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Molecular pharmaceutics [journal]
- Evaluation of a Cholecystokinin 2 Receptor-Targeted Near-Infrared Dye for Fluorescence-Guided Surgery of Cancer. [JOURNAL ARTICLE]
- Mol Pharm 2013 Dec 16.
Surgical resection of malignant disease remains one of the most effective tools for treating cancer. Tumor-targeted near-infrared dyes have the potential to improve contrast between normal and malignant tissues, thereby enabling surgeons to more quantitatively resect malignant disease. Because the cholecystokinin 2 receptor (CCK2R and its tumor-specific splice variant CCK2i4svR) is overexpressed in cancers of the lungs, colon, thyroid, pancreas, and stomach, but absent or inaccessible to parenterally administered drugs in most normal tissues, we have undertaken to design a targeting ligand that can deliver attached near-infrared dyes to CCK2R+ tumors. We report here the synthesis and biological characterization of a CCK2R-targeted conjugate of the near-infrared dye, LS-288 (CRL-LS288). We demonstrate that CRL-LS288 binds selectively to CCK2R+ cancer cells with low nanomolar affinity (Kd = 7 × 10(-9) M). We further show that CRL-LS288 localizes primarily to CCK2R-expressing HEK 293 murine tumor xenografts and that dye uptake in these xenografts is significantly reduced when CCK2R are blocked by preinjection of excess ligand (CRL) or when mice are implanted with CCK2R-negative tumors. Because CRL-LS288 is also found to reveal the locations of distant tumor metastases, we suggest that CRL-LS288 has the potential to facilitate intraoperative identification of malignant disease during a variety of cancer debulking surgeries.
- Mathematical Modeling of the in Vitro Hepatic Disposition of Mycophenolic Acid and Its Glucuronide in Sandwich-Cultured Human Hepatocytes. [JOURNAL ARTICLE]
- Mol Pharm 2013 Dec 13.
In recent years, it has become increasingly important to test the safety of circulating metabolites of novel drugs as part of drug discovery and development programs. Accordingly, it is essential to develop suitable methods for identifying the major metabolites and their disposition in animal species and in humans. Mycophenolic acid (MPA), a selective inosine-5'-monophosphate dehydrogenase (IMPDH) inhibitor, is metabolized by glucuronidation and enterohepatic circulation of MPA-glucuronides is an important factor in the continuous systemic exposure of MPA. In humans, about 90% of the administered MPA dose is finally excreted as MPA phenyl-glucuronide (MPAG) in urine. Notably, the plasma concentration of MPAG is much higher than that of MPA. These factors suggest that, after its formation in hepatocytes, MPAG is excreted into bile and is also transported across the basolateral membrane to enter the circulation. In the present study, we performed metabolic/hepatobiliary transport studies of MPA and MPAG using sandwich-cultured human hepatocytes (SCHH) and constructed mathematical models of their hepatic disposition. We also performed vesicular transport studies to identify which human multidrug resistance-associated proteins (MRPs) are involved in the transport of MPAG from hepatocytes. MPAG was a preferred substrate for the biliary excretion transporter MRP2 and the hepatic basolateral transporters MRP3 and MRP4 in conventional and metabolic/hepatobiliary transport studies using SCHH and vesicular transport studies using human MRP-expressing membrane vesicles. The resulting mathematical model suggested that the basolateral transport plays an important role in the hepatic disposition of MPAG formed in hepatocytes. Our findings suggest that mathematical modeling of metabolic/hepatobiliary transport studies using SCH will provide useful information for determining the fate of metabolites formed in hepatocytes.
- Formulation and Characterization of Nanoemulsion Intranasal Adjuvants: Effects of Surfactant Composition on Mucoadhesion and Immunogenicity. [JOURNAL ARTICLE]
- Mol Pharm 2013 Dec 13.
