Radioprotective and antineoplastic activity of polyene antibiotics combined with dimethyl sulfoxide]

Radioprotective and antineoplastic activity of polyene, its derivatives and combinations with dimethyl sulfoxide (DMSO) was studied. The most potent radioprotective effect was demonstrated by methylated levorin, original levorin and by its isomer--isolevorin. Survival rate of the animals on 12th day after X-ray exposure was 100, 60, 60 per cent, at the control group 33.6, 20 and 0 per cent consequently. Levorin and alkyl derivatives of amphotericin B--methamphocin and buthamphocin inhibited growth of ascites and solid tumors to 46.3-79.0 per cent when compared to control group. Polyen antibiotics combined with DMSO also demonstrated antineoplastic activity at the animals treated with carcinogenic agent--diethyl nitrosoamine (DENA). 5-month survival of the animals was 76 per cent at nystatin and levorin group and 35.7 per cent at the control group (animals treated with DENA only).     

An aromatic heptaene antibiotic levorin and its derivatives in muscle activity]

The review is concerned with the outlooks for the use of levorin, a membrane active and channel forming polyene antibiotic, and its alkyl derivatives in muscle activity. In complex with cholesterol and ergosterol, the aromatic heptaene antibiotic levorin forms structural ionic channels of the molecular size in the lipid and cell membranes. Levorin increases the membrane permeability for monosucrose and other neutral molecules as follows: H2O > urea > acetamide > glycerine > ribose > arabinose > glucose > saccharose. As a channel forming compound, levorin is able to induce in the cell membranes of the muscle fibres formation of additional channels permeable for the cations and to increase the flow of the energy dependent substrates to the cells and the outburst of the metabolites from them during intensive muscle activity. Levorin several times decreases the surface tension of aqueous solutions. In some models of experimental animals levorin promoted an increase of the blood fluidity and accelerated the blood stream in the blood vessels both in rest and in muscle activity. Physical load in a high power zone increases the intensity of lipid peroxidation that results in fatigue and lower physical efficiency. Possible prevention of an increase of the rate of free radical reactions by levorin and its alkyl derivatives providing higher antioxidant protection is discussed.     

Action of levorin on amino acid transport in Candida albicans]

It was shown that suppression by levorin of the leucine transport into the cells of C. albicans was due to replacement of intracellular K+ by Na+ induced by the antibiotic. The alanine transport was suppressed by levorin irrespective of the ratio of the monovalent cations concentration in the medium and inside the cell. The levorin effect on the protone escape from the cells was negligible and probably played no significant role in the mechanism of the amino acid transport suppression by the antibiotic.     

Physical and Chemical Properties of Erythrocyte Membranes in Interaction with Polyene Antibiotics in the Field of Ultrasonic Waves

Biophysics - Red blood cell hemolysis induced by ultrasound and alkyl derivatives of amphotericin B and levorin modified by the amine and carboxyl groups has been studied. A kinetic method has been...     

Effect of amigluracil and levorin on membrane permeability and protein synthesis in Candida albicans protoplasts]

It was shown that amigluracyl, a water soluble derivative of methacyl which decreased the nephrotoxic effect of polyens activated the membrane permeability in Candida albicans for a mixture of 14C-amino acids but had no significant effect on protein synthesis in this microorganism. The level of inhibition of the membrane permeability in C. albicans for the amino acids and protein synthesis in the fungus by levorin did not practically depend on the presence of amigluracyl in the incubation medium. The minimum levorin concentration inhibiting the growth of Candida albicans in the presence or absence of levorin was 0.039 gamma/ml. Therefore, amigluracyl may be used in combination with polyenic antibiotics for the treatment of mycoses.     

