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Background: Capecitabine and paclitaxel are established effective treatments, alone and combined with other cytotoxic and targeted agents, for metastatic breast cancer (MBC). Paclitaxel polyglumex (a macromolecular conjugate of paclitaxel bound to poly-L-glutamic acid) has potential advantages over conventional paclitaxel, including little alopecia, short infusion time with no premedication, enhanced tumor permeability/retention effect, and improved tolerability. We therefore examined tolerability and efficacy of paclitaxel polyglumex with capecitabine in patients with MBC., Patients and Methods: This was a single-stage phase 2 study, with interim analysis conducted with endpoints of tumor response, adverse events (toxicities), time to progression, and overall survival. The main eligibility criteria were: age >18 years, no prior MBC chemotherapy, Eastern Cooperative Oncology Group performance score <2, disease measurable by RECIST criteria, no HER2 overexpression or amplification, no brain metastases or peripheral sensory neuropathy. Treatment consisted of paclitaxel polyglumex (135 mg/m) by intravenous infusion on day 1+capecitabine (825 mg/m) orally twice daily on days 1 to 14, repeated on a 3-week cycle. Forty-one evaluable patients were required to test the null hypothesis that the complete and partial tumor response rate (CR+PR) was at the most 40% against the alternative of at least 60%. Paclitaxel polyglumex+capecitabine would be considered promising in this population if ≥21 responses were observed among first 41 evaluable patients., Results: Forty-eight patients were enrolled between April 2006 and April 2007; all patients were evaluable. The median number of treatment cycles administered was 6. Eighteen patients [38%; 95% confidence interval (CI), 24%-53%] had a confirmed tumor response (2 CR, 16 PR) by RECIST criteria. Fifteen (38%; 95% CI, 23%-53%) responses occurred in first 41 patients, falling short of prespecified goal of 21 responses. Median duration of tumor response was 13.2 months. Three of the responders were progression free at last follow-up with a median follow-up of 43 months. Median progression-free survival was 5.1 months (95% CI, 4.0-7.6 mo). Six-month progression-free survival was 42% (95% CI, 30%-58%). Median dose level administered was paclitaxel polyglumex (135 mg/m) and capecitabine (825 mg/m) for cycles 1 to 7. Most common severe (grade 3/4) toxicities (at least possibly related to study drug) were: leukopenia 9 (19%), neutropenia 8 (17%), neurosensory 4 (8%), skin reaction-hand/foot 4 (8%), and dyspnea 2 (4%). Forty-six percent (22/47) of patients experienced grade ≥3 toxicity and 8% (4/48) experienced grade ≥4 toxicity. No alopecia was reported., Conclusions: Although the trial failed to reach goal of 21 confirmed tumor responses among the first 41 evaluable patients, paclitaxel polyglumex and capecitabine is well tolerated and effective in MBC.

Increasing evidence reveals that traditional pharmacokinetics parameters based on plasma drug concentrations are insufficient to reliably demonstrate accurate pharmacological effects of drugs in target organs or cells in vivo. This underscores the increasing need to improve the types and qualities of cellular pharmacokinetic information for drug preclinical screening and clinical efficacy assessments. Here we report a whole cell-based method to assess drugs that disturb microtubule dynamics to better understand different formulation-mediated intracellular drug release profiles. As proof of concept for this approach, we compared the well-known taxane class of anti-microtubule drugs based on paclitaxel (PTX), including clinically familiar albumin nanoparticle-based Abraxane™, and a polymer nanoparticle-based degradable paclitaxel carrier, poly(L-glutamic acid)-paclitaxel conjugate (PGA-PTX, also known as CT-2103) versus control PTX. This in vitro cell-based evaluation of PTX efficacy includes determining the cellular kinetics of tubulin polymerization, relative populations of cells under G2 mitotic arrest, cell proliferation and total cell viability. For these taxane tubulin-binding compounds, the kinetics of cell microtubule stabilization directly correlate with G2 arrest and cell proliferation, reflecting the kinetics and amounts of intracellular PTX release. Each individual cell-based dose-response experiment correlates with published, key therapeutic parameters and taken together, provide a comprehensive understanding of drug intracellular pharmacokinetics at both cellular and molecular levels. This whole cell-based evaluating method is convenient, quantitative and cost-effective for evaluating new formulations designed to optimize cellular pharmacokinetics for drugs perturbing tubulin polymerization as well as assisting in explaining drug mechanisms of action at cellular levels.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that is closely related to dysregulation of the T helper type 1 and 2 (Th1)/Th2 balance. A previous study showed that high molecular mass poly-γ-glutamate (γ-PGA) isolated from Bacillus subtilis sp. Chungkookjang induces the production of IL-12 from dendritic cells (DCs). Here, we investigated the effect of γ-PGA on AD-like skin disease using an Nc/Nga mouse model. In vitro, γ-PGA activated DCs and induced IL-12 production in mice. In vivo, oral administration of γ-PGA markedly reduced the AD symptoms, similar to the response seen in the dexamethasone (Dex)-treated group. Treatment with γ-PGA also decreased the serum levels of IgG1, the skin levels of Th2 cytokines, the extent of skin inflammation, and the accumulation of mast cells. Furthermore, γ-PGA was effective against established AD, significantly decreasing serum IgE and Th2 cytokines in the inflamed tissue. Interestingly, the production of IL-17A in splenocytes was also suppressed by γ-PGA, indicating that it inhibits both Th2 and Th17 immune responses. Collectively, these results suggest that oral administration of γ-PGA could be a therapeutic strategy for treating AD via the modulation of Th2-biased immune responses in an Nc/Nga mouse model.

A novel rapid pH-responssive polymer polyethylenimine-poly(l-lysine)-poly(l-glutamic acid) (PELG) was designed as the shielding system. The zwitterionic copolypeptide PELG with negatively charged at physical pH can act as the shielding system to shield positively charged polyplexes. PELG was used to shield PEI25k/DNA to form ternary polyplex, the polyplex surface zeta potential can change from a negative to positive nearly pH value of 6.9. Because the pH value of tumor extracellular environment is about 6.5, the positive charges on the polyplexes could be restored in tumors, which is beneficial to the electrostatic interactions between positive polyplexes and negative tumor cells, leading to high cell uptake efficiency and high transfection efficiency., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A novel amphiphilic copolymer (γ-PGA-co-PLA-DPPE) containing poly(γ-glutamic acid) (γ-PGA), polylactide (PLA), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) segments has been successfully synthesized. The chemical structures of the copolymers were characterized by Fourier-transform infrared spectroscopy (FT-IR), NMR ((1)H NMR, (13)C NMR, (31)P NMR), and thermogravimetric analysis (TGA). In order to estimate the feasibility as novel drug carriers, an anti-tumor model drug, doxorubicin hydrochloride salt (DOX) was encapsulated into the copolymers nanoparticles (NPs) by double emulsion and nanoprecipitation methods. The influence of processing factors on encapsulation efficiency and particle size using double emulsion and nanoprecipitation technique were studied. In addition, the DOX-loaded NPs exhibited pH-dependent drug release profiles in vitro. The cumulative release of DOX-loaded NPs was much faster at pH 5.0 than that at pH 7.4. In vitro cytotoxicity test of DOX-loaded NPs against Hela and C666-1 cells demonstrated that DOX-loaded NPs exhibited effectively time-delayed cytotoxicity. Confocal laser scanning microscopy (CLSM) showed that DOX-loaded NPs accumulated mostly in lysosomes instead of cell nucleus, in contrast to free DOX. Therefore, the copolymer nanoparticles were proved to be an available carrier for anti-tumor drug delivery., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: Postoperative pleural adhesions lead to major problems in repeated thoracic surgery. To date, no antiadhesive product has been proven clinically effective. Previous studies of differently charged polypeptides, poly-L-lysine (PL) and poly-L-glutamate (PG) have shown promising results reducing postoperative abdominal adhesions in experimental settings. This pilot study examined the possible pleural adhesion prevention by using the PL+PG concept after pleural surgery and its possible effect on key parameters; plasmin activator inhibitor-1 (PAI-1) and tissue growth factor beta 1 (TGFb) in the fibrinolytic process., Methods: A total of 22 male rats were used in the study, one control group (n=10) and one experimental group (n=12). All animals underwent primary pleural surgery, the controls receiving saline in the pleural cavity and the experimental group the PL+PG solution administered by spray. The animals were evaluated on day 7. Macroscopic appearance of adhesions was evaluated by a scoring system. Histology slides of the adhesions and pleural biopsies for evaluation of PAI-1 and TGFb1 were taken on day 7., Results: A significant reduction of adhesions in the PL+PG group (p<0.05) was noted at day 7 both regarding the length and severity of adhesions. There were no significant differences in the concentration of PAI-1 and TGFb1 when comparing the two groups., Conclusions: PL+PG may be used to prevent pleural adhesions. The process of fibrinolysis, and fibrosis was though not affected after PLPG administration.

