(12) United States Patent
`Zamora et al.
`
`11111111111111111 11111 IIIII IIIII IIIII IIIII IIIII IIIII IIIII IIIIII IIII 11111111
`US006261536Bl
`US 6,261,536 Bl
`Jul. 17, 2001
`
`(10) Patent No.:
`(45) Date of Patent:
`
`(54) POST LABELING STABILIZATION OF
`RADIOlABELED PROTEINS AND PEIYfIDES
`
`(56)
`
`Referenct..'S Cited
`U.S. PATENT DOCUMENTS
`
`(75)
`
`Inventors: Paul 0. Zamora, Guadalajara (MX);
`Michael J. Marek, Albuquerque, NM
`(US)
`
`(73) Assignee: RhoMed Incorporated, Princeton, NJ
`(US)
`
`( *) Noiice:
`
`Subject to any disclaimer, tbe term of tbis
`patent is extended or adjusted under 35
`U.S.C. 154(b) by O days.
`
`(21) Appl. No.: 09/393,581
`
`(22) Filed:
`
`Sep. 9, 1999
`
`Related U.S. Application Data
`
`(63)
`
`(60)
`
`(51)
`(52)
`
`(58)
`
`Con1inuation of application No. 08/794,311, filed on Feb. 3,
`1997, now Pat. No. 6,066,309, which is a continuation-in(cid:173)
`part of application No. 08/794,270, filed on Jan. 31, 1997,
`now abandoned.
`Provisional application No. 60/011,027, filed on Feb. 2,
`1996.
`lnt. CJ.7
`........................... A61K 51/00; A61M 36/14
`U.S. Cl . ....................... 424/1.49; 424/1.11; 424/1.69;
`424/1.53; 424/806; 530/863
`Field of Search ................................... 424/ 1.11, 1.37,
`424/1.49, 1.53, 1.65, 1.69, 9.1, 179.1, 806,
`807, 804,805; 530/300, 311, 317,333,
`334, 338, 863, 864, 350, 866, 861, 862;
`206/223, 569, 570; 549/315; 436/129
`
`Rhodes ct al. ........ ................ ... 424/1
`Grogg ct al. .......................... 424/1.1
`Bugaj et al. .......................... 424/ 1.1
`Srinivasan et al. ................... 424/1.1
`Rhodes .................................. 424/1.1
`Rhodes .............................. 530/391.5
`Wolfangel ............................. 424/ 1.l
`Gustavson et al. ............... 530/391.5
`Rhodes ................................ 424/ 1.69
`Thakur ................................ 424/ l.49
`Deutsch et al ...................... 424/1.69
`Vanderheyden et al. ........... 424/1.53
`Zamora el al. ................... 424/1.l69
`Rhodes et al. ...................... 424/1.49
`Vanderheyden el al. ........... 424/1.11
`Zamora et al. ..................... 424/ l.69
`Rhodes ................................ 424/ 1.69
`Zamora cl al. ..................... 424/1.49
`
`8/1983
`4,401,646
`4/1985
`4,510,125
`4,707,353
`ll/1987
`4,988,496
`1/1991
`5,078,985
`1/1992
`4/]992
`5,102,990
`5,2L9,556
`6/1993
`5,250,666
`10/1993
`5,277,892
`1/1994
`5,308,603
`5/1994
`5,384,113
`1/1995
`5,393,512
`2/1995
`5,443,816
`8/1995
`5,460,785
`l0/1995
`10/1997
`5,679,318
`12/1997
`5,700,444
`6/1998
`5,759,S LS
`6,066,309
`• 5/2000
`* cited by examiner
`Primary Examiner-Dameron L. Jones
`(74) Allomey, Agent, or Firm-Darby & Darby
`ABSTRACT
`
`(57)
`
`Tbe subject invention relates to tbe use of ascorbic acid and
`derivatives tbereof in stabilizing radiolabeled proteins and
`peptides against oxidation loss of radiolabel and autoradi(cid:173)
`olysis. Ascorbic acid is added after radiolabeling, including
`any required incubation period, but prior to patient admin(cid:173)
`istration.
`
`12 C laims, 13 Drawing Sheets
`
`Evergeen Ex. 1013
`1 of 22
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`

`

`U.S. Patent
`
`Jul. 17, 2001
`
`Sheet 1 of 13
`
`US 6,261,536 Bl
`
`•
`
`(!) -LL
`
`Evergeen Ex. 1013
`2 of 22
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`

