The relative in vitro antiviral activities of three related nucleoside carboxamides, ribavirin (1-3-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), tiazofurin (2-3-D-ribofuranosylthiazole-4-carboxamide), and selenazole (2-p-D-ribofuranosylselenazole-4-carboxamide), were studied against selected DNA and RNA viruses. Although the activity of selenazole against different viruses varied, it was significantly more potent than ribavirin and tiazofurin against all tested representatives of the families Paramyxoviridae (parainfluenza virus type 3, mumps virus, measles virus), Reoviridae (reovirus type 3), Poxviridae (vaccinia virus), Herpesviridae (herpes simplex virus types 1 and 2), Togaviridae (Venezuelan equine encephalomyelitis virus, yellow fever virus, Japanese encephalitis virus), Bunyaviridae (Rift Valley fever virus, sandfly fever virus [strain Sicilian], Korean hemorrhagic fever virus), Arenaviridae (Pichinde virus), Picornaviridae (coxsackieviruses B1 and B4, echovirus type 6, encephalomyocarditis virus), Adenoviridae (adenovirus type 2), and Rhabdoviridae (vesicular stomatitis virus). The antiviral activity of selenazole was also cell line dependent, being greatest in HeLa, Vero-76, and Vero E6 cells. Selenazole was relatively nontoxic for Vero, Vero-76, Vero E6, and HeLa cells at concentrations of up to 1,000 ,g/ml. The relative plating efficiency at that concentration was over 90%. The effects of selenazole on viral replication were greatest when this agent was present at the time of viral infection. The removal of selenazole from the medium of infected cells did not reverse the antiviral effect against vaccinia virus, but there was a gradual resumption of viral replication in cells infected with parainfluenza type 3 or herpes simplex virus type 1 (strain KOS). However, the antiviral activity of ribavirin against the same viruses was reversible when the drig was removed.

Reporter, proliferation, and direct STAT activation assays using cell lines expressing transfected receptors revealed differences between the receptor complexes. IL-19 and IL-24 also exhibited growth inhibition on a cell line endogenously expressing all three receptor subunits, an effect that was seen at cytokine levels two orders of magnitude above those required for STAT activation or proliferation. These results demonstrate that, although this subclass exhibits receptor complex redundancy, there are differences in ligand/receptor interactions and in signal transduction that may lead to specificity and a distinct biology for each cytokine.

Several 3,4,6-trisubstituted pyrazolo[3,4-d]pyrimidine ribonucleosides were prepared and tested for their biological activity. High-temperature glycosylation of 3,6-dibromoallopurinol with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose in the presence of BF3 X OEt2, followed by ammonolysis, provided 6-amino-3-bromo-1-beta-D-ribofuranosylpyrazolo-[3,4-d]pyrimidin-4(5H)-on e. Similar glycosylation of either 3-bromo-4(5H)-oxopyrazolo [3,4-d]pyrimidin-6-yl methyl sulfoxide or 6-amino-3-bromopyrazolo [3,4-d]pyrimidin-4(5H)-one, and subsequent ammonolysis, also gave 7a. The structural assignment of 7a was on the basis of spectral studies, as well as its conversion to the reported guanosine analogue 1d. Application of this glycosylation procedure to 6-(methylthio)-4(5H)-oxopyrazolo[3,4-d]pyrimidine-3-carboxamide gave the corresponding N-1 glycosyl derivative. Dethiation and debenzoylation of 16a provided an alternate route to the recently reported 3-carbamoylallopurinol ribonucleoside thus confirming the structural assignment of 16a and the nucleosides derived therefrom. Oxidation of 16a and subsequent ammonolysis afforded 6-amino-1-beta-D-ribofuranosyl-4(5H)-oxopyrazolo[3, 4-d]pyrimidine-3-carboxamide. Alkaline treatment of 15a gave 6-azacadeguomycin. Acetylation of 15a, followed by dehydration with phosgene, provided the versatile intermediate 6-amino-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)-4(5H)-oxopyrazolo [3, 4-d]pyrimidine-3-carbonitrile. Deacetylation of 19 gave 6-amino-1-beta-D-ribofuranosyl-4(5H)-oxopyrazolo[3, 4-d]pyrimidine-3-carbonitrile. Reaction of 19 with H2S gave 6-amino-1-beta-D-ribofuranosyl-4(5H)-oxopyrazolo[3, 4-d]pyrimidine-3-thiocarboxamide. All of these compounds were tested in vitro against certain viruses and tumor cells. Among these compounds, the guanosine analogues 7a and 20a showed significant activity against measles in vitro and were found to exhibit moderate antitumor activity in vitro against L1210 and P388 leukemia. 6-Azacadeguomycin and all other compounds were inactive against the viruses and tumor cells tested in vitro.

