Soft X-ray microscopy for probing of topical tacrolimus delivery via micelles.

The penetration of topically applied tacrolimus formulated in micelles into murine skin is reported, measured by X-ray microscopy. Tacrolimus and micelles are probed for the first time by this high spatial resolution technique by element-selective excitation in the C 1s- and O 1s-regimes. This method allows selective detection of the distribution and penetration depth of drugs and carrier molecules into biologic tissues. It is observed that small, but distinct quantities of the drug and micelles, acting as a drug carrier, penetrate the stratum corneum. A comparison is made with the paraffin-based commercial tacrolimus ointment Protopic®, where local drug concentrations show to be low. A slight increase in local drug concentration in the stratum corneum is observed, if tacrolimus is formulated in micelles, as compared to Protopic®. This underscores the importance of the drug formulations for effective drug delivery. Time-resolved penetration shows presence of drug in the stratum corneum 100 min after formulation application, with penetration to deeper skin layers at 1000 min. High resolution micrographs give indications for a penetration pathway along the lipid membranes between corneocytes, but also suggest that the compound may penetrate corneocytes.

A novel biocompatible hyaluronic acid-chitosan hybrid hydrogel for osteoarthrosis therapy.

A conventional therapy for the treatment of osteoarthrosis is intra-articular injection of hyaluronic acid, which requires repeated, frequent injections. To extend the viscosupplementation effect of hyaluronic acid, we propose to associate it with another biopolymer in the form of a hybrid hydrogel. Chitosan was chosen because of its structural similarity to synovial glycosaminoglycans, its anti-inflammatory effects and its ability to promote cartilage growth. To avoid polyelectrolyte aggregation and obtain transparent, homogeneous gels, chitosan was reacetylated to a 50% degree, and different salts and formulation buffers were investigated. The biocompatibility of the hybrid gels was tested in vitro on human arthrosic synoviocytes, and in vivo assessments were made 1 week after subcutaneous injection in rats and 1 month after intra-articular injection in rabbits. Hyaluronic acid-chitosan polyelectrolyte complexes were prevented by cationic complexation of the negative charges of hyaluronic acid. The different salts tested were found to alter the viscosity and thermal degradation of the gels. Good biocompatibility was observed in rats, although the calcium-containing formulation induced calcium deposits after 1 week. The sodium chloride formulation was further tested in rabbits and did not show acute clinical signs of pain or inflammation. Hybrid HA-Cs hydrogels may be a valuable alternative viscosupplementation agent.

A novel oxido-viscosifying Hyaluronic Acid-antioxidant conjugate for osteoarthritis therapy: biocompatibility assessments.

To overcome the problem of fast degradation of Hyaluronic Acid (HA) in the treatment of osteoarthritis (OA), HA was protected against the oxidative stress generated by the pathology. Antioxidant conjugated HAs were synthesized and tested in vitro for their resistance in an oxidative environment mimicking OA. HA-4-aminoresorcinol (HA-4AR) displayed the interesting property of increasing in viscosity under oxidative conditions because of crosslinking induced by electron transfer. The novel HA polymer conjugate was shown to be biocompatible in vitro on fibroblast-like synoviocytes extracted from an arthritic patient. This HA conjugate was also assessed in vivo by intra-articular injection in healthy rabbits and was found to be comparable to the native polymer in terms of biocompatibility. This study suggests that HA-4AR is a promising candidate for a next generation viscosupplementation formulation.

Identification of aggregation breakers for bevacizumab (Avastin®) self-association through similarity searching and interaction studies.

