1111111111111111 IIIIII IIIII 111111111111111 11111 111111111111111 IIIII IIIII IIIIII IIII 11111111
`US 20120065365AI
`
`(19) United States
`c12) Patent Application Publication
`Chen et al.
`
`c10) Pub. No.: US 2012/0065365 Al
`Mar. 15, 2012
`(43) Pub. Date:
`
`(54) STABLE RADIOPHARMACEUTICAL
`COMPOSITIONS AND METHODS FOR
`THEffi PREPARATION
`
`(75)
`
`lnvcntors:
`
`Jianqing Chen, Bordentown. NJ
`(US); Karen E. Linder, Kingston,
`NJ (US); Edmund R. Marinelli,
`Lawrenceville, NJ (US); Edmund
`Metcalfe. Kingston, NJ (US):
`Adrian 0. Nunn, Lambertville, NJ
`(US); Rolf E. Swenson, Princeton,
`NJ (US); Michael F. Tweedle.
`Princeton. NJ (US)
`
`(73) Assignee:
`
`BRACCO ll\1AGING S.P.A.,
`Milan (IT)
`
`(21)
`
`Appl. No.:
`
`13/280,485
`
`(22)
`
`Filed:
`
`Oct. 25, 2011
`
`Related U.S. Application Data
`
`(63)
`
`Continuation of application No. 10/566,112. filed on
`Jul. 9, 2007, filed as application No. PCT/US04/23930
`Oil Jul. 23, 2004.
`
`(60)
`
`Provisional application No. 60/489,850, filed on Jul.
`24, 2003.
`
`Publication C lassification
`
`(51) Int. C l.
`C07K 7106
`C07K 141655
`C07K 7118
`C07K 141595
`C07K 7123
`C07K 14168
`C07K 7116
`C07K 14162
`C07K 141545
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.0I)
`(2006.01)
`
`(52) U.S. C l. ......... 530/303: 530/327; 530/315: 530/311;
`530/3 I 4; 530/351 ; 530/399; 530/312; 530/309
`
`(57)
`
`ABSTRACT
`
`Stabilized radiophamiaceutical formulations are disclosed.
`Methods of making and using stabiHzed radiophannaceutical
`formulations are also disclosed. The invention relates to sta (cid:173)
`bilizers that improve the radiostability of radiothcrapeutic
`and radiodiagnostic compounds and fonuulations containing
`them. In particular, it relates to stabilizers useful in the prepa(cid:173)
`ration and stabilizatiou of targeted radiodiaguostic and radio(cid:173)
`therapeutic compounds. and, in a preferred embodiment, to
`the preparation and stabilization of radiodiagnostic and radio(cid:173)
`therapeutic compounds that are targeted to tbe Gastrin
`Releasing Peptide Receptor (GRP-Receptor).
`
`Evergeen Ex. 1019
`1 of 48
`
`

`

`Patent Application Publication Mar. 15, 2012 Sheet 1 of 15
`
`US 2012/0065365 Al
`
`:z:::c ~)
`
`Evergeen Ex. 1019
`2 of 48
`
`

`

`Patent Application Publication Mar. 15, 2012 Sheet 2 of 15
`
`US 2012/0065365 Al
`
`C....>
`
`8 :::c:
`
`Evergeen Ex. 1019
`3 of 48
`
`

`

`"'0
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`
`FIG. 3A
`
`AD.c.1 A, ADC C~EL AIH:\HPLCDA .. 1\2002\DEC--1.200\12-4-02\B2372000.Dl
`
`mAU
`
`700 ~ RADIOACTIVE
`
`2.5
`
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`
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`
`15
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`
`20
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`
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`Evergeen Ex. 1019
`4 of 48
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`FIG. 38
`
`AOC1 A, ADC CHANNEL A(H:\HPLCDA~1\2002\OEC~1.200\12-9-02\B2372001.DI
`
`(Het-0 FOAHI
`
`Lu-177·A
`
`I
`
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`1401 RADIOACTIVE
`
`120
`
`100
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`
`Evergeen Ex. 1019
`5 of 48
`
`

