Combined Effect of Basic Antiherpetic Drugs with a New Inhibitor of the Terminase Complex of Herpes Simplex Virus Type 1 in Vero Cell Cultures.

Abstract-Carriers of herpes simplex virus type 1 (HSV-1) account for more than 90% of the global population. Infection manifests itself in the formation of blisters and ulcers on the face or genitals and can cause blindness, encephalitis, and generalized infection. All first- and second-line modern antiherpetic drugs selectively inhibit viral DNA polymerase. The purine-benzoxazine conjugate LAS-131 ((S)-4-[6-(purin-6-yl)aminohexanoyl]-7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine), which we have described earlier, uses the large subunit of the HSV-1 terminase complex as a biotarget and selectively inhibits HSV-1 reproduction in vitro. Basically new results were for the first time obtained to characterize the combined effect on human herpesvirus infection for LAS-131 used in combination with practically significant antiviral compounds, including the nucleoside analogs acyclovir (ACV), penciclovir (PCV), ganciclovir (GCV), brivudine (BVdU), iododeoxyuridine (IdU), and adenine arabinoside (Ara-A); the nucleoside phosphonate analog cidofovir (CDV); and the pyrophosphate analog foscarnet (FOS). A cytopathic effect (CPE) inhibition assay showed that the drug concentration that inhibited the virus-induced CPE by 50% decreased by a factor of 2 (an additive effect, FOS) or more (a synergistic effect; ACV, PCV, GCV, IdU, BVdU, Ara-A, and CDV) when the drugs were used in combination with LAS-131. Nonpermissive conditions for HSV-1 reproduction were thus created at lower drug concentrations, opening up new real possibilities to control human herpesvirus infection.

[Antiviral effect of novel purine conjugate LAS-131 against Herpes simplex virus type 1 (Herpesviridae: Alphaherpesvirinae: Simplexvirus: Human alphaherpesvirus 1) in vitro].

INTRODUCTION: Herpes simplex viruses type 1 (HSV-1) are extremely widespread throughout the world and, similar to other herpesviruses, establish lifelong persistent infection in the host. Reactivating sporadically, HSV-1 elicits recurrences in both immunocompetent and immunocompromised individuals and can cause serious diseases (blindness, encephalitis, generalized infections). The currently available antiherpetic drugs that aimed mainly at suppressing replication of viral DNA are not always effective enough, for example, due to the development of drug resistance. As we showed earlier the newly discovered compound LAS-131 exhibits the strong and highly selective inhibitory activity against HSV­1, including strain resistant to acyclovir (selective index, SI = 63). The presence of LAS-131 at a concentration of 20 µg/ml leads to a decrease in the titer of HSV-1 (strain L2) by 4 lg in a one round of HSV-1 replication. MATERIAL AND METHODS: To establish the step(s) of the virus life cycle that is sensitive to the action of LAS-131, we have applied a widely used approach, that made it possible to determine how long the addition of a compound can be postponed before it loses its antiviral activity (time-of-addition assay), and to compare this indicator with the crucial time of application of inhibitors with a well-known mechanism of action (in cell culture). RESULTS: It has been shown for the first time that LAS-131 retains a pronounced antiviral effect when introduced into the experimental system no later than 9 hours post-infection (p.i.). However, LAS-131 does not affect the release of HSV-1 from the cell. DISCUSSION: Together with published data on the termination of the synthesis of viral DNA 9-12 h after the adsorption in a cell culture infected with HSV with a high multiplicity (≥1 PFU/cell), our results suggest that LAS-131 interferes the life cycle of HSV-1 during synthesis of viral DNA. Further studies of the mechanism of action are necessary to establish definitely the biological target for this compound,.

[Factors Affecting the Labeling of NIH 3T3 Cells with Magnetic Nanoparticles].

