WO2010066051A1

WO2010066051A1 - Thiol-nota derivatives for kit 68ga radiolabeling

Info

Publication number: WO2010066051A1
Application number: PCT/CA2009/001822
Authority: WO
Inventors: Raif Schirrmacher; Carmen Waengler; Björn WAENGLER
Original assignee: The Royal Institution For The Advancement Of Learning/Mcgill University
Priority date: 2008-12-12
Filing date: 2009-12-14
Publication date: 2010-06-17

Abstract

A thiol-NOTA derivative of Formula (I): wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 is described herein. The thiol-NOTA derivatives are suitable for kit 68Ga radiolabeling of proteins and peptides.

Description

TITLE

THIOL-NOTA DERIVATIVES FOR KIT ⁶⁸Ga

RADIOLABELING

FIELD

[0001] The present disclosure broadly relates to a composition of matter for introducing ⁶⁸Ga into proteins and peptides. More specifically, but not exclusively, the present disclosure relates to novel 1 ,4,7-triazacyclononane- 1,4,7- triacetic acid (NOTA) derivatives. The present disclosure also relates to Ga radiolabeled l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA) derivatives. Moreover, the present disclosure also relates to the use of such radiolabeled derivatives for positron emission tomography (PET) imaging.

BACKGROUND

[0002] Commonly used radionuclides for PET imaging include C, F,

¹⁸O, ¹³N, and ⁷⁶Br. Bioactive molecules labeled with radionuclides suitable for molecular imaging (e.g. PET) purposes are of increasing interest. The radionuclide

Ga has gained in popularity as it enables the fast, simple and convenient labeling of peptides and other small biologically active materials such as antisense oligonucleotides. Furthermore, the labeling with ⁶⁸Ga is independent from cyclotrons and is thus highly flexible as it can be readily generated from a ⁶⁸Ge/⁶⁸Ga generator system.

[0003] Peptides labeled with the PET radionuclide ⁶⁸Ga via 1,4,7,10- tetraazacyclodocecane-iV,iV',N",N'"-tetraacetic acid (DOTA) exhibit favorable properties for nuclear imaging. In a comparative evaluation study, ⁶⁸Ga-DOTATOC, a somatostatin analog, was superior to the commonly used ⁿ 'in-labeled Octreoscan® for detecting neuroendocrine tumors, largely due to the higher mean and maximum standardized uptake values and higher tumor/non-tumor ratios in lung and skeletal manifestations. In 77.8% of discrepant osseous findings, positive PET interpretations were verified as true [I]. After elution from the Ge/ Ga generator system, ⁶⁸Ga³⁺ was mixed with the peptide-chelator-conjugate DOTATOC and kept at pH 4.8 and a temperature of 9O⁰C over a period of 15 min (conditions that would denature any protein).

[0004] Wei et al. have described the synthesis and use of ⁶⁸Ga-labeled

MSH in melanoma [2]. The synthesis Of ⁶⁸Ga labeled DOTA-ReCCMSH(Argl l) involved reactions carried out at room temperature over periods of 3 hours, at 9O⁰C over periods of 2 hours, and at 85⁰C over periods of 30 minutes at pH 3.8-4. The overall synthetic yield of unlabelled peptide-chelator-conjugate was reported to be 10-20%. Acute biodistribution and PET imaging performed on mice carrying B 16/Fl melanoma tumors showed moderate receptor-mediated tumor uptake, fast non-target organ clearance, high tumor/non-target tissue ratios, and clear visualization at all time points examined up to 2 hours following injection.

[0005] Despite its easier synthesis, ⁶⁸Ga-labeled RGD did not detect its alphavbeta3 integrin receptors as efficiently as ¹⁸F-galacto-RGD and ^{π i}In-labelled RGD [3]. In a common procedure, following the conjugation of the peptide cycloRGDfK with DOTA, the conjugate is deprotected at room temperature over a period exceeding 25 hours. Labeling with Ga occurred within 7 min at 8O⁰C with a radiochemical purity >95%. Labeling with ¹¹¹In occurred within 25 min at 95°C with a radiochemical purity >90%. Moreover, the radiochemical yield of ⁶⁸Ga- DOTA-RGD was shown to be approximately 60% as compared to approximately 30% for ¹⁸F-galacto-RGD. While tumor uptake ratios were comparable, tumor/blood ratios were higher for ¹¹¹In-DOTA-RGD and ¹⁸F-galacto-RGD than for ⁶⁸Ga-DOTA- RGD, largely due to a higher blood pool activity {i.e. higher background activity). [0006] According to Decristoforo et al, the lack of stability of ⁶⁸Ga

DOTA-peptides could be overcome by using alternative chelator systems such as p- SCN-Bn-NOTA, which could also be better suited for the clinical use of gallium [3]. While the currently used NOTA derivatives are well-suited for the derivatization of peptides, they do not yet allow the derivatization of proteins.

[0007] The cleavage of the

protecting groups of the monoreactive NOTA derived prochelator NODAGA(^BU)₃ (4-(4,7-bis(2-tert- butoxy-2-oxoethyl)- 1 ,4,7-triazonan- 1 -yl)-5-ter/-butoxy-5-oxopentanoic acid) requires concentrated trifluoroacetic acid [4]. NODAGA(tBu)₃ was synthesized in five steps in an overall yield of 21% and was subsequently coupled to the peptide [Tyr3] -octreotide (TOC) providing the peptide-chelator conjugate NODAGATOC. The conjugate was used as a SPECT and PET tracer when labeled with ¹¹¹In, ⁶⁷Ga, or ⁶⁸Ga.

