Effects of cytochalasin B in culture and in vivo on murine Madison 109 lung carcinoma and on B16 melanoma.

Cytochalasin B (CB), at 100 or at 10 mg/kg single dose s.c. in carboxymethyl-cellulose (2%)/Tween-20 (1%) 24 h after s.c. challenge of B6D2F1 mice with trocar implants of B16F10 tumor s.c., delayed the appearance of measurable tumor nodules by 157 and 93%, respectively, and extended host survival by 65 and 26%. Tumor growth was also delayed when CB treatment was given 1 day after the appearance of palpable tumor nodules. By in vivo bioassay, in vitro cloning, and dye exclusion measurements, solid tumor nodules treated in vivo with CB at either 100 or 10 mg/kg showed the same viability and tumorigenicity as did vehicle-treated nodules 4 and 6 days after drug treatment, at which time growth inhibition was still apparent. This indicates that growth inhibition by CB is not dependent on a gross cytotoxic effect. CD2F1 mice challenged s.c. with Madison 109 lung carcinoma cells and treated with CB s.c. at 100 or 150 mg/kg 24 h later showed a 66% delay in the median day of tumor nodule appearance. When administered under these conditions or at the time of nodule appearance. CB markedly inhibited the rate of tumor growth, prevented tumor invasion at day 23, extended life span by 23%, and significantly inhibited spontaneous lung metastases measured 28 days after tumor challenge. Maximum tolerated doses of CB administered i.p., s.c., or i.v. in suspension or in solution are defined. These results delineate the conditions under which CB can be tested for in vivo biological activities and establish that this microfilament-active natural product in a single-agent protocol inhibits local tumor growth and extends survival in B16F10 melanoma and Madison 109 lung carcinoma, and, in the latter model, inhibits invasion and spontaneous lung metastases by mechanisms that do not appear to depend on cytotoxicity. This work on formulation, tolerated doses in vivo, and localized peritumoral effects of CB now permits evaluation of systemic antitumor effects of cytochalasin B as a single agent. It also permits chemotherapy studies using CB as a potential amplifier of the activity of other antitumor agents in vivo.

Isoenzymic forms of NAD-linked glycerol-3-phosphate dehydrogenase from rabbit brain.

Two major enzyme forms of cytosolic NAD-linked glycerol-3-phosphate dehydrogenase in rabbit brain have been purified to apparent homogeneity. One major enzyme form designated I6.5 exhibits an iso-electric point at pH 6.5, and is indistinguishable from the major form I6.5 found in other tissues. The other major form, designated I5.9, has an isolectric point at pH 5.9, and by amino acid analysis is shown to be a true isoenzyme distinct from form I6.5. Form I5.9 appears to be closely related to or identical with the major enzyme characteristic of heart. Neither the brain enzyme form I5.9 nor the major heart isoenzyme are inhibited by antiserum to the muscle enzyme. Because of the high apparent Km for NADH, it is postulated that the brain isoenzyme I5.9 serves to maintain glycolysis when NADH levels rise under relatively anaerobic conditions especially during fetal and neonatal development.

Haloacetamido analogues of 2-amino-2-deoxy-D-mannose. Syntheses and effects on tumor-bearing mice.

Haloacetamido analogues (fluoro, chloro, and bromo) of 2-deoxy-2acetamido-D-mannose and their tetra-O-acetates were prepared from D-mannosamine hydrochloride, with either chloroacetic or bromoacetic anhydride or by dicyclohexylcarbodiimide-activated condensation with fluoroacetate followed by acetylation. Comparative specific rotations and 13C and 1H NMR spectra were consistent with a beta configuration for the tetra-O-acetylated derivatives, 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-(bromoacetamido)-beta-D-mannose and the corresponding analogue of glucose inhibited [3H]thymidine incorporation into mouse L1210 leukemia cells by 50% (IC50) at concentrations between 6 and 9 microM. 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-(chloroacetamido)-beta-D-mannose was 3-fold more active in the thymidine-incorporation assay (143 +/- 24 microM, IC50) than was the corresponding analogue in the glucose series (425 +/- 62 microM; p = 0.05). All of the haloacetamido free sugars, as well as the tetra-O-acetates of the fluoroacetamido analogues in the glucose, galactose, and mannose series, were inactive in the thymidine incorporation assay at 1mM. In the mannose series the tetra-O-acetylated chloroacetamido and bromoacetamido analogues, as well as the bromoacetamido free sugar, could be administered at relatively high in vivo tolerated doses compared to the corresponding analogues in the galactose and glucose series. These three mannose analogues produced high proportions of cures of Ehrlich tumor-bearing B6D2F1 mice, whereas in the galactose and glucose series only the tetra-O-acetylated bromoacetamido analogues had previously produced in vivo chemotherapeutic activity.

