Abstract
Purpose:
To evaluate the safety and tolerability of pooled human immune globulins, Flebogamma® 5% DIF and Flebogamma® 10% DIF, administered by topical ophthalmic instillation to New Zealand White (NZW) rabbits.
Methods:
Male NZW rabbits were used in this study. In the acute single dose tolerability study, rabbits (n = 12) received a single topical dose of Flebogamma® 5% DIF. In the two-week repeated-dose tolerability study, rabbits (n = 5 for each group) were administered either Flebogamma® 5% DIF or Flebogamma® 10% DIF by topical bilateral administration four times daily (q.i.d.) between 8 am and 6 pm for a period of two weeks. Full ophthalmic examinations were conducted to evaluate ocular tolerability at baseline, Day 7, and Day 14.
Results:
In the acute single dose study, mild hyperemia was observed in 1 out of 4 eyes at each 4 h and 24 h post-instillation of Flebogamma® 5% DIF. In the repeated dose study, no ocular signs were detected after q.i.d. topical instillation of Flebogamma® 5% DIF, while Flebogamma® 10% DIF resulted in mild hyperemia in 8 out of 10 eyes on Day 7, and 5 out of 10 eyes on Day 14. No positive corneal fluorescein staining was detected. Schirmer tear test results were unremarkable. No other ocular signs were observed. Administration of immune globulins had no effect on intraocular pressure.
Conclusions:
Flebogamma® 5% DIF and Flebogamma® 10% DIF were well-tolerated by NZW rabbits following single and repeat dose topical ophthalmic administration, supporting the future development of topical pooled human immune globulins for the treatment of ocular surface disease.
Keywords: pooled human immune globulins, Flebogamma®, tolerability, dry eye, drug repurposing
Subject classification code: Ocular Toxicology
Introduction
Pooled human immune globulins have been used since the 1950s to treat primary immune deficiency and contain unmodified purified immunoglobulin G (IgG) products manufactured from pooled human plasma of thousands or more blood donors [1]. This large donor pool ensures diversity of antibody repertoires typically present in normal human serum with antibody specificities to a broad spectrum of antigens. The distribution of IgG subclasses in pooled immune globulins is comparable with that of IgG in normal human serum, but contains only trace amounts of immunoglobulin A (IgA) or immunoglobulin M (IgM) [2], which may have undesired immunomodulatory effects. As even minor differences in the manufacturing processes, including sterilization, stabilization, container closure system, and route of administration of pooled human immune globulins can impact their physiochemical characteristics and therapeutic properties [3], the U.S. Food and Drug Administration (FDA) regards each pooled human immune globulin preparation as a different drug product with distinct FDA approval and labelling.
Intravenous immune globulins (IVIG) describe pooled human immune globulin products approved for intravenous injection. This study used Flebogamma® 5% and 10% dual inactivation and filtration development (DIF) [4] (Instituto Grifols, S.A, Barcelona, Spain), FDA-approved for the treatment of primary (inherited) immunodeficiency and chronic primary immune thrombocytopenia. Flebogamma® DIF reaches a purity of >99.5% owing to a combination of several methods that can inactivate and/or remove biological contaminants, including chemical inactivation, heat inactivation, nanofiltration, and precipitations [4].
We have previously reported the clinical safety and exploratory efficacy of ocular surface immune globulins (OSIG) comprised of Flebogamma® diluted to 0.4% for the treatment of dry eye disease [5]. Specifically, OSIG were delivered by topical instillation twice daily (b.i.d.) for eight weeks in dry eye disease patients with anti-citrullinated protein autoantibodies (ACPA) levels of ≥ 4.4 units in their ocular surface washes. OSIG were well-tolerated, and no differences in the presence of adverse events between OSIG and vehicle groups were identified [5]. Furthermore, OSIG resulted in statistically significant improvement in signs and symptoms of dry eye disease at week 8; vehicle treatment did not result in any improvement of dry eye disease [5].
Efficacy of OSIG in dry eye disease is considered to be multifactorial. Pooled human immune globulins contain anti-idiotypic antibodies and naturally-occurring autoantibodies, the concentration of which increases with the number of donors and the concentration of the formulation [6]. Efficacy of OSIG was further attributed to the presence of natural antibodies that provide a first-line defense against pathogens and the intact Fc-dependent effector functions [5].
