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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.)

The aim of this study was to investigate the impact of genetic polymorphisms on methotrexate (MTX) metabolism in Korean children and young adults with acute lymphoblastic leukemia, specifically focusing on MTX polyglutamates (MTX-PGs) in erythrocytes, which have been rarely studied. Korean children and young adult patients undergoing maintenance therapy for acute lymphoblastic leukemia, who were receiving weekly oral MTX doses of 20 mg/m²/week, were prospectively included. We investigated erythrocyte MTX-PG (PG1 to PG5) levels, MTX-PG/MTX dose ratios, and 222 genetic polymorphisms spanning 78 genes and three intergenic areas related to MTX transport, folate cycle metabolism, purine/pyrimidine pathways, and non-pathway genes (including TPMT and NUDT15 genotypes) to explore their association with MTX metabolism. MTX-PG levels were associated with MTX dose (p < 0.05), and MTX-PG3 comprised the majority of the total MTX-PGs, with a median of 39.3 %. Various polymorphisms within the same gene demonstrated differing associations with each type of MTX-PG, underscoring the complexity of MTX pharmacogenetics. Among the polymorphisms examined, 14 across 13 genes showed significant associations with MTX-PG2-5 levels, even after adjusting for the false discovery rate (ABCC5, ATG16L1, CEP72, FSTL5, GMPS, HTR3A, IMPDH1, NT5C2, SLC28A3, SLCO1B3, SUCLA2, TPMT, and TYMS). This study enhances our understanding of genetic polymorphisms in MTX metabolism and therapeutic monitoring for MTX maintenance, promoting personalized medicine in acute lymphoblastic leukemia patients., Competing Interests: Declarations of competing interest The authors have declared no conflicts of interest., (Copyright © 2024. Published by Elsevier B.V.)

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.)

Purpose: Osteonecrosis is a burdensome treatment-related toxicity that is mostly diagnosed during or soon after 6-mercaptopurine (6MP)/methotrexate (MTX) maintenance therapy for acute lymphoblastic leukemia (ALL), possibly indicating a pathogenic role of these drugs., Methods: We prospectively registered symptomatic osteonecrosis during treatment of 1234 patients aged 1.0-45.9 years treated according to the Nordic Society of Hematology and Oncology (NOPHO) ALL2008 protocol. MTX/6MP metabolites were measured as part of the NOPHO ALL2008 maintenance therapy study., Results: After a median follow-up of 5.6 years [interquartile range (IQR) 3.6-7.5], 68 patients had been diagnosed with symptomatic osteonecrosis. The cumulative incidence was 2.7% [95% confidence interval (CI) 1.6-3.8%] for patients aged < 10 years, 14.9% (95% CI 9.7-20.2%) for patients aged 10.0-17.9 years, and 14.4% (95% CI 8.0-20.8%) for patients aged ≥ 18 years. The median time from diagnosis of ALL to diagnosis of osteonecrosis in these age groups was 1.0 year (IQR 0.7-2.0), 2.0 years (IQR 1.1-2.4), and 2.2 years (IQR 1.8-2.8), respectively (p = 0.001). With 17,854 blood samples available for MTX and 6MP metabolite analysis, neither erythrocyte levels of 6-thioguanine (TG) nucleotides (p > 0.99), methylated 6MP metabolites (p = 0.37), MTX polyglutamates (p = 0.98) nor DNA-TG (p = 0.53) were significantly associated with the hazard of osteonecrosis in Cox models stratified by the three age groups and adjusted for sex., Conclusion: Maintenance therapy intensity determined by 6MP and MTX metabolites was not associated with the risk of developing osteonecrosis in the NOPHO ALL2008 cohort., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Methotrexate is the gold standard treatment in rheumatoid arthritis. Once absorbed, it is internalized in cells, where glutamate residues are added to produce polyglutamated forms, which are responsible for the effect of methotrexate. The aim of the current study is to determine the relationship between methotrexate triglutamate concentrations and the clinical evolution in rheumatoid arthritis patients, as well as to characterize the variability in both features to propose strategies for low-dose methotrexate optimization. The quantification of methotrexate triglutamate concentration in red blood cells was performed through ultra-performance liquid chromatography coupled with mass spectrometry. Polymorphisms of genes involved in the formation of polyglutamates were determined by real-time polymerase chain reaction. A multivariate regression was performed to determine the covariates involved in the variability of methotrexate triglutamate concentrations and a population pharmacokinetics model was developed through nonlinear mixed-effects modeling. Disease activity score changed according to methotrexate triglutamate concentrations; patients with good response to treatment had higher concentrations than moderate or nonresponding patients. The methotrexate triglutamate concentrations were related to time under treatment, dose, red blood cells, and body mass index. A 1-compartment open model was selected to estimate the pharmacokinetic parameters; the typical total clearance (L/day) was determined as 1.45 * (body mass index/28 kg/m 2 ) * (red blood cells/4.6 × 10 6 cells/μL) and the volume of distribution was 52.4 L, with an absorption rate of 0.0346/day and a fraction metabolized of 1.03%. Through the application of the model, the initial dose of methotrexate is proposed on the basis of stochastic simulations and considering methotrexate triglutamate concentrations found in responders patients., (© 2021, The American College of Clinical Pharmacology.)

Objectives: The aim of the study was to explore the effect of total glucosides of paeony (TGP) and Tripterygium wilfordii polyglycosides (TWP) on erythrocyte methotrexate polyglutamates (MTXPGs), the metabolites of methotrexate (MTX)., Methods: An ultra-high-performance liquid chromatography (UPLC)-tandem mass spectrometry (MS/MS) method was developed to determine MTXPGs. The effects of MTXPGs were analysed using 24 male Sprague-Dawley rats that were randomly divided into the MTX alone, MTX-TGP combined, and MTX-TWP combined groups. Rats were administered MTX at a dose of 0.9 mg/kg once a week, TGP at 0.054 g/kg and TWP at 1.8 mg/kg three times a day. Venous blood (1.0 ml) was collected at weeks 2, 4, 6, 9, 12 and 15 and then analysed using the developed UPLC-MS/MS method., Key Findings: Specificity, linear range, inter-and intra-day precision, recovery, matrix effect and stability of MTXPGs met the standard regulations. This method was successfully used for the detection of MTXPGs. After administration of MTX alone, erythrocyte MTXPGs increased and accumulated in a time- and dose-dependent manner. Compared to MTX alone, the combination with TGP significantly decreased the content of total MTXPGs and short-chain MTXPGs (Methotrexate [MTX/MTXPG1] and 4-amino-10-methylpteroyldiglutamic acid [MTXPG2], P < 0.05), but had no significant effect on long-chain MTXPGs (4-amino-10-methylpteroyltriglutamic acid [MTXPG3], P > 0.05) and very long-chain MTXPGs (4-amino-10-methylpteroyltetraglutamic acid [MTXPG4] and 4-amino-10-methylpteroylpentaglutamic acid [MTXPG5], P > 0.05) at week 15. The combination of MTX with TWP had no significant effect on the content of total MTXPGs, short-chain MTXPGs and long-chain MTXPGs (P > 0.05), but it significantly decreased the content of very long-chain MTXPGs (P < 0.05) at week 15., Conclusions: The UPLC-MS/MS method was successfully used to determine MTXPGs in rat erythrocytes. TGP and TWP in combination with MTX affected the production of MTXPGs of different chain lengths in erythrocytes., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

