Search

Aims: To develop a non-linear mixed-effects population pharmacokinetic and pharmacodynamic (PK-PD) model describing the change in the concentration of methotrexate polyglutamates in erythrocytes (ery-MTX-PG n with "n" number of glutamate, representing PK component) and how this relates to modified 28-joint Disease Activity Score incorporating erythrocyte sedimentation rate (DAS-28-3) for rheumatoid arthritis (RA), representing PD component., Methods: An existing PK model was fitted to data from a study consisting of 117 RA patients. The estimation of population PK-PD parameters was performed using stochastic approximation expectation maximisation algorithm in Monolix 2021R2. The model was used to perform Monte Carlo simulations of a loading dose regimen (50mg subcutaneous methotrexate as loading doses, then 20mg weekly oral methotrexate) compared to a standard dosing regimen (10mg weekly oral methotrexate for 2 weeks, then 20mg weekly oral methotrexate)., Results: Every 40 nmol/L increase in ery-MTX-PG 3-5 total concentration correlated with 1-unit reduction in DAS-28-3. Significant covariate effects on the therapeutic response of methotrexate included the use of prednisolone in the first 4 weeks (positive use correlated with 25% reduction in DAS-28-3 when other variables were constant) and patient age (every 10-year increase in age correlated with 3.4% increase in DAS-28-3 when other variables were constant). 4 methotrexate loading doses led to a higher percentage of patients achieving a good/moderate response compared to the standard regimen (Week 4: 87.6% vs. 39.8%; Week 10: 64.7% vs. 57.0%)., Conclusions: A loading dose regimen was more likely to achieve higher ery-MTX-PG concentration and better therapeutic response after 4 weeks of methotrexate treatment., (© 2024 The Author(s). British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Objective: High-dose methotrexate (HD-MTX) is the mainstream therapy of current acute lymphoblastic leukemia (ALL) regimens, but frequent intra- and interindividual differences in the clinical response to HD-MTX lead to chemotherapeutic interruption or discontinuation. The exact mechanism of transport across the cell membrane and the disposition of active methotrexate metabolites-methotrexate polyglutamates (MTXPGs)-are not well described in the literature. The aim of this study was to gain more insight into the plasma distribution of methotrexate and MTXPGs in pediatric patients with ALL and to clarify the obscure pathways of MTXPGs., Methods: We prospectively measured the concentrations of MTXPG 1-7 in plasma samples from three male pediatric patients treated with HD-MTX and leucovorin rescue according to the IC-BFM 2009 protocol using liquid chromatography-mass spectrometry (LC-MS). Blood samples were obtained at 24, 36, 42, and 48 h after the start of HD-MTX treatment., Results: Noticeable plasma concentrations of MTXPGs with a 2.2-fold interpatient variability were detected. The highest interindividual variability in total plasma MTXPG concentration was observed at 36 h, and ranged from 13.78 to 30.82 μmol/L. Among all patients, the predominant polyglutamate types in relation to the total plasma MTXPG concentration at each time point were MTXPG 3 (16.71-30.02%) and MTXPG 5 (26.23-38.60%), while MTXPG 7 was the least abundant MTXPG (3.22-5.02%)., Conclusion: The presence of MTXPGs in plasma of patients with ALL could be related to the action of ABC efflux transporters on blood cells and hepatocytes resulting from the administration of high doses of methotrexate. This study may not draw definitive conclusions, but it does reduce uncertainty about the dynamics of methotrexate and its active metabolites, which may be of vital importance for achieving a clinical response., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

We previously found that a nanoparticle constructed with an antigen, benzalkonium chloride (BK) and γ-polyglutamic acid (γ-PGA) showed high Th1 and Th2-type immune induction after subcutaneous administration. For prophylaxis of respiratory infections, however, mucosal immunity should be induced. In this study, we investigated the effect of pulmonary administration of a nanoparticle comprising ovalbumin (OVA) as a model antigen, BK, and γ-PGA on induction of mucosal immunity in the lungs and serum. The complex was strongly taken up by RAW264.7 and DC2.4cells. After pulmonary administration, lung retention was longer for the OVA/BK/γ-PGA complex than for OVA alone. OVA-specific serum immunoglobulin (Ig)G was highly induced by the complex. High IgG and IgA levels were also induced in the bronchoalveolar lavage fluid, and in vivo toxicities were not observed. In conclusion, we effectively and safely induced mucosal immunity by pulmonary administration of an OVA/BK/γ-PGA complex.

Vaccination is the most effective way to prevent influenza virus infections. However, conventional vaccines based on hemagglutinin (HA) have to be annually updated because the HA of influenza viruses constantly mutates. In this study, we produced a 3M2e-3HA2-NP chimeric protein as a vaccine antigen candidate using an Escherichia coli expression system. The vaccination of chimeric protein (15 μg) conferred complete protection against A/Puerto Rico/8/1934 (H1N1; PR8) in mice. It strongly induced influenza virus-specific antibody responses, cytotoxic T lymphocyte activity, and antibody-dependent cellular cytotoxicity. To spare the dose and enhance the cross-reactivity of the chimeric, we used a complex of poly-γ-glutamic acid and alum (PGA/alum) as an adjuvant. PGA/alum-adjuvanted, low-dose chimeric protein (1 or 5 μg) exhibited higher cross-protective effects against influenza A viruses (PR8, CA04, and H3N2) compared with those of chimeric alone or alum-adjuvanted proteins in vaccinated mice. Moreover, the depletion of CD4 + T, CD8 + T, and NK cells reduced the survival rate and efficacy of the PGA/alum-adjuvanted chimeric protein. Collectively, the vaccination of PGA/alum-adjuvanted chimeric protein induced strong protection efficacy against homologous and heterologous influenza viruses in mice, which suggests that it may be a promising universal influenza vaccine candidate.

Melanoma is a highly malignant skin cancer that frequently metastasizes to the lung, bone, and brain at an early phase. Therefore, noninvasive detection of metastasized melanoma could be beneficial to determine suitable therapeutic strategies. We previously reported a biocompatible ternary anionic complex composed of plasmid DNA (pDNA), polyethyleneimine (PEI), and γ-polyglutamic acid (γ-PGA) based on an electrostatic interaction, which was highly taken up by melanoma cells (B16-F10), even if it was negatively charged. Here, we developed a radiolabeled γ-PGA complex by using indium-111 ( 111 In)-labeled polyamidoamine dendrimer (4th generation; G4) instead of pDNA and iodine-125 ( 125 I)-labeled PEI instead of native PEI, and evaluated its effectiveness as a melanoma-targeted imaging probe. This ternary complex was synthesized at a theoretical charge ratio; carboxyl groups of 111 In-diethylenetriaminepentaacetic acid (DTPA)-G4 : amino groups of 125 I-PEI : carboxyl groups of γ-PGA was 1 : 8 : 16, and the size and zeta potential were approximately 29 nm and -33 mV, respectively. This complex was taken up by B16-F10 cells with time. Furthermore, a biodistribution study, using normal mice, demonstrated its accumulation in the liver, spleen, and lung, where macrophage cells are abundant. Almost the same level of radioactivity derived from both 111 In and 125 I was observed in these organs at an early phase after probe injection. Compared with the normal mice, significantly higher lung-to-blood ratios of radioactivity were observed in the B16-F10-lung metastatic cancer model. In conclusion, the radiolabeled γ-PGA complex would hold potentialities for nuclear medical imaging of lung metastatic melanoma.

Methotrexate (MTX) during maintenance therapy is essential for curing acute lymphoblastic leukemia (ALL), but dosing strategies aiming at adequate treatment intensity are challenged by interindividual differences in drug disposition. To evaluate genetic factors associated with MTX metabolism, we performed a genome-wide association study in 447 ALL cases from the Nordic Society for Pediatric Haematology and Oncology ALL2008 study, validating results in an independent set of 196 patients. The intergenic single-nucleotide polymorphism rs1382539, located in a regulatory element of DHFR, was associated with increased levels of short-chain MTX polyglutamates (P = 1.1 × 10-8) related to suppression of enhancer activity, whereas rs35789560 in FPGS (p.R466C, P = 5.6 × 10-9) was associated with decreased levels of long-chain MTX polyglutamates through reduced catalytic activity. Furthermore, the FPGS variant was linked with increased relapse risk (P = .044). These findings show a genetic basis for interpatient variability in MTX response and could be used to improve future dosing algorithms., (© 2020 by The American Society of Hematology.)

