Drug – bio-affecting and body treating compositions – Radionuclide or intended radionuclide containing; adjuvant... – In an organic compound
Reexamination Certificate
1997-11-06
2001-04-17
Rotman, Alan L. (Department: 1625)
Drug, bio-affecting and body treating compositions
Radionuclide or intended radionuclide containing; adjuvant...
In an organic compound
C424S001810, C424S001850, C530S363000, C530S387100, C530S391500, C530S395000
Reexamination Certificate
active
06217845
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to bifunctional compounds capable of linking metal ions, particularly technetium and rhenium, to biologically useful molecules.
Because of their high biological specificity, certain macromolecules (e.g., monoclonal antibodies) have been used to target radioisotopes to specific in vivo sites for the purpose of imaging and/or therapy. The use of the metastable isotope of technetium,
99m
Tc, in diagnostic nuclear medicine is well established and the beta-emitting isotopes of rhenium
186
Re,
188
Re and
189
Re can be used therapeutically. A number of methods for attaching technetium to macromolecules have been described. Some of these methods involve the reduction of disulfide groups in the macromolecule (usually an immunoglobulin) to thiols and the subsequent use of these groups to bind reduced Tc (e.g., McKenzie et al., International Publication #WO 87/04164; and Bremer et al., EPO 271 806 A2). Methods of this type have several potential disadvantages. The reduction of disulfide units can lead to protein de-naturation and a subsequent loss in biological specificity. Also, the method cannot be used to label macromolecules lacking disulfide moieties.
Alternatively,
99m
Tc can be linked to macromolecules via bifunctional chelates such as DTPA (D. Lanteigne and D. J. Hnatowich,
Int. J. Appl. Radiat. Isot.,
Vol. 35(7), p. 617 (1984), chelating thiosemicarbazones (Y. Arano et al.,
Int. J. Nucl. Med. Biol.,
Vol. 12, p. 425 (1985), and diamide-dithiol ligands (A. Fritzberg, European Patent Appl. EP 188256 2A). Problems associated with these methods include significant nonspecific binding of technetium (binding to the protein at sites other than the chelating group) and slow kinetics of Tc-labelling.
Accordingly, it is the object of the present invention to provide new bifunctional molecules having hydrazine or hydrazide groups and protein reactive groups which can be used to link metal ions, such as
99m
Tc, to macromolecules.
Another object of the present invention is to provide a method for labelling macromolecules with metal ions in which binding of the metal at sites other than the chelating group is minimal, and in which labelling occurs at a relatively fast rate (less than one hour at room temperature).
SUMMARY OF THE INVENTION
According to the invention, novel bifunctional hydrazine and hydrazide compounds, as well as conjugates thereof, are provided. Methods of labelling the conjugates with metal ions are also provided.
Broadly, the hydrazine and hydrazide compounds described herein as bifunctional aromatic hydrazines or hydrazides having a protein reactive substituent and a negative counterion. A modification of this invention is also provided in which the hydrazine or hydrazide function is protected as a lower alkyl hydrazone.
In another embodiment of the invention, conjugates are formed by reacting bifunctional hydrazine or hydrazide compounds of the invention with macromolecules such as proteins, polypeptides or glycoproteins. The bifunctional compounds react with nucleophilic groups on the macromolecules (e.g. lysine residues) to yield conjugates containing free hydrazine/hydrazide groups.
In a third embodiment, labelled macromolecules comprised of conjugates and metal ions are formed.
In a fourth embodiment, a method is provided for labelling macromolecules by reacting a conjugate of the invention with a metal species.
DETAILED DESCRIPTION OF THE INVENTION
The novel hydrazine and hydrazide compounds of the present invention are represented by one of the following formulas (I) or (II):
wherein:
A is a carbon or nitrogen atom;
B is a carbon or nitrogen atom;
D is a direct bond (to the 2-, 3-, or 4-position of the ring), CH
2
, C═O or
E is C═O or together with F forms a maleimidyl group;
F is a group readily replaced by a primary amine in neutral or basic aqueous media when E is C═O or together with E forms a maleimidyl group;
R is hydrogen or a lower alkyl group;
R′ and R″ may be the same or different and are selected from hydrogen and lower alkyl; and
X is a negative counterion.