`

`FIG. 4
`
`•ADC1 A, ADC C~EL AIH:\HPLCOA-.1\2002\SEP-.1.200\9-10-02\B2375001.Dl
`•ADC1 A, ADC C)WfH A (H: \HPLCOA~ 1 \2002\SEP-.1. 200\9-10-02\B2375007. Dl
`•ADC1 A, ADC C~EL AIH:\HPLCOA-.1\2002\SEP-.1.200\9-10-02\B2375008.Dl
`•ADC1 A. ADC C~EL AIH:\HPLCOA-.1\2002\SEP-.1.200\9-10-02\B2375009.0l
`•ADC1 A. ADC CHANNEL AIH:\HPLCOA-.1\2002\SEP-.1.200\9-10-02\B2375010.Dl
`P£P•99.9%
`
`- - - - · - · -
`
`--
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`
`RCP,,99.3%
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`600
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`
`Evergeen Ex. 1019
`6 of 48
`
`

`

`FIG. 5
`1ADC1 A, ADC CHANNEL A(H:\HPLCDA~1\2002\NJV~1.200\11-26-02\B2372000.Dl
`1ADC1 A, ADC CHANNEL A(H:\HPLCDA~1\2002\NJV~1.200\11-27-02\B2372001.Dl
`1ADC1 A, ADC CHANNEL A(H:\HPLCDA~1\2002\NJV~1.200\11-27-02\B2372004.Dl
`•ADC1 A. ADC CHANNEL AIH:\HPLCDA~1\2002\NJV~1.200\1i-27-02\02372007.D)
`•ADC1 A, ADC C~EL AIH:\HPLCDA~1\2002\NJV~1.200\11-27-02\B2372010.D)
`99.9'
`
`10()1
`
`97.Sl
`
`9B.4l
`
`96.41
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`Evergeen Ex. 1019
`7 of 48
`
`IDAU
`800
`
`700
`
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`
`600
`
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`
`500
`
`

`

`Patent Application Publication Mar. 15, 2012 Sheet 7 of 15
`
`US 2012/0065365 Al
`
`Evergeen Ex. 1019
`8 of 48
`
`

`

`Patent Application Publication Mar. 15, 2012 Sheet 8 of 15
`
`US 2012/0065365 Al
`
`Evergeen Ex. 1019
`9 of 48
`
`

`

`Patent Application Publication Mar. 15, 2012 Sheet 9 of 15
`
`US 2012/0065365 Al
`
`FIG. 7A
`
`ADC1 A. ADC CHANNEL AIH:\HPLCDA,1\2002\0CT,1.200\10-3-02\82372019.Dl
`177 Lu-A
`
`IETHIONM
`
`lflaO FOll1
`
`IIAU
`350
`300
`250
`200
`150
`100
`
`sol==:;::=;::::-::::;--;::::~::;=::::;::::::;=~;:=:;::::::;:~:::;:::::::;::::'.~::::;:::::::;:::=:::;:::::::::;::::::;:::::=;:::::::
`0
`10
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`20
`15
`
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`
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`
`0
`
`5
`
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`
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`
`20
`
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`
`COOOOL IN PBS
`
`IAU
`500
`400
`300
`200
`100
`
`IAU
`
`200
`
`150
`
`Evergeen Ex. 1019
`10 of 48
`
`

`

`Patent Application Publication Mar. 15, 2012 Sheet 10 of 15
`
`US 2012/0065365 Al
`
`FIG. 78
`
`ADC1 A. ADC ~IEL A(H:\HPLCDA,1\2002\AlXJ,1 .. 20\8-9-02\4BHSTAOS.DI
`HETHIOOINE
`
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`
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`
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`
`Evergeen Ex. 1019
`11 of 48
`
`

`

`Patent Application Publication Mar. 15, 2012 Sheet 11 of 15
`
`US 2012/0065365 Al
`
`FIG. 8
`
`ADC1 A, ADC CHANNEL A(H:\tflLCDA~1\2002\DEC~1.200\12-4-02\82372001.DI
`Lu-177 A
`
`IET=O FORH
`
`I
`
`ro
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`o
`~
`ADC1 A, ADC CHANNEL AIH:\HPLCOA~1\2002\DEC~1.200\12-S-02\B2372002.DI
`
`20
`
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`
`300 24-H
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`100
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`ADC1 A, ADC CHANNEL AIH:\HPLCOA~1\2002\DEC~1.200\12-5-02\B2372005.DI
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`100
`
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`
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`60
`
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`
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`
`10
`
`20
`
`Evergeen Ex. 1019
`12 of 48
`
`

