Note: Descriptions are shown in the official language in which they were submitted.
wo 9~/21383 PCr/US92~'04559
21 02~3
RADIOLABELLED SOMATOSTATIN DERIYATI\iES, THEIR PREPARATION AND USE
The invention relates to a metal radionuclide-labelled
polypeptide intended for diagnostic or therapeu~ic
applications.
Radionuclide-labelled compounds may be used for diagnostic
~xaminations, for example, into deviations in shape and
~5 functions of internal organs and into the presence and
location of pathological processes in the body. For t~is
p~rpose a composition in which the radioactive compound is
present is administered to the patient, for example, in the
form of an in~ection liquid. By means of a sui~able
detection apparatus, for example, a ga~ma cameral images of,
~or example, the organ or the pathological process in which
the acti~e compound is incorporated can be obtained by
detecting the emitted radiation ("scannin~").
.. ~ . . . .
Radioactive-lab~lied biological ~acromolecules, in
particular polypeptides,~present int~re ting perspec~ives
~or diagnostic applications. Certain polypeptides have a
very large target organ specificity ~nd, after ha~ing been
intxoduced into the body of the patient, can react very
selectively with biological macromolecules present therein.
Binding studies have demonstrated that certain endocrine-
related tumours comprise large numbers of binding sites with
a high affinity to somatostatin and somatostatin-related
polypeptides. Examples of such tumours having large numbers
o~ somatostatin-receptors are pituitary tumours, tumours of
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21 02~33 2
the central nervous system, malignant breast tumours, gastro
entero-pancreatic tumours, and metastases hereof.
Various metal radionuclides, provided they are bound to
tumour-selective polypeptides, may successfully be used for
controlling tumours and hence form a pOwërful tool in
radiotherapy. The polypeptides used hence serve as vehicles
to transport the desired radiation dose, viz. the metal
radionuclide, to the tumour to be exposed to the radiation.
The direct labelling of a polypeptide with a metal
radionuclide has two disadvantages. First, the biologically
active site of the peptide necessary for a good target organ
specificity or selectivity, is easily blocked by this
reaction, so that the normal behaviour of the biological
macromolecule is disturbed. In addition, the affinity
between metal-radionuclide and macromolecule often is
unsatisfactory, so that the formed bond i8 not sufficiently
stable to remain intact under physiological conditions~ The
administered material then is no longer useful - neither as
a diagnostic -it cannot be detected any more how the
polypeptide behaves in the body - nor as a therapeutic - the
radiation dose is no longer transported to the desired
target but cause~ an undesired radi~tion ~urden elsewhere!
` In or~er to m~tigate th~se disadvantages it is suggested in
European Patent Specification 237150 to tre~t proteinaceous
materials which comprise disulphide bonds first with a
disulphide reducing agent, for example, dithiothreitol, and
to react then the reduced proteinaceous substance which now
comprises free mercapto groups, specifically with
radionuclide species, for example, with Tc-99m-tartrate or -
glucoheptonate. It has been found, however, that in this
reductive treatment of the protein, in which the protein is
"unfolded" by cleavage of the disulphide bonds to the
desired mercapto groups, damage to
WO92/21383 PC~/US92/04~59
3 210263~
- the protein molecules may easily occur, as a result of which
the selertivity is disturbed.
S In the past few years a large number of publications have
.
appeared in which biological macromolecules, usually
proteins or proteinaceous substances, are described which
comprise chelating groups for a bond with the desired metal-
radionuclide. For example, International Patent Application
WO 90/06949 describes somatostatin analogues which compri~e
chelating groups, preferably derived from TDPA and the like,
for a bond with a detectable element. As an example is
describad DTPA-modified octreotide which may be labelled
radioactive with In-lll or with Y-gO, for diagnostic or
therapeutic purposes, respectively.
