Radiosynthesis and assessment of ocular pharmacokinetics of (124)I-labeled chitosan in rabbits using small-animal PET.

PURPOSE: The polysaccharide chitosan is a unique material for the design of ocular drug-delivery vehicles. The aim of this study was to radiolabel chitosan with iodine-124 ((124)I) for measurement of ocular pharmacokinetics in rabbits using small-animal positron emission tomography (PET). PROCEDURES: Crl:CHBB (HM) rabbits received one drop (35 µL) of either a formulation containing chitosan (0.5%, w/v) spiked with (124)I-labeled chitosan ([(124)I]chitosan) (n = 4) or a control solution of sodium [(124)I]iodide in buffer (n = 2) in the conjunctival sac of the right eye. Radioactivity distribution in the head region was measured at five different time points between 0 and 22 h after topical application. Regions of interest were manually defined in the reconstructed PET images, and activity concentration was quantified as percent applied dose (AD) per cubic centimeter tissue. RESULTS: Clear differences were observed in the ocular pharmacokinetics of the two formulations. At 3 h after application, ocular activity uptake was 0.5 ± 0.1%AD/cc for sodium [(124)I]iodide, compared to 4.7 ± 5.3%AD/cc for the [(124)I]chitosan formulation. CONCLUSIONS: We were able to show that ocular pharmacokinetics of (124)I-labeled ophthalmic formulations can be measured with small-animal PET and that [(124)I]chitosan had approximately a 2-fold increased ocular retention through the study period compared to sodium [(124)I]iodide.

Barrier analysis of periocular drug delivery to the posterior segment.

Periocular administration is a potential way of delivering drugs to their targets in posterior eye segment (vitreous, neural retina, retinal pigment epithelium (RPE), choroid). Purpose of this study was to evaluate the role of the barriers in periocular drug delivery. Permeation of FITC-dextrans and oligonucleotides in the bovine sclera was assessed with and without Pluronic gel in the donor compartment. Computational model for subconjunctival drug delivery to the choroid and neural retina/vitreous was built based on clearance concept. Kinetic parameters for small hydrophilic and lipophilic drug molecules, and a macromolecule were obtained from published ex vivo and in vivo animal experiments. High negative charge field of oligonucleotides slows down their permeation in the sclera. Pluronic does not provide adequate rate control to modify posterior segment drug delivery. Theoretical calculations for subconjunctival drug administration indicated that local clearance by the blood flow and lymphatics removes most of the drug dose which is in accordance with experimental results. Calculations suggested that choroidal blood flow removes most of the drug that has reached the choroid, but this requires experimental verification. Calculations at steady state using the same subconconjunctival input rate showed that the choroidal and vitreal concentrations of the macromolecule is 2-3 orders of magnitude higher than that of small molecules. The evaluation of the roles of the barriers augments to design new drug delivery strategies for posterior segment of the eye.

Low molecular weight hyaluronan shielding of DNA/PEI polyplexes facilitates CD44 receptor mediated uptake in human corneal epithelial cells.

AIM: It was the aim of this study to prepare purified DNA/PEI polyplexes, which are coated with hyaluronan to facilitate CD44 receptor mediated uptake of the DNA/PEI polyplex and to reduce unspecific interactions of the complex with negatively charged extracellular matrix components on the ocular surface. METHODS: Hyaluronans of different molecular weights (<10 kDa, 10-30 kDa and 30-50 kDa) were isolated after enzymatic degradation of high molecular weight hyaluronan via ultrafiltration by centrifugation. The influence of the different hyaluronans used for coating on the stability and transfection efficiency of the complexes was evaluated in vitro. Transfection and uptake studies were performed in human corneal epithelial (HCE) cells. CD44 receptor expression of this cell model was evaluated by immunohistochemistry. RESULTS: Coating of purified DNA/PEI polyplexes with low molecular weight hyaluronan (<10 kDa) facilitated receptor-mediated uptake via the CD44 receptor in HCE cells, increased complex stability in vitro, and effectively shielded the positive surface charges of the polyplex without decreasing its transfection efficiency. Higher molecular weights and larger amounts of hyaluronan in the complexes resulted in lesser improvements in the stability and transfection efficacy of the complexes. CONCLUSIONS: Coating of polyplexes with low molecular weight hyaluronan is a promising strategy for gene delivery to the ocular surface, where CD44 receptor mediated uptake decreased cytotoxicity and reduced non-specific interactions with the negatively charged extracellular matrix components are considered beneficial for increased transfection efficiency of non-viral vectors.