We evaluated the suppressive effects of high-gamma-polyglutamic acid ( γ -PGA) natto on postprandial blood glucose level and insulin response. After confirming the eligibility of candidates using a pre-selective test with packaged white rice, a meal loading test including low- or high- γ -PGA natto (with 57.6 mg (LPGA) and 439.6 mg (HPGA) of γ -PGA, respectively) was conducted in men aged 20 to 70 years ( n = 29) and postmenopausal women aged ≤70 years ( n = 7). On each examination day, blood samples were obtained after they fasted overnight and for 120 min after test meal loading. The primary outcome of this study was the difference between the measurements of the incremental area under the curve (IAUC) for blood glucose 0 to 30 min after loading of LPGA and HPGA meals. The IAUCs for blood glucose and insulin after the HPGA meal were lower than those after the LPGA meal within 45 min (0 to 15 and 0 to 30 min: p < 0.001, 0 to 45 min: p < 0.01) and 1 h (all p < 0.001) of loading, respectively. The suppressive effects of HPGA natto on postprandial glucose response in the early phase, which possibly relates to the risk of dysglycemia and cardiovascular disease, were clarified.

Inefficient nanoparticle accumulation in solid tumors hinders the clinical translation of cancer nanomedicines. Herein, we proposed that sildenafil, a vasodilator ampholyte, could be used to promote nanoparticle accumulation by inducing vasodilation after its tumor acidity-triggered release from the nanocarriers. To confirm this, sildenafil was first encapsulated in a cisplatin-incorporated polymeric micelle. The dense PEG shell of the micelle reduced its endocytosis by cancer cells, which in return resulted in accumulative extracellular release of protonated sildenafil in the acidic tumor microenvironment. The released sildenafil was found to be more effective in enlarging the tumor blood vessels than could be achieved without sildenafil. As a result, we demonstrated considerable improvement in the intratumoral accumulation of the sildenafil-cisplatin co-loaded nanoparticle and its enhanced cancer therapeutic efficacy over the control group. Given the generality of a dense PEG shell and a hydrophobic part in most clinically developed nanomedicines, this work implies the great potential of sildenafil as a simple and universal adjuvant to selectively promote the intratumoral accumulation of nanomedicines, thus improving their clinical translation.

Genetic variants influencing the pharmacokinetics and/or pharmacodynamics of the chemotherapeutic drugs used in Acute Lymphoblastic Leukemia (ALL) therapy often contribute to the occurrence of treatment related toxicity (TRT). In this study, we explored the association of candidate genetic variants with early hematological TRT (grade 3-4) occurring within the first 100 days of low-dose methotrexate and 6-mercaptopurine based maintenance therapy (n = 73). Fourteen variants in the following candidate genes were genotyped using allele discrimination assay by real-time PCR: ABCB1 , DHFR , GGH , FPGS , MTHFR , RFC1 , SLCO1B1 , TPMT , and NUDT15 . Methotrexate polyglutamate (MTXPG3-5) levels in red blood cells were measured by LC-MS/MS. Early hematological TRT (grade 3-4) was seen in 54.9% of patients. The NUDT15 *3 allele was associated with early TRT occurrence [HR: 3.04 (95% CI: 1.5-6.1); p = 0.007]. Sensitivity of early TRT prediction improved (from 30.7% to 89.7%) by considering FPGS variant (rs1544105) carrier status along with NUDT15 *3 allele [HR = 2.7 (1.5-4.7, p = 0.008)]. None of the considered genetic variants were associated with MTXPG3-5 levels, which in turn were not associated with early TRT. NUDT15 *3 allele carrier status could be used as a stratifying marker for Indian ALL patients to distinguish patients at high or low risk of developing early hematological TRT.

Biomaterial-associated bacterial infection is one of the major causes of implant failure. The treatment of such an implant infection typically requires the elimination of bacteria and acceleration of tissue regeneration around implants simultaneously. To address this issue, an ideal implanted material should have the dual functions of bacterial infection therapy and tissue regeneration at the same time. Herein, an enzyme-responsive nanoplatform was fabricated in order to treat implant-associated bacterial infection and accelerate tissue regeneration in vivo. Firstly, Ag nanoparticles were pre-encapsulated in mesoporous silica nanoparticles (MSNs) by a one-pot method. Then, poly-l-glutamic acid (PG) and polyallylamine hydrochloride (PAH) were assembled by the layer-by-layer (LBL) assembly technique on MSN-Ag to form LBL@MSN-Ag nanoparticles. Furthermore, the LBL@MSN-Ag nanoparticles were deposited on the surface of polydopamine-modified Ti substrates. PG is a homogeneous polyamide composed of an amide linkage, which can be degraded by glutamyl endonuclease secreted by Staphylococcus aureus. Inductively coupled plasma spectroscopy (ICP) results proved that the LBL@MSN-Ag particles show a significant enzyme responsive release of Ag ions. Furthermore, results of antibacterial experiments in vitro showed that the Ti substrates modified with an LBL@MSN-Ag nanocoating presented an excellent antibacterial effect. As for an animal experiment in vivo, in a bacterium infected femur-defect rat model, the modified Ti implants effectively treated bacterial infection. More importantly, the results of micro-CT, haematoxylin-eosin staining and Masson's trichrome staining demonstrated that the modified Ti implants significantly promoted the formation of new bone tissue after implantation for 4 weeks. The present system paves the way for developing the next generation of implants with the functions of treating bacterial infection and promoting tissue regeneration.

Ultrasound-induced microbubble sonoporation has been shown to effectively improve drug/gene delivery efficiency by enhancing tissue and cell permeability. However, the microscale size and short duration of ultrasound contrast agents limit their accumulation in target areas. Here, a kind of ultrasound-triggered phase-transitioning and size-changing cationic nanodroplet, perfluoropentane/C 9 F 17 -PAsp(DET)/miR-122/poly(glutamic acid)- g -MeO-poly(ethylene glycol) (PGA- g -mPEG) ternary nanodroplets (PFP-TNDs/miR-122), was developed to deliver microRNA-122 (miR-122) for hepatocellular carcinoma (HCC) treatment. PFP served as an ultrasound-sensitive core for ultrasound-triggered phase transition and size change from the nanoscale to the microscale. Positively charged C 9 F 17 -PAsp(DET) ensured adequate miRNA loading. PGA- g -mPEG, which served as the shell of the nanodroplet, modified the nanodroplets, enhanced their stability in serum, and protected miR-122 from degradation in vivo. The results exhibited that PFP-TNDs/miR-122 has a nanosize diameter (362 ± 15 nm) and remained stable for 24 h. After treatment with PFP-TNDs/miR-122 combined with ultrasound irradiation, the miR-122 expression level was significantly increased by approximately 600-fold in HepG2 cells, 500-fold in SMMC-7721 cells, and 30-fold in human HCC xenografts. Moreover, PFP-TNDs/miR-122 combined with ultrasound radiation effectively suppressed the growth, migration, and invasion of HCC cells, and inhibited tumor proliferation in mice. This study revealed that the biodegradable PFP-TNDs is a promising therapeutic gene carrier with functions of gene protection and effective gene delivery for clinical applications. Furthermore, PFP-TNDs/miR-122 associated with ultrasound irradiation may pave a new way to improve the prognosis of patients with HCC.

Angiogenesis plays a pivotal role in tissue regeneration following bone-grafting procedures; however, nonautogenous graft materials typically lack critical angiogenic growth factors. While much research has focused on modifying grafts with angiogenic factors, controlled delivery of these molecules remains a challenge. The current study describes a method for sustained delivery of an angiogenic peptide from hydroxyapatite (HA), a common alloplast material. Specifically, VEGF-derived "QK" peptides were synthesized with polyglutamate domains containing varying numbers of glutamates. The rate of peptide release from HA inversely correlated with glutamate number, with diglutamate-QK (E2-QK) released first, followed by tetraglutamate-QK (E4-QK), and finally, heptaglutamate-QK (E7-QK). By coating HA with a mixture of these peptides, termed, PGM-QK (polyglutamate-modified mixture), sequential peptide release was achieved, enabling gradient QK delivery. To evaluate bioactivity, HA disks were coated with PGM-QK and then placed in fresh media for 6 days. Media containing the released peptides was collected at varying time intervals and placed on human umbilical vein endothelial cells (HUVECs). Cells were evaluated for activation of angiogenic signaling pathways (ERK and Akt) and cell migration. Results showed that QK peptides were continuously released over the 6-day interval, and maintained their capacity to activate HUVECs. These findings point to a new approach for gradient delivery of an angiogenic stimulus., (© 2019 Wiley Periodicals, Inc.)

