CHEMOTHERAPEUTIC DRUG COMBINATIONS

Abstract: The invention relates to certain compositions and combinations of ATP depletion compounds and apoptosis inducing agents. The invention also relates to methods of treating antineoplastic disease by administering such combinations.

Description: CHEMOTHERAPEUTIC DRUG COMBINATIONS Field of the Invention The present invention relates to compositions comprising compounds which deplete cellular energy. The invention also relates to combinations of such compounds with compounds which induce apoptosis. The invention also relates to the use of these compositions and combinations in the treatment of anti-neoplastic disease. Background of the Invention Adenosine triphosphate, ATP, is the key energy source in major metabolic processes such as biosynthesis, active transport and DNA repair. Consequently, if ATP production is inhibited, consumption of existing ATP will result in an energy deficiency that would adversely affect the functional and morphologic integrity of the cell. Methods for selective impairment of energy metabolism have been proposed primarily involving induction of acidosiε in tumors via sustained hyperglycemia, optionally in conjunction with acid-activated prodrugs of metabolic poisons (McCarty, Med Hypotheses 16:39-60 (1985)). This approach is contingent upon particular tumors having metabolic differences relative to normal tissues sufficient to produce acidoεis, and is furthermore dependent on tumor mass being sufficiently large (and homogeneous) so that acidosis remains localized. The energy deficits thus produced are believed to be due to acidoεis-induced reductions in tumor blood flow, which are furthermore dependent on the characteristics of vascularization of a particular tumor. This approach to impairing tumor energy metabolism is therefore not SUBSTITUTESHEET universally, or even generally, applicable to treating a variety of forms of cancer. Several compounds are known to impair cellular energy metabolism; these compounds are discussed below: 6-AN The nicotinamide antagonist 6-aminonicotinamide (6-AN) (Johnson et al., Science 122:834 (1955)) is converted in vivo into the nicotinamide adenine dinucleotide (NAD) analogs, 6-ANAD and 6-ANADP. These competitive analogs of NAD and NADP cannot be reduced either chemically or enzymatically (Dietrich, Antineoplastic and Immunosuppressive Agents, ed. by A.C. Sartorelli and D.G. Johns (New York: Springer-Verlag), pp. 539-542 (1975), Dietrich et al., J. Biol. Chem. 233:964-968 (1958), and Dietrich et al, Cancer Res. 18:1272-1280 (1958)), and consequently act as potent inhibitors of NAD-dependent dehydrogenases utilized in glycolysis, in the oxidative portion of the pentose-phosphate pathway, and in mitochondrial oxidative phosphorylation (Dietrich, Antineoplastic and Immunosuppressive Agents, ed. by A.C. Sartorelli and D.G. Johns (New York: Springer-Verlag), pp. 539-542 (1975), Dietrich et al., J. Biol. Chem. 233:964-968 (1958), Dietrich et al , Cancer Res. 18:1272-1280 (1958), Woods et al, Biochem. Z. 338:381-392 (1963), Kaufman et al., J. Neurobiol. 5:391 (1974), Varnes, NCI Monographs No. 6, pp. 199-202 (1988), Ofori-Nkansah et al., Z. Krebsforsch 77:64-76 (1972), Ofori-Nkansah et al., Naunyn-Schmiedebergs Arch. Pharmacol. 272:156-168 (1972), Keller et al., Hoppe-Seyler's Z. Phyεiol. Chem. 353:1389-1400 (1972), Herken et al. ,. Biochem. Biophyε. Res. Comm. 36:93-100 (1969), Coper et al., Biochim. Biophys. Acta (Amεt. ) 82:167-170 (1964)). 6-AN has demonstrated preclinical anti-cancer activity (Martin et al., Cancer Res. 17:600-604 (1957), Hunting et al., Nature SUBSTITUTES Biochem. Pharmacol. 34:3999-4003 (1985)), but at tolerated doses, did not have antitumor efficacy as a single agent in humans (Herter et al., Cancer Research 21:31-37 (1961)). MMPR The adenosine analog 6-methylmercaptopurine riboside (MMPR) has been εhown to reεult in ATP and GTP depletion, inhibition of macromolecular εyntheεiε, and inhibition of tumor growth (Elion, Fed. Proc. 26:898-903 (1967), Elion, J. Am. Chem. Soc. 74:411-414 (1952), Shantz et al.. Cancer Res. 33:2867-2871 (1973), Woods et al., Eur. J. Cancer 14:765-770 (1978), Warnick et al., Cancer Res. 33:1711-1715 (1973), Nelson et al., Cancer Res. 32:2034-2041 (1972)). In addition, the bioεyntheεiε of NAD may be inhibited by MMPR becauεe NAD iε εyntheεized in the cell from nicotinamide mononucleotide (NMN) and ATP by an enzyme (NMN adenyltranεferase) that iε inhibited by thio-ITP (Atkinson et al., Nature 192:946-948 (1961)). The 6- N and MMPR Combination MMPR and 6-AN are εynergistic if the timing of their adminiεtration is appropriate, becauεe the lowering of NAD levelε by MMPR favorε the competition of 6-ANAD with NAD, and thereby enhances the magnitude of ATP depletion that is achieved by either drug alone. The combination of 6-AN and MMPR produces regressions of advanced murine breast tumors which cannot be obtained with either drug alone (Martin, Mt. Sinai J. Med. 52:426-434 (1985)). In addition to its antipurine action, MMPR, in high dosage, is reported to decrease pyrimidine ribonucleotide concentrations in vivo (Woods et al., Eur. J. Cancer 14:765-770 (1978), Grindey et al., Cancer Res. 36:379-383 (1976)). PALA SUBSTITUTESHEET The pyrimidine antagonist N-(phosphonacetyl)-L-aspartic acid (PALA), in low non-toxic dosage, can lower pyrimidine levels in vivo selectively in certain tumors (Martin et al., Cancer Res. 43:2317-2321 (1983)). The Triple Combination The triple combination of PALA + MMPR + 6-AN was evaluated against advanced solid tumors in mice (Martin, Biochemical modulation — Perspectives and Objectives. In: New Avenues in Developmental Cancer Chemotherapy. Ed. by Kenneth R. Harrap, London, England (1987)). Preliminary biochemical data (Martin, Metabolism and Action of Anti-cancer drugs. Ed. by Garth Powiε and Russell A. Prough (London: Taylor & Francis), pp. 91-140 (1987)), demonstrated εubεtantial depreεεion at 24 and 48 hours of NAD levels, and of the four ribonucleoside triphoεphateε of purines and pyrimidineε, including ATP. The three drug combination produced a marked antitumor effect which was not obtained with any of the individual agents or any combination of two of the agents (Martin, Mt. Sinai J. Med. 52:426-434 (1985), Martin, Biochemical modulation — Perspectiveε and Objectives. In: New Avenues in Developmental Cancer Chemotherapy. Ed. by Kenneth R. Harrap, London, England (1987)). Objects of the Invention It is an object of the invention to provide compositions which deplete cellular energy. It is a further object of the invention to provide drug combinations which are useful in treating antineoplastic diεease. SUBSTITUTESHEET Summary of the Invention The subject invention relates to chemotherapeutic drug combinations and their use in the treatment of antineoplastic diseaεe. Such drug combinations comprise: a) cellular energy depletion compounds, and b) at leaεt one apoptosis inducing agent. Advantageously, the cellular energy depletion compounds comprise: 1) an inhibitor of purine nucleotide biosynthesis, 2) a nicotinamide antagonist, and optionally 3) an inhibitor of pyrimidine nucleotide biosynthesiε. Brief Description of the Drawings Figure 1 showε changes in PCr/Pi and NTP/Pi after treatment with the triple drug combination PALA, MMPR, and 6-AN. The change in PCr/Pi and NTP/Pi at 10 hours iε statistically significant (P less than 0.01, p lesε than 0.02) . The change in NTP/Pi between the 10 and 24 hour measurement is not significant. Pretreatment values were determined prior to the administration of PALA- 00 and appear on the y intercept. At Time "0" MMPR- 50 + 6- AN. Q were administered, n * = 7 for each time point. Figure 2 shows results obtained in Example 2. CD8F1 mice bearing spontaneous, autochthonous breast tumors received three courseε of treatment on a q 10-11 day schedule with Adria alone at 11 mg/kg, or with Adria at 6 mg/kg administered 25j hrs after PALA + MMPR + 6-AN (PALA at 100 mg/kg 17 hrs before MMPR at 150 mg/kg plus 6-AN at 10 mg/kg), or with the same regimen of PALA + MMPR + 6-AN without Adria. Tumorε averaged 304 gε at initiation of therapy. SUBSTITUTESHEET Detailed Description of the Invention The subject invention relates to drug combinations comprising cellular energy depletion compounds and at least one apoptosis inducing agent. The invention also relates to the use of such drug combinations in the treatment of antineoplastic disease. Apoptosis Inducing Agents In the subject invention, apoptosiε inducing agents are used in conjunction with cellular energy depletion compounds. In the physiological context, apoptosis is a process by which cells are removed from embryonic and developing εomatic tissues, and has been implicated in terminal differentiation of myeloid cells, and in hormone-dependent tiεεue atrophy. It has also been documented in the cytoxic T-cell killing of tumor cells,-and in tumor regression (Wyllie et al., Int. Rev. Cytology 68:251-306 (1980)). Cell death via necrosis is characterized by cell swelling, chromatin flocculation and disruption of cell integrity followed by cell lyεiε (Wyllie et al., Int. Rev. Cytology 68:251-306 (1980)). Cell death induced by anti-cancer agentε may take the form of both apoptoεiε and necroεiε. Apoptotic cell death may be more apparent at low levelε of theεe agents, while at high levels (which therefore pose greater risk of toxic εide effects) necrosis may occur due to severe metabolic insult. Hence, cellε which are not killed directly, but merely injured by anti-cancer agentε, may activate a genetically programmed "suicide" mechanism (Lennon et al., Cell Prolif. 24:203-214 (1991)). SUBSTITUTESHEET A large number of agents have been demonstrated to induce apoptosis (apoptoεis inducing agents), including: Antimetabolites • ethotrexate (Barry et al., Biochem. Pharmacology 40:2353-2362 (1990) and Marks et al., Biochem. Pharmacol 42:1859-1867 (1990)), •5-fluorodeoxyuridine (Barry et al., Biochem. Pharmacology 40:2353-2362 (1990) and Kyprianou et al., Biochem. Biophys. Reε. Communications 165:73-81 (1989)), •5-fluorouracil (FUra) (Barry et al., Biochem. Pharmacology 40:2353-2362 (1990) and Kyprianou et al., Biochem. Biophys. Res. Communications 165:73-81 (1989)), •1-B-D-arabinofuranoεyl-cytoεine (Gunj et al., OOOOCancer Reε. 51:741-743 (1991)), •puro ycin (Kaufmann, Cancer Res. 49:5870-5878 (1989)), •trifluorothymidine (Kyprianou et al., Biochem. Biophys. Res. Communications 165:73-81 (1989)), DNA Damaging Agents •cisplatin (Barry et al., Biochem. Pharmacology 40:2353-2362 (1990)), •etopoεide (Barry et al., Biochem. Pharmacology 40:2353-2362 (1990), Kaufmann, Cancer Res. 49:5870-5878 (1989), Tanizawa et al., Exp. Cell. Reε. 185:237-246 (1989) and Markε et al., Biochem. Pharmacology 42:1859-1867 (1990)), SUBSTITUTESHEET •camptothecin (Kaufmann, Cancer Res. 49:5870-5878 (1989)), •cytoxan (Kaufmann, Cancer Res. 49:5870-5878 (1989) and Marks et al., Biochem. Pharmacology 42:1859-1867 (1990)), •adria ycin (adria) (Marks et al., Biochem. Pharmacology 42:1859-1867 (1990)), •tenipoεide (Kaufmann, Cancer Res. 49:5870-5878 (1989)), •podophyllotoxin (Kaufmann, Cancer Reε*. 49:5870-5878 (1989)), •aphidocolin (Barry et al., Biochem. Pharmacology 40:2353-2362 (1990) and Martin et al., Cell Tissue Kinet. 23:545-559 (1990)), •N-methy1-N*-nitro- N'mitrosoguanidine (Barry et al., Biochem. Pharmacology 40:2353-2362 (1990)), •nitrogen mustard (O'Connor et al.. Cancer Res. 51:6550-6557 (1991)), •bleomycin (Kuo et al. * , Nature 271:83-84 (1978)), •l,3-biε(2-chloroethyl)-a-nitroεourea (BCNU, Berger et al., Cancer Reε. 42:4382-4366 (1982)), •methyl glyoxal-bis-(guanylhydrazone) (MGBG, Brune et al., Exp. Cell Reε. 195(2):323-329 (1991)), •radiotherapy (Warterε, Cancer Reε. 52:883-890 (1992)). Microtubule Mod fierε •colcemid (Kaufmann, Cancer Reε. 49:5870-5878 (1989)), •vincriεtine (Martin et al., Cell Tissue Kinet. 23:545-559 (1990)), •taxol (Martin et al., Cell Tissue Kinet. 23:545-559 (1990) and Lennon et al., Cell Prolif. 