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Methods of quantifying methotrexate metabolites

a technology of methotrexate and metabolites, which is applied in the field of monitoring the efficacy and toxicity of antifolate drug therapy, can solve the problems of destroying active proliferating non-target tissues, toxicity of methotrexate treatment, and presenting a risk to patients, so as to optimize the therapeutic effect and reduce toxicity , the effect of optimizing the therapeutic

Inactive Publication Date: 2010-06-24
CYPRESS BIOSCI +1
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Benefits of technology

[0009]The present invention provides a method for efficiently converting methotrexate polyglutamates (MTXPGs) to methotrexate (MTX) in a cellular extract by contacting the cellular extract with gamma glutamyl hydrolase under conditions suitable for efficient conversion of MTXPGs to MTX. The method is useful for quantitating the level of MTXPGs in a cellular extract.
[0012]The invention provides a method of optimizing therapeutic efficacy or reducing toxicity associated with methotrexate therapy administered to an individual. The method includes the steps of: (a) converting methotrexate polyglutamates to MTX in a cellular extract from an individual under conditions suitable for efficient conversion of MTXPGs to MTX; and (b) determining a level of MTX in the cellular extract, the level of MTX correlating with a level of MTXPGs, thereby determining a level of MTXPGs in the cellular extract, wherein a drug or dosage subsequently administered to the individual is selected based on the level of MTXPGs.
[0016]A deglutamated endogenous composition of the invention can be useful for reducing toxicity associated with methotrexate therapy. A deglutamated endogenous composition of the invention also can be useful in diagnostic methods such as methods for determining an intracellular level of methotrexate or a method of optimizing therapeutic efficacy or reducing toxicity associated with methotrexate therapy.

Problems solved by technology

Despite its therapeutic efficacy for a wide variety of diseases and conditions, treatment with methotrexate can present a risk to the patient.
In particular, because MTX interferes with processes required for replication and division of normal as well as diseased cells, inappropriately high levels of the drug can lead to destruction of actively proliferating non-target tissues such as bone marrow and intestinal mucosa.
MTX consequently is associated with renal and hepatic toxicity when administered in the “high-dose regimen” that is required for some conditions.
In addition, low-dose MTX therapy can lead to toxicity and unwanted side-effects in some patients, where the dosage is not appropriate due to individual variability in pharmacokinetic parameters influencing, for example, drug uptake, targeting and clearance.
This situation is especially problematic in the treatment of chronic conditions such as rheumatoid arthritis, where methotrexate can be administered over a period of many years.
However, these plasma detection methods have not been useful in monitoring low-dose methotrexate therapy.
However, the extent of enzyme inhibition in these assays is dependent upon the number of glutamyl residues attached to MTX, rendering an accurate determination of intracellular MTXPGs levels impossible by this method.

Method used

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  • Methods of quantifying methotrexate metabolites
  • Methods of quantifying methotrexate metabolites
  • Methods of quantifying methotrexate metabolites

Examples

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example i

[0100]Quantification of Methotrexate by Fluorimetric Detection of Methotrexate-Photolytic Products

[0101]This example describes quantification of methotrexate using high performance liquid chromatography (HPLC) and post-column photolysis followed by fluorometric detection of photolytic products.

[0102]The MTX detection system was based on an HPLC system (Agilent 1100 HPLC chemstation system) equipped with a C18 reversed phase column (25 cm×4.6 mm X Terra MS C18, 5 micrometer particle size; Waters, Milford, Mass.), a post-column photochemical reactor unit (Aura Industries; New York, N.Y.) and a post-reactor unit fluorometer detector. The system also included a C18 pre-column that was changed every 200 injections.

[0103]Samples containing 20 to 1000 nmol / L MTX (Sigma, St. Louis, USA) in RBC extracts were run in the HPLC system at a flow rate of 1 ml / min, with a 15-minute linear gradient from 2% acetonitrile / 98% mobile phase A (10 mM ammonium acetate, pH 6.5, unless otherwise indicated an...

example ii

Conversion of Methotrexate Polyglutamates to Methotrexate

[0110]This example describes efficient conversion of methotrexate polyglutamates (MTXPGs) to methotrexate (MTX) and further demonstrates quantitation of methotrexate polyglutamates in a cellular extract.

[0111]MTXPGs were enzymatically converted to MTX by incubation with RBC extract and plasma as follows. RBCs were isolated from healthy donors (Blood bank; San Diego, Calif.). Hemolysates were prepared having 0.88×109 RBC per 100 μl. For spiked samples, MTXPG (Schirks Laboratories; Jona, Switzerland) was added to RBC extracts at a final concentration of 1 μM (from stock solutions containing 100 μM MTXPG in 0.1 N potassium hydroxide). Reconstituted plasma was prepared from lyophilized plasma (Sigma, St. Louis, USA; Cat. No. P9523) and 100 μl was added to 50 μl of RBC extract, mixed for 30 seconds, and 100 μl of buffer containing 100 mM potassium phosphate pH 4.5 and 150 mM mercaptoethanol was added. Because the reconstituted plas...

example iii

Monitoring Methotrexate Polyglutamate Levels in Patients Receiving Low-Dose Methotrexate Therapy

[0118]Methotrexate requires intracellular activation to methotrexate polyglutamate to exert anti-arthritic effects. This example describes a method for quantitating the levels of intracellular methotrexate polyglutamates in the RBCs of a patient following administration of low-dose MTX therapy.

[0119]Blood samples (5 ml) from polyarthritis patients receiving low-dose MTX were collected after written informed consent. The patients had received a median of 15 mg MTX (range 2.5 mg to 40 mg per week) for at least three months. Blood samples were centrifuged for 10 minutes to separate plasma and buffy coat from RBCs. After washing the RBC pellet with 2 volumes of saline, RBC count was determined with a Beckman Coulter Onyx Counter; RBCs were subsequently stored at −70° C. until analysis. Results were normalized to 10 red blood cells. Patient results were expressed as average ±SEM (range).

[0120]...

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Abstract

The present invention provides a method for efficiently converting methotrexate polyglutamates to methotrexate in a cellular extract by contacting the cellular extract with gamma glutamyl hydrolase under conditions suitable for efficient conversion of methotrexate polyglutamates to methotrexate.

Description

BACKGROUND OF THE INVENTION[0001]This invention relates generally to methods for monitoring drug therapy and, more specifically, to methods for monitoring efficacy and toxicity of anti-folate drug therapy.[0002]Folate (folic acid) is a vitamin that is essential for the life-sustaining processes of DNA synthesis, replication and repair. Folate is also important for protein biosynthesis, another process that is central to cell viability. The pteridine compound, methotrexate (MTX), is structurally similar to folate and as a result can bind to the active sites of a number of enzymes that normally use folate as a coenzyme for the biosynthesis of the purine and pyrimidine nucleotide precursors of DNA and for the interconversion of amino acids during protein biosynthesis. Despite its structural similarity to folic acid, MTX cannot be used as a cofactor by enzymes that require folate, and instead competes with the folate cofactor for enzyme binding sites, thereby inhibiting protein and DNA ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/34A61K35/18C12Q1/37G01N33/50
CPCA61K35/18G01N33/5014C12Q1/37C12Q1/34
Inventor DERVIEUX, THIERRYRICHERSON, RUSSELL
Owner CYPRESS BIOSCI
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