plates -96 microtiter wells that initially contain 800 cells per well. Cells were cultured for 1 hour in the presence of the indicated concentrations of A4 prodrug ± the indicated alkaline phosphatase units. The medium was removed, the cells washed in the medium, and the cultures were additionally cultivated in fresh medium for an additional 5 days. The clonogenetic growth was then established by the MTT test.

Referring to the drawings, figures 2A and 2B are graphical showing the effect to the exposure of the time in the cytotoxicity of the prodroga A4. The HMVEC, HDF, HL60, K562 cells were exposed for 2 hours (Fig. 2A) or five days (Fig. 2B) to the disodium phosphate of combretastatin A4 before the clonogenic cytotoxicity was estimated at 5 days. Note that the ICS0 values were similar to the cells after five days of exposure, which were 1.5 to 2.5 nM where only HMVEC showed IC5U of cytotoxicity when exposed, but limited to 2 hours.

Figures 3A and 3B are graphs showing the effect of alkaline phosphatase on the HMVEC and on the cultured HDF. The response to the dose of cytotoxicity was estimated after exposure time at various concentrations of disodium phosphate of combretastatin A4 in the presence today in the absence of 1 unit of alkaline phosphatase. Note the lack of cytotoxicity of HDF without adding alkaline phosphatase, but the cytotoxicity of prodrug A4 was the same for HMVEC and HDF when alkaline phosphatase was added.

Figures 4A and 4B are graphs showing the effect of the dose response of alkaline phosphatase added in the cytotoxicity of HMVEC and of HDF to prodrug A4. The HMVEC and e HDF were cultured for 1 hour in the presence of the indicated concentrations of disodium phosphate d combretastatin A4 + the indicated units of alkaline phosphatase added. The data clearly show the added dependence of alkaline phosphatase on cytotoxicity especially in the concentrations of the superior A4 prodrug.

EXAMPLE 1 Example 1 describes the importance of time to preferential cytotoxicity exposure of vascular endothelial cells to tubulin binding agents such as combretastatin A4 prodroga. If the clonogenic assay was adjusted to treat HMVEC, HDF, K562 and HL60 cells for five days in the presence of increasing concentrations of combretastatin A4 disodium phosphate (prodrug), all cell lines have similar IC50 values around d 1.5 to 2.5 nM (figure 2B). These data teach that there is no inherent difference in the toxicity of human cell lines despite their origin, if the exposure time is sufficiently long. However, the A4 prodrug as well as other tubulin-binding drugs, far from peripheric circulation in vivo within a few hours, and under these conditions, the A4 prodrug showed a preferential toxicity to the endothelial cells that proliferate in the tumors, in where tubulin binding agents have not been shown to possess this property (Cancer Res. 57 (10): 1829-1834, 1997). Therefore, we have limited exposure of several cell lines to prodrug A for 2 to 3 hours, removing the medium containing A4 to replace it with fresh medium, a continuous culture for an additional 5 days. These conditions showed that the HMVEC was quite sensitive to the induced cytotoxicity of prodrug A compared to HDF, K562 and HL60 cells (Fig. 2A). This data teaches that (i) an in vitro cell model can be used to demonstrate the selective induction of toxicity to vascular endothelial cells by d tubulin binding agents such as prodrug A4, (ii) these only occur under in vitro conditions. vitro mimicking the limitations regulated pharmacokinetic in vivo exposure, and (iii) either the tubulin binding parameters that regulate metabolic or cytotoxicity differences or both are responsible for the selective toxicity of A4 prodrug to vascular endothelial cells.