The conventional prophylactic vaccines for human papillomavirus (HPV) efficiently prevent infection with high-risk HPV types, but they do not promote therapeutic effects against cervical cancer. Previously, we developed HPV16 E7-expressing Lactobacillus casei ( L. casei -E7) as a therapeutic vaccine candidate for cervical cancer, which induces antitumor therapeutic effects in a TC-1 murine cancer model. To improve the therapeutic effect of L. casei -E7, we performed co-treatment with poly-gamma-glutamic acid (γ-PGA), a safe and edible biomaterial naturally secreted by Bacillus subtilis . We investigated their synergistic effect to improve antitumor efficacy in a murine cancer model. The treatment with γ-PGA did not show in vitro cytotoxicity against TC-1 tumor cells; however, an enhanced innate immune response including activation of dendritic cells was observed. Mice co-administered with γ-PGA and L. casei -E7 showed significantly suppressed growth of TC-1 tumor cells and an increased survival rate in TC-1 mouse models compared to those of mice vaccinated with L. casei -E7 alone. The administration of γ-PGA markedly enhanced the activation of natural killer (NK) cells but did not increase the E7-specific cytolytic activity of CD8 + T lymphocytes in mice vaccinated with L. casei -E7. Overall, our results suggest that oral administration of γ-PGA induces a synergistic antitumor effect in combination with L. casei -E7.

Herein, an injectable formulation composed of a low molecular weight gelator (LMWG) based hydrogel and drug-loaded polymeric nanocapsules (NCs) is described. The NCs, made of hyaluronic acid and polyglutamic acid and loaded with C14-Gemcitabine (GEM C14), showed a size of 40 and 80 nm and a encapsulation efficiency >90%. These NCs exhibited a capacity to control the release of the encapsulated drug for >1 month. GEM C14-loaded NCs showed activity against various cancer cell lines in vitro; cell growth inhibition by 50% (GI 50 ) values of 15 ± 6, 10 ± 9, 13 ± 3 and 410 ± 463 nM were obtained in HCT 116, MIA PaCa-2, Panc-1 and Panc-1 GEM resistant cell lines respectively. Nanocomposite hydrogels were prepared using the LMWG - N4-octanoyl-2'-deoxycytidine and loaded for the first time with polymeric NCs. 2% and 4% w/v nanocapsule concentrations as compared to 8% w/v NC concentrations with 2% and 3% w/v gelator concentrations gave mechanically stronger gels as determined by oscillatory rheology. Most importantly, the nanocomposite formulation reformed instantly into a gel after injection through a needle. Based on these properties, the nanocomposite gel formulation has potential for the intratumoural delivery of anticancer drugs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This study investigated the impact of seven polymorphisms in genes of folate transport and (de)glutamation pathway on methotrexate polyglutamate levels and response in patients with rheumatoid arthritis. This prospective study included patients with rheumatoid arthritis. They were treated with methotrexate (up to 25 mg per week) for 24 weeks and categorized by EULAR response criteria into responders (good and moderate) and non-responders. Using real-time Taqman discrimination assay, SNPs were genotyped-rs1045642 (ABCB1 3435C>T), rs1128503 (ABCB1 1236C>T), rs10106 (FPGS 1994A>G), rs1544105 (FPGS G>A), rs11545078 (GGH 452C>T), rs3758149 (GGH -401C>T), and rs1051266 (RFC1 80G>A). RBC methotrexate polyglutamate 1-5 (MTX-glu 1-5 ) levels were determined at 4, 8, 16, and 24 weeks using by reverse phase HPLC using C-18 column followed by post column photo-oxidation. This study included 117 patients with rheumatoid arthritis (M:F = 14:103). The mean dose of methotrexate at 24 weeks was 22.0 ± 4.0 mg, with data on DAS28(3) at 24 weeks available in 96 patients-61 responders and 35 non-responders. Minor allele of GGH 452C>T had an association with non-response (odds ratio 2.9, 95% CI 1.4-5.6) and assuming the dominance of C, the recessive genetic model found GGH 452C>T CC genotype (odds ratio 9.5, 95% CI 1.2 to 76.0) was significantly associated with response. However, there was no difference in MTX-glu 1-5 levels among the various genotypes of this SNP (p = 0.9). Other SNPs were neither associated with response nor with alteration in methotrexate polyglutamate levels. On logistic regression, GGH 452C>T CC genotype and DAS28(3) at baseline were independent predictors of response. GGH 452C>T CC genotype was associated with response to methotrexate. None of the SNPs affected MTX-glu 1-5 levels.

Purpose: The subtle impairments of the disc due to anular punctures may have an immediate effect on the functional integrity due to the altered intradiscal pressure, hence the subsequent catabolic degradation. This study evaluates functional restoration of needle puncture injured intervertebral discs with a newly developed injectable hydrogel using the quantitative discomanometry (QD) test. The proposed hydrogel is composed of gelatin and poly (γ-glutamic acid) (γ-PGA) and crosslinked with 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC)., Methods: Thirty-six bovine motion segments were distributed into six groups. Needle puncture injured discs were created in all discs except for those in the first group (intact). The second group included injured discs that received no treatment (injury). The remaining four groups included injured discs repaired with injected hydrogels fabricated with different polymer solutions and EDC concentrations including: gelatin/γ-PGA solution crosslinked with the EDC solution at a 10:1 and 40:1 ratio to form the GP/E(10:1) and GP/E(40:1) groups, respectively, and gelatin and γ-PGA solution crosslinked with the EDC solution at a 10:1 ratio to form the G/E(10:1) or P/E(10:1) groups. The QD tests were performed to evaluate disc integrity of all six groups., Results: Among all hydrogel repair groups, the GP/E(10:1) group was found to have the highest leakage and saturate pressure and was the only group comparable to the intact one., Conclusions: Restoration of disc integrity secondary to needle puncture injury can be achieved via the repair with the newly developed gelatin hydrogel incorporated with γ-PGA and EDC. These slides can be retrieved under Electronic Supplementary Material.

Enterovirus 71 (EV71) is the major causative agent of hand-foot-and-mouth disease (HFMD) and many neurological manifestations. Recently, this virus has become a serious concern because of consecutive epidemics in the Asia-Pacific region. However, no effective vaccine for EV71 has been discovered except two EV71 vaccines which are being used in local communities of China. To develop a safe and efficient EV71 vaccine candidate, we generated inactivated EV71 and evaluated its efficacy with γ-PGA/Chitosan nanoparticles (PC NPs), which are safe, biodegradable and effective as an adjuvant. The subcutaneous administration of inactivated EV71 with PC NPs adjuvant induces higher levels of virus-specific humoral (IgG, IgG1, and IgG2a) and cell-mediated immune responses (IFN-γ and IL-4). Additionally, inactivated EV71 with PC NPs adjuvant induces significantly higher virus neutralizing antibody responses compared to the virus only group, and resulted in a long lasting immunity without any noticeable side effects. Together, our findings demonstrate that PC NPs are safe and effective immunogenic adjuvants which may be promising candidates in the development of more efficacious EV71 vaccines.

Conjugation of cisplatin to macromolecular carriers has been extensively studied for reducing systemic side effects. Here, a cisplatin-stitched α-poly(glutamatic acid) nanoconjugate with reduced adverse reactions and effective anti-tumor efficacy was synthesized via ionic interaction between platinum ion of cisplatin with carboxylic groups of α-poly(glutamatic acid). The nanoconjugate exhibited good water solubility, suitable size and polydispersity, almost spherical morphologies, and a sustained release profile without burst release. In vitro cytotoxicity and cell apoptosis assays performed in MCF-7 cells showed significantly decreased cytotoxicity of nanoconjugate compared with free cisplatin, and larger ratio of early apoptosis than late apoptosis. Quantitative cellular uptake assay also supported that conjugation of cisplatin to α-poly(glutamatic acid) reduced its cytotoxicity. Further studies revealed that the unique space structure of nanoconjugate acted as a shield for cisplatin against GSH detoxification under physiological conditions. In vivo studies regarding maximum tolerated dose, hematological parameters evaluation and histopathology assay demonstrated the superior safety of nanoconjugates. Furthermore, the nanoconjugates also achieved comparable antitumor efficacy with no apparent weight loss and death at a high equivalent cisplatin dose of 25 mg/kg. Moreover, the survival rate of mice treated with nanoconjugate was greatly larger than that of free cisplatin. These findings suggest that the cisplatin-stitched α-poly(glutamatic acid) nanoconjugate may hold great potential in clinical application for cancer therapy., (Copyright © 2018. Published by Elsevier B.V.)

Poly-gamma-glutamic acid (γ-PGA), an extracellular biopolymer produced by Bacillus sp., is a non-canonical toll-like receptor 4 (TLR4) agonist. Here we show its antiviral efficacy against noroviruses. γ-PGA with a molecular mass of 2,000-kDa limited murine norovirus (MNV) replication in the macrophage cell line RAW264.7 by inducing interferon (IFN)-β and conferred resistance to viral infection-induced cell death. Additionally, γ-PGA interfered with viral entry into cells. The potent antiviral state mounted by γ-PGA was not attributed to the upregulation of TLR4 or TLR3, a sensor known to recognize norovirus RNA. γ-PGA sensing by TLR4 required the two TLR4-associated accessory factors MD2 and CD14. In ex vivo cultures of mouse ileum, γ-PGA selectively increased the expression of IFN-β in villi. In contrast, IFN-β induction was negligible in the ileal Peyer's patches (PPs) where its expression was primarily induced by the replication of MNV. Oral administration of γ-PGA, which increased serum IFN-β levels without inducing proinflammatory cytokines, reduced MNV loads in the ileum with PPs and mesenteric lymph nodes in mice. Our results disclose a γ-PGA-mediated non-conventional TLR4 signaling in the ileum, highlighting the potential use of γ-PGA as a prophylactic antiviral agent against noroviruses.

