Biological properties of dipyridyl sulphides and related compounds

Antibacterial, antifungal and antitumor activity of 15 newly synthetized compounds were investigated in vitro. These compounds were mainly 1-[4-pyridyl]-pyridinium salts, dipyridyl sulphides and corresponding sulphones. Antitumor activity was evaluated in a model of leukemia L1210, P388, Sarcoma 180, Ehrlich carcinoma, NK-lymphoma and melanoma B16. None of the compounds investigated, even at the concentration of 100 micrograms/ml, appeared to influence Gram-negative bacteria. However, at the concentration as low as 1.0 microgram/ml, three of them (No. III, VI and VII) strongly inhibited the growth of Gram positive bacteria and fungi. The compounds did not prolong the survival time of Sarcoma 880 leukemia L1210 or P388--bearing mice. Few of the compounds examined exerted marked antitumor effect on Ehrlich carcinoma and NK-lymphoma--compounds No. I and XV inhibited the growth of these tumors by ca. 70%. The strongest inhibitory effect on melanoma B16 was observed with compound No. XV, the effect being dose-dependent.     

Quantitation of camptothecin and related compounds

Camptothecin and congeners represent a clinically very useful class of anticancer agents. Proper identification and quantitation of the original compounds and their metabolites in biological fluids is fundamental to assess drug metabolism and distribution in animals and in man. In this paper we will review the recent literature available on the methods used for separation and quantitative determination of the camptothecin family of drugs. Complications arise from the fact that they are chemically labile, and the pharmacologically active lactone structure can undergo ring opening at physiological conditions. In addition, a number of metabolic changes usually occur, producing a variety of active or inactive metabolites. Hence, the conditions of extraction, pre-treatment and quantitative analysis are to be carefully calibrated in order to provide meaningful results.     

Microbial oxidation of dehydroepiandrosterone and related compounds

The oxidation of dehydroepiandrosterone (DHEA), 4-androstene-3,17-dione, and estrone with Streptomyces roseochromogenes NRRL B-1233 was studied. The oxidation products were isolated and identified as 16α-hydroxy-DHEA, 16α-hydroxy-4-androstene-3,17-dione and 16α-hydroxy-estrone. The yields of these three products were 85%, 41% and 18%, respectively. This indicates the substrate stereospecificity of 16α-hydroxylase of the organism. An interrelationship between cell growth and the formation of 16α-hydroxylated steroid was observed in any case. For formation of 16α-hydroxy-DHEA, 16α-hydroxylase showed good activity at DHEA concentration of 3.47 × 10^?4 M. In the case of DHEA, 16α-hydroxy-4-androstene-3,17-dione and 5-androstene-3β, 16α, 17β-triol were obtained after the yield of 16α-hydroxy-DHEA reached the maximum yield for about 30 hr. The oxidation pathway of DHEA is discussed.     

Comparative antioxidant properties of some GABAergic phenols and related compounds, determined for homogeneous and membrane systems

Some phenols, like propofol, thymol and related compounds, have been shown to act on the GABA(A) receptor. Several compounds with GABAergic activity have displayed neuroprotective effects attributed mainly to the potentiation of GABA(A)-mediated inhibition of synaptic transmission. It has also been found that compounds containing a phenolic OH group can scavenge reactive oxygen species, as in the case of propofol, among others. Thus, the neuroprotective action mechanism of GABAergic phenols would involve both effects, their pharmacological activity on GABA(A) and their intrinsic antioxidant ability. In this context, the study of the antioxidant properties of phenolic compounds included in the present work will enable these capacities to be correlated with their eventual pharmacological activities. The assays chosen in this study included determination of antioxidant ability in homogeneous isotropic systems (DPPH reduction, FRAP and hydrogen peroxide scavenging) and in heterogeneous membrane systems (inhibition of lipid peroxidation of phospholipid SUVs). The comparative evaluation of the results showed some differences between the relative order of antioxidant potency among all assayed compounds determined by using both types of systems. This analysis supports the conclusion that the antioxidant values obtained in homogeneous non-membrane systems, for phenols or other lipophilic compounds, should be revised according to their capacity of interaction with membranes (i.e. Log P in membrane-buffer system) in order to obtain antioxidant potency values more approximate to those actually occurring in biological systems. These results are essential to understand the actual neuroprotective action mechanism exerted by phenolic compounds involving a pharmacological activity, an antioxidant effect or both actions exerted mutually.     