`

`U.S. Patent
`
`Jul. 17, 2001
`
`Sheet 2 of 13
`
`US 6,261,536 Bl
`
`FIG. 18
`
`>-
`.._ 100
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`Column
`40
`80
`0
`PERCENT ETHANOL IN ELUTION
`
`Evergeen Ex. 1013
`3 of 22
`
`

`

`U.S. Patent
`
`Jul. 17, 2001
`
`Sheet 3 of 13
`
`US 6,261,536 Bl
`
`FIG. 2A
`
`FIG. 2B
`
`37°
`
`90°
`
`Evergeen Ex. 1013
`4 of 22
`
`

`

`U.S. Patent
`
`Jul. 17, 2001
`
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`Evergeen Ex. 1013
`5 of 22
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`

`

`U.S. Patent
`
`Jul. 17, 2001
`
`Sheet 5 of 13
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`Evergeen Ex. 1013
`6 of 22
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`

`

`U.S. Patent
`
`Jul. 17, 2001
`
`Sheet 6 of 13
`
`US 6,261,536 Bl
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`Evergeen Ex. 1013
`7 of 22
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`

`

`U.S. Patent
`
`Jul. 17, 2001
`
`Sheet 7 of 13
`
`US 6,261,536 Bl
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`
`Evergeen Ex. 1013
`8 of 22
`
`

`

`U.S. Patent
`
`Jul. 17, 2001
`
`Sheet 8 of 13
`
`US 6,261,536 Bl
`
`•
`
`(!) -LL
`
`Evergeen Ex. 1013
`9 of 22
`
`

`

`U.S. Patent
`
`Jul. 17, 2001
`
`Sheet 9 of 13
`
`US 6,261,536 Bl
`
`FIG. SA
`
`Fl G. 8 B
`
`Evergeen Ex. 1013
`10 of 22
`
`

`

`N
`0
`
`rJ)
`
`=" ~
`
`~ .
`r;,:i
`~
`~ .....
`~ = .....
`~ F--,..-...:a
`0 -
`~ .... -0
`~ -(.,.l
`
`Q
`
`A
`
`e
`r:J)
`9'(cid:173)
`N
`,__
`0-,
`'vl w
`0'I
`o=
`....
`
`FIG. 9A
`
`LEUK0-I, Lot#LEU505II-IMM-I2,ug SnT
`Cel I Binding Assay
`ITLC
`HPLC
`No Cel Is
`Pk Bound %Free %Colloid HL60
`293
`
`cone during
`labeling
`
`90.91
`
`5.75
`
`0.00
`
`70.21
`
`10.41
`
`4.95
`
`0.71 mg/ml
`
`80.00
`
`2.82
`
`0.00
`
`87.70
`
`3.02
`
`0.00
`
`77.35
`
`9.81
`
`0.00
`
`77.03
`-
`73.10
`
`7.51
`-
`13.26
`
`1.89
`
`I mg/ml
`
`-
`
`I mg/ml
`
`4.44
`
`I mg/ml
`
`77.37
`
`0.00
`
`3.58
`
`70.82
`
`7.52
`
`5.26
`
`I mg/ml
`
`78.48
`
`0.00
`
`o.oo
`
`70.54
`8l.l8(NEW)
`
`14.28
`
`10.62
`
`I mg/ml
`
`84.11
`
`6.15
`
`0.00
`
`78.04
`
`15.95
`
`7.36
`
`I mg/ml
`
`84.08
`
`5.46
`
`0 .00
`
`81.65
`
`12.59
`
`5.54
`
`lmg/rnl
`
`Test
`Date
`
`ID
`
`5/15/95
`
`5/30/95
`
`Froz
`-I l.7mCI
`Lyop
`-I3.2mCI
`Lyop
`-4.97mCI
`Lyop
`-16.6mCI
`
`6/27/95
`
`7/19/95
`
`Lyop
`-20.9mCI
`Lyop
`-I9.3mCI
`8 /4/95 RhoMed
`-I5.45mCI
`UCLA
`-I6.58mCI
`
`A
`
`Evergeen Ex. 1013
`11 of 22
`
`

