(12) United States Patent
`Saviano et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,329,644 B2
`Feb. 12, 2008
`
`USOO732964.4B2
`
`(54) PREPARATION AND USE OF CYCLIC AND
`BRANCHED PEPTIDES AND THEIR
`LABELLED DERVATIVES AS
`THERAPEUTIC AGENTS,
`CHOLECYSTOKININ AGONSTS OR
`ANTAGONISTS, AND DIAGNOSTICAGENTS
`TO DENTIFY AND LOCATE TUMIOURS
`
`(75) Inventors: Michele Saviano, Milan (IT): Stefania
`De Luca, Milan (IT): Giancarlo
`Morelli, Milan (IT); Diego Tesauro,
`Milan (IT): Carlo Pedone, Milan (IT)
`(73) Assignee: Bracco Imaging S.p.A., Milan (IT)
`
`(*) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 188 days.
`(21) Appl. No.:
`10/479,096
`
`May 21, 2002
`
`(22) PCT Filed:
`(86). PCT No.:
`S 371 (c)(1),
`(2), (4) Date:
`Jul. 16, 2004
`(87) PCT Pub. No.: WO02/094873
`PCT Pub. Date: Nov. 28, 2002
`
`(65)
`
`Prior Publication Data
`US 2004/O254339 A1
`Dec. 16, 2004
`
`Foreign Application Priority Data
`(30)
`May 22, 2001
`(IT)
`.......................... MI2OO1A1057
`
`(51) Int. Cl.
`(2006.01)
`A6 IK 38/00
`... 514/9
`(52) U.S. Cl. ......................................
`(58) Field of Classification Search ..................... None
`See application file for complete search history.
`References Cited
`
`(56)
`
`
`
`FOREIGN PATENT DOCUMENTS
`
`DE
`EP
`WO
`WO
`
`276482
`O960939
`WO92, 18627
`WO97/44341
`
`10, 1988
`1, 1999
`10, 1992
`11, 1997
`
`OTHER PUBLICATIONS
`Robert T. Jensen, “Involvement of Cholecystokinin/Gastrin-Related
`Peptides and their Receptors in Clinical Gastrointestinal Disorders.”
`Pharmacology & Toxicology, vol. 91, p. 333.*
`
`Rudinger (J. Rudinger. In: Peptide Hormones, JA Parsons, Ed.
`(1976) 1-7).*
`Sigma (Sigma. Designing Custom Peptides. http://www.sigma
`genosys.com/peptide design.asp (Accessed Dec. 16, 2004), 2
`pages).
`Berendsen (H.J.C. Berendsen. A Glimpse of the Holy Grail?
`Science (1998) 282, pp. 642-643).*
`Voet (D. Voet and J.G. Voet. Biochemistry, 2nd Edition. (1995), pp.
`235-241).*
`Robert T. Jensen, “Involvement of Cholecystokinin/Gastrin-Related
`Peptides and their Receptors in Clinical Gastrointestinal Disorders.”
`Pharmacology & Toxicology, vol. 91, p. 333.*
`Sporn et at, “Chemoprevention of Cancer.” Carcinogenesis, vol. 21
`(2000), 525-530.*
`Gura, “Cancer Models: Systems for Identifying New Drugs Are
`Often Faulty,” Science vol. 278 (1997), 1041-1042.*
`Kwekkeboom, et al., "Cholecystokinm receptor imaging using an
`octapeptide DTPA-CCK analouge in patients with medullary thy
`roid carcinoma'. European Journal of Nuclear Medicine, vol. 27.
`No. 9, Sep. 2000, pp. 1312-1317.*
`PCT International Search Report for PCT/EP02/05562 dated Mar.
`31, 2003.
`PCT International Preliminary Examination Report for PCT/EP02/
`05562 dated Jul. 4, 2003.
`Romani, S., et al.: “Synthesis of the trypsin fragment 10-25/75-88
`of mouse nerve growth factor'. Int. J. Peptide Protein Res. 29, 1987.
`101-117.
`Schaffhausen, et al., “Antibody to the Nonapeptide Glu-Glu-Glu
`Gly-Tyr-Met-Pro-Met-Glu Is specific for Polyoma Middle T Anti
`gen and Inhibits in Vitro Kinase Activity”. The Journal of Biological
`Chemistry, vol. 257, No. 21, Nov. 10, 1982, pp. 12467-12470.
`Kwekkeboom, et al., “Cholecystokinin receptor imaging using an
`octapeptide DTPA-CCK analouge in patients with medullary thy
`roid carcinoma'. European Journal of Nuclear Medicine, vol. 27.
`No. 9, Sep. 2000, pp. 1312-1317.
`Behr, et al., “Targeting of cholecystokinin-B gastrin receptors in
`vivo: preclinical and initial clinical evaluation of the diagnostic and
`therapeutic potential of radiolabelled gastrin'. European Journal of
`Nuclear Medicine, vol. 25, No. 4, Apr. 1998, pp. 424–430.
`* cited by examiner
`Primary Examiner Anish Gupta
`Assistant Examiner Thomas S. Heard
`(74) Attorney, Agent, or Firm Kramer Levin Naftalis &
`Frankel LLP
`
`(57)
`
`ABSTRACT
`
`This patent application describes the preparation of cyclic
`and branched peptides of general formula (I) and their
`conjugated derivatives labelled with a paramagnetic or
`radioactive metal. The compounds of the present invention
`are used as diagnostic agents to identify and locate primary
`tumours and their metastases which over-express type A
`and/or B cholecystokinin receptors, and as therapeutic
`agents and cholecystokinin agonists or antagonists.
`
`18 Claims, 3 Drawing Sheets
`
`
`
`
`MAIA Exhibit 1010
`MAIA V. BRACCO
`IPR PETITION
`
`