BACKGROUNDInterpatient differences in the accumulation of methotrexate's active polyglutamylated metabolites (MTXPGs) in leukemia cells influence its antileukemic effects.METHODSTo identify genomic and epigenomic and patient variables determining the intracellular accumulation of MTXPGs, we measured intracellular MTXPG levels in acute lymphoblastic leukemia (ALL) cells from 388 newly diagnosed patients after in vivo high-dose methotrexate (HDMTX) (1 g/m2) treatment, defined ALL subtypes, and assessed genomic and epigenomic variants influencing folate pathway genes (mRNA, miRNA, copy number alterations [CNAs], SNPs, single nucleotide variants [SNVs], CpG methylation).RESULTSWe documented greater than 100-fold differences in MTXPG levels, which influenced its antileukemic effects (P = 4 × 10-5). Three ALL subtypes had lower MTXPG levels (T cell ALL [T-ALL] and B cell ALL [B-ALL] with the TCF3-PBX1 or ETV6-RUNX1 fusions), and 2 subtypes had higher MTXPG levels (hyperdiploid and BCR-ABL like). The folate pathway genes SLC19A1, ABCC1, ABCC4, FPGS, and MTHFD1 significantly influenced intracellular MTXPG levels (P = 2.9 × 10-3 to 3.7 × 10-8). A multivariable model including the ALL subtype (P = 1.1 × 10-14), the SLC19A1/(ABCC1 + ABCC4) transporter ratio (P = 3.6 × 10-4), the MTX infusion time (P = 1.5 × 10-3), FPGS mRNA expression (P = 2.1 × 10-3), and MTX systemic clearance (P = 4.4 × 10-2) explained 42% of the variation in MTXPG accumulation (P = 1.1 × 10-38). Model simulations indicated that a longer infusion time (24 h vs. 4 h) was superior in achieving higher intracellular MTXPG levels across all subtypes if ALL.CONCLUSIONSThese findings provide insights into mechanisms underlying interpatient differences in intracellular accumulation of MTXPG in leukemia cells and its antileukemic effectsFUNDINGTHE National Cancer Institute (NCI) and the Institute of General Medical Sciences of the NIH, the Basque Government Programa Posdoctoral de Perfeccionamiento de Personal Investigador doctor, and the American Lebanese Syrian Associated Charities (ALSAC).

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.

In this study, an adjustable pH-responsive drug delivery system using mesoporous silica nanoparticles (MSNs) as the host materials and the modified polypeptides as the nanovalves is reported. Since the polypeptide can self-assemble via electrostatic interaction at pH 7.4 and be disassembled by pH changes, the modified poly(l-lysine) and poly(l-glutamate) are utilized for pore blocking and opening in the study. Poly(l-lysine)-MSN (PLL-MSN) and poly(l-glutamate)-MSN (PLG-MSN) are synthesized via the ring opening polymerization of N-carboxyanhydrides onto the surface of mesoporous silica nanoparticles. The successful modification of the polypeptide on MSN is proved by Zeta potential change, X-ray photoelectron spectroscopy (XPS), solid state NMR, and MALDI-TOF MS. In vitro simulated dye release studies show that PLL-MSN and PLG-MSN can successfully load the dye molecules. The release study shows that the controlled release can be constructed at different pH by adjusting the ratio of PLL-MSN to PLG-MSN. Cellular uptake study indicates that the drug is detected in both cytoplasm and nucleus, especially in the nucleus. In vitro cytotoxicity assay indicates that DOX loaded mixture nanoparticles (ratio of PLL-MSN to PLG-MSN is 1:1) can be triggered for drug release in HeLa cells, resulting in 88% of cell killing., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The functionalization of a nanoparticle surface with PEG (polyethylene glycol) is an approach most often used for extending nanomaterial circulation time, enhancing its delivery and retention in the target tissues, and decreasing systemic toxicity of nanocarriers and their cargos. However, because PEGylated nanomedicines were reported to induce immune response including production of anti-PEG antibodies, activation of the complement system as well as hypersensitivity reactions, hydrophilic polymers other than PEG are gaining interest as its replacement in nanomaterial functionalization. Here, we present the results of in vivo evaluation of polyelectrolyte nanocapsules with biodegradable, polyelectrolyte multilayer shells consisting of poly-l-lysine (PLL) and poly-l-glutamic (PGA) acid as a potential drug delivery system. We compared the effects of nanocapsules functionalized with two different "stealth" polymers as the external layer of tested nanocapsules was composed of PGA (PGA-terminated nanocapsules, NC-PGA) or the copolymer of poly-l-lysine and polyethylene glycol (PEG-terminated nanocapsules, NC-PEG)., Methods: Nanocapsules pharmacokinetics, biodistribution and routes of eliminations were analysed postmortem by fluorescence intensity measurement. Toxicity of intravenously injected nanocapsules was evaluated with analyses of blood morphology and biochemistry and by histological tissue analysis. DNA integrity was determined by comet assay, cytokine profiling was performed using flow cytometer and detection of antibodies specific to PEG was performed by ELISA assay., Results: We found that NC-PGA and NC-PEG had similar pharmacokinetic and biodistribution profiles and both were eliminated by hepatobiliary and renal clearance. Biochemical and histopathological evaluation of long-term toxicity performed after a single as well as repeated intravenous injections of nanomaterials demonstrated that neither NC-PGA nor NC-PEG had any acute or chronic hemato-, hepato- or nephrotoxic effects. In contrast to NC-PGA, repeated administration of NC-PEG resulted in prolonged increased serum levels of a number of cytokines., Conclusion: Our results indicate that NC-PEG may cause undesirable activation of the immune system. Therefore, PGA compares favorably with PEG in equipping nanomaterials with stealth properties. Our research points to the importance of a thorough assessment of the potential influence of nanomaterials on the immune system., Competing Interests: The authors report no conflicts of interest in this work., (© 2019 Karabasz et al.)

