WO2024110329A1

WO2024110329A1 - Labeling of biomolecules with actinium-225 and medical uses thereof

Info

Publication number: WO2024110329A1
Application number: PCT/EP2023/082190
Authority: WO
Inventors: Calogero D'ALESSANDRIA; Alexander WURZER; Mathias EIBER; Wolfgang Weber
Original assignee: Technische Universität München
Priority date: 2022-11-23
Filing date: 2023-11-17
Publication date: 2024-05-30

Abstract

The present invention relates to a method for radioactive labeling of a precursor with 225Ac comprising freeze-drying the precursor before labeling. The present invention further relates to the 225Ac-labeled precursor obtained in said method for use in the treatment of cancer. The present invention also relates to a kit for radioactive labeling of a precursor with 225Ac.

Description

Labeling of biomolecules with Actinium-225 and medical uses thereof

The present invention relates to a method for radioactive labeling of a precursor with ²²⁵ Ac comprising freeze-drying the precursor before labeling. The present invention further relates to the ²²⁵ Ac-labeled precursor obtained in said method for use in the treatment of cancer. The present invention also relates to a kit for radioactive labeling of a precursor with ²²⁵ Ac.

BACKGROUND OF THE INVENTION

An important prerequisite in targeted radionuclide therapy of cancer is the delivery of cytotoxic doses of radiation to malignant cells. In principle, beta-emitting-, alpha-emitting- or Auger electron-emitting nuclides, tethered to a targeted biomolecule for therapeutic purposes can be used (Boyd et al., 2006). Beta radiation is a particulate radiation consisting of high-speed electrons, which lose their kinetic energy, while their track paths become increasingly contorted due to scattering. An exemplary 2 MeV beta particle has a rather long range of 1 cm in water. The linear energy transfer (LET), which represents the total amount of energy deposited per unit track length, is comparably low for beta particles with values around 0.2 keV/pm (Boyd et al., 2006).

Alpha particles are positively charged with a mass and charge equal to a helium nucleus. Their energies are higher compared to beta particles with values of 5-9 MeV, displaying a linear track length of 20 to 80 pm in water. Moreover, their higher LET (approx. 100 keV/pm) can result in more lethal double strand DNA breaks, compared to beta particles. In combination with the short range of alphas, the high energy discharge results in a very toxic effect to a relatively small field with very limited damage to surrounding normal tissue (Scheinberg et al., 2011).

In the past, the alpha emitter Actinium-225 (²²⁵Ac) was suggested as a radionuclide for targeted alpha therapy (TAT) of cancer (Morgenstern et al, 2018). Thereby ²²⁵ Ac is complexed by a suitable chelator, connected to a biological active molecule. After intravenous injection, the radiopharmaceutical binds to cancer cells and delivers the payload, resulting in irradiation of cancer cells, while minimizing damage to healthy tissues. ²²⁵ Ac (ti/2 = 9.9 d; 6 MeV alpha-particle) decay yields 4 alpha and 3 beta disintegrations (Robertson et al., 2018). The daughter nuclides with suitable gamma emissions, i.e. ²²¹Fr and ²¹³Bi, can be exploited for imaging or measuring drug distribution. The decay chain of ²²⁵ Ac is summarized below and depicted in Figure 1 :

²²¹Fr (ti/2 = 4.8 min; 6 MeV a-particle; 218 keV y-emission)

²¹⁷At (ti/2 = 32.3 ms; 7 MeV a -particle)

²¹³Bi (ti/2 = 45.6 min; 6 MeV a -particle, 444 keV P' -particle, 440 keV y -emission)

²¹³Po (ti/2 = 4.2 ps; 8 MeV a -particle),

^2O9T1 (ti/2 = 2.2 m; 659 keV P'-particle),

²⁰⁹Pb (ti/2 = 3.25 h; 198 keV P' -particle)

²⁰⁹Bi (stable).

Despite the favorable decay properties of ²²⁵ Ac for TAT, one main challenge remains its low availability for research and clinical applications (Morgenstern et al., 2018). Currently, the primary source of ²²⁵ Ac is ²²⁹Th (ti/2 = 7340 years), originating mainly from radioactive waste of nuclear power plants. The production of ²²⁵ Actinium via this route, which still represents the most reliable source of international supply, mounts however only up to <100 GBq. This limited supply in combination with immense costs (300-600C per MBq), is still preventing ²²⁵ Ac to develop its full clinical potential (Morgenstern etal, 2018). Currently there are several research projects underway, examining alternative production strategies of ²²⁵ Ac, which mainly rely on high-energy accelerators, e.g. the irradiation of Ra-226 targets with protons, neutrons or gamma rays (Apostolidis et al., 2005).

For many years an additional main challenge was the development of suitable bifunctional chelators and standardized labeling procedures, suitable for clinical application (Thiele and Wilson, 2018). This can be explained by limited understanding of the ²²⁵ Ac coordination chemistry, which is further hampered by the fact that no non-radioactive isotopes of Actinium exist. This lack of knowledge made it difficult to develop ligands, forming stable complexes with ²²⁵ Ac and as a result, this task was and still is conducted by trial and error (Thiele and Wilson, 2018). Instability of ²²⁵ Ac-ligands in vivo results in accumulation of free ²²⁵ Ac in the liver and bone, causing potentially radiotoxic side effects (Scheinberg et al., 2011). To minimize damage to healthy tissues, ²²⁵ Ac needs to be bound kinetically inert to a chelator and labeling procedures with respective chelators need to be reliable and quantitative. In theory, free ²²⁵ Ac after incomplete labeling reactions can also be separated by a cartridge-based purification step, but due to the high costs and limited availability of this radionuclide this approach is very uneconomic.