Aggregation is a common challenge in the optimization of therapeutic antibody formulations. Since initial self-association of two monomers is typically a reversible process, the aim of this study is to identify different excipients that are able to shift this equilibrium to the monomeric state. The hypothesis is that a specific interaction between excipient and antibody may hinder two monomers from approaching each other, based on previous work in which dexamethasone phosphate showed the ability to partially reverse formed aggregates of the monoclonal IgG1 antibody bevacizumab back into monomers. The current study focuses on the selection of therapeutically inactive compounds with similar properties. Adenosine monophosphate, adenosine triphosphate, sucrose-6-phosphate and guanosine monophosphate were selected in silico through similarity searching and docking. All four compounds were predicted to bind to a protein-protein interaction hotspot on the Fc region of bevacizumab and thereby breaking dimer formation. The predictions were supported in vitro: An interaction between AMP and bevacizumab with a dissociation constant of 9.59±0.15 mM was observed by microscale thermophoresis. The stability of the antibody at elevated temperature (40 °C) in a 51 mM phosphate buffer pH 7 was investigated in presence and absence of the excipients. Quantification of the different aggregation species by asymmetrical flow field-flow fractionation and size exclusion chromatography demonstrates that all four excipients are able to partially overcome the initial self-association of bevacizumab monomers.

Advantages of bilayered vascular grafts for surgical applicability and tissue regeneration.

Nanofibrous scaffolds are part of an intense research effort to design the next generation of vascular grafts. With electrospinning, the production of micro- and nano-fiber-based prostheses is simple and cost effective. An important parameter for tissue regeneration in such scaffolds is pore size. Too small pores will impede cell infiltration, but too large pores can lead to problems such as blood leakage. In this study, bilayered grafts were made by electrospinning a high-porosity graft with a low-porosity layer on either the luminal or the adventitial side. Grafts were characterized in vitro for fiber size, pore size, total porosity, water and blood leakage, mechanical strength, burst pressure and suture retention strength, and were evaluated in vivo in the rat abdominal aorta replacement model for 3 and 12 weeks. In vitro blood leakage through these bilayered grafts was significantly reduced compared with a high-porosity graft. All grafts had an excellent in vivo outcome, with perfect patency and no thrombosis. Cell invasion and neovascularization were significantly reduced in the grafts with a low-porosity layer on the adventitial side, and there was no significant difference between the grafts in endothelialization rate or intimal hyperplasia. By tailoring the microarchitecture of biodegradable vascular prostheses, it is therefore possible to optimize the scaffold for tissue regeneration while preventing blood leakage, and thus facilitating applicability in the clinic.

Ultraviolet Resonance Raman spectroscopy used to study formulations of salmon calcitonin, a starch-peptide conjugate and TGF-ß3.

Ultraviolet Resonance Raman (UVRR) spectroscopy with excitation at 244 nm was investigated here as a possible useful tool for fast characterization of biopharmaceuticals. Studies were performed on three protein drugs: salmon calcitonin (sCT), starch-peptide conjugate, and transforming growth factor-ß3 (TGF-ß3) adsorbed onto solid granules of tricalcium phosphate (TCP). Secondary structure of sCT was investigated for solutions of 0.5mg/mL up to 200mg/mL, regardless of the turbidity or aggregation states. An increase in ß-sheet content was detected when sCT solutions aggregated. UVRR spectroscopy also detected a small amount of residual organic solvent in a starch-peptide conjugate solution containing only 40 µg/mL of peptide. UVRR spectroscopy was then used to characterize a protein, TGF-ß3, adsorbed onto solid granules of TCP at 50 and 250 µg/cm(3). This study shows that UVRR is suitable to characterize the protein formulations in a broad range of concentrations, in liquid, aggregated, and solid states.

In vivo characterisation of a novel water-soluble Cyclosporine A prodrug for the treatment of dry eye disease.

