Transport Rankings of Non-Steroidal Antiinflammatory Drugs across Blood-Brain Barrier In Vitro Models

The aim of this work was to conduct a comprehensive study about the transport properties of NSAIDs across the blood-brain barrier (BBB) in vitro. Transport studies with celecoxib, diclofenac, ibuprofen, meloxicam, piroxicam and tenoxicam were accomplished across Transwell models based on cell line PBMEC/C1-2, ECV304 or primary rat brain endothelial cells. Single as well as group substance studies were carried out. In group studies substance group compositions, transport medium and serum content were varied, transport inhibitors verapamil and probenecid were added. Resulted permeability coefficients were compared and normalized to internal standards diazepam and carboxyfluorescein. Transport rankings of NSAIDs across each model were obtained. Single substance studies showed similar rankings as corresponding group studies across PBMEC/C1-2 or ECV304 cell layers. Serum content, glioma conditioned medium and inhibitors probenecid and verapamil influenced resulted permeability significantly. Basic differences of transport properties of the investigated NSAIDs were similar comparing all three in vitro BBB models. Different substance combinations in the group studies and addition of probenecid and verapamil suggested that transporter proteins are involved in the transport of every tested NSAID. Results especially underlined the importance of same experimental conditions (transport medium, serum content, species origin, cell line) for proper data comparison.     

Respiratory Syncytial Virus Engineered To Express the Cystic Fibrosis Transmembrane Conductance Regulator Corrects the Bioelectric Phenotype of Human Cystic Fibrosis Airway Epithelium In Vitro

