Based on an animal model to improve the antitumor activity of 5fluorouracil (FUra), a Phase I study of A/-(phosphonacetyl)-i .-aspartate, methotrexate, FUra, and leucovorin was conducted on 44 patients. Meth otrexate was given in an intermediate dose (250 mg/nr) to overcome potential drug resistance, and ¿V-(phosphnnacetyl)-i.-aspartatewas given at a low dose (250 mg/nr') in order to allow escalation of FUra to toxicity. These two drugs were given 24 h before FUra to enhance maximal incorporation of FUra into RNA. Two schedules of administration were used; one every other week and one weekly for 2 weeks. The every other week schedule was well tolerated, with minimal gastrointestinal and hematological toxicity. However, the weekly for 2 weeks schedule was more toxic with increased mucositis, diarrhea requiring therapy, and decreased performance status of 20% in 4 of 6 patients. There were no responders in the every other week schedule. There was one partial response and three patients with stable disease in four évaluable patients on the weekly for 2 weeks schedule. At 24 h post-/V-(phosphonacetyl)-i.aspartate-methotrexate treatment, PRPP levels were doubled in bone marrow biopsies, and increased 2.5to 25-fold in tumor biopsies. We have currently added uridine rescue to this combination with the hope of further escalating the dose of FUra.

2096 Background: : Chemotherapy is enhanced by multi-ATP-depleting therapy (Cancer Res. 60: 6776, 2000). Malignant tumors generally increase ATP production to increase cell division. In general, tumors make and consume (deplete) more ATP than normal tissues. If ATP production is equally inhibited in normal and tumor tissues, high ATP consumption (depletion) in tumors should deplete ATP to cytocidal levels, whereas the lower ATP consumption in normal cells should reduce ATP to tolerable levels. This rationale requires multiple ATP inhibitors inhibiting multiple biochemical pathways that produce ATP. Methods: Alanosine (AL), inhibitor of AMP synthesis, + 6-methylmercaptopurine riboside (MMPR), inhibitor of purine synthesis, preceded by PALA, a pyrimidine inhibitor; acronym, PALM. F16 blocks ATP synthesis by selectively accumulating in mitochondria of responsive breast cancers (Cancer Cell 2:29, 2002). Results: F16 alone was not effective. Taxotere alone (TXT), at MTD40, affects 100% PR of tumors. The addition of PALM (P100 AL250 M150), or PALM + F16 10, effects the same 100% P.R. at half the TXT dose (TXT20), suggesting clinical benefit at reduced normal tissue toxicities. Follow-up (120 days) demonstrates substantial tumor recurrence (43–50%) in Groups 1 and 2, and minimal recurrence (17%) in Group 3. Conclusions: A multi-ATP-depleting regimen (PALM + F16) + chemotherapy augments tumoricidal capability. Chemotherapy is at markedly lower doses, strongly suggesting lower toxic side-effects in the clinic. No significant financial relationships to disclose.

The aim of this study was to examine the expression of methylthioadenosine phosphorylase (MTAP) in 21 fresh tumor samples from patients with soft tissue sarcomas (STS) and 11 human soft tissue sarcoma cell lines, and to determine if loss of expression of this enzyme was correlated with increased sensitivity to L-alanosine and/or 6-methylmercaptopurine. We used a polyclonal antibody to measure the expression of MTAP in soft tissue sarcoma cell lines and in fresh tumor samples. Transfection of the HT-1080 cell line with a plasmid containing the cDNA for the MTAP gene was also performed to generate cell lines for in vitro and in vivo comparative sensitivity studies. MTAP was not expressed in 8 of 21 fresh STS tumors. The expression of MTAP was also not detectable in 3 of the 11 soft tissue sarcoma cell lines (HT-1080, HS42, and M-9 110). These three cell lines were more sensitive to L-alanosine, a potent inhibitor of de novo AMP synthesis, and to an inhibitor of de novo purine nucleotide synthesis, 6-methylmercaptopurine riboside (MMPR). The IC50 values for L-alanosine and MMPR were >20-fold lower in MTAP-deficient cells than in MTAP-positive cells. Restoration of MTAP into HT-1080 MTAP-deficient cells also led to decreased sensitivity to L-alanosine and MMPR. An in vivo study using HT-1080 cell tumors with and without MTAP expression confirmed that tumors lacking MTAP were more sensitive to L-alanosine than tumors expressing MTAP. These results provide the basis for selective therapy using inhibitors of de novo purine nucleotide synthesis such as L-alanosine or MMPR to treat patients with STS lacking this enzyme.