`

`U.S. Patent
`
`Feb. 12, 2008
`
`Sheet 1 of 3
`
`US 7,329,644 B2
`
`
`
`Figure 1
`
`
`
`
`

`

`U.S. Patent
`
`Feb. 12, 2008
`
`Sheet 2 of 3
`
`US 7,329,644 B2
`
`20.0
`
`
`
`15.0
`
`10.0
`
`5.0
`
`4000
`
`5000
`
`O
`
`1000
`
`2000
`3000
`Lo (Mx10)
`
`Figure 2
`
`
`
`
`

`

`U.S. Patent
`
`Feb. 12, 2008
`
`Sheet 3 of 3
`
`US 7,329,644 B2
`
`
`
`pur
`g
`d
`
`G)
`d
`a
`Gld
`t
`O
`
`150
`
`--
`-- y
`O hmd
`
`50
`
`Figure 3
`
`
`
`
`

`

`US 7,329,644 B2
`
`1.
`PREPARATION AND USE OF CYCLIC AND
`BRANCHED PEPTDES AND THEIR
`LABELLED DERVATIVES AS
`THERAPEUTIC AGENTS,
`CHOLECYSTOKININ AGONSTS OR
`ANTAGONISTS, AND DIAGNOSTICAGENTS
`TO DENTIFY AND LOCATE TUMIOURS
`
`2
`(Biochemical Journal, 89, 114-123, 1963), '''In or 'In,
`used in nuclear medicine to visualise human tumours, is
`described in particular in that article and in the patent cited
`by J. C. Reubi.
`Type A receptor in particular is over-expressed in pan
`creatic and oesophageal tumours, while type B receptor has
`been found to be over-expressed in Small lung cell tumour,
`tumours of the colon and gastrointestinal tract, medullary
`thyroid tumours, astrocytomas and ovarian stromal tumours.
`Some peptides deriving from cholecystokinin modified
`with chelating agents of radioactive or paramagnetic metals
`have been studied in clinical trials. In particular, CCK8
`derivatives containing the chelating agents DTPA or DOTA
`which complex radioactive metals like 'In and 'Y, and
`their application to identify and treat tumours that over
`express type B cholecystokinin receptor, have been reported
`(M. De Jong, Journal of Nuclear Medicine, 40,2082, 1999).
`The NMR structure of the complex between the non
`sulphated peptide CCK8 and the N-terminal part of type A
`cholecystokinin receptor, responsible for the interaction
`with the peptide hormone, was recently published (M.
`Pellegrini, Biochemistry, 38, 14775, 1999). The N-terminal
`part of the receptor (receptor fragment) consists of 47 amino
`acids, and represents the extracellular N-terminal arm and
`the first part of transmembrane helix 1 of the type A receptor.
`This fragment does not contain the residue of Arg 197,
`present on the transmembrane loop, which is responsible for
`the interaction with the sulphuric group of Tyr 27 of CCK8.
`with the result that peptide CCK8 is not used in the Sul
`phated form (V. Gigoux, Protein Science, 8, 2347, 1999). In
`addition to the detailed structural information indicated in
`the NMR study, a recent study performed by observing the
`variations in fluorescence of the tryptophan residues present
`on the receptor fragment and the peptide confirmed the
`binding (R. Ragone, Biopolymers, 47-53, 56, 2001, publi
`cation pending), and enabled the affinity constant between
`non-sulphated CCK8 and the receptor fragment to be deter
`mined.
`
`DESCRIPTION OF THE INVENTION
`
`The compounds object of the present invention are cyclic
`peptides of general formula (I):
`
`(I)
`
`wherein:
`Xaa, independently of each other, is any amino acid;
`Xbb is an alpha or beta amino acid containing at least
`three functional groups selected from the group consisting
`of:
`
`—COOH, -NH2, -SH and —OH,
`n is between 0 and 15, and
`m is between 2 and 12.
`Xbb is preferably selected from the group consisting of:
`LyS, Asp, Glu, Cys, Orn, Dap, Dab, Gaba, epsilon-Aca
`and delta-Ava.
`
`5
`
`10
`
`15
`
`25
`
`30
`
