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.

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.

Permeation enhancer effect of chitosan and chitosan derivatives: comparison of formulations as soluble polymers and nanoparticulate systems on insulin absorption in Caco-2 cells.

In this study four quaternized derivatives of chitosan: trimethyl chitosan (TMC), dimethylethyl chitosan (DMEC), diethylmethyl chitosan (DEMC) and triethyl chitosan (TEC) with degree of substitution of approximately 50+/-5% were synthesized and their effect on the permeability of insulin across intestinal Caco-2 monolayers was studied and compared with chitosan both in free-soluble form and in nanoparticulate systems. Transepithelial electrical resistance (TEER) studies revealed that all four chitosan derivatives in free-soluble forms were able to decrease the TEER value in the following order TMC>DMEC>DEMC=TEC>chitosan, indicating their abilities to open the tight junctions. Recovery studies on the TEER showed that the effect of the polymers on Caco-2 cell monolayer is reversible and proves the viability of cells after incubation with all polymers. A similar rank order was also observed when measuring the zeta-potentials of the various polymers in solution form. Transport studies of insulin together with the soluble polymers across Caco-2 cell layers showed the following ranking: TMC>DMEC>DEMC>TEC>chitosan which is in agreement with the strength of the cationic charge of the polymer. In comparison to the free-soluble polymers, the nanoparticles prepared by ionic gelation of the chitosan and its quaternized derivatives had much lower effect on decreasing the TEER by opening of the tight junctions. This can be explained by the reduced available amount of positive charge at the surface of the nanoparticles. In accordance with these results, the insulin loaded nanoparticles showed much less permeation across the Caco-2 cell monolayer in comparison to the free-soluble polymers. Mass balance transport studies revealed that a substantial amount of the nanoparticles has been entrapped into the Caco-2 monolayer or attached to the cell surface. It can thus be stated that while free-soluble polymers can reversibly open the tight junctions and increase the permeation of insulin, the nanoparticles had basically only a low effect on the opening of the tight junction and the paracellular transport of insulin across the Caco-2 cell monolayer. These data convincingly show that nanoparticles consisting of chitosan and its quaternary ammonium derivatives loaded with insulin are less effective in facilitating paracellular transport across Caco-2 cell monolayers than the corresponding free polymers.

A biodegradable drug delivery system for the treatment of postoperative inflammation.

Cataract surgery is often performed in patients suffering from associated pathologies. Our goal is to develop a biodegradable drug delivery system (DDS) combined with the artificial intraocular lens (IOL). DDS were manufactured using poly(D,L-lactide-co-glycolide), or PLGA, and were loaded with triamcinolone acetonide (TA). The loading capacity was approximately 1050 microg of TA per DDS. The higher the molecular weight of PLGA (34,000, 48,000 and 80,000Da), the slower was the release of TA in vitro. Cataract surgery was performed on the right eye of rabbits. IOL was inserted with (i) no DDS, (ii) unloaded DDS PLGA48000, (iii) one loaded DDS PLGA48000, (iv) two loaded DDS. The number of inflammatory cells and the protein concentration were measured in the aqueous humor (AH). Unloaded DDS showed good ocular biocompatibility. One DDS PLGA48000 loaded with TA significantly reduced postoperative ocular inflammation. Two loaded DDS PLGA48000 was even more effective in inhibiting such inflammation. On long-term observation (days 63 and 84), reduction of inflammation could be obtained by insertion of one DDS PLGA48000 and a second DDS PLGA80000. Therefore, our "all in one" system is very promising since it could replace oral treatment and reduce the number of intraocular injections.

Biological conversion of a water-soluble prodrug of cyclosporine A.

UNIL088 is a water-soluble prodrug of cyclosporine A (CsA) designed for topical ocular delivery. The pro-moiety is grafted via an ester function to CsA and the solubilizing group is a phosphate ion. The aim of this study was to elucidate the conversion mechanisms by which UNIL088 generates CsA. UNIL088 was incubated in rabbit tears at physiological temperature to study its enzymatic and chemical conversion, respectively. Metabolites and intermediates were identified using a quadrupole-time of flight (QqTOF) mass spectrometer, which allowed biotransformation pathways to be deduced. Conversion is activated by the chemical or enzymatic hydrolysis of the terminal ester function of the pro-moiety, leading to the phospho-serine-sarcosine-cyclosporine A that spontaneously converts into CsA. In addition to the main biotransformation pathway, a secondary reaction involved hydrolysis of the phosphate ester group of the pro-moiety, probably by phosphatases present in tears.