`

`Patent Application Publication Mar. 15, 2012 Sheet 12 of 15
`
`US 2012/0065365 Al
`
`FIG. 9
`
`AOC1 A. AOC CHANtEL AIH:\HPLCDA~1\2002\:fP~1.200\9-4-02\B2375000.Dl
`
`0-H
`
`IET2 0 FORM
`
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`600
`400
`200
`
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`
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`
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`
`5
`
`10
`
`15
`
`20
`
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`
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`AOC1 A, AOC CHANtf:L AIH:\ffl.CDA~1\2002\SEP~1.200\9-4-02\B2375005.Dl
`
`20
`
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`
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`
`400
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`
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`400 120-H
`300
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`
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`
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`
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`
`Evergeen Ex. 1019
`13 of 48
`
`

`

`"'0
`
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`
`FIG. 10A
`
`15.44.5
`
`COHPOOND B (FREE LIGAND!
`AETENTI~ TitE=15.4 min.
`
`001 A, WAVELENGTH=2BO (T:\EX\2002\SF\0301-4\03012912.DI
`mAU 3
`UV TRACE AT 2B0m
`8
`7 J
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`
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`
`1in
`
`UV CHROHATOGfWt: COHPOUND B- REFERENCE STANDARD. RETENTION TIHE=15.4 HIN.
`
`Evergeen Ex. 1019
`14 of 48
`
`

`

`FIG. 108
`
`ADCA 1. AOCA CHAtffl AIT:\EX\2002\SF0301-4\03012904.Dl
`1.933
`
`GAHHA TRACE SAHPLE 1
`
`•---FREE Lu•188 IEDTA-Lu177 COMPLEX)
`
`mAU
`1400
`1200
`1000
`800
`600
`400
`200
`0-i===;::=~~=;::=::;==;::=::;==:;::=::::;==::;::=:::;;:::=::;::=:::;;:::=::;::=:::;;:::=:;:==;::=::;:==:::::=::::::==:::::=::::::=:::::::
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`UV TRACE AT 280mi
`s»PLE 1
`
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`COHPOU~ B-Lu177 COHPLEX 7 I
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`COMPOUND B FREE LIGAND~
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`
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`
`Evergeen Ex. 1019
`15 of 48
`
`

`

`"'0
`
`'CS
`
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`"t:l -;·
`~ .... o·
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`FIG. 10C
`
`ADCA 1, ADCA C~EL A(T:\EX\2002\SF0301-4\03012903.DI
`GAHHA TRACE
`SAtlPLE 2
`
`COMPOUND B-Lu177 COlflLEX
`
`5
`
`io
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`BOO j
`600
`400
`200
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`0
`
`14 _B47
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`
`14.747
`
`20
`
`11in
`
`COHPOUND B_Zinc COlfLEX
`
`001 A, WAVELENGTH=2801111 (T:\EX\2002\Sf\0301-4\0301004.Dl
`UV TRACE AT 280m
`SAMPLE 2
`EDTA
`
`AIEA: 110. 311
`COHPOU~ B-Lu177 C()fLEX-----+
`COHPOUt,i B FREE LIGAtll
`
`mAU
`
`4
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`3
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`2
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`1
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`0-i
`
`I
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`5
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`20
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`AHMONIIM SALT IN NaOAc BlfFERl ,RCP=1O0%
`
`1110
`
`Evergeen Ex. 1019
`16 of 48
`
`