Most anticancer agents effect DNA damage which initiate the cell death pathways of necrosis and apoptosis, but cancer cells of lesser sensitivity are only sublethally injured, and recover. The two death pathways and their interelationships in the presence of endogenous inhibitors of apoptosis and genetic deletions that facilitates only sublethal damage, are reviewed.Both ATP and pyrimidine levels in the sublethally injured cancer cells are reduced but not to low levels insuffient to sustain cell viability. However, this sublethal damage by the anticancer agent creates a therapeutic opportunity for further reduction of these key metabolites to lower levels that will not support life. Data in tumor-bearing animals is reviewed demonstrating that a combination of ATP-depleting agents plus a de novo pyrimidine inhibitor (PALA) administered concomitantly with each of nine different anticancer agents markedly enhances tumor regression rates, and even produces some cures. It is necessary to deplete tumor ATP levels seveerely (> 85%) by a combination of agents that block both synthesis(6-methylmercaptopurine riboside, a purine de novo synthesis inhibitor) and generation of ATP(6-aminonicotin-amide, an inhibitor of glycolysis.) Cell viability cannot be sustained if the intracellular ATP level is reduced to 15% of normal or below. In vivo data employing this novel therapeutic strategy with cisplatin is presented. The potential significance of these findings to the improvement of cancer treatment is discussed.

In the 1980s, necrosis was considered the mode of cell death induced by DNA-damaging anticancer agents because of the activity of PARP. [3][1] PARP is activated by the DNA strand breaks caused by anticancer agents and cleaves the glycolytic coenzyme, NAD+, leading to formation of poly(ADP-ribose)

Cancer cells that are sufficiently damaged by cancer chemotherapeutic agents (as well as radiotherapy) eventually die in an ordered sequential biochemical process known as apoptosis. Apoptosis is a general physiological mechanism for controlled cell deletion that is an active (i.e., an energy-dependent, at least initially), inherent (gene-directed) program of cell death, and therefore sometimes referred to as cell suicide and programmed cell death (1,2). The apoptotic biochemical events occurring after the anticancer agent's interaction with its biochemical target is the actual process of cell death and is a secondary phenomenon following the primary drug-target interaction. Thus, anticancer agents, despite having different primary biochemical targets (e.g., inhibition of thymidylate synthase, microtubule damage, topoisomerase inhibitors, DNA crosslinking agents, etc.), all ultimately kill by inducing the biochemical cascade of apoptosis (3,4). However, there is a “qualitative” and “quantitative” heterogeneity in a neoplastic cell population. “Qualitative” heterogeneity establishes the absolute need for a combination of drugs with different biochemical actions to kill all the different subpopulations of malignant cells within the tumor in order to achieve cure.

