Cross-resistance of drug-resistant murine leukemias to deoxyspergualin (NSC 356894) in vivo.

Deoxyspergualin, the 15-deoxy derivative of the antibiotic spergualin, is a novel guanidino analog structurally related to spermine. Deoxyspergualin has significant activity in selected experimental tumor models, and clinical trials have been initiated. Described here are in vivo evaluations of the therapeutic activity of deoxyspergualin against murine leukemia lines specifically resistant to eight clinically useful antitumor drugs. These were P388 lines resistant to doxorubicin, vincristine, L-phenylalanine mustard, cisplatin, ara-C, and methotrexate and L1210 lines resistant to 5-FU, L-phenylalanine mustard, and cyclophosphamide. Sensitivity to deoxyspergualin was evaluated in parallel comparisons of each resistant leukemia to the sensitive line from which it had been derived. All experiments were repeated at least once for confirmation of results. Responses were quantitated in terms of the change in tumor cell numbers from the beginning of treatment to the end of treatment as estimated from the median survival times of dying mice. The results indicated that P388 leukemia resistant to cisplatin (P388/DDPt) was cross-resistant to deoxyspergualin. No cross-resistance was observed in leukemias resistant to doxorubicin, vincristine, ara-C, methotrexate, or cyclophosphamide. L1210 resistant to 5-FU (L1210/5-FU) was collaterally sensitive to deoxyspergualin. Although cross-resistance was also observed in P388/L-PAM, L1210/L-PAM retained sensitivity to deoxyspergualin. Total glutathione concentrations in P388/L-PAM and L1210/L-PAM provided no apparent explanation for this unexpected result. It may be tentatively concluded that resistance to cisplatin, L-PAM, or other DNA alkylators or cross-linkers may increase the potential for cross-resistance to deoxyspergualin. This conclusion requires verification with additional alkylating agents, with drug-resistant human tumor cell lines, and with prospective clinical studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Response of drug-sensitive and -resistant L1210 leukemias to high-dose chemotherapy.

Alkylating agent-sensitive and -resistant L1210 leukemia cell lines were used to determine the tumor response to dose levels of drugs that exceeded conventional doses up to a factor of 10. Since those dose levels were lethal to the host mice, tumor response was based on the results of in vivo bioassays of spleen and/or tumor from drug-treated and control mice. When mice bearing about 10(8) drug-sensitive leukemic cells were treated with a single, conventional (approximately 10% lethal) dose of cis-diamminedichloroplatinum, L-phenylalanine mustard (melphalan), or 1,3-bis(2-chloroethyl)-1-nitrosourea, 10(1) to 10(4) tumor cells were recovered by bioassay. Treatment at doses that were 2 to 8 times the 10% lethal dose of either of those drugs resulted in no recoverable cells and survival of all bioassay recipient mice. Mice bearing advanced L1210 leukemia resistant to cis-diamminedichloroplatinum (L1210/DDPt), 1,3-bis-(2-chloroethyl)-1-nitrosourea (L1210/BCNU), cyclophosphamide (L1210/CPA), or melphalan(L1210/L-PAM) also were treated with a 10% lethal dose and greater doses of the drug to which the tumor line was resistant. Bioassay results indicated a direct correlation between dose intensity and tumor cell kill, the response being linear. Similarly, when mice with L1210/BCNU were treated with high doses of N-(2-chloroethyl)-N''-(2,6-dioxo-3-piperidinyl)-N-nitrosourea or 1,1',1''-phosphinothioylidynetrisaziridine (thioTEPA) and when mice with L1210/DDPt were treated with cyclophosphamide, an increasing, linear cell kill resulted throughout the high-dose range. Overall, these results indicate that resistance to these alkylating agents can be overcome by dose intensification and that the tumor response is linear in relation to increasing dose level.

Preclinical antitumor activity and pharmacological properties of deoxyspergualin.