Cystic fibrosis (CF) is the most common lethal recessive genetic disease in the Caucasian population. It is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that is normally expressed in ciliated airway epithelial cells and the submucosal glands of the lung. Since the CFTR gene was first characterized in 1989, a major goal has been to develop an effective gene therapy for CF lung disease, which has the potential to ameliorate morbidity and mortality. Respiratory syncytial virus (RSV) naturally infects the ciliated cells in the human airway epithelium. In addition, the immune response mounted against an RSV infection does not prevent subsequent infections, suggesting that an RSV-based vector might be effectively readministered. To test whether the large 4.5-kb CFTR gene could be expressed by a recombinant RSV and whether infectious virus could be used to deliver CFTR to ciliated airway epithelium derived from CF patients, we inserted the CFTR gene into four sites in a recombinant green fluorescent protein-expressing RSV (rgRSV) genome to generate virus expressing four different levels of CFTR protein. Two of these four rgRSV-CFTR vectors were capable of expressing CFTR with little effect on viral replication. rgRSV-CFTR infection of primary human airway epithelial cultures derived from CF patients resulted in expression of CFTR protein that was properly localized at the luminal surface and corrected the chloride ion channel defect in these cells.Cystic fibrosis (CF) is an autosomal recessive genetic disease that occurs with an incidence of 1 in every 3,400 live Caucasian births in the United States and is one of the most common fatal hereditary diseases in the world (47). CF is caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes a low-conductance ATP- and cyclic AMP (cAMP)-dependent chloride ion (Cl⁻) channel. More than 1,500 mutations that can lead to various degrees of CF have been found in CFTR. The most common mutation found in individuals of European descent is a deletion of 3 nucleotides in the CFTR gene resulting in the loss of phenylalanine at position 508 of the CFTR protein (ΔF508). This mutation results in the translation of a protein that folds improperly, causing it to be degraded upon exit from the endoplasmic reticulum. Since 90% of the mortality caused by CF results from lung pathology, restoring functional CFTR to the airways of CF patients remains a goal of gene replacement therapeutics for the disease. In the lung, CFTR is expressed by the respiratory epithelium that lines the lumen of the airways, where it is localized to the apical membrane of ciliated cells and the submucosal gland ductal epithelium (20, 40, 48). CFTR is responsible for the movement of Cl⁻ ions across the apical membranes of the airway epithelium and, in combination with sodium ion (Na⁺) transport, it dictates the volume of airway surface liquid that facilitates mucus transport and mucociliary clearance. Lack of functional CFTR in the cell membrane decreases Cl⁻ ion secretion; a net increase in the intracellular Cl⁻ ion concentration is then followed by increased uptake of sodium (Na⁺) ions by epithelial sodium channels (ENaCs). This additional intracellular ion concentration results in a net increase in water uptake into the cell (68). In patients with CF, the fundamental consequence of CFTR dysfunction in the airway is dehydration of the airway surface liquid (ASL) and an increase in the viscosity of the mucus secretions that coat the respiratory tract. This thickened mucus leads to plugging of the airways, in addition to decreased airway clearance, resulting in an increased susceptibility to both bacterial and viral airway pathogens.Early in vitro experiments using the available recombinant adenoviruses (AdV) and adeno-associated viruses (AAV) showed some efficacy in airway cell transduction (29, 67); however, the human clinical trials were less promising due to the low efficiency of CFTR delivery to the appropriate cells and short-lived CFTR expression, primarily as a consequence of the innate and adaptive immune responses (28, 34, 39, 90). Further studies revealed that CAR, the coxsackievirus and AdV receptor, and heparan sulfate, the AAV receptor, are both expressed on the basolateral surface of the human airway, likely providing another explanation for the poor transduction efficiency of airway cells by these vectors when introduced apically (7, 62, 77, 92). More recently, AAV serotypes that transduce the airway epithelium at a much higher rate have been identified, and additional improvements have been made by mutagenesis, capsid shuffling, and directed evolution (24, 36, 52-54, 78, 89). Lentiviral vectors for the delivery of CFTR to CF patients have also been examined, and improvements have been made, but efficiency and safety concerns persist (33, 41, 57, 72, 76, 85). Here, we suggest a potential viral vector to treat CF that naturally targets the airways.In vitro studies in which CF cells and CFTR-corrected CF cells have been mixed in measured ratios have determined that CFTR expression in 6 to 10% of respiratory cells returns Cl⁻ transport to levels similar to those measured in non-CF epithelial cell cultures (2, 42). However, this low level of correction may not repair some of the other associated defects, such as sodium hyperabsorption and mucus dehydration (40). Similar studies performed by mixing airway epithelial cells from CF and non-CF patients to create mixed well-differentiated human airway epithelial cell (HAE) cultures indicated that if 20% of the cells expressed endogenous levels of CFTR, this correlated with 70% of the Cl⁻ channel response measured in cultures made with 100% non-CF cells (25). More recently, infection of HAE cultures with a recombinant parainfluenza virus type 3 (PIV3) vector engineered to express CFTR was shown to fully correct the Cl⁻ transport defect in HAE cultures. In these studies, CFTR delivery to 25% of the surface airway epithelial cells was required to restore airway surface liquid volume and mucus transport to normal non-CF levels (93). Collectively, these in vitro experiments, in relevant airway cell models, suggest that an effective vector for CFTR delivery would need to target at least 25% of the airway surface epithelial cells.Respiratory syncytial virus (RSV) is a single-stranded negative-sense RNA virus that infects the ciliated cells of the airway epithelium of the human respiratory tract (94). Most individuals become infected with RSV during the first and second years of life; however, due to incomplete immunity, individuals can be reinfected by RSV throughout their lifetimes. In most cases, infection results in only mild, self-limited, common cold-like symptoms, although a proportion of primary infections do involve lower respiratory tract disease. Serious illness, which typically involves bronchiolitis or pneumonia, is usually restricted to young infants or the frail elderly. Although RSV infects CF patients at the same frequency that it infects their age-matched siblings, CF patients tend to develop more frequent lower respiratory tract illness. It has been shown that CF patients require more frequent hospitalization due to RSV infection when they are young, but this decreases with age, as it does for healthy children (32, 87). Since RSV can infect the lungs of CF patients, it appears that it can not only navigate through the physical barriers of the normal respiratory tract, but can also make its way through the sticky and mucus-rich environment of the CF lung. In addition, RSV has other features that suggest it might have advantages as a gene therapy vector for the delivery of CFTR to the airways of CF patients. RSV has a tropism for the luminal ciliated cells of the airway, which are a relevant target for CFTR gene therapy (40, 48), and RSV has been shown to lack the overt cytopathology of other respiratory viruses, suggesting that it will not rapidly destroy the cells that it infects (94). RSV also has the ability to reinfect, implying that multiple sequential administrations of an RSV-based vector would be possible.Here, we tested the utility of RSV as a CFTR gene transfer vector. The CFTR gene was inserted into four different sites in the RSV genome to obtain a range of expression levels. The vector was then evaluated for the ability to deliver CFTR to the ciliated cells in an in vitro model of the human airway (HAE). We show that RSV delivered CFTR to ciliated cells and resulted in sufficient transduction efficiency and functional CFTR expression to fully correct the Cl⁻ transport bioelectric defect in primary HAE cultures derived from CF patients. These data support continued efforts to explore the utility of RSV-based vectors as potential gene delivery vectors for the treatment of CF lung disease.     

