Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
Wa 95/31221 PCT/US95/06034
Somatostatin binding peptide-metal chelate conjugates
BACKGROUND OF THE INVENTION
1. Fi~ld of the Invention
This invention relates to therapeutic agents, radiotherapeutic agents,
radiodiagnostic agents, and non-radioactive diagnostic agents, and methods for
producing such diagnostic and therapeutic agents. The invention also relates
to
cyclic peptides which specifically bind to somatostatin receptors expressed at
the
surface of mammalian cells, particularly neoplastic or metastatic mammalian
cells.
The invention in one aspect relates to scintigraphic imaging agents for
imaging sites
in a mammalian body. In this aspect, the imaging agents comprise a specific-
binding
peptide that specifically binds to somatostatin receptor-expressing cells in
vivo,
labeled with technetium-99m (Tc-99m) via a radiolabel-binding moiety which
forms
a complex with Tc-99m. In another aspect, the invention provides
radioiodinated
imaging agents. The invention also provides therapeutic agents, including
radioiodinated agents, radioactive metal-reagent complexes and nonradioactive
metal-
reagent complexes, all of which have therapeutic utility. The invention
provides
reagents for preparing each of the diagnostic and therapeutic embodiments of
the
diagnostic and therapeutic agents of the invention, the radiolabeled
embodiments and
non-radioactive metal complexes thereof, methods for labeling said reagents
and kits
comprising non-radioactive embodiments of the reagents of the invention and
other
components for the convenient preparation of the radiolabeled diagnostic and
therapeutic agents of the invention.
2. D_ytnon of the Prior Art
Somatostatin is a tetradecapeptide that is endogenously produced by the
hypothalamus and pancreas in humans and other mammals. The peptide has the
formula:
Formula I
Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Tlu-Ser-Cys
I I
21901(!
WO 95/31221 PCT/US95/06034
(Single letter abbreviations for amino acids can be found in G. Zubav,
Biochemistry
(2d ed. ), 1988, (MacMillan Publishing: New York), p.33). This peptide exerts
a
wide variety of biological effects in vivo. It is known to act physiologically
on the
central nervous system, the hypothalamus, the pancreas, and the
gastrointestinal
tract.
Somatostatin inhibits the release of insulin and glucagon from the pancreas,
inhibits growth hormone release from the hypothalamus, and reduces gastric
secretions. Thus, somatostatin has clinical and therapeutic applications for
the
alleviation of a number of ailments and diseases. both in humans and other
animals.
Native somatostatin is of limited utility, however, due to its short half-life
in vivo,
where it is rapidly degraded by peptidases. For this reason, somatostatin
analogues
having improved in vivo stability have been developed in the prior art.
Freidinger, U.S. Patent No. 4,235,886 disclose cyclic hexapeptide
somatostatin analogues useful in the treatment of a number of diseases in
humans.
Coy and Murphy, U.S. Patent No. 4,485,101 disclose synthetic dodecapeptide
somatostatin analogues.
Freidinger, U.S. Patent No. 4,611.054 disclose cyclic hexapeptide
somatostatin analogues useful in the treatment of a number of diseases in
humans.
Nutt, U.S. Patent No. 4,612,366 disclose cyclic hexapeptide somatostatin
analogues useful in the treatment of a number of diseases in humans.
Coy et al., U.S. Patent No. 4,853.371 disclose synthetic octapeptide
somatostatin analoQUes.
Coy and Murphy, U.S. Patent No. 4,871,717 disclose synthetic heptapeptide
somatostatin analoQUes.
Coy et al., U.S. Patent No. 4,904,642 disclose synthetic octapeptide
somatostatin analogues.
Taylor et al., U.S. Patent No. 5,073,541 disclose a method of treating small
cell lung cancer.
Brady, European Patent Application No. 83111747.8 discloses dicyclic
hexapeptide somatostatin analogues useful in the treatment of a number of
human
diseases.
Bauer et al. , European Patent Application No. 85810617.2 disclose
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WO 95131221 ~ ~ ~ ~ ~ PCT/US95/06034
somatostatin derivatives useful in the treatment of a number of human
diseases.
Eck and Moreau, European Patent Application No. 90302760.5 disclose
therapeutic octapeptide somatostatin analogues.
Coy and Murphy, International Patent Application Serial No.
PCT/US90/07074 disclose somatostatin analogues for therapeutic uses.
Schally et al., European Patent Application Serial No. EPA 911048445.2
disclose cyclic peptides for therape~:ric use.
Bodgen and Moreau, In~Prnational Patent Application Serial No.
PCT/US92/01027 disclose compositions and methods for treating proliferative
skin
disease.
Somatostatin exerts it effects ' ~y binding to specific receptors expressed at
the
cell surface of cells comprising the cwntral nervous system, the hypothalamus,
the
pancreas, and the gastrointestinal tray ~ These hegh-aff pity somatostatin
binding sites
have been found to be abundantly e3.oressed at the cell surface of most
endocrine
active tumors arising from these tissues.
It is frequently clinically adva.itageous for a physician to be able to
localize
the site of pathological conditions in a patient using non-invasive means.
Such
pathological conditions include diseases of the lunga, heart, liver, kidneys,
bones and
brain, as well as cancer, thrombosis. ?ulmonary embolism, infection,
inflammation
and atherosclerosis.
In the field of nuclear medicine, certain pathological conditions are
localized,
or their extent is assessed, by detecting the distribution of small quantities
of
internally-administered radioactively labeled tracer compounds (called
radiotracers
or radiopharmaceuticals). Methods or detecting these radiopharmaceuticals are
known generally as imaging or radioimaging methods.
In radioimaging, the radiolabel is a gamma-radiation emitting radionuclide
and the radiotracer is located using a gamma-radiation detecting camera (this
process
is often referred to as gamma scintigraphy). The imaged site is detectable
because
the radiotracer is chosen either to localize at a pathological site (termed
positive
contrast) or, alternatively, the radiotracer is chosen specifically not to
localize at such
pathological sites (termed negative contrast). In many situations it is a
particular
advantage to use a radiolabeled specific binding compound as a
radiopharmaceutical
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which localizes specifically to the pathological site in vivo.
For example. expression of high-affinity binding sites for somatostatin is a
marker for certain tumor cells, and specific binding with somatostatin can be
exploited to locate and identify such tumor cells in vivo.
Methods for radiolabeling somatostatin analogues that have been modified so
as to contain a tyrosine amino acid (Tyr or Y) are known in the prior art.
Albert et al. , UK Patent Application 8927255.3 disclose radioimaging using
somatostatin derivatives such as octreotide labeled with '23I.
Bakker et al., 1990, J. Nucl. Med. 31: 1501-1509 describe radioactive
iodination of a somatostatin analog and its usefulness in detecting tumors in
vivo.
Bakker et al., 1991, J. Nucl. Med. 32: 1184-1189 teach the usefulness of
radiolabeled somatostatin for radioimaging in vivo.
Bomanji et al., 1992, J. Nucl. Med. 33: 1121-1124 describe the use of
iodinated (Tyr-3) octreotide for imaging metastatic carcinoid tumors.
Alternatively, methods for radiolabeling somatostatin by covalently modifying
the peptide to contain a radionuclide-chelating group have been disclosed in
the prior
art.
Albert et al., tJK Patent Application 8927255.3 disclose radioimaging using
somatostatin derivatives such as octreotide labeled with "'In via a chelating
group
bound to the amino-terminus.
Albert et al., European Patent Application No. WO 91/01144 disclose
radioimaging using radiolabeled peptides related to growth factors, hormones,
. interferons and cytokines and comprised of a specific recognition peptide
covalently
linked to a radionuclide chelating group.
Albert et al. , European Patent Application No. 92810381.1 disclose
somatostatin peptides having amino-terminally linked chelators.
Faglia et al. , 1991, J. Clin. Endocrinol. Metab. 73 : 850-856 describe the
detection of somatostatin receptors in patients.
Kwekkeboom et al., 1991, J. Nucl. Med. 32: 981 Abstract #305 relates to
radiolabeling somatostatin analogues with "'In.
Albert et al. , 1991, Abstract LM 10, 12th American Peptide Symposium: 1991
describe uses for ' "In-labeled diethylene-triaminopentaacetic acid-
derivatized
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WO 95/31221 ~~ PCT/US95/06034
somatostatin analogues.
Krenning et al., 1992, J. lVucl. Med. 33: 652-658 describe clinical
scintigraphy using ["'In][DTPA]octreotide.
A variety of radionuclides are known to be useful for radioimaging, including
6'Ga, ~'"Tc (Tc-99m), "'In, 'z'I, 'zSI, ~69Yb or '8°Re. A number of
factors must be
considered for optimal radioimaging in humans. To maximize the efficiency of
detection, a radionuclide that emits gamma energy in the 100 to 200 keV range
is
preferred. To minimize the absorbed radiation dose to the patient, the
physical half
life of the radionuclide should be as short as the imaging procedure will
allow. To
allow for examinations to be performed on any day and at any time of the day,
it is
advantageous to have a source of the radionuclide always available at the
clinical
site. Tc-99m is a preferred radionuclide because it emits gamma radiation at
140
keV, it has a physical half-life of 6 hours, and it is readily available on-
site using a
molybdenum-99/technetium-99m generator. Other radionuclides used in the prior
art are less advantageous than Tc-99m. This can be because the physical half-
life
of such radionuclides are longer, resulting in a greater amount of absorbed
radiation
dose to the patient (e.g., indium-111). Also, many disadvantageous
radionuclides
cannot be produced using an on-site generator.
Tc-99m is a transition metal that is advantageously chelated by a metal
complexing moiety. Radiolabel complexing moieties capable of binding Tc-99m
can
be covalently linked to various specific binding compounds to provide a means
for
radiolabeling such specific binding compounds. This is because the most
commonly
available chemical species of Tc-99m, pertechnetate (TcO,~), cannot bind
directly to
most specific binding compounds strongly enough to be useful as a
radiopharmaceutical. Complexing of Tc-99m with such radiolabel complexing
moieties typically entails chemical reduction of the pertechnetate using a
reducing
agent such as stannous chloride.