24:203-214 (1991)) •taxotere Hormones •dexamethasone (Barry et al., Biochem. Pharmacology, 40:2353-2362 (1990)), •retinoic acid (Piacentini et al., Eur. J. Cell Biol., 54:246-254 (1991)), •purinergic P2 receptor agonistε (Trepel et al., WO Pat. Appl. # 9116056-A) •somatostatin analogs (Pagliacci et al., Endocrinology, 129:2555-2562 (1991)) •luteinizing hormone releasing factor analogs (Szende et al., Cancer Res., 50:3716-3721 (1990)) •estrogen ablation (Kyprianou et al.. Cancer Res., 51:162-166 (1991)) •tumor necroεiε factor (Piguet et al., Am J. Pathol., 136:103-110 (1990)) Miscellaneous •turooricidal antibodieε capable of inducing apoptosis ( Krammer, Pat. W09110448-A) SUBSTITUTESHEET •cytotoxic T-cells (Ucker, Nature, 327:62-63 (1987)) •sodium azide •gossypol •lonidamine •rhodamine 123 The significance of the preceding list is that these different agentε are all capable of inducing the same final common mechanism of cancer cell death, regardless of the diverse biochemical leεionε initiated by each individual agent. Current information indicateε that the apoptotic biochemical caεcade can be activated at different pointε in different cell types (Duvall et al., Immunology Today 7(4):115-119 (1989)). The invention provideε drug combinations and methods for impairing cellular energy and nucleotide metabolism, and thereby dramatically increaεing the antitumor efficacy of a wide variety of apoptosis-inducing antineoplastic agentε, without a correεponding increaεe in host toxicity. Since apoptosis is itself an energy-requiring process (Cotter et al., Anticancer Res., 10:1153-1159 (1990)), this result iε unexpected. The triple drug combination, PALA-MMPR-6-AN, as well as all apoptosiε-inducing anti-cancer agentε, have ATP depletion aε a basic biochemical lesion, and complement each other on this basis. In addition, the PALA-MMPR-6-AN combination produces a level of wide biochemical damage in cancer cells that complements, and iε complemented by, the apoptosis-inducing biochemical effects of DNA-damaging agents, and thereby result in enhanced kill of tumor cellε. The in vivo addition of 5-fluorouracil, adriamycin, taxol, radiotherapy, mitomycin C, ciε-platinum, cytoxan, phenylalanine mustard, arid etoposide with the PALA-MMPR-6-AN triple combination demonstrate greater anticancer activity than that observed with the individual drugs alone at maximum tolerated dose (MTD) or with the PALA-MMPR-6-AN combination alone. Cellular Energy Depletion Compounds Intracellular ATP has been negelected as a primary target for cancer chemotherapy because ATP has long been considered too important a source of energy for all cellε to warrant the expectation that primarily directed anti-ATP chemotherapy could be selective for cancer cellε and εafe for hoεt tissueε. The εubject application documents novel compoεitionε and methods for impairing tumor cell energy metabolism, with striking therapeutic activity, a εafe therapeutic index, and an aεsociated depletion of ATP levels in the in vivo treated tumors. When administered in conjunction with agentε which trigger apoptoεiε (programmed cell death) in tumors, the energy-depleting compositions of the invention produce therapeutic activity against spontaneous tumorε in vivo εubεtantially greater than can be obtained by either energy depletion compositions or apoptoεiε-inducing agentε alone. Although glycolyεiε provides some of the ATP needed by the cell, the major generation of ATP occurs during oxidative phoεphorylation in the mitochondria of mammalian cells aε electrons are transferred from reduced NAD (NADH) to O, by a series of electron carriers. NADH is reconverted to NAD with concomitant conversion of ADP to ATP. In addition to their central role in energy metabolism, NAD and NADP (nicotinamide adenine dinucleotide phoεphate), are oxidizing agents which function aε coenzymes in critical biochemical reactions. Disruptions in NAD synthesis and metabolism has profoundly adverse effects on cellular integrity because of the central role played by these coenzymeε in intermediary metabolism including the generation of ATP from ADP. A limitation of adenine or NAD, or both, are keys to ATP depletion. In the subject invention, these metabolites are targets for chemotherapy designed to result in depletion of cellular levels of ATP. The cellular energy depletion compounds of the invention are typically used in combination and typically include: 1) an inhibitor of purine nucleotide biosynthesis, 2) a nicotinamide antagonist, and optionally 3) an inhibitor of pyrimidine nucleotide biosynthesis. HPLC and NMR measurementε of biochemical changes reεulting from treatment with PALA (an inhibitor of pyrimidine biosynthesis) + MMPR (an inhibitor of purine biosynthesis) + 6-AN (a nicotinamide antagonist) indicate a severe depletion of cellular energy levels in the treated tumorε. The PALA-MMPR-6-AN-induced reduction in all four individual ribonucleoεide poolε (Martin, Metabolism and Action of Aήti-cancer drugs. Ed. by Garth Powis and Rusεell A. Prough (London: Taylor & Francis), pp. 91-140 (1987)), which generally correlates with a reduction in the corresponding deoxyribonucleoside triphoεphate poolε (Hunting et al., Can. J. Biochem. 59:821-829 (1981)), appearε to not only deplete cellular energy εources and to inhibit DNA synthesis, but to inhibit the potential for DNA repair as well. Because the DNA damage produced by many chemotherapeutic drugs is subject to repair, the cytotoxic activity of these DNA-damaging drugs is increased when the DNA repair potential of the cell is decreased. A 3-drug combination, PALA-MMPR-6-AN, augments the antitumor activity of DNA-damaging drugs by virtue of its ability to deplete both the deoxyribonucleotideε, and the energy source required for the DNA repair processeε. Inhibitors of Purine Nucleotide Biosynthesis Inhibitorε of purine nucleotide biosynthesiε of the invention include the following: a) Direct Inhibitors 6-methylmercaptopurine riboside (MMPR) 6-mercaptopurine Thioguanine Thiamiprine Tiazofurin Azaserine 6-diazo-5-oxo-L-norleucine Acivicin b) Folate Antagonists Methotrexate Trimetrexate Pteropterin Denopterin Didiazotetrahydrofolate (DDATHF) Advantageous inhibitors of purine nucleotide biosynthesiε are MMPR and folate antagoniεtε which are relatively εelective inhibitors of the enzyme glycinamide ribonucleotide transformylaεe, e.g. DDATHF or DACTHF. Nicotinamide Antagonists Nicotinamide antagoniεts of the invention include the following: 6-aminonicotinamide (6-AN) SUBSTITUTESHEET Thionico inamide 2-amino-l,3,4-thiadiazole 2-ethylamino-l,3,4-thiadiazole 6-aminonicotinic acid 5-methylnicotinamide 3-acetylpyridine Inhibitors of Pyrimidine Biosynthesis Inhibitors of pyrimidine nucleotide biosynthesiε of the invention include the following: N-(phoεphonoacetyl)-L-aεpartic acid (PALA) 6-azauridine Triacetyl-6-azauridine Pyrazofuran Brequinar Acivicin While not wishing to be bound by theory, the following is offered as an explanation of the mechanism of the combinations of the invention. The triple combination of an inhibitor of purine biosynthesis, a nicotinamide antagonist and an inhibitor of pyrimidine biosynthesis (e.g., PALA + MMPR + 6-AN) was designed to produce a mutually reinforcing blockade on pyrimidine and purine de novo biosynthesis, as well as a specific attack on NAD metabolism, that in toto result primarily in a damaging depletion of high energy nucleoside triphosphates, particularly ATP. PALA is an inhibitor of de novo pyrimidine biosynthesis (Collins et al., J. Biol. Chem. 246:6599-6605 (1971), Johnson et al., Cancer Reε. 36:2720-2725 (1976)). PALA haε been found to be non-toxic to the hemopoietic SUBSTITUTESHE syεtemε in mice (Johnson et al., Cancer Reε. 36:2720-2725 (1976), Harrison et al., Cancer Chemother. Pharmacol. 2:183-187 (1978)), and when administered at a low dose (100 mg/kg) exerts selective action in CD8F1 breast tumor tissue, but not in the intestinal tissue of the host (Martin et al., Cancer Res. 43:2317-2321 (1983)). The antitumor toxicity of high dose MMPR as a single agent is known to be associated with a general depletion of purine nucleotides that results in the inhibition of macromolecular synthesis and tumor growth (Nelson et al., Cancer Reε. 32:2034-2041 (1972), Scholar et al., Cancer Reε. 32:259-269 (1972), Warnick et al., Cancer Reε. 33:1711-1715 (1973), and Woodε et al, Eur. J. Cancer 14:765-770 (1978)). Thiε thiopurine-induced decrease in tumor ATP levels haε been implicated previously as the causal mechanism in the therapeutic effect produced in tumors (Atkinson, Regulation of energy metabolism: Exploitable molecular mechanisms and neoplasia. The University of Texas M.D. Anderson Hospital and Tumor Institute at Houston, 22nd Annual Symposium on Fundamental Cancer Research, 1968. (Baltimore: Williams and Wilkins), 397-413 (1969)). The earlieεt reported studies emphasizing ATP depletion as a chemotherapeutic approach in vivo employed 6-AN as a single agent to iower ATP levels in the tumor and thereby produced effective antitumor activity (Dietrich et al., Cancer Res. 18:1272-1280 (1958) and Martin et al., Cancer Reε. 17:600-604 (1957)). In more recently conducted in vitro studies, 6-AN inhibition of tumor cell growth was accompanied by a depletion of purine and pyrimidine nucleotideε, and NAD, as well as by a reduction in the ATP to ADP ratio (Hunting et al., Biochem. Pharmacol. 34:3999-4003 (1985)). The triple combination is less toxic when the interval between courses of PALA + MMPR + 6-AN treatment SUBSTITUTESHEET iε extended from 7 dayε to 10 or 11 days, and this change in the schedule of adminiεtration reduces toxicity sufficiently to permit the safe addition of other drugε (e.g., an apoptoεiε inducing agent εuch aε FUra) every 10 or 11 days together with the three drug combination. The PALA-MMPR-6-AN-induced reduction of ATP levelε haε been demonstrated by both HPLC and the non-invasive technique of NMR spectroscopy (Fig. 1). Although the degree of εuppresεion of ATP waε εignificant at early time pointε after the triple chemotherapy, by 72 hourε the ATP levels had returned to normal. In some normal cellε (e.g., hepatocyteε), maintenance of severely depressed levels of ATP (20% of control values) for as long as 36-48 hourε does not neceεεarily compromise viability as long aε the adenine nucleotide concentrations then return to control values (Farber, Fed. Proc. 32:1534-1539 (1973)). Sustained or permanent loss of ATP is incompatible with cell survival (Schraufstatter et al., J. Clin. Inveεt. 77:1312-1320 (1986)). However, a temporary (48 hour) depletion of ATP iε never the sole determinant compromising cell viability. Any εignificant drop in ATP concentration has many metabolic consequences, and other induced perturbations to cellular biochemistry (e.g., those induced by PALA and 6-AN) act in concert with ATP loss to cause cell death (Hyslop et al., J. Biol. chem. 263:1665-1675 (1988)). For. example, the pentose phosphate shunt provides reducing equivalents in the form of NADFH for certain anabolic reactionε and also for the maintenance of reduced glutathione, GSH. GSH is a major cellular reductant (Meister et al., Pharmac. Ther. 49:125-132 (1991), Morrow et al., Cancer Cells 2:15-22 (1990), Doroshow et al., Pharm. Ther. 47:359-370 (1990), Keizer et al., Pharm. Ther. 47:219-231 (1990)), and in the proceεε of detoxification of radical species becomes oxidized glutathione, GSSG. NADPH iε required for the conversion of GSSG back to GSH by the enzyme, glutathione reductase. Thus, 6-AN'ε inhibition of the pentose SUBSTITUTESHEET phosphate shunt can lead to a lowering of NADPH levels which in turn can prevent adequate GSH reεynthesis from GSSH. And, εince ATP iε required for the initial eyntheεiε of GSH from itε conεtituent amino acids (Meister et al, Pharmac. Ther. 49:125-132, (1991)), the ATP depletion induced by the triple combination can limit supplies of GSH. Thus, the two effects of ATP and NADPH depletion complement each other in thiε regard, and when the level of GSH falls substantially after chemical injury, cell death usually ensues (Boobis et al., TIPS 10:275-280 (1989)). The term "biochemical modulation" refers to the pharmacologic manipulation of metabolic pathways by an agent (the modulating agent) to produce the selective enhancement of the antitumor effect of a second agent (the "effector" agent) (Martin, Biochemical modulation — Perspectiveε and Objectiveε. In: New Avenueε in Developmental Cancer Chemotherapy. Ed. by Kenneth R. Harrap, London, England (1987)). In this context, the triple combination, PALA + MMPR + 6-AN, may be viewed as biochemical modulation employed to establish in tumor cells a wide array of biochemical changes — i.e., a primary diminution of ATP lowering of all of the nucleoside triphosphateε and NAD, inhibition of macrocmolecular synthesis, and εupreεεion of the flux through the pentose phosphate shunt and glycolytic pathways — thereby eεtabliεhing a level of disrupted metabolism in cancer cells that complements, and is complemented by the cascade of similar biochemical derangements induced by the apoptosiε effects of DNA-damaging anticancer agentε (e.g., FUra, cisplatinum, BCNU) (Barry et al., Biochem. Pharmacol. 