Prolonging the half-life of human interferon-alpha 2 in circulation: Design, preparation, and analysis of (2-sulfo-9-fluorenylmethoxycarbonyl)(7)-interferon-alpha 2

Polypeptide drugs are generally short-lived species in circulation. In this study, we have covalently linked seven moieties of 2-sulfo-9-fluorenylmeth oxycarbonyl (FMS) to the amino groups of human interferon-alpha2. The deriv ative thus obtained (FMS7-IFN-alpha2) has approximate to4% the biological p otency and 33 +/- 4% the receptor binding capacity of the native cytokine. Upon incubation, FMS7-IFN-alpha2 undergoes time-dependent spontaneous hydro lysis, generating active interferon with t(1/2) values of 24 +/- 2 h at pH 8.5 and 98 +/- 10 h at pH 7.4. When native IFN-alpha2 is intravenously admi nistered to mice, circulating antiviral activity is maintained for a short duration and then declines with t(1/2) = 4 +/- 0.5 h, reaching undetectable values at approximate to 18 h after administration. With intravenously adm inistered FMS7-IFN-alpha2, there is a lag period of 2 h, followed by a prog ressive elevation in circulating antiviral-active protein, which peaked at 20 h and declined with t(1/2) = 35 +/- 4 h. FMS7-IFN-alpha2 is resistant to cu-chymotrypsin digest and to proteolytic inactivation by human serum prot eases in vitro. We have thus introduced here an inactive IFN-alpha2 derivat ive, which is resistant to in site inactivation and has the capability of s lowly reverting to the native active protein at physiological conditions in vivo and in vitro. Having these attributes, FMS7-IFN-alpha2 maintains prol onged circulating antiviral activity in mice, exceeding 7-8 times the activ ity of intravenously administered native cytokine.