Mechanism of polyene antibiotic inactivation. Possible ways for levorin stabilization

Various means for levorin isolation were studied with the EPR method and approaches to stabilization of the antibiotic on storage under natural conditions were discussed. It was shown that formation of the radicals begins already at the first stage of the antibiotic isolation, i.e. during extraction from the mycelium. Treatment of the solvents with an inert gas or addition of antioxidants decreased the number of free radicals in a freshly isolated product. The antibiotic inactivation rate depended on the initial concentration of the free radicals and conditions of natural storage. The levorin stability increased when oxygen was thoroughly removed from the solvents at all isolation stages and the antibiotic was subsequently stored under conditions preventing any access of the air. The stabilizing effect was also observed when the oxidative effect of the amino sugar moiety on destruction of the polyenic chromophore during the antibiotic complex formation with respect to the amino group was decreased.     

Mechanism of the effect of levorin on transport of amino acids inCandida albicans

By use of incubation media of various ionic compositions and pH's, it was possible to reveal that inhibition of the transport of neutral amino acids having long hydrophobic chains (leucine, phenylalanine) inCandida albicans by the polyene antibiotic levorin was mainly due to redistribution—caused by the antibiotic—of monovalent cations in the cells and protoplasts of the yeast. In an acidic medium, levorin was also found to induce a decrease of proton release from the cells, accompanied by some inhibition of leucine transport. Transport of neutral amino acids having short hydrophobic chains (alanine, glycine, proline) was inhibited by this polyene antibiotic, irrespective of the ionic composition or pH of the medium.     

Characteristics of diffusion of polyene antibiotic levorin

Studies were performed which provided estimation of the impact of some physico-chemical properties of levorin, a polyene antibiotic, and in particular its multicomponent nature and capacity for isomerization on formation of inhibition zones in assay of the antibiotic activity by the agar diffusion method. It was shown that the levorin complex components markedly differed in the biological activity and diffusion capacity. The diffusive properties of the highly active components A2 and A3 were better. The diffusion rate of isomerized levorin was much lower than that of the initial levorin. To provide better diffusion of the levorin components with low diffusion capacity it was recommended to use a new solution composed of ethanol, glycerol and water at a ratio of 15:25:60.     

Dependence of inhibition by levorin of amino acid incorporation into protoplasts and subcellular proteins of Candida albicans on the change of endocellular potassium concentration]

Levorin is found to decrease more efficiently potassium concentration in C. albicans protoplasts under their incubation in the presence of sodium than in the medium containing the equivalent amount of potassium. Minimal inhibitory concentration of levorin for resistant C. albicans cells incubated on potassium-depeleted medium was in 4 times lower than for cells incubated in potassium-enriched medium. The decrease of membrane permeability for 14C-amino acids and their incorporation into membrane, ribosomal and soluble proteins under the effect of levorin was more pronounced when protoplasts were cultivated in sodium-containing medium than in potassium-containing one. In both media the inhibition of 14C-amino acid incorporation by levorin into ribosomal and cytosol proteins was more efficient than into membrane proteins, but these differences were less pronounced in case of potassium-containing medium.     

Effect of the products of the vital activity of yeast-like fungi on levorin synthesis

The effect on levorin synthesis of the cells and fermentation broth filtrates of Candida tropicalis after their cultivation in the fermentation medium was studied. It was found that the yeast-like fungi belonging to Candida excreted during their development some products capable of stimulating the synthesis of levorin by 40--60 per cent. When the actinomycete producing levorin was grown on the medium containing 80 per cent of the filtrate the level of levorin synthesis was the same as that observed with mixed cultivation of the actinomycete and C. tropicalis. The study on the conditions providing accumulation of the stimulating substances showed the following: production of the stimulating substances started during the first hours of the yeast growth and reached its maximum by the 48th hour, these substances being consumed by the actinomycete during the fermentation process. Aeration is required for production of the stimulating substances but its high levels are not necessary.     

Effect of a modifying factor on the sensitivity of Crithidia oncopelti to levorin

The paper deals with possible discovery of ways for increasing sensitivity of trypanosomides to polyenic antibiotics. The following substances were tested: sodium pyruvate and acetate, calcium salts, ascorbic acid and 1-valine. The total number of the cells and the number of the viable cells in the culture and their morphological characteristics were used as the criteria for estimation of the C. oncopelti sensitivity. It was shown that sodium acetate most actively modified the levorin effect on C. oncopelti. Its addition in a concentration of 40 mg/ml to the cultivation medium with levorin in a concentration of 1 microgram/ml induced a trypanocidal effect. With the use of levorin alone such an effect was observed when the antibiotic was used in a concentration of 10 micrograms/ml. The growth rate of the protozoon was decreased by 60-80 per cent as compared to the control. The number of the viable cells was lowered 4 times. The morphology of the culture markedly changed. This indicates that the presence of sodium acetate as a modifier in the culture medium allowed one to decrease 10 times the dose of levorin and to preserve the trypanocidal effect.     