The use of chelating agents for complexing Tc-99m is known in the prior art.
Byrne et al., U.S. Patent No. 4,434,151 describe homocysteine containing
chelating agents for Tc-99m.
Fritzberg, U.S. Patent No. 4,4.44,690 describes a series of technetium-
chelating agents based on 2,3-bis(mercaptoacetamido) propanoate.
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WD 9/31221 PCT/US95/06034
Byrne et al.. U.S. Patent No. 4,571.-I30 describe homocvsteine containing
chelating agents for Tc-99m.
Byrne et al., U.S. Patent No. 4.575.556 describe homocysteine containing
chelating agents for Tc-99m.
S Nosco et al., U.S. Patent No. 4,925.650 describe Tc-99m chelating
complexes.
Kondo et al., European Patent Application, Publication No. 483704 A1
disclose a process for preparing a Tc-99m complex with a mercapto-Gly-Gly-Gly
moiety.
European Patent Application No. 84109831.2 describes bisamido, bisthiol Tc-
99m ligands and salts thereof as renal function monitoring agents.
Davison et al., 1981, Inorg. Chem. 20: 1629-1632 disclose oxotechnetium
chelate complexes.
Fritzberg et al., 1982, J. Nucl. Med. 23: 592-598 disclose a Tc-99m chelating
agent based on N, N'-bis(mercaptoacetyl)-2.3-diaminopropanoate.
Byrne et al., 1983, J. Nucl. Med. 24: P126 describe homocysteine containing
chelating agents for Tc-99m.
Bryson et al. , 1988, Inorg. Chem. 27: 2154-2161 describe neutral complexes
of technetium-99 which are unstable to excess ligand.
Misra et al., 1989, Tet. Len. 30: 1885-1888 describe bisamine bisthiol
compounds for radiolabeling purposes.
The use of chelating agents for radiolabeling specific-binding compounds is
known in the art.
Gansow et al., U.S. Patent No. 4,472,509 teach methods of manufacturing
and purifying Tc-99m chelate-conjugated monoclonal antibodies.
Stavrianopoulos, U.S. Patent No. 4,943,523 teach detectable molecules
comprising metal chelating moieties.
Fritzberg et al., European Patent Application No. 86100360.6 describe
dithiol, diamino, or diamidocarboxylic acid or amine complexes useful for
making
technetium-labeled imaging agents.
Albert et al. , UK Patent Application 8927255.3 disclose radioimaging using
somatostatin derivatives such as octreotide labeled with "'In via a chelating
group
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PCT/US95/06034
bound to the amino-terminus.
Albert et al., European Patent Application No. WO 91/01144 disclose
radioimaging using radiolabeled peptides related to growth factors, hormones,
interferons and cytokines and comprised of a specific recognition peptide
covalently
linked to a radionuclide chelating group.
Fischman et al., International Patent Application. Publication No.
W093/13317 disclose chemotactic peptides attached to chelating moieties.
Kwekkeboom et al. , 1991, J. Nucl. Med. 32: 981 Abstract #305 relates to
radiolabeling somatostatin analogues with "'In.
Albert et al. , 1991, Abstract LM 10, 12th American Peptide Symposium: 1991
describe uses for "'In-labeled diethylene-triaminopentaacetic acid-derivatized
somatostatin analogues.
Cox et al. , 1991, Abstract, 7th International Symposium on
Radiopharmacology, p. 16, disclose the use of, Tc-99m-, '3'I- and "'In-labeled
somatostatin analogues in radiolocalization of endocrine tumors in vivo by
scintigraphy.
Methods for labeling certain specific-binding compounds with Tc-99m are
known in the prior art.
Hnatowich, U.S. Patent No. 4,668,503 describe Tc-99m protein
radiolabeling.
Tolman, U.S. Patent No. 4,732,684 describe conjugation of targeting
molecules and fragments of metallothionein.
Nicolotti et al. , U. S. Patent No. 4.861, 869 describe bifunctional coupling
agents useful in forming conjugates with biological molecules such as
antibodies.
Fritzberg et al., U.S. Patent No. 4.965,392 describe various S-protected
mercaptoacetylglycylglycine-based chelators for labeling proteins.
Schochat et al., U.S. Patent No. 5,061,641 disclose direct radiolabeling of
proteins comprised of at least one "pendent" sulfhydryl group.
Fritzberg et al., U.S. Patent No. 5,091,514 describe various S-protected
mercaptoacetylglycylglycine-based chelators for labeling proteins.
Gustavson et al. , U. S. Patent No. 5,112,953 disclose Tc-99m chelating agents
for radiolabeling proteins.
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WO 95131221 ~ ~ PCT/US95/06034
Kasina et al.. U.S. Patent No. 5.175.57 describe various combinations of
targeting molecules and Tc-99m chelating groups.
Dean et al., U.S. Patent No. 5,180,816 disclose methods for radiolabeling
a protein with Tc-99m via a bifunctional chelating agent.
Sundrehagen, International Patent Application, Publication No. W085/03231
disclose Tc-99m labeling of proteins.
Reno and Bottino, European Patent Application 87300426.1 disclose
radiolabeling antibodies with Tc-99m.
Bremer et al. , European Patent Application No. 87118142.6 disclose Tc-99m
radiolabeling of antibody molecules.
Pak et al. , European Patent Application No. WO 88/07382 disclose a method
for labeling antibodies with Tc-99m.
Goedemans et al. , PCT Application No. WO 89/07456 describe radiolabeling
proteins using cyclic thiol compounds, particularly 2-iminothiolane and
derivatives.
Dean et al., International Patent Application, Publication No. W089/12625
teach bifunctional coupling agents for Tc-99m labeling of proteins.
Schoemaker et al., International Patent Application, Publication No.
W090/06323 disclose chimeric proteins comprising a metal-binding region.
Thornback et al. , EPC Application No. 90402206. 8 describe preparation and
use of radiolabeled proteins or peptides using thiol-containing compounds,
particularly 2-iminothiolane.
Gustavson et al., International Patent Application, Publication No.
W091/09876 disclose Tc-99m chelating agents for radiolabeling proteins.
Rhodes, 1974, Sem. Nucl. Med. 4: 281-293 teach the labeling of human
serum albumin with technetium-99m.
Khaw et al., 1982, J. Nucl. Med. 23: 1011-1019 disclose methods for
labeling biologically active macromolecules with Tc-99m.
Schwartz et al. , 1991, Bioconjugate Chem. 2: 333 describe a method for
labeling proteins with Tc-99m using a hydrazinonicotinamide group.
Attempts at radiolabeling peptides have been reported in the prior art.
Ege et al., U.S. Patent No. 4,832.940 teach radiolabeled peptides for imaging
localized T-lymphocytes.
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Morgan et al.. L,'.S. Patent No. 4.986.979 disclose methods for imaoino sites
of inflammation.
Flanagan et al., U.S. Patent No. 5,248.764 describe conjugates between a
radiolabel chelating moiety and atrial natiuretic factor-derived peptides.
Ranby et al. , 1988. PCT/US88/02276 disclose a method for detecting fibrin
deposits in an animal comprising covalently binding a radiolabeled compound to
fibrin.
Lees et al. , 1989, PCT/US89/01854 teach radiolabeled peptides for arterial
tmagtng.
Morgan et al. , International Patent Application, Publication No. W090/ 10463
disclose methods for imaging sites of inflammation.
Flanagan et al. , European Patent Application No. 90306428.5 disclose Tc-
99m labeling of synthetic peptide fragments via a set of organic chelating
molecules.
Stuttle, PCT Application, Publication No. WO 90/15818 describes Tc-99m
labeling of RGD-containing oligopeptides.
Rodwell et al. , 1991, PCT/US91 /03116 disclose conjugates of "molecular
recognition units" with "effector domains" .
Cox, International Patent Application No. PCT/US92/04559 discloses
radiolabeled somatostatin derivatives containing two cysteine residues.
Rhodes et al., International Patent Application, Publication No. W093/12819
teach peptides comprising metal ion-binding domains.
Lyle et al, International Patent Application, Publication No. W093/15770
disclose Tc-99m chelators and peptides labeled with Tc-99m.
Coughlin et al, International Patent Application. Publication No.
W093/21151 disclose bifunctional chelating agents comprising thiourea groups
for
radiolabelingtargeting molecules.
Knight et al. , 1990, 37th Annual Meeting of the Society of Nuclear Medicine,
Abstract #209, claim thrombus imaging using Tc-99m labeled peptides.
Babich et al., 1993, J. Nucl. Med. 34: 1964-1974 describe Tc-99m labeled
peptides comprising hydrazinonicotinamide derivatives.
Methods for directly labeling somatostatin, derivatives of somatostatin,
analogues of somatostatin or peptides that bind to the somatostatin receptor
and
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contain at least 2 cysteine residues that form a disulfide or wherein the
disulfide is
reduced to the sulfhydryl form, are disclosed in co-owned U.S. Patent No.
5,225,180, issued 3uly 6, 1993>
The use of chelating agents for radiolabeling peptides, and methods for
labeling
peptides with Tc-99m are known in the prior art and are disclosed in PCT
Application
Nos. WO 92/13572, WO 93/10747, WO 93/21962, WO 93/25244, WO 94/07918,
WO 95/00553 and WO 95/03330.
Lister-James et al. , 1994, J. Nuclear Med. 35: 257P-258P disclose Tc-99m
labeled somatostatin receptor binding peptides for imaging tumors in vivo.
There remains a need for synthetic (to make routine manufacture practicable
and to ease regulatory acceptance) somatostatin analogues having increased in
vivo
stability, to be used therapeutically, as scintigraphic agents when
radiolabeled with
Tc-99m or other detectable radioisotopes for use in imaging tumors in vivo,
and as
radiotherapeutic agents when radiolabeled with a cytotoxic radioisotope such
as
rhenium-188. Small synthetic somatostatin analogues are provided by this
invention
that specifically fulfill this need.
