The present invention relates to novel stabilizing agents (stabilizers) of radiopharmaceutical compositions used for diagnosis and therapy. In particular the invention relates to the use of an uric acid derivative to increase the shelf-life of diagnostic and therapeutic peptidic radiopharmaceuticals by reducing the side products originated from radiolysis. Introduction

Peptidic diagnostic and therapeutic radiopharmaceuticals are often not stable, particularly at high activity levels. At high radioactivity concentrations e.g. during radiolabeling the peptidic moieties are often very susceptible to decomposition or modifications by radiolytic processes. Since the target specificity of the radioisotope labeled pharmaceutical peptidic pharmaceutical is largely dependent on the integrity of the peptide motif, radiolytic decomposition may lead to decreased diagnostic and therapeutic efficacy and unwanted radiation toxicity (Liu, S. and D.S. Edwards, Bioconjugate Chemistry, 2001. 12(4): p. 554-558). The mechanism for radiolytic decomposition is thought to be caused by free radicals such as hydroxyl and superoxide radicals formed in the presence of a large amount of ionizing radiation arising from the radioisotopes. Known radical scavengers such as human serum albumin (Kishore R, Early JF, Krohn KA, et al., !nt J of Radial Appl Instrum Part B, Nucl Med and Biol 1986;4:457-459), gentisic acid, and ascorbic acid have been applied successfully as stabilizers for radiolabeled antibodies, but if they fail the strong demand of alternative stabilizers becomes evident.

Background

Galtium-68 is a metallic, generator-produced radionuclide that has become widely available through commercial generators from a number of suppliers for radiopharmaceutical equipment (e.g. Eckert & Ziegier Radiopharm AG, Berlin, Germany; Veenstra Instruments, Netherlands; Scintomics GmbH, Furstenfeldbruck, Germany). Its production is independent of an on-site cyclotron and the availability of this positron emitter has given rise to a continuously increasing number of clinical studies (Al-Nahhas, A., et al., Anticancer Research, 2007. 27(6B): p. 4087-4094). With well established procedures (Ocak, M., et al., Applied Radiation and Isotopes, 2010. 68(2): p. 297-302) to insert this radionuclide into small molecules and peptides, Ga has found access to clinical research and routine production in a GMP environment. Its half-iife (68 min) and high specific activity has proven useful to provide molecular information for a variety of applications (Al-Nahhas, A., et al., Anticancer Research, 2007. 27(6B): p. 4087-4094; Ambrosini, V., et a!., Journal of Nuclear Medicine, 2010. 51 (5): p. 669-673; Ambrosini, V., et al. ₍ European Journal of Nuclear Medicine and Molecular Imaging, 2010. 37(4): p. 722-727; Antunes, P., et al., European Journal of Nuclear Medicine and Molecular Imaging, 2007. 34(7): p. 982-993; Hofmann, M., et al., European Journal of Nuclear Medicine, 2001. 28(12): p. 1751-1757; Maecke, H.R., M. Hofmann, and U. Haberkom, Journal of Nuclear Medicine, 2005. 46: p. 172S-178S; Wild, D., et ai., European Journal of Nuclear Medicine and Molecular Imaging, 2005. 32(6): p. 724-724; Conry, B.G., et al., European Journal of Nuclear Medicine and Molecular Imaging, 2010. 37(1): p. 49-57; Pettinato, C, et al., European Journal of Nuclear Medicine and Molecular Imaging, 2008. 35(1 ): p. 72-79; Mansi, R., et al., European Journal of Nuclear Medicine and Molecular Imaging, 2011. 38(1): p. 97-107). Beside ⁶⁸ Ga a number of radionuclides are routinely employed in diagnostic use, such as Tc-99m, ln-111 , F- 8, and TI-20 . Other radionuclides are in therapeutic use, such as Y-90, 1- 31 , P-32, Sr-89, Sm-153.

With the availability of fully automated, commercial synthesis devices, the radiolabeling of small peptides (especially with ^s8 Ga) has become an easy task and is considered to be well described with respect to its chemistry (Decristoforo, C, et al., European Journal of Nuclear Medicine and Molecular Imaging, 2006. 33: p. S162-S162; Decristoforo, C, et al., Nuclear Medicine Communications, 2007. 28(11): p. 870-875; Di Pierro, D., et al., Applied Radiation and Isotopes, 2008. 66(8): p. 1091-1096; de Blois, E., et al., Applied Radiation and Isotopes, 201 . 69(2): p. 308-315).

Chemical decomposition may limit a radiopharmaceutical's shelf life by decreasing the radiochemical purity of the agent over the time. For example, the peptide or even the radionuclide itself might be susceptible to oxidation as a consequence of the exposure to ionizing radiation. In addition, the radiation emitted from a radionuclide can break chemical bonds of a peptide or other components of the composition, thus causing autoradiofysis (Cyr J., US patent 6881396).

Thus, many radiopharmaceuticals require stabilizing agent(s) to maximize shelf life. Such stabilizing agent(s) must be non-toxic and must be able to maintain the product's radiochemical purity for an acceptable shelf-life as well as during use. In addition, an acceptable radiopharmaceutical stabilizing agent should not interfere with the radiolabeling process and, if required, the stabilizing agent should be easily removable before application of the radiopharmaceutical. Problem to be solved by the invention and its solution

Radiolysis is a phenomenon in which the formation of ionized molecules leads to side products and degradation products. This is known from a number of different applications, e.g. from ¹⁸ F-labelled radiopharmaceuticals (Jacobson, M.S., H.R. Dankwart, and D.W. Mahoney, Applied Radiation and Isotopes, 2009. 67(6): p. 990- 995) or the field of metalioradiopharmaceuticals. There, where for therapeutics a high radioactivity concentration is produced, radiolysis is a known problem and different approaches have been investigated in order to avoid radiolytic side products. The use of gentisic acid and ascorbic acid as scavenger is common (Liu, S. and D.S. Edwards, Bioconjugate Chemistry, 2001. 12(4): p. 554-558) but was found to be insufficient in the studied cases. Another influential factor described in the literature is the presence of oxygen in the reactants. Liu et al. (Liu, S., C.E. Eilars, and D.S. Edwards, Bioconjugate Chemistry, 2003. 14(5): p. 1052-1056) present a radiosynthesis with exclusion of oxygen to avoid radiolytic degradation. In the studied cases due to the setting of the radiosynthesis which comprises the use of kits and cassettes, the presence of oxygen cannot be excluded.

Labeling the DOTA-RM2 peptide with ^6s Ga at high specific activities commonly used stabilizers e.g. gentisic acid and ascorbic acid failed and a high amount of side products was observed.

Thus, there is a need to provide a stabilizing agent avoiding production of side products due to radiolysis or oxidation while fulfilling the requirements for GMP compliant patient application in a clinical setting.

Summary of the invention

It has now been surprisingly found that the radiolabelling quality and shelf life of peptidic radiopharmaceutical compositions may be significantly increased by an addition of a stabilizing amount of uric acid or a derivative thereof.

In one embodiment, the invention provides a composition comprising a radiopharmaceutical precursor and a stabilizing amount of uric acid or a derivative thereof. In another embodiment, the invention provides a method of stabilizing a radiopharmaceutical comprising the steps of:

combining a precursor of said pharmaceutical with a stabilizing amount of uric acid or a derivative thereof in a container; and

adding a radionuclide to the container.

In a further embodiment, the invention provides a method for stabilizing the peptide radiopharmaceutical by adding the stabilizing amount of an uric acid derivative during the radiolabelling.

In a further embodiment, the invention provides a method for stabilizing the peptide radiopharmaceutical by adding the stabilizing amount of an uric acid derivative right after the radiolabelling.

in a further embodiment, the invention provides a kit comprising a sealed vial containing a predetermined quantity of a radiopharmaceutical precursor and a stabilizing amount of uric acid or a derivative thereof.

In a further embodiment, the present invention relates to the use of uric acid or derivatives thereof for stabilizing a peptidic radiopharmaceutical.

In a further embodiment, the present invention relates to the use of uric acid or derivatives thereof in a stabilizing amount thereof, being in the range of 1 pg/mL to 1.000 g/mL for stabilizing a peptidic radiopharmaceutical.

In a further embodiment, the present invention relates to the use of a kit comprising a sealed vial containing a predetermined quantity of a radiopharmaceutical precursor and a stabilizing amount of uric acid or a derivative thereof for stabilizing a peptidic radiopharmaceutical.

Description

In a first aspect, the invention is directed to a method for stabilizing a peptidic radiopharmaceutical,

characterized in that uric acid derivatives are used as stabilizing agent.

Sub-embodiments: Peptidic radiopharmaceutical

in a sub-embodiment, the peptidic radiopharmaceutical is a natural or synthetic peptide labeled with a radioisotope or a complex comprising a radioisotope and a chelator wherein the natural or synthetic peptide is suitable for being labeled with a radioisotope.

In a sub-embodiment, the natural or synthetic peptide is a peptide comprising of 4 to 700 amino acids wherein the amino acids may be selected from natural and synthetic amino acids and also may comprise modified natural and non-natural amino acids. Preferably, the peptide is of 4 to 50 amino acids.

