Tatsuyuki Hayashi

Improved anti-tumor activity of stabilized anthracycline polymeric micelle formulation, NC-6300

Anthracyclines have long been considered to be among the most active agents clinically available for the treatment of breast cancer despite their toxicity. To improve their pharmacological profiles, a new macromolecular prodrug, denoted NC‐6300, was synthesized. NC‐6300 comprises epirubicin covalently bound to polyethylene‐glycol polyaspartate block copolymer through an acid‐labile hydrazone bond. The conjugate forms a micellar structure spontaneously in aqueous media with a diameter of 60–70 nm. The block copolymers are partially substituted with hydrophobic benzyl groups to stabilize the micellar structure. The present study was designed to confirm that polymeric micelles incorporating epirubicin through an acid‐labile linker improve the therapeutic index and achieve a broad range of therapeutic doses. Pharmacokinetic studies in rats showed highly enhanced plasma retention of NC‐6300 compared with native epirubicin. The maximal tolerated doses in mice of NC‐6300 and native epirubicin were 25 and 9 mg/kg, respectively, when administered three times with a 4‐day interval between each dose. NC‐6300 at 15 and 20 mg/kg with the same administration schedule regressed a Hep3B human hepatic tumor with slight and transient bodyweight loss. Remarkably, NC‐6300 also inhibited growth of an MDA‐MB‐231 human breast tumor at the same dosage. In contrast, native epirubicin at 7 mg/kg administered three times with a 4‐day interval was only able to slow tumor growth. Tissue distribution studies of NC‐6300 showed efficient free epirubicin released in the tumor at 74% by area under the concentration‐time curve (AUC) evaluation, supporting the effectiveness of NC‐6300. In conclusion, NC‐6300 improved the potency of epirubicin, demonstrating the advantage of NC‐6300 attributable to the efficient drug release in the tumor. (Cancer Sci 2011; 102: 192–199)

NC-6300, an epirubicin-incorporating micelle, extends the antitumor effect and reduces the cardiotoxicity of epirubicin

Epirubicin is widely used to treat various human tumors. However, it is difficult to achieve a sufficient antitumor effect because of dosage limitation to prevent cardiotoxicity. We hypothesized that epirubicin‐incorporating micelle would reduce cardiotoxicity and improve the antitumor effect. NC‐6300 comprises epirubicin covalently bound to PEG polyaspartate block copolymer through an acid–labile hydrazone bond. The conjugate forms a micellar structure of 40–80 nm in diameter in an aqueous milieu. NC‐6300 (10, 15 mg/kg) and epirubicin (10 mg/kg) were given i.v. three times to mice bearing s.c. or liver xenograft of human hepatocellular carcinoma Hep3B cells. Cardiotoxicity was evaluated by echocardiography in C57BL/6 mice that were given NC‐6300 (10 mg/kg) or epirubicin (10 mg/kg) in nine doses over 12 weeks. NC‐6300 showed a significantly potent antitumor effect against Hep3B s.c. tumors compared with epirubicin. Moreover, NC‐6300 also produced a significantly longer survival rate than epirubicin against the liver orthotopic tumor of Hep3B. With respect to cardiotoxicity, epirubicin‐treated mice showed significant deteriorations in fractional shortening and ejection fraction. In contrast, cardiac functions of NC‐6300 treated mice were no less well maintained than in control mice. This study warrants a clinical evaluation of NC‐6300 in patients with hepatocellular carcinoma or other cancers.

Effect of combined treatment with the epirubicin-incorporating micelles (NC-6300) and 1,2-diaminocyclohexane platinum (II)-incorporating micelles (NC-4016) on a human gastric cancer model.

Anticancer agent‐incorporating polymeric micelles accumulate effectively in tumors via the enhanced permeability and retention effect to exert potent antitumor effects. However, combined use of such micelles has not been elucidated. We compared the effect of combining the epirubicin‐incorporating micelle NC‐6300 and 1,2‐diaminocyclohexane platinum (II) (oxaliplatin parent complex)‐incorporating micelle NC‐4016 (NCs) with that of epirubicin and oxaliplatin (E/O) in 44As3Luc cells using the combination index method. The in vivo antitumor activities of NCs and E/O were evaluated in mice bearing 44As3Luc xenografts. Pharmacokinetic analysis was performed by high‐performance liquid chromatography and mass spectrometry. Cardiotoxicity of NC‐6300 and epirubicin was assessed by echocardiography. Neurotoxicity of NC‐4016 and oxaliplatin was evaluated by examining the paw withdrawal response to noxious mechanical stimuli. NCs showed a highly synergistic activity equivalent to E/O. In vivo, NCs exhibited higher antitumor activity in the subcutaneous tumor model and longer overall survival in the orthotopic tumor model than E/O (p < 0.001, p = 0.015, respectively). The intratumor concentrations of epirubicin and platinum were significantly higher following NCs than following E/O administration. Moreover, the micelles showed lower cardiotoxicity and neurotoxicity than the corresponding conventional drugs. The combined use of the micelles was associated with remarkable efficacy and favorable toxicities in the human gastric cancer model, and warrants the conduct of clinical trials.

