Molecular pharmaceutics [journal]
- Image-guided radiotherapy targets macromolecules through altering the tumor microenvironment. [JOURNAL ARTICLE]
- Mol Pharm 2016 Aug 25.
Current strategies to target tumors with nanomedicines rely on passive delivery via the enhanced permeability and retention effect, leveraging the disorganized tumor microvasculature to promote macromolecule extravasation and the reduced lymphatic and venous drainage that favor retention. Nonetheless, FDA approvals and clinical use of nanomedicines have lagged, reflecting failure to display superiority over conventional formulations. Here, we have exploited image-guided X-irradiation to augment nanoparticle accumulation in tumors. A single 5 Gy dose of radiation, below that required to significantly delay tumor growth, can markedly enhance delivery of macromolecules and nanoparticles. The radiation effect was independent of endothelial cell integrity, suggesting a primary role for damage to microvascular pericytes and/or interstitial extracellular matrix. Significantly, radiation-guided delivery potentiated the therapeutic effects of PEGylated liposomal doxorubicin on experimental tumors. Applied to patients, these results suggest repurposing image-guided radiotherapy as a tool to guide cancer nanomedicine delivery, enhancing local control for primary tumors and metastatic disease while limiting systemic toxicity.
- Linking In Vitro Lipolysis and Microsomal Metabolism for the Quantitative Prediction of Oral Bioavailability of BCS II Drugs Administered in Lipidic Formulations. [JOURNAL ARTICLE]
- Mol Pharm 2016 Aug 24.
Lipidic formulations (LFs) are increasingly utilized for the delivery of drugs that belong to class II of the Biopharmaceutics Classification System (BCS). The current work proposes, for the first time, the combination of in vitro lipolysis and microsomal metabolism studies for the quantitative prediction of human oral bioavailability of BCS II drugs administered in LFs. Marinol® and Neoral® were selected as model LFs and their observed oral bioavailabilities (Fobserved) obtained from published clinical studies in humans. Two separate lipolysis buffers, differing in the level of surfactant concentrations, were used for digestion of the LFs. The predicted fraction absorbed (Fabs) was calculated by measuring the drug concentration in the micellar phase after completion of the lipolysis process. To determine first-pass metabolism (Fg∙Fh), drug depletion studies with human microsomes were performed. Clearance values were determined by applying the "in vitro half-life approach". The estimated Fabs and Fg∙Fh values were combined for the calculation of the predicted oral bioavailability (Fpredicted). Results showed that there was a strong correlation between Fobserved and Fpredicted values only when Fabs was calculated using a buffer with surfactant concentrations closer to physiological conditions. The general accuracy of the predicted values suggests that the novel in vitro lipolysis/metabolism approach could quantitatively predict the oral bioavailability of lipophilic drugs administered in LFs.
- Mechanism Of PAMAM Dendrimer Internalization In Hippocampal Neurons. [JOURNAL ARTICLE]
- Mol Pharm 2016 Aug 24.
Polyamidoamine (PAMAM) dendrimers are hyperbranched macromolecules which have been described as one of the most promising drug nanocarrier systems. A key process to understand is their cellular internalization mechanism because its direct influence on their intracellular distribution, association to organelles, entry kinetics, and cargo release. Despite that internalization mechanisms of dendrimer have been studied in different cell types, in the case of neurons they are not completely described. Considering the relevance of central nervous system (CNS) diseases and neuropharmacology, the aim of this report is to describe the molecular internalization mechanism of different PAMAM-based dendrimer systems in hippocampal neurons. Four dendrimers based on four generation PAMAM with different surface properties were studied: unmodified G4 with a positive charged surface, PP50 with a substitution of the 50% of amino surface groups with polyethylenglycol neutral groups, PAc with a substitution of the 30% of amino surface groups with acrylate anionic groups, and PFO decorated with folic acid groups in a 25% of total terminal groups. Confocal images show that both G4 and PFO are able to enter the neurons, but not PP50 and PAc. Colocalization study with specific endocytosis markers and specific endocytosis inhibitors assay demonstrate that clathrin-mediated endocytosis would be the main internalization mechanism for G4, whereas clathrin and caveolae-mediated endocytosis would be implicated in PFO internalization. These results show the existence of different internalization mechanism for PAMAM dendrimers in neurons and the possibility of control their internalization properties with specific chemical modifications.