Two copolymers are designed and synthesised for siRNA delivery based on polyethylenimine by grafting hydrophilic acrylamide segments and hydrophobic poly(γ-benzyl L-glutamate). The amphiphilic PEI-PBLG/siRNA complex demonstrates high gene silencing efficiency in the absence or presence of 10 vol% and 50 vol% sera in vitro. The anti-tumor effects in vivo are evaluated in luciferase-bearing mice expressing CT26 tumors. PEI-PBLG/siVEGF complex provides a higher and more sustained suppressive effect by reducing VEGF mRNA expression in the tumors, leading to higher tumor growth inhibition efficacy. Further studies on the potential use of the PEI-PBLG carrier system in mediating the silencing of genes other than VEGF or in other tumor models are recommended., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Conventional chemotherapeutic regimens often accompany severe side effects and fail to induce complete regression of chemoresistant or relapsing metastatic cancers. The need for establishing more efficacious anticancer strategies led to the development of a combined modality treatment of chemotherapy in conjunction with immunotherapy or radiotherapy. It has been reported that poly-gamma-glutamate (γ-PGA), a natural polymer composed of glutamic acids, increases antitumor activity by activating antigen-presenting cells and natural killer (NK) cells. Here, we investigated the antitumor effect of γ-PGA in combination with cyclophosphamide in a murine melanoma model. Whereas cyclophosphamide alone directly triggered apoptosis of tumor cells in vitro, γ-PGA did not show cytotoxicity in tumor cells. Instead, it activated macrophages, as reflected by the upregulation of surface activation markers and the secretion of proinflammatory factors, such as nitric oxide and tumor necrosis factor α. When the antitumor effects were examined in a mouse model, combined treatment with cyclophosphamide and γ-PGA markedly suppressed tumor growth and metastasis. Notably, γ-PGA treatment dramatically increased the NK cell population in lung tissues, coinciding with decreased metastasis and increased survival. These data collectively suggest that γ-PGA can act as an immunotherapeutic agent that exhibits a synergistic antitumor effect in combination with conventional chemotherapy.

We studied the effects of 2 different dosages of highmolecular- weight poly-γ-glutamic acid (hm γ-PGA) derived from Bacillus subtilis chungkookjang on lipid metabolism in a high-fructose diet-induced hypertriglyceridemic animal model. For 4 weeks, rats were fed either AIN-93 diet (normal control, NC; n = 10) or modified AIN-93 diet in which cornstarch was substituted with 63% fructose (n = 30) to induce hypertriglyceridemia. After 4 weeks, the hypertriglyceridemic rats were treated with daily oral doses of 0 mg (hypertriglyceridemic control, HC), 2.5 mg (hypertriglyceridemic, low hm γ-PGA, HL), or 5 mg·kg·bw(-1)·d(-1) (hypertriglyceridemic, high hm γ-PGA, HH) hm γ-PGA for 4 weeks. The HL and HH groups exhibited significantly lower levels of serum triglyceride, total cholesterol, LDL cholesterol, and free fatty acids than the HC group. The administration of hm γ-PGA reduced serum ALT and AST levels. The activities of lipogenic enzymes such as hepatic malic enzyme and glucose-6-phosphate dehydrogenase as well as glucose-6-phosphate dehydrogenase mRNA expression were significantly decreased by hm γ-PGA administration (p < 0.05). These results indicate that hm γ- PGA has an anti-hypertriglyceridemic effect in highfructose diet-induced hypertriglyceridemic rats.

Background: Measurement of intracellular concentrations of methotrexate (MTX) and its polyglutamated metabolites (MTXGlu(2-5)) in red blood cells (RBCs) has been suggested as a potential means of monitoring low-dose MTX treatment of rheumatoid arthritis (RA). However, a possible correlation between RBC MTX and MTXGlu2-5 concentrations and clinical outcomes of MTX treatment in RA is debated. A better understanding of the dose-concentration-time relationship of MTX and MTXGlu(2-5) in RBCs by population pharmacokinetic modelling is desirable and will facilitate assessing a potential RBC concentration-effect relationship in the future., Aim: The purpose of this analysis was to describe the pharmacokinetics of MTX and MTXGlu(2-5) in RBCs. Secondary objectives included investigation of deglutamation reactions and the loss of MTX and MTXGlu(2-5) from the RBC., Methods: A model was developed using NONMEM(®) version 7.2 based on RBC data obtained from 48 patients with RA receiving once-weekly low-dose MTX treatment. This model was linked to a fixed two-compartment model that was used to describe the pharmacokinetics of MTX in the plasma. A series of five compartments were used to describe the intracellular pharmacokinetics of MTX and MTXGlu(2-5) in RBCs. Biologically plausible covariates were tested for a significant effect on MTX plasma clearance and the intracellular volume of distribution of all MTX species in RBCs ([Formula: see text]). The developed model was used to test hypotheses related to the enzymatic deglutamation of MTXGlu(2-5) and potential loss of MTXGlu(2-5) from RBCs., Results: The final RBC pharmacokinetic model required the intracellular volumes of distribution for the parent and metabolites to be set to the value estimated for the parent drug MTX alone, and the rate constants describing the polyglutamation steps were fixed at literature values. Significant covariates included effect of body surface area-adjusted estimated glomerular filtration rate on renal plasma clearance and effect of allometrically scaled total body weight with a fixed exponent of 0.75 on non-renal plasma clearance of MTX. The only significant covariate with an effect on [Formula: see text] was mean corpuscular volume (MCV). The model supported single deglutamation steps and a single mechanism of MTX and MTXGlu(2-5) loss from RBCs., Conclusions: The developed model enabled acceptable description of the intracellular kinetics of MTX and MTXGlu(2-5) in RBCs. In the future it can form the basis of a full pharmacokinetic-pharmacodynamic model to assess the time-RBC concentration-effect relationship of low-dose MTX treatment in RA.

A novel biomaterial poly(ethylene glycol)-block-poly(γ-cholesterol-l-glutamate) (mPEG-PCHLG) was designed and synthesized by introducing cholesterol side chains into this pegylated poly(amino acid) copolymers to enlarge the core space to increase the drug capacity. Paclitaxel (PTX) loaded mPEG-PCHLG nanoparticles (PTX-mPEG-PCHLG-Nps) were developed for the first time. The preparation method of nanoparticles was screened and optimized systemically. The optimal PTX-mPEG-PCHLG-Nps with the average diameter of 213.71 nm were constructed through the O/W single-emulsion solvent evaporation method. The entrapment efficiency and drug loading was 38.02 ± 4.51% and 93.90 ± 4.56%, respectively. PTX-mPEG-PCHLG-Nps were spherical and well-dispersed and displayed a dramatic sustained-release property. The in vitro cytotoxicity experiments demonstrated that the blank mPEG-PCHLG nanoparticles had no cytotoxicities on four tumor cell lines including A549, HepG-2, MCF-7 and C26, which implied that mPEG-PCHLG might be biocompatible. PTX-mPEG-PCHLG-Nps obtained the same cell growth inhibition activities as free PTX when incubated with the above tumor cells for 48h. It can be inferred that PTX-mPEG-PCHLG-Nps could probably have higher anticancer efficacy due to the inadequate release of PTX from nanoparticles. PTX-mPEG-PCHLG-Nps achieved the highest antitumor activity in A549 rather than HepG-2, MCF-7 and C26, thus PTX-mPEG-PCHLG-Nps could have a potential application in lung cancer therapy. All the data indicated that mPEG-PCHLG was one of biocompatible biomaterials and worth being widely investigated as hydrophobic antitumor drug carrier., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cisplatin (CDDP) has been a key drug for chemotherapy in patients with head and neck squamous cell carcinoma. Nephrotoxicity is one of its adverse reactions that are dose limiting. To increase its antitumor effects and reduce such toxicity problems, polymeric micelles carrying CDDP (NC-6004) have been developed. The present study was designed to evaluate the efficacy and safety of NC-6004 for oral squamous cell carcinoma. In vitro antitumor activity was assayed in four oral squamous cell carcinoma cell lines. To investigate the antitumor and nephrotoxic effects of NC-6004, nude mice bearing OSC-19 were administered NC-6004 or CDDP. The in vitro growth-inhibitory effect of NC-6004 was significantly less than that of CDDP. However, both NC-6004 and CDDP showed equivalent antitumor effects in vivo. Mice with CDDP developed renal cell apoptosis; however, those injected with NC-6004 were almost free of renal cell injury. Moreover, in an orthotopic tongue cancer model using OSC-19, NC-6004 reduced the rate of sentinel lymph node metastasis to lower than that with CDDP. In conclusion, considering the potential advantages in terms of noticeable antitumor activity, lymphatic drug delivery and reduced nephrotoxicity, NC-6004 represents a significant structural improvement in the development of a platinum complex., (© 2012 Japanese Cancer Association.)

