Chemically stable, lipophilic prodrugs of phosphoramide mustard as potential anticancer agents.

Benzyl phosphoramide mustard (3), 2,4-difluorobenzyl phosphoramide mustard (4), and methyl phosphoramide mustard (5) were examined as lipophilic, chemically stable prodrugs of phosphoramide mustard (2). These phosphorodiamidic esters are designed to undergo biotransformation by hepatic microsomal enzymes to produce 2. The rate of formation of alkylating species, viz., 2, from these prodrugs and their in vitro cytotoxicity toward mouse embryo Balb/c 3T3 cells were comparable to or better than that of cyclophosphamide (1). Preliminary antitumor screening against L1210 leukemia in mice, however, suggests that these prodrugs are devoid of any significant antitumor activity in vivo.

Chemically stable N-methyl-4-(alkylthio)cyclophosphamide derivatives as prodrugs of 4-hydroxycyclophosphamide.

Two prototype N-methyl-4-thio-substituted cyclophosphamide (CP) derivatives (5 and 6), prodrugs of 4-hydroxycyclophosphamide (4-HO-CP), were designed to undergo oxidative N-demethylation to release the active alkylating agent. These prodrugs were chemically stable until oxidatively N-demethylated in the presence of hepatic microsomal P-450 enzymes. While the metabolism of 5 was enhanced in the presence of phenobarbital-induced microsomes, 6 was unaffected. Compound 6 was more active than 5 against L1210 leukemia cells grown in mice and exhibited statistically significant activity against the small cell lung cancer panel in the National Cancer Institute anticancer drug screen. Compound 5, like CP (1), was inactive in this screen. Thus, placement of a dithioester at the 4-position of N-methyl-HO-CP as in 6 markedly changes its spectrum of activity and has resulted in a new type of CP-based prodrug with antitumor activity against small cell lung cancer as well as leukemia cells in vitro as shown by their ability to inhibit tumor cell growth at concentrations as low as 10(-6) M.

Sulfoxide-containing aromatic nitrogen mustards as hypoxia-directed bioreductive cytotoxins.

A series of diaryl and alkylaryl sulfoxide-containing nitrogen mustards were synthesized and evaluated for their hypoxia-selective cytotoxicity against V-79 cells in vitro as well as for their metabolism profiles with the rat S-9 fractions. In general, the diaryl sulfoxides (4, 5, and 7-9) showed much greater hypoxia selectivity (11-27-fold) than the alkylaryl sulfoxides (approximately 3-fold) (1 and 3). The fused diphenyl sulfoxides (10 and 11), on the other hand, showed very low hypoxia selectivity (1.3-3-fold). Compound 10 was highly cytotoxic under both aerobic and anaerobic conditions, while 11 showed low cytotoxicity under both conditions. The bioreduction of 8 by the rat S-9 fraction under anaerobic conditions was inhibited by menadione and enhanced by benzaldehyde, acetaldehyde, or 2-hydroxypyrimidine suggesting the involvement of aldehyde oxidase in the reduction of the sulfoxides. Bioreductive metabolism studies of selected model sulfoxides suggested that diaryl sulfoxides are better substrates for aldehyde oxidase than alkylaryl sulfoxides.

Properties of a cytotoxic kidney antibody associated with human renal transplantation.

A technic for demonstrating a complement-dependent cytotoxic response specific for human kidney cells was developed. Positive and negative sera from kidney-transplant recipients were subjected to several assay and adsorption procedures. The cytotoxic antibody reaction appeared to be specific against human kidney in tests with a variety of target cells. Manifestation of the typical cytotoxic effect depended on kidney explant target-cell surface changes following attachment, outgrowth, and dispersion of the first passage monolayer. A comparison of serum responses to lymphocytes and to kidney cells indicated that the cytotoxic response by human kidney cells is not related to the lymphocytotoxic antibodies associated with the HLA system.

Anti-kidney cytotoxic antibodies in human renal allografts.