Currently, one of the most often used chelator for complexation of ²²⁵ Ac, is the 12-membered macrocyclic ligand 1,4,7, 10-Tetraazacyclododecane-l,4,7,10-tetrayltetraacetic acid (DOTA) or the glutamic-acid substituted derivative (DOTA-GA) (Thiele and Wilson, 2018). There are numerous ²²⁵Ac-DOTA constructs which have been evaluated in preclinical studies and are also under evaluation in several clinical trials, e.g. for the treatment of leukemia (Jurcic etal., 2017), multiple myeloma, and prostate cancer (Sathekge et al., 2021). Despite these studies, DOTA- based chelators are not regarded as ideal chelators for complexation of ²²⁵ Ac, because the metal ion radius is too large for the cavity of this chelator (Thiele and Wilson, 2018). Even though, the in vivo stability is considered to be sufficiently high for in vivo applications, a main challenge of this size mismatch between ²²⁵ Ac and DOTA, results in difficulties during radiolabeling. In general, complexation of DOTA with ²²⁵ Ac requires harsh reaction conditions, including elevated temperatures, micro-wave assistance and long time periods (Thiele and Wilson, 2018). Noteworthy, much research effort is currently dedicated to the development of novel chelators, which allow complexation of ²²⁵ Ac under mild reaction conditions. This resulted for example in identification of the larger MACROPA chelator, allowing radiolabeling at room temperature within 5 minutes (Thiele et al., 2017).

There is a need in the art for improved means and methods for radio-labelling of active biomolecules with ²²⁵ Ac, which in particular allow their use as radionuclides for targeted alpha therapy (TAT) of cancer.

SUMMARY OF THE INVENTION

According to the present invention this object is solved by a method for radioactive labeling of a precursor with ²²⁵ Ac, said method comprising the steps of

(1) providing a precursor dissolved in a buffer solution;

(2) freeze-drying the precursor provided in (1) and thereby obtaining a powder of the freeze-dried precursor;

(3) reconstituting the freeze-dried precursor with water;

(4) providing ²²⁵ Ac in 0.04 M to 0.2 M HC1; (5) adding the ²²⁵ Ac of (4) to the reconstituted precursor of (3);

(6) heating to at least 75°C for a period of time; and

(7) obtaining the ²²⁵ Ac-labeled precursor

According to the present invention this object is solved by the ²²⁵ Ac-labeled precursor obtained in a method according to the present invention for use in the treatment of cancer.

According to the present invention this object is solved by a kit for radioactive labeling of a precursor with ²²⁵ Ac, said kit comprising

(i) a freeze-dried precursor obtained according to steps (1) and (2) of the method of the present invention, and

(ii) ²²⁵ Ac in 0.04 M to 0.2 M HC1,

(iii) optionally, water for reconstituting the freeze-dried precursor (i).

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

Before the present invention is described in more detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. For the purpose of the present invention, all references cited herein are incorporated by reference in their entireties.

Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of "0.5 to 10" should be interpreted to include not only the explicitly recited values of 0.5 to 10, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 0.5, 1, 1.5, 2, 2.5, 3, 4, 5 .... 8, 8.5, 9, 9.5, 10 and sub-ranges such as from 2 to 8, 4 to 5, etc. This same principle applies to ranges reciting only one numerical value, such as "less than 1 mm". Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.

Method for labeling of biomolecules with ²²⁵ Ac

As outlined above, the present invention provides a method for radioactive labeling of a precursor with ²²⁵ Ac.

Said method comprises the steps of:

(1) providing a precursor dissolved in a buffer solution;

(3) reconstituting the freeze-dried precursor with water;

(4) providing ²²⁵ Ac in 0.04 M to 0.2 M HC1;;

(5) adding the ²²⁵ Ac of (4) to the reconstituted precursor of (3);

(6) heating to at least 75°C for a period of time; and

(7) obtaining the ²²⁵ Ac-labeled precursor.

Precursor

The precursor is preferably a biomolecule comprising a chelator suitable to complex ²²⁵ Ac.

The chelator is preferably connected to the biomolecule.

Suitable chelators are DOTA, DOTA-GA and MACROPA:

- DOTA:

1 ,4,7, 10-tetraazacyclododecane- 1 ,4,7, 10-tetrayltetraacetic acid

- DOTA-GA:

Glutamic-acid substituted derivative of DOTA.

- MACROPA:

6- [ [ 16-[(6-carboxypyridin-2-yl)m ethyl]- 1 ,4,10, 13 -tetraoxa-7, 16-diazacycl ooctadec-7- yl]methyl]-4-isothiocyanatopyridine-2-carboxylic acid (MACROPA).

PubChem CID: 135349208. In a preferred embodiment, the precursor is a prostate-specific membrane antigen (PSMA)- targeted ligand.

Preferred PSMA-targeted ligands are PSMA I&T, or PSMA-617.

- PSMA I&T:

Prostate-specific membrane antigen for imaging & therapy (PSMA I&T) is commercially available, such as from ITM Isotope Technologies Munich SE, Germany, or Scintomics Molecular, Applied Theranostics Technologies (SCI-att), Germany.

Synonym s : (R)-DOT AGA-(I-y )fk( Sub -KuE) .

Sequence: Suber- 1 -oyl-s-(DOTA-GA-3 -iodo-D-Tyr-D-Phe-D-Lys-OH)-8-oyl -s-(HO-Glu- ureido-Lys-OH).

Molecular Weight: 1497.5 (m.i.), 1498.4 (av.).

Molecular Formula: C63H92IN11O23.