SUMMARY OF THE INVENTION
The present invention provides somatostatin analogues that comprise cyclic
peptides for therapeutic applications, including radiotherapeutic
applications, and
diagnostic applications, including radiodiagnostic applications, in particular
scintigraphic .imaging applications. Distinct from native somatostatin, the
cyclic
peptides of the invention are not comprised of a disulfide bond. The invention
also
provides cyclic peptide reagents comprised of cyclic peptide somatostatin
analogues
wherein such peptides incorporate a covalently linked metal ion-binding
moiety. The
invention provides such cyclic peptides, cyclic peptide reagents and
radiolabeled~
cyclic peptide reagents that are scintigraphic imaging agents, radiodiagnostic
agents
and radiotherapeutic agents. Scintigraphic imaging agents of the invention
comprise
cyclic peptide reagents radiolabeled with a radioisotope, preferably
technetium-99m.
Radiotherapeutic agents of the invention comprise cyclic peptide reagents
radiolabeled with a cytotoxic radioisotope, preferably rhenium-186 or rhenium-
188.
Methods for making and using such cyclic peptides, cyclic peptide reagents and
radiolabeled embodiments thereof are also provided.
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WO 95/31221 ~ PCTlUS95I06034
The invention provides compositions of matter comprising a specific binding
peptide that specifically binds to somatostatin receptors in a mammalian body,
covalently linked to a metal ion-complexing moiety. The compositions of matter
of
the invention have formula:
S cyclo(N CH3)-Phe-Tvr-(o-Trp)-Lvs-Val-I-~I~cv~.(cH.co.x)
wherein X is (amino acid)-B-(amino acid)m-Z, wherein B is is a thiol-
containing
moiety that is cysteine, homocysteine, isocysteine, or penicillamine; (amino
acid)n
and (amino acid)m are each independently any primary a- or 13-amino acid that
does
not comprise a thiol group; Z is -OH or -NH,; and n and m are each
independently
an integer between 2 and 5 and 0 and 5, respectively.
In the formulae describing the compositions of matter of the invention, the
combination of the term "cyclo" and an underlined amino acid sequence will be
understood to mean that the underlined amino acid sequence is cyclized through
a
peptide bond linking the amino terminus of the first amino acid of the
sequence to
the carboxyl terminus of the last amino acid of the sequence. When followed by
a
parenthetical term such as "(cH.co.x), said parenthetical term will be
understood to
mean that the sequence in parentheses is covalently linked to the amino acid
sidechain sulfur atom of the thiol-containing amino acid in the underlined
amino acid
sequence, forming a sulfide bond therewith. Thus, in the above chemical
structure,
it will be understood that the -CH~CO.X group is covalently linked to the
sidechain
sulfur atom of homocysteine. An example of such a chemical formula is shown in
Example 2.
A preferred embodiment of the compositions of matter of the invention is a
compound having formula:
cyclo(N methyl)FYW~,KVHcY.(cH,co.GGCK.amide).
The compositions of matter of the invention have utility for a number of
diagnostic and therapeutic purposes.
Thus, one aspect of the invention provides reagents for preparing radiolabeled
scintigraphic imaging agents for imaging sites within a mammalian body having
an
overabundance of somatostatin receptors. In such embodiments, the metal ion-
complexing moiety comprises a radiolabel binding moiety, and the reagent forms
a
complex with the radiolabel via complex formation with said moiety. In one
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WO 95/31221 PCT/US95/06034
embodiment of this aspect of the invention, the scintigraphic imaging agent is
formed
by complexing the reagent with Tc-99m under reducing conditions.
Thus, the invention also comprises scintigraphic imaging agents that are
complexes of the reagents of the invention with Tc-99m and methods for
S radiolabeling the reagents. Tc-99m radiolabeled complexes provided by the
invention are formed by reacting the reagents of the invention with Tc-99m in
the
presence of a reducing agent. Preferred reducing agents include but are not
limited
to dithionite ion, stannous ion and ferrous ion. Complexes of the invention
are also
formed by labeling the reagents of the invention with Tc-99m by ligand
exchange of
a prereduced Tc-99m complex as provided herein.
A preferred embodiment of the reagents provided by the invention for
preparing scintigraphic imaging agents is a compound having formula:
cyclo(N methvl)F~'W V.Hc .(cH.co.GGCK.amide).
Preferred imaging agents comprise Tc-99m complexes of this reagent.
The invention also provides kits for preparing scintigraphic imaging agents
that are the reagents of the invention radiolabeled with Tc-99m. Kits for
labeling the
reagents provided by the invention with Tc-99m are comprised of a sealed vial
containing a predetermined quantity of a reagent of the invention and a
sufficient
amount of reducing agent to label the reagent with Tc-99m.
In a second aspect of the invention are provided imaging agents wherein the
compositions of matter of the invention are radiolabeled with a radioisotope
of
iodine. preferably I-123. I-125, or I-131. The invention provides
radioiodinated
imaging agents that are complexes of the reagents of the invention with
radiolabeled
iodine.
A preferred embodiment of the reagents provided by the invention for
preparing radioiodinated imaging agents is a compound having formula:
cvclo(N methyl)FYW V.Hc .(cH:co.GGCK.amide).
Preferred imaging agents comprise radioiodinated embodiments of this reagent.
In a third aspect are provided nonradioactively-labeled imaging agents,
comprising reagents that are the compositions of matter of the invention
complexed
with a paramagnetic metal ion or particle. In embodiments of this aspect of
the
invention, the paramagnetic metal ion is complexed with the metal ion-
complexing
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PCT/US95/06034
moiety. In embodiments where a particle. preferably a superparamagnetic metal
panicle, is used, the compositions of matter of the invention are linked to
the
superparamagnetic particle either covalently or associatively using methods
described
by Weissleder et al. (1992, Radioloy 182: 381-385). The invention provides
such
imaging agents for use in magnetic resonance imaging. Methods for preparing
these
non-radiolabeled imaging agents are also provided.
A preferred embodiment of the reagents provided by the invention for
preparing such non-radioactively-labeled imaging agents is a compound having
formula:
cyclo(N methyl)FYW V.Hcv.(cH,co.GGCK.amide).
Preferred non-radioactively labeled imaging agents comprise ferric ion
complexes of
this reagent.
Therapeutic agents are also provided herein. In a fourth aspect, the invention
provides the compositions of matter of the invention uncomplexed with a metal
ion
as a therapeutic agent per se. Preferred embodiments of such therapeutic
agents
provided by the invention are compounds having formula:
cyclo(N methyllFYW V.Hc .(cH,co.GGCK.amide).
In a fifth aspect, the invention provides radiolabeled therapeutic agents
comprising a reagent for preparing the therapeutic agent that is a composition
of
matter provided by the invention. In such embodiments, the compositions of
matter
of the invention are provided wherein the metal ion-complexing moiety is
complexed
with a /3-particle emitting or a conversion electron emitting radioisotope.
Preferred
embodiments of (3-particle emitting radioisotopes are rhenium-186 and rhenium-
188.
A preferred conversion electron emitting radioisotope is tin-117m. The
invention
provides such radioisotope complexes of the compositions of matter of the
invention
for radiotherapeutic treatment of somatostatin-receptor expressing
pathological cells
and tissues, particularly neoplastic and metastatic cells. Methods for
preparing such
therapeutic radioisotope complexes of the compositions of matter of the
invention are
also provided.
A preferred embodiment of the reagents of the invention provided for
preparing such radiotherapeutic agents is a compound having formula:
cyclo(N methvl)FYW V.Hc .(cH,co.GGCK.amide).
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WO 95/31221 PCTlUS95/06034
Preferred radiotherapeutic agents comprise ~3-panicle emitting or conversion
electron-
emitting metal ion complexes with this reagent.
In another aspect of the radi~therapeutic agents of the invention are provided
radiotherapeutic agents wherein the compositions of matter of the invention
are
radiolabeled with a radioisotope of iodine, preferably I-125 or I-131, or with
astatine-211. The invention also provides the radiolabeled therapeutic agents
themselves, as well as methods for radiolabeling the reagents.
Preferred embodiments of tl-~: reagents of the invention provided for
preparing
these therapeutic radioiodinated and radioastatinated agents are compounds
having
formula:
cvclo(N-methvl)FY~-;'"KV . Hcy.. (cH.co. GGCK. amide).
Preferred radiotherapeutic agents :ompris:: radiciodinated and
radioastatinated
embodiments of this reasent.
Therapeutic agents are provi~ed that are nonradioactively-labeled, metal atom-
complexed agents, comprising reagents that are the compositions of matter of
the
invention complexed with a nomad inactive metal atom, such as copper, zinc or
rhenium. In embodiments of this aapect of the invention, the nonradioactive
metal
ion is complexed with the metal ion-comolPx~ng r~toiety. The invention
provides
such therapeutic agents for use in treating conditions in which somatostatin
receptors
are overexpressed in the cells comprising certain tissues, and in treating
neoplastic
cells which hyperexpress somatostatin receptors. Methods for preparing these
non-
radiolabeled metal atom complexed therapeutic agents are also provided.
A preferred embodiment of the reagents of the invention provided for
preparing non-radioactively-labeled therapeutic agents is a compound having
formula:
cyclo(N methyl)FYWaICV.Hcy,.(cH.co.GGCK.amide).
Preferred non-radioactively labeled therapeutic agents comprise a rhenium
complex
of this reagent.
This invention provides methods for preparing peptide embodiments of the
reagents of the invention by chemical synthesis in vitro. In a preferred
embodiment,
peptides are synthesized by solid phase peptide synthesis.
This invention provides methods for using the diagnostic and radiodiagnostic
and therapeutic and radiotherpaeutic agents of the invention. For radiolabeled
- 14-
2 ~ ~~ ~ ~~~
WO 95/31221 PCT/US95/06034
embodiments of the agents of the invention, for example. Tc-99m labeled
scintigraphic imaging agents, an effective diagnostic or therapeutic amount of
the
diagnostic or radiodiagnostic or therapeutic or radiotherapeutic agent of the
invention
are administered. In radiodiagnostic embodiments, localization of the
radiolabel is
detected using conventional methodologies such as gamma scintigraphy. In non-
radioactive diagnostic embodiments, localization of sites of accumulation of
the
paramagnetic metal-labeled diagnostic agents of the invention is achieved
using
magnetic resonance imaging methodologies. The imaging agents provided by the
invention have utility for tumor imaging, particularly for imaging primary and
metastatic neoplastic sites wherein said neoplastic cells express somatostatin
receptors
(SSTR), and in particular such primary and especially metastatic tumor cells
that
have been clinically recalcitrant to detection using conventional
methodologies. In
addition, the imaging agents of the invention are useful in detecting sites of
T
lymphocyte accumulation associated with occult disease or pathology, e. g. ,
as occurs
in patients suffering prom tuberculosis.
