Rabbit Pyrogenicity Assays Demonstrate HEALON5 PRO OVD is Non-pyrogenic
To rule out the presence of any chemical pyrogens in HEALON OVD products, a rabbit pyrogenicity study was conducted based on United States Pharmacopeia 38 <151> Pyrogen Test on HEALON5 PRO OVD. HEALON5 PRO OVD was selected for testing because it has a higher viscosity than HEALON PRO OVD and is considered a “worst case” product for this category of OVDs. Typically, this standard requires the injection of 10 mLs test article per 10 kg of rabbit body weight. However, the viscosity of HEALON/HEALON PRO OVDs, and especially HEALON5/HEALON5 PRO OVDs, precludes direct injection into the ear vein as the standard calls for. Instead, a sample comprised of HEALON5 PRO OVD diluted 1:50 in saline was injected based on rabbit body weight. Approximately 30 min prior to test article injection, baseline temperatures were determined for each rabbit. The baseline temperature served as a reference point for identifying temperature shifts following test article injection. After test article injection, temperature measurements were taken at 30-min intervals. Three identical studies were conducted on separate occasions, each with three rabbits injected with test article and one rabbit injected with saline control. Evaluation of results from all three studies shows that of the nine rabbits receiving test article, five experienced very minimal temperature changes between 0.1 and 0.3 °C (Fig. 1). If no rabbit shows an individual rise in temperature of 0.5 °C or more, then the test article meets the requirements of the pyrogenicity test. Thus, all nine rabbits tested in these studies clearly demonstrate that HEALON5 PRO OVD is non-pyrogenic.
Aqueous Exchange in Rabbits Demonstrates Equivalence Between Bacterial-Derived HA and Animal-Derived HA
To conduct a head-to-head comparison between HEALON OVD comprised of bacterial-derived HA or animal-derived HA, aqueous exchange studies were conducted in New Zealand White rabbits. In this in vivo test, the safety of the OVD is evaluated by assessing ocular biocompatibility indicators including anterior and posterior segment inflammation, intraocular pressure changes and corneal pathology and pachymetry. One study compared HEALON OVD to HEALON PRO OVD, while a second compared HEALON5 OVD to HEALON5 PRO OVD. In both studies, approximately 25% of aqueous humor was removed and replaced with 0.04–0.1 mL OVD in the anterior chamber. Bacterial-derived OVD (HEALON PRO, HEALON5 PRO) was injected into the OD eye of each animal, while animal-derived OVD (HEALON, HEALON5) were injected into the OS eye.
Over the course of each study, routine health observations, gross ocular observations, and body weight measurements were taken daily. No adverse changes in weight occurred during the duration of either study. Mild, procedure-related ocular discharge and irritation were observed, as expected, on day 0 for all animals. Most animals still demonstrated procedure-related irritation in one or both eyes on days 1–3, but it was largely resolved in the following days. Incidental observations included one animal with irritation in a HEALON OVD treated eye on day 2, as well as diarrhea on day 3. Further, corneal haze was noted in one HEALON PRO OVD treated eye on day 2. These observations were considered routine and unremarkable.
Ophthalmic examinations by slit lamp were performed and scored by the McDonald-Shadduck 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 the course of the study were typical for the aqueous exchange rabbit model and were expected following an ocular surgical procedure. At 6 h post procedure (Tables 1, 2), most animal eyes showed iritis, and conjunctival congestion and swelling. HEALON5 OVD and HEALON5 PRO OVD injected eyes also displayed mild conjunctival discharge, as well as aqueous cells in two HEALON5 OVD and two HEALON5 PRO OVD injected eyes. Over the course of the study, the observed tissue swelling and inflammation resolved, until Day 7 when the scores had largely returned to 0. Two observations of note were a hemorrhage in the anterior chamber in the HEALON PRO OVD injected eye of one animal, and a fibrin streak present on the corneal endothelium in the HEALON PRO OVD injected eye of a different animal. Both adverse findings were determined to be due to the injection procedure and unrelated to the HEALON OVD that was used.
IOP measurements were taken several times on 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 were observed with all OVDs (HEALON, HEALON PRO, HEALON5, and HEALON5 PRO) at 4, 6, and 8 h following the aqueous exchange procedure before returning to baseline levels. There were no statistically significant differences in IOP between HEALON OVD and HEALON PRO OVD, with p values ranging from 0.07 (4 h) to 0.88 (7 days). Likewise, there were no statistically significant differences in IOP between HEALON5 OVD and HEALON5 PRO OVD, with p values ranging from 0.24 (4 h) to 0.90 (8 h). These IOP observations demonstrate that HEALON PRO OVD products made with bacterially-derived HA do not alter IOP in a manner different from HEALON OVD products made with animal-derived HA.
Pachymetry measurements were taken at baseline and after the procedure at 6 h, 24 h, 48 h, 72 h, and 7 days (Fig. 3a, b). As expected, there was a very slight increase in corneal thickness 6 h post procedure that was observed with all four types of HEALON OVDs. There was no statistically significant difference between HEALON PRO OVD and HEALON5 PRO OVD compared to HEALON OVD and HEALON5 OVD, respectively, at any time point. Thus, pachymetry measurements demonstrate that there were no significant differences in corneal thickness between bacteria-derived and animal-derived HEALON OVD products.
A Novel Mini-Pig Model was Utilized for Preclinical Safety Evaluation of OVD Comprised of Bacterial or Animal Sourced HA
A novel model based on the Yucatan mini-pig was developed to evaluate the potential protective effects of OVDs on endothelial cells during cataract surgery. Pigs were chosen for this model based on the limited capacity, as that in humans, to regenerate the corneal endothelium post injury . Two GLP studies were conducted in mini-pigs where the mini-pigs underwent intraocular lens implantation surgery in which the OS eye received HEALON or HEALON5 OVD and the OD eye received HEALON PRO or HEALON5 PRO OVD.