Selective Interactions of Zein Microspheres with Different Class of Drugs: An In Vitro and In Silico Analysis

In this study, we have evaluated the interactions of zein microspheres with different class of drugs (hydrophobic, hydrophilic, and amphiphilic) using in vitro and in silico analysis. Zein microspheres loaded with aceclofenac, metformin, and promethazine has been developed by solvent evaporation technique and analyzed for its compatibility. The physical characterization depicted the proper encapsulation of hydrophobic drug in the microspheres. The in vitro release study revealed the sustaining ability of the microspheres in the following order: hydrophobic > hydrophilic > amphiphilic. In silico analysis also confirmed the better binding affinity and greater interactions of hydrophobic drug with zein. The above results revealed that zein is more suitable for hydrophobic drugs in the development of sustained drug delivery systems using solvent evaporation technique. The study therefore envisages a scope for identifying the most suitable polymer for a sustained drug delivery system in accordance with the nature of the drug.KEY WORDS: hydrophilic drugs, hydrophobic drugs, in silico analysis, protein-drug interactions, solvent evaporation, zein microspheres     

In Vitro Response of Blastocystis hominis to Antiprotozoal Drugs

Ten antiprotozoal drugs were tested in vitro against four axenic strains of the intestinal parasite Blastocystis hominis. Inhibitory drugs in order of effectiveness were emetine, metronidazole, furazolidone, trimethoprim sulfamethoxazole, 5-chloro-8-hydroxy-7-iodo-quinoline (Entero-Vioform), and pentamidine. Moderately inhibitory were two quinolines other than iodochlorhydroxquin. These were chloroquine and 5, 7-diiodo-8-hydroxy-quinoline(Floraquin). Diloxanide furoate was not inhibitory. Paromomycin and other antibiotics were not inhibitory. Entero-Vioform and metronidazole have been effective in human and higher primate diarrhea caused by B. hominis.     

In Vitro Percutaneous Permeation and Skin Accumulation of Finasteride Using Vesicular Ethosomal Carriers

In order to develop a novel transdermal drug delivery system that facilitates the skin permeation of finasteride encapsulated in novel lipid-based vesicular carriers (ethosomes)finasteride ethosomes were constructed and the morphological characteristics were studied by transmission electron microscopy. The particle size, zeta potential and the entrapment capacity of ethosome were also determined. In contrast to liposomes ethosomes were of more condensed vesicular structure and they were found to be oppositely charged. Ethosomes were found to be more efficient delivery carriers with high encapsulation capacities. In vitro percutaneous permeation experiments demonstrated that the permeation of finasteride through human cadaver skin was significantly increased when ethosomes were used. The finasteride transdermal fluxes from ethosomes containing formulation (1.34 ± 0.11 μg/cm²/h) were 7.4, 3.2 and 2.6 times higher than that of finasteride from aqueous solution, conventional liposomes and hydroethanolic solution respectively (P < 0.01).Furthermore, ethosomes produced a significant (P < 0.01) finasteride accumulation in the skin, especially in deeper layers, for instance in dermis it reached to 18.2 ± 1.8 μg/cm². In contrast, the accumulation of finasteride in the dermis was only 2.8 ± 1.3 μg/cm² with liposome formulation. The study demonstrated that ethosomes are promising vesicular carriers for enhancing percutaneous absorption of finasteride.     