Prolonged circulation and in vivo efficacy of recombinant human granulocyte colony-stimulating factor encapsulated in polymeric micelles

To improve the pharmacokinetics of granulocyte colony-stimulating factor (G-CSF) and decrease dosing frequency, polyethylene glycol polyglutamate block copolymers were used as delivery carriers. The block copolymers are partially substituted with hydrophobic octyl or benzyl groups to form a micellar structure in aqueous media and encapsulate the protein. G-CSF is encapsulated in the polymeric micelles with a diameter of 60-70nm. The present study was designed to evaluate the plasma pharmacokinetics, G-CSF release and in vivo efficacy of G-CSF-encapsulating micelles. Pharmacokinetic studies in rats showed highly enhanced plasma retention of the micelles compared with native G-CSF. The AUC (area under the curve) of the octyl-based polymer formulation showed a 5-fold increase, compared with native G-CSF. Size-exclusion chromatography of the blood from rats injected with the micelles demonstrated the release of G-CSF from the micelles in the blood circulation. The pharmacokinetic behavior supports the in vivo studies showing that the micelles display a comparable efficacy to PEGylated G-CSF. Simultaneous pharmacokinetic analysis of released and encapsulated G-CSF plasma levels showed that the G-CSF release occurs with the first-order kinetics and the half-life is 4.8h. In conclusion, G-CSF is endowed by the polymeric micelles with prolonged half-life and increased efficacy without any chemical modification.

Combination therapy with dendritic cell vaccine and IL-2 encapsulating polymeric micelles enhances intra-tumoral accumulation of antigen-specific CTLs.

Dendritic cell (DC) vaccine is a promising immunotherapy for cancer due to its ability to induce antigen-specific CTLs efficiently. However, a number of clinical studies have implied insufficient therapeutic benefits with the use of MHC class 1 restricted peptide-pulsed DC vaccine. To enhance the clinical efficacy, we examined combination therapy of DC vaccine pulsed with OVA peptide and intravenous low dose unmodified IL-2 (IL-2 solution) administration against EG7 tumor-bearing mice. Unexpectedly, no additional effects of IL-2 solution were observed on CTL induction and the therapeutic effects of DC vaccine, possibly because of the short half-life of IL-2 in plasma. Therefore, we generated IL-2-encapsulating polymeric micelles (IL-2 micelle), which showed prolonged IL-2 retention in the circulation after intravenous administration compared with IL-2 solution. When mice were treated with OVA peptide-pulsed DCs in combination with IL-2 micelle, OVA-specific CTLs were efficiently induced in the spleen in comparison with DC vaccine combined with IL-2 solution or DC vaccine alone. In addition, combination therapy of DC vaccine and IL-2 micelle against EG7 tumor-bearing mice induced the efficient accumulation of antigen-specific CTLs into the tumor and marked anti-tumor effects. Thus, the administration of IL-2 micelle can significantly enhance DC vaccine efficacy against tumors.

NC-6301, a polymeric micelle rationally optimized for effective release of docetaxel, is potent but is less toxic than native docetaxel in vivo

Drug release rate is an important factor in determining efficacy and toxicity of nanoscale drug delivery systems. However, optimization of the release rate in polymeric micellar nanoscale drug delivery systems has not been fully investigated. In this study NC-6301, a poly(ethylene glycol)-poly(aspartate) block copolymer with docetaxel (DTX) covalently bound via ester link, was synthesized with various numbers of DTX molecules bound to the polymer backbone. The number of DTX molecules was determined up to 14 to achieve an optimal release rate, based upon the authors’ own pharmacokinetic model using known patient data. Efficacy and toxicity of the formulation was then tested in animals. When administered three times at 4-day intervals, the maximum tolerated doses of NC-6301 and native DTX were 50 and 10 mg/kg, respectively, in nude mice. Tissue distribution studies of NC-6301 in mice at 50 mg/kg revealed prolonged release of free DTX in the tumor for at least 120 hours, thus supporting its effectiveness. Furthermore, in cynomolgus monkeys, NC-6301 at 6 mg/kg three times at 2-week intervals showed marginal toxicity, whereas native DTX, at 3 mg/kg with the same schedule, induced significant decrease of food consumption and neutrophil count. NC-6301 at 50 mg/kg in mice also regressed a xenografted tumor of MDA-MB-231 human breast cancer. Native DTX, on the other hand, produced only transient and slight regression of the same tumor xenograft. NC-6301 also significantly inhibited growth of OCUM-2MLN human scirrhous gastric carcinoma in an orthotopic mouse model. Total weight of metastatic lymph nodes was also reduced. In conclusion, NC-6301 with an optimized release rate improved the potency of DTX while reducing its toxicity.

