Mitomycin C combination therapy against murine tumor systems. Effectiveness with cyclophosphamide and methotrexate.

Mitomycin C (MMC) has been evaluated in combination with several antitumor agents. Full dose response curves were established for all drugs and drug combinations. Synergy was shown with MMC plus either cyclophosphamide (CYC) or methotrexate (MTX). In testing MMC and CYC against P388 leukemia, the combined treatment yielded a 75% rate of long-term survivors at the optimal level, compared to no survivors at the optimal level of the best single agent, CYC, alone. There was no increased toxicity among the combination-treated animals. Large increases in lifespan were obtained against L1210 and B16. Maximally tolerated doses of the single agents could be combined without increased toxicity. The combination of MMC and MTX was synergistic against ip L1210 and P388 leukemias. The responses of mice bearing L1210 to treatment on days 1, 5, and 9 respectively, were 42% ILS for 3.0 mg/kg MMC; 96% ILS for 15 mg/kg MTX; 172% ILS with four out of ten survivors for 3.0 mg/kg MMC plus 15 mg/kg MTX. MMC and adriamycin (ADR) were found to be synergistic against B16 melanoma at one schedule but not against another schedule, or against colon carcinoma 26. No improvements over optimal nontoxic single agent therapy were seen for chlorambucil, 5-fluorouracil, dibromodulcitol, cis-diaminedichloroplatinum or 4-'(9-acridinylamino) methansulfon-M-anisidide. On the basis of these data, recommendations were made for clinical trials for MMC plus either CYC or MTX against lung, breast, and colon tumors.

Correlates of vincristine resistance in four murine tumor cell lines.

The mechanism(s) of cellular resistance to vincristine (VCR) are poorly understood. Four murine tumor cell lines with varying degrees of VCR resistance, as measured by prolonged survival of tumor-bearing mice following VCR treatment, were selected for study. These lines were P1534, P388, P388/VCR and L1210. Steady-state cellular VCR levels, bound intracellular VCR, displaceable intracellular VCR, influx velocities and efflux velocities following VCR preloading were all measured in vitro and correlated with augmentation of survival. Neither the influx velocity, efflux velocity nor the steady-state VCR level showed any apparent correlation with in vivo sensitivity. Moreover, the ratio of influx velocity to efflux velocity was highest in the most sensitive cell line (i.e. P1534) and lowest in the most resistant cell line (i.e. P388/VCR). Bound intracellular VCR correlated best with VCR sensitivity suggesting that high-affinity intracellular binding, presumably to tubulin (Ka congruent to 1 X 10(-7) M), is a critical determinant of VCR sensitivity.

18F-5-Fluorouridine, a new probe for measuring the proliferation of tissue in vivo.

(1) Increased metabolic trapping of labeled fluorouridine reflects the interaction of three parameters in rapidly proliferating tissues: increased rates of intracellular phosphorylation, increased rates of transport, and increased rates of synthesis of RNA. (2) We have taken advantage of these metabolic phenomena, demonstrating in this paper that the uptake of 18F-5-fluorouridine, a positron-emitting radiopharmaceutical, can provide a very practical means for measuring changes in proliferative states of tissues in vivo. (3) Two major changes in proliferative states have been examined: one involves changes in growth of normal mouse tissues induced by pharmacological agents; the other involves tumor growth and neoplastic infiltration in mice and rabbits. (4) We describe tracer experiments with 18F-5-fluorouridylate, prepared by enzymatic means, and with 18F-5-fluorouridine, prepared by both enzymatic means and direct radiochemical procedures. (5) Uptakes of 18F after a pulse of 18F-5-fluorouridine were increased in mouse spleen following phenylhydrazine treatment to induce increased splenic erythropoiesis. (6) Uptakes of 18F in various mouse tissues were decreased following pretreatment with actinomycin D. This finding is consistent with the known inhibitory action of actinomycin on RNA synthesis. (7) Intracerebral Zimmerman ependymoblastoma tumors showed extraordinarily high uptakes of fluorine-18 in mice injected intravenously with 18F-5-fluorouridylate or with 18F-5-fluorouridine in contrast to very low uptakes by normal brain tissue. (8) After intracerebral injection of mice with suspensions of L1210 leukemia cells, distant organs such as lung, liver, and spleen became involved. These tissues showed significant increases of radioactivity after pulse labeling with 18F-5-fluorouridylate consistent with histological evidence for infiltration of these tissues by neoplastic cells. (9) Intramuscular VX2 carcinoma tumors in rabbits showed localized uptakes of 18F significantly higher than surrounding normal muscle tissue. (10) The most important clinical implication of the present work is the promise that 18F-5-fluorouridine uptakes can be followed in humans by positron emission tomography. This would provide a direct means of measuring different rates of in vivo proliferation in neoplasms, hematologic tissues and other organs undergoing rapid growth changes.

Enhancement of antitumor activity of alkylating agents by the radiation sensitizer misonidazole.

