Plasma and cellular pharmacology of 8-chloro-adenosine in mice and rats.
OBJECTIVE
The nucleoside 8-chloro-adenosine (8-Cl-Ado) is currently being developed for treatment of multiple myeloma and leukemias. Although accumulation of the phosphorylated drug product is known to occur within cell lines, its metabolic fate in plasma or circulating cells in animals is unclear. The purpose of the present study was to determine the pharmacology of 8-Cl-Ado in rodents through examination of plasma and cellular levels of parent drug and metabolites. In addition, we sought to determine whether an inhibitor of adenosine deaminase, 2'-deoxycoformycin (dCF), could enhance intracellular formation of 8-Cl-ATP by preventing degradation of 8-Cl-Ado to 8-Cl-inosine (8-Cl-Ino).


METHODS
A validated HPLC assay permitted simultaneous determination of 8-Cl-Ado, 8-Cl-adenine (8-Cl-Ade), dCF, and 8-Cl-Ino. Radiolabeled cellular nucleotides were obtained from peripheral blood mononuclear cells (PBMC) of both mice and rats using a perchloric acid extraction procedure and were separated by HPLC.


RESULTS
Stability of 8-Cl-Ado in the presence or absence of dCF was examined in fresh plasma from mice, rats and humans. Conversion of 8-Cl-Ado to 8-Cl-Ino was only marginally affected by coincubation with dCF. In CD(2)F(1) mice given 8-Cl-Ado i.p. at 100 mg/kg, there was rapid appearance in plasma of both 8-Cl-Ade and 8-Cl-Ino. The identities of the metabolites were confirmed by mass spectrometry. The plasma [(3)H]8-Cl-Ado concentration 1 h after drug injection was 1.3 micro M in mice while the intracellular levels of [(3)H]8-Cl-AMP and [(3)H]8-Cl-ATP were 1 m M and 350 micro M, respectively. Mice that had received dCF (2 mg/ml) 30 min prior to [(3)H]8-Cl-Ado had 27% less intracellular [(3)H]8-Cl-ATP in PBMC compared to mice without dCF pretreatment. The pharmacokinetics of 8-Cl-Ado were examined in greater detail in Sprague-Dawley rats. Animals were given [(3)H]8-Cl-Ado (42.5 mg/kg, i.v.) by itself or 30 min following injection of dCF (4 mg/kg). Mononuclear cells in mice accumulated 350 or 1200 micro M [(3)H]8-Cl-ATP 1 h after injection of either 50 or 100 mg [(3)H]8-Cl-Ado, respectively. The major metabolite in these cells was the monophosphate, which was four- to sevenfold higher in concentration than the triphosphate metabolite. In rats, [(3)H]8-Cl-AMP concentrations in PBMC were similar to those of the triphosphate metabolite which achieved a peak of 90 micro M 2 h after a bolus injection of 8-Cl-Ado (40 mg/kg). Cellular clearance of 8-Cl-ATP appeared to be slow: 24 h after injection of 8-Cl-Ado the cellular concentration of 8-Cl-ATP was still 40 micro M.


CONCLUSIONS
The use of dCF did not significantly alter 8-Cl-ATP levels in PBMC and is not considered to be a useful therapeutic strategy. Even though a portion of 8-Cl-Ado is metabolically inactivated in plasma, high levels of cytotoxic 8-Cl-ATP accumulated intracellularly in these animals and were retained for a considerable length of time. Further development of 8-Cl-Ado is recommended.
Gandhi
Varsha
V
Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA.
Chen
Wei
W
Ayres
Mary
M
Rhie
Julie K
JK
Madden
Timothy L
TL
Newman
Robert A
RA
D015762:2-Chloroadenosine / Q000031:analogs & derivatives* / Q000097:blood / Q000493:pharmacokinetics* / Q000494:pharmacology; D000243:Adenosine Deaminase / Q000378:metabolism; D000248:Adenosine Kinase / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000097:blood; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000097:blood / Q000493:pharmacokinetics* / Q000494:pharmacology; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D005260:Female; D007288:Inosine / Q000031:analogs & derivatives / Q000097:blood; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D008297:Male; D051379:Mice / Q000378:metabolism*; D015649:Pentostatin / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011355:Prodrugs / Q000493:pharmacokinetics*; D051381:Rats / Q000378:metabolism*; D017207:Rats, Sprague-Dawley; D013045:Species Specificity
C465859:8-chloro-ATP; C465858:8-chloro-inosine; D000964:Antimetabolites, Antineoplastic; D011355:Prodrugs; D015762:2-Chloroadenosine; D015649:Pentostatin; D007288:Inosine; D000255:Adenosine Triphosphate; D000248:Adenosine Kinase; D000243:Adenosine Deaminase; C075440:8-chloroadenosine