A new antibiotic, deoxyspergualin (DSG), demonstrated antitumor activity against L1210 leukemia in mice. The life span of mice bearing either i.p. or s.c.-implanted L1210 increased greater than 150% following i.p. administration of 25 mg/kg DSG on days 1-9. Activity obtained with i.p. bolus treatments was schedule dependent. The tumor burden in mice bearing the s.c. implanted L1210 was reduced by 4-6 log10 units at the end of treatment when DSG was administered every 3 h for 8 injections on days 1, 5, and 9. By contrast, single injections of DSG on days 1, 5, and 9 allowed the tumor burden to increase at least 100-fold during treatment and daily single injections for 9 days reduced the tumor burden by 2 log10 units. The therapeutic advantage for i.p.-implanted L1210 of maintaining plasma concentrations of DSG was indicated further by infusion studies using s.c.-implanted Alzet osmotic pumps. Tumor burden was reduced by 3.5 and 6 log10 units following s.c. bolus treatments every 3 h on day 1 and a 24 h-infusion, respectively. The optimal infusion time for an infusion rate in mice of 179 mg/kg/day appeared to be 72 h. Pharmacokinetic studies following bolus i.v. injection revealed a rapid plasma clearance of parent drug (20.8 ml/min/kg) and a beta half-life of approximately 12 min. The bolus dose kinetics was used to predict the steady state plasma concentrations resulting from s.c. infusion; good agreement was observed between predicted values and experimental results. Based on these preclinical data, DSG has been developed to clinical trial. Initial Phase I protocols involve a 120-h infusion schedule.

Evaluation of trans-tetrachloro-1,2-diaminocyclohexane platinum (IV) in murine leukemia L1210 resistant and sensitive to cis-diamminedichloroplatinum (II).

trans-Tetrachloro-1,2-diaminocyclohexane platinum (IV) (tetraplatin) was therapeutically effective in mice bearing leukemia L1210 resistant (L1210/DDPt) or sensitive (L1210/0) to cis-diamminedichloroplatinum (II) (cisplatin). Furthermore, the sensitivity of cultured L1210/DDPt and L1210/0 cell populations to tetraplatin, cisplatin, and dichloro-trans-dihydroxyisopropylamine platinum (IV) (CHIP) was a function of the concentrations used for each compound. The relative degree of sensitivity between cultured L1210/DDPt and L1210/0 cells for each compound on the basis of the LC99 (the concentration of each compound required to reduce the number of viable cells by 99% in each cell line) was 3-fold for cisplatin, 2-fold for tetraplatin, and 3-fold for CHIP; thus the cultured L1210/0 cells exhibited a greater degree of sensitivity than the L1210/DDPt cells to the platinum compounds. The data indicate that if reduction of platinum IV compounds to platinum II compounds or metabolites is required for antitumor activity, then the cultured L1210 cells are capable of this bioreduction independently of any host factors.

Therapeutic synergism of tiazofurin and selected antitumor drugs against sensitive and resistant P388 leukemia in mice.

Tiazofurin is a synthetic "C" nucleoside analogue with a promising spectrum of experimental antitumor activity and a relatively novel mechanism of action. Previous work in our laboratories had revealed indications of collateral sensitivity and therapeutic synergism for selected murine tumor models treated with tiazofurin alone or in combination with an antimetabolite or an alkylating agent. Elucidation by others of biochemical indicators of tiazofurin activity provided the rationale for extending our studies to include the tiazofurin combinations reported here. Young, adult, female, BALB/c X DBA/2 F1 mice bearing body burdens of about 4 X 10(7) cells at the start of treatment were used. Cells were implanted either i.p. or s.c. Tiazofurin plus cisplatin or the 5'-palmitate of 1-beta-D-arabinofuranosylcytosine (ara-C) was evaluated against the parent P388/O leukemia line. Tiazofurin plus 6-thioguanine was evaluated against the ara-C-resistant P388. All drug treatments were i.p. injections given daily for 9 days. The experimental design permitted comparison of optimal nontoxic single-agent and two-drug combination regimens on the basis of the estimated log10 change in tumor cell burden at the end of treatment. Concurrent untreated control mice bearing tumor burdens ranging from approximately one to 10(7) cells permitted estimates of cells surviving treatment. Optimal treatment with each of these combinations afforded tumor burden reductions that were greater by 1 to 7 orders of magnitude than the effects of the respective single agents. Optimal single-agent and combination dosages (mg per kg per dose) were as follows: tiazofurin, 500; cisplatin, 2.0; the 5'-palmitate of ara-C, 25; 6-thioguanine, 0.8; tiazofurin, 330 plus cisplatin, 0.58; tiazofurin, 220 plus the 5'-palmitate of ara-C, 20; tiazofurin, 100 plus 6-thioguanine, 0.8. The observed therapeutic synergism of these drugs with tiazofurin in animal models suggests the possibility that treatment with tiazofurin combinations may yield clinical results superior to those obtained with the single agents alone. Therapeutic synergism can be most readily maximized when biochemical markers of drug action are available to provide appropriate clinical-laboratory correlations. Extension of these approaches to the use of tiazofurin, for which biochemical markers and experimental combination chemotherapy leads are now available, would support the rational clinical development of tiazofurin combinations.