Abstract 2648: Enhanced antitumor effect of anti tissue factor (TF) antibody-conjugated epirubicin-incorporating polymeric micelles in human cancer xenografts with high TF expression

Background: Anticancer agent-incorporating polymeric micelles, categorized as passive targeting agents, are expected to accumulate in tumors based on the enhanced permeability and retention effect. Recently, we made epirubicin-incorporating micelles containing acid-sensitive bonds (NC-6300), and reported the enhanced antitumor activity and reduced cardiotoxicity in preclinical studies; a Phase I study of NC-6300 is also under way in Japan. In order to further enhance the accumulation of NC-6300 in tumors using the active targeting strategy, we tried to attach a tumor-specific monoclonal antibody (mAb) to the terminal of polyethylene glycol comprising NC-6300. As the targeting molecule, we selected transmembrane receptor tissue factor (TF), namely, the primary initiator of coagulation. TF, which is known to play an important role in not only blood coagulation, but also in tumor growth and metastasis, is frequently overexpressed in various types of tumor. We have established a mAb against human TF, and succeeded in developing anti TF mAb-conjugated NC-6300 (anti TF-NC-6300). Here, we report the results of in vitro and in vivo studies of this immunoconjugate. Methods: Anti TF-NC-6300 was prepared based on our Antibody/Drug-Conjugated Micelle (ADCM) technology (Japan Patent No.4538666) with slight modification. In the in vitro assay, real-time cell analysis was performed using the xCELLigence system to determine the effects of anti TF-NC-6300 and NC-6300 on pancreatic tumor cell proliferation. Next, image analysis and quantification of the intracellular epirubicin concentration were performed using Array Scan VTI. For evaluating the in vivo antitumor effects, anti TF-NC-6300 (10 mg/kg), NC-6300 (10 mg/kg) and epirubicin (10 mg/kg) were administered intravenously once a week for 3 weeks to mice bearing human pancreatic cancer xenografts or human gastric cancer xenografts with high TF expression (BxPC3 or 44As3). Results: In the real-time cell proliferation assay, anti TF-NC-6300 exerted a greater degree of inhibition of cell growth than NC-6300 in BxPC3. On the other hand, there was no difference in effect between these two drugs in SUIT2, a human pancreatic cancer with low TF expression. In the imaging cytometric analysis, anti TF-NC-6300 was internalized sooner than NC-6300, resulting in higher accumulation of epirubicin in the cytoplasm and nuclei of the BxPC3 cells. On the other hand, no such difference was observed in the case of SUIT2. In the in vivo antitumor assay, as compared to both epirubicin and NC-6300, anti TF-NC-6300 showed significant antitumor activity in xenograft models of BxPC3 and 44As3. Conclusion: These data warrant a clinical evaluation of anti TF mAb-conjugated epirubicin-incorporating polymeric micelles in patients with TF-positive cancer subtypes. Citation Format: Yoshiyuki Yamamoto, Ichinosuke Hyodo, Yoshikatsu Koga, Ryo Tsumura, Ryuta Sato, Toshihumi Obonai, Hirobumi Fuchigami, Masahisa Kudo, Masahiro Yasunaga, Mitsunori Harada, Tatsuyuki Hayashi, Yasuki Kato, Yasuhiro Matsumura. Enhanced antitumor effect of anti tissue factor (TF) antibody-conjugated epirubicin-incorporating polymeric micelles in human cancer xenografts with high TF expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2648. doi:10.1158/1538-7445.AM2014-2648

