(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2007/0269375 A1
`(43) Pub. Date:
`Nov. 22, 2007
`Chen et al.
`
`US 2007026.9375A1
`
`(54) STABLE RADIOPHARMACEUTICAL
`COMPOSITIONS AND METHODS FOR
`PREPARATION
`
`(75) Inventors: Jianqing Chen, Bordentown, NJ (US);
`Karen E. Linder, Kingston, NJ (US);
`Edmund R. Marinelli, Lawrenceville,
`NJ (US); Edmund Metcalfe, Kingston,
`NJ (US); Adrian D. Nunn,
`Lamberville, NJ (US); Rolf E.
`Swenson, Princeton, NJ (US); Michael
`F. Tweedle, Princeton, NJ (US)
`Correspondence Address:
`KRAMER LEVN NAFTALIS & FRANKEL
`LLP
`INTELLECTUAL PROPERTY DEPARTMENT
`1177 AVENUE OF THE AMERICAS
`NEW YORK, NY 10036 (US)
`(73) Assignee: Bracco Imaging S.p.A., Milan (IT)
`(21) Appl. No.:
`10/566,112
`
`(22) PCT Filed:
`(86). PCT No.:
`
`Jul. 23, 2004
`PCT/USO4/23930
`
`S 371(c)(1),
`(2), (4) Date:
`
`Jul. 9, 2007
`
`Related U.S. Application Data
`(60) Provisional application No. 60/489,850, filed on Jul.
`24, 2003.
`
`Publication Classification
`
`(51) Int. Cl.
`(2006.01)
`A6II 5L/08
`(2006.01)
`A6II IO3/
`(2006.01)
`A6II IO3/30
`(2006.01)
`A6II IO3/36
`(2006.01)
`A6II IO3/40
`(2006.01)
`A6II 3L/98
`(52) U.S. Cl. ........................ 424/1.69: 424/1.65; 514/561
`
`(57)
`
`ABSTRACT
`
`Stabilized radiopharmaceutical formulations are disclosed.
`Methods of making and using stabilized radiopharmaceuti
`cal formulations are also disclosed. The invention relates to
`stabilizers that improve the radiostability of radiotherapeutic
`and radiodiagnostic compounds, and formulations contain
`ing them. In particular, it relates to stabilizers useful in the
`preparation and stabilization of targeted radiodiagnostic and
`radiotherapeutic compounds, and, in a preferred embodi
`ment, to the preparation and stabilization of radiodiagnostic
`and radiotherapeutic compounds that are targeted to the
`Gastrin Releasing Peptide Receptor (GRP-Receptor).
`
`
`
`AAA, Ex. 2003
`Evergreen Theragnostics, Inc. v. AAA SA
`PGR2021-0003
`Page 1 of 50
`
`

`

`Patent Application Publication Nov. 22, 2007 Sheet 1 of 11
`
`US 2007/026.9375 A1
`
`
`
`Fig. 1
`
`HN
`S1
`COOH
`?u?.
`o
`1.
`SR-A- ?' O ''', 's O
`NH,
`Hooc/&N) .
`l
`N
`N
`l
`HNO
`
`NH
`
`Fig. 2
`
`AAA, Ex. 2003
`Page 2 of 50
`
`

`

`Patent Application Publication Nov. 22, 2007 Sheet 2 of 11
`
`US 2007/026.9375 A1
`
`ADC1A, ADC Channal A(H:HPLCDA-20024DEC.20012.402B232COE).D)
`
`Radioactive
`
`mAU
`
`70
`
`50
`
`Fig. 3A
`
`
`
`Radioactive
`
`lu-177. A (Mel-O form)
`
`Fig. 3B
`
`AAA, Ex. 2003
`Page 3 of 50
`
`

`

`Patent Application Publication Nov. 22,2007 Sheet 3 of 11
`
`US 2007/0269375 Al
`
`*ADC4 A, ABC Channel A(HAHPLODA~1\2002\SEP™1 20012-10-02\82476001.D)
`*ADC4 A, ADC Channel A(HAHPLODA™1\2002\SEP~4,200\8-10-02\82375007.D)
`*ADC1 A, ADE Channel A(HAHPLODA~ 1\2002\8EP~4,20018-10-02\82475006.0)
`“AGT A, ADC Channel A(HAHPLODA~1Q002\SEP~1 20010-D2182375009.D)
`“ADC A, AGE Channel ACHAHPLODA~1Q00NSEPe4 2O0N0.10-02\B2978010.3
`CP=99.9%
`
`RCP=99.3%
`
`mAU
`
`
`
`
`
`
`
`
`
`Fig. 4
`
`AAA,Ex. 2003
`Page 4 of 50
`
`AAA, Ex. 2003
`Page 4 of 50
`
`

