Download Transvitreal Subretinal Injection

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.
»
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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