P-glycoprotein inhibition by glibenclamide and related compounds

 Glibenclamide is well known to interact with the sulphonylurea receptor (SUR) and has been shown more recently to inhibit the cystic fibrosis transmembrane conductance regulator protein (CFTR), both proteins that are members of the ABC [adenosine 5′-triphosphate (ATP)-binding cassette] transporters. The effect of glibenclamide and two synthetic sulphonylcyanoguanidine derivatives (dubbed BM-208 and BM-223) was examined on P-glycoprotein, the major ABC transporter responsible for multidrug resistance (MDR) in cancer cells. To this end, we employed different cell lines that do or do not express P-glycoprotein, as confirmed by Western blotting: first, a tumour cell line (VBL600) selected from a human T-cell line (CEM) derived from an acute leukaemia; second, an epithelial cell line derived from a rat colonic adenocarcinoma (CC531^mdr+) and finally, a non tumour epithelial cell line derived from the proximal tubule of the opossum kidney (OK). Glibenclamide and the two related derivatives inhibited P-glycoprotein because firstly, they acutely increased [³H]colchicine accumulation in P-glycoprotein-expressing cell lines only; secondly BM-223 reversed the MDR phenomenon, quite similarly to verapamil, by enhancing the cytotoxicity of colchicine, taxol and vinblastine and thirdly, BM-208 and BM-223 blocked the photoaffinity-labelling of P-glycoprotein by [³H]azidopine. Furthermore, glibenclamide is itself a substrate for P-glycoprotein, since the cellular accumulation of [³H]glibenclamide was low and substantially increased by addition of P-glycoprotein substrates (e.g., vinblastine and cyclosporine) only in the P-glycoprotein-expressing cell lines. We conclude that glibenclamide and two sulphonylcyanoguanidine derivatives inhibit P-glycoprotein and that sulphonylurea drugs would appear to be general inhibitors of ABC transporters, suggesting an interaction with some conserved motif. Received: 15 December 1997 / Received after revision: 10 November 1998 / Accepted: 2 December 1998     

Immune stimulatory and anti-tumour properties of haemin

IL-2 induces tumour regression in some patients with metastatic disease, but the dose of IL-2 is limited by severe toxicity. Agents that increase the expression of IL-2 receptors in the effector cells could be used to improve the effectiveness of IL-2 in mediating its anti-tumour effect. We have reported that haemin increased the expression of IL-2 receptors in human peripheral blood mononuclear cells (PBMC) and synergized with IL-2 in the induction of mitogenicity, cytotoxicity and cytokine production. We now report on haemin-induced immune stimulation and tumour regression in mice. Haemin-induced mitogenicity in mouse splenocytes was potentiated up to twofold by IL-2. The combination of haemin and IL-2 was also effective in inducing cytotoxicity for natural killer (NK)-resistant target cells. Maximal induction of cytotoxicity was attained at an optimal concentration of haemin of 10 μm. Higher concentrations were less effective. Splenocytes isolated from mice that had been treated in vivo with haemin and IL-2 incorporated twice the amount of ³H-thymidine compared with splenocytes from mice treated with either haemin or IL-2 alone. Cytotoxicity of splenocytes for NK-rcsistant target cells was not increased following in vivo administration of haemin and IL-2 when fresh splenocytes were tested. Cytotoxicity was enhanced, however, up to five-fold following 48 h in vitro incubation with IL-2. Administration of haemin and IL-2 resulted in a significant decrease (40%) of established hepatic metastases in mice. Either IL-2 or haemin alone at the dose used were ineffective. The anti-tumour effect of haemin and IL-2 was enhanced (63% decrease in metastases) by administration of the thiol compound, N-acetylcysteine. Since haemin can safely be administered to patients, it may represent a new class of biologic response modifiers that could enhance IL-2-mediated anti-tumour effects.     

Separation methods for camptothecin and related compounds

This paper reviews working procedures for the analytical determination of camptothecin and analogues. We give an overview of aspects such as the chemistry, structure-activity relationships, stability and mechanism of action of these antitumor compounds. The main body of the review describes separation techniques. Sample treatment and factors influencing high-performance liquid chromatography development are delineated. Published high-performance liquid chromatographic methods are summarized to demonstrate the variability and versatility of separation techniques and a critical evaluation of separation efficiency, detection sensitivity and specificity of these methods is reported.     

