Our laboratory has synthesized and evaluated the anticancer activity of a number of sulfonylhydrazine DNA modifying agents. As a class, these compounds possess broad spectrum antitumor activity, demonstrating significant activity against a variety of experimental murine tumors, including the P388 and L1210 leukemias, B16 melanoma, M109 lung carcinoma, and M5076 reticulum cell sarcoma, as well as against the human LX-1 lung carcinoma xenograft. The current report describes the activity of a more recently synthesized member of this class, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylamino)carbonylhydrazine (101M). 101M was active in mice against the i.p. implanted L1210 leukemia over a wide range of doses and produced long-term survivors when administered as a single i.p. bolus of 10, 20, 40, 60, or 80 mg/kg, demonstrating a wider margin of safety than the nitrosourea, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Curative therapy was achieved with doses of 101M that did not produce depression of the bone marrow. 101M was also highly effective against the L1210 leukemia when administered by the oral route. The ability of 101M to penetrate the blood-brain barrier and eradicate leukemia cells in the brain was remarkable (>6 log kill). This agent was also curative against L1210 variants resistant to cyclophosphamide, BCNU, or melphalan. Mice implanted with the murine C26 colon carcinoma were also cured by two injections of 10 or 20 mg/kg of 101M. Administration of 101M by two different well-tolerated regimens caused complete regression of established human glioblastoma U251 xenografts in 100% of treated mice, and significant responses were also obtained with 101M against advanced murine M109 lung carcinomas in mice. The broad spectrum of anticancer activity of the sulfonylhydrazine prodrug 101M coupled with the wide range of therapeutic safety exhibited by this agent, makes 101M particularly attractive for further development and clinical evaluation.

Tumor cell DNA is the primary target of many effective anticancer agents. Thus, DNA alkylating agents occupy a key position in the currently available chemotherapeutic arsenal. The cross-linking of DNA is believed to be the primary event responsible for the anticancer activity of most of the clinically useful alkylating agents (1, 2). One series of agents, shown to be capable of alkylating and cross-linking DNA (3, 4, 5) and which are highly effective in model tumor systems, are the CENUs.3 Clinically useful members of this class are BCNU and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU). These agents have been used effectively in the treatment of lymphomas (6) and a variety of human solid tumors including brain tumors (7); however, their utility is limited by serious toxicities. Studies on the decomposition of the CENUs have demonstrated the generation of several reactive species, with chloroethylating (8, 9), carbamoylating (8, 9, 10), hydroxyethylating (11), and vinylating (10) activities. Because the antitumor activity of the CENUs most likely results from the chloroethylation and subsequent cross-linking of DNA (12), the other biological reactivities may contribute little to the therapeutic value of these compounds and actually may contribute significantly to increasing the toxicity of these drugs. In fact, animal studies have shown that hydroxyethylation of DNA is largely a carcinogenic and/or mutagenic event (13, 14) and vinylation produces no known therapeutic benefit. The potential therapeutic contribution of the carbamoylating species (i.e., the isocyanate) generated by the CENUs is unclear. Isocyanates, which can react with thiol and amine functionalities in proteins, have been shown to inhibit DNA polymerase II at the enzyme level rather than acting directly on the DNA (15), thereby preventing the repair of DNA strand breaks (16). BCNU has also been shown to inhibit the thiol-containing enzymes, glutathione reductase, ribonucleotide reductase, and thioredoxin reductase (17). Some of these properties undoubtedly contribute to the toxic side effects of the CENUs (18, 19, 20, 21); however, it is possible that isocyanate generation in tumor cells may act to prevent the repair of DNA alkylations and cross-links, thereby potentiating the cytocidal activity of the chloroethylation induced by the CENUs. Thus, one can envision the potential improved usefulness of agents that possess both chloroethylating and carbamoylating properties but lack hydroxylating and vinylating activities.

Our laboratory has been involved in the design, synthesis, and biological evaluation of a new class of antitumor compounds, the 1,2-bis(sulfonyl)hydrazines (also called SHPs), which generate, through activation, the reactive electrophilic structures that are capable of alkylating and cross-linking DNA. The chloroethylating species generated by these agents are similar to, but not identical to, the ones produced by the CENUs. Penketh et al.(22) have recently compared the DNA lesions produced by SHPs with those produced by the CENUs. One of the SHPs, 101M, which was designed to retain chloroethylating and carbamoylating activities but not hydroxylating and vinylating activities, demonstrated pronounced antitumor activity in mice in preliminary studies (23). We now report the results of preclinical studies with this promising new antineoplastic agent, which include several direct comparisons of the therapeutic efficacy of 101M with that of the clinically useful CENU, BCNU.

Drugs and Mice.

101M and 90CE were synthesized according to published methodology (23, 24). The chemical structure of 101M is shown in Fig. 1. All of the other agents were purchased from Sigma Chemical Co. (St. Louis, MO).

BALB/c × DBA/2 (CD2F1) mice, 8–10 weeks of age, were used with the L1210 leukemia, the P388 leukemia, and the M109 lung carcinoma. Athymic nu/nu mice were used with the human U251 glioma xenograft. BALB/c mice were used with the colon 26 carcinoma. Mice were treated i.p. with drugs dissolved in 100% dry DMSO and delivered at a rate of 0.001 ml/g of mouse body weight or in some studies with drugs prepared in solutions of 10% DMSO-90% water and delivered at a rate of 0.01 ml/g of mouse body weight, or in pure water and delivered at rates of 0.01–0.03 ml/g of mouse body weight. For oral administration, 101M was prepared as a fine suspension in corn oil and delivered by catheter gavage.

