MXPA00001079A - Methods and compositions for overcoming resistance to biologic and chemotherapy - Google Patents

Abstract

This invention provides a method for identifying potential therapeutic agents by contacting a target cell with a candidate therapeutic agent which is a selective substrate for an endogenous, intracellular enzyme in the cell which is enhanced in its expression as a result of selection by biologic or chemotherapy. This invention also provides methods and examples of molecules for selectively killing a pathological cell by contacting the cell with a prodrug that is a selective substrate for an endogenous, intracellular enzyme. The prodrug is subsequently converted to a cellular toxin. Futher provided by this invention is a method for treating a pathology characterized by pathological, hyperproliferative cells in a subject by administering to the subject a prodrug that is a selective substrate for an endogenous, overexpressed, intracellular enzyme, and converted by the enzyme to a cellular toxin in the hyperproliferative cell.

Description

METHODS AND COMPOSITIONS FOR OVERCOMING RESISTANCE TO BIOLOGICAL AGENTS AND CHEMOTHERAPY DESCRIPTION OF THE INVENTION The present invention relates to the field of drug discovery and specifically to the design of prodrugs, which are substrates for an intracellular enzyme critical to resistance to cancer therapeutics. in pathological cells and converted to a cell toxin through the intracellular enzyme. Through this description, several publications referenced by the first author and date, patent number or publication number are presented. All bibliographic citations for each reference can be found at the end of this application, immediately preceding the claims. The descriptions of these references are incorporated herein by reference in this description to more fully describe the state of the art to which this invention pertains. Cancer cells are characterized by uncontrolled growth, de-differentiation and genetic instability. Instability expresses itself as an aberrant number of chromosomes, chromosome deletions, redispositions, loss or duplication beyond the normal diploid number. Wilson, J.D. and others (1991). This genetic instability can be caused by several factors.

One of the best characterized is the improved genomic plasticity that occurs after the loss of tumor suppression gene function (eg, Almasan, A. and others (1995)). The genomic plasticity itself leads to the adaptability of the tumor cells to their changing environment, and may allow more frequent mutation, gene amplification and the formation of extracrsmosomal elements (Smith, KA et al. (1995) and ilson, JD and others (1991)). These characteristics provide mechanisms that result in more aggressive malignancy, since it allows tumors to rapidly develop resistance to defense mechanisms to natural hosts, biological therapies (ilson, JD et al. (1991) and Shepard, HM et al. (1988)). , as well as chemotherapy. Almasan, A. and others (1995) and ilson, J. D. and others (1991). Cancer is one of the most commonly fatal human diseases worldwide. Treatment with anticancer drugs is an increasingly important option, especially for systemic malignancies or for metastatic cancer, which have passed the state of surgical cure. Unfortunately, the subgroup of human cancer types that can be managed with curative treatment at present is rather small (Haskell, C.M., eds., 81995), p. 32). Progress in the development of drugs that can cure human cancer is low. The Heterogeneity of malignant tumors with respect to their genetics, biology and biochemistry as well as primary resistance or treatment-induced therapy are mitigated against curative treatment. In addition, many anticancer drugs exhibit only a low degree of selectivity, generally causing severe or life-threatening toxic side effects, thus avoiding the application of doses high enough to destroy all cancer cells. The search for anti-neoplastic agents with improved selectivity to malignant, pathological cells resistant to treatment remains, therefore, a central task for the development of drugs. In addition, a broad resistance to antibiotics has become an important, global health issue. Segovia M. (1994) and Syndman, D. R. and others (1996). Classes of chemotherapeutic agents The main classes of agents include alkylating agents, antitumor antibiotics, plant alkaloids, antimetabolites, hormonal agonists and antagonists and a variety of various other agents. See Haskell, C.M. ed., (1995) and Dorr, - R. T. and Von Hoff, D. D. eds. (1994). Classical alkylating agents are highly reactive compounds that have the ability to substitute alkyl groups for hydrogen atoms of certain compounds The alkylation of nucleic acids, mainly DNA, is the critical cytotoxic action for most of these compounds. The damage they cause interferes with DNA replication and RNA transcription. Classical alkylating agents include mechlorethamine, chlorambucil, melphalan, cyclophosphamide, ifosfamide, thiotepa and busulfan. A number of non-classical alkylating agents also damage DNA and proteins, but through diverse and complex mechanisms, such as methylation or chloroethylation, which differ from the classical alkylating agents. Non-classical alkylating agents include dacarbazine, carmustine, lomustine, cisplatin, carboplatin, procarbazine and altretamine. The clinically useful antitumour drugs are natural products of several strains of the Streptomyces ground fungus. These produce their tumoreidal effects through one or more mechanisms. All antibiotics capable of binding DNA, usually through intercalation, with subsequent winding of the helix. This distortion damages the ability of DNA to serve as a template for DNA synthesis, RNA synthesis, or both. These drugs can also damage DNA through the formation of free radicals and the chelation of important metal ions. They can also act as inhibitors of topoisomerase II, a critical enzyme for division cell phone. Drugs of this class include doxorubicin (Adriamycin), daunorubicin, idarubicin, mitoxantrone, bleomycin, dactinomycin, mitomycin C, plicamycin and streptozocin. The plants have provided some of the most useful antineoplastic agents. Three groups of agents of this class with Vinca alkaloids (vincristine and vinblastine), epipodophyllotoxins (etoposide and teniposide) and paclitaxel (Taxol). Vinca alkaloids bind to microtubule proteins found in dividing cells and the nervous system. This union alters the dynamics -of the addition of tubulin and the loss at the ends of mitotic spindles, ultimately resulting in mitotic arrest. Similar proteins form an important part of nerve tissue, therefore these agents are neurotoxic. Epipodophyllotoxins inhibit topoisomerase II and, therefore, have profound effects on cell function. Paclitaxel has complex effects on microtubules. Antimetabolites are structural analogues of normal metabolites that are required for cell function and replication. They typically work by interacting with cellular enzymes. Among the many antimetabolites that have been developed and clinically proven are methotrexate, 5-fluorouracil (5-FU), floxuridine (FUDR), cytarabine, 6-mercaptopurine (6-MP), 6-thioguanidine, deoxicoformycin, fludarabine, 2-chlorodeoxyadenosine and hydroxyurea. Endocrine manipulation is an effective therapy for various forms of neoplastic disease. A wide variety of hormones and hormone antagonists has been developed for potential use in oncology. Examples of available hormonal agents are diethylstilbestrol, tamoxifen, megestrol acetate, dexamethasone, prednisone, amingluterimide, leuprolide, goserelin, flutamide and octreotide acetate. Setbacks of Current Chemotherapeutic Agents Among the current problems associated with the use of chemotherapeutic agents to treat cancers are the high doses of agent required; toxicity towards normal cells, that is, lack of selectivity; immunosuppression; secondary malignancies; and drug resistance. Most agents that are now used in cancer chemotherapy act by an antiproliferative mechanism. However, most human solid cancers do not have a high proportion of cells that are rapidly proliferating and are therefore not particularly sensitive to this class of agent. In addition, most antineoplastic agents have stepped dose response curves. Due to host toxicity, the treatment has to be discontinued at dose levels that are well below the dose that would be required for Destroy all viable tumor cells. Another side effect associated with therapies in the present day is the toxic effect of chemotherapy on normal host tissues that are the most rapidly dividing, such as "the bone marrow, intestinal mucosa and cells of the lymphoid system." The agent also exerts a variety of other adverse effects, including neurotoxicity, negative effects on sexuality and gonadal function, and cardiac, pulmonary, pancreatic and hepatic toxicities, vascular and hypersensitivity reactions, and dermatological reactions Haematological toxicity is the most dangerous form of toxicity for many of the antineoplastic drugs used in clinical practice.The most common form is neutropenia, with a high associated risk of infection, although thrombocytopenia and bleeding can also occur and be life threatening.Chemotherapy can also induce qualitative defects in the function of polymorphonuclear leukocytes and platelets. or hematopoietic agents have been developed to direct these important side effects. ilson, J.D. et al. (1991) and Dorr, R.T. and Von Hoff, D.D., eds. (1994). Most commonly used antineoplastic agents are capable of suppressing cellular and humoral immunity. Infections commonly lead to the death of patients with advanced cancer, and impaired immunity can contribute to such deaths. Chronic delayed immunosuppression can also result from cancer chemotherapy. The main forms of neurotoxicity are arachnoiditis; myelopathy or encephalomyelopathy; chronic encephalopathies and somnolence syndrome; acute encephalopathies; peripheral neuropathies; and acute cerebellum syndromes or ataxia. Many of the commonly used antineoplastic agents are mutagenic as well as teratogenic. Some, including procarbazine and alkylating agents, are clearly carcinogenic. This carcinogenic potential is first seen as watery delayed leukemia in patients treated with polyfunctional alkylation agents and topoisomerase II inhibitors, such as etoposide and anthracycline antibiotics. Chemotherapy has also been associated with cases of solid tumors and non-Hodgkin's lymphoma without delay. The present invention will minimize these effects since the drug will only be activated within the tumor cells. The clinical utility of the chemotherapeutic agent can be severely limited by the emergence of malignant cells resistant to that drug. A number of cellular mechanisms are probably involved in drug resistance, for example, altered drug metabolism, impermeability of the cell to activate the compound or removal of the accelerated drug from the cell, altered specificity of an inhibited enzyme, increased production of a target molecule, repair of increased cytotoxic lesions, or deviation of an inhibited reaction by alternative biochemical trajectories. In some cases, resistance to one drug may confer resistance to another, biochemically distinct drugs. The extension of certain genes is implicated in resistance to the biological agent and chemotherapy. The amplification of these genes that encode dihydrofolate reductase is related to resistance to methotrexate, while the amplification of the gene encoding the thymidylate synthase is related to the resistance to treatment with 5-fluoropyrimidines. Table 1 summarizes some prominent enzymes in resistance to biological agents and chemotherapy. Table 1 Enzymes Overexpressed in Resistance to Chemotherapy Enzyme Biological agents Referenced or Chemotherapy (Examples) Timidylate synthase Uracil based Lonn, U. et al. Folate based Kobayashi, H. et al. Quinazoline based Jackman, AL et al.

