Intravitreal drug delivery in retinal disease : are we out of our depth?

Introduction With the ever-increasing global burden of retinal disease, there is an urgent need to vastly improve formulation strategies that enhance posterior eye delivery of therapeutics. Despite intravitreal administration having demonstrated notable superiority over other routes in enhancing retinal drug availability, there still exist various significant physical/biochemical barriers preventing optimal drug delivery into the retina. A further complication lies with an inability to reliably translate laboratory-based retinal models into a clinical setting. Several formulation approaches have recently been evaluated to improve intravitreal therapeutic outcomes, and our aim in this review is to highlight strategies that hold the most promise. Areas covered We discuss the complex barriers faced by the intravitreal route and examine how formulation strategies including implants, nanoparticulate carriers, viral vectors and sonotherapy have been utilized to attain both sustained delivery and enhanced penetration through to the retina. We conclude by highlighting the advances and limitations of current in vitro, ex vivo and in vivo retinal models in use by researchers globally. Expert opinion Various nanoparticle compositions have demonstrated the ability to overcome the retinal barriers successfully; however, their utility is limited to the laboratory setting. Optimization of these formulations and the development of more robust experimental retinal models are necessary to translate success in the laboratory into clinically efficacious outcomes.

Relevance of the deletion polymorphisms of the glutathione S-transferases GSTT1 and GSTM1 in pharmacology and toxicology

Although cytosolic glutathione S-transferase (GST) enzymes occupy a key position in biological detoxification processes, two of the most relevant human isoenzymes, GSTT1-1 and GSTM1-1, are genetically deleted (non-functional alleles GSTT1*0 and GSTM1*0) in a high percentage of the human population, with major ethnic differences. The structures of the GSTT and GSTM gene areas explain the underlying genetic processes. GSTT1-1 is highly conserved during evolution and plays a major role in phase-II biotransformation of a number of drugs and industrial chemicals, e.g. cytostatic drugs, hydrocarbons and halogenated hydrocarbons. GSTM1-1 is particularly relevant in the deactivation of carcinogenic intermediates of polycyclic aromatic hydrocarbons. Several lines of evidence suggest that hGSTT1-1 and/or hGSTM1-1 play a role in the deactivation of reactive oxygen species that are likely to be involved in cellular processes of inflammation, ageing and degenerative diseases. There is cumulating evidence that combinations of the GSTM1*0 state with other genetic traits affecting the metabolism of carcinogens (CYP1A1, GSTP1) may predispose the aero-digestive tract and lung, especially in smokers, to a higher risk of cancer. The GSTM1*0 status appears also associated with a modest increase in the risk of bladder cancer, consistent with a GSTM1 interaction with carcinogenic tobacco smoke constituents. Both human GST deletions, although largely counterbalanced by overlapping substrate affinities within the GST superfamily, have consequences when the organism comes into contact with distinct man-made chemicals. This appears relevant in industrial toxicology and in drug metabolism.

Genotoxicity of inorganic mercury salts based on disturbed microtubule function

This study investigated the hypothesis that the chromosomal genotoxicity of inorganic mercury results from interaction(s) with cytoskeletal proteins. Effects of Hg2+ salts on functional activities of tubulin and kinesin were investigated by determining tubulin assembly and kinesin-driven motility in cell-free systems. Hg2+ inhibits microtubule assembly at concentrations above 1 μM, and inhibition is complete at about 10 μM. In this range, the tubulin assembly is fully (up to 6 μM) or partially (∼6-10 μM) reversible. The inhibition of tubulin assembly by mercury is independent of the anion, chloride or nitrate. The no-observed-effect- concentration for inhibition of microtubule assembly in vitro was 1 μM Hg2+, the IC50 5.8 μM. Mercury(II) salts at the IC 50 concentrations partly inhibiting tubulin assembly did not cause the formation of aberrant microtubule structures. Effects of mercury salts on the functionality of the microtubule motility apparatus were studied with the motor protein kinesin. By using a "gliding assay" mimicking intracellular movement and transport processes in vitro, HgCl2 affected the gliding velocity of paclitaxel-stabilised microtubules in a clear dose-dependent manner. An apparent effect is detected at a concentration of 0.1 μM and a complete inhibition is reached at 1 μM. Cytotoxicity of mercury chloride was studied in V79 cells using neutral red uptake, showing an influence above 17 μM HgCl2. Between 15 and 20 μM HgCl2 there was a steep increase in cell toxicity. Both mercury chloride and mercury nitrate induced micronuclei concentration-dependently, starting at concentrations above 0.01 μM. CREST analyses on micronuclei formation in V79 cells demonstrated both clastogenic (CREST-negative) and aneugenic effects of Hg2+, with some preponderance of aneugenicity. A morphological effect of high Hg2+ concentrations (100 μM HgCl2) on the microtubule cytoskeleton was verified in V79 cells by immuno-fluorescence staining. The overall data are consistent with the concept that the chromosomal genotoxicity could be due to interaction of Hg2+ with the motor protein kinesin mediating cellular transport processes. Interactions of Hg 2+ with the tubulin shown by in vitro investigations could also partly influence intracellular microtubule functions leading, together with the effects on the kinesin, to an impaired chromosome distribution as shown by the micronucleus test.