The development of effective intranasal vaccines is of great interest due to their potential to induce both mucosal and systemic immunity. Here we produced oil-in-water nanoemulsion (NE) formulations containing various cationic and nonionic surfactants for use as adjuvants for the intranasal delivery of vaccine antigens. NE induced immunogenicity and antigen delivery are believed to be facilitated through initial contact interactions between the NE droplet and mucosal surfaces which promote prolonged residence of the vaccine at the site of application, and thus cellular uptake. However, the details of this mechanism have yet to be fully characterized experimentally. We have studied the physicochemical properties of the NE droplet surfactant components and demonstrate that properties such as charge and polar headgroup geometry influence the association of the adjuvant with the mucus protein, mucin. Association of NE droplets with mucin in vitro was characterized by various biophysical and imaging methods including dynamic light scattering (DLS), zeta potential (ZP), and surface plasmon resonance (SPR) measurements as well as transmission electron microscopy (TEM). Emulsion surfactant compositions were varied in a systematic manner to evaluate the effects of hydrophobicity and polar group charge/size on the NE-mucin interaction. Several cationic NE formulations were found to facilitate cellular uptake of the model antigen, ovalbumin (OVA), in a nasal epithelial cell line. Furthermore, fluorescent images of tissue sections from mice intranasally immunized with the same NEs containing green fluorescent protein (GFP) antigen demonstrated that these NEs also enhanced mucosal layer penetration and cellular uptake of antigen in vivo. NE-mucin interactions observed through biophysical measurements corresponded with the ability of the NE to enhance cellular uptake. Formulations that enhanced antigen uptake in vitro and in vivo also led to the induction of a more consistent antigen specific immune response in mice immunized with NEs containing OVA, linking NE-facilitated mucosal layer penetration and cellular uptake to enhancement of the immune response. These findings suggest that biophysical measurement of the mucoadhesive properties of emulsion based vaccines constitutes an effective in vitro strategy for selecting NE candidates for further evaluation in vivo as mucosal adjuvants.
- Preparation and Performance of Hydroxypropyl Methylcellulose Esters of Substituted Succinates for in Vitro Supersaturation of a Crystalline Hydrophobic Drug. [JOURNAL ARTICLE]
- Mol Pharm 2013 Dec 9.
We prepared hydroxypropyl methylcellulose (HPMC) esters of substituted succinates and examined their performance for improving the aqueous solubility of crystalline hydrophobic drugs in spray-dried dispersions (SDDs). From one HPMC, we synthesized five HPMC esters using various monosubstituted succinic anhydrides. These HPMC esters along with a commercial HPMC acetate succinate (HPMCAS) were spray-dried from solutions with phenytoin. The SDDs with different matrices at 10 wt % loading had very similar bulk properties with a minimal amount of detectable crystalline phenytoin as revealed by scanning electron microscopy (SEM), powder X-ray diffraction (powder XRD), and differential scanning calorimetry (DSC). In solution, while the SDD with HPMCAS was very effective at achieving high levels of phenytoin supersaturation initially, it was not competent at maintaining such supersaturation due to the rapid crystallization of the dissolved phenytoin. Alternatively, SDDs with several synthesized HPMC esters of substituted succinates not only achieved rather high initial supersaturation but also maintained high concentrations for extended time (i.e., 1.5 h and longer). Such maintenance was largely ascribed to the inhibition of phenytoin nucleation. Structure-property relationships were established, and the most successful systems contained a high degree of substitution and a combination of a thioether with neighboring weak electron-withdrawing groups in the substituted succinic anhydrides. The effective maintenance of supersaturated solutions was only found in SDDs with rather low drug loadings, which indicates the significance of sufficiently high concentrations of polymer additives in the dissolution media.
- Thermodynamic Approaches to the Challenges of Solubility in Drug Discovery and Development. [JOURNAL ARTICLE]
- Mol Pharm 2013 Dec 9.
This paper considers fundamental aspects determining the processes of drug compound dissolution and distribution in solvents/systems modeling biological media. Special attention is paid to the complex analysis of thermodynamic functions of sublimation and solvation/hydration processes during structural modification of lead/hit compounds and their influence on dissolution processes. The paper shows that at the first stages of drug design it is necessary to develop algorithms of optimizing the properties determining absorption, distribution, metabolism, and excretion (ADME) characteristics of molecules: in particular, diffusion flux density through lipophilic membranes. Using sulfonamides and nonsteroidal anti-inflammatory drugs (NSAIDs) as an example, we have demonstrated the efficiency of approaches to manipulating thermodynamic functions of the basic processes that result from delivering compounds to the points of their operation. We have formulated several criteria of compound selection for further tests.
- Folic Acid-Conjugated, SERS-Labeled Silver Nanotriangles for Multimodal Detection and Targeted Photothermal Treatment on Human Ovarian Cancer Cells. [JOURNAL ARTICLE]
- Mol Pharm 2013 Dec 12.