Structure is shown in Figure 2 A.

- PSMA-617:

Vipivotide tetraxetan (PSMA-617) is a high potent prostate-specific membrane antigen (PSMA) inhibitor. PSMA-617 is commercially available, such as from Advanced Accelerator Applications (AAA, a Novartis company), France.

Structure is shown in Figure 2 A.

In a preferred embodiment, the precursor is a somatostatin receptor-targeted ligand.

Preferred somatostatin receptor-targeted ligands are octreotide or octreotate, more preferably DOTA-octreotide or DOTA-octreotate

Octreotide

Octreotide, sold under the brand name Sandostatin among others, is an octapeptide that mimics natural somatostatin pharmacologically. It binds predominantly to the somatostatin receptors SSTR2 and SSTR5.

CAS number: 83150-76-9

Sequence: H-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol

Molecular Weight: 1019.25 g mol-1.

Molecular Formula: C49H66N10O10S2. Structure is shown in Figure 2B.

- DOTA-octreotide (DOTA-TOC)

DOTA-TOC (DOTATOC, DOTA-octreotide, DOTA-(Tyr³)-octreotide, and DOTA(O)- Phe(l)-Tyr(3))octreotide) is a compound containing tyrosine³-octreotide, an SSR agonist, and the bifunctional chelator DOTA. Edotreotide is itse International Nonproprietary Name.

CAS number: 204318-14-9

Molecular Weight: 1421.64 g mol-1

Molecular Formula: C65H92N14O18S2.

Structure is shown in Figure 2B.

Octreotate

Octreotate or octreotide acid is a somatostatin analog that is closely related to octreotide. It is a somatostatin receptor peptide agonist.

Sequence: H-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-OH.

Molecular Weight: 1033.23 g mol-1.

Molecular Formula: C49H64N10O11S2.

Structure is shown in Figure 2C.

DOTA-octreotate (DOTA-TATE)

DOTA-TATE (DOTATATE, DOTA-octreotate, oxodotreotide, DOTA-(Tyr³)-octreotate, and DOTA-0-Tyr3-Octreotate) is a compound containing tyrosine³ -octreotate, an SSR agonist, and the bifunctional chelator DOTA (tetraxetan).

CAS number: 177943-89-4

Molecular Weight: 1435.63 g mol-1.

Molecular Formula: C65H90N14O19S2.

Structure is shown in Figure 2C.

In one embodiment, where the precursor is obtained from a commercial provider, it has been sterile filtered by the commercial provider. Furthermore, microbiological evaluations have been carried out by the commercial provider, such as bioburden control and/or the content of bacterial endotoxins has been determined.

Step (1)

In step 1, a precursor is provided. The precursor is dissolved in a buffer solution. In one embodiment, the buffer solution is a TRIS (tris(hydroxymethyl)aminomethane) buffer solution, preferably 0.1 M TRIS buffer having pH 7 to 9, more preferably 0.1 M TRIS pH 9.

In one embodiment, the buffer solution is an acetate buffer solution, preferably 0.1 M to IM acetate pH 4 to 6, more preferably 0.1 M acetate pH 5.7.

For example, preferably 0.1 M NaOAc (sodium acetate) buffer having pH 5.7.

In one embodiment, the buffer solution is an acetate buffer solution, preferably 0.1 M to IM acetate pH 3 to 6, more preferably 0.1 M acetate pH 3.7.

For example, preferably 0.1 M NaOAc (sodium acetate) buffer having pH 3.7.

In one embodiment, the buffer solution is a phosphate buffer solution, preferably 0. 1 M to 0.3 M phosphate pH 5 to 6, more preferably 0.2 M phosphate pH 5.7.

The buffer solutions can be sterile filtered before and/or after dissolving the precursor.

In a preferred embodiment, the buffer solution including the dissolved precursor is sterile filtered.

Step (2)

In step 2, the precursor is freeze-dried. A powder of the freeze-dried precursor is obtained.

In step 2, the precursor is freeze-dried while it is dissolved in buffer. The powder of the freeze-dried precursor also contains buffer salt(s).

In one embodiment, the freeze-drying is via lyophilization.

In one embodiment, the solution is sterile filtered before lyophilization.

Step (3)

In step 3, the freeze-dried precursor is reconstituted with water.

In one embodiment, the freeze-dried precursor is reconstituted with water soon after the freeze-drying, such as immediately. In one embodiment, there is a period of time between obtaining the powder of the freeze-dried precursor and reconstituting it for further use. This means the powder of the freeze-dried precursor can be stored before radiolabeling with ²²⁵ Ac.

The storage is preferably at about -20°C. The storage time depends on the shelf life of the precursor, which can be up to one year.

Said period of time can be in a range from hours to months, such as one to two months, but also up to one year.

Step (4)

In step 4, ²²⁵ Ac is provided.

²²⁵ Ac is provided in 0.04 M to 0.2 M HC1.

Preferably, ²²⁵ Ac is provided in 0.1 M HC1.

The activity or dosage of ²²⁵ Ac is usually in the range from 4 to 12 MBq.

Step (5)

In step 5, the ²²⁵ Ac of step (4) is added to the reconstituted precursor of (3).

In one embodiment, 8 MBq of ²²⁵ Ac is added.

The ratio of ²²⁵ Ac to reconstituted precursor is preferably 5 to 20 nmol per MBq ²²⁵ Ac, preferably 10 nmol per MBq ²²⁵ Ac.

Step (6)

In step 6, heating for a period of time is carried out.

Preferably, the heating is to at least about 75°C.

In a preferred embodiment, the heating in step (6) is carried out at about 95°C in a microwave reactor and the period of time is from about 5 to 10 min, preferably about 5 min.

