Inhibition of synthesis of nucleic acids, proteins and respiration in isolated thymocytes by anthracyclines

The effect of anthracycline antibiotics such as carminomycin, daunomycin (rubomycin) and adriamycin on respiration and synthesis of nucleic acids and protein was studied comparatively. The anthracyclines inhibited the processes. By their efficacy in that respect they could be arranged in the following order: carminomycin greater than rubomycin greater than adriamycin. Thus, 50 per cent inhibition of nucleic acid synthesis in the thymocytes required 0.027, 0.044 and 0,173 mM of carminomycin, rubomycin and adriamycin respectively. Protein synthesis and respiration in the thymocytes were less sensitive to the effect of the anthracyclines than synthesis of nucleic acids. The study results were compared with the literature data on the effect of the compounds on respiration and synthesis of nucleic acids and protein in tumour and bacterial cells.     

New derivatives of carminomycin and rubomycin

For preparing new semisynthetic analogs of anthracycline antibiotics, hydrolysis of 13-dimethylketals of 14-bromrubomycin and 14-brom-arminomycin in solution of diluted hydrochloric acid was studied. It was shown that such hydrolysis yielded 14-chlorrubomycin and 14-chlorcarminomycin. Conditions for separating the mixture of 14-chlor- and 14-bromrubomycins and the mixture of 14-chlor- and 14-bromcarminomycins by HPLC were developed. Interaction of 14-chlorine derivatives of rubomycin and carminomycin with potassium formate in the presence of the crown ether yielded 14-formyloxy derivatives of rubomycin and carminomycin. Interaction of rubomycin and carminomycin with formic acid in the presence of N-oxysuccinimide and dicyclohexylcarbodiimide resulted in formation of N-formyl derivatives of rubomycin and carminomycin.     

Suppression of DNA synthesis in mice by the anthracycline antibiotics daunorubicin, carminomycin and doxorubicin

Inhibition of DNA synthesis by rubomycin (daunorubicin), carminomycin and doxorubicin in the spleen, liver, kidneys and heart was studied on mice. The antibiotics were administered intravenously in a dose of 0.3 LD50. The inhibition level was estimated by incorporation of 3H-thymidine. The time courses of DNA synthesis inhibition by daunorubicin, carminomycin and doxorubicin markedly differed, whereas the patterns of their inhibition curves for all the organs were close. The maximum inhibition of DNA synthesis by carminomycin was observed in 6 hours. After that period it gradually restored. Doxorubicin induced the maximum inhibition of DNA synthesis in 24-48 hours after its administration. Daunorubicin induced two maxima in inhibition of DNA synthesis i. e. in 6 and 48 hours. Definite correlation between the levels of DNA synthesis inhibition by the antibiotics and their toxic action was shown.     

Pathomorphological picture of the liver in mice administered antitumor antibiotics intraperitoneally and its comparative assessment]

A single administration of carminomycin, ribomycin or olivomycin in LD50 or treatment of the experimental animals with these antibiotics for 10 days in the therapeutic doses equal to 10 per cent of the LD50 induced distrophic and necrobiotic changes in the liver. The use of bruneomycin in the equivalent doses induced sclerotic process in addition to the above doses resulted in a decrease in the colour intensity of DNA, RNA and protein as compared to the control, the content of glycogen and a marked increase in the amount of lipids in the hepatocyte cytoplasm. The most pronounced shifts were observed with the use of carminomycin, rubomycin and especially bruneomycin in single doses. With the use of olivomycin in a single dose the shifts were less pronounced. It should be noted that with the use of carminomycin and rubomycin the damages were of the same character by their intensity. The changes in the liver on the use of carminomycin, rubomycin and olivomycin in single doses or during the treatment course were reversible, while on the use of bruneomycin they preserved to the end of the experiment.     

