OCULAR DELIVERY OF PEPTIDES AND PROTEINS RICHARD ADDO, R.Ph., Ph.D ASSOCIATE PROFESSOR UNION UNIVERSITY SCHOOL OF PHARMACY JACKSON TENNESSEE USA OUTLINE Structure of eye and different pathways of ocular administration Challenges for ocular delivery of proteins/peptides Formulation considerations Peptide transport systems in the eye Ocular administration for topical delivery of proteins/peptides Ocular administration for systemic delivery of proteins/peptides Strategies for ocular delivery of proteins/peptides
STRUCTURE OF THE EYE Outermost coat: Clear, transparent cornea and white, opaque sclera Middle layer: iris anteriorly, choroid posteriorly and intermediate ciliary body Inner layer: retina A. Topical administration with trans-corneal permeation B. Topical administration with non-corneal permeation across the conjunctiva and sclera C. Drug distribution from the blood through the blood- aqueous barrier into the anterior chamber
D. Drug distribution from the blood-retina barrier into the posterior chamber E. Intra-vitreal drug administration route F. Sub-tenon injection BARRIERS TO ABSORPTION Basal layer, 2-3 layers of wing cells and 1-2 outermost layers of squamous cells Outermost layers Intercellular tight junctions surround the most superficial layers and restrict passage of peptides and proteins Absorption relies on transcellular passage or strategies that can modulate the tight junctions
Wing cells and basal cells Intercellular spaces are wider and permit paracellular diffusion Negatively charged corneal epithelium offers greater resistance to negatively charged compounds as compared to positively charged ones CHALLENGES TO OCULAR DELIVERY OF PROTEINS/PEPTIDES Barriers for locally delivered drugs Loss of drug from ocular surface Lacrimal-fluid barrier Blood-ocular barrier
Low drug contact time Tear production and turnover Consequent dilution Schematic presentation of the different barriers for ocular delivery of proteins and peptides FORMULATION CONSIDERATIONS Aggregation Is induced by shaking, prolonged storage, heating, freezing, lyophilization Can lead to Reduced bioactivity
Immunogenic reactions Blockage of tubing, membranes or pumps in an infusion set Unacceptable physical appearance such as opalescence Example: Insulin can undergo self-association/aggregation due to the hydrophobic regions of the molecule
Human epidermal growth factor (hEGF) undergoes pH and concentration dependent aggregation Can be prevented by Use of appropriate formulation excipients; example: mannitol, trehalose Proper care in processing of formulation Synthesizing a resistant derivative FORMULATION CONSIDERATIONS
Formulation Additives Protease Inhibitors: Used if the protein/peptide is likely to degrade upon ocular administration Aminopeptidase inhibitors: bestatin, amastatin, puromycin, p-chloromercuribenzoate Sugars: Exert a protective effect on proteins by changing the solvent structure around the protein Cyclodextrins: Act by molecular encapsulation of amino acid chains thereby preventing hydrophobic interactions PEPTIDE TRANSPORT SYSTEMS IN EYE Epithelial cells express nutrient transporters and receptors on their surface which help the movement of vitamins and amino acids across cell
membranes Proton coupled receptors help translocation of di- and tripeptides across the epithelium Transporters are classified as PepT1, PepT2 and peptide/histidine transporters (PHT1 and PHT2) Expression of PHT1 in bovine and human retinal pigment epithelial cells (BRPE and HRPE), ARPE19 cells (human RPE cell type), bovine and human neural retina cells has been reported PepT2 and PHT 2 expression reported in bovine and human retina Drugs with poor ocular bioavailability can be suitably modified by design to facilitate
recognition and uptake by peptide transporters MODES OF OCULAR DRUG ADMINISTRATION OCULAR ADMINISTRATION FOR TOPICAL DELIVERY Topical delivery is considered to be the best option for treatment of most ocular disorders Several peptides have been identified for treatment of ocular disorders like dry eye disease, age related macular degeneration, proliferative diabetic retinopathy, etc. Loss to systemic circulation must be minimized Phenylephrine used as a vasoconstrictor to minimize systemic absorption Use of mucoadhesive polymer to improve ocular absorption
Adverse physicochemical properties or enzymatic degradation of peptides might render them less effective Loading them in a carrier system like liposome or nanoparticle may limit some of these problems OCULAR ADMINISTRATION FOR TOPICAL DELIVERY Growth Factors Human Epidermal Growth Factor (hEGF) stimulates cell proliferation in the corneal epithelium thus causing epithelialization during wound healing
EGF can be produced biotechnologically in a commercially feasible manner It can thus be a suitable therapeutic agent for corneal trauma and during intraocular surgery Tissue Plasminogen Activator tPA can be used to achieve clot lysis after surgery for cataract and/or glaucoma Since tPA is present in aqueous humor and other ocular tissues, its use is like a supplementation of body
function Cyclosporin A It has immunosuppressive, anti-fungal and anti-inflammatory activity Primary use is inhibition of kidney graft rejection Instillation in eye can inhibit rejection of corneal grafts STUDIES SHOWING OCULAR DELIVERY OF EGF EGF Incorporated in Cationized Gelatin Hydrogel