Differential tumor cell targeting of anti-HER2 (Herceptin) and anti-CD20 (Mabthera) coupled nanoparticles.

Two types of antibody-labeled nanoparticles (mAb-NPs) were prepared with the aim to achieve specific tumor targeting. Anti-HER2 and anti-CD20 monoclonal antibodies (mAb) were used as model ligands. Small poly(dl-lactic acid) nanoparticles (PLA NPs) with a mean size of about 170 nm were prepared by the salting out method. Thereafter, the coating of PLA NPs with mAbs was performed in two steps. First, thiol groups (-SH) were introduced on the surface of PLA-NPs by a two-step carbodiimide reaction. The number of -SH groups on the surface of NPs increased from 150 to 400 mmol-SH/mol PLA when cystamine concentrations of 25-1518 mol cystamine/mol PLA were used during the thiolation reaction. In the second step, covalent coupling of antibodies to thiolated NPs (NPs-SH) was obtained via a bifunctional cross-linker, m-maleimidobenzoyl-N-hydroxy-sulfosuccinimide ester (sulfo-MBS). For both mAbs anti-HER2 and anti-CD20, respectively, the number of -SH functions on the NPs had no influence on the amount of mAb coupled to the NPs. Approximately, 295 anti-HER2 and 557 anti-CD20 molecules, respectively, were covalently coupled per nanoparticle. The NPs size after the coupling reactions was about 250 nm. The specific interaction between tumor cells and mAb-NPs was determined by confocal microscopy using two cell lines: SKOV-3 human ovarian cancer cells (overexpressing HER2) and Daudi lymphoma cells (overexpressing CD20). The results showed the selective targeting of mAb-NPs to tumor cells overexpressing the specific antigen. While anti-CD20 labeled NPs (anti-CD20 NPs) bound to and remained at the cellular surface, anti-HER2 labeled NPs (anti-HER2 NPs) were efficiently internalized. The mAb-NPs represent a promising approach to improve the efficacy of NPs in active targeting for cancer therapy while the choice of the antibody-target system defines the fate of the mAb-NPs after their binding to the cells.

Intraocular implants for extended drug delivery: therapeutic applications.

An overview of ocular implants with therapeutic application potentials is provided. Various types of implants can be used as slow release devices delivering locally the needed drug for an extended period of time. Thus, multiple periocular or intraocular injections of the drug can be circumvented and secondary complications minimized. The various compositions of polymers fulfilling specific delivery goals are described. Several of these implants are undergoing clinical trials while a few are already commercialized. Despite the paramount progress in design, safety and efficacy, the place of these implants in our clinical therapeutic arsenal remains limited. Miniaturization of the implants allowing for their direct injection without the need for a complicated surgery is a necessary development avenue. Particulate systems which can be engineered to target specifically certain cells or tissues are another promising alternative. For ocular diseases affecting the choroid and outer retina, transscleral or intrasscleral implants are gaining momentum.

Evaluation of a topical cyclosporine A prodrug on corneal graft rejection in rats.

PURPOSE: To assess the efficacy of a topical cyclosporine A (CsA), water-soluble prodrug, for promoting the survival of allogenic rat corneal grafts after penetrating keratoplasty (PKP). METHODS: Corneas of Brown-Norway rats (donors) were transplanted to Lewis rats (recipients). Transplanted rats were divided in three treatment groups: group I (PBS) and group II (0.26% Debio088) received drops five times per day. Group III received a daily intramuscular CsA injection (10 mg/kg/day). Blood CsA concentrations were measured on days 2 and 14. On day 4, 10, 13 after PKP, grafts were scored for corneal transparency, edema and extent of neovascularization. An opacity score of greater than or equal to 3 was considered as a nonreversible graft rejection process. On day 14, the experimental eyes were processed for histology. RESULTS: On day 13, 12 of the 18 corneal transplants (67%) in group I showed irreversible graft rejection. Three of 18 transplants (19%) in group II and 5 of 16 transplants (28%) in group III showed irreversible graft rejection (p=0.013/p=0.019, OR=0.14/0.06 versus vehicle). Each mean clinical score for edema, opacity, and neovessels in group II were significantly lower than those of the grafts in group I (respectively p=0.010, p=0.013, p=0.024) and III except for neovessels (respectively p=0.002, p=0.001, p=0.057). Histology confirmed the clinical results. The mean CsA blood levels for groups II and III were, respectively 54+/-141 mug/l and 755+/-319 mug/l on day 2 and 14+/-34 mug/l and 1318+/-463 mug/l on day 14. CONCLUSIONS: Debio088 CsA prodrug drops given five times daily are as effective as intramuscular injection of 10 mg/kg/day for the prevention of acute corneal graft rejection in rats.