The invention provides methods for using the somatostatin analogues of the
in~ ntion to alleviate diseases or other ailments in animals, preferably
humans.
These diseases and ailments include but are not limited to diabetes and
diabetes-
related retinopathy, cirrhosis of the liver and hepatitis infection, bleeding
ulcers and
other gastrointestinal bleeding, pancreatitis, central nervous system
disorders,
endocrine disorders, Alzheimer's disease, acromegaly and other diseases and
disorders related to the production of inappropriate levels of growth hormone
in vivo,
and cancer, particularly those cancers whose growth is dependent or influenced
by
growth hormone production. Non-radiolabeled therapeutic embodiments also have
utility for treating SSTR hyperexpression-related ailments, such as diarrhea
cause by
. SSTR-hyperexpressing gastrinoma. Dosages of the somatostatin analogues
provided
by the invention may be the same as those dosages of native somatostatin
routinely
used for treatment of the above or other diseases, or less of the compounds of
the
invention may be administered due to their longer in vivo half-life.
Therapeutic uses also include radiotherapeutic ablation of neoplastic and
metastatic malignant cells in patients bearing tumors the cells of which
express the
somatostain receptor. Use of the radiotherapeutic agents of the invention
includes
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WO 95/31221 ~ ~ ~ l~ ~ ~ ~'~ PCT/US95/06034
primary therapeutic use for tumors recalcitrant to more conventional therapies
and
for tumors that are inoperable, as well as adjunct therapies supplemental to
surgery.
radiation therapy or conventional chemotherapy.
The radiolabeled embodiments of the invention also have utility as surgical
guides for identifying somatostatin receptor-expressing tumor tissue during
surgery.
For such use in radioisotope guided surgery, malignant tissue otherwise
invisible to
the surgeon can be recognized and excised during otherwise conventional
surgery.
Specific preferred embodiments of the present invention will become evident
from the following more detailed description of certain preferred embodiments
and
the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows an image of ~"'Tc-labeled P587 in a tumor-bearing rat,
indicated by an arrow, showing high uptake at the tumor site in the lower leg.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention provides cyclic peptides that are somatostatin analogues
and that do not comprise of a disulfide bond. Such somatostatin analogues
thereby
possess increased in vivo stability compared with native somatostatin. These
cyclic
peptides are themselves therapeutic agents for alleviating diseases and other
ailments
in animals including humans.
Also provided by the invention are cyclic peptides that may be radioiodinated
or radioastatinated and which are thereby useful in radiotherapeutic and
radiodiagnostic applications.
Another embodiment of these cyclic peptides that is provided by this invention
are cyclic peptide reagents wherein the cyclic peptides of the invention are
covalently
linked to a metal ion-complexing moiety. Such cyclic peptide reagents are
capable
of being radiolabeled to provide radiodiagnostic or radiotherapeutic agents.
One
example of a radiodiagnostic application using the radiolabeled agents of the
invention is scintigraphic imaging, wherein the location and extent of
somatostatin
receptor-bearing tumors may be determined. The cyclic peptide reagents of the
invention can also advantageously be radiolabeled with cytotoxic radioisotopes
such
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WO 95131221 PCTIUS95/06034
as rhenium-186 or rhenium-188 for radiotherapeutic uses. The cyclic peptide
reagents of the invention are also useful in preparing complexes with non-
radioactive
metals, said complexes being useful therapeutically.
Labeling with Tc-99m is an advantage of the present invention because the
nuclear and radioactive properties of this isotope make it an ideal
scintigraphic
imaging agent. This isotope has a single photon energy of 140 keV and a
radioactive
half-life of about 6 hours, and is readily available from a y9Mo-~'''"Tc
generator.
Other radionuclides may also be used in the practice of the invention as
disclosed
herein.
The term scintigraphic imaging agent as used herein is meant to encompass
a radiolabeled agent capable of being detected with a radioactivity detecting
means
(including but not limited to a gamma-camera, a Geiger-Muller counter and a
scintillation detector probe).
Radiotherapeutic embodiments of the invention, on the other hand, are
advantageously labeled with a cytotoxic radioisotope, including but not
Timited to
copper-67, iodine-125, iodine-131, rhenium-186, rhenium-188, and astatine-211,
most preferably 'g6Re or 'eaRe. Such embodiments are useful in the treatment
of
somatostatin-related diseases or other ailments in animals, preferably humans,
including but not limited to cancer and other diseases characterized by the
growth of
malignant or benign tumors capable of binding somatostatin or somatostatin
analogues via the expression of somatostatin receptors on the cell surface of
cells
comprising such tumors.
The present invention provides reagents for preparing diagnostic and
radiodiagnostic agents, and therapeutic and radiotherapeutic agents. The
reagents
provided by the invention comprise a metal ion-complexing moiety covalently
linked
to a specific binding peptide that binds to somatostatin receptor sites within
a
mammalian body.
Small compounds, preferably having a molecular weight of less than 10,000
daltons, are of distinct commercial advantage. Such small compounds can be
readily
manufactured. Moreover, they are likely not to be immunogenic and to clear
rapidly
from the vasculature, thus allowing for better and more rapid imaging. In
contrast,
larger molecules such as antibodies or fragments thereof, or other
biologically-
- 17-
WO 95131221 ~ ~ C> ~~ ; ~ ~ PCT/US95/06034
derived peptides larger than 10.000 daltons. are costly to manufacture. and
are likely
to be immunogenic and clear more slowly from the bloodstream, thereby
interfering
with rapid diagnoses in vivo.
It is an advantage of the somatostatin analogues provided by this invention
that the non-disulfide cyclic linkage contained therein is stable under the
conditions
of radiolabeling the covalently linked radiolabel-binding moiety. In contrast,
for
example, Tc-99m conjugation to a Tc-99m binding moiety covalently linked to
native
somatostatin, or to a somatostatin analogue having a disulfide bond, can
result in
reduction of the disulfide accompanied by a loss of biological activity. Such
loss of
biological activity can also occur in vivo using native somatostatin, or to
any
somatostatin analogue having a disulfide bond. The present invention is not
subject
to similar losses in biological activity in vivo because the non-disulfide
cyclic
linkages in each of the somatostatin analogues of the invention comprise
stable
covalent bonds.
For purposes of this invention, the term "specific binding peptide" is
intended
to mean any peptide compound that specifically binds to a target site in a
mammalian
body defined by an overabundance of somatostatin receptor (SSTR) molecules.
"Specific binding" will be understood by those with skill in this art as
meaning that
the peptide localizes to a greater extent at the target site that to
surrounding tissues.
Such specific binding is advantageous in diagnostic imaging agents comprising
such
specific binding peptides because they are dispersed throughout a mammalian
body
after administration and such specific localization of reagent-bound
radioactivity
provides visual definition of the target in vivo.
Each specific-binding peptide-containing embodiment of the invention is
comprised of a sequence of amino acids. The term amino acid as used in this
. invention is intended to include all c.- and n-, primary a- and (3-amino
acids, naturally
occurring, modified, substituted, altered and otherwise. Specific binding
peptide
embodiments of the reagents of the invention comprise specific binding
peptides
having a molecular weight of less than about 5,000 daltons. Particularly
preferred
embodiments of the specific binding peptides of the invention include peptides
that
bind specifically and with high affinity to the somatostatin receptor (SSTR)
on SSTR-
expresing cells, particularly tumor cells and activated T-lymphocyte cells.
Reagents
-18-
219CJ 108
comprisii.g specific-binding peptides provided by the invention include but
are not
limited to reagents comprising peptides having the following amino acid
sequences
(the amino acids in the following peptides are L-amino acids except where
otherwise
indicated):
cyclo(N methyl)FYWDKV . Hcv. (cH2co. GGC . amide)
cyclo(N methyl)FYWoKV~Hcv.(cH2co.GGCK.amide)
cyclo~N methvl)FYWDKV~Hcv. (cH2co. GGCR. amide)
cyclolN methyl)FYWDKV~Hcv. (cHZco. GGCRD. amide)
cyclo~N methyl)FYWpKV~Hcv.(cHZco.GGCRK.amide)
cyclo~N methyl)FYWDKV~Hcv. (cHZco. GGCRR. amide)
cyclo(N methyl)FYWDKV~Hcy.(cHzco.GGCKK.amide)
cyclo(N methyl)FYWDKV~Hcv.(cHzco.GGCKKK.amide)
cyclo(N methyl)FYWDK, V~Hcv.(cHZco.GGC.Orn.amide)
cyclo(N methyl)FYWpK. V~Hcv.(cHZco.GGCKDK.amide)
cyclolN methvl)FYWDKV . Hcv. (cHZco. GGC.Orn. D. Orn. amide)
cyclo(N methyl)FYWDKV~Hcy.(cHZco.GGC.Orn.D.amide)
cyclo(N methyl)FYWDK_ V.Hcv.(cHZco.KKC.amide)
cyclo(N methyl)FYWDKV.Hcv.(cH2co.KRC.amide)
cyclolN methyl)FYWoKV.Hcv.(cH2co.RRC.amide)
cyclo(N methyl)FYWDKV.Hcy. (cHZco.KKCK.amide)
cyclo(N methyl)FYWDKV.Hcv.(cH2co.GRCK.amide)
cyclo(N methyl)FYWDKV.Hcy.(CHzco.GKCR.amide).
(Single-letter abbreviations for amino acids can be found in Zubay, 1988,
Biochemistry, (2d. ed.), MacMillan Publishing: New York, p.33; other
abbreviations
are as in the Legend to Table I). This list of reagents provided by the
invention is
illustrative and not intended to be limiting or exclusive, and it will be
understood by
those with skill in the art that reagents comprising combinations of the
peptides
disclosed herein or their equivalents may be covalently linked to any of the
chelating
moieties of the invention and be within its scope.
