(M1130-03-20) Transport Characterization of Tramadol in an Ex Vivo Blood-Brain Barrier Model Based on Microimpalement of Vessels

Purpose: The blood-brain barrier (BBB) is a highly selective membrane that limits the passage of most therapeutic compounds, making drug delivery to the brain a significant challenge. To study drug transport across the blood-brain barrier (BBB), the recently developed ISMICAP (in situ microperfusion of capillaries) model by Hanafy et al, which utilizes 2-photon microscopy for detection and quantification, has been further developed with the aim of enabling the detection of molecules without fluorophores and to measure their concentrations. Here we directly measure drug concentrations after BBB-permeation, allowing a better understanding potential, dose dependencies, adverse effects, or involved transport mechanisms. Tramadol was selected as a model substance due to its established pharmacokinetics and its ability to cross the BBB [1].

Methods: 300 µm thick acute brain slices from male C57BL/6N mice, aged 22–35 days postnatal, were used to maintain a fully functional BBB. For brain slice preparation, mice were anesthetized and rapidly decapitated. The whole brain was quickly removed from the skull and submerged in ice-cold modified artificial cerebrospinal fluid (mACSF). Horizontal slices were cut in ice-cold mACSF, oxygenated with 5% CO₂ and 95% O₂, using a vibratome (HM650 V, Thermo Fisher Scientific, Waltham, USA) and subsequently incubated for 30 min at 35°C in mACSF. For perfusion experiments, slices were transferred to standard artificial cerebrospinal fluid (sACSF). [2] Brain slices were placed in the chamber of a fluorescent microscope and constantly perfused with sACSF at a flow rate of 1-2 ml/min.  Solutions for injections were prepared by adding tramadol and biocytin tetramethylrhodamine (TMR) from stock solutions to sACSF to achieve concentrations of 0.2, 0.5, 2, 5 and 20 mmol/l for tramadol and 0.1 mmol/l for TMR. Solutions were filled into self-made, pulled and beveled pipettes, which were then used to impale and perfuse blood vessels in the brain [2]. Successful injections were confirmed by the absence of extravascular fluorescence. A second self-made pipette with a wider tip was lowered into the tissue, adjacent to the perfused vessel. Negative pressure was applied for sampling at defined time points. The collected samples were filtered and subsequently analyzed using (U-)HPLC with fluorescence detection. Analysis was performed on a LC-2040C 3D Plus system (Shimadzu, Kyoto, Japan) using a C18 reversed-phase column (Nucleodur 100-3, Macherey-Nagel).

Results: For perfusion experiments, venules with  varying size of diameter were used. The outer diameter of the perfused vessels was measured to assess its influence on tramadol permeation, potentially explained by varying transport surface areas. No correlation was found for perfusion at 0.5 and 5 mmol/l. Consequently, for following experiments, the diameter of the perfused vessels does not need to be considered, as the extravascular concentration appears to be independent of vessel diameter. Perfusion experiments demonstrate that tramadol rapidly reaches a steady-state concentration due to its efficient permeation across the blood-brain barrier. With vascular perfusions at concentrations of 0.2, 2 and 20 mmol/l, the maximum extravascular concentration was reached after 5 minutes at 1.26 ± 0.57, 1.87 ± 0,89 and 10.08 ± 6,76 µmol/l, respectively. The correlation between the perfused concentration and the local concentration in the ACSF shows dose linearity in the range of 0.2 to 20 mmol/l for perfusion times of 5, 15 min and 30 min. The correlation was determined using linear regression, which yielded correlation coefficients of 0.9938, 0.9785, and 0.9325 for the time after perfusion of 5, 15, and 30 minutes, respectively. The slopes of these correlations decrease over time, which can be explained by a reduced diffusion rate across the BBB and the diffusion of tramadol into the surrounding tissue. It is also possible that Tramadol undergoes active transport, as suggested in previous studies [3], and that these mechanisms become saturated over time.

Conclusion: This study demonstrates that the developed ex-vivo BBB model could be used for determining drug concentrations in brain tissue und it was shown that the model is a reliable tool for investigating drug transport across the BBB. For implementation, the model confirmed as expected efficient permeation of tramadol across the BBB with rapid establishment of a steady-state concentration in brain tissue. The observed dose linearity across different perfusion concentrations shows a predictable relationship between adminstered and local concentrations. Future studies will focus on exploring the role of active transport mechanisms in the BBB and the impact of different drug delivery strategies on brain drug concentrations, using a wider range of model substances.

References: [1] B. Sheikholeslami, M. Gholami, H. Lavasani, M. Rouini, Evaluation of the route dependency of the pharmacokinetics and neuro-pharmacokinetics of tramadol and its main metabolites in rats, Eur. J. Pharm. Sci. 92 (2016) 55–63. https://doi.org/10.1016/j.ejps.2016.06.021.
[2] A.S. Hanafy, P. Steinlein, J. Pitsch, M.H. Silva, N. Vana, A.J. Becker, M.E. Graham, S. Schoch, A. Lamprecht, D. Dietrich, Subcellular analysis of blood-brain barrier function by micro-impalement of vessels in acute brain slices, Nat. Commun. 14 (2023) 481. https://doi.org/10.1038/s41467-023-36070-6.
[3] A. Kitamura, K. Higuchi, T. Okura, Y. Deguchi, Transport characteristics of tramadol in the blood-brain barrier, J. Pharm. Sci. 103 (2014) 3335–3341. https://doi.org/10.1002/jps.24129.


Figure 1: Dependence of the extravascular concentration on the diameter of the perfused vessel


Figure 2: (A) Time profile of extravascular concentrations after perfusion with different tramadol concentrations and (B) concentration plots for correlation