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The spermidine conjugate of the human carcinogen and cancer therapeutic agent chlorambucil has been evaluated in the mouse bone marrow micronucleus assay. It showed a similar level of activity to that of chlorambucil, indicating that the superior ability of the conjugate to cross-link DNA in vitro is not expressed as enhanced genotoxicity in vivo.

The chlorambucil isomer 4-[3-[N,N-bis(2-chloroethyl)amino]phenyl] butanoic acid (m-chlorambucil) has been synthesized for the first time, and the two isometric nitro derivatives of both m-chlorambucil and chlorambucil itself have been prepared as potential hypoxia-selective cytotoxins. Reduction potentials (E1/2) of the two nitro compounds were determined by cyclic voltammetry, and one-electron reduction potentials (E(1] were estimated. Both the chlorambucil isomers and the derived nitro compounds crosslink DNA, as determined by their cytotoxicity ratios in DNA repair-proficient and -deficient cell lines, but neither of the nitro derivatives showed selective toxicity under hypoxic conditions, probably due to their rather low reduction potentials.

Methotrexate and chlorambucil, each covalently linked to either abrus agglutinin, abrin, ricinus agglutinin, ricin, or concanavalin A, were prepared. A single dose of the derivative injected ip into sarcoma 180-bearing noninbred N:NIH(S) white mice resulted in prolongation of the survival time and was more effective than an equivalent dose of free drug and lectin. Drug-lectin also showed a higher inhibitory effect on the DNA biosynthesis of the tumor cell than did an equivalent dose of the free drug and lectin.

A series of P-site probes, chlorambucilyl-(Pro)n-Phe-tRNAPhe, were prepared and reacted with poly(U)-directed Escherichia coli MRE 600 ribosomes. Upon binding of the probes to ribosomes, 90% of the cpm bound were not released following subsequent interaction with puromycin. In the absence of poly(U) or in the presence of poly(C), binding was limited to the amount of cpm bound if ribosomes were incubated in the presence of puromycin before adding modified tRNA and poly(U). AcPhe-tRNAPhe was a competitive inhibitor of chlorambucilyl Phe-tRNAPhe. Binding to 50S subunits was strongly stimulated by poly(U), while binding to 30S subunits was not. Crosslinked 50S proteins were analyzed by two-dimensional gel electrophoresis. Crosslinking with molecular rulers containing zero prolines led to poly(U)-dependent labeling of L1 and L27. With rulers containing five prolines, L6, L25, L28, and the group L18,23,24 were labeled. Analysis of crosslinked ribosomal RNA on sucrose density gradients revealed almost no cpm in the 16S or 23S peaks, but only in the 5S peaks. This was observed with molecular rulers containing either zero or five proline residues.

Bestrabucil (KM2210), the benzoate of an estradiol-chlorambucil conjugate, was used experimental cancer chemotherapy against 13 human tumor xenografts serially transplanted into nude mice, and its pharmacokinetics was studied. The tumors were one esophageal, two gastric, six colon, one cholecystic and three breast carcinomas. Two tumor tissue fragments approximately 3 X 3 X 3 mm were inoculated into BALB/cA nude mice, which were then treated with KM2210 at doses of 100, 200 and 300 mg/kg/day orally starting 24 hr after the transplantation or when the tumor reached a weight of 100-300 mg. The concentration of KM2210 and its derivatives in the tumor xenografts, normal muscular tissue and blood were assayed by high performance liquid chromatography. Six out of 13 xenografts were found to be sensitive to KM2210. The concentrations of KM2210 and its derivatives in the tumor tissues of the sensitive xenografts were five to 10 times higher than those in blood and muscular tissue, and the antitumor activity correlated well with the area under the curve of active metabolites of KM2210 in the tumor.

Busramustine (KM-2210), the benzoate of a 17 beta-estradiol-chlorambucil conjugate, was administered to 11 patients with chronic lymphocytic leukemia (CLL) which included eight cases of B-cell CLL and three cases of T-cell CLL. Four patients had received prior chemotherapy. Busramustine was given orally at an initial daily dose of 50-100 mg continuously, and the dose was modified according to hematological improvement. Two cases of B-cell CLL achieved clinical complete responses, six cases including two of T-cell CLL and four of B-cell CLL achieved partial responses and one case of B-cell CLL achieved improvement. The partial and complete response rate was 72.7%. Four patients showed estrogen receptor activity of CLL cells ranging from 3.5 to 57.5 fmol/mg cytosol protein, but there seemed to be no correlation between the estrogen receptor activity of the CLL cells and the therapeutic effects of busramustine. Toxic effects included diarrhea (2/11) and estrogen-related symptoms including breast pain (4/11), genital bleeding (2/5), gynecomastia (2/6) and loss of libido (2/6). The findings of this preliminary study suggest that busramustine is effective in the treatment of CLL, irrespective of the presence of the estrogen receptor.

Early assessment of the genetic toxicology of anti-tumour drugs often employs microbial assays, particularly a group of specially developed Salmonella typhimurium strains (uvrB-) which are DNA repair-deficient due to a defective uvrB gene. While such strains are more sensitive than the wild-type to the mutagenic effects of many classes of compound, the DNA cross-linking agent chlorambucil (which is a known human carcinogen) has been shown to be toxic but non-mutagenic in this assay. The mutagenic activities of chlorambucil, its half-mustard analogue and another cross-linking agent (mitomycin C) were thus evaluated in a DNA-proficient strain of the yeast Saccharomyces cerevisiae and also in two isogenic sets of four S. typhimurium strains differing in uvrB gene and plasmid pKM101 presence. In yeast, all three compounds were effective mutagens and recombinogens, while in bacteria the two cross-linking agents were significantly mutagenic in the uvrB+ (DNA repair-proficient) but not in the uvrB- strains. By comparison, the half-mustard was mutagenic in both the uvrB- and uvrB+ strains. This work suggests that it is unwise to rely on results from the commonly used S. typhimurium bacterial strains for evaluating the mutagenicity of DNA cross-linking agents destined as clinical anti-tumour drugs. The use of yeast plus at least one of the sets of four strains employed in this study would provide information more directly relevant to mammalian cells, which are DNA repair-proficient. In addition, a comparison of the patterns of bacterial mutagenicity and toxicity provides evidence for the mode of action (DNA cross-linking or monoadduct formation) of compounds within a series.