Accelerated rejection of the second transplants of immunogenic tumor in mice under inhibition of indoleamine 2,3-dioxygenase activity by ethyl pyruvate.

AIM: A recently discovered enzyme, indoleamine 2,3-dioxygenase (IDO), is expressed in placenta, dendritic cells and also in many kinds of tumors and in tumor-infiltrating macrophages. By catabolizing tryptophan, IDO causes local depletion of this essential amino acid and excess of kinurenin, and suppresses in situ proliferation and functioning of T lymphocytes. Thus, immune resistance of tumors can be overcome by inhibiting IDO activity. MATERIALS AND METHODS: C3HA mice immunized with non-syngeneic H-29 tumor were used to study the effect of the IDO inhibitor ethyl pyruvate, under systemic or local (at site of tumor cells localization) administration, on the occurrence and rate of rejection of the second transplants of this tumor. RESULTS: Both systemic and local administration of ethyl pyruvate increases the incidence of and substantially accelerates tumor regression as compared with control. CONCLUSION: IDO inhibitors impairing immune resistance of tumors may appear useful in leveraging the efficacy of antitumor therapy.

Nitroxyl antioxidant TPPA-TEMPO increases the efficacy of antitumor therapy on the model of transplantable mouse tumor.

Nitroxyl antioxidant 4-triphenylphosphonioacetamido-2,2,6,6-tetramethylpiperidine-1-oxyl (TPPA-TEMPO) was synthesized from 4-chloroacetamido-2,2,6,6-tetramethylpiperidine-1-oxyl chloride and triphenylphosphine. Systemic administration of TPPA-TEMPO in the subtoxic dose to mice with lymphosarcoma inhibited tumor growth, but did not prolong animal lifespan. Combined treatment with TPPA-TEMPO and cyclophosphamide increased the efficacy of antitumor therapy: it prolonged animal lifespan and increased the number of recovered mice.

EPR and Quantum Chemical Studies of the pH-sensitive Imidazoline and Imidazolidine Nitroxides with Bulky Substituents.

The X- and W-band electron paramagnetic resonance (EPR) spectroscopies were employed to investigate a series of imidazolidine nitroxide radicals with different number of ethyl and methyl substituents at positions 2 and 5 of a heterocycle in liquid and frozen solutions. The influence of the substituents on the line shape and width was studied experimentally and analyzed using quantum chemical calculations. Each pair of the geminal ethyl groups in the positions 2 or 5 of the imidazolidine ring was found to produce an additional hyperfine splitting (hfs) of about 0.2 mT in the EPR spectra of the nitroxides. The effect was attributed to the hfs constant of only one of four methylene hydrogen atoms of two geminal ethyl substituents not fully averaged by ethyl group rotation and ring puckering. In accordance with this assumption, the substitution of hydrogen atoms of CH(2) groups in 2,2,5,5-tetraethyl-substituted imidazolidine nitroxides by deuterium leads to the substantial narrowing of EPR lines which could be useful for many biochemical and biomedical applications, including pH-monitoring. W-band EPR spectra of 2,2,5,5-tetraethyl-substituted imidazolidine nitroxide and its 2,2,5,5-tetraethyl-d(8) deuterium-substituted analog measured at low temperatures demonstrated high sensitivity of their g-factors to pH, which indicates their applicability as spin labels possessing high stability.

Thiol-induced nitric oxide release from 3-halogeno-3,4-dihydrodiazete 1,2-dioxides.

In this work we studied the mechanism of nitric oxide (NO) release underlying the vasorelaxant and antiaggregant effect of 3,4-dihydrodiazete 1,2-dioxides (DD). Six derivatives were included in the investigations, namely, 3-bromo- and 3-chloro-3,4,4-trimethyl-DD (1a,b), 3-bromo- and 3-chloro-4-methyl-3,4-hexamethylene-DD (2a,b), 3,3,4,4-tetramethyl-DD (3), and 3-methyl-3,4-hexamethylene-DD (4), and their reactivity toward thiols was analyzed. The 3-bromo- and 3-chloro-DD derivatives were found to react with thiols; this reaction can lead to NO formation, DD 2a being the most reactive compound. 2-(Hydroxyamino)-2-methylbutan-3-one oxime (5a) and 2-hydroxy-2-methylbutan-3-one oxime (6) were the main products isolated from the reaction of 1a with cysteine. Reaction rates of DD with thiols were dependent upon pH and concentration of the reagents. Maximum rates of NO release corresponded to thiol concentrations in the range of 1 mM. Consistent with reaction kinetics data and products isolated, a reaction mechanism was proposed. Addition of 2a to bovine aortic endothelial cells led to strong NO release indicating a reaction with endogenous thiols. In rat mesenterial arteries, the vasorelaxant action of 2a was only slightly influenced by addition of thiol to the incubation medium. For the most reactive DD derivatives, cytotoxic effects were observed at concentrations roughly 2 orders of magnitude higher than those inducing vasorelaxation.