`

`Patent Application Publication Nov. 22, 2007 Sheet 4 of 11
`
`US 2007/0269375 A1
`
`"ADCA, Adc channel ACHAHPLCEArt 2002 Nove.2001:26-028237200d.
`"ADC1A, ADC Channel ACH:HPLCDA-120C2ANOVa1.2C011-27-02s2372001.D)
`*ADC1A, ACC Channel A(H:HFLCQAmv2)32.Ng1,2011-27.283720ga.)
`"At
`A. AEC channel AH HPLCDAvaO24Novacos.11-27-282372,337.t
`'Abc1A, ADC charact is estiflict A ty2002\to\ietzctii.27-(2,237:38.ts)
`100%
`
`99.9%
`98.4%
`
`97.5%
`96.4%
`
`
`
`mA
`soo
`
`Fig. 5
`
`AAA, Ex. 2003
`Page 5 of 50
`
`

`

`Patent Application Publication Nov. 22, 2007 Sheet 5 of 11
`
`US 2007/0269375 A1
`
`
`
`Fig. 6A
`
`Fig. 6B
`
`AAA, Ex. 2003
`Page 6 of 50
`
`

`

`Patent Application Publication Nov. 22, 2007 Sheet 6 of 11
`
`US 2007/026.9375 A1
`
`
`
`ADC A, ADC Channel A. (H:HPLCDArut 20.2%OC Tru?.2001-3-2B23720g.
`
`Methionire
`
`yet) form
`
`O
`5
`5
`3
`ADC1A, ADC Channal A(H:HPLCDAru?t 2002, CT. 1.2000-3-12B2372020.D)
`
`Seleromethioire
`
`2O
`5
`ADC1A, ADC Channel A (H:HPLCD Aru 12002 AU Gru 1. 208-g-O2.48 HSTAO1.)
`
`Citro if FES
`
`Fig. 7A
`
`AAA, Ex. 2003
`Page 7 of 50
`
`

`

`Patent Application Publication Nov. 22, 2007 Sheet 7 of 11
`
`US 2007/0269375 A1
`
`ADC1A, ADC Channel A(H:HPLCDArul 2002 AUGru 1208-g-O248 HSTAD5.D)
`Methionine
`
`TAU
`O
`4)
`2O
`
`5
`5
`ADC A, ADC Channel ACH:HPLCDA 1202.) ULY217-12-02'BRL3BE07.D)
`TAU Cysteine MetR) for
`d
`
`
`
`O
`
`2O
`5
`ADC A, AEC Charine A (H:HPLC Art'22'Ajn. 28-g-O2'ASH STAD4.D)
`T.All Tryptophar
`
`4)
`
`2
`
`TA
`
`2O
`
`5
`5
`ABC1 A, ADC Channa A. (H:HPLCDAr. 12002 Augrat. 208-a-024BHSTA3.)
`Histidire
`
`2.
`5
`O
`ADCi A, ADC Channel A (H:HPLCDAraf 2032AJGr. 208-g-O2.48 HSTAD2, D)
`"AUGlysine
`
`OO
`5O
`
`Fig. 7B
`
`AAA, Ex. 2003
`Page 8 of 50
`
`

`

`Patent Application Publication Nov. 22, 2007 Sheet 8 of 11
`
`US 2007/026.9375 A1
`
`ADC1A, ADC Channel A (H:HPLCDAru i2002DECrui.2GO's 12-4-O25B??720O.D)
`Lu-liff A
`
`MetRO form
`
`O
`ADC1A, ADC Channel A (H:HPLCDAru 12002DECn 1.2OO'12-5-2B2372002.D)
`
`TA
`O)
`
`d
`
`OO
`
`TAU
`
`3O)
`
`O
`
`1.
`
`2O
`5
`i
`5
`O
`ADCfA, ADC Charinal A. H:HPLCDAru 2002DECru 1.2012-5-O2B2372O5.D)
`
`TAJ
`1548-h
`
`2
`5
`?
`AECIA, ADC Channel A. (H:HPLC (Aru 202DECn 1.200 12-g-O2B232G1.D)
`
`
`
`TAU
`
`O
`
`B
`
`O
`
`Fig. 8
`
`AAA, Ex. 2003
`Page 9 of 50
`
`

`

`Patent Application Publication Nov. 22, 2007 Sheet 9 of 11
`
`US 2007/0269375 A1
`
`ADC1A, ADC Channel A (H:'HPLCDA 12002SEP.1.2000-4-02'B2375001.D)
`
`TAL
`
`
`
`BOO
`d
`
`2
`
`Mt-O for
`
`LL-E.
`N
`
`2)
`5
`5
`ADC1A, ADC Channel A (H:HPLCDAuf 2002SEPn 1.2009-4-2B2350O3.D)
`
`TAL
`
`O
`
`O 24-h
`
`4OO
`
`2.
`
`-
`
`15
`5
`ADC A, ADC Channel A (H:\HPLCDA 12002SEP.1.2000-4-02'B2375O15.D)
`
`4B-h
`
`TA
`
`al
`
`2
`
`2
`5
`O
`5
`O
`ADC A, ADC Channel A (H:HPLCDA. i2O2SEPnt.2009-4-O2B23,750 .D)
`
`TAU
`
`4. 2
`
`3.
`2O
`
`5
`
`5
`
`Fig. 9
`
`AAA, Ex. 2003
`Page 10 of 50
`
`