The effectiveness of a therapeutic agent for cancer stands in its ability to reduce and eliminate tumors without harming the healthy tissue nearby. Nanoparticles peripherally conjugated with targeting moieties offer major improvements in therapeutics through site specificity. In this study we demonstrate this approach by targeting the folate receptor of NIH:OVCAR-3 human ovary cancer cell line. Herein we used silver nanotriangles which were biocompatibilized with chitosan (bio)polymer, labeled with para-aminothiophenol (pATP) Raman reporter molecule, and conjugated with folic acid. The nanoparticles conjugation and efficient labeling was investigated by localized surface plasmon resonance (LSPR), zeta potential, and surface-enhanced Raman scattering (SERS) measurements. Conjugated particles were proven to be highly stable in aqueous and cellular medium. The targeted uptake of conjugated nanoparticles by human ovary cancer cells was confirmed by dark field microscopy and scattering spectra of the particles inside cells. Comparative studies revealed specific internalization of the conjugated nanoparticles in comparison with similar bare nanoparticles. Moreover, the SERS identity of the particles was proven to be highly conserved inside cells. Targeted cancer cell treatment conducted by irradiating the nanoparticle-treated cells with a continuous wave-nearinfrared (cw-NIR) laser in resonance with their plasmonic band proved an efficient therapeutic response. By integrating the advantages of multimodal optical imaging and SERS detection with hyperthermia capabilities through site specificity, these nanoparticles can represent a real candidate for personalized medicine.
- Co-administration of Dual-Targeting Nanoparticles with Penetration Enhancement Peptide for Antiglioblastoma Therapy. [JOURNAL ARTICLE]
- Mol Pharm 2013 Dec 2.
Chemotherapy is an indispensable auxiliary treatment for glioma but highly limited by the existence of both blood-brain barrier (BBB) and blood-brain tumor barrier (BBTB). The dysfunctional brain tumor blood vessels and high interstitial pressure in glioma also greatly hindered the accumulation and deep penetration of chemotherapeutics into the glioma. Lactoferrin (Lf), with its receptor overexpressed on both the brain endothelial cells and glioma cells, was here functionalized to the surface of poly(ethylene glycol)-poly(lactic acid) nanoparticles to mediate BBB/BBTB and glioma cell dual targeting. tLyP-1, a tumor-homing peptide, which contains a C-end Rule sequence that can mediate tissue penetration through the neuropilin-1-dependent internalization pathway, was coadministrated with Lf-functionalized nanoparticles (Lf-NP) to enhance its accumulation and deep penetration into the glioma parenchyma. Enhanced cellular association in both BCEC and C6 cells, increased cytotoxicity of the loaded paclitaxel, and deep penetration in the 3D glioma spheroids was achieved by Lf-NP. Following coadministration with tLyP-1, the functionalized nanoparticles obtained improved tumor targeting, glioma vascular extravasation, and antiglioma efficacy. The findings here suggested that the strategy by coadministrating BBB/BBTB and glioma cells dual-targeting nanocarriers with a tumor penetration enhancement peptide represent a promising platform for antiglioma drug delivery.
- Pycup-A Bifunctional, Cage-like Ligand for (64)Cu Radiolabeling. [JOURNAL ARTICLE]
- Mol Pharm 2013 Dec 11.
In developing targeted probes for positron emission tomography (PET) based on (64)Cu, stable complexation of the radiometal is key, and a flexible handle for bioconjugation is highly advantageous. Here, we present the synthesis and characterization of the chelator pycup and four derivatives. Pycup is a cross-bridged cyclam derivative with a pyridyl donor atom integrated into the cross-bridge resulting in a pentadentate ligand. The pycup platform provides kinetic inertness toward (64)Cu dechelation and offers versatile bioconjugation chemistry. We varied the number and type of additional donor atoms by alkylation of the remaining two secondary amines, providing three model ligands, pycup2A, pycup1A1Bn, and pycup2Bn, in 3-4 synthetic steps from cyclam. All model copper complexes displayed very slow decomplexation in 5 M HCl and 90 °C (t1/2: 1.5 h for pycup1A1Bn, 2.7 h for pycup2A, 20.3 h for pycup2Bn). The single crystal crystal X-ray structure of the [Cu(pycup2Bn)](2+) complex showed that the copper was coordinated in a trigonal, bipyramidal manner. The corresponding radiochemical complexes were at least 94% stable in rat plasma after 24 h. Biodistribution studies conducted in Balb/c mice at 2 h postinjection of (64)Cu labeled pycup2A revealed low residual activity in kidney, liver, and blood pool with predominantly renal clearance observed. Pycup2A was readily conjugated to a fibrin-targeted peptide and labeled with (64)Cu for successful PET imaging of arterial thrombosis in a rat model, demonstrating the utility of our new chelator in vivo.
- Modulation of Intestinal Folate Absorption by Erythropoietin in Vitro. [JOURNAL ARTICLE]
- Mol Pharm 2013 Dec 9.
