In vitro inhibition of Chikungunya and Semliki Forest viruses replication by antiviral compounds: synergistic effect of interferon-alpha and ribavirin combination.

Chikungunya virus (CHIKV) and Semliki Forest virus (SFV) were used in our laboratory to screen active antiviral compounds against viruses of the Alphavirus genus. Antiviral activity was estimated by the reduction of the cytopathic effect of each alphavirus on infected Vero cells and by virus titer reduction. Cytotoxicity was evaluated by determining the inhibition of Trypan blue exclusion in confluent cell cultures and by the evaluation of the inhibitory effect on cell growth. With CHIKV and SFV, the selectivity indices of human recombinant interferon-alpha and iota-carrageenan were much higher than that of ribavirin, which has been previously investigated for its inhibitory effect on alphavirus infections. Compared to ribavirin, 6-azauridine was more effective against CHIKV and showed a similar antiviral activity against SFV. IFN-alpha2b, glycyrrhizin, 6-azauridine, and ribavirin caused a concentration-dependent reduction in the virus yield with CHIKV and SFV. Moreover, the combination of IFN-alpha2b and ribavirin had a subsynergistic antiviral effect on these two alphaviruses and should be evaluated for the treatment of these infections.

Central depressant effects of N3-substituted 6-azauridines in mice.

Central depressant effects in mice of N3-substituted 6-azauridines (6-AzUd) (1) were examined by intracerebroventricular (i.c.v.) injection. Eleven derivatives including alkyl-, benzyl-, xylyl- and phenylethyl-substitution onto the N3-position of 1 were synthesized and their pharmacological effects were evaluated using hypnotic activity, locomotor activity, motor incoordination and pentobarbital-induced sleep prolongation as indices. Six of 12 compounds showed the hypnotic activity. At a dose of 2 mumol/mouse, the mean sleeping time induced by 1, N3-benzyl-6-AzUd (7), N3-o-xylyl-6-AzUd (8), N3-m-xylyl-6-AzUd (9), N3-p-xylyl-6-AzUd (10) and N3-alpha-phenylethyl-6-AzUd (11) was 14, 11, 45, 12, 9 and 16 min, respectively. These derivatives and N3-beta-phenylethyl-6-AzUd (12) (1.5 mumol/mouse) significantly prolonged pentobarbital-induced (40 mg/kg, i.p.) sleeping time, whereas none of the N3-alkylated derivatives (methyl-, ethyl-, n-propyl-, n-butyl- and allyl-substitution) exerted the hypnotic activity or pentobarbital-induced sleep prolongation. Nucleoside 1 and its xylyl-derivatives (1.5 mumol/mouse) significantly decreased locomotor activity of mice, their effects paralleled the hypnotic activity. These compounds (1.5 mumol/mouse) also produced motor incoordination and potentiated the effect of diazepam-induced motor incoordination. These results indicate that 1 and its benzyl-related derivatives, but not alkyl-derivatives have a depressant effect on the central nervous system.

Effects of D-glucosamine and 6-azauridine on nucleotide contents, 5-fluorouridine uptake, and cytotoxicity in TA3 mammary tumor cells.