`

`Patent Application Publication Nov. 22, 2007 Sheet 10 of 11
`
`US 2007/0269375 A1
`
`WRD 1 A, travelength=2a) nn (T:EX2002.SFO30?-4031292.D)
`UW trace G280mm
`
`Compound B (free ligand)
`retention time = 15.4 mil.
`
`All
`8
`7
`
`A.
`3
`
`2
`t
`
`2.5
`5
`s
`13
`12.5
`is
`5
`UV chromatogram: COMPOUND B reference standard, retention time = 15.4 min.
`Fig. 10A
`
`min
`
`
`
`
`
`TAU
`
`ADC1A, ADC CHANNEL.A. (T:EX2002\SFO301-4NO3G 1204.D)
`Gama trace
`Sample 1
`
`Free LU-177 (EDTA-Lul 77 complex)
`
`f
`O
`widt A. ?lavis length=280 nrn (T:EX2002's Fig301-4vaggi2Q04.D)
`UW trace G.28Onm
`Sample 1
`r Lu-177 (EDTA-Lu177 complex)
`Compound B-Zinc Complex
`Compound B free ligand
`Compound B-Lu177 compe
`
`
`
`
`
`
`
`15
`
`2
`
`mir
`
`Radiochromatogram (top) and UV chromatogram (bottom) of Sample 1 (NaOAe buffer
`control); RCP = 0%
`
`5
`
`Thin
`
`s
`
`Fig. 10B
`
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`

`

`Patent Application Publication Nov. 22, 2007 Sheet 11 of 11
`
`US 2007/026.9375 A1
`
`ADC1 A, ADC?. CHANNEL A. (T:\EX2002.SFO3(1-403012go3.D)
`Gamma trace
`Sample 2
`Compound B-Lu177 complex
`
`All
`
`B
`
`OO
`
`a.
`
`OO
`
`IAU
`al
`
`3
`
`5
`WD1A, Wavelength-280 nm (T:EX520O2SFO3D1-4NO3(12903.D)
`UW trace G.28Onm
`Sample 2
`EDTA
`Compound B-Luff complex
`Compound B free ligand
`
`s
`
`
`
`nir
`20
`Com Ound B-finC
`complex
`
`5
`O
`is
`2
`Inin
`Radiochromatogram (top) and UV chromatogram (bottom) of Sample 2 (containing
`1-Pyrrollidinecarbodithioic acid ammonium salt in NaOAc buffer); RCP = 100%
`
`Fig. 10C
`
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`