Leukemia Studies.

In studies with the parental L1210 leukemia, female CD2F1 mice were inoculated i.p. with 1 × 105 cells. For studies with drug-resistant variants of the L1210 or with the P388 leukemia, the inoculum level was increased to 1 × 106 cells. Twenty-four h after tumor inoculation, treatment was initiated on the schedule indicated. Postinoculation life span was monitored for 60 days.

To determine the effects of 101M on leukemia cells in the brain, a bioassay procedure developed by Skipper et al.(25, 26) was used. Briefly, CD2F1 mice were given i.p. injections of drug or of 0.9% NaCl 24 h after intracerebral inoculation of 1 × 105 L1210 cells. Approximately 24 h later, the mice were killed and the brains of three mice/experimental group were collected. A brei was prepared from the three pooled brains, and the equivalent of one-half of a brain was injected i.p. into each of five mice of the same strain. Mean postinoculation life spans were then determined and viable L1210 cells/brain were estimated by using concomitantly derived inoculum response data that defined the relationship between life span and inoculum size.

Solid Tumor Studies.

Three solid tumors were tested for responsiveness to treatment with 101M. For the murine M109 lung carcinoma, tumor brei were implanted s.c. into CD2F1 mice, and therapy was initiated 9 days later and continued on the schedule indicated. In studies with the human U251 glioma, fragments of the tumor were implanted s.c. in athymic mice, and therapy was initiated 21 days later and continued on the schedule indicated. For both of these models, tumor growth delay/regression was the evaluation end point; thus, tumor measurements were recorded on the initial day of treatment and at various times thereafter. In studies with the colon 26 carcinoma, tumor brei were implanted i.p., and two treatments with the test agents were administered on postimplant days 1 and 5. Life span was the evaluation end point for this experimental tumor system.

Determination of Blood Leukocyte Counts.

Peripheral blood was obtained from the retroorbital sinus of mice at selected times after i.p. treatment with 101M. After lysis of RBCs with Zapoglobin II reagent (Coulter Electronics, Miami, FL), the total blood leukocyte counts were determined using a Coulter Multisizer II particle counter.

Cell Culture Studies with the Human HT-29 and BE Colon Carcinomas.

BE and HT-29 human colon carcinoma cells were provided by Dr. Neil Gibson (Bayer Corporation, West Haven, CT). Stock cultures were grown at 37°C as monolayers in DMEM supplemented with 15% FCS and containing 100 units/ml penicillin and 0.1 mg/ml streptomycin. For the growth inhibition studies, 250 cells were seeded into each well of a 96-well tissue culture plate. Approximately 24 h later, test agents were added to the wells, and incubation was continued for 5 days. The numbers of viable cells were then estimated using the CellTiter 96 AQ cell proliferation assay (Promega, Madison, WI).

In preliminary studies in mice, of the SHPs tested, 101M was found to possess superior activity against both the L1210 leukemia and the B16F10 melanoma (23). In the present more detailed studies, 101M demonstrated activity in mice against the i.p. implanted L1210 leukemia over a wide range of doses, producing long-term survivors when administered as a single i.p. bolus dose of 10, 20, 40, 60, 80, or 100 mg/kg (Table 1). The therapeutic utility of 101M was also superior to the clinically useful CENU, BCNU, in this model of acute leukemia. Both BCNU and 101M produced highly significant increases in life span. Note that treated (T)/control (C) values greater than 150% were produced at several dosages of both agents and both compounds were capable of producing long-term survivors (i.e., cures; mice alive and tumor-free 60 days after tumor inoculation). The data presented in Table 1 also indicate that 101M is therapeutically superior to BCNU. At least some long-term survivors were produced with each of the six doses of 101M from 10 to 100 mg/kg and 100% of the mice were cured at doses of 20, 40, and 60 mg/kg. In contrast, BCNU produced long-term survivors only at doses of 40 and 60 mg/kg, with neither regimen producing 100% cures. Moreover, mice died of acute drug toxicity at the next higher dose of 80 mg/kg of BCNU. Thus, 101M exhibited a considerably wider therapeutic safety range than BCNU.

As shown in Table 2, 101M was also highly effective against the i.p. implanted L1210 leukemia when administered by the oral route, curing mice with single treatments of 60, 80, 100, 200, and 300 mg/kg. Although we do not have pharmacological bioavailability information on 101M, we can estimate by comparison of the minimum curative single doses when given i.p. with that when given p.o., that a single oral dose of 60 mg/kg has roughly the equivalent curative potential as an i.p. dose of 10 to 20 mg/kg. As gauged by body weight change, curative oral doses did not produce acute toxicity.

It should be noted from the data reported in Table 1, that the high bolus doses of 80 and 100 mg/kg of 101M were not as effective as the lower curative doses (10–60 mg/kg) in producing 60-day survivors. This was not attributable to acute toxicity, however, because the times of occurrence of death in these mice were in excess of 50 days after treatment. These late-dying mice displayed no evidence of tumor development yet showed signs of a wasting condition (loss of body weight, small spleen, and loss of inguinal fat). Evaluation of the extent of bone marrow toxicity (gauged by depression of the peripheral WBC count) of normal mice treated with single i.p. injections of 101M at doses that produced long-term survivors in the leukemia studies demonstrated that very little marrow depression occurred at the curative doses of 10 and 20 mg/kg (Fig. 2). At the higher doses, the WBC counts were depressed with a nadir at about 4 days, but blood counts, even at the 80 mg/kg dose, uniformly recovered to normal levels by 3 weeks after treatment. Thus, curative therapy of L1210 leukemia-bearing mice can be achieved with 101M at doses that produce little bone marrow toxicity (i.e., doses of 10 and 20 mg/kg).