More preferably, the peptide is of 4 to 15 amino acids.

Even more preferably, the peptide is, but is not limited to, somatostatin and derivatives thereof and related peptides, somatostatin receptor specific peptides, neuropeptide Y and derivatives thereof and related peptides, neuropeptide and the analogs thereof, bombesin and derivatives thereof and related peptides, gastrin, gastrin releasing peptide and the derivatives thereof and related peptides, epidermal growth factor (EGF of various origin), insulin growth factor (IGF) and IGF-1 , integrins (α ₃ βι , α _ν β ₃ , α _ν β ₅₎ allb ₃ ), LHRH agonists and antagonists, transforming growth factors, particularly TGF-a; angiotensin; cholecystokinin receptor peptides, cholecystokinin (CCK) and the analogs thereof; neurotensin and the analogs thereof, thyrotropin releasing hormone, pituitary adenylate cyclase activating peptide (PACAP) and the related peptides thereof, chemokines, substrates and inhibitors for cell surface matrix metalloproteinase, prolactin and the analogs thereof, tumor necrosis factor, interleukins (IL-1 , IL-2, IL-4 or IL-6), interferons, vasoactive intestinal peptide (VIP) and the related peptides thereof. Even more preferably, the peptide is selected from the group comprising bombesin and bombesin analogs, preferably those having the sequences listed herein below, somatostatin and somatostatin analogs, preferably those having the sequences listed herein below, neuropeptide Y _f and the analogs thereof, preferably those having the sequences listed herein below, vasoactive intestinal peptide (VIP) and the analogs thereof.

Even more preferably, the peptide is bombesin and the analogs thereof,

in a sub-embodiment, the peptide is bombesin and the analogs thereof.

Bombesin is a fourteen amino acid peptide that is an analog of human Gastrin releasing peptide (GRP) that binds with high specificity to human GRP receptors present in prostate tumor, breast tumor and metastasis. The invention is related to peptides as listed above but also to their analog peptides wherein analog peptides have mutated amino acids compared to original peptide and retain biological activity of the original peptide. Peptides are preferably selected from but not limited to

Seq ID 1 : D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH ₂ (peptide RM2);

Seq ID 2: D-Phe-Gln-Trp-Ala-Val-Gly-H!S-Leuψ{CHOH-CH ₂ )-(CH ₂ ) ₂ -CH3;

Seq ID 3: D- he-Gln-Trp-Ala-Val-Gly-His-Leuψ(CH ₂ NH)-Phe-NH2;

Seq ID 4: D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu (CH ₂ NH)-Cys-NH ₂ ; .

Seq ID 5: Gin-Trp-Ala-Va!-Gly-His-Sta-Leu-NH ₂ ;

Seq ID 6: ΰ\η-Ύΐρ-Α\3-^\'β^-Η5-Ιβυψ(ΟΗΟΗ-ΟΗ ₂ )-(Ο ₂ ) ₂ -ΟΗ ₃ -,

Seq ID 7: Gln-Trp-Ala-Val-Giy-Hts-Leuψ{CH ₂ NH)-Phe-NH ₂ ;

Seq ID 8: Gln-Trp-Ala-Val-Gly-His-Leu (CH ₂ NH)-Cys-NH ₂ ;.

Seq ID 9: Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH ₂ ;

Seq ID 10: ΟΙη-ΤΓρ-Αΐ3- 3ΐ- ^β ΟΙγ-Ηί8-Ιβυψ(ΟΗΟΗ-ΟΗ ₂ ΗΟΗ ₂ )2-ΟΗ ₃ ;

Seq ID 1 1 : Gln-Trp-Ala-Val-NMeGly-His-Leuψ(CH ₂ H)-Phe- H ₃ ;

Seq ID 12: 0Ιη-ΤΓρ-Α!3-ν3ΐ-ΝΜβ0Ιγ-Ηί5~ίβυψ(0Η ₂ Η)-0γ5- Η ₂ ;

Seq ID 13: D-Phe-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH ₂ ;

Seq ID 14: Ι>ΡΓΐβ-βΙη-ΤΓ -Αΐ3-ν3ΐ-Ν Θ6Ιγ-Ηϊ8-ί6υψ(0ΗΟΗ-0Η2)-(0Η ₂ ) _Γ 0Η ₃ ;

Seq ID 15: D-Phe-Gln-Trp-Aίa-Vai- MeGly-H^s-Leuψ(CH ₂ H)-Phe-NH ₂ ;

Seq ID 16: D-Phe-Gln-Trp-Ala-Val-NMeGly-His-Leu _v (CH ₂ NH)-Cys-NH ₂ ;

Seq ID 17: D-PHE-CYS-TYR-D-TRP-LYS-THR-CYS-TH -OH, (DISULFIDE

BOND) (peptide TATE);

Seq ID 18: D-Phe-cyclo[Cys-Tyr-D-Trp-Lys-Thr-Cys]-Thr(o!) (peptide TOC).

Peptidic radiopharmaceutical are preferably selected from but not limited to

[ ⁶⁸ Ga]~DOTA-Gly-aminobenzoyl-D-Phe-Gln-Trp-Ala-Val-Gly-Hi s-Sta-Leu-NH ₂ ;

[ ⁶⁸ Ga]-DOTA-4-amino-1 -carboxymethyl-piperidine-D-Phe-Gln-Trp-Aia-Val-Gly-His-Sta- Leu-NH ₂ ;

[ ⁶⁸ Ga]-DOTA-4-amino-1-piperidine-4~carboxylicacid-D-Phe-G ln-Trp-Ala-Val-Gly-His- Sta-Leu-NH ₂ ;

[ ⁶⁸ Ga]-DOTA- 5-amino-4,7, 0, 13-tetraoxapentadecanoic acid-D-Phe-Gln-Trp-Ala-Val- Gly-His-Sta-Leu-NH ₂ ; f Ga]-DOTA-( 5-amino-4,7 , 10,13-tetraoxapentadecanoic acid)-(4-amino-1 - carboxymethyl-piperidine)-D-Phe-Gln-Trp-Ala-Val-GIy-His-Sta- Leu-NH ₂ ;

[ ^e8 Ga]-DOTA-diaminobutyricacid-D-Phe-Gin-Trp-Ala-Val-Gly- His-Sta-Leu-NH ₂ ;

[ ⁶⁸ Ga]-DOTA-4-(2-aminoethyl)-1-carboxymethyl-piperazine-D -Phe-Gln-Trp-Ata-Val-Gly- Hts-Sta-Leu-NH ₂ ;

[ ⁶⁸ Ga]-LeuDOTA-(5-amino-3-oxa-penty))-succinamic acid-D-Phe-G!n-Trp-Ala-Val-Gly- His-Sta-Leu-NH ₂ ;

^Gaj-DOTA^-amino-l-carboxymethyl-piperidine-D-Phe-Gln-Trp-Ai a-Val-Gty-His- Le^(CHOH-CH ₂ HCH ₂ ) ₂ -CH ₃ ;

[ ⁶⁸ Ga]-DOTA-(15-amino-4,7,10, 13-tetraoxapentadecanoic acid-4-amino-1- carboxymethyl-piperidine-D-Phe-Gln-Trp-A[a-Val-Gly-His-Leuψ (CHOH-CH ₂ )-(CH ₂ )2- CH ₃ ;

[ ⁶⁸ Ga]-DOTA- 5-amino-4,7, 10, 13-tetraoxapentadecanoic acid -D-Phe-Gln-Trp-Ala-Val- Gly4His-Le^(CHOH-CH ₂ )-(CH ₂ )2-CH ₃ ;

[ ⁶⁸ Ga]-DOTA-4-amino-1 -carboxymet yl-piperidine-D-Phe-Gln-Trp-Aia-Val-Giy-His- Leu4J(CH ₂ NH)-Phe-NH ₂ ;

[ ⁶⁸ Ga]-DOTA-4-amino-1-carboxymethyl-piperidine-D-Phe-Gln- Trp-Ala-Vai-Gly-His- Le^(CH ₂ NH)-Cys-NH _z ;

[ ⁶⁸ Ga]-DOTA-4-arnino-1-carboxymethylpiperidine-D-Phe-Gln- Trp-Ala-Val-Gly-His-Sta- -NH ₂ , ⁶⁸ Ga-DOTA-RM2

3-cyano-4-[ ¹⁸ F]fluorobenzoyl-Ala(S0 ₃ H)-Ala(S0 ₃ H)-Ava-Gin-Trp-Ala-Val-NMeGly-His- Sta-Leu-NH ₂

-cyano-4-[ ⁸ F]fluorobenzoyl-Ala(S0 ₃ H)-Ava-G!n-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH2

Ga-DOTATATE

Preferably:

[ ^e8 Ga]-DOTA ^~ 4-amino-1-carboxymethylpiperidine-D-Phe-Gln-Trp-Afa-Va l-Gly-His-Sta- Leu~NH ₂ , ⁶⁸ Ga-DOTA-RM2

3-cyano-4-[ ¹⁸ F]fluorobenzoyl-Ala(S0 ₃ H)-Ala(S0 ₃ H)-Ava-Glri-Trp-Ala-Val-NMeGly-His- Sta-Leu-NH ₂

3-cyano-4-[ ^{1 B} F]fluorobenzoyi-Ala(S0 ₃ H)-Ava-G[n-Trp-Ala-Vai-NMeGiy-His-Sta-Leu-NH _:

8, Ga-DOTATOC

Radioisotope or complex comprising a metal radioisotope and a chelator.

