Labeled vitamin D compounds and the use thereof

• US 6,455,714 B1
• Filed: 03/19/2001
• Issued: 09/24/2002
• Est. Priority Date: 12/29/1995
• Status: Expired due to Term

• Current Assignee: A & D Bioscience Incorporated
• Original Assignee: A & D Bioscience Incorporated
• Inventors: Ray, Rahul, Holick, Michael F.
• Primary Examiner(s): GITOMER, RALPH J
• Application Number:
  US09/810,624
• Publication Number:
  US 20020107411A1
• Time in Patent Office: 554 Days
• Field of Search: 552/653, 424/9.1, 548/168
• US Class Current: 552/653
• CPC Class Codes: C07C 401/00 Irradiation products of cholesterol or its derivatives; Vitamin D derivatives, 9,10-seco cyclopenta[a]phenanthrene or analogues obtained by chemical preparation without irradiation
• CPC Class Codes: C07D 495/04 Ortho-condensed systems
• CPC Class Codes: Y10S 435/975 Kit

Abstract

Biotin, fluorescent and chemiluminescent labeled vitamin D compounds are disclosed as well as their use in assays for the presence of vitamin D, its metabolites and vitamin D analogs in biological fluids.

Claims

1. In an assay method for the presence of a vitamin D compound in a sample, the improvement comprising using as the labeled vitamin D compound, a labeled compound having the formula:

wherein

R₁ is a substituted or unsubstituted alkyl, alkenyl or alkynyl group having 1 to 15 C-atoms which may be substituted by one or more halo, hydroxy, lower alkoxy, oxo, oxime, lower alkanoyloxy, aryloxy, aryl, benzoyl, a C₄ lactone, a C₄ lactone substituted by a methyl and a hydroxy group, C₃-C₆ cycloalkyl, or C₃-C₆ cycloalkyl substituted by hydroxy, lower alkyl, or hydroxyloweralkyl;

R₂ is a methyl group and R₃ is hydrogen, or

R₂ is hydrogen and R₃ is a methyl group, or

R₂ and R₃ are both hydrogen or,

R₂ and R₃ together are a methylene group (═

CH₂),

R₄ is hydrogen, hydroxy, lower alkoxy or lower alkanoyloxy,

W is oxygen or amino;

X is carbonyl (C═

O) or methylene (CH₂);

Y is oxygen, sulfur, amino, —

C(O)O—

or —

C(O)NH—

;

Z is biotin, a fluorescent group or a chemiluminescent group; and

n₁ and n₂ are independently 1, 2, 3, 4, or 5.

2. The method of claim 1, wherein Z is biotin.

3. The method of claim 1, wherein said labeled compound is 25-hydroxy-3-aminopropoxyvitamin D₃ biotinamide, 25-hydroxy-3beta-[(5-biotinamidyl)pentanamido]-3-aminopropoxyvitamin D₃, 25-hydroxy-3beta-[(6-biotinamidyl)-hexanamido]-3-aminopropoxyvitamin D₃, 25-hydroxy-3beta-aminopropoxyvitamin D₃ fluorescein amide, 1,25-dihydroxy-3-aminopropoxyvitamin D₃ biotinamide, 1,25-dihydroxy-3beta-[(5-biotinamidyl)pentanamido]-3 -aminopropoxyvitamin D₃, 1,25-dihydroxy-3beta-[(6-biotinamidyl)-hexanamido]-3-aminopropoxyvitamin D₃, 1,25-dihydroxy-3beta-aminopropoxyvitamin D₃ fluorescein amide, 3-aminopropoxyvitamin D₃ biotinamide, 3beta-[(5-biotinamidyl)pentanamido]-3-aminopropoxyvitamin D₃, 3beta-[(6-biotinamidyl)-hexanamido]-3-aminopropoxyvitamin D₃, or 3beta-aminopropoxyvitamin D₃ fluorescein amide.

4. The method of claim 1, wherein R₁ is the side chain of vitamin D₂ or vitamin D₃.

5. The method of claim 1, wherein R₁ is the side chain of vitamin D₂ or vitamin D₃ substituted with at least one hydroxy group at the C₂₃, C₂₄ or C₂₅, positions.

6. A solid phase assay method for detecting a vitamin D, its metabolite or analog in a test sample, comprising

(a) providing a solid phase support having immobilized thereon a protein or antibody which binds to the labeled compound having the formula:

wherein;

R₁ is a substituted or unsubstituted alkyl, alkenyl or alkynyl group having 1 to 15 C-atoms which may be substituted by one or more halo, hydroxy, lower alkoxy, oxo, oxime, lower alkanoyloxy, aryloxy, aryl, benzoyl, a C₄ lactone, a C₄ lactone substituted by a methyl and a hydroxy group, C₃-C₆ cycloalkyl, or C₃-C₆ cycloalkyl substituted by hydroxy, lower alkyl, or hydroxyloweralkyl;

R₂ is a methyl group and R₃ is hydrogen, or

R₂ is hydrogen and R₃ is a methyl group, or

R₂ and R₃ are both hydrogen or,

R₂ and R₃ together are a methylene group (═

CH₂),

R₄ is hydrogen, hydroxy, lower alkoxy or lower alkanoyloxy,

W is oxygen or amino;

X is carbonyl (C═

O) or methylene (CH₂);

Y is oxygen, sulfur, amino, —

C(O)O—

or —

C(O)NH—

;

Z is biotin, a fluorescent group or a chemiluminescent group; and

n₁ and n₂ are independently 1, 2, 3, 4, or 5;

(b) contacting said solid phase support with a solution of the labeled compound for a time sufficient to allow binding of the labeled compound to said protein or antibody;

(c) washing the solid phase support obtained in step (b) for a time sufficient to remove unbound labeled compound;

(d) contacting the solid phase support obtained in step (c) with a liquid test sample suspected of containing a vitamin D, its metabolite or analog for a time sufficient to effect displacement of the labeled compound from said protein or antibody;

(e) removing the liquid obtained in step (d); and

(f) detecting the presence of the labeled compound in the liquid obtained in step (e);

wherein the amount of labeled compound in step (f) is directly proportional to the amount of the vitamin D, its metabolite or analog in said test sample.