The factors that affect the labeling of NIH 3T3 murine fibroblasts with Fe3O4-based magnetic nanoparticles (MNPs) were studied using MNPs produced by the gas condensation and solution precipitation methods and MNPs surface-modified with 3-aminopropylsilane or L-lysine. The production method, surface modifications, the particle concentration and size, the state of the cell population, and the method of MNP introduction were found to substantially affect the efficiency of MNP binding by cells. In particular, large MNP clusters may occur in MNP suspensions in DMSO, and their disruption by sonication increased the percent yield of magnetically labeled cells. Static incubation of a cell suspension led to a more efficient labeling as compared with continuous agitation. Cells attached to a plastic support could be labeled to a higher degree than cells in suspension, but required substantially longer incubations with MNPs. MNP centrifugation on cell layers (magnetic spinoculation) significantly increased the rate and efficiency of labeling. The stability of magnetic labeling was shown to depend on the MNP dose during labeling. Electron microscopy studies demonstrated that MNPs were associated with the cell surface after 20-min incubation with cells and were mostly in the cell interior after 4-h incubation. The results of the study may be useful for preparation and application of magnetized cell samples.

Effectiveness of Repeated Administration of Praziquantel with Disodium Glycyrrhizinate and Two Enantiomers of Praziquantel on Opisthorchis felineus (Rivolta, 1884).

BACKGROUND: Nowadays, it is still important to develop effective anti-opisthorchiasis agents. In this work, we tested a complex of praziquantel (PZQ) with a plant origin compound-disodium glycyrrhizinate-in the ratio 1:10 PZQ:Na2GA, containing 11-fold less of the active ingredient. Our aim was to study various ways to treat trematode Opisthorchis felineus with this complex in vitro. Additionally, an in vitro comparison of the anthelmintic action was made among racemic-PZQ, (R)-PZQ, and (S)-PZQ on juvenile and adult maritae of O. felineus. METHODS: Worms extracted from the hamsters were subjected to various regimens of administration of the complex: once a day for 3 days or three times within 1 day. Moreover, mature maritae and juvenile worms of O. felineus were subjected to the comparison the anthelmintic effectiveness of racemic-PZQ, (R)-PZQ, and (S)-PZQ. RESULTS: The O. felineus maritae that received PZQ:Na2GA (1:10) thrice within 1 day were most strongly affected by the drug. Their motility substantially decreased already on the second day after the last dose, and the percentage of live worms by the end of the experimental period was the lowest. These results indicate a cumulative anthelmintic effect of this substance under the regimen "three times within 1 day." For the first time, we report that among the three substances (racemic-PZQ and two enantiomers), (R)-PZQ has the highest anthelmintic activity, toward both juvenile and sexually mature maritae of O. felineus. CONCLUSION: These findings suggest that the development of a supramolecular complex of (R)-PZQ with disodium glycyrrhizinate and administration of this complex three times within 1 day are promising approaches.

[The synthesis of RGD peptide derivatives containing glutaric and adipic residues].

A method of the synthesis of RGD peptide derivatives containing glutaric or adipic residues linked with α-amino group of L-arginine and allowing carrying out their coupling with other biomolecules and nanoparticles.

[Cytotoxicity of lysomustine and its isomers, and their potential use for selection of cells].

N epsilon-Nitroso-N epsilon- [N'-(2-chloroethyl)carbamoyl]-L-lysine (I) and N epsilon- [N'-(2-chloroethyl)-N'-nitrosocarbamoyl]-L-lysine (II), the isomers being the constituents of antitumor agent Lysomustine, were obtained by RFHPLC. The study of cytotoxicity of the above compounds against K562 cells showed that the lesions induced by isomer (II) produce a significant cytotoxic effect but can be efficiently repaired by the action of MGMT (O6-methylaguanine DNA methyltransferase). Under similar conditions, the lesions induced by isomer (I) produce substantially smaller effect but are weakly if at all repairable by MGMT. The effects of a clinically approved agent Lysomustine, which is the mixture of isomers (I) and (II), are similar to those of isomer (II). The results obtained point to a different chemical nature of DNA lesions induced by two Lysomustine isomers. Our data indicate that Lysomustine and its isomer (II) can be used for in vitro selection of cells expressing MGMT.

