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Molecular pharmaceutics [journal]
- A preface for engineered biomimetic tissue platforms for in vitro drug evaluation. [Journal Article]
- Mol Pharm 2014 Jul 7; 11(7):1931-2.
- PET and SPECT imaging of a radiolabeled minigastrin analog conjugated with DOTA, NOTA and NODAGA and labeled with (64)Cu, (68)Ga and (111)In. [JOURNAL ARTICLE]
- Mol Pharm 2014 Jul 3.
Cholecystokinin-2 (CCK-2) receptors, overexpressed in cancer types such as small cell lung cancers (SCLC) and medullary thyroid carcinomas (MTC), may serve as targets for peptide receptor radionuclide imaging. A variety of CCK and gastrin analogues has been developed, but a major drawback is metabolic instability or high kidney uptake. The minigastrin analogue PP-F11 has previously been shown to be a promising peptide for imaging of CCK-2 receptor positive tumors and was therefore further evaluated. The peptide was conjugated with one of the macrocyclic chelators DOTA, NOTA or NODAGA. The peptide conjugates were then radiolabeled with either 68Ga, 64Cu or 111In. All (radio)labeled compounds were evaluated in vitro (IC50) and in vivo (biodistribution and PET/CT and SPECT/CT imaging). IC50 values were in the low nanomolar range for all compounds (0.79 - 1.51 nM). In the biodistribution studies, 68Ga- and 111In-labeled peptides showed higher tumor-to-background ratios than the 64Cu-labeled compounds. All tested radiolabeled compounds clearly visualized the CCK2 receptor positive tumor in PET or SPECT imaging. The chelator did not seem to affect in vivo behavior of the peptide for 111In- and 68Ga-labeled peptides. In contrast, the biodistribution of the 64Cu-labeled peptides showed high uptake in the liver and in other organs, most likely caused by high blood levels, probably due to dissociation of 64Cu from the chelator and subsequent transchelation to proteins. Based on the present study, 68Ga-DOTA-PP-F11 might be a promising radiopharmaceutical for PET/CT imaging of CCK2 receptor expressing tumors such as MTC and SCLC. Clinical studies are warranted to investigate the potential of this tracer.
- Synthesis and biological evaluation of 18F labeled fluoroethoxy tryptophan analogues as potential PET tumor imaging agents. [JOURNAL ARTICLE]
- Mol Pharm 2014 Jul 2.
As a continuation of our research efforts towards the development of tryptophan based radiotracers for tumor imaging with positron emission tomography (PET), three new fluoroethoxy tryptophan analogues were synthesized and evaluated in vivo. These new tracers (namely 4-(2-[18F]fluoroethoxy)-DL-tryptophan ([18F]4-FEHTP), 6-(2-[18F]fluoroethoxy)-DL-tryptophan ([18F]6-FEHTP) and 7-(2-[18F]fluoroethoxy)-DL-tryptophan ([18F]7-FEHTP) carry the fluoroethoxy side chain either at positions 4-, 6- or 7- of the indole core. Reference compounds and precursors were synthesized by multistep approaches. Radiosynthesis was accomplished by no-carrier-added nucleophilic 18F-fluorination following either an indirect approach (O-alkylation of the corresponding hydroxytryptophan with [18F]fluoroethyltosylate) or a direct approach using a protected mesyl precursor. Radiochemical yields (decay corrected) for both methods were in the range of 10 - 18% . Small animal PET imaging with xenograft bearing mice revealed highest tumor/background ratio for [18F]6-FEHTP which, in a direct comparison, outperformed the other two tryptophan tracers and also the well established tyrosine analogue O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET). Investigation of the transport mechanism of [18F]6-FEHTP in small cell lung cancer cells (NCI-H69) revealed that it is most probably taken up exclusively via the large neutral amino acid transporter(s) (LAT).
- Evaluation of (99m)Tc-probestin SPECT as a novel technique for noninvasive imaging of kidney aminopeptidase N expression. [JOURNAL ARTICLE]
- Mol Pharm 2014 Jul 2.