`

`US 2007/0269375 A1
`
`Nov. 22, 2007
`
`STABLE RADIOPHARMACEUTICAL
`COMPOSITIONS AND METHODS FOR
`PREPARATION
`
`0006. Some of the immediate products that form from the
`radiolysis of water are outlined below.
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`0001. This application claims benefit of U.S. Provisional
`Application No. 60/489,850 filed Jul. 24.2003, which is
`hereby incorporated by reference in its entirety.
`
`FIELD OF THE INVENTION
`0002 This invention relates to stabilizers that improve
`the radioStability of radiotherapeutic and radiodiagnostic
`compounds, and formulations containing them. In particular,
`it relates to stabilizers useful in the preparation and stabili
`Zation of targeted radiodiagnostic and radiotherapautic com
`pounds, and, in a preferred embodiment, to the preparation
`and Stabilization of radiodiagnostic and radiotherapeutic
`compounds that are targeted to the Gastin Releasing Peptide
`Receptor (GRP-Receptor).
`
`BACKGROUND OF THE INVENTION
`0003 Radiolabeled compounds designed for use as
`radiodiagnostic agents are generally prepared with a
`gamma-emitting isotope as the radiolabel. These gamma
`photons penetrate water and body tissues readily and can
`have a range in tissue or air of many centimeters. In general,
`Such radiodiagnostic compounds do not cause significant
`damage to the organ systems that are imaged using these
`agents. This is because the gamma photons given off have no
`mass or charge and the amount of radioactive material that
`is injected is limited to the quantity required to obtain a
`diagnostic image, generally in the range of about 3 to 50
`mCi, depending on the isotope and imaging agent used. This
`quantity is Small enough to obtain useful images without
`significant radiation dose to the patient. Radionuclides Such
`as "Tc, '''In
`I, 7Ga and Cu have been used for this
`purpose.
`0004. In contrast, radiolabeled compounds designed for
`use as radiotherapeutic agents are generally labeled with an
`Auger-, beta- or an alpha-emitting isotope, which may
`optionally also give off gamma photons. Radionuclides Such
`aS 90Y. 77Lu, 14°Pm, 'Sim, 109Pd, 67Cu, 166Ho, 131I. 32p,
`86/8Re, 103Rh, 21 At, 225Ac, 7Sc,
`Bi, and others, are
`potentially useful for radiotherapy. The +3 metal ions of the
`lanthanide isotopes are of particular interest, and include
`'77Lu (relatively low energy B-emitter), ''Pm, 'Sm
`(medium energy) and 'Ho (high energy). 'Yalso forms a
`+3 metal ion, and has coordination chemistry that is similar
`to that of the lanthanides. The coordination chemistry of the
`lanthanides is well developed and well known to those
`skilled in the art.
`0005 The ionizing radiation given off from compounds
`labeled with these radioisotopes is of an appropriate energy
`to damage cells and tissue in sites where the radiolabelled
`compound has localized. The radiation emitted can either
`damage cellular components in the target tissue directly, or
`can cause water in tissues to form free radicals. These
`radicals are very reactive and can damage proteins and
`DNA
`
`0010. Of the products that form, (e.g. H", OH, H*, and
`OH), the hydroxyl radical OHis particularly destructive.
`This radical can also combine with itself to form hydrogen
`peroxide, which is a strong oxidizer.
`0011 OH*+OH*->HO (strong oxidizer)
`0012. In addition, interaction of ionizing radiation with
`dissolved oxygen can generate very reactive species such as
`Superoxide radicals. These radicals are very reactive towards
`organic molecules (see, e.g. Garrison, W. M., Chem. Rev.
`1987, 87,381-398).
`0013 Production of such reactive species at the site or
`sites that the radiotherapeutic or radiodiagnostic compound
`is targeted to (e.g., a tumor, bone metastasis, blood cells or
`other targeted organ or organ system) will, if produced in
`Sufficient quantity, have a cytostatic or cytotoxic effect. The
`key factor for successful radiotherapy is the delivery of
`enough radiation dose to the targeted tissue (e.g., tumor
`cells, etc.) to cause a cytotoxic or tumoricidal effect, without
`causing significant or intolerable side effects. Similarly, for
`a radiodiagnostic, the key factor is delivery of Sufficient
`radiation to the target tissue to image it without causing
`significant or intolerable side effects.
`0014 Alpha particles dissipate a large amount of energy
`within one or two cell diameters, as their range of penetra
`tion in tissues is only ~50 um. This can cause intense local
`damage, especially if the radiolabeled compound has been
`internalized into the nucleus of the cell. Likewise, radio
`therapeutic compounds labeled with Auger-electron emitters
`Such as 'In have a very short range and can have potent
`biological-effects at the desired site of action. The emissions
`from therapeutic beta-emitting isotopes such as 'Lu or 'Y
`have somewhat longer ranges in tissue, but again, most of
`the damage produced occurs within a few millimeters or
`centimeters from the site of localization.
`0015. However, the potentially destructive properties of
`the emissions of a radiotherapeutic isotope are not limited to
`their cellular targets. For radiotherapeutic and radiodiagnos
`tic compounds, radiolytic damage to the radiolabeled com
`pound itself can be a serious problem during the preparation,
`purification, storage and/or shipping of a radiolabeled radio
`therapeutic or radiodiagnostic compound, prior to its
`intended use.
`0016 Such radiolytic damage can cause, for example,
`release of the radioisotope e.g., by dehalogenation of radio
`iodinated antibodies or decomposition of the chelating moi
`ety designed to hold the radiometal, or it can damage the
`targeting molecule that is required to deliver the targeted
`agent to its intended target. Both types of damage are highly
`undesirable as they can potentially cause the release of
`unbound isotope, e.g., free radioiodine or unchelated radio
`metal to the thyroid, bone and other organs, or cause a
`decrease or abolishment of targeting ability as a result of
`radiolytic damage to the targeting molecule. Such as a
`receptor-binding region of a targeting peptide or radiola
`
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`