Uridine diphosphoglucose (UDPG) is a precursor of uridine that can be used as a rescuing agent from 5-fluorouracil (5FU) toxicity. Four doses of UDPG (2000 mg/kg i.p. or p.o. at 2, 6, 24, and 30 h after 5FU bolus) allowed the escalation of a weekly bolus of 5FU from 100 mg/kg (5FU100) to 150 mg/kg (5FU150) in healthy and tumor-bearing BALB/c, C57/BI, and CD8F1 (BALB/c x DBA/8) mice. 5FU150 without rescuing agents is not tolerated by the animals. When followed by UDPG, on the contrary, it is possible to increase the dose of 5FU even when it is modulated by leucovorin. Toxicity was the same for 5FU100 and 5FU150 + UDPG, and the nadir values (expressed as a percentage of pretreatment values) were 83 and 85% for weight, 45 and 45% for hematocrit, and 45 and 61% for leukocytes, respectively. Platelets were not affected by treatment. A protective effect was also shown for the gastrointestinal tract. The enzymes thymidine kinase, maltase, and sucrase were measured in the intestinal mucosa at different times after 5FU treatment with or without UDPG rescue. Even if the nadir values in enzyme activities were similar in mice receiving or not receiving UDPG, the pattern of recovery showed that cell repopulation was more rapid in the group treated with UDPG. 5FU150 + UDPG had enhanced antitumor activity against CD8F1 mammary carcinoma and against the resistant tumor Colon 26 (tumor doubling time 1.9 days for controls, 8.5 days for 5FU100, 13.7 days for 5FU150 + UDPG, and 15.9 days for 5FU150 + leucovorin + UDPG). We demonstrated that UDPG administered at 2, 24, and 30 h after 5FU100 does not reduce the antitumor activity of 5FU in two sensitive tumors (Colon 38 and Colon 26-10). In conclusion, UDPG is a promising rescuing agent for 5FU; it reduces the toxic side effects and increases the therapeutic index.

The combination of N-(phosphonacetyl)-L-aspartate (PALA), 6-methylmercaptopurine riboside (MMPR), and 6-aminonicotinamide (6AN) has been shown to be an effective antineoplastic regimen and also to enhance the effects of their other antineoplastic agents (1-4). To further enhance the effect of this combination, we investigated the effects of adding adriamycin, at its maximally tolerated dose, to this regimen. The response rate (complete regression + partial regression) for the four-drug regimen was higher than for the three-drug regimen, and the tumor growth delay was also significantly higher than for treatment with PALA, MMPR, 6AN, or after treatment with maximally tolerated doses of adriamycin alone (11 mg/kg). The addition of adriamycin to PALA, MMPR, 6AN did not result in enhancement of the effect of radiation, as measured by tumor growth delay studies and tumor control (complete and partial regression rate). The mechanism of action of the combination of PALA, MMPR, and 6AN is not known definitively, but a possible mechanism previously suggested is biochemical modulation of energy metabolism and inhibition of production of tumor ATP. Treatment with PALA< MMPR, 6AN, and adriamycin (at 2.5 hr post MMPR, 6AN) resulted in a nadir NTP/Pi value, as determined by P-31 NMR spectroscopy, at approximately 10 hr post MMPR + 6AN (7.5 hr post adriamycin), which was not significantly different from the NTP/Pi value determined after treatment with the three-drug combination.

Purpose: To evaluate the effects of biochemical modulation by N-(phosphonacetyl)-L-aspartate (PALA), 6-methylmercaptopurine riboside (MMPR), and 6-aminonicotinamide (6AN), (PALA + MMPR + 6AN is referred to as PMA) on tumor radiosensitivity, and evaluate the efficacy of the addition of 5-FU to the PMA + XRT regimen for enhancement of tumor response to radiation without exceeding normal tissue tolerance.Methods and Materials: A first generation transplant of the CD8F1 spontaneous murine tumor was studied, 31P nuclear magnetic resonance spectroscopy was used to determine the interval between chemotherapy and radiation based on energy depletion. PMA was administered three times with fractionated XRT (15 Gy x 3 = 45 Gy) on days 1, 10, or 11, and 21. The addition of 5-fluorouracil (5-FU) at maximum tolerated doses was evaluated and intergroup comparisons were made for tumor growth delay, local control, and disproportionate normal tissue damage,.Results: The combination of 5-FU + XRT induced a tumor doubling time of 75.4 days (67.4-84.4) (p < 0.0001 compared to XRT), validating that in this tumor model, pretreatment with bolus i.p. 5-FU enhanced XRT. In comparison, mice treated with PMA + XRT had a tumor doubling time (TDT) > 123.2 days (109.4-138.7), (p < 0.0001 compared to 5-FU + XRT). The addition of 5-FU to PMA + XRT induced a doubling time of > 170.8 days (150.7-193.7) (p = 0.0002 compared to PMA + XRT). The doubling time for the PMA + XRT cohort and the PMA + 5-FU + XRT cohorts are underestimates since some of the tumor bearing mice continue to have a complete regression (CR). The CR rate (measured on day 250) for the PMA + 5-FU + XRT cohort was 31.7% compared to 0% for 5-FU + XRT and 10% for PMA + XRT (p < 0.05), Mortality and local effects induced by radiation in the PMA + XRT group were comparable to the toxicity for the PMA + 5-FU + XRT group indicating that the addition of 5-FU at 75 mg/kg to PMA + XRT was tolerated and induced both greater CR and tumor doubling times than XRT alone, 5-FU (150 mg/kg) + XRT, or PMA + XRT.Conclusions: PMA is superior to 5-FU as a radiosensitizer in the schedule studied. The combination of PMA + 5-FU further enhanced XRT without exceeding normal tissue tolerance. (C) 1997 Elsevier Science Inc,.

