Methods and compositions to lower plasma cholesterol levels

• US 6,887,712 B1
• Filed: 11/09/1999
• Issued: 05/03/2005
• Est. Priority Date: 11/09/1998
• Status: Expired due to Fees

• Current Assignee: Salutria Pharmaceuticals, LLC
• Original Assignee: Atherogenics, Inc.
• Inventors: Saxena, Uday, Medford, Russell M.
• Primary Examiner(s): Chin, Chris
• Assistant Examiner(s): GABEL, GAILENE
• Application Number:
  US09/436,892
• Time in Patent Office: 2,002 Days
• Field of Search: 435/2, 435/7.1, 435/7.6, 435/7.92, 435/7.8, 435/7.72, 435/7.9, 435/7.94, 435/7.93, 436/501, 436/507, 436/514, 436/515, 436/523, 436/536, 436/537, 436/546, 436/518, 436/524, 436/528, 436/55, 436/71, 436/148, 436/164, 436/172, 436/805, 436/815, 424/9.2, 530/387.3, 530/388.1, 530/388.25, 514/548, 514/712, 514/824, 514/825, 514/826, 514/855
• US Class Current: 436/71
• CPC Class Codes: A61K 31/00 Medicinal preparations containing organic active ingredients
• CPC Class Codes: G01N 2800/044 Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity
• CPC Class Codes: G01N 33/92 involving lipids, e.g. cholesterol , lipoproteins, or their receptors steroid hormones G01N33/743
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/815 Test for named compound or class of compounds
• CPC Class Codes: Y10T 436/12 Condition responsive control

Abstract

A method is disclosed for determining whether a compound binds to a lipoprotein such as LDL or VLDL in a manner which will lower plasma cholesterol. The method provided includes assessing the ability of the compound to form a complex with the lipoprotein, and then determining whether the newly formed complex causes a change in the structure of apoB-100 that results in increased binding affinity to an LDL receptor.

Claims

1. A method to determine if a compound causes a change in the structure of apolipoprotein B-100 in a cholesterol-containing low density lipoprotein thus increasing the binding of an epitope on the apolipoprotein B-100 to the LDL-receptor, comprising:

(i) mixing the compound with and allowing it to bind to cholesterol-containing low density lipoprotein forming a complex;

(ii) exposing the complex to a first capture antibody that is attached to a solid phase material and is directed to the epitope on apolipoprotein B-100 that binds to the LDL-receptor, forming a combination;

(iii) adding a second antibody which binds to the combination;

(iv) detecting the second antibody bound to the combination by the addition of a third antibody that binds the second antibody and to which is attached a label;

(v) quantifying the amount of the captured complex by quantifying the amount of label; and

(vi) comparing the amount of cholesterol-containing low density lipoprotein captured by the assay to a control, wherein an increase in the amount of cholesterol-containing low density lipoprotein captured indicates a change in the structure of apoB-100 in the cholesterol-containing low density lipoprotein, thus increasing binding to the low density lipoprotein receptor.

2. The method of claim 1, wherein the control is low density lipoprotein in the absence of test compound.

3. A method for assessing whether a compound first binds to a cholesterol-containing lipoprotein, enhancing the binding of the cholesterol-containing lipoprotein to a low density lipoprotein hepatic receptor and thus lowering plasma cholesterol, the method comprising:

(a) allowing the compound to form a complex with a cholesterol-containing lipoprotein in vivo,

(b) isolating the resulting complex, and

(c) determining whether the formation of the complex causes a change in the three dimensional conformation of apoB-100 in the cholesterol-containing lipoprotein that enhances the binding of the lipoprotein to the LDL hepatic receptor.

4. The method of claim 3, wherein the formation of the complex is determined by a sandwich immunoreactivity assay.

5. The method of claim 3, wherein the formation of the complex is determined using agarose electrophoresis.

6. The method of claim 3, wherein the cholesterol-containing low-density lipoprotein is LDL.

7. The method of claim 3, wherein the cholesterol-containing low-density lipoprotein is VLDL.

8. A method to determine if a compound causes a change in the structure of apolipoprotein B-100 in a cholesterol-containing low density lipoprotein thus increasing the binding of an epitope on the apolipoprotein B-100 to an LDL-receptor, comprising:

(i) mixing the compound with and allowing it to bind to cholesterol-containing low density lipoprotein forming a complex;

(ii) exposing the complex to a first capture antibody that is attached to a solid phase material and is directed to the epitope on apolipoprotein B-100 that binds to the LDL-receptor, forming a combination;

(iii) adding to the combination a second antibody which binds the apoB-100 in the combination and to which is attached a label;

(iv) quantifying the amount of the captured complex by quantifying the amount of label; and

(v) comparing the amount of cholesterol-containing low density lipoprotein quantified in step (iv) to a control, wherein an increase in the amount of cholesterol-containing low density lipoprotein captured indicates a change in the structure of apoB-100 in the cholesterol-containing low density lipoprotein, thus increasing binding to the low density lipoprotein receptor.

9. The method of claim 8, wherein the control is low density lipoprotein in the absence of test compound.