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Molecular pharmaceutics [journal]
- Polyethylene Glycol (PEG)-Mediated Conformational Alteration of α-Chymotrypsin Prevents Inactivation of Insulin by Stabilizing Active Intermediates. [JOURNAL ARTICLE]
- Mol Pharm 2014 Apr 10.
Proteolytic enzymes in the gut represent one of the biggest barriers against oral delivery of therapeutic proteins and peptides. In the current study, we explored the effect of polyethylene glycol 400 (PEG 400), a commonly used crowing agent, on insulin degradation mediated by α-chymotrypsin (α-CT). Without PEG 400, insulin was quickly cleaved by α-CT to generate inactive degradation products. In comparison, incorporation of PEG 400 resulted in reaction mixtures with retained biological activity. The analysis on the conformation of α-CT and the local environment of the enzyme's active site unraveled that PEG 400 altered the conformation of α-CT to prevent the inactivation of insulin via stabilization of active intermediates. These findings indicated that PEG 400 may provide a promising addition toward oral delivery of insulin.
- Comparison of Conjugation Strategies of Cross-bridged Macrocyclic Chelators with Cetuximab for Copper-64 Radiolabeling and PET Imaging of EGFR in Colorectal Tumor-bearing Mice. [JOURNAL ARTICLE]
- Mol Pharm 2014 Apr 10.
Epidermal growth-factor receptor (EGFR) is overexpressed in a wide variety of solid tumors and has served as a well-characterized target for cancer imaging and therapy. Cetuximab was the first mAb targeting EGFR approved by the FDA for the treatment of metastatic colorectal and head & neck cancers. Previous studies showed that 64Cu (T1/2=12.7 h; β+ (17.4%)) labeled DOTA-cetuximab showed promise for PET imaging of EGFR-positive tumors; however the in vivo stability of this compound has been questioned. In this study, two recently developed cross-bridged macrocyclic chelators (CB-TE1A1P and CB-TE1K1P) were conjugated to cetuximab using standard NHS coupling procedures and/or strain-promoted azide-alkyne cycloaddition (SPAAC) methodologies. The radiolabeling and in vitro/vivo evaluation of the resulting cetuximab conjugates were compared. Improved Cu-64 labeling efficiency and high specific activity (684 kBq/µg, decay corrected to the end of bombardment) were obtained with the CB-TE1K1P-PEG4-click-cetuximab conjugate. Saturation binding assays indicated that the prepared cetuximab conjugates had comparable affinity (1.13 - 2.00 nM) in the HCT116 human colorectal tumor cell membranes. In the subsequent in vivo evaluation, 64Cu-CB-TE1K1P-PEG4-click-cetuximab demonstrated more rapid renal clearance with a higher tumor/non-tumor ratio than other 64Cu-labeled cetuximab conjugates, and shows the greatest promise for imaging and therapy of EGFR-positive tumors.
- Synthesis of Doxorubicin α-Linolenic Acid Conjugate and Evaluation of Its Antitumor Activity. [JOURNAL ARTICLE]
- Mol Pharm 2014 Apr 10.
Doxorubicin (DOX) is a broad-spectrum antitumor drug used in the clinic. However, it can cause serious heart toxicity. To increase the therapeutic index of DOX and to attenuate its toxicity toward normal tissues, we conjugated DOX with either α-linolenic acid (LNA) or palmitic acid (PA) by a hydrazone or an amide bond to produce DOX-hyd-LNA, DOX-ami-LNA, DOX-hyd-PA, and DOX-ami-PA. The cytotoxicity of DOX-hyd-LNA on HepG2, MCF-7, and MDA-231 cells was higher compared to that of DOX, DOX-ami-LNA, DOX-hyd-PA, and DOX-ami-PA. The cytotoxicity of DOX-hyd-LNA on HUVECs was lower than that of DOX. DOX-hyd-LNA released significantly more DOX in pH 5.0 medium than it did in pH 7.4 medium. DOX-hyd-LNA induced more apoptosis in MCF-7 and HepG2 cells than DOX or DOX-ami-LNA. Significantly more DOX was released from DOX-hyd-LNA in both MCF-7 and HepG2 cells compared with DOX-ami-LNA. Compared to free DOX, a biodistribution study showed that DOX-hyd-LNA greatly increased the content of DOX in tumor tissue and decreased the content of DOX in heart tissue after it was intravenously administered. DOX-hyd-LNA improved the survival rate, prolonged the life span, and slowed the growth of the tumor in tumor-bearing nude mice. These results indicate that DOX-hyd-LNA improved the therapeutic index of DOX. Therefore, DOX-hyd-LNA is a potential compound for use as a cancer-targeting therapy.