The objective of this work was the development of a new drug nanocarrier intended to overcome the barriers associated to the oral modality of administration and to assess its value for the systemic or local delivery of peptides. The nanocarrier was rationally designed taking into account the nature of the intestinal barriers and was loaded with insulin, which was selected as a model peptide. The nanocarrier consisted of a complex between insulin and a hydrophobically-modified cell penetrating peptide (CPP), enveloped by a protecting polymer. The selected CPP was octaarginine (r8), chemically conjugated with cholesterol (Chol) or lauric acid (C12), whereas the protecting polymer was poly (glutamic acid)-poly (ethylene glycol) (PGA-PEG). This enveloping material was intended to preserve the stability of the nanocomplex in the intestinal medium and facilitate its diffusion across the intestinal mucus. The enveloped nanocomplexes (ENCPs) exhibited a number of key features, namely (i) a unimodal size distribution with a mean size of 200 nm and a neutral zeta potential, (ii) the capacity to associate insulin (~100% association efficiency) and protect it from degradation in simulated intestinal fluids, (iii) the ability to diffuse through intestinal mucus and, most importantly, (iv) the capacity to interact with the Caco-2 model epithelium, resulting in a massive insulin cell uptake (47.59 ± 5.79%). This enhanced accumulation of insulin at the epithelial level was not translated into an enhanced insulin transport. In fact, only 2% of insulin was transported across the monolayer, and this was correlated with a moderate response of insulin following oral administration to healthy rats. Despite of this, the accumulation of the insulin-loaded nanocarriers in the intestinal mucosa could be verified in vivo upon their labeling with 99m Tc. Overall, these data underline the capacity of the nanocarriers to overcome substantial barriers associated to the oral modality of administration and to facilitate the accumulation of the associated peptide at the intestinal level., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Hepatitis B virus (HBV) infection is a severe threat to public health, which can be prevented by prophylactic vaccination. Here, we tested nanogels carriers in the prophylactic effect of hepatitis B surface antigen (HBsAg) vaccine. HBsAg nanogels (Ng) were prepared using chitosan (CS) and poly-γ-glutamic acid (γ-PGA). Positively charged Ng (+) and negatively charged Ng (-) were prepared by adjusting the CS and γ-PGA proportion. Dendritic cells (DCs) maturation in mice immunized with HBsAg Ng (+) and HBsAg Ng (-) could be augmented in response to pAAV/HBV1.2 plasmid challenge. Single-dose immunization with HBsAg Ng (+) induced HBsAg specific-antibodies. HBsAg Ng (+) immunized mice cleared HBsAg and restored anti-HBs production after pAAV/HBV1.2 plasmid challenge. Single-dose HBsAg Ng (+) induced humoral and cellular immunity, and could induce effector memory T cells. Single-dose HBsAg Ng (-) favored the induction of cellular immunity, and induced central memory T cells and effector memory T cells. However, HBsAg elimination was similar between HBsAg Ng (+)- and HBsAg Ng (+) plus HBsAg Ng (-)-immunized mice. Zeta potential measurements showed that HBsAg Ng (+) were more stable than HBsAg Ng (-). Therefore, Ng (+) are desirable HBsAg prophylactic vaccine carriers, providing long-term protection against HBV, and are a good choice to study and apply weakly immunostimulatory antigens., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Purpose: Efficacy signals but substantial myelosuppression were demonstrated in a single arm phase II study of paclitaxel poliglumex (PPX) in combination with temozolomide (TMZ) and radiation therapy (RT) for first-line treatment of glioblastoma. The objective of this randomized phase II trial was to assess the efficacy and safety of single-agent PPX with RT (PPX/RT) versus TMZ with RT (TMZ/RT) for glioblastoma without O-methylguanine-DNA methyltransferase (MGMT) methylation., Materials and Methods: Patients with glioblastoma with unmethylated MGMT without prior chemotherapy or RT were eligible. Patients were randomly assigned 2:1 to PPX, 50 mg/m/wk for 6 weeks, or standard TMZ, with concurrent 60.0 Gy RT. One month after completion of chemoradiation all patients received standard maintenance TMZ. The primary endpoint was progression-free survival (PFS)., Results: Of the 164 patients enrolled, 86 were MGMT unmethylated. Of these, 63 patients were randomized (42 to PPX/RT and 21 to TMZ/RT). Fifty-nine patients could be analyzed. The median PFS was 9 months in the PPX/RT group and 9.5 months in the TMZ/RT group (hazard ratio in the PPX/RT group, 1.10; 95% confidence interval, 0.79-2.08; P=0.75). Median overall survival was 16 versus 14.8 months for PPX/RT and TMZ/RT groups, respectively (hazard ratio, 1.44; 95% confidence interval, 0.75-2.77; P=0.27). In the PPX and TMZ groups 44% versus 22% of patients, respectively, experienced one or more grade 3 or higher toxicities during chemoradiation., Conclusions: PPX/RT did not improve PFS or overall survival. This study provides an effective trial design for screening RT sensitizers in glioblastoma.

Purpose: Methotrexate (MTX) and adalimumab are well-recognized treatments of rheumatoid arthritis (RA), the efficacy of which may be driven by intracellular polyglutamates (PGs). The aim of this analysis was to characterize MTX PG concentrations and adalimumab pharmacokinetics in the CONCERTO trial. In addition, the relationships between MTX dose/pharmacokinetics, adalimumab pharmacokinetics, and efficacy were evaluated., Methods: CONCERTO was a double-blind, parallel-arm study in patients with early RA randomized to adalimumab 40 mg SC every other week plus blinded MTX 2.5, 5, 10, or 20 mg PO once weekly, for 26 weeks. Blood samples were obtained through week 26 for the determination of concentrations of MTX PG, adalimumab, and anti-adalimumab antibody (AAA). Clinical outcomes were also assessed., Findings: A total of 395 patients were included in the analysis (MTX, 329; adalimumab, 395). The mean time to steady-state MTX PG concentration was increased with MTX dose, from 8 to >26 weeks, depending on PG chain length. Dose proportionality changed with PG chain length. As MTX dose was increased, the percentage of short-chain PGs increased less than dose proportionally, while the percentage of long-chain PGs increased more than dose proportionally. For very-long-chain PGs, dose proportionality could not be assessed due to the nonmeasurable concentrations in the 2.5- and 5-mg MTX dose groups. As MTX dose increased, mean adalimumab concentrations also increased (P < 0.001). The percentage of patients with AAA decreased with increasing MTX dose, and at week 26, AAA + status was significantly correlated with MTX dose level (P = 0.005). In general, rates of response, defined using the 28-joint count disease activity score based on C-reactive protein (DAS28[CRP]; response, <3.2), were greater in the subgroup without AAA. The likelihood of a patient achieving a DAS28(CRP) response was related to the baseline measurement (P < 0.001) and to the concentration of adalimumab (P = 0.001), but not to the MTX regimen (P = 0.689)., Implications: The dose-response characteristics of MTX PG pharmacokinetics and the resultant effects of MTX on adalimumab exposures should be considered when determining the benefit-risk profile of MTX and adalimumab combination therapy in patients with early RA. ClinicalTrials.gov identifier: NCT01185301., (Copyright © 2018 Elsevier HS Journals, Inc. All rights reserved.)

Imbalance crosslink density and polymer concentration gradient is formed within the traditional alginate hydrogel using calcium chloride as a crosslinking agent in external gelation for instantaneously process. In this studying, type I collagen (Col I) blended calcium salt form of poly(γ-glutamic acid) (γCaPGA) was mixing with RGD-modified alginate with convenient gelation process and suitable for practical use. The hydrophilicity of the resulting hydrogels was evaluated through swelling tests, water retention capacity tests, and water vapor permeation tests. Mineralization was qualitatively evaluated by alizarin red dyeing at day 14, verifying the deposition of calcium. The in vitro osteogenic differentiation is monitored by determining the early and late osteocalcin (OCN) and osteopontin (OPN) markers with MG63 cells. Obtained results demonstrated that no extremely changes in mechanical properties. After 14 days of culture, hydrogels significantly stimulated OCN/OPN gene expressions and MG63 cell proliferation. Unusually, γCaPGA with RGD-modified alginate appeared better calcium deposition in 14 days than the other. However, addition of Col I can counterpoise RGD effect in blood coagulation and platelet adhesion made the hydrogel more flexibility and selectively in use. This studying provided that non-covalently crosslinked hydrogel by γCaPGA with alginate can be upgrading by RGD and Col I in water uptake capability, obviously effective for MG63 cells and are remarkably biocompatible and exhibited no cytotoxicity. Moreover, results also displayed the injectable process without complicated procedure, have high cost/performance ratio and have great potential for bone regeneration.