Colorectal cancer (CRC) is a major malignancy characterized by a high metastasis rate. Systematic chemotherapy is important for patients with advanced CRC. However, many limitations (e.g., side effects to normal organs, shorter circulation time, and unsatisfactory tumor inhibition results) of traditional chemotherapy restrict its further application. Thus, it is necessary to find a method to overcome these challenges and improve the efficacy of CRC treatment. In this study, 20(S)-ginsenoside (Rg3) co-loaded poly(ethylene glycol)-block-poly(L-glutamic acid-co-L-phenylalanine) (mPEG-b-P(Glu-co-Phe)) nanoparticles (Rg3-NPs) were prepared. mPEG-b-P(Glu-co-Phe)-based drug delivery systems are pH sensitive that can target cancer cells and circulate for longer in blood. Rg3 could be released rapidly from the nanoparticles within tumor cells. A subcutaneous colon cancer mouse model was developed to evaluate the anticancer efficiency of the Rg3-NPs. The in vivo study indicated that the Rg3-NPs could significantly inhibit tumor proliferation by decreasing the expressions of proliferating cell nuclear antigen, resulting in tumor apoptosis through the increased expressions of caspase-3. Our study demonstrated the marked potential of the Rg3-NPs to treat CRC.

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.

Cyclization and polymer conjugation are two commonly used approaches for enhancing the pharmacological properties of protein drugs. However, cyclization of parental proteins often only affords a modest improvement in biochemical or cell-based in vitro assays. Moreover, very few studies have included a systematic pharmacological evaluation of cyclized protein-based therapeutics in live animals. On the other hand, polymer-conjugated proteins have longer circulation half-lives but usually show poor tumor penetration and suboptimal pharmacodynamics due to increased steric hindrance. We herein report the generation of a head-to-tail interferon-poly(α-amino acid) macrocycle conjugate circ-P(EG 3 Glu) 20 -IFN by combining the aforementioned two approaches. We then compared the antitumor pharmacological activity of this macrocycle conjugate against its linear counterparts, N-P(EG 3 Glu) 20 -IFN, C-IFN-P(EG 3 Glu) 20 , and C-IFN-PEG. Our results found circ-P(EG 3 Glu) 20 -IFN to show considerably greater stability, binding affinity, and in vitro antiproliferative activity toward OVCAR3 cells than the three linear conjugates. More importantly, circ-P(EG 3 Glu) 20 -IFN exhibited longer circulation half-life, remarkably higher tumor retention, and deeper tumor penetration in vivo. As a result, administration of the macrocyclic conjugate could effectively inhibit tumor progression and extend survival in mice bearing established xenograft human OVCAR3 or SKOV3 tumors without causing severe paraneoplastic syndromes. Taken together, our study provided until now the most relevant experimental evidence in strong support of the in vivo benefit of macrocyclization of protein-polymer conjugates and for its application in next-generation therapeutics.

The formation of gold nanoparticle (Au NP)-loaded γ-polyglutamic acid (γ-PGA) nanogels (NGs) for computed tomography (CT) imaging of tumors is reported. γ-PGA with carboxyl groups activated by 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide hydrochloride is first emulsified to form NGs and then in situ chemically cross-linked with polyethylenimine (PEI)-entrapped Au NPs with partial polyethylene glycol (PEG) modification ([(Au 0 ) 200 -PEI·NH 2 -mPEG]). The formed γ-PGA-[(Au 0 ) 200 -PEI·NH 2 -mPEG] NGs with a size of 108.6 ± 19.1 nm display an X-ray attenuation property better than commercial iodinated small-molecular-contrast agents and can be uptaken by cancer cells more significantly than γ-PGA-stabilized single Au NPs at the same Au concentrations. These properties render the formed NGs with an ability to be used as an effective contrast agent for the CT imaging of cancer cells in vitro and a tumor model in vivo. The developed hybrid NGs may be promising for the CT imaging or theranostics of different biosystems.

Recently we reported the development of 100nm polyglutamic acid (PGA)-based nanocapsules, which were intended to carry anticancer drugs to the lymphatic system (Abellan-Pose et al., 2016). In this work, the objective was to further assess the potential "lympho-targeting" properties of radiolabeled 111 In-PGA and 111 In-PGA-PEG, following intravenous or subcutaneous administration. The results indicate that, following intravenous administration, both types of nanocapsules exhibit a modest accumulation in the lymph nodes (⩽2.3% ID/g). On the contrary, following subcutaneous administration, and irrespective of the presence of PEG on their surface, the nanocapsules were found to form a reservoir at the injection site, from which they drained slowly into the popliteal and the iliac lymph nodes. The significant accumulation of the radiolabeled nanocapsules in the lymph nodes was attained at 24 and 48h post-injection, reaching values comprised between 70% and 187% ID/g in the popliteal lymph nodes. Altogether, the results led us to validate our hypothesis about the ability of the PGA and PGA-PEG nanocapsules to reach the lymphatic system, especially following subcutaneous administration., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