The influence of the radiosensitizer misonidazole on the effectiveness of several alkylating agents and cis-platinum against advanced solid murine tumors was investigated. Tumor regrowth delay, frequency of tumor regressions, and animal life span were used to evaluate misonidazole in combination with cyclophosphamide, L-phenylalanine mustard, 1-(2-chloroethyl)-3-trans-4-methylcyclohexyl)-1-nitrosourea, aziridinyl-benzoquinone, and cis-platinum. In the advanced M5076 ovarian carcinoma, misonidazole enhanced the activity of cyclophosphamide, L-phenylalanine mustard, 1-(2-chloroethyl)-3-trans-4-methylcyclohexyl)-1-nitrosourea, and aziridinyl benzoquinone, but not cis-platinum. In early B16 melanoma, misonidazole plus cyclophosphamide was no more effective than cyclophosphamide alone. In advanced Lewis lung carcinoma, misonidazole enhanced the antitumor activity of cyclophosphamide but not 1-(2-chloroethyl)-3-trans-4-methylcyclohexyl)-1-nitrosourea. Misonidazole, at 1000 mg/kg, increased the antitumor effectiveness of L-phenylalanine mustard and cyclophosphamide in M5076 tumors by factors of 2.2 and 1.8, but caused only a 1.2- and 1.3-fold increase in the myelotoxicity of these agents as determined by spleen colony assay of normal bone marrow. Misonidazole also increased the toxicity of cyclophosphamide and L-phenylalanine mustard in non-tumor-bearing mice but to a lesser degree than it enhanced antitumor activity. These results indicate that misonidazole is capable of enhancing the effects not only of ionizing radiation but of alkylating agents as well.

Effect of cholesterol content on antitumor activity and toxicity of liposome-encapsulated 1-beta-D-arabinofuranosylcytosine in vivo.

1-beta-D-Arabinofuranosylcytosine (ara-C) was encapsulated in anionic multilamellar liposomes prepared with different lecithin: cholesterol (L:C) ratios. The chemotherapeutic activity of encapsulated ara-C was compared with comparable doses of ara-C in 0.85% saline solution (single- and multiple-dose schedules) in mice bearing L1210 (i.p.) leukemia. Maximum survival was obtained in animals given injections of ara-C (40 mg/kg) encapsulated in liposomes with a L:C ratio of 1:1. The effect of L:C ratio on survival was not pronounced in multiple-dose schedules. Multiple doses (every 4.5 hr for 3 separate injections) of 40 mg/kg with L:C ratios of 1:1 and 1:0.5 were toxic, resulting in 83 and 50% mortality, respectively, of mice by Day 7. This study shows that drug efflux and in vivo antitumor activity and toxicity of encapsulated ara-C is influenced by the cholesterol content of the liposomal lipid bilayer.

Combination chemotherapy against B16 melanoma: bleomycin/vinblastine, bleomycin/cis-diamminedichloroplatinum, 5-fluorouracil/BCNU and 5-fluorouracil/methyl-CCNU.

Four antitumor drug combinations which are currently in clinical use were evaluated experimentally using the murine B16 melanoma model. Bleomycin plus vinblastine produced an increase in life span over either of the two agents alone against both intraperitoneal and subcutaneous B16. This combination also resulted in a large number of long-term survivors. Bleomycin plus cis-platinum produced slight enhancement against subcutaneous B16, but showed no advantage against intraperitoneal B16. The combination of 5-fluorouracil plus methyl-CCNU significantly increased survival time against the intraperitoneal tumor, and produced long-term survivors as well. The combination of 5-fluorouracil plus BCNU was not more effective than BCNU or 5-fluorouracil alone. These data were compared with the degree of success reported from the clinics against a variety of solid human neoplasms.

Pharmacokinetic considerations in evaluating the effects of tetrahydrouridine on 5-azacytidine chemotherapy in L1210 leukemic mice.

The pharmacokinetics of 5-azacytidine (5-azaCR) and tetrahydrouridine (THU) were considered in evaluating the effect of THU on chemotherapy with 5-azaCR in L1210 leukemia mice. The administration of three different dose levels of THU and 5-azaCR ip in either a 6- or 72-hour infusion gave minimal increases in therapeutic effect. At the high-dose combinations (except in the 72-hour infusion), THU appeared to enhance toxicity. Toxicity, however, occurred only after exceeding a theoretic plasma concentration for 5-azaCR of 61 microgram/ml. THU was effective in increasing the excretion of 5-azaCR by sixfold and in altering its urinary metabolites when given simultaneously with or up to 1 hour prior to 5-azaCR.

4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone, a second-generation antineoplastic agent of the alpha-(N)-heterocyclic carboxaldehyde thiosemicarbazone series.

