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Ex vivo investigation of ocular tissue distribution following intravitreal administration of connexin43 mimetic peptide using the microdialysis technique and LC-MS/MS

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Abstract

This study aimed to develop and evaluate an ex vivo eye model for intravitreal drug sampling and tissue distribution of connexin43 mimetic peptide (Cx43MP) following intravitreal injection using the microdialysis technique and LC-MS/MS. An LC-MS/MS method was developed, validated, and applied for quantification of Cx43MP in ocular tissues. Microdialysis probes were calibrated for in vitro recovery studies. Bovine eyes were fixed in a customized eye holder and after intravitreal injection of Cx43MP, microdialysis probes were implanted in the vitreous body. Vitreous samples were collected at particular time intervals over 24 h. Moreover, 24 and 48 h after intravitreal injection ocular tissues were collected, processed, and analyzed for Cx43MP concentrations using LC-MS/MS. The LC-MS/MS method showed good linearity (r 2 = 0.9991). The mean percent recovery for lower (LQC), medium (MQC), and higher quality control (HQC) (0.244, 3.906, and 125 μg/mL) was found to be 83.83, 84.92, and 94.52, respectively, with accuracy ranges between 96 and 99 % and limits of detection (LOD) and quantification (LOQ) of 0.122 and 0.412 μg/mL. The in vitro recovery of the probes was found to be over 80 %. As per microdialysis sample analysis, the Cx43MP concentration was found to increase slowly in the vitreous body up to 16 h and thereafter declined. After 48 h, the Cx43MP concentration was higher in vitreous, cornea, and retina compared to lens, iris, and aqueous humor. This ex vivo model may therefore be a useful tool to investigate intravitreal kinetics and ocular disposition of therapeutic molecules after intravitreal injection.

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Abbreviations

ACN:

Acetonitrile

AMD:

Age-related macular degeneration

Cx43MP:

Connexin43 mimetic peptide

CMV:

Cytomegalovirus

DR:

Diabetic retinopathy

LC-MS/MS:

Liquid chromatographic tandem mass spectroscopy

RP:

Reverse phase

Octd:

Octreotide

MRM:

Multiple reactions monitoring

RSD:

Relative standard deviation

LOD:

Limit of detection

LOQ:

Limit of quantification

IPBS:

Isotonic phosphate buffer saline

LQC:

Lower quality control

MQC:

Medium quality control

HQC:

High quality control

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Acknowledgments

The authors acknowledge the University of Auckland for providing a Doctoral Scholarship to Rohit Bisht and the Department of Pharmaceutical Sciences, University of Missouri-Kansas City (UMKC), for providing the research facilities. The authors would also like to thank the Sir John Logan Campbell Medical Trust and the Postgraduate Student Association (PGSA), University of Auckland, for providing travel grants to Rohit Bisht to visit the University of Missouri-Kansas City.

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Authors and Affiliations

  1. Buchanan Ocular Therapeutics Unit (BOTU), Department of Ophthalmology, New Zealand National Eye Centre, University of Auckland, Auckland, 1142, New Zealand

    Rohit Bisht & Ilva D. Rupenthal

  2. Department of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City (UMKC), Kansas City, MO, USA

    Abhirup Mandal & Ashim K. Mitra

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  1. Rohit Bisht
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Correspondence to Ashim K. Mitra.

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There is no conflict of interest with regards to the execution of the research work undertaken as well as the preparation of the manuscript. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript.

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Bisht, R., Mandal, A., Rupenthal, I.D. et al. Ex vivo investigation of ocular tissue distribution following intravitreal administration of connexin43 mimetic peptide using the microdialysis technique and LC-MS/MS. Drug Deliv. and Transl. Res. 6, 763–770 (2016). https://doi.org/10.1007/s13346-016-0308-9

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