- Brain Uptake of a Zidovudine Prodrug after Nasal Administration of Solid Lipid Microparticles. [JOURNAL ARTICLE]
- Mol Pharm 2014 Apr 9.
Our previous results demonstrated that a prodrug obtained by the conjugation of the antiretroviral drug zidovudine (AZT) with ursodeoxycholic acid (UDCA) represents a potential carrier for AZT in the central nervous system, thus possibly increasing AZT efficiency as anti-HIV drug. Based on these results and in order to enhance AZT brain targeting, the present study focuses on solid lipid microparticles (SLMs) as a carrier system for the nasal administration of UDCA-AZT prodrug. SLMs were produced by the hot emulsion technique, using tristearin and stearic acid as lipidic carriers, whose mean diameters were 16 and 7 μm, respectively. SLMs were of spherical shape and their prodrug loading was 0.57 ±0.03% (w/w, tristearin based) and 1.84 ±0.02% (w/w, stearic acid based). The tristearin SLMs were able to control the prodrug release, whereas the stearic acid SLMs induced a significant increase of the dissolution rate of the free prodrug. The free prodrug was rapidly hydrolyzed in rat liver homogenates with a half-life of 2.7 ± 0.14 min (process completed within 30 min). The tristearin SLMs markedly enhanced the stability of the prodrug (75 % of the prodrug still present after 30 min), whereas the stabilization effect of the stearic acid SLMs was lower (14 % of the prodrug still present after 30 min). No AZT and UDCA-AZT were detected in the rat cerebrospinal fluid (CSF) after an intravenous prodrug administration (200 μg). Conversely, the nasal administration of stearic acid based SLMs induced the uptake of the prodrug in the CSF, demonstrating the existence of a direct nose - CNS pathway. In the presence of chitosan, the CSF prodrug uptake increased six times, up to 1.5 μg/mL within 150 minutes after nasal administration. The loaded SLMs appear therefore as a promising nasal formulation for selective zidovudine brain uptake.
- A Radiofluorinated Divalent Cystine Knot Peptide for Tumor PET Imaging. [JOURNAL ARTICLE]
- Mol Pharm 2014 Apr 9.
A divalent knottin containing two separate integrin binding epitopes (RGD) in the adjacent loops, 3-4A, was recently discovered in our previous report. In this study, 3-4A was radiofluorinated with a 4-nitrophenyl 2-18F-fluoropropinate (18F-NFP) group and the resulting divalent positron emission tomography (PET) probe, 18F-FP-3-4A, was further evaluated as a novel imaging probe to detect integrin αvβ3 positive tumors in living animals. Knottin 3-4A was synthesized by solid phase peptide synthesis, folded and site-specifically conjugated with 18/19F-NFP to produce the fluorinated peptide 18/19F-fluoropropinate-3-4A (18/19F-FP-3-4A). The stability of 18F-FP-3-4A was tested in both phosphate buffered saline (PBS) buffer and mouse serum. Cell uptake assay of the radiolabeled peptides was performed using U87MG cell lines. In addition, small animal PET imaging and biodistribution studies of 18F-FP-3-4A were performed in U87MG tumor-bearing mice. The receptor targeting specificity of the radiolabeled peptide was also verified by co-injecting the probe with the blocking peptide cyclo(RGDyK). Our study showed that 18F-FP-3-4A exhibited excellent stability in PBS buffer (pH 7.4) and mouse serum. Small animal PET imaging and biodistribution data revealed that 18F-FP-3-4A exhibited rapid and good tumor uptake (3.76 ± 0.59% ID/g and 2.22 ± 0.62 % ID/g at 0.5 h and 1 h, respectively). Especially, 18F-FP-3-4A was rapidly cleared from the normal tissues, resulting in excellent tumor-to-normal tissue contrasts. For example, the liver uptake was only 0.39 ± 0.07 %ID/g and the tumor to liver ratio was 5.69 at 1 h p.i.. Furthermore, co-injection of 18F-FP-3-4A with cyclo(RGDyK) significantly inhibited tumor uptake (0.41 ± 0.12 vs. 1.02 ± 0.19 %ID/g at 2.5 h) in U87MG xenograft models, demonstrating specific accumulation of the probe in tumor. Together, the divalent probe 18F-FP-3-4A is characterized by rapid and high tumor uptake, and excellent tumor-to-normal tissue ratios. It is a highly promising knottin based PET probe for translating into clinical imaging of tumor angiogenesis.
- Bioengineered 3D Brain Tumor Model to Elucidate the Effects of Matrix Stiffness on Glioblastoma Cell Behavior using PEG-based Hydrogels. [JOURNAL ARTICLE]
- Mol Pharm 2014 Apr 8.