EGF Incorporated in Beta Cyclodextrin Complex Controlled-release of epidermal growth factor from cationized gelatin hydrogel enhances corneal epithelial wound healing, Hori K, Sotozono C, Hamuro J, Yamasaki K, Kimura Y, Ozeki M, Tabata Y, Kinoshita S, J Control Release. 2007 Apr 2;118(2):169-76. rhEGF/HP-beta-CD complex in poloxamer gel for ophthalmic delivery, Kim EY, Gao ZG, Park JS, Li H, Han K, Int J Pharm. 2002 Feb 21;233(1-2):159-67. OCULAR DELIVERY OF GANCICLOVIR In vitro transcorneal permeation Concentration in aqueous humor after instillation in rabbit eye
Preparation and ocular pharmacokinetics of ganciclovir liposomes, Yan Shen, Jiasheng Tu, AAPS J. OCULAR ADMINISTRATION FOR SYSTEMIC DELIVERY Occurs because of contact of instilled solution with conjunctival and nasal mucosae Advantages: Relative ease and low cost of formulating and administering eye drops (compared to injections) Relative insensitivity of eye towards immunological reactions (compared to lung and gut)
Absence of first pass metabolism Challenges: Reproducible delivery Low bioavailability OCULAR ADMINISTRATION FOR SYSTEMIC DELIVERY Insulin: When administered to the eye, a sustained lowering of blood glucose was observed Use of absorption enhancers may often be
required to enhance absorption of peptides through the eye Absorption enhancers must be safe and non- irritating to the eye Order of efficacy: Saponin>Fusidic Acid>BL-9 = EDTA>Glycocholate>Decamethonium=Tween 20 Aminopeptidase inhibitors or peptide analogs that are resistant to enzymes also help to improve bioavailability Systemic absorption of insulin ( SEM; n=5) following the ocular instillation of a 0.25% insulin solution containing Brij-78 as an enhancer. Data generated
following a b.i.d. administration of eyedrops over a three-month period ( - blood insulin concentration; o EFFECT OF ABSORPTION ENHANCER (BRIJ 78) ON SYSTEMIC DELIVERY OF INSULIN FROM AN OCULAR INSERT DEVICE Effect of Brij-78 on systemic delivery of insulin from an ocular device, Lee YC, Simamora P, Yalkowsky SH, J Pharm Sci. 1997 Apr;86(4):430-3. OCULAR ADMINISTRATION FOR SYSTEMIC DELIVERY Glucagon Used in treatment of hypoglycemia Can be delivered by the ocular route and has been reported to increase blood glucose Mol wt. is lower than insulin; may not need absorption enhancers
Calcitonin Long term administration required for treatment of hypercalcemia Besides the ocular route, other alternative routes like nasal, rectal, transdermal have also been explored TRANS-SCLERAL DELIVERY OF IgG TO THE RETINA ery of Bioactive Protein to the Choroid and Retina, Ambati J, Gragoudas ES, Miller JW, You TT, Miyamoto K, Delori FC, Adamis AP, Invest Ophthalmol Vis Sci. 2000 STRATEGIES FOR THE OCULAR DELIVERY OF PROTEINS/PEPTIDES Prodrugs Change physicochemical properties of a drug to improve permeation across cornea and enhance bioavailability First prodrug for ocular delivery: Dipivefrin, prodrug of
epinephrine used to treat glaucoma Desirable properties Good stability High enzyme lability Most common barriers that can be overcome are A low aqueous solubility, which prevents the development of aqueous eyedrops A low lipid solubility, which results in low corneal permeation and low ophthalmic bioavailability A short duration of action due to rapid drug elimination from site of action Systemic side-effects, due to low corneal and high systemic
absorption STRATEGIES FOR THE OCULAR DELIVERY OF PROTEINS/PEPTIDES Mucoadhesive Particulate Carriers Cornea and conjunctiva have a net negative charge Cationic polymers help to increase the concentration and residence time of polymer-associated drug
Chitosan biocompatible, biodegradable, enhances the paracellular transport of drugs Conventional eye drops Anionic or polyanionic Cationic or polycationic Zeta potential (mV)
Effect of Chitosan on Zeta Potential of Microparticles 40 30 20 10 0 -10 -20 -30 -40 -50 washout BSA
BSA+CSN Electrostatic Repulsion Electrostatic Attraction DELIVERY MECHANISM OF CATIONIC NANOPARTICLES Electrostatic interaction leading to Retention at the surface Reservoir effect in : Cornea Conjunctiva Transcorneal Route Diffusion via the scleral route
Sustained release to the retina CHITOSAN NANOPARTICLES FOR CYCLOSPORIN A DELIVERY Chitosan nanoparticles: a new vehicle for the improvement of the delivery of drugs to the ocular surface. Application to cyclosporin A, De Campos AM, Snchez A, Alonso MJ. Int J Pharm. 2001 Aug 14;224(1-2):159-68 STRATEGIES FOR THE OCULAR DELIVERY OF PROTEINS/PEPTIDES Hydrogel Delivery Systems Allows slow release of drug from a hydrogel inserted beneath the eyelid Ocusert: First such device o Non-erodible ocular insert o Pilocarpine alginate core sandwiched between two
transparent, rate controlling membranes PLGA MICROSPHERES FOR DELIVERY OF VANCOMYCIN PLGA microspheres for the ocular delivery of a peptide drug, vancomycin using emulsification/spray-drying as the preparation method: in vitro/in vivo studies, Gavini E, Chetoni P, Cossu M, Alvarez MG, Saettone MF, Giunchedi P, Eur J Pharm Biopharm. 2004 Mar;57(2):207-12 STRATEGIES FOR THE OCULAR DELIVERY OF PROTEINS/PEPTIDES Absorption Enhancers Promote penetration of drugs through corneal barrier by changing integrity of epithelial cell layer Examples: EDTA, sodium glycocholate and related cholates, tween-20, saponin Miscellaneous Approaches Cell penetrating peptides: TAT (Trans-activating transcription factor from
human immunodeficiency virus) exhibit efficient penetration to the retina after topical delivery Intravitreal injections o Can cause several complications like hemorrhage and retinal displacement o Bevacizumab (Avastin): Used for the treatment of ocular vascularization Adapted from Peptide and Protein Delivery by Chris Van Der Walle
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