Cell type-specific pharmacological kinase inhibition for cancer chemoprevention.

Safety is prerequisite for preventive medicine, but non-toxic agents are generally ineffective as clinical chemoprevention. Here we propose a strategy overcoming this challenge by delivering molecular-targeted agent specifically to the effector cell type to achieve sufficient potency, while circumventing toxicity in the context of cancer chemoprevention. Hepatic myofibroblasts drive progressive fibrosis that results in cirrhosis and liver cancer. In a rat model of cirrhosis-driven liver cancer, a small molecule epidermal growth factor receptor inhibitor, erlotinib, was delivered specifically to myofibroblasts by a versatile nanoparticle-based system, targeting platelet-derived growth factor receptor-beta uniquely expressed on their surface in the liver. With systemic administration of erlotinib, tumor burden was reduced to 31%, which was further improved to 21% by myofibroblast-targeted delivery even with reduced erlotinib dose (7.3-fold reduction with equivalent erlotinib dose) and less hepatocyte damage. These findings demonstrate a strategy, cell type-specific kinase inhibition, for more effective and safer precision cancer chemoprevention.

Clinicopathological indices to predict hepatocellular carcinoma molecular classification.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is the second most lethal cancer caused by lack of effective therapies. Although promising, HCC molecular classification, which enriches potential responders to specific therapies, has not yet been assessed in clinical trials of anti-HCC drugs. We aimed to overcome these challenges by developing clinicopathological surrogate indices of HCC molecular classification. METHODS: Hepatocellular carcinoma classification defined in our previous transcriptome meta-analysis (S1, S2 and S3 subclasses) was implemented in an FDA-approved diagnostic platform (Elements assay, NanoString). Ninety-six HCC tumours (training set) were assayed to develop molecular subclass-predictive indices based on clinicopathological features, which were independently validated in 99 HCC tumours (validation set). Molecular deregulations associated with the histopathological features were determined by pathway analysis. Sample sizes for HCC clinical trials enriched with specific molecular subclasses were determined. RESULTS: Hepatocellular carcinoma subclass-predictive indices were steatohepatitic (SH)-HCC variant and immune cell infiltrate for S1 subclass, macrotrabecular/compact pattern, lack of pseudoglandular pattern, and high serum alpha-foetoprotein (>400 ng/ml) for S2 subclass, and microtrabecular pattern, lack of SH-HCC and clear cell variants, and lower histological grade for S3 subclass. Macrotrabecular/compact pattern, a predictor of S2 subclass, was associated with the activation of therapeutically targetable oncogene YAP and stemness markers EPCAM/KRT19. BMP4 was associated with pseudoglandular pattern. Subclass-predictive indices-based patient enrichment reduced clinical trial sample sizes from 121, 184 and 53 to 30, 43 and 22 for S1, S2 and S3 subclass-targeting therapies respectively. CONCLUSIONS: Hepatocellular carcinoma molecular subclasses can be enriched by clinicopathological indices tightly associated with deregulation of therapeutically targetable molecular pathways.

A genomic and clinical prognostic index for hepatitis C-related early-stage cirrhosis that predicts clinical deterioration.