[0008] The chelator p-SCN-Bn-NOTA (5'-2-(4-isothiocyanatobenzyl)- l,4,7-triazacyclononane-l,4,7-triacetic acid) was shown to achieve only moderate yields of the corresponding chelator-protein conjugates [5]. Peptide-chelator conjugates, NOTA-RGD, were prepared under standard SCN-amine reaction condition {i.e. stirring at room temperature over a period of 5 hours and isolation by semi-preparative HPLC) producing NOTA-RGDl (NOTA-c(RGDyK)) in 61% yield, NOTA-RGD2 (NOTA-E[c(RGDyK)]2) in 52 % yield, and N0TA-RGD4 (NOTA-E {E[c(RGDyK)]2}2) in 43% yield. The ⁶⁸Ga labeling procedure was conducted using 185 MBq Of ⁶⁸Ga over a period of 10 minutes at 40°C. Radioactive Ga-NOTA-RGD peptides were purified by semi-preparative HPLC leading to decay-corrected yields of 90% for NOTA-RGDl, 82% for NOTA-RGD2 and 64% for N0TA-RGD4. The specific activity of the ⁶⁸Ga-NOTA-RGD multimers was measured to be about 9.7-13.6 MBq/nmol. Similar results have been published [6]. [0009] Velikyan et al. have previously reported a straightforward labeling method for quickly introducing macrocyclic bifunctional chelators into Ga peptide-chelator conjugates at room temperature as demonstrated for NODAGA- TATE in which NOTA has been coupled to the eight amino acid residue peptide [Tyr3]Octreotate [7]. Formation kinetics of ⁶⁸Ga-NOTA, studied as a function of pH, and formation kinetics of ⁶⁸Ga-NODAGA-TATE, studied as a function of the bioconjugate concentration, indicated a nearly quantitative radioactivity incorporation of more than 95% for ⁶⁸Ga-NOTA within less than 10 minutes at room temperature and at pH 3.5, and from 90-95% for ⁶⁸Ga-NODAGA-TATE respectively. The purification of the ⁶⁸Ga-labeled products was described as being unnecessary since the radiochemical purity was >95%. Additional NOTA derivatives, such as TACN-TM (l,4,7-tris(2-mercaptoethyl)-l,4,7- triazacyclononane), NODASA (1,4,7-tri-azacyclononane-l -succinic acid-4,7- deacetic acid), NOTP (l,4,7-triazacyclononane-N,N',N"- tris(methylenephosphonic)acid), and NOTPME (l,4,7-triazacyclononane-N,N',N"- tris(methyl-enephosphonate-monoethylester)) were shown to demonstrate high thermodynamic stability and similar plasma and in vivo stability. While the authors suggest this method to be suitable for ⁶⁸Ga-labelling of proteins, the allegation has not been substantiated since the NOTA-NHS-ester can only be introduced into macromolecules at pH values of 8.5-9 and using incubation periods of several hours. This conjugation step would adversely affect the NHS-ester of NOTA itself by leading to a high rate of hydrolysis, as it would most proteins [8, 9].

[0010] Only one method has thus far been described for introducing

⁶⁸Ga into antibody fragments [10]. Although N,N'-bis[2-hydroxy-5- (carboxyethyl)benzyl]ethylene-di-amine-N,N'-diacetic acid (HBED-CC)-derivatized antibodies could be generated, the labeling efficiency and product yield dropped with increasing reaction temperature. Lower reaction temperatures however, entailed the requirement for purification steps. Moreover, the chelator is only accessible following a complex multi-step synthesis. [0011] Contrary to ⁶⁸Ga, ^-SCN-Bn-NOTA was reported as being successfully applied for introducing ⁶⁷Ga (an Auger electron emitter) into antibodies as well as into an antibody fragment [11, 12, 13, 14]. Although it allowed for introducing ⁶⁷Ga into protein molecules, p-SCN-Bn-NOTA only provided for low radiochemical yields for ⁶⁸Ga, making subsequent purification steps necessary. Moreover, reaction times ranging from 30-60 minutes are incompatible with the short half-life Of⁶⁸Ga.

[0012] The p-SCN-Bn-NOTA chelator has an additional inherent disadvantage. While only a limited number of chelators can be introduced into peptides, proteins might have more than one site capable of accepting a chelator. While an increased number of chelators per macromolecule could increase the specific activity of the chelator-protein conjugate, it has been shown that a high number of derivatization sites leads to a dramatic loss of the biological activity of the derivatized biomolecule [15, 16, 17]. In case of the isothiocyanate derivative of NOTA, the number of derivatization sites per protein molecule could only be determined by means of ¹⁴C labeled chelators or isotopic dilution titration which represent highly complex methods.

[0013] The present disclosure refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

SUMMARY

[0014] The present disclosure broadly relates to thiol-NOTA derivatives.

[0015] As broadly claimed, the present disclosure relates to a thiol-

NOTA derivative of Formula I:

[0016] wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and

[0017] m is 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0018] In an embodiment, the present disclosure relates to a thiol-

NOTA derivative of Formula Ia:

[0019] In an embodiment, the present disclosure broadly relates to Ga radiolabeled thiol-NOTA derivatives.

[0020] As broadly claimed, the present disclosure relates to a ⁶⁸Ga radiolabeled thiol-NOTA derivative of Formula II:

[0021] wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and

[0022] m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0023] In an embodiment, the present disclosure relates to a 68 G/ a radiolab led thiol-NOTA derivative of Formula Ha:

[0024] In a further embodiment, the present disclosure broadly relates to thiol-NOTA derivatives for protein and peptide labeling.

[0025] In yet a further embodiment, the present disclosure relates to the use of thiol-NOTA derivatives of Formula I, Formula Ia, Formula II and Formula Ha for protein and/or peptide labeling.

[0026] In yet a further embodiment, the present disclosure relates to the use of thiol-NOTA derivatives of Formula I, Formula Ia, Formula II and Formula Ha for protein labeling. [0027] In yet a further embodiment, the present disclosure relates to the se of thiol-NOTA derivatives of Formula I, Formula Ia, Formula II and Formula Ha for labeling hMAb 425.

[0028] In yet a further embodiment, the present disclosure relates to the use of thiol-NOTA derivatives of Formula I, Formula Ia, Formula II and Formula Ha for labeling human serum albumin (HSA).

[0029] In yet a further embodiment, the present disclosure relates to the use of thiol-NOTA derivatives of Formula I, Formula Ia, Formula II and Formula Ha for labeling rat serum albumin (RSA).

[0030] In yet a further embodiment, the present disclosure relates to the use of thiol-NOTA derivatives of Formula I, Formula Ia, Formula II and Formula Ha for labeling bovine serum albumin (BSA).

[0031] In yet a further embodiment, the present disclosure relates to the use of thiol-NOTA derivatives of Formula I, Formula Ia, Formula II and Formula Ha for labeling diphtheria toxin (CRM mutant).

