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[Cancer Research 61, 3033-3038, April 1, 2001]
© 2001 American Association for Cancer Research

Experimental Therapeutics

1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylamino)carbonylhydrazine (101M): A Novel Sulfonylhydrazine Prodrug with Broad-Spectrum Antineoplastic Activity1

Rick A. Finch, Krishnamurthy Shyam, Philip G. Penketh and Alan C. Sartorelli2

Department of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06520


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Drugs and Mice.
101M and 90CE were synthesized according to published methodology (23 , 24) . The chemical structure of 101M is shown in Fig. 1Citation . All of the other agents were purchased from Sigma Chemical Co. (St. Louis, MO).



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  		Fig. 1. Chemical structure of 101M.

 
BALB/c x 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 x 105 cells. For studies with drug-resistant variants of the L1210 or with the P388 leukemia, the inoculum level was increased to 1 x 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 x 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).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
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)Citation . 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 1Citation 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.


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  		Table 1 Comparison of the effectiveness of a single i.p. treatment of 101M or BCNU against i.p. implanted L1210 leukemia in mice

 
As shown in Table 2Citation , 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.


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  		Table 2 Effectiveness of a single oral treatment of 101M against i.p. implanted L1210 leukemia in mice

 
It should be noted from the data reported in Table 1Citation , 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)Citation . 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).



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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.

 
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 3Citation . 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)Citation , 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.


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  		Table 3 Occurrence of delayed toxicity in long-term survivors of L1210-inoculated 101M-treated mice

 

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  		Table 4 Effectiveness of aqueous 101M against i.p. implanted L1210 leukemia in mice

 
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 5Citation . 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.


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  		Table 5 Effects of various anticancer drugs on the intracranially implanted L1210 leukemia in mice

 
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)Citation , 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)Citation . 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.



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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 {approx}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. q2dx11, every 2 days for 11 doses; q4dx6, every 4 days for 6 doses.

 
101M was also highly active against an L1210 variant that is resistant to BCNU (L1210/BCNU; Table 6Citation ). 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)Citation and melphalan-resistant (Table 8)Citation L1210 sublines, making this agent an attractive alternative to these drugs.


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  		Table 6 Effectiveness of a single treatment of 101M or BCNU against i.p. implanted BCNU-resistant L1210 leukemia

 

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  		Table 7 Effectiveness of a single treatment of 101M or cyclophosphamide (CTX) against i.p. implanted CTX-resistant L1210 leukemia

 

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  		Table 8 Effectiveness of a single treatment of 101M or melphalan against i.p. implanted melphalan-resistant L1210 leukemia in mice

 
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 9Citation 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.


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  		Table 9 Effectiveness of a single treatment of 101M against i.p. implanted P388 leukemia in mice

 
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)Citation . 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)Citation . 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 {approx}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.


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  		Table 10 Comparison of the effectiveness of 101M and BCNU against the C26 colon carcinoma in mice

 


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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 {approx}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. q2dx10, every 2 days for 10 doses; q4dx5, every 4 days for 5 doses.

 
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. 5Citation , 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



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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).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
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 x 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)Citation . 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.


   FOOTNOTES
 
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. Back

2 To whom requests for reprints should be addressed, at Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520. Phone: (203) 785-4533; Fax: (203) 737-2045; E-mail: alan.sartorelli{at}yale.edu Back