`

`US 2012/0065365 Al
`
`Mar. 15, 2012
`
`STABLE RADIOPHARMACEUTICAL
`COMPOSITIONS AND METHODS FOR
`THEIR PREPARATION
`
`CROSS RE FERENCE TO RELATED
`APPLICATIONS
`
`[0001] 1llis 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 related to stabilizers that improve the
`radiostability of radiotherapeutic and radiodiaguostic com(cid:173)
`pounds, and formulations contai1ling them. In particular, it
`related to stabilizers useful in the preparation and stabiliza(cid:173)
`tion of targeted radiodiagnostic a11d radiotherapeutic com(cid:173)
`pounds, and, i11 a preferred embodiment, to the preparation
`and stabilization of radiodiagnostic and radiotherapeutic
`compounds d1at are targeted to the Gastrin Releasing Peptide
`Receptor (GRP-Reccptor).
`
`BACKGROUND OF THE INVENTION
`I 0003) Radio labeled compounds designed for use as radio(cid:173)
`diagnostic agents are generally prepared with a ga1runa-emjt(cid:173)
`ting isotope as the raruolabel. ·n1ese gamma photons pen(cid:173)
`etrate water aud body tissues readily and can have a range in
`tissue or air of many centimeters. In general. such radiodiag(cid:173)
`nostic compounds do not cause sigilificant damage to ilie
`organ systems that arc imaged using these agents. T his is
`because the gamma photons given off have no mass or charge
`and the amount of radioactive material that is i1tjected 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
`does to the patient. Radionucleotides such as 99"'Tc, 1111n,
`1231, 57Ga and 64Cu have been used for this purpose.
`(0004]
`ln 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 ofT gamma photons. Radionucleotides
`such as 90y_ 111Lu. 149Pm, 1s3sm. 109Pd. 67Cu, 166Ho. 1311.
`32P. 1s611ssRe. 1osRh, 211 At, 22s Ac. 47Sc, 2u 8 i, and others,
`are potentiaJly useful for radioil1erapy. The +3 metal ions of
`the lanthanide isotopes are of particular interest, and include
`177Lu (relatively low energy ~-emitter), 14 9Pm, 153Sm (me(cid:173)
`dium energy) and 166Ho (high energy). 90Y also forms, a +3
`metal ion, and has coordination chemistry mat is similar to
`that of me lanthanides. The coordination chemistry of ilie
`lanilrnnides is well developed and well known to those skilled
`in the art.
`(00051 The io11izing radjation given off from compounds
`labeled with these radioisotopes is of an appropriate energy to
`damage cells and tissue in sites where the radiolabeled com(cid:173)
`pound has localized. The radiation emitted can either damage
`cellular components in the target tissue directly, or can cause
`water in tissues to fonu free radicals. These radicals are very
`reactive and can damage proteins and DNA.
`(0006) Some of the immediate products that form from the
`radiolysis of water are outlined below.
`
`(0007] Of the products that fonu, (e.g. l·f.._ Ol--r. H"'. and
`OH*). the hydroxyl radical [OH*] is particularly destructive.
`This radical can also combine with itself to fonn hydrogen
`peroxide, which is a strong oxidizer.
`
`OH 0 +0H0 - H20 2(strong oxidizer)
`10008]
`ln 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).
`(0009] Production of such reactive species at the site or
`sites that me radiotherapeutic or radiodiagnostic compound is
`targeted to ( e.g .. a n1mor, bone metastasis, blood cells or other
`targeted o.rgan ororgan 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 cJTect, without causing significant or
`intolerable side effects. S imilarly. for a radiodiagnostic, the
`key factor is delivery of sullicient radiation to the target tissue
`to image it without causing significant or intolerable side
`effects.
`10010) Alpha particles dissipate a large amount of energy
`within one or two cell diameters, as their range of penetration
`in tissues is only -50 ~un. This can cause intense local dam(cid:173)
`age, especially if the radiolabeled compound has been inter(cid:173)
`nalized into tl1e nucleus of the cell. Likewise. radiotherapeu(cid:173)
`tic compounds labeled with Auger-electron emitters such as
`111 In have a very short range and can have potent biological
`effects at the desired site of action. The emissions from thera(cid:173)
`peutic beta-emitting isotopes such as 177Lu or90Y have some(cid:173)
`what longer ranges in tissue. but again, most of 1he damage
`produced occu rs within a few millimetersorcentimeters from
`the site of localization.
`(0011] However, the potentially desrn1ctive properties of
`the emissions of a radiotberapeutic isotope are not limited to
`U1eir cellular targets. For radiotherapeutic and radiodiagnos(cid:173)
`tic compounds. radiolytic damage to the radiolabeled com(cid:173)
`pound itself can be a serious problem during the preparation,
