Biodegradable Calcium Phosphate Nanoparticles As A New Vehicle For Formulation Of Methazolamide Eye Drop Solutions

BIODEGRADABLE CALCIUM PHOSPHATE NANOPARTICLES AS A NEW VEHICLE FOR FORMULATION OF METHAZOLAMIDE EYE DROP SOLUTIONSINTRODUCTIONGlaucoma, the leading cause of irreversible blindness in the world, is estimated to be encountered by 67 million people. It is the term used for a group of ophthalmic disorders characterized by an increase in intraocular pressure (IOP), which results in a damage to the optic disc and visual field disturbances. IOP increases through an imbalance between the production and drainage of aqueous humor. Agents used to treat glaucoma are designed to decrease IOR Carbonic anhydrase inhibitors (CAI) were first used to lower IOP in glaucoma in 1954, with the introduction of acetazolamide, methazolamide and diclofenamide were subsequently. Although IOP can be controlled by oral administration of CAIs, systemic side effects severely limit this mode of therapy. Topical formulations of CAIs were initially unsuccessful due to the poor ocular bioavailability of these drug agents. Under the extensive work on structural modification of CAIs resulted in the development of two topically CAIs, dorzolamide and brinzolamide. But its usage had been restricted by many factors: these agents although administered topically are absorbed systemically, ocular burning and stinging was reported, and the price was highly expensive. Thus, the need of the hour is to develop a topically effective CAI devoid of these side effects. A nanoparticlulate, sustained-release carrier system of calcium phosphate (CAP), incorporated with significant amount of drug, has been studied as a drug delivery system. Reports demonstrated that CAP, administered by oral gavage, or intramuscular and subcutaneous routes, was well tolerated and absorbed with relative absence of side effects. This system has also been implemented in the topical application of a potential ocular hypotensive agent 7-OH-DPAT and can enhance the ocular hypotension. In this study, we introduced a new formulation, which combined biodegradable calcium phosphate nanoparticles with methazolamide, and the effect and ocular toxicity of the new formulation were investigated and evaluated. METHODSPreparation and Characterization of Calcium Phosphate Nanoparticles: Biodegradable calcium phosphate nanoparticles (CAP) were made of calcium chloride, dibasic sodium phosphate and sodium citrate. Nanoparticles were prepared by precipitation method at different temperatures, buffers or mixing orders. The structure, particle size and morphology of resulting nanoparticles were characterized by scanning electron microscope(SEM), particle meter and X-ray photoelectron spectrometer(XPS).Formulation of CAP-methazolamide eye drop solutions: methazolamide dissolved in 50ml ethanol, then added the CAP solution(40mg/ml), 4℃, rotating for 2h and 8500rpm centrifugation 3 times, 15min for each time, abandoned the supernatant. The sendimentation was diluted with distilled water. The drug concentration and pH values were determined by HPLC analysis and pH meter.Intraocular Pressure Measurements: Healthy NZW and pigment rabbits without any ocular defects were selected. IOP (mmHg) was measured before and after drug administration 1,2,3,4,6,8,12h using an impression tonometer. Each experiment was designed into CAP-methazolamide eye drop solutions, 10mg/ml brinzolamide, blank nanoparticle suspention(5mg/ml) and control. At the appropriate time point IOP was measured. HPLC analysis: The aqueous humor samples were carried out at 15,30,60,90,120,180 min after 100μlCAP-methazolamide eye drop solutions administered in NZW rabbits using a 1.0ml syringe fitted with a 29-gauge needle. The samples were immediately frozen and stored at -20℃. For analysis, after centrifugation 20μl samples of the supernatant were submitted to HPLC analysis.Irritation and Toxicology study: Healthy animals were selected to this experiment. It is designed to ocular irritation, acute toxicology and allergic experiment according to SFDA.RESULTSThe size of particles ranged from 100 to 1000 nm with majority of the particles in 500 nm and 300nm, and the average diameter was 400nm. Scanning electromicrograph showed the gross surface and oval shape. The drug concentration of CAP-methazolamide eye drop solutions was 400～500μg/ml, pH was 6.8～7.5.In normotensive NZW rabbits, the maximum lowering after CAP-methazolamide eye drop solutions administration was about 6.14±0.96mmHg and the duration of the activity was about 8h, otherwise the maximum lowering after brinzolamide administration was about 2.73±1.03mmHg and the duration of the activity was about 6h.In normotensive pigmented rabbits, the maximum lowering after CAP-methazolamide eye drop solutions administration was about 4.20±1.38mmHg and the duration of the activity was about 6h, otherwise the maximum lowering after brinzolamide administration was about 2.30±1.24mmHg and the duration of the activity was about 4h.HPLC analysis results showed the drug concentration in aqueous humor after drug administration 15,30,60,90,120,180minare 0.3,0.06,0.064,0.061,0.063,0.025μg/ml. The drug peak in the aqueous humor appeared in 15 minutes which was 0.3μg/ml and the drug sustained for nearly 120minutes in the humor aqueous.Clinical and toxicity evaluation: There is no irritation and inflammation on the experimented eyes compared with the control by slit lamp. The histology examination showed no severe inflammation responses. There is no toxic effect in the acute toxicology and allergic experiment.CONCLUSION1. In comparative IOP studies with the brinzolamide(10mg/ml), the CAP—methazolamide has a better IOP effect. The ocular hypotension induced by topical administration of CAP-methazolamide eye drop solutions was more pronounced and sustained than the brinzolamide.2. Results of HPLC analysis indicated the CAP-methazolamide eye drop solutions can transfer the corneal barrier and penetrate into the anterior humor. It is worthy to do further study about it.3. The CAP-methazolamide eye drop solutions are safe and no toxicity. It will be applied in clinic glaucoma therapy.