In a sub-embodiment, the radioisotope is selected from the group comprising carbon- 11 ( ¹¹ C), nitrogen-13 ( ³ N) _S oxygen-15 ( ^1S 0), bromine-75 ( ⁷⁵ Br), bromine-76 ( ^7B Br) _r iodine-124 ( ¹²⁴ l) and ffuorine-18 ( ¹⁸ F). Preferably, the radioisotope is selected from the group comprising bromine-75 ( ⁷⁵ Br), bromine-76 ( ⁷⁶ Br), iodine-124 ( ¹²⁴ l) and fluorine-18 ( ¹⁸ F). More preferably, the radioisotope is fluorine-18 ( ¹⁸ F).

In a sub-embodiment, the metal radioisotope complexed to a chelator is selected from the group comprising ¹⁷⁷ Lu, ⁹⁰ Y, ¹³³ η, ^99m Tc, ⁶⁷ Ga, ⁵² Fe, ⁶⁸ Ga, ⁷² As, ¹¹¹ ln, ⁹⁷ Ru, ²⁰³ Pb, ⁶² Cu, ⁶⁴ Cu, ⁵¹ Cr, ^52m Mn, and ¹⁵⁷ Gd. Preferably, the metal radioisotope is selected from the group comprising ^99m Tc, ⁶⁷ Ga, ⁶⁸ Ga, and ¹¹ ln. More preferably, the metal radioisotope is ⁶⁸ Ga.

In a sub-embodiment, the chelator complexing the metal radioisotope is selected from the group comprising

DOTA-, NODASA-, NODAGA-, NOTA-, DTPA-, EDTA-, TETA-, and TRITA- based chelators, CE-DTS, DADT derivative, triamidethiol derivative, DADS derivative, hydrazinonicotinic acid, and bis(hydroxoamamide) derivative wherein

DOTA stands for 1 ,4,7,10-tetrazacyclododecane-N, Ν',Ν",^" tetraacetic acid,

DTPA stands for diethylenetriaminepentaacetic acid,

EDTA stands for ethylenediamine-N.N'-tetraacetic acid,

TETA stands for 1 ,4,8,1 1 -tetraazacyclododecane-1, 4,8, 11 -tetraacetic acid,

NOTA stands for 1 ,4,7-triazacyclononanetriacetic acid,

CE-DTS stands for 3-{4-[(5E,7E)-3,10-dithioxo-2,4,5,8,9,1 1-hexaazadodeca-5,7-dien- 6-yl]phenyl}propanotc acid,

DADT derivative stands for 4-methyl-3,4-bis[(2-methyl-2- su[fany!propyl)amino]pentanoic acid,

triamidethiol derivative stands for N-(sulfanylacetyl)glycylglycylglycine,

DADS stands for N-{sulfanylacetyl)-3-[(sulfany!acetyl)amino]alanine, and

bis(hydroxoamamide) derivative stands for 4-amino-N'-hydroxy-N-(3-{[(Z)- (hydroxyimino)methyl]amino}propyl)benzenecarboximidamide.

Preferably, the chelator is selected from the group comprising:

DOTA-, NOTA-, DTPA-, and TETA-based chelators.

DTPA n = n' = 1 DOTA NOTA n = n' = 2 TETA

Preferably, ^99m Tc or ^186/188 R _e chelators are selected from the group comprising:

bis{hydroxoamamide) derivative

Uric acid derivatives

Uric acid derivatives mean uric acid as such, derivatives thereof and/or mixture thereof. In a sub-embodiment, the uric acid derivatives are compounds of formula I

wherein

R1 is Hydrogen, substituted or unsubstituted C,-C ₃ alkyi group, CH ₃ -(CH ₂ ) _m -0-(CH ₂ - CH ₂ -0) _n -CH ₂ -CH ₂ , n = 0, 1 , 2 or 3 and m = 0 or 1 or substituted or unsubstituted acyi group,

R2 is Hydrogen, substituted or unsubstituted C C ₃ alkyl group, CH ₃ -{CH ₂ )m-0-(CH ₂ - CH ₂ -0) _n -CH ₂ -CH ₂ , n = 0, 1 , 2 or 3 and m = 0 or 1 or substituted or unsubstituted acyi group,

R3 is Hydrogen, substituted or unsubstituted C C ₃ alkyl group, CH ₃ -(CH ₂ ) _m -0-(CH ₂ - CH ₂ -0) _n -CH2-CH ₂ , n = 0, 1 , 2 or 3 and m = 0 or 1 or substituted or unsubstituted acyi group, and

R4 is Hydrogen, substituted or unsubstituted ( C ₃ alkyl group, CH ₃ -(CH ₂ ) _m -0-(CH ₂ - CH ₂ -0) _n -CH ₂ -CH _2i n = 0, 1, 2 or 3 and m = 0 or 1 or substituted or unsubstituted acyi group. in a sub-embodiment, R1 , R2, R3 and R4 are independently from each other Hydrogen, or substituted or unsubstituted C1-C3 alkyl group.

In a sub-embodiment, R1 , R2, R3 and R4 are independently from each other Hydrogen, CH ₃ -(CH ₂ ) _m -0-(CH ₂ -CH ₂ -0) _n -CH ₂ -CH ₂ , n = 1, 2 or 3 and m = 0 or 1 .

In a sub-embodiment, R1 , R2, R3 and R4 are independently from each other Hydrogen, or substituted or unsubstituted acyi group. Preferably, unsubstituted C C ₃ alkyi group is methyl or ethyl, More preferably, unsubstituted C1-C3 alkyl group is methyl.

Preferably, substituted alkyl group is substituted with one or two substituents defined as hydroxyl, hydroxy methyl, methyl, ethyl, methoxy, methoxymethyl, ethoxy, or 2- methoxyethoxy. More preferably, substituted C _r C ₃ atkyl group is 2-hydroxyethyl, 3- hydroxy propyl, 2-hydroxypropyl, 2,3-dihydroxypropyl, 2-methoxyethyl, or 2-(2- methoxyethoxy)ethyt. Preferably, CH ₃ -(CH ₂ ) _m -0-(CH ₂ -CH O) _n -CH ₂ -CH ₂ is defined such as n = 0 and m = 0 or n = 1 and m = 1.

Preferably, unsubstituted acyi group is acetyl.

Preferably, substituted acyi group is substituted with one substituent defined as hydroxyl, methyl, ethyl, methoxy, ethoxy, or 2-methoxyethoxy. More preferably, substituted acyi group is hydroxyacetyi, or methoxyacetyl.

In a sub-embodiment, R1 , R2, R3 and R4 are independently from each other Hydrogen methyl or ethyl.

In a sub-embodiment, R1 , R2, R3 and R4 are independently from each other Hydrogen or methyl optionally substituted with hydroxymethyl or methoxymethyl.

In a sub-embodiment, R1 , R2, R3 and R4 are Hydrogen, i.e. uric acid.

Preferably, compound of formula I is selected from but not limited to

uric acid:

1-methyluric acid:

methyluric acid:

-methyluric acid:

-methyluric acid:

1 ,3-Dimethyluric acid:

1,7-Dimethyluric acid:

1 ,9-Dimethyluric acid:

-Dimethyluric acid:

Tetramethyluric acid:

In a sub-embodiment, the concentrations of the uric acid derivatives are in the range of 1 pg/mL to 1.000 μ9/ιηΙ_, preferably of 10 pg/mL to 100 pg/mL uric acid derivatives and more preferably of 20 pg/mL to 60 pg/mL uric acid derivatives. Even more preferably, the concentration of the uric acid derivative is 40 pg/mL. Method of stabilizing

in a sub-embodiment, the invention is directed to a method for stabilizing a peptidic radiopharmaceutical characterized in that uric acid derivatives are used as stabilizing agent

wherein the uric acid derivatives are com ound of formula I

wherein

R1 is Hydrogen, substituted or unsubstituted Ci-C ₃ alkyl group, CH ₃ -(CH ₂ ) _m -0-(CH ₂ - CH ₂ -0) _n -CH ₂ -CH ₂ , n = 0, 1 , 2 or 3 and m = 0 or 1 or substituted or unsubstituted acyl group,

R2 is Hydrogen, substituted or unsubstituted C _r C ₃ alkyl group, CH ₃ -(CH ₂ ) _m -0-(CH ₂ - CH ₂ -0) _n -CH ₂ -CH ₂ , n - 0, 1 , 2 or 3 and m = 0 or 1 or substituted or unsubstituted acyl group,

R3 is Hydrogen, substituted or unsubstituted C^-C ₃ alkyl group, CH ₃ -(CH ₂ )m-0-(CH ₂ - CH ₂ -0) _n -CH ₂ -CH ₂ , n = 0, 1 , 2 or 3 and m = 0 or 1 or substituted or unsubstituted acyi group, and

R4 is Hydrogen, substituted or unsubstituted C C ₃ alkyl group, CH ₃ -(CH ₂ ) _m -0-(CH ₂ - 0Η ₂ -Ο) _Γ ,-0Η2-ΟΗ ₂ , n = 0, 1 , 2 or 3 and m = 0 or 1 or substituted or unsubstituted acyl group.