40:2353-2362 (1990), Berger et al., Anti-Cancer Drug Design, 2:203-210 (1987)). Thiε modulation and complementation results in enhanced cancer cell deaths which reflect in improved tumor regresεion rates. SUBSTITUTESHEE Intracellular ATP haε been neglected as a primary target for cancer chemotherapy because ATP haε long been conεidered too important a source of energy for all cellε to warrant the expectation that primarily directed anti-ATP chemotherapy could be εelective for cancer cellε and εafe for host tissues. However, the subject application documents striking therapeutic activity, a safe therapeutic index, and an associated depletion of ATP levelε in the in-vivo treated tumors. The relative selectivity of the triple therapy for the tumor over host tissues is explained by the finding that many of the enzymes affected by the antimetabolites are in lower concentration in neoplaεtic than in normal tiεεueε (e.g., NAD-dependent enzymes; Dietrich et al., Cancer Res. 18:1272-1280 (1958), Martin et al., Cancer Res. 17:600-604 (1957), Glock et al., Biochem. J. 65:413-416 (1957), Jedkien et al. , J. Biol. Chem. 213:271-280 (1955) and Morton, Nature 18:540-542 (1958)). It is possible to inhibit more selectively enzymes present in cancer tissue in small amounts, while producing much less inactivation of the same enzyme in normal tissues containing larger amounts (Ackerman et al., Proc. Soc. Biol. Med. 72:1-9 (1949)). The therapeutic activity of a widely diverse group of anticancer agents has been markedly enhanced by this biochemical modulation approach using PALA + MMPR + 6-AN in the CD8F1 murine breast tumor model. The addition of 5-fluorouracil, adriamycin, taxol, radiotherapy, mitomycin C, cis-platinum, cytoxan, phenylalanine muεtard, and etopoεide with the PALA-MMPR-6-AN triple combination have demonstrated greater anticancer activity that that observed with the individual drugs alone at MTD, or with the PALA-MMPR-6-AN combination alone. SUBSTITUTESHEET Cellular Energy Depletion Compounds Plus FUra In one embodiment of the invention cellular energy depletion compounds are administered with 5-fluoroaracil aε the apoptosiε inducing agent. In an advantageous embodiment, the subject invention relates to a highly active chemotherapeutic drug combination comprising: •N-(phosphonacetyl)-L-aspartate (PALA), •6-methylmercaptopurine riboεide (MMPR), •6-aminonicotinamide (6-AN), and •5-fluorouracil (FUra). A quadruple drug combination of PALA + MMPR + 6-AN + FUra, administered in a 10-11 day schedule, produces an impressive partial tumor regression rate. The underlying molecular mechanism of fluoropyrimidine-induced "thymineless death" haε been shown to be due to "programmed cell death" (apoptosiε) activated by DNA εtrand breakage (Kyprianou et al., Biochem. Biophyε. Reε. Communications 165:73-81, (1989)). Measurements of biochemical changes in CD8F1 breast tumors after in vivo administration of the PALA-MMPR-6-AN drug combination reveal severe ATP loss, inhibition of macromolecular εyntheεiε, inhibition of the pentose phosphate shunt, NAD depletion, reduction of ribonucleoside triphoεphateε and inhibition of protein εynthesiε, a pattern of findingε which overlap with thoεe reported in "thymineleεε death" and apoptosis. In the subject invention, the addition of an apoptoεiε-inducing anticancer agent εuch aε FUra (Kyprianou et al., Biochem. Biophyε. Res. Communications 165:73-81, (1989), Barry et al., Biochem. Pharmacol. SUBSTITUTESHEET 20 40:2353-2362 (1990)), to the triple combination results in complementary therapeutic activity. PALA-MMPR-6-AN induces elevation of PRPP levels and reduction of UTP poolε. The triple drug combination alεo increaεeε the therapeutic activity of FUra by increasing the anabolism of FUra to its nucleotides, as well as by favoring the competition of the analog over the natural pyrimidine intermediates whose levelε have been reduced by PALA. The elevation of PRPP levelε and the lowering of UTP levels by the triple combination were expected to facilitate the conversion of FUra into its active nucleotides for effective blockade of key FUra-sensitive enzymes, and this waε indeed accompliεhed (Table 5). Thus, the addition of FUra, which at 75 mg/kg as a single agent causes few (<5%) regressions and only inhibits the growth of spontaneous CD8F1 breast tumors, markedly increased the tumor regression rate of the triple combination from 38% to 67% (Table 2). Three of these drugs (PALA, MMPR, and FUra) currently are used clinically as components of various drug combinations. A combination of PALA pluε FUra has proven to be significantly more active than FUra alone in the clinical treatment of colorectal cancer (Ardalan et al., J. Clin. Oncol. 6:1053-1058 (1988), 0'Dwyer et al., J. Clin. Oncol. 8:1497-1503 (1990)). MMPR can result in the elevation of PRPP levels in human tumors such as colon, ovary and breast (Peters et al., Cancer Chemother. Pharmacol. 13:136-138 (1984), 0'Dwyer et al., J. Natl. Cancer Inst. 83:1235-1240 (1991), Wiemann et al., Med. Oncol. & Pharmacother. 5(2):113-116 (1988)). The addition of MMPR augments the metabolic activation of FUra in human tumors. S Cellular Energy Depletion Compounds Plus Adriamycin In this embodiment of the invention, cellular energy depletion compounds are administered with adriamycin as the apoptoεiε inducing agent. Another advantageouε embodiment of the invention comprises: •N-(phosphonacetyl)-L-aspartate (PALA), •6-methylmercaptopurine riboside (MMPR), •6-aminonicotinamide (6-AN), and •Adriamycin (Adria). Thiε combination yieldε εignificantly enhanced anti-cancer activity over that produced by either Adria alone at maximum tolerated doεe (MTD), or by the triple drug combination, against large, spontaneous, autochthonous murine breast tumorε. The augmented therapeutic reεultε were obtained with approximately one-half the MTD of Adria aε a εingle agent, and provides the clinical benefit of longer and more effective treatment with increased safety. The combination of an ATP-depleting drug combination administered prior to Adria resulted in a 100% tumor regression rate (12% CR; 88% PR) in the population of treated spontaneouε tumorε indicates that the energy-depleting combination overcomes resiεtance mechanisms to adriamycin. Adriamycin haε been shown to induce death by apoptosiε in cancer cells in vitro (Marks et al., Biochem. Pharmacology 42:1859-1867 (1990)). The addition of Adria to the triple drug combination significantly enhances anti-cancer activity over that produced by either Adria alone at MTD, or by the triple drug combination, against advanced εpontaneouε, autochthonous murine breast tumors. The augmented therapeutic results are obtained with SUBSTITUTESHEET approximately one-half the MTD of Adria as a single agent. The therapeutic resultε of the most effective single agent against human breast cancer, Adriamycin, are greatly enhanced by the prior adminiεtration of the triple combination. These reεultε are obtained with essentially half the dose of the MTD of Adria aε a single agent. Adriamycin cannot be given to breast cancer patients for long periods because its cumulative dosage reaches life-threatening heart damage; therefore, the use of much lower doses of Adria, offer the benefit of longer and more effective treatment with increased safety. Further, the clinical development of Adriamycin resistance is frequently based on energy-dependent mechanisms (e.g., multiple drug resiεtance (mdr) expression: p-glycoprotein) in the cancer cells (Gerlach et al.. Cancer Surveys 5:26-46 (1986) and Versantvoort et al.. Cancer Res. 52:17-23 (1992)). Results with the triple drug combination are primarily based on depletion of cancer cell energy levelε. This ATP-depleting approach in combination with Adria reverses ATP-dependent mechanisms of resistance to Adria in vivo, as has been demonstrated in vitro (Gerlach et al., Cancer Surveys 5:26-46 (1986) and Dano, Biochim. Biophyε. Acta. 323:466 (1973)). The results obtained with the 4-drug combination in CD8F1 mice bearing spontaneous, autochthonous breast tumorε repreεent a significant therapeutic breakthrough in two reεpectε (εee Example 2). Firεtly, the therapeutic effect of treatment increased from a tumor regression rate of 66% after the first course of treatment to 93 and 100% after second and third courses of treatment, respectively. In contrast, although the first course of Adria alone at itε MTD produced a respectable 43% regression rate, the therapeutic value of this drug diminished with subsequent courses of treatment (i.e., 21% regressions after the second course and 16% after the SUBSTITUTESHEET 23 third course). Thiε decrease in chemotherapeutic activity on εucceεεive courses of treatment haε been observed in preclinical and clinical trials with Adria, and with most chemotherapeutic drugs. This phenomenon has usually been ascribed to the selection by the drug of cellε that were innately resistant to the specific drug, and preferential growth of this sub-population after the drug-sensitive cellε have been killed by expoεure to the drug. Subsequent drug exposures are confronted with relatively more resistant cells, and the resulting therapeutic observation iε drug reεiεtance. Aε shown in Example 2, the appearance of increased resistance to Adria alone was observable as early as the second course of treatment (on day 10). However, no operational resiεtance to the PALA - MMPR - 6-AN combination could be detected throughout the 3 courses of treatment regimen and 28 day observation period (Fig. 2). And, importantly, the administration of PALA - MMPR - 6-AN in conjunction with Adria prevented the manifestation of reεiεtance to Adria. In fact, the regression rate observed with the 4-drug treatment increased to 100% after the last course of treatment (Fig. 2). As mentioned above, the fact that the 4-drug regimen resulted in a 100% tumor regression rate in each of the three εucceεεive experiments (a total of 42 spontaneouε tumorε) was unprecedented in thiε spontaneouε murine breast tumor εystem. Spontaneouε tumorε are unlike common tranεplantable tumor modelε, which have been repeatedly tranεplanted for years, and where all of the tumorε in a particular experiment, although somewhat heterogeneous on the cellular level, are nevertheless quite similar from one individual host to another. In contrast, spontaneous tumors in these mice, like in humans, differ in drug sensitivity from one individual to another. For example, although on average approximately 20% of the population of εpontaneouε CD8F1 breast tumorε respond with partial tumor regression on expoεure to optimal treatment with FUra S UBSTI alone (Stolfi et al., J. Natl. Cancer Inεt. 80:52-55 (1988)), 80% of thiε population of tumors does not respond to this extent. In fact, if one measures individual tumorε during and after treatment with e.g. FUra, one sees an array of responεeε in individual tumorε varying from partial regreεεion to stasis to progression. However, despite this heterogeneity in drug εusceptibility among individual tumors, the 4-drug treatment produced regression in all of the tumors in the population. Although this treatment does not overcome heterogeneity in εuεceptibility completely, as evidenced by the failure to achieve 100% cure of theεe tumors, thiε is a new level of therapeutic activity. Further, the spontaneous, autochthonus CD8F1 breaεt tumor model haε demonstrated a remarkable 100% therapeutic correlation with human breast cancer in terms of both positive and negative senεitivity to individual chemotherapeutic drugs uεing tumor regression as the criterion for evaluation (Stolfi et al., J. Natl. Cancer Inst. 80:52-55 (1988). Cellular Energy Depletion Compounds Plus Taxol In thiε embodiment of the invention cellular energy depletion compoundε are administered with taxol aε the apoptoεiε inducing agent. An advantageous embodiment of the invention comprises; •N-(phosphonacetyl)-L-aspartate (PALA), •6-methylmercaptopurine riboεide (MMPR), •6-aminonicotinamide (6-AN), and •taxol. The above combination yields significantly enhanced anti-cancer activity over that produced by either taxol alone at MTD, or by the triple drug combination, against advanced, first passage spontaneous murine breast tumorε. The augmented therapeutic results are obtained with SUBSTI approximately one-third the MTD of taxol aε a εingle agent. The latter iε an important conεideration with taxol becauεe of the severe supply problem. Further, taxol reεiεtance iε frequently based on energy-dependent mechanisms, and therefore the combination of an ATP-depleting drug combination administered in conjunction with taxol results in the inhibition of energy-dependent resiεtance mechaniεmε to taxol. The new antimicrotubule agent, taxol, an antimitotic agent and the firεt compound with a taxane ring has demonstrated significant antineoplaεtic activity in patients with refractory ovarian cancer, refractory breast cancer, non-small cell lung cancer, and other cancers (Rowinsky et al., Pharmac. Ther. 52:35-84 (1991)). Its antitumor activity, novel mechanism of action, and unique structure have generated excitement. Taxol is a plant product (obtained from the bark of the Pacific Yew tree, Taxol brevifolia), and becauεe it is obtained from a limited resource there is a supply problem critical to its widespread clinical use. Unlike Adria, taxol is not conεidered a DNA-damaging agent. Taxol iε an antimitotic agent that bindε preferentially to microtubuleε in the abεence of the cofactorε tubulin and GTP, a mechaniεm unlike that of other antϊmicrotuble agentε in cancer chemotherapeuticε (e.g., vincriεtine, and colchicine). It blockε cellε in the mitotic phaεe of the cell cycle εo that these cells are unable to replicate normally, and cell death ensues (Rowinsky et al., Pharmac. Ther. 52:35-84 (1991)). Importantly, however, the taxol-induced disruption of the microtubular network of cancer cells induces death of cancer cells by apoptosis (Martin et al., Cell Tiβsue Kinet. 23:545-559 (1991), and Lennon et al., Cell Prolif. 24:203-214 (1991)), as Adria does (Marks et al., Biochem. Pharmacol. 42:1859-1867 (1990)). SUBSTITUTE SHEET Another similarity between taxol and Adria is that cellε resistant to both agents usually display the multi-drug reεiεtant (MDR) phenotype, p-glycoprotein, an energy-dependent drug efflux pump that maintains each of these agents below an intracellular cytotoxic level (Gerlach et al. , Cancer Surveys 5:26-46 (1986), and Endicott et al., Annu. Rev. Biochem. 