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. Back

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

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. Back

Received 8/30/00. Accepted 1/26/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Teicher B. A. Antitumor alkylating agents Ed. 5 DeVita V. T., Jr. Hellman S. Rosenburg S. A. eds. . Cancer: Principles and Practice of Oncology, : 405-418, Lippencott-Raven Philadelphia 1997.
  2. Lawley P. D., Brooks P. Interstrand cross-linking of DNA by bifunctional alkylating agents. J. Mol. Biol., 25: 143-160, 1967.[Medline]
  3. Ludlum D. B., Kramer B. S., Wang J., Fenselau C. Reaction of 1,3-bis(2-chloroethyl)-1-nitrosourea with synthetic polynucleotides. Biochemistry, 14: 5480-5485, 1975.[Medline]
  4. Kohn K. W. Interstrand cross-linking of DNA by 1,3-bis(2-chloroethyl)-1-nitrosourea and other 1-(2-haloethyl)-1-nitrosoureas. Cancer Res., 37: 1450-1454, 1977.[Abstract/Free Full Text]
  5. Lown J. W., McLaughlin L. W., Chang Y-M. Mechanism of action of 2-haloethylnitrosoureas on DNA and its relation to their antileukemic properties. Bioorg. Chem., 7: 97-110, 1978.
  6. Nissen N. I., Pajak T. F., Glidewell O., Pedersen-Bjergaard J., Stutzman L., Falkson G., Cuttner J., Blom J., Leone L., Sawitsky A., Coleman M., Haurani F., Spurr C. L., Harley J. B., Seligman B., Cornell C., Jr., Henry P., Senn H., Brunner K., Martz G., Maurice P., Bank A., Shapiro L., James G. W., Holland J. F. Comparative study of a BCNU containing 4-drug program versus MOPP versus 3-drug combination in advanced Hodgkin’s disease. Cancer (Phila.), 43: 31-40, 1979.[Medline]
  7. DeVita V. T., Carbone P. P., Owens A. H., Jr., Gold G. L., Krant M. J., Edmonson J. Clinical trials with 1,3-bis(2-chloroethyl)-1-nitrosourea, NSC-409962. Cancer Res., 25: 1876-1881, 1965.[Abstract/Free Full Text]
  8. Montgomery J. A., James R., McCaleb G. S., Kirk G. S., Johnston T. P. Decomposition of N-(2-chloroethyl)-N-nitrosoureas in aqueous media. J. Med. Chem., 18: 568-570, 1975.[Medline]
  9. Montgomery J. A., James R., McCaleb G. S., Johnston T. P. The modes of decomposition of 1,3-bis(2-chloroethyl)-1-nitrosourea and related compounds. J. Med. Chem., 10: 668-674, 1967.[Medline]
  10. Weinkam R. J., Lin H-S. Reactions of 1,3-bis(2-chloroethyl)-1-nitrosourea and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea in aqueous solution. J. Med. Chem., 22: 1193-1198, 1979.[Medline]
  11. Brundrett R. B. Chemistry of nitrosoureas. Intermediacy of 4,5-dihydro-1,2,3-oxadiazole in 1,3-bis(2-chloroethyl)-1-nitrosourea decomposition. J. Med. Chem., 23: 1245-1247, 1980.[Medline]
  12. Kohn K. W. Mechanistic approaches to new nitrosourea development Carter S. K. Sakurai Y. Umezawa H. eds. . Recent Results in Cancer Research, Vol. 76: 141-152, Springer Berlin 1981.[Medline]
  13. Pelfrene A., Mirvish S. S., Gold B. Induction of malignant bone tumors in rats by 1-(2-hydroxyethyl)-1-nitrosourea. J. Natl. Cancer Inst. (Bethesda), 56: 445-446, 1976.[Medline]
  14. Swenson D. H., Frei J. V., Lawley P. D. Synthesis of 1-(2-hydroxyethyl)-1-nitrosourea and comparison of its toxicity to that of 1-ethyl-1-nitrosourea. J. Natl. Cancer Inst. (Bethesda), 63: 1469-1473, 1979.[Medline]
  15. Baril B. B., Baril E. F., Lazlo J., Wheeler G. P. Inhibition of rat liver DNA polymerase by nitrosoureas and isocyanates. Cancer Res., 35: 1-5, 1975.[Abstract/Free Full Text]
  16. Kann H. H., Kohn K. W., Lyles J. M. Inhibition of DNA repair by 1,3-bis(2-chloroethyl)-1-nitrosourea breakdown product, 2-chloroethyl isocyanate. Cancer Res., 34: 398-402, 1974.[Abstract/Free Full Text]
  17. Schallreuter K. U., Gleason F. K., Wood J. M. The mechanism of action of the nitrosourea antitumor drugs on thioredoxin reductase, glutathione reductase and ribonucleotide reductase. Biochim. Biophys. Acta, 1054: 14-20, 1990.[Medline]
