Assessment of l-Asparaginase Pharmacodynamics in Mouse Models of Cancer

Thomas D Horvath, Wai Kin Chan, Michael A Pontikos, Leona A Martin, Di Du, Lin Tan, Marina Konopleva, John N Weinstein, Philip L Lorenzi, Thomas D Horvath, Wai Kin Chan, Michael A Pontikos, Leona A Martin, Di Du, Lin Tan, Marina Konopleva, John N Weinstein, Philip L Lorenzi

Abstract

l-asparaginase (ASNase) is a metabolism-targeted anti-neoplastic agent used to treat acute lymphoblastic leukemia (ALL). ASNase's anticancer activity results from the enzymatic depletion of asparagine (Asn) and glutamine (Gln), which are converted to aspartic acid (Asp) and glutamic acid (Glu), respectively, in the blood. Unfortunately, accurate assessment of the in vivo pharmacodynamics (PD) of ASNase is challenging because of the following reasons: (i) ASNase is resilient to deactivation; (ii) ASNase catalytic efficiency is very high; and (iii) the PD markers Asn and Gln are depleted ex vivo in blood samples containing ASNase. To address those issues and facilitate longitudinal studies in individual mice for ASNase PD studies, we present here a new LC-MS/MS bioanalytical method that incorporates rapid quenching of ASNase for measurement of Asn, Asp, Gln, and Glu in just 10 µL of whole blood, with limits of detection (s:n ≥ 10:1) estimated to be 2.3, 3.5, 0.8, and 0.5 µM, respectively. We tested the suitability of the method in a 5-day, longitudinal PD study in mice and found the method to be simple to perform with sufficient accuracy and precision for whole blood measurements. Overall, the method increases the density of data that can be acquired from a single animal and will facilitate optimization of novel ASNase treatment regimens and/or the development of new ASNase variants with desired kinetic properties.

Keywords: Erwinaze; Kidrolase; asparaginase; glutaminase; pharmacodynamics; targeted metabolomics.

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
“Metaburst” of metabolic pathways modulated by ASNase, including biological reactions associated with the metabolites asparagine (Asn), aspartic acid (Asp), glutamine (Gln), and glutamic acid (Glu). All reactions are also listed in Table S1.
Figure 2
Figure 2
Screen for ASNase Activity Quenchers. The following conditions were tested for the ability to quench the conversion of Asn to Asp by ASNase: (1) 80:20 methanol (MeOH):water; (2) 80:20 MeOH containing 1% formic acid (FA):water; (3) 80:20 acetonitrile (ACN):water; (4) 80:20 ACN containing 1% FA:water; (5) 80:20 water containing 1% FA:water; (6) 80:20 water containing 10% FA:water; (7) 80:20 water containing 10% (w/v) sulfosalicylic acid (SSA):water; (8) 80:20 water containing 1% (w/v) trichloroacetic acid (TCA):water; (9) 80:20 water containing 40 mM 5-diazo-4-oxo-l-norvaline (DONV):water; (10) positive control sample containing ASNase in water; and (11) negative control sample containing water without ASNase. Data are presented as normalized response of Asn and Asp. The final activity of ASNase and solution concentration of Asn in each sample tested was approximately 20 IU/mL and 100 µM, respectively.
Figure 3
Figure 3
Amino acid concentration (µM) versus time (h) in whole blood of mice treated with ASNase. (A) asparagine (Asn), (B) aspartic acid (ASP), (C) glutamine (Gln), and (D) glutamic acid (Glu). Each data series represents an individual mouse, and the mice were arranged into the following three cohorts that were given intraperitoneal injections of either vehicle (1x PBS) or ASNase at 0, 24, and 48 h: (1) Control mice (n = 2; blackline with black squares and circles); (2) mice treated with 1000 IU/(kg·day) ASNase (n = 2; blue lines with blue squares and circles); (3) mice treated with 5000 IU/(kg·day) ASNase (n = 2red lines with red squares and circles). ASNase was administered at 0, 24, and 48 h, and whole blood (10 µL) was collected from each mouse at 0, 48, and 96 h, and was processed as described. The gray box represents the NR levels of Asn, Asp, Gln, and Glu defined by the 0 h (pre-treatment) samples.

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