Our ongoing pre-Investigational New Drug (IND) studies have revealed that Flebogamma® DIF shows a clear pharmacological dose-response with low doses of Flebogamma® DIF achieving partial, and Flebogamma® 5% DIF achieving near complete prevention of ocular surface-relevant mechanisms, including ACPA-mediated neutrophil activation and adhesion to corneal epithelial cells [7]. These data support the development of higher concentrations of human pooled immune globulins for ophthalmic use.
Therefore, the objective of the present study was to evaluate safety and tolerability of Flebogamma® 5% DIF and Flebogamma® 10% DIF administered by topical instillation to New Zealand White rabbits with the goal to advance development of OSIG formulations for the treatment of dry eye disease.
Materials and Methods
Animals
Male New Zealand White rabbits, approximately 3 - 4 months of age (~ 1.5 – 2 kg) were purchased from either Charles River Laboratories (Wilmington, MA) or Envigo Laboratories, Inc. (Hackensack, NJ) for the acute single dose tolerability study (n = 12) and the two-week repeated dose tolerability study (n = 10), respectively. Animals were single housed with enrichment (balls, bells) for the duration of the study in compliance with United States Department of Agriculture (USDA) Guidelines. No other species were kept in the same room. No fasting was required for any procedure.
Animals were housed at 16 to 22 °C with a relative humidity of 30 to 70% and on a 12 h/12 h light/dark cycle. All animals were provided Certified PMI Rabbit Diet ad libitum. Water was available ad libitum to each animal via a water bottle with sipper tube or an automated watering system. Animals were acclimated to their housing for at least 5 days following arrival into the facility prior to the study initiation.
All procedures were performed in accordance with local Standard Operating Procedures adhering to the regulations outlined in the USDA Animal Welfare Act [Code of Federal Regulations (CFR) Parts 1, 2, and 3] and the conditions specified in the Guide for the Care and Use of Laboratory Animals. The Animal Use Protocol governing the studies described herein were reviewed and approved by the Institutional Animal Care and Use Committees of Loyola University Chicago (protocol #20-060) and the University of North Texas Health Science Center (protocol #2021-0009).
Macroscopic ophthalmic exams were performed prior to the study to ensure normal ocular health.
Drug administration
Pooled human immune globulins were marketed product Flebogamma® 5% DIF (lot A04H013651) and Flebogamma® 10% DIF (lot G04H009631) obtained from the manufacturer (Instituto Grifols, S.A, Barcelona, Spain). In the acute single dose tolerability study, rabbits received a single topical dose of Flebogamma® 5% DIF administered using a micropipette (35 μL). In the two-week tolerability study, rabbits were randomly divided into two groups and administered either Flebogamma® 5% DIF (n = 5) or Flebogamma® 10% DIF (n = 5) by topical bilateral administration using a micropipette (35 μL) four times daily between 8 am and 6 pm for a period of two weeks (Figure 1).
Figure 1. Experimental Design for the repeated dose tolerability study in NZW rabbits.
Schematic representation of the experimental design. Adult NZW rabbits were randomly divided into two groups, receiving either Flebogamma® 5% DIF or Flebogamma® 10% DIF four times daily (q.i.d.) by bilateral topical ophthalmic instillation (35 μL) for two weeks. Ophthalmic exams (OE) were performed at baseline (D0), Day 7 (D7), and Day 14 (D14).
Ophthalmic exams
Ophthalmic examinations were performed in awake animals by a veterinary ophthalmologist using a slit lamp (SL-17, Kowa American Corporation, Torrance, CA) on the anterior segment. Corneal damage was assessed using fluorescein staining test (BioGlo™, HUB Pharmaceuticals, Scottsdale, AZ) and cobalt blue light using the slit lamp. An indirect ophthalmoscope (Keeler, Malvern, PA) and a 28D lens were used to assess the posterior segment. Prior to posterior examination, eyes were dilated using 1% tropicamide (Akorn Inc., Lake Forest, IL). Full ophthalmic examinations were conducted at baseline, Day 7, and Day 14 and scored using the semi quantitative preclinical ocular toxicology scoring (SPOTS) system [8].
