Radionuclide labeling of vitamin B12 and coenzymes thereof

Details Radionuclide labeling of vitamin B12 and coenzymes thereof Radionuclide labeling of vitamin B12 and coenzymes thereof

2000-07-26

2003-09-02

Jones, Dameron L. (Department: 1616)

Drug, bio-affecting and body treating compositions

Radionuclide or intended radionuclide containing; adjuvant...

In an organic compound

C424S001110, C424S001650, C424S009100, C536S026440

Reexamination Certificate

active

06613305

ABSTRACT:

BACKGROUND OF THE INVENTION
For several years after the isolation of vitamin B
12
as cyanocobalamin in 1948, it was assumed that cyanocobalamin and possibly hydroxocobalamin, its photolytic breakdown product, occurred in man. Since then it has been recognized that cyanocobalamin is an artifact of the isolation of vitamin B
12
and that hydroxocobalamin and the two coenzyme forms, methylcobalamin and adenosylcobalamin, are the naturally occurring forms of the vitamin.
The structure of these various forms is shown in
FIG. 1
, wherein X is CN, OH, CH
3
or adenosyl, respectively. Hereinafter, the term cobalamin will be used to refer to all of the molecule except the X group. The fundamental ring system without cobalt (Co) or side chains is called corrin and the octadehydrocorrin is called corrole. The Co-contg heptacarboxylic acid resulting from hydrolysis of all the amide groups without the CN and the nucleotide, is designated cobyrinic acid. The corresponding hexacarboxylic acid with D-1-amino-2-propanol side chain f is called cobinic acid and the hexacarboxylic acid with the &agr;-D-ribofuranose-3-phosphate attached to the 2-position of the amino propanol is called cobamic acid. Thus, cobamide is the hexaamide of cobamic acid, cobyric acid is the hexaamide of cobyrinic acid and cobinamide is the hexaamide of cobinic acid.
FIG. 1
is adapted from
The Merck Index
, Merck & Co. (11
th
ed. 1989), wherein X is above the plane defined by the corrin ring and nucleotide is below the plane of the ring. The corrin ring has attached six amidoalkyl (H
2
NC(O)Alk) substituents, at the 2, 3, 7, 8, 13, and 18 positions, which can be designated a-e and g, respectively. See D. L. Anton et al.,
J. Amer. Chem. Soc.,
102, 2215 (1980). The molecule shown in
FIG. 1
can be abbreviated as shown below:
wherein, e.g., X is CN, OH, CH
3
, or adenosyl.
Methylcobalamin serves as the cytoplasmic coenzyme for
5
N-methyltetrahydrofolate:homocysteine methyl transferase (methionine synthetase, EC 2.1.1.13), which catalyzes the formation of methionine from homocysteine. Adenosylcobalamin is the mitochondrial coenzyme for methylmalonyl CoA mutase (EC5.4.99.2) which interconverts methylmalonyl CoA and succinyl CoA.
All forms of vitamin B
12
(adenosyl-, cyano-, hydroxo-, or methylcobalamin) must be bound by the transport proteins, Intrinsic Factor and Transcobalamin II to be biologically active. Specifically, gastrointestinal absorption of vitamin B
12
relies upon the intrinsic factor-vitamin B
12
complex being bound by the intrinsic factor receptors in the terminal ileum. Likewise, intravascular transport and subsequent cellular uptake of vitamin B
12
throughout the body is dependent upon transcobalamin II and the cell membrane transcobalamin II receptors, respectively. After the transcobalamin II-vitamin B
12
complex has been internalized, the transport protein undergoes lysozymal degradation, which releases vitamin B
12
into the cytoplasm. All forms of vitamin B
12
can then be interconverted into adenosyl-, hydroxo-, or methylcobalamin depending upon cellular demand. See, for example, A. E. Finkler et al.,
Arch. Biochem. Biophys.,
120, 79 (1967); C. Hall et al.,
J. Cell Physiol.,
133, 187 (1987); M. E. Rappazzo et al.,
J. Clin. Invest.,
51, 1915 (1972) and R. Soda et al.,
Blood,
65, 795 (1985).
Cells undergoing rapid proliferation have been shown to have increased uptake of thymidine and methionine. (See, for example, M. E. van Eijkeren et al.,
Acta Oncologica,
31, 539 (1992); K. Kobota et al.,
J. Nucl. Med.,
32, 2118 (1991) and K. Higashi et al.,
J. Nucl. Med.,
34, 773 (1993)). Since methylcobalamin is directly involved with methionine synthesis and indirectly involved in the synthesis of thymidylate and DNA, it is not surprising that methylcobalamin as well as Cobalt-57-cyanocobalamin have also been shown to have increased uptake in rapidly dividing tissue (for example, see, B. A. Cooper et al.,