This study investigated the effects of administering poly-γ-glutamic acid (γ-PGA), isoflavones, and γ-PGA with isoflavones on the lipid, fatty liver, and gene expression levels associated with fatty acid oxidation and adipose synthesis in high-fat diet (HFD)-induced C57BL/6 mice. The results demonstrate a significant decrease in the body weight gain, food intake, food efficiency, liver weight, and epididymal adipose tissue of the experimental groups in comparison with the HFD-induced control group. The serum biochemistry indices for hepatic damage, hyperlipidemia, hyperglycemia, and lipid deposits in the liver and adipose tissue were also lower in the experimental groups than in the control group. The anti-oxidative index, and cytokine and enzyme levels associated with obesity (e.g., leptin, adiponectin, AMPK, CPT-1, PPARα, GLUT-4, and UCP-2) were enhanced in the experimental groups in comparison with the control group. These results demonstrate that γ-PGA and isoflavones improved the blood lipid level, insulin resistance, and hyperglycemia. Increased fatty acid oxidation inhibited the synthesis and accumulation of adipose tissue. The results suggest that γ-PGA and isoflavones could be used as new functional foods for preventing obesity.

Background: Diabetic wound is one of the most serious complications of diabetes mellitus. There are no significantly effective therapies for chronic non-healing diabetes ulcer so far. This study aimed to explore the feasibility of healing impaired wound using artificial dermis constructed with human adipose derived stem cells (ASCs) and poly (L-glutamic acid)/chitosan (PLGA/CS) scaffold in streptozotocin-induced diabetic mice., Methods: ASCs were isolated from fresh human lipoaspirates and expanded ex vivo for three passages, and then cells were seeded onto PLGA/CS scaffold to form artificial dermis. Expression of VEGF and TGFβ1 by ASCs presented in artificial dermis was determined. The artificial dermis was transplanted to treat the 20 mm × 20 mm full-thickness cutaneous wound created on the back of diabetic mice. Wound treated with scaffold alone and without treatment, and wound in normal non-diabetic mice served as control., Results: Cells growing within scaffold showed great proliferation potential, depositing abundant collagen matrix. Meanwhile, expression of VEGF and TGF-β1 by seeded ASCs maintained at a consistent high level. After treated with ASC based artificial dermis, diabetic wounds exhibited significantly higher healing rate compared with wounds treated with scaffold alone or without treatment. Histological examination also demonstrated an improvement in cutaneous restoration with matrix deposition and organization. Further quantitative analysis showed that there was a significant increase in dermis thickness and collagen content on artificial dermis treated wounds., Conclusion: ASC/PLGA artificial dermis can effectively accelerate diabetic wound healing by promoting angiogenic growth factors and dermal collagen synthesis.

The purpose of the present study was to investigate the usefulness of the ternary complex with protamine and γ-polyglutamic acid (γ-PGA), which are biodegradable materials for foods and medical products, as a safe gene delivery vector. We formed cationic binary complexes (plasmid DNA (pDNA)/protamine complexes) with high transfection efficiency. The binary complex showed slight toxicity probably related to its total cationic charge. Then, we formed ternary complexes (pDNA/protamine/γ-PGA complexes) by addition of anionic polymer, γ-PGA, and they showed no cytotoxicity. The transfection efficiency of the pDNA/protamine/γ-PGA complexes was as high as that of the pDNA/protamine complexes, although their zeta potentials were different. Inhibition study of the gene expressions in B16-F10 cells suggested that pDNA/protamine complexes were taken up by caveolae-mediated endocytosis and macropinocytosis. On the other hand, pDNA/protamine/γ-PGA complexes were taken up by clathrin-mediated endocytosis and macropinocytosis. Thus, we succeeded in developing the ternary complex as a safe gene delivery vector with biocompatible materials.

In this experiment, we developed a novel safe and effective gene delivery vector coated with γ-polyglutamic acid (γ-PGA-coated complexes). The γ-PGA-coated complex was composed of chiseled spherical nano-particles with anionic charges. The plasmid DNA/polyethyleneimine complex (non-coated complex) showed high transgene efficiency in the spleen and lung after intravenous administration in mice, with high liver toxicity and lethality. On the other hand, γ-PGA-coated complex selectively showed high transgene efficiency in the spleen without such toxicity. Furthermore, the γ-PGA-coated complex highly accumulated and showed high gene expression in the marginal zone of the spleen. Those results strongly indicated that γ-PGA-coated complex was suitable as a DNA vaccine vector. We therefore applied γ-PGA-coated complex to melanoma DNA vaccine, pUb-M. The γ-PGA-coated complex containing pUb-M significantly inhibited the growth and metastasis of a melanoma cell line, B16-F10 cells. In conclusion, we developed a splenic gene vector, γ-PGA-coated complex, as a novel technology for clinical vaccination.

We have developed a novel type of polymer nanogel loaded with anticancer drug based on bio-derived poly(gamma- glutamic acid) (gamma-PGA). gamma-PGA is a highly anionic polymer that is synthesized naturally by microbial species, most prominently in various bacilli, and has been shown to have excellent biocompatibility. Thiolated gamma-PGA was synthesized by covalent coupling between the carboxyl groups of gamma-PGA and the primary amine group of cysteamine. Doxorubicin (Dox)-loaded gamma-PGA nanogels were fabricated using the following steps: (1) an ionic nanocomplex was formed between thiolated gamma-PGA as the negative charge component, and Dox as the positive charge component; (2) addition of poly(ethylene glycol) (PEG) induced hydrogen-bond interactions between thiol groups of thiolated gamma-PGA and hydroxyl groups of PEG, resulting in the nanocomplex; and (3) disulfide crosslinked gamma-PGA nanogels were fabricated by ultrasonication. The average size and surface charge of Dox-loaded disulfide cross-linked gamma-PGA nanogels in aqueous solution were 136.3 +/- 37.6 nm and -32.5 +/- 5.3 mV, respectively. The loading amount of Dox was approximately 38.7 microgram per mg of gamma-PGA nanogel. The Dox-loaded disulfide cross-linked gamma-PGA nanogels showed controlled drug release behavior in the presence of reducing agents, glutathione (GSH) (1- 10 mM). Through fluorescence microscopy and FACS, the cellular uptake of gamma-PGA nanogels into breast cancer cells (MCF-7) was analyzed. The cytotoxic effect was evaluated using the MTT assay and was determined to be dependent on both the concentration and treatment time of gamma-PGA nanogels. The bio-derived gamma-PGA nanogels are expected to be a well-designed delivery carrier for controlled drug delivery applications.

