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Published August 2022 | Published + Submitted + Supplemental Material
Journal Article Open

Photoacoustic imaging reveals mechanisms of rapid-acting insulin formulations dynamics at the injection site


Objective: Ultra-rapid insulin formulations control postprandial hyperglycemia; however, inadequate understanding of injection site absorption mechanisms is limiting further advancement. We used photoacoustic imaging to investigate the injection site dynamics of dye-labeled insulin lispro in the Humalog® and Lyumjev® formulations using the murine ear cutaneous model and correlated it with results from unlabeled insulin lispro in pig subcutaneous injection model. Methods: We employed dual-wavelength optical-resolution photoacoustic microscopy to study the absorption and diffusion of the near-infrared dye-labeled insulin lispro in the Humalog and Lyumjev formulations in mouse ears. We mathematically modeled the experimental data to calculate the absorption rate constants and diffusion coefficients. We studied the pharmacokinetics of the unlabeled insulin lispro in both the Humalog and Lyumjev formulations as well as a formulation lacking both the zinc and phenolic preservative in pigs. The association state of insulin lispro in each of the formulations was characterized using SV-AUC and NMR spectroscopy. Results: Through experiments using murine and swine models, we show that the hexamer dissociation rate of insulin lispro is not the absorption rate-limiting step. We demonstrated that the excipients in the Lyumjev formulation produce local tissue expansion and speed both insulin diffusion and microvascular absorption. We also show that the diffusion of insulin lispro at the injection site drives its initial absorption; however, the rate at which the insulin lispro crosses the blood vessels is its overall absorption rate-limiting step. Conclusions: This study provides insights into injection site dynamics of insulin lispro and the impact of formulation excipients. It also demonstrates photoacoustic microscopy as a promising tool for studying protein therapeutics. The results from this study address critical questions around the subcutaneous behavior of insulin lispro and the formulation excipients, which could be useful to make faster and better controlled insulin formulations in the future.

Additional Information

© 2022 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Received 10 March 2022, Revised 20 May 2022, Accepted 24 May 2022, Available online 4 June 2022, Version of Record 14 June 2022. We thank Steven Cottle and Linh Nguyen, Eli Lilly and Company for preparing the insulin formulation solutions. We also thank Jake Anderson, Eli Lilly and Company for useful discussions. This work was funded by Eli Lilly and Company. Data availability: The data that support the conclusions are present in the main draft or the supplementary information. The data processing and quantification procedures are described in detail in the Methods section. Author contributions: L.V.W., S.S.O., J.M.B., C.D.P., and A.K. conceived the project and the ideas. C.D.P. designed the chemistry for dye labeling and analyzed the chemistry data. F.A.V. labeled the insulin molecules with the sulfo-cyanine7.5 dye and characterized them. F.A.V. and A.K. prepared the insulin and dye buffer solutions. A.K. and K.M. designed and built the scanning photoacoustic microscope. J.S. and R.C. wrote the LabVIEW software for photoacoustic data acquisition. A.K. designed and performed all the photoacoustic experiments, wrote the MATLAB codes, and analyzed all the photoacoustic data. A.K., K.M., and L.V.W. designed the photoacoustic quantification algorithm. A.K. and K.M. devised the pharmacokinetics and diffusion methodologies. A.L.C. designed and supported the pharmacokinetics study in pigs and A.E.S. performed the pharmacokinetic experiments. R.M. prepared the unlabeled insulin lispro samples, and designed and performed the SV-AUC experiments. S.A.B. designed and performed the NMR spectroscopy analyses. A.K., C.D.P., K.M., J.M.B, and L.V.W. interpreted the final data. P.B.A., E.L., and R.L.B. were involved in active discussions. L.V.W., S.S.O., and J.M.B. supervised the project. A.K. wrote the manuscript. C.D.P., K.M., R.C., J.M.B, S.S.O., and L.V.W. contributed to writing the manuscript. Competing interests: A.K., R.C., and J.S. declare no competing interests. C.D.P., F.A.V, A.E.S., A.M.C., P.L.B.A., E.L., R.L.B, R.M., SA.B., J.M.B, and S.S.O. are employees and stockholders of Eli Lilly and Company. L.V.W. and K.M. have financial interests in Microphotoacoustics, Inc., CalPACT, LLC and Union Photoacoustic Technologies, Ltd, which did not support this work. L.V.W. received a contract from Eli Lilly and Company to conduct the studies reported herein.

Attached Files

Published - 1-s2.0-S2212877822000916-main.pdf

Submitted - 2022.03.10.483309v1.full.pdf

Supplemental Material - 1-s2.0-S2212877822000916-mmc1.zip


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Additional details

August 22, 2023
November 16, 2023