Preferably, the method is defined as referring to peptidic radiopharmaceuticals selected from

[ ⁶⁸ Ga3-DOTA-4-amino-1-carboxymethylpiperidine-D-Phe-Gln-T rp-Ala-Val-Gly-His-Sta- Leu-NH ₂ , ⁶⁸ Ga-DOTA-R 2;

3-cyano-4-[ ¹⁸ F]fluorobenzoyl-Ala(S0 ₃ H)-Ala{S0 ₃ H)-Ava-Gln-Trp-Ala-Val-NMeGly-His~ Sta-Leu~NH ₂ ;

3-cyano-4-[ ¹⁸ F]fluorobenzoyl-Ala(S0 ₃ H)-Ava-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu- NH ₂ ;

[ ⁶⁸ Ga]-DOTATOC; or

[ ⁶⁸ Ga]-DOTATATE and the uric acid derivative is selected from

uric acid, 1-methyluric acid, 3-methyluric acid, 7-methyluric acid, 9-methyluric acid, 1 ,3- Dimethyluric acid, 1 ,7-Dimethyluric acid, 1 ,9-Dimethyiuric acid, 3,7-Dimethyluric acid, or Tetramethyiuric acid.

More preferably, the method is defined as referring to the peptidic radiopharmaceutical [ ⁶⁸ Ga]-DOTA-4-amino-1-carboxymethylpiperidine-D-Phe-Gln-T rp-Ala-Val-Gly-His-Sta- Leu-NH ₂ , ⁶⁸ Ga-DOTA-RM2.

In a sub-embodiment, the invention is directed to a method for stabilizing a peptidic radiopharmaceutical,

characterized in that uric acid derivative(s) is/are used as stabilizing agent

comprising the steps,

-adding uric acid derivative(s) to a peptidic radiopharmaceutical precursor,

-adding uric acid derivative(s) into the reaction mixture of a radiolabeling reaction or

-adding uric acid derivative(s) to the freshly prepared peptidic radiopharmaceutical. In a further sub-embodiment, the method for stabilizing a peptidic radiopharmaceutical, characterized in that uric acid dertvative(s) are used as stabilizing agent,

comprising the steps

-adding uric acid derivative(s) to a peptidic radiopharmaceutical precursor,

-adding uric acid derivative(s) into the reaction mixture of a radiolabeling reaction or

-adding uric acid derivative(s) to the freshly prepared peptidic radiopharmaceutical,

wherein the uric acid derivative(s) are compounds of formula I wherein

R1 is Hydrogen, substituted or unsubstituted CrC ₃ aikyi group, CH ₃ -(CH ₂ ) _m -0-(CH ₂ - CH ₂ -0) _n -CH ₂ -CH ₂ , n = 0, 1 , 2 or 3 and m = 0 or 1 or substituted or unsubstituted acyl group,

R2 is Hydrogen, substituted or unsubstituted CTC ₃ alkyi group, CH ₃ -(CH ₂ ) _m -0-(CH ₂ - CH ₂ -0) _n -CH ₂ -CH ₂ , n = 0, 1 , 2 or 3 and m = 0 or 1 or substituted or unsubstituted acy! group,

R3 is Hydrogen, substituted or unsubstituted C1-C3 aikyl group, CH ₃ -(CH ₂ ) _m -0-(CH CH ₂ -0) _n -CH ₂ -CH _2l n = 0, 1 , 2 or 3 and m = 0 or 1 or substituted or unsubstituted acyl group, and

R4 is Hydrogen, substituted or unsubstituted ^-C ₃ aikyl group, CH ₃ -(CH ₂ ) _m -0-(CH ₂ - CH ₂ -0)n-CH ₂ -CH ₂ , n = 0, 1 , 2 or 3 and m = 0 or 1 or substituted or unsubstituted acyi group.

Preferably, the method is defined as referring to peptidic radiopharmaceuticals selected from

[ ⁶⁸ Ga]-DOTA-4-amino-1-carboxymethylpiperidine-D-Phe-Gln-T rp-Ala-Val-Gly-Hfs-Sta- Leu-NH ₂ , ^6a Ga-DOTA-RM2;

3-cyano-4-[ ^ie F]fluorobenzoyl-Ala(S0 ₃ H)-Aia(S0 ₃ H)-Ava-Gin-Trp-Ala-Val-N eGly-His- Sta-Leu-NH ₂ ;

3-cyano-4-E ⁸ F]fluorobenzoyi-Ala(S0 ₃ H)-Ava-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu- NH ₂ ;

[ ⁶⁸ Ga]-DOTATOC; or

[ ^6S Ga]-DOTATATE, and

the uric acid derivative is selected from uric acid, 1-methyluric acid, 3-methyluric acid, 7-methyluric acid, 9-methyluric acid, 1 ,3- Dimethyiuric acid, 1 ,7-Dimethyluric acid, 1 ,9-Dimethyluric acid, 3,7-Dimethyluric acid, or Tetramethyluric acid.

More preferably, the method is defined as referring to the peptidic radiopharmaceutical [ ^6S Ga]-DOTA-4-amino-1-carboxymethylpipe dine-D-Phe-Gln-Trp-Ala-Vai-Gly-His-Sta- Leu-NH ₂ , ⁶⁸ Ga-DOTA-RM2.

In a further sub-embodiment, the method is directed to uric acid derivatives that are added to peptidic radiopharmaceutical precursors such peptidic radiopharmaceutical precursors are well known in the art.

In a sub-embodiment, the method is directed to uric acid derivatives that are added into the reaction mixture of a radiolabeling reaction.

In a sub-embodiment, the method is directed to uric acid derivatives that is added to the freshly prepared peptidic radiopharmaceutical, wherein freshly prepared means that the radiolabeling synthesis is substantially completed.

Preferably, the peptidic radiopharmaceutical precursor is selected from but not limited to

DOTA-Gly-aminobenzoyl-D-Phe-Gin-Trp-Ala-Val-Gly-His-Sta-Leu- NH ₂ ;

DOTA-4-amino-1-carboxymethyl-piperidine-D-Phe-Gln-Trp-AIa-Va l-Gly-His-Sta-Leu-

NH ₂ ;

DOTA-4-amino-1-piperidtne-4-carboxylicacid-D-Phe-Gln-Trp-Ala -Val-Gly-His-Sta-Leu- NH ₂ ;

DOTA-15-amino-4,7,10,13-tetraoxapentadecanoic acid-D-Phe-Gln-Trp-Ala-Val-Giy- His-Sta-Leu-NH ₂ ;

DOTA-( 5-amino-4,7,10,13-tetraoxapentadecanoic acid)-(4-amino-1-carboxymethyl- piperidine)-D-Phe-Gln-Trp-Ala-Val-Gfy-His-Sta-Leu-NH ₂ ;

DOTA-diaminobutyricacid-D-Phe-Gln-Trp-Aia-Val-Gly-His-Sta -Leu-NH ₂ ; DOTA-4-(2-aminoethyl)-1 -carboxymethyl-piperazine-D-Phe-Gln-Trp-Ala-Val-G!y-His- Sta-Leu-NH ₂ ;

LeuDOTA-{5-amino-3-oxa-pentyl)-succinamic acid-D-Phe-Gln-Trp-Aia-Val-Gly-His-Sta- Leu-NH ₂ ;

DOTA-4-amino-1-carboxymethyl-pipendine-D-Phe-Glri-Trp-Ala -Val-Gly-His- Le^(CHOH-CH ₂ )-(CH ₂ ) ₂ ~CH ₃ ;

DOTA-(15-amino-4,7, 10,13-tetraoxapeniadecanoic acid-4-amino-1 -carboxymethyl- pi eridine-D-Phe-Gln-Tφ-Ala-Va!-Gly-His-Leuψ(CHOH-CH ₂ HCH ₂ ) ₂ -CH ₃ ;

DOTA-15-amino-4,7,lO,13-tetraoxapentadecanoic acid -D-Phe-Gin-Trp-Ala-Val-Gly- His-Le^(CHOH-CH ₂ )-(CH ₂ ) ₂ -CH ₃ ;

DOTA-4-amino-1-carboxymethyl-pipendine-D-Phe-Gln-Trp-A!a-Vai -Giy-His- Le^(CH ₂ NH)-Phe-NH ₂ ;

DOTA-4-amino-1-carboxynneihyl-piperidine-D-Phe-Gln-Trp-Ala-V al-Giy-Hts- Le^{CH ₂ NH)-Cys-NH ₂ ; DOTA-4-amino-l-carboxymethylpiperidine-D-Phe-Gln-Trp-Ala-Vai -Gly-His-Sta-Leu- -RM2

