Synthesis of new 3,4,5-trisubstituted isothiazoles as effective inhibitory agents of enteroviruses.

The synthesis and evaluation of 3,4,5-trisubstituted isothiazoles as antiviral agents led to the discovery of several compounds effective in vitro against enteroviruses polio 1 and ECHO 9. Structure-activity relationship studies revealed that a short thioalkyl chain in the 3-position and a methyl ester group in the 4-position are the structural components that, to a large extent, contribute to the positive biological profile in terms of both selectivity and low cytotoxicity. Under one-step growth conditions, methyl 3-methylthio-5-phenyl-4-isothiazolecarboxilate caused the greatest activity if added within 1 h after poliovirus adsorption. These data suggest interference with early events of viral replication.

Synthesis and antiviral activity of a new series of 4-isothiazolecarbonitriles.

A series of 4-isothiazolecarbonitriles was synthesized and screened for in vitro antiviral activity. The effect of various substituents on the phenyl ring, as well as the substitution of the phenyl for other aromatic and heteroaromatic rings, was examined to establish the requirements for optimum activity. The most active member of the series, 3methylthio-5-phenyl-4-isothiazolecarbonitrile, exhibited a high level of activity against enteroviruses polio 1 and ECHO 9. Preliminary studies on its mechanism of action indicated that this compound had an effect on an early event in the replication of poliovirus type 1.

Antipoliovirus activity of isothiazole derivatives: mode of action of 5,5'-diphenyl-3,3'-diisothiazole disulfide (DID).

In this report we describe the antiviral activity of 5,5'-diphenyl-3,3'-diisothiazole disulfide (DID) and discuss its mode of action. DID selectively inhibits the replication of poliovirus type 1 (therapeutic index = 255) by affecting some early process of the virus growth cycle. The compound does not interfere with adsorption and internalization of virus to HEp-2 cells, nor with uncoating of the viral RNA. However, no viral RNA synthesis occurs after 2 h post-infection in the presence of 50 microM DID. Thus, we investigated some molecular events in poliovirus replication occurring between uncoating and viral RNA synthesis. In our experimental design, we studied the activity of RNA polymerase complex isolated from HEp-2 infected cells in the presence or absence of DID. Our results showed that the RNA polymerase complex was formed in the presence of DID. On the contrary, DID markedly inhibited poliovirus RNA synthesis in a cell-free system using RNA polymerase complex isolated from infected cells. These findings indicate that DID may exert its antiviral activity by preventing viral RNA chain elongation via the inhibition of replicase activity and/or interfering with viral RNA polymerase complex.

In vitro antiviral activity of four isothiazole derivatives against poliovirus type 1.

The in vitro effects of four isothiazoles [5,5'-diphenyl-3,3'-diisothiazole disulfide, 5-phenyl-3-mercapto-isothiazole, 5,5'-(4-chlorophenyl)-3,3'- diisothiazole disulfide, and 5-(4-chlorophenyl)-3-mercapto-isothiazole] on poliovirus type 1 were studied. The derivatives tested demonstrated remarkable viral inhibition, with a higher selectivity index than the previously studied iminodithiole precursors. Under one-step growth conditions, all the isothiazole derivatives caused the greatest activity if added during or after (within 1 h) poliovirus adsorption. These data suggest interference with early events of viral replication. [5-3H]Uridine incorporation into RNA showed that the compounds tested reduced poliovirus RNA synthesis, which was completely shut off after 2 h of incubation and reduced by 50-60% after 4 h. Also, pretreatment of the cell cultures with the compounds for 24 h caused a substantial inhibition of viral replication. The data suggest that the four isothiazole derivatives may have a multi-step antiviral mode of action different from their iminodithiole precursors.

Some pharmacokinetic data on miocamycin II. Concentrations in gynaecological tissues.

The gynaecological tissue levels of miocamycin were studied in ten female patients after pre-operative administration. The patients were divided into two groups on the basis of the scheduled sampling time. All the patients received 4200 mg of the drug, divided in 7 tablets of 600 mg each every 8 h, last administration 2 or 3 h before surgery. The tissue levels, obtained by means of the microbiological method (Sarcina lutea 9341), were frequently higher than those obtained in the serum at the same time. Miocamycin reached the highest concentration in the endometrium (2.5 micrograms/g) and the lowest in the vagina (1.1-0.8 micrograms/g).