To document the occurrence of “late nontumor deaths” in the leukemia studies, mice that were “cured” were observed for extended periods of time (i.e., longer than 60 days). This information is summarized in Table 3. Late deaths occurred only in mice treated with relatively high bolus doses of 101M (≥80 mg/kg i.p. and ≥100 mg/kg p.o.). It is important to note that these doses far exceed those required to cure L1210 leukemia-bearing mice. In an experiment designed to test the efficacy of repeated administration of aqueous formulations of 101M against the L1210 leukemia (Table 4), no late toxic deaths occurred when mice were treated at a dosage of once every day for 6 doses, days 1–6, with 18 mg/kg/day (a cumulative dosage of 108 mg/kg). This result suggests that the apparent delayed toxicity was attributable to excessively large acute bolus exposure (≥80 mg/kg) and that toxicity is probably not cumulative.

Because the CENUs such as BCNU, largely because of their capacity to traverse the BBB, are among the most effective chemotherapeutic agents available for the treatment of brain tumors, we were interested in the potential of the SHPs against tumors of the CNS. We have used a quantitative bioassay system (25, 26) to compare the ability of chemotherapeutic agents to cross the BBB and kill tumor cells in the brain. Comparative results of 101M and clinically established agents (27) in the brain bioassay system are shown in Table 5. Drugs such as methotrexate are unable to cross the BBB as evidenced by the lack of a reduction in the tumor cell burden in the brain tissue. Other drugs such as 6-mercaptopurine and doxorubicin are marginally effective against tumor cells in the CNS. Cyclophosphamide is able to reach and kill tumor cells in the brain, producing nearly a one log reduction in the number of tumor cells. Several of the SHPs, including 101M, were effective in crossing the BBB and eradicating leukemia cells in the CNS (>6 log kill), rivaling the efficacy of BCNU, which is one of the most effective agents for the treatment of experimental neoplasms in the CNS.

Because curative therapy of the L1210 leukemia was possible with doses of 101M that did not produce severe depression of the bone marrow (Fig. 2), multidose scheduling of 101M should allow effective treatment of tumors with various growth characteristics. In addition, because 101M was effective at crossing the BBB, we were interested in whether it would be effective against a primary brain tumor. This was demonstrated with the U251 human glioma xenograft model. When administered to athymic nude mice implanted with this tumor using two dosage regimens that were well tolerated by the host, 101M caused complete regression of tumors in 16 of 16 treated mice (Fig. 3). Both treatment regimens of 101M resulted in cumulative doses exceeding 100 mg/kg yet produced no manifestations of the delayed wasting condition. Thus, it appears that treatment schedules can be designed that provide effective therapy for experimental leukemias as well as for solid tumors without the imposition of excessive host toxicity. The relatively wide range of safety in dosing makes this agent particularly attractive for further development.

101M was also highly active against an L1210 variant that is resistant to BCNU (L1210/BCNU; Table 6). Although BCNU was marginally effective against L1210/BCNU, 101M was considerably more active, producing 100% long-term survivors with single treatments at two dosage levels. BCNU also had a low therapeutic index against this tumor. It is important to note that L1210/BCNU has multiple mechanisms of resistance, possessing a >3-fold increase in the levels of the DNA repair enzyme, AGT, as well as increases in the levels of glutathione S-transferase, glutathione reductase activities, and increased reduced glutathione levels compared with the parental L1210 leukemia (28, 29). Pharmacological doses of 101M do not impact the pool of reduced glutathione and do not inhibit glutathione reductase.4 Thus, 101M may be an attractive alternative to BCNU, not only because of its greater therapeutic index, but also because it possesses superior activity against nitrosourea-resistant tumors. 101M also exhibited pronounced activity in mice against cyclophosphamide-resistant (Table 7) and melphalan-resistant (Table 8) L1210 sublines, making this agent an attractive alternative to these drugs.

Because one of the most valuable predictors of clinical activity of a potential anticancer drug is broad-spectrum activity in preclinical model tumor systems, we examined the efficacy of 101M in several additional model systems. Table 9 shows the results of an experiment with the murine P388 lymphoid leukemia. In this dose-ranging study, 101M was highly effective, producing long-term survivors at all of the six dose levels used between 10 and 100 mg/kg.

Mice implanted with the murine C26 colon carcinoma were cured by two injections of 10, 20, 40, or 60 mg/kg of 101M (Table 10). Although BCNU was also effective in this model system, the CENU demonstrated a narrower safety range than 101M. Significant activity by 101M was also demonstrated against the advanced M109 murine lung carcinoma in mice (Fig. 4). In previously published studies from our laboratory, when treatment was initiated 24 h after tumor implantation, cyclophosphamide produced a tumor growth delay (time to reach a mass of 1 gram) of 8.8 days (23). In the current study, the s.c. tumor implant was allowed to grow for nine days, at which time tumors were easily palpable, before initiating treatment with 101M. With the average starting size of these established tumors at ≈150 mg, 101M produced significant growth delays of 8 and 14 days for the 10 and 20 mg/kg/injection regimens, respectively. Thus, the growth delay produced by the 20 mg/kg/injection was more pronounced than that produced by cyclophosphamide in previous studies with far-less-well-established tumors.