Mutagenicities of 61 flavonoids and 11 related compounds

The mutagenicities of 61 flavonoids (naturally occurring flavonoid aglycones and flavonal glycosides and synthetic flavonoids) and those of 11 compounds structurally related to flavonoids were tested with Salmonella typhimurium strains TA100 and TA98. Among the 22 flavone derivatives tested, only wogonin was strongly mutagenic, while five derivatives, apigenin triacetate, acacetin, chrysoeriol, pedalitin, and pedalitin tetraacetate, were only weakly mutagenic. Two bisflavonyl derivatives, neither of which has a 3-hydroxyl group, were not mutagenic. Of the 16 flavonol derivatives tested, all except 3-hydroxyflavone and the tetra- and penta-methyl ethers of quercetin were mutagenic. Of the five flavanone derivatives tested, only 7,4-dihydroxyflavanone was mutagenic, showing weak activity. Of the four flavanonol derivatives tested, hydrorobinetin and taxifolin were weakly mutagenic. Of the six isoflavone derivatives tested, tectorigenin was weakly mutagenic. Of the 11 compounds in the miscellaneous group structurally related to flavonoids, only iso-liquiritigenin was mutagenic, showing weak activity. For the emergence of strong mutagenicity, the double bond between positions 2 and 3 and the hydroxyl group at position 3 are required, except in wogonin, which does not have a hydroxyl group at position 3 but is strongly mutagenic to TA100. The 3-O-acetyl ester of flavonol, quercetin, was mutagenic with S9 mix, but 3-O-methyl ethers were not. Six flavonol glycosides, three quercetin glycosides and three kaempferol glycosides were mutagenic after preincubation with “hesperidinase,” a crude extract of Aspergillus niger. Of 66 flavonoid agylcones and compounds structurally related to flavonoids, quercetin was the strongest mutagen. The carcinogenicity of this compound should be clarified because it is ubiquitously found in vegetables.     

Unspecific reactions of HLA-B antisera on fibroblasts from patients and carriers of Duchenne muscular dystrophy

Skin fibroblasts of patients with Duchenne muscular dystrophy were analysed for HLA tissue antigens. For the examination of the antigens two modified methods were used: the microdroplet cytotoxicity assay on fibroblast suspension and the cytotoxicity assay on fibroblast monolayer cultures using fluorochromasia. The HLA determination on the fibroblast suspensions of the patients showed a variety of unspecific HLA expressions which were not found in fibroblasts of healthy controls. These reactions were not apparent in the test in monolayer cultures. The exact cause has not been elucidated yet, but it appears to have a strong relation to some membrane abnormality in cells of DMD patients. The reproducible lack of unspecific reactions on monolayer cultures lead us to propose that these expressions arise from artificial injuries to membranes of these patient cells during trypsination or centrifugation. The validity of this test has been confirmed by a blind study.     

Mutagenic potency of chlorofuranones and related compounds in salmonella

One of the mutagenic byproducts associated with chlorinated humic waters and kraft pulp bleaching effluents was recently identified as 3-chloro-4-(dichloromthyl)-5-hydroxy-2(5H)-furanone. This compound and several related chlorofuranones and precursors were synthesized and evaluated for direct-acting mutagenicity in Salmonella typhimurium tester strain TA100. Mutagenicity was greatest for 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone, its 5-methoxy derivative, and the precursor in their synthesis, 3-(dichloromethyl)-2,4,4-trichloro-2-butenoic acid. Several of the compounds were tested in the presence of added rat liver homogenate S9 fraction, and in all cases mutagenicity was substantially reduced. An important structural feature which may govern the mutagenic response in these instances appears to be the cis arrangement of CHCl₂ and Cl substituents on a carbon-carbon double bond. These compounds may also be transformed in vitro to the same acyclic chlorine substituted alpha, beta-unsaturated aldehyde derivative, which is proposed to be the agent responsible for the observed mutagenicity.     

Bacteriocidal properties of a class of quinoid compounds related to sporulation in the mushroom, Agaricus bisporus

A small phenolic compound, γ-L-glutamyl-4-hydroxybenzene, and several quinoid derivatives appear in the gill tissues of the mushroom, Agaricus bisporus, during the prodromal period of sporulation. These quinoids markedly inhibit respiratory enzymes and protein synthesis. The temporal relationship of their appearance to sporulaton and their properties as metabolic inhibitors indicate that they initiate and maintain the dormant or cryptobiotic state of the spore. In very low concentrations, these quinoids show significant bacteriocidal action against a variety of microorganisms. The therapeutic potential of these antibiotic properties is considered.     

Synthesis and antiviral evaluation of bisnoradamantane sulfites and related compounds

The reaction of a series of 1,2-diols with thionyl chloride led to bisnoradamantane sulfites in very good yields. The reaction has also been applied to related polycyclic scaffolds. The compounds have been tested for antiviral activity but none of them showed to be active. Several attempts to generate and trap SO from these polycyclic sulfites have been unsuccessful.     