Dihydrofolate Folate based Banerjée, D. et al reductasa Bertino, J.R. et al. Tyrosine kinases TNF-alpha Rudziak, R.M. et al. Resistance to Stühlinger, M. et multifármaco al. Associated proteins Resistance to Simón, S.M. and with multidrug MDR Schindler, M. (ABC P-Gottesman, M.M., and gp proteins) al. CAD * PALLA ** Smith, K.A. et al. Dorr, R.T. and Vo, Hoff, D.D., eds.

Ribonucleotti? O Hi? Roxiurea ^ Ñettergren, Y. et reductase al.

Yen, Y, et al. * CAD = carbamyl-P synthase, aspartate transcarbamylase, dihydroorotate ** PALA = N- (phosphonacetyl) -L-aspartate. Use of pro-drugs as a solution to improve the selectivity of a chemotherapeutic agent Poor selectivity of anticancer agents has been recognized for a long time and attempts to improve the selectivity and allow large doses to be administered have been numerous. One approach has been the development of prodrugs. Prodrugs are compounds that are toxicologically inert, but which can be converted in vivo to activate toxic products. In some cases, activation occurs through the action of a non-endogenous enzyme supplied to the target cell by an antibody ("ADEPT" or an antibody-dependent pro-drug enzyme therapy (U.S. Patent No. 4,975,278)) or a PGDEPT target gene "or a gene-dependent prodrug therapy (Melton, RG and Sherwood, RF (1996).) These technologies have several limitations with respect to their ability to draw blood and penetrate tumors. TA and Knox, RJ (1995) .Therefore, this is a need for more selective agents which can penetrate the tumor and inhibit proliferation and / or destroy cancer cells that have developed resistance to therapy. This need and provides related advantages also This invention provides a method for the identification of potential therapeutic agents by contacting a target cell or test cell with an agent. the therapeutic candidate or prodrug which is a substrate selective for an objective enzyme in the cell. In one embodiment, the target enzyme is an endogenous intracellular enzyme, which is overexpressed and confers resistance to biological and chemotherapeutic agents. In a separate mode, the activity of the e? Z. M.aa \ a s do ^ xa ?. em? .e. improved in a tumor cell as a result of the loss of a tumor suppressor function (Smith, KA, et al. (1995) and Li,., et al. (1995)) and / or selection resulting from the previous exposure to the tumor. chemotherapy, (Melton, RG and Sherwood, RF (1996)). In a separate embodiment the target enzyme is a product of expression of a foreign gene in the cell, wherein the foreign gene encodes an objective enzyme. After the cell is in in vitro and / or in vivo contact with the candidate prodrug, the cell is analyzed for the effectiveness of an agent by noting whether the agent caused a reduction in cell proliferation or whether the agent destroys the cell. In one aspect of this invention, the prodrug destroys the cell or inhibits cell proliferation by the release of a toxic prodrug byproduct from the target enzyme. In a further aspect of this invention, one or more "target enzymes" can be used to activate the prodrug in a manner that releases the toxic by-product. Another aspect of this invention includes equipment for use in assays for novel prodrugs having the characteristics described herein against target enzymes. The Teams include the reagents and instructions necessary to complete the trial and analyze the results. This invention also provides methods and examples of molecules to selectively destroy a pathological cell by contacting the cell with a prodrug that is a selective substrate for a target enzyme, for example, an intracellular endogenous enzyme as defined above. The substrate is specifically converted to a cellular toxin by the intracellular target enzyme. In another aspect of this invention, the product of an initial preparative reaction is subsequently activated by a common cellular enzyme such as an acylase, phosphatase or other "maintenance" enzyme. Voet, et al. (1995) to release the toxic byproduct of the prodrug. Further provided for this invention is a method for treating a pathology characterized by pathological cells, hyperproliferatives in a subject by administering to the subject a prodrug that is a selective substrate for an objective enzyme, and selectively converted by the enzyme to a cellular toxin in the hyperproliferative cell. The prodrugs of this invention may be used alone or in combination with other chemotherapeutics or alternative anti-cancer therapies such as radiation. A further aspect of this invention is the preparation of a medicament for use in the treatment of a pathology characterized by pathological, hyperproliferative cells in a subject by administering to the subject a prodrug that is a selective substrate for an objective enzyme, and selectively converted by the enzyme to a cellular toxin in the hyperproliferative cell. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the development of resistance to anti-cancer modalities in cells, and the consequences. Figure 2 schematically shows activation of trajectories of the prodrugs of this invention. Figure 3 schematically shows the High Crossing Classification for prodrugs activated by intracellular enzymes important in drug resistance. Figure 4 schematically shows how the targeted human thymidylate synthase (TS) prodrugs are analyzed using TS-negative E. coli as the target cell. Figure 5 shows an example of how to use this classification to simultaneously optimize the prodrug by reactivity to two target enzymes. The invention is achieved by exploring some of the key genomic and phenotypic changes intimately linked to the resistance to biological agents and chemotherapy of Carcinogenic cells . The invention provides a means for selectively inhibiting in vivo the growth and / or destruction of the cells having selection experienced by exposure to cancer therapy (including biological therapy such as a tumor necrosis factor (TNF) or chemotherapy). (Reference to Table 1). As a result, certain enzymes which have been activated by mutation or gene amplification are resistant to additional therapy by the agent. Unlike prior art therapies aimed at creating more potent inhibitors of endogenous, intracellular enzymes, this invention explores the high enzymatic activity associated with resistance to the therapy of diseased cells and tissues against normal cells and tissues and does not rely on or inhibit enzyme. In one aspect, the tumor cells successfully treated by the prodrugs of this invention are characterized by enhancing the activity of the target enzyme and therefore have a higher potential to convert the prodrug to its toxic form which makes normal cells which do not overexpress the target enzyme. The term "target enzyme" is used herein to define enzymes that have one or more of the characteristics noted above. As used herein, the terms "host cells", "target cells" and "hyperproliferative cells" encompass cells characterized by activation by mutation. genetic or endogenous overexpression of an intracellular enzyme. In some embodiments, overexpression of enzymes is related to drug resistance or genetic instability associated with the pathological phenotype. A number of cellular mechanisms are involved in drug resistance, for example altered metabolism of the drug, impermeability of the cell with respect to the active compound or removal of the accelerated drug from the cell, altered specificity of an inhibited enzyme, increased production of a target molecule, increased repair of cytotoxic lesions, or the deviation of an inhibited reaction by alternative biochemical trajectories. Enzymes activated or overexpressed and related to drug resistance include, but are not limited to thymidylate synthase (TS) (Lónn, U. et al. (1996); Kobayashi, H. et al. (1995); Jackman, AL et al (1995)), dihydrofolate reductase (Banerjee, D. et al. (1995) and Bertino, JR et al- (1996)), tyrosine kinases (TNF-a, Hudziak, RM et al. (1998) and multidrug resistance (Stühlinger, M. et al. (1994)); Akdas, A. et a. (teen ty six); and (Tannock, I.F. (1996)); and resistance to the ATP-dependent multidrug associated with proteins (Simón, S.M. and Schindler, M. (1994)). Alternatively, resistance to a drug can confer resistance to other biochemically distinct drugs. While this application is specifically targeted to cancer, a similar approach can be applied to enzymes encoded by human and animal pathogens, and in which the inhibitors have failed due to the development of resistance. The amplification of certain genes is implicated in resistance to chemotherapy. The extension of dihydrofolate reductase (DHFR) is related to resistance to methotrexate, while the amplification of the gene encoding the thymidylate synthase is related to resistance to tumor treatment with 5-fluoropyrimidines. The amplification of genes associated with drug resistance can be detected and monitored by a modified polymerase chain reaction (PCR) as described in Kashini-Sabet, et al. (1998) or North American Patent No. 5,085,983. The resistance of the acquired drug can be monitored by the detection of cytogenic abnormalities, such as homogenous chromosome staining regions and double-minute chromosomes of which are associated with gene amplification. Alternative assays include direct and indirect enzymatic activity assays and both of which are associated with gene amplification (e.g., Carreras &Santi (1995)); other methodologies (eg, polymerase chain reaction, Houze, T.A. et al. (1997) or immunohistochemistry (Johnson, P.G. et al. (1997)) - Alternatively, the target cell is characterized as having inactivated tumor suppressor function, eg, loss or inactivation of retinoblastoma (RB), or p53, known to improve the expression of TS (Li,., et al. (1995)) or DHFR (Bertino, et al. (1996) and Li, W. et al. (1995)). The prodrugs of this invention are useful for treating or ameliorating any disease in which the associated diseased enzyme is associated with drug resistance to chemotherapy and in some embodiments wherein the enzyme is overexpressed, over-accumulated or activated in pathological cells against normal cells, for example, the TS enzyme. Particularly excluded is the enzyme glutathione-S-transferase which has been shown to be elevated in some human tumors. Morgan, A.S. et al. (1998). The prodrugs of the subject invention are distinguishable in the bases that the objective enzymes of this invention are commonly overexpressed, overaccumulated or activated in pathological cells against normal cells. The most important principle that distinguishes the current invention for other approaches are: (1) This invention describes the synthesis of substrates for enzymes similar to thymidylate synthase. The overexpressed enzyme will generate toxin, preferentially in diseased cells. The above methods have relied on inhibition. The inhibitors lead to the expanded expression of the enzyme, and subsequent resistance to treatment (see, for example, Lonn, U. et al., (1996). (2) The current method is also distinguishable from other "substrate-prodrug" methods, eg, enzymes glutathione-S-transferase (see, for example, Morgan, AS et al. (1998).) Enzymes of the GST family are expressed at increased levels in response to toxic injury to the cell.The GST family of enzymes have substrate specificities overlapping, which make it impossible to design a substrate reagent with only a single enzyme species with high expression in a cancer cell (Morgan, AS et al., (1998).) Because each of the enzymes of the current invention ( for example, thymidylate synthase, dihydrofolate reductase and thymidine kinase) is unique with respect to its structure and specific substrate, it is easy to design unique substrates Several examples of substrates for thymidylate synthase are provided in the specifications of this application (3) In some cases the gene encoding the target enzyme (eg, thymidylate synthase) may undergo mutation to give resistance to inhibitors, but it would still be able to carry out the reaction with substrates without inhibition. Barbour, K.W. et al. (1992) and Dicken, A.P. et al. (1993). Drug Test This invention provides a method for identifying agents which have therapeutic potential for the treatment of hyperproliferative or neoplastic disorders, for example, cancer. The method also identifies agents that inhibit cell growth or cyclization of hyperproliferative cell cells, such as cancer cells. Other cells that are included are bacteria, yeasts and parasitic cells which cause diseases as a result of inappropriate proliferation in the patient. The agent is considered a potential therapeutic agent if cell proliferation, replication or cell cyclization is reduced relative to cells in a control sample. More preferably, the cells are eliminated by the agent. The cells can be prokaryotic (bacteria such as E. coli) or eukaryotic. The cells can be mammalian or non-mammalian cells, for example, mouse cells, rat cells, human cells, fungi (eg, yeast) or parasites (eg, Pneumocys tis or Leishmania) which causes disease. As used herein, a "hyperproliferative cell" is intended to include cells that are de-differentiated, immortalized, neoplastic, malignant, metastatic, or transformed. Examples of such cells include, but are not limited to, sarcoma cell, a leukemia cell, a carcinoma cell, or a cancer cell. adenocarcinoma More specifically, the cell can be a breast cancer cell, a hepatoma cell, a colorectal cancer cell, a pancreatic carcinoma cell, an oesophageal carcinoma cell, a bladder cancer cell, an ovarian cancer cell, a skin cancer cell, a liver cancer cell, or a gastric cancer cell. In an alternative embodiment, the target cell can be resistant to a drug or compound used to prevent or destroy a cell infected with an infectious agent which is resistant to conventional antibiotics. Infectious agents include bacteria, yeast and parasites, such as trypanosomes. Specific examples of target enzymes that are the subject matter of this invention are listed in Table 1 (above) or Table 2 (following). These enzymes are involved in resistance to chemotherapy, are endogenously activated, overexpressed or overaccumulated in a cell characterized by resistance to cancer therapy and associated with a disease or disease included, but not limited to enzymes, such as a member of the tyrosine kinase superfamily or an associated MDR-dependent ATP protein, CAD, thymidylate synthase, dihydrofolate reductase, and ribonucleotide reductase. Table 2 provides a list of enzymes which can be targeted by this approach in infectious disease.