Another embodiment of the invention includes compounds of the formula (III):
where R, R′, R″, E and F have the values given above.
When E is carbonyl C═O, F is any group which, in combination with the attached carbonyl group, forms an active ester or active amide. Examples of suitable species for F include such diverse groups as N-oxysuccinimidyl, tetrafluorophenolate, N-oxybenztriazole and imidazolate. These examples are not intended to be construed as limiting the scope of the invention.
Suitable groups for R, R′ and R″ include, but are not limited to, the following: H, CH
3
, C
2
H
5
, and C
3
H
7
.
Examples of useful X ions are halides, nitrate, trifluoroacetate, tetrafluoroborate and sulfate. These examples are not intended to limit the scope of suitable counterions.
The above-described compounds are stable, isolable derivatives of molecules that contain two cross-reactive moieties: a hydrazine/hydrazide group and a protein reactive group such as an active ester, active amide or maleimido group.
In the synthesis of these stable derivatives, an acid labile protecting group such as t-butoxycarbonyl (t-BOC) is removed from the hydrazine/hydrazide under anhydrous acidic conditions, leaving the protein reactive group unchanged and the hydrazine/hydrazide group in an unreactive, protonated form. Alternatively, the hydrazine/hydrazide group can be protected as a lower alkyl hydrazone.
When a bifunctional compound having a protonated (or hydrazone protected) hydrazine/hydrazide function is then combined with a macromolecule such as a protein, polypeptide or glycoprotein in neutral or slightly basic media, preferably a pH of about 7-8.5, the protein-reactive part of the compound will react with nucleophilic groups on the protein, polypeptide or glycoprotein (e.g., amine groups such as lysine residues) to yield a conjugate containing free hydrazine/hydrazide groups. In the case of hydrazone conjugates, the free hydrazine/hydrazide is formed by dialysis into an acidic (pH 5.6) buffer. Because this type of conjugate includes a hydrazine or hydrazide, a strong metal binding group, it will then readily react when mixed with a suitable metal species in acidic media to yield a labelled protein, polypeptide or glycoprotein.
The metal species may be, for example, a reduced Tc species formed by reacting TcO
4
−
with a reducing agent, for example, stannous ion, in the presence of a chelating oxygen ligand (e.g. glucoheptonate). Examples of suitable reduced Tc species include Tc-glucoheptonate, Tc-gluconate, Tc-2-hydroxyisobutyrate, Tc-lactate and Tc-4,5-dihydroxy 1,3-benzene disulfonate. Other metals and ligands are also within the scope of the invention.
A Tc labelling process can be conveniently performed in an aqueous buffer, preferably at a pH of about 4.5-6.5, in one hour or less. Reaction with other suitable metal species occurs in a similar manner under similar conditions.
Radiochemical yield as determined by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC) using Tc is ≧90%. Treatment of protein with nonlinkable analogs, (i.e., compounds without a protein reactive carbonyl group, such as 4-hydrazinobenzoic acid or 6-hydrazinopyridine-3-carboxylic acid) does not yield protein capable of significant Tc binding, thus demonstrating the high specificity of this technique.
The technetium atoms are believed to be bound to the conjugate via a hydrazide or diazenido linkages:
Wherein: L is an ancillary dioxygen ligand.
Examples of this type of linkage have been described for Mo and Re (
Comprehensive Coordination Chemistry,
Vol. 2, G. Wilkinson, ed., Pergamon (Oxford) 1987, p. 130-151) and several analogous complexes of
99
Tc have been prepared by the reaction of an organohydrazine derivative and a Tc(V) oxo species.
The above labelling scheme has been used to label polyclonal human IgG and the Fc region of human IgG. The Tc-conjugates have been used
Abrams Michael J.
Giandomenico Christen M.
Schwartz David A.
Zubieta Jon A.
AnorMed Inc.
Rotman Alan L.
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