Immunostimulatory properties of ethylene-2,2' -bis(dithio)bis(ethanol) and related compounds in vivo

ADA 202-718, as well as HEDS and higher homologues of ADA 202-718, were found to profoundly stimulate the delayed type hypersensitivity reaction in mice when given i.p. or orally in a dose range of 0.1 - 10 mg/kg. While even a single application of ADA 202-718 at the time of sensitization resulted in a stimulation of the hypersensitivity reaction, administration of the compound at the time of challenge was without effect. When ADA 202-718 was given to animals which were subjected to immunosuppressive therapy by cyclosporine, the suppressed hypersensitivity reaction was restored to normal. At much higher doses (50-200 mg/kg) ADA 202-718 enhanced the local graft-vs-host reaction in the rat. ADA 202-718 did not interfere with the suppressed graft-vs-host reaction obtained by immunosuppressive treatment with cyclosporine nor with the immunosuppressed skin transplant rejection. Single applications of HEDS or ADA 202-718 enhanced the humoral response of mice to sheep erythrocytes as well as to haptenized sheep or chicken erythrocytes. Although antibody levels at the time of maximal antibody production (day 4 for IgM) were only moderately enhanced, elevated antibody titres (IgM and IgG) were found even 23 days after sensitization. The age-dependent decreased humoral response of mice to sheep erythrocytes tended to be partially restored by twice weekly oral applications of HEDS or ADA 202-718 (0.1 to 1 mg/kg for 4 weeks). ADA 202-718 did not decrease the swelling in the Freund's adjuvant induced arthritis in the rat, but reduced the pain in this model. Swelling in a locally induced oedema was reduced in a dose-independent fashion. ADA 202-718 was more effective than acetyl salicylic acid in alleviating the oedema-associated pain.     

壳聚糖／魔芋葡甘聚糖共混膜细胞毒性和机械性能研究

目的利用天然高分子材料壳聚糖（CS）和魔芋葡甘聚糖（KGM）制备两者含量不同比例的生物膜,测定其细胞毒性和机械性能。方法流延法制备CS与KGM含量不同比例的混合膜。并将其与人脐带间充质干细胞联合培养,观察其细胞毒性：测定其断裂伸长率、拉伸强度及吸水率。结果CS/KGM含量比例分别为3：0、2：1、1：1和1：2制备的8种共混膜,具有良好的细胞毒性：膜平均厚度0．110mm,拉伸强度为35．84～48．17MPa,断裂伸长率为4．84％-6．25％,吸水率均随着CS含量增加而增加。结论CS／KGM含量比为3：0、2：1、1：1和1：2制备的共混膜,都具有良好的细胞毒性,初步具备了引导组织再生膜的基本性能。     

Resting muscle glucose metabolites and related compounds in hypercapnia

Summary Glucose metabolites (lactate, pyruvate, citrate, malate), alanine, glutamate and adenosine triphosphate (ATP) were determined in the resting anterior tibial muscle of dogs. The muscle was sampled in anesthetized animals first breathing air, and secondly after an hour of breathing a hypercapnic mixture, FICO₂=0.10 (experimental subjects n=6) or air (control subjects n=6). A decrease in concentration of glucose metabolites (lactate: –34%; pyruvate: –24%; Citrate: –34%; malate: –54%), glutamate (–43%), alanine (–35%) and ATP (–8%) was observed in the resting muscle during acute hypercapnic acidosis. This was not the case in control animals breathing air.     

Neuroprotection by (-)-deprenyl and related compounds

There is an increasing number of data by in vitro and in vivo experiments, indicating that (-)-deprenyl is neuroprotective to dopamine neurons, even though detailed mechanism remains to be clarified. In this paper neuroprotection by (-)-deprenyl and structurally related compounds was examined in concern with the suppression of apoptosis induced by a reactive oxygen species, peroxynitrite generated from SIN-1. The apoptotic DNA damage was quantitatively determined using dopaminergic SH-SYSY cells and by a single cell gel electrophoresis (comet) assay. DNA damage induced by peroxynitrite was proved to be apoptotic by prevention of the damage by cycloheximide or actinomycin-D. (-)-Deprenyl and other propargylamines protected the cells from apoptosis in a dose-dependent way. (-)-Deprenyl protected the cells even after it was washed out, suggesting that it may initiate the intracellular process to repress the apoptotic death program. The study on the structure-activity relationship of (-)-deprenyl analogues revealed that a N-propargyl residue with adequate size of hydrophobic structure is essentially required for the anti-apoptotic activity. These results suggest that (-)-deprenyl and related compounds may protect neurons from apoptosis and be applicable to delay the deterioration of neurons during advancing ageing and in neurodegenerative disorders.     

Cell culture test system for express analysis of cytotoxic and growth-stimulating effects of bioactive compounds

Cultures of human and mammalian cells presenting 4 types of differentiation (normal human fibroblasts and myoblasts, human and Syrian hamster hepatoma cells, and mouse/mouse hybridoma cells) were used in a panel biotest system. This system allowed to evaluate the cytotoxic and stimulatory effect of bioactive compounds by determining the dose-effect relationships and some quantitative parameters including LD₅₀. Examination of some biolactive compounds of different nature (sangviritrin, escin, deltostim, cycloheximide, dexamethasone) confirmed high efficacy of this biotest system.