In one embodiment, the heating in step (6) is carried out at about 75°C to about 85°C and the period of time is from about 20 to 30 min, preferably 75°C for about 20 min. Step (7)

In step 7, the ²²⁵ Ac-labeled precursor is obtained.

Step (7) preferably comprises cooling.

In one embodiment, the obtained ²²⁵ Ac-labeled precursor is formulated, preferably in isotonic saline, and/or sterile filtered.

The ²²⁵ Ac-labeled precursor obtained in step (7) can be formulated in isotonic saline. The formulation optionally contains one or more stabilizing agents and/or a co-chelator.

Examples for suitable stabilizing agents are:

- ascorbic acid, such as with a concentration of 10 to 100 mg per mL of the total formulation,

- gentisic acid (such as with a concentration of 1 to 5 mg per mL of the total formulation),

- methionine (such as with a concentration of 1 to 5 mg per mL of the total formulation)

An example for a suitable co-chelator is diethylenetriamine pentaacetate (DTP A), such as with a concentration of 0.01 to 0.03 mg per mL of the total formulation. Said co-chelator is capable of chelating radioactive daughter nuclides of Actinium -225.

In one embodiment, the formulated ²²⁵ Ac-labeled precursor can be sterile filtered.

Preferably, the radioactive labeled precursor obtained in step (7) has a radiochemical purity of more than about 95%, more preferably more than 97%. The radiochemical purity can be 99% or more than 99%.

In one embodiment, a sample from the obtained ²²⁵ Ac-labeled precursor solution or the formulated ²²⁵ Ac-labeled precursor solution is subjected to a bacterial endotoxin test, such as the Limulus amebocyte lysate (LAL) assay.

Microbiological examinations

The presence of microorganisms may affect the stability of drug substances due to their propensity to degrade/metabolize peptides. Microbiological examinations involve the bioburden control (see European Pharmacopeia, Ph. Eur 2.6. 12) and content of bacterial endotoxins (see European Pharmacopeia, Ph Eur. 2.6.14). The microbial enumeration tests for total aerobic microbial counts (TAMC) and total yeast and mold counts (TYMC) must adhere to the acceptance criteria of 103 CFU/g and 102 CFU/g for bulk material and 102 CFU/g and 101 CFU per container for chemical precursors packed in single and multi-dose containers, respectively. Bacterial endotoxin can be determined by the gel-clot or photometric methods (turbidimetric and chromogenic techniques) and acceptance criteria are limited to a maximum 100 lU/g for bulk material or maximum 10 IU per container for chemical precursors packed in single-dose and multidose containers.

In one embodiment, the method of the present invention further comprises the step

(8) quality control of the ²²⁵ Ac-labeled precursor.

The quality control preferably comprises radio thin layer chromatography (radio TLC) and/or radio high-performance liquid chromatography (HPLC) analysis.

Medical uses of the ²²⁵ Ac-labeled precursors

As outlined above, the present invention provides the ²²⁵ Ac-labeled precursor obtained in a method of the present invention for use in the treatment of cancer.

The treatment preferably comprises targeted alpha therapy (TAT) of cancer.

Targeted alpha-particle therapy (TAT) is a method of targeted radionuclide therapy of various cancers. It employs radioactive substances which undergo alpha decay to treat diseased tissue at close proximity. TAT has the potential to provide highly targeted treatment, especially to microscopic tumor cells.

In a preferred embodiment, the ²²⁵ Ac-labeled precursor is a prostate-specific membrane antigen (PSMA)-targeted ligand, preferably ²²⁵ Ac-labeled PSMA-617 or ²²⁵ Ac-labeled PSMA I&T, and the cancer is prostate cancer.

In one embodiment, the ²²⁵ Ac-labeled precursor is a somatostatin receptor-targeted ligand, preferably ²²⁵ Ac-labeled DOTA-octreotate (²²⁵ Ac-labeled DOTA-TATE) or ²²⁵ Ac-labeled DOTA-octreotide (²²⁵ Ac-labeled DOTA-TOC), and the cancer are neuroendocrine tumors. Kits for radioactive labeling

As outlined above, the present invention provides a kit for radioactive labeling of a precursor with ²²⁵ Ac.

Said kit comprises

(ii) ²²⁵ Ac in 0.04 M to 0.2 M HCL, preferably 0.1 M HC1,

(iii) optionally, water for reconstituting the freeze-dried precursor (i).

The precursor is preferably a biomolecule comprising a chelator suitable to complex ²²⁵ Ac, which is preferably connected to the biomolecule.

As discussed above, suitable chelators are DOTA, DOTA-GA or MACROPA.

As discussed above, the precursor is preferably a prostate-specific membrane antigen (PSMA)-targeted ligand, such as PSMA I&T or PSMA-617.

As discussed above, in one embodiment, the precursor is preferably a somatostatin receptor- targeted ligand, such as DOTA-TOC or DOTA-TATE.

Preferred embodiments

The ²²⁵ Ac-labeled prostate-membrane antigen (PSMA)-targeted ligands PSMA-617 and PSMA I&T are applied for targeted alpha-therapy (TAT) of prostate cancer. The current state-of-the- art labeling procedure was previously published (Hooijman et al, 2021) and is also applied in a slightly modified form in magnetic resonance imaging (MRI) for patient care:

In said prior method, the precursor (PSMA I&T) is dissolved in an organic solvent with a concentration of 10 nmol per MBq ²²⁵ Ac. An aqueous solution of a TRIS (tris(hydroxymethyl)aminomethane, 0.1 M, pH 9) is added. ²²⁵ Ac (dissolved in 0.1 M aqueous HC1) is added. The solution is heated for 5 min at 95°C in a microwave. Quality control is performed by radio-thin layer chromatography (radio-TLC). The product is sterile filtered and formulated in isotonic saline.