`The present invention relates to cyclic and branched
`peptides of general formula (I) and their derivatives conju
`gated with a spacer molecule Y and a chelating agent C.
`labelled with a paramagnetic or radioactive metal.
`The compounds of the invention are used as diagnostic
`agents to identify and locate primary human tumours and
`their metastases which over-express type A and/or type B
`cholecystokinin receptors, and as therapeutic agents and
`cholecystokinin agonists or antagonists.
`Cholecystokinins (CCKs) are a family of peptide mol
`ecules whose biological action is performed as a hormone
`and a neurotransmitter. All the CCKs originate from a
`process of fragmentation which takes place on a pre-hor
`mone consisting of 115 amino acid residues, followed by a
`post-translational process of alpha-amidation of the C-ter
`minal phenylalanine residue and sometimes, Sulphation of
`the tyrosine residue contained in the C-terminal portion. The
`cholecystokinins therefore exist in various molecular forms;
`the most important ones have a sequence of 58, 39, 33 or 8
`amino acid residues, and they all have the same C-terminal
`sequence of 8 amino acid residues:
`Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-amide.
`The form containing this sequence only is known as
`CCK8.
`The biological activity of cholecystokinin depends on the
`type of receptor with which it interacts. Two types of
`receptor are known: type A and type B. In non-pathological
`35
`situations, type A receptor is present in the tissues of
`peripheral organs such as the stomach, gallbladder, intestine
`and pancreas. The most important physiological actions due
`to the interaction of the CCK peptide hormone with type A
`receptor are contraction of the gall bladder, secretion of
`40
`pancreatic enzymes, regulation of secretion, and absorption
`into the gastrointestinal tract. Type B receptor is mainly
`present in the central nervous system, where the interaction
`with cholecystokinin causes analgesia, Satiety and anxiety,
`and regulates the release of dopamine.
`45
`Both the cholecystokinin receptors belong to the class of
`G-Protein Coupled Receptors (GPCRs), membrane recep
`tors with seven transmembrane helixes joined by intra- and
`extra-cellular loops with an extracellular N-terminal arm and
`an intracellular C-terminal part. Both receptors have high
`affinities for the various forms of cholecystokinin; however,
`type A receptor has a greater affinity for the Sulphated forms
`of cholecystokinin, namely the ones which contain a Sul
`phuric group on the Tyr 27 residue, while type B receptor
`has a high affinity for the various forms of non-sulphated
`cholecystokinin and for gastrin. A series of peptide and
`non-peptide cholecystokinin-analog molecules with agonist
`or antagonist activity for type A and type B receptors are
`known (P. De Tullio, Current Medicinal Chemistry, 6, 433,
`1999; F. Noble, Progress in Neurobiology, 58, 349, 1999).
`No pharmacological application has been found for any of
`the known molecules due to their low bioavailability and
`low solubility or high enzymatic degradation.
`Cholecystokinin receptors have recently been identified in
`primary human tumours and metastases (J. C. Reubi, Cancer
`Research, 57, 1377, 1997, WO9731657). The use of func
`tional peptides labelled with radioactive metals such as 'I
`
`50
`
`55
`
`60
`
`65
`
`
`
`
`