[0032] In yet a further embodiment, the present disclosure relates to the use of thiol-NOTA derivatives of Formula I, Formula Ia, Formula II and Formula Ha for labeling human annexin V.

[0033] In yet a further embodiment, the present disclosure relates to thiol-NOTA-protein conjugates comprising a thiol-NOTA derivative of Formula I or Formula Ia. [0034] In yet a further embodiment, the present disclosure relates to thiol-NOTA-protein conjugates comprising a thiol-NOTA derivative of Formula I or Formula Ia and hMAb 425.

[0035] In yet a further embodiment, the present disclosure relates to thiol-NOTA-protein conjugates comprising a thiol-NOTA derivative of Formula I or Formula Ia and human serum albumin (HSA).

[0036] In yet a further embodiment, the present disclosure relates to thiol-NOTA-protein conjugates comprising a thiol-NOTA derivative of Formula I or Formula Ia and rat serum albumin (RSA).

[0037] In yet a further embodiment, the present disclosure relates to thiol-NOTA-protein conjugates comprising a thiol-NOTA derivative of Formula I or Formula Ia and bovine serum albumin (BSA).

[0038] In yet a further embodiment, the present disclosure relates to thiol-NOTA-protein conjugates comprising a thiol-NOTA derivative of Formula I or Formula Ia and diphtheria toxin (CRM mutant).

[0039] In yet a further embodiment, the present disclosure relates to thiol-NOTA-protein conjugates comprising a thiol-NOTA derivative of Formula I or Formula Ia and human annexin V.

[0040] In yet a further embodiment, the present disclosure relates to

⁶⁸GGaa rraaddiioollaabbeelleedd tthhiiooll--NNOOTTAA--pprrootteeiin conjugates comprising a thiol-NOTA derivative of Formula II or Formula Ha. [0041] In yet a further embodiment, the present disclosure relates to

⁶⁸Ga radiolabeled thiol-NOTA-protein conjugates comprising a thiol-NOTA derivative of Formula II or Formula Ha and hMAb 425.

[0042] In yet a further embodiment, the present disclosure relates to

⁶⁸Ga radiolabeled thiol-NOTA-protein conjugates comprising a thiol-NOTA derivative of Formula II or Formula IIa and human serum albumin (HSA).

[0043] In yet a further embodiment, the present disclosure relates to

⁶⁸Ga radiolabeled thiol-NOTA-protein conjugates comprising a thiol-NOTA derivative of Formula II or Formula Ha and rat serum albumin (RSA).

[0044] In yet a further embodiment, the present disclosure relates to

Ga radiolabeled thiol-NOTA-protein conjugates comprising a thiol-NOTA derivative of Formula II or Formula Ha and bovine serum albumin (BSA).

[0045] In yet a further embodiment, the present disclosure relates to

Aft

Ga radiolabeled thiol-NOTA-protein conjugates comprising a thiol-NOTA derivative of Formula II or Formula IIa and diphtheria toxin (CRM mutant).

[0046] In yet a further embodiment, the present disclosure relates to

Ga radiolabeled thiol-NOTA-protein conjugates comprising a thiol-NOTA derivative of Formula II or Formula IIa and human annexin V.

[0047] In yet a further embodiment, the present disclosure relates to the use of thiol-NOTA derivatives of Formula I, Formula Ia, Formula II and Formula IIa for peptide labeling. [0048] In yet a further embodiment, the present disclosure relates to the use of ⁶⁸Ga-radiolabelled thiol-NOTA derivatives of Formula II and Formula Ha for positron emission tomography (PET) imaging.

[0049] In yet a further embodiment, the present disclosure relates to a

⁶⁸Ga protein labeling kit comprising a thiol-NOTA derivative of Formula I or Formula Ia.

[0050] In yet a further embodiment, the present disclosure relates to a

⁶⁸Ga protein labeling kit comprising a thiol-NOTA derivative of Formula I or Formula Ia and a maleimide-based linker for conjugating the thiol-NOTA derivative of Formula I or Formula Ia to a target protein.

[0051] In yet a further embodiment, the present disclosure relates to a

⁶⁸Ga protein labeling kit including a thiol-NOTA-protein conjugate comprising a thiol-NOTA derivative of Formula I or Formula Ia and a target protein.

[0052] In yet a further embodiment, the present disclosure relates to a

Ga protein labeling kit including a thiol-NOTA-protein conjugate comprising a thiol-NOTA derivative of Formula I or Formula Ia and hMAb 425.

[0053] In yet a further embodiment, the present disclosure relates to a

Ga protein labeling kit including a thiol-NOTA-protein conjugate comprising a thiol-NOTA derivative of Formula I or Formula Ia and human serum albumin (HSA).

[0054] In yet a further embodiment, the present disclosure relates to a

⁶⁸Ga protein labeling kit including a thiol-NOTA-protein conjugate comprising a thiol-NOTA derivative of Formula I or Formula Ia and rat serum albumin (RSA). [0055] In yet a further embodiment, the present disclosure relates to a

⁶⁸Ga protein labeling kit including a thiol-NOTA-protein conjugate comprising a thiol-NOTA derivative of Formula I or Formula Ia and bovine serum albumin (BSA).

[0056] In yet a further embodiment, the present disclosure relates to a

⁶⁸Ga protein labeling kit including a thiol-NOTA-protein conjugate comprising a thiol-NOTA derivative of Formula I or Formula Ia and diphtheria toxin (CRM mutant).

[0057] In yet a further embodiment, the present disclosure relates to a

⁶⁸Ga protein labeling kit including a thiol-NOTA-protein conjugate comprising a thiol-NOTA derivative of Formula I or Formula Ia and human annexin V.

[0058] In yet a further embodiment, the present disclosure relates to a method for ⁶⁸Ga radiolabeling of a target protein, the method comprising:

[0059] reacting the target protein with a maleimide containing reagent to afford a maleimide-modified target protein;

[0060] reacting the maleimide modified protein with a thiol-NOTA derivative of Formula I or Formula Ia to afford a thiol-NOTA-protein conjugate; and

[0061] reacting the thiol-NOTA-protein conjugate with a source of Ga to afford a Ga-radiolabeled thiol-NOTA-protein conjugate.