The biochemical death cascade of apoptosis is separate from, although induced by, the anticancer drug-target interaction. The failure of many of our chemotherapeutic agents reflects an inability of anticancer drugs to induce apoptosis. Understanding the basic cellular mechanisms that control apoptosis will greatly increase our ability to treat cancer. Identification of the components of the apoptotic biochemical cascade will present new targets for complementary enhancement of chemotherapeutically induced cancer cell death. One factor that has been directly implicated in apoptosis is adenosine triphosphate (ATP). Nevertheless, in this regard, ATP is controversial. This commentary takes issue with dogma, and points to the need for additional thought and research in this field. ATP-depleting therapy of tumor-bearing mice has been shown to induce a marked therapeutic result with minimal mortality, and this effect can be further enhanced when combined with chemotherapy. The definitive mechanism of action is still controversial, although several mechanisms for ATP depletion have been implicated in the process. These include reduction in the mitochondrial transmembrane potential, activation of poly (ADP-ribose) polymerase (PARP) and depletion of the coenzyme nicotinamide adenine dinucleotide (NAD(+)). Even though the definitive experiments have yet to be carried out, the identification of ATP depletion as a critical determinant in apoptosis should allow for the development of new therapeutic strategies in the treatment of human cancer.

The drug combination N-(phosphonacetyl)-l-aspartic acid (PALA), methylmercaptopurine riboside (MMPR) and 6-aminonicotinamide (6AN), referred to as PMA, induces regressions of advanced CD8F1 murine mammary carcinomas in vivo. We demonstrated that CD8F1 tumor regressions were preceded by the appearance of apoptotic bodies, as observed by microscopic examination of morphology and TUNEL end-labeling, and fragmentation of DNA into nucleosomal “ladder” patterns. These indications of apoptosis were present as early as 6 h after simultaneous administration of MMPR and 6AN and further increased by over fivefold during the next 3 to 6 h, then remained at 7 to 12.8% (0.6 to 2.4% in saline-treated controls) of the cell population for at least 24 h after MMPR + 6AN administration. The 5′-phosphate derivative of MMRP, MMPR-5P, which inhibits de novo purine biosynthesis, was present at a “steady-state” level, and significant (40%) depletion of ATP had occurred by 3 h and both of these events preceded the onset of apoptosis. In addition, MMPR-5P was retained in CD8F1 tumors at a high level over a prolonged period (>96 h) even as tumors were undergoing regression. The prolonged presence of MMPR-5P was important for optimal chemotherapeutic effect, since treatment with iodotubercidin (IodoT), an inhibitor of MMPR/adenosine kinase, 6 h after MMPR + 6AN administration prevented the prolonged accumulation of MMPR-5P and reversed the regression of CD8F1 tumors. In addition, compared to the PMA-treated group, there was a significant restoration of ATP levels after treatment with IodoT. In individual PMA-treated CD8F1 tumors the degree of ATP depletion was found to correlate with the degree of tumor shrinkage at 24 h, after tumors had sufficient time to respond to treatment. These results define the time-course of drug-induced apoptosis in CD8F1 tumors, show that ATP depletion occurs prior to apoptosis and demonstrate that prolonged retention of MMPR-5P is associated with optimal chemotherapy. Collectively, these results suggest that the depletion of ATP by PMA treatment may be a component of the biochemical apoptotic cascade in the CD8F1 tumor.