OBJECTIVE: The number of patients with HCV-related cirrhosis is increasing, leading to a rising risk of complications and death. Prognostic stratification in patients with early-stage cirrhosis is still challenging. We aimed to develop and validate a clinically useful prognostic index based on genomic and clinical variables to identify patients at high risk of disease progression. DESIGN: We developed a prognostic index, comprised of a 186-gene signature validated in our previous genome-wide profiling study, bilirubin (>1 mg/dL) and platelet count (<100,000/mm(3)), in an Italian HCV cirrhosis cohort (training cohort, n=216, median follow-up 10 years). The gene signature test was implemented using a digital transcript counting (nCounter) assay specifically developed for clinical use and the prognostic index was evaluated using archived specimens from an independent cohort of HCV-related cirrhosis in the USA (validation cohort, n=145, median follow-up 8 years). RESULTS: In the training cohort, the prognostic index was associated with hepatic decompensation (HR=2.71, p=0.003), overall death (HR=6.00, p<0.001), hepatocellular carcinoma (HR=3.31, p=0.001) and progression of Child-Turcotte-Pugh class (HR=6.70, p<0.001). The patients in the validation cohort were stratified into high-risk (16%), intermediate-risk (42%) or low-risk (42%) groups by the prognostic index. The high-risk group had a significantly increased risk of hepatic decompensation (HR=7.36, p<0.001), overall death (HR=3.57, p=0.002), liver-related death (HR=6.49, p<0.001) and all liver-related adverse events (HR=4.98, p<0.001). CONCLUSIONS: A genomic and clinical prognostic index readily available for clinical use was successfully validated, warranting further clinical evaluation for prognostic prediction and clinical trial stratification and enrichment for preventive interventions.

Transcriptome profiling of archived sectioned formalin-fixed paraffin-embedded (AS-FFPE) tissue for disease classification.

BACKGROUND: Archived tissues from previously completed prospective trials represent invaluable resource for biomarker development. However, such specimens are often stored as sections on glass slides, in which RNA is severely degraded due to prolonged air exposure. We evaluated whether a proportion of archived sectioned formalin-fixed paraffin-embedded (AS-FFPE) tissues yield transcriptome profiles comparable to freshly cut (FC) FFPE tissues, which can be used for retrospective class prediction analysis. METHODS: Genome-wide transcriptome profiles of 6 to 7-year-old AS-FFPE tissue sections (generated from 5 to 16-year-old blocks) of 83 hepatocellular carcinoma (HCC) and 47 liver cirrhosis samples were generated by using whole-genome DASL assay (Illumina) and digital transcript counting (nCounter) assay (NanoString), and gene signature-based prediction of HCC subclasses and prognosis was compared with previously generated FC-FFPE profiles from the same tissue blocks. RESULTS: RNA quality and assay reproducibility of AS-FFPE RNA were comparable to intermediate to poor quality FC-FFPE samples (RNA Integrity Number: up to 2.50, R-square for technical replicates: up to 0.93). Analyzable transcriptome profiles were obtained in 64 (77%) HCC and 36 (77%) cirrhosis samples. Statistically more confident predictions based on random resampling-based method (nearest template prediction) were obtained in 37 (58%) HCC and 13 (36%) cirrhosis samples. Predictions made in FC-FFPE profiles were reproduced in 36 (97%) HCC and 11 (85%) cirrhosis AS-FFPE profiles. nCounter assay was tested in 24 cirrhosis samples, which yielded confident prediction in 15 samples (63%), of which 10 samples (67%) showed concordant predictions with FC-FFPE profiles. CONCLUSIONS: AS-FFPE tissues yielded poorer quality RNA and transcriptome profiles compared to FC-FFPE tissues. Statistically more confident class prediction was feasible in 37 of 83 HCC samples and 13 of 47 cirrhosis samples. These results suggest that AS-FFPE tissues can be regarded as a resource for retrospective transcriptome-based class prediction analysis when they are the only available materials.

Pharmaceutical and toxicological properties of engineered nanomaterials for drug delivery.

Novel engineered nanomaterials (ENMs) are being developed to enhance therapy. The physicochemical properties of ENMs can be manipulated to control/direct biodistribution and target delivery, but these alterations also have implications for toxicity. It is well known that size plays a significant role in determining ENM effects since simply nanosizing a safe bulk material can render it toxic. However, charge, shape, rigidity, and surface modifications also have a significant influence on the biodistribution and toxicity of nanoscale drug delivery systems (NDDSs). In this review, NDDSs are considered in terms of platform technologies, materials, and physical properties that impart their pharmaceutical and toxicological effects. Moving forward, the development of safe and effective nanomedicines requires standardized protocols for determining the physical characteristics of ENMs as well as assessing their potential long-term toxicity. When such protocols are established, the remarkable promise of nanomedicine to improve the diagnosis and treatment of human disease can be fulfilled.