Because expression of the DNA repair protein, AGT, by tumor cells limits the effectiveness of the CENUs, we were interested in whether the activity of 101M would be affected by the AGT status of the tumor. We used two human colon tumor cell lines with different levels of AGT expression in cell culture studies to address this question (30). Cells that express AGT activity and, therefore, have the capacity to repair O6-alkylguanine lesions in the DNA are termed Mer+, and those with very low or negligible AGT expression are termed Mer. As shown in Fig. 5, 101M was found to be nearly as effective in inhibiting the growth of Mer+ HT-29 colon carcinoma cells as Mer BE colon carcinoma cells, which express very low levels of AGT. This is in sharp contrast to the differential sensitivity of these two tumor lines to 90CE, a SHP that generates the identical alkylating species as 101M but that does not generate methyl isocyanate. This result implicates methyl isocyanate in overcoming the AGT-dependent resistance to chloroethylation-induced cytotoxicity. This concept has been strengthened by recent studies in our laboratory that have shown that methyl isocyanate can inhibit AGT activity in simple in vitro chemical systems.5

The SHPs represent a new class of agents that possess broad spectrum antineoplastic activity against a variety of experimental murine and human tumors (22, 23, 24). The discovery of numerous agents in this class with significant activity in the preclinical setting has necessitated additional studies aimed at the identification of an agent or agents most likely to be of benefit in the clinic and, therefore, to merit further development. We have used the i.p. implanted L1210 lymphocytic leukemia in mice as an initial test system to provide quantitative comparisons of the therapeutic potential of the various SHPs among themselves, as well as comparisons with proven clinically useful agents. This system was selected because the results obtained are highly reproducible, readily quantifiable, and rapidly generated. Furthermore, a single L1210 cell has the malignant potential to replicate and kill the host mouse in about 18 days, and a control mouse inoculated i.p. with 1 × 105 cells will die about 7 days after inoculation. In addition, because i.p. inoculated cells disseminate rapidly throughout the body and are found in tissues within 3 h after implantation, candidate drugs must reach and eradicate every leukemia cell in tissues to effect a cure (25). The use of an animal tumor:host system also provides valuable information about the comparative therapeutic utility of compounds of interest. Of the many SHPs with activity in this system (examples can be found in Refs. 23 and 24), 101M demonstrated the widest margin of safety, curing mice with a single treatment over a wide range of doses. Moreover, the ability of 101M to cure mice at levels that did not produce severe weight loss or depression of the bone marrow indicates that this agent may be used effectively on a variety of scheduling regimens, thereby being applicable to the treatment of tumors with diverse growth characteristics.

Although 101M has limited solubility in water, effective scheduling regimens using aqueous solutions of 101M can be designed to produce curative therapy for L1210 bearing mice (Table 4). Improved formulations containing polyethylene glycol, ethanol, and water are planned for use in future clinical trials.

The ability of 101M to penetrate the BBB of mice, with its activity against human tumors of CNS origin, coupled with its exceptional therapeutic properties, suggests that this agent may be clinically superior to the CENUs in the treatment of brain tumors. The importance of the potential role of 101M in the treatment of these tumors can be appreciated by an understanding of the immensity of the CNS tumor problem. The combined incidence of primary intracranial and spinal axis tumors is between 2 and 19 in 100,000 persons per year, depending on age, with CNS neoplasms being the most common solid tumors of childhood (31). Brain metastases from primary tumors originating in other tissues are the most prevalent intracranial tumors among adults, with as many as 170,000 new cases of brain metastases occurring each year in the United States and between 20 and 40% of all cancer patients developing CNS involvement (32). Because 101M was capable of readily crossing the BBB and was active against BCNU-, cyclophosphamide-, and melphalan-resistant tumor cells, and was highly active against a human glioblastoma xenograft, as well as against tumors of various tissue types, this new agent may have an immediate niche in the treatment of CNS tumors.

An additional aspect favoring 101M as a clinically useful alternative to presently available alkylating agents is the limited responsiveness of human tumors, especially brain tumors, to treatment with CENUs because of the expression of the DNA repair protein, AGT. This enzyme protects the genome from the toxicity of various CENUs by removing the alkyl group transferred by the alkylating agent onto the O6 position of guanine prior to the formation of the lethal DNA cross-link (33, 34). AGT is significantly overexpressed in numerous human cancers including many brain tumors (35) and, therefore, plays an important role in the sensitivity of these tumors to alkylating agents such as the CENUs. The carbamoylating species (i.e., the isocyanate) generated by the CENUs can react with thiol and amine functionalities in proteins. We postulated that the methyl isocyanate generated by 101M could inhibit the DNA repair activity of AGT, an enzyme containing a cysteine thiol in its active site (33, 34), and could, therefore, be beneficial in the treatment of AGT-expressing tumors. This notion is supported by the work of Gibson and Hickman (21) with the TLX5 lymphoma, which is naturally resistant to alkylating agents of the 2-chloroethylamine type, yet is sensitive to the CENUs; these authors postulated that intracellular release of isocyanates from the CENUs may overcome the resistance mechanism. We indirectly addressed this possibility using two human colon tumor cell lines with very different levels of AGT expression. 101M was found to be nearly as effective in inhibiting the growth of Mer+ HT-29 colon carcinoma cells as it was with Mer BE colon carcinoma cells, which express very low levels of AGT. This is in sharp contrast to the differential sensitivity of these two tumor lines to 90CE, a SHP that generates the identical alkylating species as 101M but that does not generate methyl isocyanate. Thus, when the DNA alkylating/cross-linking capacity of the sulfonylhydrazines is coupled with the capacity to generate an isocyanate to inhibit AGT, the spectrum of activity of the SHPs is extended to Mer+ tumor cells.