Cation conductance and efflux induced by polyene antibiotics in the membrane of skeletal muscle fiber.

Cation conductance and efflux induced by polyene antibiotics amphotericin B (AMB), amphotericin B methyl ester (AME), nystatin, mycoheptin, and levorin on frog isolated skeletal muscle fibers and whole sartorius muscles were investigated. Conductance was measured under current-clamp conditions using a double sucrose-gap technique. Cation efflux was studied using flame emission photometry. Some new data were obtained concerning the effects of levorin and mycoheptin on biological membranes. The power dependence of polyene-induced cation transport on antibiotic concentration in muscle membrane was lower than that in bilayers. The decline in the equilibrium conductance caused by polyene removal (except for levorin) was very fast. There was reverse temperature dependence of AMB- and nystatin-induced conductances. Both induced conductance and efflux values demonstrated a correlation with the order of antifungal activities: levorin > AMB, mycoheptin > AME > nystatin, except for AME, which was more potent on yeastlike cells. These effects were interpreted in terms of possible differences in the kinetics of channel formation in biological and model membranes and in light of the role of nonconducting antibiotic forms in biological membranes.     

Doses levorin display an antiandrogenic action?

The antiandrogenic effect of levorin on immature castrated rats treated with exogenic testosterone was studied. In a dose of 200 mg/kg levorin lowered the cholesterin blood levels in the rats, inhibited the testosterone-induced increase in RNA concentration in the ventral and dorsal prostate and the seminal vesicles and to a less extent suppressed the growth of the accessory sexual glands. However, the antiandrogenic effect was observed with the use of levorin in the dose producing a pronounced toxic action evident from death of a part of the animals and a marked decrease in the animal body weight. This fact casts doubt on specificity of the levorin effect. Apparently, in high doses levorin impairs metabolism as a whole which cannot but affect the response of the sexual glands to administration of testosterone.     

Sensitivity of fungi of the genus Candida to antifungal drugs

Seventy two Candida strains isolated from patients with candidiosis of the oral mucosa were studied with respect to their sensitivity to nystatin, levorin, decamine, ethonium, sanguiritrin and clotrimazole. At concentrations of 0.5 to 5 micrograms/ml all the Candida species i.e. C. albicans, C. tropicalis, C. krusei and C. quilliermondii were highly sensitive to clotrimazole. Fungistatic action of levorin, nystatin and sanguiritrin was observed in 91, 67 and 38 per cent of the strains respectively. The Candida strains were resistant to decamine and ethonium used in the above concentrations.     

Biochemical analysis of the microvillose membranes of the small intestine mucosa of dogs after levorin administration

The effect of oral levorin used for a prolonged period of time on the lipid composition and activity of alkaline phosphatase and invertase of the microvilli membranes of the small intestinal enterocytes of old dogs was studied. Higher ratios of cholesterol/phospholipids in the membranes and inactivation of alkaline phosphatase and invertase were noted in the old dogs as compared to the young ones. Exposure of the old dogs to levorin had a significant effect on the microvilli membranes of the intestinal epithelial cells. It was evident from a lower ratio of cholesterol/phospholipids in the membranes and stimulation of the alkaline phosphatase activity. It is supposed that the changes in the state of the microvilli membranes of the small intestinal mucosa due to levorin play a definite role in the mechanism of its hypercholesterolemic action.     