[0062] In yet a further embodiment, the present disclosure relates to a method for Ga radiolabeling of a target protein, the method comprising: [0063] reacting a thiol-NOTA derivative of Formula I or Formula Ia with a maleimide containing reagent to afford a maleimide-modified thiol-NOTA conjugate;

[0064] reacting the maleimide-modified thiol-NOTA conjugate with a target protein to provide a thiol-NOTA-protein conjugate; and

[0065] reacting the thiol-NOTA-protein conjugate with a source of ⁶⁸Ga to afford a ⁶⁸Ga-radiolabeled thiol-NOTA-protein conjugate.

[0066] The foregoing and other objects, advantages and features of the present disclosure will become more apparent upon reading of the following non- restrictive description of illustrative embodiments thereof, given by way of example only.

BRIEF DESCRIPTION OF THE DRAWINGS

[0067] In the appended drawings:

[0068] FIG. 1 shows HPLC chromatograms illustrating the radioactivity traces for labeling mixtures comprising ⁶⁸Ga-labeled thiol-NOTA- annexin (A), ⁶⁸Ga-labeled thiol-NOTA-diphtheria toxin CRM mutant (B) and ⁶⁸Ga- labeled thiol-NOTA-RSA (C). The ⁶⁸Ga-labeled conjugates were produced using thiol-NOTA derivative 3 (Formula Ia) and linker 5.

[0069] FIG. 2 shows a maximum intensity projection image of a blood pool scan of a healthy rat with Ga-labeled thiol-NOTA-RSA acquired over 30 min, demonstrating radiotracer accumulation in large arteries, the liver, and the heart. Atria and ventricles can be easily identified. RA, right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle; RK, right kidney; LK, left kidney; B, bladder. [0070] FIG. 3 shows a maximum intensity projection image of an anterior myocardial infarction in a rat with [¹⁸F] Fluorodeoxyglucose (FDG) (leftside image) and with ⁶⁸Ga-labeled thiol-NOTA-Annexin V acquired over 60 min (right-side image). The middle image is an overlay of the left and right-side images. The right-side image demonstrates the superiority of direct labeling of apoptotic tissue via apoptosis specific Annexin-V versus negative demonstration by the use of FDG which shows no uptake into apoptotic/necrotic tissue.

DETAILED DESCRIPTION

[0071] In order to provide a clear and consistent understanding of the terms used in the present specification, a number of definitions are provided below. Moreover, unless defined otherwise, all technical and scientific terms as used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains.

[0072] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one". Similarly, the word "another" may mean at least a second or more.

[0073] As used in this specification and claim(s), the words

"comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "include" and "includes") or "containing" (and any form of containing, such as "contain" and "contains"), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps. [0074] The term "about" is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value.

[0075] The term "derivative" as used herein, is understood as being a substance similar in structure to another compound but differing in some slight structural detail.

[0076] The present description refers to a number of chemical terms and abbreviations used by those skilled in the art. Nevertheless, definitions of selected terms are provided for clarity and consistency.

[0077] The present disclosure relates to thiol-NOTA derivatives for protein and peptide labeling. In an embodiment, the thiol-NOTA derivatives of the present disclosure function as chelators for Ga.

[0078] In an embodiment of the present disclosure, the thiol-NOTA derivatives of Formula I or Formula Ia are coupled to a target protein using a maleimide-based linker. In a further embodiment of the present disclosure, a target protein is modified using 4-(N-maleimidomethyl)cyclohexane-l-carboxylic acid 3- sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-SMCC) to produce a maleimide modified protein. Other suitable maleimide-based linkers are known in the art and are within the capacity of a skilled technician. A non-limiting example of such other suitable maleimide-based linkers includes 1 ,2-bis(maleimido)ethane. The maleimide modified proteins exhibited a modified molecular size as well as a different structure when compared to the starting protein. The maleimide modified proteins are subsequently coupled with the thiol-NOTA derivatives of Formula I or Formula Ia. [0079] High protein coupling (i.e. conjugation) efficiencies of the thiol-

NOTA derivatives of Formula I or Formula Ia are important. This observation is further compounded when working with proteins of limited availability and high cost. In an embodiment of the present disclosure, deprotection steps, prior and following protein coupling, are eliminated. Moreover, the number of derivation sites per protein should be controlled such that coupling does not entail loss of biological activity.

[0080] The thiol-NOTA derivatives of the present disclosure afford high coupling rates at ambient temperatures with maleimide modified proteins leading to protein conjugates of high chemical purity. In an embodiment of the present disclosure, the thiol-NOTA-protein conjugates are obtained in yields ranging from 85-99%.

[0081] The maleimide-modified proteins comprise a well-defined number of derivatization sites for coupling with the thiol-NOTA derivatives of Formula I or Formula Ia. The thiol-NOTA protein conjugates (labeling precursors) are readily labeled with Ga to provide Ga-radiolabelled thiol-NOTA-protein conjugates. The radiolabeling process does not affect the biological activity of the target protein. In an embodiment of the present disclosure, the thiol-NOTA-protein conjugates are labeled with Ga within a period of time ranging from about 5 to about 10 minutes. In a further embodiment of the present disclosure, the thiol- NNOOTTAA--pprrootteeiinn ccoonnjjuuggaatteess aarree llaabbeled with ⁶⁸Ga within a period of time ranging from about 7 to about 10 minutes.

[0082] In an embodiment of the present disclosure, the thiol-NOTA- protein conjugates are labeled with ⁶⁸Ga in high specific radioactivities ranging from about 10 to about 50 GBq/μmol. In a further embodiment of the present disclosure, the thiol-NOTA-protein conjugates are labeled with ⁶⁸Ga in radiochemical yields >95%. The ⁶⁸Ga-radiolabeled thiol-NOTA-protein conjugates of the present disclosure are stable (i.e. radiochemical yields remain substantially constant).

[0083] The synthesis of a thiol-NOTA derivative in accordance with an embodiment of the present disclosure is illustrated hereinbelow in Scheme 1. A protected commercially available NOTA-GA(tBu)₃ (1) is coupled to S-trityl- mercaptoethanolamine-trifiuoroacetate to provide adduct 2. Subsequent deprotection using concentrated TFA afforded thiol-NOTA derivative 3 (Formula Ia) in moderate overall yield (22%).