During the 14-day study period, routine health observations and gross ocular observations were taken daily. Mild ocular swelling, discharge and irritation related to the procedure were observed, as expected, on day 0 for all animals. In the study comparing HEALON OVD to HEALON PRO OVD, four of six animals continued to demonstrate intermittent swelling and irritation in one or both eyes throughout the duration of the study. In the study comparing HEALON5 OVD to HEALON5 PRO OVD, one of six animals had excessive discharge and swelling in the HEALON5 PRO OVD treated eye. Furthermore, fibrin was noted in the HEALON5 OVD eye of one animal, the HEALON5 PRO OVD eye of one animal, and both eyes in two animals. Several eyes, both HEALON5 OVD and HEALON5 PRO OVD treated, demonstrated corneal cloudiness (4/6 HEALON5 PRO OVD eyes), and haze (4/6 HEALON5 PRO OVD eyes; 4/6 HEALON5 OVD eyes). The body weight was measured at day 0 prior to surgery and after final examinations on day 14. No adverse changes in weight occurred during the duration of either study. Damage to the corneal endothelium during surgery, causing endothelial cell loss, may lead to an increased measurement of central corneal thickness, or pachymetry. Thus, pachymetry measurements indirectly informs on the health and integrity of the endothelial cell layer. In these studies, pachymetry measurements demonstrate no apparent changes over the course of the study for any OVD (Fig. 4a, b). All these observations were considered routine and unremarkable. This data suggests all four OVD products were able to protect the integrity of the endothelial cell layer.
Ophthalmic examinations by slit lamp were performed and scored by the McDonald-Shadduck scoring system at baseline as well as after the procedure on days 1, 2, 3, 7, and 14. All baseline scores were reported as 0. Following the procedure, during day 1 of ophthalmic examinations by slit lamp, mild to severe conjunctival discharge, congestion, and swelling were observed in both eyes of all pigs (Tables 3, 4). Additional observations on Day 1 include mild to severe anterior chamber flare, fibrin, and inflammatory cells present in the anterior capsule in all eyes. Mild to severe aqueous flare was noted in both eyes of all but two animals (unable to observe posterior portion of the eye). Loss of corneal transparency was noted in the HEALON PRO OVD injected eye in three of six animals, HEALON5 PRO OVD in one of six animals, and in both eyes in one of six animals in the HEALON/HEALON PRO OVD study, and five of six animals in the HEALON5/HEALON5 PRO OVD study. In animals where HEALON5 OVD and HEALON5 PRO OVD were used, all animals also experienced mild conjunctival hyperemia. Stromal cloudiness was observed in two HEALON5 PRO OVD injected eyes and two HEALON5 OVD injected eyes. Over the course of the 14 days, the animal eyes continued to recover from procedural inflammation and irritation. On day 14, mild to moderate conjunctival congestion and swelling were still present in two of six HEALON PRO OVD injected eyes and one of six HEALON5 PRO OVD injected eyes. Loss of corneal transparency was present in 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 of six HEALON PRO OVD injected eyes and one of six HEALON5 PRO OVD injected eyes. Fibrin was present in the HEALON PRO OVD injected eye in five of six animals, in the HEALON OVD injected eye in three of six animals, in the HEALON5 PRO OVD injected eyes of two of six animals, and in the HEALON5 OVD injected eyes of one of six animals. Overall, ophthalmic observations noted were incidental, procedure related findings and not dependent on the type of viscoelastic device that was utilized.
IOP measurements were taken at baseline and on Days 1, 3, 7, and 14 (Fig. 5a, b). An initial variability in IOP at early time points, followed by a return to baseline levels at later time points was observed and is consistent with typical IOL surgical procedures. Figure 5a compares HEALON OVD with HEALON PRO OVD and HEALON5 OVD with HEALON5 PRO OVD, respectively. There were no statistically significant differences in IOP between HEALON OVD and HEALON PRO OVD, with p values ranging from 0.186 (Day 3) to 0.657 (Day 14). There were also no statistically significant differences in IOP between HEALON5 OVD and HEALON5 PRO OVD, with p values ranging from 0.476 (baseline) to 1 (Day 14) (Fig. 5b). These IOP observations demonstrate that OVD with bacterially-derived HA behave similar to OVD with animal-derived HA regarding IOP changes.
Corneal endothelial cell density was measured at baseline and on Days 3, 7, and 14 following the surgical procedure. To decrease postoperative damage and cell loss, OVDs are used to protect the integrity of the endothelial cell layer . Corneal endothelial cell density measurements allow investigation of the effectiveness of this protective property of OVDs. In the studies evaluating HEALON OVD and HEALON PRO OVD or HEALON5 OVD and HEALON5 PRO OVD, all four OVD demonstrated corneal endothelial cell protection. There was not a statistically significant difference between HEALON OVD and HEALON PRO OVD at any time point, with p values of 0.705, 0.594, and 0.668 for baseline, day 3, and day 14 (p value of day 7 could not be calculated due to small n) (Fig. 6a). There was a slight reduction in endothelial cell density on day 7 in HEALON OVD injected eyes (p = 0.046) and on day 14 in HEALON PRO OVD injected eyes (p = 0.045). This very slight reduction did not appear to have a physiological impact and the cell numbers were not significantly different (data not shown). Likewise, there was not a statistically significant difference between HEALON5 OVD and HEALON5 PRO OVD at any time 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 a slight reduction in endothelial cell density at day 14 in HEALON5 PRO OVD eyes, and at days 3, 7, and 14 in HEALON5 OVD eyes, but without significant differences in cell numbers, except at day 3.