`purification, storage and/or shipping of a radio labeled radio(cid:173)
`its
`therapeutic or radiodiag nostic co111potu1d, prior to
`intended use.
`(0012) Sucb radiolytic damage can cause, for example,
`release of the radioisotope [ e.g .. by dehalogenation of radio(cid:173)
`iodinated antibodies or decomposition of the chelating moi(cid:173)
`ety designed to hold the radiometal]. or it can damage the
`targeting molecule that is required ro deliver the targeted
`agent to its intended target. Both types of damage are highly
`undesirable as they ca11 potentially cause the release of
`unbound isotope. e.g., free radloiodine or unchelated radio(cid:173)
`metal to tbe thyroid, bone and other organs. or cause a
`decrease or abolishment of targeting ability as a result of
`radiolytic damage to ilie targeting molecule, such as a recep(cid:173)
`tor-binding region of a targeting peptide or radiolabeled anti(cid:173)
`body. Radioactivity that does not become associated with its
`target tissue may be responsible for unwanted side effects.
`(0013] For example. DOTA-Gly-ACA-Gln-Trp-Ala-Val(cid:173)
`Gly-ms-Leu-Met-N1l2(ACA =3-Amino-3-deoxycholic acid)
`and DOTA-Gly-Abz4-Gln-Trp-Ala-Val-Gly-His-Leu-Met(cid:173)
`NH2 (Abz4=4-am.inobenzoic acid) the two chelatiug ligands
`shown iu FIGS. 1 and 2. respectively, have been shown to
`specifically target the Gastrin Releasing Peptide (GRP)
`Receptors. ln the examples U1at follow, these have been
`
`Evergeen Ex. 1019
`17 of 48
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`
`2
`
`described as CompoUllds A and Compound B respectively.
`Other GRP receptor-binding ligands are described in U.S.
`Pat. No. 6,200,546. to Hoffman et al.. published U.S. appli(cid:173)
`cation US. 2002/0054855, and in copending application Ser.
`No. J0/341,577, tiled Jan. 13, 2003, the entire contents of
`which are incorporated by reference.
`10014) When radiolabeled with diagnostic and radiothera(cid:173)
`peutic radionuclides such as 1111n and 177Lu, Compounds A
`and B have been shown to have high affinity for GRP recep(cid:173)
`tors. both in vitro and in vivo. However, these compounds can
`undergo significant radiolytic damage that is induced by the
`radioactive label ifll1ese radio labeled complexes are prepared
`without concomitant or subsequent addition of one or more
`radiostabilizers (compounds that protect against radiolytic
`damage). This result is not surprising. as the hydroxyl and
`superoxide radicals generated by the interaction of ~-par(cid:173)
`ticles with water are highly oxidizing. Radiolytic damage to
`the methionine (Met) residue in these peptides is the most
`facile mode of decomposition, possibly resulting in a
`methionine sulfoxide derivative.
`10015) CeU binding results show that the resulting radi(cid:173)
`olytically damaged derivatives are devoid of GRP-receptor
`binding activity OC50 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(cid:173)
`olytic decomposition ro utes in radiodiagnostic and radio(cid:173)
`therapeutic compounds.
`[0016) Preventing such radiolytic damage is a major chal(cid:173)
`lenge in the formulation of radiodiagnostic and radiothera(cid:173)
`peutic compounds. For this purpose, compounds known as
`radical scavengers or antioxidants are typically \ISed. These
`are compoUllds that react rapidly with, e.g., hydroxyl radicals
`a nd superoxide, thus preveming them from reaction with the
`radiophamiaceutical of interest or reagents for its prepara(cid:173)
`tion.
`10017] There bas been extensive research in this area. Most
`of it bas focused on the prevention of radiolytic damage in
`radiod iagnostic formulations, and several radical scavengers
`have been proposed for such use. However, it bas been found
`in the studies described herein that the stabilizers reported to
`be effective by others, provide insuJTicient radiostabiliz.ation
`to protect ' 77Lu-A and 177Lu-B. the Lutetium complexes of
`Compounds A and B, respectively, from radiolytic damage,
`especially when high concentrations and large amounts of
`radioactivity are used.
`(0018] For example, Cyr and Pearson [Stabiliz.ation of
`radiopbarmaceutical compositions using hydrophilic thioet(cid:173)
`hers and hydrophilic 6-hydroxy chromans. Cyr, John E.:
`Pearson, Daniel A. (Diatide, lnc., USA), PCT Int. Appl.
`(2002). WO 200260491 A2 20020808) state that diagnostic
`and therapeutic radiophannaceutical compositions radiola(cid:173)
`beled with 1251. !JI 1, 211 At, 47Sc. 67Cu, 72Ga, 90y, ISJSm,
`159Gd, l65Dy, 166Ho, 175Yb, t77Lu, 212Bi, 213Bi, 6SGa, 99"'Tc.
`11n and 123
`] can be stabilized by the addition of a hydro(cid:173)
