Efficacy of HPMA Copolymer Bound Mce6

We have previously demonstrated the efficacy of photodynamic therapy utilizing the hematoporphyrin derivative, Photofrin II on the human ovarian carcinoma cell line, OVCAR-3, xenografted in nude mice. [47] Microcirculatory damage caused by arteriolar constriction and venous thrombosis are the notable histopathologic markers of cellular damage attributed to photodynamic therapy. [48,49] Cell membrane and mitochondrial damage are the primary cellular mechanisms of cytotoxicity resulting from singlet oxygen formation. [50] In our Photofrin II study, the nonselective uptake of the photoactivatable hematoporphyrin derivative through simple diffusion resulted tumor ablation, but was also associated with significant toxicity and morbidity.

Mesochlorin eé monoethylene diamine (Mce6) is a photoactivatable chemical which exhibits pharmacologic and photophysical properties better suited to PDT. [51] In vitro studies using HPMA copolymer delivery of documented, as with doxorubicin, a reduced specific toxicity compared to free drug. [32] The need for higher HPMA copolymer bound Mce6 concentrations compared to free [Mce6] to achieve equivalent inhibition of cell growth after light administration is consistent with the different mechanisms of cellular entry of the two forms of Mce6, respectively: passive or simple diffusion for low molecular weight drugs in contrast to endocytosis for the higher molecular weight HPMA copolymer-drug conjugates. Increasing concentrations of HPMA copolymer-Mce6 with light (up to 100X free [Mce6] with light) demonstrated increased cytotoxicity in all assays consistent with a toxicity which was not restricted by cleavage from the HPMA copolymer tetrapeptide spacer for activity. Thus, photosensitizing agents in combination with known cytotoxic agents may be carried via the HPMA copolymer vehicle to reduce nonspecific toxicities and improve the therapeutic safety margins of both agents. HPMA copolymer delivery therefore allows: photosensitizer action which is independent of release from the HPMA copolymer carrier; a longer intravascular half life; higher accumulation in tumors; and a degree of targetability through the controlled administration of light. Figure 4 demonstrates the in vivo activities of free and HPMA copolymer delivered Mce6. In nude mice xenografted with OVCAR-3 a significant reduction of HPMA copolymer bound Mce6 toxicity compared to free was noted.

To summarize, HPMA copolymer-drug conjugates significantly broaden the therapeutic window, in vivo, of diverse anticancer agents possessing different cytotoxic pathways. The HPMA carrier system also allows one to design single agent and multiple drug combinations which maximize efficacy through capitalizing upon cellular processing requirements, control of light administration for targeting, and specific toxicities unique to each anticancer agent. Wider therapeutic indices for photosensitizers and many other potential drug combinations are expected to enhance response and cure rates without a parallel increase in nonspecific toxicity.

No human clinical and pharmacokinetics trials have been performed with this photosensitizer pending the results of the preclinical investigations proposed.

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Figure 4. Panel A. Percentage of tumor volume in nude mice treated with Mce6 and light (650 nm, 220 J/cm2): 1.25 mg/kg (- 0-), 2.5 mg/kg (- □-), 5 mg/kg (- +-), and lOmg/kg (- ■-) i.v., 2.5 mg/kg with no light (- ▲-) and controls (- A-). The lack of a significant response to 1.25 mg/kg Mce^ and light (- 0-) by human ovarian epithelial carcinoma is demonstrated. With Mce6 and light at 2.5 mg/kg (-□-), the human OVCAR-3 tumor was destroyed (all P< 0.01 compared with controls). This dose caused mortality (shock syndrome) to one of six mice (17%). Eighty percent of tumors responded to therapy, with regression of tumor volume. Continued growth in nonresponding tumors became evident by day 21, but mean tumor volumes remained significantly less than control tumors (all P< 0.01). Mce6 with light at 5 mg/kg(- +-) and 10 mg/kg (- ■-) caused mortality in five of six (83%) mice. A shock syndrome developed within 24 h after irradiation. Autopsy specimens revealed Mce6

Figure 4. Panel A. Percentage of tumor volume in nude mice treated with Mce6 and light (650 nm, 220 J/cm2): 1.25 mg/kg (- 0-), 2.5 mg/kg (- □-), 5 mg/kg (- +-), and lOmg/kg (- ■-) i.v., 2.5 mg/kg with no light (- ▲-) and controls (- A-). The lack of a significant response to 1.25 mg/kg Mce^ and light (- 0-) by human ovarian epithelial carcinoma is demonstrated. With Mce6 and light at 2.5 mg/kg (-□-), the human OVCAR-3 tumor was destroyed (all P< 0.01 compared with controls). This dose caused mortality (shock syndrome) to one of six mice (17%). Eighty percent of tumors responded to therapy, with regression of tumor volume. Continued growth in nonresponding tumors became evident by day 21, but mean tumor volumes remained significantly less than control tumors (all P< 0.01). Mce6 with light at 5 mg/kg(- +-) and 10 mg/kg (- ■-) caused mortality in five of six (83%) mice. A shock syndrome developed within 24 h after irradiation. Autopsy specimens revealed Mce6

aggregation in the liver and lungs without evidence of acute toxicity or hemorrhagic necrosis. The surviving mice (5 and 10 mg/kg) had complete ablation of treated tumors. Bars, SE. Panel B. Mortality plot for Mce« administered at 1.25 mg/kg (- ■-), 2.5 mg/kg (- 0-) ,5 mg/kg(-□-), and 10 mg/kg (- A-) i.v. and 2.5 mg/kg (- •-) given i.p. followed by light (650 nm, 220 J/cm2). Mortality (shock syndrome) was noted in doses of >2.5 mg/kg. Panel C. Inhibition of OVCAR-3 tumors heterotransplanted in nude mice treated with P-C and light (650 nm, 220 J/cm2) at 12.5 mg/kg (1.5 mg/kg Mce« equivalent; (- •-); 25 mg/kg (2.9 mg/kg Mcei equivalent, (- ■-); and 75 mg/kg (8.7 mg/kg Mce« equivalent, (- A-). Tumor volumes for 12.5 mg/kg P-C (1.5 mg/kg Mce6 equivalent) were not significantly different compared with controls (-□-). Tumor volumes for 25 mg/kg (2.9 mg/kg Mce$ equivalent) and 75 mg/kg (8.7 mg/kg Mce« equivalent) P-C were significantly less than controls (P < 0.003) but not significantly different from each other. Tumor recurrence became evident after day 20 for PC at 25 mg/kg (2.9 mg/kg Mce6 equivalent) and 75mg/kg (8.7 mg/kg Mce« equivalent). Two toxic deaths were noted with P-C at 25 mg/kg (2.9 mg/kg Mce« equivalent), but none was noted at 75 mg/kg (8.7 mg/kg Mce« equivalent). Bars, SE. [33]

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