ORIGINAL RESEARCH

Preclinical Safety Evaluation of OphthalmicViscosurgical Devices in Rabbits and a Novel Mini-PigModel

Ronika S. Leang . Lisa J. Kloft . Brad Gray . Arlene E. Gwon .

Ling C. Huang

Received: December 5, 2018 / Published online: February 18, 2019� The Author(s) 2019

ABSTRACT

Introduction: To establish the preclinical safetyand equivalency of ophthalmic viscosurgicaldevices (OVDs) comprised of bacterially sourcedsodium hyaluronate (HA) to animal sourced HAusing pyrogenicity and aqueous exchangemodels in rabbits and a novel mini-pig model toevaluate corneal endothelial cell protectionin vivo.Methods: HEALON OVD and HEALON5 OVDcontaining animal-derived HA and HEALONPRO OVD and HEALON5 PRO OVD containingbacterial-derived HA were used. Two rabbitaqueous exchange studies were conductedwhere aqueous humor was exchanged withOVDs in six animals each to observe potentialocular inflammation, intraocular pressure (IOP)response, corneal health and pachymetry until7 days post procedure, as well as overall assess-ment of the OVDs. Endothelial cell protectionwas evaluated in a Yucatan mini-pig cataract

surgery model where HEALON PRO and HEA-LON5 PRO OVDs were compared to HEALONand HEALON5 OVDs, respectively. Followingcataract surgery with use of OVDs in six animalsper study, animals were evaluated for ocular andgeneral health, IOP, corneal thickness, ocularinflammation, and endothelial cell protectionon days 1, 3, 7 and 14 post-surgery.Results: All rabbit studies demonstrated equiv-alence between bacterial-derived and animal-derived OVDs. Mild, post-surgical irritation, IOPincrease, and corneal thickness measurementswere not significantly different in HEALON PROOVD and HEALON5 PRO OVD compared toHEALON and HEALON5 OVDs, respectively.The mini-pig model developed to investigateendothelial cell protection was successful indemonstrating equivalence between the OVDsstudied. Changes in IOP mirrored actual surgi-cal procedures, while corneal pachymetry andendothelial cell density remained constant forall OVDs used. Slit lamp observations showedexpected inflammation following surgical pro-cedures, likely due to challenges encounteredduring surgical procedures.Conclusion: Rabbit pyrogenicity and aqueousexchanged paired with a novel simulated cat-aract surgery mini-pig model demonstrateequivalence of OVDs regardless of HA source.Albeit with challenges, the mini-pig model wasshown to be a promising tool for endothelialcell evaluation during the development of newOVDs for ophthalmic use.

Enhanced Digital Features To view enhanced digitalfeatures for this article go to https://doi.org/10.6084/m9.figshare.7637171.

R. S. Leang � L. J. Kloft � B. Gray � L. C. Huang (&)Biological Sciences, Johnson & Johnson SurgicalVision, Inc., Santa Ana, CA, USAe-mail: lhuang64@its.jnj.com

A. E. GwonGavin Herbert Eye Institute, University ofCalifornia, Irvine, CA, USA

Ophthalmol Ther (2019) 8:101–114

https://doi.org/10.1007/s40123-019-0167-9

Funding: Johnson & Johnson Surgical Vision,Inc.

Keywords: Cataract surgery; Endothelial cell;HEALON OVD; HEALON PRO OVD; Oph-thalmic viscosurgical device; Sodium hyal-uronate

INTRODUCTION

Since the invention of ophthalmic viscosurgicaldevices (OVDs) and the introduction of HEA-LON OVD (sodium hyaluronate 1%), the safetyand clinical outcome of cataract surgery, themost frequently performed surgical procedure,has vastly improved [1, 2]. Decades ago, cataractsurgery was performed with the aid of an airbubble or balanced salt solution injected intothe anterior chamber, however these materialsprovided poor tissue protection and did little tocreate adequate space maintenance duringintraocular lens (IOL) implantation [3, 4]. Inaddition to maintenance of anterior chamberspace during IOL implantation, another criti-cally important function of an OVD is the pro-tection of the corneal endothelium [4, 5]. Thecorneal endothelium protects the inner layersof the cornea maintaining its hydration andtransparency [6]. During cataract surgery, dam-age from surgical instruments, turbulence fromirrigation fluids, or oxidative tissue damagefrom phacoemulsification can contribute tocorneal endothelial cell loss [7, 8]. In humans,the corneal endothelium does not have a sig-nificant capacity for regeneration, thus wheninjury occurs, corneal endothelium cell func-tion may be affected leading to corneal opacityand loss of vision [6]. OVDs also protect theendothelium by providing anti-free radicaleffects and reducing the temperature rise duringphacoemulsification and aspiration stages ofcataract surgery [9, 10]. Overall, OVDs havesignificantly increased the safety of cataractsurgery, thus allowing tremendous improve-ments in surgical technique with less compli-cations [5].

A key ingredient in OVDs is a viscoelasticmaterial. Several viscoelastic materials arecommercially available, including sodium

hyaluronate (HA), chondroitin sulfate, andhydroxypropyolmethylcellulose (HPMC) [11].HA, the most widely used viscoelastic material,is a naturally-occurring molecule identified inseveral human tissues including the vitreousand aqueous humor of the eye [7]. Historically,OVDs were developed with HA derived fromrooster-combs [1]. Several currently availableOVDs, including HEALON and HEALON5OVDs, contain HA sourced from rooster combs.However, increasing concerns of environmentalimpact, animal welfare, and cost of productionhas led to increased popularity of OVDs andother consumer and medical devices comprisedof HA derived from microbial sources [1, 12].