The chemotherapeutic regimen of N-(phosphonacetyl)-L-aspartate (PALA) followed 17 h later by 6-methylmercaptopurine riboside (MMPR) and 6-aminonicotanamide (6AN) has been shown to be a potent sensitizer of anti-neoplastic therapy We undertook this study to compare the therapeutic and metabolic effects of this triple drug combination vs one of its components, 6AN, in a murine mammary carcinoma, After treatment with PALA, MMPR and 6AN, a new peak was detected which was assigned to 6-phosphogluconate (6PG), which is a marker of inhibition of the pentose phosphate pathway at the B-phosphogluconate dehydrogenase step, Treatment with PALA, MMPR and 6AN also induced a decrease in the ratios of nucleoside triphosphate/inorganic phosphate (NTP/P-i) and phosphocreatine/inorganic phosphate (PCr/P-i) similar to previous results with a different tumor model, These effects were most pronounced at 6 and 10 h. In addition, an increase in PME/phosphocholine (PME'=downfield peak in the phosphomonoester region) was detected, which was expected because of the cytotoxic effect of this regimen, Treatment with 6AN alone also resulted in the detection of 6PG with a maximum intensity at 6 h post-6AN. Treatment with 6AN alone induced a smaller change in 6PG and failed to cause a decrease in PCr/P-i or NTP/P-i at 6 and 10 h. The enhanced response to the combination of PALA, MMPR and 6AN vs 6AN alone, both with regard to cytotoxicity and radiosensitization, may be due to energy depletion.

An inhibitor of poly (ADP-ribose) polymerase, 1,5-dihydroxyisoquinoline (DHIQ), evaluated in vivo against a murine advanced breast cancer, significantly improved by 20% the PR rate of tumor-regressing chemotherapy. A detailed sequential biochemical cascade is proposed for chemotherapy-induced apoptosis, and the rationale for the utilization of the inhibitor is explained.

BACKGROUND Dose intensification of 5-fluorouracil (5-FU) is complicated by increased toxicity. 5-FU is a fluorine-substituted pyrimidine analog of uracil. In preclinical studies, administration of oral uridine (Ur) has been shown to allow for dose intensification of 5-FU with enhancement of its antitumor activity. Therefore, a Phase I trial was designed aimed at dose intensification of 5-FU as a component of a modified 5-FU-doxorubicin-methotrexate (FAMTX) regimen using oral Ur rescue. METHODS Methotrexate (MTX) was administered to all patients at a fixed dose of 1.5 g/m2. MTX was followed 24 hours later by escalating doses of 5-FU starting at 800 mg/m2 with leucovorin rescue. Cycles of 5-FU and MTX were repeated every 15 days. Every other cycle, patients received doxorubicin (“Adria cycles”) at a dose of 30 mg/m2. Oral Ur was administered at a dose of 8 gm/m2 every 6 hours for 12 doses. In the first phase of the study, patients received Ur only if they developed Grade 3 or 4 hematologic toxicity. In the second phase, all patients received Ur 24 hours after 5-FU on all cycles. RESULTS Without Ur rescue, the maximum tolerated dose (MTD) of 5-FU was 900 mg/m2 on the Adria cycles and 1.1 gm/m2 on the non-Adria cycles. With Ur, the MTD of 5-FU increased to 1.2 gm/m2 on the Adria cycles and to 1.6 gm/m2 on the non-Adria cycles. CONCLUSIONS In this modified FAMTX regimen, oral Ur administration allowed for dose-intensification of 5-FU, with a 33% increase in the MTD of 5-FU on the Adria cycles and a 45% increase in the MTD of 5-FU dose on the non-Adria cycles. Cancer 1997=6;78:1988-95.

