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Molecular pharmaceutics [journal]
- Cellular uptake of coumarin 6 under microfluidic conditions into HCE-T cells from nanoscale formulations. [JOURNAL ARTICLE]
- Mol Pharm 2014 Nov 20.
In vitro studies of ocular bioavailability of active pharmaceutical ingredients (API) from colloidal drug delivery systems do not consider physiological shear stress generated by eyelid wiping and tear flow. The present study introduces a live cell imaging approach which enables the investigation of model drug uptake from various formulations under shear stress by using custom-made microchannels for the cultivation of human corneal epithelial cells (HCE-T). Coumarin-6 (C-6) was used as a model API incorporated into solid lipid nanoparticles and liposomes, and as an aqueous crystalline suspension. Confocal laser scanning microscopy visualized C-6 uptake into HCE-T cells in a time-resolved manner with an applied shear stress of 0.1 Pa. Static conditions were also studied for comparative purposes. Additionally, solid lipid nanoparticles (SLN) were labeled with a fluorescent phospholipid to check whether C-6 uptake was associated with SLN incorporation into the cells. Results: Intact SLN were not incorporated into the cells, i.e., C-6 was passively redistributed from SLN to lipophilic cellular compartments. C-6 was enriched up to a given limit in HCE-T cells within 5 minutes of contact with the dispersions both under static and under flow conditions. The C-6 delivery rate from liposomes was superior to that from SLN whereby the suspension exhibited the lowest rate. C-6 release rates were comparable for static and flow conditions. Alternate flushing with formulations and buffer revealed that cells accumulated C-6. The results suggest that combining microfluidics with live cell imaging provides a valuable option for in vitro studies of ocular drug delivery.
- Nucleolar Targeting by Platinum. p53-independent Apoptosis Follows rRNA Inhibition, Cell-cycle Arrest and DNA Compaction. [JOURNAL ARTICLE]
- Mol Pharm 2014 Nov 18.
TriplatinNC is a highly positively charged, substitution-inert derivative of the phase II clinical anticancer drug, BBR3464. Such substitution-inert complexes form a distinct subset of polynuclear platinum complexes (PPCs) interacting with DNA and other biomolecules through "non-covalent" interactions. Rapid cellular entry is facilitated via interaction with cell surface glycosoaminoglycans and is a mechanism unique to PPCs. Nano-scale secondary ion mass spectrometry (NanoSIMS) showed rapid distribution within cytoplasmic and nucleolar compartments, but not the nucleus. In this paper, the downstream effects of nucleolar localization are described. In human colon carcinoma cells, HCT116, the production rate of 47S rRNA precursor transcripts was dramatically reduced as an early event after drug treatment. Transcriptional inhibition of rRNA was followed by a robust G1 arrest, and activation of apoptotic proteins caspase-8, -9, -3, and PARP-1 in a p53-independent manner. Using cell synchronization and flow cytometry, it was determined that cells treated while in G1 immediately arrest , but cells treated in S or G2 successfully complete mitosis. Twenty-four hours after treatment, the majority of cells finally arrest in G1, but nearly one-third contained highly compacted DNA; a distinct biological feature that cannot be associated with mitosis, senescence, or apoptosis. This unique effect mirrored the efficient condensation of tRNA and DNA in cell-free systems. The combination of DNA compaction and apoptosis by TriplatinNC treatment conferred striking activity in platinum-resistant and/or p53 mutant or null cell lines. Taken together, our results support that the biological activity of TriplatinNC reflects reduced metabolic deactivation (substitution-inert compound not reactive to sulfur nucleophiles), high cellular accumulation and novel consequences of high-affinity non-covalent DNA binding - producing a new profile and a further shift in the structure-activity paradigms for anti-tumor complexes.
- Improving the Therapeutic Index in Cancer Therapy by Using Antibody-Drug Conjugates Designed with a Moderately-Cytotoxic Drug. [JOURNAL ARTICLE]
- Mol Pharm 2014 Nov 17.
