Use of stable label compounds with accelerator mass spectrometry

Abstract

Disclosed herein is a method for evaluating and measuring the performance, efficacy and safety of candidate new chemical entities. This method comprises employing a target compound having one or more .sup.12C atoms in the molecule wherein at least one of the .sup.12C atoms is substituted with a .sup.13C atom, The stable labelled target compound is then administered to a test subject following which the target compound and/or one or more of its metabolites are recovered using conventional separation techniques and purified. The resulting isolated material of interest is then combusted in the presence of a petrochemical based carrier, the .sup.12C content of such carrier with respect to naturally occurring .sup.13C being in excess of 99.9 percent. The CO.sub.2 resulting from such combustion is then graphitized and the graphitized material is analyzed employing techniques, such as Accelerator Mass Spectrometry (AMS), capable of differentiating and counting the carbon atom isotopes (.sup.13C vs. .sup.12C), thus allowing quantification of the compound/metabolite of interest.

Claims

1. A process for the tracing and quantification of metabolites or other breakdown products of target compounds without the use of radiolabels which comprises: employing a target compound on which one or more of .sup.12C carbons has been substituted with .sup.13C carbons, administering the .sup.13C labeled target compound to a test subject, separating and purifying the parent compound and one or more metabolites of interest from the test subject by conventional separation techniques, combusting the purified compound of interest in the presence of a carrier having a .sup.12C purity of at least 99.9% and graphitizing the resultant CO.sub.2 and subjecting the graphitized material to analysis by Accelerator Mass Spectrometry to quantify the amount of .sup.13C present in the sample.

2. A process as in claim 1, wherein greater than one .sup.12C carbon has been substituted with a .sup.13C carbon.

3. A process as in claim 1 wherein the carrier is benzoic acid.

4. A process as in claim 1 wherein the carrier has a .sup.12C purity of at least 99.99%.

5. A process as in claim 1 wherein the carrier has a .sup.12C purity of at least 99.999%.

Description

DETAILED DESCRIPTION

(1) The science of attaching labels, both stable and radioactive, is highly specialized owing to the diversity of materials that are candidates for such labelling. Generally speaking however, the use of stable labels such as .sup.13C, presents fewer problems. In connection with the drug industry for example, a wide variety of candidates, large and small molecule, and intermediates are being proposed and each requires special considerations, for example regarding the location and number of labels to be attached without altering the chemical structure of the molecule. Teams of specially trained and experienced scientists both within the pharma industry and in custom radiolabelled synthesis contract laboratories have come into being to meet the growing requirements for such services. Those skilled in the art will understand that the scope of this invention does not relate to the various synthesis techniques that are known or must be developed, rather to the process that follows and allows the testing and quantification of the target material, once labelled with one or more .sup.13C atoms.

(2) Once synthesized, the labelled material will generally be employed by in vivo administration to a test subject with the objective of potentially determining virtually every aspect of the material's behavior in vivo or in any other applied environment (plants, soil, etc.). In this manner, a variety of safety and efficacy information may be obtained. For example a drug candidate may be used in preclinical studies to evaluate the absorption, distribution, metabolism, excretion and toxicology (ADMET) profile.

(3) Following application of the labelled material, depending upon the nature of the evaluation being conducted, the material and/or its metabolites may be separated from the test subject by conventional means such as chemical extraction and chromatographic separation specific to the nature of the substrate and the material to be further analyzed. Conventionally the target analyte containing the .sup.14C labelled compound or metabolite would then be analyzed, for example by decay counting, to determine the amount of .sup.14C present, for comparison to total .sup.14C in the applied labelled material.

(4) Recently, an analytical platform known as Accelerator Mass Spectrometry (AMS) has been developed which, owing to its high sensitivity (that is, its power to separate a minor amount of one isotope from an abundant neighboring mass, e.g., .sup.14C from .sup.12C), allows the use of increasingly small amounts of .sup.14C radiolabelled material while still obtaining meaningful results. Indeed it has been proposed that the sensitivity of the AMS technology may allow relatively risk free microdosing of human subjects to allow the study of the candidate material in the ultimately intended target. Simply put, the AMS platform entails combusting (oxidizing) the purified target analyte in the presence of relatively large quantities of a petrochemical based organic carrier such as benzoic acid. The resultant CO.sub.2 is then graphitized by known methods and the AMS instrument ionizes the graphitized sample. The .sup.12C, .sup.13C (naturally occurring) and .sup.14C ions are separated and counted, using separate detectors the most sensitive of which is able to count individual .sup.14C ions. These results can then be compared to the specific radioactivity of the target analyte and the total carbon content of the graphitized sample. However, the problems associated with radiolabelled materials, such as synthesis, expense, storage, handling and disposal, continue.

(5) The present invention builds upon the conventional process by recognizing that, with the application of appropriate improvements, the advantages provided by the sensitivity of the advanced AMS technology can be used to allow the use of stable labelled isotopes, particularly .sup.13C, in place of the radiolabel(s), such as .sup.14C. A simple substitution of labels is not possible owing to the fact that, while .sup.14C is quite rare in nature (less than one atom in a million), .sup.13C is much more prevalent being naturally present on the order of 1.1%. While still small relative to the .sup.12C present in organic compounds, the amount of carbon attributed to the .sup.13C naturally present in the carrier and the test material, would obscure that contributed by a .sup.13C stable label. However, it has now been found that use of a carrier with the AMS platform that has a .sup.12C purity in excess of 99.9%, preferably more than 99.99% and for some applications 99.999% or more, coupled with the sensitivity realized by the AMS technology, allows detection of the stable label with sufficient sensitivity for many purposes now only capable of being realized with the use of radioisotopes. Further, the degree of purity of .sup.12C required can be controlled by increasing the number of .sup.13C labels incorporated in the material to be evaluated, thus increasing the ability to detect and differentiate the .sup.13C attributable to the labelled material versus background.

(6) Although the disclosure has been described with respect to specific embodiments, it is not intended to be limited thereto. It will be understood by those skilled in the art that various changes in the steps, details, materials and the like which have been herein described in order to explain the nature of the invention may be made while remaining within the spirit of the disclosure and the scope of the claims.