Aminopeptidase N (APN; CD13; EC 220.127.116.11) is a zinc-dependent membrane-bound exopeptidase that catalyzes the removal of N-terminal amino acids from peptides. APN is known to be highly expressed on renal cortical proximal tubules. APN expression levels are markedly decreased under the influence of nephrotoxins and in the tumor regions of renal cancers. Thus, molecular imaging of kidney APN expression could provide pathophysiological information about kidneys noninvasively. Probestin is a potent APN inhibitor and binds to APN. Abdominal SPECT imaging was conducted at 1 hr post-injection of (99m)Tc-probestin in a group of twelve UPII-SV40T transgenic and wild-type mice. UPII-SV40T mice spontaneously develop urothelial carcinoma in situ and invasive transitional cell carcinoma (TCC) that invade kidneys. Histopathology and immunohistochemistry analysis were used to confirm the presence of tumor and to evaluate APN expression in kidney. Radioactivity in normal tissue regions of renal cortex was clearly visible in SPECT images, whereas tumor regions of renal cortex displayed significantly lower or no radioactivity uptake. Histopathological analysis of kidney sections showed normal morphology for both renal pelvic and cortical regions in wild-type mice and abnormal morphology in some transgenic mice. Proliferating cell nuclear antigen staining confirmed the presence of tumor in those abnormal regions. Immunohistochemical analysis of kidney sections using anti-CD13 antibody showed significantly lower APN expression in tumor regions compared to normal regions. Results obtained in this study demonstrates the potential use of (99m)Tc-probestin SPECT as a novel technique for noninvasive imaging of kidney APN expression.
- Digestion of phospholipids after secretion of bile into the duodenum changes the phase behaviour of bile components. [JOURNAL ARTICLE]
- Mol Pharm 2014 Jul 2.
Bile components play a significant role in the absorption of dietary fat, by solubilizing the products of fat digestion. The absorption of poorly water-soluble drugs from the gastrointestinal tract is often enhanced by interaction with the pathways of fat digestion and absorption. Thus the phase behaviour of bile components and digested lipids is of great interest to pharmaceutical scientists who seek to optimise drug solubilisation in the gut lumen. This can be achieved by dosing drugs after food or preferably by formulating the drug in a lipid-based delivery system. Phase diagrams of bile salts, lecithin and water have been available for many years but here we investigate association structures that occur in dilute aqueous solution, in concentrations that are present in the gut lumen. More importantly we have compared these structures with those that would be expected to be present in the intestine soon after secretion of bile. Phosphatidylcholines are rapidly hydrolysed by pancreatic enzymes to yield equimolar mixtures of their monoacyl equivalents and fatty acids. We constructed phase diagrams that model the association structures formed by the products of bile digestion. The micelle-vesicle phase boundary was clearly identifiable by dynamic light scattering and nephelometry. The data indicate that a significantly higher molar ratio of lipid to bile salt is required to cause a transition to lamellar phase, i.e. liposomes in dilute solution. Mixed micelles of digested bile have a higher capacity for solubilisation of lipids and fat digestion products and can be expected to have a different capacity to solubilize lipophilic drugs. We suggest that mixtures of lysolecithin, fatty acid and bile salts are a better model of molecular associations in the gut lumen and such mixtures could be used to better understand the interaction of drugs with the fat digestion and absorption pathway.
- PEG-Farnesyl Thiosalicylic Acid Telodendrimer Micelles as An Improved Formulation for Targeted Delivery of Paclitaxel. [JOURNAL ARTICLE]
- Mol Pharm 2014 Jul 2.
We have recently designed and developed a dual-functional drug carrier that is based on polyethylene glycol (PEG)-derivatized farnesylthiosalicylate (FTS, a nontoxic Ras antagonist). PEG5K-FTS2 readily form micelles (20~30 nm) and hydrophobic drugs such as paclitaxel (PTX) could be effectively loaded into these micelles. PTX formulated in PEG5K-FTS2 micelles showed an antitumor activity that was more efficacious than Taxol in a syngeneic mouse model of breast cancer (4T1.2). In order to further improve our PEG5K-FTS2 micellar system, four PEG-FTS conjugates were developed that vary in the molecular weight of PEG (PEG2K vs PEG5K) and the molar ratio of PEG/FTS (1/2 vs 1/4) in the conjugates. These conjugates were characterized including CMC, drug loading capacity, stability, and their efficacy in delivery of anti-cancer drug PTX to tumor cells in vitro and in vivo. Our data showed that the conjugates with four FTS molecules were more effective than the conjugates with two molecules of FTS and that FTS conjugates with PEG5K were more effective than the counterparts with PEG2K in forming stable mixed micelles. PTX formulated in PEG5K-FTS4 micelles was the most effective formulation in inhibiting the tumor growth in vivo.
- Mitomycin C-Soybean Phosphatidyhlcholine Complex-Loaded Self-Assembled PEG-Lipid-PLA Hybrid Nanoparticles for Targeted Drug Delivery and Dual-Controlled Drug Release. [JOURNAL ARTICLE]
- Mol Pharm 2014 Jul 2.
