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Molecular pharmaceutics [journal]
- (44)Sc: An Attractive Isotope for Peptide-Based PET Imaging. [JOURNAL ARTICLE]
- Mol Pharm 2014 Jul 23.
The overexpression of integrin αvβ3 has been linked to tumor aggressiveness and metastasis in several cancer types. Because of its high affinity, peptides containing the arginine-glycine-aspartic acid (RGD) motif have been proven valuable vectors for noninvasive imaging of integrin αvβ3 expression and for targeted radionuclide therapy. In this study, we aim to develop a (44)Sc-labeled RGD-based peptide for in vivo positron emission tomography (PET) imaging of integrin αvβ3 expression in a preclinical cancer model. High quality (44)Sc (t1/2, 3.97 h; β(+) branching ratio, 94.3%) was produced inexpensively in a cyclotron, via proton irradiation of natural Ca metal targets, and separated by extraction chromatography. A dimeric cyclic-RGD peptide, (cRGD)2, was conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and radiolabeled with (44)Sc in high yield (>90%) and specific activity (7.4 MBq/nmol). Serial PET imaging of mice bearing U87MG tumor xenografts showed elevated (44)Sc-DOTA-(cRGD)2 uptake in the tumor tissue of 3.93 ± 1.19, 3.07 ± 1.17, and 3.00 ± 1.25 %ID/g at 0.5, 2, and 4 h postinjection, respectively (n = 3), which were validated by ex vivo biodistribution experiments. The integrin αvβ3 specificity of the tracer was corroborated, both in vitro and in vivo, by competitive cell binding and receptor blocking assays. These results parallel previously reported studies showing similar tumor targeting and pharmacokinetic profiles for dimeric cRGD peptides labeled with (64)Cu or (68)Ga. Our findings, together with the advantageous radionuclidic properties of (44)Sc, capitalize on the relevance of this isotope as an attractive alternative isotope to more established radiometals for small molecule-based PET imaging, and as imaging surrogate of (47)Sc in theranostic applications.
- Efficient decoration of nanoparticles intended for intracellular drug targeting with targeting residues, as revealed by a new indirect analytical approach. [JOURNAL ARTICLE]
- Mol Pharm 2014 Jul 20.
In our previous studies, we developed a nano-drug delivery system (nano-DDS) based on poly(lactic-co-glycolic acid) PLGA nanoparticles encapsulating antigenic peptide and fluorescent marker and 3-stage approach for its decoration with peptide targeting residues. The objectives of this study were: a) to develop methods for quantitative analysis of efficiency of individual conjugation steps, and b) to determine, based on these methods, the efficiency of our 3-stage approach of nano-DDS decoration. We prepared antigenic peptide-loaded PLGA-based nano-DDSs and sequentially decorated them with specific residues using carbodiimide and Click (azide-alkyne Huisgen cycloaddition using copper (I) catalysis) reactions. The extent of cargo encapsulation and release kinetics were analyzed using HPLC-based and colorimetric analytical methods. The efficiency of residues conjugation to the nano-DDSs was analyzed using FTIR spectroscopy and by quantifying the unreacted residues in the reaction mixture (i.e., by indirect analysis of reaction efficiencies). We revealed that copper, the catalyst of the Click reactions, formed complexes with unreacted targeting residues and interfered with the analysis of their conjugation efficiency. We used penicillamine (a chelator) to disrupt these complexes, and to recover the unreacted residues. Quantitative analysis revealed that 28,800-34,000 targeting residues (corresponding to 11-13 nm2 surface area per residue) had been conjugated to a single nano-DDS using our 3-stage decoration approach, which is much higher than previously reported conjugation efficiencies. We conclude that the applied analytical tools allow quantitative analysis of nano-DDSs and the efficiency of their conjugation with targeting residues. The 3-stage decoration approach resulted in dense conjugation of nano-DDSs with targeting residues. The present decoration and analytical approaches can be effectively applied to other types of delivery systems and other targeting residues.
- Insights into Hydrate Formation and Stability of Morphinanes from a Combination of Experimental and Computational Approaches. [JOURNAL ARTICLE]
- Mol Pharm 2014 Jul 18.