Synthetic adhesive oligopeptides with rigid polyhydroxylated amino acids.

Synthetic oligopeptides containing polyhydroxylated bicyclic dipeptide (Glc=Tap) are investigated for their adhesion properties. The non-natural amino acid building block composed of Glc=Tap is derived from glucuronic acid and mimics the hydroxyl-amino acids of the natural proteins. Peptide oligomers of Glc=Tap flanked by the amino acids Tyr and Lys were synthesized and characterized. Solution structural studies performed by circular dichromism spectroscopy suggests that poly(Lys-Glc=Tap-Tyr) and poly(Glc=Tap-Tyr) adopts extended helical structures. Adhesion of these oligomers to the mica surface is shown by atomic force microscopy spectroscopy. Studies indicate that extended polyproline II polyhydroxylated peptide chains, which bear additional phenolic as well as cationic side chains, can mimic some of the adhesion properties of the natural protein models. Furthermore, obtained data suggest that poly(Glc=Tap-Tyr) and poly(Lys-Glc=Tap-Tyr) as outstanding adhesive compounds, which combine efficient synthetic accessibility with promising adhesive properties.

Biodistribution and renal clearance of biocompatible lung targeted poly(ethylene glycol) (PEG) nanogel aggregates.

A novel stabilized aggregated nanogel particle (SANP) drug delivery system was prepared for injectable passive lung targeting. Gel nanoparticles (GNPs) were synthesized by irreversibly cross-linking 8 Arm PEG thiol with 1,6-hexane-bis-vinylsulfone (HBVS) in phosphate buffer (PB, pH 7.4) containing 0.1% v/v Tween™ 80. Aggregated nanogel particles (ANPs) were generated by aggregating GNPs to micron-size, which were then stabilized (i.e., SANPs) using a PEG thiol polymer to prevent further growth-aggregation. The size of SANPs, ANPs and GNPs was analyzed using a Coulter counter and transmission electron microscopy (TEM). Stability studies of SANPs were performed at 37°C in rat plasma, phosphate buffered saline (PBS, pH 7.4) and PB (pH 7.4). SANPs were stable in rat plasma, PBS and PB over 7 days. SANPs were covalently labeled with HiLyte Fluor™ 750 (DYE-SANPs) to facilitate ex vivo imaging. Biodistribution of intravenous DYE-SANPs (30 µm, 4 mg in 500 µL PBS) in male Sprague-Dawley rats was compared to free HiLyte Fluor™ 750 DYE alone (1mg in 500 µL PBS) and determined using a Xenogen IVIS® 100 Imaging System. Biodistribution studies demonstrated that free DYE was rapidly eliminated from the body by renal filtration, whereas DYE-SANPs accumulated in the lung within 30 min and persisted for 48 h. DYE-SANPs were enzymatically degraded to their original principle components (i.e., DYE-PEG-thiol and PEG-VS polymer) and were then eliminated from the body by renal filtration. Histological evaluation using H & E staining and broncho alveolar lavage (BAL) confirmed that these flexible SANPs were not toxic. This suggests that because of their flexible and non-toxic nature, SANPs may be a useful alternative for treating pulmonary diseases such as asthma, pneumonia, tuberculosis and disseminated lung cancer.

Doxycycline hydrogels with reversible disulfide crosslinks for dermal wound healing of mustard injuries.

