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Molecular pharmaceutics [journal]
- Simultaneously Targeting Tissue Transglutaminase and Kidney Type Glutaminase Sensitizes Cancer Cells to Acid Toxicity and Offers New Opportunities for Therapeutic Intervention. [JOURNAL ARTICLE]
- Mol Pharm 2014 Nov 26.
Most cancer cells undergo characteristic metabolic changes that are commonly referred to as the Warburg effect, with one of the hallmarks being a dramatic increase in the rate of lactic acid fermentation. This leads to the production of protons, which in turn acidifies the microenvironment surrounding tumors. Cancer cells have acquired resistance to acid toxicity, allowing them to survive and grow under these detrimental conditions. Kidney type glutaminase (GLS1), which is responsible for the conversion of glutamine to glutamate, produces ammonia as part of its catalytic activities, and has been shown to modulate cellular acidity. In this study, we show that tissue, or type 2, transglutaminase (TG2), a γ-glutamyl transferase which is highly expressed in metastatic cancers and produces ammonia as a byproduct of its catalytic activity, is up-regulated by decreases in cellular pH, and helps protect cells from acid-induced cell death. Since both TG2 and GLS1 can similarly function to protect cancer cells, we then proceeded to demonstrate that treatment of a variety of cancer cell types with inhibitors of each of these proteins results in synthetic lethality. The combination doses of the inhibitors induce cell death, while individual treatment with each compound shows little or no ability to kill cells. These results suggest that combination drug treatments that simultaneously target TG2 and GLS1 might provide an effective strategy for killing cancer cells.
- The Role of Drug-Polymer Hydrogen Bonding Interactions on the Molecular Mobility and Physical Stability of Nifedipine Solid Dispersions. [JOURNAL ARTICLE]
- Mol Pharm 2014 Nov 26.
We investigated the influence of drug-polymer hydrogen bonding interactions on molecular mobility and the physical stability in solid dispersions of nifedipine with each of the polymers polyvinylpyrrolidone (PVP), hydroxypropylmethyl cellulose (HPMCAS), and poly(acrylic acid) (PAA). The drug-polymer interactions were monitored by FT-IR spectroscopy, the molecular mobility was characterized using broadband dielectric spectroscopy, and the crystallization kinetics was evaluated by powder X-ray diffractometry. The strength of drug-polymer hydrogen bonding, the structural relaxation time, and the crystallization kinetics were rank ordered as PVP > HPMCAS > PAA. At a fixed polymer concentration, the fraction of the drug bonded to the polymer was the highest with PVP. Addition of 20% w/w polymer resulted in ∼65-fold increase in the relaxation time in the PVP dispersion and only ∼5-fold increase in HPMCAS dispersion. In the PAA dispersions, there was no evidence of drug-polymer interactions and the polymer addition did not influence the relaxation time. Thus, the strongest drug-polymer hydrogen bonding interactions in PVP solid dispersions translated to the longest structural relaxation times and the highest resistance to drug crystallization.
- On the Injection Site Radioactivity of 99mTc-Labeled Mannosylated Dextran for Sentinel Lymph Node Mapping. [JOURNAL ARTICLE]
- Mol Pharm 2014 Nov 25.
The high and persistent radioactivity at the injection site hinders the accuracy and expansion of sentinel lymph node (SLN) mapping. We investigated the mechanism underlying the undesirable radioactivity after subcutaneous injection of 99mTc-labeled mannosylated dextran (99mTc(CO)3-DCM20), a SLN mapping agent targeting mannose receptors on macrophages and dendritic cells, in a mouse model. Biodistribution studies were performed 1 h after subcutaneous injection of 99mTc(CO)3-DCM20 from the rear footpad of mice in the presence of varying molar amounts of DCM20 or DC15, a modified dextran without mannose. Biodistribution studies were also conducted after subcutaneous injection of [125I]radioiodinated mannosyl-neoglycoalbumin (125I-NMA) from the rear footpad. The distribution of fluorescence-labeled DCM20 and DC15 at the injection site was also compared 1 h after subcutaneous injection by immunofluorescent histochemistry. The radioactivity levels of 99mTc(CO)3-DCM20 at the injection site and popliteal lymph node, a SLN in this model, decreased with an increase in molar amounts of DCM20, whereas no significant changes in biodistribution were observed after injection of 99mTc(CO)3-DCM20 with varying molar amounts of DC15. 125I-NMA exhibited rapid elimination of radioactivity from both the popliteal lymph node and the injection site. The fluorescence-labeled DCM20 co-localized well with CD68-positive cells such as macrophages and dendritic cells at the injection site. While partial co-localization was observed between DC15 and CD68-positive cells, the signal intensity was very weak. These findings suggest that specific binding of 99mTc(CO)3-DCM20 to the mannose receptor on macrophages and dendritic cells would be responsible for the sustained radioactivity levels at the injection site. These results also imply that discriminated blockage of 99mTc(CO)3-DCM20 binding to mannose receptors at the injection sites would reduce the radioactivity at the injection site.