3-cyano-4-trimethy[amnnoniobenzoyl-Ala(S0 ₃ H)-Ala(S0 ₃ H)-Ava-GIn-Trp-Ala-Val-

NMeGly-His-Sta-Leu-NH ₂

trifluoroacetate

3-cyano-4-trimethytammoniobenzoyl-Ala{S0 ₃ H)-Ava-Gln-Trp-Ala-Val-NMeGly-His-Sta- Leu-NH ₂ trifluoroacetate

Preferably:

DOTA-4-amino~1-carboxymethylpipertdine-D-Phe-Gln-Trp-Ala-Val -Gly-His-Sta-Leu~ NH _2l DOTA-RM2

3-cyano-4-trimethylammoniobenzoyl-Ala(S0 ₃ H)-Ala(S03H)-Ava-Gln-Trp-Ala-Val-

3-cyano-4-trimethylammoniobenzoyl-Ala(S0 ₃ H)-Ava-Gin-Trp-Aia-Val-N eGly-His-Sta- Leu-NH ₂ trifluoroacetate

DOTATOC

More preferably, DOTA-4-amino-1 -carboxymeihylpiperidine-D-Phe-GIn-Trp-Ala-Val- Gi -His-Sta-Leu-NH ₂ , DOTA-RM2

The invention is directed to a method defined as above and by the scope of all or some of the preferred features and sub-embodiments as combined to each other . [n a second aspect, the invention is directed to compositions comprising

-a peptidic radiopharmaceutical and

-uric acid derivatives used as stabilizing agent.

The peptidic radiopharmaceutical and uric acid derivatives are as disclosed above.

In a third aspect, the invention is directed to compositions comprising

-a peptidic radiopharmaceutical precursor and

-uric acid derivatives used as stabilizing agent.

The peptidic radiopharmaceutical precursor and uric acid derivatives are as disclosed above.

In a fourth aspect, the invention is directed to a kit comprising

-a peptidic radiopharmaceutical precursor and

-uric acid derivatives used as stabilizing agent.

The peptidic radiopharmaceutical precursor and uric acid derivatives are as disclosed above.

The preferred features and sub-embodiments disclosed in the first aspect are herein incorporated in the second, third and fourth aspects.

In a fifth aspect, the invention is directed to the use of uric acid or derivatives thereof for stabilizing a peptidic radiopharmaceutical comprising

- the use of uric acid or derivatives thereof in a stabilizing amount, being in the range of 1 g/mL to 1.000 μg/mL for stabilizing a peptidic radiopharmaceutical

- the use of a kit comprising a sealed vial containing a predetermined quantity of a radiopharmaceutical precursor and a stabilizing amount of uric acid or a derivative thereof for stabilizing a peptidic radiopharmaceutical. The peptidic radiopharmaceutical precursor and uric acid derivatives are as disclosed above.

The preferred features and sub-embodiments disclosed in the first aspect are herein incorporated in the fifth aspect.

Definitions

The term "stabilizing agent" or "stabilizer" refers to a chemical which inhibits radiolysis reactions.

The term "radiopharmaceutical" refers to a radioactive compound used in radiotherapy or diagnosis in the field of nuclear medicine wherein the radioactive compound is used as tracer in the radiotherapy or diagnosis of diseases. The term "peptidic radiopharmaceutical" refers to a radiopharmaceutical as defined above comprising a natural or synthetic peptide containing two or more natural or synthetic amino acids linked by the carboxyi group of one amino acid to the amino group of another including modifications of these peptide bonds such as methylation or reduction. The natural or synthetic peptide of the radiopharmaceutical is interfering with mammal cell structures such as receptors or enzymes.

As used hereinafter in the description of the invention and in the claims, the term "amino acid" means any molecule comprising at least one amino group and at least one carboxyi group, but which has no peptide bond within the molecule. In other words, an amino acid is a molecule that has a carboxylic acid functionality and an amine nitrogen having at least one free hydrogen, preferably in alpha position thereto, but no amide bond in the molecule structure. Thus, a dipeptide having a free amino group at the N-terminus and a free carboxyi group at the C-terminus is not to be considered as a single "amino acid" in the above definition. The amide bond between two adjacent amino acid residues which is obtained from such a condensation is defined as "peptide bond". Optionally, the nitrogen atoms of the polyamide backbone (indicated as NH above) may be independently alkylated, e.g., with d-Ce-alkyl, preferably CH ₃ ,

An amide bond as used herein means any covalent bond having the structure

wherein the carbonyl group is provided by one molecule and the NH-group is provided by the other molecule to be joined. The amide bonds between two adjacent amino acid residues which are obtained from such a polycondensation are defined as "peptide bonds". Optionally, the nitrogen atoms of the poiyamide backbone (indicated as NH above) may be independently alkylated, e.g., with -CrCe-alkyl, preferably -CH ₃ .

As used hereinafter in the description of the invention and in the claims, an amino acid residue is derived from the corresponding amino acid by forming a peptide bond with another amino acid.

As used hereinafter in the description of the invention and in the claims, an amino acid sequence may comprise naturally occurring and/or synthetic / artificial amino acid residues, proteinogenic and/or non-proteinogenic amino acid residues. The non- proteinogenic amino acid residues may be further classified as (a) homo analogues of proteinogenic amino acids, (b) β-homo analogues of proteinogenic amino acid residues and (c) further non-proteinogenic amino acid residues.

Accordingly, the amino acid residues may be derived from the corresponding amino acids, e.g., from e proteinogenic amino acids, namely Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie,

Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val; or

• non-proteinogenic amino acids, such as

o homo analogues of proteinogenic amino acids wherein the sidechain has been extended by a methylene group, e.g., homoalanine (Hal), homoarginine (Har), homocysteine (Hey), homoglutamine (Hgl), homohistidine (Hhi), homoisoleucine (Hil), homoleucine (Hie), homolysine (Hly), homomethionine (Hme), homophenylalanine (Hph), homoproline (Hpr), homoserine (Hse), homothreonine (Hth), homotryptophane (Htr), homotyrosine (Hty) and homovaline (Hva);

o β-homo analogues of proteinogenic amino acids wherein a methylene group has been inserted between the ot-carbon and the carboxyl group yielding β-amino acids, e.g., β-homoalanine (βΙ-lal), β-homoarginine {βΙ-lar), β-homoasparagine (βΐ-ias), β-homocysteine (pHcy), β-homoglutamine (pHgl), β-homohistidine (βΗηί), β-homotsoleucine (βΗΗ), β-homoleucine (βΗΙθ), β-homoiysine (βΗΙγ), β- homomethionine (βΗπηβ), β-homophenylalanine (βΗρη), β-homoproline (βΙ-fpr), β-homoserine (βΗεε), β-homothreonine (βΗΐίη), β-homotryptophane (βΗίΓ), β- homotyrosine (βΗΐγ) and β-homovaline (βΙ-iva);

ο further non-proteinogenic amino acids, e.g., α-aminoadipic acid (Aad), β- aminoadipic acid (pAad), a-aminobutyric acid (Abu), a-aminoisobutyric acid (Aib), β-alanine ( Ala), 4-aminobutyric acid (4-Abu), 5-aminovaleric acid (5-Ava), 6- aminohexanoic acid (6-Ahx), 8-aminooctanoic acid (8-Aoc), 9-aminononanoic acid (9-Anc), 10-aminodecanoic acid (10-Adc), 12-aminododecanoic acid (12-

Ado), a-aminosuberic acid (Asu), azetidine-2-carboxylic acid (Aze), β- cyclohexy!aianine (Cha), aitrulline (Cit), dehydroalanine (Dha), γ-carboxyglutamic acid (Gla), -cyclohexylglycine (Chg), propargylglycine (Pra), pyroglutamic acid (Gip), a-tert-butylglycine (Tie), 4-benzoylphenyia!anine (Bpa), δ-hydroxylysine (Hyl), 4-hydroxyproline (Hyp), allo-isoleucine (aile), lanthionine (Lan), (1- naphthyl)alanine (1~Nal), (2-naphthyl)alanine (2-Nal), norieucine (Nle), norvaline (Nva), ornithine (Orn), phenylglycin (Phg), pipecolic acid (Pip), sarcosine (Sar), selenocysteine (Sec), statine (Sta), β-thienylalanine (Thi), 1 ,2,3,4- tetrahydroisochinoline-3-carboxyIic acid (Tic), allo-threonine (aThr), thtazolidine- 4-carboxylic acid (Thz), γ-aminobutyric acid (GABA), iso-cysteine (iso-Cys), diaminopropionic acid (Dpr), 2,4-diaminobuiyric acid (Dab), 3,4-diaminobutyric acid ^Dab), biphenylalanine (Bip), phenylalanine substituted in para-position with -C Ce alkyl, -halide, -NH ₂ , -C0 ₂ H or Phe(4-R) (wherein = -d-C _e alky!, - halide, -NH ₂ , or -C0 ₂ H); peptide nucleic acids (PNA, cf., P.E. Nielsen, Ace. Chem. Res., 32, 624-30);

e or their N-alkylated analogues, such as their N-methylated analogues.