Besides the direct stimulation of erythropoiesis, erythropoietin (EPO) therapy in renal anemia may also play a regulatory role in maintaining the homeostasis of hematopoietic nutrients. It has been reported that EPO can stimulate intestinal iron absorption. However, the involvement of EPO in intestinal folate absorption remains elusive. The objective of this study was to investigate the effect of EPO on intestinal transport of folate in vitro and to elucidate the possible mechanism(s) involved in this regulation. Transport assays of folic acid were performed in Caco-2 monolayers treated with EPO. The effect of EPO on the expression of transporters involved in the folate absorption was investigated. The possible involvement of three main EPO signaling pathways, the janus protein tyrosine kinase 2 (JAK-2) pathway, extracellular signal regulated kinases (ERK) pathway, and phosphatidylinositol 3 kinase/Akt (PI3K/Akt) pathway, in the transporter regulation was explored. The absorptive flux (apical to basolateral) of folic acid was enhanced by EPO treatment in a dose-dependent manner, which was companied with the significant up-regulation of reduced folate carrier (RFC) and apical proton coupled folate transporter (PCFT). The efflux (basolaterial to apical) of folic acid was enhanced only by the high dose of EPO treatment, which was associated with the significant up-regulation of apical multidrug resistance-associated protein 2 (MRP2). The expression levels of all of these transporters were up-regulated by EPO treatment in a dose- and time-dependent manner. Transporter expression in response to blocking EPO induced activation of JAK-2, ERK, and PI3K/Akt was changed to a different extent. As a conclusion, intestinal folate absorption was enhanced by EPO treatment in vitro. Our findings provided direct evidence to establish the correlation between EPO and folate homeostasis.
- Delivery of Chemically Glycosylated Cytochrome c Immobilized in Mesoporous Silica Nanoparticles Induces Apoptosis in HeLa Cancer Cells. [JOURNAL ARTICLE]
- Mol Pharm 2013 Dec 10.
Cytochrome c (Cyt c) is a small mitochondrial heme protein involved in the intrinsic apoptotic pathway. Once Cyt c is released into the cytosol, the caspase mediated apoptosis cascade is activated resulting in programmed cell death. Herein, we explore the covalent immobilization of Cyt c into mesoporous silica nanoparticles (MSN) to generate a smart delivery system for intracellular drug delivery to cancer cells aiming at affording subsequent cell death. Cyt c was modified with sulfosuccinimidyl-6-[3'-(2-pyridyldithio)-propionamido] hexanoate (SPDP) and incorporated into SH-functionalized MSN by thiol-disulfide interchange. Unfortunately, the delivery of Cyt c from the MSN was not efficient in inducing apoptosis in human cervical cancer HeLa cells. We tested whether chemical Cyt c glycosylation could be useful in overcoming the efficacy problems by potentially improving Cyt c thermodynamic stability and reducing proteolytic degradation. Cyt c lysine residues were modified with lactose at a lactose-to-protein molar ratio of 3.7 ± 0.9 using mono(lactosylamido)-mono(succinimidyl) suberate linker chemistry. Circular dichroism (CD) spectra demonstrated that part of the activity loss of Cyt c was due to conformational changes upon its modification with the SPDP linker. These conformational changes were prevented in the glycoconjugate. In agreement with the unfolding of Cyt c by the linker, a proteolytic assay demonstrated that the Cyt c-SPDP conjugate was more susceptible to proteolysis than Cyt c. Attachment of the four lactose molecules reversed this increased susceptibility and protected Cyt c from proteolytic degradation. Furthermore, a cell-free caspase-3 assay revealed 47% and 87% of relative caspase activation by Cyt c-SPDP and the Cyt c-lactose bioconjugate, respectively, when compared to Cyt c. This again demonstrates the efficiency of the glycosylation to improve maintaining Cyt c structure and thus function. To test for cytotoxicity, HeLa cells were incubated with Cyt c loaded MSN at different Cyt c concentrations (12.5, 25.0, and 37.5 μg/mL) for 24-72 h and cellular metabolic activity determined by a cell proliferation assay. While MSN-SPDP-Cyt c did not induced cell death, the Cyt c-lactose bioconjugate induced significant cell death after 72 h, reducing HeLa cell viability to 67% and 45% at the 25 μg/mL and 37.5 μg/mL concentrations, respectively. Confocal microscopy confirmed that the MSN immobilized Cyt c-lactose bioconjugate was internalized by HeLa cells and that the bioconjugate was capable of endosomal escape. The results clearly demonstrate that chemical glycosylation stabilized Cyt c upon formulation of a smart drug delivery system and upon delivery into cancer cells and highlight the general potential of chemical protein glycosylation to improve the stability of protein drugs.