Unlabelled: The linear, Y-shaped, and linear-dendritic block copolymers of methoxy poly(ethylene glycol)-block-polyamidoamine-block-poly(l-glutamic acid) (MPEG-b-PAMAM-b-PGA) with one, two, four, and eight PGA arms but similar MPEG/PGA weight ratios (W/W) (named as P1PA, P2PA, P4PA and P8PA, respectively) were synthesized and comparatively investigated for doxorubicin hydrochloride (DOX) delivery. All the obtained block copolymers were highly biocompatible and could efficiently load DOX into nanoparticles (NPs) through electrostatic interaction. The NPs formed by linear (P1PA) or Y-shaped (P2PA) block copolymers and DOX were spherically shaped with smaller sizes, while the NPs formed from linear-dendritic block copolymers (P4PA and P8PA) were irregular in shape and larger in size. The P1PA/DOX and P2PA/DOX NPs exhibited better DOX protection and slower DOX release profile. However, cell cytotoxicity assays indicated that all the DOX-loaded NPs exhibited similar cytotoxicities with free DOX, indicating effective DOX release after cellular uptake. The NPs from linear and Y-shaped block copolymers greatly extended the blood circulation time, and displayed more accumulation in tumor site and less accumulation in the liver and kidney compared with the linear-dendritic counterparts. In addition, the P1PA/DOX and P2PA/DOX NPs also exhibited higher anti-tumor efficacy and less toxicity than the other DOX formulations. All these results indicated that the linear and Y-shaped MPEG-b-PAMAM-b-PGA block copolymers displayed better DOX delivery ability in anti-tumor treatment than the linear-dendritic copolymers., Statement of Significance: Polymeric NPs derived from block copolymers have emerged as effective vehicles for drug delivery. However, the majority of the researches in this field have involved simple linear block copolymers and there are very few comparative studies on the self-assembly, in vitro, and in vivo drug delivery by the block copolymers with similar composition but different architectures. In this study, a series of linear, Y-shaped, and linear-dendritic polypeptide-based block copolymers were prepared and thoroughly investigated for DOX delivery. These block polymers loaded DOX into NPs with different sizes and morphologies, and exhibited different anti-tumor capabilities both in vitro and in vivo. The results indicated that the architecture of the block copolymers played an important role in their drug delivery behaviors., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Purpose: A pharmacokinetic (PK) model is available for describing the time course of the concentrations of methotrexate (MTX or MTXGlu1) and its active polyglutamated metabolites (MTXGlu2-5) in red blood cells (RBCs). In this study, we aimed to simplify the MTX PK model and to optimise the blood sampling schedules for use in future studies., Methods: A proper lumping technique was used to simplify the original MTX RBC PK model. The sum of predicted RBC MTXGlu3-5 concentrations in both the simplified and original models was compared. The sampling schedules for MTXGlu3-5 or all MTX polyglutamates in RBCs were optimised using the Population OPTimal design (POPT) software., Results: The MTX RBC PK model was simplified into a three-state model. The maximum of the absolute value of relative difference in the sum of predicted RBC MTXGlu3-5 concentrations over time was 6.3 %. A five blood sample design was identified for estimating parameters of the simplified model., Conclusions: This study illustrates the application of model simplification processes to an existing model for MTX RBC PK. The same techniques illustrated in our study may be adopted by other studies with similar interest.

Encapsulating chemotherapy drugs in targeted nanodelivery systems is one of the most promising approaches to tackle cancer disease, avoiding side effects of common treatment. In the last decade, several nanocarriers with different nature have been tested, but polypeptide-based copolymers have attracted considerable attention for their biocompatibility, controlled and slow biodegradability as well as their low toxicity. In this work, we synthesized, characterized and evaluated poly(trimethylene carbonate)-bock-poly(L-glutamic acid) derived polymersomes, targeted to epidermal growth factor receptor (EGFR), loaded with plitidepsin and ultimately tested in HT29 and LS174T colorectal cancer cell lines for specificity and efficacy. Furthermore, morphology, physico-chemical properties and plitidepsin loading were carefully investigated. A thorough in vitro cytotoxicity analysis of the unloaded polymersomes was carried out for biocompatibility check, studying viability, cell membrane asymmetry and reactive oxygen species levels. Those cytotoxicity assays showed good biocompatibility for plitidepsin-unloaded polymersomes. Cellular uptake and cytotoxic effect of EGFR targeted and plitidepsin loaded polymersome indicated that colorectal cancer cell lines were.more sensitive to anti-EGFR-drug-loaded than untargeted drug-loaded polymersomes. Also, in both cell lines, the use of untargeted polymersomes greatly reduced plitidepsin cytotoxicity as well as the cellular uptake, indicating that the use of this targeted nanocarrier is a promising approach to tackle colorectal cancer disease and avoid the undesired effects of the usual treatment. Furthermore, in vivo assays support the in vitro conclusions that EGFR targeted polymersomes could be a good drug delivery system. This work provides a proof of concept for the use of encapsulated targeted drugs as future therapeutic treatments for cancer.

There is a need to develop new and innovative polymer carriers to be used as drug delivery systems and/or imaging agents owing to the fact that there is no universal polymeric system that can be used in the treatment of all diseases. Additionally, limitations with existing systems, such as a lack of biodegradability and biocompatibility, inevitably lead to side effects and poor patient compliance. New polymer therapeutics based on amino acids are excellent candidates for drug delivery, as they do not suffer from these limitations. This article reports on a simple yet powerful methodology for the synthesis of 3-arm star-shaped polyglutamic acid with well-defined structures, precise molecular weights (MW), and low polydispersity (Đ = <1.3). These were synthesized by ring-opening polymerization (ROP) of N-carboxyanhydrides (NCA) in a divergent method from novel multifunctional initiators. Herein, their exhaustive physicochemical characterization is presented. Furthermore, preliminary in vitro evaluation in selected cell models, and exhaustive in vivo biodistribution and pharmacokinetics, highlighted the advantages of these branched systems when compared with their linear counterparts in terms of cell uptake enhancement and prolonged plasma half-life.

The term amyloidosis describes neurological diseases where an abnormal protein is misfolded and accumulated as deposits in organs and tissues, known as amyloid, disrupting their normal function. In the most common familial amyloid polyneuropathy (FAP), transthyretin (TTR) displays this role primarily affecting the peripheral nervous system (PNS). Advanced stages of this inherited rare amyloidosis, present as fibril deposits that are responsible for disease progression. In order to stop disease progression, herein we designed an efficient family of nanoconjugates as fibril disrupters. These polymer conjugates are based on doxycycline (doxy), already in phase II trials for Alzheimer's disease, covalently linked to poly-l-glutamic acid (PGA). The conjugates were rationally designed, looking at drug loading and drug release rate by adequate linker design, always considering the physiological conditions at the molecular target site. Conjugation of doxycycline exhibited greater potential towards TTR fibril disaggregation in vitro compared to the parent drug. Exhaustive physico-chemical evaluation of these polymer-drug conjugates concluded that drug release was unnecessary for activity, highlighting the importance of an appropriate linker. Furthermore, biodistribution studies through optical imaging (OI) and the use of radiolabelled polymer-drug conjugates demonstrated conjugate safety profile and renal clearance route of the selected PGA-doxy candidate, settling the adequacy of our conjugate for future in vivo evaluation. Furthermore, preliminary studies in an FAP in vivo model at early stages of disease development showed non-organ toxicity evidences. This nanosized-system raises a promising treatment for advanced stages of this rare amyloidotic disease, and also presents a starting point for possible application within other amyloidosis-related diseases, such as Alzheimer's disease., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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.)

