RU2795398C2 - Complex containing a psma-targeting compound bound to a lead or thorium radionuclide - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a compound comprising a PSMA-targeted unit and a chelating unit, wherein the PSMA-targeted unit is bonded via a carbon backbone to the chelating unit, and wherein the compound is characterized by the following formula

where X represents either -NH₂ or -OH. Also a complex for use in radioligand therapy is proposed that contains the above compound complexed with a radionuclide selected from the group consisting of ²¹²Pb, ²¹²Bi, ²¹³Bi, ²²⁵Ac or ²²⁷Th, a pharmaceutical composition containing the complex, the use of a pharmaceutical composition, a method for treating a disease characterized by PSMA expression, and kits.

EFFECT: invention makes it possible to obtain complex compounds with high radiobiological efficiency to treat a disease characterized by PSMA expression.

18 cl, 2 dwg, 7 tbl, 17 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to complexes containing a PSMA-targeting compound associated with a radionuclide such as ²¹² Pb or ²²⁷ Th. These compounds and pharmaceutical compositions containing them can be used in medical applications. Such applications include the treatment of prostate cancer, and the complexes allow dual targeting of different forms of cancer. PSMA-targeting peptide and peptidomimetic urea derivatives are conjugated to chelators to form a complex with ²¹² Pb and ²²⁷ Th for use in radiotherapy. They can be used for single and dual targeting.

BACKGROUND OF THE INVENTION

Prostate cancer is one of the most common causes of cancer-related death in men. There is a great need for new and effective treatments, especially for advanced hormone resistant disease. Skeletal metastases are a common problem in advanced disease, and therefore, for patients suffering from advanced prostate cancer with skeletal metastases, a ^223Ra alpha particle emitter (Xofigo) has been introduced as a bone-specific therapy.

Although ^223Ra has shown significant clinical efficacy as an osteotrope in patients, its activity is limited to bone metastases and does not target soft tissue metastases.

There are several carrier molecules that target the radioligand to prostate-specific membrane antigen (PSMA). Lutetium-177-labeled PSMA-617 ( ¹⁷⁷ Lu-PSMA-617) is a compound in the latest stage of clinical development for use in radionuclide therapy.

This molecule functions appropriately and provides appropriate values for the ratio of accumulation in tumor and normal tissues for longer-lived (i.e., with a half-life of several days) radionuclides, including ¹⁷⁷ Lu and ²²⁵ Ac, but at the same time at early time points ( usually a few hours after injection) is characterized by high accumulation in the kidneys. In the case of shorter lived radionuclides such as ²¹² Pb (half life 10.6 hours), initial accumulation in the kidneys presents a potential toxicity problem.

Therefore, it is preferable to use a PSMA ligand with less accumulation in the kidneys, and this should not interfere with the accumulation in the tumor. The PSMA ligand molecules consist of (1) a PSMA binding region, (2) a linker region, and (3) a chelator, with the linker region connecting (1) and (3). The linker region is also used to control the size of the molecule.

and polarity, etc., in order to influence in vivo distribution properties. The PSMA-binding region (motif) used in PSMA-617 is a structure that can be found in several molecules of this class developed by several different inventors and researchers, including PSMA-11 and PSMA I&T, as well as labeled through ¹³¹ I and ²¹¹ At PSMA-binding ligands.

New compounds are required that contain the PSMA region, as all ligands currently tested have disadvantages, including relatively low radiobiological efficacy (RBE) and suboptimal biodistribution.

There is also a need for an improved alpha particle emitter that can target both bone and soft tissue metastases.

The present invention relates to compounds that provide overcoming these problems.

BRIEF DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to the complex of the present invention, where the compound X is associated with a radionuclide, such as ²¹² Pb or ²²⁷ Th, using a chelating fragment Z.

In one embodiment of the present invention, the radionuclide is ²¹² Pb.

In another embodiment of the present invention, the radionuclide is ²²⁷ Th.

The chelating moiety Z may be selected from the group consisting of acyclic chelators, cyclic chelators, cryptands, crown ethers, porphyrins, or cyclic or non-cyclic polyphosphonates, DOTMP, EDTMP, bisphosphonate, DOTA, a DOTA derivative, DOTA-conjugated pamidronate, TCMC, a TCMC, TCMC-conjugated pamidronate, antibody-conjugated DOTA, antibody-conjugated TCMC, HBED-CC, NOTA, NODAGA, TRAP, NOPO, PCTA, DFO, DTPA, CHX-DTPA, AAZTA, DEDPA, and oxo-Do3A.

In one embodiment of the present invention, the linker is DOTA or a derivative of DOTA.

In another embodiment of the present invention, the linker is TCMC or a derivative of TCMC.

In the case of ²²⁷ Th, octadentate hydroxypyridinone-containing ligands, such as 3,2-HOPO, are particularly suitable.

One aspect of the present invention relates to a radiopharmaceutical composition according to the present invention for use as a drug.

One aspect of the present invention relates to a radiopharmaceutical composition according to the present invention for use in the treatment of soft tissue and/or skeletal disease.

In one embodiment of the present invention, the skeletal disease is selected from the group consisting of skeletal metastases in forms of breast, prostate, kidney, lung, bone, or multiple myeloma or non-cancer diseases causing unwanted calcification, including ankylosing spondylitis.