`US 2007/0269375 A1
`
`Nov. 22, 2007
`
`beled antibody. Radioactivity that does not become associ
`ated with its target tissue may be responsible for unwanted
`side effects.
`0017 For example, DOTA-Gly-ACA-Gln-Trp-Ala-Val
`Gly-His-Leu-Met-NH.
`(ACA=3-Amino-3-deoxycholic
`acid) and DOTA-Gly-Abz4-Gln-Trp-Ala-Val-Gly-His-Leu
`Met-NH. (Abz4=4-aminobenzoic acid) the two chelating
`ligands shown in FIGS. 1 and 2, respectively, have been
`shown to specifically target the Gastrin Releasing Peptide
`(GRP) Receptors. In the examples that follow, these have
`been described as Compounds A and Compound B respec
`tively. Other GRP receptor-binding ligands are described in
`U.S. Pat. No. 6,200,546, to Hoffmnan et al., published U.S.
`application U.S. 2002/0054855, and in copending applica
`tion Ser. No. 10/341,577, filed Jan. 13, 2003, the entire
`contents of which are incorporated by reference.
`0018 When radiolabeled with diagnostic and radiothera
`peutic radionuclides such as 'In and '77Lu, Compounds A
`and B have been shown to have high affinity for GRP
`receptors, both in vitro and in vivo. However, these com
`pounds can undergo significant radiolytic damage that is
`induced by the radioactive label if these radiolabeled com
`plexes are prepared without concomitant or Subsequent
`addition of one or more radioStabilizers (compounds that
`protect against radiolytic damage). This result is not Sur
`prising, as the hydroxyl and Superoxide radicals generated
`by the interaction of B-particles with water are highly
`oxidizing. Radiolytic damage to the methionine (Met) resi
`due in these peptides is the most facile mode of decompo
`sition, possibly resulting in a methionine Sulfoxide deriva
`tive.
`Cell binding results show that the resulting radi
`0.019
`olytically damaged derivatives are devoid of GRP-receptor
`binding activity (ICso values greater than micromolar).
`Hence, it is critical to find inhibitors of radiolysis that can be
`used to prevent both methionine oxidation and other radi
`olytic decomposition routes in radiodiagnostic and radio
`therapeutic compounds.
`0020 Preventing such radiolytic damage is a major chal
`lenge in the formulation of radiodiagnostic and radiothera
`peutic compounds. For this purpose, compounds known as
`radical scavengers or antioxidants are typically used. These
`are compounds that react rapidly with, e.g., hydroxyl radi
`cals and Superoxide, thus preventing them from reaction
`with the radiopharmaceutical of interest or reagents for its
`preparation.
`0021. There has been extensive research in this area.
`Most of it has focused on the prevention of radiolytic
`damage in radiodiagnostic formulations, and several radical
`Scavengers have been proposed for Such use. However, it has
`been found in the studies described herein that the stabilizers
`reported to be effective by others, provide insufficient radio
`stabilization to protect '77Lu-A and '77Lu-B, the Lutetium
`complexes of Compounds A and B, respectively, from
`radiolytic damage, especially when high concentrations and
`large amounts of radioactivity are used.
`0022. For example, Cyr and Pearson Stabilization of
`radiopharmaceutical compositions using hydrophilic thioet
`hers and hydrophilic 6-hydroxy chromans. Cyr, John E.;
`Pearson, Daniel A. (Diatide, Inc., USA). PCT Int. Appl.
`(2002), WO 200260491 A2 20020808 state that diagnostic
`
`and therapeutic radiopharmaceutical compositions radiola
`beled with 125I, II, At, 7Sc, 7Cu, 7°Ga, OY, SSm,
`15°Gd, 165Dy, 16Ho, 175Yb, 77Lu, 212Bi, 213Bi,
`Ga,
`"Tc, '''In and 'I can be stabilized by the addition of a
`hydrophilic thioether, and that the amino acid methionine, a
`hydrophilic thioether, is especially useful for this purpose.
`0023. A study was therefore performed wherein L-me
`thionine (5 mg/mL) was added to ''Lu-A, to evaluate its
`ability to serve as a radical scavenger. As will be described
`in more detail below, reverse phase HPLC shows that after
`five days, almost complete decomposition of '77Lu-A had
`occurred, indicating that the radioStabilizer used was insuf
`ficient to prevent radiolytic damage. In vitro binding results
`indicate that Such decomposition can dramatically decrease
`the potency and targeting ability, and hence the radiothera
`peutic efficacy, of the compound thus damaged. To attain the
`desired radiotherapeutic effect, one would need to inject
`more radioactivity, thus increasing the potential for toxicity
`to normal organs.
`0024. In order to identify suitable antioxidant radical
`scavengers that might be useful for the radiostabilization of
`GRP-receptor binding radiodiagnostic and radiotherapeutic
`compounds, several studies were performed. One or more
`potential radiostabilizers was added after complex formation
`(a two-vial formulation) or they were added directly to the
`reaction mixture prior to complexation with a radiometal (or
`both). Ideally, the radiostabilizer should be able to be added
`directly to the formulation without significantly decreasing
`the radiochemical purity (RCP) of the product, as such a
`formulation has the potential to be a single-vial kit.
`0025 The radical scavengers identified as a result of
`these studies have general utility in formulations for the
`preparation of compounds used for a variety of radiodiag
`nostic and radiotherapeutic applications, and may be useful
`to stabilize compounds radiolabeled with a variety of iso
`topes, e.g., 99mTc, 186 188Re,
`In, 90y, 77Lu, 213Bi, 225Ac,
`Ho and others. The primary focus of the examples in this
`application is the radiostabilization of GRP-binding pep
`tides, and in particular, the radioprotection of methionine
`residues in these molecules. However, the stabilizers iden
`tified should have applicability to a wide range of radiola
`beled peptides, peptoids, Small molecules, proteins, antibod
`ies, and antibody fragments and the like. They are useful for
`the radioprotection of any compound that has a residue or
`residues that are particularly sensitive to radiolytic damage,
`Such as, for example, tryptophan (oxidation of the indole
`ring), tyrosine (oxidative dimerization, or other oxidation),
`histidine, cysteine (oxidation of thiol group) and to a lesser
`extent serine, threonine, glutamic acid, and aspartic acid.
`Unusual amino acids commonly used in peptides or drugs
`that contain sensitive functional groups (indoles, imidazoles,
`thiazoles, furans, thiophenes and other heterocycles) could
`also be protected.
`
`SUMMARY OF THE INVENTION
`It is the aim of this invention to provide stabilizers
`0026.
`and stabilizer combinations that slow or prevent radiolytic
`damage to targeted radiotherapeutic and radiodiagnostic
`radiolabeled compounds, especially compounds labeled
`with radiometals, and thus preserve the targeting ability and
`specificity of the compounds. It is also an aim to present
`formulations containing these stabilizers. As described by
`
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`Page 14 of 50
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`