`

`~ .
`r;,:i
`~
`~ .....
`~ = .....
`~ F--,,.....:a
`0 -
`~ .... --0
`~ -(M
`
`N
`0
`
`rJ)
`
`=" ~
`
`e
`r:J)
`9'(cid:173)
`N
`,__
`0-,
`'vl w
`0'I
`o=
`....
`
`FIG. 98
`
`A
`
`8/21/95
`
`9/7/95
`
`Vial# 18
`l9.5mCI
`
`9/7/95
`
`Vial#l9
`19.5mCI
`
`With
`Sscorbic
`Acid
`
`t-30 min
`-19.00mCI
`t-2.5 hrs
`-19.00mCI
`
`83.56
`
`86.70
`
`t-30 min 90.12
`
`t-2.5hrs 92.81
`
`t-23 hrs 86.36
`
`t-30 min
`Jfil Ascorb
`
`92. 16
`
`t-1 hr
`
`82.87
`
`t-2.5 hrs 89.13
`
`t-24 hrs 80.23
`
`3.16
`
`4.36
`
`0.00
`9.68%**
`0.00
`3.20%•
`3.30
`0.00
`*8.18
`*4.48
`7. 34
`0.00
`*10.49 *7.75
`5.84
`3.51
`*11.09 *6.87
`0.00
`4.62
`*3.00
`*4.30
`0.00
`7.89
`*5.80
`*7.75
`0.00
`2.32
`*8.78
`*7.15
`11.79
`0.00
`•14.16
`*12.61
`
`A
`
`74.28
`
`17.08
`
`8.50
`
`lmg/ml
`
`77.66
`
`11.38
`
`4.90
`
`lmg/ml
`
`72.63
`
`16.14
`
`6.56
`
`I mg/ml
`
`82.56
`
`10.47
`
`7.39
`
`lmg/ml
`
`80.13
`
`6.10
`
`3.45
`
`I mg/ml
`
`79.54
`
`19.78
`
`8.60
`
`lmg/ml
`
`77.00
`
`8.47
`
`3.96
`
`75.29
`
`7.77
`
`6.41
`
`74.03
`
`9.83
`
`7.81
`
`lmg/ml
`during
`labeling
`0.25mg/ml
`after
`addition of
`Ascorbic
`acid
`
`*or.,.* Average of strips counted on Gamma Counter and Dose Calibrator
`
`Evergeen Ex. 1013
`12 of 22
`
`

`

`~ .
`r;,:i
`~
`~ .....
`~ = .....
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`0 -
`~ .... -N
`~ -(,,.l
`
`N
`0
`
`rJ)
`
`=" ~
`
`FIG.
`
`IOA
`
`2hrs post labeling
`
`2 hrs post labeling
`
`24 hrs post labeling
`
`Sample ID
`
`HPLC
`
`ITLC
`
`ITLC
`
`% protein
`Bound
`
`Standard SOP
`
`86.59
`
`HPLC
`Yield
`
`60.63
`
`w/ ascorbic acid
`during labeling
`
`73.53
`
`37.87
`
`% Free % Colloid
`
`% Free % Colloid
`
`1.82
`
`3.47
`
`3.18
`
`2.55
`
`11 .89
`
`13.53
`
`17.11
`
`8 .42
`
`Q
`
`e
`r:J)
`9'(cid:173)
`N
`,__
`0-,
`'vl w
`o=
`....
`
`0-,
`
`Evergeen Ex. 1013
`13 of 22
`
`

`

`U.S. Patent
`
`Jul. 17, 2001
`
`Sheet13of13
`
`US 6,261,536 Bl
`
`FIG. 108
`
`FIG.
`
`IOC
`
`Evergeen Ex. 1013
`14 of 22
`
`