`J1
`philic thioether. and that the amino acid methionine. a hydro(cid:173)
`philic ll1ioether, is especially useful for this purpose.
`[0019] A study was therefore performed wherein L-me(cid:173)
`thionine (5 mg/mL) was added to 177Lu-A, to evaluate its
`ability to serve as a radical scavenger. As will be described i11
`more detail below, reverse phase HPLC shows that after five
`days, almost complete decomposition of 177Lu-A had
`occurred, indicating that the radiostabilizer used was insuffi(cid:173)
`cient to prevent radiolytic damage. In vitro binding results
`indicate that such decomposition can drama tically decrease
`
`the potency and targeti11g ability, and hence the radiothera(cid:173)
`peutic efficacy. of the compound thus damaged. To attain the
`desired radiotherapeutic effects one would need to inject
`more radioactivity, thus increasing the potential for toxicity to
`nonual organs.
`[0020)
`In order to identify suitable antioxidant radical scav(cid:173)
`engers that might be useful for the radiostabilization ofGRP(cid:173)
`receptor binding radiodiagnostic and radiotberapeutic com(cid:173)
`pounds, several st11dies were performed. One or more
`potential radiostabilizers was added after complex formation
`(a two-vial fonnulat ion) or they were added di rectly to ll1e
`reaction mixture prior to complexation will1 a racliometal (or
`both). JdeaJly. the radiostabilizer should be able to be added
`directly to the formulation without significantly decreasing
`the radiochemical parity (RCP) of the product. as such a
`formulation bas the potential to be a single-vial kit.
`10021] Tue radical scavengers identified as a result of these
`studies have general utility in formulations for the preparation
`of compounds used fora variety ofradiodiagnostic and radio(cid:173)
`therapeutic applications. and may be useful to stabilize com(cid:173)
`pounds radiolabeled with a variety of isotopes. e.g., 99"'Tc,
`1s6/1ssRe, 1111n. 90Y, 1n Lu, 2uBi, 225 Ac. 1661:-Jo, and others.
`The prin1ary focus of the examples in this application is the
`radiostabilization ofGRP-bindingpeptides, and in partkular,
`the radioprotection of methionine residues in these mol(cid:173)
`ecules. However, ll1e stabilizers identified should have appli(cid:173)
`cability to a wide range of radiolabeled peptides. peptoids,
`small molecules. proteins. antibodies, and antibody frag(cid:173)
`ments and the like. They are useful for the radioprotection of
`any compotmd that has a residue or residues that are particu(cid:173)
`larly sensitive to radiolytic damage. such as, for example,
`tryptophan (oxidation of the iJJdole ring), tyrosine (oxidative
`dimerizatio11, or other oxidation), histidine, cysteine (oxida(cid:173)
`tion of thiol group) and to a lesser exte nt serine, threonine,
`glutamic acid, and aspartic acid. Unusual amino acids com(cid:173)
`monly used in peptides or drugs ll1at contain sensitive nmc(cid:173)
`imidazolcs.
`thiazoles.
`furans,
`(indo les.
`tional groups
`thiophenes and other heterocycles) could also be protected.
`
`SUMMARY OF THE INVE NTION
`
`10022]
`It is the aim of this invention to provide stabilizers
`and stabilizer combinations that slow or prevent radiolytic
`dan1age to targeted radiotherapeutic and radiodiagnostic
`radio labeled compounds, especially compounds labeled with
`radiometals, and thus preserve the targeting ability and speci(cid:173)
`ficity of the compounds. Ji is also an aim to present formula(cid:173)
`tions containing these stabilizers. As described by the
`examples below. many stabilizers have been identified that,
`alone or in combination, inhibit radiolytic damage to radio(cid:173)
`labeled compounds. At this time. four approaches are particu(cid:173)
`larly preferred. Ju the first approach, radiolysis stabilizing
`solution contaioi11g a mixtwe of the following ingredients is
`added to the radiolabeled compound immediately following
`the radiolabeling reaction: gentisic acid, ascorbic acid,
`human semm a lbumin, benzyl alcohol, a physiologically
`accep table bulferorsalt solution at a pl-I o f about 4.5 to about
`8.5, and one or more amino acids selected from methionine,
`selenomethionine, selenocysteine, or cysreine).
`10023) The physiologically acceptable bufrer or salt solu(cid:173)
`tion is preferably selected from phosphate, citrate, or acetate
`buffers or physiologicalJy acceptable sodium chloride solu(cid:173)
`tions or a mixture thereof at a molarity of from about 0.02M
`to about 0.2M. The reagent benzyl alcohol is a key component
`in this fonuulation and serves two purposes. For compounds
`
`Evergeen Ex. 1019
`18 of 48
`
`