`

`3
`The invention also relates to compounds of formula (I)
`which are labelled, either with the use of a chelating group
`or directly, with radioactive or paramagnetic metals or
`radioactive halogens and the salts thereof with physiologi
`cally acceptable organic or inorganic bases or with anions of
`physiologically acceptable organic or inorganic acids.
`At least one of Xaa amino acids will preferably be a
`residue of methionine (Met), tyrosine (Tyr) or tyrosine-m-
`sulphonate (SOH-Tyr).
`If the Sulphate group is present on the tyrosine residue
`(Tyr or SO3H-Tyr), interaction with type A cholecystokinin
`receptor will be aided; conversely, a non-Sulphated tyrosine
`residue (Tyr or SO3H-Tyr) sometimes may promote the
`interaction with type B cholecystokinin receptor.
`The term “any amino acid used above refers to the Land
`D isomers of the natural amino acids and “non-protein’
`amino acids commonly used in peptide chemistry to prepare
`synthetic analogs of natural peptides, such as alpha amino
`acids substituted and not substituted at the alpha and beta
`positions of the L and D configurations, and unsaturated
`alpha/beta amino acids.
`Examples of “non-proten' amino acids are norleucine,
`norvaline, alloisoleucine, allothreonine, homoarginine, thio
`proline, dehydroproline, hydroxyproline, pipecolic acid,
`aZetidine acid, homoserine, cyclohexylglycine, alpha
`amino-n-butyric acid, cyclohexylalanine, aminophenylbu
`tyric acid, phenylalanine mono and di -substituted at the
`positions ortho, meta and para of the aromatic ring, O-alky
`lated derivatives of serine, threonine and tyrosine, S-alky
`lated cysteine, epsilon-alkylated lysine, delta-alkylated orni
`thine, aromatic amino acids, Substituted at the positions meta
`or para of the ring Such as phenylalanine-nitrate, -sulfate,
`-phosphate, -acetate, -carbonate, -methylsulfonate, -meth
`ylphosphonate, tyrosine-Sulfate, -phosphate, -sulfonate,
`-phosphonate, para-amido-phenylalanine, C-alpha,alpha-di
`alkylated, amino acids such as alpha,alpha-dimethylglycine
`(Aib), alpha-aminocyclopropane-carboxylic acid (Ac3c),
`alpha-aminocyclobutane-carboxylic acid (Ac4c), alpha-ami
`nocyclopentanecarboxylic acid (Ac5c), alpha-aminocyclo
`hexanecarboxylic acid (Acóc), diethylglycine (Deg), dipro
`pylglycine (Dpg), diphenylglycine (Dph). Examples of beta
`amino acids are beta-alanine (beta-Ala), cis and trans 2.3-
`diaminopropionic acid (Dap).
`Other non-protein amino acids are identified on the web
`50
`site http://CHFMLIBRARY BRI.NRC.CA/.
`Preferred compounds of formula (I) are those wherein:
`a) m is an integer between 4 and 8, n is an integer between
`3 and 5, and the Xaa amino acids include at least one
`methionine residue and one tyrosine residue in the
`Sulphonated or non-Sulphonated form;
`b) m is an integer between 4 and 8, n is an integer between
`3 and 5, and the Xaa amino acids include at least one
`methionine residue, one tyrosine residue in the Sulpho
`nated or non-Sulphonated form, and one lysine residue;
`c) m is an integer between 4 and 8, n is an integer between
`3 and 5, and the Xaa amino acids include at least one
`methionine residue, one tyrosine residue in the Sulpho
`nated or non-Sulphonated form, one lysine residue or
`one amino acid selected from ornithine, aspartic acid
`and glutamic acid.
`
`4
`Particularly preferred compounds are peptides of general
`formula (II):
`
`(II)
`
`wherein
`m is 4,
`n is an integer between 3 and 5;
`Xaa1 and/or Xaa2 may be absent;
`Xaa3 is Asp or Glu;
`Xaa-4 is Tyr or SO3H-Tyr;
`Xaa10 is Phe or an amino acid selected from Leu, Ile, Val,
`Ala, Trp, Gly and Pro.
`Even more particularly preferred are peptides of formula
`(III-VIII):
`
`-
`H-Glu-Tyr-Met-Dap-Trp-Met-Asp-Phe-Lys
`
`-
`H-Asp-Tyr-Met-Dap-Trp-Met-Asp-Phe-Lys
`
`(III)
`
`(IV)
`
`(V)
`-
`H-Asp-SO3HTyr-Met-Dap-Trp-Met-Asp-Phe-Lys
`
`(VI)
`-
`H-Asp-SO3HTyr-Met-Dap-Trp-Met-Asp-Val-Lys
`
`(VII)
`-
`H-Asp-SO3HTyr-Met-Dap-Trp-Met-Asp-Phe-Lys
`
`(VIII)
`-
`H-Gly-Ala-Glu-Tyr-Met-Dap-Trp-Met-Asp-Ala-Lys
`
`The compounds of the invention are synthetised by
`known techniques such as Solid-phase peptide synthesis,
`peptide synthesis in solution, organic chemistry synthesis
`methods, or any combination of those techniques. Synthesis
`methods based on appropriate combinations of Solid-phase
`techniques and conventional methods in Solution, which
`involve low production costs, especially on an industrial
`scale, will preferably be used. These methods involve solid
`phase synthesis of the peptide, including branching with the
`use of protected amino acids with orthogonal functions,
`possibly solid-phase conjugation of the macrocycle, cleav
`age of the protective peptide from the resin, Solution cycli
`sation in diluted concentrations, and purification of the
`compound.
`The compounds prepared according to general formula (I)
`can be labelled with radioactive or paramagnetic metals or
`radioactive halogens, either directly or using a chelating
`group.
`A further object of the present invention are the com
`pounds of general formula (IX):
`A-Y-C
`wherein
`A is a peptide of general formula (I);
`Z is an integer between 0 and 5;
`Y is a spacer chain respectively bonded to one of the
`functionalities present on the side chains of the individual
`amino acids present in peptide A, or to an N-terminal
`(—NH) group or a C-terminal (-COH) group of A, and
`to C; when Z is an integer between 2 and 5, units Y may be
`the same or different from each other;
`
`(DX)
`
`US 7,329,644 B2
`
`10
`
`15
`
`25
`
`30
`
`35
`
`40
`
`45
`
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`
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`
`65
`
`
`
`
`

