Antiarthritic and suppressor cell inducing activity of azaspiranes: structure-function relationships of a novel class of immunomodulatory agents.

Spirogermanium (1; 8,8-diethyl-N,N-dimethyl-2-aza-8- germaspiro[4.5]decane-2-propanamine dihydrochloride) is a potent cytotoxic agent in vitro which has demonstrated limited activity in experimental animal tumor models. Subsequently, it has been reported that spirogermanium has antiarthritic and suppressor cell-inducing activity. We have synthesized a series of substituted 8-hetero-2-azaspiro[4.5]decane and 9-hetero-3-azaspiro[5.5]undecane analogues of spirogermanium to identify the heteroatom requirements for in vivo antiarthritic and suppressor cell-inducing activity. This structure-activity relationship study has identified that appropriately substituted silicon and carbon analogues of spirogermanium retain both antiarthritic and immunosuppressive activity, with the 8,8-dipropyl (carbon) analogue being among the most active. Following the identification of N,N-dimethyl-8,8-dipropyl-2-azaspiro[4.5]decane-2-propanamine++ + dihydrochloride (9) as a more active analogue than spirogermanium, a series of 8,8-dipropyl analogues with various amine substituents were synthesized. A number of these analogues had activity similar to that of 9. A correlation between activity in the adjuvant arthritic rat and the ability to induce suppressor cells (r = 0.894, p less than 0.001) suggests an association between the two pharmacologic effects. While the precise biochemical mechanism(s) for the pharmacological activity is unclear, these data suggest that compounds within this series, e.g., N,N-dimethyl-8,8-dipropyl-2-azaspiro[4.5]decane-2-propanamine++ + dihydrochloride, may provide effective therapy in diseases of autoimmune origin and/or the prevention of rejection in tissue transplantation.

Pharmacological activities of spirogermanium and other structurally related azaspiranes: effects on tumor cell and macrophage functions.

Spirogermanium is a germanium containing azaspirane which has been shown to have activity in experimental models of cancer and immune dysfunction. A series of analogs of the parent compound were synthesized and evaluated in a number of in vitro and in vivo biological assays to define the structure-activity relationships of this class of compounds relative to their potential therapeutic activities. In a colony-forming assay using HT-29 human colon carcinoma cells various analogs in which carbon replaced germanium (e.g. carbon) retained the potent cytotoxic activity in vitro seen with spirogermanium. Increased cytotoxic potency within the group of carbon containing analogs was directly related to increase in the length of the alkyl group(s) attached to the carbon atom opposite the azaspirane ring structure. DNA and protein synthesis by HT-29 cells was inhibited by these compounds. However, inhibition occurred only at supralethal concentrations or after long exposure times with the drug. None of the azaspiranes demonstrated in vivo anti-tumor activity against P388 leukemia or ADJ-PC6 plasmacytoma. The effect of these compounds on macrophage cell function was evaluated in vitro by their ability to modulate superoxide (O2-) production by macrophages. Spirogermanium inhibited the production of O2- by activated macrophages with an IC50 of 5 microM. Although macrophage viability did not appear to be decreased at the respective IC50 concentrations, the rank order potency for the analogs in the O2- production assay was directly proportional to that measured for their cytotoxic potency in the HT-29 colony formation assay. The results demonstrate that, within this class of compounds, (1) potent biological activity does not require the presence of germanium in the structure; (2) in vitro cytotoxic activity does not appear to be a direct result of the inhibition of macromolecular synthesis, and (3) macrophage function can be modulated in vitro at non-cytotoxic concentrations. These results are discussed in context with the reported anti-tumor activity of spirogermanium and the potential anti-arthritic and immunomodulatory activity of this class of compounds.

Effect of auranofin and other gold complexes on the activity of phospholipase C.

