Chelators that predominantely form a single stereoisomeric...

Details Chelators that predominantely form a single stereoisomeric... Chelators that predominantely form a single stereoisomeric...

1997-12-24

2002-01-01

Dees, Jose′ G. (Department: 1616)

Drug, bio-affecting and body treating compositions

Radionuclide or intended radionuclide containing; adjuvant...

In an organic compound

C424S001110, C424S001650, C424S009100, C530S300000, C530S328000, C530S329000, C530S330000, C534S014000

Reexamination Certificate

active

06334996

ABSTRACT:

TECHNICAL FIELD
This invention relates to chelators that form a mixture enriched for a single stereoisomeric species upon coordination to a metal center.
BACKGROUND OF THE INVENTION
The current interest in the radiolabeling of biologically important molecules (proteins, antibodies, and peptides) with
99m
Tc stems from the desire to develop a target specific diagnostic radiopharmaceutical.
1-10
The advantages of using
99m
Tc in diagnostic nuclear medicine are well known
11-15
and a number of techniques have been developed for the
99m
Tc labeling of biologically important molecules.
16-20
One obvious approach is to coordinate a
99m
Tc metal directly with the targeting molecule. This approach is known as the direct labeling method and it involves the use of a reducing agent to convert disulfide linkages into free thiolates, which then bind to the
99m
Tc metal. A major disadvantage of this method is the lack of control over the coordination of the
99m
Tc metal and the stability of the resulting metal complex. In addition, the lack of suitable or accessible coordination sites in some proteins and peptides exclude direct labeling as a viable technique. Two common alternatives to direct labeling are the final step labeling method and the pre-formed chelate approach. Both techniques involve the use of a bifunctional chelator, which provides the site of
99m
Tc coordination. The difference between the two approaches lies in the order in which the
99m
Tc complex is formed. In the final step labeling method, complexation occurs after the chelator has been attached onto the targeting molecule. With the pre-formed chelate method, the
99m
Tc complex is initially prepared and purified before being attached to the targeting molecule. In both techniques, the bifunctional chelator must coordinate to
99m
Tc to form a complex that is stable in vivo and the chelator must have an active moiety that can react with a functional group on the targeting molecule.
A number of bifunctional chelators have been used in the labeling of proteins, peptides and monoclonal antibodies.
2,9,10,17,21-28
Depending on the chelator, the labeling of biologically important molecules with bifunctional chelators often results in the formation of multiple species and/or isomeric complexes. An example is the
99m
Tc labeling of molecules using the hydrazinonicotinamide (HYNIC) system. Since the HYNIC group can only occupy one or two sites of Tc coordination, co-ligand are required to complete the coordination sites. Glucoheptonate
29-30
, tris(hydroxymethyl)methylglycine (tricine)
25
, ethylenediamine-N, N′-diacetic acid (EDDA)
9
, water soluble phosphines
25
[trisodium triphenylphosphine-3,3′,3″-trisulfonate (TPPTS); disodium triphenylphosphine-3,3′disulfonate (TPPDS); and sodium triphenylphosphine-3-monosulfonate (TPPMS)] and polyamino polycarboxylates
9
have all been used as co-ligand in the HYNIC system. It has been clearly shown the Tc-99m labeling of molecules via the HYNIC/co-ligand system produces multiple species, which is due to the different coordination modalities of the hydrazine moiety and the co-ligands. The number of species, the type, the stability and the properties of the species vary greatly from one co-ligand to another. In the labeling of chemotatic peptide using the HYNIC system, the nature of the co-ligand also greatly affects the biodistribution of the labeled peptide.
31
Another example of a bifunctional chelator producing multiple species is dithiosemicarbazone (DTS) system. It has been shown that the DTS bifunctional chelator produces at least four complexes with technetium.
32
Two of the complexes are known to be charged; hence they have different biodistribution from the uncharged species.