Doxycycline hydrogels containing reversible disulfide crosslinks were investigated for a dermal wound healing application. Nitrogen mustard (NM) was used as a surrogate to mimic the vesicant effects of the chemical warfare agent sulfur mustard. An 8-arm-poly(ethylene glycol) (PEG) polymer containing multiple thiol (-SH) groups was crosslinked using hydrogen peroxide (H(2)O(2) hydrogel) or 8-arm-S-thiopyridyl (S-TP hydrogel) to form a hydrogel in situ. Formulation additives (glycerin, PVP and PEG 600) were found to promote dermal hydrogel retention for up to 24 h. Hydrogels demonstrated high mechanical strength and a low degree of swelling (< 1.5%). Doxycycline release from the hydrogels was biphasic and sustained for up to 10-days in vitro. Doxycycline (8.5 mg/cm(3)) permeability through NM-exposed skin was elevated as compared to non vesicant-treated controls at 24, 72 and 168 h post-exposure with peak permeability at 72 h. The decrease in doxycycline permeability at 168 h correlates to epidermal re-epithelialization and wound healing. Histology studies of skin showed that doxycycline loaded (0.25% w/v) hydrogels provided improved wound healing response on NM-exposed skin as compared to untreated skin and skin treated with placebo hydrogels in an SKH-1 mouse model. In conclusion, PEG-based doxycycline hydrogels are promising for dermal wound healing application of mustard injuries.

Enhanced passive pulmonary targeting and retention of PEGylated rigid microparticles in rats.

The current study examines the passive pulmonary targeting efficacy and retention of 6µm polystyrene (PS) microparticles (MPs) covalently modified with different surface groups [amine (A-), carboxyl (C-) and sulfate (S-)] or single (PEG(1)-) and double (PEG(2)-) layers of α,ω-diamino poly(ethylene glycol) attached to C-MPs. The ζ-potential of A-MPs (-44.0mV), C-MPs (-54.3mV) and S-MPs (-49.6mV) in deionized water were similar; however PEGylation increased the ζ-potential for both PEG(1)-MPs (-18.3mV) and PEG(2)-MPs (11.5mV). The biodistribution and retention of intravenously administered MPs to male Sprague-Dawley rats was determined in homogenized tissue by fluorescence spectrophotometry. PEG(1)-MPs and PEG(2)-MPs demonstrated enhanced pulmonary retention in rats at 48h after injection when compared to unmodified A-MPs (59.6%, 35.9% and 17.0% of the administered dose, respectively). While unmodified MPs did not significantly differ in lung retention, PEGylation of MPs unexpectedly improved passive lung targeting and retention by modifying surface properties including charge and hydrophobicity but not size.

Doxycycline hydrogels as a potential therapy for ocular vesicant injury.

PURPOSE: The goals of this study were (1) to compare the injury at the basement membrane zone (BMZ) of rabbit corneal organ cultures exposed to half mustard (2 chloroethyl ethyl sulfide, CEES) and nitrogen mustard with that of in vivo rabbit eyes exposed to sulfur mustard (SM); (2) to test the efficacy of 4 tetracycline derivatives in attenuating vesicant-induced BMZ disruption in the 24-h period postexposure; and (3) to use the most effective tetracycline derivative to compare the improvement of injury when the drug is delivered as drops or hydrogels to eyes exposed in vivo to SM. METHODS: Histological analysis of hematoxylin and eosin­stained sections was performed; the ultrastructure of the corneal BMZ was evaluated by transmission electron microscopy; matrix metalloproteinase-9 was assessed by immunofluorescence; doxycycline as drops or a hydrogel was applied daily for 28 days to eyes exposed in vivo to SM. Corneal edema was assessed by pachymetry and the extent of neovascularization was graded by length of longest vessel in each quadrant. RESULTS: Injury to the BMZ was highly similar with all vesicants, but varied in degree of severity. The effectiveness of the 4 drugs in retaining BMZ integrity did not correlate with their ability to attenuate matrix metalloproteinase-9 expression at the epithelial­stromal border. Doxycycline was most effective on organ cultures; therefore, it was applied as drops or a hydrogel to rabbit corneas exposed in vivo to SM. Eyes were examined at 1, 3, 7, and 28 days after exposure. At 7 and 28 days after SM exposure, eyes treated with doxycycline were greatly improved over those that received no therapy. Corneal thickness decreased somewhat faster using doxycycline drops, whereas the hydrogel formulation decreased the incidence of neovascularization. CONCLUSIONS: Corneal cultures exposed to 2-chloroethyl ethyl sulfide and nitrogen mustard were effective models to simulate in vivo SM exposures. Doxycycline as drops and hydrogels ameliorated vesicant injury. With in vivo exposed animals, the drops reduced edema faster than the hydrogels, but use of the hydrogels significantly reduced neovascularization. The data provide proof of principle that a hydrogel formulation of doxycycline as a daily therapy for ocular vesicant injury should be further investigated.

