Carboxylesterase inhibitors

a technology of carboxylesterase and inhibitors, applied in the field of carboxylesterase inhibitors, can solve the problems of delayed diarrhea, high toxicity of compounds, cell death, etc., and achieve the effect of increasing the effectiveness of insecticides and pesticides

Inactive Publication Date: 2005-03-10
ST JUDE CHILDRENS RES HOSPITAL INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

Further embodiments are directed to a method for treating or ameliorating the toxic effects of drugs (such as the cancer therapy drug CPT-11) administered to a patient, to treating or ameliorating the effects of a drug overdose, and to the use of the compounds for increasing the effectiveness of insecticides and pesticides.

Problems solved by technology

These compounds are regarded as highly toxic poisons.
SN-38 prevents DNA unwinding, and thus inhibits critical cellular processes in tumor cells, resulting in cell death.
The toxicities associated with this agent include a cholinergic syndrome due to direct inhibition of acetylcholinesterase, and delayed diarrhea due to gastrointestinal toxicity.
Following deposition into the small intestine via the bile, SN-38G can be cleaved by bacterial glucuronidases to yield the toxic metabolite SN-38, resulting in local irritation and toxicity to the gut.
Hence very high, local concentrations of SN-38 will be produced, resulting in cytotoxicity and hence diarrhea.
The delayed diarrhea associated with CPT-11 administration can be life-threatening and is the dose limiting toxicity for this agent.
Hence, animal models designed to predict tumor responses in humans may overestimate the efficacy of the drug due to the increased plasma activation of CPT-11.
Therefore, it appears that the activation of CPT-11 in the human intestine by hiCE results in local toxicity, and hence produces the unwanted toxic side effects such as diarrhea.

Method used

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Examples

Experimental program
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Effect test

example 1

Carboxylesterase Inhibition Assay

(a) ortho-nitrophenyl Acetate (o-NPA) as the Substrate

Carboxylesterase (CE) inhibition was determined by a spectroscopic assay using o-nitrophenyl acetate (o-NPA) as a substrate. Recombinant CEs produced from expression in Spodoptera frugiperda Sf9 insect cells via baculovirus were purified from serum-free culture media for the enzyme inhibition studies. Enzymes were incubated in 200 μl of 50 mM Hepes (pH 7.4) containing 3 mM o-NPA. Conversion of the o-NPA to nitrophenol was monitored by measuring the change in the absorbance at 420 nm. Absorbance readings were taken every 15 seconds for 2 minutes, and data transferred to a computer data spreadsheet. Inhibitors were dissolved in dimethylsulfoxide (DMSO) and inhibition of CE activity determined by the addition of the inhibitor solution to the o-NPA reaction mixture at a concentration of 100 μM. The final DMSO concentration was always 1% or less. The Ki value (i.e., the concentration of inhibitor t...

example 2

Inhibition of Acetylcholinesterase and Butyrylcholinesterase

The activity of the hiCE selective carboxylesterase inhibitors identified in Table 3 toward acetylcholinesterase (AcChE) and butyrylcholinesterase metabolism was investigated. Purified AcChE was purchased from Sigma Biochemicals (St. Louis, Mo.) and substrate metabolism was monitored using a spectrophotometric assay. 1 mM acetylthiocholine was mixed with 0.5 mM of 5,5′-dithio-bis-(2-nitrobenzoic acid) in 50 mM Hepes (pH 7.4) in the presence of the inhibitor (100 μM). The reaction was initiated by the addition of 0.22 U / ml AcChE (where 1 U is the amount of enzyme that hydrolyzes 1 μmol of acetylthiocholine iodide per min at pH 7.4 at 37° C.), and the reaction monitored by measuring the change of the absorbance at 405 nM every 15 seconds 2 minutes. Data was transferred to GraphPad Prism software, and Ki values were calculated as described in the Example above.

Purified BuChE was purchased from Sigma Biochemicals (St. Louis...

example 3

Ability of the Selective hiCE Inhibitors to Cross Cell Membranes

To monitor the ability of the compounds to cross the cell membrane and inhibit carboxylesterase activity intracellularly, an assay using human tumor cells expressing hiCE was devised. U373MGhiCE (a human glioma cell line transfected with a plasmid expressing hiCE) were plated in T25 flasks and allowed to grow to confluency (approximately 2-5×105 cells). Inhibitors were then added at a final concentration of 10 μm, and incubation allowed to continue for 1 hour. At this time, the cells were washed extensively with 5 ml of complete media and 2×5 ml of Hanks balanced salt solution, and cell extracts were prepared. Carboxylesterase activity was then determined using o-NPA as a substrate.

Table 8 sets forth the level of intracellular enzyme inhibition following incubation of human glioblastoma tumor cells expressing human intestinal carboxylesterase with 10 μM of each compound identified in Table 3.

TABLE 8Inhibition of i...

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Abstract

This disclosure relates to amides, aryl sulphonamides, aryl ureas, and α,β-diketones derivatives useful as carboxylesterase esterase inhibitors. The disclosure is also directed to the use of these compounds as selective human intestinal carboxylesterase inhibitors and insect carboxylesterase inhibitors. The disclosure is also directed to pharmaceutical compositions and pesticide formulations containing these compounds, and to methods for treating or ameliorating the toxic effects following administration of drugs such as cancer therapy drugs, treating or ameliorating the effects of a drug overdose, and to the use of the compounds for increasing the effectiveness of insecticides and pesticides.

Description

FIELD OF THE INVENTION This invention is directed to the use of amide, aryl sulphonamide, aryl urea, and α,β-diketone compounds as carboxylesterase inhibitors, and particularly to the use of these compounds as selective human intestinal carboxylesterase inhibitors. The compounds are also useful as insect carboxylesterase inhibitors. The invention is also directed to pharmaceutical compositions and pesticide formulations containing the amide, aryl sulphonamide, aryl urea, and α,β-diketone esterase inhibitors, and to methods for treating or ameliorating the toxic effects of drugs, such as cancer therapy drugs, treating or ameliorating the effects of a drug overdose, and to the use of the compounds for increasing the effectiveness of insecticides and pesticides. BACKGROUND OF THE INVENTION Carboxylesterase (CE) is one of a sub-class of enzymes known collectively as hydrolases. Hydrolase enzymes catalyze the hydrolysis of various bonds, with carboxylesterase specific for the hydrolysi...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K31/165A61K31/40A61K31/44
CPCA61K31/165A61K31/44A61K31/40
Inventor POTTER, PHILIP M.HYATT, JANICE L.MORTON, CHRISTOPHER L.BEROZA, PAUL P.DAMORADAN, KOMATH V.
Owner ST JUDE CHILDRENS RES HOSPITAL INC
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