Establishment of cross-resistance profiles for new agents.

Sublines of murine leukemias (L1210 and P388) and solid tumors selected for resistance to representatives of all of the chemical and functional classes of clinically useful anticancer drugs have been isolated and established in serial in vivo passage and, in some cases, in vitro culture. Extensive resistance, cross-resistance, and collateral-sensitivity patterns have been established with most of the sublines of the drug-resistant murine leukemias under treatment with greater than 100 different established and clinically useful anticancer drugs or new candidate anticancer drugs currently under study. Patients selected for inclusion in phase I-II trials usually have tumors that have failed to respond to treatment with established clinically useful drugs, either from the start of treatment or during continuing treatment after initial useful response. These treatment failures are no doubt due, in many cases, to drug-resistant tumors if initially unresponsive or to the overgrowth of drug-resistant mutant tumor stem cells in initially responding patients who ultimately failed under continuing treatment. Therefore, the cross-resistance profiles of drug-resistant murine tumors to treatment with new drugs going into phase I-II trials should provide useful guides for patient selection for those trials. Also, these cross-resistance profiles will provide useful information indicating likely biochemical mechanism of action of new drugs with promising anticancer activity, thus guiding drug selection for combination chemotherapy trials in animals or man. Numerous examples of all of the above indications for useful application of such information derived from chemotherapy trials with drug-resistant murine tumors are reported.

Isophosphoramide mustard, a metabolite of ifosfamide with activity against murine tumours comparable to cyclophosphamide.

Isophosphoramide mustard was synthesized and was found to demonstrate activity essentially comparable to cyclophosphamide and ifosfamide against L1210 and P388 leukaemia. Lewis lung carcinoma, mammary adenocarcinoma 16/C, ovarian sarcoma M5076, and colon tumour 6A, in mice and Yoshida ascitic sarcoma in rats. At doses less than, or equivalent to, the LD10, isophosphoramide mustard retained high activity against cyclophosphamide-resistant L1210 and P388 leukaemias, but was less active against intracerebrally-implanted P388 leukaemia while cyclophosphamide produced a 4 log10 tumour cell reduction. It was also less active (one log10 lower cell kill) than cyclophosphamide against the B16 melonoma. Metabolism studies on ifosfamide in mice identified isophosphoramide mustard in blood. In addition, unchanged drug, carboxyifosfamide, 4-ketoifosfamide, dechloroethyl cyclophosphamide, dechloroethylifosfamide, and alcoifosfamide were identified. The latter 4 metabolites were also identified in urine from an ifosfamide-treated dog. In a simulated in vitro pharmacokinetic experiment against L1210 leukaemia in which drugs were incubated at various concentrations for various times, both 4-hydroxycyclophosphamide and isophosphoramide mustard exhibited significant cytoxicity at concentration times time values of 100-1000 micrograms X min ml-1, while acrolein was significantly cytotoxic at 10 micrograms X min ml-1. Treatment of mice with drug followed by L1210 cells demonstrated a shorter duration of effective levels of cytotoxic activity for isophosphoramide mustard and phosphoramide mustard in comparison with cyclophosphamide and ifosfamide. Isophosphoramide mustard and 2-chloroethylamine, a potential hydrolysis product of isophosphoramide mustard and carboxyifosfamide, were less mutagenic in the standard Ames test than the 2 corresponding metabolites of cyclophosphamide [phosphoramide mustard and bis(2-chloroethyl)amine].

Some biochemical characteristics of L1210 cell lines resistant to 6-mercaptopurine and 6-thioguanine and with increased sensitivity to methotrexate.