Nature,
191, 393 (1961); H. Flodh,
Acta Radiol. Suppl.
284, 55 (1968); L. Bloomquist et al.,
Experientia,
25, 294 (1969)). Additionally, upregulation in the number of transcobalamin II receptors has been demonstrated in several malignant cell lines during their accelerated thymidine incorporation and DNA synthesis (see, J. Lindemans et al.,
Exp. Cell. Res.,
184, 449 (1989); T. Amagasaki et al.,
Blood,
26, 138 (1990) and J. A. Begly et al.,
J. Cell Physiol.
156, 43 (1993).
Vitamin B
12
has several characteristics which potentially make it an attractive in vivo tumor imaging agent. Vitamin B
12
is water soluble, has no known toxicity, and in excess is excreted by glomerular filtration. In addition, the uptake of vitamin B
12
could potentially be manipulated by the administration of nitrous oxide and other pharmacological agents (D. Swanson et al.,
Pharmaceuticals in Medical Imaging
, MacMillan Pub. Co., New York (1990) at pages 621-628).
Bacteria naturally insert Cobalt-59 into the corrin ring of vitamin B
12
. Commercially this has been exploited by the fermentative production of Co-56, Co-57, Co-58, and Co-60 radiolabeled vitamin B
12
. For example, see Chaiet et al.,
Science,
111, 601 (1950). Unfortunately Cobalt-57, with a half life of 270.9 days, makes Co-57-cyanocobalamin unsuitable for clinical tumor imaging. Other metal ions (cobalt, copper and zinc) have been chemically inserted into naturally occurring descobaltocorrinoids produced by Chromatim and
Streptomyces olivaceous
. Attempts to chemically insert other metal ions in these cobalt free corrinoid rings has been unsuccessful. The placement of metals (cobalt, nickel, palladium, platinum, rhodium, zinc, and lithium) into a synthetic corrin ring has not presented any major difficulties. However, their instability and cost to produce makes them impractical for biological assays. Although Co-59 is a weakly paramagnetic quadrapolar nuclei in the 2
+
oxidation state, Co-59 exists in the 3
+
oxidation state within the corrin ring of vitamin B
12
and is diamagnetic. Therefore, insertion of either a radioactive or paramagnetic metal ion other than cobalt into the corrin ring does not seem feasible at this time.
A process for preparing
125
I-vitamin B
12
derivatives is described in Niswender et al. (U.S. Pat. No. 3,981,863). In this process, vitamin B
12
is first subjected to mild hydrolysis to form a mixture of monocarboxylic acids, which Houts, infra, disclosed to contain mostly the (e)-isomer. The mixture is then reacted with a p-aininoalkyl)phenol to introduce a phenol group into the B
12
acids (via reaction with one of the free carboxylic acid groups). The mixed substituent B
12
derivatives are then iodinated in the phenol-group substituent. This U.S. patent teaches that the mixed
125
I-B
12
derivatives so made are useful in the radioimmunoassay of B
12,
using antibodies raised against the mixture.
T. M. Houts (U.S. Pat. No. 4,465,775) reported that the components of the radiolabelled mixture of Niswender et al. did not bind with equal affinity to IF. Houts disclosed that radioiodinated derivatives of the pure monocarboxylic (d)-isomer are useful in assays of B
12
in which IF is used. However, although Houts generally discloses that the monocarboxylic (d)-isomer can be labelled with fluorophores or enzymes and used in competitive assays for vitamin B
12
in fluids, a continuing need exists for labelled vitamin B
12
derivatives suitable for tumor and organ imaging and therapy.
SUMMARY OF THE INVENTION
The present invention provides detectable compounds of the general formula (I):
wherein the moiety
is cobalamin, X is CN, OH, methyl or adenosyl,
is the residue of a monocarboxylic acid of the cobalamin, derived from a corrin propionamide group, and is preferably the essentially pure (b)-, (d)-, or (e)-monocarboxylic acid; Y is a linking group and Det is a chelating group comprising a detectable metal, such as a radionuclide or paramagnetic metal ion. Preferably, the linking group is —N(H)(CH
2
)
2-6
NH—.
For example, compounds of formula (I) derived from the (b)-monocarboxylic acid, wherei

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