The antibody-drug conjugate (ADC), IMMU-130, of the moderately-cytotoxic topoisomerase I inhibitor, SN-38, and the CEACAM5-targeted humanized antibody (MAb), labetuzumab, was evaluated in model systems of human colon carcinoma and in Phase I clinical trials of heavily-pretreated patients with metastatic colorectal cancer. The conjugate, designed with a near-homogeneous drug substitution of 78 SN-38/MAb and with a linker that released 50% of the drug in ~ 20 h, showed significant antitumor effects compared to a non-targeted ADC in human tumor xenografts, which could be augmented in combination with bevacizumab. The advantage of fractionated dosing was demonstrated, with potential implications for the clinical dosing schedule. Biodistribution comparing IMMU-130 with labetuzumab showed that the conjugate cleared somewhat faster from the blood, but this did not affect tumor uptake and retention. The use of an ultra-stable linker in the conjugate design abrogated antitumor effects. A tolerability study in rabbits showed a high safety margin, with no-observed-adverse-effect level (NOAEL) corresponding to a cumulative human-equivalent protein dose of 40-60 mg/kg. The preclinical findings appear to be corroborated in two Phase I clinical trials, with high tolerability and evidence of antitumor activity, including objective responses. The impact of the ADC design on the utility of IMMU-130, tailored to a poorly-internalizing target, is discussed.
- Serial in vivo imaging using a fluorescence probe allows identification of tumor early response to cetuximab immunotherapy. [JOURNAL ARTICLE]
- Mol Pharm 2014 Nov 14.
Cetuximab is an anti-epidermal growth factor receptor monoclonal antibody that has received the approval of the Food and Drug Administration (FDA) for cancer treatment. However, most clinical studies indicate that cetuximab can only elicit positive effects on a subset of cancer patients. In this study, we investigated whether near-infrared fluorescence (NIRF) imaging of tumor vascular endothelial growth factor (VEGF) expression could be a biomarker for tumor early response to cetuximab therapy in preclinical wild-type and mutant tumor models of the KRAS gene. The treatment efficacy of cetuximab was determined in both HT-29 (wild-type KRAS) and HTC-116 (mutant KRAS) human colon cancer models. A VEGF-specific optical imaging probe (Dye755-Ran) was synthesized by conjugating ranibizumab (an anti-VEGF antibody Fab fragment) with a NIRF dye. Serial optical scans with Dye755-Ran were performed in HT-29 and HTC-116 xenograft models. By using longitudinal NIRF imaging, we were able to detect early tumor response on day 3 and day 5 after initiation of cetuximab treatment in the cetuximab-responsive HT-29 tumor model. Enzyme-linked immunosorbent assay (ELISA) confirmed that cetuximab treatment inhibited VEGF expression in the KRAS wild-type HT-29 tumor but not in the KRAS mutant HCT-116 tumor. We have demonstrated that the antitumor effect of cetuximab can be noninvasively monitored by serial fluorescence imaging using Dye755-Ran. VEGF expression detected by optical imaging could serve as a sensitive biomarker for tumor early response to drugs that directly or indirectly act on VEGF.
- Comparing Multi-Label Classification Methods for Provisional Biopharmaceutics Class Prediction. [JOURNAL ARTICLE]
- Mol Pharm 2014 Nov 14.
The biopharmaceutical classification system (BCS) is now well established and utilised for the development and biowaivers of immediate oral dosage forms. The prediction of BCS class can be carried out using multi-label classification. Unlike single label classification, multi-label classification methods predict more than one class labels at the same time. This paper compares two multi-label methods, binary relevance and classifier chain, for provisional BCS class prediction. Large datasets of permeability and solubility of drug and drug-like compounds were obtained from the literature and were used to build models using decision trees. The separate permeability and solubility models were validated and an BCS validation set of 127 compounds where both permeability and solubility were known was used to compare the two aforementioned multi-label classification methods for provisional BCS class prediction. Overall, the results indicate that the classifier chain method, which takes into account label interactions, performed better compared to the binary relevance method. This work offers a comparison of multi-label methods and shows the potential of the classifier chain multi-label method for improved biological property predictions for use in drug discovery and development.
- Trantinterol, a novel β2-adrenoceptor agonist non-competitively inhibits P-glycoprotein function in vitro and in vivo. [JOURNAL ARTICLE]
- Mol Pharm 2014 Nov 12.
