Yasuki Kato

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)

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.

Effect of Iontophoretic Patterns on in Vivo Antidiuretic Response to Desmopressin Acetate Administered Transdermally

The effects of concentration, amperage and duration on the antidiuretic response induced by iontophoretic delivery of desmopressin acetate (DDAVP) were examined using a diabetes insipidus model in rats. A higher current density brought about a larger and longer antidiuretic response. Prolonged iontophoretic duration caused an overdose. Repeated short iontophoretic treatments with lower current density maintained a constant response with a short lag time and a rapid disappearance of pharmacological response immediately after cessation of the final treatment. This type of iontophoresis substantially reduced the inter-subject variability of response as compared to the response using an intranasal route of administration.

Vaccination with OVA-bound nanoparticles encapsulating IL-7 inhibits the growth of OVA-expressing E.G7 tumor cells in vivo.

Immunotherapy has gained special attention due to its specific effects on tumor cells and systemic action to block metastasis. We recently demonstrated that ovalbumin (OVA) conjugated to the surface of nanoparticles (NPs) (OVA‑NPs) can manipulate humoral immune responses. In the present study, we aimed to ascertain whether vaccination with OVA-NPs entrapping IL-7 (OVA-NPs-IL-7) are able to induce antitumor immune responses in vivo. Pretreatment with a subcutaneous inoculation of OVA-NPs delayed the growth of thymic lymphoma cells expressing a model tumor antigen OVA (E.G7-OVA), and OVA-NPs-IL-7 substantially blocked the growth of E.G7-OVA tumor cells, although NPs-IL-7 alone had a meager effect, as assessed by the mean tumor size and the percentage of tumor-free mice. However, pretreatment with OVA-NPs-IL-7 failed to reduce the growth of parental thymic tumor cells, suggesting that the antitumor effect was antigen-specific. A tetramer assay revealed that vaccination with OVA-NPs-IL-7 tended to enhance the proportion of cytotoxic T cells (CTLs) specific for OVA. When the tumor-free mice inoculated with OVA-NPs-IL-7 plus EG.7 cells were rechallenged with E.G7-OVA cells, they demonstrated reduced growth compared with that in the control mice. Thus, a single subcutaneous injection of OVA-NPs-IL-7 into mice induced tumor-specific and also memory-like immune responses, resulting in regression of tumor cells. Antigens on NPs entrapping IL-7 would be a promising carrier to develop and enhance immune responses, including humoral and cellular immunity as well as a method of drug delivery to a specific target of interest.

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

Effect of drug lipophilicity and polar surface area on iontophoretic transdermal absorption.

Iontophoretic transdermal absorption of a series of β-biockers (atenolol, pindolol, metoprolol, acebutolol, oxprenolol, propranolol) was investigated by measuring the drug concentration in the skin. The objective of this study was to determine the effects of drug properties on the enhancement of drug concentration in the skin by iontophoretic delivery. Anodal iontophoresis of β-blocker was carried out by applying a constant direct current (0.5 mA/0.8 cm2, 30 min) to the drug solution (1 mg/mL), placed on the abdominal skin of anesthetized rat, and the drug concentration in the skin was measured by HPLC analysis. The enhancement effect of iontophoresis, represented as a ratio of drug concentration in the skin after iontophoresis to that after passive diffusion (without current), was exponentially attenuated with an increase in drug lipophilicity (the logarithm of 1-octanol/water partition coefficient at pH 7.4). On the other hand, when plotted against polar surface area (PSA) of drug molecule, the iontophoretic enhancement was linearly increased with increasing the PSA. The latter result implied that the transdermal absorption of drug having a strong interaction with water molecule was raised by iontophoresis. As a result, this study demonstrated that the PSA might be a useful parameter for clarifying the mechanism of iontophoretic enhancement and for optimizing the molecular structure of drug candidate to the iontophoretic delivery.