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WO 95131221 g ~' ~ ~PCTIUS95/06034
In a preferred embodiment, the reagent of the invention has formula:
H=
H
li=N
Specific-binding peptides comprising the reagents of the present invention can
be chemically synthesized in vitro. Such peptides can generally advantageously
be
prepared on a peptide synthesizer. The peptides of this invention can be
synthesized
wherein the metal ion-complexing moiety is covalently linked to the peptide
during
chemical synthesis in vitro, using techniques taught herein (see, for example,
Example 2, subsection O).
In scintigraphic imaging embodiments of the invention, a complex of
technetium-99m is formed with the reagents of this invention. To accomplish
this,
Tc-99m, preferably as a salt of Tc-99m pertechnetate, is reacted with the
reagents
of this invention in the presence of a reducing agent. Preferred reducing
agents are
dithionite, stannous and ferrous ions; the most preferred reducing agent is a
stannous
salt such as stannous chloride. In an additional preferred embodiment, the
reducing
agent is a solid-phase reducing agent. Complexes and means for preparing such
complexes are conveniently provided in a kit form comprising a sealed vial
containing a predetermined quantity of a reagent of the invention to be
labeled and
a sufficient amount of reducing agent to label the reagent with Tc-99m.
Alternatively, the complex may be formed by reacting a reagent of this
invention
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219 0 i 0 8 pCT~S95/06034
WO 95/31221
with a pre-formed labile complex of technetium and another compound known as a
transfer ligand (such as tartrate, citrate, gluconate or mannitol, for
example>. This
process is known as ligand exchange and is well known to those skilled in the
arc.
Among the Tc-99m pertechnetate salts useful with the present invention are
included
the alkali metal salts such as the sodium salt, or ammonium salts or lower
alkyl
ammonium salts.
Technetium-99m labeled scintigraphic imaging agents according to the present
invention can be prepared by the addaion of an appropriate amount of Tc-99m or
Tc-
99m complex into the vials and reac~ion under conditions described in Example
3
hereinbelow. The kit may also con~.a~r. conventional pharmaceutical adjunct
materials
such as, for example, pharmaceu. ically acceptable salts to adjust the osmotic
pressure, buffers, preservatives and the like. The components of the kit may
be in
liquid, frozen or dry form. In a pre f~rred embodiment, kit components are
provided
in lyophilized form. Radiolabeled ~,:inti~raphic imaging reagents according to
the
present invention may be prepared by reaction under conditions described in
Example
3 hereinbelow.
Radioactively labeled reagents provided by the present invention are provided
having a suitable amount of radioactivity. In forming, for example, Tc-99m
radioactive complexes, it is generally preferred to form radioactive complexes
in
solutions containing radioactivity at concentrations of from about 0.01
millicurie
(mCi) to 100 mCi per mL.
The imaging reagents provid;,d by the present invention can be used for
visualizing organs such as the kidney for diagnosing disorders in these
organs, and
tumors, in particular gastrointestinal rumors, my~lomas, small cell lung
carcinoma
and other APUDomas, endocrine tumors such as medullary thyroid carcinomas and
pituitary tumors, brain tumors such as meningiomas and astrocytomas, and
tumors
of the prostate, breast, colon, and ovaries can also be imaged. In accordance
with
this invention, the Tc-99m labeled peptide reagents are administered in a
single unit
injectable dose. The Tc-99m labeled peptide reagents provided by the invention
may
be administered intravenously in any conventional medium for intravenous
injection
such as an aqueous saline medium, or in blood plasma medium. Generally, the
unit
dose to be administered has a radioactivity of about 0.01 mCi to about 100
mCi,
-21 -
CA 02190108 2000-06-20
preferably 1 mCi to 20 mCi. The solution to be injected at unit dosage is from
about
O.OI mL to about 10 mL. After intravenous administration, imagine in vivo can
take
place in a matter of a few minutes. However, imaging can take place, if
desired,
in hours or even longer, after the radiolabeled peptide is injected into a
patient. In
most instances, a sufficient amount of the administered dose will accumulate
in the
area to be imaged within about 0.1 of an hour to permit the taking of
scintiphotos.
Any conventional method of scintigraphic imaging for diagnostic purposes can
be
utilized in accordance with this invention.
The somatostatin receptor-binding cyclic peptides and non-radioactive metal
complexes of the cyclic peptide reagents of the invention may be used
clinically as
therapeutic agents to promote regression of certain types of tumors,
particularly those
that express somatostatin receptors. The somatostatin analogue cyclic peptides
of the
invention can also be used to reduce the hormonal hypersecretion that often
accompanies certain cancers, such as the APUDomas. Peptides of the invention
used
as therapeutic agents may be administered by any appropriate route, including
intravenous, intramuseular or by mouth, arid in any acceptable pharmaceutical
carrier; in doses ranging from about 0.1 to about 49 mg/kg body weightJday.
This invention also provides peptides radiolabled with cytotoxic radioisotopes
such as rhenium-186 or rhenium-188 that may be used for radiotherapy of
certain
tumors as described above. For this purpose, an amount of radioactive isotope
from
about lOmCi to about 200mCi may be administered via any suitable clinical
route,
preferably by intravenous injection.
The methods for making and labeling these compounds are more fully
illustrated in the following Examples. These Examples illustrate certain
aspects of
the above-described method and advantageous results, and are shown by way of
illustration and not limitatian.
E~LANfPLE 1
Solid Phase Peptide Synthesis
Solid phase peptide synthesis (SPPS) was carried out on a 0.25 millimole
TM
(mmole) scale using an Applied Biosystems Model 431A Peptide Synthesizer and
using 9-fluorenylmethyloxycarbonyl (Fmoc) amino-terminus protection, coupling
with
-22-
219010
dicyclohexyl-carbodiimide/hydroxybenzotriazole or 2-(1H-benzo-triazol-1-yl)-
1,1,3,3-
tetramethyluronium hexafluorophosphate/ hydroxybenzotriazole (HBTU/HOBT), and
using p-hydroxymethyl-phenoxymethylpolystyrene (HMP) or Sasrin'~ resin for
carboxyl-terminus acids or Rink amide resin for carboxyl-terminus amides.
Fmoc.Hcy(S-trityl) and Fmoc.penicillamine(S-trityl) were prepared from the
appropriate amino acid by tritylation with triphenylmethanol in
trifluoroacetic acid,
followed by Fmoc derivitization as described by Atherton et al. (1989, Solid
Phase
Peptide~nthesis, IRL Press: Oxford) and further in Example 2, subsection J
below.
Fmoc-S-(3-(t-butoxycarbonyl (Boc)-aminopropyl)cysteine was prepared from L-
cysteine and Boc-aminopropyl bromide in methanolic sodium methoxide followed
by
treatment with O-9-fluorenylmethyl-O'-N succcinimidyl carbonate (FmocOSu) at
pH
10.
2-haloacetyl groups were introduced either by using the appropriate 2-
haloacetic acid as the last residue to be coupled during SPPS or by treating
the N-
terminus free amino peptide bound to the resin with either 2-haloacetic acid/
diisopropylcarbodiimide/N hydroxysuccinimide in N methyl-2-pyrrolidinone (NMP)
of 2-haloacetic anhydride/ diisopropylethylamine in NMP.
Thiol-containing peptides were reacted with chloroacetyl-containing, thiol-
protected Tc-99m complexing moieties at pH 10 for 0.5-24 hours at room
temperature, optionally followed by acetic acid acidification and evaporation
of the
solution to give the corresponding peptide-sulfide adduct, as described in
more detail
in Example 2, subsection O below. Deprotection and purification were routinely
performed as described to yield the chelator-peptide conjugate.
Sasrin"' resin-bound peptides were cleaved using a solution of 1 % TFA in
dichloromethane to yield the protected peptide.
Where appropriate, protected peptide precursors were cyclized between the
amino- and carboxyl-termini by reaction of sidechain-protected, amino-terminal
free
amine and carboxyl-terminal free acid using diphenylphosphorylazide, as
described
in more detail in Example 2, subsection M below.
HMP or Rink amide resin-bound products were routinely cleaved and
protected cyclized peptides deprotected using a solution comprised of
trifluoroacetic
acid (TFA), or TFA and methylene chloride, optionally comprising water,
thioanisole, ethanedithiol, and triisopropylsilane in ratios of 100 : 5 : 5 :
2.5 : 2, for
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r~~~ ~ f~
._
CA 02190108 2000-06-20
0.5 - 3 hours at room temperature. Where appropriate, products were re-S-
trovlated
in triphenoimethanol/TF.A, and lV-Boc groups re-introduced into the
peptide.usine
(Boc)~O.
Crude peptides were purified by preparative high pressure liquid
chromatography (HPLC) using a Waters Delta-Pak CI8'column and gradient elution
with 0.1 % TFA in water moditied with acetonirrile. After column elution,
acetonitrile was evaporated from the eluted fractions, which were then
lyophilized.
The identity of each product so prenuced and purified was confirmed by fast
atom
bombardment mass spectroscopy ( 1"ABMS) or electrospray mass spectroscopy
(ESMS).
~':'~AMPLP, 2
Synthesis of cyclo(N-CH,.,)phen _'al_anvil-tvrosvl-~-trvntonhanvi-lvsvt-valyl
homocysteine. S-2-acet l~-Q,!~~~v__h~IYCVI-cvsteinv!-lvsinamide 1P~8~
I5 A. Synthesis of N a-cart.ubenzoxy-~'V-E-ten-butoxyc~rbonyl-lysine, N
hydroxy-succinirnide ester
To a mixture of N a-carbo~enzoxy-N-e-ten-butoxycarbonyl lysine (23g,
60.5mmol) and N hydroxysuccin~-nide (7, lg, 6I.7mmo1) in 180mL dry
tetrahydrofuran (THF) cooled in a coed water bath was added
diisopropylcarbodiimide (9.66mL, 61.7mmol). This reaction mixture was stirred
overnight and then filtered and the filtrate evaporated. The residue of the
filtrate
was then redissolved in minimal ethyl acetate. 200mL ether and 200mL hexanes.