Comparative study of the effect of daunorubicin and its nitroxyl derivative on in vitro function of rat liver mitochondria

The effect of rubomycin (daunorubicin) and its nitroxyl derivative, ruboxyl, on respiration and oxidative phosphorylation of the rat liver mitochondria was comparatively studied. It was shown that ruboxyl had a more pronounced uncoupling effect than rubomycin, especially during respiration in the presence of the glutamate mixture with malate. Unlike rubomycin, ruboxyl in concentrations of 0.05 to 0.5 mM induced stimulation under metabolic conditions rather than respiration. When the antibiotic concentration increased ruboxyl started to inhibit the respiration as compared to the control and the inhibition level appeared to be higher than that induced by rubomycin. Possible mechanisms for decreasing rubomycin toxicity by its modification with the nitroxyl radical are discussed.     

Interaction with DNA of semisynthetic carminomycin and rubomycin derivatives

The apparent binding constants and the effect of semisynthetic derivatives of carminomycin and rubomycin (anthracycline antibiotics) on DNA fusion were studied. The following semisynthetic derivatives were used. 13-dihydrocarminomycin, 14-hydroxycarminomycin, 13-(4-methylpiperazinyl) imine carminomycin, 13-benzoylhydrazone carminomycin (carminazone), 13-tret-butoxycarbonyl hydrazone rubomycin, 13-(4-methylpiperazinyl) imine rubomycin, 14-(1-hydroxyl-2,2,6,6-tetramethylpiperidyl-4)-acetoxyrubomycin (spin-labeled rubomycin). The above derivatives slightly differed from the initial antibiotics by their affinity to DNA. The binding constants of methylpiperazinyl imines was 2-3 times higher than those of the respective antibiotics.     

Effect of rubomycin and carminomycin on antibody formation during the immunization of mice with various antigens

The maximum immunodepressive effect of rubomycin and carminomycin was observed when the antibiotics were administered intravenously 24 hours after the immunization. The immune response induced by the sheep erythrocytes or the lipopolysaccharide was equally inhibited by rubomycin. Carminomycin in a dose of 0.5 mg/kg (0.1 of the LD50) to a larger extent inhibited the immune response stimulated by the lipopolysaccharide. Dependence of the immunodepressive effect of the antibiotics on their dose was found when the drugs were administered intravenously or orally.     

Effect of the anthracycline group antibiotics, mitomycin C and bruneomycin, on the transduction of drug resistance in staphylococci]

The effect of various concentrations of antitumor antibiotics, such as carminomycin, rubomycin, adriamycin, mitomycin C and bruneomycin on transduction of erythromycin resistance from the donor strain 8325 P II/de of Staph, aureus to the recipient strain 8325-I in different transduction systems was studied. It was shown that the above antibiotics inhibited the transduction in the systems with constant presence of the drugs. Preliminary treatment of the recipient cells with the drugs in the subbacteriostatic doses did not decrease the transfer frequency. The preliminary treatment of the donor cells resulted in an increase in the phase titer and the transfer frequency in the "preliminary-treated donor + recipient" system.     

Semisynthetic derivatives of daunorubicin and carminomycin: inhibition of DNA synthesis after intravenous administration to mice

Inhibition of DNA synthesis in the liver, kidneys, spleen and heart of mice after intravenous administration of 0.1 and 0.3 LD50 of semisynthetic derivatives of rubomycin (daunorubicin) and carminomycin was studied. The level of DNA synthesis inhibition was estimated by a decrease in incorporation of (methyl-3H) thymidine. Under the action of 13-trebutoxycarbonyl hydrazone and 14-salicyloiloxy derivatives of rubomycin and carminomycin maximum inhibition of DNA synthesis was reached later while its recovery started earlier as compared to the initial antibiotics.     

Effect of rubomycin (daunorubicin) and its nitroxyl analog on the functioning of rat heart mitochondria

Changes in the functional parameters of the rat heart mitochondria were studied in time after a single intraperitoneal administration of rubomycin, the rubomycin combination with 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO-OH) and ruboxyl, a nitroxyl derivative of rubomycin. The administration of rubomycin resulted in inhibition of the heart mitochondria bioenergetic functions (a decrease in the respiration control coefficient, RCC, and the respiration rate, RR, on phosphorylation) during respiration in the presence of NAD(+)-dependent substrates 6 to 24 hours after the administration. Later the mitochondria functions recovered while in 2 to 3 weeks a secondary decrease in the RCC and RR was observed. During respiration in the presence of succinate the inhibitory effect on the antibiotics was higher. The combined administration of rubomycin and TEMPO-OH eliminated the primary inhibition. In the presence of ruboxyl the inhibitory effect in regard to the NAD(+)-dependent substrates was not detected. The mechanisms of the toxic action of the anthracycline antibiotics are discussed.     