Morphine, codeine and ethylmorphine, are important drug compounds, whose free bases and hydrochloride salts form stable hydrates. These compounds were used to systematically investigate the influence of the type of functional groups, the role of water molecules and the Cl- counterion on molecular aggregation and solid state properties. Five new crystal structures have been determined. Additionally, structure models for anhydrous ethylmorphine and morphine hydrochloride dihydrate, two phases existing only in a very limited humidity range, are proposed on the basis of computational dehydration modelling. These match the experimental powder X-ray diffraction patterns and the structural information derived from infrared spectroscopy. All twelve structurally characterized morphinane forms (including structures from the Cambridge Structural Database) crystallize in the orthorhombic space group P212121. Hydrate formation results in higher dimensional hydrogen bond networks. The salt structures of different compounds exhibit only little structural variation. Anhydrous polymorphs were detected for all compounds except ethylmorphine (one anhydrate) and its hydrochloride salt (no anhydrate). Morphine HCl forms a trihydrate and dihydrate. Differential scanning and isothermal calorimetry were employed to estimate the heat of the hydrate ↔ anhydrate phase transformations, indicating an enthalpic stabilization of the respective hydrate of 5.7 to 25.6 kJ mol-1 relative to the most stable anhydrate. These results are in qualitative agreement with static 0 K lattice energy calculations for all systems except morphine hydrochloride, showing the need for further improvements in quantitative thermodynamic prediction of hydrates having water∙∙∙water interactions. Thus, the combination of a variety of experimental techniques, covering temperature and moisture dependent stability, and computational modelling allowed us to generate sufficient kinetic, thermodynamic and structural information to understand the principles of hydrate formation of the model compounds. This approach also led to the detection of several new crystal forms of the investigated morphinanes.
- Effects of a Novel Archaeal Tetraether-based Colipid on the In Vivo Gene Transfer Activity of Two Cationic Amphiphiles. [JOURNAL ARTICLE]
- Mol Pharm 2014 Jul 16.
Gene therapy for treating inherited diseases like cystic fibrosis might be achieved using multi-modular non-viral lipid-based systems. To date, most optimizations have concerned cationic lipids rather than colipids. In this study, an original archaeal tetraether derivative was used as a colipid in combination with one or the other of two monocationic amphiphiles. The liposomes obtained, termed archaeosomes, were characterized regarding lipid self-assembling properties, macroscopic/microscopic structures, DNA condensation/neutralization/relaxation abilities, and colloidal stability in presence of serum. In addition, gene transfer experiments were conducted in mice with lipid/DNA complexes being administered via systemic or local delivery routes. Altogether, the results showed that the tetraether colipid can provide complexes with different in vivo transfection abilities depending on the lipid combination, the lipid/colipid molar ratio, and the administration route. This original colipid appears thus as an innovative modular platform endowed with properties possibly beneficial for fine-tuning of in vivo lipofection and other biomedical applications.
- Effect of Surfactants, Gastric Emptying, and Dosage Form on Supersaturation of Dipyridamole in an in Vitro Model Simulating the Stomach and Duodenum. [JOURNAL ARTICLE]
- Mol Pharm 2014 Jul 15.
The purpose of this study was to investigate the influence of gastric emptying patterns, surfactants, and dosage form on the supersaturation of a poorly soluble weakly basic drug, dipyridamole, using an in vitro model mimicking the dynamic environment of the upper gastrointestinal tract, and, furthermore, to evaluate the usefulness of this model in establishing correlations to in vivo bioavailability for drugs with solubility/dissolution limited absorption. A simulated stomach duodenum model comprising four compartments was used to assess supersaturation and precipitation kinetics as a function of time. It integrates physiologically relevant fluid volumes, fluid transfer rates, and pH changes of the upper GI tract. Monoexponential gastric emptying patterns simulating the fasted state were compared to linear gastric emptying patterns simulating the fed state. The effect of different surfactants commonly used in oral preparations, specifically, sodium lauryl sulfate (SLS), poloxamer-188, and polysorbate-80, on dipyridamole supersaturation was investigated while maintaining surface tension of the simulated gastric fluids at physiological levels and without obtaining artificial micellar solubilization of the drug. The supersaturation behavior of different dose strengths of dipyridamole was explored. Significant levels of dipyridamole supersaturation were observed in the duodenal compartment under all the different in vivo relevant conditions explored. Dipyridamole supersaturation ratios of up to 11-fold have been observed, and supersaturation has been maintained for up to 120 min. Lower duodenal concentrations of dipyridamole were observed under linear gastric emptying patterns compared to mononexponential gastric emptying. The mean duodenal area under concentration-time curves (AUC60min) for the dipyridamole concentration profile in the duodenal compartment is significantly different for all the surfactants explored (P < 0.05). Our investigations with the different surfactants and comparison of dosage form (solution versus suspension) on the precipitation of dipyridamole revealed that crystal growth, rather than nucleation, is the rate-limiting step for the precipitation of dipyridamole. A linear dose-response relationship was found for the mean in vitro duodenal area under concentration-time curves (AUC∞) in the dose range of 25 mg to 100 mg (R(2) = 0.886). This is in agreement with the pharmacokinetic data of dipyridamole reported in the literature. The simulated stomach duodenum model can provide a reliable and discriminative screening tool for exploring the effect of different physiological variables or formulations on the supersaturation/precipitation kinetics of weakly basic drugs with solubility limited absorption. The amount of drug in solution in the duodenal compartment of the SSD correlates to bioavailability for the weakly basic drug, dipyridamole, which has solubility limited absorption and undergoes supersaturation/precipitation.