Table 2 Enzymes Overexpressed in infectious diseases, and contributing to drug resistance Enzyme Provides Increased resistance to: Beta-lactamases Penicillin and other beta-lactam containing antibiotics Aminoglycosidase, or Aminoglycoside aminoglycoside antibiotics that modifies (eg, streptomycin, enzymes gentamicin) Chloramphenicol transacetylase Chloramphenicol Trimetroprim Dihydrofolate reductase Reference: Mechanisms of Microbial Disease, second edition., M. Schaechter, G. Medloff, Bl Eisenstein. Editor TS Satterfield, Publ. Williams and Wilkins, pp. 973 (1993). The potentially therapeutic agent identified by the method of this invention is a prodrug that is a substrate for the enzyme and is converted intracellularly to an intracellular toxin. As used herein, a "prodrug" is a precursor or derivative form of a pharmaceutically active agent or substance that is less cytotoxic to target or hyperproliferative cells as compared to the drug metabolite and is capable of being enzymatically activated or converted into the most active form (see Connors, T.A. (1986) and Connors, T.A. (nineteen ninety six)). The toxicity of the agent is directed to cells that are produced to convert the enzyme to an effective amount to produce a therapeutic concentration of the cellular toxin in the diseased cell. This invention also provides a rapid and simple classification assay which will be capable of initial identification of the compounds with at least some of the desired characteristics. For purposes of this current invention, the general scheme of a modality is shown in Figure 3. This drawing describes how the test is arranged and the materials necessary for its processing. As shown in Figure 3, the assay requires two cell types, the first being a control cell in which the target enzyme is not expressed, or is expressed at a low level. The second type of cell is the test cell, in which the target enzyme is expressed at a detectable level, for example, a high level. For example, a prokaryotic E. coli that does not endogenously express the TS target cell is a suitable host cell or target cell. The cell may have a counterpart of control (lacking the target enzyme), or in a separate mode, a genetically modified counterpart to differentially express the target enzyme, or enzymes (containing the appropriate species of the target enzyme). More than one species of enzyme can be used to separately transduce separate host cells, either the effect of the candidate drug or on a target enzyme can be simultaneously compared to its effect on another enzyme or a corresponding enzyme of other species. In another embodiment, transformed cell lines, such as ras-transformed NIH 3T3 cells (ATCC, 10801 University Blvd., Manassas, VA 20110-2209, USA) are designed with variable expression and increased amounts of the target enzyme of interest to from the cloned cDNA encoding the enzyme. The transfection is either transient or permanent using procedures well known in the art and described in Chen, L. et al. (1996), Hudziak, R.M. et al. (1988) or Carter, P. et al. (1992). Suitable vectors for cDNA insertion are commercially available from Stratagene, La Jolla, CA and other vendors. The level of expression of the enzyme in each transfected cell line can be monitored by immunoabsorption and enzyme assay in cell lysates, using monoclonal or polyclonal antibodies previously rinsed against the enzyme for immunodetection. See for example, as described by Chen, L. et al. (nineteen ninety six) . The amount of expression can be regulated by the number of copies of the expression cassette introduced into the cell or by the variation of the use of the promoter. Enzymatic assays can be performed as reviewed by Carreras, C.W. and Santi, D.V. (nineteen ninety five) . As noted above, cells containing the desired genetic deficiencies can be obtained from Cold Spring Harbor, the Agricultural Research Service Culture Collection, or the American Type Culture Collection. Appropriate strains can also be prepared by inserting into the cell a gene encoding the target enzyme using standard techniques as described in Miller (1992), Sambrook, et al. (1989) and Spector, et al. (1997). Growth assays can be performed by standard methods as described by Miller (1992) and Spector, et al. (1997). It should be understood by those skilled in the art that the classification shown in Figure 3 can be applied widely for the discovery of antibiotics. For example, yeast thymidylate synthase could be substituted for that of E. coli in Figure 4. This could allow the discovery of pathogen-specific antifungal antibiotics related to the target yeast. In addition, other enzymes may be subjected to this treatment. For example, prodrugs that are specifically targets for the dihydrofolate reductase activity of infectious agents, such as Pneumocystis carnii, could be selected. These agents will be selected for specificity by the target enzyme, and can be displayed without the activation of the enzyme of the natural host using the classification assay described in Figure 3. The constructed cellular control could contain the corresponding normal human enzyme, for show the lack of toxicity when the normal human enzyme is present. For example and as shown in Figure 4, a foreign gene, eg, a gene encoding TS, can be inserted into the host cell such that human TS is expressed. This genetically coupled cell is shown as the "test cell" in Figure 3. The "control cell" does not express the target enzyme. In some embodiments it may be necessary to supplement the culture medium with the protein product of the target enzyme. In a separate embodiment, the wild-type host cell is deficient or expresses no more than an enzyme of interest. As shown in Figure 4, the host cell does not endogenously produce thymidine kinase (TK) or thymidylate synthase (TS). The genes encoding the human counterpart of these enzymes are introduced into the host cell to obtain the desired level of expression. The level of expression of the enzyme in each transfected cell line can be verified by methods described herein, for example, by immunoabsorption and enzyme assay in cell lysates, using monoclonal or polyclonal antibodies previously raised against the enzyme for immunodetection. See, for example as described by Chen, L. et al. (nineteen ninety six) . Enzymatic assays can also be performed as reviewed by Carreras, C.W. and Santi, D.N. (1995) using detectably labeled substituents, for example, labeled substituents of tritium. The test cell is augmented in small multi-well plates and is used to detect the biological activity of test prodrugs. For the purpose of this invention, the successful candidate drug will block the growth or elimination of the test cell type, but will leave the cell-type control undamaged. The candidate prodrug can be directly added to the cell culture medium or previously conjugated to a specific ligand to a cell surface receptor and then added to the medium. Methods of conjugation for specific cell delivery are well known in the art, see for example, U.S. Patent Nos. 5,459,127; 5,264,618; and published patent specification WO 91/17424 (published November 14, 1991). The leaving group of the candidate prodrug can be detectably labeled, for example, with tritium. The target cell or culture medium is then analyzed for the amount of label released from the candidate prodrug. Alternatively, cell consumption can be improved by packaging the prodrug into liposomes using the method described in Lasic, D.D., (1996) or combined with cytofectins as described in Lewis, J.G. et al. (nineteen ninety six). "In a separate embodiment, cultured human tumor cells that overexpress the enzyme of interest, ie, target enzyme, are identified as described above.The cells are contacted with the potential therapeutic agent under conditions which favor the incorporation of the agent in the intracellular compartment of the cell The cells are then analyzed by inhibition of cell proliferation or cell destruction The following provision is a brief summary of cells and target enzymes which are useful for activating the prodrugs of this invention. overexpression of thymidylate synthase is associated with colon cancer, breast cancer, gastric cancer, head and neck cancer, liver cancer and pancreatic cancer.These diseases are currently treated by antimetabolite drugs (based on uracil, based on folate , based on quinazoline (see Table 1)). In each of these It is likely that 5-fluorouracil therapy can lead to extended TS activity, or selected from drug-resistant forms of the enzyme, and therefore leads to drug resistance of relapse in the disease. Lonn, U. et al. (1996) reports that the expansion of the TS gene occurs in breast cancer patients who previously received adjuvant chemotherapy (cyclophosphamide, methotrexate, 5-fluorouracil [CMF]) after surgery. The main reaction normally performed by TS is the synthesis of deoxythymidine monophosphate (dTMP) and dihydrofolate (DHF) of deoxyuridine monophosphate (dUMP) and N (5), N (10) -methylene-tetrahydrofolate (THF). In one embodiment, a derivative of uracil or THF is provided to cells expressing TS. For purposes of this invention, "uracil" (base only) and "uridine" (base and sugar) are used interchangeably and synonymously. The derivative or "prodrug" is converted by the enzyme into highly toxic metabolites. The low level of TS expressed in normal cells will not produce a toxic amount of the converted toxin. High levels of TS expressed in diseased tissues generate more toxin and therefore lead to an inhibition of cell proliferation and / or cell death. For example, the current therapy uses 5-fluorodeoxyuridylate to inhibit TS activity. During the reaction with substrate, the fluoro atom irreversibly binds to the TS enzyme and inhibits it. In contrast to one embodiment of the present invention, the Prodrug allows TS to complete the reaction, but generates a modified product that, when incorporated into the DNA, causes a toxic effect. The enzyme product can also block other critical cellular functions (e.g., protein synthesis or metabolic energy). The conversion of the prodrug can also release a metabolite, such as Br "or I" or CN "which is toxic to the cell.The derivatives of uracil / dUMP and N (5) (10) -THF can be synthesized, all the which have the potential to generate toxic product after catalyzing metabolically by TS The synthesis of 5-substituted pyrimidine nucleosides and 5-substituted pyrimidine nucleoside monophosphates can be achieved by methods that are well known in the art. of 5-chloromercury-2'-deoxyuridine with haloalkyl compounds, haloacetates or haloalkenes in the presence of Li2PdCl results in the formation, through an organopalladium intermediate, of the 5-alkyl, 5-acetyl or 5-alkene derivative, respectively. et al. (1979) and Bergstrom, et al. (1981) . Another example of the C5 modification of pyrimidine nucleosides and nucleotides is the formation of C5-trans-styryl nucleotide derivatives deprotected with mercury acetate followed by the addition of styrene or substituted ring styrenes in the presence of Li2PdCl4 Bigge, et al. (1980). The pyrimidine deoxyribonucleoside triphosphates were derived with mercury at the 5-position of the pyrimidine ring by treatment with acetate of mercury in acetate buffer at 50 ° C for 3 hours. Dale, et al. (1973). Such treatment could also be expected to be effective by modification of monophosphates; alternatively, a modified triphosphate could be converted enzymatically to a modified monophosphate, for example, by controlled treatment with alkaline phosphatase followed by the purification of monophosphate. Other portions, organic or non-organic, with molecular properties similar to mercury, but with preferred pharmacological properties could be substituted. For general methods for synthesis of substituted pyrimidines, for example, US Patent Nos. 4,247,544, 4,267,171; and 4,948,882; and Bergstrom et al. (1981) . The above methods could also be applicable to the synthesis of the 5-substituted pyrimidine nucleoside derivatives and nucleotides containing different sugars of ribose or 2'-deoxyribose, for example 2'-3'-dideoxyiribosa, arabinose, furanose, lixose, pentose, hexose, heptose, and pyranose. An example of such substituents are halovinyl groups, for example, E-5- (2-bromovinyl) -2'-deoxyuridylate. Barr, P.J. et al. (1983). In this reference, the authors demonstrated that the normally inert substituent at position 5 (bromovinyl) is convertible to a chemical reactive group as a result of enzyme-mediated nucleophilic attack to position 6 of the uridine heterocycle, leading to the production of a reactive alkylating agent. This compound is not useful from the point of view of the current application because it can not be activated by endogenous thymidine kinase, and due to its conversion by thymidylate synthase leading to the activation of thymidylate synthase (Balzarini, et al., 1987) . However, the improved substituents will be synthesized and compared for the reactivity with TS and specific cytotoxicity to TS-superproduced tumor cells. Alternatively, 5-bromodeoxyuridine, 5-iododeoxyuridine, and their monophosphate derivatives are commercially available from Glen Research, Sterling, VA (USA), Sigma-Aldrich Corporation, St. Louis, MO (USA), Moravek Biochemicals, Inc., Brea, CA (USA), ICN, Costa Mesa, CA (USA), and New England Nuclear, Boston, MA (USA). Commercially available 5-bromodeoxyuridine and 5-iododeoxyuridine can be converted to their monophosphates either chemically or enzymatically, through the action of a kinase enzyme using reagents commercially available from Glen Research, Sterling, VA (USA) and ICN, Costa Mesa, AC (USES) . These halogen derivatives could be combined with other substituents to create new and more potent antimetabolites.

