OPHTHALMIC DRUG FORMULATIONS OF NSAIDs WITH
POLYMERIC NANOPARTICLES HAVING HIGHER BIOAVAILABILITY.
Amarnath Maitra Department of chemistry, University of Delhi, delhi-110007 (1) DETAILS OF THE PRODUCT Medication of the eyes is done for the two purpose: to treat the outside of the year for such infections as conjunctivits , blephritis, keratitis sicca etcand to provide intraocular treatment through the cornea for diseases such as glaucoma or uveitis. Most ocular diseases are treated with topical application of solution administered as eye drop .In India these conventional dosage forms account for more than 99% of the currently accessible marketed formulations, which are mostly high, concentrated aqueous solution. One of the major problems encountered with the topical delivery of ophthalmic drugs is the rapid and extensive precorneal loss caused b drainage and high tear fluid turnover. After instillation of an eye drop, typically less than 2-3% of the applied drugs penetrates the cornea and reaches the intracular tissues, while a major fraction of theinstilled dose is often absorbed systemically via the conjunctiva and nasolacrimal duct. Another limitation is relatively impermeable corneal barrier that limits ocular adsorption. Topically applied drugs reach bloodstream mainly via absorption across the mucosa in the nasal cavity, which is contiguous with the conjunctival sac. Physicochemical properties of the administered drug such as lipphilicity, solubility, size and shape, charge and degree of ionization affect the route and rate of permeation. Because of the inherent problems associated with the conventional eye drops, there is significant effort directed towards new drug delivery system such as hydrogels, micro and Nanoparticles, liposomes, and collagen shields. Ocular drug delivery is an approach to controlling and ultimately optimizing delivery of drug to its target tissue absorption through prolongation of the drug residence time in the cornea and conjunctival sac as well as to slow drug release from the delivery system and minimizes precorneal drugs loss. Keterolac, indomethacin and nimesulide are Non- Steroidal Anti-Inflammatory drugs (NSAIDs), Which have potent analgesic and anti- inflammatory activity due to prostaglandin inhibitory effect of these drugs. Keterolac (free acid) is not solubale in water and therefore, it is available in the market in the form of tromethamine salt (KT) which increase its solubility in water. KT is effective in inhibiting postoperative inflammation of eyes. It is also reducing conjunctivitis with no alteration of corneal opacity. It does not facilitate Herpes Simplex, bacterial or fungal infection of the eye. KT (0.5& w/v) eye drop are available in the market. KT (0.5%) solution applied topically to eyes is non-irritant and does not increase intraocular pressure. But only a small amount of instilled does (1-3%) from such a formulation penetrates the cornea and reaches intraocular tissue. This is due to complex tear fluid dynamics such as tear turn over, lachrymal drainage and drug dilution by tears. We have encapsulated various NSAIDs in the mucoadhesive, thermosensensitive polymeric Nanoparticles of size less than 50nm diameter and studied the in vitro corneal permeation studies through the goat/rabbit cornea. In vivo ocular inflammation was also studied in the rabbit eyes and the therapeutic efficacy of the drug-loaded Nanoparticles was compared with the various formulations of the drugs available in the market. Various formulations have been designated. Ketorolac loaded Nanoparticles formulation: PMNK Indomethacin loaded Nanoparticles formulation: PMNI Nimesulide loaded Nanoparticles formulation: PMNN OBJECTIVES:
The size and size distribution of the polymeric Nanoparticles were measured by means of dynamic light scattering method. Fig.1 shows the typical size distribution of the Nanoparticles. From DLS measurement the average size was found to be less than 50nm diameter at 250 C with narrow size distribution and unimodal pattern. Since this polymer is a thermosensitive polymer, the size of the Nanoparticles was found to be temperature dependent as shown in Fig.2. A quasisudden increase of size at a particles temperature depends on the composition of the polymer particularly on the amount of thermosensitive polymer component present in the copolymer. Polymeric particles formed have shown a raid increase of size from 35nm diameter at 250 C to more than 300nm diameter at 370 C. At beyond this critical temperature the particles dispersed in aqueous buffer become turbid. Interesting, the particle size of the Nanoparticles (at 250 C) does not change significantly when the particles are loaded with NSAIDs. TEM picture of the stained samples of polymeric Nanoparticles loaded with Ketorolac is shown in Fig.3. The picture shows that the particles are sphricaland nearly monodispersed with an approximate size of around 40nm diameter, which is comparable to the size, obtain from DLS measurements.
The Nanoparticles (lyophilized powder) was dispersed in water and ethanolic solution of NSAIDs like Ketorolac, indomethacin or nimesulide was dissolved. The clear solution were kept at room temperature till tubidity appears due to sufficient amount of drug coming uot of the particles. The duration for different amount of Ketorolac loaded Nanoparticles was noted. The formulation was found to be stable for more than 7 days at room temperature. 
In vitro release Kinetic studies: The in vitro profiles of the loaded ketorolac from drug loaded Nanoparticles at pH 5.0, 7.2, and 10.0 were determined at 250 C and the results obtained are shown Fig.4. It is evident from the figure that the release rate was slow at acidic pH as compared to that in the alkaline pH and the release rate was found to increase exponentially with time in alkaline pH. Ketorolac free acid is insoluble in water, but as the pH of the solution is increased, the free acid is turned into salt and its solubility is increased. As a results, the release of ketorolac from the Nanoparticles becomes faster. Thus in a given time, the release of ketorolac from the Nanoparticles becomes faster in alkaline medium than that in the acidic medium. In vitro release kinetic profiles for the indomethacin and nimesulide were found to follow the same pattern as of ketorolac. The temperature dependent release profile of ketorolac from Nanoparticles at pH 7.2 also shows that the release is highly temperature dependent. The release is slower at 250 C (below LCST) and faster at 370 C (Fig.5) Transcorneal permeation studies: Transcorneal permeation studies through excised rabbit cornea indicated about 2-fold increase in drug permeation from Nanoparticles formulation compared to that of an aqueous suspension of drug of same concentration.   
