(T1230-01-01) Development and Validation of a Novel LC-MS/MS Method for the Simultaneous Quantification of Alvocidib and Its Glucuronide: Application to Pharmacokinetic and Tissue Distribution Study in Rats

Purpose: Alvocidib (Flavopiridol) is a potent cyclin-dependent kinase (CDK) 9 inhibitor that has been approved for the treatment of acute myeloid leukemia (AML). However, clinical use of alvocidib has been associated with side effects such as dose-limiting secretory diarrhea, hypotension, fatigue, vomiting, neutropenia, cytokine release syndrome, and tumor lysis syndrome, all of which make treatment less effective. A study linked a lower incidence of diarrhea in patients who are extensive glucuronidators of the drug. Thus, it is necessary to understand the pharmacokinetics of alvocidib and monitor alvocidib glucuronide (Alvo-G) in vivo exposure for a better understanding of its efficacy and toxicity. Currently, there is no known method to simultaneously quantify alvocidib and its glucuronide in literature. The aim of this study is to develop a robust and sensitive LC-MS/MS method to simultaneously quantify alvocidib and its metabolite, Alvo-G, and apply this method in a pharmacokinetic (PK) and tissue distribution study in rats.

Methods: A UHPLC system coupled to a QTrap 4000 triple quadrupole mass spectrometer was used for the analysis. The rat plasma and tissue samples were processed using an organic-based protein precipitation method. The analytes were separated using an Ultra Biphenyl 5 µm column (50 × 2.1 mm) with 0.1% formic acid in water (A) and acetonitrile (B) as the mobile phases, employing a gradient elution method. They were then quantified under positive electrospray ionization (ESI) mode and a multiple reaction monitoring (MRM) approach. The method was validated per ICH M10 guidelines for linearity, accuracy, precision, recovery, matrix effect, carry-over, and stability [4]. Pharmacokinetic studies were conducted in male Sprague-Dawley rats (n=5) dosed with 10 mg/kg alvocidib P.O, with plasma samples collected at multiple time points and analyzed to determine PK parameters C_max and T_max and AUC and t_½. The liver, small intestine, colon, and kidneys were collected 24 hours post-dose and processed to assess the distribution of the alvocidib and Alvo-G in these tissues.

Results: The method showed a good linearity within the range of 2.4 nM – 5000.00 nM for both analytes, exhibiting correlation coefficient values, R > 0.99. The method was reproducible, with intra- and inter-day accuracy and precision ranging from 84-98% and < 13%, respectively, for all analytes in rat plasma. The extraction recovery and matrix effect of rat plasma using acetonitrile (100% v/v) achieved acceptable ranges (82-105%). Stability studies carried out on the analytes in plasma demonstrated that they were stable under bench-top (91-99%), freeze-thaw (83-110%), and long-term storage conditions (80-106%). This validated method was successfully applied to quantify alvocidib and Alvo-G in rats. The pharmacokinetic study results showed that after oral administration of alvocidib, both alvocidib and its glucuronide were detected in plasma, with the parent drug showing the highest distribution in the colon and lowest in the small intestine. In contrast, the glucuronide showed the highest distribution in the small intestine and the least amount in the colon as compared to the other tissues.

Conclusion: This study established a novel, robust, and sensitive LC-MS/MS method for the simultaneous quantification of alvocidib and its metabolite, Alvo-G, and successfully applied it to pharmacokinetic and tissue distribution studies in rats. Pharmacokinetic studies showed that alvocidib can be rapidly converted into its glucuronide form. The levels of alvocidib and Alvo-G were distinct in the different tissues, suggesting that both alvocidib and its glucuronide should be taken into consideration in efficacy and toxicity studies. This method will enable future pharmacokinetic studies and interventions to prevent the toxicities of Alvocidib by potentially modulating its glucuronidation to avoid systemic toxicity.

References: 1. Joshi, H., et al., The Pharmacological Implications of Flavopiridol: An Updated Overview. Molecules, 2023. 28(22).
2. Wiernik, P.H., Alvocidib (flavopiridol) for the treatment of chronic lymphocytic leukemia. Expert Opin Investig Drugs, 2016. 25(6): p. 729-34.
3. Innocenti, F., et al., Flavopiridol metabolism in cancer patients is associated with the occurrence of diarrhea. Clin Cancer Res, 2000. 6(9): p. 3400-5.
4. Center for Drug Evaluation and Research, F. M10 Bioanalytical Method Validation and Study Sample Analysis. 2022 [cited 2025; Available from: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/m10-bioanalytical-method-validation-and-study-sample-analysis.

Acknowledgements: This study was funded in part by the Cancer Prevention & Research Institute of Texas (CPRIT) Core Facilities Support Awards (RP240430), R16 grant GM149425, and the NIH/NIMHD grant U54MD007605.

Chemical Structures of alvocidib and alvocidib Glucuronide (Alv-G) and representative chromatogram of a PK sample.

Plasma concentration versus time profiles of the analytes after oral administration of Alvocidib. Each symbol represents the mean, and the error bars indicate the S.D. (n = 5)

The liver, kidney, small intestine, and colon concentration of Alvocidib and Alv-G at 24 hours after 10 mg/kg oral administration of Alvocidib. Each symbol represents the mean, and the error bars indicate the S.D. (n = 5).