The primary sequences showed that TS is one of the most highly conserved enzymes. Perry, K. et al. (1990) . The TS crystal structures of several prokaryotic species, Lactobacillus casei (Hardy, L.W. et al. (1987), Finer-Moore, J. et al. (1993)) and Escherichia coli (Perry, K. et al. (1990)); and eukaryote Leishmania major (Knighton, E.R. et al. (1994)); and fag T4 (Finer-Moore, J.S. et al., (1994)) have been determined and indicate that the tertiary structure is also well conserved. The sequence alignment of TS species whose three dimensional structures have been determined and shown in Schiffer, C.A. et al. (nineteen ninety five). Of these amino acid sequences, the DNA sequences can be deduced or isolated using methods well known to those skilled in the art. Sambrook, et al. (1989). Alternatively, some of the 29 TS sequences from the different organisms have been cloned and deposited into the DNA databases as described in Carreras, C.W. and Santi, D.V. (nineteen ninety five) . The sequence of the human thymidylate synthase gene, its cloning, expression and purification is provided in Takeishi, K. et al. (1985), Davisson, V.J. et al. (1989) and Davisson, V.J. et al. (1994). The genes encoding the TS protein and containing the necessary regulatory sequences are constructed using methods well known to those of skill in the art. He The gene encoding TS is introduced into the target cells by electroporation, transformation or transfection methods. Sambrook et al. (1989). Alternatively, the gene is inserted into an appropriate expression vector by methods well known in the art, for example, as described in Carreras, C.W. and Santi, D.V. (1995), Miller (1992) and Spector et al. (1997). The expression vector inserts the TS gene in the cells. The cells are then grown under conditions that favor the expression and production of TS protein. Human gastric cancer cell lines MKN-74, MKN-45, MKN-28 and KATO-III can be used in the assay described above to identify potential therapeutic agents which are selective substrates for TS. MKN-74 and MKN-45 are well established and poorly differentiated adenocarcinomas, respectively. These cell lines and culture conditions are described in Osaki, M. et al. (1997) and the references cited therein. Alternatively, cell tumor lines such as those described by Copur, S. et al. (1995), who have been selected for 5-FU to overexpress thymidylate synthase. The quantification of TS can be performed using enzymatic biochemical assays that are well known to those skilled in the art. For quantify the TS protein level and TS gene expression of human tumor tissue samples, methods as reported by Johnston, P.G. et al., (1991) and Horikoshi, T. et al. (1992) provide sensitive tests. Alternatively, the PCR method of Lónn, U. et al. (1996) is used for TS gene amplification assays and identifies cells that are useful in the method for identifying therapeutic agents as described herein. As is apparent to those skilled in the art, the drug-free cell control culture systems and separately with referenced drug such as exemplified in the following, are also analyzed. A preferred embodiment of the prodrugs is one which preferably removes the target cells with approximately 2-fold and preferably about 3-fold or greater activity than normal cells. This invention also provides the agents identified by the methods described herein. In another aspect, this invention provides a method for inhibiting the proliferation of a hyperproliferative cell, to drive the above assay first. A prodrug identified by this assay is in contact with the cell and converted to a toxic metabolite in the cell by an endogenous intracellular enzyme as described above.

