(W0930-05-28) LC-MS/MS Method Development and Validation for Evaluation of Brain Pharmacokinetic Parameters of Metformin after an Intravenous Bolus Administration: Application in Ischemic Stroke

Purpose: Metformin, a widely prescribed anti-diabetic drug, has been shown by our lab to attenuate post-ischemic brain injury promoted by stroke [1, 2]. Additionally, metformin’s effect on age-related neurological diseases is being studied in pre-clinical investigations [3]. However, most published studies have not established dose/concentration and brain response effects because of the lack of metformin quantification in the brain [4, 5]. Therefore, our purpose is to evaluate the brain pharmacokinetic parameters of metformin utilizing a therapeutically relevant dose using LC-MS/MS quantification.

Methods: In our study, we developed and validated an LC-MS/MS method using the US-FDA bioanalytical method guidelines that involved selectivity, accuracy and precision, recovery, and freeze-thaw stability.2-3 months old male C57BL/6J mice with 23-27g body weight were used for the pharmacokinetic study, where they were intravenously administered 30 mg/kg metformin, and brains were collected at 10-, 20-, and 30-minutes time points. We estimated % of the injected dose reaching the brain (Cbr) and unidirectional blood-to-brain influx constant (kin).

Results: Our study showed that we could accurately quantify metformin in brain and plasma samples from the developed and validated LC-MS/MS method. The plasma profile showed a biexponential decline until 30 minutes time point, while the Cbr for 10-, 20- and 30-minutes values were found to be 0.027 ± 0.003, 0.030 ± 0.003, and 0.033 ± 0.004, respectively. Furthermore, the kin value was calculated to be 1.75 ± 0.112, 0.74 ± 0.023, and 0.78 ± 0.09 for 10, 20 and 30 minutes, respectively (mean ± S.D, n=3-4).

Conclusion: LC-MS/MS method for metformin in brain and plasma was developed and validated under the US-FDA guidelines. The preliminary Cbr and kin values for metformin after an intravenous administration were determined. High kin values at earlier time points suggest the involvement of active transporter-mediated uptake from blood to the brain. For future studies, we will evaluate Cbr and kin at more sampling time points and assess these parameters during an in-vivo stroke by performing transient middle cerebral artery occlusion surgery that is routinely utilized in our lab.

References:
1. Prasad, S., et al., Role of Nrf2 and protective effects of Metformin against tobacco smoke-induced cerebrovascular toxicity. Redox biology, 2017. 12: p. 58-69.
2. 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.
3. Sharma, S., et al., Repurposing metformin to treat age-related neurodegenerative disorders and ischemic stroke. Life Sciences, 2021. 274: p. 119343.
4. Song, R., Mechanism of metformin: a tale of two sites. Diabetes care, 2016. 39(2): p. 187-189.
5. Chaudhari, K., et al., Determination of metformin bio-distribution by LC-MS/MS in mice treated with a clinically relevant paradigm. PloS one, 2020. 15(6): p. e0234571.

Acknowledgements:The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding: NIH R01NS117906


Figure 1: Panel A, mass spectra of metformin demonstrating the precursor and product ions considered in validation. Panel B, chromatograms of blank brain matrix spiked with the internal standard metformin d-6. Panel C, chromatograms for metformin and metformin d-6 in plasma matrix. Panel D, chromatograms for metformin and metformin d-6 in brain matrix.


Figure 2: 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 brain matrix.


Figure 3: Panel A, Plasma and brain pharmacokinetic profile reported as percentage injected dose of metformin. Panel B, Brain concentrations (Cbr) of metformin at different time points until 30 minutes. Panel C, unidirectional blood-to-brain influx constant (kin) using single-time point analysis. (mean ± S.D, n=3-4 animals)