(T1430-05-32) Co-Modeling of Re (CO)₃-Phenformin and Phenformin in Whole Blood and Tissues after Dosing of Re (CO)₃-Phenformin in Mice with Validated LC-MS/MS Assay

Purpose: Phenformin (N-phenylethyl biguanide) exhibits higher antitumor potency than metformin due to increased lipophilicity and enhanced tumor cell uptake. However, its clinical application is limited by systemic toxicity and poor tumor selectivity. Re (CO)₃-Phenformin (Re-Phen), a rhenium tricarbonyl complex of phenformin, was synthesized to enhance tumor targeting and induce mitochondrial membrane disruption through a mechanism distinct from classical oxidative phosphorylation inhibition. This study focuses on investigating the pharmacokinetics and systemic metabolic conversion of Re-Phen to phenformin in mice using a parent–metabolite co-modeling approach. A sensitive and validated LC-MS/MS method was established to support quantification of both compounds in whole blood and tissues, enabling mechanistic interpretation and future tissue distribution analysis of converted phenformin.

Methods: Re-Phen and phenformin were quantified using an optimized LC-MS/MS method. Chromatographic separation was performed on a Kinetex XB-C₁₈ column (2.1 mm × 100 mm, 1.7 µm) with a mobile phase of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) at a flow rate of 0.3 mL/min. Mass spectrometric detection was conducted in positive ion mode using multiple reaction monitoring (MRM): Re-Phen (m/z 476 → 375/105) and phenformin (m/z 207.2 → 105). D₅-phenformin (m/z 210.7→ 60/110) was used as the internal standard. Sample extraction from mouse whole blood and tissues was carried out by protein precipitation using acetonitrile with 0.1% formic acid. To accurately quantify converted phenformin following Re-Phen dosing, three calibration curves were constructed in pre-spiked mouse whole blood based on peak area (PA) versus molar concentration (mol/L): (1) a calibration curve for Re-Phenformin (m/z 375), (2) a calibration curve for the 105 ion derived specifically from Re-Phenformin (fragment contribution), and (3) a calibration curve for pure phenformin (m/z 105) (Figure 1). To calculate the concentration of converted phenformin, the molarity of Re-Phenformin was first determined using the m/z 375 calibration curve. From this, the expected PA at m/z 105 due to Re-Phenformin fragmentation was calculated. This PA was subtracted from the total observed PA at m/z 105 to isolate the signal corresponding to converted phenformin. The resulting PA was then converted to molarity using the phenformin calibration curve. The method was validated per FDA bioanalytical guidelines, including assessment of linearity (0.25–100 ng/mL), accuracy, precision (intra-/inter-day), recovery, matrix effect, and stability in mouse whole blood. Tissue samples were also harvested for planned quantification of Re-Phen and converted phenformin, currently under analysis. Mice (n=4) were dosed intravenously with Re-Phen (25 mg/kg), and serial whole blood samples were collected at defined time points. Phoenix WinNonlin^® software was used for parent–metabolite co-modeling based on two-compartment models for Re-Phen and phenformin. The model included a metabolic link to represent in vivo conversion kinetics.

Results: The method achieved LLOQ of 0.25 ng/mL and showed excellent linearity (R² > 0.99) for both analytes. Mean recovery in whole blood was 93.9% for Re-Phen and 91.95% for phenformin. ME ranged from 86.6% to 91.3% for Re-Phen and 87.3% to 102.5% for phenformin. Intra- and inter-day precision and accuracy values for 5 QC levels were within ±15% CV for both analytes (Figures 2 and 3: validation data tables for each compound). Following IV administration, phenformin was detected in whole blood, confirming in vivo conversion. The co-modeling framework captured parent–metabolite kinetics, enabling estimation of key PK parameters and visualizing the temporal appearance of phenformin post Re-Phen administration. Tissue concentrations of both Re-Phen and converted phenformin will be reported upon ongoing quantification.

Conclusion: A sensitive and selective LC-MS/MS assay was developed and validated for simultaneous quantification of Re-Phen and phenformin in whole blood. The method successfully resolved analytical interference from shared daughter ions through a three-curve strategy. Application of this method enabled parent–metabolite co-modeling in whole blood and confirmed systemic conversion of Re-Phen to phenformin. The approach provides a robust platform for mechanistic PK evaluation and supports the continued preclinical development of Re-Phen as a targeted anticancer agent for PDAC.

Acknowledgements: This work is supported in part by the John S. Dunn Foundation and a National Cancer Institute grant (R01CA258540).

Figure 1. (A) Fragment ions of Re(CO)₃-Phenformin and phenformin showing shared m/z 105. (B) Calibration curves in pre-spiked blood for Re-Phen (m/z 375), Re-Phen-derived 105, and phenformin (m/z 105).

Figure 2. Validation of Re (CO)₃-Phenformin in Mouse Whole Blood: Recovery, Matrix Effect, and Intra-/Inter-Day Accuracy and Precision by LC-MS/MS

Figure 3. Validation of Phenformin in Mouse Whole Blood: Recovery, Matrix Effect, and Intra-/Inter-Day Accuracy and Precision by LC-MS/MS