Johnson & Johnson Surgical Vision, Inc. hasrecently added two new formulations contain-ing bacterial-sourced HA, HEALON PRO OVDand HEALON5 PRO OVD to its OVD portfolio.Prior to clinical testing, new OVD formulationsundergo thorough preclinical evaluation ofbiocompatibility that allow scientists to observeOVD performance during surgery as well asidentify possible immunogenic or inflammatoryresponses [3]. Although the derivation for theHA raw material for HEALON PRO OVD andHEALON5 PRO OVD was changed to a bacterialsource, the final viscosity ranges of the two newOVD formulations closely overlapped those ofthe original formulations, HEALON OVD andHEALON5 OVD. The rheologic properties of theoriginal and new formulations are so similarthat during wet lab evaluations, surgeons couldnot distinguish a performance difference whencomparing the original and new HEALON PROOVD products [13].

Preclinical ocular biocompatibility testinghas traditionally been performed in the rabbitmodel given the similarity of ocular anatomy tohumans, ease of handling and maintenance,and the wealth of experimental data that existson several rabbit species [14]. Additionally,rabbits have been widely used in cornealendothelium studies as the rabbit and humanshare similar corneal endothelium characteris-tics such as diameter, repair mechanisms,thickness, and composition [6]. In developmentof an OVD, a key endpoint to consider is theprotection of endothelial cell layer integrity.Given the ability of rabbits to regenerate the

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endothelium, evaluation of cell loss at the pointof injury and over time would be limited [6, 15].Thus, this paper summarizes the preclinicalevaluation of these two new OVD formulationsin the traditional rabbit aqueous exchangemodel and in a novel Yucatan minipig model.Minipigs are increasingly being used in oculartoxicology studies due to their anatomical sim-ilarities with human eyes and as a substitute fornonhuman primates [16]. Minipigs, likehumans, do not have the ability to regeneratethe corneal endothelium post injury [17]. Dueto this physiological similarity, the minipig waschosen as an additional model specifically toevaluate the corneal endothelium.

METHODS

All institutional and national guidelines for thecare and use of laboratory animals were fol-lowed. These studies were carried out in strictaccordance with the Guide for the Care and Useof Laboratory Animals of the Public HealthService and Johnson & Johnson CorporateHuman Animal Care and Use Policy. Theintraocular implantation studies in rabbits, andthe endothelial protection studies in mini-pigswere performed at Absorption Systems, Inc.(San Diego, CA). The rabbit pyrogenicity testwas performed at Toxikon Corporation (Bed-ford, MA). Both laboratories are AAALACaccredited contract research organizations(CRO). During the studies, the care and use ofanimals was conducted in accordance withregulations of the USDA Animal Welfare Act. Allstudy protocols were reviewed and approved bythe CRO’s Institutional Animal Care and UseCommittee (IACUC) prior to the initiation ofstudy procedures. All studies were conducted inaccordance with US FDA 21 CFR Part 58, GoodLaboratory Practice for Nonclinical LaboratoryStudies. Data analysis was performed usingGraphPad Prism software with unpaired t-tests.The discovery was determined using the Origi-nal FDR method of Benjamini and Hochberg.Each time point was analyzed individually,without assuming a consistent standarddeviation.

Rabbit Pyrogen Test

Four New Zealand White (NZW) rabbits at least10 weeks old and weighing between 2.7 and3.2 kg were used per study. A total of threestudies were conducted to test three differentlots of the investigational OVD. Rabbits wererestrained for the course of the study with nofood or water during the 3-h test period. Rectaltemperature measuring probes were insertedthroughout the testing period and the rabbitswere placed in light-fitting stocks that allowed anatural resting position. Rabbits were weighedprior to the test to determine dose volume. TheOVD was diluted 1:50 with 0.9% SodiumChloride for Injection, which also served as thenegative control. Diluted OVD (three rabbits)and control articles (one rabbit) were warmed toa temperature of 37 ± 2 �C and injected intothe marginal ear vein at a volume of 10 mL/kg.Baseline temperatures were taken within 30 minprior to injection of the test article, then at30-minute intervals between 1 and 3 h follow-ing the injection. Prior to actual pyrogen test-ing, a sham test was conducted within 7 daysprior to pyrogen testing which included allsteps except for injection of test or controlarticles.

Aqueous Exchange

Surgical ProcedureSix male naı̈ve NZW rabbits approximately11–13 weeks of age and weighing 2.7–3.0 kg atstudy initiation were used for each study. Sur-gical procedures were performed by a board-certified ophthalmologist. Rabbits were anes-thetized with an intramuscular injection ofketamine hydrochloride (30 mg/kg) and xyla-zine (5 mg/kg). Both eyes were topically dosedwith one drop of proparacaine hydrochloride(0.5%) and then cleaned with betadine andrinsed with balanced salt solution. A wire lidspeculum was inserted to retract the lids. A 30Gneedle attached to a 1 mL syringe was insertedinto the anterior chamber of each eye, andwithdrawal of approximately 100 lL of aqueoushumor was attempted. The amounts of aqueoushumor withdrawn were recorded by weight,

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averaging 0.06 g, with a standard deviation of0.015. Based on withdrawal amount,0.04–0.1 mL of OVD was then injected into theanterior chamber to replace the aqueous humorthat was withdrawn. The bacterially-derivedOVD was injected into the right eye (OD) andthe control, animal-derived OVD was injectedinto the left eye (OS). Three lots of bacterially-derived OVD were tested (two animals per lot),and one lot of animal-derived OVD was used asthe control article. No incisions were required.The OVD remained in the eye for the durationof the study.