Biodegradable poly(ethylene glycol) hydrogels based on a self-elimination degradation mechanism.

Two vinyl sulfone functionalized crosslinkers were developed for the purpose of preparing degradable poly(ethylene glycol) (PEG) hydrogels (EMXL and GABA-EMXL hydrogels). A self-elimination degradation mechanism in which an N-terminal residue of a glutamine is converted to pyroglutamic acid with subsequent release of diamino PEG (DAP) is proposed. The hydrogels were formed via Michael addition by mixing degradable or nondegradable crosslinkers and copolymer {4% w/v; poly[PEG-alt-poly(mercapto-succinic acid)]} at room temperature in phosphate buffer (PB, pH = 7.4). Hydrogel degradation was characterized by assessing diamino PEG release and examining morphological changes as well as the swelling and weight loss ratio under physiological conditions (37 degrees C). Degradation of EMXL and GABA-EMXL hydrogels occurred by surface erosion (confirmed by SEM). GABA-EMXL degradation was significantly faster (approximately 3-fold) than EMXL; however, the degradation of both hydrogels in mouse plasma was 12-times slower than in PBS. The slower degradation rate in plasma as compared to buffer is consistent with the presence of gamma-glutamyltransferase, gamma-glutamylcyclotransferase and/or glutaminyl cyclase (QC), which have been shown to suppress pyroglutamic acid formation. The current studies suggest that EMXL and GABA-EMXL hydrogels may have biomedical applications where 1-2 week degradation timeframes are optimal.

Threshold size for optimal passive pulmonary targeting and retention of rigid microparticles in rats.

The relationship between microparticle (MP) size and lung targeting efficiency, intra-lung distribution and retention time was systematically studied after intravenous administration of rigid fluorescent polystyrene MPs of various sizes (2, 3, 6 and 10 microm) to Sprague Dawley rats. Total fluorescence was assessed and it was found that 2 microm and 3 microm MPs readily passed through the lung to the liver and spleen while 10 microm MPs were completely entrapped in the lung for the one-week duration of the study. Approximately 84% of 6 microm MPs that were initially entrapped in the lung were cleared over the next 2 days and 15% were cleared over the remaining 5 days. A Caliper IVIS 100 small animal imaging system confirmed that 3 microm MPs were not retained in the lung but that 6 microm and 10 microm MPs were widely distributed throughout the lung. Moreover, histologic examination showed MP entrapment in capillaries but not arterioles. These studies suggest that for rigid MPs the optimal size range required to achieve transient but highly efficiently targeting to pulmonary capillaries after IV injection is >6 microm but <10 microm in rats and that systemic administration of optimally sized MPs may be an efficient alternative to currently used inhalation-based delivery to the lung.

A series of alpha-amino acid ester prodrugs of camptothecin: in vitro hydrolysis and A549 human lung carcinoma cell cytotoxicity.

The objective of the present study was to identify a camptothecin (CPT) prodrug with optimal release and cytotoxicity properties for immobilization on a passively targeted microparticle delivery system. A series of alpha-amino acid ester prodrugs of CPT were synthesized, characterized, and evaluated. Four CPT prodrugs were synthesized with increasing aliphatic chain length (glycine (Gly) (2a), alanine (Ala) (2b), aminobutyric acid (Abu) (2c), and norvaline (Nva) (2d)). Prodrug reconversion was studied at pH 6.6, 7.0, and 7.4 corresponding to tumor, lung, and extracellular/physiological pH, respectively. Cytotoxicity was evaluated in A549 human lung carcinoma cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The hydrolytic reconversion rate to parent CPT increased with decreasing side chain length as well as increasing pH. The Hill slope of 2d was significantly less than CPT and the other prodrugs tested, indicating a higher cell death rate at lower concentrations. These results suggest that 2d is the best candidate for a passively targeted sustained release lung delivery system.

