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Methods for Detection of Promoter Polymorphism in UGT Gene Promoter

a technology of promoter polymorphism and promoter, which is applied in the direction of heterocyclic compound active ingredients, enzymology, transferases, etc., can solve the problems of major toxicity associated with tas-103 therapy, major problem of increased susceptibility to drug toxicity, and mild intermittent jaundi

Inactive Publication Date: 2007-04-26
DI RIENZO ANNA +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention provides methods for detecting promoter polymorphisms in a UGT gene by measuring the number of TA repeats in the UGT promoter. These polymorphisms are associated with altered gene expression and can affect drug metabolism and sensitivity. The invention also provides methods for screening individuals for these polymorphisms to optimize drug dosages and predict an individual's sensitivity to xenobiotics. The invention further provides a kit for detecting these polymorphisms and a method for detecting glucuronidation activity by measuring the number of TA repeats in the UGT promoter."

Problems solved by technology

The higher than normal levels of bilirubin in the blood often result in episodes of mild intermittent jaundice.
On the other hand, the major toxicity associated with TAS-103 therapy is leukopenia.
In patients with GS or CN, where glucuronidation activity is low, increased susceptibility to drug toxicity is a major problem.

Method used

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  • Methods for Detection of Promoter Polymorphism in UGT Gene Promoter

Examples

Experimental program
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example 1

SN-38 Glucuronidation Assay

[0031] To investigate which UGT enzymes are responsible for SN-38 glucuronidation, the in vitro metabolism of SN-38 was measured using normal human and rat liver microsomes, as well as microsomes from Gunn rats, Gunn, C. H., J. Hered; 29:137-139 (1938), and patients with Crigler-Najjar type I (CN-1) syndrome. Liver microsomes were prepared from normal humans, CN-1 patients, normal rats, and Gunn rats and assayed for SN-38 glucuronidation. Microsomes from human liver samples medically unsuitable for liver transplantation were acquired under the auspices of the Washington Regional Transplant Consortium. In addition, microsomes were prepared by differential centrifugation methods. Purba, H. S., et al., Br. J. Clin. Pharmacol. 23: 447-453, using human liver samples obtained with the approval of the Institutional Review Boards of institutions involved through the Liver Tissue Procurement and Distribution System. Investigations were performed with pooled micros...

example 2

AZT Glucuronidation Assay

[0035] To ensure the viability and functional enzymatic activity of the microsomal preparations, the UGT activity toward another substrate, AZT (3′-azido-3′-deoxythymidine) was investigated as a control. Liver microsomes from normal humans, CN-1 patients, normal rats, and Gunn rats were prepared as described above. Investigations were performed in pooled microsomes from normal rats, normal humans and Gunn rats, while microsomes from CN-1 patients were used individually.

[0036] Microsomes were preincubated with oleolyl lysophosphatidylcholine (OLPC) (0.8% wt / wt. OLPC / microsomal protein) at 4° C. for 20 minutes. The final reaction mixture contained 1 mg / ml preincubated microsomes, AZT (5 mM), MgCl2 (10 mM), 0.2 M Tris-HCl buffer (pH 7.3), and UDPGA (10 mM) in a final volume of 300 μl. After an incubation period of 1 hour at 37° C., the reaction was stopped with the addition of 30 μl of HCl. Proteins were removed after centrifugation at 10,500 g for 5 minutes ...

example 3

Glucuronidation Correlation Assay

[0038] To determine which UGT1 isoform is responsible for SN-38 glucuronidation, the correlation between the rates of formation of SN-38G and glucuronidation of bilirubin & PNP was investigated. PNP was chosen because planar phenols such as PNP and acetaminophen are glucuronidated predominantly by UGT1A6, See Burchell, et al., DNA cell biol., 10:487-494 (1991); Harding, et al., Proc. Natl. Acad Sci. USA, 85:8381-8385 (1988), although several other isoforms such as UGT1A1, UGT1A4, and UGT2B15 have been reported to play a role in PNP glucuronidation in humans. See King, et al., Arch. Biochem. Biophys., 332:92-100 (1996); Green, et al., Drug Metab. Dispos., 23:299-302 (1995). Bilirubin was chosen because bilirubin has been previously shown to be glucuronidated by two isozymes of UGT1: UGT1A1 and UGT1A4. Ritter, et al., J. Biol. Chem., 266:1043-1047 (1991).

[0039] The formation of bilirubin mono- and diglucuronides was quantified using 14C-labeled UDPGA...

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Abstract

The present invention is directed to methods for detecting the presence of genetic polymorphisms that correlate with altered gene expression. More specifically, the present invention is directed to methods for detecting the genetic polymorphisms located in the UGT1A1 promoter. The invention also provides methods for optimizing drug dosages based upon the presence of the polymorphisms. The invention further provides methods of predicting sensitivity to xenobiotics and diagnostic kits for detecting genetic polymorphisms.

Description

BACKGROUND [0001] Researchers have invested considerable effort attempting to identity the pharmacogenetic basis of idiosyncratic adverse drug reactions, particularly hypersensitivity reactions. There is clear evidence for pharmacogenetic influence on susceptibility to hypersensitivity reactions. One such pharmacogenetic influence is genetic polymorphism. Genetic polymorphisms involve the regular and simultaneous existence in the same population of two or more discontinuous variants or genotypes in frequencies that cannot be due to recurrent mutations. Probably, the best known example of genetic polymorphism involves the different human blood groups. Genetic polymorphisms at loci which encode enzymes involved in metabolism of toxic or carcinogenic compounds can have clinical implications in drug metabolism. The pharmacokinetic and pharmacodynamic consequences of the activity of a polymorphic enzyme depend upon whether it mediates metabolism of the parent drug, the metabolites or bot...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12P19/34A61K31/55C12N9/24C12Q1/6844C12Q1/6883C12Q1/6886
CPCA61K31/55C12Q1/6844C12Q1/6883C12Q1/6886C12Q2600/106C12Q2600/156C12Q2600/158C12N9/1051C12Y204/01017
Inventor DI RIENZO, ANNAIYER, LALITHARATAIN, MARK J.
Owner DI RIENZO ANNA
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