Observations/MeasurementsGeneral health assessment and gross ocularobservations consisted of body weight mea-surements and a visual appraisal of the eye forswelling, discharge, and/or irritation. Prior tostudy placement and during the in-life phase,rabbits underwent an ophthalmic examinationby a board-certified veterinary ophthalmologistusing a slit-lamp biomicroscope (Haag-Streit BP900) and an indirect ophthalmoscope (KeelerVantage Plus). Ocular findings were scored fol-lowing a modified McDonald–Shadduck scoringsystem. Rabbits were acclimated to intraocularpressure (IOP) measurement procedures onceper day for two days prior to study initiation todetermine baseline IOP levels. Prior to taking allIOP measurements, a 0.5% proparacaine solu-tion was applied as a topical anesthetic and IOPmeasurements were performed using a ReichertModel 30 Classic pneumatonometer. Cornealpachymetry measurements were taken pre-treatment with a Sonogage Corneo-Gage Plus 4Spachymeter to determine baseline cornealthickness.

Endothelial Cell Protection Test

Surgical ProcedureSurgical procedures were performed by a board-certified ophthalmologist. Six male naı̈veYucatan mini-pigs 17–18 weeks of age andweighing 19–23 kg at study initiation were usedfor each study. Animals were fasted (food only)approximately 12 h prior to the use of anes-thesia. Animals were anesthetized using a

combination of ketamine (10 mg/kg), xylazine(1 mg/kg), and atropine (0.04 mg/kg) via anintramuscular injection. Upon loss of respon-siveness and spontaneous movement, animalswere intubated and maintained on isoflurane(1–4%) in oxygen (1–3 L/min) as necessary. Asingle drop of topical proparacaine hydrochlo-ride anesthetic (0.5%) was placed on each eyeprior to the procedure. Following anesthesia,the eyes were cleaned with betadine and thenrinsed with balanced salt solution (BSS). A wirelid speculum was inserted to retract the lids. Acorneal incision of 2–3 mm was made approxi-mately at the 12 o’clock position with a ker-atome. The bacterially-derived OVD wasinjected into the right eye (OD) and the control,animal-derived OVD was injected into the lefteye (OS). The OVD was injected into the eye tomaintain anterior chamber depth and a 5–6 mmcontinuous curvilinear capsulorrhexis was per-formed. Hydrodissection was executed to sepa-rate the capsule bag from the lens cortex. A 19or 20-gage phacoemulsification tip was insertedthrough the corneal wound and an endocap-sular lens extraction was performed utilizingphacoemulsification and irrigation/aspirationprocedures with BSS, 5% heparin, and1:1,000,000 epinephrine. Low-vacuum suctionof both the anterior and posterior capsule wasperformed and viscoelastic was injected to dee-pen the anterior chamber and separate theanterior and posterior surfaces of the capsularbag. TECNIS intraocular lenses (IOLs) (Johnson& Johnson Surgical Vision, Inc., Santa Ana, CA)were implanted in all eyes and the viscoelasticwas removed from the capsule bag. At thecompletion of the implantation procedure, thecorneal incision was closed via sutures, and theprocedure was repeated on the contralateraleye. Following surgery, 20 mg/0.25 mL of gen-tamicin and 2 mg/0.1 mL of dexamethasonewas injected subconjunctivally and tripleantibiotic ophthalmic solution was placed inboth eyes. Prednisolone Acetate (1%) oph-thalmic suspension was topically administeredin both eyes of each animal up to 4 times a dayfor 13 days post-implantation.

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Observations/MeasurementsGeneral health assessment and gross ocularobservations consisted of body weight mea-surements and a visual appraisal for swelling,discharge, and/or irritation of the eye. Oph-thalmic examinations by slit-lamp biomi-croscopy (Haag-Streit BP 900) were performedby a board-certified veterinary ophthalmologistfollowing the McDonald–Shadduck scoringsystem. IOP measurements were performedusing a pneumatonometer (Reichert Model 30Classic) on unanesthetized animals placed inslings at approximately the same time of day foreach reading. Prior to all IOP measurements, a0.5% proparacaine solution was applied as atopical anesthetic. Endothelial cell counts (3–5scans per eye at different locations) and pachy-metry measurements for corneal thickness weretaken in parallel on anesthetized animals usinga Konan Specular Microscope Model 7700. Priorto these measurements, 1–2 drops of 0.5%proparacaine topical anesthetic solution wasapplied to the eye.