For a prior art method, see also Kratochwil et al. (2016). The inventors herein disclose a new and easy method to facilitate the radio-labeling procedure of PSMA I&T, as an example for other small biomolecules, with ²²⁵ Ac by employing freeze- dried kits, which can promote widespread of Ac-225 labeling procedure.

For the inventors’ protocol, the precursor is dissolved in a suitable buffer and then freeze-dried. The freeze-dried precursor is then reconstituted with water, whereupon ²²⁵ Ac is added. After heating for 5 min at 95°C in a micro-wave reactor, quality control is performed.

The method as disclosed herein has the following advantages:

1) Faster and minimized protocol. Only water and ²²⁵ Ac is required for reconstitution of the kit.

2) This bears potential advantages with respect to GMP-guidelines. Solutions can be sterile filtered before lyophilization, resulting in a low risk of contamination.

3) No sophisticated methods. The radiolabeling can also easily be performed by technicians.

4) The freeze-dried kits (i.e. freeze-dried precursors) can be stored over long time-periods

5) The radiolabeling can be performed on-site. Since the stability of ²²⁵ Ac-radiopharmaceuti cals is limited, this could hamper their widespread use (Hooijman et al., 2021). Therefore, there might be a huge demand for sites to produce their ²²⁵Ac-radiopharmaceuticals on-site in an easy and fast way.

6) Kits have a long tradition in nuclear medicine. They also had a huge impact, facilitating the production of e.g. "^mTc-labeled compounds and lately ⁶⁸Ga-based radiopharmaceuticals (Lepareur, 2022).

The method disclosed herein can also be applied to other precursors, such as somatostatin receptor-targeted ligands, e.g. DOTA-TATE or DOTA-TOC.

Somatostatin analogues have been investigated for both diagnostic and treatment purposes in tumors that are known to express somatostatin receptors (SSTR). In the past the macrocyclic peptide-based ligands DOTATATE ((DOTA°-Tyr³)octreotate) and DOTATOC ((DOTA⁰- Tyr³)octreotide) demonstrated excellent characteristics for ⁶⁸Ga-imaging and targeted therapy with lutetium-177, yttrium-90 and lately with actinium-225 in some types of neuroendocrine tumors. Neuroendocrine tumors (NETs) are a class of slow-growing tumors that arise from cells distributed mainly in the lungs, gastrointestinal tract, or pancreas. A unique feature of NETs is their overexpression of SSTR on the tumor cells, which has established the basis for both diagnostic imaging and peptide receptor radionuclide therapy. The following examples and drawings illustrate the present invention without, however, limiting the same thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Decay chain of ²²⁵ Ac.

The scheme was adapted from Thiele and Wilson (2018).

Figure 2. Biomolecules as precursors.

A) The prostate-membrane antigen (PSMA)-targeted ligands PSMA-617 and PSMA I&T radiolabeled with ²²⁵ Ac.

B) The somatostatin receptor-targeted ligand octreotide and DOTA-TOC which is radiolabeled with ²²⁵ Ac.

C) The somatostatin receptor-targeted ligand octreotate and DOTA-TATE which is radiolabeled with ²²⁵ Ac.

Figure 3. Quality control: Radio-TLC of²²⁵Ac-PSMA I&T after Kit-Labeling.

Figure 4. Exemplary radio-HPLC analysis of 225Ac-PSMA I&T.

Performed on a Shimadzu system, equipped with a variable wavelength detector (both Shimadzu) and a gamma-detector Gabi Star (Elysia-raytest, Straubenhardt, Germany). A) shows detection at the radiochannel and B) detection at 220 nm. Water/0. 1% TFA (solvent A) and MeCN/0.1% TFA (solvent B) served as mobile phases, a Nucleosil 100-5 C18 column of 125 x 4 mm was used as stationary phase. 15 pl of product solution were injected and the following linear solvent gradient was applied: 10-70% B in 15 min, 95 % B for 5 min (flow rate = 1 ml/min). Chemical impurities were monitored at 220 nm.

Figure 5. Quality control: Radio-TLC of²²⁵Ac-DOTATOC after Kit-Labeling.

Figure 6. Exemplary radio-HPLC analysis of 225Ac-DOTATOC.

Performed on a Shimadzu system, equipped with a variable wavelength detector (both Shimadzu) and a gamma-detector Gabi Star (Elysia-raytest, Straubenhardt, Germany). A) shows detection at the radiochannel and B) detection at 220 nm. EXAMPLES

EXAMPLE 1 Materials and Methods

Actinium-225 was provided by JRC Karlsruhe and then dissolved in 0.1 M HC1. The precursor PSMA I&T was purchased by ITM Isotope Technologies Munich SE (Munich Germany). All necessary solvents and other organic reagents were purchased from either, Alfa Aesar (Karlsruhe, Germany), Sigma-Aldrich (Munich, Germany) or VWR (Darmstadt, Germany). High-performance liquid chromatography (HPLC) was performed on a Prominence HPLC system (Shimadzu, Kyoto, Japan) with a Photo Diode Array detector (Shimadzu) and a GABI Star detector (Raytest, Straub enhardt, Germany). Eluents for all HPLC operations were water (solvent A) and acetonitrile (solvent B), both containing 0.1% trifluoroacetic acid. ANucleosil 100 C18 (125 x 4.6 mm, 5 pm particle size) column (CS Chromatographic Service, Langerwehe, Germany) was used for analytical measurements at a flow rate of 1 mL/min. Radio-Thin layer chromatography (radio-TLC) was performed using 0.5 M aqueous sodium citrate pH 5 and ITCL-SG paper (0.5 x 6 cm). TLC was then analyzed using a radio-TLC scanner from Bioscan (Eckert and Ziegler, Brussels, Belgium) and the Bio-Chrom Lite software. Activity quantification was performed using a 2480 WIZARD2 automatic gamma counter (PerkinElmer, Waltham, United States). Labeling experiments are performed in sealed and crimped glass vials (0.5-2.0 mL, Biotage Uppsala, Sweden), using a Biotage, Initiator⁺ microwave reactor.