Forkhead box protein 3 (FoxP3(+)) regulatory T (T(reg)) cells and interleukin (IL)-17-producing T helper 17 (Th17) cells have opposing effects on autoimmunity, as the former are crucial for maintaining self-tolerance while the latter play a key role in precipitating inflammatory autoimmune diseases. Here we report that Bacillus-derived poly-γ-glutamic acid (γ-PGA) signals naive CD4(+) T cells to promote the selective differentiation of T(reg) cells and to suppress the differentiation of Th17 cells. The γ-PGA inducibility of FoxP3 expression was due partially to transforming growth factor (TGF)-β induction through a Toll-like receptor (TLR)-4/myeloid differentiating factor 88 (MyD88)-dependent pathway. However, this pathway was dispensable for γ-PGA suppression of Th17 differentiation. γ-PGA inhibited IL-6-driven induction of Th17-specific factors including signal transducer and activator of transcription-3 (STAT-3) and retinoic acid-related orphan receptor γt (RORγt) while up-regulating the STAT-3 inhibitor suppressor of cytokine signalling 3 (SOCS3). Importantly, in vivo administration of γ-PGA attenuated the symptoms of experimental autoimmune encephalomyelitis and at the same time reduced Th17 cell infiltrates in the central nervous system. Thus, we have identified the microbe-associated molecular pattern, γ-PGA, as a novel regulator of autoimmune responses, capable of promoting the differentiation of anti-inflammatory T(reg) cells and suppressing the differentiation of proinflammatory Th17 cells. These findings draw attention to the potential of γ-PGA for treating Th17 cell-mediated autoimmune diseases., (© 2012 British Society for Immunology.)

In addition to development of vaccines and synthetic antiviral drugs, recent studies have advocated the use of natural substances that inhibit or prevent viral infections. High-molecular-weight poly-γ-glutamate (HM-γ-PGA) produced by Bacillus subtilis chungkookjang was evaluated for anti-influenza virus activity. HM-γ-PGA induced type I interferons (IFNs), which in turn stimulated expression of Myxovirus resistant 1 protein and IFN-related proteins in vitro. In the B6.A2G-Mx1 mouse model, which mimics the innate immune system of humans, treatment with HM-γ-PGA enhanced the antiviral state of mice and protected them against highly pathogenic influenza A virus. Naturally synthesized HM-γ-PGA has potent anti-influenza activity and may be a useful means for control of influenza virus., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Purpose: To evaluate the pathologic complete response (CR) rate and safety of paclitaxel poliglumex (PPX), cisplatin, and concurrent radiation for patients with esophageal cancer., Patients and Methods: Patients with adenocarcinoma or squamous cell carcinoma of the esophagus or gastroesophageal junction with no evidence of distant metastasis received PPX (50 mg/m(2)/wk) and cisplatin (25 mg/m(2)/wk) for 6 weeks with 50.4 Gy concurrent radiation. Six to eight weeks after completion of chemoradiotherapy, patients underwent surgical resection., Results: Forty patients were enrolled, 37 patients with adenocarcinoma and 3 patients with squamous cell cancer. The treatment-related grade 3 nonhematologic toxicities included esophagitis (7%), nausea (7%), and fatigue (5%). Three patients with clinical endoscopic CR (2 with squamous cell cancer) refused surgery. Twelve of the remaining 37 patients (32%) had a pathologic CR. The 12 patients with pathologic CR all had adenocarcinoma., Conclusion: PPX, cisplatin, and concurrent radiation are well tolerated, easily administered regimen for esophageal cancer with a low incidence of significant esophagitis and a high pathologic CR rate consistent with the preclinical data of PPX and radiation.

Nanocarriers potentially reduce or prevent chemotherapy-induced side effects, facilitating the translation of nanocarrier formulation into the clinic. To date, organ-specific toxicity by nanocarriers remains to be clarified. Here, we studied the potential of polymeric micelle nanocarriers to prevent the ototoxicity, which is a common side effect of high-dose cisplatin (CDDP) therapy. In this study, we evaluated the ototoxicity of CDDP-incorporating polymeric micelles (NC-6004) in guinea pigs in comparison with that of cisplatin. Their auditory brainstem responses (ABRs) to 2, 6, 12, 20, and 30kHz sound stimulation were measured before and 5 days after the drug administration. Groups treated with NC-6004 showed no apparent ABR threshold shifts, whereas groups treated with CDDP showed dose-dependent threshold shifts particularly at the higher frequencies. Consistent with the ABR results, groups treated with NC-6004 showed excellent hair-cell preservation, whereas groups treated with CDDP exhibited significant hair-cell loss (P<0.05). Synchrotron radiation-induced X-ray fluorescence spectrometry imaging demonstrated that the platinum distribution and concentration in the organ of Corti were significantly reduced (P<0.01) in guinea pigs treated with NC-6004 compared with guinea pigs treated with CDDP. These findings indicate that micellization of CDDP reduces its ototoxicity by circumventing the vulnerable cells in the inner ear., (Copyright © 2011 Elsevier B.V. All rights reserved.)

To maintain immunity against Japanese encephalitis virus (JEV), a formalin-inactivated Japanese encephalitis (JE) vaccine should be administered several times. The repeated vaccination is not helpful in the case of a sudden outbreak of JEV or when urgent travel to a high-JEV-risk region is required; however, there are few single-injection JE vaccine options. In the present study, we investigated the efficacy of a single dose of a new effective JE virus-like particle preparation containing the JE envelope protein (JE-VLP). Although single administration with JE-VLP protected less than 50% of mice against lethal JEV infection, adding poly(γ-glutamic acid) nanoparticles (γ-PGA-NPs) or aluminum adjuvant (alum) to JE-VLP significantly protected more than 90% of the mice. A single injection of JE-VLP with either γ-PGA-NPs or alum induced a significantly greater anti-JEV neutralizing antibody titer than JE-VLP alone. The enhanced titers were maintained for more than 6 months, resulting in long-lasting protection of 90% of the immunized mice. Although the vaccine design needs further modification to reach 100% protection, a single dose of JE-VLP with γ-PGA-NPs may be a useful step in developing a next-generation vaccine to stop a JE outbreak or to immunize travelers or military personnel.

Lansoprazole sodium is a proton pump inhibitor used in treating gastroesophageal reflux disease (GERD). It is highly acid-labile and presents many formulation challenges. Therefore, this drug needs to be protected from the harsh environment in the stomach. In order to achieve this, a pH-sensitive microparticle system composed of chitosan and γ- poly-(glutamic acid) was prepared and loaded with Lansoprazole. The prepared microparticles were not stable in gastric pH. To overcome this problem microparticles were freez-dried and filled in an enteric-coated capsule. Upon oral administration, the enteric-coated capsule remained intact in the acidic environment of the stomach, but dissolved rapidly in the distal segment of the GIT. Consequently, all the microparticles loaded in the capsule were brought into the intestine, thus enhancing the intestinal absorption of drug. Drug encapsulation efficiency of formulation F3 was found to be 82.82 % and in vitro release of prepared formulation F3 was found to be 94% after 8 h of dissolution in 7.4 pH phosphate buffer. FTIR and DSC studies showed no interaction between the drug and polymer. The formulation showed good swelling property. SEM photographs showed that microparticles are spherical and lies in size range of 300-400 μm. From the above, it can be concluded that the prepared chitosan/ γ-poly-(glutamic acid) microparticles can be used as carriers for the intestinal delivery of acid liable drugs such as lansoprazole.

The in vivo efficacy of doxorubicin (DOX)-loaded poly(γ-benzyl l-glutamate)-block-hyaluronan (PBLG(23)-b-HYA(10))-based polymersomes (PolyDOX) was evaluated. Samples were efficiently labeled with technetium-99m radionuclide with good stability for in vivo studies. PolyDOX enhanced circulation time compared to free DOX. Biodistribution studies revealed selective accumulation of PolyDOX in the Ehrlich ascites tumor (EAT) as a result of passive accumulation and active targeting (CD44-mediated endocytosis) in EAT-bearing mice. Toxicity studies demonstrated PolyDOX is a safe drug carrier, and no hemolysis was observed with PolyDOX equivalent to 200 μg/mL of free DOX. PolyDOX dominantly controlled tumor growth by delaying doubling time of EATs compared to free DOX over 30 days after treatment. PolyDOX also increased life span six times more than free DOX. Hence, it is reasonable to expect that higher DOX levels attributable to PolyDOX improve the therapeutic index and reduce side effects due to site-specific drug accumulation., From the Clinical Editor: In this preclinical project, doxorubicin loaded polymersomes enhanced intracellular uptake of doxorubicin in a murine model of Ehrlich Ascites Tumor (EAT) through CD44 receptor mediated endocytosis, resulting in prolonged Tumor Doubling Time and increase in life span of mice., (2012 Elsevier Inc. All rights reserved.)