RESULTS

Rabbit Pyrogenicity Assays DemonstrateHEALON5 PRO OVD is Non-pyrogenic

To rule out the presence of any chemical pyro-gens in HEALON OVD products, a rabbit pyro-genicity study was conducted based on UnitedStates Pharmacopeia 38\151[Pyrogen Test onHEALON5 PRO OVD. HEALON5 PRO OVD wasselected for testing because it has a higher vis-cosity than HEALON PRO OVD and is consid-ered a ‘‘worst case’’ product for this category ofOVDs. Typically, this standard requires theinjection of 10 mLs test article per 10 kg ofrabbit body weight. However, the viscosity ofHEALON/HEALON PRO OVDs, and especiallyHEALON5/HEALON5 PRO OVDs, precludesdirect injection into the ear vein as the standardcalls for. Instead, a sample comprised of HEA-LON5 PRO OVD diluted 1:50 in saline wasinjected based on rabbit body weight. Approxi-mately 30 min prior to test article injection,baseline temperatures were determined for eachrabbit. The baseline temperature served as a

reference point for identifying temperatureshifts following test article injection. After testarticle injection, temperature measurementswere taken at 30-min intervals. Three identicalstudies were conducted on separate occasions,each with three rabbits injected with test articleand one rabbit injected with saline control.Evaluation of results from all three studiesshows that of the nine rabbits receiving testarticle, five experienced very minimal tempera-ture changes between 0.1 and 0.3 �C (Fig. 1). Ifno rabbit shows an individual rise in tempera-ture of 0.5 �C or more, then the test articlemeets the requirements of the pyrogenicity test.Thus, all nine rabbits tested in these studiesclearly demonstrate that HEALON5 PRO OVD isnon-pyrogenic.

Aqueous Exchange in RabbitsDemonstrates Equivalence BetweenBacterial-Derived HA and Animal-DerivedHA

To conduct a head-to-head comparison betweenHEALON OVD comprised of bacterial-derivedHA or animal-derived HA, aqueous exchangestudies were conducted in New Zealand Whiterabbits. In this in vivo test, the safety of theOVD is evaluated by assessing ocular biocom-patibility indicators including anterior andposterior segment inflammation, intraocularpressure changes and corneal pathology andpachymetry. One study compared HEALONOVD to HEALON PRO OVD, while a secondcompared HEALON5 OVD to HEALON5 PROOVD. In both studies, approximately 25% ofaqueous humor was removed and replaced with0.04–0.1 mL OVD in the anterior chamber.Bacterial-derived OVD (HEALON PRO, HEA-LON5 PRO) was injected into the OD eye ofeach animal, while animal-derived OVD (HEA-LON, HEALON5) were injected into the OS eye.

Over the course of each study, routine healthobservations, gross ocular observations, andbody weight measurements were taken daily.No adverse changes in weight occurred duringthe duration of either study. Mild, procedure-related ocular discharge and irritation wereobserved, as expected, on day 0 for all animals.

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Most animals still demonstrated procedure-re-lated irritation in one or both eyes on days 1–3,but it was largely resolved in the following days.Incidental observations included one animalwith irritation in a HEALON OVD treated eyeon day 2, as well as diarrhea on day 3. Further,corneal haze was noted in one HEALON PROOVD treated eye on day 2. These observationswere considered routine and unremarkable.

Ophthalmic examinations by slit lamp wereperformed and scored by the McDonald-Shad-duck scoring system at baseline as well as post-surgically at 6 h, 24 h, 48 h, 72 h, and 7 days.Overall, all observations noted during thecourse of the study were typical for the aqueousexchange rabbit model and were expected fol-lowing an ocular surgical procedure. At 6 h postprocedure (Tables 1, 2), most animal eyesshowed iritis, and conjunctival congestion andswelling. HEALON5 OVD and HEALON5 PROOVD injected eyes also displayed mild con-junctival discharge, as well as aqueous cells intwo HEALON5 OVD and two HEALON5 PROOVD injected eyes. Over the course of the study,the observed tissue swelling and inflammationresolved, until Day 7 when the scores had lar-gely returned to 0. Two observations of notewere a hemorrhage in the anterior chamber inthe HEALON PRO OVD injected eye of one

animal, and a fibrin streak present on the cor-neal endothelium in the HEALON PRO OVDinjected eye of a different animal. Both adversefindings were determined to be due to theinjection procedure and unrelated to the HEA-LON OVD that was used.

IOP measurements were taken several timeson the day of the procedure (baseline, 2, 4, 6, 8,12 h), then at 24 h and 7 days (Fig. 2a, b).Anticipated increases in IOP levels wereobserved with all OVDs (HEALON, HEALONPRO, HEALON5, and HEALON5 PRO) at 4, 6,and 8 h following the aqueous exchange pro-cedure before returning to baseline levels. Therewere no statistically significant differences inIOP between HEALON OVD and HEALON PROOVD, with p values ranging from 0.07 (4 h) to0.88 (7 days). Likewise, there were no statisti-cally significant differences in IOP betweenHEALON5 OVD and HEALON5 PRO OVD, withp values ranging from 0.24 (4 h) to 0.90 (8 h).These IOP observations demonstrate that HEA-LON PRO OVD products made with bacterially-derived HA do not alter IOP in a manner dif-ferent from HEALON OVD products made withanimal-derived HA.