  18. Colvin M., Brundrett R. B., Cowens J. W., Jardine I., Ludlum D. B. A chemical basis for the antitumor activity of the chloroethylnitrosoureas. Biochem. Pharmacol., 25: 695-699, 1976.[Medline]
  19. Wheeler G. P., Bowdon B. J., Grimsley J., Lloyd H. H. Interrelationships of some chemical, physiochemical and biological activities of several 1-(2-haloethyl)-1-nitrosoureas. Cancer Res., 34: 194-200, 1974.[Abstract/Free Full Text]
  20. Panasci L. C., Green D., Nagourney R., Fox P., Schein P. S. A structure-activity analysis of chemical and biological parameters of chloroethylnitrosoureas in mice. Cancer Res., 37: 2615-2618, 1977.[Abstract/Free Full Text]
  21. Gibson N. W., Hickman J. A. The role of isocyanates in the toxicity of antitumor haloalkylnitrosoureas. Biochem. Pharmacol., 31: 2795-2800, 1982.[Medline]
  22. Penketh P. G., Shyam K., Sartorelli A. C. Comparison of DNA lesions produced by tumor inhibitory 1,2-bis(sulfonyl)hydrazines and chloroethylnitrosoureas. Biochem. Pharmacol., 59: 283-291, 2000.[Medline]
  23. Shyam K., Penketh P. G., Loomis R. H., Rose W. C., Sartorelli A. C. Antitumor 2-(aminocarbonyl)-1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazines. J. Med. Chem., 39: 796-801, 1996.[Medline]
  24. Shyam K., Penketh P. G., Divo A. A., Loomis R. H., Rose W. C., Sartorelli A. C. Synthesis and evaluation of 1,2,2-tris(sulfonyl)hydrazines as antineoplastic and trypanocidal agents. J. Med. Chem., 33: 2259-2264, 1990.[Medline]
  25. Skipper H. E., Schabel F. M., Trader M. W., Thompson J. R. Experimental evaluation of potential anticancer agents. XI. Anatomical distribution of leukemic cells and failure of chemotherapy. Cancer Chemother. Rep., 21: 1154-1164, 1961.
  26. Skipper H. E., Schabel F. M., Wilcox W. S., Laster W. R., Trader M. W., Thompson S. A. Experimental evaluation of potential anticancer agents. XVIII. Effects of therapy on viability and rate of proliferation of leukemic cells in various anatomic sites. Cancer Chemother. Rep., 47: 41-64, 1965.[Medline]
  27. Avery T. L., Finch R. A., Vasquez K. M., Radparvar S., Hanna N. B., Revankar G. R., Robins R. K. Chemotherapeutic characterization in mice of 2-amino-9-·-D-ribofuranosylpurine-6-sulfinamide (sulfinosine), a novel purine nucleoside with unique antitumor properties. Cancer Res., 50: 2625-2630, 1990.[Abstract/Free Full Text]
  28. Gorbacheva L. B., Kukushkina G. V., Elksne S. J., Mikiner S. J., Melnik S. Y., Preobrazhenskaya M. N. O6-Methylguanine as a modulator of antitumor activity of N-alkyl-N-nitrosoureas in vivo. Anticancer Drugs, 2: 185-189, 1991.[Medline]
  29. Ribrag V., Massaad L., Janot F., Bissery M. C., Parise O., Gouyette A., Chabot G. G. Principal drug-metabolizing enzyme systems in L1210 leukemia sensitive or resistant to BCNU in vivo. Leukemia Res., 18: 829-835, 1994.[Medline]
  30. Pratviel G., Shyam K., Sartorelli A. C. Cytotoxic and DNA-damaging effects of 1,2-bis(sulfonyl)hydrazines on human cells of the Mer+ and Mer- phenotype. Cancer Biochem. Biophys., 10: 365-375, 1989.[Medline]
  31. Levin V. A., Leibel S. A., Gutin P. H. Neoplasms of the central nervous system Ed. 5 DeVita V. T., Jr. Hellman S. Rosenberg S. A. eds. . Cancer: Principles and Practice of Oncology, : 2022-2082, Lippincott-Raven Philadelphia 1997.
  32. Posner J. B. Management of brain metastases. Rev. Neurol., 148: 477-487, 1992.[Medline]
  33. Pegg A. E., Dolan M. E., Moschel R. C. Structure, function, and inhibition of O6-alkylguanine-DNA alkyltransferase. Prog. Nucleic Acid Res. Mol. Biol., 51: 167-223, 1995.[Medline]
  34. Mitra S., Kaina B. Regulation and repair of alkylation damage in mammalian genomes. Prog. Nucleic Acid Res. Mol. Biol., 44: 109-142, 1995.
  35. Silber J. R., Mueller B. A., Berger M. S. Comparison of alkyltransferase activity in brain tumors and adjacent brain. Cancer Res., 53: 3416-3420, 1993.[Abstract/Free Full Text]



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