An investigation of the biological effect of structural modifications of isothiosemicarbazones and their cyclic analogues.

Several arylideneisothiosemicarbazones and arylidenehydrazothiazoles have been synthesised to obtain new antimicrobial agents. Their activity against both bacteria and fungi has been tested and some interesting informations about their biological activity have been obtained.

Synthesis and anti-microbial activity of isothiosemicarbazones and cyclic analogues.

It is known that some derivatives of both thiourea and thiosemicarbazide exhibit potent anti-microbial activity. In order to investigate the effects on the biological properties of structural modifications of such structures, we have synthesised and studied some arylidenisothiosemicarbazones. In this paper we report on the synthesis and structure-activity relationships of some isothiosemicarbazones, where the arylidene group has been replaced with a cycloalkyl group and the sulfur atom has been either differently substituted or enclosed in a thiazole ring.

Comparison of two rapid colorimetric methods for determining resistance of mycobacterium tuberculosis to rifampin, isoniazid, and streptomycin in liquid medium.

The usefulness of two colorimetric methods for the determination of the susceptibility or resistance of Mycobacterium tuberculosis to rifampin, streptomycin, and isoniazid in liquid medium based on the reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was investigated. The agar proportion method was used as the reference method. Results obtained indicate that the sensitivity of the XTT reduction assay for the detection of rifampin resistance was comparable to that observed, and previously described, for the MTT assay. However, the reduction of XTT yields a water-soluble formazan that can be easily quantified without performing additional steps such as addition of lysing buffer and solubilization. Furthermore, the colorimetric assays, based on the reduction of XTT and MTT for the detection of isoniazid and streptomycin resistance in Mycobacterium tuberculosis, were standardized. The inhibition of MTT and

Liposome-incorporated santolina insularis essential oil: preparation, characterization and in vitro antiviral activity.

The effect of liposomal inclusion on the stability and in vitro antiherpetic activity of Santolina insularis essential oil was investigated. In order to study the influence of vesicle structure on the liposome properties, multilamellar and unilamellar vesicles were prepared by the film method and sonication, respectively. Vesicles were obtained from hydrogenated soya phosphatydilcholine and cholesterol. Formulations were examined for their stability for over one year monitoring the drug leakage from vesicles and the average size distribution. The stability of the incorporated oil was verified by studying its quali-quantitative composition. The antiviral activity was studied against Herpes simplex virus type 1 (HSV-1) by plaque reduction and yield reduction assays. Results showed that Santolina insularis essential oil can be incorporated in high amounts in the prepared liposomes, which successfully prevented its degradation. Moreover, stability studies pointed out that vesicle dispersions were stable for at least one year and neither oil leakage nor vesicle size alteration occurred during this period. Antiviral activity assays demonstrated that Santolina insularis essential oil is effective in inactivating HSV-1 and that the activity is principally due to direct virucidal effects. Free essential oil proved to be more effective than liposomal oil and a different activity was discovered which related to the vesicular structure. The ED(50) values, significantly lower when cells were pre-incubated with the essential oil before the virus adsorption, indicate an intracellular mechanism in the antiviral activity of Santolina insularis. Moreover, liposomal Santolina essential oil is non toxic in the range of the concentration tested.

Prevention by L-alpha-phosphatidylcholine of antifungal activity in vitro of liposome-encapsulated imidazoles determined by using time-killing curves.

The antifungal activity of the imidazole derivatives miconazole and ketoconazole was reduced when they were entrapped in liposomal structures and significant differences were detected between small unilamellar vesicles (SUV) and multilamellar vesicles (MLV). To understand which component of liposomes interfered with the antifungal activity of miconazole and ketoconazole, we examined the influence of pure egg and soy L-alpha-phosphatidylcholine and cholesterol on activity against Candida albicans ATCC E10231 by time killing curves. Association of phospholipids-cholesterol-imidazole leads to an inhibitory effect on the antifungal activity comparable to that shown when miconazole or ketoconazole were entrapped in SUV liposomes or when miconazole and ketoconazole were incubated in the presence of L-alpha-phosphatidylcholine. The antifungal activity determined in the presence of cholesterol was comparable to that observed with the free drugs. Inhibition of the antifungal activity of miconazole and ketoconazole by phospholipids is dependent on the phospholipid concentration but is independent of the source of phospholipids (egg or soy). Cholesterol had no influence on the antifungal activity of the imidazoles, unlike the effect on other antifungal drugs, such as amphotericin B.

Synergistic interactions of antibodies in rate of virus neutralization.