In one embodiment, the endogenous intracellular enzyme is thymidylate synthase and the cell is selected from the group consisting of a colorectal cell, a head and neck cancer cell, a breast cancer cell, or a gastric cancer cell. In a further aspect, the prodrug contacted with the cell is exposed to TIMI? YY-O s t?. ASci SS \ YT compound L- or D- of the formulas: which may be any of its enantiomeric, diastereomeric, or stereoisomeric forms, including, for example, D- or L- forms, and for example, α- or β-anomeric forms. In the above formulas, Ri (at position 5) is, or contains a leaving group which is a chemical entity that has a molecular dimension and electrophilicity compatible with the extraction of the pyrimidine ring by thymidylate synthase, and which is released from the ring pyrimidine by thymidylate synthase, has the ability to inhibit cell proliferation or destroy the cell.

In one embodiment, Ri is or contains a chemical entity selected from the group consisting of: -Br -I, -0-alkyl, -O-aryl, -O-heteroaryl, -S-alkyl, -S-aryl, - S-heteroaryl, -CN, -OCN, -SCN, -NH2, -NH-alkyl, - (alkyl) 2, -NHCHO, -NHOH, -NHO-alkyl, NH2C0NH0-, NHNH2, and -N3. Other Example of Ri is derived from cis-platin: In the above formulas for compounds L- or D-, Q is a chemical entity selected from the group consisting of sugar groups, thio-sugar groups, carbocyclic groups, and derivatives thereof. Examples of sugar groups include, but are not limited to, cyclic monosaccharide sugar groups such as those derived from oxetanes (4-membered ring sugars), furanoses (5-membered ring sugars), and pyranose (6-membered ring sugars). Examples of furanoses include threo-furanosyl (from threo, or a four-carbon sugar); erythro-furanosyl (from erythrose, a four-sugar carbons); ribo-furanosyl (from ribose, a five-carbon sugar); ara-furanosyl (also frequently referred to as arabino-furanosyl; arabinose, a five-carbon sugar); xylo-furanosyl (from xylose, a five-carbon sugar); and lixo-furanosyl (from lixose, a five-carbon sugar). Examples of derivatives of the sugar group include "deoxy", "keto" and "dehydro" derivatives as well as substituted derivatives. Examples of sugar thio groups include the sulfur analogs of the above sugar groups, in which the oxygen ring has been replaced with a sulfur atom. Examples of carbocyclic groups include carbocyclic C4 groups, carbocyclic C5 groups and carbocyclic groups of C? which may additionally have one or more substituents, such as -OH groups. In one embodiment, Q is a furanosyl group of the formula: wherein R2 and R3 are the same or different and are independently H or -OH. In one modality, R? and R3 are H. In one embodiment, R2 is OH and R3 is H. In one embodiment, R2 is H and R3 is OH. In one embodiment, where R2 and R3 are OH. In one embodiment, Q is a β-D-ribofuranosyl group of the formula: - wherein R2 and R3 are the same or different and are independently H or -OH. In some embodiments, the hydroxymethyl group (e.g., the 4'-hydroxymethyl group of β-D-ribofuranosyl) can be phosphorylated. Modifications of current alkylating agents attached to the 5- position of pyrimidine and which fix the spherical constraints as described above can be employed (Haskell, C.M., eds. (1995), pp. 55-58). The free cell, or cell based, sorting assays for release of the constituent to the 5-position of uracil are described by Roberts. D. (1966) and Hashimoto, Y. et al. (1987). In the case where Ri comprises CN ", the CN portion" highly toxic "is the therapeutically active species.

Due to the highly non-specific toxic nature of CN ", it can not normally be used in a therapeutic mode This problem is overcome by supplying the toxin in the form of a prodrug that will be significantly activated only in cells overexpressing thymidylate synthase. The prodrug can be converted to a toxic metabolite by an intracellular enzyme which, in some embodiments, can be further modified by an intracellular "maintenance" enzyme.An example is shown as follows: The description of the "partial" reaction of dUMP and TS, as well as relevant assays are described in Garrett, C. et al. (1979). Assays for other products, ie, wherein a complete reaction product is an anti-metabolite of bromovinyl derivatives of dUMP, are described by Barr, P.J., et al. (1983). The salts of the prodrugs of the present invention can be derived from inorganic or organic acids and bases. Examples of acids include hydrochloric, hydrobromic, sulfuric, nitric, perchloric acids, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulphonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic and benzenesulfonic. Other acids, such as oxalic, in themselves are not pharmaceutically acceptable, can be used in the preparation of salts used as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts. Examples of bases include alkali metal (eg, sodium) hydroxides, alkaline earth metal (eg, magnesium), hydroxides, ammonia and compounds of formula NW4 +, wherein W is C? _ Alkyl. Examples of salts include: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorrate, camphorsulfonate, cyclopentanpropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide. , iodohydrate, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate. Other examples of salts include anions of the compounds of the present invention compounds with a suitable cation such as Na +, NH + and NW4 + (wherein W it is an alkyl group of C? -4). For therapeutic use, the salts of the compounds of the present invention will be pharmaceutically acceptable. However, salts of acids and bases that are not pharmaceutically acceptable can also be found in use, for example, in the preparation or purification or a pharmaceutically acceptable compound. Esters of the prodrugs or compounds identified by the method of this invention include carboxylic acid esters (ie, -0-C (= 0) R) obtained by esterification of the 2'-, 3'- and / or 5-groups. '-hydroxy, wherein R is selected from (1) straight or branched chain alkyl (e.g., n-propyl, t-butyl or n-butyl), alkoxyalkyl (e.g., methoxymethyl), aralkyl (e.g. benzyl), aryloxyalkyl (e.g., phenoxymethyl), aryl (e.g., phenyl optionally substituted by, for example, halogen, C? -4 alkyl or C? - or amino alkoxy); (2) sulfonate esters, such as alkylsulfonyl (for example, methanesulfonyl) or aralkylsulfonyl; (3) amino acid esters (e.g., L-valil or L-isoleucyl); (4) phosphonate esters and (5) mono-, di- or triphosphate esters. The phosphate esters can be further esterified by, for example, a C? _ Alcoholo alcohol or reactive derivative thereof, or by a glycerol 2,3-di- (C6-24) acyl. On such esters, unless otherwise specified, any advantageously present alkyl portion contains from 1 to 18 carbon atoms, particularly from 1 to 6 carbon atoms, more particularly from 1 to 4 carbon atoms. Any portion of cycloalkyl present in such esters advantageously contains from 3 to 6 carbon atoms. Any aryl moiety present in such esters advantageously comprises a phenyl group. Examples of lixo-furanosyl prodrug derivatives of the present invention include, for example, those with chemically protected hydroxyl groups (e.g., with O-acetyl groups), such as 2'-O-acetyl-lixo-furanosyl; 3'-O-acetyl-lixo-furanosyl; 5'-O-acetyl-lixo-furan-silo; 2 ', 3' -di-O-acetyl-lixo-furanosyl and 2 ', 3', 5 '-tri-O-acetyl-lixo-furanosyl. Ethers of the compounds of the present invention include methyl, ethyl, propyl, butyl, isobutyl, and sec-butyl ethers. In a further embodiment, the substrate may not be chemically related to pyrimidines or folates, but rather synthesized based on the known parameters of the rational drug design. See Dunn, W.J. et al. (nineteen ninety six) . As is apparent to those skilled in the art, the drug-free cell control culture systems and separately with a named drug such as one exemplified in the following, are also analyzed. Compounds that preferably destroy target cells with about 2 times and preferably 3 times or greater activity than normal cells are preferred. This invention also provides the agents identified by the methods described herein.