Purpose: To investigate the effects of gelatin hydrogel (GH) containing a chymase inhibitor (CI) on intraocular pressure (IOP) and conjunctival scarring in a canine model of glaucoma surgery., Methods: Glaucoma surgery models were made in beagles. As the first experiment, GH was implanted. IOP was measured for 4 weeks, followed by histologic evaluation. As the second experiment, GH containing a CI or GH alone was implanted. IOP and bleb features were evaluated for 12 weeks. The densities of proliferative cell nuclear antigen (PCNA)-positive cells, fibroblasts, and mast cells were quantified. The mRNA levels of transforming growth factor-β (TGF-β) and chymase were determined by real-time PCR., Results: In the first experiment, IOP was significantly lower in the eyes treated with GH than that in the control eyes. The conjunctival area normalized by the scleral area was reduced in the treated eyes. In the second experiment, IOP reduction was maintained for 12 weeks in the eyes treated with GH containing a CI, but not in the eyes treated with GH alone. In addition, the bleb score was larger, whereas the adhesion score and densities of PCNA-positive cells, fibroblasts, mast cells, and chymase-positive cells were lower in the eyes treated with GH containing a CI. The mRNA levels of TGF-β and chymase were significantly decreased in the eyes treated with GH containing a CI., Conclusions: Implanting GH alone maintained IOP reduction, whereas GH containing a CI enhanced the IOP-reducing effect by suppressing cell proliferation. This drug delivery system might be useful for maintaining filtering blebs for a longer duration after glaucoma surgery.

We investigated the effect of high molecular weight polygamma- glutamic acid (hm gamma-PGA) on adiposity and lipid metabolism of rats in the presence of an obesity-inducing diet. Thirty-two Sprague-Dawley rats were fed either a normal-fat (11.4% kcal fat, NFC) or high-fat (51% kcal fat, HFC) diet. After 5 weeks, half of each diet-fed group was treated with hm gamma-PGA (NFP or HFP) for 4 weeks. The HFC group had significantly higher body weight, visceral fat mass, fasting serum levels of total cholesterol, LDL cholesterol, and leptin, and lower serum HDL cholesterol level compared with those of the NFC group (p < 0.05). Treatment with hm gamma-PGA decreased body weight gain and perirenal fat mass (p<0.05), fasting serum total cholesterol, and mRNA expression of glucose-6- phosphate dehydrogenase (G6PD), regardless of dietary fat contents (p < 0.01). However, hm gamma-PGA increased serum HDL cholesterol in the HFC group (p < 0.05). In vitro, 3-hydroxy-3-methylglutaryl coenzyme-A (HMGCoA) reductase activity was suppressed by the addition of hm gamma-PGA. In agreement with observations in animal study, the supplementation of hm gamma-PGA (150 mg/day) to 20 female subjects in an 8-week double-blind, placebocontrolled study resulted in a tendency to decrease total cholesterol and LDL cholesterol concentrations. We thus conclude that dietary supplementation of hm gamma-PGA may act as a hypocholestrolemic agent, secondary to its inhibitor effect on HMG-CoA reductase, and decrease abdominal adiposity by decreasing hepatic lipogenesis. The present study is an important first step in establishing the effect of hm gamma-PGA on cholesterol levels in rats and humans.

Angiogenesis plays a prominent role in cancer progression. Anti-angiogenic therapy therefore, either alone or in combination with conventional cytotoxic therapy, offers a promising therapeutic approach. Paclitaxel (PTX) is a widely-used potent cytotoxic drug that also exhibits anti-angiogenic effects at low doses. However, its use, at its full potential, is limited by severe side effects. Here we designed and synthesized a targeted conjugate of PTX, a polymer and an integrin-targeted moiety resulting in a polyglutamic acid (PGA)-PTX-E-[c(RGDfK)(2)] nano-scaled conjugate. Polymer conjugation converted PTX to a macromolecule, which passively targets the tumor tissue exploiting the enhanced permeability and retention effect, while extravasating via the leaky tumor neovasculature. The cyclic RGD peptidomimetic enhanced the effects previously seen for PGA-PTX alone, utilizing the additional active targeting to the α(v)β(3) integrin overexpressed on tumor endothelial and epithelial cells. This strategy is particularly valuable when tumors are well-vascularized, but they present poor vascular permeability. We show that PGA is enzymatically-degradable leading to PTX release under lysosomal acidic pH. PGA-PTX-E-[c(RGDfK)(2)] inhibited the growth of proliferating α(v)β(3)-expressing endothelial cells and several cancer cells. We also showed that PGA-PTX-E-[c(RGDfK)(2)] blocked endothelial cells migration towards vascular endothelial growth factor; blocked capillary-like tube formation; and inhibited endothelial cells attachment to fibrinogen. Orthotopic studies in mice demonstrated preferential tumor accumulation of the RGD-bearing conjugate, leading to enhanced anti-tumor efficacy and a marked decrease in toxicity as compared with free PTX-treated mice., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The objectives of this study were to evaluate the efficiency in treatment of lead-induced intoxication in mice with γ-PGA as chelating agent and compare with the drug (meso-2,3-dimercaptosuccinic acid). The results showed the incorporation of γ-PGA at 200 and 400 mg/kg could reduce the accumulation of lead in the liver, heart, and testis; however, the latter was more effective in decreasing the lead content in the kidney and spleen. Nevertheless, both doses failed to inhibit the lead accumulation in the lung and brain. Additionally, both doses of γ-PGA could reduce TBARs in the kidney and brain, as well as elevate δ-aminolevulinic acid dehydrase (δ-ALAD) activity in blood and decrease glutamic pyruvic transaminase (GPT) and lactic dehydrogenase (LDH) activities in the serum. For hematological parameters, both white blood cells (WBCs) and hematocrite (HCT) were raised by 400 mg/kg of γ-PGA, while for both doses of γ-PGA, a slight decline in hemoglobin (HGB), mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC) was observed, with the red blood cells (RBCs) being unaffected.

The purpose of this study was to develop a novel, highly water-soluble poly(L-γ-glutamyl-glutamine)-paclitaxel nanoconjugate (PGG-PTX) that would improve the therapeutic index of paclitaxel (PTX). PGG-PTX is a modification of poly(L-glutamic acid)- paclitaxel conjugate (PGA-PTX) in which an additional glutamic acid has been added to each glutamic side chain in the polymer. PGG-PTX has higher water-solubility and faster dissolution than PGA-PTX. Unlike PGA-PTX, PGG-PTX self-assembles into nanoparticles, whose size remains in the range of 12-15 nm over the concentration range from 25 to 2,000 μg/mL in saline. Its critical micellar concentration in saline was found to be ~25 μg/mL. The potency of PGG-PTX when tested in vitro against the human lung cancer H460 cell line was comparable to other known polymer-PTX conjugates. However, PGG-PTX possesses lower toxicity compared with PGA-PTX in mice. The maximum tolerated dose of PGG-PTX was found to be 350 mg PTX/kg, which is 2.2-fold higher than the maximum tolerated dose of 160 mg PTX/kg reported for the PGA-PTX. This result indicates that PGG-PTX was substantially less toxic in vivo than PGA-PTX.

The transfection efficiency of cationic polymers decreases dramatically in the presence of serum, which hampers the in vivo application of these polymers for gene delivery. Due to its shielding effect of poly(alpha-glutamic acid) (PGA) from negatively charged serum proteins, it was introduced into DNA polyplexes to overcome the serum inhibitory effect. In the present studies, the transfection efficiency of DNA/PEI/PGA terplex system was compared to PEI 25 kDa and Lipofectamine 2000 in the presence of serum. The successful formation of DNA/PEI/PGA terplexes was confirmed by their near-neutral surface charge. Interaction between components in the terplex system demonstrated that PGA was competing with DNA to combine with PEI. PEI/PGA combined carriers were not cytotoxic at a C/N ratio higher than 0.3. The in vitro transfection efficiency of DNA/PEI/PGA terplexes was not significantly different from those of DNA/PEI25kDa in serum-free medium. Importantly, in serum-containing medium, the DNA terplexes at their optimal C/N ratios maintained the same level of transfection efficiency as that of serum-free medium, even though the transfection efficiency of PEI 25 kDa and Lipofectamine 2000 was significantly decreased under serum-containing conditions. CLSM results confirmed that the cellular import of pDNA delivered by PEI/PGA combined carriers was more efficient than PEI 25 kDa alone under serum-containing conditions. Therefore, PGA could be used as a versatile serum-resistant reagent to overcome the serum inhibitory effect of polycations for gene delivery., (Copyright 2010 Elsevier B.V. All rights reserved.)