3-Fluoro-1-hydroxypropan-2-one (fluorohydroxyacetone) and some esters. Syntheses and effects in BDF mice.

1-(Benzoyloxy), 1-(4-nitrobenzoyloxy), and 1-(3,5-dinitrobenzoyloxy) derivatives of 3-fluoro-, 3-chloro-, and 3-bromopropan-2-one were prepared by oxidation of the 1-benzoyloxy-3-halopropan-2-ols in turn prepared from the appropriate benzoyl chloride and 3-halo-1,2-propanediols, 1-Benzoyloxy-3-fluoropropan-2-one was allowed to react with acidic trimethyl orthoformate to yeild 1-benzoyloxy-2,2-dimethoxy-3-fluoropropane which upon basic hydrolysis afforded 2, 2-dimethoxy-3-fluoropropan-1-ol (fluorohydroxyacetone dimethyl ketal). This was deketalized with aqueous HCL to afford 3-fluoro-1-hydroxypropan-2-one (fluorohydroxyacetone), the title compound. By reacting 1-chloro-3-fluoropropan-2-one and 1, 3-dichloropropan-2-one with potassium acetate, 1-acetoxy-3-fluoropropan-2-one and 1-acetoxy-3-chloropropan-2-one (fluoro- and chlorohydroxyacetone acetate, respectively) were obtained. Similarly, sodium benzoate and 1-chloropropan-2-one produced 1-benzoyloxypropan-2-one. Stucture-activity relationships are discussed which relate chemical structure, alkylating ability, toxicity, and antitumor effects. Comparative toxicities in mice showed decreasing toxicity, on a molar basis, in the 1-benzoyloxy-3-halopropan-2-one series of bromo greater than fluoro greater than chloro. Ketones were much more toxic than the corresponding alcohols. In general the phosphate and benzoyloxy derivatives are more toxic than acetoxy compounds, with nitro-substituted benzoyloxy derivatives being much less toxic.

Deoxyfluoroketohexoses: 4-deoxy-4-fluoro-D-sorbose and -tagatose and 5-deoxy-5-fluoro-L-sorbose.

4-Deoxy-4-fluoro-alpha-D-sorbose (6) was prepared in crystalline form by the action of potassium hydrogen fluoride on 3,4-anhydro-1,2-O-isopropylidene-beta-D-psicopyranose (3) followed by deacetonation. Under identical conditions, 3,4-anhydro-1,2-O-isopropylidene-beta-D-tagatopyranose (7) underwent epoxide migration to give 4,5-anhydro-1,2-O-isopropylidene-beta-D-fructopyranose (12), which after deacetonation yielded 4-deoxy-4-fluoro-D-tagatose (15) and 5-deoxy-5-fluoro-alpha-L-sorbopyranose (16), the latter as the crystalline, free sugar. The action of glycol-cleavage reagents on the isopropylidene acetals of the deoxyfluoro sugars was consistent with the assigned structures. The structures were established by 13-C n.m.r. studies of the free deoxyfluoro sugars 6 and 16 and of the isopropylidene acetal 13, and by 1-H n.m.r. studies on the acetylated isopropylidene acetals 5 diacetate, 13 diacetate, and 14 diacetate. 5-Deoxy-5-fluoro-L-sorbose (16) was biologically active, producing in mice effects characteristic of deoxyfluorotrioses and of fluoroacetate. 4-Deoxy-4-fluoro-D-tagatose (15) and 4-deoxy-4-fluoro-D-sorbose (6) produced no apparent effects in mice up to a dose of 500mg/kg. The implications of these findings with respect to transport, phosphorylation, and the action of aldolase on ketohexoses are discussed.