EXAMPLE 2 The combretastatins are a family of naturally occurring tubulin binding agents comprising a series of structures A-, B-, C- and D- (U.S. Patent Nos. 4,940,726, 4,996,237, 5,409,953, and 5,569,786). In example 2, for the IC 50 values of the clonogenétic toxicity induced by a selection of these compounds in in vivo culture of HDF, HMVEC and HL60. The compounds are added to the micro-cultures in DMSO (for example, <0.5%) and the toxicity was evaluated by MTT test after 5 to 7 days in the culture The data in table 1 shows that the combretastatin analogies varied considerably in their total clonogenic toxicity between the various analogies as well as between the different types of human cells that are evaluated. A has the most toxic mechanism of tubulin binding in all types of cells tested, and showed no preference for the clonogenic toxicity among the cell types. However, the cytotoxicity of the other combretastatins can generally be classified according to the clonogenétic toxicity from most to least toxic such as: HL-60 > HDF > HMVEC These data established the prerequisites of tubulin binding drugs to have a property where toxicity to normal cells is not much greater than HMVEC, s phosphate prodrugs are not to be used in the vascular approach of toxicity antimitótica.

Table 1 Clonogenetic toxicity values IC50 in nM Combretastatin HDF HMVEC HL-60 A4 1-2 1-2 1-2 A3 8-10 > 12 5 A2 25-35 30-40 15 Al 20 500 n.d. Bl 200-300 200-300 500 B2 1100 800-1000 125 K-228 40-90 90-120 90 K-332 800-900 > 1000 500 The combretastatins were kindly provided by Professor G.R. Pettit from Arizona State University. HDF = human diploid fibroblastomas; HMVEC = human microvessel endothelial cell; HL-60 = human myeloid leuchymal cells.

EXAMPLE 3 The effect of time exposure on the clonogenic toxicity induced by a variety of uni d tubulin drugs are presented in Figure 5. Taxol, taxotera, vincristine, and combretastatins Al and A4 were added to the micro-cultures of HMVEC and of HDF for 1 and 6 hours, washed with saline and continuously and incubated in complete medium for more days before estimating the clonogenic toxicity by means of the MTT assay. The data in this example show that the kinetics of the binding of various tubulin binding drugs influenced their cytotoxicities under conditions that are similar to the exposure in vivo (eg 1 hour). For example, taxol, taxotera and combretastatin Al did not induce maximum toxicity to HMVEC after 1 hour of exposure for 6 hours, and additionally, the degree of cytotoxic regulated kinetic responses were also different in HDF compared to the HMVEC.

Therefore, in order to focus microvessel toxicity in humans the tubulin binding cytotoxic mechanism needs to be completed within a period of 1 to hours after treatment in a manner that allows toxicity to the HMVEC to be comparable to HDF or other normal cells. When this is the case then the phosphate prodrugs are able to focus on the microvessel toxicity because they have high alkaline phosphatase compared to normal cells to transform the prodrug into its cytotoxic form.

EXAMPLE 4 Both emphasize the damage and the presence of invading tumor cells can induce microvessels to produce up to 50 parts increased levels of alkaline phosphatase (J. Invest, Dermatol, 109 (4): 597-603, 1997, FEBS Lett. (1): 99 103, 1994). The alkaline phosphatases present in the membrane of the cells and the circulation can hydrolyze the organic phosphate containing compounds by endorsing or releasing the phosphate part (eg, calcium phosphate) from the part of the organic molecule. The physiological need of the microvessel to repair the damage to themselves by elevating the alkaline phosphatase is a part of the normal wound healing process that leads to an increased deposition of calcium deposition in the damaged area. One consequence of this metabolic specificity may be that cytotoxic modified tubulin binding agents in a phosphate salt (eg, A4 prodrug) may also be a substrate for alkaline phosphatase. This process then can in turn lead to an increase in cytotoxic sensitivity of tubule microbial d tubulin binding drugs, which do not bind tubulin in a phosphorodilated form and are not cytotoxic to the phosphorodilated form that binds tubulin and is cytotoxic. This example shows that this is certainly the case. The HDF and HMVEC exposed to in vitro culture for 2 hours to increase the concentrations of A4 prodrug in the presence or absence of 1 unit of added alkaline phosphatase, demonstrates a high degree of selective cytotoxicity to HMVEC without added alkaline phosphates, but the HDF becomes identically cytotoxic as the HMVEC to the A4 prodrug in the presence of added alkaline phosphates (Figures 3A and 3B). It was concluded that vascular targeted destruction was directly dependent on the presence of high levels of alkaline phosphatase in HMVEC, and the lack thereof in other tumor and normal cells such as HDF.