Anterior segment evaluations after single dose instillation were graded according to a modified SPOTS system for hyperemia (0 - 3), chemosis (0 – 4), discharge (0 – 3), and blepharitis (0 - 3). In the acute single dose tolerability study, anterior segment evaluations were performed 30 minutes, 2 hours, 4 hours, 24 hours, 48 hours, and 72 hours.
Schirmer Tear Test (STT) using Schirmer test strips (Merck Animal Health, Rahway, NJ) placed in the lower fornix for 60 seconds and measurements were recorded immediately upon its removal. STT was performed at baseline, Day 7, and Day 14.
Intraocular pressure (IOP) measurements were performed using a rebound tonometer (Tonovet, Icare Finland Oy, Vantaa, Finland). IOP measurements were performed at baseline, Day 7, and Day 14.
Results
Tolerability of pooled human immune globulins (Flebogamma® DIF) in rabbits after single topical ophthalmic instillation
Tolerability of Flebogamma® 5% DIF after single topical ophthalmic instillation was evaluated at six time points between 30 min and 72 h post-dose by anterior segment evaluation. Animals did not show any behavioral signs of discomfort or pain as determined by a qualitative behavioral assessment.
Ophthalmic anterior segment evaluation revealed mild hyperemia (score 1) in one eye at 4 h and 24 h post-instillation and resolved at later timepoints. No other ophthalmic signs were noted, specifically no presence of chemosis, discharge, or blepharitis was observed (Table 1).
Table 1.
Anterior segment findings graded using a modified SPOTS scoring system after single dose topical ophthalmic administration of Flebogamma® 5% DIF.a
| Hyperemia | Chemosis | Discharge | Blepharitis | |
|---|---|---|---|---|
| 30 min | 0 (4/4) | 0 (4/4) | 0 (4/4) | 0 (4/4) |
| 2 h | 0 (4/4) | 0 (4/4) | 0 (4/4) | 0 (4/4) |
| 4 h | 0 (3/4) 1 (1/4) |
0 (4/4) | 0 (4/4) | 0 (4/4) |
| 24 h | 0 (3/4) 1 (1/4) |
0 (4/4) | 0 (4/4) | 0 (4/4) |
| 48 h | 0 (4/4) | 0 (4/4) | 0 (4/4) | 0 (4/4) |
| 72 h | 0 (4/4) | 0 (4/4) | 0 (4/4) | 0 (4/4) |
Data are presented as “score (number of eyes with score / total number of eyes)”
Tolerability of pooled human immune globulins (Flebogamma® DIF) in rabbits after repeated topical ophthalmic instillation
Repeated instillation of Flebogamma® 5% DIF did not result in ophthalmic signs, and both anterior and posterior ophthalmic exams were unremarkable (Table 2).
Table 2.
Full ophthalmic examinations performed on Day 7 and Day 14 and scored using the SPOTS system.b
| Semiquantitative preclinical ocular toxicology scoring (SPOTS) |
Flebogamma® 5% DIF Score (eyes/total eyes) |
Flebogamma® 10% DIF Score (eyes/total eyes) |
||
|---|---|---|---|---|
| Day 7 | Day 14 | Day 7 | Day 14 | |
| Anterior Segment | ||||
| Conjunctiva | ||||
| - Hyperemia | 0 (10/10) | 0 (10/10) | 0 (2/10); 1 (8/10) | 0 (5/10); 1 (5/10) |
| - Chemosis | 0 (10/10) | 0 (10/10) | 0 (10/10) | 0 (10/10) |
| - Discharge | 0 (10/10) | 0 (10/10) | 0 (10/10) | 0 (10/10) |
| Cornea | ||||
| - Opacity (severity) | 0 (10/10) | 0 (10/10) | 0 (10/10) | 0 (10/10) |
| - Opacity (area) | 0 (10/10) | 0 (10/10) | 0 (10/10) | 0 (10/10) |
| - Vascularization | 0 (10/10) | 0 (10/10) | 0 (10/10) | 0 (10/10) |
| A.C. | ||||
| - Flare | 0 (10/10) | 0 (10/10) | 0 (10/10) | 0 (10/10) |
| - Cells | 0 (10/10) | 0 (10/10) | 0 (10/10) | 0 (10/10) |
| Fluorescein staining | 0 (10/10) | 0 (10/10) | 0 (10/10) | 0 (10/10) |
| Iris | 0 (10/10) | 0 (10/10) | 0 (10/10) | 0 (10/10) |
| Posterior Segment | ||||
| Vitreous Haze | 0 (10/10) | 0 (10/10) | 0 (10/10) | 0 (10/10) |
| Degraded fundus view | 0 (10/10) | 0 (10/10) | 0 (10/10) | 0 (10/10) |
| Retinal perivascular sheathing | 0 (10/10) | 0 (10/10) | 0 (10/10) | 0 (10/10) |
Data are presented as “score (number of eyes with score / total number of eyes)”
A.C. = anterior chamber
Repeated instillation of Flebogamma® 10% DIF showed mild hyperemia (score 1) in 80% of eyes on Day 7. This phenotype partially resolved with only 50% of eyes showing mild hyperemia (score 1) on Day 14. No other ophthalmic signs were noted (Table 2). Notably, topical administration of OSIG did not cause any sign of inflammation, and corneal fluorescein staining was normal (score 0) in all eyes.