Peribulbar poloxamer for ocular drug delivery.

PURPOSE: The use of biopolymers in peribulbar injection for controlled drug delivery provides alternative options to eyedrops and intravitreal or surgical methods. Polymerizable biopolymers are especially likely to have a role because of their particular properties. In liquid form, they can be easily injected into the target site and, after polymerization, they provide a prolonged and controlled release of the drug. This study was undertaken to demonstrate the suitability of a thermopolymerizable biopolymer poloxamer (Lutrol F127) for peribulbar injections and controlled drug release. METHODS: The toxicity of injected poloxamer compounds was evaluated by visual inspection and histological and immunohistochemical tissue evaluation. The release of marker substances such as 5(6)-carboxyfluorescein (376 Da) or fluorescein isothiocyanate-dextran (FITC-dextran) (4-40 kDa) from poloxamer was used to simulate drug release and penetration into the eye using in vivo fluorometry. RESULTS: According to our clinical and pathological analyses, poloxamer was well tolerated in peribulbar injections and did not cause acute toxicity at the site of injection. The marker compounds were released from the site of injection during the first 24 hours. CONCLUSIONS: Although poloxamer appears to be suitable for peribulbar injections, a more prolonged period of dissolution is desirable for clinical purposes.

Corneal epithelium as a platform for secretion of transgene products after transfection with liposomal gene eyedrops.

BACKGROUND: The first objective of the study was to evaluate the transfection of corneal epithelium with non-viral vectors to secrete transgene products into the tear fluid and aqueous humor. The second goal was to evaluate the differentiated corneal epithelial cell culture for transfection studies. METHODS: The human corneal epithelial (HCE) cell line was cultured to different stages of differentiation and transfected with complexes of pCMV-SEAP2 with DOTAP/DOPE, DOTAP/DOPE/protamine sulfate (PS) and polyethylenimine (PEI). The complexes of DOTAP/DOPE with plasmid (CMV-SEAP2 or pCMV-Luc4) were subsequently applied topically to the rabbit eyes. Secreted alkaline phosphatase (SEAP) was analyzed using chemiluminescent assay. Luciferase (Luc) was detected at the mRNA level in cornea and conjunctiva using a qRT-PCR. RESULTS: The transfection levels decreased with differentiation of HCE cells. PEI was effective in transfecting both the dividing and partly differentiated cells, but ineffective in differentiated cells. DOTAP/DOPE showed high activity in differentiated cell cultures, while added PS did not improve transfection. Significant SEAP expression was observed for three days after in vivo transfection in the tear fluid and aqueous humor. The luciferase mRNA was found both in the cornea and conjunctiva. The rates of SEAP secretion from both the basolateral side of differentiated HCE cells and cornea in vivo were within the same range. CONCLUSIONS: Corneal epithelium can be transfected topically to secrete gene products to the tear fluid and aqueous humor. The differentiated HCE model is a useful tool in the evaluation of non-viral carriers for corneal transfection.

Cell culture models of the ocular barriers.

The presence of tight barriers, which regulate the environment of ocular tissues in the anterior and posterior part of the eye, is essential for normal visual function. The development of strategies to overcome these barriers for the targeted ocular delivery of drugs, e.g. to the retina, remains a major challenge. During the last years numerous cell culture models of the ocular barriers (cornea, conjunctiva, blood-retinal barrier) have been established. They are considered to be promising tools for studying the drug transport into ocular tissues, and for numerous other purposes, such as the investigation of pathological ocular conditions, and the toxicological screening of compounds as alternative to in vivo toxicity tests. The further development of these in vitro models will require more detailed investigations of the barrier properties of both the cell culture models and the in vivo ocular barriers. It is the aim of this review to describe the current status in the development of cell culture models of the ocular barriers, and to discuss the applicability of these models in pharmaceutical research.