In TA3 mammary carcinoma cells in suspension culture, D-glucosamine X HCl (GlcN) induced a diversion of uridylate from UTP into UDP-N-acetylhexosamines, reducing the intracellular pool of UTP and eliciting an increased rate of de novo uridylate synthesis. This rise in de novo synthesis was completely suppressed by addition of 6-azauridine (6-AzaUrd) to the cell suspension in vitro or in the solid TA3 mammary tumor in NMRI mice in vivo. A synergistic depletion of UTP pools to less than 6% of the UTP in controls was observed in TA3 cell suspensions exposed to GlcN and 6-AzaUrd. In solid TA3 tumors in vivo, UTP was reduced by this combination to 19% of the control value. A high sensitivity of the solid tumor to inhibition of pyrimidine synthesis de novo was indicated by the reduction of the UTP content after administration of 6-AzaUrd alone. UTP deficiency in TA3 tumor cells was accompanied by CTP deficiency. In addition, 6-AzaUrd caused a lowering of GTP in the neoplastic tissue. Host liver was resistant to 6-AzaUrd but responded to treatment with GlcN with a decrease in UTP to 67%. Uridine-cytidine kinase was less inhibited in the presence of lowered UTP and CTP, which are potent feedback inhibitors of the enzyme, and enabled an enhanced formation of phosphorylated derivatives of 5-fluorouridine (FUrd). Aside from the formation of 5-fluoro-UTP, we have identified 5-fluoro-UDP-N-acetylhexosamines (FUDPHexNAc), which accumulated when FUrd and GlcN were sequentially administered. Treatment of TA3 cells with FUrd after a pretreatment with 6-AzaUrd and GlcN resulted in a 2.5-fold increase in [14C]FUrd uptake and a duplication of 5-fluorouridylate incorporation into the RNA. The proportion of FUDPHexNAc increased to 58% of the phosphorylated FUrd metabolites, as compared to 6% in TA3 cells exposed exclusively to FUrd. In vivo chemotherapy of mice bearing TA3 ascites tumors was most effective with respect to tumor growth inhibition and animal survival when GlcN and FUrd were combined.

Rationally designed combination chemotherapy for the treatment of patients with recalcitrant psoriasis.

In an effort to improve clinical response and reduce systemic toxicity, nine patients with recalcitrant psoriasis were treated with rational combinations of chemotherapeutic agents. Five patients received methotrexate by injection, 7.5 or 10 mg, followed 1 hour later by intravenous 5-fluorouracil, 170 to 562 mg/m2, on a weekly schedule. Four patients received oral triacetyl-azauridine, 2 to 4 gm daily, in combination with intravenous 5-fluorouracil, 225 to 600 mg/m2, administered every week. Three patients experienced greater than 75% clearing of disease, five patients experienced greater than 50% clearing, and only one patient failed to respond. Response rates did not differ between the two treatment groups. Adverse effects of these therapies were mild and infrequent. We conclude that 5-fluorouracil in combination with either methotrexate or triacetyl-azauridine is a relatively safe and effective alternative for the therapy of patients with severe psoriasis.

Embryotoxicity of transplacentally and intraamniotically administered 6-azauridine in mice.

Embryotoxic effects were compared of intramuscularly (im) and intraamniotically (ia) administered 6-azauridine (Riboazauracil Spofa) in random-bred mice H-Velaz. Effects of single doses (0.25 mg, 2.5 mg, 25.0 mg and 250.0 mg for im and 0.0025 mg, 0.025 mg and 2.5 mg for ia administration) on days 11, 12, 13 and 14 were evaluated as a sum of dead fetuses and fetuses with cleft lip and/or palate, fetuses with limb deformities and fetuses with deformities constituting the syndrome of caudal regression (hypoplasia of the caudal part of the trunk, absent tail, short tail, curled tail). Considering the sensitivity peaks of the morphogenetic processes which were observed, the dose-response relationships, the transformation of the teratogenic to a lethal effect and critical period extension with increasing doses, it was found that the effects of ia and im administered 6-azauridine did not differ. It was concluded that ia administered 6-azauridine had direct effect on embryonic morphogenetic processes and that this, too, was the essential mechanism of embryotoxicity of im administered 6-azauridine. The value of the intraamniotic technique for establishing the direct embryotoxic effect is discussed.

The thrombogenicity of 6-azouridine.

The antimetabolite 6-azauridine blocks the de novo synthesis of pyrimidines and causes increased serum levels of several amino acids including homocystine. 6-Azauridine was was withdrawn from clinical use for the treatment of psoriasis because of the occurence of arterial and venous thromboembolic episodes in some psoriatic patients. Utilizing a standard animal model for the recognition of venous and arterial thrombosis, 6-azauridine was demonstrated in this study to cause thrombosis without producing homocystinemia when administered orally or intravenously.

