Redox state affects fecundity and insecticide susceptibility in Anopheles gambiae.

Redox reactions play a central role in the metabolism of an organism. It is vital to maintain redox homeostasis in response to the fluctuation of redox shift in various biological contexts. NADPH-dependent reducing capacity is one of the key factors contributing to the redox homeostasis. To understand the redox capacity and its impact on mosquito fecundity and susceptibility to insecticides in Anopheles gambiae, we examined the dynamics of elevated oxidative state via induction by paraquat (PQ) and the inhibition of NADPH regeneration by 6-aminonicotinamide (6AN). In naïve conditions, inherent oxidative capacity varies between individuals, as measured by GSSG/GSH ratio. The high GSSG/GSH ratio was negatively correlated with fecundity. Both PQ and 6AN feeding increased GSSG/GSH ratio and elevated protein carbonylation, a marker of oxidative damage. Both pro-oxidants lowered egg production. Co-feeding the pro-oxidants with antioxidant lycopene attenuated the adverse effects on fecundity, implying that oxidative stress was the cause of this phenotype. Pre-feeding with 6AN increased insecticide susceptibility in DDT resistant mosquitoes. These results indicate that oxidative state is delicate in mosquitoes, manipulation of NADPH pool may adversely affect fecundity and insecticide detoxification capacity. This knowledge can be exploited to develop novel vector control strategies targeting fecundity and insecticide resistance.

Controlled release of 6-aminonicotinamide from aligned, electrospun fibers alters astrocyte metabolism and dorsal root ganglia neurite outgrowth.

Following central nervous system (CNS) injury, activated astrocytes form a glial scar that inhibits the migration of axons ultimately leading to regeneration failure. Biomaterials developed for CNS repair can provide local delivery of therapeutics and/or guidance mechanisms to encourage cell migration into damaged regions of the brain or spinal cord. Electrospun fibers are a promising type of biomaterial for CNS injury since these fibers can direct cellular and axonal migration while slowly delivering therapy to the injury site. In this study, it was hypothesized that inclusion of an anti-metabolite, 6-aminonicotinamide (6AN), within poly-l-lactic acid electrospun fibers could attenuate astrocyte metabolic activity while still directing axonal outgrowth. Electrospinning parameters were varied to produce highly aligned electrospun fibers that contained 10% or 20% (w/w) 6AN. 6AN release from the fiber substrates occurred continuously over 2 weeks. Astrocytes placed onto drug-releasing fibers were less active than those cultured on scaffolds without 6AN. Dorsal root ganglia placed onto control and drug-releasing scaffolds were able to direct neurites along the aligned fibers. However, neurite outgrowth was stunted by fibers that contained 20% 6AN. These results show that 6AN release from aligned, electrospun fibers can decrease astrocyte activity while still directing axonal outgrowth.

Radiosensitization of murine Ehrlich ascites tumor by a combination of 2-deoxy-D-glucose and 6-aminonicotinamide.

Enhanced radiosensitizing effects of a combination of 2-deoxy-D-glucose (2-DG), a glycolytic inhibitor and 6-aminonicotinamide (6-AN) an analogue of nicotinamide, which inhibits hexose monophosphate shunt (HMP) have been demonstrated in vitro. The purpose of the present studies is to investigate in vivo effects of this combination in Ehrlich ascites tumor (EAT) bearing mice. EAT tumor was grown in Swiss albino strain A mice. Treatment induced growth delay and tumor free animal survival were evaluated as parameters of radiation response. Focal irradiation of the tumor with a single fraction of 10 Gy induced a moderate delay in tumor growth but did not lead to complete regression of the tumor. Intravenous administration of either 6-AN or 2-DG immediately before irradiation enhanced radiation-induced growth delay with a cure rate of 45%. However, administration of a combination of 2-DG (2 g/kg b.wt.) and 6-AN (2 mg/kg b.wt.) immediately before irradiation led to complete regression of tumor in 80% animals resulting in survival of more than 300 days. A similar response (approximately 80%) was observed when 2-DG dose was reduced to 1 g/kg in combination with 6-AN. It is concluded that 6-AN enhances the radiosensitizing effects of 2-DG and the combination may have potential application in improving radiotherapy of tumors.

Contribution of oxidative stress to radiosensitization by a combination of 2-DG and 6-AN in human cancer cell line.

