Method for producing rapamycin-specific antibodies

a technology of rapamycin and specific antibodies, which is applied in the field of producing rapamycin-specific antibodies, can solve the problems of not being able to establish a consensus regarding the identity or steady state concentration of whole blood

Inactive Publication Date: 2004-10-07
YATSCOFF RANDALL W +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, no consensus has been established concerning the identity or steady state concentrations in whole blood after oral administration.

Method used

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  • Method for producing rapamycin-specific antibodies
  • Method for producing rapamycin-specific antibodies
  • Method for producing rapamycin-specific antibodies

Examples

Experimental program
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Effect test

example 2

Synthesis of Rapamycin-42-succinate and Conjugation to a Protein Carrier

[0021] Preparation of rapamycin-42-O-hemisuccinate: Dimethylaminopyridine (11.8 mg, 97 .mu.mol) was added to a solution of rapamycin (80.0 mg, 88 .mu.mol) and succine anhydride (30.7 mg, 307 .mu.mol) in 2 mL dry pyridine and the mixture stirred at room temperature for 23 hours. The pyridine was evaporated and the residue dissolved in ethyl acetate. The ethyl acetate solution was washed twice with water and finally with brine before drying over magnesium sulfate and evaporating the solvent. The residue was eluted through a silica gel column using methanol / chloroform (1:19) and then methanol / chloroform (1:9) as eluent to give 20.0 mg (23%) of the product as a colorless solid.

[0022] Preparation of 42-O-succinimidooxysuccinyl rapamycin: N-hydroxysuccinimide (2.3 mg 19.7 .mu.mol) was added to a solution of rapamycin-42-0-hemisuccinate (20.0 mg, 19.7 .mu.mol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) (...

example 3

Synthesis of Rapamycin-27-Oxime-Divinyl Sulfone and Conjugation to a Protein Carrier

[0025] Preparation of rapamycin-27-oxime: Hydroxylamine hydrochloride (3.0 mg, 44 .mu.mol) in 100 mL of water was added to a solution of rapamycin (20.0 mg, 22 .mu.mol) and pyridine (40 mL) in 4 mL of ethanol and the reaction mixture stirred at room temperature for 24 hours. The reaction mixture was diluted with ethyl acetate and washed sequentially with water, dilute aqueous hydrochloric acid, and brine. The organic phase was dried over magnesium sulfate and the solvent evaporated to give 20 mg of crude product.

[0026] Analysis of rapamycin-27-oxime-divinyl: LC / MS analysis (Gradient condition: 25 / 25 / 50 water / acetonitrile / methanol at 0 minutes up to 20 / 30 / 50 water / acetonitrile / methanol at 18 minutes. Column: SPHERISORB.TM. C-8 (octyl bonded spherical silica packing, Waters) semi-prep. Temperature was 35.degree. C. and the flow rate set at 3.5 mL / min. The UV signal was monitored at 276 nm) of the crude...

example 4

Synthesis of Rapamvcin-3 1-Divinyl Sulfone and Conjugation to a Protein Carrier

[0034] Preparation of a rapamycin-31-divinyl sulfone hapten (Rapa-DVS): Vinyl sulfone (82.6 mg, 0.7 .mu.mol) was added to a mixture of rapamycin (5.0 mg, 5.5 .mu.mol) and dried anhydrous potassium carbonate (30 mg) in 3 mL of dry acetone at room temperature under a. nitrogen atmosphere. The mixture was stirred for 19 hours. Passing a stream of nitrogen through the flask evaporated the solvent and the resulting residue was immediately quenched with 5 mL of a solution of 10 drops of acetic acid in 10 mL methanol. The clear solution was then decanted off the potassium carbonate granules and the solution concentrated. The residue was passed through a silica gel column using a gradient of methanol / chloroform (1% to 2% methanol) as eluent to separate the reaction products from excess vinyl sulfone. The combined reaction products were then purified and analyzed as follows.

[0035] Analysis of a rapamycin-31-diviny...

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Abstract

This invention relates to the production of polyclonal and monoclonal antibodies to specific sites of rapamycin (Sirolimus). The reactivity of these poly and monoclonal antibodies make them particularly useful for immunoassays for therapeutic drug monitoring (TDM). These immunoassays or TDM kits may include polyclonal or monoclonal antibodies to specific sites of rapamycin. These kits may also include various combinations of polyclonal antibodies, polyclonal and monoclonal antibodies or a panel of monoclonal antibodies. Rapamycin conjugate immunogens are prepared for the immunization of a host animal to produce antibodies directed against specific regions of the rapamycin molecule. By determining the specific binding region of particular antibody, immunoassays which are capable of distinguishing between the parent molecule, active metabolites, inactive metabolites and other structurally similar immunosuppressant compounds are developed. The use of divinyl sulfone (DVS) as the linker arm molecule for forming rapamycin-protein conjugate immunogens is described. DVS-linked rapamycin-protein conjugates were found to elicit antibodies with greater specificity to the rapamycin molecule than succinate linked conjugates.

Description

[0001] This application is a divisional application of U.S. patent application Ser. No. 09 / 419,877, filed Oct. 15, 1999, now co-pending, which application is a continuation-in-part of U.S. patent application Ser. No. 09 / 325,994 filed Jun. 4, 1999, now abandoned, which application is a continuation of U.S. patent application Ser. No. 09 / 101,309 filed Jul. 7, 1998, now abandoned, which application was filed as international patent application PCT / CA98 / 00361 on Apr. 9, 1998 which claims priority to U.S. provisional patent application 60 / 043,215 filed Apr. 9, 1997. The disclosure of each of the above-referenced applications is incorporated herein by reference in its entirety.[0002] This invention relates to the production of polyclonal and monoclonal antibodies to specific sites of rapamycin and / or rapamycin metabolites, derivatives and analogues. The reactivity of these polyclonal and monoclonal antibodies makes them particularly useful for immunoassays for therapeutic drug monitoring ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/14
CPCC07K16/14
Inventor YATSCOFF, RANDALL W.MALCOLM, ANDREW J.NAICKER, SELVARAJ
Owner YATSCOFF RANDALL W
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