[Stability of high molecular weight anticancer agent SMANCS and its transfer from oil-phase to water-phase. Comparative study with neocarzinostatin].

SMANCS is a conjugate protein of copolymer of styrene-maleic acid [SMA] (molecular weight: 1,500) and an antitumor protein neocarzinostatin [NCS] (molecular weight: 11,700). It has an approximate molecular weight of 15,000. We report here stability of SMANCS in oil and in water, and NCS in water, under various physical conditions such as exposure to heat, UV, pH, and ultrasonic treatment. Then, we carried out an experiment of transfer of SMANCS in lipid contrast medium [lipiodol] (oil phase) to water phase (blood and physiological saline) in vitro. Results are summarized as follows: In aqueous condition, SMANCS is far more stable than NCS against the exposure to heat and UV, though it is inactivated by excessive exposures. SMANCS in an oily medium was found much more stable even at higher temperatures than in the aqueous phase. Both SMANCS and NCS are the most stable at pH 4.9-6.0. SMANCS dissolved in oil transferred to water phase slowly, having T1/10 of 24 hours (in case of lipiodol). This helps maintaining the anticancer effect of the drug in vivo for a long period of time. SMANCS in lipiodol was found to exert its action against cultured tumor cells as in an aqueous solution.

Poly(deoxyadenylic-deoxythymidylic acid) damage by radiolytically activated neocarzinostatin.

The anaerobic reaction of poly(deoxyadenylic-deoxythymidylic acid) with neocarzinostatin activated by the carboxyl radical CO2-, an electron donor generated from gamma-ray radiolysis of nitrous oxide saturated formate buffer, has been characterized. DNA damage includes base release and strand breaks. Few strand breaks are formed prior to alkaline treatment; they bear 3'-phosphoryl termini. In contrast, most (66%) of the base release occurs spontaneously. DNA damage is highly (95%) specific for thymidine sites. Neither DNA-drug covalent adduct nor nucleoside 5'-aldehyde, which are major products in the DNA-nicking reaction initiated by mercaptans and oxygen, is formed in this reaction. Data are presented to show that the CO2(-)-activated neocarzinostatin intermediate is a short-lived free radical able to abstract hydrogen atoms from the C-1' and C-5' positions of deoxyribose. Attack occurs mostly (68%) at the C-1' position, producing a lesion whose properties are consistent with those of (oxidized) apyrimidinic sites.

Gamma-radiolysis study of the reductive activation of neocarzinostatin by the carboxyl radical.

The activation of the antitumor protein antibiotic neocarzinostatin (NCS) by the carboxyl radical CO-2, a one-electron donor obtained selectively from gamma-ray irradiation of nitrous oxide-saturated formate buffer, has been investigated in the presence and in the absence of DNA at pH 4.7 and pH 7.0. The reaction of NCS with CO-2 in the absence of DNA is followed by a marked red shift (420----441 nm) and a pronounced increase (X 8.8) of the fluorescence emission corresponding to the naphthalene moiety of the NCS chromophore. The light absorption spectrum shows in parallel a hypochromic change with considerable fine structure throughout the 250-400 nm wavelength range. When DNA is present, the fluorescence intensity at completion of the reaction is slightly reduced (by 5 to 15 per cent) and the maximum emission wavelength shifted to 436-438 nm. However, the bulk rate of reaction is not altered by DNA and is independent of the pH, of the temperature and of the concentration of NCS. The NCS concentration-independence of the reaction rate is consistent with a high intrinsic rate (k greater than 10(8)M-1 . s-1) for the reaction of CO-2 with the NCS chromophore. Complete reduction of the NCS chromophore involves a total of three electron-equivalents. The final product does not react with oxygen, shows no odd electron spin, and is unable to induce DNA strand scission. Its molecular state, however, is fundamentally different when gamma-ray irradiations are performed with DNA. This bears evidence of short-lived one electron or two-electrons reduced intermediates decaying via non-identical routes depending on the presence of the acceptor DNA. Actually, dose-related strand breaks appear in DNA exposed to the action of NCS and CO-2. Some NCS chromophore-DNA covalent adducts are also found. DNA strand breakage by CO-2-activated NCS is correlated with thymine release and is inhibited by a redox-stable intercalating agent. The DNA-nicking process thus bears resemblance to that reported by other authors using mercaptans to initiate reductive activation of the NCS chromophore. However, some spectral differences are observed between the CO-2-reacted and the thiol-treated chromophores. Moreover, thymine release and strand scission in DNA incubated with CO-2 and NCS proceed under anaerobic conditions. It is proposed that the strict oxygen requirement for DNA damage by NCS in the presence of mercaptans is due, at least partly, to competition between oxygen and thiols for reaction with the same primary deoxyribose radical resulting from DNA attack by the reductively activated NCS chromophore.