Bispecific antibodies (bsAbs) present an attractive opportunity to combine the additive and potentially synergistic effects exhibited by combinations of monoclonal antibodies (mAbs). Current challenges for engineering bsAbs include retention of the binding affinity of the parent mAb or antibody fragment, the ability to bind both targets simultaneously, and matching valency with biology. Other factors to consider include structural stability and expression of the recombinant molecule, both of which may have significant impact on its development as a therapeutic. Here, we incorporate selection of stable, potent single-chain variable fragments (scFvs) early in the engineering process to assemble bsAbs for therapeutic applications targeting the cytokines IL-17A/A and IL-23. Stable scFvs directed against human cytokines IL-23p19 and IL-17A/A were isolated from a human Fab phage display library via batch conversion of panning output from Fabs to scFvs. This strategy integrated a step for shuffling V regions during the conversion and permitted the rescue of scFv molecules in both the V(H)V(L) and the V(L)V(H) orientations. Stable scFvs were identified and assembled into several bispecific formats as fusions to the Fc domain of human IgG1. The engineered bsAbs are potent neutralizers of the biological activity of both cytokines (IC(50) < 1 nM), demonstrate the ability to bind both target ligands simultaneously and display stability and productivity advantageous for successful manufacture of a therapeutic molecule. Pharmacokinetic analysis of the bsAbs in mice revealed serum half-lives similar to human mAbs. Assembly of bispecific molecules using stable antibody fragments offers an alternative to reformatting mAbs and minimizes subsequent structure-related and manufacturing concerns.

A novel nucleoside analog, 4(5H)-oxo-l1-,-D-ribofuranosylpyrazolo [3,4-d]pyrimidine-3-thiocarboxamide (N10169), was evaluated in cell culture and in animals for antiviral activity against DNA and RNA viruses. The compound was highly active against strains of adeno-, vaccinia, influenza B, paramyxo-, picorna-, and reoviruses, with 50% inhibition of virus-induced cytopathology at 1 to 10 ,uM. Lesser or no antiviral effects were observed against herpes simplex, cytomegalo-, corona-, influenza A, vesicular stomatitis, and visna viruses. Drug potency against certain viruses was highly cell line dependent (N10169 was highly active in HeLa cells but was much less potent in Vero cells). This was correlated, in part, to differences in levels of adenosine kinase activity in these cell lines, since adenosine kinase appears to phosphorylate N10169 to its active form.N10169 was inhibitory to proliferating cells at antiviral concentrations, whereas stationary-phase monolayers tolerated higher concentrations (5100 ,uM). Exogenous uridine was able to reverse the virus-inhibitory effects of the compound, leading to the discovery that N10169 5'-monophosphate is a potent inhibitor of cellular orotidylate decarboxylase. N10169 was evaluated in mice that were infected intraperitoneally with banzi virus or inoculated intranasally with influenza B virus, and in hamsters that were infected intranasally with vaccinia virus. In each model, intraperitoneal injection of N10169 (100 to 300 mg/kg per day for 7 days) twice daily was ineffective, whereas intraperitoneal injection of ribavirin showed some benefit in the influenza B and banzi virus infection models.In a recent report (3) a series of pyrazolo [3,4-d]pyrimidine nucleoside analogs possessing antiviral and antitumor properties was described. Additional studies of these compounds were undertaken to determine the spectrum of viruses which would be inhibited and to ascertain any therapeutic potential in animals. One compound, 4(5H)-oxo-1-P-D-ribofuranosylpyrazolo[3,4-d]pyrimidine-3-thiocarboxamide (N10169; Fig. 1), although similar in structure to the broad-spectrum antiviral agent ribavirin (18), was found to have an unusual spectrum of antiviral activity, degree of potency, and mode of action. Although the compound is related to purine nucleosides, it was found to be a potent inhibitor of pyrimidine biosynthesis. As the work proceeded it was determined that the properties of N10169 were remarkably similar to those of 6-azauridine (23). The compounds related to N10169 (3) did not exhibit the same degree of potency and spectrum of activity that N10169 did in vitro. In this report we summarize the results of cell culture studies and evaluate the antiviral activity of this novel compound in appropriate animal infection models.