Cyclic amino acids may be proteinogenic or non-proteinogenic, such as Pro, Aze, GIp, Hyp, Pip, Tic and Thz.

For further examples and details reference can be made to, e.g., J.H. Jones, J. Peptide ScL, 2003, 9.

As used herein, the term "alkyl" refers to a C C ₁₀ straight chain or branched chain alkyl group such as, for example methyl, ethyl, propyl, isopropyl, butyl, isobutyi, terf-butyl, penty!, isopenty!, neopentyl, heptyi, hexyl, decyl. Preferably, alkyl is C _r C ₆ straight chain or branched chain alkyl or C ₇ -C ₁₀ straight chain or branched chain alkyl.

Whenever the term "substituted" is used, it is meant to indicate that one or more hydrogens on the atom indicated in the expression using "substituted" is / are replaced by one ore multiple moieties from the group comprising halogen, hydroxyl, nitro, C _r C ₆ - alkylcarbonyi, cyano, thfiuoromethyl, d-Ce-aikylsulfonyl, C _r C ₆ -alkyl, C-i-C ₆ -alkoxy and (VCe-alkylsulfanyl, provided that the regular valency of the respective atom is not exceeded, and that the substitution results in a chemically stable compound, /. e. a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into a pharmaceutical composition.

As used herein, C _n -C _m indicates the range of number of carbon atoms the respective moiety may feature, illustrated by but not limited to e.g. C C ₆ -alkyl or C C ₆ alkoxy, which may feature 1 , 2, 3, 4, 5, or 6 carbon atoms not covering optional additional substitution.

As used herein, the term "carrier" refers to microcrystalline cellulose, lactose, mannitol.

As used herein, the term "solvents" refers to liquid polyethylene glycols, ethanol, corn oil, cottonseed oil, glycerol, isopropanol, mineral oil, oleic acid, peanut oil, purified water, water for injection, sterile water for injection and sterile water for irrigation. eta! radioisotopes such as ^98m Tc or ^186/188 Re can be bound to peptides using two methods. The direct coordination of these metals is applicable in particular to proteins (high molecular weight peptides). The other approach involves the linkage of a bifunctional chelating agent (Arano Y., Annals of Nuclear Medicine Vol. 16, No. 2, 79- 93, 2002) such as exemplarily depicted above. The expression "stabilizing amount" with the context of the present invention denotes concentrations of the uric acid or derivatives thereof in the range of 1 pg/rnL to 1.000 pg/mL, preferably of 10 pg/mL to 100 pg/mL uric acid derivatives and more preferably of 20 g/mL to 60 pg/mL uric acid derivatives. Even more preferably, the concentration of the uric acid derivative is 40 g/mL

Abbreviations

EOS End of synthesis

ESI-TOF Electrospray-lonization Time-of-Fltght

GMP Good Manufacturing Practice

HPLC High Performance Liquid Chromatography

LA Low Activity

LC-MS Liquid Chromatography / Mass Spectrometry

LOD Limit of detection

RCP Radiochemical purity

RP Reversed phase

RCY Radiochemical yield

TIC Total ion current

TFA Trifluoroacetic acid

UV Ultra violet

VWD Variable wavelength detector

Experimental Section

Tab!e 1 : List of chemicats and further consumables Chemical Purity Provider

TFA For spectroscopy Merck

Uric acid 99% ABCR

Water Trace Select Sigma Aldrich

Fresenius

Water for Injection Ph. Eur Kabi

1 Radiolabeling of Peptides

1.1 General Synthesis of ⁶⁸ Ga-DOTA-RM2 {compound 2)

Most of the ⁶⁸ Ga labeling syntheses described below were performed starting with compound 1 which is a DOTA-conjugated Bombesin derivative (Ga-DOTA-RM2) for radiolabeling. Precursor i is stored at -20°C in fractions of 28 ig dissolved in 40 Mi- water. The stability of 1 was determined by HPLC over a period of 5 months. No degradation was observed.

RM2 is a modified sequence of peptide Bombesin. The peptide sequence of RM2 contains 9 amino acid as described below

D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH ₂

Radiochemical yields are corrected for decay unless stated otherwise.

Figure 1 shows the radiolabeling of DOTA-RM2 (compound 1) with 68Ga to give ⁶⁸ Ga- DOTA-RM2 (compound 2).

1.2 Gallium-68 generators

Two ^BB Ge ⁸ Ga generators (IGG 100, Eckert & Ziegier Eurotope GmbH) of different activities (generator A = 740 MBq; generator B = 1850 MBq) were used for the labeling experiments. ⁶⁸ Gallium was obtained by elution with 0.1 M HCI (7 ml_).

The syntheses were performed using an automated synthesis module (Modular-Lab PharmTracer, Eckert & Ziegier Eurotope GmbH) and disposable synthesis cassettes (C4-Ga68-PP or C4-Ga68-FR; Eckert & Ziegier Eurotope GmbH).

Two general methods for the preparation of ⁶⁸ Ga are described in the literature (Decristoforo, C, et ai., Nuclear Medicine Communications, 2007. 28(11): p. 870-875; de Blots, E., et a!., Applied Radiation and Isotopes, 2011. 69(2): p. 308-315; Breeman, W.A.P., et al., European Journal of Nuclear Medicine and Molecular Imaging, 2005. 32(4): p. 478-485; Zhernosekov, K.P., et al. Journal of Nuclear Medicine, 2007. 48(10): p. 1741-1748). Programs for the fractionation method and the pre-purification method were provided by Eckert & Ziegler Eurotope GmbH. The impact of both methods on the impurity profile was tested.

1.3 No-Carrier-Added ⁶⁸ Ga Syntheses 1.3.1 Syntheses of ⁶⁸ Ga-DOTA-RM2 Using the Pre-purification Method

The ⁶⁸ Ga eluate was pre-purified using a cation exchange cartridge (Strata-X-C, Phenomenex). A solution of 98% acetone / 0.02 M HCI (0.4 mL) was used to elute ⁶⁸ Ga from the cartridge. Sodium acetate buffer (0.2 ; 2 mL; pH 4) and peptide 1 (28 pg dissolved in 40 μί water) was pre-filled into the reactor vessel. The reaction mixture was heated to 95 °C for 400 s.

After radiolabe!ing, the reaction mixture was cooled down to approx. 50 °C and diluted with saline (3 mL). The mixture was passed through a pre-conditioned SepPak C18 cartridge (pre-conditioned with 6 mL ethanol (50%), 6 mL saline) in order to immobilize the drug substance on the cartridge. Educts and impurities were washed away with saline (3 mL). The product was eiuted from the cartridge with 1 mL ethanol (50%) and passed through a 0.22 μιη sterile filter. The final product (2) was collected in a vial and formulated with saline (9 mL) to yield a final batch volume of 10 mL

1.3.2 Syntheses of Ga-DOTA-RM2 Using the Fractionation Method

The ⁶⁸ Ga eluate was collected in three fractions (fraction 1 with 2.4 mL and fraction 3 with 2 mL were discarded). Only fraction 2 (1.7 mL) containing the main radioactivity (approx. 80% of the total radioactivity) was transferred into the reactor vessel. The reactor was prefilled with HEPES buffer (2.5 M; 350 pL), water (450 \iL) and peptide 1 (28 g) dissolved in water (40 pL). The reaction mixture was heated to 95 °C for 400 s. The purification steps of the crude product were the same as described in the pre- purification method.

After radiolabeling, the reaction mixture was cooled down to approx. 50 °C and diluted with saline (3 mL). The mixture was passed through a pre-conditioned SepPak C18 cartridge (pre-conditioned with 6 mL ethanol (50%), 6 mL saline) in order to immobilize the drug substance on the cartridge. Educts and impurities were washed away with saline (3 mL). The product was eluted from the cartridge with 1 mL ethanol (50%) and passed through a 0.22 pm sterile filter. The final product was collected in a vial and formulated with saline (9 mL) to yield a final batch volume of 10 mL.

1.3.3 Syntheses of DOT AT ATE and DOTATOC Using the Pre-purification

Method

The ⁶⁸ Ga eiuate was pre-purified using a cation exchange cartridge (Strata-X-C, Phenomenex). A solution of 98% acetone / 0.02 M HCI (0.4 mL) was used to elute ^6S Ga from the cartridge. Sodium acetate buffer (0.2 M; 2 mL; pH 4) and peptide DOTATATE and DOTATOC, respectively, (50 pg peptide dissolved in 50 pL water) was pre-filled into the reactor vessel. The reaction mixture was heated to 95 °C for 400 s.

After radiolabeltng, the reaction mixture was cooled down to approx. 50 °C and diluted with saline (3 mL). The mixture was passed through a pre-conditioned SepPak C18 cartridge (pre-conditioned with 6 mL ethanol (50%), 6 mL saline) in order to immobilize the drug substance on the cartridge. Educts and impurities were washed away with saline (3 mL). The product was eluted from the cartridge with 1 mL ethanol (50%) and passed through a 0.22 pm sterile filter. The final product was collected in a vial and formulated with saline (9 mL) to yield a final batch volume of 10 mL.