Periodic comparisons were made of sera from two groups of patients, ten who rejected their renal transplants within a year after transplantation and ten who successfully maintained their transplanted kidneys for five years or more. What appeared to be anti-kidney cytotoxic antibodies were found in much higher levels in the sera of those patients with the short-lived transplants, the difference in titer levels between the two groups being significant at the .0001 levels. This antibody showed no correlation with either the presence or the absence of lymphocytotoxic antibodies, nor did it appear to have any relationship to the HL-A antigens or the previous renal disease of the recipient patient. While it was cytotoxic to tissue cultures of cells obtained from random human kidneys, human kidney tumor cells (Wilms), and human embryonic kidneys, this antibody did not react with non-renal human tissues (lung, spleen, deltoid muscle, foreskin). It did not react with nonhuman (simian) kidney tissue culture cells. The findings suggest the appearance of an organ-specific, cytotoxic anti-kidney antibody in patients undergoing renal homograft rejection.

Antibodies to synovial-derived cells in patients undergoing artificial prosthesis implants.

In this preliminary study, the authors have found cytotoxic complement-dependent synovial antibodies in the serum of patients undergoing prosthesis implantation; these were particularly evident in those patients undergoing revision surgery. In order to demonstrate the synovial antibodies, it was necessary that the authors modify the method they had previously used in their studies of cytotoxic lung and kidney antibodies. While conventional trypsinization dispersion produced synovial target cells that would not react in the test system, mechanical dispersion successfully produced target cells sensitive to complement-dependent cytotoxic antibodies. In this study, synovial antibodies reacted similarly to cells derived from the synovium of different individuals, but they did not react to cells derived from tissues other than synovium. This tissue specificity was confirmed by absorption studies that indicated the synovial antibodies to be organ specific. Whether these cytotoxic synovial antibodies can be predictive of the impending loss of an artificial prosthesis, or somehow be directly involved in the failure mechanism of that prosthesis, will require additional studies.

Cocarcinogenic and tumor-promoting capabilities of anthralin.

Numerous chemicals to which humans are exposed either therapeutically or as a result of living in an industrial environment constitute a potential threat as carcinogens, mutagens, and/or tumor promoters and cocarcinogens. Anthralin, and antipsoriatic agent, acts as a tumor promoter for Balb/c-3T3 mouse embryo cell cultures that were previously exposed to a low dose of either benzo-a-pyrene (BaP), an indirect-acting carcinogen needing metabolic conversion for its carcinogenic action, or beta-propiolactone (BPL), a direct-acting carcinogen which needs no metabolic conversion. As a cocarcinogen, i.e., when exposure of cells to anthralin was simultaneous with exposure to the carcinogen, anthralin enhanced neoplastic transformation only when the carcinogen was BaP. Several explanations are explored. The possibility that cocarcinogens and tumor promotion occur by separate mechanisms is suggested.

Pyrene acts as a cocarcinogen with the carcinogens benzo[a]pyrene, beta-propiolactone and radiation in the induction of malignant transformation in cultured mouse fibroblasts; soybean extract containing the Bowman-Birk inhibitor acts as an anticarcinogen.

Pyrene was found to act as a cocarcinogen in the induction of transformation of cultured Balb/c3T3 cells by three different types of carcinogens: a direct acting chemical carcinogen, beta-propiolactone, a chemical carcinogen requiring metabolic activation, benzo[a]pyrene, and a physical carcinogen (60Co) gamma radiation. Since pyrene enhanced transformation in vitro by approximately the same amount for all the carcinogens tested, these results suggest that the carcinogenic action of pyrene is not related to carcinogen metabolism or uptake in vitro. An extract of soybeans containing the Bowman-Birk protease inhibitor was shown to reduce transformation induced by beta-propiolactone, benzo[a]pyrene and gamma-rays, both with and without the cocarcinogenic effect of pyrene, to background levels; the magnitude of the reduction in transformation by the protease inhibitor preparation was unrelated to the concentration of carcinogen. Neither the mechanism for the cocarcinogenic action of pyrene not the anticarcinogenic effect of the soybean extract is known, but several hypotheses are discussed.

In vitro reduction of peroxidation in UVC-irradiated cell cultures by concurrent exposure with Bowman-Birk protease inhibitor.