Purpose: NC-6004, a novel cisplatin nanoparticle developed using micellar technology exhibits sustained release of cisplatin and selective distribution to tumors. Preclinical data demonstrated a favorable tolerability profile and preserved or improved antitumor activity compared with cisplatin across animal models. We evaluated the safety and tolerability of NC-6004 and gemcitabine using a Bayesian continual reassessment model (N-CRM) to determine the optimal dose. Experimental Design: Patients with advanced solid tumors received NC-6004 at 60 to 180 mg/m 2 on day 1 and gemcitabine at 1,250 mg/m 2 on days 1 and 8 every 3 weeks. Dose escalation of NC-6004 began with a single patient run-in until a dose-limiting toxicity occurred at 180 mg/m 2 Cohorts of four patients were enrolled at doses predicted by the N-CRM. The maximum tolerated dose (MTD) was defined as having the greatest probability of target toxicity <25%. Quality of life was assessed using EORTC-QLQ-C30. Results: Among 22 patients, the most common grade III/IV hematologic adverse events were leukopenia (68%) and thrombocytopenia (59%). Of 20 pretreated patients evaluable for response, half were previously exposed to a platinum agent. The MTD was 135 mg/m 2 Nine patients were treated at the MTD with median treatment duration of 15 weeks (range, 3-50). Tumor shrinkage occurred in 11 (55%), partial responses in 3 (15%), and stable disease in 14 (70%). Most patients reported stable or improved EORTC QLQ-C30 scores. Conclusions: Greater cisplatin equivalent doses were achieved with no clinically significant neuro-, oto-, or nephrotoxicity. These data demonstrate tolerability and promising activity of NC-6004 in combination with gemcitabine. Clin Cancer Res; 24(1); 43-51. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Introduction: Platinum anticancer drugs are effective against a wide range of tumors, though their severe adverse effects and resistance remain unresolved. A breakthrough in these drawbacks could be achieved by using polymeric micelles, i.e. nanoassemblies having a drug loaded core and a protective hydrophilic shell, incorporating platinum drugs for tumor-targeted delivery. Areas covered: The development of cisplatin- (NC-6004) and DACHPt-loaded micelles (NC-4016) has been reviewed, particularly, from the viewpoint of the effect of their structural features on the tumor-targeting efficacy. We have also described new approaches for molecular targeting by installing ligand moieties on the surface of micelles. Moreover, small platinum drugs and platinum drug-loaded nanocarriers are introduced to explain the context for developing platinum drug-loaded polymeric micelles. Expert opinion: NC-6004 and NC-4016 micelles meet critical structural and functional requirements for achieving safe and effective tumor targeting, enhancing efficacy even against drug resistant cancer cells, and have been translated into human studies, aiming to be the first-of-their-kind in the clinic. Due to their flexible design, polymeric micelles could readily integrate new features based on the knowledge arising from the human clinical trials toward the development of innovative formulations with superior performance.

The microcarrier system offers an attractive method for cellular amplification and phenotype enhancement in the field of bone tissue engineering. However, it remains a challenge to fabricate porous microcarriers with osteoinductive activity for speedy and high-quality osseointegration in regeneration of serious complication of bone fracture, like nonunion. Here, we present a facile method for the first time manufacture microcarriers with osteogenic effects and properties based on well controlled and long-term Sr 2+ release. At first, strontium-substituted hydroxyapatite was prepared (Sr-HA) and a novel Sr-HA-graft-poly(γ-benzyl-l-glutamate) (Sr-HA-PBLG) nanocomposite was synthesized. Then, the microcarriers with highly interconnected macropores were fabricated by a double emulsion method, which allowed cells to adhere and proliferate and secrete extracellular matrix. Besides, the microcarriers with a relatively uniform diameter of 271.5 ± 45.0 μm are feasible for injection. The Sr-HA-PBLG microcarriers efficiently promoted osteogenic gene expression in vitro. With injection of the Sr-HA-PBLG microcarriers loading adipose derived stem cells (ADSCs) into the nonunion sites, bone regeneration was observed at 8 weeks after injection in a mice model.

Computational techniques are useful to predict interaction models and molecular properties for the design of drug delivery systems, such as dendrimers. This work evaluated the impact of surface modifications of mannosamine-conjugated multifunctional poly(glutamic acid) (PG)-dendrimers as nanocarriers of the tumour associated antigens (TAA) MART-1, gp100:44 and gp100:209. Molecular dynamics simulations and docking studies were performed. Nitrobenzoxadiazole (NBD)-PG-G4-dendrimer displayed 64 carboxylic groups, however, the Frontier Molecular Orbital Theory study evidenced that only 32 of those were available to form covalent bonds. When the number of mannosamines conjugated to dendrimer was increased from 16 to 32, the dendrimer interacted with the receptor with higher affinity. However, 16 mannosamines-NBD-PG-G4-dendrimer was chosen to conjugate TAA for added functionality as no carboxylic end groups were available for further conjugation in the 32 mannosamines-dendrimer. Docking results showed that the majority of TAA-conjugated NBD-PG-G4-dendrimer demonstrated a favourable interaction with mannosamine binding site on mannose receptor, thus constituting a promising tool for TAA targeted delivery. Our in silico approach effectively narrows down the selection of the best candidates for the synthesis of functionalised PG-dendrimers with desired functionalities. These results will significantly reduce the time and efforts required to experimentally synthesise modified dendrimers for optimal antigen delivery.

A novel hyaluronan (HA) derivative, poly(L-glutamic acid)-grafted hyaluronan (PGA-g-HA), was synthesized to improve the durability of conventional HA products for intra-articular injection. The purpose of this study was to investigate the characteristics of the novel HA derivative in terms of viscoelasticity, degradation behavior, non-immunogenicity, and bioactivity using preliminary in vitro and in vivo experiments. The storage modulus (G') and loss modulus (G″) of PGA-g-HA were similar to those of HA80 (approximately 8.0 × 10 5 Da) rather than those of original HA200 (approximately 2.0 × 10 6 Da). PGA-g-HA showed strong resistance against hyaluronidase hydrolysis compared to unmodified HA200. The immunogenicity resulting from grafting PGA to HA200 was not detected in bone marrow derived dendritic cells. The anti-inflammatory activity of PGA-g-HA was confirmed in IL-1β-stimulated chondrocytes. In addition, compared to unmodified HA200, the intra-articular injection of PGA-g-HA produced greater chondroprotective effects on a monoiodoacetic acid-induced model of rat knee osteoarthritis at two weeks after a single treatment. Therefore, PGA-g-HA is expected to be a promising medicine and biomedical device for intra-articular injection. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3006-3016, 2017., (© 2017 Wiley Periodicals, Inc.)