58:137-171 (1989)). Theεe MDR cellε are croεε-reεiεtant to both Adria and Taxol (Rowinsky et al., Pharmac. Ther. 52:35-84 (1991)) and Gerlach et al., Cancer Surveys 5:26-46 (1986)), and, εince inhibitorε of energy production (e.g., azide) when added to such resiεtant cellε in vitro increaεe the net accumulation of drug in MDR-cellε (Gerlach et al., Cancer Surveys 5:26-46 (1986) and Dano, Biochim. Biophys. Acta. 323:466-483 (1973)), the ATP-depleting effects of the PALA-MMPR-6-AN triple combination reverses Adria-and taxol-resistant cells to chemosensitivity. Still another similarity is that both Adria,. and taxol (Holmes et al., Proc. Am. Soc. Clin. Oncol. 10:113 (1991) and Holmes et al., J. Natl. Cancer Inεt. 83:1797-1807 (1991)), are effective against human breast cancer. Since the CD8F1 murine breast tumor model has demonstrated a remarkable 100% therapeutic correlation with human breast cancer in terms of both positive and negative senεitivity to individual chemotherapeutic drugε uεing tumor regreεεion as the criterion for evaluation (Stolfi et al., J. Natl. Cancer Inst. 80:52-55 (1988)), it waε deemed likely that taxol, like Adria, also is effective as a single agent against the CD8F1 murine breast tumor. Taxol is similarly enhanced in therapeutic activity by the prior adminiεtration of the PALA-MMPR-6-AN triple combination. A markedly lower dose of taxol is necessary in the quadruple combination. Although taxol had been previously reported to be ineffective against the CD8F1 murine mammary carcinoma (Rowinsky et al., Pharmac. Ther. 52:35-84 (1991)), it was considered that the high degree of chemotherapeutic correlation that had been SUBSTITUTESHEET observed between thiε model and the human disease warranted re-assessment in the same CD8F1 εyεtem. Example 3 εhowε that taxol alone is active against the CD8F1 tumor, that the administration of the PALA-MMPR-6-AN triple drug combination prior to taxol significantly enhanced antitumor activity over that produced by either taxol alone at MTD, or the triple drug combination alone, and that only one-third the dose of taxol (in combination) is required for this greater antitumor activity. Importantly, the antitumor effects of taxol were significantly enhanced by the prior administration of the triple combination, PALA + MMPR + 6-AN, and these therapeutic results were obtained with one-third the MTD of taxol as a single agent (Table 2 and 3). The potential benefit, in addition to enhanced clinical responses with taxol in human breast cancer, is that the ability to employ lower effective doseε of taxol εhould alleviate the taxol εupply problem. An additional benefit of thiε particular drug combination iε that, εince acquired taxol reεiεtance involveε the mdr phenotype with p-glycoprotein aε an energy-dependent drug effux pump (Rowinsky et al., Pharmac. Ther. 52:35-84 (1991)), this ATP-depleting approach in combination with taxol overcomeε ATP-dependent mechanisms of resistance to taxol. Cellular Energy Depletion Compounds Plus Radiation In thiε embodiment of the invention, cellular energy depletion compounds are administered with radiation aε the apoptosiε inducing agent. The PALA-MMPR-6-AN combination εenεitizes tumorε to ionizing radiation therapy (εee Example IV). SUBSTITUTESHEET 28 Therapeutic Uses of the Compositions of the Invention The drug combinations of the subject invention are useful in treating a wide variety of neoplastic diseaseε. For a particular type of cancer, the apoptosis inducing agent of the combination of the invention (which comprises the cellular energy depletion compounds plus an apoptosiε inducing agent), iε selected on the basiε of demonεtrated antitumor activity when it iε used without the cellular energy depleting combination. The cellular energy depletion compounds (e.g. PALA-MMPR-6-AN) are primarily intended to sensitize tumorε to apoptoεis inducing agentε to which the tumors are already suεceptible to some degree. For example, fluorouracil (and therefore, the combination of the invention comprising fluorouracil and the cellular energy depletion compounds, e.g. PALA-MMPR-6-AN) iε useful for treating tumors of the colon, stomach, breast, head-and-neck, and pancreas. Taxol (and therefore the combination of ** taxol with the cellular energy depletion compounds) is useful for treating cancers of the ovary and breast. Adriamycin (and therefore the combination of adriamycin with the energy depletion compounds of the subject invention) is useful in a wide variety of tumorε: acute leukemiaε, malignant lymphomas, cancers of the ovary, breast, lung, bladder, thyroid, endometrium, testes, prostate, cervix, head-and-neck, and in osteogenic and soft tissue sarcomas (The Pharmacological Basiε of Therapeutics, Seventh Edition (1985), ed.A.G. Gilman, L.S. Goodman, T.W. Rail, and F. Murad, Macmillan Publishing Company, New York, Ny, pp. 1283-1285). Ionizing radiation (and therefore the combination of ionizing radiation with the cellular energy depletion compoundε of the invention) is useful for treating a variety of tumor types, including, lymphomas, and cancers of the breast, pelvis, and lung. SUBSTIT T Furthermore, since the energy depleting combinations of the subject invention render tumors more εenεitive to each apoptoεiε inducing agent, the range of tumorε εuεceptible to treatment with a given apoptosis inducing agent in combination with the cellular energy depleting combinations of the invention is broader than the range of tumor types treatable with the apoptosis inducing agent alone. A combination of two or more apoptosiε inducing agentε iε optionally administered in conjunction with an energy depleting compoεition of the invention. The cellular energy depleting combination of the invention iε also useful for overcoming multiple drug reεiεtance in patientε. "Multiple drug reεistance" (MDR) is a condition in which tumor cellε become insensitive to a variety of cytotoxic antineoplatic agents. MDR is typically due to the presence of an energy-requiring pump system which removes cytotoxic agentε including adriamycin, taxol, and vinca alkaloids from cells (or critical regions of cellε, such aε the nucleus) (Gervasoni et al., Cancer Research, 51:4955-4963 (1991)). In Example 2, in mice treated with adriamycin alone, the reεponεe to adriamycin decreaεeε over time during the three courεeε of drug treatment, indicating that the tumorε are developing reεiεtance to the drug. In contrast, in mice treated with adriamycin plus an energy depleting combination of the invention, the responεe rate actually improveε over time during the three courεes of treatment, indicating that the tumors are not developing reεistance to adriamycin, and in fact, an unprecedented 100% partial regresεion rate waε observed in these animals (Figure 2). Administration and Formulation of the Compositions of the Invention The compounds of the invention are administered in SUBSTITUTESHEET therapeutically effective amounts. The term "therapeutically effective amount" aε uεed herein refers to that amount which provideε therapeutic effectε for a given condition and adminiεtration regimen. The compoundε and compoεitionε of the invention are administered orally, by parenteral injection, intravenously, or topically, depending on the condition being treated. The timing and εequence of administration of the compounds and compoεitionε of the invention affect the efficacy of the treatment. Typically, an inhibitor of pyrimidine biosynthesis (e.g. PALA) iε administered 10 to 24 hourε prior to adminiεtration of an inhibitor of purine biosynthesis and a nicotinamide antagonist. An inhibitor of purine nucleotide biosynthesiε (e.g. MMPR) and a nicotinamide antagonist (e.g. 6-AN) are typically administered at approximately the same time. The apoptosiε inducing agent(ε) iε administered after the cellular energy depleting combination, typically about 2 to 3 hours afterwards (although the timing may be modified for particular drugs according to observed clinical benefit). The doses of the particular agentε are determined according to clinical reεponse, alterations in biochemical indices of efficacy, and observed signs of toxicity. PALA is typically administered in a dose of 250 mg/square meter; this dose has been found to be suitable in clinical εtudieε in which PALA waε administered as a modulator of fluorouracil. A typical single dose of 6-AN is 10 to 50 mg/square meter. The optimum dose range for 6-AN is determined by taking tissue biopsies before and after adminiεtration of increasing doεeε of 6-AN and determining activity of enzymes requiring pyridine 31 courεeε of treatment. A typical εingle doεe of MMPR iε 200 to 400 mg/square meter; a doεe of 225 mg/kg haε been εafely adminiεtered to humanε in combination with PALA. The compoundε and compoεitionε of the invention are formulated in pharmaceutically acceptable carrierε. The doses of the components of the energy-depleting combinations are chosen with the intention of providing significant sensitization of tumorε to apoptosis-inducing agents. If undue toxicity due to one or more of the energy-depleting compounds is encountered, subsequent doεeε or the interval between courεeε of treatment are modified. Alternatively, appropriate antidoteε are adminiεtered: Niacin or niacinamide ameliorate toxicity due to 6-AN (or other nicotinamide antagoniεtε; uridine, prodrugs of uridine, or other pyrimidine nucleotide precursors reverse the biochemical deficits produced by PALA or other inhibitors of pyrimidine nucleotide biosynthesis; and cellular purine nucleotide poois affected by MMPR (or other inhibitors of purine nucleotide biosynthesis) are replenished by administering appropriate purine nucleotide precursors, e.g., inosine, AICAR, adenosine, or hypoxanthine, with or without an inhibitor of purine degradation, such as allopurinol. Doεes of apoptoεiε-inducing agentε are chosen to optimize the therapeutic index. Since the energy-depleting combination εenεitizeε tumorε to apoptosis-inducing agentε, doses of apoptosiε-inducing agentε are typically less than or equal to the doses that would be adminiεtered in the abεence of the energy-depleting compoεition. Aε iε demonεtrated in Exampleε II and III, pretreatment with PALA-MMPR-6-AN permitε εubεtantial reductionε in the doεes of adriamycin and taxol required to produce optimum benefit. This iε very important, εince adriamycin produceε cumulative cardiotoxicity, limiting the total amount that a patient can εafely receive. Supplies of taxol are currently S UBSTIT 32 limited (εince it is obtained from the bark of the Pacific Yew, a relatively uncommon tree). A substantial reduction in the dose of taxol needed to produce an optimum clinical effect (as iε demonεtrated in Example III), permitε more patientε to be effectively treated with taxol (or other scarce but effective apoptosis-inducing agents). A course of treatment (PALA-MMPR-6-AN followed by an apoptoεis-inducing agent) iε repeated about every 10 dayε. Patientε typically receive three or more conεecutive courseε of treatment. The precise number of courses of treatment and the interval between is adjusted by the skilled person according to clinical determination of efficacy and toxicity. For parenteral administration by injection or intravenous infusion, the compounds and compositions of the invention are dissolved or suεpended in aqueous medium such as sterile physiological saline. In the case of poorly-soluble compounds (e.g. taxol), solubilizing agentε like ethanol, propylene glycol, or polyoxyethylated caεtor oil are used. The following examples are offered to more fully illustrate the invention, but are not to be construed as limiting the scope thereof. THE EXAMPLES Example I: PALA, MMPR, 6-AN and FUra Murine Breast Tumor System: Spontaneous, Autochthonous Mammary Carcinoma — CD8F1 hybrid mice bearing single εpontaneouε, autochthonous breast tumors arising during the preceding week were selected from a colony which has been described previously (Stolfi et al., SUBSTIT 33 Cancer Chemother. Rep. 55:239-251 (1971), Martin et al., Cancer Chemother. Rep., Part 2, 5:89-109 (1975)). All tumorε were meaεured with caliperε, and the mice were diεtributed among experimental groupε so that mice carrying tumors of approximately equal weight were represented in each treatment group. Individual tumorε ranged in εize from 100 to 500 mg, and the average tumor weight in all groupε was 260 mg at the beginning of treatment. Murine Breast Tumor System: First Passage CD8F1 Mammary Carcinoma — For each experiment, the spontaneously arising CD8F1 breast tumors were tranεplanted into syngeneic three-month old mice. As in all εpontaneouε tumorε, whether human or murine, each individual cancer haε a heterogeneouε neoplaεtic cell population. The firεt generation transplants of CD8F1 breast tumors are obtained from a tumor cell brei made by pooling 3-4 spontaneously-arising tumorε. Thus, the individual tranεplantε in each experiment develop from a εingle brei that, although common to all