Glioblastoma (GBM) is the most common and aggressive form of primary brain tumor with a median survival of 12-15 months, and the mechanisms underlying GBM tumor progression remains largely elusive. Given the importance of tumor niche signaling in driving GBM progression, there is a strong need to develop in vitro models to facilitate analysis of brain tumor cell-niche interactions in a physiologically relevant and controllable manner. Here we report the development of a bioengineered 3D brain tumor model to help elucidate the effects of matrix stiffness on GBM cell fate using poly(ethylene-glycol) (PEG)-based hydrogels with brain-mimicking biochemical and mechanical properties. We have chosen PEG given its bioinert nature and tunable physical property, and the resulting hydrogels allow tunable matrix stiffness without changing the biochemical contents. To facilitate cell proliferation and migration, CRGDS and a MMP-cleavable peptide were chemically incorporated. Hyaluronic acid (HA) was also incorporated to mimic the concentration in the brain extracellular matrix. Using U87 cells as a model GBM cell line, we demonstrate that such biomimetic hydrogels support U87 cell growth, spreading, and migration in 3D over the course of 3 weeks in culture. Gene expression analyses showed U87 cells actively deposited extracellular matrix and continued to upregulate matrix remodeling genes. To examine the effects of matrix stiffness on GBM cell fate in 3D, we encapsulated U87 cells in soft (1 kPa) or stiff (26 kPa) hydrogels, which respectively mimics the matrix stiffness of normal brain or GBM tumor tissues. Our results suggest that changes in matrix stiffness induce differential GBM cell proliferation, morphology, and migration modes in 3D. Increasing matrix stiffness led to delayed U87 cell proliferation inside hydrogels, but cells formed denser spheroids with extended cell protrusions. Cells cultured in stiff hydrogels also showed upregulation of HA synthase 1 and matrix metalloproteinase-1 (MMP-1), while simultaneously downregulating HA synthase 2 and MMP-9. This suggests that varying matrix stiffness can induce differential ECM deposition and remodeling by employing different HA synthases or MMPs. Furthermore, increasing matrix stiffness led to simultaneous upregulation of Hras, RhoA and ROCK1, suggesting a potential link between the mechanosensing pathways and the observed differential cell responses to changes in matrix stiffness. The bioengineered 3D hydrogel platform reported here may provide a useful 3D in vitro brain tumor model for elucidating the mechanisms underlying GBM progression, as well as for evaluating the efficacy of potential drug candidates for treating GBM.
- Combinational delivery of Hydrophobic and Hydrophilic Anticancer Drugs in Single Nanoemulsions to Treat MDR in Cancer. [JOURNAL ARTICLE]
- Mol Pharm 2014 Apr 8.
In the study, we developed the core-matched nanoemulsions (NEs) functionalized by Vitamin E (VE) and tocopherol polyethylene glycol succinate (TPGS) to co-deliver hydrophobic and hydrophilic drugs, paclitaxel (PTX) and 5-fluoroucacil (5-FU), in order to achieve synergistic effects and overcome PTX resistance in a multidrug resistant (MDR) human epidermal carcinoma cell line KB-8-5. Antitumor effect of the combination therapy based on core-matched technology (CMT) was evaluated in vitro and in vivo in mice. The core-matched NEs showed entrapment efficiency of > 90 %, and were of nanoscale particle size and negative zeta potential. The combined core-matched NEs exhibited concentration and time-dependent cytotoxicity against PTX sensitive KB-3-1 cells and PTX resistant KB-8-5 cells as well as an obviously increased G2/M phase block. The improvements in therapeutic response over either PTX-VE or 5FU-TPGS therapy alone were demonstrated by the ability to effectively induce the apoptosis of tumor cells via up-regulation of tumor suppressor p53 and β-tubulin and by the significant inhibition of cell cycle progression. The combination therapy led to dramatic inhibition of tumor growth with little toxicity in vivo, especially in the PTX resistant KB-8-5 tumors, whereas Taxol had little therapeutic effect. This was mainly ascribed to the synergism of PTX and 5-FU and the reverse of MDR by the inhibition of ATPase activity by VE and TPGS. Co-encapsulation of two chemotherapeutic agents with different mechanisms allows simultaneous interruption of diverse anti-cancer pathways, resulting in increased therapeutic response and low toxicity. The CMT markedly facilitated the long circulation of PTX and 5-FU, which was closely associated with the high accumulation of chemotherapeutic agents within the tumors and the improvement of anti-tumor efficacy. The current study demonstrated the feasibility of incorporating PTX and 5-FU targeting to different pathways into a single core-matched NE for the reversal of MDR and synergism in cancer therapy.
