(M1330-04-28) Permeability of Metformin across the Blood-Brain Barrier: Repurposing a Potential Neurotherapeutic for Ischemic Stroke

Purpose: Our lab previously established that metformin (MF), a first-line type 2 diabetes treatment, activates the Nrf2 pathway and improves post-stroke recovery [1]. More recently, we determined that the in vitro blood-to-brain permeability of MF suggests that it is a highly permeable molecule [2]. Moreover, we found altered Oct1 transporter expression and increased MF permeability during both normoxic and in vitro stroke conditions. Additionally, we found MF does not interact with the efflux transporter, plasma glycoprotein. For the present study, we characterize MF’s in vivo brain pharmacokinetic (PK) parameters, such as the blood-to-brain flux constant (Kin) and estimation of brain concentration (Cbr), using a pharmacologically relevant dose. Our premise is that the integrity of BBB is compromised during in vivo ischemic stroke, and thus, consideration of the dosage is required for the safety and therapeutic utility of metformin for cerebroprotective action.

Methods: We developed and validated a highly sensitive LC-MS/MS method for metformin’s quantification in plasma and brain matrices US-FDA bioanalytical method. Then, we administer 30 mg/kg MF intravenously in mice and perfuse the brains, and collected at 5, 10, 20, 30, and 60 minutes. Lastly, we estimated the % injected dose Cbr and Kin. We will utilize an in vivo ischemic stroke model using transient middle cerebral artery occlusion (tMCAO) with 1-hour occlusion and 3 hours reperfusion.

Results: We were able to accurately quantify MF in brain and plasma samples from the developed and validated LC-MS/MS method. The plasma profile showed a bi-exponential decline until 60 minutes time point. The Cbr for 5, 10, 20, 30, and 60 minutes was found to be 0.034 ± 0.005, 0.027 ± 0.003, 0.033 ± 0.003, 0.028 ± 0.005, and 0.032 ± 0.005, respectively. There was no significant difference in the Cbr values for the time points (n= 4-5 animals, mean ± SD). Furthermore, the Kin value was calculated to be 1.87 ± 0.266, 1.7 ± 0.057, 0.68 ± 0.089, 0.87 ± 0.107, and 0.67 ± 0.089 for 5, 10, 20, 30, and 60 minutes, respectively. There are no significant differences in the Kin values for 5 mins and 10 minutes. However, there is a significant decline from 10 minutes to 20 minutes time points and no further decline in the Kin up to 60 minutes.

Conclusion: For our in vivo studies, the mean Cbr in terms of µM for 60 minutes was evaluated to be ~ 1.69 µM. Our value at 60 minutes is similar to a previous study that reported ~1.47 µM brain concentration using I.P administration in rats [3]. A significant decline in Kin value from 10 to 20 minutes suggests that MF is rapidly getting eliminated at early times. However, the Kin has no further significant changes from 20 to 60 minutes. Ongoing studies involve simulation of ischemic stroke by performing tMCAO surgery with subsequent physiologic PK analysis. This will provide us with the necessary in vitro-in vivo correlation (IVIVC) values that will inform future stroke efficacy studies that will determine the necessary dosage and time window for safe and effective drug therapy.

References: 1. Kaisar, M.A., et al., Offsetting the impact of smoking and e-cigarette vaping on the cerebrovascular system and stroke injury: Is Metformin a viable countermeasure? Redox biology, 2017. 13: p. 353-362.
2. Sharma, S., et al., Permeability of Metformin across an In Vitro Blood–Brain Barrier Model during Normoxia and Oxygen-Glucose Deprivation Conditions: Role of Organic Cation Transporters (Octs). Pharmaceutics, 2023. 15(5): p. 1357.
3. Zhang, G., et al., Development and evaluation of novel metformin derivative metformin threonate for brain Ischemia treatment. Frontiers in Pharmacology, 2022. 13.

Acknowledgements:

Funding: National Institute of Neurological Disorders and Stroke: R01 NS#117906. The authors have no conflict of interest.

Figure 1: Panel A, inter-run and intra-run accuracy and precision values of lowest, low, middle, and highest concentrations of metformin in standard curves for plasma and brain matrices, respectively (mean ± S.D, n=4). Panel B, Recoveries (mean ± S.D, n=4) of metformin in plasma and brain matrices at low, middle, and high concentrations. Panel C, freeze-thaw stability of metformin (mean ± S.D, n=3) by subjecting low, middle, and high concentrations to three freeze-thaw cycles in plasma matrix. Panel D, freeze-thaw stability of metformin (mean ± S.D, n=3) by subjecting low, middle, and high concentrations to three freeze-thaw cycles in the brain matrix.

Figure 2: Plasma and brain pharmacokinetic profile reported as percentage injected dose of metformin


Figure 3: Unidirectional blood-to-brain influx constant (kin) using single-time point analysis. (mean ± S.D, n=4-5 animals)