Quantification of azacitidine incorporation into human DNA/RNA by accelerator mass spectrometry as direct measure of target engagement.
J Pharm Biomed Anal
; 202: 114152, 2021 Aug 05.
Article
en En
| MEDLINE
| ID: mdl-34051483
ABSTRACT
We report an accelerator mass spectrometry (AMS) assay to quantify azacitidine (Aza) incorporation into DNA and RNA from human acute myeloid leukemia (AML) cells, mouse bone marrow (BM) and peripheral blood mononuclear cells (PBMCs). Aza, a cytidine nucleoside analogue, is a disease modifying pharmacological agent used for treatment of myelodysplastic syndromes (MDS) and AML. Our assay was able to directly quantify the complex of Aza incorporated into DNA/RNA, via isolation of DNA/RNA from matrix (i.e., cancer cells, BM and PBMC) and subsequent measurement of total radioactivity (i.e., 14C-Aza) by using AMS. The sensitivity of the method was able to quantify as little as a single Aza molecule incorporated into DNA with approximately 2 × 107 nucleotides from PBMCs. An in vivo mouse model was used for establishing the lower limits of quantification (LLOQs) for Aza incorporated into DNA/RNA in mouse PBMCs (â¼ 3.7 × 105) and BM (â¼27.8 mg) collected 24 h post-dose after total exposure of 18 nCi/mouse (Aza 1 mg/kg). The LLOQs for PBMC analysis were 2.5 picogram equivalents per microgram (pgEq/µg) DNA and 0.22 pgEq/µg RNA, and for BM analysis were 1.7 pgEq/µg DNA and 0.22 pgEq/µg RNA. A linear relationship (i.e., â¼10-fold) was established of radioactive dose from 14C-Aza 17 nCi/mouse to 188 nCi/mouse and AMS response (i.e., 14C/12C ratio ranging from 2.45 × 10-11 to 2.50 × 10-10), as Aza was incorporated into DNA in mouse BM. The current method enables the direct measurement of Aza incorporation into DNA and RNA from patient PBMCs and BM to provide dosing optimization, and to assess target engagement with as little as â¼5 mL whole blood and â¼3 mL of BM from patients.
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Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Azacitidina
/
Leucemia Mieloide Aguda
Límite:
Animals
/
Humans
Idioma:
En
Revista:
J Pharm Biomed Anal
Año:
2021
Tipo del documento:
Article