Antibodies and antibody combinations are often evaluated only by their potency in inactivating a known quantity of virus in dose-effect assays. However, a crucial additional parameter is the rate at which neutralization takes place, or kinetics. Synergism of certain antibody combinations in dose-effect assays has been previously demonstrated. In the present report, using a battery of murine monoclonal antibodies to herpes simplex virus (HSV), we investigated whether antiviral antibodies can also synergize in neutralization kinetics. To determine whether synergism in dose-effect assays can predict synergism in neutralization rate, the ability of neutralizing antibodies to synergize in neutralization rate (kinetics) was compared to their ability to synergize in dose-effect assays (potency) in cell-free assays. Although certain antibody combinations synergized in both neutralization rate and potency, combinations that did not clearly synergize in potency could still significantly synergize in neutralization rate. Weak neutralizing antibodies could also greatly increase the neutralization rate of more potent antibodies. These results suggest that evaluating antibody combinations in dose-effect assays but not in neutralization kinetics provides a partial picture of neutralizing antibody dynamic interactions and may prevent the identification of certain favorable antibody combinations. These findings also support the importance of establishing defined antibody cocktails for prophylactic and therapeutic purposes. A simple strategy to evaluate antibody interactions in neutralization kinetics is proposed in which a quantitative prediction of additivity is made on the basis of the neutralization rate constants of the individual antibodies in the combination.

Inactivation of HSV-1 and HSV-2 and prevention of cell-to-cell virus spread by Santolina insularis essential oil.

The essential oil obtained in toto from Santolina insularis was investigated for its antiviral activity on herpes simplex type 1 (HSV-1) and type 2 (HSV-2) in vitro. The IC(50) values, determined by plaque reduction assays, were 0.88 and 0.7 microg/ml for HSV-1 and HSV-2, respectively, while the CC(50) determined by the MTT test on Vero cells was 112 microg/ml, indicating a CC(50)/IC(50) ratio of 127 for HSV-1 and 160 for HSV-2. Results obtained by plaque reduction assays also indicated that the antiviral activity of S. insularis was principally due to direct virucidal effects. Antiviral activity against HSV-1 and HSV-2 was not observed in a post-attachment assay, and attachment assays indicated that virus adsorption was not inhibited. Up to 80% inhibition of HSV-1 was achieved at the concentration of 40 microg/ml by yield reduction assay. Furthermore, reduction of plaque formation assays also showed that S. insularis essential oil inhibits cell-to-cell transmission of both HSV-1 and HSV-2.

pFab-CMV, a single vector system for the rapid conversion of recombinant Fabs into whole IgG1 antibodies.

We have constructed a single vector system for the rapid conversion of recombinant Fabs into whole IgG1 antibodies and their expression in eukaryotic cells. This vector, named pFab-CMV, utilizes the same unique cloning sites present on the pComb3 phagemid thus allowing for the direct subcloning of light chains and heavy chain Fd regions. pFab-CMV also allows for the expression of recombinant Fabs in eukaryotic cells by removal of a cassette containing part of the hinge, CH2 and CH3 sequences. Stable cell lines are rapidly obtained with pFab-CMV by NEO selection without the need for co-transfection of heavy and light chain expressing vectors.

Characterization of a type-common human recombinant monoclonal antibody to herpes simplex virus with high therapeutic potential.

We report the characterization of a type-common human recombinant monoclonal antibody previously isolated by antigen selection from a phage-displayed combinatorial antibody library established from a herpes simplex virus (HSV)-seropositive individual. Competition with well-characterized murine monoclonal antibodies and immunodetection of gD truncations revealed that this antibody recognizes the group Ib antigenic site of glycoprotein D, a highly conserved and protective type-common determinant. To our knowledge, this is the first human group Ib monoclonal antibody ever described. The antibody also displayed first-order neutralization kinetics and a high neutralization rate constant, was capable of completely inhibiting syncytium formation by a fusogenic strain of HSV type 1, and efficiently neutralized low-passage clinical isolates of both HSV serotypes. Taken together with our earlier observations of the in vivo antiviral activities of this human recombinant antibody in animal models of HSV infection, the present results support the high therapeutic potential of this antibody.

Effects of in-vitro activity of miconazole and ketoconazole in phospholipid formulations.

Antifungal agents are often used in liposomal formulations in order to improve their pharmacological activity, but how vesicle inclusion can actually affect this is still not fully understood. We report here the results obtained from evaluation of the in-vitro activity against Candida albicans ATCC E10231 of miconazole and ketoconazole in various vesicular and non-vesicular preparations, obtained from egg and soya phospholipids, using time-kill curves. In most cases inclusion of miconazole or ketoconazole in liposomes led to a delayed and decreased activity of the drugs, with detectable differences among the various phospholipid concentrations and different liposomal preparations (small unilamellar vesicle, liposomes, multilamellar aggregates and broken liposomal structures). The results obtained may be helpful in the study of new preparations of antifungal agents entrapped in liposomal structures.

Topically applied human recombinant monoclonal IgG1 antibody and its Fab and F(ab')2 fragments protect mice from vaginal transmission of HSV-2.