EXAMPLE 2 Quality Control of ²²⁵ Ac-labeled radiopharmaceuticals

After the labeling reaction, radiochemical purity is determined by radio-TLC (ITLC-SG paper, 0.5 M sodium citrate pH 5) in triplicate. After analysis the TLC-stripe is cut at a relate-to-front value (Rf-value) of 0.7. The origin represents complexed ²²⁵ Ac whereas the front represents non-complexed ²²⁵ Ac. To ensure formation of an equilibrium between ²²⁵ Ac and daughter nuclides, the samples are analyzed after at least 30 minutes in a gamma-counter quantifying the gamma-energy of Francium -221. Additionally, one TLC-analysis is performed without cutting the stripe using a radio-TLC-scanner.

In order to determine potential degradation of the radiotracer, HPLC-analysis is performed using the following parameters: Water/0.1% TFA (solvent A) and MeCN/0.1% TFA (solvent B) served as mobile phases, a Nucleosil 100-5 C18 column of 125 x 4.6 mm was used as stationary phase. 5-50 pl of product solution were injected and the following linear solvent gradient was applied: 10-70 % B in 15 min, 95 % B for 5 min, and re-equilibration at 10% B for 5 min (flow rate = 1 ml/min). Chemical impurities were monitored at 220 and 254 nm Please note that analysis of ²²⁵ Ac-labeled radiopharmaceuticals results in an injection peak at the radioactivity channel. This injection peak is a result of the daughter nuclides and do not represent free ²²⁵ Ac.

EXAMPLE 3 State-of-the-Art labeling reaction

A 1 mM solution of PSMA-I&T is prepared using EtOH/water (1 : 1, v/v) or DMSO. In the reaction vial, 500 pL of TRIS buffer (0. 1 M, pH 9) are mixed with 50 pL PSMA I&T (1 mM, 50 nmol), and 5.0 MBq of ²²⁵ Ac is added (10 pL, 0.1 M HC1). The reaction vial is sealed and heated in a microwave reactor for 5 minutes. After cooling of the reaction vial, quality control is performed as described in Example 2. The radiochemical purity by radio-TLC was determined to be 98.7±0.3% (n=3). Radiochemical purity as determined by radio-HPLC was >97%.

EXAMPLE 4 Freeze drying experiments

As the pH is crucial for the efficient labeling reaction, and it can change during freeze-drying if volatile buffers are used, several kits were prepared and the pH was measured after reconstitution in 500 pL of water. Moreover, after reconstitution, 10 and 20 pL of 0.1 M HC1 was added, simulating the addition of ²²⁵ Ac.

In a first set of experiment TRIS buffer (0.1 M, pH 9.0) was used, as it represents the most- often used buffer for ²²⁵Ac-labeling of PSMA-targeted ligands. Recently, also NaOAc buffer (0.1 M, pH 5.7) was described as a promising buffer for ²²⁵Ac-labeling, which suits better to current GMP-guidelines (Pretze et al., 2021). However, acetate/acetic acid is known from literature (see e.g. Baheti et al., 2010) to be removed in some parts during freeze-drying.

Results are shown in Table 1.

Table 1

EXAMPLE 5

A kit was prepared, by lyophilisation of 500 pL of TRIS buffer (0.1 M, pH 9) and 50 nmol of PSMA I&T. After reconstitution in 500 pL of water, ²²⁵ Ac (5.0 MBq, 20 pL, 0.1 M HC1) was added. The reaction vial was sealed and heated for 5 min at 95°C in a microwave reactor. After cooling of the reaction vial, quality control was performed as described above (see Example 2). The radiochemical purity by radio-TLC was determined to be 99.3±0.1% (n=3). Radiochemical purity as determined by radio-HPLC was >97%.

EXAMPLE 6

Kits were prepared, by lyophilisation of 30 nmol of PSMA I&T, together with (A) 500 pL NaOAc, 0.1 M, pH 3.7; (B) 500 pL NaOAc, 0.1 M, pH 4.7 and (C) 500 pL NaOAc, 0.1 M, pH 5.7, in three separate vials. After reconstitution in 500 pL of water, 225Ac (3.0 MBq, 3.0 pL, 0.1 M HC1) was added. The reaction vials were sealed and heated for 5 min at 95°C in a microwave reactor. After cooling quality control is performed as described above (see Example 2).

The radiochemical purity by radio-TLC was:

(A) 99.5±0.1% (n=3),

(B) 99.8±0.0% (n=3),

(C) 99.6±0.1% (n=3).

Short summary of Examples 4 to 6:

The pH value of TRIS buffer was stable after freeze-drying, reconstitution and addition of 10 or 20 pL of 0.1 M HC1, as shown in Table 1. The pH value of acetate buffer changed after freeze-drying, nevertheless labeling of the kits with ²²⁵ Ac was successful, see Table 1.

Kit labeling of PSMA I&T with ²²⁵ Ac was feasible using different buffers and pH values, as shown in Table 1.