Biodegradable scleral implants as new triamcinolone acetonide delivery systems.

The goal of this study was to develop ocular scleral implants able to release triamcinolone acetonide (TA) overall several months. Scleral discs were manufactured by a compression-molding method using a new synthetic polymer, poly(methylidene malonate) (PMM2.1.2), as matrix. Implants with good mechanical properties adapted for in vivo implantation have been obtained when using high M(w) PMM2.1.2 (100,000 - 150,000 Da) associated with ethoxylated derivatives of stearic acid (Simulsol) or oligomers of methylidene malonate as plasticizer. After implantation in rabbit eyes, scleral implants showed a good ocular biocompatibility. Indeed, the clinical follow-up and ocular inflammation parameters, such as inflammatory cell number and protein content in aqueous humor, demonstrated that implants were well tolerated and did not provoke abnormal inflammation. Implants were able to release significant concentrations of TA in the vitreous and the sclera throughout 5 weeks.

An open pharmacokinetic study of hexylaminolevulinate-induced photodiagnosis after intravesical administration.

BACKGROUND AND OBJECTIVES: Hexylaminolevulinate-mediated photodiagnosis of superficial bladder cancer recently gained marketing authorisation in Europe and is therefore being used increasingly, especially to visualise flat tumours such as carcinoma in situ. Although no significant adverse effects related to the drug have been reported to date, precise information with respect to the safety of this procedure in clinical practice has not yet been published. In the present study, we investigated the possible systemic absorption of different hexylaminolevulinate concentrations and characterised the adverse effects after bladder instillation. METHODS: Twelve patients with known bladder cancer received a 2-hour administration of 50 mL of phosphate-buffered saline containing 4 mmol/L, 8 mmol/L or 16 mmol/L of hexylaminolevulinate. Safety was primarily evaluated by monitoring adverse effects. Standard haematology and clinical biochemistry were assessed by the local hospital laboratory. Measurements of hexylaminolevulinate, 5-aminolevulinate and protoporphyrin IX in plasma and solution collected after instillation were performed, as were fluorescence measurements in the urothelium and the skin by using an optical fibre-based spectrofluorometer. RESULTS: No hexylaminolevulinate was found in the plasma. Furthermore, 5-aminolevulinate and protoporphyrin IX showed no significant increases in plasma after intravesical instillation of hexylaminolevulinate 4 mmol/L, 8 mmol/L or 16 mmol/L for 2 hours. This implies a very low systemic absorption at the administered doses with a hexylaminolevulinate uptake from the bladder of about 5%. Neither skin sensitivity nor adverse reactions that could be attributed to the drug were reported. Renal and liver function were not affected by the hexylaminolevulinate doses used in this study. CONCLUSION: We demonstrated for the first time that hexylaminolevulinate-mediated photodiagnosis is a safe procedure for a patient undergoing this examination, the drug being only minimally systemically absorbed after intravesical instillation. Therefore, no or only minimal adverse effects compared with those seen with conventional photosensitising agents can be expected.

In vitro and in vivo activities of verteporfin-loaded nanoparticles.