4-Methyl-5-amino-1-formylisoquinoline thiosemicarbazone (MAIQ-1) was studied to determine its potential for clinical trail as a second-generation antineoplastic agent of the alpha-(N)-heterocyclic carboxaldehyde thiosemicarbazone class. MAIQ-1 was shown to be among the most potent known inhibitors of the major target for the expression of antineoplastic activity by this class of agents, the enzyme ribonucleoside diphosphate reductase, requiring only 0.06 micronM for 50% inhibition. This potency at the enzymatic level was consistent with its antineoplastic activity against the murine neoplasms Sarcoma 180, Leukemia L1210, Leukemia P388, and the B16 melanoma. The acetylation of the 5-amino group of the model substrate 5-amino-1,4-dimethylisoquinoline was lower than that of 5-amino-1-methylisoquinoline when incubated with acetyl-coenzyme A and rat liver homogenate. This finding suggests that the presence of the 4-methyl function offers steric hinderance to enzymatic substitution of the adjacent 5-amino group. In vivo metabolism of MAIQ-1 in mice, studied with [3'-14C]MAIQ-1 showed that relatively slow excretion of this agent occurred, since the cumulative urinary excretion of radioactivity was only 35% in 48 HR. About 51% of excreted urinary radioactivity was present in chromatograms in an area corresponding to the iron chelate of MAIQ-1, and only a minor quantity of material migrating like acetylated MAIQ-1 was present in urine, a finding consistent with enzymatic data with liver homogenates. The results indicate that MAIQ-1 has the antineoplastic activity, enzyme inhibitory potency, and relative resistance to metabolic inactivation required of an agent of this class for clinical trials.

L-Asparagine synthetase in serum as a marker for neoplasia.

L-Asparagine synthetase appears in serum approximately 7 days after the s.c. implantation of 1 X 10(5) cells of Leukemia 5178Y/AR (resistant to L-asparaginase) and increases in activity as the neoplasm grows and metastasizes. The principal source of the enzyme is the primary tumor. After intravranial inoculation of tumor, the rate of leakage of the enzyme is more pronounced than when the subcutaneous, intramuscular, or intraperitoneal routes are used. 1-(2-Chloroethyl)-3-cyclohexyl-1-nitrosourea (NSC 79037), a nitro-sourea effective in the palliation of L5178Y/AR, temporarily halts the influx of enzyme into the blood stream, as does surgical excision of the s.c. tumor nodules. Treatment of mice with L-asparaginase within 24 hr of inoculation of the tumor markedly augments both tumor growth and the rate of penetration of L-asparagine synthetase into the circulation. Several other L-asparagine synthetase into the circulation. Several other L-asparaginase-resistant tumors also were found to spill L-asparagine synthetase into the serum, but the correlation between this phenomenon and the specific activity of the enzyme in homogenates of the tumor was imperfect.

Cyclophosphamide (NSC-26271)-related phosphoramide mustards- recent advances and historical perspective.

The antitumor activity of three of the most phosphoramide mustards, NSC-69947, NSC-72505, and NSC-72510, was compared with that of phosphoramide mustard (NSC-69945), the apparent active metabolite of cyclophosphamide (CP), against the L1210, P388, B16, and Lewis lung tumor systems. This comparison did not reveal any significant differences in the patterns of inhibitory activity predictive of significant advantages in the clinic of any of these compounds over CP or NSC-69945. Attempts to prepare aldophosphamide, the key intermediate in CP metabolism, by oxidation of hydroxyphosphamide under the Sarett reaction conditions lead primarily to 4-ketocyclophosphamide. Under milder conditions the product isolated appears to be the elusive aldophosphamide on the basis of positive alkylating and aldehyde tests, Rf and infrared data, and the formation and characterization of a semicarbazone. The possibility that this product is an equilibrium mixture with the tautomeric 4-hydroxycyclophosphamide has not been as yet defintely ruled out. Sarett oxidation of homohydroxyphosphamide straightforwardly gives the stable analog, homoaldophosphamide. Biologic testing of this putative aldophosphamide in direct comparison with homoaldophosphamide, CP, and NSC-69945 reveals that aldophosphamide is a potent antitumor agent indistinguishable in activity from CP and NSC-69945, whereas homoaldophosphamide is inactive. These results provide confirmatory evidence for the postulated role of aldophosphamide as an intermediate in CP metabolism and suggest, furthermore, that aldophosphamide itself is not active in vivo, requiring transformation to NSC-69945 via beta-elimination of acrolein to exert its antitumor effects. An historical account of the development of the phosphoramide mustard field is also given.

Antitumor activity of amygdalin MF (NSC-15780) as a single agent and with beta-glucosidase (NSC-128056) on a spectrum of transplantable rodent tumors.

Experiments are described in which four transplantable rodent tumors (L1210 lymphoid leukemia, P388 lymphocytic leukemia, B16 melanoma, and Walker 256 carcinosarcoma) were used to investigate the antitumor activity of amygdalin MF. Amygdalin MF was given alone and in combination with beta-glucosidase which was administered 1/2 hour prior to amygdalin MF, starting 24 hours after tumor implantation. No antitumor activity was observed in any of the four tumor systems tested with the drug alone or in combined therapy. The combined therapy showed potentiation of toxicity with doses of amygdalin MF greater than or equal to 100 mg/kg.