In the present studies, the role of oxidative stress in radiosensitization by a combination of 2-DG and 6-aminonicotinamide (6-AN) was examined in a human glioma cell line (BMG-1: wild type p53). Presence of 2-DG or 6-AN for 4 hr after irradiation (gamma ray 2.5 Gy) significantly enhanced the radiation-induced cell death by 18% and the combination (2-DG + 6-AN) enhanced the cell death by 35%. Neither 2-DG nor 6-AN had any further significant effect on the glutathione levels in irradiated cells. However, the combination (2-DG + 6-AN) caused a significant decrease in GSH content, increase in GSSG levels, and enhanced the superoxide radical generation under these conditions. The enhanced cell death caused by the combination (2-DG + 6-AN) mainly resulted by the process of apoptosis as revealed by annexin V binding and was associated with elevated levels of Cyclin B1. However, no significant change was observed in the levels of Bcl-2. Thus, for the first time, our results have demonstrated that the radiosensitizing effects of these modifiers could also be mediated through alterations in the oxidative stress besides energy limited inhibition of repair and recovery processes.

Effects of ATP on the stability of enzymes against heat treatment in 6-aminonicotinamide treated quail.

The stabilities of liver and pectoral muscle enzymes in 6-aminonicotinamide (6-AN) treated quail against heat treatment in the presence and absence of added ATP were investigated. Only ATP level in the brain and pectoral muscle of 6-AN treated group was significantly reduced compared to the control group whereas ADP and AMP levels were not affected. In the thermal stability (55 degrees C) of liver enzymes, the activity of acetylcholinesterase (AChE) was not affected whereas the activities of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH) were significantly lowered (P<0.01). The addition of 1mM ATP to liver enzyme extracts of 6-AN group afforded 4- and 1.7-fold more protection for GAPDH and LDH, respectively (P<0.01). In liver, LDH appeared to be more protected by ATP than GAPDH. In muscle, however, GAPDH and AChE activity were significantly affected but not LDH. The addition of 1mM ATP to muscle enzyme extracts of 6-AN group afforded 1.7-fold more protection for GAPDH (P<0.01) but rather inactivated AChE. A marked reduction in ATP levels in muscle did not affect specifically muscle enzyme activities only since liver enzyme activities were also affected to the same degree as muscle.

Effects of neurotoxin 6-aminonicotinamide on levels of enzyme activities and metabolites in quail plasma.

Effects of 6-aminonicotinamide (6-AN) on the levels of proteins, metabolites and enzyme activities in the plasma of Japanese quail were investigated. The concentrations of soluble proteins in the pectoral and hindlimb muscle of the 6-AN treated and the pair-fed groups were significantly reduced compared to the control group. In the plasma, the levels of total proteins and albumin were not affected, but the levels of globulin were significantly lower than those of the control and pair-fed groups. In contrast, the levels of glucose and creatine were significantly elevated. Cellulose acetate gel electrophoresis showed that 6-AN induced a new synthesis of prealbumin and also increased the levels of beta-globulin relative to the control and pair-fed groups. In contrast, the levels of gamma-globulin were markedly lower than those of the control group, whereas the levels of alpha-globulin were not affected. The specific activity of alkaline phosphatase of the 6-AN group was significantly lower than that of the control and pair-fed groups and that of aspartate aminotransferase only lower than that of the control group but not the pair-fed group. The specific activities of creatine phosphokinase and lactate dehydrogenase of the 6-AN group were the greatest among the three groups, whereas those of the pair-fed group were greater than those of the control group. The results suggest that 6-AN may interfere with the proper maintenance of energy charges and the immune system function.

Murine pharmacokinetics of 6-aminonicotinamide (NSC 21206), a novel biochemical modulating agent.