Selection of genetically modified hematopoietic cells in vitro and in vivo using alkylating agent lysomustine.

Efficient gene transfer into hematopoietic stem cells is vital for the success of gene therapy of hematopoietic and immune system disorders. An in vivo selection system based on a mutant form of the O(6)-methylguanine-DNA-methyltransferase gene (MGMTm) is considered one of the more promising strategies for expansion of hematopoietic cells transduced with viral vectors. Here we demonstrate that MGMTm-expressing cells can be efficiently selected using lysomustine, a nitrosourea derivative of lysine. K562 and murine bone marrow cells expressing MGMTm are protected from the cytotoxic action of lysomustine in vitro. We also show in a murine model that MGMTm-transduced hematopoietic cells can be expanded in vivo on transplantation into sublethally irradiated recipients followed by lysomustine treatment. These results indicate that lysomustine can be used as a potent novel chemoselection drug applicable for gene therapy of hematopoietic and immune system disorders.

Synthesis of ( 2S, 4S)-4-phenylamino-5-oxoproline derivatives.

The paper describes the synthesis of ( 2S, 4S)-4-(N-Ts)- and ( 2S, 4S)-4-(N-Boc)-phenylamino-5-oxoprolines (pyroglutamic acid). These derivatives have been shown to be useful for synthesis of their amides and peptides in spite of steric hindrances caused by bulky groups adjacent to the reaction centre. Under the conditions applied no lactam ring opening and no loss of stereochemical integrity of any of the chiral centres were observed, which has been confirmed by NMR techniques.

[Chemobiokinetics of sarcolysin and its peptides with glutaminic acid]. / Khemobiokinetika sarkolizina i ego peptidnykh analogov s glutaminovoi kislotoi.

A 14C study of chemobiokinetics of sarcolysin and its peptides of glutaminic acid, dosage and routes of administration was conducted in intact rats and those bearing Walker's carcinoma. Similar in shape for peptides, kinetic curves differed from those found for sarcolysin. The rates of absorption and excretion of sarcolysin peptides in intraperitoneal and, particularly, oral administration were lower than those of sarcolysin. Tumor appeared to play a role in a higher rate of peptide excretion. While sarcolysin and its peptides distribution in organs and tissues was generally identical, time of peak radioactive concentration build-up was different. Time needed for accumulation and excretion of peptides from tumor was much longer than from other organs or tissues. Sarcolysin went chiefly to urine while peptides--to faeces.

[Reactions of decarboxylated and side transamination during interaction of glutamate decarboxylase from Escherichia coli with substrate analogs, modified through C3 and C4 atoms]. / Reaktsii dekarboksilirovaniia i pobochnogo transaminirovaniia pri vzaimodeistvii glutamatdekarboksilazy iz Escherichia coli s analogami substrata, modifitsirovannymi po atomam C3 i C4.

The interaction of glutamate decarboxylase with the aspartate and glutamate analogues modified at C3 and C4 was studied. 3-Arsonoalanine, 3-phosphonoalanine, 2-amino-4-arsonobutyric acid, 2-amino-4-phosphonobutyric acid, a mixture of diastereoisomers of 4-(methylthio) glutamic acid and erythro-4-(methylthio) glutamic acid were shown to be poor substrates for the enzyme. Their decarboxylation was accompanied by transamination of the coenzyme (PLP) to pyridoxamine phosphate (PMP) which reversibly inactivated the enzyme. With arsonoalanine only part of PLP was converted into PMP and another part irreversibly formed a complex. 4-(Methylsulfonyl)-L-glutamic and 4-[(phenyl)(hydroxy)phosphoryl]-L-glutamic acids did not react with the glutamate decarboxylase.

The interaction of glutamate decarboxylase from Escherichia coli with substrate analogues modified at C-3 and C-4.