However, better suitable isotopes for these applications are
radioact~ve technetium, ~n particular Tc-99m, and
radioactive rhenium, in particular Re-186 and Re-188,
becau~e these radioisotopes have better radiation
characteristics and are more readily available. However, it
has 80 far not succeeded to label the somatostatin analogues
mentioned hereinbe~ore with these radioisotopes to
compositions wh`ich are ~uitable for in vivo applications and
which are~-sufficiently stable. Somatostatin it~elf is
~r~pi~ly biologically converted in the body and i8 therefore
generally considered not su~table for xadioactiva labelling.
Therefore, one has so far resorted to stabilised
somatostatin derivatives in which the two cysteine amino
acid radicals, which apparently ar~ responsible for the
instability, are oxidised together to a cystine group. ~oth
the commercially available somatostatin, and the octreotide
described in Patent Application W0 90/06949 mentioned
hereinbefore, comprise cystine bridges, formed by oxidation
from two cysteine amino acid radicals.
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210263~ 4
It is the object of the present invention to provide an
easily accessible radioactive-technetium-labelled or radio-
active-rhenium-labelled polypeptide for the selective
detection/localisation or for the selective therapeutic
treatment of tumours with somatostatin r~ceptors, which is
sufficiently stable for in ViVQ application. This object can
be achieved with a labelled polypeptide which according to
the present invention is characterised by the general
formula
Rl~Sl~Al~A2~(D)TrP-R3-A4-A5-Thr-s2-~2 (I~
wherein
Rl is a hydrogen atom or a Cl-C4 alkylcarbonyl group,
R2 is an amino group, a hydroxy group or a Cl-C4
alkoxy group,
Al and A5 each independently are Phe, MePhe, EtPhe,
Tyr, Trp, Nal or Cys,
A2 is Phe, MePhe, EtPhe, Tyr, Trp or Nal,
A3 is Lys, MeLys or (-Me) Lys,
is Thr or Val,
Sl ~s an amino acid ~equence of 1 to 6 amino acids,
selected from the group consisting of Ala, Cys,
. Asn, Phe, MePhe, EtPhe, Tyr, Trp, Nal, Gly, Lys,
25 - MeLys, (-Me)Lys, Thr, Val, Asn and Sar, and
S2 i8 an a~no ~cid~sequence of 0 to 3 ~ino acids,
selected from the group mentioned ~ub Sl, with the
pro~iso that the polypeptide comprises two cysteine
amino acid radicals,
the metal-radionuclide being selected from a radioactive Tc-
isotope or Re-isotope which as a cation is bound covalently
to the mercapto groups of the cysteine amino acid radicals.
(Nal = naphthylalanyl).
It has been found surprisingly that the labelled polypeptide
can simply be prepared and is sufficiently stable in ViYo
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5 21~ 33
for performing the desired examinations and the desired
therapeutic treatment, respectively. It has been established
that the labelled compound remained stable at least cne hour
after injection. The selectivity is not adversely influenced
by the labelling with radioactive technetium ~ rhenium. For
example, Tc-99m-labelled polypeptide according to the
invention is bound specifically to som~tostatin receptor-
sites.
Suitable labelled polypeptides according to the invention
are derived from the previously mentioned octreotide and
analogues thereof, and may then be represented by the
general formula
Rl-A6-cys-A2-(D)Trp-A3-A4-cys-Thr-R4 (II)
wherein Rl, R2, A2, A3 and A4 have the meanings given
here~nbefore, and
A6 is Phe, MePhe, EtPhe, Tyr, Trp or Nal,
the met~l radionuclide being selected from the group
consisting of Tc-99m, Re-186 and Re-188 wh~ch as a cation is
bound covalently to the mercapto groups of the cysteine
amino acid rad~cals.
``l
Other excellently suitable labelled polypeptides according
to the invention are derived from so~atostatin and analogues
thereof, and may be represented by the general formula
Rl S 1 A l~A2~(D)TrP~A3~A4-A'5-Thr-s'2-R (III)
wherein Rl, A2, A3, A4 and R2 have the meanings given
hereinbefore,
A'l and A'5 each independently represent Phe,
MePhe, EtPhe, Tyr, Trp or Nal,
S'1 is an amino acid sequence of 1 to 6 amino acids,
preferably of 5 amino acids, selected from the group
~. . .