Pachymetry measurements were taken atbaseline and after the procedure at 6 h, 24 h,48 h, 72 h, and 7 days (Fig. 3a, b). As expected,

Fig. 1 Rabbit pyrogenicity study conducted on HEA-LON5 PRO OVD demonstrates HEALON OVD prod-ucts are non-pyrogenic. Three separate studies wereconducted on three different lots of HEALON5 PROOVD. Each study was conducted with n = 4, one control

‘‘C’’ and three experimental animals (- 1, - 2, - 3).Temperatures were taken every 30 min for a total of 3 h.Bars from left to right in each grouping are baseline, 1 h,1.5 h, 2 h, 2.5 h, and 3 h. Data from all three studies aregraphed together

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there was a very slight increase in cornealthickness 6 h post procedure that was observedwith all four types of HEALON OVDs. There wasno statistically significant difference betweenHEALON PRO OVD and HEALON5 PRO OVDcompared to HEALON OVD and HEALON5

OVD, respectively, at any time point. Thus,pachymetry measurements demonstrate thatthere were no significant differences in cornealthickness between bacteria-derived and animal-derived HEALON OVD products.

Table 1 Clinical ophthalmic observations for rabbit aqueous exchange at 6 h post procedure in eyes injected withHEALON (OS) and HEALON PRO (OD) OVDs

Animal 1 2 3 4 5 6

Eye OD OS OD OS OD OS OD OS OD OS OD OS

Conjunctival congestion 1 1 1 1 1 1 2 2 1 1 2 1

Conjunctival swelling 0 0 0 0 1 1 2 2 0 0 3 1

Cornea 0 0 0 0a 0 0 0 0 0 0 0 0

Corneal clarity 0 0 0 0 1 0 0 0 0 0 1 0

Aqueous cells 0 0 0 0 0 0 0 0 0 0 1 2

Aqueous fibrin 0 0 0 0 0 0 0 0 0 0 1 2

Aqueous flare 0 0 0 0 0 0 0 0 0 0 1 2

Iritis 0 1 1 1 1 1 0 1 1 1 1 0

Lens clarity 0 0 0 0 0 0 0 0 0 0 0 2

Both eyes in each animal also had a score of 0 for the following parameters not included in this table: pannus, pupillaryresponse, lens, vitreous, vitreal hemorrhage, retinal detachment, retinal hemorrhage, choroidal/retinal inflammationa Injection tract noticeable

Table 2 Clinical ophthalmic observations for rabbit aqueous exchange at 6 h post procedure in eyes injected withHEALON5 (OS) and HEALON5 PRO (OD) OVDs

Animal 1 2 3 4 5 6

Eye OD OS OD OS OD OS OD OS OD OS OD OS

Conjunctival congestion 1 2 2 2 2 2 2 2 3 3 2 2

Conjunctival swelling 1 3 2 3 3 2 2 2 3 3 3 3

Cornea 0 0 0 0 0 0 0 0 0 0 0 0

Corneal clarity 0 0 0 0 0 0 0 0 0 0 0 0

Aqueous cells 0 0 0 0 0 0 1 0 1 1 0 1

Aqueous fibrin 0 0 0 0 0 0 0 0 0 0 0 0

Aqueous flare 0 0 0 0 0 0 0 0 0 0 0 0

Iritis 1 2 2 2 1 1 2 2 1 2 1 1

Lens clarity 0 0 0 0 0 0 0 0 0 0 0 0

Both eyes in each animal also had a score of 0 for the following parameters not included in this table: pannus, pupillaryresponse, lens, vitreous, vitreal hemorrhage, retinal detachment, retinal hemorrhage, choroidal/retinal inflammation

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A Novel Mini-Pig Model was Utilizedfor Preclinical Safety Evaluation of OVDComprised of Bacterial or Animal SourcedHA

A novel model based on the Yucatan mini-pigwas developed to evaluate the potential

protective effects of OVDs on endothelial cellsduring cataract surgery. Pigs were chosen forthis model based on the limited capacity, as thatin humans, to regenerate the corneal endothe-lium post injury [17]. Two GLP studies wereconducted in mini-pigs where the mini-pigsunderwent intraocular lens implantation

Fig. 2 IOP measurements in aqueous exchange rabbitmodel demonstrate similarity between OVDs with bacte-rial derived versus animal derived HA. Average IOPmeasurement is graphed from n = 6, error bars are 1standard deviation. No statistically significant differenceswere found between HEALON/HEALON5 OVD

compared with HEALON PRO/HEALON5 PROOVD. a OD eye injected with HEALON PRO OVD,OS eye injected with HEALON OVD. p values =0.07–0.88, using unpaired t test; b OD eye injected withHEALON5 PRO OVD, OS eye injected with HEALON5OVD. p values = 0.24–0.90, using unpaired t test

Fig. 3 Corneal thickness in aqueous exchange rabbitmodel is comparable between OVDs with bacterial-derivedand animal-derived HA. Average pachymetry measurementis graphed from n = 6, error bars are 1 standard deviation.No statistically significant differences were found betweenHEALON/HEALON5 OVD compared with HEALON

PRO/HEALON5 PRO OVD. a OD eye injected withHEALON PRO OVD, OS eye injected with HEALONOVD. p values = 0.250–1, using unpaired t test; b OD eyeinjected with HEALON5 PRO OVD, OS eye injectedwith HEALON5 OVD. p values = 0.527–0.911, usingunpaired t test

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surgery in which the OS eye received HEALONor HEALON5 OVD and the OD eye receivedHEALON PRO or HEALON5 PRO OVD.