Interactions of ingested food, beverage, and tobacco components involving human cytochrome P4501A2, 2A6, 2E1, and 3A4 enzymes

Human cytochrome P450 (P450) enzymes are involved in the oxidation of natural products found in foods, beverages, and tobacco products and their catalytic activities can also be modulated by components of the materials. The microsomal activation of aflatoxin B1 to the exo-3,9-epoxide is stimulated by flavone and 7,8-benzoflavone, and attenuated by the flavonoid naringenin, a major component of grapefruit. P4502E1 has been demonstrated to play a potentially major role in the activation of a number of very low-molecular weight cancer suspects, including ethyl carbamate (urethan), which is present in alcoholic beverages and particularly stone brandies. The enzyme (P4502E1) is also known to be inducible by ethanol. Tobacco contains a large number of potential carcinogens. In human liver microsomes a significant role for P4501A2 can be demonstrated in the activation of cigarette smoke condensate. Some of the genotoxicity may be due to arylamines. P4501A2 is also inhibited by components of crude cigarette smoke condensate. The tobacco-specific nitrosamines are activated by a number of P450 enzymes. Of those known to be present in human liver, P4501A2, 2A6, and 2E1 can activate these nitrosamines to genotoxic products.

Disturbed microtubule function and induction of micronuclei by chelate complexes of mercury(II)

Interactions of mercury(II) with the microtubule network of cells may lead to genotoxicity. Complexation of mercury(II) with EDTA is currently being discussed for its employment in detoxification processes of polluted sites. This prompted us to re-evaluate the effects of such complexing agents on certain aspects of mercury toxicity, by examining the influences of mercury(II) complexes on tubulin assembly and kinesin-driven motility of microtubules. The genotoxic effects were studied using the micronucleus assay in V79 Chinese hamster fibroblasts. Mercury(II) complexes with EDTA and related chelators interfered dose-dependently with tubulin assembly and microtubule motility in vitro. The no-effect-concentration for assembly inhibition was 1 μM of complexed Hg(II), and for inhibition of motility it was 0.05 μM, respectively. These findings are supported on the genotoxicity level by the results of the micronucleus assay, with micronuclei being induced dose-dependently starting at concentrations of about 0.05 μM of complexed Hg(II). Generally, the no-effect-concentrations for complexed mercury(II) found in the cell-free systems and in cellular assays (including the micronucleus test) were identical with or similar to results for mercury tested in the absence of chelators. This indicates that mercury(II) has a much higher affinity to sulfhydryls of cytoskeletal proteins than to this type of complexing agents. Therefore, the suitability of EDTA and related compounds for remediation of environmental mercury contamination or for other detoxification purposes involving mercury has to be questioned.

Synthesis and toxicological evaluation of a chitosan‑L‑leucine conjugate for pulmonary drug delivery applications

Herein are reported the synthesis of a conjugate of chitosan with L-leucine, the preparation of nanoparticles from both chitosan and the conjugate for use in pulmonary drug delivery, and the in vitro evaluation of toxicity and inflammatory effects of both the polymers and their nanoparticles on the bronchial epithelial cell line, BEAS-2B. The nanoparticles, successfully prepared both from chitosan and the conjugate, had a diameter in the range of 10−30 nm. The polymers and their nanoparticles were tested for their effects on cell viability by MTT assay, on trans-epithelial permeability by using sodium fluorescein as a fluid phase marker, and on IL-8 secretion by ELISA. The conjugate nanoparticles had a low overall toxicity (IC50 = 2 mg/mL following 48 h exposure; no induction of IL-8 release at 0.5 mg/mL concentration), suggesting that they may be safe for pulmonary drug delivery applications.

Colloidal drug probe : Method development and validation for adhesion force measurement using Atomic Force Microscopy

This study demonstrates a novel technique of preparing drug colloid probes to determine the adhesion force between a model drug salbutamol sulphate (SS) and the surfaces of polymer microparticles to be used as carriers for the dispersion of drug particles from dry powder inhaler (DPI) formulations. Model silica probes of approximately 4 lm size, similar to a drug particle used in DPI formulations, were coated with a saturated SS solution with the aid of capillary forces acting between the silica probe and the drug solution. The developed method of ensuring a smooth and uniform layer of SS on the silica probe was validated using X-ray Photoelectron Spectroscopy (XPS) and Scanning Electron Microscopy (SEM). Using the same technique, silica microspheres pre-attached on the AFM cantilever were coated with SS. The adhesion forces between the silica probe and drug coated silica (drug probe) and polymer surfaces (hydrophilic and hydrophobic) were determined. Our experimental results showed that the technique for preparing the drug probe was robust and can be used to determine the adhesion force between hydrophilic/ hydrophobic drug probe and carrier surfaces to gain a better understanding on drug carrier adhesion forces in DPI formulations.

