Preferential inhibition of xanthine oxidase by 2-amino-6-hydroxy-8-mercaptopurine and 2-amino-6-purine thiol.
BACKGROUND
The anticancer drug, 6-mercaptopurine (6MP) is subjected to metabolic clearance through xanthine oxidase (XOD) mediated hydroxylation, producing 6-thiouric acid (6TUA), which is excreted in urine. This reduces the effective amount of drug available for therapeutic efficacy. Co-administration of allopurinol, a suicide inhibitor of XOD, which blocks the hydroxylation of 6MP inadvertently enhances the 6MP blood level, counters this reduction. However, allopurinol also blocks the hydroxylation of hypoxanthine, xanthine (released from dead cancer cells) leading to their accumulation in the body causing biochemical complications such as xanthine nephropathy. This necessitates the use of a preferential XOD inhibitor that selectively inhibits 6MP transformation, but leaves xanthine metabolism unaffected.


RESULTS
Here, we have characterized two such unique inhibitors namely, 2-amino-6-hydroxy-8-mercaptopurine (AHMP) and 2-amino-6-purinethiol (APT) on the basis of IC50 values, residual activity in bi-substrate simulative reaction and the kinetic parameters like Km, Ki, kcat. The IC50 values of AHMP for xanthine and 6MP as substrate are 17.71 +/- 0.29 microM and 0.54 +/- 0.01 microM, respectively and the IC50 values of APT for xanthine and 6MP as substrates are 16.38 +/- 0.21 microM and 2.57 +/- 0.08 microM, respectively. The Ki values of XOD using AHMP as inhibitor with xanthine and 6MP as substrate are 5.78 +/- 0.48 microM and 0.96 +/- 0.01 microM, respectively. The Ki values of XOD using APT as inhibitor with xanthine and 6MP as substrate are 6.61 +/- 0.28 microM and 1.30 +/- 0.09 microM. The corresponding Km values of XOD using xanthine and 6MP as substrate are 2.65 +/- 0.02 microM and 6.01 +/- 0.03 microM, respectively. The results suggest that the efficiency of substrate binding to XOD and its subsequent catalytic hydroxylation is much superior for xanthine in comparison to 6MP. In addition, the efficiency of the inhibitor binding to XOD is much more superior when 6MP is the substrate instead of xanthine. We further undertook the toxicological evaluation of these inhibitors in a single dose acute toxicity study in mice and our preliminary experimental results suggested that the inhibitors were equally non-toxic in the tested doses.


CONCLUSIONS
We conclude that administration of either APT or AHMP along with the major anti-leukemic drug 6MP might serve as a good combination cancer chemotherapy regimen.
Kalra
Sukirti
S
Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector 67, Phase X, S.A.S Nagar, Mohali, Punjab, India. skalra11@yahoo.com
Jena
Gopabandhu
G
Tikoo
Kulbhushan
K
Mukhopadhyay
Anup Kumar
AK
D000225:Adenine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006900:Hydroxylation / Q000187:drug effects; D007700:Kinetics; D015122:Mercaptopurine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D008956:Models, Chemical; D015394:Molecular Structure; D013379:Substrate Specificity; D013866:Thioguanine; D014527:Uric Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D019820:Xanthine / Q000737:chemistry / Q000378:metabolism; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014970:Xanthines / Q000737:chemistry / Q000378:metabolism
C520946:2-amino-6-hydroxy-8-mercaptopurine; D004791:Enzyme Inhibitors; D014970:Xanthines; D019820:Xanthine; C054700:6-thioxanthine; D014527:Uric Acid; C010352:6-thiouric acid; D015122:Mercaptopurine; D014969:Xanthine Oxidase; D013866:Thioguanine; D000225:Adenine