Kazuya Okamoto

Doxorubicin-loaded poly(ethylene glycol)-poly(β-benzyl-L-aspartate) copolymer micelles : Their pharmaceutical characteristics and biological significance

Doxorubicin (DOX) was physically loaded into micelles prepared from poly(ethylene glycol)-poly(beta-benzyl-L-aspartate) block copolymer (PEG-PBLA) by an o/w emulsion method with a substantial drug loading level (15 to 20 w/w%). DOX-loaded micelles were narrowly distributed in size with diameters of approximately 50-70 nm. Dimer derivatives of DOX as well as DOX itself were revealed to be entrapped in the micelle, the former seems to improve micelle stability due to its low water solubility and possible interaction with benzyl residues of PBLA segments through pi-pi stacking. Release of DOX compounds from the micelles proceeded in two stages: an initial rapid release was followed by a stage of slow and long-lasting release of DOX. Acceleration of DOX release can be obtained by lowering the surrounding pH from 7.4 to 5.0, suggesting a pH-sensitive release of DOX from the micelles. A remarkable improvement in blood circulation of DOX was achieved by use of PEG-PBLA micelle as a carrier presumably due to the reduced reticuloendothelial system uptake of the micelles through a steric stabilization mechanism. Finally, DOX loaded in the micelle showed a considerably higher antitumor activity compared to free DOX against mouse C26 tumor by i.v. injection, indicating a promising feature for PEG-PBLA micelle as a long-circulating carrier system useful in modulated drug delivery.

Development of the polymer micelle carrier system for doxorubicin

We show the result of pre-clinical study of NK911, a polymeric micelle carrier system for doxorubicin (DOX). The NK911 micelle carrier consists of polyethyleneglycol and conjugated doxorubicin-polyaspartic acid. It has high hydrophobic inner core, and therefore can entrap the sufficient amount of DOX. NK911 has a small particle size of about 40 nm in diameter that accumulates in tumor tissue by EPR effect showing much stronger activity than the free DOX. We plan to perform a clinical trial at National Cancer Center Hospital, Japan from 2001.

composition-dependent in vivo antitumor activity of adriamycin-conjugated polymeric micelle against murine colon adenocarcinoma 26

AbstractMicelle-forming block copolymer–drug conjugates, Adriamycin-conjugated polyethylene glycol–poly(aspartic acid) block copolymers (PEG-P[Asp(ADR)]), were synthesized in eight compositions with varying chain lengths of the segments constituting the block co-polymer. The antitumor activity of this micelle-forming polymeric anticancer drug against murine colon adenocarcinoma 26 (C 26) was evaluated by injection into the tail vein. The in vivo activity of the micelle-forming polymeric anticancer drug was revealed to be strongly dependent on the composition, while the in vitro cytotoxic activity was found to be in the same range regard-less of the composition. One composition of PEG–P[Asp(ADR)] was observed to significantly suppress tumor growth and to prolong survival of the treated mice in a wide dose range. These results indicated that selective delivery of the micelle-forming polymeric anti-cancer drug to the tumor was achieved by the appropriate composition of the block copolymer–drug conjugates.

NK314 potentiates antitumor activity with adult T-cell leukemia-lymphoma cells by inhibition of dual targets on topoisomerase IIα and DNA-dependent protein kinase

Adult T-cell leukemia-lymphoma (ATL) is an aggressive disease, incurable by standard chemotherapy. NK314, a new anticancer agent possessing inhibitory activity specific for topoisomerase IIα (Top2α), inhibited the growth of various ATL cell lines (50% inhibitory concentration: 23-70nM) with more potent activity than that of etoposide. In addition to the induction of DNA double-strand breaks by inhibition of Top2α, NK314 induced degradation of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), resulting in impaired DNA double-strand break repair. The contribution of DNA-PK to inhibition of cell growth was affirmed by the following results: NK314 inhibited cell growth of M059J (a DNA-PKcs-deficient cell line) and M059K (a cell line with DNA-PKcs present) with the same potency, whereas etoposide exhibited weak inhibition of cell growth with M059K cells. A DNA-PK specific inhibitor, NU7026, enhanced inhibitory activity of etoposide on M059K as well as on ATL cells. These results suggest that NK314 is a dual inhibitor of Top2α and DNA-PK. Because ATL cells express a high amount of DNA-PKcs, NK314 as a dual molecular targeting anticancer agent is a potential therapeutic tool for treatment of ATL.

