Labelled peptide compounds

Details Labelled peptide compounds Labelled peptide compounds

1999-06-16

2001-02-27

Huff, Sheela (Department: 1642)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S014800, C514S017400

Reexamination Certificate

active

06194386

ABSTRACT:

The present invention relates to labelled peptide compounds, to a method of preparing these compounds, to a pharmaceutical composition comprising these compounds, to the use of this composition for diagnosis and therapy, and to a kit for preparing a radiopharmaceutical composition.
Neurotensin is a thirteen amino acid peptide, in 1973 isolated from bovine hypothalamus. It has the following structure:
pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH (SEQ ID NO:1)
High concentrations of neurotensin are found in discrete regions of the mammalian central nervous system- In addition, neurotensin interacts with specific receptors in the periphery. During the last decade, neurotensin receptors were found in several tumour cells, like small cell lung carcinoma, human colon carcinoma and human meningiomas.
Two radioiodinated neurotensin derivatives are mentioned in the literature. Because of the existence of two tyrosine residues in the neurotensin sequence, iodination of neurotensin yields a complex mixture of radioiodinated peptides, that possess very different biological properties and, moreover, are difficult to purify. Therefore, Mazella et al. (J. Biol. Chem. 1983, 258, 3476-3481) synthesized a neurotensin analogue in which the tyrosine-11 is replaced by a tryptophan residue: [Trp
11
]neurotensin- lodination (with 125-iodide under electrophilic conditions on the aromatic ring of tyrosine-3) results in a mono-iodo derivative showing a K
d
of 0.1 nM for binding on rat brain synaptic membranes. Preliminary experiments from the same group of researchers (Sadoul et al.; Biochem. Biophys. Res. Commun. 1984, 120, 812-819) indicated that neurotensin receptors in human brain showed a low affinity for monoiodo-[Trp
11
]-neurotensin, making this compound unsuitable for binding experiments. The same group of researchers succeeded later on in preparing a mono-iodo derivative of neurotensin itself; only tyrosine-3 was iodinated with 125-iodide in this method. This radioiodinated analogue has a K
d
of 0.2 nM for binding on rat brain synaptic membranes and a K
d
of 0.26 nM for binding to human brain neurotensin receptors.
The labelled natural neurotensin as well as the labelled tryptophan-11 neurotensin analogue, however, suffers from an enzymatic breakdown due to cleavage of peptide bonds, resulting in an in vivo half-life of only a few minutes.
Structure-activity studies (Granier et al.; Eur. J. Biochem. 1982, 124, 117-125) revealed that the right-hand part of the neurotensin molecule fulfills the structural requirements for mimicking the entire sequence, provided its a-amino end group is protected by acetylation. The binding affinities of this analogue are comparable with those of neurotensin in two binding assays, viz. the binding assay on rat brain synaptic membranes and that on HT 29 cells.
This analogue contains one remaining tyrosine residue which can be readily radioiodinated using electrophilic substitution of the hydrogen in the ortho position of the phenolic group. Structure-activity studies revealed, however, that iodination in the 3-position of said tyrosine residue of this analogue results in a remarkable loss of receptor affinity, viz. with a factor of 20 (Mazella et al.; see above).
It is the objective of the present invention to provide a labelled peptide compound which has a selective affinity to neurotensin receptors, comparable with that of neurotensin itself, and which hasia sufficient enzymatic resistance to allow its use in diagnosis and therapy.
This objective can be achieved by a labelled peptide compound, wherein the peptide has a selective neurotensin receptor affinity and is represented by the general formula
R
1
—(
1
Pro)
n
—
2
Xaa—
3
Xbb—4Xcc—
5
Xdd—
6
Xee—
7
Leu—OH(SEQ ID NO:2)  (I)
wherein:
R
1
is a (C
1
-C
3
)alkanoyl group, an arylcarbonyl group, an aryl-(C
1
