Effects of a selective glucocorticoid receptor agonist on experimental keratoplasty.

BACKGROUND: New insights into the molecular mechanisms of corticosteroid-mediated actions have revealed new substances, such as selective glucocorticoid receptor agonists (SEGRA), for the treatment of inflammatory diseases. We set out to evaluate the effect of a SEGRA compound following topical application on the course of experimental orthotopic corneal grafts. METHODS: A total of 42 female Lewis rats received 3.5-mm MHC I/II-incompatible corneal grafts from DA donors. Recipients were randomly assigned to receive either no therapy, 0.25% cyclodextrin-encapsulated SEGRA compound in a new microemulsion formulation or carrier system only. All treatments started on the day of surgery and were given five times daily for 35 days. Grafts were graded every day and a rejection score was generated based on cornea clarity and edema. In addition, intragraft mRNA expression of CD3, IFN-gamma, TNF-alpha, IL-10 and IL-4 was analyzed using real-time RT-PCR analysis at day 7 after transplantation before rejection occurred in additional control animals. RESULTS: Topical application of a SEGRA compound was highly effective in prolonging the mean survival time of corneal grafts (42.2+/-4.0 days) compared with untreated controls (11.7+/-1.2 days, p=0.00003) or animals that received the vehicle only (15.0+/-1.5 days, p=0.114). In addition, real-time RT-PCR analysis of SEGRA-treated grafts revealed lower mRNA expression of intragraft cytokines; the difference was significant for IL-4 (p<0.05). CONCLUSIONS: Our results indicate that topical application of a SEGRA compound significantly prolongs corneal graft survival in an experimental keratoplasty model. It further suggests that SEGRA can be a potentially useful drug to suppress the immune response.

In vitro permeation studies comparing bovine nasal mucosa, porcine cornea and artificial membrane: androstenedione in microemulsions and their components.

The components of a carrier formulation can interact with an added drug as well as with the membrane surface they were applied on. Therefore, they can influence permeability of the membrane and permeation of the drug. The particular membrane structure might lead to different drug permeation out of one and the same carrier formulation. In this study, in vitro permeability of androstenedione (AD) as a highly lipophilic substance was investigated in excised bovine nasal mucosa, porcine cornea and the artificial cellulose membrane Nephrophan. Two microemulsions (ME) with either the addition of the co-surfactants hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD; ME-CD) or propylene glycol (PG, ME-PG) were tested in order to be used as carrier systems. Both MEs also consisted of 5% isopropyl myristate (IPM), 20% Cremophor EL (CrEL), and water. Buffer solution (EBS) with 0.0025% AD served as control solution and was furthermore compared to 0.0025% AD/buffer-solutions containing the ME components HP-gamma-CD in different concentrations (0.012, 0.024, 9%) as well as 20% CrEL. The AD-permeation behaviour through the three tissues was differently influenced by the MEs. The apparent permeability coefficients (Papp) of nasal mucosa for both ME systems did not differ from the Papp of the AD/buffer solution. In case of the other two barriers (cornea, Nephrophan, ME-PG as well as ME-CD provoked extended time lags for AD to permeate, so the Papp could not be calculated or declined to zero. Papp of AD/buffer solution without any additives resulted for cornea, nasal mucosa and Nephrophan in a ratio of 1:3:4. CrEL and 9% HP-gamma-CD diminished the Papp, except HP-gamma-CD in molar AD/HP-gamma-CD-ratios of 1:1 (0.012%) and 1:2 (0.024%). It seems that the composition of lipophilic and hydrophilic structures of the carrier systems or the additives had a higher impact on the Papp of cornea than on the Papp of the other tissues. Structure and character of the different membranes are considered to be mainly responsible for the differentiated permeation behaviour.

Intraocular availability of topically applied mycophenolate mofetil in rabbits.

The efficacy of mycophenolate mofetil (MMF), a prodrug of mycophenolic acid (MPA), in high risk keratoplasties and ocular immune-mediated diseases has been shown in recent years. As peroral administration of MMF can cause various side effects, topical application should be considered. This study investigates the intraocular availability of MMF and MPA in the rabbit eye. An eye drop solution (MMF-CD; 1% MMF/10% hydroxypropyl-beta-cyclodextrin (HP-beta-CD)) or a 1% aqueous suspension (MMF-SP) was instilled into the lower cul-de-sac of the right eye of each rabbit. Rabbits (each group: n = 4) were put down after 30, 60 and 240 min. Aqueous humor, vitreous, cornea, sclera, conjunctiva, iris-ciliary-body, and plasma were isolated. Several extraction procedures were performed in order to quantify the drug by HPLC. The aqueous humor concentration of the active metabolite MPA was 24 microg/mL after 30 min for both preparations. The ratio of the MPA concentrations after 30, 60, and 240 min was 1 : 2 : 0.07 for MMF-CD and 1 : 0.6 : 0.04 for MMF-SP, respectively. MPA levels in the cornea were 90.78 / 56.90 / 4.08 x 10(-6) microg/microg for MMF-CD, whereas MMF-SP resulted in MPA levels of 102.65 / 31.18 / 2.59 x 10(-6) microg/microg at the three time points. As a high concentration of the active drug MPA in cornea and aqueous humor is desired, e.g. following corneal transplantation, the MMF/HP-beta-CD formulation could be an useful topical treatment. Furthermore, the present study shows that MMF-CD is superior to MMF-SP by increasing intraocular availability.

Pilocarpine permeability across ocular tissues and cell cultures: influence of formulation parameters.

In vitro permeation studies of drugs across biological barriers are promising tools for estimating the quality and quantity of drug transport in vivo. The objective of this work was to compare the permeability of the hydrophilic model drug pilocarpine-HCl (P-HCl) through different ocular tissues and cell cultures: isolated pig cornea (PCr) and sclera (PSc), rabbit conjunctiva (RCo), and rabbit conjunctival (RCoEC) or corneal epithelial cell culture (RCrEC). Furthermore, the study included investigations about the influence of the excipients benzalkonium chloride (BAC) and ethylene diamine tetra acetic acid disodium salt (EDTA) on the permeability of the small drug. In general, BAC caused a facilitated drug transport, while EDTA hardly influenced the P-HCl concentration on the acceptor side, except for RCoEC. Additionally, the impact of variation in buffer solution pH and tonicity on drug transport in both cell cultures was tested. The higher the tonicity of the buffer solution (80, 300, and 600 mOsm/kg) the lower the permeability coefficient (P(eff)). At different pH values (6.4, 7.4, and 8.4) the P(eff) showed a directly proportional demeanor. In summary, a good correlation between the isolated tissues and cell cultures with regard to P-HCl transport could be observed.

Ocular drug permeation following experimental excimer laser treatment on the isolated pig eye.

Excimer laser photorefractive keratectomy (PRK) is a well-established procedure which is frequently applied to correct myopia. Since structural alterations of the corneal epithelium occur after the treatment, a different drug permeation can be assumed. To investigate the effects of PRK on drug permeation, excimer laser ablations with varying depths were performed on isolated pig eyes. The permeation of lipophilic (diclofenac-sodium; D-Na) and hydrophilic (pilocarpine-hydrochloride; P-HCl model drugs were studied in vitro. Under these experimental conditions, P-HCl demonstrated a significant (p < 0.05) enhancement of permeation in relation to the ablation depth. In contrast, corneal epithelial thickness scarcely influenced the permeation rate of D-Na. Not until removing the entire epithelium did a significantly increased permeability occur, when compared to untreated cornea. These results suggest that PRK may significantly reduce the corneal barrier function and alter pharmacokinetics of topical medication.