the mice in that experiment, haε a neoplastic cell composition that iε likely εlightly different from that in another experiment. Therefore, quantitative measurements of any individual parameter (e.g., TSase activity, or average tumor size) may be somewhat different from experiment to experiment, but the findings will be quantitatively relevant within individual experimentε, as will similar trends among experiments. The CD8F1 firεt generation breast tumor iε included in the murine tumor teεting panel of the National Cancer Drug Screening Program (Goldin et al., Eur. J. Cancer 17:129-142 (1981)). In approximately three to four weekε, when tranεplanted tumors were measurable, the tumor-bearing mice were diεtributed among experimental groups so that mice carrying tumorε of approximately equal weight were SUBSTITUTESHE represented in each treatment group. The average tumor weight was close to 125 mg at the beginning of treatment. Tumor Measurements — Two axeε of the tumor (the longeεt axis, L, and the shorteεt axiε, W) were meaεured with the aid of a Vernier caliper. Tumor weight waε estimated according to the formula: tumor weight (mg) - L (mm) x (W(MM) 2 )/2. Chemotherapeutic Agents — MMPR, 6-AN and FUra were obtained from Sigma Chemical Co., St. Louis, MO. Each of these agents was dissolved in 0.85% NaCl solution immediately before use. PALA was obtained from the Department of Health, Education, and Welfare, USPHS of the National Cancer Institute, Bethesda, MD. PALA was dissolved in 0.85% NaCl solution, and the pH was adjusted to 7.2 to 7.5 with IN NaOH before adjustment to final volume. All agents were administered i.p. so that the desired dose was contained in 0.1 ml/10 g of mouse body weight. These drugs were administered in a timed sequence, with PALA administered 17 hours before the simultaneous administration of MMPR + 6-AN, and with 5-FUra administered 2_ $ hours after MMPR + 6-AN. Throughout thiε application, the timing of biochemical measurements is given in relation to the injection of the last chemotherapy (i.e., MMPR + 6-AN) in the chemotherapeutic sequence. Determination of Chemotherapy-Induced Tumor Regression Rate — The initial size of each tumor in each treatment group was recorded prior to the initiation of treatment. Tumor εize was recorded weekly during treatment and again at 7-9 days after the last course of treatment. For each experiment a εingle observer made all measurements in order to avoid variation in caliper measurements from individual to individual. By SUBSTITUTESHEE 35 convention, partial tumor regression iε defined as a reduction in tumor volume of 50% or greater compared to the tumor volume at the time of initiation of treatment. The partial regression rate obtained from a particular treatment is expressed aε a percentage; i.e., number of partial regreεsionε per group/total number of animals per group x 100. Statistical Evaluation -- Differenceε in the number of partial tumor regreεsions between treatment groupε were compared for εtatiεtical significance by chi-square analysiε. Studentε t test was used for evaluation of measured biochemical differences between treatment groups. Differenceε between groups with p ≤ 0.05 were considered to be significant. Incorporation of Precursors into RNA and DNA — Radiolabeled precursorε, 32P and (3H)-L-Leucine were administered i.p.; the labeling period was 2 hrs; At the end of the labeling period, animals were sacrificed by cervical dislocation. Tumor tissues were homogenzied in TNE buffer containing 1% Triton-X 100 (TNE: 0.01 M Tris-HCl, pH 7.6; 0.15M NaCl; 0.001 M EDTA). The homogenate was .briefly sonicated, and then digeεted with Pronase for 60 min. at 37°C (0.2 mg/ml for 2 hr. at 37°C) and finally, this material waε extracted with chloroform/iεoamyl alcohol (24:1 vol/vol). Samples of extracted material were precipitated with trichloroacetic acid to determine total radioactivity. Other sampleε are firεt treated with alkali (0.4M NaOH, for 90 min at 37°C) to determine alkali-εtable, trichloroacetic acid-precipitable radioactivity. The difference between the total and alkali-stable radioactivity was assumed to 3 represent radioactivity in RNA. (5- H)Fluorouracil (20 Ci/mmol), (5- 3H), thymidine and (5-3H)deoxyuridine monophoεphate (20 Ci/mmol) were purchased from Moravek. SUBSTITUTESHEET Substrate Accumulations — Cellular levels of 6-phoεphogluconate, glucoεe-6-phosphate and fructoεe-6-phosphate were measured on perchloric acid extracts by publiεhed methodε (6-phosphogluconate (Haid, Methods of Enzymatic Analysis (Bergmeyer, H.U., ed). New York: Academic Preεε pp. 1248-1250 (1974)); glucoεe-6-phoεphate and fructose-6-phoεphate (Lang et al., Methodε of enzymatic analyεiε, Vol. 2 (ed. H.U. Bergmeyer)). 14 C-Orotic Acid Assay for PRPP(5-phosphoribosvl pyrophosphate) — The assay is based on the conversion of 14 C-orotic acid to uridine monophosphate with release of 14 CO, by orotidine-5-phosphate- pyrophoεphorylase + orotidine-5-phoεphate decarboxylaεe (Houghton et al., Mol. Pharmacol. 22:771-778 (1982)). An aliquot of the homogenate waε aεsayed for protein content by the method of Lowry et al., J. Biol. Chem. 193:265-275 (1951). Processing of Samples for NTP Content — Frozen tumor specimens were homogenized in ice-cold 1.2 N perchloric acid. The acid-insoluble fraction was removed by centrifugation (7000 rpm for 15 min). The acid-soluble fraction aε neutralized by extraction with a mixture of freon and tri-N-octylamine (2:1). The extract waε then filtered through a 0.22 u Millipore membrane filter prior to HPLC analysis. NTP contents in tumor were normalized to the protein content of the acid-insoluble fraction. Measurements of NTP Levelε in Tumor — HPLC analyεiε in tumor waε performed on a Waterε 840 HPLC system with a WISP automatic sampler. NTP levels were analyzed by ion-exchange gradient chromatography using a Waters SAX column starting with 3 mM NH-H^PO., pH 3.5, proceeding in two εtepε to 70% 0.5 M NH 4 H 2 0 4 , pH 5.0, plus 30% starting buffer. The run time for each 100 ul of extracted sample was 60 min. Tumor NTP levels are expressed as microgramε nucleotide triphoεphate per milligram of protein. 31P NMR (Nuclear Magnetic Reeonance) Spectra — 31P NMR εpectra were obtained uεing techniques described previously (Koutcher et al., Cancer Reε. 50:7252-7256 (1990)). Briefly, εpectra were obtained on a General Electric NT-300 wide bore spectrometer operating at 121.5 MHz. Experimental parameters included a spectral width of +/- 12,000 Hz, 60° tip angle, recycle delay of 2 seconds, 512-1024 averaged free induction decays (FID'ε), and 1024 data pointε. The εpectra are partially εaturated uεing theεe experimental conditionε. Four turn solenoid coils with a Faraday εhield (Ng et al., J. Magn. Reson. 49:526 (1982)), positioned between the body of the mouse and the coil were used to detect the NMR signal. Control experiments verified that no signal was obtained from non-tumor bearing animalε mounted in an identical manner. Spectra were analyzed uεing 25 Hz exponential multiplication followed by Fourier tranεformation. Spectral peak areaε were eεtimated by fitting the spectra to a series of Lorentzian peaks, using a program (GEMCAP) available on the spectrometer, after fitting the baseline to a third order polynomial (using standard General Electric software). Since other peaks overlap the alpha- and gamma-nucleotide triphosphate (NTP) peaks, the beta-NTP waε used for calculating NTP peak area ratioε. Thvmidine kinase and Thymidylate svnthaεe assays — Tisεueε were homogenzied (Potter-Elveh em homogenizer) aε a 20% (wt/vol) solution in Tris-Cl (100 mM, pH 7.6), 2-mercaptoethanol (20 mM) and sodium fluoride (100 mM) . Homogenateε were centrifuged at 4 (100,000 x g, 60 min. or 10,000 x g, 30 min) and the εupernatant fractions retained on ice. Enzyme asεayε were performed either on individual εampleε or on pooled tiεsues from three animals. Tumorε were between 300-500 mg. Thymidine kinase was measured immediately after cytosol preparation by means of a DE81-filter-binding assay (Ives et al.. Anal. Biochem. 28:192-205 (1969)). The aεεay mixture contained Tris-Cl (100 mM, pH 7.6), ATP (5 mM), MgCl 2 and (5-C 3 H 3 )thymidine SUBSTITUTESHEET (25 uM, 1.0 Ci/mmol) and cytoεolic protein. Thymidylate εynthase activity waε measured by the release of tritium from (5- 3 H)dUMP (10 uM, 1.0 Ci/mmol), CH 2 H 4 PteGlu (100 uM) and cytosolic fraction (25 uL) (Roberts, Biochemiεtry 5:3546-3548 (1966)). Reactionε were terminated by the addition of perchloric acid (10 uL, 0.7M). Protein waε determined by the method of Lowry et al., J. Biol. chem. 193:265-275 (1951). Incorporation of FUra into RNA — Incorporation of FUra into RNA waε determined by isolating tumor RNA by the acid-guanidine isothiocyanate procedure (Chomczynski et al., Anal. Biochem. 162:156-159 (1987)), after treatment 3 with (5- H)FUra (2.0 mCi/mmol). Tisεues were harvested 4-5 hr after treatment, immediately frozen in liquid nitrogen and stored at -70. Tissues were homogenized (80 mg/ml) in the denaturing solution (citrate-sarcosyl laurate-2 mercaptoethanol) and extracted with one volume of phenol:chloroform:isoamyl alcohol (50:40:2). .After extraction the RNA was precipitated with alcohol, washed, redisεolved and quantitated by uv, and then precipitated with perchloric acid. This final precipitate was collected on GF-C filters and the radioactivity waε determined. Chemotherapeutic Effectε on Breast Tumor Transplants In a series of 5 similar experiments, a group (Group 1) of CD8F1 mice bearing advanced, firεt passage spontenaouε CD8F1 breaεt tumor tranεplants were treated with the triple combination of PALA + MMPR + 6-AN. PALA (100 mg/kg) was administered 17 hours before MMPR (150 mg/kg) plus 6-AN (10 mg/kg) (Table 1). A second group (Group 2) received the same treatment with PALA + MMPR + 6-AN, followed 2% hours later by FUra (75 mg/kg). Treatment was repeated at 10 or 11 day intervals for a total of 3 courses, and observations were recorded 7 days after the last course of treatment. SUBSTITUTE Table 1 Therapeutic Comparison of PλLλ + MMPR + 6-AN chemotherapy Hith and Without the Addition of 5-Fluorouracil (FUra) in CD8F1 Mice Bearing First Passage Syngeneic Breast Tumors* Percent Body Partial Treatment Height C ang DeadTotal Regress jgnt CO 1. PA A 100 -17 hr->MMPR 1M + 6-AN, 0 -16 1/49 (21) 8 (171) 5 2. PALA 100 -17 hr->MMPR 'I l SO ft + 6-AN '1 ι 0 n -2 hr->FUra 75 -22 0/50 (01) 37 (741) H 3. FUra ?5 0/88 Pooled data: Experiments 2055F, 2076M; 2091F, 2102F, 2113F. First passage CDBPl breast tumors averaging 125 mgs at the time of initiation of treatment. Three courses of the indicated treatment were administered with a 10 or 11 day interval between courses. Subscripts refer to doses in mg/kg. Observations were recorded 7 days after the third course of treatment. Partial tumor regreεsionε were obεerved in 8 of the εurviving 48 mice (17%), treated with PALA + MMPR + 6-AN (Group 1). The range of regression rate in individual experimentε varied from 0 to 30%. The addition of FUra to the εame regimen of PALA + MMPR + 6-AN (Group 2) produced a significant and meaningful increase in therapeutic activity. Thirty-seven of the 50 treated mice (i.e., 74%) experienced partial tumor regressionε (with a range in the individual experimentε of 60-90%). Thiε level of therapeutic activity waε found to be εignificantly better than that achieved with the three-drug combination (without FUra, Group 1, p < 0.001), and most importantly, thiε increase in antitumor activity was achieved without mortality. It εhould be noted that FUra alone at 75 mg/kg did not produce any tumor regreεsionε after 3 courses of treatment in nine separate experiments (0/88 mice, not shown). In 3 separate experiments the therapeutic activity of the PALA + MMPR + 6-AN + FUra combination was compared with that of the combination of PALA + MMPR + FUra (i.e., without 6-AN) at the same doses and adminiεtration schedule. One week after three courses of treatment the four-drug combination yielded a 71% tumor regresεion rate (20 tumor regresεionε in 28 εurviving mice) whereas the three-drug combination without 6-AN yielded a regresεion rate of only 41% (12 tumor regreεεionε in 29 εurviving mice). The difference in regreεsion rate between these two treatments was found to be statiεtically εignificant, p < 0.05, indicating that the low-doεe 6-AN was contributing to the therapeutic activity in the four-drug combination. Chemotherapeutic Effects on Spontaneous, Autochthonouε Breaεt Tumors The PALA + MMPR + 6-AN regimen, with or without FUra, was administered at 10-11 day intervals in a series of six experiments to large numbers (a total of 66 or 67 mice in each treatment arm) of CD8F1 mice bearing advanced, spontaneouε, autochthonouε breaεt tumorε averaging 260 mgε. Reεults were observed at 6 weekε after the initiation of treatment (i.e., 9 days after the fourth course of treatment) (Table 2). SUBSTITUTESHEET Table 2 Therapeutic Comparison of PALA + MMPR + 6-AN Chemotherapy With and Without the Addition of 5-Fluorouracil (FUra) in Mice Bearing Spontaneous, Autochthonous CD8F1 Breast Tumors* Percent Body Partial CO Treatment' Heig t Change