`

`US 7,329,644 B2
`
`5
`C is a chelating agent, bonded covalently to spacer chain
`Y or directly to peptide A, or to more than one amino acid
`units of peptide A, which is able to complex a paramagnetic
`metal or a radioisotopes.
`Y is preferably a group of formula:
`
`11"r
`
`wherein
`r, 1, and q are each independently 0 or 1, and p can vary
`between 0 and 10, provided that at least one of 1, r and q is
`other than Zero;
`X is an O atom, or a NR group wherein R is an H atom
`or an alkyl group (C-Cs);
`K is a benzene nucleus, Substituted or non-substituted, or
`a —CHR group, wherein R is a hydrogen atom or a
`—COOH or - SOH group:
`W is a —CO— or —CS group.
`Preferred compounds of formula (IX) are those in which
`the spacer chains Y have the following formulae (X), (XI)
`and (XII).
`
`(X)
`
`10
`
`15
`
`25
`
`30
`
`6
`ethoxyphenyl)propyl-N-2-bis(carboxymethyl)aminoeth
`ylglycine (EOB-DTPA), N,N-bis(2-(carboxymethyl)(me
`thylcarbamoyl)methylaminoethyl-glycine (DTPA-BMA),
`2-methyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraace
`tic acid (MCTA), (C.O.'.O.".C.")-tetramethyl-1,4,7,10-tet
`raazacyclododecane-1,4,7,10-tetracetic acid (DOTMA); or
`is the residue of a polyaminophosphate acid ligand or
`derivatives thereof, in particular N,N'-bis-(pyridoxal-5-
`phosphate)ethylenediamine-N,N'-diacetic acid (DPDP) and
`ethylenedinitrilotetrakis(methylphosphonic) acid (EDTP);
`or is the residue of a polyaminophosphonic acid ligand and
`derivatives thereof, or polyaminophosphinic acid and
`derivatives thereof, in particular 1,4,7,10-tetraazacy
`clododecane-1,4,7,10-tetrakismethylene(methylphospho
`nic) acid and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tet
`rakismethylene-(methylphosphinic) acid; or is the residue
`of macrocyclic chelants such as texaphrines, porphyrins,
`phthalocyanines; or is N,N-bis(2-bis(carboxymethyl)
`aminoethyl L-glutamic acid (DTPA-GLU) or DTPA conju
`gated with Lys (DTPA-Lys).
`The cyclic and/or branched peptide A can be linked to the
`chelating group C, directly with a covalent bond between
`two functional groups of A and C, or by the spacer chain Y
`and this can be obtained, for example, with the acid groups
`of the ligand, or by a Suitable reactive group present in the
`starting ligand, for example an amino group, or a functional
`group present on a phenyl, etc.
`Particularly preferred reactive groups present on C or Y.
`are selected from the group consisting of —COH, -NH2,
`NCS,
`NHCSNHNH,
`NHCSNH(CH)NH,
`NCO, NHNH, -NHCONHNH, CHO.
`Particularly preferred are the compounds of formula (IX)
`in which C is a residue of the ligand DTPA, DO3A or DOTA.
`Further examples of chelating groups C, which can be
`used in particular as chelating agents for radionuclides (such
`as Tc, Re, Cu, Ga) are amino?amide thiol derivatives that can
`be represented by the general formula (XIII), wherein J is
`included within the range 0-2 and has generally unitary
`value.
`
`(Namino?amido). (S thiol derivative),
`(XIII)
`Preferred chelating groups of formula (XIII) include N.
`aminopropylene oximes, diaminedioximes, hydrazines, NS
`triamide monothiols, NS diamido dithiols, diamine dithi
`ols, monoamide monoaminedithiols and monoamine
`monoamidedithiols.
`A large number of applications of the chelating com
`pounds of formula (XIII), labelled with Technetium or
`Rhenium are known in literature.
`Preferred chelating agents C of metal ions and in particu
`lar of rhenium or technetium of formula (XIV a), (XIV b).
`(XIV c) and (XV) are disclosed in EP 629,617, EP544,412,
`U.S. Pat. No. 5,663,307 and U.S. Pat. No. 5,651,954,
`respectively, the content of which is incorporated herein by
`reference, in particular as far as the definition of the groups
`Q, R. R. G1, G2 and R1 are concerned.
`
`R
`
`Q
`1 N.
`1.
`
`H
`
`(XIV a)
`
`R
`
`R
`
`R
`R1SN
`OH
`
`N21 NR
`OH
`
`R
`
`N
`
`R
`
`R
`
`N
`
`CH
`
`p
`
`s
`
`O
`
`c
`
`Z
`
`CH
`
`p
`
`s
`
`!"
`
`p
`
`R
`
`O
`
`c
`
`z-l
`
`CH:
`
`p
`
`R
`
`N
`
`R
`
`CH2
`
`p W
`c
`
`(XI)
`
`35
`
`40
`
`(XII)
`
`45
`
`s
`
`z-2
`
`50
`Particularly preferred are the compounds (IX) in which Y
`represents one of the following groups:
`
`C preferably represents a chelating group selected from
`the group consisting of:
`a residue of a polyaminopolycarboxylic acid and the
`derivatives thereof, in particular selected from diethylenetri
`aminopentaacetic acid (DTPA), 1,4,7,10-tetraazacyclodode
`cane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7,10-tetraazacy
`clododecane-1,4,7-triacetic
`acid
`(DO3A),
`10-(2-
`hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-
`triacetic
`acid (HPDO3A), 4-carboxy-5,8,11
`-tris
`(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazatridecan-13
`oic acid (BOPTA), N-(2-bis(carboxymethyl)amino-3-(4-
`
`55
`
`60
`
`65
`
`
`
`
`