An excellent radiochemical purity of >99% was reached in all experiment for different kitpreparations (the cut-off for patient-application is 97%). The radiochemical purity was similar to the state-of-the-art procedure, as described in Example 3.

The identity of ²²⁵Ac-PSMA I&T could be confirmed by radio-HPLC, after kit-labeling.

EXAMPLE ?

We developed a kit-based approach for labeling the somatostatin receptor-targeted ligands DOTATOC and DOTATATE with actinium-225 for application in targeted alpha therapy of neuroendocrine tumors. The technical equipment, reagents and methods are identical to the ones described above for actinium-labeling of PSMA-ligands, see Examples 1-6.

Kits were prepared by lyophilisation of 30 nmol of DOTATOC (30 pL, 1 mM in water), together with aqueous buffers of:

(A) 500 pL NaOAc (1.0 M, pH 3.7),

(B) 500 pL TRIS (0.1 M, pH 9.0),

(C) 500 pL phosphate buffer (0.2 M, pH 5.7).

The radiochemical purity by radio-TLC was:

(A) 99.7±0.0% (n=3)

(B) 80.7±1.0% (n=3)

(C) 81.0±0.2% (n=3)

In radio-HPLC analysis, no degradation of the ligands was determined after labeling, see Figure 6. The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

REFERENCES

Apostolidis C, Molinet R, McGinley J, Abbas K, Mollenbeck J, Morgenstern A. Cyclotron production of Ac-225 for targeted alpha therapy. Appl Radiat Isot. 2005;62(3):383-7.

Baheti A, Kumar L, Bansal A K. Excipients used in lyophilization of small molecules. Journal of Excipients and Food Chemicals, [S.I.], v. 1, n. 1, p. 41-54, June 2010. ISSN 21502668.

Boyd M, Ross SC, Dorrens J, Fullerton NE, Tan KW, Zalutsky MR, et al. Radiation-induced biologic bystander effect elicited in vitro by targeted radiopharmaceuticals labeled with alpha- , beta-, and auger electron-emitting radionuclides. J Nucl Med. 2006;47(6): 1007-15.

Hooijman EL, Chalashkan Y, Ling SW, Kahyargil FF, Segbers M, Bruchertseifer F, et al. Development of [(225)Ac]Ac-PSMA-I&T for Targeted Alpha Therapy According to GMP Guidelines for Treatment of mCRPC. Pharmaceutics. 2021 ; 13(5).

Jurcic J, Levy M, Park J, Ravandi F, Perl A, Pagel J, et al. Phase I trial of alpha-particle immunotherapy with ²²⁵Ac-lintuzumab and low-dose cytarabine in patients age 60 or older with untreated acute myeloid leukemia. Journal of Nuclear Medicine. 2017;58(supplement 1):456-.

Kratochwil C, Bruchertseifer F, Giesel FL, Weis M, Verburg FA, Mottaghy F, Kopka K, Apostolidis C, Haberkron U, Morgenstern A. 225Ac-PSMA-617 for PSMA targeting alpharadiation therapy of patients with metastatic castration-resistant prostate cancer. J Nucl Med., 2016, 1-20.

Lepareur N. Cold Kit Labeling: The Future of (68)Ga Radiopharmaceuticals? Front Med (Lausanne). 2022;9:812050.

Morgenstern A, Apostolidis C, Kratochwil C, Sathekge M, Krolicki L, Bruchertseifer F. An Overview of Targeted Alpha Therapy with (225)Actinium and (213)Bismuth. Curr Radiopharm. 2018;l l(3):200-8. Pretze M, Kunkel F, Runge R, Freudenberg R, Braune A, Hartmann H, et al. Ac-EAZY! Towards GMP-Compliant Module Syntheses of (225)Ac-Labeled Peptides for Clinical Application. Pharmaceuticals (Basel). 2021;14(7).

Robertson AKH, Ramogida CF, Schaffer P, Radchenko V. Development of (225)Ac Radiopharmaceuticals: TRIUMF Perspectives and Experiences. Curr Radi opharm. 2018;l l(3):156-72.

Sathekge MM, Bruchertseifer F, Vorster M, Morgenstern A, Lawai IO. Global experience with PSMA-based alpha therapy in prostate cancer. Eur J Nucl Med Mol Imaging. 2021;49(l):30- 46.

Scheinberg DA, McDevitt MR. Actinium-225 in targeted alpha-particle therapeutic applications. Curr Radi opharm. 2011;4(4):306-20.

Thiele NA, Brown V, Kelly JM, Amor-Coarasa A, Jermilova U, MacMillan SN, et al. An Eighteen-Membered Macrocyclic Ligand for Actinium-225 Targeted Alpha Therapy. Angew Chem Int Ed Engl. 2017;56(46): 14712-7.

Thiele NA, Wilson JJ. Actinium-225 for Targeted a Therapy: Coordination Chemistry and Current Chelation Approaches. Cancer Biother Radiopharm. 2018;33(8):336-48.

Zielinska B, Apostolidis C, Bruchertseifer F, Morgenstern A (2007): An Improved Method for the Production of Ac-225/Bi-213 from Th-229 for Targeted Alpha Therapy, Solvent Extraction and Ion Exchange, 25:3, 339-349.

Claims

1. A method for radioactive labeling of a precursor with ²²⁵ Ac, said method comprising the steps of:

(1) providing a precursor dissolved in a buffer solution;

(3) reconstituting the freeze-dried precursor with water;

(4) providing ²²⁵ Ac in 0.04 M to 0.2 M HC1;

(5) adding the ²²⁵ Ac of (4) to the reconstituted precursor of (3);

(6) heating to at least 75°C for a period of time; and

(7) obtaining the ²²⁵ Ac-labeled precursor.