The title compound precipitated from this mixture and was recovered by
filtration,
washed with cold hexanes and dried to give 28.18 (58.9mmol, 97% yield).
zs
B. Synthesis of N-a-carbobenzoxy-N-E-tent-buto~rycarbonyl-lysyl-
violins, methyl ester
To a solution of violins methyl ester (8.388, 50mmo1) and
diisopropylethylamine (12.7mL. SOmmo!) in 150mL THF was added N a
carbobenzoxy-N e-ten-butoxycarbonyl lysine. N hydroxysuccinimide ester (23.98,
50mmo1), followed by an additional SOmL THF. After 2h the solvent was removed
by evaporation and 50mL ethyl acetate added to the residue. This solution was
washed sequentially with 200mL S % citric acid. 200mL saturated sodium
bicarbonate
-24-
CA 02190108 2001-12-05
and 200mL saturated brine. dried over anhydrous magneswm sulfate, filtered and
evaporated. The resulting oil was dissolved in minimal ethyl acetate. 200mL
ether
and 200mL hexanes. The title compound was isolated by filtration and dried to
yield
20g (40.Smmol. 81 % yield).
C. Synthesis of N E-tent-butoxycarbonyl-lysyl-valise, methyl ester
To a solution of N a-carbobenzoxy-N e-ten-butoxycarbonyl-lysyl-valise,
methyl ester (19g, 38.Smmo1) and acetic acid (1mL) in 100mL ethyl acetate was
added 10% palladium on carbon catalyst (0.19g) and stirred under a hydrogen
atmosphere overnight. The reaction mixture was then filtered over CeliteTM,
the filtrate
evaporated and the residue redissolved in 100mL methanol containing 1mL acetic
acid. To this solution was added 10% palladium on carbon (0.19g) and the
mixture
was hydrogenated under a pressure of 45 pounds per square inch for 2h using a
Pan
hydrogenator. The reaction mixture was again filtered over Celite and the
filtrate
evaporated to give 13.56g of the title compound (37.7mmol, 98% yield).
D. Synthesis of fluorenylmethoxycarbonyl-o-tryptophan, N-
hydroxysuccinimide ester
To a solution of N a-fluorenylmethoxycarbonyl-tryptophan hemihydrate (25g,
82. lmmol) and N hydroxysuccinimide (9.78g, 85mmol) in 250mL dry T'HF cooled
in a cold water bath was added diisopropylcarbodiimide (13.3mL. 85mmol). The
reaction mixture was stirred overnight, filtered and the filtrate evaporated.
The
residue was then redissolved in toluene and hexanes. The title compound was
isolated by filtration and dried to yield 34g (66.Smmol. 81 % yield).
E. Synthesis of fluorenylmethoxycarbonyl-o-tryptophanyl-N e-tat-
butoxy-carbonyl-lysyl-valise, methyl ester
To a mixture of N E-ten-butoxycarbonyl-lysyl-valise, methyl ester (13g,
36.1mmo1) in 200mL THF was added fluorenylmethoxycarbonyl-v-tryptophan. N
hydroxysuccinimide ester (18.98, 36.1mmo1). Diisopropylethylamine was added to
adjust the pH of the reaction mixture to pH 8, and the reaction stirred for 2
days.
The solvent was then removed by evaporation and the residue redissolved in
ethyl
acetate, washed with 5 % citric acid, saturated bicarbonate, and saturated
brine, and
-25-
~.21901n~
WO 95/31221 PCT/US95/06034
then dried over sodium sulfate. The solution was then filtered and evaporated
and
the residue redissolved in ethyl acetate. The title compound was recovered
from
several crystallizations of this solution to give 17.3g of product (22.5mmol,
62%
yield).
F. Synthesis of N fluorenylmethoxycarbonyl-O-tent-butyltyrosine, lf-
hydroxy-succinimide ester
To a solution of N a-fluorenylmethoxycarbonyl-O-ten-butyltyrosine (25g,
54.3mmol) and N hydroxysuccinimide (6.628, 57.5mmo1) in 250mL dry THF cooled
in a cold water bath was added diisopropylcarbodiimide (9.OmL, 57.5mmo1). The
reaction mixture was stirred overnight, filtered and the filtrate evaporated.
The
residue was then redissolved in toluene and hexanes. The title compound was
isolated by filtration and dried to yield 27.7g (49.7mmol, 92 % yield).
G. Synthesis of N-fluorenylmethoxycarbonyl-O-tent-butyltyrosyl-o-
tryptophanyl-N E-tent-butoxycarbonyl-lysyl-valine, methyl ester
Fluorenylmethoxycarbonyl-n-tryptophanyl-N-E-ten-butoxycarbonyl-lysyl-
valine, methyl ester (17g, 22. lmmol) was treated with 40mL diethylamine and
40mL
THF at room temperature for 1.5h. The reaction mixture was then evaporated,
resuspended in 100mL THF and re-evaporated three times. The residue was taken
up in 120mL dry THF and N fluorenylmethoxycarbonyl-O-ten-butyltyrosine, N
hydroxysuccinimide ester (12.3g, 22. lmmol) was added, followed by 20mL dry
THF
and 4mI. diisopropylethylamine resulting in a solution having a pH of 9. After
stirring overnight, the reaction was evaporated and the residue taken up in
ethyl
acetate. The ethyl acetate solution was washed with 5 % citric acid, saturated
sodium
bicarbonate and saturated brine, then dried over magnesium sulfate and
evaporated.
The residue was taken up in ethyl acetate, ether and hexanes and the title
compound
precipitated. The precipitated compound was isolated by filtration and dried
to yield
14.6g of the title compound ( 14.8mmol, 67 ~7 yield).
H. Synthesis of N fluorenylmethoxycarbonyl-N methylphenylalanine,
N hydroxysuccinimide ester
To a solution of N-a-fluorenylmethoxycarbonyl-N-methylphenylalanine (25g,
-26-
,~ r.
wn QSmt22t ~ ~ ~ 1 / 1 ~ ~ ~ PCT/US95/06034
62.3mmo1) and N-hydroxysuccinimide (7.5g. 65mmol) in 180mL dry THF cooled
in a cold water bath was added diisoprogylcarbodiimide ( IOmL. 64.2mmo1). The
' reaction mixture was stirred overnight, filtered and the filtrate
evaporated. The
residue was then redissolved in toluene and hexanes. The title compound was
isolated by filtration and dried to yield 28.3g (57mmol, 91 % yield).
I. Synthesis of N-fluorenylmethoxycarbonyl-N-methylphenylalanine-
O-tert-butyl-tyrosyl-v-tryptophanyl-N-E-tent-butoxyearbonyl-lysyl-
valine, methyl ester
Fluorenylmethoxycarbonyl-O-tert-butyl-tyrosyl-n-tryptophanyl-N-e-tert-
butoxycarbonyl-lysyl-valine, methyl ester ( 14g, 14.2mmo1) was treated with
35mL
diethylamine and 35mL THF at room temperature for lh. The reaction mixture was
then evaporated, resuspended in 100mL THF and re-evaporated three times. The
residue was taken up in ethyl acetate and hexanes. The product, O-tent-
burylryrosyl-
n-tryptophanyl-N e-tert-butoxycarbonyl-lysyl-valine, methyl ester was
precipitated,
isolated by filtration and dried to yield 9.7g (12.7mmol, 90% yield). The O-
tert-
burylryrosyl-o-tryptophanyl-N e-ten-butoxycarbonyl-lysyl-valine, methyl ester
was
dissolved in 35mL dry THF and N fluorenylmethoxycarbonyl-N
methylphenylalanine,
N hydroxysuccinimide ester (6.35g, 14.2mmo1) was added, followed by 3mL
diisopropyl-ethylamine resulting in a solution having a pH of 9. After
stirring
overnight, the reaction was evaporated and the residue taken up in ethyl
acetate. The
ethyl acetate solution was washed with 5 % citric acid, saturated sodium
bicarbonate
and saturated brine, then dried over magnesium sulfate and evaporated. The
residue
was taken up in ethyl acetate, ether and hexanes and the title compound
precipitated.
The precipitated compound was isolated by filtration and dried to yield 12.4g
of the
title compound (11.5mmol, 81% yield).
J. Synthesis of N-fluorenylmethoxycarbonyl-S-tritylhomocysteine
a. To about 400mL liquid ammonia cooled to -78°C in a dry icelethanol
bath was added small amounts of elemental sodium followed by a sufficient
amount
of homocysteine to quench the resulting blue color of the sodium/ammonia
solution
until 5.7g (248mmol) sodium and 9.75g (36.3mmo1) homocysteine had been
consumed and the blue color of the sodium/ammonia solution persisted for about
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WO 95/31221
PCT/US95/06034
l5min. Ammonium chloride (O.Sg) was added to quench the final blue color. and
then the reaction was removed from the cooling bath and the ammonia allowed to
evaporate under an argon stream. The flask was warmed slightly to drive off
essentially all residual ammonia.
To the residue was added triphenylmethanol (23.6g, 9lmmol) and the reaction
flask cooled in an ice/water bath. 250mL trifluoroacetic acid (TFA) was added,
and
after 30min the mixture was evaporated and the residue redissolved and re-
evaporated three times with chloroform. The residue was then redissolved in
300mL
water and the pH adjusted to pH 4 with 5% citric acid and 1M KOH. The product
precipitated as a gum and was collected by filtration. The residue was then
triturated
with ether to yield S-triryl-homocysteine (7g, 18.6mmol, 26% yield). A second
crop
was isolated from the filtrate by crystallization from dimethylformamide
(DMF)/water to give a combined yield of 24.2g (64mmo1, 89%).
b. To a solution of S-triryl-homocysteine (20g, 53mmol) in 150mL
acetone/100mL water was added sodium carbonate (ll.Sg, 109mmo1) and then O
fluorenylmethyl-O'-(N succinimidyl)carbonate (l7.Sg, 52mmol) dissolved in
200mL
acetone, these additions being made over the course of about lh. The reaction
mixture was then stirred for about 2 days and then the organic solvents were
evaporated. To the aqueous residue was added 300mL ethyl acetate and the
mixture
was acidified with 1M HCI. The organic phase was separated and washed
sequentially with 1M HCI, O.SM HC1, and 0.25M HC1, then dried over magnesium
sulfate, filtered and evaporated. The crude product was chromatographed on
silica
gel (100% chloroform -~ 3% methanol in chloroform) to yield the title compound
(19.4g, 32mmo1, 62% yield).
