Poly[N-(2-hydroxypropyl)methacrylamide] conjugates of bovine seminal ribonuclease. Synthesis, physicochemical, and preliminary biological evaluation

The synthesis of three conjugates of poly(HPMA) with bovine seminal ribonuclease (BS-RNase) differing in their structure is described. Two conjugates contained BS-RNase conjugated with the polymer via functional group situated at the end of the polymer chain (star-shaped conjugate I) or attached to the poly(HPMA) carrier via biodegradable oligopeptide spacers randomly distributed along the polymer chain ('classic' conjugate II). These two conjugates differ in structure, molecular weight, and molecular weight distribution. In addition, a conjugate combining the activity of two compounds, BS-RNase and doxorubicin, both attached to the same polymer chain via biodegradable spacers was synthesized ('classic' conjugate III). Biological activity of all BS-RNase conjugates was compared with that of free BS-RNase and to the polymer-bound anticancer drug doxorubicin (conjugate IV). Unlike the bovine pancreatic ribonuclease (RNase A), BS-RNase displays a potent antitumor activity when tested in vitro and, if administered intratumorally, also in vivo. BS-RNase in its polymer-conjugated forms (conjugates I, II and III) tested on various human tumor cell lines has lost at least part of its antitumor activity. In in vivo experiments (nude mice bearing human melanoma), intratumoral (i.t.) therapy with BS-RNase or with its conjugates II and III showed a significant antitumor effect. Intravenous (i.v.) application of free BS-RNase was totally ineffective, while both BS- RNase conjugates II and III caused significant inhibition of tumor growth. BS-RNase bound to a star-shaped polymer (conjugate I) and administered i.t. or i.v. at the same concentration showed very high toxicity. Our results demonstrate that modification of BS-RNase with poly(HPMA) can prevent it from degrading or inactivating events occurring in blood vessels after intravenous application, significantly enhancing its potential for therapeutical application.