Mucociliary Clearance Defects in a Murine In Vitro Model of Pneumococcal Airway Infection

Mucociliary airway clearance is an innate defense mechanism that protects the lung from harmful effects of inhaled pathogens. In order to escape mechanical clearance, airway pathogens including Streptococcus pneumoniae (pneumococcus) are thought to inactivate mucociliary clearance by mechanisms such as slowing of ciliary beating and lytic damage of epithelial cells. Pore-forming toxins like pneumolysin, may be instrumental in these processes. In a murine in vitro airway infection model using tracheal epithelial cells grown in air-liquid interface cultures, we investigated the functional consequences on the ciliated respiratory epithelium when the first contact with pneumococci is established. High-speed video microscopy and live-cell imaging showed that the apical infection with both wildtype and pneumolysin-deficient pneumococci caused insufficient fluid flow along the epithelial surface and loss of efficient clearance, whereas ciliary beat frequency remained within the normal range. Three-dimensional confocal microscopy demonstrated that pneumococci caused specific morphologic aberrations of two key elements in the F-actin cytoskeleton: the junctional F-actin at the apical cortex of the lateral cell borders and the apical F-actin, localized within the planes of the apical cell sides at the ciliary bases. The lesions affected the columnar shape of the polarized respiratory epithelial cells. In addition, the planar architecture of the entire ciliated respiratory epithelium was irregularly distorted. Our observations indicate that the mechanical supports essential for both effective cilia strokes and stability of the epithelial barrier were weakened. We provide a new model, where - in pneumococcal infection - persistent ciliary beating generates turbulent fluid flow at non-planar distorted epithelial surface areas, which enables pneumococci to resist mechanical cilia-mediated clearance.     

Mechanisms of Acid and Base Secretion by the Airway Epithelium

One of the main functions of the airway epithelium is to inactivate and remove infectious particles from inhaled air and thereby prevent infection of the distal lung. This function is achieved by mucociliary and cough clearance and by antimicrobial factors present in the airway surface liquid (ASL). There are indications that airway defenses are affected by the pH of the ASL and historically, acidification of the airway surfaces has been suggested as a measure of airway disease. However, even in health, the ASL is slightly acidic, and this acidity might be part of normal airway defense. Only recently research has focused on the mechanisms responsible for acid and base secretion into the ASL. Advances resulted from research into the airway disease associated with cystic fibrosis (CF) after it was found that the CFTR Cl⁻ channel conducts HCO ₃ ⁻ and, therefore, may contribute to ASL pH. However, the acidity of the ASL indicated parallel mechanisms for H⁺ secretion. Recent investigations identified several H⁺ transporters in the apical membrane of the airway epithelium. These include H⁺ channels and ATP-driven H⁺ pumps, including a non-gastric isoform of the H⁺-K⁺ ATPase and a vacuolar-type H⁺ ATPase. Current knowledge of acid and base transporters and their potential roles in airway mucosal pH regulation is reviewed here.     

Effect of Formulation Components on the In Vitro Permeation of Microemulsion Drug Delivery System of Fluconazole

The purpose of this study was to evaluate the effect of formulation components on the in vitro skin permeation of microemulsion drug delivery system containing fluconazole (FLZ). Lauryl alcohol (LA) was screened as the oil phase of microemulsions. The pseudo-ternary phase diagrams for microemulsion regions were constructed using LA as the oil, Labrasol (Lab) as the surfactant and ethanol (EtOH) as the cosurfactant. The formulation which showed a highest permeation rate of 47.15 ± 1.12 μg cm⁻² h⁻¹ and appropriate physicochemical properties was optimized as containing 2% FLZ, 10% LA, 20% Lab/EtOH (1:1), and 68% double-distilled water (w/w). The efficiency of microemulsion formulation in the topical delivery of FLZ was dependent upon the contents of water and LA as well as Lab/EtOH mixing ratio. It was concluded that the percutaneous absorption of FLZ from microemulsions was enhanced with increasing the LA and water contents, and with decreasing the Lab/EtOH ratio in the formulation. Candida albicans was used as a model fungus to evaluate the antifungal activity of the best formula achieved, which showed the widest zone of inhibition as compared to FLZ reference. The studied microemulsion formulation showed a good stability for a period of 3 months. These results indicate that the studied microemulsion formulation might be a promising vehicle for topical delivery of FLZ.     