: :
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.. ..
~2~33 6
consisting of Ala, Cys, Asn, Phe, MePhe, EtPhe, Tyr,
Trp, Nal, Gly, Lys, NeLys, (~-Me)Lys, ~hr, Val, Asn
and Ser, with the proviso that S'l comprises a
cysteine amino acid radical, and
S'2 is an amino acid sequence of 1 to 3 am~n~ acids,
preferably of 2 amino acids, selected from the group
mentioned sub Sl', with the proviso that S2' comprises a
cysteine amino acid radical, the metal radionuclide being
selected from the group consisting of Tc-99m, Re-186 and R~-
188 which as a cation is bound covalently to the mercapto
groups of the cysteine amino acid radicals.
In connection with the excellent properties of the .labelled
product and the ready availability of comatostatin as a
starting peptide, a labelled polypeptide is preferred of the
general formula
Rl-Ala-Gly-Cy~-Lys-Asn-Phe-Phe-(D)Trp-Lys-Thr-Phe-
Thr-Ser-Cys-R2 (IV)
the metal radionuclide being selected from the group
consisting of Tc-99m, Re-186 and Re-188 which as a cation is
bound co~alentl~y to the mercapto groups of the cysteine
amino ac~d radicals.
The invention also relates to a method of preparing a metal-
radionuclide-labelled po~ypeptide according to the invention
by startin~ from a cyclised polypeptide, in which the
cysteine amino acid radicals together are oxidised to a
cystine group. Examples of such cyclised polypeptides are
the already mentioned somatostatin commercial product and
analogues thereof. Analogues are to be understood to mean
polypeptides having corresponding biological activity, i.e.
specific somatostatin-receptor binding afinity, but with
modifications in the amino acid sequence. It has been found
." ., , - .
..
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- 21021;~.~3
.
that the said cyclised polypeptides can be excellently
reduced and may then be labelled under reducing conditions
without the polypeptide molecule being attacked.
S AS a reducing agent are preferably chosen5zinc ions or
metallic zinc, the latter, for example, in the form of zinc
powder, because such reducing agents are suitable both for
the preparation of the polypeptide from the cyclised
material, and also for the reduction of pertechnetate or
perrhenate. An excellent example of use of the metalllc
zinc powder in the so-called SPED (Solid Phase Electron
Donor) technique, in which the cyclised polypeptide is
incubated by means of zinc powder, for example, on a 0.22
~ filter, after which addition of a Tc-99m pertechnetate
solution immediately provides a solution of the pure, Tc-
99m-labelled polypeptide in the filtrate. Contaminations
and non-reacted starting material remain on the filter, so
that the filtrate is immediately ready for use. Metallic
zinc may also be provided excellently as a zinc mirror on
the inner wall of a tube or other reaction vessel and thus
produce the desired conversions in the tube or reaction
vessel.
., :
~. The. invention~- further .relates to a pharmaceutical
composition::which comprises the metal-radionuclide-labelled
polypeptide according to the invention, and to the use of
the said c.omposition for diagnostic or therapeutic
purposes. For diagnostic purposes, i.e. for detecting and
localising certain tumour tissues, as defined hereinbefore,
the active substance should be labelled with radioactive
technetium, for therapeutic
purposes, the active substance should be labelled with
radioactive rhenium. All this is defined in more detail in
Claims 8 and 9.
The invention finally relates to a kit for preparing a
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radiopharmaceutical composition, comprising, in an
optionally dry condition, a cyclised polypeptide, as
defined hereinbefore, a reducing agent, preferably metallic
zinc or zinc ions, and directives for reacting the
ingredients of the kit and of the resulti~g~-product with
Tc-99m pertechnetate or with Re-186 or Re-188 perrhenate.
