A Possible Antineoplastic Potential of Selective,Irreversible Proteasome Inhibitor,Carfilzomib on Chemically Induced Hepatocarcinogenesis in Rats

The antineoplastic effect of carfilzomib (CFZ) against chemically induced hepatocarcinogenesis was studied. A total of 60 male Wistar albino rats were divided into six groups with 10 animals in each group. Rats in group 1 (control group) were given dimethylsulphoxide (DMSO) (0.4 mL/kg i.p) twice a week for 3 weeks from week 8 to week 10. Animals in groups 2 and 3 were given CFZ (2 and 4 mg/kg i.p) twice a week from week 8 to week 10, respectively. Rats in group 4 were given diethylnitrosamine (DENA) at a dose of 0.01% in drinking water for 10 weeks and received a DMSO (0.4 mL/kg i.p) twice a week from week 8 to week 10. Animals in groups 5 and 6 were given DENA at a dose of 0.01% in drinking water for 10 weeks and treated with CFZ (2 and 4 mg/kg i.p) twice a week from week 8 to week 10, respectively. CFZ succeeded in suppressing the elevated serum tumor marker α‐fetoprotein and carcinoembryonic antigen. The antineoplastic effect of CFZ was also accompanied by normalization of elevated hepatic tissue growth factors, matrix metalloproteinase‐2 and tissue inhibitor of metalloproteinase‐1, and augmentation of hepatic endostatin and metallothionein. A histopathological examination of liver samples treated with CFZ after DENA intoxication correlated with the biochemical observation. Treatment with CFZ confers an antineoplastic activity against chemically induced hepatocarcinogenesis. These findings suggest that CFZ plays a pivotal role in the treatment of hepatocarcinogenesis.     

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.     

The role of succinic acid in the biosynthesis of levorin

The influence of succinic acid as a component of media for biosynthesis of levorin, a polyenic antibiotic was studied. It was shown that with the use of the soybean-corn medium supplemented with succinic acid (0.05-0.4 per cent) the antibiotic content in the fermentation broth was higher than that in the control. The highest stimulating effect (135 per cent) was observed with addition of 0.1 per cent of succinic acid. For providing optimal antibiotic production in the synthetic medium supplemented with succinic acid (0.4 per cent) addition of acetic acid (0.05 per cent) was required. Studies with the soybean-corn medium with and without succinic acid revealed differences in the level of p-aminoacetophenone, an aromatic fragment of the levorin molecule. Under the conditions of the medium with succinic acid the content of p-aminoacetophenone in the mycelium was higher by 10 to 18 per cent as compared to that in the control and depended on the fermentation period. The role of succinic acid in biosynthesis of levorin is discussed.     

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.     

Action of polyene antibiotics on actinomycetes with a varying lipid makeup]

The effect of polyenic antibiotics, such as nystatin, levorin, amphotericin B and mycoheptin on 93 representatives of various species of the ray fungi was studied. It was shown that resistance of the actinomycetes to the polyens was connected with the absence or insufficient content of sterols (0.001--0.008 per cent in the dry mycelium). On addition of cholesterol to the nutrient media (100 microgram/ml) it was included into the membranes of some cultures and their sensitivity increased 2--60 times. Resistance of Actinomyces sp. LIA 0775 grown on the media with fats differing in their composition decreased 2--4 times. In these cases the culture lipids were characterized by lower content of phospholipids (35--45 per cent from the total lipids as compared to 70--80 per cent when grown on the control medium without fats) and significantly increased content of unsaturated fatty acids (3--4 times).     

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.     

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.     

The effect of dimethyl sulfoxide on cholesterol and bile acid metabolism in rats

The effect of DMSO on cholesterol and bile acid metabolism was studied in rats. Male Sprague-Dawley rats were randomly assigned to one of two groups and given either tap water or 2% DMSO (v/v) in tap water to drink for 9 days. Both food (stock rat diet) and water were available ad libitum. Animals in both groups gained weight equally throughout the study. They also had similar liver weights (g/100 g body wt) at the end of the study (control: 5.0 +/- 0.1 (N = 6) vs DMSO: 4.9 +/- 0.1 (N = 6]. The activity of hepatic cholesterol 7 alpha-hydroxylase (pmole/mg/min), the rate-limiting enzyme of bile acid biosynthesis, was significantly (P less than 0.005) reduced in the treated animals (control: 9.7 +/- 1.0 (N = 6) vs DMSO: 4.3 +/- 0.7 (N = 6)). Plasma cholesterol (mg/dl) was significantly (P less than 0.005) elevated in the treated animals (control: 90 +/- 3 (N = 6) vs DMSO: 107 +/- 4 (N = 6)), a finding consistent with the reduced CH-7 alpha hydroxylase activity in this group. DMSO treatment did not affect either microsomal cholesterol content or hepatic glutathione content. Thus, this study has shown that DMSO treatment per se can affect cholesterol and bile acid metabolism. However, the precise mechanisms whereby DMSO exerts the observed effects are not known.     