Controlled Release Hydrophilic Matrix Tablet Formulations of Isoniazid: Design and In Vitro Studies

The aim of the present investigation was to develop oral controlled release matrix tablet formulations of isoniazid using hydroxypropyl methylcellulose (HPMC) as a hydrophilic release retardant polymer and to study the influence of various formulation factors like proportion of the polymer, polymer viscosity grade, compression force, and release media on the in vitro release characteristics of the drug. The formulations were developed using wet granulation technology. The in vitro release studies were performed using US Pharmacopoeia type 1 apparatus (basket method) in 900 ml of pH 7.4 phosphate buffer at 100 rpm. The release kinetics was analyzed using Korsmeyer–Peppas model. The release profiles were also analyzed using statistical method (one-way analysis of variance) and f ₂ metric values. The release profiles found to follow Higuchi’s square root kinetics model irrespective of the polymer ratio and the viscosity grade used. The results in the present investigation confirm that the release rate of the drug from the HPMC matrices is highly influenced by the drug/HPMC ratio and viscosity grade of the HPMC. Also, the effect of compression force and release media was found to be significant on the release profiles of isoniazid from HPMC matrix tablets. The release mechanism was found to be anomalous non-Fickian diffusion in all the cases. In the present investigation, a series of controlled release formulations of isoniazid were developed with different release rates and duration so that these formulations could further be assessed from the in vivo bioavailability studies. The formulations were found to be stable and reproducible.     

Effects of GABAergic Drugs In Vivo on High-Affinity Choline Uptake In Vitro in Mouse Hippocampal Synaptosomes

The effects of several gamma-aminobutyric acid (GABA)-ergic drugs on sodium-dependent high-affinity choline uptake (HACU) were investigated in the hippocampus. HACU was measured in vitro after in vivo administration of the drug to mice. HACU was inhibited by those drugs that enhance GABA transmission. The convulsant 3-mercaptopropionic acid, which decreases GABA levels, stimulated HACU. From these results and previous findings, it appears that GABA mediates a tonic inhibitory effect on the septal-hippocampal cholinergic system.     

Evaluation of Paeonol Skin-Target Delivery from Its Microsponge Formulation: In Vitro Skin Permeation and In Vivo Microdialysis

The aim of the present study was to design a novel topical skin-target drug-delivery system, the paeonol microsponge, and to investigate its drug-release patterns in dosage form, both in vitro and in vivo. Paeonol microsponges were prepared using the quasi-emulsion solvent-diffusion method. In vitro release studies were carried out using Franz diffusion cells, while in vivo studies were investigated by microdialysis after the paeonol microsponges were incorporated into a cream base. In vitro release studies showed that the drug delivered via microsponges increased the paeonol permeation rate. Ex vivo drug-deposition studies showed that the microsponge formulation improved drug residence in skin. In addition, in vivo microdialysis showed that the values for the area under the concentration versus time curve (AUC) for the paeonol microsponge cream was much higher than that of paeonol cream without microsponges. Maximum time (T_max) was 220 min for paeonol microsponge cream and 480 min for paeonol cream, while the half-life (t_1/2) of paeonol microsponge cream (935.1 min) was almost twice that of paeonol cream (548.6 min) in the skin (n = 3). Meanwhile, in the plasma, the AUC value for paeonol microsponge cream was half that of the paeonol cream. Based on these results, paeonol-loaded microsponge formulations could be a better alternative for treating skin disease, as the formulation increases drug bioavailability by lengthening the time of drug residence in the skin and should reduce side-effects because of the lower levels of paeonol moving into the circulation.     

Comparison of In Vitro Release Rates of Acyclovir from Cream Formulations Using Vertical Diffusion Cells

Acyclovir, indicated in the treatment of herpes labialis (“cold sores”), is formulated as semisolid topical dosage forms and marketed in numerous countries. Since the formulations of the various acyclovir products may differ from country to country, this study was undertaken to compare the in vitro release of acyclovir from various generic cream products available on the South African and Indian markets using the respective brand/innovator product as the reference product. The in vitro studies were carried out using vertical diffusion cells with a diffusional surface area of 1.767 cm² and various commercially available membranes. Normal saline was used as receptor fluid and the temperature maintained at 32 ± 0.5°C. The in vitro release comparisons were based on the recommendations described in the US Food and Drug Administration Draft Guidance for acyclovir ointment and the SUPAC-SS Guidance for non-sterile semisolid dosage forms. The release rates (slope) of the test (T) and the relevant reference product (R) were monitored and compared. The comparative release of acyclovir from the various generic formulations compared with the reference product was found to be within the limits of 75–133.33% with a 90% confidence interval. These experiments indicate that the generic acyclovir cream formulations exhibited release rates that were comparable to the innovator product and could be considered to be bioequivalent.KEY WORDS: acyclovir cream, FDA Guidance, in vitro release, membranes, vertical diffusion cells     