T ± ros ± na. Kinases The tyrosine kinase superfamily comprises the EGF receptor (EGFR), the stimulated colony macrophage factor (CSF-1) receptor (v-fms), and the insulin receptor, which shows 30 to 40% identity with the product of the oncogen ros. More specifically, the members of this superfamily include v-src, c-src, EGFR, HER2, the CSF-1 receptor, c-fms, v-ros, insulin receptor, and c-mos. See Figure 8.5 Burck, K.B. et al., eds. (1988). Overexpression of type 1 members of the tyrosine kinase receptor superfamily has been documented in many types of cancers (Eccles, S.A. et al. (1994-95)). Overexpression of tyrosine kinases is linked to the exposure of the a-cancer biologic agent TNF-a (Hudziak, RM et al., (1988) and Hudziak, RM et al- (1990)) and to chemotherapy (Stühlinger et al. 1994)). The transforming gene of sarcoma virus Rous, v-src, encodes an enzyme that phosphorylates tyrosine residues in proteins. The proto-oncogene c-src is found on chromosome 20. The tissues and cell lines derived of tumors of neuroectodermal origin have a neural phenotype expressing high levels of c-src accompanied by high specific kinase activity. Several groups of researchers have reported overexpression of c-erbB-2 / neu oncogene ("HER2") in cancer cells. Brison (1993) observed that erbB proto-oncogene is amplified in human tumors with overexpression resulting in more cases. The extension of the c-erbB-2 / neu oncogene has been reported in human mammary tumors (Slamon, et al. (1987), van de Vijver et al. (1987), Pupa et al. (1993), and Andersen et al. (1995) and in bladder tumors (Sauter et al. (1993)), and in each case the enlargement was accompanied by overexpression.c-erbB-2 / neu overexpression has also been reported in cancer tissue samples from ovary (Slamon, et al. (1989), Meden et al. (1994) and Felip et al. (1995)) and tumors derived from the peripheral nervous system, Sukumar and Barbacid, (1990). drug, the tumor cell lines will be analyzed by expression of the oncogene or they will be designed to express levels of tyrosine kinase variation.The selected cell lines are cultures and candidate drugs are aggregated in varying concentrations.The cells are analyzed by cell destruction or inhibition of cell proliferation, as described in Hudziak, RM , et al. (1988) and Hudziak, R.M. et al. (1990) . Dihydro olate reductase Methotrexate is a potent inhibitor of dihydrofolate reductase, an enzyme necessary for the metabolism of intracellular folate. Dihydrofolate reductase functions to regenerate dihydrofolate tetrahydrofolate, a product of the thymidylate synthase reaction (Voet, et al., Eds. (1995), p.813). It will be established that an important mechanism of cell resistance to methotrexate is an increase in DHFR activity due to the extension of the DHFR gene. Banerjee, D. et al. (1995), Schimke, R.T. et al. (1988). Lónn, U. et al. (1996) reported that the extension of the DHFR gene occurs in patients with breast cancer who previously received adjuvant chemotherapy (cyclophosphamide, methotrexate, 5-fluorouracil [CMF]) after surgery. The lack of retinoblastoma (Rb) can also lead to improved MTX resistance as a consequence of an increase in DHFR mRNA expression activity without gene widening. Li, W. W. et al. (nineteen ninety five) . The cell lines with mutated p53 have been shown to undergo gene amplifications, and the resistant cells are selected by chemotherapy. Banerjee, D. et al. (nineteen ninety five). Yin, Y. et al. (1992) and Livingston ,. L.R. et al. (1992). For the purposes of performing the assay of this invention, Schimke, R.T. et al. (1988) describes several lines Cells in mouse, hamster and human. Alternatively, the PCR method of Lónn U. et al. (1996) is used to analyze the extension of the DHFR gene and identify cells that are useful in the method of identifying therapeutic agents as described herein. The nucleotide sequence of the cDNA encoding human dihydrofolate reductase is provided in Masters, J.N. and Attardi, G. (1983) and the cells can be designed to express varying levels of the enzymes as observed herein. Dicken, A.P. et al. (1993) describes a mutant DHFR gene selected by chemotherapy. The purification of DHFR and assays related to enzyme function are described in Nakano, T. et al. (1994). Alternatively, the cDNA encoding DHFR is transfected into NIH 3T3 cells. Candidate drugs are aggregated in varying concentrations and the cells of destruction and inhibition of proliferation are analyzed. Antimetabolites that depend on the activity of dihydrofolate reductase can be synthesized by the binding of, for example, an alkylation group of either N5 or the C6 position of dihydrofolate. Reduction of the N5-C6 bond by DHFR will result in the release of the alkylating agent. In addition to the alkylation groups, any portion whose release by DHFR results in the production of a toxin or an antimetabolite will be useful in the practice of the invention.

Methylthioma Resistant Tumors Multidrug resistance (MDR) is a generic term for the variety of tumor strategy cells used to evade the cytotoxic effects of anticancer drugs. MDR is characterized by a decreased sensitivity of tumor cells not only to the drug used by chemotherapy, but also to a broad spectrum of drugs without any obvious structural homology or common goals. This pleitropic resistance is one of the biggest obstacles to the successful treatment of tumors. MDR can result from structural or functional changes in the plasma membrane or within the cytoplasm, cell compartments, or nuclei. Molecular mechanisms of MDR are discussed in terms of modifications in detoxification and DNA repair trajectories, changes in cell sites of drug sequestration, decrease in affinity of the target drug, synthesis of specific drug inhibitors within cells, altered or inappropriate targets of proteins, and accelerated removal or drug secretion. One of the mechanisms involved in MDR results from the amplification and overexpression of a gene known as the multidrug-resistant ATP-dependent drug-associated protein (MRP) in the drug selected from cell lines. For a review of the MDR mechanism, see Gottesman, M.M. et al. (1995) and Noder et al. (nineteen ninety six) . To stabilize the MDR cell lines, the drug selections are conducted in either single-step or multi-step as described in Gottesman, M.M. et al. (1995) and Simón, S.M. and Schindler, M. (1994), and references cited therein. Isolation of DNA sequences encode MDR of several mammalian species as described in Gros, P. et al. (1986), Gudkov, A.V. et al. (1987), and Roninson, I.B. et al. (1984), and reviewed in Gottesman, M.M. et al. (1995), and the cells can be designed to express varying levels of this enzyme as described above. The MDR target prodrug will be based on the ATPase activity of this transporter. Ribonucleotide reductase Ribonucleotide reductase reduces the ribonucleoside diphosphates to the corresponding deoxyribonucleoside diphosphates. The enzyme is a tetramer made of two a-subunits and two β-subunits. Hydroxyurea specifically blocks this reaction by interaction with the tyrosyl free radial (Tyr-122) of the β2-substrate complex. Voet et al. (nineteen ninety five) . The goal in the objective of this reaction is to allow the accumulation of the free radical product 02; which is highly cytotoxic. Application of Technology to Other Diseases While the primary focus of this application is on cancer, it must be recognized that the technology is broadly applicable to other diseases, especially bacterial infections resistant to antibiotics. Β-lactam antibiotics encounter resistance in bacteria as a result of overexpression of β-lactamases. Hamilton-Miller, J.M.T. and Smith, J.T. eds. (1979) p. 443. Other enzymes, such as the aminoglycoside phosphotransferase Type III, are induced and selected by the following treatment with aminoglycoside antibiotics, such as kanamycin, McKay, G.A. et al. (1994). For the purposes of this application, substrates of prodrugs derived from known substrates will be prepared which will not block enzymatic activity, but instead will take advantage of the elevated enzymatic activity to generate intracellular toxins in the infectious agents. In Vivo Administration In vitro tests are confirmed in animal models supported by human tumors or infected with an antibiotic-resistant microorganism to determine efficacy in vivo. Another aspect of this invention is a method for treating a pathology characterized by hyperproliferative cells in a subject comprising administering to the subject a therapeutic amount of a prodrug which is converted to a toxin in a hyperproliferative cell by an endogenous intracellular enzyme as defined herein. When the prodrug is administered to a subject such as a mouse, a rat or a human patient, the agent can be added to a pharmaceutically acceptable carrier and systemically or topically administered to the subject. To determine patients who can be beneficially treated, a tumor sample is extracted from the patient and the cells are analyzed by the level of expression of the enzyme of interest. If the expression is earlier than that expressed in normal cells and an amount of prodrug effective to destroy or inhibit the cell can be administered without undesirable side effects, then the prodrug is a preferred embodiment. The therapeutic amounts can be empirically determined and will vary with the pathology being treated, the subject will be treated and the toxicity of the converted prodrug or cellular toxin. When supplied to an animal, the method is useful to further confirm the efficacy of the prodrug. As an example of an animal model, groups of nude mice (Balb / c NCR nu / nu females, Simonsen, Gilroy, CA) are each subcutaneously inoculated with about 105 to about 109 hyperproliferative, cancer or target cells as defined in I presented. When the tumor is established, the prodrug it is administered, for example, by subcutaneous injection around the tumor. Tumor measurements to determine tumor size reduction are made in two dimensions using venier gauges twice a week. Other animal models can also be used as appropriate. Lovejoy, et al. (1997) and Clarke, R. (1996). In vivo administration can be effected in one dose, continuously or intermittently through the course of treatment. Methods for determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out without the dose level and standard being selected by physical treatment. Suitable dosage formulations and methods for administering the agents can be found as follows. The agents and compositions of the present invention can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration according to conventional procedures, such as an active ingredient in pharmaceutical compositions.