- Multifunctional Cationic Lipid-Based Nanoparticles Facilitate Endosomal Escape and Reduction-Triggered Cytosolic siRNA Release. [JOURNAL ARTICLE]
- Mol Pharm 2014 Jul 14.
Small interfering RNA (siRNA) has garnered much attention in recent years as a promising avenue for cancer gene therapy due to its ability to silence disease-related genes. Effective gene silencing is contingent upon the delivery of siRNA into the cytosol of target cells and requires the implementation of delivery systems possessing multiple functionalities to overcome delivery barriers. The present work explores the multifunctional properties and biological activity of a recently developed cationic lipid carrier, (1-aminoethyl)iminobis[N-(oleicylcysteinyl-1-amino-ethyl)propionamide]) (ECO). The physicochemical properties and biological activity of ECO/siRNA nanoparticles were assessed over a range of N/P ratios to optimize the formulation. Potent and sustained luciferase silencing in a U87 glioblastoma cell line was observed, even in the presence of serum proteins. ECO/siRNA nanoparticles exhibited pH-dependent membrane disruption at pH levels corresponding to various stages of the intracellular trafficking pathway. It was found that disulfide linkages created during nanoparticle formation enhanced the protection of siRNA from degradation and facilitated site-specific siRNA release in the cytosol by glutathione-mediated reduction. Confocal microscopy confirmed that ECO/siRNA nanoparticles readily escaped from late endosomes prior to cytosolic release of the siRNA cargo. These results demonstrate that the rationally designed multifunctionality of ECO/siRNA nanoparticles is critical for intracellular siRNA delivery and the continuing development of safe and effective delivery systems.
- Computational Predictions of Glass-Forming Ability and Crystallization Tendency of Drug Molecules. [JOURNAL ARTICLE]
- Mol Pharm 2014 Jul 11.
Amorphization is an attractive formulation technique for drugs suffering from poor aqueous solubility as a result of their high crystal lattice energy. Computational models that can predict the material properties associated with amorphization, such as glass-forming ability (GFA) and crystallization behavior in the dry state, would be a time-saving, cost-effective, and material-sparing approach compared to traditional experimental procedures. This paper presents predictive models of these properties developed using support vector machine (SVM) algorithm. The GFA and crystallization tendency were investigated by melt-quenching 131 drug molecules in situ using differential scanning calorimetry. The SVM algorithm was used to develop computational models based on calculated molecular descriptors. The analyses confirmed the previously suggested cut-off molecular weight (MW) of 300 for glass-formers, and also clarified the extent to which MW can be used to predict the GFA of compounds with MW <300. The topological equivalent of Grav3_3D, which is related to molecular size and shape, was a better descriptor than MW for GFA; it was able to accurately predict 87% of the dataset regardless of MW. The potential for crystallization was predicted using chemical descriptors reflecting Hückel pi atomic charges and the number of hydrogen bond acceptors. The models developed could be used in the early drug development stage to indicate whether amorphization would be a suitable formulation strategy for improving the dissolution and/or apparent solubility of poorly soluble compounds.
- The impact of inter and intramolecular interactions on physical stability of Indomethacin dispersed in acetylated saccharides. [JOURNAL ARTICLE]
- Mol Pharm 2014 Jul 10.