Broad spectrum activity in transplantable tumor models is one of the most valuable predictors of clinical potential. In these studies, 101M demonstrated significant activity in each of nine different model tumor systems, including several leukemias and solid tumors originating from colon, lung, and brain. In previous evaluations, 101M was also active against the murine B16 melanoma (23). Taken together, with the superiority of 101M over BCNU, its broad-spectrum antitumor activity in preclinical models, the avoidance of the mutagenic/carcinogenic activities of hydroxyethylation, the demonstrated activity against tumors resistant to other clinically useful alkylating agents, attractive therapeutic properties including oral activity, and its effectiveness against tumor cells in the CNS, suggest that 101M exhibits sufficient properties to merit consideration for further development and clinical trial.

Fig. 1.

Chemical structure of 101M.

Fig. 1.

Chemical structure of 101M.

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Fig. 2.

Effects of a single i.p. bolus dose of 101M on the peripheral WBC count of female CD2F1 mice. Data are average values of three mice per dosage level.

Fig. 2.

Effects of a single i.p. bolus dose of 101M on the peripheral WBC count of female CD2F1 mice. Data are average values of three mice per dosage level.

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Fig. 3.

Effects of multiple dose regimens of 101M on the growth of the U251 human glioma in athymic nude mice. Treatment on the indicated schedule was initiated 21 days after s.c. implantation of tumor fragments near the flank. The average tumor volume was ≈300 mm3 at the time of the first treatment. Tumor size was estimated from caliper measurements made every 4 days using the formula for the volume of a prolate ellipsoid. Data are average values of eight mice per treatment group. q2d×11, every 2 days for 11 doses; q4d×6, every 4 days for 6 doses.

Fig. 3.

Effects of multiple dose regimens of 101M on the growth of the U251 human glioma in athymic nude mice. Treatment on the indicated schedule was initiated 21 days after s.c. implantation of tumor fragments near the flank. The average tumor volume was ≈300 mm3 at the time of the first treatment. Tumor size was estimated from caliper measurements made every 4 days using the formula for the volume of a prolate ellipsoid. Data are average values of eight mice per treatment group. q2d×11, every 2 days for 11 doses; q4d×6, every 4 days for 6 doses.

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Fig. 4.

Growth of the murine M109 lung carcinoma in mice treated with 101M. Treatment on the indicated schedule was initiated 9 days after s.c. implantation of tumor fragments. The average tumor volume was ≈150 mm3 at the time of the first treatment. Tumor size was estimated from caliper measurements made every 2 days. Data are average values of five mice per treatment group. q2d×10, every 2 days for 10 doses; q4d×5, every 4 days for 5 doses.

Fig. 4.

Growth of the murine M109 lung carcinoma in mice treated with 101M. Treatment on the indicated schedule was initiated 9 days after s.c. implantation of tumor fragments. The average tumor volume was ≈150 mm3 at the time of the first treatment. Tumor size was estimated from caliper measurements made every 2 days. Data are average values of five mice per treatment group. q2d×10, every 2 days for 10 doses; q4d×5, every 4 days for 5 doses.

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Fig. 5.

Growth inhibitory capacity of 90CE and 101M for cultured colon carcinoma cell lines with differential expression of AGT activity. Colon carcinoma cells were subcultured at a density of 200 cells per well in 96-well plates. Twenty-four h later, various concentrations of either 90CE or 101M were added to the wells. Five days thereafter, the number of viable cells was determined using the CellTiter 96 AQ cell proliferation assay (Promega).

Fig. 5.

Growth inhibitory capacity of 90CE and 101M for cultured colon carcinoma cell lines with differential expression of AGT activity. Colon carcinoma cells were subcultured at a density of 200 cells per well in 96-well plates. Twenty-four h later, various concentrations of either 90CE or 101M were added to the wells. Five days thereafter, the number of viable cells was determined using the CellTiter 96 AQ cell proliferation assay (Promega).

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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This research was supported in part by USPHS Grant CA-74970 from the National Cancer Institute. R. A. F. is a Special Fellow of the Leukemia and Lymphoma Society.

3

The abbreviations used are: 101M, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylamino)carbonylhydrazine; CENU, (chloroethyl)nitrosourea; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; SHP, sulfonylhydrazine prodrug; i.p., intraperitoneal; BBB, blood-brain barrier; CNS, central nervous system; AGT, O6-alkylguanine-DNA alkyl transferase; 90CE, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine.

4

P. G. Penketh et al., unpublished observations.

5

P. G. Penketh, K. Shyam, R. P. Baumann, and A. C. Sartorelli. Role of O6-alkylguanine-DNA alkyltransferase in the inhibition of the cross-linking of T7 DNA by 1,2-bis(sulfonyl)hydrazines, manuscript in preparation.

Table 1

Comparison of the effectiveness of a single i.p. treatment of 101M or BCNU against i.p. implanted L1210 leukemia in mice