Female genital tissue concentrations of roxithromycin.

A new macrolide, roxithromycin, appears to have some interesting pharmacokinetic characteristics dissimilar to those of erythromycin, e.g. it has the unusual property of a long serum half-life and the possibility of once daily dosing. Moreover tissue levels are higher than those obtained with other macrolides, and the drug is still present in the tissues up to 24 h. after dosing. The pharmacokinetics of roxithromycin were studied in 36 women undergoing gynecological surgery, and divided by means of the time of sampling into six different groups. Each group received an initial 300 mg dose followed by 8 successive doses of 150 mg at 12 h intervals. Blood and tissues samples were taken during surgery at 2, 6, 9, 12, 18 and 24 h after the last dose of roxithromycin and the patients were then allocated to one of the six groups by means of the sampling time. Tissue fragments were obtained from ovary, fallopian tubes, endometrium, myometrium, cervix and vagina immediately after the surgical resection of the organs. Tissue and serum concentrations were determined by the microbiological method using Sarcina lutea ATCC 9341; the lowest limit of detection was 0.01 microg/ml or 0.01 microg/g. Roxithromycin reached the highest concentration at the 9th hour after last administration and its tissue levels would encourage a wide use of this drug as a satisfactory alternative to tetracyclines for the therapy of some gynecological infections.

Inhibitory effects of 3-imino-5-phenyl-3H-1,2-dithiole on poliovirus type 1 replication in vitro.

The effect of 3-imino-5-phenyl-3H-1,2-dithiole (PDTI) on different steps of the replicative cycle of poliovirus type 1 in HEp-2 cells was studied. This compound inhibited the replication of poliovirus type 1 as shown by cytopathic effect and virus yield reduction. This inhibitory action was not due to a virucidal effect, nor did the cells to have been pretreated. Under one-step growth conditions 3-imino-5-phenyl-3H-1,2-dithiole caused the greatest inhibition if added within 1 h after poliovirus adsorption. [5-3H]uridine incorporation into RNA showed that PDTI reduced poliovirus RNA synthesis. In fact, in the presence of PDTI viral RNA synthesis was shut off completely at 2 h post infection, and at 4 h post infection viral RNA synthesis was reduced by 50%. The compound may have an inhibitory effect on the early transcriptional and/or replicative functions of the poliovirus genome.

Antioxidant enzymatic systems in neuronal and glial cell-enriched fractions of rat brain during aging.

The activities of Cu,Zn superoxide dismutase, glutathione peroxidase, catalase and glutathione reductase in neuronal and glial cell-enriched fractions obtained from the cerebral cortex of rat brain during aging (15, 30, 90, 350, 750 days of age) were assayed. Our results showed that glutathione peroxidase, catalase and glutathione reductase activities varied little during the examined periods. Only the Cu,Zn superoxide dismutase activity decreased notably from 15th to 750th day of age in both neuronal and glial cells, moreover the activities of all enzymes studied were always detected at lower levels in neuronal cells with respect to glial cells. In agreement with diminished SOD activity, the lipid peroxidation showed an elevated increase with aging; this fact is more evident in neuronal than in glial cells. In conclusion our data show that Cu,Zn superoxide dismutase is the most affected antioxidant enzymatic system of brain aging and it could be responsible for the increased lipid peroxidation in both cell types examined.

Serum IgA antibodies in HSV asymptomatic genital infections.

Cervico-vaginal swabs and serum samples from 81 women without herpes-associated clinical symptoms were examined for virus isolation and for the presence of IgA antibodies to herpes simplex virus (HSV) using the indirect fluorescent antibody technique. Nineteen of the 81 women were followed up for 2 years. Blood samples and swabs from cutaneous lesions were obtained from another group of 20 women (medical staff) with herpes labialis. By analysing the specific anti-HSV IgA antibodies, a higher geometric mean titre (GMT) was found in women with positive HSV-2 isolation (GMT = 380.03), as opposed to the GMT observed in women with negative HSV-2 (GMT = 63.43) or positive HSV-1 isolation (GMT = 55.15). Results from the longitudinal study also demonstrated that positive virus isolation always correlated with a marked rise in serum IgA antibody to HSV.