`

`US 6,261,536 Bl
`
`1
`POST LABELING STABILIZATION OF
`RADIOLABELEO PROTEINS AND PEPTIDES
`
`Tbis is a continuation application of U.S. patent appli(cid:173)
`cation Ser. No. 08/794,311 filed Feb. 3, 1997, now U.S. Pat. 5
`No. 6,066,309, wbicb is a continuation-in-part of U.S. patent
`application Ser. No. 08/794,270 filed Jan. 31, 1997,
`abandoned, and wbicb claimed priority under 35 U.S.C. §
`119(e) of U.S. Provisional Patent Application Ser. No.
`60/011,027, filed Feb. 2, 1996.
`
`BACKGROUND
`
`1. Field of the Invention
`This invention relates to stabilizers for radiopbarmaceu(cid:173)
`tical compositions wbich are added after radiolabeling but
`prior to administration. Ascorbic acid (and/or a derivative
`thereof), alone or in combination with other stabilizers, is
`used to inhibit oxidation loss and autoradiolysis of radiola(cid:173)
`beled peptides and proteins.
`2. Background Art
`A large number of protein-based radiopharmaceuticals are
`under clinical investigation, and a smaller number have been
`approved in the United States and other countries. Similarly,
`peptide-based radiophaanaceutieals are also 11nder clinical
`investigation, witb several approved for clinical use. 'Thera(cid:173)
`peutic and diagnostic uses of botb protein- and peptide(cid:173)
`based radiopharmaceuticals continue to be developed. Typi(cid:173)
`cal therapeutic and diagnostic applications are described in
`U.S. Pat. Nos. 5,078,985; 5,102,990; 5,277,893; 5,443,816;
`and 5,460,785; and in U.S. patent Applications Ser. Nos.
`08/087,219; 08/269,929 and 08/651,179, incorporated by
`reference in their entirety.
`Protein- or peptide-based radiopharmaceuticals are pri(cid:173)
`marily based upon use of monoclonal antibodies (or frag(cid:173)
`ments thereof) as a targeting vehicle, but other peptides or
`proteins can also be used, inclucling albumins and hormones.
`Both intact antibodies (monoclonal and polyclonal) and
`fragments, made by any method known to the art, as well as
`peptide mimics o( fragments or antibody binding sites can
`be radiolabeled and used as imaging, diagnostic or tbera(cid:173)
`peutic agents.
`A variety of peptide-based radiopharmaceuticals have
`been investigated, including those in which the peptide is
`derived from somatostatin. Radiolabeled peptide analogues
`of somatostatin used for diagnostic imaging include 12 3I(cid:173)
`labeled Tyr-3-octreotide and rnl n-cliethylene tetraamine(cid:173)
`pentaacetic acid 99"'TPA)-octreotide imaging agents.
`Research is underway o n a variety of 99"'Tc-Jabeled soma(cid:173)
`tostatin analogues, incl11ding direct-labeled peptide soma(cid:173)
`tostatin analogues. An l llJn-DTPA-octrcotide product is
`commercially available in the United States and European
`countries, and is distributed by Mallinckrodt Medical, Inc.
`Both protein- and peptide-based radiopharmaceuticals
`may be radiolabeled by a variety of means. Botb peptides
`and proteins can be directly radioiodinated, through elec(cid:173)
`trophilic substitution at reactive aromatic amino acids. Iodi(cid:173)
`nation may also be accomplisbed via prelabeled reagents, in
`which tbe reagent is iodinated and purified, and then linked
`to the peptide or protein.
`The utility of OTPA and EDTA chelates covalently
`coupled to proteins, polypeptides and peptides is well
`known in tbe art. DTPA bas been used as a bifunctional
`chelating agent for radiolabeling a variety of peptides with
`111In, including somatoslatin analogues for cancer imaging,
`a-melanocyte-simulating hormone for imaging melanoma,
`
`30
`
`2
`chemotactic peptides for infection imaging, laminin frag(cid:173)
`ments for targeting tumor-associated laminin receptors and
`atrial natriuretic peptides for imaging atrial natriuretic recep-
`tors in tbe kidney.