Anticancer immune responses depend on efficient presentation of tumor antigens and co-stimulatory signals provided by antigen-presenting cells (APCs). However, it is described that immature dendritic cells (DCs) and macrophages at the tumor site may have an immunosuppressive profile, which limits the activity of effector T cells and supports tumor progression. Therapeutic targeting of these innate immune cells, either aiming at their elimination or re-polarization towards an immunostimulatory profile, has been pointed as an attractive approach to control tumor progression. In the present work, we assessed the potential of Chitosan (Ch)/Poly(γ-glutamic acid) (γ-PGA) nanoparticles (NPs) to modulate macrophages and DCs inflammatory profile and to impair their ability to promote cancer cell invasion. Interestingly, Ch/γ-PGA NPs, prepared by co-acervation method, induced an immunostimulatory DCs phenotype, enhancing the expression of the co-stimulatory molecules CD86, CD40 and HLA-DR, and the secretion of the pro-inflammatory cytokines TNF-α, IL-12p40 and IL-6. Furthermore, Ch/γ-PGA NPs re-educated IL-10-stimulated macrophages towards a pro-inflammatory profile, decreasing the expression of CD163 and promoting the secretion of IL-12p40 and TNF-α. These alterations in the immune cells phenotype promoted CD4 + and CD8 + T cell activation/proliferation and partially inhibited APCs' ability to induce colorectal cancer cell invasion. Overall, our findings open new perspectives on the use of Ch/γ-PGA NPs as an immunomodulatory therapy for antigen-presenting cells reprogramming, providing a new tool for anticancer therapies., Statement of Significance: The immune system is responsible to detect and destroy abnormal cells preventing the development of cancer. However, the immunosuppressive tumor microenvironment can compromise the immune response favoring tumor progression. Thus, immune system modulation towards an immunostimulatory profile can improve anticancer therapies. This research focus on the development of chitosan/poly(γ-glutamic acid) nanoparticles (NPs) to modulate human antigen-presenting cells (APCs) phenotype and to counteract their pro-invasive capacity. Interestingly, Ch/γ-PGA NPs had a prominent effect in inducing macrophages and dendritic cells immunostimulatory phenotype, thus favoring T cell proliferation and inhibiting colorectal cancer cell invasion. We propose that their combination with other immunomodulatory drugs or conventional anticancer therapies can improve patients' outcome., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Combretastatin A4 (CA4) is a leading agent in vascular disrupting strategies for tumor therapy. Although many small-molecule prodrugs of CA4 have been developed to improve its solubility, the overall therapeutic efficiency is moderate. A key reason for this is the reversible effect that CA4 has on tubulin as well as its rapid clearance from plasma and tissues. In this study, we proposed a poly(l-glutamic acid)-CA4 conjugate (PLG-CA4) nanomedicine to fulfill the requirements for fully liberating the potential of CA4 on tumor therapy. Enhanced accumulation and retention of CA4 in tumor tissue, especially, high distribution and gradual release around tumor blood vessels resulted in prolonged vascular disruption and markedly enhanced therapeutic efficiency. We examined and compared the therapeutic effect of PLG-CA4 and commercial combretastatin-A4 phosphate (CA4P) in a murine colon C26 tumor. PLG-CA4 showed significantly prolonged retention in plasma and tumor tissue. Most importantly, the PLG-CA4 was mainly distributed around the tumor vessels because of its low tissue penetration in solid tumor. Pathology tests showed that PLG-CA4 treatment resulted in persistent vascular disruption and tumor damage 72h after a single injection, this in contrast to CA4P treatment, which showed quick relapse at an equal dose. Tumor suppression tests showed that PLG-CA4 treatment resulted in a tumor suppression rate of 74%, which indicates a significant advantage when compared to tumor suppression rate of the CA4P group, which was 24%. This is the first time that an advantage of the polymeric CA4 nanomedicine with low tissue penetration for solid tumor therapy has been shown. Thus, the results presented in this study provide a new idea for enhancing the tumor therapeutic effect of vascular disrupting agents., Statement of Significance: Nanomedicine usually has low tissue penetration in solid tumors, which limits the efficacy of nanomedicine in most cases. But herein, we demonstrate a nanosized vascular disruptive agent (VDA) PLG-CA4 has supper advantages over small molecular combretastatin-A4 phosphate (CA4P) because the PLG-CA4 was mainly distributed around the tumor vessels due to its low tissue penetration in solid tumor., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background: Adjustment of mercaptopurine and methotrexate maintenance therapy of acute lymphoblastic leukaemia by leucocyte count is confounded by natural variations. Cytotoxicity is primarily mediated by DNA-incorporated thioguanine nucleotides (DNA-TGN). The aim of this study was to establish whether DNA-TGN concentrations in blood leucocytes during maintenance therapy are associated with relapse-free survival., Methods: In this substudy of the NOPHO ALL2008 phase 3 trial done in 23 hospitals in seven European countries (Denmark, Estonia, Finland, Iceland, Lithuania, Norway, and Sweden), we analysed data from centralised and blinded analyses of 6-mercaptopurine and methotrexate metabolites in blood samples from patients with non-high-risk childhood acute lymphoblastic leukaemia. Eligible patients were aged 1·0-17·9 years; had been diagnosed with non-high-risk precursor B-cell or T-cell leukaemia; had been treated according to the Nordic Society of Pediatric Hematology and Oncology ALL2008 protocol; and had reached maintenance therapy in first remission. Maintenance therapy was (mercaptopurine 75 mg/m 2 once per day and methotrexate 20 mg/m 2 once per week, targeted to a leucocyte count of 1·5-3·0 × 10 9 cells per L). We measured DNA-TGN and erythrocyte concentrations of TGN nucleotides, methylated mercaptopurine metabolites, and methotrexate polyglutamates. The primary objective was the association of DNA-TGN concentrations and 6-mercaptopurine and methotrexate metabolites with relapse-free survival. The secondary endpoint was the assessment of DNA-TGN concentration and 6-mercaptopurine and methotrexate metabolites during maintenance therapy phase 2., Findings: Between Nov 26, 2008 and June 14, 2016, 1509 patients from the NOPHO ALL2008 study were assessed for eligibility in the DNA-TGN substudy, of which 918 (89%) of 1026 eligible patients had at least one DNA-TGN measurement and were included in the analyses. Median follow-up was 4·6 years (IQR 3·1-6·1). Relapse-free survival was significantly associated with DNA-TGN concentration (adjusted hazard ratio 0·81 per 100 fmol/μg DNA increase, 95% CI 0·67-0·98; p=0·029). In patients with at least five blood samples, erythrocyte concentrations of TGN, methylated mercaptopurine metabolites, and methotrexate polyglutamates were associated with DNA-TGN concentration (all p<0·0001)., Interpretation: Our results suggest the need for intervention trials to identify clinically applicable strategies for individualised drug dosing to increase DNA-TGN concentration, and randomised studies to investigate whether such strategies improve cure rates compared with current dose adjustments based on white blood cell counts., Funding: Danish Cancer Society, Childhood Cancer Foundation (Denmark), Childhood Cancer Foundation (Sweden), Nordic Cancer Union, Otto Christensen Foundation, University Hospital Rigshospitalet, and Novo Nordic Foundation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: It is unclear whether erythrocyte methotrexate polyglutamate levels (MTX-glun) are associated with response or adverse effects to methotrexate in rheumatoid arthritis. This preliminary study evaluated their utility in Asian Indian patients over 24 weeks., Methods: Rheumatoid arthritis patients were started on oral methotrexate at a dose of 15 mg/wk, which was escalated to 25 mg by 12 weeks and continued till 24 weeks. Erythrocyte (RBC) MTX-glu1 to MTX-glu5 levels (nmol/L RBC) were determined at 4, 8, 16, and 24 weeks by using reverse-phase high-performance liquid chromatography. Area under the concentration curve (AUC) of MTX-glu1-5, MTX-glu3-5, and MTX-glu3 levels was compared between groups with regards to response and adverse effects., Results: This study included 117 patients with mean (SD) age of 42.7 (±11.9) years and disease duration of 2.0 (1.7) years. Mean (SD) RBC MTX-glu1-5 levels at 4, 8, 16, and 24 weeks were 93 (±29), 129 (±46), 143 (±49), and 159 (±65) nmol/L RBC; the highest individual polyglutamate was MTX-glu3 (40%). There was significant correlation between MTX-glu1-5 (r = 0.38, P < 0.001) and MTX-glu3 (r = 0.49, P < 0.001) with methotrexate dose. There was no significant difference of AUC MTX-glun between responders and nonresponders. However, AUC MTX-glu3 was significantly (P = 0.03) higher in patients with adverse effects. On logistic regression, AUC of MTX-glu3 [odds ratio = 1.004 (95% confidence interval 1.002-1.007)] and methotrexate dose at 24 weeks were independent predictors of adverse effects., Conclusions: In this preliminary study, higher levels of RBC MTX-glu3 were found to be the independent predictors for adverse effects in rheumatoid arthritis patients.