Differential Scanning Calorimetry (DSC), Broadband Dielectric (BDS), Fourier Transform Infrared (FTIR) spectroscopies and theoretical computations were applied to investigate inter- as well as intramolecular interactions between active pharmaceutical ingredient (API) indomethacin (IMC) and a series of acetylated saccharides. It was found that solid dispersions formed by modified glucose and IMC are the least physically stable of all studied samples. Dielectric measurements showed that this finding can be related to neither the global nor local mobility that were fairly the same in each case. On the other hand combined studies with the use of Density Functional Theory (DFT) and FTIR methods indicated that, in contrast to acetylated glucose, modified disaccharides (maltose and sucrose) interact strongly with indomethacin. As a result, internal H-bonds between IMC molecules become very weak or are eventually broken. Simultaneously, strong H-bonds between the matrix and the API are formed. This hint was furthermore used to explain the physical stability of the investigated solid dispersions. Finally solubility measurements revealed that the solubility of IMC can be enhanced by the use of acetylated carbohydrates, although the observed improvement is marginal due to strong interactions.
- PET Imaging of Fatty Acid Amide Hydrolase with [18F]DOPP in Non-Human Primates. [JOURNAL ARTICLE]
- Mol Pharm 2014 Jul 8.
Fatty acid amide hydrolase (FAAH) regulates endocannabinoid signaling. [11C]CURB, an irreversibly binding FAAH inhibitor, has been developed for clinical research imaging with PET. However no fluorine-18 labeled radiotracer for FAAH has yet advanced to human studies. [18F]DOPP ([18F]3-(4,5-dihydrooxazol-2-yl)phenyl (5-fluoropentyl)carbamate) has been identified as a promising 18F-labeled analog based on rodent studies. The goal of this work is to evaluate [18F]DOPP in non-human primates to support its clinical translation. High specific activity [18F]DOPP (5 - 6 Ci•µmol-1) was administered intravenously (i.v.) to three baboons (2M/1F, 3 - 4 yr). The distribution and pharmacokinetics were quantified following a 2 h dynamic imaging session using a simultaneous PET/MR scanner. Pretreatment with the FAAH-selective inhibitor, URB597, was carried out at 200 or 300 µg/kg i.v., 10 min prior to [18F]DOPP administration. Rapid arterial blood sampling for the first 3 min was followed by interval sampling with metabolite analysis to provide a parent radiotracer plasma input function that indicated ~95% baseline metabolism at 60 min and a reduced rate of metabolism after pretreatment with URB597. Regional distribution data were analyzed with 1-, 2-, and 3-tissue compartment models (TCMs), with and without irreversible trapping since [18F]DOPP is believed to covalently link to the active site of FAAH. Consistent with previous findings for [11C]CURB, the 2TCM with irreversible binding was found to provide the best fit for modeling the data in all regions. The composite parameter λk3 was therefore used to evaluate whole brain (WB) and regional binding of [18F]DOPP. Pretreatment studies showed inhibition of λk3 across all brain regions (WB baseline: 0.112 mL/cm3/min; 300 µg/kg URB597: 0.058 mL/cm3/min) suggesting that [18F]DOPP binding is specific for FAAH, consistent with previous rodent data.
- Targeting Breast Tumors with pH (Low) Insertion Peptides. [JOURNAL ARTICLE]
- Mol Pharm 2014 Jul 8.
Extracellular acidity is associated with tumor progression. Elevated glycolysis and acidosis promote the appearance of aggressive malignant cells with enhanced multidrug resistance. Thus, targeting of tumor acidity can open new avenues in diagnosis and treatment of aggressive tumors and targeting metastatic cancers cells within a tumor. pH (low) insertion peptides (pHLIPs) belong to the class of pH-sensitive agents capable of delivering imaging and/or therapeutic agents to cancer cells within tumors. Here, we investigated targeting of highly metastatic 4T1 mammary tumors and spontaneous breast tumors in FVB/N-Tg (MMTV-PyMT)634Mul transgenic mice with three fluorescently labeled pHLIP variants including well-characterized WT-pHLIP and, recently introduced, Var3- and Var7-pHLIPs. The Var3- and Var7-pHLIPs constructs have faster blood clearance than the parent WT-pHLIP. All pHLIPs demonstrated excellent targeting of the above breast tumor models with tumor accumulation increasing over 4 h postinjection. Staining of nonmalignant stromal tissues in transgenic mice was minimal. The pHLIPs distribution in tumors showed colocalization with 2-deoxyglucose and the hypoxia marker, Pimonidazole. The highest degree of colocalization of fluorescent pHLIPs was shown to be with lactate dehydrogenase A, which is related to lactate production and acidification of tumors. In sum, the pHLIP-based targeting of breast cancer presents an opportunity to monitor metabolic changes, and to selectively deliver imaging and therapeutic agents to tumors.