Most present drug-phospholipid delivery systems were based on water-insoluble drug-phospholipid complex but rarely water-soluble drug-phospholipid complex. Mitomycin C (MMC) is a water-soluble anticancer drug extensively used in first-line chemotherapy, but limited by its poor aqueous stability in vitro, rapid elimination from the body and lack of target specificity. In this paper, we report the MMC-soybean phosphatidyhlcholine complex-loaded PEG-lipid-PLA hybrid NPs with FA functionalization (FA-PEG-PE-PLA NPs@MMC-SPC) for targeted drug delivery and dual-controlled drug release. The FA-PEG-PE-PLA NPs@MMC-SPC was comprised of a hydrophobic core (PLA) loaded with MMC-SPC, an amphiphilic lipid interface layer (PE), a hydrophilic shell (PEG) and a targeting ligand (FA) on the surface, with a spherical shape, a nanoscaled particle size and high drug encapsulation efficiency of almost 95%. The advantage of the new drug delivery systems is the early-phase controlled drug release by the drug-phospholipid complex and the late-phase controlled drug release by the pH-sensitive polymer-lipid hybrid NPs. In vitro cytotoxicity and hemolysis assays demonstrated that the drug carriers were cytocompatible and hemocompatible. The pharmacokinetics study in rats showed that the FA-PEG-PE-PLA NPs@MMC-SPC significantly prolonged the blood circulation time than the free MMC. More importantly, the FA-PEG-PE-PLA NPs@MMC-SPC presented the enhanced cell uptake/cytotoxicity in vitro and superior tumor accumulation/therapeutic efficacy in vivo while reducing the systemic toxicity. A significant accumulation of MMC in the nuclei as the site of MMC action achieved in the FA-PEG-PE-PLA NPs@MMC-SPC made them ideal for MMC drug delivery. This study may provide an effective strategy for the design and development of the water-soluble drug-phospholipid complex-based targeted drug delivery and sustained/controlled drug release.
- Immuno-PET Imaging of Tumor Endothelial Marker 8 (TEM8). [JOURNAL ARTICLE]
- Mol Pharm 2014 Jul 1.
Tumor endothelial marker 8 (TEM8) is a cell surface receptor that is highly expressed in a variety of human tumors and promotes tumor angiogenesis and cell growth. Antibodies targeting TEM8 block tumor angiogenesis in a manner distinct from the VEGF receptor pathway. Thus development of a TEM8 imaging agent could aid in patient selection for anti-angiogenic therapies and for response monitoring. In these studies, L2, a therapeutic anti-TEM8 monoclonal IgG antibody (L2mAb) was labeled with (89)Zr and evaluated in vitro and in vivo in TEM8 expressing cells and mouse xenografts (NCI-H460, DLD-1), respectively, as a potential TEM8 immuno-PET imaging agent. Methods (89)Zr-df-L2mAb was synthesized using a desferioxamine-L2mAb conjugate (df-L2mAb); (125)I-L2mAb was labeled directly. In vitro binding studies were performed using human derived cell lines with high [HEK-293F+ (TEM8 transfected)], moderate (NCI-H460) and low/undetectable [DLD-1, HEK-293(parental)] TEM8 expression. (89)Zr-df-L2mAb in vitro autoradiography studies and CD31 IHC staining were performed with cryosections from human tumor xenografts (NCI-H460, DLD-1, MKN-45, U87-MG, T-47D, and A-431). Confirmatory TEM8 Western blots were performed with the same tumor types and cells. (89)Zr-df-L2mAb biodistribution and PET imaging studies were performed in NCI-H460 and DLD-1 xenografts in nude mice. Results (125)I-L2mAb and (89)Zr-df-L2mAb exhibited specific and high affinity binding (nM) to TEM8 that was consistent with TEM expression levels in the cells. In NCI-H460 and DLD-1 mouse xenografts non-target tissue uptake of (89)Zr-df-L2mAb was similar with the liver and spleen exhibiting the highest uptake at all time points. (89)Zr-L2mAb was highly retained in NCI-H460 tumors with < 10% losses from day 1 (5.2% ID/g) to day 3 (4.7% ID/g) with the highest tumor to muscle ratios (T:M= 13:1) occurring at day 3. DLD-1 tumors exhibited similar pharmacokinetics but tumor uptake and T:M ratios were decreased ~ 2 fold in comparison to NCI-H460 at all time points. Both NCI-H460 and DLD-1 tumors were easily visualized in PET imaging studies despite their low in vitro TEM8 expression indicating in vivo expression might be higher in DLD-1 tumors. From in vitro autoradiography studies (89)Zr-df-L2mAb specific binding was found in 6 tumor types (U87-MG, NCI-H460, T-47D MKN-45, A-431 and DLD-1) which highly correlated to vessel density (CD31 IHC; P=0.0055). Westerns blots confirmed the presence of TEM8 in the 6 tumor types but found undetectable TEM8 levels in DLD-1 and MKN-45 cells. Overall this data would indicate that TEM8 is associated with the tumor vasculature rather than the tumor tissue thus, explaining the increased TEM8 expression in DLD-1 tumors compared to DLD-1 cell cultures. Conclusion (89)Zr-df-L2mAb specifically targeted TEM8 in vitro and in vivo although the in vitro expression was not necessarily predictive of in vivo expression which seemed to be associated with the tumor vasculature. In mouse models, (89)Zr-df-L2mAb tumor uptakes and T:M ratios were sufficient for visualization during PET imaging. These results would suggest that a TEM8 targeted PET imaging agent, such as (89)Zr-df-L2mAb , may have potential clinical, diagnostic and prognostic applications by providing a quantitative measure of tumor angiogenesis and patient selection for future TEM8 directed therapies.