Scheme 1

[0084] The synthesis of a thiol-NOTA protein conjugate in accordance with an embodiment of the present disclosure is illustrated hereinbelow in Scheme 2. Target protein (4) is derivatized using 4-(N-maleimidomethyl)cyclo-hexane-l- carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-SMCC) (5) to provide maleimide-modified protein (6). Subsequent conjugation using thiol- NOTA derivative 3 (Formula Ia) provides the thiol-NOTA-protein conjugate labeling precursor (7).

Scheme 2

[0085] The synthesis of a ⁶⁸Ga-radiolabelled thiol-NOTA protein conjugate in accordance with an embodiment of the present disclosure is illustrated hereinbelow in Scheme 3. Thiol-NOTA-protein conjugate (7) is labeled with ⁶⁸Ga at room temperature and at a pH ranging from about 3.5 to 4 to provide ⁶⁸Ga-labeled thiol-NOTA-protein conjugate (8).

7 8

Scheme 3

[0086] The synthesis of a maleimide modified thiol-NOTA derivative in accordance with an embodiment of the present disclosure is illustrated hereinbelow in Scheme 4.

Scheme 4

[0087] The synthesis of a ⁶⁸Ga-radiolabelled thiol-NOTA protein conjugate in accordance with an embodiment of the present disclosure is illustrated hereinbelow in Scheme 5.

Scheme 5

[0088] GENERATION OF MALEIMIDE-MODIFIED PROTEINS

[0089] Prior to coupling the thiol-NOTA derivatives of Formula I or

Formula Ia, the target protein was modified using 4-(N- maleimidomethyl)cyclohexane- 1 -carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-SMCC) to produce a maleimide modified protein. The maleimide group provides for covalent thiol-maleimide coupling at ambient temperatures within a period of time ranging from about 5 to about 10 minutes. The number of maleimide groups introduced into the target protein for subsequent thiol- NOTA coupling could be quantified using Ellman's assay. Target proteins (e.g. annexin, diphtheria toxin (CRM mutant) and BSA) were subjected to a seven-fold excess of 4-(N-maleimidomethyl)cyclohexane-l-carboxylic acid 3-sulfo-N- hydroxysuccinimide ester sodium salt (Sulfo-SMCC) affording maleimide modified proteins comprising from about 1.4 to about 1.7 maleimide groups/protein. This results in only minor alterations of the biological activity of the target protein [15, 16, 17]. The maleimide-modified proteins were purified using size-exclusion gel chromatography (recovery rates of 95-98%).

[0090] The modification of recombinant target proteins with free cysteine residues provides an additional improvement. The introduction of cysteine residues into the target protein, at positions which are not essential for its biological function, leads to an optimization between labeling and optimal biological activity, determining the diagnostic or therapeutic value of a ⁶⁸Ga labeled radiochemical. The newly introduced cysteine residue(s) allow(s) for the preparation of a maleimide modified protein with a site specific instead of a limited but random modification site.

[0091] GENERATION OF THIOL-NOTA-PROTEIN-

CONJUGATES

[0092] The maleimide-modified proteins were coupled with a thiol-

NOTA derivative of Formula I or Formula Ia in an aqueous medium at pH 7.2 over a period of 30 minutes. Purification by ultrafiltration afforded the labeling precursors (thiol-NOTA-protein conjugates) at yields ranging from 85 to 95% (Scheme 2). Maleimide-modified proteins were generated based on human Monoclonal Antibody 425 (IgG, MW 150 IcDa), RSA (MW 69 kDa), HSA (MW 68 kDa), BSA (MW 66 kDa), diphtheria toxin (CRM mutant) (MW 63 kDa) and human annexin V (MW 36 kDa).

[0093] LABELING OF THE THIOL-NOTA-PROTEIN-

CONJUGATES WITH ⁶⁸Ga

[0094] The thiol-NOTA-protein conjugates were radiolabeled with ⁶⁸Ga by mixing each protein conjugate (6.9 nmol) with 1 mL of Ga (240-340 MBq, eluted from a ⁶ Ge/⁶⁸Ga generator) in an acetate-buffered aqueous medium at ambient temperature (pH ranging from 3.5 to 4.0) over a period ranging from 5 to 10 minutes (Scheme 3). All labeling reactions were complete after a period of time ranging from 7-10 minutes affording the corresponding Ga radiolabeled thiol- NOTA-protein-conjugates in suitable radiochemical purities. A completed labeling reaction afforded the ⁶⁸Ga radiolabeled thiol-NOTA-protein-conjugate in a radiochemical purity of more than 95% (FIG. 1) and specific radioactivities ranging between 10 and 50 GBq/μmol. The high radiochemical purity of the ⁶⁸Ga- radiolabeled thiol-NOTA-protein-conjugates makes a subsequent purification step redundant Moreover, the thiol-NOTA-protein-conjugates were shown to be stable in aqueous solution for at least 4 weeks at 4°C as they could be labeled with unvarying radiochemical yields. The thiol-NOTA derivative of Formula Ia was demonstrated to be stable for 2 years at room temperature.

[0095] MATERIALS AND METHODS

[0096] GENERAL REAGENTS

[0097] All commercially available chemicals were of analytical grade and used without further purification. Sulfo-SMCC and NODA-GA(tBu)₃ were purchased from Pierce (Bonn, Germany) and CheMatech (Dijon, France), respectively. S-trityl-mercaptoethanolamine-trifluoroacetate was synthesised according to published procedures [18].

[0098] An Agilent 1200 analytical and semi -preparative HPLC system was used, together with a Chromolith Performance (RP-18e, 100-4.6 mm, Merck, Germany) and a Chromolith SemiPrep (RP- 18e, 100-10 mm, Merck, Germany) column, respectively. ESI mass spectra were obtained using a Finnigan MAT95Q mass spectrometer. NMR spectra were recorded using a Jeol AS500 spectrometer. Thin-layer chromatography was performed using Polygram SIL G/UV254 TLC plates (Macherey-Nagel, Duren, Germany). Size-exclusion gel chromatography was carried out using NAP-5 or NAP-10 columns (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and size-exclusion fast protein liquid chromatography (FPLC) was performed using a Superdex 200 10/30 GL column (Amersham Biosciences AB, Uppsala, Sweden). Ultrafiltration was performed using Vivaspin 6 centrifugal concentrators (Sartorius, Gόttingen, Germany) with a MWCO of 10 or 100 kDa.