Type I Collagen as an Extracellular Matrix for the In Vitro Growth of Human Small Intestinal Epithelium

Background

We previously reported in vitro maintenance and proliferation of human small intestinal epithelium using Matrigel, a proprietary basement membrane product. There are concerns over the applicability of Matrigel-based methods for future human therapies. We investigated type I collagen as an alternative for the culture of human intestinal epithelial cells.

Methods

Human small intestine was procured from fresh surgical pathology specimens. Small intestinal crypts were isolated using EDTA chelation. Intestinal subepithelial myofibroblasts were isolated from a pediatric sample and expanded in vitro. After suspension in Matrigel or type I collagen gel, crypts were co-cultured above a confluent layer of myofibroblasts. Crypts were also grown in monoculture with exposure to myofibroblast conditioned media; these were subsequently sub-cultured in vitro and expanded with a 1∶2 split ratio. Cultures were assessed with light microscopy, RT-PCR, histology, and immunohistochemistry.

Results

Collagen supported viable human epithelium in vitro for at least one month in primary culture. Sub-cultured epithelium expanded through 12 passages over 60 days. Histologic sections revealed polarized columnar cells, with apical brush borders and basolaterally located nuclei. Collagen-based cultures gave rise to monolayer epithelial sheets at the gel-liquid interface, which were not observed with Matrigel. Immunohistochemical staining identified markers of differentiated intestinal epithelium and myofibroblasts. RT-PCR demonstrated expression of α-smooth muscle actin and vimentin in myofibroblasts and E-Cadherin, CDX2, villin 1, intestinal alkaline phosphatase, chromogranin A, lysozyme, and Lgr5 in epithelial cells. These markers were maintained through several passages.

Conclusion

Type I collagen gel supports long-term in vitro maintenance and expansion of fully elaborated human intestinal epithelium. Collagen-based methods yield familiar enteroid structures as well as a new pattern of sheet-like growth, and they eliminate the need for Matrigel for in vitro human intestinal epithelial growth. Future research is required to further develop this cell culture system for tissue engineering applications.     

Solid-State Stability Issues of Drugs in Transdermal Patch Formulations

The transdermal patch formulation has many advantages, including noninvasiveness, an ability to bypass the first-pass metabolism, low dosage requirements, and prolonged drug delivery. However, the instability of solid-state drugs is one of the most critical problems observed in transdermal patch products. Therefore, a well-characterized approach for counteracting stability problems in solid-state drugs is crucial for improving the performance of transdermal patch products. This review provides insight into the solid-state stability of drugs associated with transdermal patch products and offers a comprehensive update on the various approaches being used for improving the stability of the active pharmaceutical ingredients currently being used.     

Characterization of Cell Surface Lectin-binding Patterns of Human Airway Epithelium

Glycosylated structures on the cell surface have a role in cell adhesion, migration, and proliferation. Repair of the airway epithelium after injury requires each of these processes, but the normal cell surface glycosylation of non-mucin producing airway epithelial cells is unknown. We examined cell surface glycosylation in human airway epithelial cells in tissue sections and in human airway epithelial cell lines in culture. Thirty-eight lectin probes were used to determine specific carbohydrate residues by lectin-histochemistry. Galactose or galactosamine-specific lectins labeled basal epithelial cells, lectins specific for several different carbohydrate structures bound columnar epithelial cells, and fucose-specific lectins labeled all airway epithelial cells. The epithelial cell lines 1HAEo– and 16HBE14o– bound lectins that were specific to basal epithelial cells. Flow cytometry of these cell lines with selected lectins demonstrated that lectin binding was to cell surface carbohydrates, and revealed possible hidden tissue antigens on dispersed cultured cells. We demonstrate specific lectin-binding patterns on the surface of normal human airway epithelial cells. The expression of specific carbohydrate residues may be useful to type epithelial cells and as a tool to examine cell events involved in epithelial repair.     