Therefore, this example teaches a preferential destruction method focused on microvessels, where cytotoxic agents such as d tubulin binding compounds, which when converted to the prodrog form by for example form a phosphonic hydroxid phosphate salt it can not induce cytotoxicity, it can be selectively metabolized by alkaline phosphatase, which is present in high amounts only in vascular endothelial cells, back to its cytotoxic form.

EXAMPLE 5 Example 5 also establishes and verifies the description presented in Example 4. Here, the experimental design was designed to demonstrate the dose dependence of alkaline phosphates in regulating the cytotoxicity of the A4 prodrug. The data clearly show how the amount of alkaline phosphatase determines the clonogenic cytotoxicity of disodium phosphate d combretastatin A4 in both the HMVEC and the HDF (Figures 4A and 4B). The results are taught in which more phosphatase to the cabinet should be added before the HDF can be killed by the A4 prodrog, while the HMVEC directly expresses clonogenic toxicity to the A4 prodrug without or after adding the low alkaline phosphatase levels , but at high added levels of alkaline phosphatase the toxicities become equal for both cell lines.

It is therefore demonstrated that the in vivo approach to the vascular destruction of the tumor is directly dependent on alkaline phosphatase, and that this knowledge should be useful in designing agents and methods for the treatment of cancer other disorders, which proliferate vascular, not malignant EXAMPLE 6 The compounds presented in Table 2 represent examples of how toxicity can be focused on microvasel cells by converting the cytotoxic forms into phosphate prodrug, which in turn are not cytotoxic until they are converted back into the cytotoxic form by cell phosphates such as alkaline phosphatase, which has a higher concentration - > 50 parts of microvessel endothelial cells that proliferate than other normal cells. In general, tubulin binding drugs can not bind tubulin in the phosphate salt form, and therefore represent a preferred cytotoxic mechanism as a cytotoxic mechanism for vascular focus. All compounds were evaluated for toxicity after 1 hour of exposure in the microculture and tested for cytotoxicity by the MTT assay after an additional 5 days of incubation in the culture. Under these conditions the kinetics of the tubulin binding were sufficiently rapid to cause toxicity in both HMVEC and in HDF which proliferate normally. The data reported in table 2 states that (i) phosphate prodrugs in general condone the normal HD of toxicity while not affecting the toxicity to HMVEC as shown by the higher IC 50 values for the prodrugs in the HDF but not in the the HMVEC, (ii) if the cytotoxic agent is more toxic to the HDF than the HMVEC, even though the prodrug pardons the toxicity in the HDF it can not complete the difference in the inherent cytotoxicities between the HDF and HMVEC, (iii) not all the amine or metal salts of the phosphate carry over are equally effective since the combretastatin Al piperazine phosphate was only marginally effective in protecting the HDF from cytotoxicity, and (iv) why pancratistatin is not known to bind the tubulin, compounds that have other cytotoxic mechanisms can also be targeted by the phosphatase mechanism. In summary, this data shows that cytotoxic agents can focus microvessel cell destruction through the construction of phosphate extension, if there is protection for normal cells that have little alkaline phosphatase to metabolize enough of the phosphate prodrug to its cytotoxic form within of one hour of exposure (for example, mimics in vivo conditions).

Table 2 Evidence to focus on microvaso cell toxicity mediant to convert cytotoxic compounds into cytotoxic phosphate prodrugs (Note: "Figure No. 1" in the left column refers to the structure identification number in Figures IA, IB, and 1C The drawings Compounds I to VIII were provided by Professor GR Pettit of the Arizona State University and compounds X to XVI by Dr. Kevin G. Pinney of Baylor University in Waco, Texas).

