# US 2014/0154706 A1 · Compounds and Methods for Determination of FKBP-Binding Immunosuppressant Drugs

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- Number: US 2014/0154706 A1
- Document ID: US20140154706A1
- Status: Published application
- Kind: A1
- Application number: 13/693,229
- Filed: 2012-12-04
- Published: 2014-06-05
- Earliest claimed: 2012-12-04
- Assignees: Siemens Healthcare Diagnostics Inc.
- Applicants: Siemens Healthcare Diagnostics Inc.
- Inventors: Yi Feng Zheng; Tie Q. Wei
- Web page: https://patentgrep.com/patent/US20140154706A1

## CPC

- G01N 33/9493: {Immunosupressants}
- A61K 31/436: the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
- G01N 33/5308: {for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

Compositions are disclosed for releasing an FKBP-binding immunosuppressant drug from endogenous binding substances in a sample suspected of containing the FKBP-binding immunosuppressant drug. The compositions include sirolimus derivatives that are modified with a bulky organic radical in the triene portion of the sirolimus molecule. The compositions may be employed in conjunction with assays for an FKBP-binding immunosuppressant drug in a sample suspected of containing the drug.

## Claims

### Claim 1

A composition for releasing an FKBP-binding immunosuppressant drug from endogenous binding substances in a sample suspected of containing the FKBP-binding immunosuppressant drug, the composition comprising:
(a) 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; and
(b) a buffered medium.

### Claim 2 (depends on 1)

The composition according to claim 1 further comprising a hemolytic agent.

### Claim 3 (depends on 1)

The composition according to claim 1 wherein the compound has the formula:
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.

### Claim 4 (depends on 1)

The composition according to claim 1 wherein the compound has the formula:
wherein:
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.

### Claim 5 (depends on 1)

The composition according to claim 1 wherein:
R1 is H and R2 and R3 are each independently lower alkyl;
R4 and R5 are taken together to form a double bond to O;
R6 and R7 are taken together to form a double bond to O;
R8 and R9 are taken together to form a double bond to O;
R10 and R11 are taken together to form a double bond to O;
R12 is aryl;
p is 1;
a is 1;
D is N.

### Claim 6 (depends on 1)

The composition according to claim 1 wherein the compound has the formula:

### Claim 7 (depends on 1)

The composition according to claim 1 wherein the compound has the formula:

### Claim 8 (depends on 2)

The composition according to claim 2 wherein the hemolytic agent is selected from the group consisting of detergents, low ionic strength aqueous solutions, bacterial agents, and antibodies that cause complement dependent lysis.

### Claim 9 (depends on 2)

The composition according to claim 2 further comprising one or more of an anti-blood clotting agent and a preservative.

### Claim 10 (depends on 1)

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 a composition according to claim 1 in an amount sufficient to release the FKBP-binding immunosuppressant drug from endogenous binding substances and
incubating the combination under conditions sufficient to release the FKBP-binding immunosuppressant drug from endogenous binding substances.

### Claim 11 (depends on 5)

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 a composition according to claim 5 in an amount sufficient to release the FKBP-binding immunosuppressant drug from endogenous binding substances and
incubating the combination under conditions sufficient to release the FKBP-binding immunosuppressant drug from endogenous binding substances.

### Claim 12

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.

### Claim 13 (depends on 12)

The method according to claim 12 wherein the compound is 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, 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.

### Claim 14 (depends on 13)

The method according to claim 13 wherein in the formula for the compound:
R1 is H and R2 and R3 are each independently lower alkyl;
R4 and R5 are taken together to form a double bond to O;
R6 and R7 are taken together to form a double bond to O;
R8 and R9 are taken together to form a double bond to O;
R10 and R11 are taken together to form a double bond to O;
R12 is aryl;
a is 1;
D is N.

### Claim 15

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.

### Claim 16 (depends on 15)

The method according to claim 15 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.

### Claim 17 (depends on 15)

The method according to claim 15 wherein in the compound of the releasing agent:
R1 is H and R2 and R3 are each independently lower alkyl;
R4 and R5 are taken together to form a double bond to O;
R6 and R7 are taken together to form a double bond to O;
R8 and R9 are taken together to form a double bond to O;
R10 and R11 are taken together to form a double bond to O;
R12 is aryl;
a is 1;
D is N.

### Claim 18 (depends on 15)

The method according to claim 15 wherein the compound of the releasing agent has the formula:
or the formula:

### Claim 19 (depends on 15)

The method according to claim 15 wherein the reagents in step (c) further comprises an analog of the FKBP-binding immunosuppressant drug that comprises a label.

### Claim 20 (depends on 15)

The method according to claim 15 wherein in step (c) a second specific binding member is added to the medium wherein the second specific binding member binds to the complex.