Thiolated chitosans.

The derivatization of the primary amino groups of chitosan with coupling reagents bearing thiol functions leads to the formation of thiolated chitosans. So far, three types of thiolated chitosans have been generated: chitosan-cysteine conjugates, chitosan-thioglycolic acid conjugates and chitosan-4-thio-butyl-amidine conjugates. Various properties of chitosan are improved by this immobilization of thiol groups. Due to the formation of disulfide bonds with mucus glycoproteins, the mucoadhesiveness is 6--100-fold augmented (I). The permeation of paracellular markers through intestinal mucosa can be enhanced 1.6--3-fold utilizing thiolated instead of unmodified chitosan (II). Moreover, thiolated chitosans display in situ-gelling features, due to the pH-dependent formation of inter- as well as intra-molecular disulfide bonds (III). This latter process provides a strong cohesion and stability of carrier matrices being based on thiolated chitosans (IV). Consequently, thiolated chitosans can guarantee a prolonged controlled release of embedded therapeutic ingredients (V). The potential of thiolated chitosans has meanwhile also been demonstrated in vivo. A significant pharmacological efficacy of 1.3% of orally given salmon calcitonin, for instance, could be achieved utilizing thiolated chitosan as polymeric drug carrier matrix, while no effect was reached using unmodified chitosan. According to these results thiolated chitosans represent a promising new category of polymeric excipients in particular for the non-invasive administration of hydrophilic macromolecules. Further applications such as their use as scaffold materials in tissue engineering or as coating material for stents seem feasible.

Mucoadhesive thiolated chitosans as platforms for oral controlled drug delivery: synthesis and in vitro evaluation.

The aim of the present study was to evaluate the influence of the degree of modification and the polymer chain length on the mucoadhesive properties and the swelling behavior of thiolated chitosan derivatives obtained via a simple one-step reaction between the polymer and 2-iminothiolane. The conjugates differing in molecular mass of the polymer backbone and in the amount of immobilized thiol groups were compressed into tablets. They were investigated for their mucoadhesive properties on freshly excised porcine mucosa via tensile studies and the rotating cylinder method. Moreover, the swelling behavior of these tablets in aqueous solutions was studied by a simple gravimetric method. The obtained results demonstrated that the total work of adhesion of chitosan-TBA (=4-thio-butyl-amidine) conjugates can be improved by an increasing number of covalently attached thiol groups; a 100-fold increase compared to unmodified chitosan was observed for a medium molecular mass chitosan-TBA conjugate exhibiting 264 microM thiol groups per gram polymer. Also, the polymer chain length had an influence on the mucoadhesive properties of the polymer. The medium molecular mass polymer displayed a fourfold improved adhesion on the rotating cylinder compared to the derivative of low molecular mass. These results contribute to the development of new delivery systems exhibiting improved mucoadhesive properties.

Thiolated polymers--thiomers: synthesis and in vitro evaluation of chitosan-2-iminothiolane conjugates.

The aim of this study was to improve the properties of chitosan as excipient in drug delivery systems by the covalent attachment of thiol moieties. This was achieved by the modification of chitosan with 2-iminothiolane. The resulting chitosan-4-thio-butyl-amidine conjugates (chitosan-TBA conjugates) displayed up to 408.9+/-49.8 micromol thiol groups per gram polymer. Because of the formation of disulfide bonds based on an oxidation process of the immobilized thiol groups under physiological conditions, chitosan-TBA conjugates exhibit in situ gelling properties. After less than 2h, 1.5% (m/v) chitosan-TBA conjugate solutions of pH 5.5 formed covalently cross-linked gels. The viscosity increased in positive correlation with the amount of thiol groups immobilized on chitosan. In addition, also the mucoadhesive properties were strongly improved by the covalent attachment of thiol groups on chitosan. The adhesion time of tablets based on the unmodified polymer on freshly excised porcine intestinal mucosa spanned on a rotating cylinder in an artificial intestinal fluid was extended more than 140-fold by using the thiolated version. Drug release studies out of tablets comprising the chitosan-TBA conjugate demonstrated that an almost zero-order release kinetic was achieved for the model drug clotrimazole within the first 6h. The modification of chitosan with 2-iminothiolane leads, therefore to thiolated polymers, which represent a promising tool for the development of in situ gelling and/or mucoadhesive drug delivery systems.