During the 14-day study period, routinehealth observations and gross ocular observa-tions were taken daily. Mild ocular swelling,discharge and irritation related to the procedurewere observed, as expected, on day 0 for allanimals. In the study comparing HEALON OVDto HEALON PRO OVD, four of six animalscontinued to demonstrate intermittent swellingand irritation in one or both eyes throughoutthe duration of the study. In the study com-paring HEALON5 OVD to HEALON5 PRO OVD,one of six animals had excessive discharge andswelling in the HEALON5 PRO OVD treated eye.Furthermore, fibrin was noted in the HEALON5OVD eye of one animal, the HEALON5 PROOVD eye of one animal, and both eyes in twoanimals. Several eyes, both HEALON5 OVD andHEALON5 PRO OVD treated, demonstratedcorneal cloudiness (4/6 HEALON5 PRO OVDeyes), and haze (4/6 HEALON5 PRO OVD eyes;4/6 HEALON5 OVD eyes). The body weight wasmeasured at day 0 prior to surgery and afterfinal examinations on day 14. No adversechanges in weight occurred during the durationof either study. Damage to the cornealendothelium during surgery, causing endothe-lial cell loss, may lead to an increased mea-surement of central corneal thickness, orpachymetry. Thus, pachymetry measurementsindirectly informs on the health and integrity ofthe endothelial cell layer. In these studies,pachymetry measurements demonstrate noapparent changes over the course of the studyfor any OVD (Fig. 4a, b). All these observationswere considered routine and unremarkable.This data suggests all four OVD products wereable to protect the integrity of the endothelialcell layer.

Ophthalmic examinations by slit lamp wereperformed and scored by the McDonald-Shad-duck scoring system at baseline as well as afterthe procedure on days 1, 2, 3, 7, and 14. Allbaseline scores were reported as 0. Following theprocedure, during day 1 of ophthalmic exami-nations by slit lamp, mild to severe conjunctivaldischarge, congestion, and swelling wereobserved in both eyes of all pigs (Tables 3, 4).

Additional observations on Day 1 include mildto severe anterior chamber flare, fibrin, andinflammatory cells present in the anterior cap-sule in all eyes. Mild to severe aqueous flare wasnoted in both eyes of all but two animals (un-able to observe posterior portion of the eye).Loss of corneal transparency was noted in theHEALON PRO OVD injected eye in three of sixanimals, HEALON5 PRO OVD in one of sixanimals, and in both eyes in one of six animalsin the HEALON/HEALON PRO OVD study, andfive of six animals in the HEALON5/HEALON5PRO OVD study. In animals where HEALON5OVD and HEALON5 PRO OVD were used, allanimals also experienced mild conjunctivalhyperemia. Stromal cloudiness was observed intwo HEALON5 PRO OVD injected eyes and twoHEALON5 OVD injected eyes. Over the courseof the 14 days, the animal eyes continued torecover from procedural inflammation and irri-tation. On day 14, mild to moderate conjunc-tival congestion and swelling were still presentin two of six HEALON PRO OVD injected eyesand one of six HEALON5 PRO OVD injectedeyes. Loss of corneal transparency was presentin two of six HEALON PRO OVD injected eyes,four of six HEALON5 PRO OVD injected eyes,and one of six HEALON5 OVD injected eyes.Anterior chamber flare was observed in one ofsix HEALON PRO OVD injected eyes and one ofsix HEALON5 PRO OVD injected eyes. Fibrinwas present in the HEALON PRO OVD injectedeye in five of six animals, in the HEALON OVDinjected eye in three of six animals, in theHEALON5 PRO OVD injected eyes of two of sixanimals, and in the HEALON5 OVD injectedeyes of one of six animals. Overall, ophthalmicobservations noted were incidental, procedurerelated findings and not dependent on the typeof viscoelastic device that was utilized.

IOP measurements were taken at baselineand on Days 1, 3, 7, and 14 (Fig. 5a, b). Aninitial variability in IOP at early time points,followed by a return to baseline levels at latertime points was observed and is consistent withtypical IOL surgical procedures. Figure 5a com-pares HEALON OVD with HEALON PRO OVDand HEALON5 OVD with HEALON5 PRO OVD,respectively. There were no statistically signifi-cant differences in IOP between HEALON OVD

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and HEALON PRO OVD, with p values rangingfrom 0.186 (Day 3) to 0.657 (Day 14). Therewere also no statistically significant differencesin IOP between HEALON5 OVD and HEALON5PRO OVD, with p values ranging from 0.476(baseline) to 1 (Day 14) (Fig. 5b). These IOP

observations demonstrate that OVD with bac-terially-derived HA behave similar to OVD withanimal-derived HA regarding IOP changes.

