Human respiratory syncytial virus (RSV) a paramyxovirus is a major cause

Human respiratory syncytial virus (RSV) a paramyxovirus is a major cause of acute upper and lower respiratory tract infections in infants young children and adults. in the monkey model. Mechanism of action studies indicate that RFI-641 HESX1 blocks viral F protein-mediated fusion and cell syncytium formation. Human respiratory syncytial virus (RSV) a member of the family (18) is a major cause of acute upper and lower respiratory tract infections in infants young children and adults. Serological evidence indicates that approximately 95% of children have been exposed to RSV by 2 years of age and 100% of children have been exposed by the time they reach adulthood (8). In a given year around 91 0 infants are hospitalized with RSV infection in the United States. These infections are responsible for 40 to 50% of hospitalizations for pediatric bronchiolitis and 25% Ursolic acid of hospitalizations for pediatric pneumonia (8 15 Since the immune response to RSV infection is not protective RSV infections reoccur Ursolic acid throughout adulthood. In adults and older children RSV infection has been associated with upper respiratory infection tracheobronchitis and otitis media. However RSV in the institutionalized elderly can be more serious and is characterized by severe pneumonia and mortality rates of up to 20 and 78% respectively (5 6 Adults with a previous history of heart or lung conditions are at a high risk for RSV infection. The infection has been linked to exacerbation of patients with chronic obstructive pulmonary disease. Significant mortality has been observed in immunocompromised patients particularly those undergoing bone marrow transplantation. Regular outbreaks of RSV are well characterized and predictable occurring between October and May each year with peak occurrences in January and February. Ribavirin is the only commercially available agent used to treat RSV infection (2). The utilization of ribavirin is limited due to efficacy and toxicity concerns as well as the very long treatment regimen required for its delivery by aerosol inhalation (19). Protective antibodies (14 27 indicated for prophylaxis in high-risk children are administered intravenously (RespiGam) or intramuscularly (Synagis). A number of small-molecule inhibitors of RSV have been identified but to date none are clinically approved (7 23 RFI-641 is the result of a chemical optimization of “type”:”entrez-nucleotide” attrs :”text”:”CL387626″ Ursolic acid term_id :”51439586″ term_text :”CL387626″CL387626 (1 4 28 a compound that inhibits RSV fusion and demonstrates antiviral activity in vitro and in vivo. We report here on the in vitro activity mechanism and in vivo activity of RFI-641. MATERIALS AND METHODS Viral strains. RSV strains A2 and Long (American Type Culture Collection Rockville Md.) were grown in human foreskin fibroblast (HFF) cells to contain approximately 107 PFU/ml. Cultures were aliquoted and kept frozen at ?70°C until required. Recent human isolates collected Ursolic acid from 1992 to 1995 (Baylor College of Medicine Houston Tex.) were received at passage no. 2 expanded to a larger stock (passage no. 3) and tested for sensitivity to RFI-641. The cold-passaged RSV deletion mutant cp-52 was previously described (3 10 Compound. RFI-641 (4 4 6 3 5 triazin-2-ylamino}-biphenyl-2 2 acid) (Fig. ?(Fig.1)1) was synthesized at Wyeth-Ayerst Research Pearl River N.Y. (18a). The compound was solubilized in water at a concentration appropriate to the dose to be administered. FIG. 1. Structure of RFI-641 (4 4 6 3 5 2 Molecular mass 1 684 Da. Antiviral activity and cytotoxicity assays. The antiviral activity of RFI-641 was evaluated by measuring the amount of RSV protein with an enzyme-linked immunosorbent assay (ELISA). Vero or HFF cells were infected with RSV at a multiplicity of infection (MOI) of 0.004 and 50% inhibitory concentrations (IC50s) were determined over a range by using 5 to 10 concentrations of the compound. Infected cells were incubated for 4 days before the cells were fixed by treatment with 50% methanol-50% acetone washed with buffer and developed by an ELISA with antibody to F protein. Cytotoxicity assays were performed with the same cell line incubated with serially diluted RFI-641 for 4 days. {At the end of the.|At the final end of the.}