`99"'Tc is a preferred isotope for diagnostic imaging, due lo
`its low cost, ready availability, excellent imaging properties
`and high specific activities. Two approaches have been
`described for radiolabeling proteins and peptides with
`9 9"'Tc: direct labeling a(cid:143) d bifunctional chelates. Direct
`l O labeling methods are generally described in U.S. Pat. Nos.
`5,078,985; 5,102,990; 5,277,893; 5,443,816; and 5,460,785
`referenced above, in wbich a variety of methods of direct
`labeling of peptides and proteins tbrougb sulf-ur-, oxygen(cid:173)
`and nitrogen-containing amino acid sequences available for
`l 5 binding are disclosed.
`A variety of high affinity chelates to bind 99"'Tc to specific
`sites on peptides have bee(cid:143) developed. In one approach, the
`bifunctional reagent is first labeled with 99"'Tc, and tben
`conjugated to the peptide. However, multiple species can
`20 result, and post-labeling purification is generally required. In
`another approach, a chelating agent is covalently attached to
`the peptide prior to radiolabeling. Cbelates which have been
`employed include a variety of N2S2 and N3S ligands,
`DTPA, 6-hyd.razinonicotioate groups, metallothionein and
`25 metallothionein fragments.
`Isotopes of rhenium, principa!Jy ' 86Re and 188Re, have
`been used to radiolabel proteins and peptides for investiga(cid:173)
`tion as tberapeutic agents. The chemistry of l 86Re and l 88Re
`is similar to that of 9 9"'Tc, though not identical, and botb
`direct and chelate labeling approaches have been used in
`radiolabeling proteins and peptides with rhenium.
`Protein and peptide radiopharmace utical compositions are
`known to degrade after racliolabeling, primarily by oxidation
`35 losses and by autoradiolysis. Some radiopharmaceuticals,
`s uch as 99"'1c, and especially 186Re and 188Re labeled
`compounds, are particularl y susceptible to oxidation Jos.ses
`if the isotope is not maintained in a suitable oxidation state.
`Both technetium and rhenium isotopes norma!Jy exist in
`40 their bighest or + 7 oxidation state, which is the stable slate,
`until reduced with stannous or other reducing agents. A
`technetium or rhenium mliol~beled compound can becoIDe
`unstable if the complexed reduced isotope is oxidized lo a
`bigber oxidation state, releasing the bound isotope as free or
`45 unbound pertechnetate + 7 or free perrhenate + 7.
`The tem1 "auloradiolysis" includes chemical decomposi(cid:173)
`tion of a radiolabeled peptide o r protein by the action of
`radiation emilted from the radioisotope coupled lo the
`peptide or protein. Autoradiolysis may be caused by the
`50 formation of free radicals in the water or other medium due
`to the effect of radiation emitted from the radioisotope. Free
`radicals are molecules or atoms containing a single impaired
`electron, wbich exhibit bigb cbemical reactivity. Autoradi(cid:173)
`olysis is a significant problem witb high energy ~-emitting
`55 isotopes, such as rhenium isotopes, and with a -emitting
`isotopes, but is typically somewhat less of a problem with
`y-emilling isotopes, such as 9 9"'Tc.
`A variety of methods have been employed to stabilize
`radiopharmaceuticals in general, including addition of I-ISA
`60 (human serum albumin) to a composition or keeping ii
`frozen between preparation and use. However, these meth(cid:173)
`ods are not reliably effective or practical for use with many
`radiolabeled peptides and proteins. Substances sucb as
`ascorbic acid and gentisic acid have also been used to inhibit
`65 the oxidation of the radioisotope, and to limit autoradiolysis
`by acting as "free radica I scavengers" which donate reactive
`hydrogen atoms to the free radical intermediates yielding a
`
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`