Tumor Suppressor Function of Syk in Human MCF10A In Vitro and Normal Mouse Mammary Epithelium In Vivo

The normal function of Syk in epithelium of the developing or adult breast is not known, however, Syk suppresses tumor growth, invasion, and metastasis in breast cancer cells. Here, we demonstrate that in the mouse mammary gland, loss of one Syk allele profoundly increases proliferation and ductal branching and invasion of epithelial cells through the mammary fat pad during puberty. Mammary carcinomas develop by one year. Syk also suppresses proliferation and invasion in vitro. siRNA or shRNA knockdown of Syk in MCF10A breast epithelial cells dramatically increased proliferation, anchorage independent growth, cellular motility, and invasion, with formation of functional, extracellular matrix-degrading invadopodia. Morphological and gene microarray analysis following Syk knockdown revealed a loss of luminal and differentiated epithelial features with epithelial to mesenchymal transition and a gain in invadopodial cell surface markers CD44, CD49F, and MMP14. These results support the role of Syk in limiting proliferation and invasion of epithelial cells during normal morphogenesis, and emphasize the critical role of Syk as a tumor suppressor for breast cancer. The question of breast cancer risk following systemic anti-Syk therapy is raised since only partial loss of Syk was sufficient to induce mammary carcinomas.     

Development and Evaluation of Ethyl Cellulose-Based Transdermal Films of Furosemide for Improved In Vitro Skin Permeation

Transdermal films of the furosemide were developed employing ethyl cellulose and hydroxypropyl methylcellulose as film formers. The effect of binary mixture of polymers and penetration enhancers on physicochemical parameters including thickness, moisture content, moisture uptake, drug content, drug–polymer interaction, and in vitro permeation was evaluated. In vitro permeation study was conducted using human cadaver skin as penetration barrier in modified Keshary–Chein diffusion cell. In vitro skin permeation study showed that binary mixture, ethyl cellulose (EC)/hydroxypropyl methylcellulose (HPMC), at 8.5:1.5 ratio provided highest flux and also penetration enhancers further enhanced the permeation of drug, while propylene glycol showing higher enhancing effect compared to dimethyl sulfoxide and isopropyl myristate. Different kinetic models, used to interpret the release kinetics and mechanism, indicated that release from all formulations followed apparent zero-order kinetics and non-Fickian diffusion transport except formulation without HPMC which followed Fickian diffusion transport. Stability studies conducted as per International Conference on Harmonization guidelines did not show any degradation of drug. Based on the above observations, it can be reasonably concluded that blend of EC–HPMC polymers and propylene glycol are better suited for the development of transdermal delivery system of furosemide.     

Assessment of the Therapeutic Potential of Metallothionein-II Application in Focal Cerebral Ischemia In Vitro and In Vivo

Metallothionein-II (MT-II) is an ubiquitously expressed small-molecular-weight protein and highly induced in various species and tissues upon stress, inflammation, and ischemia. MT-deficiency exacerbates ischemic injury in rodent stroke models in vitro and in vivo. However, there is conflicting data on the potential neuroprotective effect of exogenously applied metallothionein. Thus, we applied MT-II in an in vitro stroke model and intraperitoneally (i.p.) in two in vivo standard models of transient middle cerebral artery occlusion (MCAO) (a ‘stringent’ one [60min MCAO/48h reperfusion] and a ‘mild’ one [30min MCAO/72h reperfusion]), as well as i.v. together with recombinant tissue plasminogen activator (rtPA) to evaluate if exogenous MT-II-application protects against ischemic stroke. Whereas MT-II did not protect against 60min MCAO, there was a significant reduction of direct and indirect infarct volumes and neurological deficit in the MT-II (i.p.) treated animals in the ‘mild’ model at 3d after MCAO. Furthermore, MT-II also improved survival of the mice after MCAO, suppressed TNF-α mRNA induction in ischemic brain tissue, and protected primary neuronal cells against oxygen-glucose-deprivation in vitro. Thus, exogenous application of MT-II protects against ischemic injury in vitro and in vivo. However, long-term studies with different species and larger sampling sizes are required before a clinical use can be envisaged.