Cyclosporine A (CsA) has been demonstrated to be effective for the treatment of a variety of ophthalmological conditions, including ocular surface disorders such as the dry eye disease (DED). Since CsA is characterised by its low water solubility, the development of a topical ophthalmic formulation represents an interesting pharmaceutical question. In the present study, two different strategies to address this challenge were studied and compared: (i) a water-soluble CsA prodrug formulated within an aqueous solution and (ii) a CsA oil-in-water emulsion (Restasis, Allergan Inc., Irvine, CA). First, the prodrug formulation was shown to have an excellent ocular tolerance as well as no influence on the basal tear production; maintaining the ocular surface properties remained unchanged. Then, in order to allow in vivo investigations, a specific analytical method based on ultra high pressure liquid chromatography coupled with triple quadrupole mass spectrometer (UHPLC-MS/MS) was developed and optimised to quantify CsA in ocular tissues and fluids. The CsA ocular kinetics in lachrymal fluid for both formulations were found to be similar between 15 min and 48 h. The CsA ocular distribution study evidenced the ability of the prodrug formulation to penetrate into the eye, achieving therapeutically active CsA levels in tissues of both the anterior and posterior segments. In addition, the detailed analysis of the in vivo data using a bicompartmental model pointed out a higher bioavailability and lower elimination rate for CsA when it is generated from the prodrug than after direct application as an emulsion. The interesting in vivo properties displayed by the prodrug solution make it a safe and suitable option for the treatment of DED.

Stability of seasonal influenza vaccines investigated by spectroscopy and microscopy methods.

The stability of different seasonal influenza vaccines was investigated by spectroscopy and microscopy methods before and after the following stress-conditions: (i) 2 and 4 weeks storage at 25°C, (ii) 1 day storage at 37°C and (iii) one freeze-thaw cycle. The subunit vaccine Influvac (Solvay Pharma) and the split vaccine Mutagrip (Sanofi Pasteur) were affected by all stresses. The split vaccine Fluarix (GlaxoSmithKline) was affected only by storage at 25°C. The virosomal vaccine Inflexal V (Berna Biotech) was stable after the temperature stresses but aggregated after one freeze-thaw cycle. This study provides new insights into commercial vaccines of low antigen concentration and highlights the importance of using multiple techniques to assess vaccine stability.

It is all about proteases: from drug delivery to in vivo imaging and photomedicine.

Clinical studies provide overwhelming evidence for the importance of proteolytic imbalance and the upregulation of diverse protease classes in diseases such as cancer and arthritis. While the complex nature of proteolytic networks has hampered the development of protease inhibitors for these indications, aberrant enzyme activity could be successfully exploited for the development of proteasesensitive drug delivery systems and fluorescent in vivo imaging agents. More recently, these concepts have also been translated into photomedical applications to develop dual modality prodrugs for the simultaneous treatment and imaging of disease. After an introductory overview of proteases and their role in cancer, we present and discuss different strategies to exploit upregulated protease activity for the development of drug delivery systems, fluorescent in vivo reporter probes, and photosensitizer-prodrugs with respect to their potential and limitations. The main approaches used for targeting proteases in all three areas can be roughly divided into peptide-based and macromolecular strategies. Both involve the use of a short, peptide-based protease substrate, which is either directly tagged to the therapeutic agent or dye/quencher pair, or alternatively, serves as a linker between the polymeric carrier and a functional unit. In the latter case, the pharmacokinetic properties of peptide-based protease-sensitive prodrugs and imaging probes can be further ameliorated by the passive targeting capacity of macromolecular drug delivery systems for neoplastic and inflammatory lesions.

Colloidal systems for the delivery of cyclosporin A to the anterior segment of the eye.

Due to the eye's specific anatomical and physiological conformation, the treatment of eye diseases is a real challenge for pharmaceutical therapy. The presence of efficient protective barriers (i.e., the conjunctival and corneal membranes) and protective mechanisms (i.e., blinking and nasolachrymal drainage) makes this organ particularly impervious to local drug therapy. To overcome these issues, numerous strategies have been envisioned using pharmaceutical technology. Many formulations currently on the market or still under development are emulsions or colloidal systems intended to enhance precorneal residence time and corneal penetration, causing a consequent increase in drug bioavailability after instillation. After a review of some recent developments in the field of cyclosporin A formulations for the eye, a novel micellar formulation of cyclosporine A based on a diblock methoxy-poly(ethylene glycol)-hexysubstituted poly(lactides) (MPEG-hexPLA) is described.