N-1-carboxyalkyl derivatives of LSD

• US 5,843,682 A
• Filed: 11/20/1995
• Issued: 12/01/1998
• Est. Priority Date: 11/20/1995
• Status: Expired due to Fees

• Current Assignee: Roche Diabetes Care Inc. (Roche Holding AG), Microgenics Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Boehringer Mannheim Corporation
• Inventors: Davoudzaheh, David, Sigler, Gerald F., Rouhani, Riaz
• Primary Examiner(s): Achutamurthy, Ponnathapura
• Assistant Examiner(s): PONNALURI, PADMASHRI
• Application Number:
  US08/560,871
• Time in Patent Office: 1,107 Days
• Field of Search: 530/387.1, 530/403, 435/7.6, 435/7.9, 436/545, 436/546, 546/61, 546/69, 546/70, 546/77
• US Class Current: 435/7.6
• CPC Class Codes: C07D 457/06 Lysergic acid amides
• CPC Class Codes: G01N 33/946 CNS-stimulants, e.g. cocaine, amphetamines

Abstract

Novel derivatives of LSD having the formula ##STR1## wherein R₁ is an alkyl, cycloalkyl or aryl group having 1 to 10 carbon atoms, preferably an alkyl group having 1 carbon atom;

R₂ is a bond or ##STR2## wherein R₃ is alkyl, cycloalkyl or aryl group having 2 to 10 carbon atoms, preferably an alkyl group having 2 or 5 carbon atoms; Z is an immunogenic carrier substance, an enzyme donor polypeptide or a label selected from the group consisting of an enzyme, a substance having fluorescent or luminescent properties and a radioactive substance; and n is 1 to p where p equals MW of Z/1000. The derivatives include maleimide conjugates of an immunogenic poly(amino acid), an enzyme donor polypeptide or a labeling substance such as an enzyme, a fluorescent substance or a radioactive substance. Novel activated hapten intermediates useful in the preparation of the conjugates and methods for synthesis of the hapten intermediates and their conjugate derivatives are also disclosed.

Claims

1. An enzyme immunoassay method for determining LSD in a sample comprising:

(a) contacting said sample with

(i) an enzyme donor polypeptide conjugate of the form;

##STR8## wherein Z is an enzyme donor polypeptide of β

-galactosidase;

R₁ =an alkyl, cycloalkyl or aryl group having 1 to 10 carbon atoms;

R₂ =a bond or ##STR9## wherein R₃ =an alkyl, cycloalkyl, or aryl group having 2 to 10 carbon atoms; and

n=1 to p where p=MW of Z/1000;

(ii) an enzyme acceptor polypeptide wherein said enzyme acceptor polypeptide is characterized by forming with said enzyme donor polypeptide conjugate an active enzyme complex having β

-galactosidase activity in the absence of an antibody to LSD;

(iii) an antibody specific for LSD, wherein said enzyme donor conjugate is capable of competitively binding to said antibody, thereby inhibiting the formation of active enzyme complex; and

(iv) a substrate for β

-galactosidase; and

(b) measuring the rate of conversion of said substrate by said active enzyme complex as a measure of the amount of LSD in said sample.

2. An enzyme immunoassay method for determining LSD in a sample comprising:

(a) contacting said sample with

(i) an enzyme donor polypeptide conjugate of β

-galactosidase;

(ii) an enzyme acceptor polypeptide wherein said enzyme acceptor polypeptide is characterized by forming with said enzyme donor polypeptide conjugate an active enzyme complex having β

-galactosidase activity in the absence of an antibody to LSD;

(iii) an antibody to a compound of the form;

##STR10## wherein Z is an immunogenic carrier substance;

R₁ =an alkyl, cycloalkyl or aryl group having 1 to 10 carbon atoms;

R₂ =a bond or ##STR11## wherein R₃ =an alkyl, cycloalkyl, or aryl group having 2 to 10 carbon atoms; and

n=1 top where p=MW of Z/1000,

wherein said enzyme donor conjugate is capable of competitively binding to said antibody thereby inhibiting the formation of active enzyme complex; and

(iv) a substrate for β

-galactosidase; and

(b) measuring the rate of conversion of said substrate by said active enzyme complex as a measure of the amount of LSD in said sample.

3. A set of reagents for determining the presence of LSD or its metabolites in a urine sample by enzyme immunoassay, comprising:

a first reagent containing a conjugate of LSD and an enzyme or enzyme donor polypeptide; and

a second reagent containing an antibody capable of binding, the conjugate of the first reagent in a manner that is inhibitable by LSD.

4. The set of reagents of claim 3, wherein the antibody is a monoclonal antibody.

5. The set of reagents of claim 3, wherein the conjugate of the first reagent is LSD conjugated to an enzyme donor of β

-galactosidase.

6. The set of reagents of claim 3, wherein the enzyme or enzyme donor polypeptide is conjugated to the N-1position of the LSD.

7. The set of reagents of claim 3, wherein the conjugate is of the form:

##STR12## wherein Z is enzyme or enzyme donor polypeptide;

R₁ =an alkyl, cycloalkyl or aryl group having 1 to 10 carbon atoms;

R₂ =a bond or ##STR13## wherein R₃ =an alkyl, cycloalkyl, or aryl group having 2 to 10 carbon atoms; and

n=1 to p where p=MW of Z/1000.

8. The set of reagents of claim 7, wherein R₂ is ##STR14## wherein R₃ =an alkyl group having 2 to 5 carbon atoms.

9. The set of reagents of claim 4, wherein the antibody of the second reagent is capable of binding the conjugate of the first reagent in the immunoassay in a manner that is inhibitable by LSD in the sample at a dose of 0.3 ng/ml.

10. The set of reagents of claim 9, wherein the antibody of the second reagent is capable of binding the conjugate of the first reagent in a manner that is inhibitable by the LSD, wherein 50% maximal inhibition occurs at an LSD dose below 2.0 nM.

11. The set of reagents of claim 3, wherein the antibody of the second reagent is capable of binding the conjugate of the first reagent in a manner that is inhibitable by unconjugated LSD when used in a homogeneous immunoassay.

12. An enzyme immunoassay method for determining the presence of LSD or LSD metabolites in a sample, comprising

a) contacting an antibody capable of binding LSD with any LSD or LSD metabolites in the sample;

b) contacting the antibody with a conjugate of LSD and an enzyme or enzyme donor polypeptide; and

c) measuring any complexes formed between the conjugated and the antibody.

13. The method of claim 12, wherein the antibody is a monoclonal antibody.

14. The method of claim 12, wherein the conjugate is LSD conjugated to an enzyme donor of β

-galactosidase.

15. The method of claim 12, wherein the enzyme or enzyme donor polypeptide is conjugated to LSD through the N-1 position of the LSD.

16. The method of claim 12, wherein the sample is a urine sample.

17. The method of claim 12, which is a homogeneous assay method.

18. The method of claim 12, which is a competitive binding assay method.

19. The method of claim 12, which is an automated assay method.

20. The method of claim 12, which is a cloned enzyme donor immunoassay method.

21. The method of claim 12, wherein the enzyme polypeptide is an enzyme donor, and the measuring in step c) comprises contacting the enzyme donor with an enzyme acceptor that complexes with the enzyme donor to form an enzyme complex when the enzyme donor is not bound to the antibody, contacting the enzyme complex with a substrate;

and measuring the rate of conversion of the substrate to a product by the enzyme complex.

22. The method of claim 12, wherein the contacting of step a) is performed before the contacting of step b).

23. The method of claim 12, comprising preparing a reaction mixture comprising the antibody, the sample, and the conjugate.