In this manner the user of the kit himself can prepare in
a clinical laboratory the labelled polypeptide according to
the in~ention in the form of a composition to be
administered: reduction of the cyclised polypeptide to the
polypeptide to be labelled, as well as the required
labelling. The use of one reaction agent for the two
reactions simplifies the method of preparation.
The invention will now be described in greater detail with
reference to the following examples.
Exam~le 1
Commercially available somatostatin is treated for 30
minutes at a pH~of 8 on a 0.22~ filter by means of the so-
called SPED technique as descrîbed hereinbefore. A freshly
2S ;eluted sodiwm~pertèchnetate solution is then added and the
mixture is incubated at room temperature for 15 minutes.
The labelled polypeptide may be obtained as a filtrate.
Precipitate and liquid can be separated without a filter by
decanting and extracting the liquid by means of a syringe.
A labelling efficiency of 90% is obtained. Free technetium
is bound to the SPED and cannot contaminate the product.
- Labelling is confirmed by means of thin-layer
chromatography and ion exchange column chromatography. The
labelled compound is stable in vitro up to 4 hours.
A quantity of 22 MBq of the labelled compound is
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9,
administered t~ rats suffering from colorectal carcinoma
and the biodistribution is determined by dynamic gamma
camera scintigraphy up to one hour after injection.
The tumour take-up reaches a maximum at a,pproximately 4
minutes after injection. During the determination period
no sig~ificant acti~ity reduction from the tumour can be
observed. The labelled compound is stable in vivo during
the whole of the determination period, as appears from the
absence of thyroid gland and stomach activities.C
Scintigrams of the tumours are made four minutes after
injection. The uptake ratios tumour:muscle t~ssue are
favourable, namely 5:1.
That the tumour accummulation of technetium is related to
the somatostatin binding to receptors in the tumour has
been checked by treating one group of experîmental animals
having tumours prior to administration of the labelled
compound with Sursamine~ to block the receptor sites. No
tumour uptake is observed in these animals, so that it is
confirmed that the binding takes place at somatostatin
receptors in the tumour.
; , .
25 : Examole 2;
.
A group of adult female rats were implanted with CC531
Colon Carcinoma which is known to have somatostatin
receptors. When the subcutaneous tumour implants had
reached a size of ~1.5cm diameter, one group was injected
with Technetium Somatostatin complex by intravenous
injection and a second group was injected in a similar
manner with Indium-111 Octreotide complex for comparison.
The animals were anaesthetised by means of nembutal and
serial scintigrams were made. The Technetium Somatostatin
- ~; . .
WO92/21383 PCTtUS92/04~59
21 ~js33 ` `~
was clearly visible in the tumour within ~-5 minutes post
injection. At six minutes post injection the animals were
sacrificed by cervical dislocation and tissue samples were
taken and counted. Approximately ll~ of the injected dose
was found in the total tumour tissue with ~ tumour to soft
tissue uptake ratio of 4.2/l. The blood concentration was
relatively high tumour to blood ratios being 0.23. The
bulk of the activity was recovered in the stomach, liver,
spleen and kidneys.
In the case of animals injected with indium Octreotide
complex the visualisation of the tumour was similar in
pattern to that of the technetium complex. At 24 hours
post injection, the optimal scanning time in humans, the
animals were sacrificed and tissue samples were counted.
An average concentration of 4.9% of the injected dose was
found in the total tumour with a tumour to soft tissue
ratio of 9.8/l this higher value in relation to the
technetium somatostatin was prim~rily due to the low blood
concentration. As with the technetium complex the bulk of
the activity was recovered from the liver, spleen, GI tract
and kidneys.
The results show a slight difference in the biodistribution
characteristics of the two complexes but the kinetics
during the first lS minutes post injection were similar,
and during ~his period good scintigraphic images of the
tumour could be obtained with the technetium complex using
a gamma camera.