`

`US 2007/0269375 A1
`
`Nov. 22, 2007
`
`the examples below, many stabilizers have been identified
`that, alone or in combination, inhibit radiolytic damage to
`radiolabeled compounds. At this time, four approaches are
`particularly preferred. In the first approach, a radiolysis
`stabilizing solution containing a mixture of the following
`ingredients is added to the radiolabeled compound imme
`diately following the radiolabeling reaction: gentisic acid,
`ascorbic acid, human serum albumin, benzyl alcohol, a
`physiologically acceptable buffer or salt solution at a pH of
`about 4.5 to about 8.5, and one or more amino acids selected
`from methionine, selenomethionine, selenocysteine, or cys
`teine).
`0027. The physiologically acceptable buffer or salt solu
`tion is preferably selected from phosphate, citrate, or acetate
`buffers or physiologically acceptable sodium chloride solu
`tions or a mixture thereof, at a molarity of from about 0.02M
`to about 0.2M. The reagent benzyl alcohol is a key compo
`nent in this formulation and serves two purposes. For
`compounds that have limited Solubility, one of its purposes
`is to solubilize the radiodiagnostic or radiotherapeutic tar
`geted compound in the reaction Solution, without the need
`for added organic solvents. Its second purpose is to provide
`a bacteriostatic effect. This is important, as Solutions that
`contain the radiostabilizers of the invention are expected to
`have long post-reconstitution stability, so the presence of a
`bacteriostat is critical in order to maintain sterility. The
`amino acids methionine, selenomethionine, cysteine, and
`selenocysteine play a special role in preventing radiolytic
`damage to methionyl residues in targeted molecules that are
`stabilized with this radiostabilizing combination.
`0028. In the second approach, stabilization is achieved
`via the use of dithiocarbamate compounds having the fol
`lowing general formula:
`
`wherein R1 and R2 are each independently H, C1-C8 alkyl,
`—OR3, wherein R3 is C1-C8 alkyl, or benzyl (Bn) (either
`unsubstituted or optionally substituted with water solubiliz
`ing groups),
`or wherein R1R2N combined=1-pyrrolidinyl-, piperidino
`morpholino-, 1-piperazinyl- and M=H, Na', K", NH,
`M-methylglucamine, or other pharmaceutically acceptable
`+1 ions.
`0029. Alternatively, compounds of the form shown below
`may be used, wherein M is a physiologically acceptable
`metal in the +2 oxidation state, such as Mg" or Ca", and
`R1 and R2 have the same definition as described above.
`
`0030 These reagents can either be added directly into
`reaction mixtures during radiolabeled complex preparation,
`or added after complexation is complete, or both.
`0031. The compound 1-Pyrrolidine Dithiocarbamic Acid
`Ammonium salt (PDTC) proved most efficacious as a sta
`bilizer, when either added directly to the reaction mixture or
`added after complex formation. These results were unex
`pected, as the compound has not been reported for use as a
`stabilizer for radiopharmaceuticals prior to these studies.
`Dithiocarbamates, and PDTC in particular, have the added
`advantage of serving to Scavenge adventitious trace metals
`in the reaction mixture.
`0032. In the third approach, formulations contain stabi
`lizers that are water Soluble organic selenium compounds
`wherein the selenium is in the oxidation state +2. Especially
`preferred are the amino acid compounds selenomethionine,
`and selenocysteine and their esters and amide derivatives
`and dipeptides and tripeptides thereof, which can either be
`added directly to the reaction mixture during radiolabeled
`complex preparation, or following complex preparation. The
`flexibility of having these stabilizers in the vial at the time
`of labeling or in a separate vial extends the utility of this
`invention for manufacturing radiodiagnostic or radiothera
`peutic kits.
`0033. It is highly efficacious to use these selenium com
`pounds in combination with sodium ascorbate or other
`pharmaceutically acceptable forms of ascorbic acid and its
`derivatives.
`0034. The ascorbate is most preferably added after com
`plexation is complete. Alternatively, it can be used as a
`component of the stabilizing formulation described above. A
`fourth approach involves the use of water soluble sulfur
`containing compounds wherein the Sulfur in the +2 oxida
`tion state. Preferred thiol compounds include derivatives of
`cysteine, mercaptothanol, and dithiol threotol. These
`reagents are particularly preferred due to their ability to
`reduce oxidized forms of methionine residues (e.g.,
`methionine oxide residues) back to methionyl residues, thus
`restoring oxidative damage that has occurred as a result of
`radiolysis. With these thiol compounds, it is highly effica
`cious to use these stabilizing reagents in combination with
`Sodium ascorbate or other pharmaceutically acceptable
`forms of ascorbic acid and its derivatives. The ascorbate is
`most preferably added after complexation is complete.
`0035. The stabilizers and stabilizer combinations may be
`used to improve the radiolytic stability of targeted radiop
`harmaceuticals, comprising peptides, non-peptidic Small
`molecules, radiolabeled proteins, radiolabeled antibodies
`and fragments thereof. These stabilizers are particularly
`useful with the class of GRP-binding compounds described
`herein.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`0036 FIG. 1 shows the structure of Compound A.
`0037 FIG. 2 shows the structure of Compound B.
`0038 FIG.3 illustrates the results of an HPLC analysis of
`a mixture of ''Lu-A with 2.5 mg/mL L-Methionine over 5
`days at room temperature at a radioconcentration of 25
`mCi/mL. 50 mCi total). FIG. 3A is a radiochromatogram of
`a reaction mixture for the preparation of 'Lu-A, which was
`
`AAA, Ex. 2003
`Page 15 of 50
`
`