K. SynthesisofN-fluorenylmethoxycarbonyl-S-trityl-homocysteine-N
methyl-phenylalanine-O-tent-butyltvrosyl-o-tryptophanyl-N-E-tert-
butoxycarbonyl-lysyl-valise, methyl ester
Fluorenylmethoxycarbonyl-N-methylphenylalanine-O-ten-butyl-tyrosyl-o
tryptophanyl-N e-tent-butoxycarbonyl-lysyl-valise, methyl ester (9.8g, S.Smol)
was
treated with 28mL diethylamine and 30mL THF at room temperature for 1 h. The
reaction mixture was then evaporated, resuspended in 100mL THF and re-
evaporated
three times. The residue was taken up in ethyl acetate and hexanes. The
product,
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WO 95/31221 PCT/US95106034
. N-methylphenylalanine-O-ten-butyltyrosvl-o-tryptophanyl-N-E-ten-
butoxvcarbonvl-
lysyl-valine, methyl ester was precipitated. isolated by filtration and dried
to yield
4.9g (8. lmmol, 95 % yield).
To a solution of N fluorenylmethoxycarbonyl-S-trityl-homocysteine in SOmL
dry THF cooled in a -15°C cooling bath was added N, N'-bis(2-oxo-3
oxazolidinyl)phosphinic chloride (BOP-C1; 2.47g, 9.7mmo1) and l.7mL
diisopropylethylamine (9.7mmo1) and the reaction mixture was stirred for
30min.
N methylphenylalanine-O-ten-butyltyrosyl-n-tryptophanyl-N E-tent-
butoxycarbonyl
lysyl-valine, methyl ester (7.48g) in SOmL dry THF was added, followed by an
additional l.7mL diisopropylethylamine. The reaction volume was reduced 50% by
evaporation and then stirred for 2 days at room temperature. The solvent was
removed by evaporation and the residue redissolved in ethyl acetate, washed
with 5
citric acid, saturated sodium bicarbonate and saturated brine, and dried over
magnesium sulfate. The residue was taken up in ethyl acetate, ether and
hexanes and
the title compound precipitated. The precipitated compound was isolated by
filtration
to give 10.2g (6.77mmo1, 84 % yield).
L. Synthesis of S-trityl-homocysteine-N-methylphenylalanine-O-tert-
butyl-tyrosyl-n-tryptophanyl-N e-tert-butoxycarbonyl-lysyl-valine
A solution of N fluorenylmethoxycarbonyl-S-trityl-homocysteine-N
methylphenylalanine-O-ten-butyl-tyrosyl-n-tryptophanyl-N e-tent-butoxycarbonyl-
lysyl-valine, methyl ester ( lOg, 6.6mmo1) and LiOH . 2H~0 in SOmL THF and 4mL
water was stirred for 3 days. An additional 25 mol % LiOH . 2H,0 was added,
and
two hours later the solvent was evaporated and the residue redissolved in
ethyl
acetate. This solution was then washed with 5 % citric acid, saturated sodium
bicarbonate, and saturated brine, dried over magnesium sulfate, filtered and
evaporated. The residue was redissolved in ethyl acetate, ether and hexanes
and the
title compound precipitated and was isolated by filtration and dried. The
resulting
impure product was flash chromatographed in silica gel using chloroform/10%
methanol in chloroform to yield 3.46 g of the pure title compound (47mmol, 41
yield).
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9 ri ~ ~~~ C~
WO 95/31221 PCT/US95106034
M. Synthesis of cyclo-N-methvlnhenvlalanine-O-tert-butvitvrosvi o
trvntoohanvl-~'-E-tert-butoxvcarbonvl-lvsvl valvl S tritvl
homocvsteine
A solution of S-trityl-homocysteine-N methylphenylalanine-O-ten-buryl-
ryrosyl-n-tryptophanyl-N E-ten-butoxycarbonyl-lysyl-valine (3.42g, 2.69mmol)
dissolved in 1740mL DMF was cooled in an ice water bath, and 1.16mL
diisopropylethylamine and diphenyl phosphorylazide (DPPA; 5.4g, 1. l6mmol)
were
added. The reaction was incubated at -20°C for 5 days and an additional
25 mol
DPPA was added, followed by an additional 100 mol % diisopropylethylamine. The
DMF solvent was then removed by evaporation and the crude title compound
crystallized from ethyl acetate, ether and hexanes to yield 2.43g (l.9mmol,
69%).
N. Synthesis of cyclo-N methvlphenvlalanvl-tvrosyl-v-trvptophanyl-
Iysvl-valvl-S-tritvl-homocvsteine
cvclo-S-tritvl-homocvsteine-N-methylphenvlalanine-O-ten-butyl tyrosyl ~
trvptophanvl-N e-ten-butoxycarbonyl-1 ~svl-valine (2.348, 1.9mmo1) was treated
with
18.7mL TFA, 2mL dichloromethane, 0.94mL water, 0.47mL ethanedithiol and
0.37mL triisopropylsilane for lh at room temperature. TFA was removed by
evaporation and the residue redissolved and re-evaporated from chloroform
three
times. The residue was then redissolved in IOmL chloroform and poured into
400mL cold ether. The crude product precipitate was collected by filtration
and
purified by C 18 preparative reverse phase HLPC (using a gradient of 30 %
acetonitrile -~ 60% acetonitrile in water, all solvents containing 0.1 % TFA)
to give
the title compound (lg, l.2mmol, 62% yield). Fast atom bombardment mass
spectrometry (FABMS) analysis gave a MH' of 855, compared with the theoretical
(average) predicted value of 855.09.
O. Synthesis of cyclo-N methvinhen l~~~osvl-n-trvptoohanvl
Iysvl-valvl-S-trityl-homocysteine~-2-acetyl-glycyl-glycyl-cysteinyl
lysinamide (P58'n
cyclo-N-methylphenylalanyl-tyrosyl-o-tryptophanyl-lysyl-valyl-homocysteine
(250mg, 0.29mmol) and 2-chloroaceryl-glycyl-glycyl-S-triryl-cysteinyl-
lysylamide
(prepared by SPPS as described in Example l: 238mg, 0.35mmol) were dissolved
in 7mL acetonitrile and 7mL of a solution of 100mM sodium carbonate/ O.SmM
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WO 95/31221 PCT/US95l06034
EDTA, pHlO and stirred overnight. The reaction mixture was then evaporated to
dryness and deprotected in TFA containing triisopropylsilane as described
above in
Example 1. The title compound was then purified by reverse phase HLPC to yield
92.4 of the expected theoretical yield. Fast atom bombardment mass
spectrometry
analysis gave a MHf of 1258, compared with the theoretical (average) predicted
value of 1257.70. The structure of tL~is product, termed P587, is represented
by the
formula:
CH~CO-Gly-Gly-Cys-Lys-amide
I(N-CH3)Phe-Tyr-D-Trp-Lys-Val-Hcyl
H2N
~~O
'~S
HN
c.~
NH NH NH ~O O NH
O O
HN
O 'S H
NH ~~ ~N\ O NH
HN
OH
H2N
EXAMPLE 3
General Methods for Radiolabeling
0.1 mg of a peptide reagent prepared as in Example 1 was dissolved in
O.ImL of water, or 0.9~ sodium chloride, or 10~ hydroxypropylcyclodextrin
-31-
CA 02190108 2000-06-20
. (HPCD), or 50:50 ethanol:water, or phosphate-buffered saline (PBS), or
50mlrt
potassium phosphate buffer (pH = ~, 6 or 7.4). Tc-99m gluceptate was prepared
by reconstituting a Glucoscan vial (E.I. DuPont de Nemours, Inc., Wilmington,
DE)
with l.OmL of Tc-99m sodium pertechnetate containing up to 200mCi and allowed
to stand for I5 minutes at room temperature. 25uL of Tc-99m gluceptate was
then
added to the reagent and the reaction allowed to proceed at room temperature
or at
100°C for S-30 min and then filtered through a 0.2 ,um filter.
The Tc-99m labeled peptide reagent purity was determined by HPLC using
the following conditions: a Waters Delta-Pak RP-18 analytical column, having
dimensions of S~cm x 4.dmm x 220mm, was loaded with each radiolabeied peptide,
which were then eluted at a solvent flow rate of 1mL/min. Gradient elution was
performed over 10-20 min using a linear gradient beginning with 100% Solvent A
(0. i %a TFA/water) and ending with 100% Solution B (0.1 % TFA/90%
acetonitrile/water). Radioactive components were detected by an in-line
radiometric
detector linked to an integrating recorder. Tc-99m gluceptate and Tc-99m
sodium
pertechnetate elute between I and 4 minutes under these conditions, whereas
the Tc-
99m labeled peptide eluted after a much greater amount of time.
The following Table illustrates successful Tc-99m labeling of peptides
prepared according to Example 1 using the method described herein.
-32-
~ 19~~ 1 (~
WO 95131221 PCT/US95/06034
J:
N ~ L..
U ~ c
~ o ....
~ .. z
o
~
xi ~ ~ v
~ ~ ~ c
'
L
v
O
r
r
J >,
J :~ _ .
. _
C y >,
~ .
V p
00 C~ ~..00 ~ .T.
V
~ ~ 41
U Z .
'
J
.~ , G ..
S C
,v C tC
cb
U
C ~ C_
r~
M E V
C~ N v7 ~ a Q d
"
", 00 O .
~ 0
~ ~ ~ N ~ N
Q ... O O
.L
G O
1
a N v
_ c > va
.
c >
.v II
.., C o
w
o = .~ o a
c .~ o c ~,
'fl ~ ~ V '~ eCC
~ O ~
.~i.~.1 J U ~ ~ ~7 a.
y
U U U U U ,
"
~
~ ~ o
V V V V V v ~ a
a~
V O J O O U v E G . :C
- ~ ' ~O ' C
_ . ~~ = r ~ v =
a
a ~ ~ ~ = L ~ V Q
V V V V V 3 ~ $ d C
'~ ~ O
W i z .n w C 4.. ~ C v
r.