Background and Objectives: Therapeutic drug monitoring in patients with rheumatoid arthritis (RA) receiving methotrexate (MTX, MTXGlu1) has not been established. In this study, we aim to explore the relationship between red blood cell (RBC) concentrations of MTX and its polyglutamate metabolites (MTXGlu(n); n = 2, 3, 4, 5) and clinical response in RA patients commencing MTX., Methods: The binding activity of MTXGlu(n) to three putative enzymes involved in the MTX mechanism of action—dihydrofolate reductase, thymidylate synthase, and 5-aminoimidazole-4-carboxamide ribonucleotide transformylase—was simulated. RBC MTXGlu(n) concentrations that gave the highest inhibition activity across all three enzymes were linked with the disease activity score DAS28-3v (C-reactive protein [CRP]). A population pharmacokinetic-pharmacodynamic model was developed to describe the relationship between RBC MTX polyglutamate concentrations and clinical response in 12 RA patients commencing MTX., Results: The highest inhibition activity was with RBC MTXGlu(3-5). These polyglutamates were further evaluated for their relationship with DAS28-3v (CRP). Three of the 12 patients had a high DAS28-3v (CRP) at baseline (mean = 6.1) and showed a delayed response to MTX treatment. The remaining nine patients with a lower DAS28-3v (CRP) baseline (mean = 3.6) showed an immediate response. The developed MTX pharmacokinetic-pharmacodynamic model provided an acceptable description of the observed DAS28-3v (CRP) across all patients., Conclusions: The developed model describes a longitudinal relationship between RBC MTXGlu(3-5) concentrations and DAS28-3v (CRP) in patients with RA commencing MTX. Further work is required to determine whether measurement of RBC MTX polyglutamates might be useful for dose individualisation in patients with RA.

We established a ternary anionic complex constructed with polyamidoamine dendrimer (4th generation; G4) modified with chelating agents (diethylenetriamine pentaacetic acid (DTPA) derivative), polyethyleneimine (PEI), and γ-polyglutamic acid (PGA) as a safe nano-platform for molecular imaging. We prepared indium-111-labeled DTPA-G4/PEI/γ-PGA, and evaluated the effectiveness as a nuclear imaging probe for sentinel lymph node (LN), the first LN that drains the primary tumor. (111)In-DTPA-G4/PEI with strong cationic charge agglutinated with erythrocytes and showed extremely high cytotoxicity. By contrast, the anionic (111)In-DTPA-G4/PEI/γ-PGA had little agglutination activity with erythrocytes and no cytotoxicity, indicating their high biocompatibility. (111)In-DTPA-G4/PEI/γ-PGA was highly taken up by macrophage cells (high populations in LNs) comparable to (111)In-DTPA-G4/PEI. The uptake mechanisms of (111)In-DTPA-G4/PEI/γ-PGA were suggested to be both phagocytosis and γ-PGA-specific pathway. Upon administration of each (111)In-labeled nano-platform into rat footpads intradermally, significantly higher radioactivity of (111)In-DTPA-G4/PEI/γ-PGA was observed in the first draining popliteal LN when compared with that of (111)In-DTPA-G4/PEI. Moreover, (111)In-DTPA-G4/PEI/γ-PGA clearly visualized the sentinel LN with single photon emission computed tomography (SPECT) compared with (111)In-DTPA-G4/PEI. Thus, (111)In-DTPA-G4/PEI/γ-PGA can be useful as a nano-platform for molecular imaging including sentinel LN imaging., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objective: Low-dose methotrexate (MTX) is the anchor drug in the treatment for rheumatoid arthritis (RA). Response to MTX is related to the intracellular MTX-polyglutamate (MTX-PG) levels and little is known about its determinants. We aimed to define the determinants of erythrocyte MTX-PG concentrations in 2 prospective cohorts of patients with RA., Methods: Patients with RA treated with MTX from 2 longitudinal cohorts were included: 93 from the MTX-R study (Rotterdam, the Netherlands derivation cohort), and 247 from the treatment in Rotterdam Early Arthritis Cohort study (validation cohort). MTX-PG concentrations were measured at 3 months of treatment using liquid chromatography/mass spectrometry. The MTX-PG were used as outcome measure. Various sociodemographic, clinical, biochemical, and genetic factors were assessed at baseline. Associations with MTX-PG levels were analyzed using multivariate regression analysis., Results: Age was positively associated with MTX-PG1 (stβ 0.23, p=0.033) and total MTX-PG (stβ 0.23, p=0.018) in the derivation cohort, and with all MTX-PG in the validation cohort (MTX-PG1: stβ 0.13, p=0.04; MTX-PG2: stβ 0.21, p=0.001; MTX-PG3: stβ 0.22, p<0.001; MTX-PG4+5: stβ 0.25, p<0.001; and total MTX-PG: stβ 0.32, p<0.001). Erythrocyte folate levels were positively associated with MTX-PG3 (stβ 0.3, p=0.021) and total MTX-PG levels (stβ 0.32, p=0.022) in the derivation cohort, which was replicated for MTX-PG3 (stβ 0.15, p=0.04) in the validation cohort. Patients with the folylpolyglutamate synthase (FPGS) rs4451422 wild-type genotype had higher concentrations of MTX-PG3 (p<0.05), MTX-PG4+5 (p<0.05), and total MTX-PG (p<0.05) in both cohorts. In the combined cohort, MTX dose was positively associated with levels of MTX-PG3 (stβ 0.23, p<0.001), MTX-PG4+5 (stβ 0.30, p<0.001), and total MTX-PG (stβ 0.20, p=0.002), but negatively associated with MTX-PG2 levels (stβ -0.22, p<0.001)., Conclusion: Our prospective study shows that higher age, higher MTX dose, higher erythrocyte folate status, and the FPGS rs4451422 wild-type genotype are associated with higher MTX-PG concentrations. While only up to 21% of interpatient variability can be explained by these determinants, this knowledge may aid in the development of personalized treatment in RA.

