Minor immunophilin binding of tacrolimus and sirolimus metabolites.
OBJECTIVE
We have previously identified three minor immunophilins of molecular weights 37 kDa, 14 kDa, and 5-8 kDa capable of binding tacrolimus and sirolimus.


METHODS
When tested against pure preparations of five sirolimus metabolites, the 14 kDa protein had almost no cross-reactivity, the 37 kDa protein cross-reacted from a high of 23.2% to <10% and the 5-8 kDa protein cross-reacted from <10% to 46.4%. When the 5-8 kDa immunophilin was tested in whole blood samples to assess interference in clinical samples, the demethylated sirolimus metabolites showed about 25% less cross-reactivity while the hydroxylated metabolites reacted about the same.


RESULTS
Since MLC data on sirolimus metabolites to date indicates that their pharmacologic potencies are < or =10% of the parent, the 14 kDa immunophilin appears to be the best candidate for a sirolimus radioreceptor assay. The 5-8 kDa immunophilin is newly identified and its cross-reactivity with tacrolimus metabolites had not been assessed. Binding of the 5-8 kDa immunophilin to pure preparations of three tacrolimus metabolites showed virtually no binding of the protein to 13-O-demethyl and 31-O-demethyl tacrolimus and binding to 15-O-demethyl tacrolimus at 121% relative to parent tacrolimus. Cross-reactivity of 15-O-demethyl tacrolimus with the 5-8 kDa protein was then assessed in whole blood samples, and it bound at a level of 163%. MLC data indicates that 31-O-demethyl tacrolimus is equipotent to parent tacrolimus in immunosuppressive activity, while the 13-O-demethyl and 15-O-demethyl have negligible immunosuppressive activity.


CONCLUSIONS
Therefore, the 5-8 kDa immunophilin would have limitations in a radioreceptor assay for tacrolimus. In addition, we have evidence that the 5-8 kDa immunophilin is a subunit of a 52 kDa immunophilin previously identified by our group, and the cross-reactivity of the 5-8 kDa immunophilin with these metabolites is similar to that found previously with the 52 kDa, indicating that the two proteins could be related.
Davis
D L
DL
Department of Laboratory Medicine, Children's National Medical Center, Washington, DC 20010, USA.
Murthy
J N
JN
Gallant-Haidner
H
H
Yatscoff
R W
RW
Soldin
S J
SJ
D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism; D002417:Cattle; D003429:Cross Reactions; D006801:Humans; D020104:Immunophilins / Q000302:isolation & purification / Q000378:metabolism*; D007166:Immunosuppressive Agents / Q000378:metabolism; D007959:Lymphocyte Culture Test, Mixed; D008970:Molecular Weight; D011485:Protein Binding; D011869:Radioligand Assay; D020123:Sirolimus / Q000031:analogs & derivatives / Q000378:metabolism*; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D007166:Immunosuppressive Agents; D020104:Immunophilins; D020123:Sirolimus; D016559:Tacrolimus