-C
3
)alkanoyl group, or a chelating group attached by an amide bond or through a spacing group to the peptide molecule;
Xaa and Xbb are each individually Arg or Lys;
Xcc is an unsubstituted or substituted cyclic amino acid, preferably selected from Pro and Hyp;
Xdd is Tyr, Trp or Phe;
Xee is Leu, Ile or t.-butylalanine; and
n is 0 or 1;
and wherein said peptide is labelled with (a) a metal isotope selected from the group consisting of
99m
Tc,
203
Pb,
67
Ga,
68
Ga,
72
As,
111
In,
113m
In,
97
Ru,
62
Cu,
64l Cu,
52
Fe,
52m
Mn,
51
Cr,
186
Re,
188
Re,
77
As,
90
Y,
67
Cu,
169
Er,
121
Sn,
127
Te,
142
Pr,
143
Pr,
198
Au,
199
Au,
161
Tb,
109
Pd,
165
Dy,
149
Pm,
151
Pm,
153
Sm,
157
Gd,
159
Gd,
166
Ho,
172
Tm,
169
Yb,
175
Yb,
177
Lu,
105
Rh and
111
Ag, or
(b) with a radioactive halogen isotope;
on the understanding that:
(i) if the label is a metal isotope, R
1
represents a chelating group for chelating said isotope; and
(ii) if the label is a radioactive halogen isotope, said halogen isotope is attached to
4
Tyr in the 2-position of the phenyl ring, to
4
Trp, or to the aryl group of substituent R
1
.
Suitable examples of aryl groups in R
1
are: phenyl, halo-substituted phenyl or indolyl; preferably phenyl, 4-fluorophenyl, 2- or 4-bromo-phenyl, 2-iodophenyl, 4-fluoro-2-bromophenyl and 4-fluoro-2-iodophenyl. Suitable examples of radioactive halogen isotopes are:
123
I,
124
i,
125
I,
131
I,
75
Br,
76
Br,
77
Br and
82
Br.
In the above labelled peptide compounds one or more of the amino acids may have the D-configuration instead of the normal L-configuration. The labelled peptide compounds of the invention may also comprise so-called pseudo peptide bonds, viz. —CH
2
—NH— bonds, in addition to the natural amide bonds, viz. —CO—NH— bonds. Such modifications of the amino acids naturally occurring in peptides are within the scope of the present invention.
Peptide compounds which, according to the invention, are labelled with a metal isotope as indicated above, are provided, directly or through a spacing group, with a chelating group, said chelating group being attached by an amide bond to an amino group of said peptide compound.
Said chelating group is preferably derived from ethylene diamine tetra-acetic acid (EDTA), diethylene triamine penta-acetic acid (DTPA), cyclohexyl 1,2-diamine tetra-acetic acid (CDTA), ethyleneglycol-O,O′-bis(2-aminoethyl)-N,N,N′,N′-tetra-acetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid (HBED), triethylene tetramine hexa-acetic acid (TTHA), 1,4,7,10-tetraazacyclododecane-N,N′-,N″,N″′-tetra-acetic acid (DOTA), hydroxyethyldiamine triacetic acid (HEDTA), 1,4,8,11-tetra-azacyclotetradecane-N,N′,N″,N″′-tetra-acetic acid (TETA), substituted DTPA, substituted EDTA, or from a compound of the general formula
wherein R is a branched or non-branched, optionally substituted hydrocarbyl radical, which may be interrupted by one or more hetero-atoms selected from N. O and S and/or by one or more NH groups, and Y is a group which is capable of reacting with an amino group of the peptide and which is preferably selected from the group consisting of carbonyl, carbimidoyl, N-(C
1
-C
6
)alkylcarbimidoyl, N-hydroxycarbimidoyl and N-(C
1
-C
6
)alkoxycarbimidoyl
Examples of suitable chelators of the general formula III are unsubstituted or, substituted 2-iminothiolanes and 2-iminothiacyclohexanes, in particular 2-imino-4-mercaptomethylthiolane. Said optionally present spacing group has preferably the general formula
wherein R
5
is a C
1
-C
10
alkylene group, a C
1
-C
10
alkylidene group or a C
2
-C
10
alkenylene group, and X is a thiocarbonyl group or a methylcarbonyl group.
In connection with their biological properties, in particular their binding affinity to neurotensin receptors and their in vivo stability, and with their synthetic accessibility, the following labelled peptide compounds according to the invention are preferred:
(1) (2-″I-phenyl)acetyl-
1
Arg-
2
Arg-
3
Pro-
4
Tyr-
5
Ile-
6
Leu-OH (SEQ ID NO:4)
(2) (4-″I-phenyl)acetyl-
1
Arg-
2
Arg-
3
Pro-
4
Tyr-
5
Ile-
6
Leu-OH (SEQ ID NO:4)
(3) 2-″I-b

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