ead/TotaI RgrooB gno c CD 1. PALA |00 -17hr->MMPn 150 + 6-AN 10 -10 3/67 (41) 24 (38%) 3 2. PALA, 00 -17hr->MMPR |50 + 6-AN 10 -2 ->FUra„ -10 5/66 (7t) 41 (67%) Pooled data: Experiments R536; R537; R53Θ; R539 R540; R541. CO Spontaneous, autochthonous CDBPl breast tumors averaging 260 mgs at the time of Initiation m of treatment. The indicated treatment was administered at 10-11 day Intervals. Subscripts refer to doses in mg/kg. Observations were recorded 6 weeks after initiation of treatment (i.e., approximately 9 days after the fourth course of treatment). The PALA + MMPR + 6-AN regimen (group 1) produced a partial tumor regression rate of 38% in mice bearing spontaneouε, autochthonouε breaεt tumorε (24 partial tumor regreεεionε in 64 surviving mice) with an acceptable mortality rate (4%, 3 deaths in 67 treated mice). The addition of FUra to the three-drug regimen (Group 2) resulted in 41 partial regressions in the 61 surviving mice, or 67% (with a range of 50-90% in the individual experiments) without an increase in the mortality rate (only 7%), and with only a 10% body weight loss. Aε in the previous experimentε in mice bearing firεt passage tumors, the addition of FUra to the PALA, MMPR, 6-AN regimen resulted in a εignificant increaεe in tumor regreεεionε (p < 0.01) in mice bearing εpontaneous, autochthonous breaεt tumorε. Again, it should be noted that FUra alone at 75 mg/kg produced < 5% regressionε of εpontaneouε, autochthonouε CD8F1 breaεt tumorε (1 regreεεion in 24 treated tumorε), and spontaneous regressions of these tumors haε not been observed. BIOCHEMICAL FINDINGS Macromolecular Synthesis —■ Administration of PALA-MMPR-6-AN resulted in significant inhibition of macromolecular εyntheεiε in firεt paεsage CD8F1 breaεt tumorε detectable at the earlieεt time point examined, 2.5 hourε after drug adminiεtration, and progreεsing to 80% inhibition of DNA syntheεiε, 85% inhibition of RNA εyntheεiε and 70% inhibition of protein εyntheεis at 48 hourε. Pentoεe Shunt and Glycolvtic Intermediates 6-AN has been reported to inhibit the pentose phosphate pathway by producing a metabolic block on 6-phosphogluconate (6-PG) dehydrogenaεe (Herken et al., Biochem. Biophyε. Reε. Comm. 36:93-100 (1969)). The SUBSTITUTESHEET accumulation of thiε substrate resultε in feed-back inhibition of phosphoglucoεe isomerase and preventε the formation of fructoεe-6-phoεphate from glucoεe-6-phosphate (G-6-P) (Racker, In: Mechaniεmε in Bioenergeticε. Academic Preεε, New York-London, p. 207 (1965) , Horecker, B.C. In: Carbohydrate Metabolism and Itε Disorders. Vol. I. Academic Press, New York-London (1968)). Aε iε evident in Table 3, adminiεtration of 6-AN did, in fact, result in significant elevation of 6-PG (167-fold increase above εaline-treated control), and G-6-P (3-fold increase above control) in CD8F1 breast tumors. Similar resultε were obtained in tumorε from mice treated with the 6-AN-containing 3-drug combination (PALA-MMPR-6-AN). The 3-drug combination reduces NAD levels in this tumor (Martin, Metabolism and Action of Anti-cancer drugε. Ed. by Garth Powiε and Russell A. Prough (London: Taylor & Francis), pp. 91-140 (1987)). SUBSTITUTESHEET Table 3 Effects of PALA 100 —17 hrs—> MMPR 150 + 6-AN 10 On Substrate Concentrations (umol/g frozen weight) Of Pentose Shunt and Glycolytic Intermediates 46 Estimation of Cellular Energy Levels by NMR (See Fig. 1) — NMR spectra were obtained from firεt passage CD8F1 breast tumorε prior to treatment, and at 2, 10, and 24 hours post treatment with PALA-MMPR-6-AN. Baseline spectra were similar to those obtained in previous studies ( outcher et al., Magnetic Resonance in Medicine 19:113-123 (1991)). Post-treatment, a decrease in the B-NTP peak relative to inorganic phosphate was noted. The results obtained from 7 tumor-bearing animals treated in thiε manner are summarized in Fig. 1 which shows that both phoεphocreatine/inorganic (PCr/Pi) and NTP (nucleoside triphosphate)/Pi ratios were decreased after treatment with this drug combination, which iε indicative of energy depletion. The changeε in PCr/Pi and NTP/Pi at 10 hours were found to be εtatiεtically εignificant. Biochemical Meaeurement of ATP Levels Energy depletion in drug-treated tumors also was manifeεt in decreased ATP pools as shown in Table 4. At 6 and 24 hours after adminiεtration of MMPR alone (Group 2), 6-AN alone (Group 3), or the 3-drug combination, PALA-MMPR-6-AN (Group 4), ATP levelε in first passage CD8F1 breaεt tumorε were εignificantly depreεsed, reaching a level of 32% of control in tumorε from mice treated with the 3-drug combination at 24 hours post treatment. Table 4 Effect of PAIA 100 17 hr MMPR 150 + «-AN 10 on Tumor ATP Pools* in CD8F1 Female Mice (ug/mg protein ± S.E.) ua AT /mo protein f • _» £Z__2_2S Treatment i hr 24 hr 1 Saline 7.1 7.1 ± 0.32 ± 0.32 MMPR ISO 3.3* (47%) 3.3* (47%) ± 0.32 ± 0.96 6-AN 10 5.5 * (77%) 5.3 * (75%) ± 0.21 ± 0.13 2%) Mean ± S.E. of 10 tuaors/group (11 separate experiments) at indicated times after last chemotherapy. Subscripts - mg/kg body weight; i.p. injection. Significant - p value lass than or equal to 0.05; ( ) ■» % of saline control (Group 1) . SUBSTITUTE SHEET Biochemical Changes Favoring the Activation and Competitive Activity of FUra — As a consequence of the block of de novo purine synthesis, the administration of MMPR has been shown to result in the accumulation of PRPP in CD8F1 tumor cellε, and when MMPR iε adminiεtered appropriately before FUra, the increaεed levelε of PRPP reεult in enhanced activation of FUra (Martin et al., Nucleoεideε and Cancer Treatment (M.H.N. Tatterεal and R.M. Fox, edε.), Academic Press, Australia, pp. 339-392 (1981)). Because of the possibility of complex interactions among drugε in combination, it waε necessary to verify that the MMPR-containing 3-drug combination also was capable of producing this elevation of PRPP in tumor cells. Accordingly, PRPP levelε were measured in untreated firεt passage CD8F1 tumor, and in CD8F1 tumorε at 3 and 24 hours after treatment with PALA-MMPR-6-AN. Measurements from 4 untreated tumors in each of 3 separate experiments yielded an average PRPP level of 288 pmol/mg with a standard error of 19 pM/mg. PRPP levels rose to 490 ± 63 pmol/mg (i.e., 2.2-fold increase, p < 0.01) and 833 ± 153 pmol/mg (i.e., 3.7-fold increase, p < 0.05) at 3 and 24 hours, respectively, after adminiεtration of PALA-MMPR-6-AN. Aε a conεequence of the inhibition of aεpartate transcarbamylase, the administration of PALA alone has been demonstrated to result in depletion of UTP pools in CD8F1 tumors, and when administered appropriately before FUra, this reεultε in an augmentation of the activity of the competitive FUTP analog (Martin et al., Cancer Reε. 43:2317-2321 (1983)). Again, UTP levelε were measured in firεt passage CD8F1 breast tumorε at 24 hours after administration of PALA-MMPR-6-AN and found statistically significant depression of UTP pools compared to saline-treated control tumorε (data not shown). Therefore, two of the drugs (MMPR and PALA).in the 3-drug combination produce the same biochemical alterations in CD8F1 breaεt tumor cellε when adminiεtered in the 3-drug combination as they did when adminiεtered aε εingle agentε, and theεe alterations have been demonstrated to result in the augmentation of subsequently adminiεtered FUra. Biochemical Measurements of FUra Activity when Administered Two- and One-half Houre after PALA-MMPR-6-AN Since the inhibition of RNA synthesis is one of the earliest measurable eventε after the adminiεtration of the triple combination, the effect of the combination upon the incorporation of FUra into RNA iε of interest. The amount of tumor (FU)RNA in the group which received PALA + MMPR + 6-AN + FUra waε appreciable (355 ± 127 cpm/mg RNA). Thuε, although RNA εyntheεiε waε εignificantly inhibited following treatment with PALA-MMPR-6-AN, the incorporation of FUra into reεidual newly εyntheεized RNA waε not inhibited. Three groupε of 5 CD8F1 mice bearing equi-sized firεt passage breaεt tumorε were treated with saline, PALA-MMPR-6-AN, or PALA-MMPR-6-AN followed 2.5 hours later with FUra. Measurementε of thymidine kinase and thymidylate synthase activity 24 hours after treatment are shown in Table 5. SUBSTITUTESHEET Table 5 Tumor Thymidine Kinase and Thymidylate Synthase Activities 24 Hours After* Treatment % of Control Activity TKase TSa β e Treatment 100. 0 0 1 22.30 100.00 ± 18.31 1. saline DJ 2. PALA 100 17 hr MMPR 150 + 6-AN 10 19.63 ± 4.28 53.40 ± 13.61 S. 3. PALA 100 I? hr MMPR 150 + 6-AN 10 2 .5 r.. FU ?5 6.26 ± 1.61 23.60 ± 3.07 sQ *Tumors harvested 24 hours after MMPR + 6-AN treatment; M = 5. πϊ *avg. control activity - 46.0 pmol/min/mg protein rπ **avg. control activity » 12.71 pmol/min/mg protein Thymidylate synthaεe activity was decreased by nearly 50% after treatment with PALA-MMPR-6-AN, even without the addition of FUra (Group 2, Table 5). This depression of enzyme activity is likely due to the general inhibition of protein syntheεiε following treatment with the 3-drug combination deεcribed above. However, the addition of FUra 2.5 hourε following treatment with the 3-drug combination (Group 3, Table 5) reεulted in more than 76% inhibition of thiε enzyme. This increased level of inhibition is believed to be a result of FdUMP inhibition of residual thymidylate εynthaεe activity. Nord et al., Biochem. Pharmacol. 42:2369-2375 (1991), reported that thymidine kinaεe activity iε lost over 24 hour in CD8F1 tumorε from mice treated with the maximum tolerated dose (100 mg/kg) of FUra alone. The triple combination (Group 2, Table 5) produced approximately 80% inhibition of TKase (apparently due to inhibition of RNA εynthesis), while the addition of FUra (75 mg/kg) to the triple combination (Group 3, Table 5) provided further inhibition (93.7%) of TKaεe activity. Therapeutic activity haε been measured in these εtudieε uεing stringent clinical criteria of tumor regreεεion (i.e., 50% or greater drug-induced decrease in tumor size), rather than the more conventional animal model criteria of tumor growth inhibition. It εhould also be noted that the εpontaneouε, autochthonouε CD8F1 breaεt tumor model haε demonstrated a remarkable correlation with human breast cancer in termε of both positive and negative eenεitivity to individual chemotherapeutic drugε uεing tumor regreεεion aε the criterion for evaluation (Stolfi et al., J. Natl. Cancer Inεt. 80:52-55 (1988)). Thiε example εhowε an impresεive increaεe in tumor regreεεion rates when FUra waε adminiεtered in conjunction with PALA, MMPR and 6-AN in the therapy of either advanced firεt passage, or spontaneouε, murine breast tumorε, and SUBSTITUTESHEET shows the results of measurements of biochemical parameters affected by treatment. Example 2: PALA, MMPR, 6-AN and Adriamycin Murine Breast Tumor Svεtem Spontaneous, Autochthonouε Mammary Carcinoma CD8F1 hybrid mice bearing εingle εpontaneouε, autochthonouε breast tumors arising during the preceeding week were selected from a colony which has been described previously (Stolfi et al., Cancer Chemother. Rep. 55:239-251 (1971) and Martin et al., Cancer Chemother. Rep., Part 2, 5:89-109 (1975)), and iε included in the murine tumor teεting panel of the National Cancer Drug Screening Program (Goldin et al., Eur. J. Cancer 17:129-142 (1981)). All tumorε were measured with caliperε, and the mice were diεtributed among experimental groupε so that mice carrying tumors of approximately equal weight were represented in each treatment group. Individual tumors ranged in εize from 100 to 500 mg, and the average tumor weight in all groupε waε 304 mg at the beginning of treatment. Aε in all εpontaneous tumorε, whether human or murine, each individual cancer has a heterogeneous cell population and therefore, unlike long-transplanted tumor lines, one εpontaneous tumor may differ from another of the same histiotype in εuεceptibility to a given drug treatment. Tumor Measurement Two axeε of the tumor (the longeεt axis, L and the shorteεt axis, ) were meaεured with the aid of a Vernier caliper. Tumor weight waε estimated according to the 2 formula: tumor weight (mg) = L (mm) x (W(mm) )/2. Chemotherapeutic Agents SUBSTITUTESHEET MMPR and 6-AN were obtained from Sigma Chemical Co., St. Louis, MO. Adria waε obtained from Adria Laboratorieε, Columbuε, Ohio. Each of these agentε waε diεεolved in 0.85% NaCl εolution immediately before use. PALA was obtained from the Department of Health, Education, and Welfare, USPHS of the National Cancer Institute, Bethesda, MD. PALA waε diεεolved in 0.85% NaCl solution, and the pH waε adjuεted to 7.2 to 7.5 with IN NaOH before ad uεtment to final volume. All agentε were adminiεtered εo that the deεired dose was contained in 0.1 ml/10 g of mouse body weight. Adria waε adminiεtered i.v. and all other agentε were adminiεtered i.p. Theεe drugε were adminiεtered in a timed sequence, with PALA administered 17 hourε before MMPR + 6-AN, and Adria adminiεtered 2 1/2 hourε after MMPR + 6-AN. Determination of Chemotherapy-Induced Tumor Reqreεεion Rate The initial location and εize of each tumor in each treatment group waε recorded prior to the initiation of treatment. Tumor εize waε recorded weekly during treatment