P-glycoprotein (P-gp)-mediated drug-drug interactions are important factors causing adverse effects of drugs in clinical use. The aim of this study was to determine whether trantinterol (also known as SPFF), a novel β2-adrenoceptor agonist, was a P-gp inhibitor or substrate. The results showed that trantinterol was not a substrate of P-gp but increased rhodamine 123 (Rho 123) uptake by MDCK-MDR1 cells and decreased the efflux transport of both Rho 123 and cyclosporine A (CsA) in bi-directional transport studies across MDCK-MDR1 cell monolayers. This suggested that trantinterol was a P-gp inhibitor but not a P-gp substrate. The mechanism of inhibition was investigated in the P-gp-Glo assay system where it was found that trantinterol inhibited P-gp ATPase activity in a dose-dependent manner. A subsequent study using the antibody binding assay with the conformation-sensitive P-gp-specific antibody UIC2 confirmed that trantinterol decreased UIC2 binding at 10 μM in contrast to the competitive inhibitor, verapamil. This suggested that trantinterol was a non-competitive inhibitor of P-gp. Finally, a pharmacokinetic study in rat showed that trantinterol significantly increased the area under the plasma concentration-time curve (AUC) and maximum plasma concentration (Cmax) of digoxin and paclitaxel (PAC), and the Cmax of cyclosporine A (CsA). In summary, trantinterol is a potent non-competitive P-gp inhibitor which may increase the bioavailability of other P-gp substrate drugs co-administered with it.
- Specific Ion and Buffer Effects on Protein-Protein Interactions of a Monoclonal Antibody. [JOURNAL ARTICLE]
- Mol Pharm 2014 Nov 12.
Better predictive ability of salt and buffer effects on protein-protein interactions requires separating out contributions due to ionic screening, protein charge neutralization by ion bind- ing, and salting-in(out) behaviour. We have carried out a systematic study by measuring protein-protein interactions for a monoclonal antibody over an ionic strength range of 25 to 525 mM at 4 pH values (5, 6.5, 8, 9) in solutions containing either sodium chloride, calcium chlo- ride, sodium sulfate, or sodium thiocyante. The salt ions are chosen so as to represent a range of affinities for protein charged and non-charged groups. The results are compared to effects of various buffers including acetate, citrate, phosphate, histidine, succinate, or tris. In low ionic strength solutions, anion binding affinity is reflected by the ability to reduce protein-protein repulsion, which follows the order thiocyanate > sulfate > chloride. The sulfate specific effect is screened at the same ionic strength required to screen the pH dependence of protein-protein interactions indicating sulfate binding only neutralizaes protein charged groups. Thiocyanate specific effects occur over a larger ionic strength range reflecting adsorption to charged and non-charged regions of the protein. The latter leads to salting-in behaviour and, at low pH, a non-monotonic interaction profile with respect to sodium thiocyanate concentration. The ef- fects of thiocyanate can not only be rationalized in terms of neutralizing double layer forces indicating the presence of an additional short-ranged protein-protein attraction at moderate ionic strength. Conversely, buffer specific effects can be explained through a charge neutral- ization mechanism, where buffers with greater valency are more effective at reducing double layer forces at low pH. Citrate binding at pH 6.5 leads to protein charge inversion and the for- mation of attractive electrostatic interactions. Throughout the report, we highlight similarities in the measured protein-protein interaction profiles with previous studies providing evidence that the behaviour is universal to all proteins.
- Putative Transmembrane Domain 6 of the Human Organic Anion Transporting Polypeptide 1A2 (OATP1A2) Influences Transporter Substrate Binding, Protein Trafficking, and Quality Control. [JOURNAL ARTICLE]
- Mol Pharm 2014 Nov 20.
The human organic anion transporting polypeptides (OATPs) are a family of important membrane proteins that mediate the cellular influx of various anionic substances including clinically important drugs. Transmembrane domain 6 (TM6) is a distinctive consensus "signature" common to all OATPs. Two naturally occurring variants were previously identified in TM6 of the important transporter OATP1A2; these variants may be associated with suboptimal drug influx into cells. Because of the potential importance of TM6 in drug efficacy, this study investigated its role in substrate uptake by OATP1A2. Single amino acid replacements were introduced into TM6 of OATP1A2 (residues 245-266) by alanine-scanning mutagenesis. Uptake assays, biotinylation and immunoblotting were used to assess the function and expression of OATP1A2 and its mutants after overexpression in HEK293 cells. Uptake of the model substrates estrone-3-sulfate and methotrexate by OATP1A2 mutants carrying amino acid replacements within the TM6 subregions of 245-248 and 261-266 was impaired, while transport function was largely retained by other mutants. From kinetic, biotinylation, and immunoblot analysis the diminished function of the 245-248 and 261-266 mutants was due primarily to decreased plasma membrane and total cell expression and also to a less extent, impacted by altered substrate binding. Further experiments with proteasomal or lysosomal inhibitors were consistent with impaired maturation and impaired plasma membrane insertion of several mutants of OATP1A2 within the subregions of 245-248 and 261-266. In addition, the finding that total cellular expression, but not plasma membrane expression, was less impaired for the W245A and W246A mutants suggests that these two TM6 residues might be involved in membrane targeting of OATP1A2. These findings implicate the TM6 subregions of 245-248 and 261-266 in substrate binding, protein trafficking, and quality control of OATP1A2.