Neural cell adhesion molecule (NCAM) expression is known to be associated with an aggressive biological behavior, increased metastatic capacity and expression of stem-cell markers in several tumor types. NCAM was also found to be expressed on tumor endothelial cells while forming new capillary-like tubes, but not on normal endothelial cells. An NCAM-targeted polymer-drug conjugate can be used both to target tumors expressing high levels of NCAM as well as the angiogenic vessels and cancer stem cells populations characterized by NCAM expression within tumors. Here, we describe the design, synthesis, physico-chemical characterization and the biological evaluation of an NCAM-targeted conjugate of polyglutamic acid with paclitaxel that was developed and evaluated on neuroblastoma, a high NCAM-expressing tumor. This conjugate inhibited tumor growth to a higher extent compared to the control conjugates and was less toxic than free paclitaxel. The dose of the conjugate could be increased at least twice than the maximum tolerated dose of paclitaxel to achieve better activity without aggravating toxicity. This work presents evidence that NCAM targeting can highly increase the efficacy of nanomedicines in the appropriate tumor models., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objectives: This study was an open-label phase I study to confirm the safety and tolerability of NC-6004 in combination with gemcitabine in Japanese patients with advanced solid tumors and to assess the PK effects of NC-6004 monotherapy., Methods: This phase I study used a 3 + 3 design to determine the maximum tolerated dose (MTD) and recommended dose of NC-6004 combined with gemcitabine. Safety and pharmacokinetics were assessed. The administration of NC-6004 alone was started at 60 mg/m 2 every treatment cycle (21 days per cycle). From the second through eighth cycles, patients received NC-6004 in combination with 1000 mg/m 2 of gemcitabine that was administered on day 1 and day 8 of each cycle, except for the first treatment cycle., Results: Twelve patients with advanced solid tumors received 60 or 90 mg/m 2 NC-6004. Both MTD and RD were determined to be 90 mg/m 2 . The most common drug-related adverse events were neutrophil decrease (66.7%) and white blood cell count decrease (41.7%). Population pharmacokinetic (PK) analysis revealed that NC-6004 PK profile in Japanese study was not significantly different from that in a previous Caucasian study., Conclusions: Both MTD and RD of NC-6004 were determined to be 90 mg/m 2 . The pharmacodynamic (PD) model well explained the time course of estimated glomerular filtration rate (eGFR) and amplitude of decrease in eGFR. The decrease in eGFR appeared to reach saturation at >100 mg/m 2 with NC-6004. Estimated probability of acute kidney injury on this PK/PD simulation was 30% with NC-6004 and 70% with cisplatin, which may better explain the renal toxicity profile.

Prebiotics improve the growth or activities of specific microbial genera and species in the gut microbiota in order to confer health benefits to the host. In this study, we investigated the effect of poly-gamma-glutamate (γ-PGA) as a prebiotic on the gut microbiota of mice and the organ distributions of γ-PGA in mice. Pyrosequencing analysis for 16S rRNA genes of bacteria indicated that oral administration of γ-PGA increased the abundance of Lactobacillales while reducing the abundance of Clostridiales in murine guts. It is suggested that oral administration of γ-PGA can be helpful for modulating the gut microbiota as a prebiotic.

Mesenchymal stem cells (MSCs) can ameliorate renal injury and accelerate repair of acute kidney injury. Herein, we developed a collagen/poly(γ-glutamic acid) (γ-PGA) hydrogel as an injectable scaffold for the delivery of mouse MSCs (mMSCs) and anti-oxidant drugs into injured sites. By the introduction of γ-PGA into conventional collagen, the viscosity of collagen was reduced at ambient temperature for easy handling, while the elastic and viscous moduli of collagen were increased and a new porous structure was generated near body temperature. When in situ gel-forming collagen/γ-PGA hydrogels loaded with mMSCs and α-lipoic acid (LA) were administered to a mouse model of renal dysfunction, they significantly attenuated the level of blood urea nitrogen and creatinine, which resulted from the increased retention of therapeutic mMSCs and the controlled release of anti-oxidant drugs at the injured site. These findings suggested that this novel type of hydrogel could be applied as an injectable scaffold for use in regenerative medicine.

Introduction: Methotrexate (MTX) is a known teratogenic drug used off-label in the treatment of ectopic pregnancies (EP). As MTX polyglutamated derivatives remains into the cells during several weeks, it is recommended to avoid conception during 3 to 6 months following MTX therapy. We report the follow-up of pregnancies after preconceptional exposure to MTX for EP., Material/methods: Prospective cases of pregnancy occurring within 3 months after MTX injection for an EP recorded in the Terappel database were analyzed., Results: Data were obtained on 52 pregnant women. The median age of patients was 28 (18-38), and the median gestational age at inclusion was 7 weeks after last menstrual period (3-22). The time between the last MTX injection and conception ranged from 12 days to 13 weeks and the total MTX dose was between 40 to 210mg. Out of 45 pregnancies with known outcome, there were 39 live births (87%), 3 spontaneous abortions (6.7%) occurring 63 to 94 days after MTX administration, 2 elective terminations, and 1 medical termination after premature rupture of membranes, oligohydramnios and arthrogryposis (48mg of MTX 9 and 8 weeks before conception). Two additional cases of major malformations were observed among 40 examinable babies or fetuses: tetralogy of Fallot (MTX 6 weeks before conception), and cerebral ventriculomegaly with normal karyotype (50mg of MTX 9 to 13 weeks before conception). The resulting rate of major malformations was 7.5% (95% CI: 1.6-20.4)., Discussion/conclusion: Although this prospective study shows a major malformation rate higher than expected in the general population, the observed malformations are not consistent with the typical pattern of methotrexate embryopathy. However, the case of tetralogy of Fallot is reminiscent of previously published cases with MTX exposure during early pregnancy. Owing to the small sample size, more powerful studies are needed to confirm or refute these findings., (Copyright © 2016 Société française de pharmacologie et de thérapeutique. Published by Elsevier Masson SAS. All rights reserved.)

This study was conducted to determine if humoral antibody response of foot-and-mouth disease (FMD) vaccine improved in 8-week-old growing pigs born to well-vaccinated sows pre-treated with 60 mg of poly-γ-glutamic acid (γ-PGA) three days before vaccination. Antibody against FMD virus serotype O was measured 0, 2, 4 and 6 weeks post-vaccination, using a PrioCHECK FMDV type O ELISA kit. The results showed that positive antibody reactions against FMDV serotype O antigen among a component of the vaccine significantly increased in response to pre-injection with γ-PGA.

Diabetes mellitus is a high prevalence and one of the most severe and lethal diseases in the world. Insulin is commonly used to treat diabetes in order to give patients a better life condition. However, due to bioavailability problems, the most common route of insulin administration is the subcutaneous route, which may present patients compliance problems to treatment. The oral administration is thus considered the most convenient alternative to deliver insulin, but it faces important challenges. The low stability of insulin in the gastrointestinal tract and low intestinal permeation, are problems to overcome. Therefore, the encapsulation of insulin into polymer-based nanoparticles is presented as a good strategy to improve insulin oral bioavailability. In the last years, different strategies and polymers have been used to encapsulate insulin and deliver it orally. Polymers with distinct properties from natural or synthetic sources have been used to achieve this aim, and among them may be found chitosan, dextran, alginate, poly(γ-glutamic acid), hyaluronic acid, poly(lactic acid), poly(lactide-co-glycolic acid), polycaprolactone (PCL), acrylic polymers and polyallylamine. Promising studies have been developed and positive results were obtained, but there is not a polymeric-based nanoparticle system to deliver insulin orally available in the market yet. There is also a lack of long term toxicity studies about the safety of the developed carriers. Thus, the aims of this review are first to provide a deep understanding on the oral delivery of insulin and the possible routes for its uptake, and then to overview the evolution of this field in the last years of research of insulin-loaded polymer-based nanoparticles in the academic and industrial fields. Toxicity concerns of the discussed nanocarriers are also addressed., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: The global outbreak of a novel swine-origin strain of the 2009 H1N1 influenza A virus and the sudden, worldwide increase in oseltamivir-resistant H1N1 influenza A viruses highlight the urgent need for novel antiviral therapy., Methods: Here, we investigated the antiviral efficacy of poly-gamma glutamate (γ-PGA), a safe and edible biomaterial that is naturally synthesized by Bacillus subtilis, against A/Puerto Rico/8/1934 (PR8) and A/California/04/2009 (CA04) H1N1 influenza A virus infections in C57BL/6 mice., Results: Intranasal administration of γ-PGA for 5 days post-infection improved survival, increased production of antiviral cytokines including interferon-beta (IFN-β) and interleukin-12 (IL-12), and enhanced activation of natural killer (NK) cells and influenza antigen-specific cytotoxic T lymphocytes (CTL) activity., Conclusions: These results suggest that γ-PGA protects mice against H1N1 influenza A virus by enhancing antiviral immune responses.

The focus of this study is to disclose a new delivery carrier intended to improve the pharmacokinetic characteristics of the anticancer drug plitidepsin and to favor its accumulation within the tumor. These nanocarriers named as nanocapsules, consist of an oily core surrounded by a highly PEGylated polyglutamic acid (PGA-PEG) shell loaded with plitidepsin. They showed a size of around 190 nm, a zeta potential of -24 mV and were able to encapsulate a high percentage (85%) of plitidepsin. In vivo studies, following intravenous injection in healthy mice, indicated that the encapsulation of the drug within PGA-PEG nanocapsules led to an important increase in its area under the curve (AUC) which is related to the important decrease of the clearance, as compared to the values observed for the drug dissolved in a Cremophor(®) EL solution. This improvement of the pharmacokinetic profile of the encapsulated plitidepsin was accompanied by a high increase (2.5-fold) of the maximum tolerated dose (MTD) in comparison to that of plitidepsin Cremophor(®) EL solution. The efficacy study performed in a xenograft tumor mice model evidenced the capacity of PGA-PEG nanocapsules to significantly reduce tumor growth. These promising results highlight the potential of PGA-PEG nanocapsules as an effective drug delivery system for cancer therapy., (Copyright © 2015. Published by Elsevier B.V.)