Flupirtine enhances the anti-hyperalgesic effects of morphine in a rat model of prostate bone metastasis

Objective Current treatments for cancer pain are often inadequate, particularly when metastasis to bone is involved. The addition to the treatment regimen of another drug that has a complementary analgesic effect may increase the overall analgesia without the necessity to increase doses, thus avoiding dose-related side effects. This project investigated the synergistic effect of the addition of the potassium channel (KCNQ2–3) modulator flupirtine to morphine treatment in a rat model of prostate cancer-induced bone pain. Design Syngeneic prostate cancer cells were injected into the right tibia of male Wistar rats under anesthesia. This led to expanding tumor within the bone in 2 weeks, together with the concurrent development of hyperalgesia to noxious heat. Paw withdrawal thresholds from noxious heat were measured before and after the maximum non-sedating doses of morphine and flupirtine given alone and in combinations. Dose-response curves for morphine (0.13–5.0 mg/kg ip) and flupirtine (1.25–10.0 mg/kg ip) given alone and in fixed-dose combinations were plotted and subjected to an isobolographic analysis. Results Both morphine (ED50 = 0.74 mg/kg) and flupirtine (ED50 = 3.32 mg/kg) caused dose-related anti-hyperalgesia at doses that did not cause sedation. Isobolographic analysis revealed that there was a synergistic interaction between flupirtine and morphine. Addition of flupirtine to morphine treatment improved morphine anti-hyperalgesia, and resulted in the reversal of cancer-induced heat hyperalgesia. Conclusions These results suggest that flupirtine in combination with morphine may be useful clinically to provide better analgesia at lower morphine doses in the management of pain caused by tumors growing in bone.

Menstrual effluent induces epithelial-mesenchymal transitions in mesothelial cells

Background Menstrual effluent affects mesothelial cell (MC) morphology. We evaluated whether these changes were consistent with epithelial-mesenchymal transitions (EMT). Methods Monolayer cultures of MC were incubated overnight in conditioned media, prepared from cells isolated form menstrual effluent, with or without kinase and ATP inhibitors. Changes in cell morphology were monitored using time-lapse video microscopy and immunohistochemistry. Effects on the expression of EMT-associated molecules were evaluated using real-time RT-PCR and/or Western blot analysis. Results Incubation in conditioned media disrupted cell-cell contacts, and increased MC motility. The changes were reversible. During the changes the distribution of cytokeratins, fibrillar actin and α-tubulin changed. Sodium azide, an inhibitor of ATP production, and Genistein, a general tyrosine kinase inhibitor, antagonized these effects. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, and SU6656, an Src tyrosine kinase inhibitor, only partially antagonized the effect. The expression of Snail and vimentin was markedly up-regulated, whereas the expression of E-cadherin was decreased and cytokeratins were altered. Conclusions In MC, menstrual effluent initiates a reversible, energy-dependent transition process from an epithelial to a mesenchymal phenotype. Involvement of the (Src) tyrosine kinase signalling pathway and the changes in the expression of cytokeratins, Snail, vimentin and E-cadherin demonstrate that the morphological changes are EMT.

Engineered microenvironments provide new insights into ovarian and prostate cancer progression and drug responses

Tissue engineering technologies, which have originally been designed to reconstitute damaged tissue structure and function, can mimic not only tissue regeneration processes but also cancer development and progression. Bioengineered approaches allow cell biologists to develop sophisticated experimentally and physiologically relevant cancer models to recapitulate the complexity of the disease seen in patients. Tissue engineering tools enable three-dimensionality based on the design of biomaterials and scaffolds that re-create the geometry, chemistry, function and signalling milieu of the native tumour microenvironment. Three-dimensional (3D) microenvironments, including cell-derived matrices, biomaterial-based cell culture models and integrated co-cultures with engineered stromal components, are powerful tools to study dynamic processes like proteolytic functions associated with cancer progression, metastasis and resistance to therapeutics. In this review, we discuss how biomimetic strategies can reproduce a humanised niche for human cancer cells, such as peritoneal or bone-like microenvironments, addressing specific aspects of ovarian and prostate cancer progression and therapy response.

Observing the mother-infant feeding interaction

Early parenting is critical to effective attachment and a range of positive developmental outcomes for children. Feeding is a key task of early parenting and increasing evidence indicates that early feeding practices are important for the development of self-regulation of intake and food preferences which in turn are predictors of later obesity risk. However, relatively little is known about the mother-infant interaction at the transition to solids among typically developing children. This study aimed to describe parenting strategies used by mothers at the transition from milk feeding to solid food. Twenty mother-infant dyads were video-taped during a feeding interaction and data was analysed to describe maternal use of parenting strategies. It was predicted that positive feeding strategies would be correlated with lower levels of Infant Food Refusal (IFR), higher maternal sensitivity, and better overall parenting scores. The opposite was predicted for negative feeding strategies. It was found that positive strategy use and general parenting scores were significantly correlated in the predicted direction, however maternal instruction, aversive contact and ineffective strategy use were significantly correlated with and predictive of IFR. Additionally, it was hypothesised that maternal strategy use would deteriorate towards the end of the interaction, and this hypothesis was partially supported: significantly more negative strategy use was observed in the last third of the interaction, whilst positive strategy use remained consistent through the feeding interaction. The findings have important implications for early feeding parent education and intervention programs.