- Targeted Decationized Polyplexes for siRNA Delivery. [JOURNAL ARTICLE]
- Mol Pharm 2014 Nov 20.
The applicability of small interfering RNA (siRNA) in future therapies depends on the availability of safe and efficient carrier systems. Ideally, siRNA delivery requires a system that is stable in the circulation but upon specific uptake into target cells can rapidly release its cargo into the cytoplasm. Previously, we evaluated a novel generation of carrier systems ("decationized" polyplexes) for DNA delivery, and it was shown that folate targeted decationized polyplexes had an excellent safety profile and showed intracellular triggered release upon cell specific uptake. Targeted decationized polyplexes consist of a core of disulfide cross-linked poly(hydroxypropyl methacrylamide) (pHPMA) stably entrapping nucleic acids and a shell of poly(ethylene glycol) (PEG) decorated with folate molecules. In the present study, the applicability of folate targeted decationized polyplexes for siRNA delivery was investigated. This required optimization of the carrier system particularly regarding the cross-linking density of the core of the polyplexes. Stable and nanosized siRNA decationized polyplexes were successfully prepared by optimizing the cross-link density of their core. Upon incubation in human plasma, a significant portion of siRNA remained entrapped in the decationized polyplexes as determined by fluorescence correlation spectroscopy (FCS). When tested in a folate receptor overexpressing cell line stably expressing luciferase, Skov3-luc, sequence specific gene silencing was observed. As expected, neither interference on the intrinsic luciferase expression nor on the cell metabolic activity (determined by XTT) was induced by the free-polymer or the siRNA polyplexes. In conclusion, targeted decationized polyplexes are safe and stable carriers that interact with the targeted cells and rapidly disassemble upon cell entry making them promising siRNA delivery systems.
- Highly Viscous Antibody Solutions Are a Consequence of Network Formation Caused by Domain-Domain Electrostatic Complementarities: Insights from Coarse-Grained Simulations. [JOURNAL ARTICLE]
- Mol Pharm 2014 Nov 20.
Therapeutic monoclonal antibody (mAb) candidates that form highly viscous solutions at concentrations above 100 mg/mL can lead to challenges in bioprocessing, formulation development, and subcutaneous drug delivery. Earlier studies of mAbs with concentration-dependent high viscosity have indicated that mAbs with negatively charged Fv regions have a dipole-like quality that increases the likelihood of reversible self-association. This suggests that weak electrostatic intermolecular interactions can form transient antibody networks that participate in resistance to solution deformation under shear stress. Here this hypothesis is explored by parametrizing a coarse-grained (CG) model of an antibody using the domain charges from four different mAbs that have had their concentration-dependent viscosity behaviors previously determined. Multicopy molecular dynamics simulations were performed for these four CG mAbs at several concentrations to understand the effect of surface charge on mass diffusivity, pairwise interactions, and electrostatic network formation. Diffusion coefficients computed from simulations were in qualitative agreement with experimentally determined viscosities for all four mAbs. Contact analysis revealed an overall greater number of pairwise interactions for the two mAbs in this study with high concentration viscosity issues. Further, using equilibrated solution trajectories, the two mAbs with high concentration viscosity issues quantitatively formed more features of an electrostatic network than the other mAbs. The change in the number of these network features as a function of concentration is related to the number of pairwise interactions formed by electrostatic complementarities between antibody domains. Thus, transient antibody network formation caused by domain-domain electrostatic complementarities is the most probable origin of high concentration viscosity for mAbs in this study.