[Antiviral activity of experimental combination of 5-iodo-2-deoxyuridine with 6-azauridine and its effectiveness in the treatment of herpetic infection with lesions of the skin and mucous membranes]. / Protivovirusnaia aktivnost' kombinatsii 5-iod-2-dezoksiuridina v éksperimente i ee éffektivnost' pri lechenii gerpeticheskoi infektsii s porazheniem kozhi i slizistykh obolochek.

The use of the combination of 5-iodo-2-dioxyuridine (IUDR) and 6-azauridine drugs differing in their mechanisms of action in an experimental herpetic infection demonstrated its definite antiviral activity, not only not inferior to the effect of each of the drugs alone, but also remaining sufficiently high when the concentration of IUDR was decreased 2-fold. In clinical trials in relapsing genital herpes all the drugs under study showed statistically significant therapeutic activity. The combined use of the drugs exerted a certain synergistic effect.

Treatment of psoriatic arthritis with azaribine.

Thirty-two patients with psoriatic arthritis unresponsive to conventional therapy were treated wtih the antipyrimidine azaribine. For the group, improvement was highly significant (P less than 0.01) when average painful joint count, ring size, grip strength, pain, and morning gel were compared with baseline measurements. The spectrum of response of individual patients varied from total remission or arthritis in 5 patients to no improvement in 7. Dermatitis improved more than 50% in 26 patients. Reduction in hematocrit, gastrointestinal irritation, and occasional central nervous system toxicity were readily reversed by decreasing or discontinuing dosage. Azaribine appears to be a useful agent for trial in the treatment of refractory psoriatic arthritis.

Double-blind controlled crossover high-dose study of Azaribine in psoriasis.

Azaribine used in high doses of 200 mg/kg a day is an effective agent in inducing temporary remissions in patients with severe psoriasis but potentially serious neurotoxicity may occur. Therapy should be initiated at lower doses of 125 mg/kg a day and advanced carefully only if clinical response does not ensue at the lower level.

The treatment of psoriasis with azaribine.

An open study was made of 25 patients with severe, recalcitrant psoriasis treated with azaribine (6-azauridine triacetate). Most patients received 125 mg/kg/day for a period of 8 weeks. A good to excellent response with 60-100% clearing of lesions was observed in 14 patients and a fair response with 40-60% improvement in another 6 patients. Thus 20 patients (80% of the series) exhibited a favorable clinical response. 16 of these 20 patients relapsed to approximately pretreatment status within 1 month after stopping therapy. The most frequently observed side-effects were mild reversible anemia, fatigue and mild transient gastrointestinal symptons. 8 patients (32% of the series) exhibited sufficient toxicity to necessitate the discontinuance of therapy. 1 patient experienced an unexplained femoral arterial thrombotic episode while on the drug. Azaribine may find a place in the therapy of severe psoriasis particularly in patients with hepatic disease. However, further studies of its potential for toxicity are indicated.

Changes in preference for NaCl following administration of 6-azauridine and 6-azauridine triacetate.

Preliminary evidence in man has suggested that 6-azauridine triacetate (6-AzUrdTA) might adversely affect taste acuity. The production of measurable serum concentrations of homocysteine in rabbits treated with 6-AzUrdTA further suggested a mechanism for the possible adverse effects of this drug on taste, since administration of other thiol-containing drugs in man and animals had been shown to decrease taste acuity. Since changes in preferences for NaCl solutions have been shown to reflect changes in taste acuity in the rat, preference for NaCl solutions in rats treated with 6-AzUrdTA were measured in a two-bottle test. Detection and recognition thresholds were also measured in patients with scleroderma before and after the administration of 6-AzUrdTA. Serum copper and zinc concentrations were measured in both rats and man. Rats given 6-AzUrdTA exhibited a significantly greater intake of 0.30-M NaCl than control rats, who avoided this solution. This change was accompanied by a significant decrease in serum zinc concentrations. No significant changes in taste acuity occurred in the patients. These data suggest that administration of 6-AzUrdTA affects taste acuity in rats either through the addition of thiols or the depletion of zinc.