`

`US 6,261,536 Bl
`
`3
`non-reactive molecule. Use of gentisic acid and its deriva(cid:173)
`tives to stabilize radiolabeled proteins and peptides is
`described in U.S. Pat. No. 5,384,113, incorporated herein by
`reference, and use of ascorbic acid to stabilize some
`chemical-based radiolabeled compounds, but not protein- or 5
`peptide-based radiolabeled compounds, is described in 1bfe,
`A. J. and Francis, M. D.,J. Nucl. Med., 17, 820---825 (1976).
`However, ascorbic acid bas been recognized in the art as
`unsuitable for use as a stabilizing agent with many chemical(cid:173)
`based radiolabeled compounds, presumably because it com- JO
`petes for the 99""fc and forms a 99"'Tc-ascorbale complex.
`Ballinger, J., Der, M., and Bowen, B., Eur. J. Nuc/. Med., 6,
`154-154 (1981). In fact, because of the stability of
`Tc-ascorbate complex, ascorbic acid has been labeled with
`technetium by numerous investigators for use as a potential l 5
`renal imaging agent. In addition, use of ascorbic acid prior
`to and during radiolabeling has been described in U.S. Pat.
`No. 5,011,676, and has been described
`in
`Radiopharnaceuticals, G. Subramanian, B. A. Rhodes, J. F.
`Cooper and V. J. Sodd, eds, Society of Nuclear Medicine, 20
`New York, 1975, pp. 37-38, as an agent, used either singly
`or in combination with Fe(III), in technetium labeling of
`HSA. Despite tbe promise shown by a number of newly(cid:173)
`developed proteins and peptides for diagnostic and thera(cid:173)
`peutic appbcations, susceptibility to oxidation loss, autora- 25
`dialysis and other impurities may limit use. Therefore, tbe
`development of means for the effective stabilization of
`radiolabeled compounds, without loss due to the stabiJiz.ing
`agent, is a significant and mucb-needed advancement in the
`art.
`
`SUMMARY OF THE INVENTION
`
`4
`agents 10 the pre-radiolabeled composition in an amount
`effective to prevent degradation of the radiolabeled
`substance, such as that cause<! by autoradiolysis of the
`labeled composiiion and oxidation loss of the radioJabel.
`Tbere is also provided a method of preparing a stabilized
`peptide or protein radiopharmaceutical composition, com(cid:173)
`prising the ordered steps of labeling a peptide or protein with
`a radioisotope to form a radiolabeled pharmaceutical
`product, said radiolabeled pharmaceutical product being
`substantially free of any stabilizing agents, and then adding
`a stabilizing agent consisting of ascorbic acid, or one or
`more derivatives thereof (such as salts, esters, and mixtures
`tbereot) to the radiolabeled pharmaceutical product. In one
`embodiment, the radioisotope may be an isotope of rhenium
`or technetium, and particularly 99"'Tc, 186 Re or 188Re.
`There is also provided a method of preparing a stabilized
`rhenium-labeled peptide-based somatostatin analogue
`radiopharmaceutical composition, comprising the ordered
`steps of labeling said peptide with an isotope of rhenium to
`form a radiolabeled pharmaceutical product, said radiola(cid:173)
`beled pharmaceutical product being heretofore substantially
`free of any stabilizing agents, and then adding a stabilizing
`agent consisting of ascorbic acid, its salts, esters, derivatives
`and mixtures thereof to the radiolabcled pharmaceutical
`product. In one embodimeut, the radioisotope may be 186 Re
`or 188Re.
`There is also provided a method of preparing a stabilized
`technetium-labeled anti-SSEA-1 IgM antibody-based
`30 radiopharmaceutical composition, comprising the ordered
`steps of labeling said anli-SSEA-1 antibody with an isotope
`of technetium 10 form a radiolabeled pharmaceutical
`product, said radiolabeled pharmaceutical product being
`heretofore substantially free of any stabilizing agents, and
`35 then adding a stabilizing agent consisting of ascorbic acid,
`its salts, esters, derivatives and mixtures thereof to the
`radiolabeled pharmaceutical product. In one embodiment,
`tbe radioisotope may be 9 9"'Tc.
`Additionally, the present invention provides stabilized
`40 compositions containing a radiolabeled protein or peptide,
`which compositions have been stabilized by the addition of
`a stabilizing agent containing ascorbic acid or a derivative
`thereof 10 a composition containing said protein or peptide
`already radiolabeled.
`
`Accordingly, it is an object of the present invention to
`provide a composition containing a radiolabeled protein or
`peptide having improved stability (e.g., against oxidation,
`autoradiolysis and more generally degradation). Another
`object is to provide novel methods for stabilizing a radio(cid:173)
`labeled protein peptide or polypeptide.
`It is a further object of the invention to provide a diluent
`and a bodying agent, providing volume to the radiolabeled
`protein or peptide preparation which is helpful in a phar(cid:173)
`maceutical or clinical setting for case of manipulation and
`administration. It is a further object of the invention to
`provide a stabilizing agent which further stabilizes a radio- 45
`labeled protein or peptide preparation containing excess
`stannous or stannic ions, preventing formation of
`Sn-colloids or otber radiochemical impurities. It is a further
`object of the invention to provide a radiolabeled protein or
`peptide composition wbich uses a relatively non-toxic and 50
`readily obtainable stabilizing agent. It is a further object of
`the invention to provide stabilized racliopharmaceutical
`products made by the various methods disclosed herein(cid:173)
`above.
`Other objects, advantages, and novel features, and further
`scope of applicability of the present invention will be set
`forth in part in the detailed description to follow, and in part
`will become apparent to tbose skilled in the art upon
`examination of the following, or may be learned by practice
`of tbe invention. Tbe objects and advantages of tbe invention
`may be realized and attained by means of the instrumen(cid:173)
`talities and combination particularly pointed out in the
`appended claims.
`The present invention provides a method for stabilizing
`radiopharmaceutical compositions, including compositions 65
`based on peptides and proteins, including antibodies. The
`method comprises adding ascorbate or other stabilizing
`
`55
`
`FIG. 1. Elution profile of Rc-188-RC-160 labeled with 65
`mCi of Re-188 at 6 hours post-labeling from a C18-reverse(cid:173)
`phase HPLC column. The y-axis in arbitrary units. The
`figure inset is an elution profile of Re-188-RC-160 from a
`C18-SepPak column using a step-gradient of acidified etha(cid:173)
`nol.
`FIG. 2. Comparative elutions profiles of Re-188- RC-160
`radiolabeled at either 90° C. or 37° C.
`FIG. 3. Displacement of Re-188 from Re-188- RC-160
`using increasing concentrations of cysteine as a challenge
`agent.
`FIG. 4. Comparative biodistribution of Re-188-RC-160
`60 racliolabeled at either 90° C. or 37° C. in normal female
`mice.
`FIG. 5. Comparison of Tc-99m- or Re-188-
`labeled
`RC-160 over Lime as analyzed by TLC. Saline was used as
`a mobile phase. Similar amounts (20 mCi) of each radio(cid:173)
`nuclide were used, and the same formulation (optimized for
`Re-labeling) was used in U1e comparison. No post-labeling
`stabilizer was used.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
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`16 of 22
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`