Abstract 4525: Combined antitumor activity of the epirubicin-incorporating polymeric micelle NC-6300 and the DACHPt-incorporating polymeric micelle NC-4016 in mice bearing human gastric cancer xenografts.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Several anticancer agent (ACA)-incorporating polymeric micelles are known to accumulate effectively in tumors and exert sufficient antitumor effects via the enhanced permeability and retention (EPR) effect. These agents are now under clinical evaluation. However, there have been few reports of the antitumor effects of combinations of these agents. We evaluated the effects of a combination of the epirubicin-incorporating micelle NC-6300 and the DACHPt (oxaliplatin parent complex)-incorporating micelle NC-4016 on human gastric cancer 44As3Luc xenografts. Methods: First, we evaluated the cytotoxic effects of a combination of NC-6300+NC-4016 or epirubicin+oxaliplatin in vitro in 44As3Luc cells by using the combination index method. Next, to evaluate the antitumor effects, NC-6300 (8 mg/kg) and NC-4016 (4 mg/kg) or epirubicin (8 mg/kg) and oxaliplatin (4 mg/kg) were intravenously administered once a week for 3 weeks to mice bearing 44As3Luc xenografts implanted subcutaneously or orthotopically. Drug distribution was analyzed by high-performance liquid chromatography or inductively coupled plasma mass spectrometry. To evaluate the typical cumulative toxicity of each drug, we examined cardiotoxicity and neurotoxicity in the mice by using echocardiography and by measuring the latency of paw withdrawal in response to a noxious mechanical stimulus following long-term administration of each drug. Results: In our in vitro assay the combination of NC-6300+NC-4016 had more synergistic activity than that of epirubicin+oxaliplatin. Compared with the conventional anticancer drug combination, the combination of micelles had significantly greater antitumor activity against 44As3Luc cells in the subcutaneous tumor model and prolonged overall survival in the orthotopic tumor model. A high drug concentration of each ACA was detected in the tumor tissue in the case of co-administration of the two micelle formulations. Both cardiotoxicity and neurotoxicity were significantly lower in the micelle treatment group than in the conventional group. Conclusion: These data warrant the clinical evaluation of combination therapy with ACA-incorporating micelles. Citation Format: Yoshiyuki Yamamoto, Ichinosuke Hyodo, Misato Takigahira, Yoshikatsu Koga, Masahiro Yasunaga, Mitsunori Harada, Tatsuyuki Hayashi, Yasuki Kato, Yasuhiro Matsumura. Combined antitumor activity of the epirubicin-incorporating polymeric micelle NC-6300 and the DACHPt-incorporating polymeric micelle NC-4016 in mice bearing human gastric cancer xenografts. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4525. doi:10.1158/1538-7445.AM2013-4525

Abstract LB-11: Reduced cardiotoxicity and improved antitumor effect of the epirubicin-incorporating micelle NC-6300 in a hepatocellular carcinoma model

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Epirubicin, an anthracycline antitumor drug, has been approved for the treatment of various human tumors. However, epirubicin has significant dose-limiting cardiotoxicity, and there are no effective therapies to prevent or reduce this cardiotoxicity. Therefore, we investigated whether the epirubicin-incorporating micelle NC-6300 was more effective than epirubicin alone in reducing cardiotoxicity and improving the antitumor effect. Method: NC-6300 comprises epirubicin covalently bound to the polyaspartate chain of the polyethylene glycol-polyaspartate block copolymer via an acid-labile hydrazone bond. The conjugate spontaneously forms a micellar structure in an aqueous milieu. The diameter of NC-6300 is 60-70 nm, and the micellar formulation is stable under physiological condition. For evaluating the antitumor effects, NC-6300 (10 or 15 mg/kg) and epirubicin (10 mg/kg) were intravenously administered once a week for 3 weeks to C57BL/6 mice bearing human hepatocellular carcinoma (HCC) Hep3B xenografts implanted subcutaneously or orthotopically (n = 6-10). Cardiotoxicity was evaluated by using echocardiography to the mice that were given NC-6300 (10 or 15 mg/kg) or epirubicin (10 mg/kg) on days 1, 8, and 15 every 4 weeks, to a total of 9 administrations for 12 weeks (n = 6 for each evaluation point). Results: Compared to epirubicin, NC-6300 showed significant antitumor activity against Hep3B. Moreover, the survival rate in the orthotopic tumor model of Hep3B was significantly higher with NC-6300 treatment than with epirubicin treatment. With respect to cardiotoxicity, epirubicin-treated mice showed significantly deteriorated fractional shortening (FS) and ejection fraction (EF) 21 days after the beginning of administration. In contrast, no mice treated with NC-6300 experienced cardiotoxicity in terms of EF or FS during the observation period. Conclusion: The findings of this study imply that NC-6300 has a strong antitumor effect against HCC. Furthermore, NC-6300 significantly reduces the cardiotoxicity of epirubicin and warrants a clinical evaluation in patients with advanced solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-11. doi:1538-7445.AM2012-LB-11