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.     

Increase in efficacy of antibiotic therapy of anthrax under experimental conditions]

The results of experimental therapy of antraxis infected mice with cefazoline (kefzol) and ampicillin incapsulated into liposomes are presented. Protective activity of the same free antibiotics combinated with amixine and leukinferone was evaluated also. Treatment with liposomal cefazoline enhanced mice survival upto 60 per cent, and life period upto 1.3 +/- 0.3 days. After liposomal ampicillin administration for 3 times the same indices were 60 per cent and 6.5 +/- 0.9 days, after 2 times administration--80 per cent and 14 +/- 1.8 days when compared to the groups of the animals treated with free antibiotics. It was shown that administration of ampicillin with amixine or with leukinferone provided enhanced mice survival upto 20 per cent, administration of cefazoline with leukinferone--upto 30 per cent.     

Stabilization of standard samples of levorin and nystatin with antioxidants]

To prolong the storage period of the reference samples of levorin and nystatin, polyenic macrolide antibiotics, their effect of 23 antioxidants was studied by using rapid inactivation. The antioxidants belonged to compounds of different classes. The inactivation was performed at 20, 37 and 50 degrees C in the presence of 1, 2 or 5 per cent of the antioxidants. An antioxidant of the class of partially hydrated oxyquinolines was shown to have the highest stabilizing action on levorin and nystatin. It may be recommended for stabilizing the levorin and nystatin reference samples.     

Site of OH radical attack on dihydrouracil and some of its methyl derivatives

The site of attack of OH radicals on dihydrouracil and five of its methylated derivatives was determined by pulse radiolysis using N,N,N',N'-tetramethylphenylenediamine (TMPD) to detect oxidizing radicals and tetranitromethane (TNM) as well as K3Fe(CN)6 to detect reducing radicals. In the case of dihydrouracil OH radicals abstract preferentially an H atom at C(6) to give the 6-yl radical (greater than or equal to 90 per cent) which at pH approximately 6.5 reduces TNM and K3Fe(CN)6 at almost diffusion-controlled rates. Only a small fraction of OH radicals abstract the H atom at C(5) (less than or equal to 10 per cent). The resulting 5-yl radical oxidizes TMPD to TMPD+ at pH 7-8. With the methylated derivatives of dihydrouracil, OH radicals react less selectively, especially in the case of N(1)-methyl derivatives. This methyl group is activated to a similar degree as the methylene group at C(6). In 1-Medihydrouracil the yield of N(1)-CH2 radicals is about 29 per cent, which has been deduced from the yield of formaldehyde formed after oxidation of this radical by TNM at pH approximately 6.5 and the subsequent hydrolysis. Radicals at the other methyl substituents are generated to a lesser extent (less than or equal to 10 per cent) and are relatively unreactive towards oxidizing agents such as TNM and K3Fe(CN)6 as well as towards the reducing agent TMPD. Although methyl substitution opens new routes for OH attack the preferred site of H abstraction remains C(6) (greater than 60 per cent).     

Differential spectrophotometric method of analysing levorin in the culture broth and in the mycelium]

It was shown on model experiments that the microbiological method was not applicable for determination of levorin content in industrial intermediate products containing in addition levoristatin, since the presence of the latter made higher the results of the microbiological assay. Because of this till to the present date the quantitative content of levorin in the industrial intermediate products was determined photometrically using alcohol (pure solvent) as the reference solution. Still, this method also made higher the results of the assay, especially when the content of levorin was determined in the fermentation broth. In the solid phase levorin is contained in the mycelium which occupies only 1 to 2 per cent of the fermentation broth, while the liquid phase or the fermentation broth filtrate occupies 98 to 99 per cent. It was found that the fermentation broth filtrate contained protein admixtures which coagulated on addition of alcohol to the fermentation broth and formed fine colloid solutions. As a result the absorption values became higher. In the present study not the pure solvent but an extract of the fermentation broth filtrate containing neither levorin, nor levoristatin was used as the reference solution. Such a differential method provided elimination of all errors due to the presence of various metabolites in the fermentation broth filtrate which varied both qualitatively and quantitatively.     

Effect of inorganic phosphate on levorin biosynthesis and on the mycelial makeup of Streptomyces levoris]

The effect of inorganic phosphate on biosynthesis of the polyenic antibiotic levorin by Streptomyces levoris and composition of the culture mycelium was studied. It was found that the synthetic medium with 0.4 mM of phosphate was optimal for growth of Str. levoris. When the concentration of phosphate was higher, the biomass increased, while the synthesis of levorin appeared to be inhibited and morphological changes in the culture were observed. Phosphate had a significant effect on the mycelium composition. When its concentration was increased 10 times as compared to the optimal one, the amounts of protein, RNA, total phosphorus and polyphosphates increased 1.3--1.4, 1.6--1.7, 2--3 and 10 times respectively, while the synthesis of levorin decreased 5 times. Changes in the lipid component of the mycelium were also observed. In the absence of inorganic phosphate in the medium the acetone precipitating fraction of the lipids contained 20--40 per cent of the phosphoruless compounds. During cultivation their portion increased up to 70--77 per cent. However, in the presence of its excess the polar lipids were represented only by phospholipids during the whole life cycle. The fatty acid spectrum of the lipids did not depend on the phosphate concentration and was represented mainly by saturated fatty acids with a branched chain of a series of iso- and anteiso-structures containing 14--18 carbon atoms.     

