Compounds and Methods for Determination of FKBP-Binding Immunosuppressant Drugs

A method of releasing an FKBP-binding immunosuppressant drug from endogenous binding substances in a sample suspected of containing the FKBP-binding immunosuppressant drug, the method comprising:

• providing in combination the sample and, in an amount sufficient to release the FKBP-binding immunosuppressant drug from endogenous binding substances, a compound of the formula:
• or of the formula:
• or a mixture thereof;
• wherein:
• R1, R2 and R3 are each independently H or lower alkyl;
• R4 and R5 are each independently H, lower hydrocarbyl or lower hydrocarbyloxy or taken together to form a double bond to O, CH2, N-alkyl, or N—O-alkyl;
• R6 and R7 are each independently H, lower hydrocarbyl or lower hydrocarbyloxy or taken together to form a double bond to O, CH2, or N-alkyl, or N—O-alkyl;
• R8 and R9 are each independently H, non-bulky organic radical or a bulky organic radical, or are taken together to form a double bond to O or CH2;
• R10 and R11 are each independently H, non-bulky organic radical or a bulky organic radical, or are taken together to form a double bond to O or CH2;
• R12 is H, H non-bulky hydrocarbyl or a bulky organic radical;
• wherein at least one of R8, R9, R10, R11 or R12 is a bulky organic radical;
• p is 1, 2 or 3;
• a is 0 or 1;
• and
• D is N, O, or CH, with the proviso that a is 0 when D is O;
• and
• incubating the combination under conditions sufficient to release the FKBP-binding immunosuppressant drug from endogenous binding substances.

A method of determining one or both of the presence and amount of an FKBP-binding immunosuppressant drug in a sample suspected of containing the FKBP-binding immunosuppressant drug, the method comprising:

• (a) providing in combination in a medium:
• (i) the sample, and
• (ii) a releasing agent for releasing the FKBP-binding immunosuppressant drug from endogenous binding substances, wherein the releasing agent is a compound of the formula:
• or of the formula:
• or a mixture thereof;
• wherein:
• R1, R2 and R3 are each independently H or lower alkyl;
• R4 and R5 are each independently H, lower hydrocarbyl or lower hydrocarbyloxy or taken together to form a double bond to O, CH2, N-alkyl, or N—O-alkyl;
• R6 and R7 are each independently H, lower hydrocarbyl or lower hydrocarbyloxy or taken together to form a double bond to O, CH2, N-alkyl, or N—O-alkyl;
• R8 and R9 are each independently H, non-bulky organic radical or a bulky organic radical, or are taken together to form a double bond to O or CH2;
• R10 and R11 are each independently H, non-bulky organic radical or a bulky organic radical, or are taken together to form a double bond to O or CH2;
• R12 is H, H non-bulky hydrocarbyl or a bulky organic radical;
• wherein at least one of R8, R9, R10, R11 or R12 is a bulky organic radical;
• p is 1, 2 or 3;
• a is 0 or 1;
• and
• D is N, O, or CH, with the proviso that a is 0 when D is O;
• (b) incubating the medium under conditions for releasing the FKBP-binding immunosuppressant drug from endogenous binding substances,
• (c) adding to the medium reagents for determining the presence and/or amount of the FKBP-binding immunosuppressant drug in the sample wherein the reagents comprise at least one specific binding member for the FKBP-binding immunosuppressant drug, and
• (d) examining the medium for the presence of a complex comprising the FKBP-binding immunosuppressant drug and the specific binding member for the FKBP-binding immunosuppressant drug, the presence and/or amount of the complex indicating the presence and/or amount of the FKBP-binding immunosuppressant drug in the sample.