Cisplatin (cis-diaminodichloroplatinum, CDDP) loaded methoxy poly (ethylene glycol)-block-poly (glutamic acid-co-phenyl alanine) [mPEG-b-P (Glu10 -co-Phe10 ) (PGlu10 ) and mPEG-b-P (Glu20 -co-Phe10 ) (PGlu20 )] nanoparticles with two different formulations (CDDP/PGlu10 and CDDP/PGlu20 ) are successfully developed in uniformly sizes. In 190 h, the CDDP/PGlu10 shows 30% release at physiological pH and 39% at lysosomal pH. Similarly, the CDDP/PGlu20 shows 60% release at physiological pH and 90% release at lysosomal pH. The sustained and controlled release of both formulations evidences the in vitro longevity of the nanoparticles. The cell proliferation inhibition of nanoparticles against human breast cancer cell line ZR-75-30 is dose and time dependent. Both CDDP/PGlu10 and CDDP/PGlu20 show excellent hemo compatibility as evaluated by hemolysis experiments. The in vivo fate of CDDP and CDDP loaded nanoparticles are evaluated by pharmacokinetics studies. Free CDDP underwgoes instant platinum concentration decrease after intravenous administration with 1.0 wt% left in 24 h while the CDDP loaded nanoparticles show prolonged blood circulation time with 5 wt% (CDDP/PGlu20 ) to 14 wt% (CDDP/PGlu10 ) left in 24 h. This prolonged blood circulation of CDDP loaded nanoparticles makes them as promising nanocarriers for tumor targeting delivery., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

Objective: The combination of methotrexate (MTX) with infliximab can modify infliximab pharmacokinetics and lower the incidence of antibodies against infliximab (ATIs). We hypothesised that the pharmacokinetic interaction between MTX and infliximab is related to activation of MTX to immunosuppressive MTX polyglutamates (MTXPGs)., Methods: Adult patients with rheumatoid arthritis receiving weekly MTX with infliximab for more than 3 months were enrolled in a cross-sectional study. Blood was collected at trough before the infusion of infliximab. Red blood cell (RBC) MTXPGs were measured using liquid chromatography, and circulating levels of infliximab were measured using a cell-based assay. ATIs were measured using enzyme immunoassays. Statistical analyses consisted of multiple regression and Wilcoxon tests., Results: In the 61 patients enrolled in the study, ATIs were detected in 11 (18%). Regression analyses revealed that lower infliximab levels (median 3.3 µg/ml) were associated with the presence of ATIs and lower RBC MTXPG levels (median 28 nmol/l) (p<0.05). Logistic regression revealed that RBC MTXPG levels above 25 nmol/l were associated with a 4.7-fold lower likelihood of having ATIs (OR=4.7; 95% CI 1.1 to 20.8; p=0.02). None of the 12 patients with RBC MTXPG levels above 50 nmol/l tested positive for ATIs., Conclusions: These hypothesis-generating data indicate that MTXPGs are associated with infliximab pharmacokinetics and ATI formation.

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.

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.

Novel smart microgel particles made of poly (L-glutamic acid-2-hydroxylethyl methacrylate) (PGH) and hydroxypropyl cellulose-acrylic acid (HPC-AA) have been successfully prepared via emulsion polymerization. The dynamic light scattering measurement reveals that the average hydrodynamic radius 〈Rh〉 and hydrodynamic radius distributions f (Rh) of the microgel particles depend on the temperature and pH value thus the microgel particles exhibit both pH- and temperature-sensitivity. In vitro release study shows that the amount of insulin released from microgels in the gastric juice (at pH 1.2) is significantly less than that in the intestinal fluid (at pH 6.8). These results indicate that the resultant microgels are of potential for use in intelligent oral drug delivery systems., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objectives: Methotrexate (MTX) is a cornerstone in the treatment of rheumatoid arthritis (RA). Although in general MTX is very effective, the major drawback is the large inter-patient variability in clinical response. The circulating levels of MTX polyglutamates (MTXPGs) are supposed to correlate with clinical efficacy, therefore having a potential role in drug monitoring. However, there is a controversial discussion about the importance of methotrexate polyglutamates as outcome parameters in the therapy of rheumatoid arthritis. The aim of the present study was to investigate the formation and pharmacokinetics of MTXPGs and to correlate their concentration with clinical response in MTX-naïve patients., Methods: The pharmacokinetics of erythrocyte MTXPGs was determined in samples of nineteen MTX-naïve patients by high pressure liquid chromatography (HPLC) using post-column photo-oxidation and fluorimetric detection. The relationship between erythrocyte concentrations of MTXPGs and the primary outcome parameter DAS-28 was assessed using the Spearman's correlation coefficient., Results: The short-chain polyglutamate MTXPG2 revealed to be a potential marker for clinical outcome in rheumatoid arthritis with a statistically significant positive correlation of MTXPG2 Cmax levels and improvement in DAS-28 (+0.518, p=0.023) over 16 weeks. Furthermore, Cmax levels of MTXPG2 negatively correlated with basophils (-0.478, p=0.038) and eosinophils (-0.531, p=0.019), both pro-inflammatory cells involved in the disease., Conclusions: MTXPG2 seems to be a potential indicator for clinical response and may serve as a marker for drug monitoring.

Methotrexate (MTX) is the first-line treatment for rheumatoid arthritis and is frequently used in the management of other forms of inflammatory arthritis. It is currently challenging to predict which patients will achieve adequate disease control and which patients will develop adverse effects while taking MTX. As an analog of dihydrofolic acid, MTX enters cells through the reduced folate carrier-1 protein, and is polyglutamated. MTX polyglutamates inhibit key enzymes in the folate pathway to produce an anti-inflammatory effect. It has been suggested that genetic polymorphisms in the folate pathway may influence intracellular folate and MTX polyglutamates pools, and thus MTX response. However, studies to identify genetic predictors have yielded inconclusive results. Nonreplication across studies has been attributed to insufficient statistical power as well as pharmacological and clinical confounders. Prospective studies, standardizing the definitions of response and toxicity, and application of genome-wide approaches may advance the search for genetic predictors of MTX response.

Poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) with surface poly-(γ-glutamic acid) (γ-PGA) were applied to enhance the transport of saquinavir (SQV) across the blood-brain barrier (BBB). PLGA NPs encapsulated SQV and grafted with γ-PGA to form drug carriers (γ-PGA/SQV-PLGA NPs) for crossing through a monolayer of human brain-microvascular endothelial cells (HBMECs) regulated with human astrocytes. The results revealed that a lower molecular weight of γ-PGA yielded a higher grafting efficiency of γ-PGA on PLGA NPs. In addition, γ-PGA with a low molecular weight accelerated the dissolution of SQV from γ-PGA/SQV-PLGA NPs. A higher grafting efficiency (more didecyl dimethylammonium bromide) and a lower molecular weight of γ-PGA increased the permeability of SQV across the BBB, in general. When the grafting efficiency was 85.2% at 6kDa of γ-PGA, γ-PGA/SQV-PLGA NPs reached about 6 times the permeability of free SQV (the maximal permeability). γ-PGA could also promote the endocytosis of NPs and expression of ornithine decarboxylase by HBMECs. γ-PGA/SQV-PLGA NPs are efficacious nanoparticulate carriers in delivering antiretroviral drug across the BBB., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Introduction: Despite an 80% initial response rate to the standard primary regimen of carboplatin and paclitaxel, most women with ovarian cancer will experience recurrence with incurable disease within five years and will be treated with several successive palliative regimens. Consequently, a significant need exists for chemotherapeutic agents, which are not only clinically efficacious, but have acceptable side-effect profiles. Paclitaxel poliglumex (PPX) is a recently developed taxane in which paclitaxel is conjugated to poly(l-glutamic acid), which renders it water soluble, reduces hypersensitivity reactions and preferentially targets it to the tumor., Areas Covered: This review covers pre-clinical pharmacokinetic data and key Phase I and II clinical trial results in ovarian cancer., Expert Opinion: While PPX is active in ovarian cancer, it is unclear at present whether it offers significant benefit in terms of its side-effect profile or outcomes over a standard taxane-based regimen as first-line therapy, or what role it will have in maintenance therapy as studies are ongoing.