`

`US 2012/0065365 Al
`
`Mar. 15, 2012
`
`3
`
`that have limited solubility, one of its purposes is lo solubilize
`the radiodiagnostic or radiotherapeutic targeted compound in
`the reaction solution. without the need for added organic
`solvems. !is second purpose is 10 provide a bacteriostatic
`effect. This is important, as solutions that contain the radio(cid:173)
`stabilizers of the invention are expected to have long post(cid:173)
`reconstruction stability, so the presence of a bacteriostat is
`critical iu order to maintain sterility. The amino acids
`methionine, seleuomethionine, cysteine, and selenocysteine
`play a special role in preventing radiolytic damage to methio(cid:173)
`nyl residues in targeted molecules that are stabilized with this
`radiostabilizing combination.
`[0024]
`ln the second approach, stabilization is achieved via
`the use of dithiocarbarnate compounds having the following
`general fomrnla:
`
`sl
`\ -{
`
`Rt
`
`[
`
`R2/
`
`S M
`
`wherein Rl and R2 are each independently H, Cl-C8 all-..-yl,
`- OR3, wherein R3 is Cl-C8 alkyl, or benzyl (Bu) (either
`unsubstituted or optionally substituted with water solubiliz(cid:173)
`ing groups).
`or wherein RI R2N combined= 1-pyrrolidinyl-, piperidino-,
`morpholino-, 1-piperazinyl- and M=l-J•, Na•, K+. Nl-14 • ,
`M-mcthylglucamine, or other phannaceutically acceptable
`+I ions.
`[0025) Alternatively, compounds oftbe form shown below
`may be used. wherein Mis a physiologically acceptable metal
`in the+2 oxidation state. such as Mg2 + orCa2
`• , and RI and R2
`have the same definition as described above.
`
`sl >-<
`
`R1
`
`[
`
`R2
`
`S 2M
`
`[0026] 171ese reagents can either be added directly into
`reaction mixtures during radiolabeled complex preparation.
`or added after complexation is complete, or both.
`[0027] The compound 1-Pyrrolidine Dithiocarbamic Acid
`Ammonium salt (PDTC) proved most efficacious as a stabi(cid:173)
`lizer, when either added directly to the reaction mixn1re or
`added alter complex fomrntion. 111cse results were unex(cid:173)
`pected, as the compound has not been reported for use as a
`stabilizer for radiopharmaceuticals prior to these sn1dies.
`Dithiocarbamates, and PDTC in particular, have the added
`advantage of serviug to scavenge adveutitious trace metals in
`the reaction mixture.
`[0028) 1n the third approach, formulations contain stabiliz(cid:173)
`ers that are water soluble organic selenium compom1ds
`wherein the selenium is in the oxidation state +2. Especially
`preferred are the amino acid compounds selcnomethioni11e,
`and selenocysteine and their esters and amide derivatives and
`di peptides and tri peptides thereof, which can e ither be added
`directly to the reaction mixture during radiolabeled complex
`preparation. or following complex preparation. The flexibil(cid:173)
`ity of having these stabilizers in the vial at the time oflabeling
`or in a separate via l extends ihe utility of this invention for
`manufacturing radiodiagnostic o r radiotherapcutic kits.
`
`[0029]
`It is highly efficacious to use these selenium com(cid:173)
`pounds i11 combination with sodium ascorbate or oilier phar(cid:173)
`maceutically acceptable forms of ascorbic acid and its deriva(cid:173)
`tives.
`[0030] 1l1e ascorbate is most preferably added after com(cid:173)
`plexation is complete. Alternatively. it can be used as a com(cid:173)
`ponelll of the stabilizing fomrnlation described above. A
`fourth approach involves the use of water soluble sulfur(cid:173)
`containing compounds wherein the sulfur i11 the +2 oxidation
`state. Preferred thiol compounds include derivatives of cys(cid:173)
`teine, mercaptothanol, and dithiolthreotol. These reagents are
`particularly preferred due to their ability to reduce oxidized