1.4 Carrier Added Syntheses of ⁶⁸ Ga-DOTA-RM2 Using the Fractionation Method

The carrier added radiolabeltng experiment was performed with the fractionation method. The fraction that was used for the labeling contained a starting activity of 930 MBq. The reactor vial was pre-filled with peptide i (3 mg) dissolved in water (490 pL) and HEPES buffer (2.5 M; 350 pL). After 100 s of heating the solution at 95 °C, Ga(N0 ₃ ) ₃ - n H ₂ 0 (1.04 mg) dissolved in HCI (0.1 M; 10 pL) was added to the reaction mixture and kept stirring for another 300 s at 95 °C. The purification steps were the same as mentioned above.

1.4.1 LC-MS Measurements

The products of the carrier added syntheses were allowed to decay and were analyzed within 1 week after synthesis by LC-MS (ESI-TOF, LCT Premier, Waters; column: ACE 3 C18; 3 pm 100A, 50 mm χ 4.6 mm; gradient method from 17% B, to 25% B within 20 min; eluent A: water with 0.1 % HCOOH; eluent B; MeCN with 0.1 % HCOOH).

The LC- S measurements allowed the structure assignment of the unknown radiochemical impurities .

Figure 2 shows a LC-MS TIC of the reaction mixture obtained from a carrier added radiosynthesis without addition of a stabilizer.

After decay of the carrier added Ga-DOTA-RM2 sample, LC-MS analysis was performed (cf. Figure 2). The main peak at a retention time R _t = 6.53 min represents ^{69 7} Ga-DOTA-RM2 (M = 1 06.6 g/mol) (detected as [M+2H] ²⁺ /2; m/= 853.3). The m/z ratios of the more polar side products in the range of 4.2 to 5.6 min were determined to be m/z = 861.4 and m/z = 869.4, respectively. The mass difference of m/z = +8 is interpreted as the detection of [M+2H+0] ²⁺ /2, whereas m/z = +16 indicates the detection of the molecule after insertion of two oxygen atoms as [M+2H+20] ² 72. The structure assignment was realized by using the isotope pattern calculator of the MassLynx 4.1 software. The calculated isotope patterns of the detected compounds mentioned above are in perfect agreement with the experimental data (cf. Figure 3.1 , 3.2, 3.3 and 3.4).

Figure 3.1 , 3.2, 3.3 and 3.4 shows calculated isotope patterns of Ga-DOTA-RM2 derivatives with two {m/z = 869) and one (m/z = 861) additional oxygen atoms introduced by radiolytic processes, and unchanged Ga-DOTA-RM2 (m/z = 853) (first three spectra 3.1 - 3.3,) as well as the corresponding experimental isotope patterns of all three Ga-DOTA-RM2 species (3.4).

The data support the hypothesis of the formation of radiolytically oxidized products of ^6s Ga-DOTA-RM2 with one and two additional oxygen atoms, respectively.

1.5 Quality Contro! - Analytical HPLC

1.5.1 Determination of Chemical and Radiochemical Purity

Anafytical HPLC was performed with a gradient RP-HPLC system, equipped with an UV-detector and a Nal(TI) well-type scintillation detector for radioactivity detection. Data acquisition and interpretation were carried out with associated software. An analytical column ACE 3 C18 (3 μιτι 100A, 50 mm * 4,6 mm) was used with a flow rate of 2 mL/min, a wavelength of 230 nm for method 1 and 2 and a wavelength of 225 nm for methods 3-5, respectively. The eluents used for method 1 and 2 were acidified with TFA, methods 3-5 with H ₂ S0 ₄ .

Method 1

Mobile phase A: water with 0.027% H ₂ S0 ₄ ; B: MeCN with 0.027% H ₂ S0 ₄

0 min, 0% B, 0 min 40% B, 10.1 min 90% B, 12.1 min 90% B, 12.3 min 0% B, 16 min 0% B

Method 2

Mobile phase A: water with 0.027% H ₂ S0 ₄ ; B: MeCN with 0.027% H ₂ S0 ₄

0 min 15% B, 40 min 25% B

Method 3

Mobile phase A: water with 0.027% H ₂ S0 ₄ ; B: MeCN with 0.027% H ₂ S0 ₄

0 min, 17% B; 12 min, 25% B; 14 min, 75% B; 15 min, 17% B, 17 min, 17% B.

1.5.2 Determination of Uric Acid

The quantification of uric acid in the final product was performed using an analytical HPLC with a gradient RP-HPLC system (Agilent 1100 Serie) equipped with an UV-detector (VWD). Data acquisition and interpretation were carried out with associated software (ChemStation Software). An analytical column Phenomenex Luna 5 Mm C18 (5 μιπ, 100 A, 250 mm * 4.6 mm) was used with a flow rate of 1 mUmin. The detector wavelength was set to 288 nm for 0 - 12 min and to 225 nm for 12.01 - 18 min. Method 4

Mobile phase A: 10 mM NaH ₂ P0 ₄ , mobile phase B; acetonitrile

0 min, 0% B, 12 min 0% B, 13 min 50% B, 15 min 50% B, 17 min 0% B, 18 min 0% B

1.6 Influence of the Starting Radioactivities of ⁶⁸ Ga on the RCP of ^6B Ga- DOTA-RM2

1.6.1 Experiments with Low Starting Radioactivity (up to 240 MBq ^6S Ga)

Low radioactivity batches were conducted by two different methods (LA1 and LA2, respectively). For method LA1 , the generator was eluted as usual, and then the program was stopped after drawing up ail the activity in the syringe. The ⁶⁸ Ga was allowed to decay to the desired starting radioactivity. For method LA2, the generator was efuted short time after the last elution. Due to this short regeneration period, low starting radioactivities of ⁶⁸ Ga were obtained.

The low activity batches were carried out with starting activities ranging from 170 to 240 MBq obtained from generator A.

In Figure 4, the HPLC chromatograms of a low activity batch with a starting activity of 230 MBq and a product activity of 84 MBq (RCY 46%) are depicted. Radiochemical purity varies depending on the HPLC method used: 95% (method 1) and 93% (method 2), respectively. Method 2 with a shallower gradient is able the separate the unknown radiochemical impurities from the main product.

Figure 4 shows HPLC radiochromatograms of ⁶⁸ Ga-DOTA-RM2 (low activity batch). The RCP was determined to be of 95% (HPLC method 1 , top) and 93% with the optimized HPLC method 2 (bottom).

Nine low radioactivity syntheses were conducted with radiochemical yields of (69 ± 9)% using the fractionation as well as pre-purifi cation method. The radiochemical purities were determined to be (94 ± 2)% using HPLC method 1.

1.6.2 Experiments with High Starting Radioactivity (> 720 MBq ⁶⁸ Ga)

High radioactivity batches were performed with starting radioactivities ranging from 720 to 1300 MBq obtained from generator B.

The high radiochemical purities obtained with the generator A could not be reproduced with generator B. Three batches showed a radiochemical purity of 89% (method 1). As an example, Figure 5 shows the HPLC chromatogram of a batch produced with a starting radioactivity of 900 MBq. The radioactivity of the product at EOS was 445 MBq (61 % RCY). A radiochemical purity of 83% (method 2) and 89% (method 1), respectively, were determined for this batch.

Seven syntheses with high starting radioactivities were performed with radiochemical yields of (64 ± 9)% using the fractionation as well as the pre-purification method. The radiochemical purities were determined to be (82 + 13)% using HPLC method 1 or method 3.

Figure 5 shows HPLC radiochromatograms from a ^e8 Ga-DOTA-RM2 high radioactivity batch. With HPLC method 1 (top), the RCP was determined to be of 89% in contrast to method 2 with a better resolution and separation of impurities (83% RCP, bottom).

Based on HPLC method 2, the method 3 was developed with a shorter total run time, but with a similar shallow gradient (cf. Figure 6).

Figure 6 shows the HPLC radiochrotnatogram of ^e3 Ga-DOTA-RM2 obtained with a shallow gradient but shorter run time (method 3). The radiochemical impurities are completely separated from the product peak.

The data clearly show a correlation between starting radioactivity and radiochemical purity: the lower the starting radioactivity, the better the radiochemical purity. These results were independent of the use of either method LA1 or LA2 and independent of the used buffer (HEPES or sodium acetate).

1.7 ⁶⁸ Ga-Labeling in Presence of Putative Stabilizers 1.7.1 Ascorbic Acid, Gentisic Acid and Sodium Thiosulfate

The use of ascorbic acid completely inhibited the formation of oxygenated ⁶⁸ Ga-DOTA- RM2 side products. However, a new radiochemical impurity was detected as an unresolved peak after the ⁶⁶ Ga-DOTA-RM2 peak (cf. Figure 7). Hence, the radiochemical purity decreased to (78 ± 1)%, n = 3. Furthermore, the RCY decreased remarkably to (34 ± 1)%, n = 3. However, based on these results obtained from experiments with three different concentrations of ascorbic acid (0.1 mg, 1 mg, and 10 mg), no dependence of the RCP and RCY, respectively, on the concentration was observed. Figure 7 shows HPLC radiochromatogram (method 3) of Ga-DOTA-RM2 after addition of 0.1 mg ascorbic acid as stabilizer to the reaction mixture. Besides the successful suppression of the formation of oxygenated side products, an additional unknown radiochemical impurity at a retention time of 7.6 min was detected.