As in the development of a pharmaceutical based on organic molecules, the stereochemistry or isomerism of a metal complex is also very important in the development of a radiopharmaceutical or metallodrug. It is well known that isomers can often have different lipophilicities, biodistribution and biological activities. An example of this is the
99m
Tc complex of 3,6,6,9-tetramethyl-4,8-diazaundecane-2,10-dione dioxime (
99m
Tc-d,1-HMPAO or Ceretec), which is a cerebral perfusion imaging agent.
14,33-35
Though
99m
Tc-d,1-HMPAO is active, it has been shown that the meso analogs of the
99m
Tc HM-PAO
14,36
complex and the
99m
Tc complex of 3,3,9,9-tetramethyl-4,8-diazaundecane-2,10-dione dioxime
14,37
(PnAO) does not possess the properties necessary for use as a cerebral perfusion imaging agent.
A type of Tc and Re coordination modality common in Tc and Re radiopharmaceuticals is the coordination of a tetradentate N
4−x
S
x
chelator to a metal oxo moiety to form a square pyramidal or octahedral metal oxo complex. A host of bifunctional chelators have been developed based on the tetradentate N
4−x
S
x
coordination motif. Examples include N
4
propylene amine oxime
38
, N
3
S triamide thiols
9, 39-43
, N
2
S
2
diamide dithiols
9, 44-46
, N
2
S
2
monoamide monoaminedithiols
47-49
and N
2
S
2
diamine dithiols
50-55
. Functionalization of the chelator backbone enable these chelators to be attached to biologically interesting molecules. The labeling of these bifinctional chelators with TcO
3+
or ReO
3+
often produce isomers or epimers.
39-43, 46-55
The isomers or epimers (syn and anti) arise from the configuration of the metal oxo group relative to the functional group on the chelator backbone. It has been clearly shown that the biodistribution and biological activity of the syn and anti isomers are often different.
39-43, 46, 56
The Tc complex of mercaptoacetylglycylglycylglycine (MAG
3
), a renal imaging agent, exists in the syn and anti isomers. The biological activities of the syn and anti isomers are known to be different.
39,40
The syn and anti isomers of the Tc complex of 2,3-bis(mercaptoacetamide)propanoate (map) was also shown to have different biological activity.
46
It was reported that in humans, 58% of the syn isomers was excreted at 30 minutes as compared to only 19% of the anti isomer. Another example of the isomers exhibiting a difference in biological behaviour is the
99m
Tc labeled diamino dithiol piperidine conjugate, which were investigated as a brain perfusion imaging agents. It was shown that the two isomeric complexes exhibit widely disparate brain uptake.
55
At 2 minute post-administration in rats, uptake of the anti isomer in the brain was 1.08% dose/g, while the uptake of the syn isomer was 2.34% dose/g. The brain/blood ratio at 2 minute post-administration was 2.09 for the anti isomer and 5.91 for the syn isomer.
The peptide dimethylglycine-serine-cysteine-glycine is a bifunctional chelator that can be use to label biologically important molecules.
61,62
It has been shown that dimethylglycine-serine-cysteine-glycine coordinates to TcO
3+
and ReO
3+
via a monoamine diamide monothiol coordination modality.
61
The resulting Tc and Re complexes exist as two isomers; the serine CH
2
OH side chain is in the syn and anti conformations with respect to the metal oxo bond. The presence of the syn and anti isomers are very evident from the NMR spectral data. In the
1
H NMR spectrum of the Re complex, there were two pairs of singlets associated with the nonequivalent methyl groups in the dimethylglycine residue. Each pair of singlets corresponded to either the syn or anti isomers. The
1
H and
13
C NMR spectral data for the Re oxo complex of dimethylglycine-sercine-cysteine-glycine-NH
2
(RP294) were obtained. The presence of the two isomers are clearly evident from the NMR data. In the coordination of dimethylglycine-isoleucine-cysteine-glycine (RP349) to ReO
3+
, two isomers (syn and anti) were also observed. The
99m
Tc labeling of RP294 and RP349 produced syn and anti isomers; two peaks were observed in the HPLC using the radiometric detector. The
99m
Tc labeling of biotin with dimethylglycine-lysine-cysteine-NH
2
(RP332) also produced syn and anti isomers; two peaks were observed in t

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