Mucoadhesive ocular insert based on thiolated poly(acrylic acid): development and in vivo evaluation in humans.

The aim of the study was to develop a mucoadhesive ocular insert for the controlled delivery of ophthalmic drugs and to evaluate its efficacy in vivo. The inserts tested were based either on unmodified or thiolated poly(acrylic acid). Water uptake and swelling behavior of the inserts as well as the drug release rates of the model drugs fluorescein and two diclofenac salts with different solubility properties were evaluated in vitro. Fluorescein was used as fluorescent tracer to study the drug release from the insert in humans. The mean fluorescein concentration in the cornea/tearfilm compartment as a function of time was determined after application of aqueous eye drops and inserts composed of unmodified and of thiolated poly(acrylic acid). The acceptability of the inserts by the volunteers was also evaluated. Inserts based on thiolated poly(acrylic acid) were not soluble and had good cohesive properties. A controlled release was achieved for the incorporated model drugs. The in vivo study showed that inserts based on thiolated poly(acrylic acid) provide a fluorescein concentration on the eye surface for more than 8 h, whereas the fluorescein concentration rapidly decreased after application of aqueous eye drops or inserts based on unmodified poly(acrylic acid). Moreover, these inserts were well accepted by the volunteers. The present study indicates that ocular inserts based on thiolated poly(acrylic acid) are promising new solid devices for ocular drug delivery.

In vitro evaluation of the viscoelastic properties of chitosan-thioglycolic acid conjugates.

The aim of this study was to evaluate the viscoelastic properties of chitosan-thioglycolic acid conjugates with different amounts of thiol groups immobilized on the polymer. The modification of chitosan was achieved via the covalent attachment of thioglycolic acid mediated by a carbodiimide. Chitosan-thioglycolic acid conjugates displaying 120, 209 and 439 microM thiol groups per gram of polymer were synthesized. The rheological properties of the three different conjugates were investigated. The elastic properties of the gels were found to increase significantly at pH 5.5. After 6 h the elastic modulus G' of chitosan-TGA 120, chitosan-TGA 209 and chitosan-TGA 439 gels increased 7-, 32- and 168-fold, respectively. In parallel the formation of disulfide bonds was observed. Accordingly, proof of principle that chitosan modified by the introduction of thiol groups has in situ gelling properties due to the formation of inter- and intramolecular disulfide bonds at physiological pH values is provided. Based on their in situ gelling properties, chitosan-thioglycolic acid conjugates seem to be very promising new excipients for liquid or semisolid formulations which should stabilize themselves once applied on the site of drug delivery.

In vitro evaluation of the permeation enhancing effect of polycarbophil-cysteine conjugates on the cornea of rabbits.

It was the aim of this study to investigate the permeation enhancing effect of thiolated polycarbophil on the cornea of rabbits in vitro. The proposed reaction mechanism involves the opening of the tight junctions in the corneal epithelium. The modification of polycarbophil was achieved via covalent attachment of L-cysteine mediated by a carbodiimide. Transcorneal permeation studies were performed in Ussing-type diffusion chambers. As model compounds, sodium fluorescein, as a marker for paracellular transport, and dexamethasone phosphate were used. To evaluate potential corneal damage the corneal hydration level of each cornea was determined. Polycarbophil-cysteine was found to increase the permeation of sodium fluorescein 2.2-fold and that of dexamethasone phosphate 2.4-fold in comparison to the unmodified polymer. The concentration of dexamethasone in the acceptor medium was 1.5-fold increased. As evidenced by the corneal hydration level, polycarbophil-cysteine did not damage the corneal tissues. Therefore, polycarbophil-cysteine conjugates seem to be promising excipients for ocular drug delivery systems where they might be used as safe permeation enhancers.