The advantage of polymeric drug carriers lies in the uptake of the polymer nanoparticles by cancer cells before they release the drug, thereby reducing its toxic effects on healthy cells. A poly(gamma-glutamic acid)-b-poly(epsilon-caprolactone)-b-poly(gamma-glutamic acid) block copolymer was synthesized to encapsulate the anti-cancer drug doxorubicin in the treatment of wild type human breast cancer cells (MCF-7/WT). This pH-controllable carrier is negatively-charged in the presence of healthy tissues leading to lower cellular uptake. On the other hand, it becomes more hydrophobic in the acidic environment of cancer tissues, increasing its cellular uptake through the lipid bilayer. The block copolymer was characterized using Fourier transform infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, differential scanning calorimetry and dynamic light scattering. The micelles formed at a critical concentration range of 62-130 microg/mL depending on the composition of poly(gamma-glutamic acid) and poly(epsilon-caprolactone) chains. The nano-sized micelles were found to have pH-dependent sizes in the range of 90-200 nm. The role of poly(gamma-glutamic acid) was to increase the hydrophilicity and decrease the particle size of the copolymer. The structures of micelles that were more compact and less anionic showed better stability in plasma. It was found that the drug loading content and drug loading efficiency were 12.14% and 97.22% respectively. The copolymer showed shrinking and aggregation at low pH which led to a slower drug release. These nano-sized micelles showed potential as effective drug delivery carriers for doxorubicin because of its accumulation and slow release inside the MCF-7/WT cells.

Implant-associated infection is a serious complication in orthopedic surgery, and endowing implant surfaces with antibacterial properties could be one of the most promising approaches for preventing such infection. In this study, we developed cefazolin loaded biodegradable polypeptide multilayer nanofilms on orthopedic implants. We found that the amount of cefazolin released could be tuned. A high local concentration of cefazolin was achieved within the first a few hours and therefore may inhibit bacterial colonization in the critical postimplantation period. The developed cefazolin loaded nanofilms showed their in vitro efficacy against Staphylococcus aureus; the more antibiotics loaded, the longer the nanocoated implant had antibacterial properties. More interestingly, antibiotic-loaded polypeptide multilayer nanofilms also improved osteoblast bioactivity including cell viability and proliferation. These findings suggested that biodegradable polypeptide multilayer nanofilms as antibiotic carriers at the implant/tissue interface are compatible with human cells such as osteoblasts and bactericidal to bacteria such as S. aureus. These characteristics could be promising for preventing implant-associated infection and potentially improving bone healing., (Copyright 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Poly-gamma-glutamic acid (gamma-PGA) prepared by fermentation of microbe was used as drug carrier for vanadium sulfate to obtain vanadyl-poly-gamma-glutamic acid (VO-gamma-PGA) complex. The FI-IR spectrum of the complex demonstrated that the expected VO-gamma-PGA complex is formed by the coordination of VO(2+) through the side chain carboxylic groups of the gamma-PGA. Studies of the complex in treating type I diabetes were carried out on alloxan induced diabetes rats. The results of treating the rats in 2 weeks and then stopping administration for 10 days showed that VO-gamma-PGA can effectively lower blood glucose levels of diabetic rats during administration. But after ceasing treatment there were no differences between groups in blood glucose level and water intake. The results of oral glucose tolerance and some serum parameters also demonstrated that VO-gamma-PGA was more effective than vanadium sulfate in treating diabetic rats., ((c) 2010 Wiley-Liss, Inc. and the American Pharmacists Association)

To enhance the stability of the anticancer drug gemcitabine (2'-deoxy-2',2'-difluorocytidine), it was conjugated to poly-l-glutamic acid (PG-H) via a carbodiimide reaction. The synthesised poly-l-glutamic acid-gemcitabine (PG-G) was purified and characterised by using SDS-PAGE to estimate its molecular weight, HPLC to determine its purity and degree of drug loading, and NMR to elucidate the structure. In vitro aqueous hydrolytic studies showed that the gemcitabine release from the polymeric drug conjugate was pH dependent, and that the conjugation to PG-H improved its stability in human plasma. The release of the bound gemcitabine from PG-G in plasma was mediated by a hydrolytic process. It began with a lag phase, followed by linear release between 12 and 48h, and reached equilibrium at 72h with 51% of the gemcitabine released. In vitro cytotoxicity studies using MCF-7 and MDA-MB-231 human mammary cancer cells, as well as human dermal fibroblasts (HDF), showed that PG-G displayed a lower dose dependent cytotoxic effect with respect to the parent drug gemcitabine. On the other hand, in 4T1 mouse mammary tumour cells, PG-G and gemcitabine showed similar toxicities. Gemcitabine was more than likely released hydrolytically from PG-G and taken up by MCF-7, MDA-MB-231 and HDF, whereas both released gemcitabine and PG-G were taken up by 4T1 to mediate the observed cytotoxicities. The improved stability and extended sustained release profile may render PG-G a potential anticancer prodrug., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

The prognosis of liver cancer remains poor, but recent advances in nanotechnology offer promising possibilities for cancer treatment. Novel adjuvant, amphiphilic nanoparticles (NPs) composed of L: -phenylalanine (Phe)-conjugated poly(gamma-glutamic acid) (gamma-PGA-Phe NPs) having excellent capacity for carrying peptides, were found to have the potential for use as a peptide vaccine against tumor models overexpressing artificial antigens, such as ovalbumin (OVA). However, the anti-tumor potential of gamma-PGA-Phe NPs vaccines using much less immunogenic tumor-associated antigen (TAA)-derived peptide needs to be clarified. In this study, we evaluated the effectiveness of immunization with EphA2, recently identified TAA, derived peptide-immobilized gamma-PGA-Phe NPs (Eph-NPs) against mouse liver tumor of MC38 cells (EphA2-positive colon cancer cells). Immunization of normal mice with Eph-NPs resulted in generation of EphA2-specific type-1 CD8+ T cells. Immunization with Eph-NPs tended to provide a degree of anti-MC38 liver tumor protection more than that observed for immunization with the mixture of EphA2-derived peptide and complete Freund's adjuvant (Eph + CFA). Neither Eph-NPs nor Eph + CFA vaccines inhibited tumor growth of BL6, EphA2-negative melanoma cells. Splenocytes isolated from MC38-bearing mice treated with Eph-NPs showed strong and specific cytotoxic activity against MC38 cells. Immunization with Eph + CFA induced liver damage as evidenced by elevation of serum alanine aminotransferase, while Eph-NPs vaccination did not exhibit any toxic damage to the liver. These results demonstrated that immunization with Eph-NPs displayed anti-tumor effects against liver tumor by generating acquired immunity equivalent to the toxic adjuvant CFA, suggesting that safe gamma-PGA-Phe NPs could be applied clinically for the vaccine treatment of liver cancer.

A specific and sensitive liquid chromatography-tandem mass spectrometric method for quantitative determination of paclitaxel in rat plasma was developed and validated using docetaxel as an internal standard. Liquid-liquid extraction using tert-butyl methyl ether was used to extract the drug and the internal standard from plasma. The separation of paclitaxel was performed on a C(18) column with a mobile phase of acetonitrile:water:formic acid (65:35:0.1, v/v/v) over 5min. The assay was based on the selected reaction monitoring transitions at m/z of the precursor-product ion transitions m/z 854.2-->286.1 for paclitaxel and 808.3-->527.2 for internal standard. The lower limit of quantification was 0.5ng/mL based on 100microL of plasma. Intra- and inter-day assay variations were less than 15%, and the accuracy values were between 95.4 and 105.4%. The extraction recoveries ranged from 96.7 to 103.7% across the calibration curve range. The method was successfully applied to measurement of low concentrations of paclitaxel or regenerated paclitaxel in plasma after intravenous administration of a single dose (10mg/kg) of a poly(l-glutamic acid)-alanine-paclitaxel conjugate to rats., (Copyright 2009 Elsevier B.V. All rights reserved.)