6‐aminonicotinamide (6AN) has been shown to enhance radio‐sensitivity in vitro , although previous in vivo studies failed to show an effect. 31 P NMR spectra were obtained by using a one‐dimensional chemical shift imaging technique on a first generation transplant of the CD8FI spontaneous mammary carcinoma tumor model. Spectra were obtained both before and 10 h after treatment with 6AN (20 mg/kg). Changes in pH, nucleoside triphosphate/inorganic phosphate, and phosphocreatine/inorganic phosphate measured at 10 h post‐6AN were not significant. A new peak was detected 10 h post‐6AN, which was assigned to 6‐phosphogluconate (6PG), indicating inhibition of the pentose phosphate pathway (PPP). Based on the spectral data demonstrating inhibition of the PPP at 10 h post‐6AN, tumor‐bearing mice were irradiated (15 Gy × 3 fractions) on Days 1, 10 or 11, and 21 10 h after administration of 6‐aminonicotinamide (20 mg/kg). Tumor‐bearing mice receiving 6AN alone (20 mg/kg × 3), radiation alone (15 Gy × 3), or saline were also studied. Tumor growth delay studies indicated that 6AN alone induced a small but significant tumor growth delay (4.3 ± 0.8 days). Radiation alone induced a tumor growth delay of 34.5 ± 2.7 days. Treatment with 6AN followed by radiation induced a tumor growth delay of 57.0 ± 3.8 days. This was significantly greater than the TGD values for treatment with 6AN alone or radiation ( P < 0.01). No complete regressions were noted after treatment with 6AN or radiation alone. Concomitant therapy with 6AN plus radiation yielded 6/28 complete regressions (21%), which was significantly greater than radiation ( P < 0.05) or 6AN alone ( P < 0.01) on this mammary carcinoma.

Treatment with a combination (PMA) of (N-phosphonacetyl)-L-aspartic acid (PALA), methylmercaptopurine riboside (MMPR), and 6-aminonicotinamide (6AN) induced partial regressions of CD8F1 murine mammary tumors and provided for tumor growth inhibition without regression of Colon 38 tumors. HPLC-nucleotide pool analysis of CD8 mammary tumors obtained at various times after treatment with PMA revealed that MMPR-5′-phosphate, which inhibits de novo purine nucleotide biosynthesis, was constant at levels of approximately 2.5 nmol/mg protein for 72 hr after treatment. In contrast, the MMPR-5′-phosphate levels of C38 tumors decreased from 24-hr levels at 1.5 nmol/mg protein with a half-time of about 24 hr. Treatment of CD8 tumor-bearing mice with iodotubercidin, a potent inhibitor of adenosine/MMPR kinase, at various times after PMA, reversed both the accumulation of high levels of MMPR-5′-phosphate and the number of partial tumor regressions These data demonstrate that a cycle of MMPR rephosphorylation is active in the CD8 mammary tumor and suggest that this recycling of MMPR is important for the optimal effect of PMA treatment.

Paclitaxel alone Is active against the CD8F1 murine spontaneous mammary cancer, and when administered following an ATP-depleting combination of N-(phosphonacetyl)- L-aspartate (PALA) + 6-methylmercaptopurine riboside (MMPR) + 6-aminonicotinamide (6-AN) (PMA) produced significantly enhanced partial tumor regressions over that produced by either paclitaxel alone at the maximal tolerated dose (MTD), or by the PMA drug combination alone, against advanced, first passage spontaneous murine breast tumors. The anticancer activity of paclitaxel is due to enhancement and stabilization of microtubule polymerization. Pertinently, microtubule disassembly (an ATP-dependent process) is known to sharply decrease in the presence of ATP depletion. Thus, the dramatic therapeutic enhancement observed with paclitaxel in combination with PMA is in agreement with biochemical expectations, since PMA has been shown to deplete ATP in CD8F1 tumor cells. The augmented therapeutic results were obtained with approximately one-third the MTD of paclitaxel as a single agent and suggest the potential clinical benefit of more effective treatment with lesser amounts of drug.