A recombinant human anti-herpes simplex virus monoclonal IgG1, antibody and the corresponding Fab and F(ab')2 fragments were tested for efficacy in preventing vaginal transmission of HSV-2 infection in a well-established mouse model for genital herpes. IgG1, Fab, and F(ab')2 were approximately equally protective; vaginal delivery of 1-5 ng provided approximately 50% protection, and vaginal delivery of 400 ng completely protected mice from genital herpes infection (P < 0.001). These results suggest that topical applications of human monoclonal antibodies may be useful in developing new methods for preventing sexually transmitted disease.

Protection of nude mice by passive immunization with a type-common human recombinant monoclonal antibody against HSV.

Herpes simplex viral disease is an important cause of morbidity and mortality in man. Although the development of very effective nucleoside analogs with a high therapeutic index has greatly improved the clinical management of herpetic infections, the emergence of drug-resistant viral strains has become a cause of serious concern both because of its clinical implications and in terms of viral ecology. The present report is the first demonstration of the in vivo protective activity of a type-common human recombinant monoclonal antibody derived from a combinatorial antibody library. Athymic nude mice were infected with HSV type 1 either intracutaneously in the flank or by corneal scarification. Beside reducing morality rates when administered before infection, the antibody dramatically and significantly prolonged survival times (P < 0.0001) when administered up to 24 hr postinfection, a time when the virus had already reached the peripheral nervous system. This suggests that the antibody may act, at least in part, by interfering with axonal transport of the virus and/or with viral expression. These results indicate that human recombinant antibodies isolated by antigen selection from combinatorial libraries can be effective in vivo. Such antibodies could complement antiviral chemotherapy and represent valuable tools for the prophylaxis of infections by the herpes simplex viruses.

Rapid assay of phage-derived recombinant human fabs as bispecific antibodies.

Specific anti-tumor and anti-viral activities can be conferred on lymphocytic and myeloid effector cells by retargeting them with bispecific antibodies. These are antibodies which possess an anti-target binding region and a region capable of binding specific effector cell surface markers. For the rapid evaluation of recombinant human Fabs as bispecific antibodies, we have constructed a vector that allows for the conversion of Fabs into protein A fusion proteins. These can be used to generate bispecific antibodies when complexed to appropriate anti-effector cell immunoglobulins. As a model system, a protein A fusion derivative of a human recombinant anti-herpes simplex virus (HSV) Fab was constructed and complexed to OKT3, a T cell-activating antibody specific for CD3. This complex reduced HSV-2 yields in infected cells by about three logs relative to controls when incubated on HSV-2-infected cell monolayers in the presence of IL-2-activated lymphocytes. The system described allows for the rapid evaluation of recombinant human Fabs as bispecific antibodies for therapeutic applications. In addition, Fab-protein A fusion proteins can be used in ELISA and other immuno-assays with increased sensitivity.

Directed selection of recombinant human monoclonal antibodies to herpes simplex virus glycoproteins from phage display libraries.

Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

New thioxopyrimidines. Synthesis and evaluation for antimicrobial activity.

A convenient and simple synthesis of some new thioxopyrimidines was developed starting from 3-amino-3-(dialkylamino)propenethioamide derivatives. The prepared compounds were assayed in vitro for antimicrobial activity and found practically inactive.

Synthesis and antimicrobial activity of new 3,5-diaminoisothiazole derivatives.

The 3,5-diaminoisothiazole derivatives 23-42 were synthesized in excellent yields by oxidative cyclization of 3-amino-3-(dialkylamino)propenethioamide derivatives. These intermediates and the isothiazole derivatives were tested in vitro for their antimicrobial activity.

Synthesis and antimicrobial activity of some pyrrole derivatives. V: 5-Aryl-3-ethoxycarbonyl-2-(4-substituted piperazino)pyrrole derivatives.

The synthesis of new 5-aryl-3-ethoxycarbonyl-2-piperazinopyrrole derivatives is reported. The new compounds were tested in vitro for their antimicrobial activity. Compound 11 was the most active against the Gram-positive microorganisms.

1-Acylaminoimidazoles synthesis and antimicrobial activity.

Several new 1-acylamino-2-alkyl-4-arylimidazoles were synthesized. The compounds were obtained by reaction of N1-acylacetamidrazones and alpha-bromoketones. The antimicrobial activity of the prepared compounds was tested.

Synthesis and anti-microbial activity of benzo-condensed heterocyclic derivatives.

The synthesis of 2,4-dione derivatives of 1,5-benzodithiepine, 1,5-benzodiazepine and 1,5-benzothiazepine and the anti-microbial activity in vitro of these derivatives and of analogous of 1,5-benzodioxepine, 1,5-benzoxathiepine and 1,5-benzoxazepine, previously prepared, are reported. Some of these compounds showed a good activity against some Gram positive microorganisms and blastomycetes.

Antimicrobial activity of some isothiosemicarbazones.

The microbiological activity of a series of isothiosemicarbazones is reported. The experimental data relating to the microbiological screening show an interesting antibacterial activity associated to a good antifungal activity.