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Discovery bioanalysis and in vivo pharmacology as an integrated process: a case study in oncology drug discovery.

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Method. The article is a realistic description of the comprehensive work flow implemented within our drug discovery department in oncology to obtain preliminary exposures data in mice. Many different parameters were considered: i/ the overall drug discovery objective of identifying as quickly and safely as possible a candidate drug; ii/ the ethical view of getting as much information as possible from a single animal sampling, with both reduction and refinement of the protocols (the “3Rs” principle); iii/ the chemist request of getting PK data to build the Structure Activity Relationship; iv/ the pharmacologist constraints in terms of work load, sampling strategy, ergonomy and animal welfare; v/ the bioanalyst perspective, at the interface between chemistry and pharmacology, with specific validation steps, methodologies, devices; vi/ and the compromise between cost and data generation.

Hybrid high resolution mass spectrometers (HR/MSMS) were found to be the most versatile instruments for implementing Quan / Qual bioanalytical approaches[1]. In vivo pharmacologists considered the dried blood spot (DBS) microsampling technique as the least invasive in their daily workload. Whole blood matrix instead of plasma matrix was chosen. To overcome spot homogeneity issue, or the heterogeneous distribution profile of analyte across the DBS, which can have a significant impact on quantitative results, accurate sample volumes (10µl) and whole spot extraction were carried out[2]. The Figure 1 illustrates our overall process of serial tail fixed-volume micro-sampling of whole blood on DBS with HR/MSMS bioanalysis. The method of serial microsampling implemented in our laboratory provided also opportunities to perform new and informative studies with a minimum number of mice per experiment. The Figure 2 shows results from a bioequivalence study carried out for a vehicle screening before in vivo pharmacology studies.

Case Study. Acute myeloid leukemia (AML) represents a significant unmet medical need. Progress in treatment to reduce relapse and induce long term remission has been reduced so far. Therefore the development of more effective drugs for AML is a high priority. Approximately 30% of patients with AML have activating mutations in the FLT3 gene. Although several FLT3 inhibitors currently ongoing clinical trials have shown encouraging results in terms of response rate, responses are only transient and resistance develops rapidly. The bone marrow microenvironment can diminish sensitivity to FLT3 inhibitors. Based on published data[3], we hypothesized that the inhibition of JAK2 kinase in addition to FLT3 may be effective in overriding drug resistance in AML. Our compound PFX shows potent anti-leukemic effects on AML models driven by mutant FLT3. After multiple oral administrations of PFX at various doses, a marked increase of lifespan (50-80%) was observed in MV4-11-bearing mice as shown on Figure 3.

A PK/PD study showed that the anti-leukemic activity of PFX correlated with the inhibition of STAT5 phosphorylation in both circulating and bone marrow leukemic cells from PFX blood concentration of 100ng/ml (Figure 4). STAT5 was used as pharmaco-dynamic biomarker (PD) of FLT3 inhibition. These results demonstrated that PFX exhibited a marked preclinical anti-leukemic activity. Moreover the combined inhibition of FLT3 and JAK2 can override tumor resistance (unpublished data) and might result in prolonged clinical responses. Furthermore, potential combinations with immune-checkpoint inhibitors could open new perspectives of FTL3/JAK2 inhibitors.

Conclusion. This article has put in evidence the importance of integrating bioanalysis to pharmacology in order to respond to specific biological and pharmacological questions. New technologies like HRMS and serial microsampling were discussed and the most appropriate one for each activity was selected and implemented in our laboratory. Results were liberated faster to both medicinal chemists for SAR and pharmacologists for optimising in vivo experiment studies.

References:

[1] King L, Kotian A, Jairaj M. Introduction of a routine quan/qual approach into research DMPK: experiences and evolving strategies. Bioanalysis 6(24), 3337–3348 (2014).

[2] Wagner M, Tonoli D, Varesio E, Hopfgartner G. The use of mass spectrometry to analyze dried blood spots. Mass Spectrom. Rev. 35, 361–438 (2016).

[3] Leung A, Man C, Kwong Y. FLT3 inhibition: a moving and evolving target in acute myeloid leukaemia. Leukemia 27, 260-268 (2013)