The pyridine nucleotide 6-aminonicotinamide (6AN) was shown recently to sensitize a number of human tumor cell lines to cisplatin in vitro. The present studies were undertaken to compare the drug concentrations and length of exposure required for this sensitization in vitro with the drug exposure that could be achieved in mice in vivo. Human K562 leukemia cells and A549 lung cancer cells were incubated with 6AN for various lengths of time, exposed to cisplatin for 1-2 hr, and assayed for Pt-DNA adducts as well as the ability to form colonies. K562 cells displayed progressive increases in Pt-DNA adducts and cisplatin sensitivity during the first 10 hr of 6AN exposure. An 18-hr 6AN exposure was likewise more effective than a 6-hr 6AN exposure in sensitizing A549 cells to cisplatin. HPLC analysis of 6AN and its metabolite, 6-amino-NAD+, permitted assessment of exposures achieved in vivo after i.v. administration of 10 mg/kg of 6AN to CD2F1 mice. 6AN reached peak serum concentrations of 80-90 microM and was cleared rapidly, with T1/2alpha and T1/2beta values of 7.4 and 31.3 min, respectively. Bioavailability was 80-100% with identical plasma pharmacokinetics after i.p. administration. At least 25% of the 6AN was excreted unchanged in the urine. The metabolite 6-amino-NAD+ was detected in perchloric acid extracts of brain, liver, kidney, and spleen, but not in serum. Efforts to prolong systemic 6AN exposure by administering multiple i.p. doses or using osmotic pumps resulted in lethal toxicity. These results demonstrated that 6AN exposures required to sensitize tumor cells to cisplatin in vitro are difficult to achieve in vivo.

Radiation enhancement by biochemical modulation and 5-fluorouracil.

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.

Apoptosis induced in advanced CD8F1-murine mammary tumors by the combination of PALA, MMPR and 6AN precedes tumor regression and is preceded by ATP depletion.

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 endlabeling, 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.

Evaluation of chemotherapy and radiation enhancement and 31P NMR spectral changes induced by biochemical modulation.

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 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 31P 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.

Chemotherapeutically induced DNA damage, ATP depletion, and the apoptotic biochemical cascade.

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.

Marked enhancement in vivo of paclitaxel's (taxol's) tumor-regressing activity by ATP-depleting modulation.

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.

In vivo detection by 31P NMR of pentose phosphate pathway block secondary to biochemical modulation.

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 6-phosphogluconate dehydrogenase step. Treatment with PALA, MMPR and 6AN also induced a decrease in the ratios of nucleoside triphosphate/inorganic phosphate (NTP/Pi) and phosphocreatine/inorganic phosphate (PCr/Pi) 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 PME'/PC and failed to cause a decrease in PCr/Pi or NTP/Pi 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.

The effect of 6-aminonicotinamide on testicular development in the rat.

6-Aminonicotinamide (6-AN), a niacin antagonist, was administered sc to pregnant female (1.0, 3.0, or 6.0 mg 6-AN/kg body weight) and neonatal male (1.5, 3.0, 6.0 or 12.0 mg 6-AN/kg body weight) Sprague-Dawley rats on the 15th, 17th and 19th days of gestation or the 5th, 7th and 9th days of life, respectively, to determine the effects of the antimetabolite on testicular morphology and development. In prenatal males, microscopic alterations were present in testes of fetuses from females treated with 6.0 mg 6-AN/kg and consisted of necrosis and loss of gonocytes, and vacuolation of interstitial cells. Histologic changes in testes of neonatal rats treated with 3.0, 6.0 or 12.0 mg 6-AN/kg were qualitatively similar with necrosis and loss of spermatogonia and supporting cells, and increased cross-sectional areas of affected tubules. Quantitation of the number of nuclei/cm2 of seminiferous tubule indicated 6-AN caused a significant reduction in the numbers of supporting cells and spermatogonia/tubular cross-section.

Correlation of retention of tumor methylmercaptopurine riboside-5'-phosphate with effectiveness in CD8F1 murine mammary tumor regression.

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.

Biochemical modulation of tumor cell energy. IV. Evidence for the contribution of adenosine triphosphate (ATP) depletion to chemotherapeutically-induced tumor regression.

DNA-damaging agents, e.g. Adriamycin (ADR), are reported to cause tumor regression by induction of apoptosis. A reduction in the intracellular content of ATP is part of the biochemical cascade of events that ultimately results in programmed death of the cell, or apoptosis. A chemotherapeutic three-drug combination (PMA) consisting of N-(phosphonacetyl)-L-aspartate (PALA) + 6-methylmercaptopurine riboside (MMPR) + 6-aminonicotinamide (6AN) significantly lowers levels of ATP in CD8F1 murine breast tumors in vivo and produces tumor regression by apoptosis. Addition of the DNA-damaging antitumor agent ADR to PMA was found to further significantly deplete ATP in CD8F1 murine breast tumors in vivo with a concomitant significant increase in the number of tumor regressions. The correlative biochemical and therapeutic results are consistent with, and support, the hypothesis that ATP depletion is a significant factor and, therefore, is a worthy therapeutic target in the production of apoptosis.

