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US 7,884,186 B2Granted
Isotopically labeled trapping agent and method for identifying reactive metabolites
AssigneeJanssen Pharmaceutica NV
InventorsNorman D. Huebert, Zhengyin Yan
Life of this patent
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Abstract
The present invention is directed to an isotopically labeled trapping agent, methods for detecting reactive metabolites and methods of identifying drug candidates. More specifically, the isotopically labeled trapping agent and methods for detecting reactive metabolites may be used to detect both “hard” and “soft” reactive metabolites, thereby eliminating false positives.
Claims39
An isotopically labeled compound of formula (I)
- wherein one or more of the carbon, nitrogen, oxygen, sulfur or non-exchangeable hydrogen atoms on the starred groups are isotopically enriched.
An isotopically enriched compound of formula (I-IS)
A method for detecting reactive metabolites of a test compound comprising
- (a) incubating a test compound with an isotopically labeled compound of formula (I)
- wherein one or more of the carbon, nitrogen, oxygen, sulfur or non-exchangeable hydrogen atoms on the starred groups are isotopically enriched;
- and a drug metabolizing enzyme;
- to yield a product mixture comprising one or more adducts;
- and
- (b) detecting the one or more adducts of Step (a).
3 more elements
A method for detecting reactive metabolites of a test compound comprising
- (a) incubating a test compound with a mixture comprising a compound of formula (I)
- an isotopically labeled compound of formula (I)
- wherein one or more of the carbon, nitrogen, oxygen, sulfur or non-exchangeable hydrogen atoms on the starred groups are isotopically enriched;
- and a drug metabolizing enzyme;
- to yield a product mixture comprising one or more adducts;
- and
- (b) detecting the one or more adducts of Step (a).
4 more elements
A method for detecting reactive metabolites of a test compound comprising
- (a) incubating a test compound with a mixture comprising a compound of formula (I)
- an isotopically labeled compound of formula (I)
- wherein one or more of the carbon, nitrogen, oxygen, sulfur or non-exchangeable hydrogen atoms on the starred groups are isotopically enriched;
- and a drug metabolizing enzyme;
- to yield a product mixture comprising one or more adducts;
- (b) detecting one or more isotopic doublets in a neutral loss mass spectrum of the one or more adducts produced in Step (a), wherein the doublet differs in mass by the difference in mass between the compound of formula (I) and the isotopically labeled compound of formula (I).
3 more elements
A mixture comprising a compound of formula (I)
- and an isotopically labeled compound of formula (I)
- wherein one or more of the carbon, nitrogen, oxygen, sulfur or non-exchangeable hydrogen atoms on the starred groups are isotopically enriched.
A mixture comprising
- (a) a covalently bonded complex of a reactive metabolite and a compound of formula (I)
- and (b) a covalently bonded complex of a reactive metabolite and an isotopically labeled compound of formula (I)
- wherein one or more of the carbon, nitrogen, oxygen, sulfur or non-exchangeable hydrogen atoms on the starred groups are isotopically enriched.
A method for detecting reactive metabolites of a test compound comprising
- (a) incubating a test compound with a mixture comprising a compound of formula (I),
- an isotopically-labeled compound of formula (I-IS)
- and a drug metabolizing enzyme;
- to yield a product mixture comprising one or more adducts;
- (b) measuring a neutral loss mass spectrum of the one or more adducts produced in step (a);
- and
- (c) detecting one or more isotopic doublets in the neutral loss mass spectrum of step (b), wherein the isotopic doublets differ in mass by 8 Da.
4 more elements
A method for detecting reactive metabolites of a test compound comprising
- Step A:
- incubating a mixture comprising
- (a) a test compound;
- (b) a compound of formula (I)
- (c) an isotopically labeled compound of formula (I-IS)
- wherein the molar ratio of the compound of formula (I) to the isotopically labeled compound of formula (I-IS) is about 1:1;
- and
- (c) a drug metabolizing enzyme selected from the group consisting of human liver microsomes, cytochrome P450 enzymes in combination with NADPH co-factor and cytochrome P450 enzymes in combination with NADPH regenerating system;
- to yield a product mixture comprising one or more incubation products selected from the group consisting of
- (a) a non-reactive metabolite;
- (b) an adduct formed between the compound of formula (I) and a reactive metabolite;
- and
- (c) an adduct formed between the isotopically labeled compound of formula (I-IS) and a reactive metabolite;
- Step B:
- measuring a neutral loss mass spectrum of the product mixture produced in Step (A);
- and
- Step C:
- detecting one or more isotopic doublets in a neutral loss mass spectrum of Step (B), wherein the isotopic doublets differ in mass by 8 Da.
15 more elements
A method for detecting reactive metabolites of a test compound comprising
- (a) incubating a test compound with a mixture comprising a non-isotopically labeled compound of formula (I)
- an isotopically labeled compound of formula (I)
- wherein one or more of the carbon, nitrogen, oxygen, sulfur and/or non-exchangeable hydrogen atoms on the starred groups are isotopically enriched;
- and a drug metabolizing enzyme to yield a product mixture comprising one or more adducts;
- (b) separating the one or more adducts of step (a);
- (c) measuring a neutral loss mass spectrum of said separated adducts;
- (d) detecting one or more isotopic doublets in the neutral loss mass spectrum of said separated adducts, wherein the doublet differs in mass by the difference in mass between the non-isotopically labeled compound of formula (I) and the isotopically labeled compound of formula (I).
4 more elements
A method for detecting reactive metabolites of a test compound comprising
- (a) incubating a test compound with a mixture comprising a non-isotopically labeled compound of formula (I)
- an isotopically labeled compound of formula (I-IS)
- wherein the molar ratio of the compound of formula (I) to the isotopically labeled compound of formula (I-IS) is about 1:1;
- and a drug metabolizing enzyme to yield a product mixture comprising one or more adducts;
- (b) separating the one or more adducts of step (a);
- (c) measuring a neutral loss mass spectrum of said separated adducts;
- (d) detecting one or more isotopic doublets in the neutral loss mass spectrum of said separated adducts, wherein the doublet differs in mass by 8 Da.
4 more elements
A method for identifying a drug candidate comprising
- (a) incubating a test compound with a mixture comprising a non-isotopically labeled compound of formula (I)
- an isotopically labeled compound of formula (I)
- wherein one or more of the carbon, nitrogen, oxygen, sulfur or non-exchangeable hydrogen atoms on the starred groups are isotopically enriched;
- and a drug metabolizing enzyme;
- to yield a product mixture;
- (b) measuring a neutral loss mass spectrum of the product mixture produced in Step (a);
- and
- (c) detecting the absence of isotopic doublets in the neutral loss mass spectrum of Step (b).
5 more elements
A method for identifying a drug candidate comprising
- (a) incubating a test compound with a mixture comprising a non-isotopically labeled compound of formula (I)
- an isotopically labeled compound of formula (I-IS)
- and a drug metabolizing enzyme;
- to yield a product mixture;
- (b) measuring a neutral loss mass spectrum of the product mixture produced in Step (a);
- and
- (c) detecting the absence of isotopic doublets in the neutral loss mass spectrum of Step (b).
4 more elements
Dependencies are inferred from the claim text.