- Peritoneal Macrophage-Specific TNFα Gene Silencing in LPS-Induced Acute Inflammation Model using CD44 Targeting Hyaluronic Acid Nanoparticles. [JOURNAL ARTICLE]
- Mol Pharm 2016 Aug 23.
The main goal of this study was to evaluate tumor necrosis factor-alpha (TNFα) gene silencing in peritoneal macrophages upon activation with lipopolysaccharide (LPS) using CD44-targeting hyaluronic acid (HA)-based nanoparticles encapsulating TNFα-specific small interfering RNA (siTNFα). HA nanoparticles were formulated by blending hyaluronic acid-poly(ethylene imine) (HA-PEI), hyaluronic acid-hexyl fatty acid (HA-C6), and hyaluronic acid-poly(ethylene glycol) (HA-PEG) in 3:2:1 weight ratio, encapsulating siTNFα to form spherical particles of 78-90 nm diameter. Following intraperitoneal (IP) administration in LPS-treated C57BL/6 mice, the nanoparticles were actively taken up by macrophages and led to a significant downregulation of peritoneal TNFα levels. Downregulation of peritoneal macrophage-specific TNFα also had a significant impact on other pro-inflammatory cytokine and chemokine levels in the serum. The C57BL/6 group of mice challenged with 5mg/kg LPS had a significantly higher survival rate when they were treated with 3 mg/kg siTNFα, either prior or simultaneously with the LPS administration, as compared to the LPS-challenged mice, which were treated with controls, including the scrambled siRNA formulation. Overall, the results of this study demonstrate that CD44 targeting HA nanoparticles can selectively deliver siTNFα to peritoneal macrophages leading to downregulation of pro-inflammatory cytokines in the peritoneal fluid and in the serum. This RNAi strategy could potentially provide an important therapeutic modality for acute inflammatory diseases, such as septic shock.
- Reduction sensitive PEG hydrogels for co-delivery of antigen and adjuvant to induce potent CTLs. [JOURNAL ARTICLE]
- Mol Pharm 2016 Aug 23.
Educating our immune system via vaccination is an attractive approach to combat infectious diseases. Eliciting antigen specific cytotoxic T cells (CTLs), CD8+ effector T cells, are essential in controlling intracellular infectious diseases such as influenza (Flu), tuberculosis (TB), hepatitis, HIV/AIDS, as well as tumors. However, vaccination utilizing subunit peptides to elicit a potent CD8+ T cell response with antigenic peptides are typically ineffective due to poor immunogenicity. Here we have engineered a reduction sensitive nanoparticle (NP) based subunit vaccine for intracellular delivery of an antigenic peptide and immunostimulatory adjuvant. We have co-conjugated an antigenic peptide (ovalbumin-derived CTL epitope [OVA257-264 -SIINFEKL]) and an immunostimulatory adjuvant (CpG ODNs, TLR9 agonist) to PEG hydrogel NPs via a reduction sensitive linker. Bone-marrow derived dendritic cells (BMDCs) treated with the SIINFEKL-conjugated NPs efficiently cross-presented the antigenic peptide via MHC-I surface receptor and induced proliferation of OT-I T cells. CpG ODN-conjugated NPs induced maturation of BMDCs as evidenced by the overexpression of CD80 and CD40 co-stimulatory receptors. Moreover, co-delivery of NP conjugated SIINFEKL and CpG ODN significantly increased the frequency of IFN- producing CD8+ effector T cells in mice (∼6 fold improvement over soluble antigen and adjuvant). Furthermore, the NP subunit vaccine-induced effector T cells were able to kill up to 90% of the adoptively transferred antigenic peptide-loaded target cell. These results demonstrate that the reduction sensitive NP subunit vaccine elicits a potent CTL response and provide compelling evidence that this approach could be utilized to engineer particulate vaccines to deliver tumor- or pathogen-associated antigenic peptides to harness the immune system to fight against cancer and infectious diseases.
- The role of N-glycosylation in maintaining the transporter activity and expression of human Oligopeptide transporter 1 (hPepT1). [JOURNAL ARTICLE]
- Mol Pharm 2016 Aug 22.