Corneal endothelial cell density was mea-sured at baseline and on Days 3, 7, and 14 fol-lowing the surgical procedure. To decrease

Fig. 4 Corneal thickness in the pig model is comparablebetween OVDs with bacterial-derived and animal-derivedHA. Average pachymetry measurement is graphed fromn = 6, error bars are 1 standard deviation. No statisticallysignificant differences were found between HEALON/HEALON5 OVD compared with HEALON PRO/HEALON5 PRO OVD. a OD eye injected with

HEALON PRO OVD, OS eye injected with HEALONOVD. Pachymetry measurements were not taken on day 1post surgery. p values = 0.195–0.872, using unpaired t test;b OD eye injected with HEALON5 PRO OVD, OS eyeinjected with HEALON5 OVD. p values = 0.196–0.938,using unpaired t test

Table 3 Clinical ophthalmic observations for mini-pig model at 1 day post procedure in eyes injected with HEALON (OS)and HEALON PRO (OD) OVDs

Animal 1 2 3 4 5 6

Eye OD OS OD OS OD OS OD OS OD OS OD OS

Conjunctival congestion 2 2 2 2 4 1 3 2 3 2 4 3

Conjunctival swelling 2 2 2 2 4 1 3 2 3 2 4 2

Cornea 2 2 2 1 3 1 3 2 3 2 3 3

Aqueous flare 2 3 2 3 NA 1 3 2 3 2 4 3

Anterior Capsule (Cells) 2 3 2 3 4 1 3 2 3 2 4 3

Anterior chamber flare 2 3 2 3 4 1 3 2 3 2 4 3

Fibrin in anterior chamber 1 1 2 0 4 1 3 NA 3 2 4 3

Both eyes in each animal also had a score of 0 for the following parameters not included in this table: pannus, pupillaryresponse. Evaluation was not possible regarding lens, vitreous, vitreal hemorrhage, retinal detachment, retinal hemorrhage,choroidal/retinal inflammation, anterior capsule (cell growth), posterior synechiae, IOL position, posterior capsule haze, andposterior capsule growthNA not available for evaluation

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postoperative damage and cell loss, OVDs areused to protect the integrity of the endothelialcell layer [18]. Corneal endothelial cell densitymeasurements allow investigation of the effec-tiveness of this protective property of OVDs. Inthe studies evaluating HEALON OVD andHEALON PRO OVD or HEALON5 OVD andHEALON5 PRO OVD, all four OVD demon-strated corneal endothelial cell protection.There was not a statistically significant

difference between HEALON OVD and HEALONPRO OVD at any time point, with p values of0.705, 0.594, and 0.668 for baseline, day 3, andday 14 (p value of day 7 could not be calculateddue to small n) (Fig. 6a). There was a slightreduction in endothelial cell density on day 7 inHEALON OVD injected eyes (p = 0.046) and onday 14 in HEALON PRO OVD injected eyes(p = 0.045). This very slight reduction did notappear to have a physiological impact and the

Table 4 Clinical ophthalmic observations for mini-pig model at 1 day post procedure in eyes injected with HEALON5(OS) and HEALON5 PRO (OD) OVDs

Animal 1 2 3 4 5 6

Eye OD OS OD OS OD OS OD OS OD OS OD OS

Conjunctival congestion 1 0 2 2 2 3 3 1 4 2 3 1

Conjunctival swelling 1 0 2 2 2 3 2 1 3 1 2 0

Cornea 2 0 2 1 1 1 1 2 3 1 2 1

Aqueous flare 1 1 1 1 1 1 1 0 NA 1 1 1

Anterior Capsule (Cells) 1 1 1 1 1 1 1 1 NA 1 1 1

Anterior Chamber Flare 1 1 1 1 1 1 1 1 NA 1 1 1

Fibrin in Anterior chamber 1 1 1 1 1 1 2 1 NA 1 1 1

Both eyes in each animal also had a score of 0 for the following parameters not included in this table: pannus, pupillaryresponse, lens, vitreous, vitreal hemorrhage, retinal detachment, retinal hemorrhage, choroidal/retinal inflammation, anteriorcapsule (cell growth), posterior synechiae, IOL position, posterior capsule haze, posterior capsule growthNA not available for evaluation

Fig. 5 IOP measurements in the pig model demonstratesimilarity between OVDs with bacterial derived versusanimal derived HA. Average IOP measurement is graphedfrom n = 6, error bars are 1 standard deviation. Nostatistically significant differences were found betweenHEALON/HEALON5 OVD compared with HEALON

PRO/HEALON5 PRO OVD. a OD eye injected withHEALON PRO OVD, OS eye injected with HEALONOVD. p values = 0.186–0.657, using unpaired t test; b ODeye injected with HEALON5 PRO OVD, OS eye injectedwith HEALON5 OVD. p values = 0.276–1, usingunpaired t test

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cell numbers were not significantly different(data not shown). Likewise, there was not astatistically significant difference betweenHEALON5 OVD and HEALON5 PRO OVD at anytime point, with p values of 0.287, 0.538, 0.745,and 0.828 for baseline, day 3, day 7 and day 14,respectively (Fig. 6b). Again, we observed aslight reduction in endothelial cell density atday 14 in HEALON5 PRO OVD eyes, and at days3, 7, and 14 in HEALON5 OVD eyes, but with-out significant differences in cell numbers,except at day 3.

DISCUSSION

Advancements in cataract surgical techniquesand OVD materials continuously create theneed for more specialized OVD formulationswith unique physio-chemical and rheologicfeatures [1]. The International StandardizationOrganization (ISO), the US FDA, and theAmerican National Standards Institute (ANSI)all have implemented guidelines for preclinical

testing [14] including ISO 15798: OphthalmicImplants-Ophthalmic Viscosurgical Devices andISO 10993: Biological Evaluation of Medical Devi-ces which apply to ophthalmic viscosurgicaldevice development.