Cytochalasin B: preparation, analysis in tissue extracts, and pharmacokinetics after intraperitoneal bolus administration in mice.

Cytochalasin B (CB) was prepared by methanol extraction of dehydrated mold (Drechslera dematioidea) matte, reverse-phase C18 silica gel batch adsorption, selective elution with 1:1 (v/v) hexane:tetrahydrofuran (THF), crystallization, preparative TLC, and recrystallization. Unit gravity silica gel normal phase chromatography afforded additional CB. Yield per liter of medium was 300 mg of CB greater than 95% pure by NMR, HPLC (60:40 hexane:THF, Lichrosorb Si60 silica gel, 230 nm), and TLC. CB added exogenously to mouse organs at 1 and 5 micrograms/organ was recovered 70 to 100% by methanol extraction, adsorption to C18 silica gel Sep-Pak cartridges, elution with ethyl acetate, and analysis by TLC and/or HPLC. Limiting sensitivity (micrograms/extract) was 0.5 TLC; 1.0 HPLC. Quantitative extraction was confirmed with 3H-labeled CB. CB ip in mice at 50 mg/kg (LD10) distributed rapidly into liver, renal fat, kidney, intestines, mesentery, pancreas, spleen, and blood cells and was cleared from all but liver within 24 h. CB was below detectable levels in thymus, lymph nodes, heart, brain, bone marrow, and lungs. Cytochalasin A is fixed to tissues and not extractable. This work affords a source of CB in quantities permitting in vivo study, provides methods for extraction and analysis, and reveals the pharmacokinetics of ip bolus CB.

1-Halo analogs of dihydroxyacetone 3-phosphate. The effects of the fluoro analog on cytosolic glycerol-3-phosphate dehydrogenase and triosephosphate isomerase.

Fluoro-o-hydorxyacetone phosphate (fluoroacetol phosphate) has been prepared by oxidation of 1-fluoro-3-chloro-2-propanol to 1-fluoro-3-chloroacetone, phosphorylation with silver dibenzylphosphate, and the intermediate isolation of 1-fluoro-3-hydroxyacetone phosphate dibenzyl ester, followed by catalytic hydrogenation and preparation of the stable monosodium salt. The chloro analog as the pure, stable monosodium salt has been prepared by a similar route from 1,3-dichloroacetone. 1-Fluoro-3-hydroxyacetone-P is substrate for cytosolic NAD+-linked glycerol-3-P dehydrogenese (EC 1.1.1.8) from rabbit skeletal muscle with an apparent Km of 50 mM under conditions in which dihydroxyacetone-P exhibits an apparent Km of 0.15 mM. Under these conditions the fluoro analog is 85% hydrated wheras dihydroxyacetone-P has been shown by others to be 44% hydrated. The turnover numbers are 49,000 molecules of NADH oxidized per minute per molecule of enzyme at 25 degrees with the fluoro analog as substrate, and 60,000 with dihydrocyacetone-P as substrate. The product of the reduction of the fluoro analog has been identified as 1-fluorodeoxyglycerol-3-P. 1-Fluoro-3-hydroxyacetone-P is comparatively weak irreversible inhibitor at 4 degrees of rabbit muscle triosephosphate isomerase (EC 5.3.1.1) with second-order rate constant of 2.6 M minus 1 sec minus 1. Inhibition by pyrazole in vivo of alcohol dehydrogenese catalyzed oxidation of 1-fluorodeoxyglecerol-3-P indicates in mice the reduction of 1-fluoro-3-hydroxyacetone-P to -l-1-fluorodexoxyglycerol-3-P is not significant metabolic route, or that an alternative route exists when the alcohol dehydrogenase dependent pathway is inhibited.