2. The method of claim 1, wherein the precursor is a biomolecule comprising a chelator suitable to complex ²²⁵ Ac, which is preferably connected to the biomolecule, such as l,4,7,10-tetraazacyclododecane-l,4,7,10-tetrayltetraacetic acid (DOTA), a glutamic- acid substituted derivative of DOTA (DOTA-GA), or 6-[[16-[(6-carboxypyridin-2- yl)methyl]-l,4, 10, 13-tetraoxa-7, 16-diazacyclooctadec-7-yl]methyl]-4-isothiocyanatopyridine- 2-carboxylic acid (MACROP A).

3. The method of claim 1 or 2, wherein the precursor is a prostate-specific membrane antigen (PSMA)-targeted ligand, such as PSMA I&T, or PSMA-617, or wherein the precursor is a somatostatin receptor-targeted ligand, such as DOTA-octreotide (DOTA-TOC), or DOTA-octreotate (DOTA-TATE).

4. The method of any one of claims 1 to 3, wherein the buffer solution in step (1) is a TRIS buffer solution, preferably 0.1 M TRIS pH 7 to 9, more preferably 0.1 M TRIS pH 9, an acetate buffer solution, preferably 0.1 M to 1 M acetate pH 3 to 6, more preferably 0.1 M acetate pH

5.7 or 1.0 M acetate pH 3.7, or a phosphate buffer solution, preferably 0.1 M to 0.3 M phosphate pH 5 to 6, more preferably 0.2 M phosphate pH 5.7.

5. The method of any one of claims 1 to 4, wherein the ratio of ²²⁵ Ac to reconstituted precursor is 5-20 nmol per MBq ²²⁵ Ac, preferably 10 nmol per MBq ²²⁵ Ac, and/or wherein ²²⁵ Ac is provided in 0.1 M HC1.

6. The method of any one of claims 1 to 5, wherein the heating in step (6) is carried out at about 95°C in a microwave reactor and the period of time is from about 5 to 10 min, preferably about 5 min, and/or wherein the heating in step (6) is carried out at about 75°C to about 85°C and the period of time is from about 20 to 30 min, preferably 75°C for about 20 min.

7. The method of any one of claims 1 to 6, wherein the radioactive labeled precursor obtained in step (7) has a radiochemical purity of more than about 95%, preferably more than 97%, and/or wherein the ²²⁵ Ac-labeled precursor obtained in step (7) is formulated, preferably in isotonic saline.

8. The method of any one of claims 1 to 7, further comprising the step

(8) quality control of the ²²⁵ Ac-labeled precursor, wherein, preferably, the quality control comprises radio thin layer chromatography (radio TLC) and/or high-performance liquid chromatography (HPLC) analysis.

9. The method of any one of the preceding claims, wherein between obtaining the powder of the freeze-dried precursor in (2) and reconstituting it in step (3) is a period of time, such as in a range from hours to months.

10. The method of any one of the preceding claims, comprising sterile filtering of the solutions, such as of the buffer solution of (1), the precursor dissolved in the buffer solution in step (1), and/or the precursor of (1), and/or comprising microbiological examinations, including bioburden control and/or content of bacterial endotoxins, such as of the precursor of (1) or the ²²⁵ Ac-labeled precursor obtained in step (7).

11. The ²²⁵ Ac-labeled precursor obtained in a method of any one of claims 1 to 10 for use in the treatment of cancer.

12. The ²²⁵ Ac-labeled precursor for use according to claim 11, wherein the treatment comprises targeted alpha therapy (TAT) of cancer.

13. The ²²⁵ Ac-labeled precursor for use according to claim 11 or 12, wherein the ²²⁵ Ac- labeled precursor is a prostate-specific membrane antigen (PSMA)-targeted ligand, preferably ²²⁵ Ac-labeled PSMA-617 or ²²⁵ Ac-labeled PSMA I&T, and wherein the cancer is prostate cancer, or wherein the ²²⁵ Ac-labeled precursor is a somatostatin receptor-targeted ligand, preferably ²²⁵ Ac-labeled DOTA-octreotate (DOTA-TATE) or ²²⁵ Ac-labeled DOTA-octreotide (DOTA- TOC), and wherein the cancer are neuroendocrine tumors.

14. A kit for radioactive labeling of a precursor with ²²⁵ Ac, said kit comprising

(i) a freeze-dried precursor obtained according to steps (1) and (2) of the method of any one of claims 1 to 10, and

(ii) ²²⁵ Ac in 0.04 M to 0.2 M HCL, preferably 0.1 M HC1,

(iii) optionally, water for reconstituting the freeze-dried precursor (i).

15. The kit of claim 14, wherein the precursor is a biomolecule comprising a chelator suitable to complex ²²⁵ Ac, which is preferably connected to the biomolecule, such as l,4,7,10-tetraazacyclododecane-l,4,7,10-tetrayltetraacetic acid (DOTA), a glutamic- acid substituted derivative of DOTA (DOTA-GA), or 6-[[16-[(6-carboxypyridin-2- yl)methyl]-l,4, 10, 13-tetraoxa-7, 16-diazacyclooctadec-7-yl]methyl]-4-isothiocyanatopyridine- 2-carboxylic acid (MACROP A), and/or wherein the precursor is preferably a prostate-specific membrane antigen (PSMA)- targeted ligand, such as PSMA I&T, or PSMA-617, or a somatostatin receptor-targeted ligand, such as DOTA-TOC or DOTA-TATE.