Roles of adriamycin and adriamycin dimer in antitumor activity of the polymeric micelle carrier system

Abstract Adriamycin (ADR) dimer was prepared and its antitumor activity was evaluated with mouse colon adenocarcinoma 26 (C 26). As compared with original ADR, the dimer did not show significant antitumor activity, either in vitro or in vivo. Furthermore, polymeric micelles containing varied ratios of the dimer to the original ADR were prepared. Polymeric micelles with a higher dimer/ADR ratio (9.7) showed significant antitumor activity, but the effective dose shifted higher. Effective doses were found to largely depend on the concentration of the original ADR, rather than that of the dimer at the tumor sites. Therefore, it was presumed that the original ADR played a major role in antitumor activity, and the dimer played a supplementary role to contribute selective delivery of ADR to the tumor sites.

Antimyeloma activity of NK012, a micelle‐forming macromolecular prodrug of SN‐38, in an orthotopic model

NK012 is a micelle‐forming macromolecular prodrug of 7‐ethyl‐10‐hydroxy camptothecin (SN‐38), an active metabolite of irinotecan. It is accumulated and retained in tumor tissues and gradually releases SN‐38 in an enzyme‐independent manner. NK012 was previously demonstrated to have stronger antitumor activity than irinotecan in a broad range of human solid‐tumor xenograft models. In our study, we used an orthotopic multiple myeloma (MM) model created by injecting CD138‐positive U266B1, a myeloma cell line that produces human IgE lambda light chain (monoclonal protein, M protein), into immunodeficient NOD/Shi‐scid, IL‐2Rγcnull mice. This model shows typical bone marrow infiltration by the human myeloma cells. We evaluated the antimyeloma activity of intravenously administered NK012 in this model and showed that it suppressed the M protein concentration in the plasma and proliferation of myeloma cells in the bone marrow in a dose‐dependent manner. NK012 suppressed the progression of hind‐leg paralysis and prolonged the survival time of the mice compared to the untreated control group. In combination with bortezomib (BTZ), NK012 increased the median survival time compared to that with BTZ alone. In conclusion, these results suggest that NK012 is a potential candidate for use—alone and in combination—in the treatment of MM in humans.

Abstract 4435: Anti-multiple myeloma activity of NK012, a micelle-forming macromolecular prodrug of SN-38, in an orthotopic model

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL NK012 is a micelle-forming macromolecular prodrug of SN-38, an active metabolite of irinotecan (CPT-11). It accumulates in tumor tissue and gradually releases SN-38 in an enzyme-independent manner. We previously reported that NK012 showed stronger antitumor activity in a broad range of human solid tumor xenograft models than CPT-11 (2006 AACR Annual Meeting, #3062). Currently, phase II clinical trials of NK012 in patients with triple-negative breast cancer, small cell lung cancer, and colorectal cancer are ongoing in the United States and Japan. In this study, we evaluated the antitumor activity of NK012 in an orthotopic multiple myeloma model that shows typical infiltration of human myeloma cells into murine bone marrow tissue. A suspension of 2 × 106 cells of CD138 positive U266 human myeloma cells was injected into immunodeficient NOG (NOD/Shi-scid, IL-2Rγnull) mice via tail vein. The transplanted mice produced plasma M protein (human IgE) and developed lytic bone lesions. In addition, hind-leg paralysis caused by myeloma progression in spinal cord was observed. These symptoms were similar to complications observed in the patients with multiple myeloma. The intravenous administration of NK012 at 3.75, 7.5, and 15 mg/kg/day by q4d × 3 suppressed M protein concentration in plasma and proliferation of myeloma cells in bone marrow in a dose-dependent manner. Also observed were decrease in the area of osteolytic lesions and recovery of decreased total bone mineral density in the tibias. To examine the efficacy of NK012 in survival time, the myeloma-transplanted mice were treated with 9.4 mg/kg/day (28.2 mg/m2/day), q7d × 6, equivalent to the clinical recommended dose of 28 mg/m2/day as a single dose. NK012 significantly prolonged survival time of the mice compared with control group (78.5 days vs. 40 days, P<0.001). We next examined the combination effect of NK012 with bortezomib, which is commonly used for multiple myeloma treatment. The combined treatment of NK012 (9.4 mg/kg/day, q7d × 3) with bortezomib (0.5 mg/kg/day, 2 times/week × 2, i.v.) exhibited increase in median survival time over bortezomib alone (83.5 days vs. 68 days). In conclusion, these results suggested NK012 is a promising candidate for human multiple myeloma treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4435. doi:10.1158/1538-7445.AM2011-4435

Abstract 5513: Synergistic antitumor activity of NK012, a micelle-forming macromolecular prodrug of SN-38, combined with platinum compounds in human cancer xenograft models