L1210 cells resistant to 6MP and 6TG exhibit increased sensitivity to MTX compared to the parent line. The differential response of parent and purine analog-resistant cell lines to MTX is not due to host influences, for both L1210/6MP and L1210/6TG cell lines are cross-resistant to 6-MeMPR, an inhibitor of de novo synthesis, and cultured L1210/6MP cells are more sensitive to MTX than the parent cell line. Following treatment of tumor-bearing mice with MTX, the drug concentration in L1210/6TG cells was about 50% greater than in L1210/0 cells for 24 hr and may account, wholly or in part, for the increased sensitivity of the L1210/6TG cell line to MTX. L1210/6MP cells, however, accumulated less MTX than L1210/0 cells, indicating that an equivalent mechanism is not operative in these cells. DHFR activity in L1210/6TG cells was the same as that in L1210/0 cells, but activity in L1210/6MP cells was lower by 60%. Cultured L1210/6MP cells also exhibited a deficiency in DHFR activity as compared to the parent cell line. The sensitivity of the enzyme to MTX was the same for all three cell lines propagated in vivo. Therefore, the increased sensitivity of the L1210/6MP cell line to MTX may be due, in part, to decreased DHFR activity. Significantly lower levels of GTP + GDP and CTP in 6TG-resistant cells than in parent cells 4 hr after the administration of MTX to tumor-bearing mice may be related to the increased MTX sensitivity of these cells. Our results indicate that the observed alterations in drug sensitivity are associated with more than one biochemical change and that these changes are different in the two purine analog-resistant cell lines.

Response of transplantable tumors of mice to anthracenedione derivatives alone and in combination with clinically useful agents.

Other investigators have demonstrated that dihydroxyanthracenedione (DiOHA) and anthracenedione acetate (AA) are active against a broad spectrum of transplantable mouse tumors. DiOHA and AA are in clinical trial in the US; AA is in clinical trial in Europe. Because of the broad spectrum of activity of these DNA binders against murine tumors and due to their promising clinical utility, we have evaluated these agents in combination with a variety of clinically useful antitumor drugs. Studies were carried out against three colon adenocarcinomas (38, 06/A, and 11/A), three mammary adenocarcinomas (13/C, 16/C, and 14), and two lymphocytic leukemias (P388 and L1210). The therapeutic synergism of one of these combinations, DiOHA and cisplatin, has been previously reported. Four additional combinations which were found to have confirmed therapeutic synergism are reported here: DiOHA and palmO-ara-C, DiOHA or AA and 5-FU, DiOHA or AA and vincristine, and DiOHA and decarbazine. The combination toxicity indices (CTI; a measure of the degree of overlap in dose-limiting toxic effects) were obtained for all the following combinations: DiOHA and palmO-ara-C = 1.25-1.6; DiOHA or AA and 5-FU = 1.2-1.3; DiOHA or AA and vincristine = 1.6; and DiOHA and dacarbazine = 1.3-1.5. A CTI of 1.0 indicates complete overlap in dose-limiting toxic effects, eg, only 50% of the maximum tolerated dose of each agent can be used in combination. On the other hand, a CTI of 2.0 indicates no overlap in toxicity, and 100% of the maximum tolerated dose of each agent can be used in combination.

Synergistic antileukemic activity of combinations of two nitrosoureas.

A rationale based on known chemical, biochemical, and biologic properties of nitrosoureas led to the testing of combinations of CCNU with chlorozotocin for in vivo activity against L1210 leukemia. Several combinations yielded synergistic antileukemic activity.

cis-Dichlorodiammineplatinum(II): combination chemotherapy and cross-resistance studies with tumors of mice.

cis-Dichlorodiammineplatinum(II) (cis-platinum) has no more than additive, and often much less than additive, lethal toxicity for mice when given in combination with other anticancer agents representing several of the major functional classes of clinically useful anticancer drugs. The previously reported broad spectrum of anticancer activity of cis-platinum against tumors in laboratory animals has now been extended to promisingly useful therapeutic synergism in combination with other active anticancer drugs, including advanced-staged tumors in mice; eg, cis-platinum plus cyclophosphamide against advanced Ridgway osteogenic sarcoma and advanced P388 leukemia, and as surgical adjuvant chemotherapy against advanced colon tumor 26; cis-platinum plus Adriamycin against advanced P388; and cis-platinum plus VP-16-213 against advanced P388. Therapeutic synergism was also seen with cis-platinum plus carminomycin (an Adriamycin analog) against early colon tumor 26. Resistance and cross-resistance studies using sublines of L1210 and P388 selected for resistance to various alkylating agents (cyclophosphamide, melphalan, BCNU, or cis-platinum) indicate a variety of resistance and cross-resistance patterns which further support the growing body of evidence that wide differences in mechanism of cytotoxic activity exist among alkylating agents having experimentally and clinically useful anticancer activity. These data support the observed therapeutic synergisms with combinations of alkylating agents seen against a broad spectrum of murine tumors, and they suggest other drug combinations that might be considered for experimental and clinical trial based on a growing number of logical differences in biochemical mechanism of action of alkylating agent anticancer drugs that have been reported.

