Purpose: BI-X is therapeutic protein that is being developed to treat human ocular disease via intravitreal administration. In addition to binding to its therapeutic targets, BI-X binds to endogenous albumin in plasma and in tissues and forms a BI-X:albumin complex with a molecular weight of ~110 KDa, which is expected to increase the drug half-life (t1/2).
Monkey ocular PK indicated that BI-X has a biphasic pharmacokinetic profile, with a short initial t1/2 of ~3 days, and a long terminal t1/2 of ~ 14 days. The initial monkey t1/2 was shorter than predicted based on previous rat and rabbit ocular PK studies (J. Ocular Pharmacology and Therapeutics (2017) 33: 115-122). Based on those studies, albumin binding was expected to extend BI-X t1/2 to at least ~ 10 days. Literature reports showed that albumin concentration in monkey vitreous humor was estimated at ~80 µg/mL (BBA. (1983) 758: 17-23). Vitreous albumin concentration is reported to be 300 µg/mL for healthy human eyes and 1600 µg/mL for diabetic retinopathic human eyes (Acta Ophthalmol.Scand. (2007) 85:618-622). Since the vitreous albumin level is critical in the understanding of the ocular PK data of BI-X, the goal of this investigation was to measure albumin in the vitreous humor of monkeys used for the ocular PK study with a sandwich ELISA method to help understand the unexpected monkey ocular PK. Concentrations of albumin in human vitreous humor from different donors were also measured to predict human ocular PK of BI-X and its intravitreal injection frequency.
Methods: 14 Cynomolgus monkeys were administered 0.25mg of BI-X in each eye via intravitreal (IVT) injection. Terminal sampling was performed at 1, 2, 4, 6, 8 and 10 weeks post-administration. Plasma samples were obtained at multiple time points during the study. Concentrations of BI-X in vitreous humor and plasma were determined using a previously published single and double immunocapture LC/MS method (JAB. (2017) Vol.3, No.5). The concentration-time data of BI-X were analyzed by non-compartmental method using composite ocular concentration-time curves. Vitreous albumin concentrations in the monkey study samples were determined with an ELISA kit (Abcam, Cat#ab190533). The same ELISA kit was used to measure human vitreous albumin from healthy donors. Both assays were qualified prior to data analysis using as calibrator a species specific albumin purified from serum.
Results: The ocular pharmacokinetics of BI-X in monkey showed a biphasic decline. In the initial phase (up to 2 – 4 weeks post-dosing), vitreous concentrations declined with a t1/2 of 3 days. In the terminal phase, vitreous BI-X concentrations could be followed up to 10 weeks post-dosing with a t1/2 of 13 days. In addition, plasma concentrations of BI-X in the monkey study were 1.2% of that of the concentration in the vitreous humor one week post-administration. (Figure 1)
The mean vitreous albumin concentration was 34 µg/mL with a range from 17 to 79.1 µg/mL. (Table 1) In contrast, albumin vitreous concentrations in healthy human samples ranged from 423.3 µg/mL to 1583.3 µg/mL with a mean of 1018.0 µg/mL. (Table 2)
Conclusion: The concentrations of vitreous albumin in the monkey ocular PK study were on average slightly lower than the previously reported 80 μg/mL. In contrast, the concentrations of vitreous albumin in healthy human vitreous samples were within the previously reported range of 80 µg/mL -1900 μg/mL. The pharmacokinetics of BI-X in the monkey ocular PK study showed a different PK profile in comparison to what was previously observed in rat and rabbits when BI-X was co-dosed with human albumin at a ratio of 1:1. This could be explained by the different molar ratio of BI-X:albumin in the monkey eye in comparison to the equimolar ratio in the rat and rabbit ocular PK studies. Considering the initial concentration of BI-X in the vitreous humor and the molecular weight difference between BI-X and albumin, the expected molar ratio in the monkey of BI-X:albumin during the initial phase is 5.6:1. This indicates that the monkey vitreous albumin concentration is insufficient to extend t1/2. The molar ratio of BI-X:albumin decreases during the terminal phase and reaches equimolarity, leading to t1/2 extension. High albumin concentrations in the human eye are expected to result in a low ratio of BI-X:albumin (~0.4:1). At this molar ratio, BI-X will exist mostly in the BI-X:albumin bound complex form. As a result, the t1/2 of BI-X in the human eye is likely to be extended to reduce intravitreal dosing frequency. Taken together these results highlight the importance of understanding mechanisms of t1/2 extension in animal species and translation to human PK predictions.
Lin-Zhi Chen– Sr.Research Fellow, Boehringer Ingelheim Pharmaceuticals, Inc.
Holger Fuchs– Lab Head, Cardio-Metabolic Diseases Research, Boehringer Ingelheim Pharma GmbH & Co. KG
Christine Grimaldi– Director of Biotherapeutic Bioanalysis, DMPK, Boehringer Ingelheim Pharmaceuticals, Inc., Connecticut
Elisa Oquendo– Principal Scientist, Boehringer Ingelheim Pharmaceuticals, Inc.
Shirin Pagels– Scientist III, Boehringer Ingelheim Pharmaceuticals, Inc.
Elsy Philip– Scientist IV, Boehringer Ingelheim Pharmaceuticals, Inc.
David Roos– Scientist IV, Boehringer Ingelheim Pharmaceuticals, Inc.
Hongbin Yu– Director, Bioanalytical Mass Spec, DMPK, Boehringer Ingelheim Pharmaceuticals, Inc.