Surface modifications of nanocarriers for effective intracellular delivery of anti-HIV drugs.

A variety of nanocarriers such as bioconjugates, dendrimers, liposomes, and nanoparticles have been widely evaluated as potential targeted drug delivery systems. Passive targeting of nanoscale carriers is based on a size-flow-filtration phenomenon that is usually limited to tumors, the reticular endothelial system, and possibly lymph nodes (LNs). In fact, targeting the delivery of drugs to pivotal physiological sites such as the lymph nodes has emerged as a promising strategy in treating HIV disease. Ligands for specific cell surface receptors can be displayed on nanocarriers in order to achieve active targeting. The approach has been extensively used preclinically in cancer where certain receptors are over-expressed at various stages of the disease. Unfortunately, markers of HIV infection are lacking and latently infected cells do not show any signs of infection on their surface. However, the disease naturally targets only a few cell types. The HIV receptor CD4, coreceptors (CCR5 and CXCR4), and some receptors relatively specific for macrophages provide potentially valuable surface targets for drug delivery to all susceptible cells in patients infected by HIV. This review focuses on nanoscale targeting with an emphasis on surface modifications of drug delivery nanocarriers for active targeting. A number of related issues, including HIV biology, targets, pharmacokinetics, and intracellular fate as well as literature-cited examples of emerging surface-modified targeted carrier systems are discussed.

Pulmonary targeting microparticulate camptothecin delivery system: anticancer evaluation in a rat orthotopic lung cancer model.

Large (>6 microm) rigid microparticles (MPs) become passively entrapped within the lungs after intravenous (i.v.) injection making them an attractive and highly efficient alternative to inhalation for pulmonary delivery. In this study, PEGylated 6 microm polystyrene MPs with multiple copies of the norvaline (Nva) alpha-amino acid prodrug of camptothecin (CPT) were prepared. Surface morphology was characterized using a scanning electron microscope. CPT was released from the CPT-Nva-MPs over 24 h in rat plasma at 37 degrees C. In-vivo CPT plasma concentrations were low (approximately 1 ng/ml or less) and constant over a period of 4 days after a single i.v. injection of CPT-Nva-MPs as compared with high but short-lived systemic exposures after an i.v. injection of free CPT. This suggests that sustained local CPT concentrations were achieved in the lung after administration of the MP delivery system. Anticancer efficacy was evaluated in an orthotopic lung cancer animal model and compared with a bolus injection of CPT. Animals receiving free CPT (2 mg/kg) and CPT-Nva-MPs (0.22 mg/kg CPT and 100 mg/kg MPs) were found to have statistically significant smaller areas of lung cancer (P<0.05 and 0.01, respectively) than untreated animals. In addition, 40% of the animals receiving CPT-Nva-MPs were found to be free of cancer. The CPT dose using targeted MPs was 10 times lower than after i.v. injection of free CPT, but was more effective in reducing the amount of cancerous areas. In conclusion, CPT-Nva-MPs were able to achieve effective local lung and low systemic CPT concentrations at a dose that was 10 times lower than systemically administered CPT resulting in a significant improvement in anticancer efficacy in an orthotopic rat model of lung cancer.

Nonenzymatic, self-elimination degradation mechanism of glutathione.

In the present in vitro studies, evidence is provided showing that glutathione (GSH) can undergo spontaneous, nonenzymatic auto-degradation. The initial cleavage of the Glu-Cys bond involves nucleophilic attack of the N-terminal amino group of GSH at the gamma-carbonyl side chain, followed by an unusual cleavage of the Cys-Gly amide bond that is complete within 3-5 weeks in the physiological pH range. These observations may be useful for the development of novel biodegradable polymers and releasable prodrugs for sustained and targeted drug delivery applications.