and again at 7 dayε after the laεt courεe of treatment. For each experiment a εingle obεerver made all meaεurementε in order to avoid variation in caliper meaεurementε from individual to individual. By convention, partial tumor regression is defined aε a reduction in tumor volume of 50% or greater compared to the tumor volume at the time of initiation of treatment. The partial regreεεion rate obtained from a particular treatment iε expressed aε a percentage; i.e., Number of partial regreεεionε per group/Total number of animalε per group x 100. Complete tumor regreεεion was defined aε the inability to detect tumor by palpation at the initial site of tumor appearance. Statistical Evaluation SUBSTITUTESHEET Differences in the number of partial tumor regresεionε between treatment groupε were compared for statistical significance by chi-square analyεiε. Differenceε between groupε with p - 0.05, or lesε, were considered significant. Chemotherapeutic Effectε of the Triple Drug Combination and Adria, Alone and in Quadruple Combination (PALA+MMPR+6-AN+Adria) on Spontaneous, Autochthonous CD Q F- Breast Tumors The PALA + MMPR + 6-AN regimen, with (Group 2) or without (Group 1) Adria, was administered at 10-11 day intervals in a series of three separate experiments to a total of 44 CD8F1 mice bearing advanced, spontaneouε, autochthonouε breaεt tumorε averaging 304 mgs in each group. In each of these experimentε, a comparable group of mice was treated with Adria alone at 11 mg/kg which had been determined previously to be the MTD of Adria alone when administered in thiε 10-11 day treatment schedule. Reεultε were observed 7 dayε after the third course of treatment. SUBSTITUT Table 6 Therapeutic Comparison of PALA + MMPR + 6-AN Chemotherapy With and Without The Addition of Adriamycin (Adria) in CD8F1 Mice Bearing Spontaneous, Autochthonous Breast Cancers* Partial Toner ■ Pooled data: Experiments R559, R560, R561. Tumor weight averaged 304 mgs at the ttøe of initiation of treatment. b Three courses of the indicated treatment were administered with a 10 or 11 day interval between courses. Subscripts refer to doses in mg/kg. Adria was administered i.v. and all other drugs were administered i.p. Observations were recorded 7 days after the third course of treatment. β Adria at 11 eg/kg iβ the MTD of Adria alone in a 10-11 day treatment schedule. The PALA + MMPR + 6-AN regimen (Group 1) produced a partial tumor regression rate of 76% in mice bearing spontaneouε, autochthonouε breaεt tumorε (32 partial tumor regreεεionε in 42 surviving mice with a range of 50-92% in the individual experimentε) with no toxic deaths in the treated mice. The addition of Adria to the three-drug regimen (Group 2) resulted in 42 partial tumor regreεsionε in the 42 surviving mice, or 100% (i.e., with no range in the individual experimentε), without a εignificant increaεe in the mortality rate (only 5%), and with only a 16% body weight loεs. The addition of Adria to the PALA, MMPR, 6-AN regimen was found to result in a statistically εignificant increase in tumor regreεεionε (p < 0.01) in mice bearing εpontaneouε, autochthonouε breast tumorε. Moreover, 5 of the 42 tumor regreεεionε in Group 2 were complete aε opposed to partial regressions, and it should be noted that complete regressions were not observed in the other two groupε. It εhould be noted that Adria alone at 11 mg/kg produced only 16% regressionε in 42 treated tumors, and spontaneous regreεεions of these tumorε haε not been observed. A critically important feature of the results of these experimentε in mice bearing εpontaneous, autochthonouε breaεt tumorε can be εeen in Figure 1 where the percent of tumorε in regreεεion in each of the treatment groupε iε plotted at 7 dayε after each of the 3 courses of treatment. Note the diminishing therapeutic effect in mice treated with Adria alone at it's MTD of 11 mg/kg every 10-11 days. After the first course, 43% of the tumors had regressed to 50% or less of their initial size. However, 7 days after the second course, only 21% were partially regresεed, and 7 dayε after the third courεe, only 16% were still in partial regression. In contrast, in mice treated with PALA + MMPR + 6-AN followed by Adria at 6 mg/kg, the regresεion rate was 66% after the first course, and then it increased to 93% after the SUBSTITUTESHE εecond courεe and to 100% after the third course. - Note that none of the tumors in the 42 mice εurviving three courses of treatment escaped from the regression-inducing activity of the quadruple-drug regimen. This is an unprecedented response rate in this εpontaneouε, and highly heterogeneous tumor model, and because of the high degree of chemotherapeutic correlation that has been observed between thiε model and the human disease we believe that thiε 4-drug regimen may be a therapeutic breakthrough. Example 3: PALA, MMPR, 6-AN and Taxol Murine Breaεt Tumor System CD8F1 hybrid mice bearing εingle εpontaneouε, autochthonouε breaεt tumors arising during the preceding week were selected from a colony (Stolfi et al., Cancer Chemother. Rep. 55:239-251 (1971) and Martin et al., Cancer Chemother. Rep., Part 2, 5:89-109 (1975)). For each experiment, a tumor cell brei, prepared by pooling 3-4 εpontaneouεly ariεing CD8F1 breaεt tumorε, waε tranεplanted into εyngeneic three-month old mice. In approximately three to four weekε, when transplanted tumorε were measurable, the tumor-bearing mice were diεtributed among experimental groupε εo that mice carrying tumorε of approximately equal weight were repreεented in each treatment group. Therapy waε begun when the tumors were advanced and relatively large; the average tumor weight was close to 130 mg at the beginning of treatment. As in all εpontaneouε tumorε, whether human or murine, each individual cancer haε a heterogeneouε cell population. The firεt generation transplants of CD8F1 breaεt tumors are obtained from a tumor cell brei made by pooling 3-4 spontaneously arising tumors. Thus, the individual transplantε in each experiment develop from a single brei that, although common to all the mice in that experimentε, has a neoplastic cell composition that iε likely slightly different from that in another experiment. Therefore, quantitative measurement of any individual parameter (e.g., average tumor size) maybe somewhat different from experiment to experiment, but the findings will be quantitatively relevant within individual experimentε, aε will εimilar trends among experimentε. The CD8F1 firεt generation breaεt tumor iε included in the murine tumor teεting panel of the National Cancer Drug Screening Program (Goldin et al. , Eur. J. Cancer 17:129-142 (1981)). Tumor Measurement Two axes of the tumor (the longeεt axiε, L and the εhorteεt axiε, W) were meaεured with the aid of a Vernier caliper. Tumor weight was estimated according to the 2 formula: tumor weight (mg) = L (mm) x (W(mm) )/2.. Chemotherapeutic Agents MMPR and 6-AN were obtained from Sigma Chemical Co. , St. Louiε, MO. Each of theεe agentε waε diεsolved in 0.85% NaCl solution immediately before use. PALA and taxol were obtained from the Department of Health, Education, and Welfare, USPHS of the National Cancer Institute, Bethesda, MD. PALA was dissolved in 0.85% NaCl solution, and the pH waε adjuεted to 7.2 to 7.5 with IN NaOH before adjuεtment to final volume. Taxol waε received already εoluabilizxed in polyoxyethylated castor oil and dehydrated alcohol. Because of the known toxicity of thiε diluent, the Taxol εtock waε diluted, depending upon the dose to be administered, a minimum of 6-fold in saline before injection. For doses below 10 mg/kg, taxol was administered in 0.1 ml/10 g of bodyweight. For doses above 10 mg/kg, an appropriate additional volume was administered. All other agents were administered so that the desired doεe was contained in 0.1 ml/10 g of mouse body weight, with the exception of taxol. SUBSTITUTE These drugs were adminiεtered in a timed sequence, with PALA adminiεtered 17 hourε before MMPR + 6-AN, and taxol adminiεtered 2 1/2 hourε after MMPR + 6-AN. In one experiment (Exp. 2536, Table 6), Taxol was administered in a fractionated schedule aε follows: Taxol (4 mg/kg) simultaneously with MMPR + 6-AN then, 1 1/2 hours later, Taxol (4 mg/kg) q 3 hours x 7. Determination of Chemotherapy-Induced Tumor Regreεεion Rate The initial εize of each tumor in each treatment group waε recorded prior to the initiation of treatment. Tumor εize waε recorded weekly during treatment and again at 7 dayε after the laεt courεe of treatment. For each experiment a εingle observer made all measurementε in order to avoid variation in caliper meaεurements from individual to individual. By convention, partial tumor regression is defined as a reduction in tumor volume of 50% or greater compared to the tumor volume at the time of initiation of treatment. The partial regresεion rate obtained from a particular treatment iε expressed as a percentage; i.e., Number of partial regressions per group/total number of animals per group x 100. Statistical Evaluation Differenceε in the εize of tumorε between treatment groupε were compared for statistical significance by the Student'ε t-teεt. Differenceε between groupε with p = 0.05, or leεε, were conεidered εignificant. Chemotherapeutic Effect of Taxol Alone In the Treatment of Firεt Paεεaqe CD8F1 Murine Advanced Breaεt Tumors Table 7 reports a serieε of four experiments in CD8F1 mice bearing firεt paεεage εpontaneouε CD8F1 advanced breaεt tumor tranεplants. Each individual experiment compared a group of saline-treated controlε with a εecond group that received the maximal tolerated doεe (MTD) of SUBSTITUTESHEET taxol alone (80 mg/kg) in a q 10-11 day adminiεtration schedule for a total of 3 courseε, and obεervationε were recorded 6 dayε after the laεt courεe of treatment. SUBSTITUTESHEET TAB B 7 ACTIVITY OF TAIO A Q E TH TOE TREATMENT OT T RET TAB8AGE CDSri MURINE ADVANCED* BREAST TϋMORβ Tumor Wt. Peg-a/Trta! f%) f" g » 9/10 (90%) 7,159 0/10 ( 0%) 1,749 Advanced: Tumor weight averaged 130 (Exp. 2540) , 80 (Exp. 2542) , 115 (Exp. 2544) , and 155 (Exp. 2546) mg. at the time of initiation of treatment. Three courses of the indicated treatment (i.p) were administered with a 10-11 day interval between courses. Observations were recorded 6 days after the third course of treatment. Subscript is dose at 80mg/kg, the maximum tolerated dose of Taxol on the 10-11 day schedule,. SUBSTITUTE SHEET At the end of the observation period, the untreated (saline-treated) controls averaged an 80% mortality due to unrestricted tumor growth. In contrast, the four taxol-treated groups evidenced little toxicity (average weight loss = 5%; average mortality - 5%), and there was marked inhibition of tumor growth. Taxol alone clearly haε εtrong anti-tumor activity in the CD8F1 breaεt tumor model. Chemotherapeutic Effects of the Addition of Taxol to the Triple Combination of PALA * MMPR + 6-AI * . + Taxol In Eirst Passage CD8F1 Advanced Breast Tumors The triple drug regimen, with (Group 2) or without taxol (Group 1), waε adminiεtered at 10-11 day intervals in a series of three separate experiments, and observations were recorded 6 days after the third course of treatment. SUBSTITUTESHE EHKAMCEP ACTTVTTY OT TλXOt, WHBI APKTtnSTERgD WTTH THE TRIPLE COMBiyλTTON OF PXIA ■* g-M *** THYTJπ__RC_--PTOPURTKg fMMPR- 4- g-MTTMOWtCOTpAKIDe f -XWl ιw THE TREXTMPΓΓ QΪ FIRST PXSSXG* man WORTH* xbvxncro- BREXST TUMORS Percent Body Dead/Total Tumor «t. CΓOUP TRgftnmπ* If tight Chiiwi fw.) 2____& 1. PAXA. g p-17 hr- -MHFRuo "10 -21 0/10 (0%) 1301 2. PAIA. β0 -17 hr->KKPR. ll + «-ΛN lβ ♦ Taxol 4 -I i . β 5hr-> Taxol 4 q3hr x 7 -25 0/10 (0%) 472* 1. PALA ωβ -17 hr->MKPR uβ -23 0/10 (0%) 051 2. PAIA. βp -17 hr-> MPR , β + «-A χo -2.5 hr-> Taxol 3β -23 2/10 (20%) 31»* ________ 1. PAIA, nft -17 hr->HMPR + C 3s?; *' ISO "10 -22 !