Female CD2F1 mice were inoculated i.p. with 1 × 105 leukemia cells. A single i.p. treatment was administered 24 h later. Drugs were dissolved in DMSO and delivered at a rate of 0.001 ml/g of mouse body weight. Data represent at least five mice per treatment group.
Dosage (mg/kg)101MBCNU
 Δ wt.a (%) T/Cb (%) Long-termc survivors (%) Δ wt.a (%) T/Cb (%) Long-termc survivors (%) 
1.25 +7.4 117 +10.5 103 
2.5 +4.6 131 +8.9 114 
−1.0 160 +3.5 137 
10 +1.0 229 60 −1.8 209 
20 −0.8  100 +0.0 170 
40 −0.9  100 −3.2 200 80 
60 −2.8  100 −7.4 429 80 
80 −6.7 800 80 Toxic 77 
100 −17.4 814 33 ndd nd nd 
Female CD2F1 mice were inoculated i.p. with 1 × 105 leukemia cells. A single i.p. treatment was administered 24 h later. Drugs were dissolved in DMSO and delivered at a rate of 0.001 ml/g of mouse body weight. Data represent at least five mice per treatment group.
Dosage (mg/kg)101MBCNU
 Δ wt.a (%) T/Cb (%) Long-termc survivors (%) Δ wt.a (%) T/Cb (%) Long-termc survivors (%) 
1.25 +7.4 117 +10.5 103 
2.5 +4.6 131 +8.9 114 
−1.0 160 +3.5 137 
10 +1.0 229 60 −1.8 209 
20 −0.8  100 +0.0 170 
40 −0.9  100 −3.2 200 80 
60 −2.8  100 −7.4 429 80 
80 −6.7 800 80 Toxic 77 
100 −17.4 814 33 ndd nd nd 
a

Δwt, change in weight from onset of therapy to day 6.

b

T/C (%), mean life span of treated (T) mice/mean life span of control (C) mice × 100. T/C calculations do not include long-term survivors.

c

Long-term survivors, mice alive and tumor-free 60 days after inoculation.

d

nd, not determined.

Table 2

Effectiveness of a single oral treatment of 101M against i.p. implanted L1210 leukemia in mice

Female CD2F1 mice were inoculated i.p. with 1 × 105 leukemia cells. A single oral treatment was administered 24 h later. 101M was suspended in corn oil and delivered at a rate of 0.01 ml/g of mouse body weight. Data represent five mice per treatment group.
Dosage (mg/kg) Δ wt.a (%) T/Cb (%) Long-termc survivors (%) 
40 +2.6 214 
60 +1.8  100 
80 +0.9  100 
100 −3.9  100 
200 −1.9 457 80 
300 −2.7 314 60 
Female CD2F1 mice were inoculated i.p. with 1 × 105 leukemia cells. A single oral treatment was administered 24 h later. 101M was suspended in corn oil and delivered at a rate of 0.01 ml/g of mouse body weight. Data represent five mice per treatment group.
Dosage (mg/kg) Δ wt.a (%) T/Cb (%) Long-termc survivors (%) 
40 +2.6 214 
60 +1.8  100 
80 +0.9  100 
100 −3.9  100 
200 −1.9 457 80 
300 −2.7 314 60 
a

Δwt, change in weight from onset of therapy to day 6.

b

T/C (%), mean life span of treated (T) mice/mean life span of control (C) mice × 100. T/C calculations do not include long-term survivors.

c

Long-term survivors, mice alive and tumor-free 60 days after inoculation.

Table 3

Occurrence of delayed toxicity in long-term survivors of L1210-inoculated 101M-treated mice

RouteScheduleaDosage (mg/kg)Long-term survivorsDelayed toxic deathsTime of death (days after treatment)
i.p. qd×1; d1 10 3/5 0/5 ndb 
  20 5/5 0/5 nd 
  40 5/5 0/5 nd 
  60 5/5 0/5 nd 
  80 8/8 3/8 55, 56, 84 
  100 6/6 5/6 58, 59, 96, 100, 100 
p.o. qd×1; d1 40 5/5 0/5 nd 
  60 5/5 0/5 nd 
  80 5/5 0/5 nd 
  100 5/5 3/5 93, 102, 119 
  200 4/5 1/4 97 
  300 3/5 3/3 87, 100, 101 
RouteScheduleaDosage (mg/kg)Long-term survivorsDelayed toxic deathsTime of death (days after treatment)
i.p. qd×1; d1 10 3/5 0/5 ndb 
  20 5/5 0/5 nd 
  40 5/5 0/5 nd 
  60 5/5 0/5 nd 
  80 8/8 3/8 55, 56, 84 
  100 6/6 5/6 58, 59, 96, 100, 100 
p.o. qd×1; d1 40 5/5 0/5 nd 
  60 5/5 0/5 nd 
  80 5/5 0/5 nd 
  100 5/5 3/5 93, 102, 119 
  200 4/5 1/4 97 
  300 3/5 3/3 87, 100, 101 
a

qd×1, every day for one dose; d1, day 1.

b

nd, no delayed toxic deaths occurred.

Table 4

Effectiveness of aqueous 101M against i.p. implanted L1210 leukemia in mice

Female CD2F1 mice were inoculated i.p. with 1 × 105 leukemia cells. Treatment was initiated 24 h later. 101M was dissolved in water at its maximum solubility (0.6 mg/ml) and delivered at 0.01, 0.02, or 0.03 ml/g of mouse body weight to achieve doses of 6, 12, or 18 mg/kg, respectively. Data represent five mice per treatment group.
Schedule of administration Dosage (mg/kg) Δ wt.a (%) T/Cb (%) Long-termc survivors (%) 
qd × 1; d1 +4.0 203 
 18 +0.0 219 
qd × 6; d1–6 +0.0 333 40 
 12 +2.9  100 
 18 −4.6  100 
Female CD2F1 mice were inoculated i.p. with 1 × 105 leukemia cells. Treatment was initiated 24 h later. 101M was dissolved in water at its maximum solubility (0.6 mg/ml) and delivered at 0.01, 0.02, or 0.03 ml/g of mouse body weight to achieve doses of 6, 12, or 18 mg/kg, respectively. Data represent five mice per treatment group.
Schedule of administration Dosage (mg/kg) Δ wt.a (%) T/Cb (%) Long-termc survivors (%) 
qd × 1; d1 +4.0 203 
 18 +0.0 219 
qd × 6; d1–6 +0.0 333 40 
 12 +2.9  100 
 18 −4.6  100 
a

Δwt, change in weight from onset of therapy to day 6; qd, every day; d1, day 1; d1–6, days 1–6.

b

T/C (%), mean life span of treated (T) mice/mean life span of control (C) mice × 100. T/C calculations do not include long-term survivors.

c

Long-term survivors, mice alive and tumor-free 60 days after inoculation.