[0099] SYNTHESIS OF røRr-BUTYL 2.2'-(7-(1-!TERr-BUTOXY- l,5-DIOXO-5-(2-(TRITYLTHIO) ETHYLAMINO)PENTAN-2-YLM.4.7-

TRIAZONANE-1.4-DIYL) DIACETATE (S-TRITYL-TRIS- !TERT-BUTYL- THIOL-NOTA) (2)

[00100] A solution of dicyclohexylcarbodiimide (DCC) (189 mg; 919 μmol) in pyridine (5 mL) was added dropwise to a solution consisting of 4-(4,7- bis(2-tert-butoxy-2-oxoethyl)- 1 ,4,7-triazonan- 1 -yl)-5-tert-butoxy-5-oxopentanoic acid (1, NOTA-GA(tBu)₃) (500 mg; 919 μmol) and S-trityl-mercaptoethanolamine- trifluoroacetate (293 mg; 919 μmol) in 40 mL water/MeCN 1 : 1 at room temperature. Following overnight reaction, the solvents were evaporated and the product was purified via semi-preparative HPLC using 0-30% MeCN + 0.1% TFA (R, = 3.9 min). The product was isolated as a white solid (343 mg; 406 μmol; 44%) after lyophilisation. ¹H NMR (DMSO-d₆) (δ, ppm; J, Hz): 7.35-7.20 (m, 15H); 3.97-3.85 (m, 2H); 3.79-3.65 (m, 3H); 3.41 (t, IH, J³=7.3); 3.16-2.82 (bm, 12H); 2.69-2.65 (m, 2H); 2.19 (t, 2H, J³=7.2); 2.12-2.09 (m, 2H); 1.43+1.40+1.39 (3s, 27H). ¹³C NMR (DMSO-J₆) (δ, ppm; J, Hz): 172.08; 171.82; 169.47; 144.99; 129.62; 128.60; 127.32; 82.19; 81.65; 66.47; 63.91; 55.45; 55.27; 51.61; 50.34; 49.47; 48.82; 46.91; 44.97; 38.02; 32.37; 32.01; 28.34; 28.32; 28.27; 25.96. ESI-MS (m/z) for [M + H]⁺ (calculated): 816.49 (816.14).

[00101] SYNTHESIS OF MERCAPTOETHYLAMINO-NODA-GA

(THIOL-NOTA) (3)

[00102] S-trityl-tris-tert-butyl-thiol-NOTA (2, 343 mg; 406 μmol) was dissolved in a mixture of TFA (5 mL) and TIS (triisopropylsilane; 200 μL) and reacted for 3 hours at room temperature. The volatile components were evaporated and the product was purified using semi-preparative HPLC with 0-20% MeCN + 0.1% TFA (R, = 2.3 min). The product was isolated as a yellow hardening oil (90 mg; 207 μmol; 51%) after lyophilisation. ¹H NMR (DMSO-J₆) (δ, ppm; J, Hz): 3.97-3.89 (m, 4H); 3.66-3.63 (m, IH); 3.36 (t, 2H, J³=6.4); 3.31-3.11 (m, 12H); 2.64 (t, 2H, J³=6.4); 2.47 (t, 2H, J³=7.7); 2.19-2.01 (m, 2H); 1.29 (s, IH). ¹³C NMR (DMSO-J₆) (δ, ppm; J, Hz): 175.56; 175.35; 175.22; 63.94; 55.62; 50.97; 49.86; 49.84; 49.71; 49.38; 45.97; 45.92; 42.23; 32.78; 24.52; 23.38. ESI-MS (m/z) for [M + H]⁺ (calculated): 435.2 (435.5).

[00103] GENERAL PROCEDURE FOR THE MALEIMIDE

MODIFICATION OF THIOL-NOTA DERIVATIVES: PREPARATION OF 2,2'-(7-α-CARBOXY-4-(2-α-(2-(2.5-DIOXO-2.5-DIHYDRO-lH-PYRROL-l- YLVETHYL)-2.5-DIOXOPYRROLIDIN-3-YLTHIO)ETHYLAMINOV4- OXO-BUTYLyIAy-TRIAZONANE-M-DIYL)DIACETIC ACID [00104] A solution of 1,1 '-(ethane- l,2-diyl)bis(l//-pyrrole-2,5-dione)

(4.7 mg; 21.6 μmol) in phosphate buffer (0.1M, pH 6.0):MeCN 1 :1 (1 mL) was added to thiol-NOTA 3 and the pH of the solution adjusted to 7.2 by the addition of phosphate buffer (0.5 M, pH 7.2, 200 μL). After a period of 5 minutes, the reaction was complete and the crude product purified by semi-preparative HPLC using 0- 30% MeCN + 0.1% TFA (R, = 3.4 min). The product was isolated as a lightly yellow solid (11.9 mg, 18.1 μmol; 84%) after lyophilisation. ¹H NMR (DMSO-J₆) (S, ppm; J, Hz): 7.64 (bs, IH); 6.88 (s, 2H); 4.14 (s, IH); 4.01-3.98 (m, IH); 3.93- 3.82 (m, 2H); 3.75-3.61 (m, 4H); 3.54-3.13 (m, 16H); 3.06-2.98 (m, IH); 2.85-2.77 (m, IH); 2.52-2.48 (m, 2H); 2.46-2.40 (m, 2H); 2.23-2.08 (m, 2H). ¹³C NMR (DMSO-J₆) (S, ppm; J, Hz): 177.76; 175.52; 173.80; 171.67; 171.67; 160.03; 135.28; 64.01; 55.89 + 55.85 + 55.76 + 55.48 + 55.41 + 52.12 + 52.07 + 52.04 + 51.19 + 51.04 + 50.84 + 50.76 + 49.23 + 49.13 + 49.11 + 47.65 + 47.49 + 47.45 + 47.33 + 45.92 + 45.80 + 45.57; 39.86; 39.23; 39.11; 36.71 ; 35.95; 33.05; 32.15; 26.03. ESI-MS (m/z) for [M + H]⁺ (calculated): 656.0 (655.7). MALDI-MS (m/z) for [M + H]⁺ (calculated): 656.0 (655.7).