The studies reported here were designed todemonstrate the equivalence of animal-derivedand bacterial-derived HA. Due to the inherentnature and purpose of this medical device, inaddition to standard guidelines, the resultsreported here describe custom studies designedto address additional questions unique toOVDs. In our investigation, we first relied onrabbit models for pyrogenicity and aqueousexchange. The viscosity of HEALON OVDproducts precluded direct injection for pyro-genicity tests, thus studies were designed usingdiluted OVD. Prior to conducting this work,approval was needed from regulatory bodiesrecognizing the validity of the data. In ourrabbit aqueous exchange model, the surgeonevaluated OVD performance parametersincluding ease of injection and removal, as wellas anterior chamber space creation and

Fig. 6 Endothelial cell density demonstrates equivalencein protective potential of OVDs with bacterial derived HAand animal derived HA. Average endothelial cell densitymeasurement is graphed, error bars are one standarddeviation. No statistically significant differences werefound between HEALON/HEALON5 OVD comparedwith HEALON PRO/HEALON5 PRO OVD. a OD eyeinjected with HEALON PRO OVD, OS eye injected withHEALON OVD. Six animals were in the study, butendothelial cell density was not obtainable in every animal.For HEALON PRO OVD, n for baseline, day 3, day 7,

day 14 are 6, 3, 1, 4, respectively. For HEALON OVD, nfor baseline, day 3, day 7, day 14 are 6, 3, 4, 5, respectively.p values = 0.595–0.705, using unpaired t test; b OD eyeinjected with HEALON5 PRO OVD, OS eye injectedwith HEALON5 OVD. Six animals were in the study, butendothelial cell density was not obtainable in every animal.For HEALON5 PRO OVD, n for baseline, day 1, day 3,day 7, day 14 are 6, 4, 4, 4, 4, respectively. For HEALON5OVD, n for baseline, day 1, day 3, day 7, day 14 are 6, 3, 6,6, 6 respectively. p values = 0.286–0.951, using unpairedt test

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maintenance. Post-surgery, ocular examina-tions and measurements for signs of ocularbiocompatibility of the investigational OVDformulations (with bacterial-sourced HA)demonstrated equivalence to clinicallyapproved OVDs (with animal-sourced HA).

One key attribute of OVD is the protectionof corneal endothelial cells during cataractsurgery. A useful animal model to investigatethe protective properties of OVD has yet to beestablished. This study presents the use of anovel Yucatan mini-pig model for the firsttime, because like humans, mini-pigs havedemonstrated a limited ability to proliferateand regenerate the corneal endothelium[6, 17]. In this model, we were able to performa lens exchange to mimic cataract surgerywhile utilizing HEALON OVD products forprotection of endothelial cells, among itsother functions. The data gathered on generaland ocular health, IOP, endothelial cell den-sity, and corneal pachymetry were able toconclusively demonstrate equivalence of OVDcomprised of animal-derived or bacterial-derived HA. However, use of the mini-pigmodel is not without its limitations andcomplications. Maintaining appropriate anes-thesia throughout the procedure was a con-siderable challenge. When animals weredeeply sedated, the eyes would roll out ofposition making it difficult for the surgeon toobtain an unimpaired view of the eye. If thesedation was lightened, animals were notcompletely physically restrained and mayattempt to cough out the intubation tube.Both situations led to difficult surgeries and ahigher level of post-operative inflammationthan would normally occur.

CONCLUSIONS

Even considering the challenges that wereencountered, through the use of both the rabbitpyrogenicity and aqueous exchange models andthe mini-pig endothelial cell model, we estab-lish the equivalence of HEALON OVDs regard-less of their HA source.

ACKNOWLEDGEMENTS

Funding. These studies and article process-ing charges were funded by Johnson & JohnsonSurgical Vision, Inc. All authors had access to allof the data in this study and take completeresponsibility for the integrity of the data andaccuracy of the data analysis.

Authorship. All named authors meet theInternational Committee of Medical JournalEditors (ICMJE) criteria for authorship for thisarticle, take responsibility for the integrity ofthe work as a whole, and have given theirapproval for this version to be published.

Disclosures. Ronika S. Leang is an employeeof Johnson & Johnson Surgical Vision, Inc. LisaJ. Kloft is an employee of Johnson & JohnsonSurgical Vision, Inc. Brad Gray is an employeeof Johnson & Johnson Surgical Vision, Inc.Arlene E. Gwon is a paid consultant of Johnson& Johnson Surgical Vision, Inc. Ling C. Huangis an employee of Johnson & Johnson SurgicalVision, Inc.

Compliance with Ethics Guidelines. Allinstitutional and national guidelines for thecare and use of laboratory animals werefollowed.

Data Availability. The datasets generatedduring and/or analyzed during the currentstudy are available from the correspondingauthor on reasonable request.

Open Access. This article is distributedunder the terms of the Creative CommonsAttribution-NonCommercial 4.0 InternationalLicense (http://creativecommons.org/licenses/by-nc/4.0/), which permits any noncommer-cial use, distribution, and reproduction in anymedium, provided you give appropriate creditto the original author(s) and the source, providea link to the Creative Commons license, andindicate if changes were made.

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