Method of labeling amine-containing compounds with a metal

Details Method of labeling amine-containing compounds with a metal Method of labeling amine-containing compounds with a metal

1986-07-10

1989-07-04

Warden, Robert J.

Chemistry: analytical and immunological testing

Involving production or treatment of antibody

Monoclonal antibody

436545, 436804, 424 11, 530389, 530402, 534 10, G01N 3353

Patent

active

048450391

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

This invention is in the field of chemistry and particulary relates to the labeling of amine-containing compounds, such as proteins, with metals.


BACKGROUND OF THE INVENTION

Proteins are used widely in bio-medical science. For example, antibodies are particularly useful in many diagnostic or therapeutic applications because of their special biological activity and their specific affinity for certain antigenic determinants found on specialized cells in tissue and organs.
The ability to detect the extent to which an antibody has accumulated within certain tissue or organs often depends upon the detection of a label bound to the antibody. Labels which emit radioactivity, fluorescent light under excitation or signals detectible by NMR measurements have been employed.
Although certain metal radioisotopes have been employed as labels, there have been limitations on such use to date. For example, the methods heretofore available for labeling proteins with metal radioisotopes have been of limited utility and have been limited to specific metals. A need has existed, for example, for a better method for labeling antibodies, particularly monoclonal antibodies, with technetium-99m (.sup.99m Tc), a particularly desirable metal radioisotope.


DESCRIPTION OF THE INVENTION

This invention provides a method for labeling proteins and other amine-containing compounds with metals. The method involves reacting metal ions in an acidic medium with a compound represented by the formulas: ##STR1## wherein R.sub.1, R.sub.2 and R.sub.3 are individually selected from hydrogen, alkyl, substituted alkyl, aryl, alkaryl, etc. An example of an amine compound of formula I is diethylamine. Specific examples of amide compounds of formula II are dimethyl formamide (DMF), diethylformamide and monomethyl formamide. The result of this first reaction is a reactive intermediate compound containing the metal and which is subsequently reacted with the compound to be labeled in a solution having a pH selected for the particular compound being labeled.
This method allows the labeling of proteins with metal radioisotopes to produce labeled proteins having beneficial properties for immuno-diagnostic and therapeutic applications. Labeled proteins can be produced which are non-toxic, non-pyrogenic and sterile. These are important properties, particularly where the labeled protein will be used in vivo. Very importantly, the metal labels are tightly bound to the proteins and do not significantly interfere with the biological activity of the proteins.
This method is particularly suitable for labeling monoclonal antibodies (MAb) with the radioisotope technetium-99m (.sup.99m Tc). For example, DMF can be employed in a two-step method for labeling MAb with .sup.99m Tc by the following procedure. In a first step, DMF is reacted with technetium pertechnetate under acidic conditions for about four hours at a temperature of about 140.degree. C. to form a reactive .sup.99m Tc intermediate compound which precipitates as a crystalline white powder. In a second step, the reactive .sup.99m Tc compound and MAb are reacted at higher pH (e.g., 7.0) which is maintained with a buffer for about one hour to produce the monoclonal antibody labeled with technetium-99m. This reaction scheme can be illustrated as follows: ##STR2##
A high yield (e.g., 90%) of the reactive intermediate compound can be obtained by careful selection of the elevated temperature employed in the first reaction. The specific temperature depends, of course, on the specific reactants and other reaction conditions. When using DMF, it has been found that temperatures between about 135.degree. C. and 145.degree. C. are preferred.
The H.sup.+ ions required for the desired acidity can be obtained by addition of an acid. Hydrochloric acid is one satisfactory acid in this respect. Good results have been obtained, in particular, by using a ten-fold molecular excess of the amine or amide with respect to HCl.
The pH of the solution which is used in the second reaction stage is chosen as a

REFERENCES:
patent: 4238589 (1980-12-01), Cleeland, Jr. et al.
patent: 4472509 (1984-09-01), Garison et al.

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