STT was performed to evaluate any potential effect of Flebogamma® DIF instillation on tears. No toxicologically relevant or statistically significant effects on tears were identified, and STT values were in the normal range for rabbits (Table 3).
Table 3.
Tear volume (in mm) as assessed by STT after repeated dose topical ophthalmic administration of Flebogamma® 5% and 10% DIF.
| Flebogamma® 5% DIF (mean ± S.D. [min, max]) |
Flebogamma® 10% DIF (mean ± S.D. [min, max]) |
|
|---|---|---|
| Baseline | 8.4 ± 0.5 [6, 11] | 9.7 ± 2.1 [7, 14] |
| Day 7 | 10.6 ± 3.3 [6, 16] | 9.2 ± 2.0 [7, 14] |
| Day 14 | 8.1 ± 0.5 [5, 10] | 8.6 ± 1.7 [6, 11] |
IOP measurements were performed by rebound tonometry in awake animals. IOP was within normal range for NZW rabbits [9] and was unaffected by topical OSIG treatment (Table 4).
Table 4.
Intraocular pressure (in mm Hg) after repeated dose topical ophthalmic administration of Flebogamma® 5% and 10% DIF.
| Flebogamma® 5% DIF (mean ± S.D. [min, max]) |
Flebogamma® 10% DIF (mean ± S.D. [min, max]) |
|
|---|---|---|
| Baseline | 11.7 ± 2.2 [8, 15] | 11.2 ± 1.4 [10, 13] |
| Day 7 | 11.8 ± 2.2 [9, 15] | 11.8 ± 1.9 [9, 14] |
| Day 14 | 11.5 ± 1.8 [8, 14] | 12.3 ± 1.6 [10, 15] |
Discussion
This is the first report evaluating the tolerability and safety of topical instillation of high-dose Flebogamma® DIF pooled immune globulins to the rabbit eye. Flebogamma® 5% DIF and Flebogamma® 10% DIF were well-tolerated following four times daily topical instillation with no clinically or toxicologically relevant signs, supporting the ongoing development of Flebogamma® 5% DIF for the treatment of dry eye disease.
Based on in vitro dose-range studies that evaluated the efficacy of pooled human immune globulins against dry eye disease-relevant mechanisms, including neutrophil activation and adhesion to corneal epithelial cells, we identified Flebogamma® 5% DIF as the most relevant concentration for the ongoing development of OSIG [7]. Furthermore, based on our initial clinical experience, a twice-daily topical administration by eyedrop is the most desirable clinical dosing regimen [5]. Therefore, we designed our initial non-GLP ocular tolerability study to include both a dose and dosing regimen escalation of Flebogamma® 5% DIF and Flebogamma® 10% DIF administered four times daily (q.i.d.).
Topically administered macromolecules, such as antibodies, typically do not penetrate the cornea, as demonstrated in ex vivo and in vivo systems [10, 11]. While this poses drug delivery challenges for posterior segment diseases, limited ocular biodistribution of OSIG predicts an excellent ophthalmic safety profile and makes them uniquely suited for the treatment of ocular surface diseases, including dry eye disease.