Fis. No. 1 Cytotoxic Form Non Cytotoxic Form Values IC < n (prodroqa) HMVEC HDF I Combretastatin A4 75-150nM 50nM II Combretastatin A4 Na2P04 75-150nM > 500nM III Combretastatin At 10-15μM > 0. 5-lμM IV Combretastatin Al Na2P04 10-15μM 5-10μM v Combretastatin Al 10-15μM > 0. 5-lμM VI Combretastatin To piperazine P04 10-15μM l-2μM VII Combretastatin At 10-15μM > 0. 5-lμM VIII Combretastatin To nicotinamide P04 10-15μM > 10μM X Nine Combretastatin K4 phosphoroamidate 8-10μM 15-20μM XI Di idronaftaleno 0. 5-lμM 0. 5-lμM XII Dihydronaf Tandofosphenamidamide 5-7μM > 50μM XIII Pancratistatina 20-25μM 20μM XIV Pancrat istat ina Na2P04 20-25μM 60-80μM XV Benzo (a) thiophene 5-10μM 5-10μM XVI Benzo (a) thiophene Na2P04 8-10μM 30-40μM EXAMPLE 7 To stimulate pathogenic ocular angiogenesis, ocular neovascularization was introduced by the administration of lipid hydroperoxide (LHP) by intracorneal injection at a dose of 30μg to rabbit eyes. Seven to 14 days later, the eye vessels formed in the eyes injected due to the LHP discharge. The subjects were divided into two groups; those of a group were supplied with disodium phosphate of combretastatin A4 by intravenous administration in a dose of 40 milligrams po kilogram once a day for five days, while a vehicle without disodium phosphate of combretastatin A4 was administered to another group by intravenous administration as a dosi of water for the same period of time. The eyes of both groups were examined seven days later. A step reduction of 40% or more was observed in the group treated with phosphate d disodium of combretastatin A4, but not in the other group.

It is to be understood that the invention is not limited to the characteristics and to the incorporations specifically described above, but can be carried out in other forms without departing from its spirit.

Claims (26)

1. A method for treating a warm-blooded animal having a vascular proliferative disorder, comprising administering to the animal a quantity of a prodrug other than combretastatin A4 disodium phosphate effective to achieve targeted vascular destruction at a locality of a proliferating vasculature, wherein the Prodrug is essentially non-cytotoxic but is convertible to an essentially cytotoxic drug by the action of an endothelial enzyme selectively induced increased levels in vascular proliferation sites.

2. A method as claimed in clause 1, characterized in that the prodrug is a phosphate within the class of compound having the general formula. Z ll R ^ X-P-Y R2 and R3 where X is O, NH, or S; And it is O, NH, S, O ", NET OR S"; Z is 0 or S; each of R2 and R3 is an alkyl group, H, a mono- or divalent cationic sa, or a cationic ammonium salt, and YR3 may be the same or different; Y R1 is defined by the formula R1-R representing a compound that contains at least one group (designated Ra) and which is a group, which contains X, which can form a phosphate another salt that serves as a substrate for vascular endothelial phosphatases not specific, and thus becomes a relatively non-cytotoxic form of phosphate to a R1-cytotoxic form.

3. A method as claimed in clause 2, characterized in that the compound with the formula R1-Ra is a tubulin binder.

4. A method as claimed in clause 3, characterized in that the tubulin binder can be a compound selected from the group consisting of combretastatins, taxanes, vinca alkaloids, colchicine doblastatins, podofylatoxins, steganacines, flavonoid amphetamines, rhizoxins, curacin A, epothilones A and B welwistatins, phe- tistatins, 2-estrilquinazoline-4 (3H) - stilbenes, 2-aryl-l, 8-naphthyridine-4 (1H) - ones, 5,6 dihydroindolo- (2, 1-a) isoquinolines, 2 , 3-benzo (b) thiophenes, 2,3-benzo (b) thiophenes, 2,3-benzo (b) furans, substituted, substituted 2,3 -indoles and 2-methoxyestradiol.