Table 5

Effects of various anticancer drugs on the intracranially implanted L1210 leukemia in mice

CD2F1 mice were given a single i.p. injection of the drug 1 day after intracranial inoculation with 4 × 105 L1210 cells. Viable L1210 cells per brain were estimated from inoculum response data that defined the relationship between life span and inoculum size. Doxorubicin was dissolved in water and delivered to mice at a rate of 0.01 ml/g of body weight. BCNU and 101M were dissolved in DMSO and delivered to mice at a rate of 0.001 ml/g of body weight. Data for Doxorubicin, BCNU, and 101M represent five mice per treatment group. Data for methotrexate, 6-mercaptopurine, and cyclophosphamide were taken from Ref. 27.
Compound Dosage (mg/kg) Log kill of leukemia cells in the brain 
Methotrexate 12 0.01 
6-Mercaptopurine 160 0.43 
Cyclophosphamide 140 0.94 
Doxorubicin 0.23 
BCNU 60 >5.97 
101M 80 >6.54 
CD2F1 mice were given a single i.p. injection of the drug 1 day after intracranial inoculation with 4 × 105 L1210 cells. Viable L1210 cells per brain were estimated from inoculum response data that defined the relationship between life span and inoculum size. Doxorubicin was dissolved in water and delivered to mice at a rate of 0.01 ml/g of body weight. BCNU and 101M were dissolved in DMSO and delivered to mice at a rate of 0.001 ml/g of body weight. Data for Doxorubicin, BCNU, and 101M represent five mice per treatment group. Data for methotrexate, 6-mercaptopurine, and cyclophosphamide were taken from Ref. 27.
Compound Dosage (mg/kg) Log kill of leukemia cells in the brain 
Methotrexate 12 0.01 
6-Mercaptopurine 160 0.43 
Cyclophosphamide 140 0.94 
Doxorubicin 0.23 
BCNU 60 >5.97 
101M 80 >6.54 
Table 6

Effectiveness of a single treatment of 101M or BCNU against i.p. implanted BCNU-resistant L1210 leukemia

Female CD2F1 mice were inoculated i.p. with 1 × 106 leukemia cells. A single i.p. treatment was administered 24 h later. Drugs were dissolved in DMSO and delivered at a rate of 0.001 ml/g of mouse body weight. Data represent five mice per treatment group.
Compound Dosage (mg/kg) Δ wt.a (%) T/Cb (%) Long-termc survivors (%) 
101M 20 +14.8 133 
 40 +2.0 176 
 60 +0.0  100 
 80 −8.2  100 
BCNU 20 +14.7 129 
 40 −14.5 157 
 60 Toxic 105 
Female CD2F1 mice were inoculated i.p. with 1 × 106 leukemia cells. A single i.p. treatment was administered 24 h later. Drugs were dissolved in DMSO and delivered at a rate of 0.001 ml/g of mouse body weight. Data represent five mice per treatment group.
Compound Dosage (mg/kg) Δ wt.a (%) T/Cb (%) Long-termc survivors (%) 
101M 20 +14.8 133 
 40 +2.0 176 
 60 +0.0  100 
 80 −8.2  100 
BCNU 20 +14.7 129 
 40 −14.5 157 
 60 Toxic 105 
a

Δwt, change in weight from onset of therapy to day 6.

b

T/C (%), mean life span of treated (T) mice/mean life span of control (C) mice × 100. T/C calculations do not include long-term survivors.

c

Long-term survivors, mice alive and tumor-free 60 days after inoculation.

Table 7

Effectiveness of a single treatment of 101M or cyclophosphamide (CTX) against i.p. implanted CTX-resistant L1210 leukemia

Female CD2F1 mice were inoculated i.p. with 1 × 106 leukemia cells. A single i.p. treatment was administered 24 h later. 101M was dissolved in DMSO and delivered at a rate of 0.001 ml/g of mouse body weight. CTX was dissolved in water and delivered at a rate of 0.01 ml/g of mouse body weight. Data represent five mice per treatment group.
Compound Dosage (mg/kg) Δ wt.a (%) T/Cb (%) Long-termc survivors (%) 
101M 20 +1.9 343 60 
 40 −1.9  100 
 60 −7.0  100 
 80 −10.1 557 60 
CTX 140 +7.0 114 
 280 +5.0 114 
Female CD2F1 mice were inoculated i.p. with 1 × 106 leukemia cells. A single i.p. treatment was administered 24 h later. 101M was dissolved in DMSO and delivered at a rate of 0.001 ml/g of mouse body weight. CTX was dissolved in water and delivered at a rate of 0.01 ml/g of mouse body weight. Data represent five mice per treatment group.
Compound Dosage (mg/kg) Δ wt.a (%) T/Cb (%) Long-termc survivors (%) 
101M 20 +1.9 343 60 
 40 −1.9  100 
 60 −7.0  100 
 80 −10.1 557 60 
CTX 140 +7.0 114 
 280 +5.0 114 
a

Δwt, change in weight from onset of therapy to day 6.

b

T/C (%), mean life span of treated (T) mice/mean life span of control (C) mice × 100. T/C calculations do not include long-term survivors.

c

Long-term survivors, mice alive and tumor-free 60 days after inoculation.

