Sustained Release of an Anti-Glaucoma Drug: Demonstration of Efficacy of a Liposomal Formulation in the Rabbit Eye

Topical medication remains the first line treatment of glaucoma; however, sustained ocular drug delivery via topical administration is difficult to achieve. Most drugs have poor penetration due to the multiple physiological barriers of the eye and are rapidly cleared if applied topically. Currently, daily topical administration for lowering the intra-ocular pressure (IOP), has many limitations, such as poor patient compliance and ocular allergy from repeated drug administration. Poor compliance leads to suboptimal control of IOP and disease progression with eventual blindness. The delivery of drugs in a sustained manner could provide the patient with a more attractive alternative by providing optimal therapeutic dosing, with minimal local toxicity and inconvenience. To investigate this, we incorporated latanoprost into LUVs (large unilamellar vesicles) derived from the liposome of DPPC (di-palmitoyl-phosphatidyl-choline) by the film hydration technique. Relatively high amounts of drug could be incorporated into this vesicle, and the drug resides predominantly in the bilayer. Vesicle stability monitored by size measurement and DSC (differential scanning calorimetry) analysis showed that formulations with a drug/lipid mole ratio of about 10% have good physical stability during storage and release. This formulation demonstrated sustained release of latanoprost in vitro, and then tested for efficacy in 23 rabbits. Subconjunctival injection and topical eye drop administration of the latanoprost/liposomal formulation were compared with conventional daily administration of latanoprost eye drops. The IOP lowering effect with a single subconjunctival injection was shown to be sustained for up to 50 days, and the extent of IOP lowering was comparable to daily eye drop administration. Toxicity and localized inflammation were not observed in any treatment groups. We believe that this is the first demonstration, in vivo, of sustained delivery to the anterior segment of the eye that is safe and efficacious for 50 days

Drug loading values before and after synthesis of liposome vesicles.

<p>Drug loading values before and after synthesis of liposome vesicles.</p

Partition coefficient of latanoprost.

<p>Partition coefficient of latanoprost was calculated by taking the ratio of drug concentrations in lipid bilayer and the aqueous buffer. The drugs to lipid loading concentrations (mole ratios) are 0.04, 0.104, 0.14, 0.18, and 0.32 in DPPC. Each partition coefficient value was obtained from the mean of three MLV formulations made and the standard deviations are reported in error bars.</p

Latanoprost release rate from 100 µl of DPPC LUVs with two different drug/lipid mole ratios compared with 1 drop of commercial Xalatan® solution (1.5 µg/drop).

<p>In vitro drug release rate (µg/day) measured from (a) drug/lipid, 0.04 and (b) drug/lipid, 0.11, (c) commercial eye drop (Xalatan®, 1.5 µg/drop). The release rates are reported based on mean values of at least two batches and standard deviations are reported in error bars.</p

In vitro dialysis study of latanoprost from drug loaded DPPC LUVs after extrusion.

<p>Cumulative latanoprost release (%) from DPPC LUVs loaded with varying amount of drug/lipid mole ratios: (a) 0.044, (b) 0.11, (c) 0.14, (d) 0.18 and (e) 0.37. Each value is the mean (standard deviations is plotted as error bars, which are always less than 3% for nearly all batches) of the results obtained from at least three independent experiments.</p

Rabbit eyes condition after subconjunctival injection.

<p>Serial slit-lamp microscopy (group A) revealed no significant increase in vascularity and inflammation of all rabbit eyes (I and II: Day 0 after subconjunctival injection; III and IV: Day 30 after subconjunctival injection). AS-OCT scans (group B) revealed no abnormal scarring, scleral or conjunctival thinning in all rabbit eyes (Photos I and II). *SC = Liposome injection site; C = Cornea.</p

Comparison of intraocular pressure (IOP) between topical latanoprost eyedrop, subconjunctival latanoprost liposome and latanoprost-free blank liposomes.

<p>Group A: topical latanoprost eye drop. Group B: subconjunctival latanoprost liposome. Group C: latanoprost-free blank liposomes.</p

Histology of rabbit eyes.

<p>Group A: topical latanoprost eyedrops. Group B: subconjunctival latanoprost liposomes. Group C: subconjunctival latanoprost-free liposomes. Histology revealed no abnormal scarring or damage to the collagen layers in both H&E stain (Photos I and II) and Picrosirius red stain (Photos III and IV; Grade 1 for all eyes). S = Sclera; C = Conjunctiva.</p

DSC analysis of pure and latanoprost loaded DPPC MLVs.

<p>The liposome was made by PBS buffer hydration of anhydrous drug-lipid layer. 10 µl of the liposomal formulation (100 mg/ml) was heated at 1°C/min between −20°C to 65°C. Three heating and two cooling cycles were carried out with each sample and the last reproducible heating cycle was considered for analysis. The drug/lipid mole ratios and their corresponding enthalpy change during transition are reported within parentheses (a) 0.0 (18.9 J/g), (b) 0.03 (8.7 J/g), (c) 0.086 (8.1 J/g), (d) 0.114 (5.2 J/g) and (e) 0.14 (1.6 J/g).</p

Size measurement of liposome formulation (varying d/l mole ratio) during storage at 4°C and after in vitro drug release in PBS buffer (pH 7.4) at 37°C.

<p>Size measurement of liposome formulation (varying d/l mole ratio) during storage at 4°C and after in vitro drug release in PBS buffer (pH 7.4) at 37°C.</p