Indeed, ophthalmic exams were unremarkable with the exception of hyperemia in the Flebogamma® 10% DIF group, which represents both an escalated dose and dosing regimen. Given the reactivity of the rabbit eye and the mild and transient nature of the hyperemia this was deemed not toxicologically relevant. Furthermore, hyperemia is commonly observed with many approved topically instilled ophthalmic drugs [12]. Absence of any corneal fluorescein staining (Table 2) supports the choice of dry eye disease as indication for Flebogamma® DIF.
In our previous work, we have demonstrated the safety of low-dose OSIG (0.4%; Flebogamma® 5% DIF compounded to 4 mg/mL in physiological saline) in primary human corneal epithelial cells in vitro, and after repeated-dose instillation for 28 days in mice [5]. This led to an Investigator-initiated first-in-human, double-masked, vehicle-controlled, randomized clinical trial to determine the safety and preliminary efficacy of OSIG eye drops (0.4%) used two times a day for eight weeks to treat DED patients who had high ACPA levels in their ocular surface washes ([5]; ClinicalTrials.gov Identifier: NCT03992482). In this trial, OSIG were well-tolerated and the incidence of adverse effects was lower compared to the vehicle group that received preservative-free saline solution. Of particular relevance, OSIG resulted in a statistically significant improvement in both signs and symptoms of DED. Corneal staining was reduced by 1.5 units in the OSIG group and concomitant with a 20-unit reduction in the Ocular Surface Disease Index (OSDI, [13]) score [5]. Biomarker analysis in ocular surface washes revealed a 50% reduction in IL-8 and a 24% reduction in extracellular DNA [5].
Recently, others evaluated a different pooled human immune globulin formulation, VIG-SN™ (IV-Globulin SN™, 10%, Green Cross Corporation, KPharm, Seoul, Korea), for efficacy in a rabbit model for DED [14]. Specifically, 0.4% VIG-SN™ delivered twice daily for 20 days in New Zealand White rabbits with benzalkonium chloride-induced ocular surface disease resulted in a statistically significant improvement of tear break-up time and corneal fluorescein staining [14]. Previously, Gammagard® S/D (Baxter Healthcare) reconstituted with water had shown good ocular tolerability in NZW rabbits at concentrations of 6, 30, and 100 mg/mL following four times daily instillation for five consecutive days [15].
Rabbits are the preferred preclinical laboratory species for tolerability and safety assessments of topically delivered agents and are commonly used in the existing safety data for ophthalmic drugs [16]. This is in part due to the size of the rabbit eye, which allows for meaningful assessments of acute and chronic toxicity. However, despite its utility, tolerability assessments in a rabbit model have limitations. While we here included STT to evaluate potential effects on the tear film, we acknowledge that findings on tear film in rabbits offer limited translatability to humans. Firstly, rabbits do not produce a large amount of tears and that tear volume is highly variable [17]. Typical mean STT wetting lengths are around 5 mm (corresponding to an approximate 3 μL volume; [17]), with a range of 3 – 15 mm for a 60 s test. Secondly, rabbits have a much more stable tear film, resulting in long inter-blink times. Previous studies have estimated an inter-blink time of around ~10 min in rabbits, compared with humans 5 - 8 s in humans [18]. This explains the significantly longer tear break up times in rabbits versus humans (~30 min vs. 30 s, respectively; [18]).
Furthermore, despite the high degree of immunogenicity of rabbit ocular tissues, we did not observe any immune responses following topical instillation of human immune globulins for two weeks. Nonetheless, long-term IND-enabling ocular toxicology studies will have to evaluate anti-drug antibodies. These ongoing long-term studies will also identify any other potential toxicological effects, such as effects on limbal stem cells, that may only manifest after prolonged administration.
Conclusions
Flebogamma® 5% DIF and Flebogamma® 10% DIF were well-tolerated by NZW rabbits following repeated dose topical ophthalmic administration four times daily for two weeks. These critical safety and tolerability data support the future development of topical Flebogamma® DIF pooled human immune globulins for the treatment of ocular surface disease.
Acknowledgements
This research was funded, in part, by NIH/R24 EY032440 (S.J., S.K.), Dr. John P. and Therese E. Mulcahy Endowed Professorship in Ophthalmology (S.K.), and Grifols.
Footnotes
Declaration of interest statement:
SMZ and BPL are employees of Grifols. SJ holds equity interest in Selagine, Inc. and is an inventor on a patent pertaining to the use of pooled human immune globulins for dry eye disease (WO 2020/097528 A1).
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