Bowman-Birk protease inhibitor (BBI), extracted from soybean flour, has been shown to protect cells from damage after UVC irradiation. BBI also reduces the number of transformed foci following exposure to the same doses of UVC irradiation. Proteins have been reported to act as antioxidants by their sheer intracellular bulk. In this study we assessed BBI's ability to attenuate UVC-irradiation-induced peroxidation. Balb c/3T3 cells, clone A31, subcloned by this laboratory were used. Treatment groups consisted of cells suspended in Hanks' Balanced Salt Solution with and without 100 micrograms/ml of a crude soybean extract containing the Bowman-Birk protease inhibitor. Cells were exposed to irradiation in suspension by using GE (G8T5) germicidal lamps which deliver predominantly 254 nm light at 0.385 J/m2/s as measured at the cell surface. Aliquots of UVC-exposed cells were homogenized in HBSS in the presence and absence of 100 micrograms/ml BBI with a motor-driven Teflon pestle and assayed for peroxidation. The results showed significant (P less than 0.0001) protection afforded by BBI against peroxidation at 1.16 (65%), 1.93 (60%, and 2.70 (48%) J/m2 of UVC irradiation. Transformation was reduced to 0.33 foci per dish at all levels of radiation exposure.

Effects of amiodarone on elastin biosynthesis in primary hamster lung cell cultures.

Amiodarone is a Class III antiarrhythmic agent that has been implicated as a cause of human pulmonary fibrosis. Pulmonary fibrosis is associated with increased levels of connective tissue proteins such as collagen and elastin. The purpose of this investigation was to determine whether elastin synthesis would be altered by in vitro amiodarone administration. Primary hamster lung cell cultures were utilized. Cultures were treated with 2, 10, and 20 micrograms/ml amiodarone. Following treatment, elastin synthesis was monitored by a biochemical tracer assay based on the presence of the cross-linking amino acids: desmosine/isodesmosine. These cross-links are found only in elastin. Addition of [14C] lysine to cultures results in uptake of the radiolabel into the cross-links. Cross-links were isolated and identified using chromatography and electrophoresis. At all doses of amiodarone, elastin synthesis was seen to increase above control levels. Light and electron microscopy confirmed the presence of an extracellular matrix. The morphologic studies also revealed the presence of cytoplasmic inclusion bodies and vacuoles that are often associated with cationic, amphiphilic drugs such as amiodarone.

Induction of in vitro transformation by near-u.v. light and its interaction with beta-propiolactone.

The ability of u.v.-A light (320-400 nm) to induce cellular transformation in vitro and to modify chemical carcinogen-induced cellular transformation was investigated in BALB/c 3T3 cell cultures. When administered as a series of nontoxic exposures, u.v.-A alone was found to induce cellular transformation as a linear function of the numbers of u.v-A exposures. Possible interactions of u.v.-A with environmentally encountered chemical carcinogens were studied by examining the effects of u.v.-A light exposures on cellular transformation in cells exposed to the direct acting carcinogen, beta-propiolactone (BPL), an alkylating agent, with a standard initiation/promotion protocol. Twenty-four hours after a single treatment with 2.5 micrograms/ml of beta-propiolactone, cells were exposed to 3.0 kJ/m2 of u.v.-A light. U.v.-A exposures were repeated weekly for up to 5 weeks, after which cells were fixed, stained and dishes were scored for type III transformed foci. Weekly exposures to u.v.-A alone for 5 weeks induced approximately 3 foci/dish. Treatment with BPL alone induced approximately 1 focus/dish (background was 0.17 foci/dish). A combination of the two treatments resulted in a marked increase in the yield of transformed foci/dish, with the u.v.-A enhancement increasing with increasing numbers of exposures (approximately 10 foci/dish after a single exposure to BPL and five u.v.-A exposures). These results suggest a synergistic interaction between BPL and subsequent u.v.-A exposures in the induction of in vitro neoplastic transformation.

Anti-lung antibodies associated with human lung allografts.

Serum specimens from 4 patients who had had a lung allograft were examined for anti-lung antibodies. Two patients developed antibodies after transplantation, and in 2, the antibodies increased in titer after an allograft. The absorption of the anti-lung antibody by fetal lung cell cultures, in contrast to the failure of absorption by kidney cell cultures from the same fetus, indicates that the antibody is lung specific.