Purpose: To determine whether shortening the infusion duration of high-dose methotrexate (HDMTX; 1 g/m(2)) affects the in vivo accumulation of active methotrexate polyglutamates (MTXPG(1-7)) in leukemia cells and whether this differs among major acute lymphoblastic leukemia (ALL) subtypes., Methods: From June 2000 through October 2007, 356 children with ALL were randomly assigned to receive initial single-agent treatment with HDMTX (1 g/m(2)) as either a 24-hour infusion or a 4-hour infusion at two pediatric hospitals in the United States. The primary outcome measures were the accumulation of MTXPG(1-7) in leukemia cells and the antileukemic effects (eg, inhibition of de novo purine synthesis in bone marrow ALL cells, and decrease in circulating ALL cells)., Results: The 24-hour infusion resulted in significantly higher amounts of MTXPG(1-7) in bone marrow leukemia cells (median: 1,695 v 1,150 pmol/10(9) cells, P = .0059), and better antileukemic effects. The 24-hour infusion had the greatest effect on MTXPG(1-7) accumulation in hyperdiploid ALL (median: 3,919 v 2,417 pmol/10(9) cells, P = .0038); T-cell ALL exhibited smaller differences in MTXPG(1-7) but greater antileukemic effects with the longer infusion (median decrease in leukemia cells: 88.4% v 51.8%, P = .0075). In contrast, infusion duration had no significant impact on MTXPG(1-7) accumulation or antileukemic effects in ALL with the t(12;21)/(ETV6-RUNX1) chromosomal translocation., Conclusion: Shortening the infusion time of HDMTX reduces accumulation of active methotrexate in leukemia cells and decreases antileukemic effects, with differing consequences among major ALL subtypes.

Poly(γ-benzyl-L-glutamate) (PBLG) derivatives are synthetic polypeptides for preparing nanoparticles with well controlled surface properties. The aim of this paper was to investigate the biodistribution of pegylated PBLG in rats. For this purpose, nanoparticles were prepared by a nanoprecipitation method using mixtures of different PBLG derivates, including a pegylated derivate to avoid mononuclear phagocyte system uptake. The morphology, size distribution, and surface charge of the nanoparticles were investigated as a function of the amount of polymer employed for the preparation. Moderately polydispersed nanoparticles (polydispersity index less than 0.2) were obtained. Their size increased with polymer concentration. The zeta potential values were negative whatever the formulations. The availability of polyethylene glycol chains on the nanoparticles' surface was confirmed by measuring the decrease in bovine serum albumin adsorption. For in vivo distribution studies, pegylated and nonpegylated nanoparticles were prepared with polymer mixtures containing PBLG-fluorescein isothiocyanate and imaged by fluorescence microscopy to measure their accumulation in liver and spleen tissues of rats after intravenous administration. Injection of stealth formulations resulted in negligible fluorescence in liver and spleen compared with nonpegylated formulations, which suggests that these nanoparticles are promising candidates as a stealth-type long-circulating drug carrier system and could be useful for active targeting of drugs while reducing systemic side effects.

Methotrexate (MTX) is widely used for the treatment of childhood acute lymphoblastic leukemia (ALL). The accumulation of MTX and its active metabolites, methotrexate polyglutamates (MTXPG), in ALL cells is an important determinant of its antileukemic effects. We studied 194 of 356 patients enrolled on St. Jude Total XV protocol for newly diagnosed ALL with the goal of characterizing the intracellular pharmacokinetics of MTXPG in leukemia cells; relating these pharmacokinetics to ALL lineage, ploidy and molecular subtype; and using a folate pathway model to simulate optimal treatment strategies. Serial MTX concentrations were measured in plasma and intracellular MTXPG concentrations were measured in circulating leukemia cells. A pharmacokinetic model was developed which accounted for the plasma disposition of MTX along with the transport and metabolism of MTXPG. In addition, a folate pathway model was adapted to simulate the effects of treatment strategies on the inhibition of de novo purine synthesis (DNPS). The intracellular MTXPG pharmacokinetic model parameters differed significantly by lineage, ploidy, and molecular subtypes of ALL. Folylpolyglutamate synthetase (FPGS) activity was higher in B vs T lineage ALL (p<0.005), MTX influx and FPGS activity were higher in hyperdiploid vs non-hyperdiploid ALL (p<0.03), MTX influx and FPGS activity were lower in the t(12;21) (ETV6-RUNX1) subtype (p<0.05), and the ratio of FPGS to γ-glutamyl hydrolase (GGH) activity was lower in the t(1;19) (TCF3-PBX1) subtype (p<0.03) than other genetic subtypes. In addition, the folate pathway model showed differential inhibition of DNPS relative to MTXPG accumulation, MTX dose, and schedule. This study has provided new insights into the intracellular disposition of MTX in leukemia cells and how it affects treatment efficacy.

Methotrexate (MTX) is one of the most commonly prescribed and most effective drugs for the treatment of rheumatoid arthritis (RA). Given the partial response of many patients and the side effect profile of the drug, there is considerable interest in identifying biomarkers to guide MTX therapy in RA. Upon entering cells, MTX is polyglutamated. Measuring MTX polyglutamate (MTX PG) levels in circulating red blood cells has been proposed as an objective measure to help optimize MTX therapy in RA. Data are conflicting with regard to the clinical utility of MTX PG measurements as a predictor of the efficacy or toxicity of low-dose MTX effects in RA. Should large, randomized clinical trials of this assay show consistent, reproducible, long-term correlations between MTX PG levels and efficacy or toxicity, this test could become a prominent tool for clinicians to optimize the use of MTX in treating RA.