Therapeutic potentiation of nitrosoureas using chlorpromazine and caffeine in the treatment of murine tumors.

The therapeutic usefulness of chlorpromazine (CPZ) and caffeine (CAF) in combination with selected nitrosoureas was investigated in mice bearing L1210 leukemia, Lewis lung carcinoma, and B16 melanoma. We found that using BCNU with either CAF or CPZ was therapeutically superior to using either agent alone to treat mice bearing L1210 leukemia. Administering all three drugs in combination did not improve upon the therapeutic responses obtained with the two-drug combinations. In mice implanted with Lewis lung carcinoma or B16 melanoma, responses to treatment with the triple combination of methyl-CCNU, CAF, and CPZ suggested, but did not clearly establish, superiority over each two-drug combination or methyl-CCNU alone.

Comparative antitumor activity of actinomycin analogs in mice bearing Ridgway osteogenic sarcoma or P388 leukemia.

Actinomycin (Act) analogs, differing in the chemical substitution(s) made at various positions in either their pentapeptide chain(s) or chromophore ring, were evaluated for their antitumor activity in mice bearing either Ridgway osteogenic sarcoma (ROS) or P388 leukemia. Of the analogs tested against advanced (2--3-g) ROS tumors, azetomicin I and Act III caused therapeutic responses which, although variable, were nevertheless indicative of antitumor activities greater than was found using Act D. Several other analogs, Act C2, 2-N-(gamma-hydroxypropyl)-Act D, Act X0delta, and azetomicin II, displayed antitumor activity in ROS-bearing mice which varied, in different experiments, from comparable to superior to that achieved using Act D. Additionally, Act Pip1beta and 3'-(4-cisCl-Pro)-Act were comparable to, and Act-2-hydroxy-C3 inferior to, Act D in activity against ROS. Both azetomicin I and II were as effective as Act D in mice bearing P388 leukemia. Moreover, a subline of P388 that is resistant to Act D was cross-resistant to both azetomicin I and II.

Patterns of resistance and therapeutic synergism among alkylating agents.

Alkylating anticancer drugs are varied in chemical structure, alkylating moieties, and likely mechanisms of cytotoxic activity for vital normal cells and sensitive tumor cells. This has been objectively documented by numerous examples illustrating: (1) different in vitro and in vivo reaction products; (2) greater than additive, additive, and less than additive cytotoxicity of drug combinations for vital normal cells in the mouse; (3) readily reproducible and often marked therapeutic synergism between a variety of 2-drug combinations of alkylating agents against a wide variety of histologic types of murine tumors, and (4) observed resistance and cross-resistance of a variety of murine tumors, selected for resistance to specific alkylating agents, compatible with recognized chemical and functional differences between these drugs. The most important observations on resistance and cross-resistance reported are: (a) L1210 cells selected for complete resistance to cyclophosphamide (CPA) retain full sensitivity to selected nitrosoureas (BCNU, CCNU, MeCCNU), chlorozotocin), dianhydrogalactitol, and cis-DDPt, while retaining marked but somewhat reduced sensitivity to L-PAM, piperazinedione, and thioTEPA. (B) L1210 cells selected for resistance to BCNU retain full sensitivity to CPA, L-PAM, and dianhydrogalactitol. They show complete cross-resistance to BIC and variable cross-resistance to other selected nitrosoureas and piperazinedione. (c) L1210/L-PAM has incomplete but marked resistance to L-PAM. It is similar to the parent drug-sensitive line (L1210/0) in response to BCNU, CCNU, MeCCNU, and BIC. It is variably (usually moderately) cross-resistant to CPA, chlorozotocin, dianhydrogalactitol, and thioTEPA, but is completely cross-resistant to cis-DDPt. These resistance and cross-resistance patterns, which are consistent with most other biological and chemical principles established with these alkylating agents, may be useful in selecting alkylating drug combinations for inclusion in chemotherapy protocols in man which, on the basis of diverse observations in animal tumor systems, appear to be clearly indicated.