`forms of methionine residues (e.g., methionine oxide resi(cid:173)
`dues) back to methionyl residues, thus restoring oxidative
`damage that has occurred as a result of radiolysis. With these
`thiol compounds, ifis highly efficacious to use these stabiliz(cid:173)
`ing reagents iu combination with sodium ascorbate or other
`phamiaceutically acceptable forms of ascorbic acid and its
`derivatives. The ascorbate is most preferably added after
`complexation is complete.
`[0031] The stabilizers and stabilizer combinations may be
`used to improve the radiolytic stability of targeted radiophar(cid:173)
`maceuticals, comprising peptides. 11011-peptidic small mol(cid:173)
`ecules, radiolabeled proteins. radiolabeled antibodies and
`fragments thereof. These stabilizers are particularly useful
`with the class ofGRP-binding compow1ds described herein.
`
`BRJEF DESCRIPTION OF THE DRAWINGS
`
`[0032] FJG. l shows the srmc rure of Compound A.
`[0033) FIG. 2 shows the structure of Compound B.
`[0034] FIG. 3 illustrates the results of a n f-lPLC analysis of
`a mixture of 177Lu-A with 2.5 mg/mL L-Methionine over 5
`days at room temperan1re at a radioconcentration of25 mCi/
`mL. (50 mCi total]. FIG. 3A is a radiocluomatogram of a
`reaction mixn1re for the preparation o f 177Lu-A. which was
`initially formed in >98% yield. FIG. 38 is radiochromato(cid:173)
`gram of[ 177Lu-A], 25 mCi/mL, after live days at room tem(cid:173)
`perature, demonstrating complete radio lytic destruction of
`the desired compound. The radiostabilizer added (5 mg/mL.
`L-Methionine) was clearly insufficient for the level of radio(cid:173)
`protection required
`[0035] FIG. 4 is an HPLC trace (radiodetection] showing
`that 177Lu-B (]04 mCi) has >99% RCP for 5 days when
`diluted I: I with radiolysis protecting solution that was added
`after the complex was fom1ed.
`[0036) FIG. 5 is an HPLC trace [radiodetection] showing
`that 177Lu-A bas >95% RCP for 5 days at a concelllration of
`55 mCi/2 ml if l mLofradiolysis protecting solution is added
`after the complex was formed.
`[0037] FIG. 6A and FIG. 6B show the structure of the
`methionine sulfoxide derivative of 177Lu-A (FIG. 6A) and
`methionine sulfoxide derivative of 111ln-B (FIG. 68).
`[0038] FIG. 7A and FIG. 7B show stabilizer sn1dies
`177Lu-A (FIG. 7A) a nd m Lu-8 (FTG. 78). Radioactivity
`traces are shown from a study to compare the radiostabilizing
`effect of different amino acids, when added to 177Lu-A (FIG.
`7A) and 177Lu-B (FIG. 7B) at au amino acid concentration of
`6.6 mwmL in 10 mM Dul becco's phosphate buffered saline,
`pH 7.0 [PBS], and a radioactivity concentration of-20 mCi/
`mL. after 48 hours of storage at room temperature. A total of
`3.5 mCi of 177Lu was added 10 each vial. A fu ll description of
`the experimental procedure is given in Example I.
`
`Evergeen Ex. 1019
`19 of 48
`
`

`

`US 2012/0065365 Al
`
`Mar. 15, 201 2
`
`4
`
`[0039] FJG. 8 shows an HPLC trace [radiodetection) show(cid:173)
`ing the radiostability of 177Lu-A over 5 days at room tem(cid:173)
`perat11re at a radioconcentration of25 mCi/mL in presence of
`2.5 mg/mL L-methionine (50 mCi total). The details of this
`study are given in Example 2.
`[0040] FIG. 9 shows an Ff PLC trace [radiodetection] show(cid:173)
`ing the stability of 177Lu-B at a concentration of 50 mCi/2 mL
`in a radiolysis protectillg solution contailling L-metbionine.
`The details ofthls sn1dy are given in Example 4.
`(0041) FIGS. l0A-C show radiochromatograms and UV
`chromatograms comparing samples with and without J-pyr(cid:173)
`rolidine di thiocarbamic acid ammonium salt in the reaction
`bu !fer and containing zinc as a contaminant metal during the
`reaction of 177Lu-B. The experimental procedure for this
`study is given iJ1 Example 20.
`