Effect of intracarotid administration of 6-aminonicotinamide on cerebral blood flow in cats.

BACKGROUND AND PURPOSE: We evaluated the effects of an adenosine triphosphate blocker, 6-aminonicotinamide (6-ANA), on the cerebral blood flow (CBF), cerebral metabolism, and electroencephalogram of cats. METHODS: Catheters were inserted into the common carotid artery of 16 adult cats anesthetized with ketamine via the lingual artery. We measured CBF in the infused area by the inhaled hydrogen gas clearance method and analyzed the electroencephalogram frequency. Cerebral metabolism was estimated by oxygen extraction (vol/%) and glucose utilization (millimoles) using data arterial (aorta) and sagittal sinus blood samplings. A solution of 6-ANA (6.0 mg/mL) (n = 8) or saline (n = 8) was infused via catheter at 2.0 mL/min for 3 minutes followed by a 60-minute observation of CBF, cerebral metabolism, vascular resistance, and the electroencephalogram components, alpha-2 ratio [= alpha-2/(alpha-1+alpha-2)]. The effect of 6-ANA on capillaries was evaluated by extravasation of Evans blue dye and electron microscopic findings. RESULTS: Moderate reductions in CBF, cerebral metabolism, and the alpha-2 ratio were observed during the infusion of 6-ANA versus saline infusion (P < .05 by paired t test and ANOVA). Vascular resistance was significantly increased (P < .05). No abnormalities were observed in the capillaries of the infused hemisphere. CONCLUSIONS: Results indicated that 6-ANA produced a downregulation of cerebral blood flow in cats.

Biochemical modulation of tumor cell energy in vivo: II. A lower dose of adriamycin is required and a greater antitumor activity is induced when cellular energy is depressed.

A quadruple drug combination--consisting of a triple-drug combination of N-(phosphonacetyl)-L-aspartate (PALA) + 6-methylmercaptopurine riboside (MMPR) + 6-amino-nicotinamide (6-AN), designed to primarily deplete cellular energy in tumor cells, + Adriamycin (Adria)--yielded significantly enhanced anticancer activity (i.e., tumor regressions) over that produced by either Adria alone at maximum tolerated dose (MTD) or by the triple-drug combination, against large, spontaneous, autochthonous murine breast tumors. The adenosine triphosphate (ATP)-depleting triple-drug combination administered prior to Adria resulted in a 100% tumor regression rate (12% complete regression; 88% partial regression) of spontaneous tumors. Histological examination of treated tumors demonstrated that the treatment-induced mechanism of cancer cell death was by apoptosis. The augmented therapeutic results (100% tumor regressions) were obtained with approximately one-half the MTD of Adria as a single agent and suggest the potential clinical benefit of longer, more effective, and safer treatment by low doses of Adria when combined with the triple-drug combination. Two likely mechanisms of action are discussed: (1) prevention of DNA repair; (2) complementary disruption of biochemical pathways by both the triple-drug combination and the biochemical cascade of apoptosis that is induced by a DNA-damaging anticancer agents such as Adria.

Potentiation of a three drug chemotherapy regimen by radiation.

The combination of N-(phosphonacetyl)-L-aspartate, 6-methylmercaptopurine, and 6-aminonicotinamide has been shown to be an effective antineoplastic regimen and also to enhance the effects of other chemotherapeutic agents. The mechanism of action of this combination of drugs is not known definitively, but one possible mechanism is biochemical modulation of energy metabolism and inhibition of production of tumor ATP. Tumor-bearing mice were treated with N-(phosphonacetyl)-L-aspartate, followed 17 h later by 6-methylmercaptopurine and 6-aminonicotinamide. 31P nuclear magnetic resonance spectroscopic studies demonstrated a significant depletion of high energy phosphates at 10 h post-6-methylmercaptopurine and 6-aminonicotinamide. The addition of radiation at this time was shown to induce a significantly longer tumor growth delay and a greater number of regressions (including durable complete regressions) than either chemotherapy or radiation alone. The combination of chemotherapy and radiation was found to be supra-additive compared to the antineoplastic effects of either modality administered separately, without a measurable increase in host toxicity.