- Roughness-controlled Self-assembly of Mannitol/LB Agar Microparticles by Polymorphic Transformation for Pulmonary Drug Delivery. [JOURNAL ARTICLE]
- Mol Pharm 2014 Nov 25.
Novel roughness-controlled mannitol/LB Agar microparticles were synthesized by polymorphic transformation and self-assembly method using hexane as the polymorphic transformation reagent and spray-dried mannitol/LB Agar microparticles as the starting material. As-prepared microparticles were characterized by Fourier transform infrared spectra (FTIR), X-ray diffraction spectra (XRD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), thermal gravimetric analysis (TGA), and Andersen Cascade Impactor (ACI). The XRD and DSC results indicate that after immersing spray-dried mannitol/LB Agar microparticles in hexane, β-mannitol was completely transformed to α-mannitol in 1 h, and all the δ-mannitol was transformed to α form after 14 days. SEM shows that during the transformation, the nanobelts on the spray-dried mannitol/LB Agar microparticles become more dispersed and the contour of the individual nanobelts becomes more noticeable. Afterwards, the nanobelts self-assemble to nanorods and result in rod-covered mannitol/LB Agar microparticles. FTIR indicates new hydrogen bonds were formed among mannitol, LB Agar and hexane. SEM images coupled with image analysis software reveal that different surface morphology of the microparticles have different drug adhesion mechanisms. Comparison of ACI results and image analysis of SEM images shows that an increase in the particle surface roughness can increase the fine particle fractions (FPFs) using the rod-covered mannitol microparticles as drug carriers. Transformed microparticles show higher FPFs than commercially available lactose carriers. An FPF of 28.6±2.4% was achieved by microparticles transformed from spray-dried microparticles using 2% mannitol(w/v)/LB Agar as feed solution. It is comparable to the highest FPF reported in the literature using lactose and spray-dried mannitol as carriers.
- A novel inhaled combination powder containing amorphous colistin and crystalline rifapentine with enhanced antimicrobial activities against planktonic cells and biofilm of Pseudomonas aeruginosa for respiratory infections. [JOURNAL ARTICLE]
- Mol Pharm 2014 Nov 25.
Colistin has been increasingly used for the treatment of respiratory infections caused by Gram-negative bacteria. Unfortunately parenteral administration of colistin can cause severe adverse effects. This study aimed to develop an inhaled combination dry powder formulation of colistin and rifapentine for the treatment of respiratory infections. The combination formulation was produced by spray-drying rifapentine particles suspended in an aqueous colistin solution. The combination dry powder had enhanced antimicrobial activities against planktonic and biofilm cultures of Pseudomonas aeruginosa, with both minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC) values (2 and 4 mg/L, respectively) being half that of pure colistin (MIC 4 mg/L and MBIC 8 mg/L) and one-sixteenth that of pure rifapentine (MIC 32 mg/L and MBIC 64 mg/L). High aerosol performance, as measured via an Aerolizer device, was observed with emitted doses > 89% and FPFtotal fine particle fraction (FPF) total > 76%. The proportion of submicron particles of rifapentine particles was minimised by the attachment of colistin which increased the overall particle mass and aerodynamic size distribution. Using the spray-drying method described here, stable particles of amorphous colistin and crystalline rifapentine were distributed homogenously in each stage of the impinger. Unlike the colistin alone formulation, no deterioration in aerosol performance was found for the combination powder when exposed to a high relative humidity of 75%. In our previous study, surface coating of by rifampicin contributed to the moisture protection of colistin.; while hHere, a novel approach with a new mechanism was proposed whereby: moisture protection was attributed to the carrier effect of elongated crystalline rifapentine particles which minimised contacts between hygroscopic colistin particles. This inhaled combination antibiotic formulation with enhanced aerosol dispersion efficiency and in vitro efficacy could become a superior treatment for respiratory infections.