Tumor-associated macrophages (TAMs) are diverse population containing multiple subtypes. M2 macrophages promote tumor growth and metastasis, in part by secreting a wide range of proangiogenic factors and growth factors. Selective depletion of M2 macrophages has been evaluated as a novel approach to anti-cancer therapy. In this study, a dual magneto-optical imaging probe, PG-Gd-NIR813 was synthesized and evaluated for non-invasive assessment of TAMs after intravenous injection. PG-Gd-NIR813 injected in nude rats bearing C6 tumors showed high uptake of the polymeric contrast agent in the tumor at 1 and 48 h after injection both in vivo and ex vivo optical imaging. T(1)-weighted MR imaging results showed accumulation of PG-Gd-NIR813 into the tumor necrotic area, which was confirmed by TUNEL staining of resected tumors. The uptake of PG-Gd-NIR813 within tumor necrosis decreased after animals were treated by the macrophage-depleting agent. Immunohistochemical staining demonstrated that PG-Gd-NIR813 colocalized with CD68 (marker for macrophages) and CD169 (marker for activated macrophages), but not with CD163 (residential macrophages). Using combined near-infrared fluorescence imaging and magnetic resonance imaging (MRI), we demonstrated that the accumulation of PG-Gd-NIR813 in tumors was mediated through M2 TAMs. Therefore, poly(L-glutamic acid) based reagents could be potentially used to image response to antitumor therapies targeted at M2 TAMs. Furthermore, poly(L-glutamic acid) is a promising carrier for candidate immunotherapeutics targeting M2 TAMs., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

The clinical relevance of the concept of bioavailability rests on two main principles. First, that measurement of the active component at the site of action is generally not possible and, secondly, that a relationship exists between on the one hand efficacy and/or safety and on the other hand concentration of the active compound or its active metabolite(s) in the systemic circulation. Applying these principles to the current knowledge on methotrexate (MTX), it is clear that bioavailability of MTX is an important parameter for optimal dosing. In this manuscript the current knowledge on MTX bioavailability is reviewed. This review reveals that bioavailability of MTX in higher oral doses is decreased, most probably by limitation of absorption from the gastro-intestinal tract. It is suggested that higher doses can be given either by splitting the oral dose or by parenteral administration. Both will result in improved bioavailability as compared with one higher oral dose. However, larger, prospective studies directly comparing the efficacy and safety of the splitted oral dose strategy and the switch to parenteral MTX are needed.

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.)

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.)

Methotrexate is used widely in the pharmacotherapy of juvenile idiopathic arthritis. Polyglutamates of methotrexate are active metabolites which accumulate in cells including erythrocytes. Their intracellular concentration may reflect methotrexate bioavailability and, at the same time, may serve as a bioindicator for optimization of methotrexate therapy and drug monitoring. Therefore, a simple and selective isocratic reversed phase chromatographic method with fluorescence detection (excitation/emission wavelengths of 370/463 nm) was developed which quantifies the sum of all methotrexate polyglutamates in erythrocytes as methotrexate after their enzymatic conversion with gamma-glutamylhydrolase. Separation was carried out on a Phenomenex GEMINI C18 column using a mobile phase flowing at a rate of 0.6 ml/min and consisting of a mixture (110:890:0.25 v/v) of acetonitrile, ammonium acetate buffer (0.05 M, pH=5.5) and hydrogen peroxide 30% (w/w). The method was found linear over the concentration range of 25-400 nmol/l. Its intra- and inter-day precision and accuracy were characterized by coefficients of variation and relative errors less than 20%. The limits of detection and quantification achieved 10.9 and 32.9 nmol/l, respectively. The method was proved suitable for monitoring the concentration of methotrexate polyglutamates in erythrocytes of patients with juvenile idiopathic arthritis.

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 study contrast-enhanced MRI guided photodynamic therapy with a pegylated bifunctional polymer conjugate containing an MRI contrast agent and a photosensitizer for minimally invasive image-guided cancer treatment., Methods: Pegylated and non-pegylated poly-(L-glutamic acid) conjugates containing mesochlorin e6, a photosensitizer, and Gd(III)-DO3A, an MRI contrast agent, were synthesized. The effect of pegylation on the biodistribution and tumor targeting was non-invasively visualized in mice bearing MDA-MB-231 tumor xenografts with MRI. MRI-guided photodynamic therapy was carried out in the tumor bearing mice. Tumor response to photodynamic therapy was evaluated by dynamic contrast enhanced MRI and histological analysis., Results: The pegylated conjugate had longer blood circulation, lower liver uptake and higher tumor accumulation than the non-pegylated conjugate as shown by MRI. Site-directed laser irradiation of tumors resulted in higher therapeutic efficacy for the pegylated conjugate than the non-pegylated conjugate. Moreover, animals treated with photodynamic therapy showed reduced vascular permeability on DCE-MRI and decreased microvessel density in histological analysis., Conclusions: Pegylation of the polymer bifunctional conjugates reduced non-specific liver uptake and increased tumor uptake, resulting in significant tumor contrast enhancement and high therapeutic efficacy. The pegylated poly(L-glutamic acid) bifunctional conjugate is promising for contrast enhanced MRI guided photodynamic therapy in cancer treatment.

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.

Objective: Natural and synthetic retinoids such as N-(4-hydroxyphenyl)retinamide (4HPR) have been used for prevention and treatment of a variety of cancers; however, relapse usually occurs after treatment is stopped. Furthermore, the retinoid analogues are insoluble in water, making it difficult for systemic administration. The purpose of this study was to develop and evaluate a water-soluble polymeric formulation of 4HPR that can release 4HPR over a period of a few days., Methods: 4HPR was bound to a synthetic polyamino acid poly(L-glutamic acid) (PG). PG-4HPR was evaluated for its release kinetics and in vitro anti-proliferative and in vivo antitumor activities against ovarian cancer cell lines., Results: The release profile of PG-4HPR in phosphate buffered saline at 37 degrees C followed a first order kinetic, with a rate constant of 8.8x10(-3) h(-1). Approximately 60% of 4HPR was released over a period of 100 h. In vitro, both 4HPR and PG-4HPR inhibited proliferation of three ovarian cancer cells lines (SKOV3, OVCA420, and OVCA433) and an immortalized human ovarian epithelium cell line (IOSE) in a time- and dose-dependent manner. Increasing the exposure time of SKOV3 cells to both agents from 1 to 5 days resulted in an increased apoptotic response. In vivo, PG-4HPR demonstrated significantly enhanced antitumor activities compared to 4HPR in both early treatment and later treatment protocols. Treatments with PG-4HPR suppressed the expression of VEGF and reduced blood flow into the tumor., Conclusions: PG-4HPR may have potential applications in the prevention and therapy of ovarian cancers.