Comparison of the action of carminomycin and rubomycin on the dynamics of the primary and secondary immune responses

The effect of rubomycin and carminomycin on the dynamics of the primary and secondary immune response and formation of the immunologic memory to sheep red cells in mice was studied. Differences in the character of the antibiotics effect indicative of the higher selective action of carminomycin on multiplying cells, precursors of the antibody-forming plasmids, were found. Theoretically interesting discrepancies in the effect of the antibiotics on the content of the antibodies in the serum and the antibody-producing cells in the spleen were shown. It was demonstrated that carminomycin had no effect on formation of the immunologic memory inspite of a noticeable decrease in the total number of the spleen nuclear cells and the number of the antibody-forming cells at the moment of immunization under the effect of the antibiotic.     

Antiviral action of carminomycin and some of its derivatives]

Carminomycin was shown to inhibit the development of both the DNA-containing variolovaccine virus and the RNA-containing grippe virus in chick embryos. Comparison of the effects of rubomycin, carminomycin, 14-oxy-carminomycin and carminomycin complex with bovine serum albumin in experiments with chick embryos showed that the inhibitory effect of carminomycin and its derivatives on the development of the grippe virus was much higher than that of rubomycin. The carminomycin derivatives proved to be much more active in this respect than the initial antibiotic. Carminomycin and its derivatives had a therapeutic effect on mice with experimental grippe pneumonia also on their oral use.     

Transformation of anthracycline antibiotics and their semisynthetic derivatives as affected by the liver aldo-keto reductase of rats

Formation of 13-dihydro derivatives of rubomycin (daunorubicin), carminomycin, doxorubicin and some of their semisynthetic derivatives under the effect of pure aldo-keto reductase from the rat liver was studied. Attachment of an oxy group to C-14 markedly retarded formation of the 13-dihydro derivatives while attachment of the bulky radicals to the same position prevented their formation. Binding of the anthracycline antibiotics to human serum albumin had no impact on the fermentative reaction rate. Rubomycin, carminomycin and doxorubicin significantly differed in their lipophilic properties and capacity for binding to serum albumin.     

Oxidation of anthracycline antibiotics

The quantum-chemical estimation of the highest occupied molecular orbital energy for the aglycons of carminomycin, rubomycin, adriamycin and aclacinomycin A in the neutral and ionized states was performed with a semiempirical method. It was shown that the aglycon ionization amplified the electron donor properties of the antibiotics. On the basis of the difference in the absorption spectra of the neutral and ionized chromophores their ionization constants were determined spectrophotometrically. For comparison of the electron donor properties of the anthracyclines at the physiological pH value the reaction of their oxidation with potassium ferricyanide accompanied by decoloration of the solutions was studied. On the basis of the quantum-chemical and experimental data it was concluded that the electron donor properties amplified as follows: aclacinomycin A less than adriamycin-rubomycin less than carminomycin. At the same time their acute toxicity increased (a decrease in the LD50). Therefore, the toxicity of the anthracycline antibiotics could be also due to formation of the radicals with high reactivity on the monoelectronic oxidation.     

The effect of anthracyclines on heart mitochondria respiration during the action of creatine phosphokinase

It was shown that during glutamate+malate oxidation in the presence of creatine, antitumour anthracycline antibiotics strongly inhibit the rate of oxygen uptake by rat heart mitochondria; ADP excess activated the respiration up to the initial level, i.e., that observed after the first addition of ADP. Carboxyatractyloside addition to a system containing creatine (or hexokinase+glucose) results in the stimulation of rubomycin-induced mitochondrial respiration. Substitution of carboxyatractyloside by oligomycin gives very similar results. It is supposed that anthracycline antibiotics exert a manyfold effect on heart mitochondrial membranes which results in impaired compartmentation of enzymatic systems providing for oxidative phosphorylation.     

