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Molecular pharmaceutics [journal]
- The Low/High BCS Permeability Class Boundary: Physicochemical Comparison of Metoprolol and Labetalol. [JOURNAL ARTICLE]
- Mol Pharm 2014 Apr 15.
Although recognized as overly conservative, metoprolol is currently the common low/high BCS permeability class boundary reference compound, while labetalol was suggested as a potential alternative. The purpose of this study was to identify the various characteristics that the optimal marker should exhibit, and to investigate the suitability of labetalol as the permeability class reference drug. Labetalol's BCS solubility class was determined, and its physicochemical properties and intestinal permeability were thoroughly investigated, both in vitro and in vivo in rats, considering the complexity of the whole of the small intestine. Labetalol was found to be unequivocally a high-solubility compound. In the pH range throughout the small intestine (6.5-7.5), labetalol exhibited pH-dependent permeability, with higher permeability at higher pH values. While in vitro octanol-buffer partitioning (Log D) values of labetalol were significantly higher than those of metoprolol, the opposite was evident in the in vitro PAMPA permeability assay. The results of the in vivo perfusion studies in rats lay between the two contradictory in vitro studies; metoprolol was shown to have moderately higher rat intestinal permeability than labetalol. Theoretical distribution of the ionic species of the drugs was in corroboration with the experimental in vitro and the in vivo data. We propose three characteristics that the optimal permeability class reference drug should exhibit: (1) fraction dose absorbed in the range of 90%; (2) the optimal marker drug should be absorbed largely via passive transcellular permeability, with no/negligible carrier-mediated active intestinal transport (influx or efflux); and (3) the optimal marker drug should preferably be nonionizable. The data presented in this paper demonstrate that neither metoprolol nor labetalol can be regarded as optimal low/high-permeability class boundary standard. While metoprolol is too conservative due to its complete absorption, labetalol has been shown to be a substrate for P-gp-mediated efflux transport, and both drugs exhibit significant segmental-dependent permeability along the gastrointestinal tract. Nevertheless, the use of metoprolol as the marker compound does not carry a risk of bioinequivalence: Peff value similar to or higher than metoprolol safely indicates high-permeability classification. On the other hand, a more careful data analysis is needed if labetalol is used as the reference compound.
- Relationship between Crystallization Tendencies during Cooling from Melt and Isothermal Storage: Toward a General Understanding of Physical Stability of Pharmaceutical Glasses. [JOURNAL ARTICLE]
- Mol Pharm 2014 Apr 14.
The lack of protocols to predict the physical stability has been one of the most important issues in the use of amorphous solid dispersions. In this paper, the crystallization behaviors of pharmaceutical glasses, which have large variations in their crystallization tendencies, have been investigated. Although each compound appears to have a wide variation in their crystallization time, the initiation time for crystallization could be generalized as a function of only Tg/T, where Tg and T are the glass transition temperature and storage temperature, respectively. All compounds in which crystallization was mainly governed by temperature had similar activation energies for crystallization initiation, ca. 210-250 kJ/mol, indicating that physical stability at any temperature is predictable from only Tg. Increased stability is expected for other compounds, where crystallization is inhibited by an large energetic barrier, and stochastic nucleation plays an important role in initiating crystallization. The difference in the dominant factor, either temperature or pressure, appeared to correlate with the nucleation mechanism, and this could be determined by a cool-heat cycle after melting, using thermal analysis. This conclusion should make prediction of physical stability of amorphous formulations easier, although the investigation was conducted under ideal conditions, which eliminated surface effects.
- Avidity Mechanism of Dendrimer-Folic Acid Conjugates. [JOURNAL ARTICLE]
- Mol Pharm 2014 Apr 11.
Multivalent conjugation of folic acid has been employed to target cells overexpressing folate receptors. Such polymer conjugates have been previously demonstrated to have high avidity to folate binding protein. However, the lack of a monovalent folic acid-polymer material has prevented a full binding analysis of these conjugates, as multivalent binding mechanisms and polymer-mass mechanisms are convoluted in samples with broad distributions of folic acid-to-dendrimer ratios. In this work, the synthesis of a monovalent folic acid-dendrimer conjugate allowed the elucidation of the mechanism for increased binding between the folic acid-polymer conjugate and a folate binding protein surface. The increased avidity is due to a folate-keyed interaction between the dendrimer and protein surfaces that fits into the general framework of slow-onset, tight-binding mechanisms of ligand/protein interactions.
- Passive Lipoidal Diffusion and Carrier-Mediated Cell Uptake Are Both Important Mechanisms of Membrane Permeation in Drug Disposition. [JOURNAL ARTICLE]
- Mol Pharm 2014 Apr 11.
Recently, it has been proposed that drug permeation is essentially carrier-mediated only and that passive lipoidal diffusion is negligible. This opposes the prevailing hypothesis of drug permeation through biological membranes, which integrates the contribution of multiple permeation mechanisms, including both carrier-mediated and passive lipoidal diffusion, depending on the compound's properties, membrane properties, and solution properties. The prevailing hypothesis of drug permeation continues to be successful for application and prediction in drug development. Proponents of the carrier-mediated only concept argue against passive lipoidal diffusion, however, the arguments are not supported by broad pharmaceutics literature. The carrier-mediated only concept lacks substantial supporting evidence and succesful applications in drug development.
- Poly(ethylene glycol)-Mediated Conformational Alteration of α-Chymotrypsin Prevents Inactivation of Insulin by Stabilizing Active Intermediates. [JOURNAL ARTICLE]
- Mol Pharm 2014 Apr 21.
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 poly(ethylene glycol) 400 (PEG 400), a commonly used crowding 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 18.
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 an 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 min 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.