Nitroxides increase the detectable amount of nitric oxide released from endothelial cells.

Nitroxides are known to exert superoxide dismutase-mimetic properties and to decrease O-2- and H2O2-mediated cytotoxicity. However, the effect of nitroxides on .NO homeostasis has not been studied yet. The present study investigates the effect of nitroxides on the detectable amount of .NO released by 3-morpholinosydnonimine (SIN-1) and cultured endothelial cells. Cultured bovine aortic and atrial endothelial cells stimulated with 10 microM A23187 released a stable flux of .NO, as detected by .NO chemiluminescence. Addition of 100 units/ml SOD or 10 microM of the nitroxides 4-hydroxy-2,2,6, 6-tetramethylpiperidine-N-oxyl (TEMPOL), 3-carboxy-proxyl, and 3-ethoxycarbonyl-proxyl, increased the chemiluminescence signal. The effect of these nitroxides on the amount of .NO released from cell monolayers was dose-dependent, with the highest efficacy between 30 and 100 microM. EPR spin trapping in SIN-1 solutions revealed the formation of .OH adducts from spontaneous dismutation of O-2 and concomitant reaction with H2O2. Both SOD and TEMPOL increased the signal intensity of the .OH adduct by accelerating the dismutation of O-2. The results of this study demonstrate that the SOD-mimetic activity of nitroxides increases the amount of bioavailable .NO in vitro.

Synthesis and spin trapping applications of 2,2-dimethyl-d6-4-methyl-2H-imidazole-1-oxide-1-15N.

A new spin trap, 2,2-dimethyl-d6-4-methyl-2H-imidazole-1-oxide-1-15N (lTMIO), was synthesized and characterized. Hyperfine splitting (HFS) constants of spin adduct ESR spectra of this compound with oxygen-centered, carbon-centered, thiyl and sulfite-derived radicals were determined and compared with the data of the unsubstituted compound. The increase in ESR spectral intensity and the accompanying decrease of the spectral linewidth result in resolution of the HFS due to interaction with alpha-protons of alkyl radicals trapped by lTMIO. Trapping of the formate radical in deoxygenated aqueous solution revealed a very low spectral linewidth (delta Bpp = 0.028 mT) of the corresponding adduct. A strong dependence of the ESR spectra on pH was observed when the autoxidation product of sulfite, SO3-, was trapped. The pKa was found to be 5.8 +/- 0.3. In comparison to other nitrones, application of this spin trap provides more detailed information on the structure of the species trapped, especially for carbon-centered radicals.

Superoxide-mediated reduction of the nitroxide group can prevent detection of nitric oxide by nitronyl nitroxides.

Nitronyl nitroxides (NN), a class of compounds which react with nitric oxide forming imino nitroxides, were applied in different systems for the detection of nitric oxide. Addition of a NN to planar monolayers of bovine aortic endothelial cells (BAEC) activated by Ca2+ ionophore A23187 immediately resulted in a strong decrease of the ozone-mediated .NO chemiluminescence. Simultaneously, a rapid diminution of the electron spin resonance (ESR) signal intensity of the NN (without detectable formation of the corresponding imino nitroxide) was observed; superoxide dismutase partially inhibited this decrease in the NN concentration. Model experiments using hypoxanthine/xanthine oxidase in aqueous solution and KO2 in dimethylsulfoxide as sources of O2.- revealed that there is a rapid reduction of nitronyl nitroxides by superoxide. The second order rate constant for the reaction of the water soluble NN with O2.- was determined to be 8.8 x 10(5) M-1s-1, which is more than two orders of magnitude higher than the value reported previously for reaction with .NO (Woldman et al., BBRC 202, 195-203, 1994). Reduction of the nitronyl nitroxide was also observed in the presence of glutathione, ascorbic acid or rabbit liver microsomes. Incorporation of both nitronyl and imino nitroxides into liposomes strongly decreased reduction by superoxide and other reductants, however, in the presence of microsomes, there was no protective effect by liposomal encapsulation of NN. The results indicate that in biological systems (in addition to other reducing agents) the presence of superoxide can prevent the detection of nitric oxide using nitronyl nitroxides.