/• (11%) 271 PAlA. ββ -17 hr->MKPR j -ό ■* • C-λN 10 -2.5 hr-> Taaaxolso -21 0/0 (0%) 13i c * Advanced: Tumor weight averaged 160 (Exps. 2536, 2537) and 07 ag. (Exp. 2539) at the time of initiation of treatment. * Three courses of the indicated treatment (I.p.) vβrβ administered with a 10-11 day interval between courses. Observations were recorded β days after the third course of treatment. * p < 0.05 (Exp. 2536) or < 0.01 (Exp. 2537) versus group 1. ' NS vs group 1 (Exp. 2539) , but group 2 had 5/9 (55%) partial tumor regressions compared to only 1/9 (11%) in group 1. SUBSTITUTESHEET Pooling the toxicity data of the three groups receiving only the triple drug regimen, there waε an average weight loss of 22% and a mortality rate of only 3%. In one of the experimentε (Exp. 2536), Taxol waε added to the triple regimen in the indicated fractionated εchedule (Group 2). The weight loεε (25%) and absent mortality (0%) in the taxol-containing four drug combination, Group 2, were identical to itε three-drug-treated control without taxol, Group 1, but the therapeutic activity waε significantly better (p < 0.05) than that achieved by the three-drug combination without Taxol. In two of the experimentε (Exp. 2537 and 2539), a single boluε dose of taxol waε added to the three-drug combination (Group 2). The pooled toxicity data in theεe two taxol-containing groupε (Group 2) averaged 22% weight loεε and 11% mortality, essentially no different than the average of their two three-drug control groupε (-23% weight loss; 5% mortality). The therapeutic activity of the taxol-containing combination (Group 2) in Exp. 2537 was significantly better (p < 0.01) than that of itε three-drug control group without taxol, Group 1. In Exp. 2539, the average tumor weight (138 mg) of the three-drug regimen + Taxol, Group 2, although much smaller than the average tumor weight (271 mg) of its 3-drug control without taxol, Group 1, nevertheless waε not significantly different from that of its control (Group 1). But there were 5/9 partial tumor regressions, or a 55% PR, compared to only 1/9 (11%PR) in its three-drug control. Group 1. The data of Table β indicate that the level of therapeutic activity achieved by the quadruple drug combination containing taxol waε εignificantly better in all three experimentε than that in the triple drug combination without taxol, and that thiε improvement in anti-tumor activity was achieved without increaεe in toxicity. S Chemotherapeutic Effects of the Triple-Drug Combination and Taxol at MTD, Alone and in Quadruple Combination (PALA + MMPR + 6-AN + Taxol), In First Passage CD8F1 Murine Advanced Breast Tumors Two experiments are presented in Table 9, each consisting of four groupε of ten tumor-bearing animals each, as follows: Group 1, saline-treated controls; Group 2, Taxol at 80 mg/kg; Group 3, PALA + MMPR + 6-AN; and Group 4, PALA + MMPR + 6-AN + Taxol at 25 mg/kg. Treatment waε repeated at 10 or 11 day intervalε in all groupε for a total of 3 courses, and observationε were recorded 6 days after the last course of treatment. SUBSTITUTESHEET ™« Q ~- ~3-τϋ-re ICTTVTTT or TXXO aroiπ- * **» _»TP con-pxagp TO -ΠLTQI. appro TO m MΪA + e-icgRC yro OTTire T__O_IP_ t-S-π * *. ♦ <-xxτno_r_;eoττ-i --rrpg _«-«τ. coκ»τ * f*τrow nr π» TKg Timrf or TCTBT w cg cpan iπn n xpvmcro* IKPJT WIIOU 2SΛ± 1. Saline - 5 5/10 (50%) 4,152 2. Taxol, to - < 2/10 (20%) 1,140* 3. PA1A 100 -17hr-»ffR 150 +β-AH 10 -15 0/10 ( 0%) 312** 4. PAlA lβ0 -17hr-MΪPr IL S0+ i-AK lβ -2.5hx-Taxo 31 21 -20 0/10 ( 0%) S3* * Advanced: Tumor weight averaged SO (Exp.2542) and 115 mg (Exp.2544) at the time of initiation of treatment. * Three courses of the indicated treatment (i.p.) were administered with 10-11 day interval between courses. Observations were recorded S days after the third course of treatment. ' Croups 2 and 3 (Exp. 2542) cannot be statistically compared to ' group 1, Saline controls, where only 1 tumor-bearing mouse is alive. 90% aortality due to untreated tumor growth. * p<0.05, when group 4 is compared to groups 2 S 3 (Exp. 2542), and when group 2 is compared to group 1 (Exp. 2544). •*p<0.001 when group 3 is compared to group 1 (Exp. 2544). 4 In Exp. 2544, group 4 has a 40% partial tumor regressionrate, whereas there axe no partial turner regress÷.ons in the other groups. SUBSTITUTESHEET In Exp. 2542, Group 1, saline, had a 90% mortality due to the unreεtricted growth of untreated tumorε. Therefore, the average tumor size in Group 2, Taxol gQ alone, and in Group 3, the triple combination, cannot be compared statistically to the single tumor-bearing mouse in Group 1, saline. However, from the differences in tumor-induced mortality rates between these three groups, it iε clear that the tumors of groupε 2 and 3 were markedly inhibited by their respective treatmentε. Group 4, the taxol-containing four drug combination, had tumorε that were significantly inhibited compared to the tumorε in the three drug combination without taxol, Group 3, and to taxol alone, Group 2, and this waε achieved with little toxicity (13% weight loss; 0% mortality). It should be noted that the superior anti-tumor activity of Group 4 waε achieved with a dose of taxol (25 mg/kg) that waε lesε than one-third that of taxol alone (80 mg/kg), Group 2. In Exp. 2544, taxol alone at 80 mg/kg (Group 2), and the triple drug combination (Group 3), significantly inhibited tumor growth over that of the saline-treated tumorε (Group 1). The anti-tumor activity of the three-drug combination with taxol (Group 4) iε clearly superior to all other groups becauεe a 40% PR rate waε induced (see d, legend, Table 9), and there were no partial tumor regressions produced in any of the other groups. Again, it should be noted that the superior anti-tumor activity by the taxol-containing quadruple combination, Group 4, was achieved with a dose of taxol (25 mg/kg) approximately one-third that of the MTD dose (80 mg/kg) of Taxol alone, Group 2, and this increase in anti-tumor activity was achieved without mortality. Example IV: PALA, MMPR, 6 -AN and Radiation The PALA-MMPR-6-AΝ combination also sensitizes tumorε to ionizing radiation therapy. SUBSTITUTESHEET Mice with advanced tranεplanted CD8F1 breast tumors (initial tumor weight 150mg) were divided into four treatment groups: 1) Saline Control 2) PALA-MMPR-6-AN 3) PALA-MMPR-6-AN + radiation (15 Gy, localized) 4) Radiation (15 Gy, localized) Three courseε of theεe treatments were adminiεtered, with a 10-11 day interval between courεeε. Reεultε and detailε of treatment timing and drug doses are indicated in Table 10. While radiation therapy alone (group 4) significantly improved survival and retarded tumor growth, no tumor regressions were observed. In contrast, when mice were treated before irradiation with PALA-MMPR-6-AN, 8 out of 10 mice had regresεionε (tumor size less than 50% of original weight), and 3 of those regressions were complete. * * * While the present invention haε been described in terms of preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations which come within the scope of the invention as claimed. The features disclosed in the foregoing description, in the following claims and/or in the accompanying drawings may, both εeparately and in any combination thereof, be material for realizing the invention in diverεe forms thereof. SUBSTITUTESHEET TABLE 10 Therapy of Advanced First Passage CD8F1 Breast Tumors with PALA + MMPR + 6-AN In Conjunction with Radiation 4. Radiation 0/10 ++ 424 0/10 Exp. 2525M; tumor w't. = 150 mg when treatment initiated. Three courses of the indicated treatment were administered with a 10-11 day interval between courses. Subscripts refer to doses in mg/kg. Radiation was administered at 15 Gy. Observations are recorded at 36 days after the third course of treatment. ** p < 0.01 when compared to group 1. p < 0.01 when compared to group 3.

Claims: WHAT IS CLAIMED IS: 1. A pharmaceutical composition comprising: aa)inhibitor of purine nucleotide biosynthesis, and b) a nicotinamide antagonist. 2. A pharmaceutical composition as in claim 1 wherein said inhibitor of purine nucleotide biosynthesiε iε selected from the group consisting of MMPR, 6-mercaptopurine, thioguanine, thia iprine, tiazofurin, azaserine, 6-diazo-5-oxo L-norleucine, methotrexate, trimetrexate, pteropterin, denopterin and DDATHF. 3. A pharmaceutical composition as in claim 1 wherein εaid nicotinamide antagoniεt iε selected from the group consiεting of 6-AN, thionicotinamide, 2-amino-l,3,4-thiadiazole, 2-ethylamino-l,3, -thiadiazole, 6-aminonicotinic acid, 5-methylnicotinamide and 3-acetylpyridine. 4. A pharmaceutical composition aε in claim 1 further comprising a pharmaceutically acceptable carrier. 5. A pharmaceutical composition as in claim 1 comprising: a) MMPR, and b) 6-AN. 6. A kit comprising: a) a vial containing an inhibitor of purine biosynthesis and a niacin antagonist, and b) a vial containing an apoptosiε inducing agent. 7. A kit comprising: a) a vial containing an inhibitor of purine biosynthesis, S b) a vial containing a nicotinamide antagonist, and c) a vial containing an apoptoεiε inducing agent. 8. A kit aε in claim 6 or 7 further comprising a vial containing an inhibitor of pyrimidine biosyntheεiε. 9. A kit as in claim 6 or 7 wherein said inhibitor of purine biosyntheεis is selected from the group consisting of MMPR, 6-mercaptopurine, thioguanine, thiamiprine, tiazofurin, azaserine, 6-diazo-5-oxo L-norleucine, methotrexate, trimetrexate, pteropterin, denopterin and DDATHF. 10. A kit as in claim 6 or 7 wherein εaid nicotinamide antagoniεt is selected from the group consisting of 6-AN, thionicotinamide, 2-amino-l,3,4-thiadiazole, 2-ethylamino-l,3,4-thiadiazole, 6-aminonicotinic acid, 5-methylnicotinamide and 3-acetylpyridine. 11. A kit as in claim 6 or 7 wherein said apoptosis inducing agent is selected from the group consisting of methotrexate, 5-fluorodeoxyuridine, 5-fluorouracil, 1-B-D-arabinofuranosyl- cytoεine, puromycin, trifluorothymidine, ciεplatin, etopoεide, camptothecin, cytoxan, adriamycin, tenipoεide, podophyllotoxin, aphidocolin, eodium azide, . N-methyl-N'-nitro-N'nitroεoguanidine, nitrogen mustard, bleomycin, l,3-biε(2-chloroethyl)-a-nitroεourea, methyl glyoxal-biε-(guanylhydrazone), colcemid, vincristine, taxol, taxotere, dexamethasone, retinoic acid, purinergic F2 receptor agoniεtε, so atostatin analogs, luteinizing hormone releasing factor analogs, and antibodies capable of inducing apoptosis. 12. A kit as in claim 8 wherein said inhibitor of pyrimidine biosynthesiε iε selected from the group SUBSTITUTESHEET 93/04775 72 consisting of PALA, 6-azauridine, triacetyl-6-azauridine, pyrazofuran, brequinar and acivicin. 13. A method of treating antineoplastic disease in an animal comprising the steps of: a) administering a therapeutically effective amount of an inhibitor of purine biosynthesis, b) administering a nicotinamide antagonist, and c) administering an apoptoεis inducing agent. 14. A method aε in claim 13 further comprising the step of d) administering a therapeutically effective amount of an inhibitor of pyrimidine biosynthesis. 15. A method as in claim 13 wherein said inhibitor of purine biosynthesis iε selected from the group consisting of MMPR, 6-mercaptopurine, thioguanine, thiamiprine, tiazofurin, azasevine, 6-diazo-5-oxo L-norleucine, methotrexate, trimetrexate, pteropterin, denopterin and DDATHF. 16. A method aε in claim 13 wherein said nicotinamide antagonist is selected from the group consisting of 6-AN, thionicotinamide, 2-amino-l,3, -thiadiazole, 2-ethylamino-l,3,4-thiadiazole, 6-aminonicotinic acid, 5-methylnicotinamide and 3-acetylpyridine. 17. A method as in claim 13 wherein said apoptoεis inducing agent is selected from the group consisting of methotrexate, 5-fluorodeoxyuridine, 5-fluorouracil, 1-B-D-arabinofuranosyl- cytosine, puromycin, trifluorothymidine, ciεplatin, etoposide, camptothecin, cytoxan, adriamycin, teniposide, podophyllotoxin, aphidocolin, sodium azide, N-methyl-N'-nitro-N'hitrosoguanidine, nitrogen mustard, bleomycin, l,3-bis(2-chloroethyl)-a-nitrosourea, methyl glyoxal-bis-(guanylhydrazone), colcemid, vincriεtine, taxol, taxotere, dexamethasone, retinoic acid, purinergic P2 receptor agonists, εomatostatin analogs, luteinizing hormone releasing factor analogs, antibodieε capable of inducing apoptoεiε, and cytotoxic T-cellε. 18. A method as in claim 14 wherein said inhibitor of pyrimidine biosynthesis iε selected from the group consisting of PALA, 6-azauridine, triacetyl-6-azauridine, pyrazofuran, brequinar and acivicin. 19. A method as in claim 13 wherein step c) occurs after steps a) and b). 20. A method as in claim 14 wherein εaid administering a therapeutically effective amount of an inhibitory of pyrimidine biosynthesis step occurs before steps a), b) and c). 21. A method as in claim 13 wherein said inhibitor of purine biosynthesiε is MMPR, said nicotinamide antagoniεt is 6-AN, and said apoptoεiε inducing agent iε FUra. 22. A method aε in claim 13 wherein εaid inhibitor of purine bioεyntheεiε iε MMPR, εaid micotinamide antagoniεt is 6-AN, and εaid apoptosis inducing agent is FUra. 23. A method as in claim 13 wherein εaid inhibitor of purine bioεynthesiε iε MMPR, εaid micotinamide antagoniεt iε 6-AN, and εaid apoptosis inducing agent is taxol. 24. A method of treating antineoplastic disease in an animal comprising the steps of: a) administering a therapeutically effective amount of an inhibitor of purine biosynthesis, b) administering a nicotinamide antagonist, and SUBSTITUTESHEET c) adminiεtering radiotherapy. 25. A method as in claim 24 further comprising the step of administering a therapeutically effective amount of an inhibitor of pyrimidine biosynthesis. 26. A method of treating multiple drug resistance in an animal comprising the steps of: a) administering a therapeutically effective amount of an inhibitor of purine biosynthesis, b) adminiεtering a nicotinamide antagonist, and c) adminiεtering an apoptosis inducing agent. 27. A method as in claim 26 further comprising the step of adminiεtering a therapeutically effective amount of an inhibitor pyrimidine biosynthesis. S B

Inventors: COLOFIORE JOSEPH R, MARTIN DANIEL S, NORD L D, STOLFI ROBERT L, COLOFIORE, JOSEPH, R., MARTIN, DANIEL, S., NORD, L., D., STOLFI, ROBERT, L.
Applicants: COLOFIORE JOSEPH R, MARTIN DANIEL S, NORD L D, STOLFI ROBERT L, COLOFIORE, JOSEPH, R., MARTIN, DANIEL, S., NORD, L., D., STOLFI, ROBERT, L.
Application year: 1993
Main classification: A61K9
IPC codes: A61K31, A61K33, A61K38, A61K45, A61P35, A61P43