5. A method as claimed in clause 1, characterized in that the animal has microvessel cells in the locality of vascular proliferation wherein the animal also has other cells which are not malignant and where the essentially cytotoxic drug is not essentially more toxic to said non-malignant cells than to said microvessel cells.

6. A method as claimed in clause 5, characterized in that the prodrug is converted to the essentially cytotoxic drog by the action of the enzyme endothelium within a period of no more than 3 hours.

7. A method as claimed in clause 1, characterized in that the prodrug is converted to the essentially cytotoxic drog by the action of the enzyme endothelium within a period of no more than about 3 hours.

8. A method for treating a warm-blooded animal having a non-malignant vascular proliferative disorder, which comprises administering to the animal the amount of effective prodrug to achieve targeted vascular destruction at a locality of the proliferating vasculature, where the prodrug is essentially non-cytotoxic but it is convertible to an essentially cytotoxic drog by the action of selectively induced endothelial enzymes at increased levels and sites of vascular proliferation.

9. A method as claimed in clause 8, characterized in that the prodrug is a phosphate within the class of compounds having the general formula. Z II R ^ X-P-Y R2 and R3 where X is O, NH, or S; And it is O, NH, S, 0", NH" or S "; Z is O or S; each of R2 and R3 is an alkyl group, H, a mono- or divalent cationic sa, or a cationic ammonium salt, and R and R3 may be the same or different; Y R1 is defined by the formula R1-Ra representing a compound containing at least one group (designated Ra) and which is a group containing X, which can form a phosphate another salt that serves as a substrate for non-specific vascular endothelial phosphatases , and is therefore converted from a relatively non-cytotoxic phosphate form to a R1-cytotoxic form.

10. A method as claimed in clause 9, characterized in that the compound between the formula R1-Ra is a tubulin binder.

11. A method as claimed in clause 10, characterized in that the tubulin binder can be a compound selected from the group consisting of combretastatins, taxanes, vinca alkaloids, colchicine doblastatins, podofylatoxins, steganacines, flavonoid amphetamines, rhizoxins, curacin A, epothilones A and B welwistatins, phe- tistatins, 2-estrilquinazoline-4 (3H) - stilbenes, 2-aryl-l, 8-naphthyridine-4 (1H) - ones, 5,6 dihydroindolo- (2, 1-a) isoquinolines, 2 , 3-benzo (b) thiophenes, 2,3-benzo (b) thiophenes, 2,3-benzo (b) furans, substituted, substituted indo 2,3 -indoles and 2-methoxyestradiol.

12. A method as claimed in clause 8, characterized in that the animal has microvessel cells in the locality of vascular proliferation, where the animal also has other cells, and in which the essentially cytotoxic drug is essentially no more toxic to the animal. said other cells than said microvessel cells.

13. A method as claimed in clause 12, characterized in that the prodrug is converted to the essentially cytotoxic drog by the action of the enzyme endothelium within a period of no more than about 3 hours.

14. A method as claimed in clause 8, characterized in that the prodrug is converted to an essentially cytotoxic drog by the action of the enzyme endothelium within a period of no more than about 3 hours.

15. A composition for treating a warm blooded animal that has a vascular proliferative disorder to achieve vascular destruction focused on a vascular proliferation site, which comprises a prodrug distinct from combretastatin A4, pancratistatin and taxol phosphate prodrugs which is essentially non-cytotoxic but it is convertible to a cytotoxic drug essentially by the action of a selectively induced endothelial enzyme at increased levels and sites of vascular proliferation.

16. A composition as defined in clause 15, characterized in that the prodrug is a phosphat within the class of compounds having the general formula Z II R'-X-P-Y R2 and R3 where X is 0, NH, or S; And it is O, NH, S, O ", NH" or S; Z is O or S; Each of R2 and R3 is an alkyl group, H, a mono- or divalent cationic sa, or a cationic ammonium salt, and and R3 may be the same or different; Y R1 is defined by the formula R1-Ra representing a compound that contains at least one group (designated Ra) and which is a group containing X, which can form a phosphate another salt that serves as a substrate for non-specific vascular endothelial phosphatases and is therefore converted from a relatively non-cytotoxic phosphat to a cytotoxic form of R1-Ra.