Human Oligopeptide transporter 1 (hPepT1) mediates the absorption of dietary peptides and a range of clinically relevant drugs. According to the predicted topological structure hPepT1 contains multiple asparagine residues in putative N-glycosylation sites. This study investigated the influence of the six putative N-glycosylation sites within the extracellular region between transmembrane domains 9 and 10 on hPepT1 transporter function and expression in HEK-293T cells. Our study confirmed that hPepT1 is N-glycosylated in HEK-293T cells, with the glycosylated and fully deglycosylated isoforms exhibiting apparent molecular masses of ~78 and ~55 kDa, respectively. Transport uptake of Glycylsarcosine (Gly-sar) by the hPepT1-N562Q variant, but not by other single mutants, was moderately impaired. We also constructed multiple N-glycosylation mutants based on the hPepT1-N562Q mutant by mutagenizing the additional asparagine residues N404Q, N408Q, N439Q, N509Q and N514Q. Transport function showed a graded decrease as the number of mutagenized residues increased and simultaneous removal of all six asparagine residues essentially abolished transport activity. Kinetic studies indicated that the Vmax values for Gly-sar transport by low activity mutants were decreased compared to wild type, which suggested that the cell surface expression and/or turn-over rate of hPepT1 mutants was impaired; Km values were unchanged in most cases. Using immunoblotting and immunofluorescence, the plasma membrane and total cellular expression of the mutant transporters were decreased in accordance with functional impairments. In summary, we provide the first molecular evidence that hPepT1 is modified by N-glycosylation and that all six asparagine residues in the large extracellular loop between transmembrane domains 9 and 10 are subject to N-glycosylation. This information enhances our understanding of the role of the large extracellular loop in hPepT1 regulation and could facilitate the development of new hPepT1 substrate drugs with improved bioavailability.
- Human serum albumin and HER2-binding affibody fusion proteins for targeted delivery of fatty acid-modified molecules and therapy. [JOURNAL ARTICLE]
- Mol Pharm 2016 Aug 21.
Human epidermal growth factor receptor 2 (HER2) is a well-studied therapeutic target as well as a biomarker of breast cancer. HER2-targeting affibody (ZHER2:342) is a novel small scaffold protein with an extreme high affinity against HER2 screened by phage display. However, the small molecular weight of ZHER2:342 has limited its pharmaceutical application. Human serum albumin (HSA) and ZHER2:342 fusion protein may not only extend the serum half-life of ZHER2:342 but also preserve the biological function of HSA to bind and transport fatty acids, which can be used to deliver fatty acid-modified therapeutics to HER2-positive cancer cells. Two HSA and ZHER2:342 fusion proteins, one with a single ZHER2:342 domain fused to the C terminus of HSA (rHSA-ZHER2) and another with two tandem copies of ZHER2:342 fused to the C terminus of HSA (rHSA-(ZHER2)2), have been constructed, expressed, and purified. Both fusion proteins possessed the HER2 and fatty acid (FA) binding abilities demonstrated by in vitro assays. Interestingly, rHSA-(ZHER2)2, not rHSA-ZHER2, was able to inhibit the proliferation of SK-BR-3 cells at a relatively low concentration and the increase of HER2 and ERK1/2 phosphorylation followed by rHSA-(ZHER2)2 treatment has been observed. HSA fusion proteins are easy and economical to express, purify, and formulate. As expected, HSA fusion proteins and fusion protein-bound fatty acid-modified FITC could be efficiently taken up by cells. These results proved the feasibility of using HSA fusion proteins as therapeutic agents as well as carriers for targeted drug delivery.
- Impact of Metallic Stearates on Disproportionation of Hydrochloride Salts of Weak Bases in Solid-State Formulations. [JOURNAL ARTICLE]
- Mol Pharm 2016 Aug 19.
Excipient-induced salt disproportionation (conversion from salt form to free form) in the solid state during storage or manufacturing is a severe formulation issue that can negatively influence product performance. However, the role of excipient properties on salt disproportionation and mechanisms of proton transfer between salt and excipients are still unclear. Moreover, knowledge about the formation of disproportionation products and the consequent impact of these reactions products on the disproportionation process is still inadequate. In the present study, three commonly used lubricants (sodium stearate, calcium stearate, and magneisum stearate) were mixed with a hydrochloride salt as binary mixtures to examine their different capabilities for inducing salt disproportionation at a stressed storage condition (40°C 65% RH). The overall objective of this research is to explore factors influencing the kinetics and extent of disproportionation including surface area, alkalinity, hygroscopicity, formation of new species etc. In addition, we also aim to clarify the reaction mechanism and proton transfer between the model salt and stearates to provide insight into the in-situ formed reaction products. We found that the properties of stearates significantly affect the disproportionation process in the initial stage of storage, while properties of the reaction products negatively affect the hygroscopicity of the powder mixture promoting disproportionation during longer-term storage. In addition, lubrication difference among three stearates was evaluated by performing compaction studies. The findings of this study provide an improved understanding of the proton transfer mechanism between the ionized form of an API and excipients in solid dosage forms. It also provides pragmatic information for formulation scientists to select appropriate lubricants and other excipients, and to design robust formulations.