Similar results with the detection of a well pronounced radiochemical impurity were obtained using gentisic acid (52% RCP, 20% RCY) and sodium thiosulfate (61% RCP, 35% RCY), respectively (cf. Figure 8).

Figure 8 shows HPLC radiochromatogram (method 3) of ^6S Ga-DOTA-RM2 stabilized with 1 mg gentisic acid as stabilizer (top) and 20 uL 10% sodium thiosulfate solution (bottom), respectively.

1.7.2 Caffein and Xanthine

6 ^s Ga-DOTA-RM2 syntheses were performed using 0.1 mg caffeine and xanthine, respectively.

By the addition of caffeine to the reaction mixture, ⁶⁸ Ga-DOTA-RM2 was obtained in low RCY (14%) and with a low RCP (33%). Analytical HPLC of the drug product revealed the formation of radiolysis products as well as the unknown radiochemical impurity at R _t = 7 min. This impurity was already observed during the experiments with ascorbic acid (cf. Figure ).

Figure 9 shows the HPLC radiochromatogram (method 3) of ^6e Ga-DOTA-RM2 using caffeine as a putative stabilizer.

Using xanthine as potential stabilizer, the obtained RCY (2.3%) was extremely low. Therefore, the RCP could not be determined. 19% of the starting activity was found in the reactor, 48 % of starting activity was lost in the waste. 1.7.3 Uric Acid

1.7.3.1 Stabilizing Effect in the ⁶⁸ Ga-DOTA-RM2 Synthesis

By addition of 0.1 mg uric acid, the radiolysis reactions of ⁶⁸ Ga-DOTA-RM2 could be inhibited without negative influence on the radiochemical yield (62 ± 8)%, n = Z. The radiochemical purity increased significantly to (96 ± 1 )% (cf. Figure 10).

Figure 10 shows typical HPLC radiochromatogram (method 3) of ⁶⁸ Ga-DOTA-RM2 obtained with high radiochemical purity using 0.1 mg uric acid as stabilizer.

1.7.3.2 Stabilizing Effect in the in ⁶⁸ Ga-DOTATOC Syntheses

By addition of 0.1 mg uric acid to the reaction mixture, ⁶³ Ga-DOTATOC was obtained in high RCY (77%) and with excellent RCP (99%). Manufacturing of ⁶⁸ Ga-DOTATOC without a stabilizer resulted in a RCP of only (84 + 10)% and RCY of (73 ± 4)%, n = 2 (cf. Figure 11). Figure 11 shows the HPLC radiochromatograms (method 3) of ⁶⁸ Ga-DOTATOC manufactured without stabilizer (top) and with high radiochemical purity (99%) using uric acid as stabilizer (bottom).

1.7.3.3 StabiSizing Effect in the ⁶⁸ Ga-DOTATATE Syntheses

B ⁸ Ga-DOTATATE was obtained in high RCY (79%) and with excellent RCP (>99%). Without stabilizer, the RCP of ⁶⁸ Ga-DOTATATE (77% RCY) was 86%, n = 1 (cf. Figure 12).

Figure 12 shows HPLC radiochromatograms (method 3) of ⁶⁸ Ga~DOTATOC manufactured without stabilizer (86% RCP, top) and with high radiochemical purity (>99%) using uric acid as stabilizer (bottom).

1.7.3.4 Quantification of Uric Acid in the Drug Product

The concentrations of uric acid in the final product were determined using the validated analytical method 4. After cleaning step on the solid phase cartridge the concentrations of uric acid in three decayed ⁶⁸ Ga-DOTA-RM2 batches were below the LOD (< 0.1 pg/mL). 1.7.4 1 -Methyl uric Acid

Performing a ^es Ga-DOTA-RM2 synthesis, with a starting radioactivity of 1248 MBq the obtained RCY (40%, 406MBq) and RCP (93.2%) are lower than using uric acid. The RCP of 93.3% was much higher than using ascorbic acid, gentisic acid or sodium thiosulfate but lower than using uric acid (cf. Figure 13).

Figure 13 shows the HPLC radiochromatogram (method 3) of Ga-DOTA-R 2 with a RCP of 93.3% achieved by adding 1-Methyluric acid to the reaction mixture.

1.7.5 1 ,3-Dimethyluric Acid

Using 1 ,3-Dimethyluric acid as stabilizer in a ⁶⁸ Ga-DOTA-RM2 synthesis, the obtained RCY (19.9%, 191.6MBq) with a starting radioactivity of 1182 MBq) was significantly lower than using uric acid. The RCP of 88.5% was much higher than using ascorbic acid, gentisic acid or sodium thiosulfate but lower than using uric acid (cf. Figure ).

Figure 14 shows the HPLC radiochromatogram (method 3) of ⁶⁸ Ga-DOTA-RM2 with a radiochemical purity of 88.5% achieved by adding 1,3-Dimethyluric acid to the reaction mixture. 1.7.6 Tetram ethyl uric Acid

Performing a ⁶⁸ Ga-DOTA-RM2 synthesis, the RCY (57%, 466 MBq) with

Tetramethyluric acid as stabilizer using a starting radioactivity of 1005 MBq was higher compared to the yields obtained with 1-methyluric acid and 1 ,3-dimethyluric acid. However, the RCP (91.7 %) is lower than using uric acid (cf. Figure 10 and Figure 15 ). Figure 15 shows the HPLC radiochromatogram (method 3) of ^e8 Ga-DOTA-RM2 with a RCP of 91.7% achieved by adding Tetramethyluric acid to the reaction mixture.

2 Description of the Figures

Figure 1 : Radioiabeiirig of DOTA-RM2 (compound 1) with ^6e Ga to give ⁶⁸ Ga-DOTA- R 2 (compound 2)

Figure 2: LC-MS TIC of the reaction mixture obtained from a carrier added

radiosynthesis without addition of a stabilizer

Figure 3.1 , 3.2, 3.3 and 3.4: Calculated isotope patterns of Ga-DOTA-RM2 derivatives with two (m/z = 869) and one (m/z = 861) additional oxygen atoms introduced by radtolytic processes, and unchanged Ga-DOTA-RM2 (m/z = 853) (first three spectra, 3.1 , 3.2 and 3.3) as well as the corresponding experimental isotope patterns of all three Ga-DOTA-RM2 species (3.4).

Figure 4: HPLC radiochromaiograms of ⁶⁸ Ga-DOTA-RM2 (low radioactivity batch). The RCP was determined to be of 95% (HPLC method 1 , top) and 93% with the optimized HPLC method 2 (bottom).

Figure 5: HPLC radiochromaiograms from a ⁶⁸ Ga-DOTA-RM2 high radioactivity batch.

With HPLC method 1 (top), the RCP was determined to be of 89% in contrast to method 2 with a better resolution and separation of impurities (83% RCP, bottom).

Figure 6: HPLC radiochromatogram of ⁶⁸ Ga-DOTA-RM2 obtained with a shallow

gradient but shorter run time (method 3). The radiochemical impurities are completely separated from the product peak.

Figure 7: HPLC radiochromatogram (method 3) of ^B8 Ga-DOTA-RM2 after addition of 0.1 mg ascorbic acid as stabilizer to the reaction mixture. Besides the successful suppression of the formation of oxygenated side products, an additional unknown radiochemical impurity at a retention time of 7.6 min was detected

Figure 8: HPLC radiochromatogram (method 3) of ^e8 Ga-DOTA-RM2 stabilized with 1 mg gentisic acid as stabilizer (top) and 20 uL 0% sodium thiosulfate solution (bottom), respectively. Figure 9: HPLC radiochromaiogram (method 3) of Ga-DOTA-RM2 using caffeine as a putative stabilizer

Figure 10: HPLC radiochromatogram (method 3) of ^e8 Ga-DOTA-RM2 obtained with high radiochemical purity using 0.1 mg uric acid as stabilizer

Figure 11 : HPLC radiochromatograms (method 3) of ^es Ga-DOTA-TOC manufactured without stabilizer (top) and with high radiochemical purity (99%) using uric acid as stabilizer (bottom)

Figure 12: HPLC radiochromatograms (method 3) of ^e8 Ga-DOTA-TOC manufactured without stabilizer (86% RCP, top) and with high radiochemical purity (>99%) using uric acid as stabilizer (bottom)

Figure 13: HPLC radiochromatogram (method 3) of ^e8 Ga-DOTA-RM2 with a RCP of 93.3% achieved by adding 1-Methyluric acid to the reaction mixture

Figure 14: HPLC radiochromatogram (method 3) of ⁶⁸ Ga-DOTA~RM2 with a

radiochemical purity of 88.5% achieved by adding 1 ,3-Dimethyluric acid to the reaction mixture

Figure 15: HPLC radiochromatogram (method 3) of ⁶⁸ Ga-DOTA-RM2 with a RCP of 91.7% achieved by adding Tetramethyluric acid to the reaction mixture