Irinotecan (CPT-11) is clinically used to treat various tumors, but low enzymatic conversion to its active metabolite, SN-38, limits its therapeutic effect. NK012, a micelle-forming macromolecular prodrug of SN-38, accumulates in tumor tissue and gradually releases SN-38 in enzyme-independent manner. NK012 showed much stronger growth inhibitory effects than CPT-11 at maximum tolerated dose (MTD) against all tested xenografted human tumors. Furthermore, NK012 showed much stronger antitumor activities than other cytotoxic agents clinically used for cancer treatment (taxanes, doxorubicin, carboplatin etc.). Here, we report combination antitumor effects of NK012 with platinum compounds in human breast cancer, non-small cell lung cancer, colorectal cancer and hepatocellular cancer xenografts in nude mice. The dose of NK012 was set at 1.9-3.8 mg/kg/day to be sub-optimum for complete regression depending on the cell line. NK012 and carboplatin were administered by q7dx3. Although both of NK012 and carboplatin were ineffective as single agent (minimum T/C values >50%), their combination was effective (minimum T/C value = 8%) to cause tumor regression in MC-05-JCK breast cancer. In MC-05-JCK breast cancer, the TGDs (Tumor Growth Delay; tumor tripling time over control group delayed by treatment / tumor tripling time of control group × 100) by NK012 1.9 mg/kg/day, carboplatin 50 mg/kg/day (67% of MTD), and their combination were 41%, 29%, and 286%, respectively. In NCI-H460 non-small cell lung cancer, the TGDs by NK012 3.8 mg/kg/day, carboplatin 75 mg/kg/day (MTD), and their combination were 90%, 71%, and 403%, respectively. In Co-3 colorectal cancer, the TGDs by NK012 3.8 mg/kg/day, carboplatin 75 mg/kg/day, and their combination were 358%, 97%, and >489%, respectively. Thus, combination of NK012 with carboplatin showed remarkably synergistic effects, and it didn9t accompany toxicity exacerbation. Similarly, combination of NK012 with cisplatin exhibited remarkable synergistic effects in Li-07-JCK hepatocellular cancer. The TGDs by NK012 3.8 mg/kg, one shot, cisplatin 10 mg/kg, one shot, and their combination were 58%, 136%, and 455%, respectively. These results indicate that NK012 and platinum compounds combination regimen would be effective for treatment of patients with solid tumors including breast, non-small cell lung, colorectal and hepatocellular carcinomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5513.

Abstract 5522: Comparison of pharmacokinetics/pharmacodynamics of NK012, a micelle-forming prodrug of SN-38, and CPT-11 in human breast cancer xenograft model

Introduction: NK012 is a novel micelle-forming macromolecular prodrug of SN-38, an active metabolite of irinotecan (CPT-11), which is gradually released from the molecule in an enzyme-independent manner at physiological pH. We have demonstrated that antitumor activity of NK012 at 3.8 mg/kg/day by q4d×3 (1/8 MTD) and above was superior to that of CPT-11 at its MTD (67 mg/kg by q4d×3) against human breast cancer MC-05-JCK, and a phase II clinical trial of NK012 is ongoing against triple-negative breast cancer in the US. In the present study, we compared pharmacokinetics and pharmacodynamics of NK012 with those of CPT-11 in human breast cancer MC-05-JCK-bearing nude mice. Methods: NK012 (3.8 and 30 mg/kg) and CPT-11 (67 mg/kg) were given intravenously to nude mice bearing MC-05-JCK, and microdialysis probe was placed in the tumor tissue to determine free SN-38 concentration in tumor extracellular fluid (ECF). The drug concentrations in the plasma and tumor tissue were determined by HPLC system. Intratumoral distribution of NK012 was also investigated by observing auto-fluorescence of the drug with a fluorescence microscope. Cytotoxic effects of the drugs were examined immunohistochemically using anti-phosph γH2AX and anti-phospho histone H3 antibodies to detect cleaved DNA and M-phase cells, respectively. Results: After a single NK012 administration, polymer-bound SN-38 and polymer-unbound SN-38 (released SN-38) showed remarkably prolonged blood circulation. On the other hand, CPT-11 and its active metabolite SN-38 were promptly removed from the circulation. NK012 also retained in the tumor tissue for a long period as compared with CPT-11. Microdialysate experiment revealed that the free SN-38 from NK012 in the tumor ECF was eliminated more slowly than that converted from CPT-11. NK012-originated fluorescence in the tumor tissue was mainly observed in the stroma, necrotic area and perivascular region. The phosphorylation of γH2AX due to DNA breakage and consequent reduction in M-phase cells were observed for a longer period in NK012-treated tumor tissue than that treated with CPT-11. HE staining of MC-05-JCK revealed that apoptotic tumor cells appeared 3 days after administration of both NK012 and CPT-11 injection, but fibrosis in the tumor stroma was only observed with NK012 treatment (day 10). These histopathological changes caused by the drugs were associated with the elimination rate of SN-38 in the tumor tissue. Conclusion: The superior antitumor efficacy of NK012 to CPT-11 was associated with the sustained release of free SN-38 from the NK012 accumulated in tumor tissue as well as the prolonged circulation of NK012 in the bloodstream. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5522.