`

`7
`
`-continued
`QS /
`
`N 1N N
`H
`
`R
`R
`
`R
`
`R
`
`R
`R
`
`R
`
`R
`
`S
`
`S
`
`QS /
`N 1. N N
`
`S
`
`R
`
`S
`
`R
`
`R
`
`R
`
`R
`
`R
`
`R
`R
`
`R
`
`R
`
`R
`
`Q
`N 1. N R
`H
`R
`
`R
`R* n
`G1
`
`r R*
`G2
`
`A further example of a compound which can be used as
`a chelating group C of formula (XIII), is dimethylglycine
`L-serine-L-cysteinylglycinamide (XVI), which has proved
`to be of considerable interest and suitable for use as a
`bifunctionalised ligand for conjugation to peptides and pro
`teins and for complexing of the radionuclide "Tc or 'Re.
`Other derivatives which can be used as chelating agents C
`are disclosed in WO993.3863, the content of which is
`incorporated herein by reference.
`
`30
`
`35
`
`OH
`
`O
`
`H
`
`O
`
`D
`N S
`O
`ls l
`M/ Me
`
`N
`H
`
`Me
`
`O
`
`NH2
`
`(XVI)
`
`40
`
`45
`
`50
`
`55
`
`The functionalisation of the chelating groups of formula
`(XIII) enables them to be used as conjugates bonded with
`covalent bonds for a wide range of biologically active
`molecules.
`In particular, in the preparation of compounds of formula
`(IX) used in nuclear medicine diagnostics, chelating com
`pounds C are selected in Such a way as to obtain corre
`sponding stable complexes with radioisotopes that emit
`gamma, beta or positron radiation, using for example the
`radioisotopes of Tc, Re, T1, In, Cu, Ga, Rb or Y.
`The use of "Tc in nuclear medicine diagnostics is
`known, and it offers a number of advantages which make it
`one of the most commonly used radio nuclides. Its 6-hour
`half-life is short enough for the administration of a dose of 65
`radiation that provides high-quality images without risk to
`the patients health.
`
`60
`
`US 7,329,644 B2
`
`(XIVb)
`
`(XIV c)
`
`10
`
`15
`
`(XV)
`
`25
`
`8
`Numerous techniques have also been developed which
`enable Tc to be bonded to various molecules with biological
`activity, Such as antibodies, proteins and peptides.
`The binding between the molecule and the radionuclide is
`usually obtained by one of the following methods:
`a) direct binding of the radionuclide to the molecule con
`cerned, which is effected, for example, with the use of a
`reducing agent that reduces the disulphide bridges of a
`peptide or a protein to two hydroSulphide groups, which
`directly bind Tc(V):
`b) the use of a chelating agent C of the type described above,
`generally bifunctionalised, in which one functionality is
`used for the direct binding to the peptide (or to the
`compound, in accordance with general formula Y. conju
`gated to A) and the other is used for complexing with the
`radionuclide, which is performed before or after binding
`with the biologically active molecule, as the case may be
`(preformed-chelate or final-step-labelling method).
`The radioactive complex is prepared by methods
`described in the literature, such as by reaction of the
`functionalised chelating compound with a salt, in which
`Tc-99m pertechnetate is preferably used, in the presence of
`a suitable reducing agent.
`The reducing agents used include those reported in the
`literature. Such as dithionite, ferrous and Stannous ions (e.g.
`tartrate, chloride and fluoride), or other solid-state reducing
`agents.
`This type of complex is usually prepared with an inactive
`diagnostic kit, previously prepared under aseptic conditions,
`which contains a predefined amount of the compound con
`jugated with the chelating agent in the form of a freeze-dried
`powder, and of the reducing agent, both formulated in the
`presence of Suitable stabilising agents, Surfactants and/or
`buffers which can be used to prepare the pharmaceutical
`bulk product.
`The Solution containing the chelating agent is usually
`suitably formulated and subsequently distributed into vials
`which are freeze-dried and closed under nitrogen atmo
`sphere to ensure that the properties of the reducing agent
`(e.g. Stannous chloride) present in the composition are
`maintained.
`The inactive kits thus prepared are Subsequently recon
`stituted, for example with a sodium pertechnetate solution,
`to form the corresponding complexes with "Tc, which are
`used in radiological diagnostics for functional and morpho
`logical examinations of organs of the human body, and in
`particular, for the compounds of the invention, which are
`used for imaging of tumors that over-express cholecystoki
`nin receptors.
`The use of radioactive rhenium isotopes as an alternative
`to technetium isotopes has been proposed by Wong et al.
`(Wong, Inorganic Chemistry, vol. 36, 5799-5808, 1997),
`especially in the coordination state (V), and isotopes Re
`and 'Rehave proved to be of particular interest in nuclear
`medicine, having a large number of applications in radiop
`harmaceutical therapy.
`Another class of chelating compounds C, which are
`Suitable for conjugation with the compounds of the inven
`tion and can be used not only as chelators of paramagnetic
`metals (MRI) but also of radioactive metals (radiotherapy
`and radiodiagnostics), consists of polyazamacrocycles of
`formula (XVII). These chelating compounds contain at least
`one amine, thiol, carboxyl or carboxyl derivative group or a
`thiocarboxylate, present as free functionality and suitable for
`use in the conjugation reaction to spacer chain Y or peptide
`A, and according to the general formula (IX).
`
`
`
`
`