- In Vivo Performance of a Novel Fluorinated Magnetic Resonance Imaging Agent for Functional Analysis of Bile Acid Transport. [JOURNAL ARTICLE]
- Mol Pharm 2014 Apr 7.
A novel trifluorinated cholic acid derivative, CA-lys-TFA, was designed and synthesized for use as a tool to measure bile acid transport non-invasively using magnetic resonance imaging (MRI). In the present study, the in vivo performance of CA-lys-TFA for measuring bile acid transport by MRI was investigated in mice. Gallbladder CA-lys-TFA content was quantified using MRI and liquid chromatography/tandem mass spectrometry. Results in wild-type (WT) C57BL/6J mice were compared to those in mice lacking expression of Asbt, the ileal bile acid transporter. 19F signals emanating from the gallbladders of WT mice 7 h after oral gavage with 150 mg/kg CA-lys-TFA were reproducibly detected by MRI. Asbt-deficient mice administered the same dose had undetectable 19F signals by MRI, and gallbladder bile CA-lys-TFA levels were 30-fold lower compared to WT animals. To our knowledge, this represents the first report of in vivo imaging of an orally absorbed drug using 19F MRI. Fluorinated bile acid analogues have potential as tools to measure and detect abnormal bile acid transport by MRI.
- Prolongation of Life in Rats with Malignant Glioma by Intranasal siRNA/Drug Codelivery to the Brain with Cell-Penetrating Peptide-Modified Micelles. [JOURNAL ARTICLE]
- Mol Pharm 2014 Apr 16.
New therapeutic strategies are required to develop candidate drugs and ensure efficient delivery of these drugs to the brain and the central nervous system (CNS). Small interfering RNA (siRNA)-based therapies have been investigated as potential novel approaches for the treatment of brain disorders. Previously, we showed that Tat, a cell-penetrating peptide derived from HIV-Tat, and the modified block copolymers (MPEG-PCL-Tat) can form stable complexes with siRNA or can be loaded with an anticancer drug and efficiently deliver the drugs to the brain tissue via intranasal delivery. In this study, to develop a novel, efficient, and safe therapeutic strategy for managing brain disorders, we used MPEG-PCL-Tat micelles with a nose-to-brain delivery system to investigate its therapeutic effects on a rat model of malignant glioma using siRNA with a Raf-1 (siRaf-1)/camptothecin (CPT) codelivery system. MPEG-PCL-Tat and CPT-loaded MPEG-PCL-Tat can form a stable complex with siRNA with a particle size from 60 to 200 nm and a positive charge at N/P ratios up to 5. Additionally, MPEG-PCL-Tat/siRaf-1 and CPT-loaded MPEG-PCL-Tat/siRaf-1 have fostered cell death in rat glioma cells after the high cellular uptake of siRaf-1/drug by the MPEG-PCL-Tat carrier. Furthermore, compared to the unloaded MPEG-PCL-Tat/siRaf-1 complex, a CPT-loaded MPEG-PCL-Tat/siRaf-1 complex achieved the high therapeutic effect because of the additive effects of CPT and siRaf-1. These results indicate that drug/siRNA codelivery using MPEG-PCL-Tat nanomicelles with nose-to-brain delivery is an excellent therapeutic approach for brain and CNS diseases.
- Assessing the Efficiency of Polymeric Excipients by Atomistic Molecular Dynamics Simulations. [JOURNAL ARTICLE]
- Mol Pharm 2014 Apr 16.
We have performed all-atom molecular dynamics simulations of aqueous solutions of model oligomers of hydroxypropyl methylcellulose (HPMC) and hydroxypropyl methylcellulose acetate succinate (HPMCAS) excipients interacting with a representative poorly soluble active pharmaceutical ingredient (API), phenytoin. Simulations reveal formation of excipient-API complexes for some of the oligomers, which results in a reduction of API aggregation. API aggregation and diffusivity decreased with an increase in excipient content. Excipients form a "gel-like" phase spanning the simulation box beyond ∼10 wt %; API diffusivity within this gel phase is much smaller than API diffusivity without excipient, and decreases exponentially, by 5 orders of magnitude, with increased polymer concentration. Substantial differences are observed with variations in methyl, hydroxypropyl, acetate, and succinate substitution levels in the model oligomers and with the deprotonation state of succinate groups, with strongest interactions with hydrophobic phenytoin observed in the case of acetate substitution. These are used to develop quantitative measures of excipient-API interactions and excipient efficiency in the inhibition of API aggregation. We also find that for model oligomers based on Methocel E (manufactured by Dow Pharma & Food Solutions) chemistry, oligomers of length 10 monomers and simulation boxes of size 7 nm give results similar to those for longer oligomers and bigger boxes. The quantitative measures developed in this study are expected to prove useful as computational screening tools in excipient design.