Table 8

Effectiveness of a single treatment of 101M or melphalan against i.p. implanted melphalan-resistant L1210 leukemia in mice

Female CD2F1 mice were inoculated i.p. with 1 × 106 leukemia cells. A single i.p. treatment was administered 24 h later. 101M was dissolved in DMSO and then further diluted with water for a final DMSO concentration of 10%. Melphalan was suspended in corn oil. Both drugs were delivered at a rate of 0.01 ml/g of mouse body weight. Data represent five mice per treatment group.
Compound Dosage (mg/kg) Δ wt.a (%) T/Cb (%) Long-termc survivors (%) 
101M 20 +8.1 183 
 40 +2.1  100 
 60 −7.9  100 
 80 −12.4  100 
Melphalan  100 
 10  100 
 20 −2.1 123 
 40 Toxic 91 
 60 Toxic 100 
Female CD2F1 mice were inoculated i.p. with 1 × 106 leukemia cells. A single i.p. treatment was administered 24 h later. 101M was dissolved in DMSO and then further diluted with water for a final DMSO concentration of 10%. Melphalan was suspended in corn oil. Both drugs were delivered at a rate of 0.01 ml/g of mouse body weight. Data represent five mice per treatment group.
Compound Dosage (mg/kg) Δ wt.a (%) T/Cb (%) Long-termc survivors (%) 
101M 20 +8.1 183 
 40 +2.1  100 
 60 −7.9  100 
 80 −12.4  100 
Melphalan  100 
 10  100 
 20 −2.1 123 
 40 Toxic 91 
 60 Toxic 100 
a

Δwt, change in weight from onset of therapy to day 6.

b

T/C (%), mean life span of treated (T) mice/mean life span of control (C) mice × 100. T/C calculations do not include long-term survivors.

c

Long-term survivors, mice alive and tumor-free 30 days after inoculation.

Table 9

Effectiveness of a single treatment of 101M against i.p. implanted P388 leukemia in mice

Female CD2F1 mice were inoculated i.p. with 1 × 106 leukemia cells. A single i.p. treatment was administered 24 h later. 101M was dissolved in DMSO and then further diluted with water for a final DMSO concentration of 10%. The drug was delivered at a rate of 0.01 ml/g of mouse body weight. Data represent five mice per treatment group.
Dosage (mg/kg) Δ wt.a (%) T/Cb (%) Long-termc survivors (%) 
+0.0 158 
10 −2.7 188 80 
20 +0.0  100 
40 −3.6  100 
60 −2.8  100 
80 −3.9  100 
100 −19.1 125 20 
Female CD2F1 mice were inoculated i.p. with 1 × 106 leukemia cells. A single i.p. treatment was administered 24 h later. 101M was dissolved in DMSO and then further diluted with water for a final DMSO concentration of 10%. The drug was delivered at a rate of 0.01 ml/g of mouse body weight. Data represent five mice per treatment group.
Dosage (mg/kg) Δ wt.a (%) T/Cb (%) Long-termc survivors (%) 
+0.0 158 
10 −2.7 188 80 
20 +0.0  100 
40 −3.6  100 
60 −2.8  100 
80 −3.9  100 
100 −19.1 125 20 
a

Δwt, change in weight from onset of therapy to day 6.

b

T/C (%), mean life span of treated (T) mice/mean life span of control (C) mice × 100. T/C calculations do not include long-term survivors.

c

Long-term survivors, mice alive and tumor-free 60 days after inoculation.

Table 10

Comparison of the effectiveness of 101M and BCNU against the C26 colon carcinoma in mice

Female Balb/c mice were inoculated i.p. with brei of C26 colon carcinoma. On postinoculation days 1 and 5, mice were injected with the indicated dosage of either 101M or BCNU. Drugs were dissolved in DMSO and delivered at a rate of 0.001 ml/g of mouse body weight. Data represent five mice per treatment group.
Dosage (mg/kg)101MBCNU
 Δ wt.a (%) T/Cb (%) Long-termc survivors (%) Δ wt.a (%) T/Cb (%) Long-termc survivors (%) 
10 −4.6  100 −3.8 166 20 
20 −4.1  100 −12.2  100 
40 −6.8  100 −13.4 81 20 
60 −9.7 84 60 Toxic 25 
80 Toxic 39 ndd nd nd 
Female Balb/c mice were inoculated i.p. with brei of C26 colon carcinoma. On postinoculation days 1 and 5, mice were injected with the indicated dosage of either 101M or BCNU. Drugs were dissolved in DMSO and delivered at a rate of 0.001 ml/g of mouse body weight. Data represent five mice per treatment group.
Dosage (mg/kg)101MBCNU
 Δ wt.a (%) T/Cb (%) Long-termc survivors (%) Δ wt.a (%) T/Cb (%) Long-termc survivors (%) 
10 −4.6  100 −3.8 166 20 
20 −4.1  100 −12.2  100 
40 −6.8  100 −13.4 81 20 
60 −9.7 84 60 Toxic 25 
80 Toxic 39 ndd nd nd 
a

Δwt, change in weight from onset of therapy to day 6.

b

T/C (%), mean life span of treated (T) mice/mean life span of control (C) mice × 100. T/C calculations do not include long-term survivors.

c

Long-term survivors, mice alive and tumor-free 100 days after inoculation. Control mice lived 22.0 ± 2.0 days.

d

nd, not determined.

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