A Radiolabeled Self-assembled Nanoparticle Probe for Diagnosis of Lung-Metastatic Melanoma.

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 (111In)-labeled polyamidoamine dendrimer (4th generation; G4) instead of pDNA and iodine-125 (125I)-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 111In-diethylenetriaminepentaacetic acid (DTPA)-G4 : amino groups of 125I-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 111In and 125I 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.

Targeted delivery of 20(S)-ginsenoside Rg3-based polypeptide nanoparticles to treat colon cancer.

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.

Macrocyclization of Interferon-Poly(α-amino acid) Conjugates Significantly Improves the Tumor Retention, Penetration, and Antitumor Efficacy.

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(EG3Glu)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(EG3Glu)20-IFN, C-IFN-P(EG3Glu)20, and C-IFN-PEG. Our results found circ-P(EG3Glu)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(EG3Glu)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.

Facile Formation of Gold-Nanoparticle-Loaded γ-Polyglutamic Acid Nanogels for Tumor Computed Tomography Imaging.

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 ([(Au0)200-PEI·NH2-mPEG]). The formed γ-PGA-[(Au0)200-PEI·NH2-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.

Well-Defined Star-Shaped Polyglutamates with Improved Pharmacokinetic Profiles As Excellent Candidates for Biomedical Applications.

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 (D = <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.

Simplification of a pharmacokinetic model for red blood cell methotrexate disposition.

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.

A cell-based pharmacokinetics assay for evaluating tubulin-binding drugs.

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.

Methotrexate polyglutamation in relation to infliximab pharmacokinetics in rheumatoid arthritis.

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.

A short-chain methotrexate polyglutamate as outcome parameter in rheumatoid arthritis patients receiving methotrexate.

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.

Plasma Distribution of Methotrexate and Its Polyglutamates in Pediatric Acute Lymphoblastic Leukemia: Preliminary Insights.

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 MTXPG1-7 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 MTXPG3 (16.71-30.02%) and MTXPG5 (26.23-38.60%), while MTXPG7 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.

Modeling mechanisms of in vivo variability in methotrexate accumulation and folate pathway inhibition in acute lymphoblastic leukemia cells.

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 Triglutamate as a Determinant of Clinical Response in Mexican Patients With Rheumatoid Arthritis: Pharmacokinetics and Dose Recommendation.

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/m2 ) * (red blood cells/4.6 × 106 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.

Effect of total glucosides of paeony and Tripterygium wilfordii polyglycosides on erythrocyte methotrexate polyglutamates in rats, analysed using ultra-high-performance liquid chromatography-tandem mass spectrometry.

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.

Maintenance therapy and risk of osteonecrosis in children and young adults with acute lymphoblastic leukemia: a NOPHO ALL2008 sub-study.

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.

Methotrexate bioavailability.

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.

Measurement of erythrocyte methotrexate polyglutamate levels: ready for clinical use in rheumatoid arthritis?

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.

Pharmacogenomics of intracellular methotrexate polyglutamates in patients' leukemia cells in vivo.

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).

An Adjustable pH-Responsive Drug Delivery System Based on Self-Assembly Polypeptide-Modified Mesoporous Silica.

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.

Enhanced nanoparticle accumulation by tumor-acidity-activatable release of sildenafil to induce vasodilation.

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.