`

`FIG. 6. Comparative TLC of Re-188-RC-160 with and
`without post-labeling stabilization with ascorbate.
`FIG. 7. Elution profile ofRe-188-RC-160 labeled with 65
`mCi of Re-188 at 30 hours post-labeling from a C18-reverse(cid:173)
`phase 1-IPLC column. The y-axis in arbilrary units.
`FIG. 8. Comparative elution profiles of Re-188-RC-160
`with ascorbate added after the radiolabeliog (top) or before
`the radiolabeling (boltom). Both preparations were radiola(cid:173)
`beled with 10 mCi (370 Mbq) of Re-188.
`FIG. 9. Labeling of a RhoMed radiophamaceutical prod- JO
`ucl koowo as LeuTec-MTM, a 9 9"'Tc-labeled anti-SSEA-1
`antibody. (This product is referred to as " Leuko-1" in FIG.
`9, which is a summary of the test results from a lot of
`LeuTec-MTM manufactured by RhoMed.) This data shows
`the difference in radiolabeling yields between unstabilized l S
`and stabilized (with ascorbate) product.
`FIG. 10. 1-IPLC profile on 99"'Tc-labeled IgM samples
`showing the difference in yield over time with labeling
`occurring in the presence of ascorbate or with postlabcling
`addition of ascorbate.
`
`20
`
`DETAILED DESCRIPTION
`
`25
`
`The present invention is directed to radiolabeled protein-
`or peptide-containing compositions with a stabilizing agent
`added subsequent to radiolabeling but prior to use to prevent
`oxidation and autoradiolysis. One class of stabilizing agent
`which is effective at preventing oxidation and autoradiolysis
`is ascorbic acid (and derivatives thereoJ). With many
`protein- and peptide-based radiolabeled compositions, 30
`ascorbic acid and its derivatives interfere with radiolabcling
`if included in the composition prior to or during radiolabel(cid:173)
`ing. However, the inventors have found that, unexpectedly,
`when added subsequent to radiolabeling (and subsequent to
`any incubation period), ascorbic acid and its derivatives 35
`result in a radiolabeled substance of superior stability and
`body (volume). The radiolabeled proteins and peptides
`which are effeaively stabilized according to the present
`invention include radiopharmaceutical drugs having diag(cid:173)
`nostic or therapeutic applications.
`While the methods of this invention are particularly
`applicable to stabilizing compositions including isotopes of
`technetium and rhenium, such as 99"'Tc, 186Re and 188Re,
`because of the particular susceptibility of such isotopes to
`oxidation and/or autoradiolysis, the methods of this ioven- 45
`tion can be used with compositions including a wide variety
`of isotopes, including those found in the group consisting of
`elements 26-30 (Fe, Co, Ni, Cu, Zn), 33-34 (As, Se), 42-50
`(Mo, Tc, Ru, Rh, Pd, Ag, Cd, In, So) and 75-85 (Re, Os, Ir,
`Pt, Au, Hg, Ti, Pb, Bi, Po, At) of the Periodic Table of 50
`Elements. Radioisotopes with diagnostic or therapeutic
`applications include 62Cu, 64Cu, 67Cu, 97Ru, 105Rh,
`109Pd, 180Re, 188Re, 198Au, 199Au, 203Pb, 211Pb and
`212Bi.
`The radiolabeled peptide compositions stabilized by 55
`means of this invention include proteins, peptides and
`polypeptides which are naturally-occurring, as well as those
`produced by chemical synthesis, by recombinant DNA
`technology, by biochemical or enzymatic fragmentation of
`larger molecules, or by any other means for producing them.
`For example, peptides stabilized by the present method
`include peptide fragments, polypeptides and other structu,res
`derived therefrom, generally consisting of a sequence of
`amino acids. Representative examples of peptides include
`those derived from laminin, fibronectin, cytokines, 65
`lymphokines, serum albumin, fibrinogen, enzymes,
`hormones, somatostatin, urokinase, tissue plasminogen
`
`US 6,261,536 Bl
`
`s
`
`6
`activator, and protease inhib.itors. Peptides will generally
`comprise fewer than 100 amino acids, and preferably fewer
`than 60 amino acids, and most preferably comprise between
`about 4 and about 20 amino acids. The amino acids forming
`5 all or a part of the peptide may be naturaJly occurring amino
`acids, isomers and modifications of such amino acids, non(cid:173)
`protein amino acids, posl-lranslationally modified amino
`acids, enzymatically synthesized amino acids, isosteric
`amino acid analogues and the like, so that the LeCTJl "peptide"
`includes pseudopeptides and peptidomimetics. The term
`"peptide" also includes cyclic peptides, bridged peptides
`(e.g., disulfide-bridged peptides), dimers or rnultimers of
`peptides. Radiolabeled protein compositions stabilized by
`means of this invention include such proteins, whether
`natural or synthetic, as human seru rn albumin, fibrinogen,
`urokinase, gamma globulin, laminin, fibronectin, cytokines,
`lymphokines, enzymes, enzyme inhibitors, hormones,
`glycoproleins, and irnmunoglobulins. The term "protein" as
`used throughout the specification and claims is intended to
`include all of the foregoing substances. The protein is
`typically of marnmaJian origin, but also includes proteins of
`plant origin and proteins from prokaryotic cells. Methods of
`attaching or complexing proteins to other molecules, such as
`lipids and carbohydrates, including liposomes, are known to
`those skilled in the art.
`Immunoglobulins, a type of protein, include antibodies
`and antibody fragments (including fragments consisting
`essentially of an antigenic determinant or antigen binding
`site), of any species, ai1d include both polyclonaI and
`monoclonal antibodies made by any means, as well as
`chimeric and genetically engineered antibodies, hybrids, and
`fragments of all of the foregoing. 111is includes immuno(cid:173)
`globulins of any class, such as IgG, lgM, lgA, lgO or IgE,
`of any species origin, including human beings, chimeric
`antibodies or hybrid antibodies with dual or multiple antigen
`or epitope specificities, and fragments of all of the
`foregoing, including F(ab)2, F(ab)2, Fab, Fab and other
`fragments, including hybrid fragments, and further includes
`any irnrnunoglobulin or any natural, synthetic or genetically
`40 engineered protein that functionally acts like an antibody by
`binding specifically to a given antigen to form a complex,
`including single ctiain anti bodiC$. Toe terms "antibody" or
`·' antibodies" as used throughout the specification and claims
`are intended to include all such antibodies and antibody
`fragments.
`The products stabilized by means of the invention set
`forth herein can be used for both medical applications and
`veterinary applications. Typically, the product is used in
`humans, but may also be used in other mammals. The term
`"patient" is intended to include a mammal, and is so used
`throughout the specification and in the claims. The primary
`applications o( the invention involve human patients, but the
`invention may be applied to laboratory, farm, zoo, wildlife,
`pct or sport animals.
`The products stabilized by means of the invention set
`forth herein can be used as imaging agents, for example, to
`view tissues in order 10 monitor normal or abnormal meta(cid:173)
`bolic events, lo localize normal or abnormal tissues, lo
`localize diseases, to diagnose or treat diseases, and to bind
`60 to blood constituents, including blood cells, such as
`lymphocytes, for subsequent localization of diseases,
`infections, and abnormal tissues. The application and medi(cid:173)
`cal use of the product depends on the type of protein or
`peptide and the type of radioisotope used.
`The protein or peptide is first labeled with a radioisotope
`which can be accomplished using known techniques, and
`once labeling i<; complete, it is then stabilized according to
`
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`