- Angiopep-2 and Activatable Cell-Penetrating Peptide Dual-Functionalized Nanoparticles for Systemic Glioma-Targeting Delivery. [JOURNAL ARTICLE]
- Mol Pharm 2014 Jul 1.
Gliomas are hard to treat because of the two barriers involved: the blood-brain barrier and blood-tumor barrier. In this study, a dual-targeting ligand, angiopep-2, and an activatable cell-penetrating peptide (ACP) were functionalized onto nanoparticles for glioma-targeting delivery. The ACP was constructed by conjugating RRRRRRRR (R8) with EEEEEEEE through a matrix metalloproteinase-2 (MMP-2)-sensitive linker. ACP modification effectively enhanced the C6 cellular uptake because of the high expression of MMP-2 on C6 cells. The uptake was inhibited by batimastat, an MMP-2 inhibitor, suggesting that the cell-penetrating property of the ACP was activated by MMP-2. By combining the dual-targeting delivery effect of angiopep-2 and activatable cell-penetrating property of the ACP, the dual-modified nanoparticles (AnACNPs) displayed higher glioma localization than that of single ligand-modified nanoparticles. After loading with docetaxel, a common chemotherapeutic, AnACNPs showed the most favorable antiglioma effect both in vitro and in vivo. In conclusion, a novel drug delivery system was developed for glioma dual targeting and glioma penetrating. The results demonstrated that the system effectively targeted gliomas and provided the most favorable antiglioma effect.
- Enhanced Cellular Uptake of Short Polyarginine Peptides through Fatty Acylation and Cyclization. [JOURNAL ARTICLE]
- Mol Pharm 2014 Jun 30.
Many of the reported arginine-rich cell-penetrating peptides (CPPs) for the enhanced delivery of drugs are linear peptides composed of more than seven arginine residues to retain the cell penetration properties. Herein, we synthesized a class of nine polyarginine peptides containing 5 and 6 arginines namely R5 and R6. We further explored the effect of acylation with long chain fatty acids (i.e., octanoic acid, dodecanoic acid, hexadecanoic acid), and cyclization on the cell penetrating properties of the peptides. The fluorescence-labeled acylated cyclic peptide dodecanoyl-[R5] and linear peptide dodecanoyl-(R5) showed approximately 13.7- and 10.2-fold higher cellular uptake than that of control 5,6-carboxyfluorescein, respectively. The mechanism of the peptide internalization into cells was found to be energy-dependent endocytosis. Dodecanoyl-[R5] and dodecanoyl-[R6] enhanced the intracellular uptake of a fluorescence-labeled cell impermeable negatively charged phosphopeptide (F'-GpYEEI) in human ovarian cancer cells (SK-OV-3) by 3.4-fold and 5.5-fold, respectively, as shown by flow cytometry. The cellular uptake of F'-GpYEEI in the presence of hexadecanoyl-[R5] was 9.3- and 6.0-fold higher than that of in the presence of octanoyl-[R5] and dodecanoyl-[R5], respectively. Dodecanoyl-[R5] enhanced the cellular uptake of the phosphopeptide by 1.4-2.5 fold higher than the corresponding linear peptide dodecanoyl-(R5) and those of representative CPPs, such as hepta-arginine (CR7) and TAT peptide. These results showed that a combination of acylation by long chain fatty acids and cyclization on short arginine-containing peptides can improve their cell-penetrating property, possibly through efficient interaction of rigid positively charged R and hydrophobic dodecanoyl moiety with the corresponding residues in the cell membrane phospholipids.