Auranofin (AF) is an orally active chrysotherapeutic agent used for the treatment of rheumatoid arthritis, a self-perpetuating inflammatory disease. Because of reports suggesting that AF and other gold complexes can, under certain circumstances, exacerbate rheumatoid inflammatory lesions in humans and adjuvant arthritic rats and that phospholipase C (PLC) and phospholipase A2 activities are increased in rheumatoid patients, the effects of AF and a related gold complex on in situ mammalian and purified Bacillus cereus PLC were examined. Results of our studies show that 1) AF and triethylphosphine gold chloride (TEPG), an AF analog, stimulated PLC activity in the sonicate of RAW 264.7 macrophages; 2) AF and TEPG stimulated B. cereus PLC activity in a concentration-dependent manner, but the pattern of stimulation and concentrations of drugs required to stimulate the purified enzyme differ from those seen with the macrophage PLC; 3) metals (cobalt and zinc) and sulfhydryl reagents (N-ethylmaleimide, iodoacetic acid, and glutathione), tested at the same concentrations of AF that enhanced PLC activity, had no effect on the enzyme. These data suggest that stimulation of PLC may be a generic phenomenon since two divergent PLCs are affected by gold complexes. Additionally, these studies may provide one potential explanation for rheumatoid lesion flares seen in patients and animals on chrysotherapy.

Application of a tissue culture microtiter test for the detection of cytotoxic agents from natural products.

A method is described by which the growth inhibitory effects of cytotoxic compounds and fermentation broth cultures on adherent tumor cell lines can be quantitated. Cells are seeded into 96-well microtiter plates and 16 hours later the test compounds or broths are added to the wells. Cell growth is measured after three days (B16 mouse melanoma cells) or six days (HT-29, human colon carcinoma cells) by first fixing adherent cells, staining with Giemsa stain, washing away excess stain, then solubilizing stained cells with HCl. Absorbance is determined using a microELISA spectrophotometer and the data are transferred to and analyzed by a computer. The assay is rapid and reproducible and can be used to identify fermentation broths with cytotoxic components. Addition of DNA into the assay mixture (cells plus compound) inhibits the cytotoxic activities of certain DNA-reactive agents. The results of this study demonstrate the application of this assay system for primary and secondary evaluation of fermentation broths for in vitro antitumor activity.

Evaluation of the in vivo antitumor activity and in vitro cytotoxic properties of auranofin, a coordinated gold compound, in murine tumor models.

The coordinated gold compound, 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranosato-S-triethyl phosphine gold (auranofin; Ridaura), was evaluated for antitumor activity in a variety of mouse tumor models. Of the 15 tumor models evaluated, auranofin was found to be active only against i.p. P388 leukemia. A number of dose schedules was used to measure activity against P388 with optimal activity observed at 12 mg/kg given daily, i.p., on Days 1 to 5. Auranofin was active against i.p. P388 leukemia only when administered i.p.; the drug was completely inactive when administered i.v., s.c., or p.o. on Days 1 to 5. Evaluation of the effects of auranofin in vitro demonstrated that survival curves for B16 melanoma cells as measured by the clongenic and dye exclusion assays were exponential and monophasic; cell cycle distribution was not altered, and auranofin displayed no preferential cytotoxicity to logarithmic or plateau growth phase cell populations; auranofin inhibited DNA, RNA, and protein synthesis at cytotoxic concentrations but showed no selective effect; the cytotoxic activity and cellular association of gold from auranofin were dose, time, and temperature dependent; and binding of auranofin gold to serum proteins markedly decreased cellular uptake of gold and cytotoxicity of auranofin in vitro.

Improved genetically modified Escherichia coli strain for prescreening antineoplastic agents.

Clinical experience suggests that drugs that interact with and damage DNA are useful in cancer chemotherapy (H. Umezawa , p. 43-72, in V. T. DeVita , Jr., and H. Busch [ed.], Methods in Cancer Research; Cancer Drug Development, vol. XVI, 1979). Prescreening systems for antitumor agents in natural products require assays that are exquisitely sensitive, since the active components are often produced in quantities of micrograms per milliliter or less. One assay used to identify agents that interact with DNA is the biochemical induction assay, utilizing Escherichia coli BR 513 (R. K. Elespuru and R. J. White, Cancer Res. 43:2819-2830, 1983). In this paper we describe a genetic modification of strain BR 513 that displays an expanded spectrum of activity. This strain may provide an improved prescreen for detecting natural products that interact with DNA.