Does the treatment of primary hyperaldosteronism influence glucose tolerance?

Primary hyperaldosteronism (PH) is frequently considered to be a secondary form of diabetes mellitus (DM). In our previous study we attempted to evaluate the prevalence of DM among patients with PH compared to control subjects with essential hypertension (EH). We have noted a relatively high prevalence of DM and impaired glucose tolerance in PH, but the differences between the PH and EH groups did not reach statistical significance. We performed this study to assess whether the effective treatment of PH (surgical and conservative) would improve the glucose tolerance. We have studied 24 patients with PH of the following two subtypes: aldosterone-producing adenoma (APA) treated with adrenalectomy and idiopathic hyperaldosteronism (IHA) treated with spironolactone. No significant changes of glucose levels were found in the 60th and 120th min of the oral glucose tolerance test (OGTT) in the APA group. On the other hand, fasting glucose levels were decreased significantly after adrenalectomy. Plasma glucose levels were significantly increased in the 60th min, but no differences were found in fasting values and in the 120th min in the IHA group. There was a significantly higher incidence of impaired glucose tolerance (36 per cent before, 45 per cent after treatment) and DM (9 per cent, 18 per cent) in the IHA group compared to the APA group (8 per cent, 32 per cent; DM 0 per cent, 0 per cent). In conclusion, the treatment of PH does not improve glucose tolerance. Mild worsening of glucose tolerance after treatment could be explained by an increase of the body mass index. These data, in accordance with our previous study, do not support the idea that PH is a secondary form of diabetes mellitus.     

Determining the conditions for stimulation of levorin biosynthesis in the presence of succinic acid

Conditions promoting the stimulating effect of succinic acid on biosynthesis of levorin were determined. The optimal concentration of succinate was shown to be equal to 0.05-0.3 per cent and the best time for the addition was before inoculation. The most pronounced stimulating effect was observed when the initial pH value of the fermentation medium was equal to 7.0. The important role of ammonium nitrogen in manifestation of the succinic acid stimulating effect on biosynthesis of levorin was suggested.     

Freezing Characteristics of Cultured Catharanthus roseus (L). G. Don Cells Treated with Dimethylsulfoxide and Sorbitol in Relation to Cryopreservation

The freezing behavior of dimethylsulfoxide (DMSO) and sorbitol solutions and periwinkle (Catharanthus roseus) cells treated with DMSO and sorbitol alone and in combination was examined by nuclear magnetic resonance and differential thermal analysis. Incorporation of DMSO or sorbitol into the liquid growth medium had a significant effect in the temperature range for initiation to completion of ice crystallization. Compared to the control, less water crystallized at temperatures below −30°C in DMSO-treated cells. Similar results were obtained with sorbitol-treated cells, except sorbitol had less effect on the amount of water crystallized at temperatures below −25°C. There was a close association between the per cent unfrozen water at −40°C and per cent cell survival after freezing for 1 hour in liquid nitrogen. It appears that, in periwinkle suspension cultures, the amount of liquid water at −40°C is critical for a successful cryopreservation. The combination of DMSO and sorbitol was the most effective in preventing water from freezing. The results obtained may explain the cryoprotective properties of DMSO and sorbitol and why DMSO and sorbitol in combination are more effective as cryoprotectants than when used alone.     

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.     

Stimulation of tyrosine protein kinase activity by dimethyl sulfoxide

Dimethyl sulfoxide (DMSO) stimulated the activity of a partially purified tyrosine protein kinase from rat lung. The stimulation was concentration dependent with a maximum stimulation (about 2 fold) observed at 10 per cent (V/V) DMSO. On the other hand, acetone (10 percent, V/V), did not exert any stimulatory effect on the enzyme activity. The stimulation was associated with a decrease in the Km for the substrate and an increase in the Vmax. In contrast, the Km for ATP was not affected by DMSO. Under identical assay conditions, DMSO did not significantly alter the activities of phosphorylase kinase, catalytic subunit of cAMP-dependent protein kinase and Ca2+-phospholipid-dependent protein kinase. It may be speculated that stimulation of tyrosine protein kinase may be one of the mechanisms by which DMSO exerts its biological effects.     

Synthesis and biological activity of aminophenylphosphonic derivatives of levorin

Reactions of levorin, a polyenic macrolide antibiotic, with aromatic aldehydes and hypophosphorous acid resulted in formation of its amino phenylphosphonium derivatives. Physicochemical and biological properties of the derivatives were studied. The levorin amino phenylphosphonium derivatives were shown to be low toxic and have antifungal and antiviral activities.