The main goal of this study was to develop a dispersed polymeric drug delivery system for verteporfin, suitable for intravenous administration and capable of improving its phototherapeutic index and minimizing the side effects. To achieve this objective, two types of verteporfin-loaded nanoparticles (167 and 370 nm in diameter) based on poly(D,L-lactide-co-glycolide) were prepared using the salting-out technique and were first tested on EMT-6 mammary tumor cells in comparison with an aqueous solution (DMSO/PBS). It was observed that small nanoparticles exhibited greater photocytotoxicity compared to large nanoparticles or DMSO/PBS, and the photocytotoxic efficiency was graded as small nanoparticles>DMSO/PBS>large nanoparticles. Furthermore, verteporfin, entrapped into small nanoparticles transferred to serum proteins more rapidly than when dissolved in DMSO/PBS. Drug clearance, measured by skin phototoxicity investigated in mice exposed to simulated sunlight 15 to 150 min after the injection of small nanoparticles was modest at early light exposure times with the small nanoparticles and diminished rapidly with later exposure times. Tumor bioassay results indicated that verteporfin incorporated into small nanoparticles effectively controlled tumor growth for 20 days in mice with early light irradiation times following drug administration.

Conversion of cyclosporine A prodrugs in human tears vs rabbits tears.

The aim of this study was to evaluate the rate and mechanism of conversion of two water-soluble prodrugs of cyclosporine A (CsA) intended for topical delivery to the eye. The new molecules were designed according to the double prodrug concept: a solubilizing moiety was grafted onto CsA via an ester function, which could be hydrolysed via a two-step process (enzymatic and chemical). Prodrug solutions were prepared extemporaneously in an isotonic and neutral aqueous medium compatible with ophthalmic use. The rates of conversion into the parent molecule were determined by incubating the prodrugs in fresh rabbit or human tears or in a phosphate buffer solution (PBS) at pH 7.4. Both prodrugs were converted into CsA within the first minute in the presence of rabbit tears with rate constants of k=5.9x10(-3)min(-1) and k=3.8x10(-3)min(-1), respectively, for UNIL088 and UNIL089, whereas chemical conversion in PBS was negligible (k=0.5x10(-3)min(-1) for both molecules). Incubation of UNIL088 in human tears showed a significantly high conversion rate. It is concluded that the developed double prodrugs underwent a bioconversion in physiological media and thus represent promising candidates for topical delivery of CsA to the eye.

Pseudo-thermosetting chitosan hydrogels for biomedical application.

To prepare transparent chitosan/beta-glycerophosphate (betaGP) pseudo-thermosetting hydrogels, the deacetylation degree (DD) of chitosan has been modified by reacetylation with acetic anhydride. Two methods (I and II) of reacetylation have been compared and have shown that the use of previously filtered chitosan, dilution of acetic anhydride and reduction of temperature in method II improves efficiency and reproducibility. Chitosans with DD ranging from 35.0 to 83.2% have been prepared according to method II under homogeneous and non-homogeneous reacetylation conditions and the turbidity of chitosan/betaGP hydrogels containing homogeneously or non-homogeneously reacetylated chitosan has been investigated. Turbidity is shown to be modulated by the DD of chitosan and by the homogeneity of the medium during reacetylation, which influences the distribution mode of the chitosan monomers. The preparation of transparent chitosan/betaGP hydrogels requires a homogeneously reacetylated chitosan with a DD between 35 and 50%.

Pseudo-thermosetting chitosan hydrogels for biomedical application.

To prepare transparent chitosan/beta-glycerophosphate (betaGP) pseudo-thermosetting hydrogels, the deacetylation degree (DD) of chitosan has been modified by reacetylation with acetic anhydride. Two methods (I and II) of reacetylation have been compared and have shown that the use of previously filtered chitosan, dilution of acetic anhydride and reduction of temperature in method II improves efficiency and reproducibility. Chitosans with DD ranging from 35.0 to 83.2% have been prepared according to method II under homogeneous and non-homogeneous reacetylation conditions and the turbidity of chitosan/betaGP hydrogels containing homogeneously or non-homogeneously reacetylated chitosan has been investigated. Turbidity is shown to be modulated by the DD of chitosan and by the homogeneity of the medium during reacetylation, which influences the distribution mode of the chitosan monomers. The preparation of transparent chitosan/betaGP hydrogels requires a homogeneously reacetylated chitosan with a DD between 35 and 50%.

A novel water-soluble cyclosporine A prodrug: ocular tolerance and in vivo kinetics.