(2) CLINICAL SIGNIFICANCE OF THE PRODUCT: Our Nanoparticles formulation consists of biodegradable polymer that provide sustained release of drugs into the eyes at therapeutic, non fluctuating levels for long period of time and hence can eliminate systemic and local drug toxicity problems. Also, the Nanoparticles are pH and temperature sensitive, so that the release of the drug in the eye can be controlled. These Nanoparticles are mucoadhesive and hence can adhere at the mucin membrane of the cornea and as a result increase the residence time of the drug at cornea membrane. Moreover, due to the ultralow size of the particles (<50nm diameter), the formulation when applied topically to eyes is non-irritating and does not cause any cornea damage in the eyes. Eye drops currently available in the market are used for post-surgical treatment of inflammatory, administered to the eye five times per day for a period of weeks or months. We have developed a Nanoparticles based drug delivery system that delivers an anti-inflammatory drug in the eye, which can reduce inflammation within days using a once-a-day application, rather than the weeks it takes eye drops to reduce post-surgical inflammation. We have performed the following in vivo test with our product. IN VIVO OCULAR ANTI-INFLAMMATION ACTIVITY: Topical instillation prostaglandin (PG) or arachidonic acid induces ocular inflammation and polymorphonuclear (PMN) leukocytes migration in tear fluid. Hence either PGE2or arachodonic acid induced eye lid closure, redness in eye and PMN in rabbit were used to evaluate anti-inflammatory effect of Nanoparticles formulation of NSAIDs. The results indicated that the lid closure was prominent up to 3 hours after which it subsided. The lid closure score was found to be more an all control eyes as compared to the eyes treated with drug loaded Nanoparticles formulations. Also the lid closure in case of eyes treated aqueous suspension of NSAIDs of same concentration. PMN counts in the tears of rabbits increased up to 3 hours and afterward it decreased.  Figure7: Comparison of effect of Nanoparticles formulation of nimesulide with aqueous suspension drug of same concentration (0.5mg/ml) on PMN leukocyte migration in tears of rabbit eye. In case of nanoparticle formulations, the PMN counts were observed to be less than the control throughout the 6 hours study. The percent inhibition of PMN migration with Nanoparticles formulations was found to be much higher and longer lasting than that observed with aqueous suspension containing equivalent amount NSAIDs. The results suggest enhanced residence time of the drug on the ocular surface as well as sustained release of the drug from Nanoparticles. (3) OCULAR DRUGS WITH WHICH STUDIES HAVE BEEN CONDUCTED: Various Non-steroidal anti-inflammatory drugs were encapsulated in these Nanoparticles. These drugs were:
Hydration levels of the cornea were determined to see whether the nanoparticle drug formulation applied to cornea causes any damage to the membrane or not. It is already known that whenever there is any damage to the topmost epithelium layer, the water uptake of the stroma increase and as a results the hydration level of cornea increases enormously from its normal level of hydration. Table-1 shows the hydration levels of the cornea after treatment with aqueous suspension of NSAIDs as well as their nanoparticle formulation. From table corneal damage has taken place due to the topical application of these formulations. Also, these Nanoparticles do not cause any irritation to eyes.
Cyclodextrin (CD) were introduced in ocular drug formulations initially with the aim of increasing the solubility of drugs in solution. They form inclusion complex with many lipophilic drugs by taking up a drug molecule within the lumen, resulting in an solubility. Researchers have observed toxicity of the drug-cyclodextrins complex compared with the usual drug formulation. Finally, they have shown increased corneal permeability and increased bioavailability despite the assumption that the complexes do not penetrate across the biological membranes. However, cyclodextrin derivation possess their own toxicity and these observations vary depending on the studies. Also, they do not seem afford real improvement for hydrophilic drugs except for eliminating ocular irritation, when the drug is responsible for this damage. The improvement of ocular bioavailability is also limited by the very slow dissolution of the drug-CD complexes in the lachrymal fluid. Nanoparticles are colloidal polymeric ranging in size from 10-1000nm and are exceptionally stable entity. They consist of macromolecular materials in which the drug is entrapped to increase the specificity of the action of the drugs towards a specific target and also to protect a drug against enzyme inactivation. Sustained release of drugs in the eye can be modulated through these Nanoparticles. Nanoparticles have been shown to improve and prolong the corneal penetration of ocular drugs. Furthermore, these Nanoparticles are significantly taken up by the corneal epithelium cells without damaging the cell membrane. This interesting result has rendered these Nanoparticles as promising carriers for the corneal transport of the drug. Moreover, residence time of the drug at the ocular surface can be increased by the use of mucoadhesive particles, which have ability to adhere at the mucin membrane of the cornea. (6) Patent status: A process for Preparing Sustained Release and Long Residing Ophthalmic Formulation: Inventors: Amarnath Maitra, Ajay Kumar Gupta, Dipak Mazumdar and Sumit Madan,
Inventors: Amarnath Maitra, Ajay Kumar Gupta, Dipak Mazumdar and Sumit Madan, Indian WTO Patent application No: 871/del/2000 Dated 26th September, 2000 
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