Taxanes remain the only agents to extend survival in castration-resistant metastatic prostate cancer, but their impact on the natural history of this disease is modest. We sought to test the hypothesis that increased delivery of taxane chemotherapy to the tumor through the use of a macromolecular polymer-drug conjugate of paclitaxel modulated by estradiol could extend the utility of this class of drugs. Patients with metastatic adenocarcinoma of the prostate who progressed despite standard hormonal therapy and after docetaxel-containing chemotherapy were treated with transdermal estradiol (0.2 mg/24 h) for 4 weeks followed by the same dose of transdermal estradiol and paclitaxel poliglumex (PPX; 150 mg/m intravenous) every 28 days. The primary objective was to determine the level of activity of the regimen measured using a fraction of patients who experienced a confirmed decline in serum prostate-specific antigen (PSA) of 50% or more. A two-stage phase II study designed to identify a response rate of > or =25% required three responders among 21 patients in the first stage. Twenty-one patients who received a median of two earlier chemotherapy regimens were enrolled in the trial between March 2007 and May 2008. During the estradiol-only treatment phase, no patient had a PSA decline in excess of 50% and lesser PSA declines that ranged from 8.8 to 34.1% were seen in five patients. No patients achieved a > or =50% PSA decline following the addition of PPX and there were no responses in measurable disease. The median time to progression was 4 weeks. In conclusion, this regimen of low-dose transdermal estradiol induction followed by PPX does not have activity in taxane pretreated patients with castration-resistant prostate cancer.

Objective: There are limited data suggesting that methotrexate polyglutamate (MTXGlu) concentrations can guide MTX dosing in patients with rheumatoid arthritis (RA). The aim of this study was to define a therapeutic range of red blood cell (RBC) MTXGlu(n) concentrations (where n refers to the number of glutamate groups), including threshold values for efficacy and adverse effects in patients receiving long-term oral MTX treatment., Methods: A cross-sectional study of 192 patients receiving oral MTX was undertaken. Disease activity was assessed by the swollen and tender joint counts, the C-reactive protein level, and the Disease Activity Score in 28 joints (DAS28). High disease activity was defined as a DAS28 of >3.2. A standardized questionnaire regarding common MTX adverse effects was completed., Results: The MTX dosage was significantly higher in patients in whom the swollen joint count and DAS28 were higher. The MTXGlu(4), MTXGlu(5), MTXGlu(3-5), and MTXGlu(1-5) concentrations were significantly higher in patients with high disease activity. After correction for age, the estimated glomerular filtration rate, and the MTX dosage, the association remained significant for MTXGlu(5). RBC folate concentrations were significantly higher in the group with high disease activity. There was no association between any MTXGlu(n) concentration and adverse effects., Conclusion: In contrast to other studies, the results of the present study did not show a relationship between the MTXGlu(n) concentration and reduced disease activity in patients with RA who were receiving long-term MTX therapy. However, disease activity was influenced by the RBC folate level, which may be a more important factor than MTXGlu(n) concentrations for disease control. In accordance with the findings of previous studies, we were unable to show a relationship between MTXGlu(n) concentrations and adverse effects. Prospective studies will be important to determine whether there is a role for measuring MTXGlu(n) concentrations in patients receiving long-term treatment with MTX.

Previously, we reported that the oral administration of high molecular mass poly-gamma-glutamate (gamma-PGA) induced antitumor immunity but the mechanism underlying this antitumor activity was not understood. In the present study, we found that application of high molecular mass gamma-PGA induced secretion of tumor necrosis factor (TNF)-alpha from the bone-marrow-derived macrophages of wild type (C57BL/6 and C3H/HeN) and Toll-like receptor 2 knockout (TLR2(-/-)) mice, but not those of myeloid differentiation factor 88 knockout (MyD88(-/-)) and TLR4-defective mice (C3H/HeJ). Production of interferon (IFN)-gamma-inducible protein 10 (IP-10) in response to treatment with gamma-PGA was almost abolished in C3H/HeJ mice. In contrast to LPS, gamma-PGA induced productions of TNF-alpha and IP-10 could not be blocked by polymyxin B. Furthermore, gamma-PGA-induced interleukin-12 production was also impaired in immature dendritic cells (iDCs) from MyD88(-/-) and C3H/HeJ mice. Downregulation of MyD88 and TLR4 expression using small interfering RNA (siRNA) significantly inhibited gamma-PGA-induced TNF-alpha secretion from the RAW264.7 cells. Gamma-PGA-mediated intracellular signaling was markedly inhibited in C3H/HeJ cells. The antitumor effect of gamma-PGA was completely abrogated in C3H/HeJ mice compared with control mice (C3H/HeN) but significant antitumor effect was generated by the intratumoral administration of C3H/HeN mice-derived iDCs followed by 2,000 kDa gamma-PGA in C3H/HeJ. These findings strongly suggest that the antitumor activity of gamma-PGA is mediated by TLR4.

Background: Paclitaxel poliglumex (PPX, also called Xyotax or CT-2103) is a water soluble macromolecular drug conjugate that links paclitaxel with a biodegradable polymer, poly-L-glutamic acid. The recommended phase II dose of PPX every 3 week is 235 mg/m(2) administered over a 10-min infusion without premedication. This study was designed to determine the MTD and pharmacology of PPX administered weekly to patients with solid malignancies., Methods: The starting dose of weekly PPX was 20 mg/m(2). Each cycle consists of 6 weekly treatments with pharmacokinetics of PPX (the conjugated paclitaxel) and unconjugated paclitaxel obtained after the first and sixth dose. Three to six patients were enrolled at each dose level. Toxicity and response were assessed by the NCI Common Toxicity criteria version 2 and RECIST criteria, respectively., Results: Twenty-six patients were treated with PPX at the following dose levels: 20 mg/m(2) (five patients), 40 mg/m(2) (four patients), 60 mg/m(2) (four patients), 70 mg/m(2) (eight patients) and 80 mg/m(2) (five patients). Dose-limiting toxicities, consisting of grade 3 neutropenia, occurred in the 80 mg/m(2) cohort during cycle 1. Therefore, the dose recommended for phase II studies was 70 mg/m(2). In this cohort, a single dose-limiting event, consisting of diarrhea, was seen. Neuropathy and fatigue were the most common toxicities. No objective responses were noted. Pharmacokinetics was dose-proportional, and the degree of neutropenia related to drug exposure, but not to peak plasma concentration. There was no significant accumulation of conjugated or unconjugated paclitaxel with this dosing schedule., Conclusions: The recommended dose of PPX for subsequent disease-directed studies is 70 mg/m(2) weekly.

Purpose: Determine the toxicity, maximum tolerated dose (MTD), and pharmacokinetics of paclitaxel poliglumex (PPX; CT-2103) in combination with cisplatin administered every 3 weeks., Patients and Methods: Forty-three patients with advanced solid tumors were treated at escalating doses of PPX with a fixed dose of cisplatin at 75 mg/m(2). Conjugated and unconjugated paclitaxel were measured in plasma and urine. Cisplatin, as total platinum content in urine, was also assayed., Results: Dose-limiting toxicities included neutropenia and neuropathy with a cycle 1 MTD of 210 mg/m(2). Conjugated taxanes had a prolonged half-life of >100 h. Nine patients had partial responses, and 19 had stable disease., Conclusions: PPX is a water-soluble paclitaxel-polymer conjugate with a prolonged half-life and a limited volume of distribution. PPX/cisplatin showed good activity in a refractory patient population; however, cumulative neuropathy was a significant issue at high doses, suggesting that a lower dose may be appropriate for prolonged therapy.

In developing new insulin-mimetic zinc(II) complexes with various ligands including a biodegradable polymer, we prepared and characterized a Zn(gamma-pga) complex in solution as well as in solid, and investigated its in vitro insulin-mimetic activity and in vivo antidiabetic effect in type-2 diabetic KKA(y) mice. The in vitro insulin-mimetic activity of the Zn(gamma-pga) complex was considerable better than that of ZnSO(4). The Zn(gamma-pga) complex normalized the hyperglycemia in KKA(y) mice within 21 d when administrated orally at doses of 10-20 mg (0.15-0.31 mmol) Zn per kg body mass for 30 d. In addition, the impaired glucose tolerance, elevated HbA(1c) levels and metabolic syndromes were significantly improved in Zn(gamma-pga)-treated KKA(y) mice relative to those administrated with saline and ZnSO(4).

