Efficacy Vitro and In Vivo models

The efficacy and reduced nonspecific toxicity of HPMA copolymer bound doxorubicin for the treatment of ovarian cancer has been demonstrated in numerous preclinical in vitro and in vivo investigations. In the OVCAR-3 cell line, doxorubicin bound to the HPMA copolymer demonstrated a ten-fold increase in the drug concentration required compared to free doxorubicin in order to obtain equivalency in cell growth inhibition in vitro. [32] The reduced specific toxicity of HPMA copolymer doxorubicin was consistent with the slower pinocytotic uptake mechanism required by higher molecular weight drugs and predicted a reduced nonspecific toxicity which has been subsequently documented in vivo. [33] A greater reduction in thymidine incorporation over mitochondrial respiration in free and HPMA copolymer bound doxorubicin was consistent with a primary activity on cellular DNA in these in vitro studies. [32] HPMA copolymer bound doxorubicin also demonstrated a 15-fold longer intravascular half-life and 100-fold lower peak initial cardiac level compared to free drug, resulting in a notable 15-fold reduction in nonspecfic toxicities during in vivo studies. [14] In mouse models of ovarian carcinoma, HPMA copolymer-doxorubicin conjugate at 2.2 mg/kg of free doxorubicin equivalent caused no toxic deaths and an 8% maximal weight loss which was regained completely within three weeks. [33] This dosage of HPMA copolymer bound doxorubicin (2.2 mg/kg of free doxorubicin equivalent)

provided tumor response equivalency to a dose of 1 mg/kg of free doxorubicin in this nude mouse model. Free doxorubicin administered at doses over 1.5 mg/kg was uniformly fatal in the nude mouse model. To summarize, HPMA copolymer-doxorubicin demonstrates efficacy and reduced nonspecific toxicity in experimental models of ovarian carcinoma and other tumor models. [33, 34] The efficacy and safety of HPMA copolymer bound doxorubicin in the nude mouse model is demonstrated in Figure 2.

Figure 2. Figure 2. Panel A. Inhibition of OVCAR-3 tumors heterotransplanted in nude mice by doxorubicin (1 mg/kg; O) and HPMA copolymer-doxorubicin or P-A (30 mg/kg, 2.2mg/kg doxorubicin equivalent; •) compared with controls (A). Tumor volumes from day 10 forward were significantly less than controls (all P<0.045). There was no significant difference between P-A (30 mg/kg, 2.2 mg/kg doxorubicin equivalent) and doxorubicin (1 mg/kg). Although tumor growth was inhibited, there were no cures. Bars, SE. Panel B. Weight changes in mice receiving free doxorubicin (1 mg/kg; A) and P-A (30 mg/kg; 2.2 mg/kg doxorubicin equivalent; ▲). Weight losses were less than 8% for both drugs. There were no statistically significant differences between the drug forms. Bars, SE. [33]

Figure 2. Figure 2. Panel A. Inhibition of OVCAR-3 tumors heterotransplanted in nude mice by doxorubicin (1 mg/kg; O) and HPMA copolymer-doxorubicin or P-A (30 mg/kg, 2.2mg/kg doxorubicin equivalent; •) compared with controls (A). Tumor volumes from day 10 forward were significantly less than controls (all P<0.045). There was no significant difference between P-A (30 mg/kg, 2.2 mg/kg doxorubicin equivalent) and doxorubicin (1 mg/kg). Although tumor growth was inhibited, there were no cures. Bars, SE. Panel B. Weight changes in mice receiving free doxorubicin (1 mg/kg; A) and P-A (30 mg/kg; 2.2 mg/kg doxorubicin equivalent; ▲). Weight losses were less than 8% for both drugs. There were no statistically significant differences between the drug forms. Bars, SE. [33]

Presently, HPMA copolymer bound doxorubicin is undergoing very promising Phase I and II clinical trials in the U.K. for the treatment of solid tumors. [35] Quantitative signature gene expression studies between free and HPMA copolymer bound drugs are underway to document the enhancement derived from HPMA copolymer subcellular localization, and to reveal specific modes of action on a molecular level which may be further amplified or augmented by design features of the HPMA copolymer. One such example of the specific advantages of HPMA copolymer bound doxorubicin is that free doxorubicin up-regulates genes encoding the ATP driven efflux pumps (MDR1, MRP) while HPMA copolymer bound doxorubicin overcomes these existing pumps and downregulates the MRP gene. Furthermore, free doxorubicin activates the expression of genes responsible for detoxification and DNA repair which are suppressed or activated to a lesser degree by HPMA copolymer bound doxorubicin (glutathione-GST-pi, UDP transferase-BUDP, Topoisomerase II- alpha and beta, thymidine kinase isoform 1- TK-1, heat shock protein 70-HSP-70). Finally, both free and HPMA copolymer bound doxorubicin induce the p53 gene central death signal as well as the c-fos and c-jun pathways activating cell death. To complete cell death, bcl-2, the inhibitor of apoptosis, is also down-regulated by HPMA copolymer bound doxorubicin. [36] Figure 3 details the findings described above.

Figure 3. Typical images of gel electrophoresis of RT-PCR products form A2780 sensitive (left panel) and A2780/AD doxorubicin (DOX) resistant (right panel) human ovarian carcinoma cells. Identical numbers of cells, amounts of RT products and volumes of PCR products were used for each analysis. Primers and PCR regimens used for individual genes are listed in the reference [36]. RT-PCR products from cells indicated below were loaded and electrophoresed in 0.6-4.0% MetaPhor agarose gell: Lane 1, control cells; Lane 2, cells after 24 hr incubation with an IX IC50 dose of free DOX; Lane 3, cells after 48 hr incubation with a 1X IC50 dose of free DOX; Lane 4, cells after a 72 hr incubation with a 1 X 1C50 of free DOX; Lane 5, cells after a 24 hr incubation with 10 X IC5o dose of free DOX; Lane 6, cells after a

48 hr incubation with a 10 X ICSo dose free DOX; Lane 7, cells after a 72 hr incubation with a 10 X ICjo free DOX; Lane 8, cells after a 24 hr incubation with a 1 X IC50 dose of P(GFLG)-DOX; Lane 9, cells after a 48 hr incubation with a 1 X IC50 dose of P(GFLG)-DOX; Lane 10, cells after a 72 hr incubation with a 1 X IC50 dose of P(GFLG)-DOX;Lane 11, cells after a 24 hr incubation with a 10 X IC5o dose of P(GFLG)-ADR; Lane 12, cells after a 48 hr incubation with a 10 X IC5o dose of P(GFLG)-DOX; Lane 13, cells after a 72 hr incubation with a 10 X ICjo dose of P(GFLG)-DOX. P(GFLG) = HPMA copolymer with GFLG spacer/attachments sites. [36]

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