17. A composition as claimed in clause 16, characterized in that the compound with the formula R1 Ra is a tubulin binder.

18. A composition as claimed in clause 17, characterized in that the tubulin binder can be a compound selected from the group consisting of combretastatins, taxanes, vinca alkaloids, colchicinoids doblastatins, podofylatoxins, steganacines, flavonoid amphetamines, rhizoxins, curacin A, epothilones A and B welwistatins, phe- tistatins, 2-estrilquinazoline-4 (3H) - stilbenes, 2-aryl-l, 8-naphthyridine-4 (1H) - ones, 5,6 dihydroindolo- (2, 1-a) isoquinolines, 2 , 3-benzo (b) thiophenes, 2,3-benzo (b) thiophenes, 2,3-benzo (b) furans, substituted, substituted indo 2,3 -indoles and 2-methoxyestradiol.

19. A composition as claimed in clause 15, characterized in that the animal has microvasel cells in the locality of a vascular proliferation where the animal also has other cells which are not malignant and where the essentially cytotoxic drug is not essential. more toxic to said other non-malignant cells than to the microvessel cells.

20. A method as claimed in clause 19, characterized in that the prodrug is converted to a drog essentially cytotoxic by the action of the enzyme endothelium within a period of no more than 3 hours.

21. A method as claimed in clause 15, characterized in that the prodrug is converted to the essentially cytotoxic drog by the action of the enzyme endothelium within a period of no more than about 3 hours.

22. A method for identifying prodrugs suitable for use in the aforementioned methods and compositions, such a method comprises the steps of culturing endothelial proliferating cells, and other cells which are n malignant, in the presence of a prodrug other than disodium phosphate combretastatin A4 for a limited period of time; compare the respective cultures afterwards to determine whether the culture of the proliferating endothelial cells exhibits a significantly greater cytotoxic effect than the culture of the other cells; and if so, it cultivates the other cells in the presence of the prodrug and of a selectively induced endothelial enzyme at increased levels and sites of vascular proliferation, the cytotoxic effect increased with respect to the other cells in the presence of the enzyme in comparison to the cytotoxic effect in the initial culture of other cells indicating the adequacy of the prodrog for such methods and compositions.

23. A process as claimed in clause 22, characterized in that the prodrug is a phosphat within the class of compounds having the general formula R ^ X- R2 wherein X is O, NH, or S; And it is O, NH, S, O ", NH" or S "; Z is O or S; each of R2 and R3 is an alkyl group, H, a mono- or divalent cationic sa, or a cationic ammonium salt and and R3 may be the same or different; and R1 is defined by the formula R1-Ra representing a compound containing at least one group (designated Ra) which is a group, containing X that can form a phosphate or other salt that serves as a substrate for vascular endothelial phosphatases not specific, and s thus converts from a relatively cytotoxic phosphate form to a cytotoxic R1-Ra form.

24. The process as claimed in clause 23 characterized in that the compound with the formula R1 Ra is a tubulin binder.

25. The process as claimed in clause 24, characterized in that the tubulin binder can be a compound selected from the group consisting of combretastatins, taxanes, vinca alkaloids, colchicine doblastatins, podophyllotoxins, steganacines, flavonoid amphetamines, rhizoxins, curacin A, epothilones A and B welwistatins, phe- nistatins, 2-estrilquinazoline-4 (3H) - stilbenes, 2-aryl-l, 8-naphthyridine-4 (1H) - ones, 5,6 dihydroindolo- (2, 1-a) isoquinolines, 2 , 3-benzo (b) thiophenes, 2,3-benzo (b) thiophenes, 2, 3-benzo (b) furans, substituted, substituted indo-2,3-indoles and 2-methoxyestradiol.

26. A method as claimed in clause 22, characterized in that said other malignant n-cells are fibroblasts.