> we " :v U >.
o ' c
C
3 3 3 x o w o ~ ~ ~ cn > " L
3 3 ~
~ 3~ ~ o
r
~r ~, ~w u ( ~
~ R
~ ~ .-. r . L O G .
.~ ~ ~ ~ C
s ~ .c .r a~ U .~ U V
~: ~ z ~ >_
~ >: ~ ~ ~y "~ C p O ~ y ~ aJ
-
z ~ z z z w ~~~ ~ _ v
0 0 0 0 ~ o00 ~
v
c U L
. . . . >,
II II
II
- -- 3
o ~, o
N
- 33 -
PCTIUS95106034
WO 95/31221
Radioiodination and radioastatination are performed as described by Seevers
et al. ( 1982, Chem. Rev. 8~: 575-590).
Non-radioactive rhenium complexes were prepared by co-dissolving each of
the peptide reagents of the invention with about one molar equivalent of
tetraburylammonium oxotetra-bromorhenate (+5), prepared as described by Cotton
et al. (1966, Inorg. Chem. 5: 9-16) in dimethylformamide or acetonitrilelwater
and
stirred for 0.5-5 days. The rhenium complexes were isolated by reverse phase
HPLC as described above for Tc-99m labeled peptides and were characterized by
FABMS or ESMS.
Radioactive rhenium complexes, using for example Re-186 or Re-188, are
prepared from the appropriate perrhenate salts using the same protocol as for
Tc-99m
labeling, or by adding a reducing agent to a solution of the peptide and
perrhenate,
or optionally using a ligand transfer agent such as citrate and incubating the
reaction
at a temperature between room temperature and 100 ° C for between 5 and
60 min.
EXAMPLE 4
Inhibition of [lzsI-Tyr"]Somatostatin-14 Binding to
AR42,1 Rat Pancreatic Tumor Cell Membranes
The ability of various somatostatin receptor-binding reagents of the invention
to bind to somatostatin receptors in vitro was demonstrated in an assay of
peptide
reagent-mediated inhibition of binding of a radiolabeled somatostatin analogue
to
somatostatin receptor-containing cell membranes.
The rat pancreatic tumor cell line AR42J expressing the somatostatin receptor
was cultured in Dulbecco's modified essential media (DMEM) supplemented with
10% fetal calf serum (FCS) and 8mM glutamine in a humidified 5% CO~ atmosphere
at 37°C. Harvested cells were homogenized in cold buffer (50mM Tris-
HCI, pH
7.4), and the homogenate was then centrifuged at 39,OOOg for lOmin at
4°C. Pellets
were washed once with buffer and then resuspened in ice-cold IOmM Tris-HCI
buffer (pH 7.4). Equal aliquots of this cell membrane preparation were then
incubated with ['uI-Tyr")somatostatin-14 (Amersham, Arlington Heights, IL) at
a
final concentration of 0.5nM at 750,OOOcpm/mL, specific activity 2000Ci/mmol
and
either a peptide or peptide-rhenium complex of the invention ( at a final
concentration
-34-
2. ~ ~9
WO 95/31221 PCT/US95I06034
ranging from 10-"M to 10~6M in SOmM HEPES buffer, pH 7.4, containins 1 ','~
bovine serum albumin, 5mM MgCI~. 0.02mg/mL bacitracin, 0.02mg/mL
phenylmethyl-sulfonylfluoride and 200.000 IU Trasylol for 25min at
30°C.
After incubation, this membrane mixture was filtered through a
polyethyleneimine-washed GC/F filter (Whatman Ltd., Maidstone, England) using
a filtration manifold, and the residue remaining on the filter was washed
three times
with SmL cold HEPES buffer. The filter and a sample of the filter washings
were
then counted on a gamma counter. To assess non-specific binding, the assay was
also performed essentially as described in the presence of 200mn unlabeled
somatostatin-14. Data analysis included Hill plots of the data to yield
inhibition
constants as described by Bylund and Yamamura (1990, Methods in Neuro-
transmitter Receptor Analysis, Yamamura et al., eds., Raven Press: N.Y.). The
results obtained using this assay with the reagents of the invention are as
follows:
TABLE II
peptide K
cvclo(N methyl)FYW V.Hc .(cH,co.GGCKK.amide) 0.26
cvclo(N methyl)FYWr,,KV.H~.(cH,co.GGCK.amide) 2.5
cyclo(N methyl)FYWnKV.Hcv.(cH,co.GGC.amide) 2.6
These results demonstrate that peptide reagents of the invention bind with
high affinity to somatostatin receptors in vitro.
EXAMPLE 5
Localization and In Vivo Imaging of Somatostatin Receptor (SSTR)
Expressing Tumors in Rats
In vivo imaging of somatostatin receptors expressed by rat tumor cells was
performed essentially as described by Bakker et al. ( 1991, Life Sciences 49:
1593-
1601).
CA20948 rat pancreatic tumor cells, thawed from frozen harvested tumor
brei, were implanted intramuscularly in a suspension of 0.05 to 0.1 mL/animal,
into
the right hind thigh of b week old Lewis rats. The tumors were allowed to grow
to
approximately 0.5 to 2g, harvested, and tumor brei was used to implant a
second,
naive set of Lewis rats. Passaging in this fashion was repeated to generate
successive generations of tumor-bearing animals. The tumor-bearing animals
used
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WO 95/31221 ~ PCT/US95/06034
for the in vivo studies were usually from the third to fifth passage and
carried 0.2 to
2g tumors.
For studies of the specificity of radiotracer localization in the tumors,
selected
animals were given an subcutaneous SSTR-blocking dose (4 mg/kg) of octreotide
30
minutes prior to injection of the radiotracer. (This protocol has been shown
by
Bakker et al. to result in a lowering of "'In-[DTPA]octreotide tumor uptake by
40%.)
Third- to fifth-passage CA20948 tumor-bearing L.ewis rats were restrained
and injected intravenously via the dorsal tail vein with a dose of 0.15-0.20
mCi
~'"Tc-labeled peptide corresponding to 3 to 8 ~.g peptide in 0.2 to 0.4 mL.
At selected times, the animals were sacrificed by cervical dislocation and
selected necropsy was performed. Harvested tissue samples were weighed and
counted along with an aliquot of the injected dose in a gamma well-counter.
The 90-minute biodistribution results of selected radiolabeled peptides are
presented in Table I. Notably, ~"'Tc-P587, ~'"Tc-P617, ~"'Tc-P726, and ~'"'Tc-
P736
showed very high tumor uptake and tumor/blood ratios demonstrating their high
specific uptake in target (tumor) tissue.
Figure 1 shows an image of ~"''Tc-P587 in a tumor-bearing rat. The high
uptake in the tumor in the lower leg (arrow) is clearly visible.
~"'Tc-P587 uptake in tumors in rats was compared with and without pre-
injection treatment with octreotide, a somatostatin analogue known to bind to
the
somatostatin receptor in vivo. In these experiments, receptor-blocking by
administration of octreotide prior to administration of ~"'Tc-P587 reduced
specific
tumor uptake of the radiolabeled peptide by 76 % . These results confirmed
that
binding of ~"'Tc-P587 in vivo was SSTR-specific.
-36-
~,'"O 95131221 219 010 8 pCT/US95/06034
0
m
v ov ~ o, 00
0
E
E"
do
I
p
0
~ N
b o vc t',
f
O O O O
..., ~' [w V~
N M ~p N
~ . .
e ~ ~ "G
C
, c3 eC
.~.,
_ ea
V
V V
Q
O O O V
S S S
U G U U
H ~ ~ 'r ~r
'J U U U
~. r
w ,.~,~,.~
r~ Nr
a > > > >
vo
G~. Ate., G~. 0~.
~n O
'-' - 37 -
WO 95/31221 ~ 9 ~ ~ ~ C) PCT/US95/06034
EXAI1~IPLE 6
In Vivo Imaging of Human Somatostatin Receptor (SSTR)
Expressing Tumors with Tc-99m Labeled P587
In a clinical trial, scintigraphic imaging of human patients bearing SSTR-
expressing tumors was achieved using the Tc-99m labeled P587 reagent.
A total of 10 patients, four females and six males ranging in age from 27 to
69 years, had been previously diagnosed with growth hormone-secreting
pituitary
adenoma (4 patients), melanoma (1 subject), medullary thyroid cancer (1
subject),
small cell lung carcinoma (SCLC; 1 patient), non-Hodgkin's lymphoma ( 1
patient)
or gastric carcinoid (1 patient). Each of these patients were administered Tc-
99m
labeled P587 at a dose of 10-22mCi per 0.2-O.Smg by intravenous injection.
Scintigraphic imaging was them performed as described herein on each patient
for
4 hours post-injection.
Gamma camera imaging is started simultaneously with injection. Anterior
images were acquired as a dynamic study ( 10 sec image acquisitions) over the
first
10 min, and then as static images at 1, 2, 3 and 4h post-injection. Anterior
images
were acquired for 500,000 counts or 20 min (whichever is shorter), at
approximately
10-20 min, and at approximately 1, 2, 3 and 4h post-injection.
The scintigraphic imaging agent was found to clear rapidly from the
bloodstream, resulting in less than 10% of the injected dose remaining in the
circulation within 30 minutes of injection. This allowed image acquisition of
tumor
sites to be achieved as early as 15-30 min after injection of scintigraphic
imaging
agent. All known tumors were detected in this study, as well as two previously-
undetected metastatic lesions which were later confirmed using computer-
assisted
tomography (CAT scan).
These results demonstrated that the scintigraphic imaging agents of this
invention were highly effective in detecting SSTR-expressing primary and
metastatic
tumors in humans in vivo.
It should be understood that the foregoing disclosure emphasizes certain
specific embodiments of the invention and that all modifications or
alternatives
equivalent thereto are within the spirit and scope of the invention as set
forth in the
appended claims.
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