`

`US 7,329,644 B2
`
`10
`Compounds (I) possess particular characteristics which
`make them suitable for the purposes described above, in
`which:
`a) they take on a structure in Solution Such that they can
`interact specifically with type A and/or type B cholecys
`tokinin receptors, and have at least comparable affinity for
`CCK8:
`b) as a result of the presence of the cycle, they are particu
`larly stable to enzymatic degradation under physiological
`conditions;
`c) they take on a conformation Such that the presence of a
`chelating substituent does not interfere with binding to the
`receptor.
`Compounds of formula (IX) can be prepared by conven
`tional synthetic methods. In particular, a compound of
`formula (IX) can be obtained with convergent synthesis,
`which involves:
`1) Synthesis of a functionalised ligand, namely a ligandable
`to coordinate a paramagnetic metal ion or the isotope of
`a radioactive metal, which can also bind stably to the
`peptide, either directly or through a suitable functional
`group;
`2) synthesis of the peptide;
`3) coupling reaction between the two different synthons,
`including with the use of spacer unit Y:
`4) cleavage of any protective groups;
`5) complexing with a paramagnetic or radioactive metalion.
`The two synthons are conjugated by various known
`coupling methods widely used in synthesis (see, for
`example, Brinkley, M., Bioconjugate Chem. 1992, 3, 2),
`which involve for example the formation of an amide, a
`thiourea or an ester.
`Radioactive halogens used in therapy and diagnosis are
`known. For example, 'I is known for its use in imaging,
`while 'I can be used not only for imaging, but preferably
`in therapy. The bromine radionuclides Br and 'Br are
`used for diagnosis, while 77Br is used in radiotherapy. 'F
`and 'At are used in diagnosis and radiotherapy.
`If the radionuclide is an isotope of a radioactive halogen,
`it can be bonded directly to peptide (I) by reacting with a Trp
`or Tyr residue.
`The methods of labelling with iodine isotopes include not
`only direct iodination with oxidative methods, but also the
`nucleophilic Substitution reaction and the isotope exchange
`reaction. The choice of labelling method in this case depends
`on the structure of the precursor, the problems associated
`with the purification techniques, and the cost-effectiveness
`of the process used.
`An example of an indirect labelling method with radio
`active halogens which can be used with compounds of
`formula (I) is described in U.S. Pat. No. 5.290,937, incor
`porated herein by reference, which enables radio-labelled
`proteins of formula (XVIII) to be obtained:
`
`(XVIII)
`
`O
`
`A
`
`O-alkyl
`
`OH
`
`10
`
`15
`
`25
`
`30
`
`35
`
`For a complete description of the compounds of formula
`(XVII), see U.S. Pat. No. 6,093,382, which is incorporated
`herein by reference.
`Compounds (I) and (IX) can form chelates with the
`bi-trivalent ions of the metal elements having atomic num
`ber ranging between 20 and 31, 39, 42, 43, 44, 49, and
`between 57 and 83, with radioactive isotopes of metals or
`halogens (I, I,
`I, 7Br 7°Br, 7Br 77Br and Br) or
