Purpose: 5-azacytidine (5-AZA) is being evaluated as a potential treatment for primary lung cancer, along with the potential to treat metastases as they develop. The metastatic nature of late-stage lung cancer leads to a very low survival rate (generally accepted as 4 percent for a 5 year survival rate) in those that do have lung cancer that has metastasized. The need for an accurate and selective method in multiple matrices for 5-AZA was required to evaluate dose delivery following targeted drug delivery. The drug levels in different target tissues would be used to evaluate potential to treat both the primary tumors in lung as well as the potential metastases as they develop. The primary sites of lung metastases are brain and liver, therefore, this program includes methods to quantitate 5-AZA in plasma, as well as lung, brain, and liver tissues.
Methods: An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed for the determination of 5-AZA in rat plasma, lung, brain, and liver using 15N5-Deoxyadenosine as the internal standard. The plasma samples were prepared with a protein precipitation extraction using 0.1% formic acid in acetonitrile followed by dilution with ammonium acetate buffer. The tissue samples were prepared by a 1:4 dilution (1g tissue:4mL 1XPBS) followed by homogenization. Tissue homogenates were prepared by a protein precipitation extraction using 10% trichloroacetic acid in water, followed by dilution with phosphate buffer. Chromatography was performed on a Waters HSS T3 (100Å, 3.5 µm, 3 mm X 100 mm) using a gradient with mobile phase A consisting of 10mM ammonium acetate and mobile phase B consisting of 80/20 methanol/acetonitrile. The molecular weight of 5-AZA is 244.2 g/mol and the precursor → product ion transition (m/z 113.0 → 86.0) was monitored on a Sciex 5000 triple quadrupole mass spectrometer, operating in multiple reaction monitoring and positive electrospray ionization mode. The method was developed over a dynamic concentration range of 50 to 10,000 ng/mL in plasma and 50 to 5000 ng/mL in lung, brain, and liver tissues. The methods were applied to determine the levels of 5-AZA in each matrix.
Results: Due to the nucleosidic nature of 5-azacytidine, a great deal of interference was found with any of the parent compounds’ MRM mass transitions. Many published methods were attempted without success, primarily due to lack of chromatographic retention and a large amount of interference seen from the matrix. It was noticed that a considerable amount of the typically reported MRM product ion was seen in the Q1 scan. For greater selectivity, the primary product ion was chosen as the precursor ion and a subsequent product ion was then selected as the product ion for the quantitation MRM. Using this MS/MS/MS technique, the need for greater chromatographic separation was eliminated and the greatly reduced background allowed for even greater sensitivity. This novel approach proved successful with each of the matrices described in this presentation.
Conclusion: The novel method was utilized to evaluate 5-Aza in each tissue following IP, three doses of dry powder via inhalation and aqueous nebulizer via inhalation. Plasma levels for all groups peaked in a dose dependent manner around 0.5 to 1 hour with detectable 5-AZA out to ~ 8 hours. Lung tissue exposure was increased via inhalation exposure with peak levels early for all delivery routes and detectable 5-AZA out to 12 hours in the inhalation groups and 6 hours in the systemic delivery route. Liver and brain exposure reflected that it was a secondary or tertiary compartment of the distribution of 5-AZA following dosing. 5-AZA was detectable in both liver and brain out to 6 hours (or more) in a dose dependent manner. These results will further be analyzed to develop dosing schedule and dose level within efficacy and toxicity studies as a part of the drug development of 5-AZA.