The pharmaceutical compositions can be administered orally, intranasally, parenterally or by inhalation therapy, and can take the form of tablets, pills, granules, capsules, pills, ampules, suppositories or aerosol form. They can also be taken to form suspensions, solutions and emulsions of the active ingredient in aqueous or non-aqueous diluents, syrups, granules or powders. In addition to a compound of the present invention, the pharmaceutical compositions may also contain other pharmaceutically active compounds or a plurality of compounds of the invention. More particularly, a compound of the formula of the present invention also referred to herein as the active ingredient, can be administered by therapy through any suitable route including oral, rectal, nasal, topical (including transdermal, aerosol, buccal, and sublingual), vaginal, parental (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease being treated. In general, a suitable dose for each of the aforementioned compounds is in the range of about 1 to about 100 mg per kilogram of body weight of the container per day, preferably in the range from about 1 to about 50 mg per kilogram per body weight per day and more preferably in the range from about 1 to about 25 mg per kilogram per body weight per day. Unless otherwise indicated, all weights of the active ingredient are calculated as parent compounds of the formula of the present invention for salts or esters thereof, the weights will be proportionally increased. The desired dose is preferably presented as two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in dosage unit forms, for example, containing about 1 to about 100 mg, preferably about 1 to about 25 mg, and more preferably about 5 to about 25 mg of the active ingredient per unit form. of dose. It will be appreciated that appropriate doses of the compounds and compositions of the invention may depend on the type and severity and condition of the disease and may vary from patient to patient. The determination of the optimal dose will generally involve the balance of the level of therapeutic benefit against any risk or adverse side effects of the treatments of the present invention. Ideally, the prodrug should be administered to achieve peak concentrations of the active compound at disease sites. This can be achieved, for example, by intravenous injection of the prodrug, optionally in saline or administered orally, for example, as a tablet, capsule or syrup containing the active ingredient. The desirable blood levels of the prodrug can be maintained by a continuous infusion to provide a therapeutic amount of the active ingredient without diseased tissue. The use of operational combinations is contemplated to provide therapeutic combinations that require a low total dose of each component of antiviral agent that may be required when each individual therapeutic compound or drug is used alone, thereby reducing adverse effects. While it is possible for the prodrug ingredient to be administered alone, it is preferred to present it as a pharmaceutical formulation comprising at least one active ingredient, as defined above, together with one or more pharmaceutically acceptable carriers, therefore and optionally other therapeutic agents. . Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation not harmful to the patient. The formulations include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal, parenteral administration (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary. The formulations can conveniently be presented in dosage unit form and can be prepared by any methods well known in the pharmacy art. Such methods include the step of implementing the active ingredient in association with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. Formulations of the present invention suitable for oral administration can be presented as discrete units such as capsules, wafers or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient can also be presented in boluses, remedy or paste. A tablet can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compression in a machine suitable for the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g., povidone, gelatin, hydroxypropylmethylcellulose), lubricant, inert diluent, preservative , disintegrating (for example, sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethylceulose), active surface agent or dispersant. The molding tablets can be made by molding in a suitable machine a mixture of powder composed of moisture with an inert liquid diluent. The tablets may optionally be coated or labeled and may be formulated either to provide slow or controlled release of the active ingredient herein using, for example, hydroxypropylmethylceulose in varying proportions to provide the desired release profile. The tablets may optionally be provided with an enteric coating, to provide release in parts of the intestine other than the stomach. Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in flavor bases, usually sucrose and acacia or tragacanth; Pills comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and elixirs comprising the active ingredient in a suitable liquid carrier. Pharmaceutical compositions for topical administration according to the present invention can be formulated as an ointment, cream, suspension, lotion, powder, solution, paste, gel, spray, aerosol or oil. Alternatively, a formulation may comprise a patch or a bandage such as a plaster band or adhesive impregnated with active ingredients and optionally one or more excipients or diluents. For eye disease or other external tissues, for example, mouth and skin, the formulations are preferably applied as a topical ointment or cream containing the active ingredient in an amount of, for example, from about 0.075 to about 20% w / w, preferably, about 0.2 to about 25% w / w and more preferably about 0.5 to about 10% w / w. When formulated into an ointment, the prodrug can be employed with either a paraffin or water-miscible ointment base. Alternatively, the prodrug ingredients can be formulated in a cream with an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example, at least about 30% w / w of a polyhydric alcohol, ie, an alcohol having two or more hydroxyl groups such as propylene glycol, butan- 1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. Topical formulations may desirably include a compound that improves the absorption or penetration of the prodrug ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethyl sulfoxide and the like related The oily phase of the emulsions of this invention can be constituted of known ingredients in a known manner. While this phase may merely comprise an emulsifier (otherwise known as an emulsifier), it may, if desired, comprise a mixture of at least one emulsifier, a fat or an oil or a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include an oil and a fat. Together, the emulsifier with or without the stabilizer makes the so-called emulsification wax, and the wax together with the oil and / or grease makes the base of so-called emulsification ointment, which forms the dispersed oil phase of the formulations of cream. Emulsifier and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, ketostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in more oils likely to be used in pharmaceutical emulsion formulations is lower. So the cream will preferably be a non-greased, non-dyed and washable product with adequate consistency to prevent the escape of tubes or other containers. The straight or branched chain of mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or A mixture of branched chain esters known as Crodamol CAP can be used, the last of three esters being preferred. These can be used alone or in combination depending on the required properties. Alternatively, the high melting point of lipids such as white soft paraffin and / or liquid paraffin or other mineral oils can be used. Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially in an aqueous solvent for the prodrug ingredient. The prodrug ingredient is preferably present in such a formulation in a concentration of about 0.5 to about 20%, advantageously about 0.5 to about 10%, particularly about 1.5% w / w. Formulations for rectal administration can be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate. Formulations suitable for vaginal administration can be presented as an intrauterine device, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the prodrug ingredient, such carriers are known in the art to be appropriate. Formulations suitable for nasal administration, wherein the carrier is a solid, include a rough powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the form in which it sniffs. taken, that is, by rapid inhalation through the nasal passage of a container of dust caught closed to the nose. Suitable formulations wherein the carrier is a liquid for administration such as, for example, nasal spray, nasal drops, or by aerosol administration by nebulizer, include aqueous or oily solutions of the prodrug ingredient. Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which deliver the isotonic formulation with the blood of the intended recipient; and sterile aqueous and aqueous suspensions that they may include suspending agents and thickening agents, and liposomes or other microparticulate systems that are designed to target the compound for blood components or one or more organs. The formulations can be presented in a dose unit or sealed multi-dose containers, for example, ampoules or flasks, and can be stored in a freeze-dried (lyophilized) condition that requires only the addition of the sterile liquid carrier, for example water for injections, immediately before the sterile powders, granules and tablets of the previously described consideration. Preferred dosage unit formulations are those containing a daily dose or unit, daily sub-dose, as recited herein above, or an appropriate fraction thereof, or a prodrug ingredient. It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention can be included other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration can include such additional agents as sweetening agents, thickeners and flavorings. The prodrugs and compositions of the formula of the present invention may also be presented for use in the form of veterinary formulations, which may be prepared, for example, by methods that are conventional in the art. EXAMPLES The following examples are specifically directed to the TS target enzyme. It is apparent to those skilled in the art that the following methods can be modified for the discovery of other prodrugs to target enzymes as defined herein. Cell and Chemical Assays The pyrimidine-based prodrugs are chosen based on the ability to react with intracellular thymidylate synthase, and release the toxin in the medium without inactivation of the enzyme. The candidate drugs are classifications in reaction mixtures containing human thymidylate synthase with and without N5N10-methylenetetrahydrofolate and the candidate prodrug. The leaving group of the candidate prodrug (eg, position 5 of pyrimidine) is labeled, for example, with tritium using methods well known in the art. The control substrate is similarly labeled (eg, 5-3H) dUMP, under the same reaction conditions. The tests are carried out similarly to the description provided in Carreras, C.W. and Santi, D.v. (1995) and references cited herein. Human thymidine synthase can be purified from E. coli containing human thymidylate synthase voiced. See Davisson, V.J. et al. (1989) and Davisson, V.J. et al. (1994). This approach provides a scalable trial capable of classifying large numbers of candidate compounds. A high classification performance to identify biologically active compounds is underlined in Figures 3, 4 and 5. The basis of the test is the easy genetic manipulation and growth of E. coli and similar single cell organisms (eg, yeast), see Miler (1992) and Spector, et al. (1997). The key step is removing the endogenous enzymatic activity that corresponds to an enzyme target for prodrug design. This can be done by any of the methods described by Miller (1992), Sambrook (1989) or Spector et al. (1997). These methods include chemical and biological mutagenesis (eg, viral or transponson insertion) followed by an appropriate selection procedure. The negative TS cell (TS ") then became a negative control for the identification of prodrugs that, when acting above the thymidylate synthase, become cellular toxins.A similar approach can be made with other cell types, for example, other bacteria, yeasts, or other select cell organisms alone In the assay, both control and recombinant organisms are compared for sensitivity to the test compounds, as will be understood by those skilled in the art, the prodrugs that are distinguish between enzyme species can also be derived from this procedure. For example, another form of identical cells expressing human and yeast enzymes can be used to detect antibiotic pro-drugs that are preferably toxic only to cells expressing the yeast enzyme. In this way, novel and specific antibiotics can be discovered. Examples of cell lines are NIH 3T3 ras-transformed cells (obtained from ATCC) and are designed to express increased amounts of human thymidylate synthase (HuTS) from the cloned cDNA. Transfection is performed on a transient or permanent basis (see Chen L. et al. (1996), Hudziak, RM et al. (1988), and Carter, P. et al. (1992). NIH-000 (line ras-transformed paternal cellular), NIH-001 (low HuTS express), NIH-002 (Hu TS express intermediate), NIH-003 (elevated HuTS expressor), the level of TS expression in each cell line is verified by immunoabsorption and enzyme assay in cell lysates, using antibody directed against HuTS protein for immunodetection (eg, as described in Chen, L. et al. (1996)) Enzymatic assays are performed as reviewed by Carreras, CW and Santi, DN (1995) .The colorectal and breast tumor cell lines are classified by HuTS enzyme expression.The cell lines express low, moderate and high levels of HuTS. they will be exposed to candidate drugs as described above for the NIH 3T3 cell lines. Inhibition of growth and cytotoxicity are verified as described above. Similar tests can be carried out for each of the enzymes listed in Table 1. In vivo tests The ras-transformed NIH 3T3 cell lines are transplanted subcutaneously into immunodeficient mice. Initial therapy can direct intratumoral injection. The expected result is that it increases the level of expression of HuTS or an objective enzyme that leads to improve the antitumor activity by the candidate prodrug. Similar studies are performed with human tumors that express increased levels of HuTS or a target enzyme, and demonstrating that the efficacy in response to the drug correlated with their expression level of HuTS or target enzyme. Optionally, the experiments are performed as above, except the drug that will be administered intravenously in the animals to address issues related to the efficacy, toxicity and pharmacobiology of the candidate drug. The in vivo studies will be conducted as described in Harris, MP et al. (1996) and Antelman, D. et al. (nineteen ninety five) . 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Claims (38)

