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Intravitreal and Subretinal Administration in Minipigs Mark Vézina, Scientific Director, Ocular and Neuroscience Introduction • Intravitreal injection is now a common out-patient procedure and is the current standard route of administration for therapeutics targeting the internal posterior ocular structures. • Subretinal dosing is relatively more recent and is usually reserved for targeted delivery of gene or cell therapy products. With increased interest in these therapies the clinical relevance is becoming more important. Why the Minipig? • Largest eye of the common non-clinical species while maintaining reasonable body size. • Approximately 3/4 scale compared to human*. – Rabbits and NHP are approximately 1/2 scale. • Allows for more clinically relevant doses • Allows for more clinically relevant ocular distribution • Allows for use of devices that are too large for smaller eyes (surgical or drug delivery) • Allows for evaluation of surgical methods considered for clinical use • * Göttingen minipig or Yucatan Micropig Minipig Eye Size Comparison NHP Rabbit Dog Minipig Rat Minipig Ocular Anatomy • Retinal vascular architecture and innervation have similarities to humans • No tapetum lucidum • Lacks macula/fovea – Has visual streak Intravitreal Injection • Bypass blood-retinal barrier • Direct contact with target tissue • Most of the time • Low dose levels • Possible pharmacological activity at subtoxic dose levels • Local depot delivery • Most products remain concentrated near injection site and diffuses into vitreous in a gradient Intravitreal Injection • Dose Volume – Typical IVT dose in humans is 50 μL – Minipig eyes can handle up to 150-200 μL – Transient IOP increase even at 50 μL in human • Dosing Apparatus – 30G needle connected directly to 1 mL syringe • Target Dosing Location within Vitreous – Study dependent – Mostly mid vitreous Intravitreal Injection • General Vitreous Humor Characteristics – Gel-like – Mostly water with collagen, hyaluronic acid, proteins, sugars, salts and some cells – Collagen fibril ultrastructure – Non-replenishing. If removed, replaced by AH – Convective and/or saccadic flow pattern – Becomes more aqueous with age Intravitreal Injection • Factors that affect distribution of material in the vitreous humor: – Size of molecule – Charge of molecule – Vitreous macrostructure • Liquid vs gel component • Channels within vitreous – Number of previous injections – Injection location • Temperature gradients • Consistency may not be uniform • Flow patterns Intravitreal Injection • The larger eye of the minipig may be more representative of distribution of therapeutics in human vitreous. Intravitreal Dosing With/Without Vitrectomy Vitrectomy No Vitrectomy Intravitreal Injection • Incidence of In-Vivo background changes • Minimal to slight severity • Saline or PBS injection • N = 200 injections Day post Dose 2 3 4 7 14 Aqueous flare 2% 3% 2% 0% 0% Conjunctival hyperemia 30% 80% 60% 0% 0% Chemosis 15% 30% 10% 0% 0% Focal vitreous hemorrhage 5% 10% 5% 5% 1% Intravitreal Injection • Post mortem microscopic background changes – minimal to mild mixed cell infiltration in the conjunctiva and/or sclera (the injection site) Intravitreal Injection • Immunological Response in the Minipig Eye – Is the minipig eye more or less responsive to a biologic compared to NHP or rabbit? • Definite maybe • The reasons for inflammatory or immune response are multifactorial and not a general species or strain issue. » » » » » What’s being administered How much How frequently Homology Activity Subretinal Injection Subretinal = between photoreceptors and RPE. In-vivo OCT image of subretinal injection Subretinal Injection • Why Subretinal? – Local delivery to a specific target area • Particularly useful for cell or gene therapy – Bypass barriers such as the inner limiting membrane – Containment of material in one location (theoretically) Subretinal Injection • Methods of subretinal injection include: • Transvitreal injection with or without vitrectomy • Catheterization methods without vitrectomy • Both methods have a surgeon and dosing assistant and are similar to the clinical methods • Dose Volume: – Up to 250 µL. More is possible – Human average is 150-200 µL. One center has done up to 450 and another 1000 µL. Subretinal Injection • Transvitreal injections performed with • 41G teflon tip cut to facilitate injection for aqueous formulations • 30G bent stainless needle for viscous formulations or cell suspensions Subretinal Injection • The Role of Vitrectomy – Originally conducted to mimic the exact clinical procedure. – Also thought that vitrectomy would facilitate bleb formation and allow larger volumes to be injected. – Additional studies demonstrated this was not the case. – Unless specifically necessary to replicate a clinical situation, there is no advantage of vitrectomy in most preclinical studies. Transvitreal Subretinal Injection • Creation of the Subretinal Bleb: – The retina is not an elastic tissue. – Retinotomy remains open » Possibility for reflux – Retina overlying the bleb is stretched » Structural