- Influence of Arginine Salts on the Thermal Stability and Aggregation Kinetics of Monoclonal Antibody: Dominant Role of Anions. [JOURNAL ARTICLE]
- Mol Pharm 2016 Aug 19.
Thermal stability of the CH2 domain for an IgG1 monoclonal antibody and its aggregation kinetics were systematically studied at pH 4.8, below its pI of 8.8 in individual solutions of Arginine salts with acetate, glutamate (Glu-), chloride, and sulfate as the anion, in comparison to sodium chloride and sodium sulfate. Thermal unfolding temperature, Tm, an indicator of thermal stability, was measured by both differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF). The aggregation kinetics was determined by assessing reversibility for the CH2 domain in the DSC repetitive scans and then cross-examined by the isothermal aggregation study measured by size exclusion chromatography. The effect of Arg+ on the thermal stability and aggregation kinetics of the antibody is shown to be strongly anion-dependent: both ArgAceate and ArgGlu improve the stability while both Arg2SO4 and ArgCl decrease it. Furthermore, the addition of ArgCl and Arg2SO4 accelerates the aggregation kinetic, but to a less extent than the respective Na+ salt, suggesting that Arg+ binds to the antibody more strongly than Na+. However, the binding of Arg+ did not lead to more destabilization of the CH2 domain by the Arg+ salts at low concentrations, comparing to the respective Na+ salt. This finding indicates that Arg+ prefers the protein surface, rather than the exposed backbone upon unfolding. Furthermore, the change in the ranking for affecting the thermal stability and aggregation kinetics as the salt concentration increases implies the presence of other multiple mechanisms, e.g. cluster formation through the homo-ion pairing between Arg+ molecules and their preferential exclusion from the protein surface, and hetero-ion pairing between Arg+ and SO42-.
- Inflammation Caused by Nanosized Delivery Systems: Is There a Benefit? [JOURNAL ARTICLE]
- Mol Pharm 2016 Aug 19.
Secondary macrophage cytotoxicity induced by nanoparticles was described before. The study aim was to investigate the role of secondary cytotoxic effect in a macrophage-lung cancer coculture model after nanoparticle treatment in the presence and absence of anti-inflammatory drugs. An in vitro coculture model composed of confluent alveolar macrophage MH-S and A-549 lung cancer cells separated by a 0.4 μm porous membrane was used in the study. Macrophages were treated with two sizes of gelatin nanoparticles and two sizes of poly(isobutyl cyanoacrylate) (PIBCA) nanoparticles, with and without doxorubicin as a chemotherapeutic drug. The treatment effect with and without the presence of anti-inflammatory drug was studied using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The model drugs were ibuprofen, celecoxib, prednisolone, dexamethasone, and methotrexate. Different nanoparticles in different sizes were synthesized with a range of physicochemical characteristics. Doxorubicin loaded nanoparticles were prepared with an entrapment efficiency of 82-83% for PIBCA and 39-42% for gelatin. Nanoparticle treatment of macrophages showed a secondary cytotoxic effect on A-549 cancer cells at 24 and 36 h, with a drop in cell viability of 40-62%. However, this effect was significantly reduced to 10-48% if the macrophages were exposed to anti-inflammatory drugs. When ibuprofen and celecoxib were used the cell viability rebounded between 24 and 36 h. For prednisolone, dexamethasone, and methotrexate the cell viability dropped further between 24 and 36 h. Macrophages exposed to nanoparticles show secondary cytotoxicity, which has a significant antitumor effect in the microclimate of the coculture model. The beneficial nanoparticle treatment effect was significantly reduced if nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, or methotrexate was given at the same time. The data suggest that anti-inflammatory treatments can decrease the carrier-induced macrophage cytotoxicity and its antitumor effectiveness with chemotherapy.