One of the most important aspects in vaccine development is to induce potent antigen-specific immune responses. In this study, we examined the immunological activities of antigen-carrying biodegradable poly(gamma-glutamic acid) (gamma-PGA) nanoparticles (NPs) in mice. The immunization with ovalbumin (OVA)-carrying gamma-PGA NPs (OVA-NPs) could induce significant expansion of antigen-specific CD8(+) T cells. Unlike complete Freund's adjuvant, subcutaneous (s.c.) inoculation of OVA-NPs to footpad did not generate injection site swelling. Although OVA-NPs could induce both antigen-specific cellular and humoral immune responses, the dominant induction of either cellular or humoral immunity was found to depend on their administration routes. Strong antibody production was observed by s.c. immunization, yet no antibody was identified by intranasal immunization. Thus, gamma-PGA NPs are a safe and efficient antigen carrier with unique immunological properties.

The purpose of drug delivery systems in cancer chemotherapy is to achieve selective delivery of anti-cancer agents to cancer tissue at an effective concentrations for the appropriate duration of time, so that we may be able to reduce the adverse effects of a drug and simultaneously enhance the anti-tumor effect. Polymeric micelles were expected to increase the accumulation of drugs in tumor tissues utilizing the enhanced permeability and retention effect and to incorporate various kinds of drugs into the inner core by chemical conjugation or physical entrapment with relatively high stability. The size of the micelles can be controlled within the diameter range of 20-100 nm, to ensure that the micelles do not pass through normal vessel walls; therefore, a reduced incidence of the side effects of the drugs may be expected due to the decreased volume of distribution. There are several anti-cancer agent-incorporated micelle carrier systems under clinical evaluation. Phase 1 studies of a cisplatin-incorporated micelle, NC-6004 and an SN-38-incorporated micelle, NK012, are now underway. A Phase 2 study of a paclitaxel-incorporated micelle, NK105, against stomach cancer is also underway.

The effects of poly-gamma-glutamic acid (gamma-PGA) on calcium (Ca) bioavailability and Ca content in bone were examined in female rats. Increases in in vitro Ca solubility was observed with increases in the amount of gamma-PGA. Acute studies showed that gamma-PGA increased the amount of soluble Ca in the small intestine. In a plasma-Ca kinetic experiment, gamma-PGA stimulated Ca absorption 20 min after administration, and the duration of absorption was approximately 2 h. In an acute intestinal transit experiment, gamma-PGA markedly increased intestinal transit in mice. In a chronic Ca-balance experiment, the results showed that gamma-PGA significantly increased the apparent Ca absorption, apparent Ca balance, bone density, and bone Ca content in rats. In the same experiment, gamma-PGA was also found to decrease the amount of soluble Ca in the small intestine, especially in the proximal small intestine, and to increase calbindin-D9k mRNA expression in the proximal small intestine in rats. In a chronic intestinal transit experiment, gamma-PGA decreased intestinal transit in mice. These results suggest that gamma-PGA increased Ca solubility, thereby enhancing Ca absorption in rats in the acute phase. In addition, chronic administration of gamma-PGA to rats increased the apparent Ca balance and bone Ca content. The active transcellular pathway in the proximal small intestine was the major mechanism by which these effects were exerted.

Objective: There is evidence supporting a therapeutic range for methotrexate polyglutamate (MTXGlu) concentrations in the treatment of rheumatoid arthritis (RA). Knowledge of the pharmacokinetics of MTXGlu1-5 is required for optimal timing of blood sampling. The aim of this study was to determine the time to steady state and the half-life of accumulation of red blood cell (RBC) MTXGlu1-5 in patients with RA commencing oral MTX, and the time for RBC MTXGlu1-5 to become undetectable and the half-life of elimination of RBC MTXGlu1-5 in patients ceasing treatment with oral MTX., Methods: Ten patients beginning treatment and 10 patients stopping treatment with low-dose oral MTX were recruited. Blood samples were initially collected weekly, with gradual extension to monthly collection over the study period. RBC MTXGlu1-5 concentrations were assayed by high-performance liquid chromatography. Results were analyzed using a first-order exponential method., Results: The median times to reach steady state in RBCs (defined as 90% of the maximum concentration) were 6.2, 10.6, 41.2, 149, and 139.8 weeks, respectively, for MTXGlu1, MTXGlu2, MTXGlu3, MTXGlu4, and MTXGlu5. The median half-life of accumulation for RBC MTXGlu1-5 ranged from 1.9 weeks to 45.2 weeks. The median times for MTXGlus to become undetectable in RBCs were 4.5, 5.5, 10, 6, and 4 weeks, respectively, for MTXGlu1, MTXGlu2, MTXGlu3, MTXGlu4, and MTXGlu5. The median half-life of elimination for RBC MTXGlu1-5 ranged from 1.2 weeks to 4.3 weeks., Conclusion: There is wide interpatient variability of RBC MTXGlu1-5 accumulation and elimination in adults with RA. These data also suggest that after a dose change, >6 months are required for RBC MTXGlu1-5 to reach steady state. Such delays in achieving steady state suggest that more rapid dose escalation or subcutaneous administration from the outset should be considered.

Purpose: To estimate the maximum tolerated dose (MTD) of paclitaxel poliglumex (PPX) in combination with carboplatin in patients with chemotherapy-naive ovarian, primary peritoneal or fallopian tube cancer, and to assess the feasibility of administering multiple cycles of this regimen., Methods: The first 11 patients were treated in a standard 3 + 3 dose-seeking design, with carboplatin held constant at area under the curve (AUC) of 6 and PPX at 225, 175 or 135 mg/m(2). Pharmacokinetics of PPX and carboplatin were evaluated during this dose-seeking component of the trial. MTD was defined by acute dose-limiting toxicities (DLT) in the first cycle. Twenty additional evaluable patients were treated at the estimated MTD to assess the feasibility of this regimen over >or=4cycles., Results: PPX at 225 mg/m(2) resulted in DLT in 2/3 patients, and was de-escalated first to 175 mg/m(2) and then to 135 mg/m(2). PPX slowly hydrolyzed to paclitaxel and did not alter the pharmacokinetics of carboplatin. DLT within the first 4-cycles were observed in 3 patients (15%) treated at the MTD: neutropenia > 2weeks (2), febrile neutropenia (1). Nineteen patients (95%) experienced grade 4 neutropenia. Sixteen patients (80%) had at least one episode of grade 3 thrombocytopenia. Three patients (15%) had grade 2 and one had grade 3 peripheral neuropathy. Complete response by CA-125 was 75%., Conclusions: The recommended dose of PPX of 135 mg/m(2) with carboplatin (AUC = 6) in newly diagnosed ovarian cancer was feasible for multiple cycles, but hematologic toxicity was greater compared with standard carboplatin and 3-hour paclitaxel.

Introduction: Performance status (PS) 2 patients with non-small cell lung cancer (NSCLC) experience more toxicity, lower response rates, and shorter survival times than healthier patients treated with standard chemotherapy. Paclitaxel poliglumex (PPX), a macromolecule drug conjugate of paclitaxel and polyglutamic acid, reduces systemic exposure to peak concentrations of free paclitaxel and may lead to increased concentrations in tumors due to enhanced vascular permeability., Methods: Chemotherapy-naive PS 2 patients with advanced NSCLC were randomized to receive carboplatin (area under the curve = 6) and either PPX (210 mg/m/10 min without routine steroid premedication) or paclitaxel (225 mg/m/3 h with standard premedication) every 3 weeks. The primary end point was overall survival., Results: A total of 400 patients were enrolled. Alopecia, arthralgias/myalgias, and cardiac events were significantly less frequent with PPX/carboplatin, whereas grade > or =3 neutropenia and grade 3 neuropathy showed a trend of worsening. There was no significant difference in the incidence of hypersensitivity reactions despite the absence of routine premedication in the PPX arm. Overall survival was similar between treatment arms (hazard ratio, 0.97; log rank p = 0.769). Median and 1-year survival rates were 7.9 months and 31%, for PPX versus 8 months and 31% for paclitaxel. Disease control rates were 64% and 69% for PPX and paclitaxel, respectively. Time to progression was similar: 3.9 months for PPX/carboplatin versus 4.6 months for paclitaxel/carboplatin (p = 0.210)., Conclusion: PPX/carboplatin failed to provide superior survival compared with paclitaxel/carboplatin in the first-line treatment of PS 2 patients with NSCLC, but the results with respect to progression-free survival and overall survival were comparable and the PPX regimen was more convenient.