changes – Bleb area determined by how well retina is attached to RPE. – High individual variation in bleb area/bleb size with same dose volume. – Potential for sub-RPE dose – Rare, but only micron distance with a handheld needle Transvitreal Subretinal Injection • Creation of the Subretinal Bleb (cont): – They type of injectate determines how the bleb is created • The bleb can be created directly with aqueous solutions or suspensions. • Viscous formulations require more pressure to generate the force to elevate the retina. • Cell suspensions are more delicate and care must be taken not to generate enough force to shear the cells. • The solution: A small preliminary bleb created with an aqueous vehicle (eg. BSS) can be used in both of the latter cases to loosen the retina and create a space to inject the product. Transvitreal Subretinal Injection • Consequences of Reflux through the Retinotomy – Lower subretinal dose level. – Potentially immunogenic material more widely dispersed in the eye • Cell Therapy. – Active cells differentiating in the vitreous. – Membrane formation, traction/retinal detachment, tumor formation, etc. • Gene Therapy – Unintended transfection of off-target ocular tissues. – Ciliary body, iris etc. Transvitreal Subretinal Injection Instruments are passed through the eye from the front to administer the dose from outside of the retina The 41G needle is used to make a prebleb and then the 30 G needle is inserted to inject the dose formulation. Transvitreal Subretinal Injection • The direction of bleb formation from the retinotomy site is variable. • The fluid resorbs and the bleb reattaches in 24-48 hours. • At that time there is no way to know how much of the injectate is actually absorbed and how much has leaked out of the retinotomy. Transvitreal Subretinal Injection • Background In-Vivo Changes – Ophthalmology Examinations: – Transient inflammation • Resolves in 3-15 days – Occasional vitreal or subretinal hemorrhage. • Resolves within several weeks – Retina/choroid pigment changes in bleb area • Sometimes resolving by 3 months – Fibrosis at retinotomy site – Retinal folds or incomplete reattachment • More common with viscous formulation or cell admin. Transvitreal Subretinal Injection • Background In-Vivo Changes - cSLO/OCT – Retinotomy closure by Day 7. – Retinal folds primarily in outer layers. • May resolve over several months or remain for duration of study – Retina/choroid pigment changes in bleb area • Usually visible in infrared or blue reflectance imaging modes even when no longer visible during OE Transvitreal Subretinal Injection • Background Post Mortem Changes – No proliferative changes detected microscopically as determined by longitudinal studies over 1 year. Changes limited to bleb area. • Slight atrophy in photoreceptor layer – Related to the stretching/folding observed with OCT in this layer? • Retinal folds/rosettes • State of retinal reattachment • Presence of material in the bleb site Transvitreal Subretinal Injection • Background Post Mortem Changes – These changes correlated well with the OCT observations – Microscopic findings beyond resolution of OCT • Slight loss of cellularity in ONL and photoreceptor layer • Inflammatory cell infiltration into retinal and /or choroid (unless clumped) • Minimal focal hypertrophy of the choroid (injection site) • Minimal focal fibrosis of the sclera (entry site of dosing needle and other ports) Transvitreal Subretinal Injection • Repeat Injections – Additional injections into a different geographic area or the same geographic area as the original injection do not appear to exacerbate the procedural-related effects of the initial injection. Subretinal Injection via Catheter • Objective to deliver the subretinal dose without retinotomy – iScience iTrack-275TM – 162 procedures in minipigs, single or repeat dosing – Peripheral bleb created to allow catheter access to the subretinal space – Catheter advanced to desired injection location. – Small leading air bubble used to elevate retina prior to injection of formulation – No vitrectomy Subretinal Injection via Catheter Subretinal Injection via Catheter • Overall less traumatic than transvitreal approach • However, visualization at time of catheter entry is challenging • 17% incidence of retinal perforations or stitching during positioning • Includes subretinal entry point and during advancement Subretinal Injection via Catheter • 100% successful delivery of subretinal dose • Occasional flow of injectate along catheter tract • OE and histopathological findings similar to transvitreal approach • Catheter tract remains detectable in-vivo and histologically • Slight to moderate RPE hypertrophy, fibrosis, variable photoreceptor loss • Repeat dosing does not exacerbate in-vivo or post mortem findings • Except that there is an additional catheter tract/dose Summary • The minipig eye provides a size and anatomical relevance for the conduct of intravitreal or subretinal preclinical safety studies in support of clinical trials. • The minipig eye is also suitable for the development and refinement of new methods of intraocular dosing. Thank You