The purpose of this study is to demonstrate that a novel water-soluble prodrug of cyclosporine A (CsA) intended for topical ocular administration, does not induce eye irritation in a rabbit model and is able to generate therapeutic concentrations of CsA in the precorneal area immediately after administration. The eye irritancy of the prodrug and CsA control solution was assessed by the Draize test and by confocal laser ophthalmoscopy (CLSO). Residence time and tear concentrations of prodrug and CsA in the rabbit eye were assessed by HPLC. The Draize test showed an excellent tolerance for the prodrug solution while the reference CsA oil solution induced lachrymation and irritation. The CLSO-measured corneal lesions, subsequent to treatment with the prodrug and reference solutions, were 3% and 9%, respectively. The prodrug transformed rapidly, leading to relatively stable CsA concentrations in tears with a maximal concentration of 94 microg ml(-1) over the observation period. This study demonstrated that the prodrug solution was well tolerated and that clinically significant CsA tear concentrations were achieved. UNIL088 is a promising molecule in the treatment of immune-related disorders of the eye.

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.

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.

Therapeutic applications of viscous and injectable poly(ortho esters).

Poly(ortho esters) (POE) are hydrophobic and bioerodible polymers that have been investigated for pharmaceutical use since the early 1970s. Among the four described generations of POE, the third (POE III) and fourth (POE IV) are promising viscous and injectable materials which have been investigated in numerous biomedical applications. POE III has been extensively studied for ophthalmic drug delivery, it presents an excellent biocompatibility and is currently being investigated as a vehicle for sustained drug delivery to treat diseases of the posterior segment of the eye. POE IV is distinguishable by a highly reproducible and controlled synthesis, a higher hydrophobicity, and an excellent biocompatibility. It is currently under development for a variety of applications, such as ocular delivery, periodontal disease treatment and applications in veterinary medicine. This review will also focus on new perspectives for this promising family of polymers, such as guided tissue regeneration, treatment of osteoarthritis, as well as peptide and protein delivery.

A new poly(ortho ester)-based drug delivery system as an adjunct treatment in filtering surgery.

PURPOSE: Pharmacologic modulation of wound healing after glaucoma filtering surgery remains a major clinical challenge in ophthalmology. Poly(ortho ester) (POE) is a bioerodible and biocompatible viscous polymer potentially useful as a sustained drug delivery system that allows the frequency of intraocular injections to be reduced. The purpose of this study was to determine the efficacy of POE containing a precise amount of 5-fluorouracil (5-FU) in an experimental model of filtering surgery in the rabbit. METHODS: Trabeculectomy was performed in pigmented rabbit eyes. An ointmentlike formulation of POE containing 1% wt/wt 5-FU was injected subconjunctivally at the site of surgery, during the procedure. Intraocular pressure (IOP), bleb persistence, and ocular inflammatory reaction were monitored until postoperative day 30. Quantitative analysis of 5-FU was performed in the anterior chamber. Histologic analysis was used to assess the appearance of the filtering fistula and the polymer's biocompatibility. RESULTS: The decrease in IOP from baseline and the persistence of the filtering bleb were significantly more marked in the 5-FU-treated eyes during postoperative days 9 through 28. Corneal toxicity triggered by 5-FU was significantly lower in the group that received 5-FU in POE compared with a 5-FU tamponade. Histopathologic evaluation showed that POE was well tolerated, and no fibrosis occurred in eyes treated with POE containing 5-FU. CONCLUSIONS: In this rabbit model of trabeculectomy, the formulation based on POE and containing a precise amount of 5-FU reduced IOP and prolonged bleb persistence in a way similar to the conventional method of a 5-FU tamponade, while significantly reducing 5-FU toxicity.

Bioerodible polymers for ocular drug delivery.

Development of ophthalmic drug-delivery systems has always been challenging. The commonly used route for drug delivery to the anterior segment of the eye has been the conjunctival cul-de-sac. Because of drawbacks associated with this route, new approaches have been investigated for delivery of drugs to the eye by means of polymeric delivery systems. Development of controlled drug-release devices has been a major step forward in this respect. Bioerodible polymers have been at the forefront of such systems. They are very important because they eliminate the need for removing the implant after complete drug release. Bioerodible polymers have been divided into three classes based on their mechanism of hydrolysis: Type I--hydrolysis of crosslinked hydrogels; Type II--solubilization by ionization or hydrolysis of linear polymers; and Type III--biodegradation by backbone cleavage. Polymers from all three classes are discussed in detail in this review.