`

`US 6,261,536 Bl
`
`7
`the invention. The radioisotope may generate gamma rays,
`beta particles, or positrons wbich arc converted into gamma
`rays upon collision witb electrons. Tbe radioisotope may be
`used in diagnostic imaging procedures including gamma
`scintigraphy, specific pboton emission computerized
`tomograpby, or positron emission tomograpby, or may be
`used tbera peu ti call y.
`With 99"''fc, 186Re or 188Re labeled peptides or proteins,
`regardless of the particular method of radiolabeling
`employed, tbe addition of ascorbic acid to acbieve concen(cid:173)
`trations between about 0.001 wt % and 5 wt % to tbe
`composition post-labeling increases the stability of the
`radiolabeled composition, with apparent increased resis(cid:173)
`tance to oxidation ancVor radiolytic decomposition. If excess
`stannous or stannic ions are present in tbe composition of tbe
`labeled peptide or protein, the addition of ascorbic acid also
`prevents formation of Sn-colloids and other radiochemical
`impurities. This thus allows sufficient stannous ion to be
`added to a preparation to be radiolabeled to insure complete
`reduction of the pertechnetate or perrhenate, even if there is
`some oxidation loss of stannous to stannic ion, without
`formation of undesirable radiocbemical impurities fre(cid:173)
`quently found with excess tin. As staied above, presence of
`ascorbic acid or a derivative tbereof in a composition
`containing peptides and proteins, and radiolabeling agents
`before the completion of radiolabeling, regardless of tbe
`method of radiolabeling, inclusion of ascorbic acid prior to
`ordinary labeling has a detrimental effect on radiolabeling
`yields. When derivatives are used an equivalent amount (on
`a molar basis) can be used.
`Addition of ascorbic acid or a derivative thereof post(cid:173)
`la