- Phenotype of TPBG Gene Replacement in the Mouse and Impact on the Pharmacokinetics of an Antibody-Drug Conjugate. [JOURNAL ARTICLE]
- Mol Pharm 2014 Nov 25.
The use of predictive preclinical models in drug discovery is critical for compound selection, optimization, preclinical to clinical translation, and strategic decision-making. Trophoblast glycoprotein (TPBG), also known as 5T4, is the therapeutic target of several anti-cancer agents currently in clinical development, largely due to its high expression in tumors and low expression in normal adult tissues. In this study, mice were engineered to express human TPBG under endogenous regulatory sequences by replacement of the murine Tpbg coding sequence. The gene replacement was considered functional since the hTPBG knock-in (hTPBG-KI) mice did not exhibit clinical observations or histopathological phenotypes that are associated with Tpbg gene deletion, except in rare instances. The expression of hTPBG in certain epithelial cell types and in different microregions of the brain and spinal cord was consistent with previously reported phenotypes and expression patterns. In pharmacokinetic studies, the exposure of a clinical-stage anti-TPBG antibody-drug conjugate (ADC), A1mcMMAF, was lower in hTPBG-KI versus wildtype animals, which was evidence of target-related increased clearance in hTPBG-KI mice. Thus the hTPBG-KI mice constitute an improved system for pharmacology studies with current and future TPBG-targeted therapies and can generate more precise pharmacokinetic and pharmacodynamic data. In general the strategy of employing gene replacement to improve pharmacokinetic assessments should be broadly applicable to the discovery and development of ADCs and other biotherapeutics.
- Intestinal Permeability Study of Minoxidil: Assessment of Minoxidil as a High Permeability Reference Drug for Biopharmaceutics Classification. [JOURNAL ARTICLE]
- Mol Pharm 2014 Nov 25.
The purpose of this study is to evaluate minoxidil as a new high permeability reference drug for Biopharmaceutics Classification System (BCS). The permeability of minoxidil was determined in in situ intestinal perfusion studies in rodents and permeability studies across Caco-2 cell monolayers. The permeability of minoxidil was compared with that of metoprolol, an FDA reference drug for BCS classification. In rat perfusion studies, the permeability of minoxidil was 2.6-fold higher than that of metoprolol in the jejunum while minoxidil showed less permeability than metoprolol in the ileum. The permeability of minoxidil was independent on intestinal segment but the permeability of metoprolol was dependent on it. In mouse perfusion study, the permeability of minoxidil was 2.5-fold higher than that of metoprolol. Minoxidil and metoprolol showed similar permeability in Caco-2 study at apical pH 6.5 and basorateral pH 7.4. The permeability of minoxidil was independent on pH while metoprolol showed pH dependent transport in Caco-2 study. Minoxidil exhibited similar permeability in absorptive direction (AP-BL) in comparison with secretory direction (BL-AP), while metoprolol had higher efflux ratio (ER>2) at apical pH 6.5 and basolateral pH 7.4. No concentration-dependent transport was observed for both minoxidil and metoprolol transport in Caco-2 study. Verapamil didn't alter the transport of both compounds across Caco-2 cell monolayers. The permeability of minoxidil was independent on both pH and intestinal segment in intestinal perfusion studies and Caco-2 studies. Caco-2 studies also showed no involvement of carrier mediated transport in absorption process of minoxidil. These results for minoxidil suggest it to be a good reference drug for BCS high permeability classification. However, minoxidil exhibited a higher permeability than metoprolol and, thus, to use minoxidil as a reference drug would raise the permeability criteria for BCS high permeability classification.