[00105] GENERAL PROCEDURE FOR THE MALEIMIDE

MODIFICATION OF PROTEINS WITH SULFO-SMCC

[00106] A freshly prepared solution of sulfo-SMCC (4-(N- maleimidomethyl)cyclohexane- 1 -carboxylic acid 3 -sulfo-N-hydroxysuccinimide ester sodium salt) (92μg; 210 nmol) in 150 μL of DMF:water (1 :1) was added to a solution of the target protein (30 nmol) in 500 μL PB (0.1 M, pH 7.2). After one hour of incubation, the maleimide-modified protein was purified via size-exclusion chromatography using NAP-5 columns. The maleimide-modified proteins were obtained in high yields (85-99%). The number of maleimide groups per protein was determined using MESNA and Ellman's assay and was found to be ranging between 1.4 and 1.7. [00107] GENERAL PROCEDURE FOR GENERATING THIOL-

NOTA-PROTEIN CONJUGATES

[00108] A freshly prepared solution of thiol-NOTA (3, 131 μg; 300 nmol) was added to a solution comprising a maleimide-modified protein in phosphate buffer (0.1M, pH 7.2) and stirred over a period of 30 minutes. The proteinaceous solution was subsequently diluted with HEPES buffer (0.025 M, pH 4.0) and purified by ultrafiltration applying Vivaspin 6 centrifugal concentrators (MWCO 10 IcDa for annexin V, HSA, BSA, RSA and diphtheria toxin (CRM mutant); MWCO 100 kDa for hMAb 425). The ultrafiltration was repeated four times. The thiol-NOTA-protein conjugates were recovered at high yields (85-99%).

[00109] GENERAL PROCEDURE FOR THE COUPLING OF

MALEIMIDE-MODIFIED THIOL-NOTA DERIVATIVES WITH THIOL CONTAINING PROTEINS

[00110] To a solution of SDF-I α [chemokine stromal-derived factor- lα; a small protein (MW HkDa) shown to accumulate in mycard infarct tissue] (1 mg, 91 nmol) in phosphate buffer (0.1 M, pH 6.0, 500 μL) was added a solution of TCEP (Tris(2-Carboxyethyl) phosphine Hydrochloride) (5 eq., 455 nmol, 0.13 mg) in water (100 mL). The resulting mixture was reacted over a period of 10 minutes followed by the addition of a solution comprising NOTA-maleimide (10 eq., 909 nmol, 0.6 mg) in phosphate buffer (0.1 M, pH 6.0, 200 μL) (Scheme 5). The pH of the solution was adjusted to 7.2 by the addition of phosphate buffer (0.5 M, pH 7.2, 50 μL). After 30 minutes, the crude NOTA-protein-conjugate was purified by semi- preparative HPLC using 0-50% MeCN + 0.1% TFA. The product was isolated as a white solid (1.02 mg, 89 nmol, 98%) after lyophilisation. Radiolabeling was carried out as described hereinbelow. [00111] LABELING OF THIOL-NOTA-PROTEIN-CONJUGATES

WITH ⁶⁸Ga

[00112] A solution of a thiol-NOTA-protein conjugate (6.9 nmol) in

HEPES buffer (0.025M, pH 4.0) was added to 240-340 MBq of ⁶⁸Ga in a sodium acetate solution (1 mL; 0.11 M; pH 3.5-4.0) and incubated for 5 to 10 minutes. The reaction mixtures were subsequently analyzed by analytical HPLC. The Ga- radiolabeled thiol-NOTA-protein conjugates were found to be 95-99% pure and obtained in specific radioactivities ranging from 10 and 50 GBq/μmol.

[00113] IN VIVO BIODISTRIBUTION OF ⁶⁸Ga RADIOLABELED

THIOL-NOTA-RSA IN HEALTHY RATS

[00114] CD rats were housed according to German animal protection laws and protocols of the local committee. Experiments were performed under inhalation anesthesia (oxygen 1.2 L/min, isofluran 1.5 vol %) and appropriate warming to prevent hypothermia.

[00115] 20 to 60 MBq of ⁶⁸Ga radiolabeled thiol-NOTA-RSA were intravenously administered via the lateral tail vein of CD rats anesthetized by inhalation (oxygen 1.2 L/min, isofluran 1.5 vol %). List mode data were acquired over 30 minutes using a Siemens Inveon 120 small animal PET system (Siemens Preclinical Imaging, Knoxville, TN) and divided into temporal frames of increasing length varying from 20 s to 5 min for the assessment of temporal changes in regional tracer accumulation. Tomographic volumes were created using 3D iterative reconstruction, yielding a transaxial spatial resolution of approximately 1.4 mm. Rat biodistribution data showed nearly constant whole blood radioactivity from minute 2 over the next 25 min after injection. In previous experiments with ¹⁸F-labeled RSA, it was demonstrated that its uptake by the liver was as high as in the blood pool and its uptake by the kidneys in a range similar to the arterial blood volume [19]. In contrast to these results, Ga labeled thiol-NOTA-RSA showed a lower uptake especially in the liver in the first 60 minutes following tracer administration. This can be attributed to a lower lipophilicity of the Ga-labeled albumin resulting from a derivatization leading to the less lipophilic derivatization..

[00116] As shown in FIG. 2, ⁶⁸Ga-radiolabeled thiol-NOTA-RSA provides a blood pool image of a healthy rat including a clear illustration of the heart, aorta, arteries, and the liver. In addition, radioactivity accumulation can be seen in the kidneys and the bladder due to metabolization and excretion processes.

[00117] IN VIVO BIODISTRIBUTION OF ⁶⁸GA RADIOLABELED

THIOL-NOTA-ANNEXIN V IN A RAT INFARCT MODEL

[00118] 30-40 MBq of ⁶⁸Ga-radiolabeled annexin V were intravenously administered via the lateral tail vein of the rats anesthetized by inhalation (oxygen 1.2 L/min, isofiuran 1.5 vol %). List mode data were acquired over 60 minutes using a Siemens Inveon 120 small animal PET system (Siemens Preclinical Imaging, Knoxville, TN) and divided into temporal frames of 10 min for the assessment of temporal changes in regional tracer accumulation. Tomographic volumes were created using 3D iterative reconstruction, yielding a transaxial spatial resolution of approximately 1.4 mm.