Cytochalasin-B-induced immunosuppression of murine allogeneic anti-tumor response and the effect of recombinant human interleukin-2.

Cytochalasin B (CB), administered i.p. to C57B1/6 mice in a single dose as a suspension in carboxymethylcellulose 2%/Tween 20 1%, inhibits in a dose-dependent and time-dependent manner the ability of spleen cells to respond to allogeneic P815 mastocytoma tumor cells in vitro. Spleen cells from CB-treated animals sensitized to X-irradiated P815 cells in 4-day cultures at a 50:1 responder:stimulator ratio and tested for specific cytotoxicity against 51Cr-labelled P815 target cells showed strong inhibition 3 h after CB treatment at a dose of 50 mg/kg. A dose of 25 mg/kg showed measureable but not statistically significant inhibition at 3 h, whereas 10 mg/kg produced only slight inhibition, and 5 mg/kg and 2 mg/kg were noninhibitory. None of the doses produced significant suppression 19 h or 72 h after CB treatment. Addition to the sensitization cultures of human recombinant interleukin-2 (rhIL-2) at 350 BRMP units/ml completely restored tumor lytic capacity. C57B1/1 mice treated with CB 50 mg/kg, i.p. and challenged i.p. with 3 x 10(7) allogeneic P815 mastocytoma cells showed a brief, time-dependent, statistically significant abrogation of allogeneic responsiveness consistent with transient reversible immunosuppression within 3-12 h following CB treatment. No such inhibition of host allogeneic responsiveness in vivo was observed when CB was administered 24 h prior to, simultaneously with, or 1, 2, or 4 days after tumor challenge. Thus CB at the highest tolerated i.p. dose in vivo causes only a transient inhibition of anti-allo-responsiveness measured in culture, and rhIL-2 used in vitro restores lytic capacity. The anti-allo effect of CB is also seen to be transient directly in vivo since allogeneic tumor outgrowth is permitted for only a brief period following administration of CB. These results indicate that the use of CB in vivo in anti-tumor chemotherapy protocols will not be complicated by profound or prolonged immunosuppressive effects.

Isolation and characterization of flavin-linked glycerol-3-phosphate dehydrogenase from rabbit skeletal muscle mitochondria and comparison with the enzyme from rabbit brain.

Mitochondrial glycerol-3-P dehydrogenase (EC 1.1.99.5) has been purified in 20% yield from both rabbit skeletal muscle and brain using a four step procedure involving osmotic shock, solubilization with Triton X-100, hydrophobic chromatography, gel filtration, and preparative column isoelectrofocusing. The active muscle and brain enzymes were found to be 95% and 80% homogeneous, respectively. Final purification was performed on the denatured subunit. The active enzyme from each of the tissues focused at pH 5.25 +/- 0.12 and each produced similar biphasic thermal inactivation plots at 50 degrees C. Mixtures of the purified brain and muscle enzymes co-migrated in discontinuous electrophoresis gels and each enzyme exhibited a single polypeptide component on sodium dodecyl sulfate (SDS) gels either when run separately or in mixtures. The subunit molecular weight was shown to be 76,000 +/- 3,000 by SDS-gel electrophoresis and gel filtration in 6 M guanidine HCl. One mole of noncovalently bound FAD and 1 mole of iron were measured per Mr = 100,000. The amino acid composition was determined based on the assumption of 70 aspartate residues per subunit to give a Mr = 76,000. The absorption spectrum has a maximum at 416 nm and a shoulder at 450 to 460 nm which is bleached on treatment with sodium dithionite. The maximum at 416 nm is removed by treatment with mersalyl.

Effects of halo analogs of glycerol 3-phosphate and dihydroxyacetone phosphate upon Escherichia coli.

The fluoro and chloro analogs of glycerol 3-phosphate inhibit the growth of Escherichia coli and affect bacterial enzymes involved in lipid synthesis.