1. CLAIMS 1. A method for identifying potential therapeutic agents, characterized in that it comprises: (a) contacting a target cell with a candidate therapeutic agent that is a selective substrate for an objective enzyme, under conditions that favor the incorporation of the agent into the cell objective; and (b) analyzing the target cell for inhibition of cell proliferation or cell destruction.
2. 2. The method for identifying potential therapeutic agents, characterized in that it comprises: (a) contacting a target cell with a candidate therapeutic agent having a detectably labeled toxic leaving group and being a selective substrate for an objective enzyme, under conditions that they favor the incorporation of the agent in the target cell; and (b) analyzing the culture medium for the labeled release amount.
3. 3. The method according to claim 1 or 2, characterized in that the target cell is characterized as resistant to a chemotherapeutic drug.
4. 4. The method according to claim 1 or 2, characterized in that the target enzyme is amplified as a result of the in vivo selection by chemotherapy.
5. 5. The method according to claim 1 or 2, characterized in that the target enzyme is an endogenous intracellular enzyme that is overexpressed in the target cell.
6. 6. The method according to claim 5, characterized in that the endogenous overexpression of an intracellular enzyme is the result of the amplification of the gene coding for the enzyme.
7. 7. The method according to claim 1 or 2, characterized in that the target enzyme is thymidylate synthase.
8. 8. The method according to claim 1 or 2, characterized in that the candidate therapeutic agent is a compound L- or D- of the formula: wherein Ri is or contains a chemical entity that has a molecular dimension and electrophilicity compatible with the extraction of the pyrimidine ring by thymidylate synthase, and which releases the pyrimidine ring by thymidylate synthase which has the ability to inhibit proliferation of the cell or destroy the cell; and wherein Q is selected from the group consisting of sugar groups, thio-sugar groups, carbocyclic groups, and derivatives thereof.
9. 9. The method of compliance with the claim 8, characterized in that Q is a furanosyl group of the formula: wherein R2 and R3 are the same or different and are independently H or -OH.
10. 10. The method of compliance with the claim 8, characterized in that Q is a β-D-ribqfuranosyl group of the formula: wherein R2 and R3 are the same or different and are independently H or -OH.
11. 11. The method in accordance with the claim 8, characterized in that R], is selected from the group consisting of -Br, -I, -O-alkyl, -O-aryl, O-heteroaryl, -S-alkyl, -S-aryl, -S-hetaroaryl, - CN, -OCN, -SCN, -NH2, -NH-alkyl, -N (alkyl) 2, -NHCHO, -NHOH, -NHO-alkyl, NH2CONHO-, NHNH2 and -N3.
12. 12. The method in accordance with the claim 9, characterized in that R ± is selected from the group consisting of -Br, -I, -O-alkyl, -O-aryl, O-heteroaryl, -S-alkyl, -S-alkyl, -S-aryl, -S -heteroaryl, -CN, -OCN, -SCN, -NH2, -NH-alkyl, -N (alkyl) 2, -NHCHO, -NHOH, -NHO-alkyl, NH2CONHO-, NHNH2 and -N3.
13. 13. The method according to the claim 10, characterized in that Ri is selected from the group consisting of -Br, -I, -O-alkyl, -O-aryl, O-heteroaryl, -S-alkyl, -S-aryl, -S-heteroaryl, -CN, -OCN, -SCN, -NH2, -NH-alkyl, -N (alkyl) 2, -NHCHO, -NHOH, -NHO-alkyl, NH2CONHO-, NHNH2, and -N3.
14. 14. The agent identified by the method according to claim 1 or 2.
15. 15. A method for inhibiting the proliferation of a hyperproliferative cell, characterized in that it comprises contacting the cell with a prodrug that is selectively converted to a toxin in the cell. cell by an endogenous intracellular enzyme.
16. 16. The method according to claim 15, wherein the hyperproliferative cell is characterized by the endogenous overexpression of an intracellular enzyme.
17. 17. The method of compliance with the claim 15, wherein the hyperproliferative cell is characterized as resistant to a chemotherapeutic drug.
18. 18. The method of compliance with the claim 16, characterized in that the endogenous overexpression of an intracellular enzyme is the result of the amplification of the gene coding for the enzyme.
19. 19. The method according to claim 15, characterized in that the enzyme is amplified as a result of the in vivo selection by chemotherapy.
20. 20. The method according to claim 15, characterized in that the enzyme is thymidylate synthase.
21. 21. The method according to claim 15, characterized in that the prodrug is a compound L- or D- of the formula: where Ri is, or contains a chemical entity that has a molecular dimension and electrophilicity compatible with the extraction of the pyrimidine ring by thymidylate synthase, and which is released from the pyrimidine ring by thymidylate synthase has the ability to inhibit cell proliferation or destroy the cell; and wherein Q is selected from the group consisting of sugar groups, thio-sugar groups, carbocyclic groups, and derivatives thereof.
22. 22. The method according to claim 21, characterized in that Q is a furanosyl group of the formula: wherein R2 and R3 are the same or different and are independently H or -OH.
23. 23. The method according to claim 21, characterized in that Q is a β-D-ribofuranosyl group of the formula: - wherein R2 and R3 are the same or different and are independently H or -OH.
24. The method according to claim 21, characterized in that Ri is selected from the group consisting of -Br, -I, -O-alkyl, -O-aryl, O-heteroaryl, -S-alkyl, -S-aryl , -S-hetaroaryl, -CN, -OCN, -SCN, -NH2, -NH-alkyl, - (alkyl) 2, -NHCHO, -NHOH, -NHO-alkyl, NH2CONHO-, NHNH2 and -N3.
25. 25. The method according to claim 22, characterized in that Ri is selected from the group consisting of -Br, -I, -O-alkyl, -O-aryl, O-heteroaryl, -S-alkyl, S-alkyl, S-aryl, -S-heteroaryl, -CN, -OCN, -SCN, -NH2, -NH-alkyl, -N (alkyl) 2, -NHCHO, -NHOH, -NHO-alkyl, NH2C0NH0-, NHNH2 and - N3
26. 26. The method of compliance with the claim 23, characterized in that Ri is selected from the group consisting of -Br, -I, -O-alkyl, -O-aryl, O-heteroaryl, -S-alkyl, -S-aryl, -S-heteroaryl, -CN, -OCN, -SCN, -NH2, -NH-alkyl, - (alkyl) 2, -NHCHO, -NHOH, -NHO-alkyl, NH2CONHO-, NHNH2, and -N3.
27. 27. A method for treating a condition characterized by hyperproliferative cells in a subject comprising administering to the subject a prodrug that is converted to a toxin in a hyperproliferative cell by an intracellular enzyme that is endogenously overexpressed or over-accumulated in the cell.
28. 28. The method according to claim 27, wherein the target cell is characterized by endogenous overexpression of an intracellular enzyme related to resistance to chemotherapy.
29. 29. The method according to claim 27, wherein the target cell is characterized as resistant to a chemotherapeutic drug.
30. 30. The method according to claim 21, wherein the target cell is characterized in that it has an inactivated function of the tumor suppressor.
31. 31. The method according to claim 27, characterized in that the endogenous overexpression of an intracellular enzyme is the result of the amplification of the gene coding for the enzyme.
32. 32. The method according to claim 27, characterized in that the enzyme is amplified as a result of in vivo selection by chemotherapy.
33. 33. The method according to claim 27, characterized in that the enzyme is thymidylate synthase.
34. 34. The method according to claim 1 or 2, characterized in that the target cell expresses a foreign gene coding for the target enzyme.
35. 35. The method according to claim 1 or 2, characterized in that it further comprises contacting a control cell with the candidate therapeutic agent, under conditions that favor the incorporation of the agent into the cell.
36. 36. The method according to claim 1 or 2, characterized in that the target cell expresses two or more foreign genes, each foreign gene coding for a target enzyme.
37. 37. The method according to claim 35, characterized in that the control cell does not express the target enzyme or expresses the target enzyme at a low level
38. 38. The method according to claim 35, characterized in that the control cell is normal cell type, not neoplastic and the target cell is a cancer cell.