- Cellular Uptake Mechanism of TCTP-PTD in Human Lung Carcinoma Cells. [JOURNAL ARTICLE]
- Mol Pharm 2014 Nov 25.
We reported previously that human translationally controlled tumor protein (TCTP) contains, at its NH2-terminus, a protein transduction domain (PTD), which we called TCTP-PTD, with the amino acid sequence MIIYRDLISH. In this report we describe how TCTP-PTD penetrates A549 human lung cancer cell membranes and promotes protein internalization. Cellular uptake of fluorescent TCTP-PTD and a recombinant fusion protein consisting of TCTP-PTD and GFP (green fluorescent protein) was analyzed by confocal fluorescence microscopy and flow cytometry. Inhibitor assays using several agents that perturb the internalization process revealed that TCTP-PTD transduces the cells partly via lipid-raft/caveola-dependent endocytosis and partly by macropinocytosis in a dynamin/actin/microtubule-dependent pathway. To trace the pathway followed by the penetration of TCTP-PTD, the localization of PTDs was investigated in the lipid-raft, subcellular, and ER fractions. We found that, after entry, TCTP-PTD is localized in the cytoplasm and cytoskeleton, but not in the nucleus, and is transported into endoplasmic reticulum (ER). Expression levels of caveolin-1 in A549 and HeLa cells are different, and these differences appear to contribute to the sensitivity of TCTP-PTD uptake inhibition, against lipid-raft depleter, nystatin. This elucidation of the underlying mechanism of TCTP-PTD translocation may help the design of approaches that employ TCTP-PTD in the cellular delivery of bioactive molecules.
- Relating Toxicity to Transfection: Using Sphingosine to Maintain Prolonged Expression In Vitro. [JOURNAL ARTICLE]
- Mol Pharm 2014 Nov 24.
Cationic reagents are commonly used to facilitate DNA delivery, and transfection experiments are typically initiated in cell culture where the optimal charge ratio is determined. While transfection rates are often enhanced at higher +/- charge ratios, the cellular toxicity associated with the greater amounts of cationic components at elevated charge ratios is often not considered. In addition, the prolonged effects of cationic lipid uptake on cell viability are not evident in a typical 24 - 48 h transfection experiment. In this study, we compare the transfection efficiency of cationic lipoplexes to effects on viability of cultured cells in both the short- and long-term (7 days). Our results indicate that while minimal toxicity is evident 24 h after exposure to DOTAP-based lipoplexes, cell viability continues to decline and ultimately compromises reporter gene expression at longer times. Substitution of a naturally-occurring cationic amphiphile, sphingosine, for DOTAP greatly reduces toxicity and allows high expression to be maintained over prolonged periods.
- Active targeting docetaxel-PLA nanoparticles eradicate the circulating lung cancer stem like cells and inhibit the liver metastasis. [JOURNAL ARTICLE]
- Mol Pharm 2014 Nov 24.
Lung cancer is the major cause of cancer related lethality worldwide and the metastases to distant organs is the pivotal cause of death for the vast majority of lung cancer patients. Accumulated evidences indicate lung cancer stem cells (CSCs) play important roles in metastagenesis and these circulating CSCs may be important targets to inhibit the subsequent metastasis. Present study was aimed at establishing a CSCs-targeting polylactic acid (PLA) encapsulated docetaxel nanoparticles for anti-metastatic therapy. Cyclic binding peptides were screened on CSCs in vitro and the peptide CVKTPAQSC exhibiting high specific binding ability to pulmonary adenocarcinoma tissue was subsequently conjugated to the nanoparticles loaded with docetaxel (NDTX). Anti-metastatic effect of CSCs-targeting nanoparticles loaded with docetaxel (TNDTX) was evaluated in a nude mice model of liver metastasis. Results showed that in the absence of targeting peptide, NDTX hardly exhibited any anti-metastatic effect. However TNDTX treatment significantly decreased the metastatic tumor area in the nude mice liver. Histopathological and serological results also confirmed the anti-metastatic efficacy of TNDTX. To our knowledge, this is the first report on establishing a CSCs-based strategy for lung cancer metastatic treatment and we hope this would offer a potential therapeutic approach for management of metastatic lung cancer.