Isolation, purification and characterization of levorin produced by Streptomyces levoris 99/23

Levorin produced by Streptomyces levoris 99/23 was isolated, purified and characterized. It was established that 80% of the levorin was localized in the mycelium and only 20% was in the cell-free supernatant. Amorphous yellow levorin with activity of 24 000 IU/mg and 96% purity was obtained. The preparation exhibited three absorption maxima: at λ 362, 382 and 404 nm. The antibiotic contained seven components: A₀, A₁, A₂, A₃, A₄, and two unidentified ones. According to its composition, the preparation corresponded to the levorin used for medicinal purposes. However, the levorin produced by S. levoris 99/23 contained half as much levorin A₂ and a more than 100 times larger quantity of the more active and less toxic component levorin A₃.     

Production in an Actinomyces levoris culture under the action of an actinophage specific to it of variants with stable preservation of its resistance to the phage]

Act. levoris 28, an organism producing levorin was treated with an actinophage virulent to it. Variants of the organism were isolated from the secondary growth of the culture. As a result of lysogenization with the above phage the variants acquired stability to it which was preserved during the further generations. In the previous experiments carried out by the authors the variants isolated from the secondary growth of the culture after its exposure to the same phage lost their stability to the phage as a result of loosing the prophage by it during the subsequent passages. The phage stable variants did not differ from the initial culture either in the activity of levorin or the levorin composition. The phages found in the initial culture 28, and the virulent mutant were identical with respect to the particles morphology and antigenic properties which confirmed their relation.     

Effect of the cultivation conditions of Candida species yeast-like fungi on their sensitivity to levorin]

The effect of levorin on yeast-like fungi under (different) cultivation conditions was studied. It was shown that the medium composition and incubation temperature affected the results of determination of the test-culture sensitivity to levorin. There was correlation between the time of the population lag-phase and the critical time of the zone formation and these values depended on the temperature of the test-microbe vegetation. Strain 037 of C. utilis was more sensitive to levorin and had a longer lag-phase as compared to the other species of the yeast-like fungi, which provided favourable conditions for formation of the growth inhibition zones.     

Effect of a biostimulant formed by yeastlike fungi on the dynamics of the accumulation of CoA, biotin and levorin in the process of growth of S. levoris

Regularities of the effect of a biostimulator produced by years-like fungi on accumulation of CoA, biotin and levorin in a developing culture of S. levoris were studied. It was shown that addition of the biostimulator to the fermentation medium resulted in increased accumulation of CoA and biotin in the mycelium of the levorin-producing culture within the first 48 hours of the growth and in their more intensive consumption at the final stages of the fermentation process. The rate of the levorin synthesis in the medium with the biostimulator markedly exceeded that in the control.     

The Properties of Ion Channels in Lipid Membranes Modified by the Aromatic Antibiotic Levorin А2

It has been shown that the main components of levorin A, that is, A₀, A₁, A₂, or A₃, that contain an aromatic group increase the permeability of membranes in the series A₃ > A₂ > A₁ > A₀ when they are on the same side of the membrane. All levorin components have cationic selectivity. The most studied levorin, А₂, promotes the almost ideal permeability of membranes to potassium ions. The membrane potential for a ten-fold change in the KCl concentration gradient is 56 ± 2 mV. It has been shown that the injection of the same concentration of levorin А₂ into one side of the membrane and then, after achieving the typical membrane permeability, into the other side of the membrane generates a two-fold increase in the total membrane permeability. This means that independent levorin-induced conductive semi-pores are formed on each side of the membrane. It has been found that the injection of levorin А₂ only into one side of the membrane enhances the membrane permeability to monosaccharides and other neutral molecules. The presence of levorin А₂ in cholesterol-, ergosterol-, and stigmasterol-containing phospholipid membranes has been shown to lead to the single-channel conductivity of typical ion channels of 0.2–0.5 pS. The properties of these channels have been studied. The levorin channels exist in two states, open and closed. Most of the time, the channel remains in the open state in the KBr solution. In solutions of different salts of the same concentration, the conductivity value of the levorin channels is approximately the same (0.4–0.5 pS). An increase in the dimethyl sulfoxide concentration in aqueous solutions facilitates the transition of polyene antibiotic molecules from dispersed to monomolecular form. The molecules of polyene antibiotics in the associated form exhibit high membrane activity.