`paramagnetic metals ("Tc, Pb, 7 Ga, Ga., 7As, '''In,
`Illn, 90Y, 7Ru, 82Rb, 62Cu, Cu, 52Fe, 52"Mn, 140La,
`175Yb, 'Sim, 16Ho, 149Pm, 77Lu, 142Pr 15°Gd, 212Bi,
`*7Sc, 149Pm, 67Cu, '''Ag, 19 Au, 188Re, 186Re, 161Tb and
`'Cr), possibly in the form of salts with physiologically
`compatible bases or acids.
`Particularly preferred are the complexes with Fe(*).
`Fe(t), Cu(), Cr(t), Gd(*), Eu(), Dy(t), La(t), Yb(t)
`or Mn(*) or with radioisotopes such as 'Cr, 'Ga, Ga.
`In, 99mTc, 140La, 175Yb, Sm, 16Ho, 90Y. 149Pm, 77Lu,
`7Sc, “Pr, Gd and 21°Bi.
`Preferred cations of inorganic bases suitable for salifying
`the complexes of the invention comprise, in particular, alkali
`or alkaline-earth metal ions such as potassium, Sodium,
`calcium, magnesium.
`Preferred cations of organic bases comprise those of
`40
`primary, secondary and tertiary amines, such as ethanola
`mine, diethanolamine, morpholine, glucamine, N-methyl
`glucamine, N,N-dimethylglucamine.
`Preferred anions of inorganic acids comprise, in particu
`lar, the ions of halo acids such as chlorides, bromides,
`iodides or other ions such as Sulfate.
`Preferred anions of organic acids comprise those of acids
`conventionally used in pharmaceutical technique for the
`Salification of basic Substances, such as acetate. Succinate,
`citrate, fumarate, maleate, oxalate.
`Preferred cations and anions of amino acids comprise, for
`example, those of taurine, glycine, lysine, arginine or omith
`ine or of the aspartic and glutamic acids.
`Compounds (I) and (IX) are also useful as cholecystoki
`nin agonists or antagonists and, after labelling, either with
`the use of a chelating group or directly with radioactive or
`paramagnetic metals or radioactive halogens, as therapeutic
`and diagnostic agents to identify and locate primary human
`tumors and their metastases which over-express type A
`60
`and/or B cholecystokinin receptors. Binding to the receptor
`may be followed by a process of internalisation in the cells.
`In particular, compounds of formula (IX), labelled with
`paramagnetic metals, have proved to be useful contrast
`media for magnetic resonance, especially for imaging of
`animal tumour cells which over-express type A and/or type
`B cholecystokinin receptors.
`
`45
`
`50
`
`55
`
`65
`
`
`
`
`

`

`US 7,329,644 B2
`
`s
`
`s
`
`s
`
`12
`cystokinin receptor, responsible for interaction with the
`peptide hormone, is deposited in the Protein Data Bank
`(http://www.rcsb.org) with the code “pdb1D6G.ent'. The
`N-terminal part of the receptor (receptor filament) consists
`of 47 amino acid residues, and represents the extracellular
`N-terminal arm and the first part of transmembrane helix 1
`of the type A receptor.
`The study of the structure of the complex (fragment 1-47
`of type ACCK receptor and peptide CCK8), performed with
`NMR spectroscopy, and the Subsequent binding study per
`formed with fluorescence spectroscopy, were carried out
`with peptide CCK8 in the non-sulphated form.
`List of Abbreviations
`For the nomenclature and abbreviations of the amino
`acids, reference should be made to the recommendations of
`the