`

`US 2007/0269375 A1
`
`Nov. 22, 2007
`
`initially formed in >98% yield. FIG. 3B is radiochromato
`gram of '77Lu-A), 25 mCi/mL, after five days at room
`temperature, demonstrating complete radiolytic destruction
`of the desired compound. The radiostabilizer added (5
`mg/mL L-Methionine) was clearly insufficient for the level
`of radioprotection required.
`0039 FIG. 4 is an HPLC trace radiodetection showing
`that '77Lu-B (104 mCi) has >99% RCP for 5 days when
`diluted 1:1 with radiolysis protecting solution that was
`added after the complex was formed.
`0040 FIG. 5 is an HPLC trace radiodetection showing
`that '77Lu-A has >95% RCP for 5 days at a concentration of
`55 mCi/2 mL if 1 mL of radiolysis protecting solution is
`added after the complex was formed.
`0041 FIG. 6A and FIG. 6B show the structure of the
`methionine sulfoxide derivative of 'Lu-A (FIG. 6A) and
`methionine sulfoxide derivative of '''In-B (FIG. 6B).
`0042 FIG. 7A and FIG. 7B show stabilizer studies
`'77Lu-A (FIG. 7A) and '77Lu-B (FIG. 7B). Radioactivity
`traces are shown from a study to compare the radioStabiliz
`ing effect of different amino acids, when added to '77Lu-A
`(FIG. 7A) and ''Lu-B (FIG. 7B) at an amino acid concen
`tration of 6.6 mg/mL in 10 mM Dulbecco's phosphate
`buffered saline, pH 7.0 PBS), and a radioactivity concen
`tration of 20 mCi/mL, after 48 hours of storage at room
`temperature. A total of 3.5 mCi of 77Lu was added to each
`vial. A full description of the experimental procedure is
`given in Example 1.
`0043 FIG. 8 shows an HPLC trace radiodetection
`showing the radiostability of '77Lu-A over 5 days at room
`temperature at a radioconcentration of 25 mCi/mL in pres
`ence of 2.5 mg/mL L-methionine (50 mCi total). The details
`of this study are given in Example 2.
`0044 FIG. 9 shows an HPLC trace radiodetection
`showing the stability of 'Lu-B at a concentration of 50
`mCi/2 mL in a radiolysis protecting Solution containing
`L-methionine. The details of this study are given in Example
`4.
`004.5 FIGS. 10A-C show radiochromatograms and UV
`chromatograms comparing samples with and without 1-pyr
`rolidine dithiocarbamic acid ammonium salt in the reaction
`buffer and containing Zinc as a contaminant metal during the
`reaction of '77Lu-B. The experimental procedure for this
`study is given in Example 20.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`In the following description, various aspects of the
`0046.
`present invention will be further elaborated. For purposes of
`explanation, specific configurations and details are set forth
`in order to provide a thorough understanding of the present
`invention. However, it will also be apparent to one skilled in
`the art that the present invention may be practiced without
`the specific details.
`0047. Furthermore, well known features may be omitted
`or simplified in order not to obscure the present invention.
`
`1. Metal Chelator
`0.048. In some radiopharmaceuticals, the isotope is a
`non-metal, such as
`I, "'I or 'F, and is either coupled
`
`directly to the rest of the molecule or is bound to a linker.
`However, if the radioisotope used is a metal, it is generally
`incorporated into a metal chelator. The term “metal chelator
`refers to a molecule that forms a complex with a metal atom.