[00119] The left coronary artery (LCA) was ligated to induce transmural myocardial infarction. Under anesthesia with intramuscular administration of midazolam (0.1 mg/kg), fentanyl (1 mg/kg), and medetomidin (10 mg/kg) (MMF) and mechanical ventilation, the chest was opened to expose the heart. A 7-0 polypropylene suture on a small curved needle was passed through the LCA and ligated to occlude the LCA. To assess the relationship of in vivo and ex vivo measurements in acute and subacute phases, animals were allowed 2 days of recovery before PET imaging.

[00120] Annexin V was used as an indicator for apoptotic tissue, as can be found in infarcted tissue. While the imaging of a healthy heart with [¹⁸F] Fluorodeoxyglucose (FDG) would demonstrate a V-shaped structure, this shape is interrupted in an infarcted heart due to apoptotic tissue that can no longer absorb any nutrients, (i.e. glucose). Contrary to the lack of FDG in the apoptotic infarcted tissue, the use of annexin V as a marker for apoptosis can directly depict the infarcted site.

[00121] FIG. 3 shows the overlay of two maximum intensity projection images of an anterior myocardial infarction in a rat. The image covers a cross section through the coronary level of the heart. Negative landmarking of the infarct region was obtained by the use of [¹⁸F] Fluorodeoxyglucose (FDG) while ⁶⁸Ga- radiolabelded annexin V allows positive infarct imaging. FDG landmarking indicates the infarct region by the lack of accumulating FDG depicted in colors ranging from yellow to red, i.e. the bright apical dotted structure and the interior light and medium grey horizontal C-shaped structure. The direct accumulation of the Ga-radiolabeled annexin V in the apoptotic and necrotic infarct heart tissue is characterized by the structure in violet. Ga-radiolabeled annexin V was - as can be seen - also taken up by the liver. The images were obtained two days after infarct and were acquired over one hour.

[00122] It is to be understood that the invention is not limited in its application to the details of construction and parts as described hereinabove. The invention is capable of other embodiments and of being practiced in various ways. It is also understood that the phraseology or terminology used herein is for the purpose of description and not limitation. Hence, although the present invention has been described hereinabove by way of illustrative embodiments thereof, it can be modified, without departing from the spirit, scope and nature of the subject invention as defined in the appended claims.

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Claims

WHAT IS CLAIMED IS;

1. A thiol-NOTA derivative of Formula I:

wherein: n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

2. The thiol-NOTA derivative as defined in claim 1, wherein n and m are 2.

3. A ⁶⁸Ga radiolabeled thiol-NOTA derivative of Formula II:

4. The thiol-NOTA derivative as defined in claim 3, wherein n and m are 2.

5. Use of the thiol-NOTA derivative as defined in claims 1 or 2 for protein or peptide labelling.

6. The use as defined in claim 5, wherein the proteins are selected from the group consisting of hMAb 425, HSA, RSA, BSA, diphtheria toxin (CRM mutant), and human annexin V.

7. Use of the thiol-NOTA derivative as defined in claims 3 or 4 for protein or peptide labelling.

8. The use as defined in claim 7, wherein the proteins are selected from the group consisting of hMAb 425, HSA, RSA, BSA, diphtheria toxin (CRM mutant), and human annexin V.

9. A thiol-NOTA protein conjugate comprising a thiol-NOTA derivative as defined in claims 1 or 2 and a target protein.

10. The thiol-NOTA protein conjugate as defined in claim 9, wherein the target protein is selected from the group consisting of hMAb 425, HSA, RSA, BSA, diphtheria toxin (CRM mutant), and human annexin V.

11. A ⁶⁸Ga radiolab led thiol-NOTA protein conjugate comprising a thiol-NOTA derivative as defined in claims 3 or 4 and a target protein.

12. The thiol-NOTA protein conjugate as defined in claim 11, wherein the target protein is selected from the group consisting of hMAb 425, HSA, RSA, BSA, diphtheria toxin (CRM mutant), and human annexin V.

13. A Ga protein labeling kit comprising a thiol-NOTA derivative as defined in claims 1 or 2.

14. The ⁶⁸Ga protein labeling kit as defined in claim 13, further comprising and a maleimide-based linker for conjugating the thiol-NOTA derivative to a target protein.

15. The ⁶⁸Ga protein labeling kit as defined in claim 14, wherein the target protein is selected from the group consisting of consisting of hMAb 425, HSA, RSA, BSA, diphtheria toxin (CRM mutant), and human annexin V.

16. A Ga protein labeling kit comprising a thiol-NOTA-protein conjugate, wherein the conjugate is obtained by coupling of a maleimide-modified protein and a thiol-NOTA derivative as defined in claims 1 or 2.

17. The Ga protein labeling kit as defined in claim 16, wherein the maleimide-modified protein is selected from the group consisting of consisting of hMAb 425, HSA, RSA, BSA, diphtheria toxin (CRM mutant), and human annexin V.

18. A method for Ga radiolabeling of a target protein, the method comprising: a) reacting the target protein with a maleimide containing reagent to afford a maleimide-modified target protein; b) reacting the maleimide modified protein with a thiol-NOTA derivative as defined in claims 1 or 2 to afford a thiol-NOTA- protein conjugate; and c) reacting the thiol-NOTA-protein conjugate with a source of ⁶⁸Ga to afford a ⁶⁸Ga-radiolabeled thiol-NOTA-protein conjugate.

19. The method as defined in claim 18, wherein the target protein is selected from the group consisting of hMAb 425, HSA, RSA, BSA, diphtheria toxin (CRM mutant), and human annexin V.

20. A method for ⁶⁸Ga radiolabeling of a target protein, the method comprising: a) reacting a thiol-NOTA derivative as defined in claims 1 or 2 with a maleimide containing reagent to afford a maleimide-modified thiol-NOTA conjugate; b) reacting the maleimide-modified thiol-NOTA conjugate with a target protein to provide a thiol-NOTA-protein conjugate; and c) reacting the thiol-NOTA-protein conjugate with a source of Ga to afford a ⁶⁸Ga-radiolabeled thiol-NOTA-protein conjugate.

21. The method as defined in claim 20, wherein the target protein is selected from the group consisting of hMAb 425, HSA, RSA, BSA, diphtheria toxin (CRM mutant), and human annexin V.