Mitochondrial respiration in heart, liver, and kidney of copper-deficient rats

Morphological observations in some tissues indicate that dietary copper deficiency results in structural damage to mitochondria. The purpose of this study was to determine whether mitochondrial function is impaired as well. Male, weanling Sprague-Dawley rats were fed diets deficient or sufficient in copper for 4 weeks. Mitochondria were isolated from heart, liver, kidney cortex, and kidney medulla. P/O ratio, state 3 and state 4 respiration rates (oxygen consumed in the presence and absence of ADP, respectively), and acceptor control index (ratio of state 3:state 4) were determined using succinate or pyruvate/malate as substrate. State 3 respiration rate in mitochondria from copper-deficient hearts and livers was lower than in mitochondria from copper-sufficient hearts. Copper deficiency reduced the state 4 respiration rate only in cardiac mitochondria. Neither respiration rate was affected by copper deficiency in mitochondria from kidney medulla or cortex. P/O ratio was not significantly affected by copper deficiency in any tissue examined. Acceptor control index was reduced only in liver mitochondria. The observed decreases in respiration rates are consistent with decreased cytochrome c oxidase activity, shown by others to occur in mitochondria isolated from hearts and livers of copper-deficient rats.     

Anthracycline antibiotic, beromycin. The formation of complexes with DNA and the suppresion of nucleic acid biosynthesis]

Beromycin, an antitumor anthracycline antibiotic formed in vitro complexes with native and denaturated DNA and ribosomal RNA. Beromycin had a comparatively low constant of DNA binding and to a less extent increased the melting temperature and viscosity of DNA than the other anthracycline antibiotics. A peculiar property of beromycin was very slow binding with DNA, the complex formation was completed in 60 minutes. Beromycin had a selective inhibitory effect on synthesis of nucleic acids in bacterial and tumor cells. Beromycin inhibited synthesis of RNA in the DNA-dependent RNA-polymerase reaction when both the native and denaturated DNA were used as the template. A lower biological activity of beromycin as compared to the other anthracycline antibiotics, such as rubomycin or carminomycin may be explained by lower affinity of this antibiotic to DNA.     

Effect of the molecular structure of natural anthracycline antibiotics on their relative hydrophobicity

Relative hydrophobicities of anthracycline antibiotics, adriamycin, rubomycin and carminomycin, have been measured by the two-phase distribution method. Two different biphasic systems were used for this purpose. Possible reasons of discrepancies between results obtained and other authors, data are discussed. It was established that the relative hydrophobicities of the compounds investigated contradict the theory of increment additivity. The results are compared with quantum-chemical calculations.     

Study of toxicity and antineoplastic activity of 14-substituted derivatives of rubomycin and carminomycin

Toxicity and antitumor activity of five derivatives of rubomycin and carminomycin were studied in animals. The derivatives were prepared by modification of the methyl C-14 group. These were the following: 14-chlorrubomycin, 14-chlorcarminomycin, 14-salicyloyloxyrubomycin, 14-salicyloyloxycarminomycin and 14-quinaldinoyloxyrubomycin. The chemotherapeutic study revealed that, in their activity, all the compounds were inferior to the starting antibiotics. Unlike the other derivatives, 14-chlorcarminomycin induced a significant inhibition of leukemia P-388 development (the average lifespan of the mice amounted to 165 per cent as compared to the control). However, in the magnitude of its effect, the derivative was inferior to carminomycin.     

Respiration-stimulated activation of DNAase I associated with mitochondria]

The influence of respiration and Ca++ transport in the liver mitochondria on the activation of DNAase I, associated with these organelles, was studied. It was shown that 96% of the total activity of this enzyme in mitochondria is in the latent state. Aeration of the mitochondrial suspension leads to a sharp increase in the enzyme activity. The activation of DNAase I is inhibited by EGTA addition (optimal pH 8.0), and stimulated in mitochondria, releasing Ca++. It is concluded that the activation of DNAase I is dependent on the state of cellular energetics. Participation of mitochondrial phospholypase A, activated by the Ca++ release from mitochondria during DNAase I activation is suggested.