`For radiodiagnostic and radiotherapeutic applications it is
`generally preferred that said complex is stable under physi
`ological conditions. That is, the metal will remain com
`plexed to the chelator backbone in vivo. In a preferred
`embodiment, a metal chelator is a molecule that complexes
`to a radionuclide metal to form a metal complex that is stable
`under physiological conditions and which also has at least
`one reactive functional group for conjugation with a target
`ing molecule, a spacer, or a linking group, as defined below.
`The metal chelator M may be any of the metal chelators
`known in the art for complexing a medically useful metalion
`or radionuclide. The metal chelator may or may not be
`complexed with a metal radionuclide. Furthermore, the
`metal chelator can include an optional spacer Such as a
`single amino acid (e.g., Gly) which does not complex with
`the metal, but which creates a physical separation between
`the metal chelator and the linker.
`0049. The metal chelators of the invention may include,
`for example, linear, macrocyclic, terpyridine, and NS,
`NS, or N. chelators (see also, U.S. Pat. No. 4,647.447.
`U.S. Pat. No. 4,957,939, U.S. Pat. No. 4,963,344, U.S. Pat.
`No. 5,367,080, U.S. Pat. No. 5,364,613, U.S. Pat. No.
`5,021,556, U.S. Pat. No. 5,075,099, U.S. Pat. No. 5,886,142,
`the disclosures of which are incorporated by reference
`herein in their entirety), and other chelators known in the art
`including, but not limited to, HYNIC, DTPA, EDTA,
`DOTA, TETA, and bisamino bisthiol (BAT) chelators (see
`also U.S. Pat. No. 5,720.934). For example, macrocyclic
`chelators, and in particular Na chelators are described in U.S.
`Pat. Nos. 4,885,363; 5,846,519; 5,474,756; 6,143,274;
`6,093,382; 5,608, 110; 5,665,329; 5,656,254; and 5,688,487,
`the disclosures of which are incorporated by reference
`herein in their entirety. Certain NS chelators are described
`in PCT/CA94/00395, PCT/CA94/00479, PCT/CA95/00249
`and in U.S. Pat. Nos. 5,662,885; 5,976,495; and 5,780,006,
`the disclosures of which are incorporated by reference
`herein in their entirety. The chelator may also include
`derivatives of the chelating ligand mercapto-acetyl-glycyl
`glycyl-glycine (MAG3), which contains an NS, and NS
`systems such as MAMA (monoamidemonoaminedithiols),
`DADS (NS diaminedithiols), CODADS and the like. These
`ligand systems and a variety of others are described in Liu
`and Edwards, Chem Rev. 1999, 99, 2235-2268; Caravan et
`al., Chem. Rev. 1999,99, 2293-2352; and references therein,
`the disclosures of which are incorporated by reference
`herein in their entirety.
`0050. The metal chelator may also include complexes
`known as boronic acid adducts of technetium and rhenium
`dioximes, such as those described in U.S. Pat. Nos. 5,183,
`653: 5,387.409; and 5,118,797, the disclosures of which are
`incorporated by reference herein, in their entirety.
`0051
`Examples of preferred chelators include, but are
`not limited to, derivatives of diethylenetriamine pentaacetic
`acid (DTPA), 1,4,7,10-tetraazacyclotetradecane-1,4,7,10
`tetraacetic acid (DOTA), 1-substituted 14.7-tricarboxym
`ethyl 1,4,7,10 tetraazacyclododecane triacetic acid (DO3A),
`derivatives of the 1-1-(1-carboxy-3-(p-nitrophenyl)propyl
`1,4,7,10 tetraazacyclododecane triacetate (PA-DOTA