We investigated the effect of ultra-high molecular weight poly-γ-glutamic acid (UHMW γ-PGA) on hair loss in vitro and in vivo. 5-Alpha reductase is an enzyme that metabolizes the male hormone testosterone into dihydrotestosterone. By performing an in vitro experiment to analyze the inhibitory effects of UHMW γ-PGA on 5-alpha reductase activity, we determined that UHMW γ-PGA did in fact inhibit 5-alpha reductase activity, indicating the use of UHMW γ-PGA as a potential 5-alpha reductase inhibitor in the treatment of men with androgenetic alopecia. To evaluate the promotion of hair growth in vivo, we topically applied UHMW γ-PGA and minoxidil on the shaved dorsal skin of telogenic C57BL/6 mice for 4 weeks. At 4 weeks, the groups treated with UHMW γ-PGA showed hair growth on more than 50% of the shaved skin, whereas the control group showed less hair growth. To investigate the progression of hair follicles in the hair cycle, hematoxylin and eosin staining was performed. Histological observations revealed that the appearance of hair follicles was earlier in the UHMW γ-PGA-treated group than in the control group. The number of hair follicles on the relative area of shaved skin in the UHMW γ-PGA-treated group was higher than that observed on the shaved skin in the control group. These results indicate that UHMW γ-PGA can promote hair growth by effectively inducing the anagen phase in telogenic C57BL/6 mice.

Polymers are widely applied to drug delivery systems because polymers are generally excreted from the body more slowly than small molecules. Subcutaneous injection is one plausible means of administration. In this study, the in vivo behaviors of subcutaneously injected polymers, linear poly(glutamic acid) (Poly-Glu), acetylated dendrimer (Ac-den) and collagen peptide-conjugated dendrimer (CP-den), were investigated. Single photon emission computed tomography (SPECT) imaging was used to noninvasively monitor the in vivo behaviors. Diethylenetriaminepentaacetic acid (DTPA) was conjugated to these polymers, which were labeled with radioactive (111)In. These (111)In-DTPA-bearing polymers (Poly-Glu-DTPA, Ac-den-DTPA and CP-den-DTPA) and unconjugated DTPA were subcutaneously injected into tumor-bearing mice, which were subjected to SPECT imaging. These (111)In-DTPA-bearing polymers were largely retained at the injection site for at least 1 day, whereas the unconjugated DTPA was rapidly cleared from the whole body through excretion. Poly-Glu-DTPA and Ac-den-DTPA were partly accumulated in the kidney (and the liver), but the CP-den-DTPA was not. However, these (111)In-DTPA-bearing polymers were accumulated in the liver and the kidney following intravenous administration. These results indicate that the subcutaneously injected polymers did not largely gain substantial access to the systemic circulation, which is useful for a depot of drug around the injection site., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Protein therapeutics have become an important class of medicines for a large variety of diseases. However, they have disadvantages such as rapid elimination/metabolism leading to the need for repeated doses, immunogenicity/antigenicity, and aggregation/degradation during formulation and storage. The concept of polymer masked-unmasked protein therapy (PUMPT) makes use of polymer-protein multivalent conjugation with biodegradable carriers, which mask the protein activity during transport and increase its stability, but is capable of specifically triggering an unmasking effect at the disease site, allowing its therapeutic action. The aim of this study was to widen the PUMPT concept by designing reduction sensitive poly-l-glutamic acid (PGA)-based conjugates, in which the protein release and unmasking effect takes place in the reducing environments found intracellularly as well as in the tumor microenvironment. Lysozyme was used as the model protein to achieve proof of concept. Overall, the synthesized platform showed to be promising for the delivery of anticancer proteins as well as for enzyme replacement therapeutic approaches aiming to treat lysosomal storage disorders.

Objectives: Paclitaxel poliglumex (PPX), a drug conjugate that links paclitaxel to poly-L-glutamic acid, is a potent radiation sensitizer. Prior studies in esophageal cancer have demonstrated that PPX (50 mg/m/wk) can be administered with concurrent radiation with acceptable toxicity. The primary objective of this study was to determine the safety of the combination of PPX with temozolomide and concurrent radiation for high-grade gliomas., Methods: Eligible patients were required to have WHO grade 3 or 4 gliomas. Patients received weekly PPX (50 mg/m/wk) combined with standard daily temozolomide (75 mg/m) for 6 weeks with concomitant radiation (2.0 Gy, 5 d/wk for a total dose of 60 Gy)., Results: Twenty-five patients were enrolled, 17 with glioblastoma and 8 with grade 3 gliomas. Seven of 25 patients had grade 4 myelosuppression. Hematologic toxicity lasted up to 5 months suggesting a drug interaction between PPX and temozolomide. For patients with glioblastoma, the median progression-free survival was 11.5 months and the median overall survival was 18 months., Conclusions: PPX could not be safely combined with temozolomide due to grade 4 hematologic toxicity. However, the favorable progression-free and overall survival suggest that PPX may enhance radiation for glioblastoma. A randomized study of single agent PPX/radiation versus temozolomide/radiation for glioblastoma without MGMT methylation is underway.

Paclitaxel and doxorubicin are potent anticancer drugs used in the clinic as mono-therapies or in combination with other modalities to treat various neoplasms. However, both drugs suffer from side effects and poor pharmacokinetics. These two drugs have dissimilar physico-chemical properties, pharmacokinetics and distinct mechanisms of action, toxicity and drug resistance. In order to target both drugs selectively to the tumor site, we conjugated them at a synergistic ratio to a biocompatible and biodegradable polyglutamic acid (PGA) backbone. Drugs conjugation to a nano-sized polymer enabled preferred tumor accumulation by passive targeting, making use of the enhanced permeability and retention (EPR) effect. The rational design presented here resulted in co-delivery of combination of the drugs and their simultaneous release at the tumor site. PGA-paclitaxel-doxorubicin nano-sized conjugate exhibited superior anti-tumor efficacy and safety compared to the combination of the free drugs or a mixture of the drugs conjugated to separate polymer chains, at equivalent concentrations. This novel polymer-based multi-drug nano-sized conjugate allowed for true combination therapy since it delivered both drugs to the same target site at the ratio required for synergism. Using mice bearing orthotopic mammary adenocarcinoma, we demonstrate here the advantage of a combined polymer therapeutic bearing two synergistic drugs on the same polymer backbone, compared to each drug bound separately to the backbone., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We developed novel gene vectors composed of dendrigraft poly-L-lysine (DGL). The transgene expression efficiency of the pDNA/DGL complexes (DGL complexes) was markedly higher than that of the control pDNA/poly-L-lysine complex. However, the DGL complexes caused cytotoxicity and erythrocyte agglutination at high doses. Therefore, γ-polyglutamic acid (γ-PGA), which is a biodegradable anionic polymer, was added to the DGL complexes to decrease their toxicity. The resultant ternary complexes (DGL/γ-PGA complexes) were shown to be stable nanoparticles, and those with γ-PGA to pDNA charge ratios of >8 had anionic surface charges. The transgene expression efficiency of the DGL/γ-PGA complexes was similar to that of the DGL complexes; however, they exhibited lower cytotoxicity and did not induce erythrocyte agglutination at high doses. After being intravenously administered to mice, the DGL6 complex demonstrated high transfection efficiency in the liver, lungs, and spleen, whereas the DGL6/γ-PGA8 complex only displayed high transfection efficiency in the spleen. Future studies should examine the utility of DGL and DGL/γ-PGA complexes for clinical gene therapy., (Copyright © 2014 Elsevier B.V. All rights reserved.)

It is expected that the incidence of various adverse effects of anticancer agents maybe decreased owing to the reduced drug distribution in normal tissue. Anticancer agent incorporating nanoparticles including micelles and liposomes can evade non-specific capture by the reticuloendothelial system because the outer shell of the nanoparticles is covered with polyethylene glycol. Consequently, the micellar and liposomal carrier can be delivered selectively to a tumor by utilizing the enhanced permeability and retention effect. Presently, several anticancer agent-incorporating nano-carrier systems are under preclinical and clinical evaluation. Several drug delivery system formulations have been approved worldwide. Regarding a pipeline of clinical development of anticancer agent incorporating micelle carrier system, several clinical trials are now underway not only in Japan but also in other countries. A Phase 3 trial of NK105, a paclitaxel incorporating micelle is now underway. In this paper, preclinical and clinical studies of NK105, NC-6004, cisplatin incorporating micelle, NC-6300, epirubicin incorporating micelle and the concept of cancer stromal targeting therapy using nanoparticles and monoclonal antibodies against cancer related stromal components are reviewed., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)