The interaction of glutamate decarboxylase with the aspartate analogues 3-arsonoalanine and 3-phosphonoalanine, with the glutamate analogues 2-amino-4-arsonobutyric acid and 2-amino-4-phosphonobutyric acid, and with 4-(methylthio)-L-glutamic acid, both as a mixture of diastereoisomers and as the (2S,4R)-form, was studied. All these analogues were poor substrates for the enzyme and only weak inhibitors. Their decarboxylation was accompanied by transamination of the enzyme-bound pyridoxal phosphate (PLP) to pyridoxamine phosphate (PMP), thus inactivating the decarboxylase. With arsonoalanine only part of the PLP was converted into PMP.

[Comparison of the level of epithelial cell proliferation in the liver, islands of Langerhans and pancreatic acini of mice from the age and individual viewpoints]. / Sopostavlenie urovnia proliferatsii épitelial'nykh kletok pecheni, ostrovkov Langergansa i atsinusov podzheludochnoi zhelezy myshei v vozrastnom i individual'nom aspektakh.

The index of labelled nuclei (ILN) was determined in the mouse liver and pancreas epithelium within 24 hrs following five injections of 3H-thymidine. Five age groups were studied with the mean weight 15, 19, 20.4, 24.3, and 28.2 g. The ILN was equal in hepatocytes, respectively, to 5.2, 0.6, 0.03, 0.13 and 0.02%, in pancreatic islet cells to 4.6, 4.1, 1.7, 1.5, and 0.7%, and in acinar cells to 0.4, 1.1, 0.07, 0.30, and 0.10%. Marked individual ILN variations were observed. In hepatocytes the highest ILN in each age group correlated positively with maximal absolute liver weight and, in four groups, with its maximal relative weight. The maximal ILN indices did not coincide for the three cell types. A suggestion is put forward on relationship between ILN fluctuations and unequal character or organ growth and asynchrony of growth of different organs.

[Localization of labelled cells in the islets, acini and periinsular zone of the pancreas of mice undergoing resection]. / Lokalizatsiia mechennykh kletok v ostrovkakh, atsinusakh i periinsuliarnoi zone podzheludochnoi zhelezy myshei v usloviiakh rezektsii.

Thymidine-3H was injected 5 times during 24 hours to mice CBA X C57BL/6 with resected pancreas (about 40 per cent of the organ tissue). The number of labeled cells in the islets and the acini failed to change with the prolongation of the interval after the last injection of thymidine-3H (5 to 18 days). Localization of labeled cells in the pancreatic islets at later periods showed not much difference from the same localization two hours after the isotope injection. Examination of large andmedium-sized islets at the maximal distance from the wound revealed a similar number of labeled cells per square unit in the central, middle andperipheral region of the islets 2 hours and 18 days after the last injection of the isotope. It is concluded that under conditions of this experiment no islet cells are formed due to external sources.

[Proliferation of the exocrine and endocrine portions of the pancreas after its resection]. / O proliferatsii ékzokrinnogo i éndokrinnogo otdelov podzheludochnoi zhelezy posle ee rezektsii.

A weak regenerative capacity of the pancreas was revealed after the resection of about 40 per cent of the tissue of this organ in mice-hybrids CBA X C57BL/6, weighing 20--28 g. During the 21 days of the experiment there was no significant restoration of the pancreas weight. An increase of the proliferative activity (the number of mitoses and daily fraction of the thymidine-3H-labeled cells were counted) was brief and failed to spread over the whole organ. The maximal number of the labeled nuclei in the epithelium of the acini and the islets was found in the area near the wound where the organ tissue was somewhat edematous. In the areas near the wound surface, but unaffected, the labeled cells count was increased during the early post-operative period only. The number of labeled cells of the islets and acini of the pancreas in the areas distant from the wound (in the loop of the duodenum) was no different from the control. The number of labeled cells in the islets was greater than in the acini.