BRIEF DESCRIPTION OF GRAPHICS

Description of figures

Figure 1 shows the biodistribution of ²¹² Pb 2 hours after injection of ²¹² Pb labeled p-SCN-Bn-TCMC-PSMA Ligand 1 and PSMA-617, respectively, in the presence of ²²⁴ Ra.

Figure 2 shows survival of mice with PSMA-positive C4-2 xenograft after treatment with saline, 52 MBq ¹⁷⁷ Lu-OSMA-617, and 0.32 MBq ²¹² Pb-p-SCN-Bn-TCMC-PSMA Ligand 1.

DETAILED DESCRIPTION OF THE INVENTION

Some used abbreviations

A peptide mimetic, also called a peptidomimetic, is a small, similar protein chain designed to mimic a peptide. As a rule, they are formed either as a result of modification of an existing peptide, or as a result of the development of similar systems that mimic peptides, such as peptoids and β-peptides. Regardless of the approach, the modified chemical structure is designed to provide preferential control of molecular properties such as stability or biological activity. This may be of value in the development of drug-like compounds based on existing peptides. These modifications include changes to the peptide that will not occur naturally (such as altered backbones and the inclusion of non-naturally occurring amino acids). Based on their similarity to the precursor peptide, peptidomimetics can be grouped into four classes (A–D), where A is the most similar and D is the least similar. Classes A and B include peptide-like backbones, while classes C and D include small molecules.

PSMA is a prostate-specific membrane antigen. Synonyms of PSMA are prostate-specific cancer antigen, PSM, FGCP, FOLH, GCP2, mGCP, GCPII, NAALAD1, NAALA-dase, FOLH1, glutamate carboxypeptidase 2, glutamate carboxypeptidase II, membrane glutamate carboxypeptidase, N-acetylated-alpha-linked acid dipeptidase I, petroylpoly -gamma-glutamate carboxypeptidase, folylpoly-gamma-glutamate carboxypeptidase, folate hydrolase 1, prostate-specific membrane antigen, cell growth-inhibiting protein 27.

DOTMP - 1,4,7,10-tetraazacyclododecan-1,4,7,10-tetra(methylenephosphonic acid).

EDTMP - ethylenediaminetetra (methylenephosphonic acid).

EDTA is ethylenediaminetetraacetic acid.

p-SCN-Bn-DOTA is 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraacetic acid.

DOTA - 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, also used to denote benzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (for example, conjugated with a monoclonal antibody).

p-SCN-Bn-TCMC is 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamonylmethyl)cyclododecane.

TCMC - 1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamonylmethyl)cyclododecane, also used to denote benzyl-1,4,7,10-tetraaza-1,4, 7,10-tetra-(2-carbamonylmethyl)cyclododecane (eg conjugated to a monoclonal antibody).

mAb is a monoclonal antibody.

HOPO - Me-3,2-HOPO, octadentate-hydroxypyridinone to form a complex with 227Th, 4-((4-(3-(bis(2-(3-hydroxy-1-methyl-2-oxo-1,2- dihydropyridine-4-carboxamido)ethyl)amino)-2-((bis(2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)methyl)propyl) phenyl)amino)-4-oxobutanoic acid and derivatives.

Ligand 1 - p-SCN-Bn-TCMC-PSMA-ligand 1, p-SCN-Bn-TCMC-PSMA, etc.

Ligand 2 - p-SCN-Bn-DOTA-PSMA-ligand 2, p-SCN-Bn-DOTA-PSMA, etc.

The same abbreviations are used hereafter for acids, salts, or partially or fully dissociated versions of chelators.

The present invention relates to solutions with single or dual targeting properties based on a low molecular weight urea derivative as a targeting agent for prostate cancer cells designed to carry ²¹² Pb.

It can be used with purified ²¹² Pb or in a dual targeting solution where ²²⁴ Ra will act as a skeletal treatment and ²¹² Pb urea will act as a systemic therapy against cells expressing the PSMA antigen, which is bound primarily with advanced metastatic prostate cancer and to some extent also with other types of cancer.

It is known in the art that urea-based compounds conjugated to a chelator group can help target radionuclides to cells expressing PSMA. Radionuclides that have been evaluated for radiotherapeutic targets using PSMA targeting include ¹⁷⁷ Lu, ²¹¹ At, ²¹³ Bi, and ²²⁵ Ac.

The present invention relates to the field of radionuclide labeled therapeutic agents. In accordance with the present invention, radionuclide-labelled derivatives of urea-based prostate-specific membrane antigen (PSMA) inhibitors are disclosed.

Thus, the present invention relates to a complex containing Compound X coupled to a radionuclide such as ²¹² Pb or ²²⁷ Th, wherein Compound X is a peptidic or peptidomimetic urea derivative suitable for targeting PSMA expressing cells and tissues.

Linker

One aspect of the present invention relates to the complex of the present invention, where the compound X is associated with a radionuclide, such as ¹⁷⁷ Lu, ²¹³ Bi, ²²⁵ Ac, ²¹² Pb or ²²⁷ Th, using a Z chelating moiety.

In one embodiment of the present invention, the radionuclide is ²¹² Pb.

In another embodiment of the present invention, the radionuclide is ²²⁷ Th.

In another embodiment of the present invention, the radionuclide is ¹⁷⁷ Lu.

In a further embodiment of the present invention, the radionuclide is ²¹³ Bi or ²¹² Bi.

In yet another embodiment of the present invention, the radionuclide is ²²⁵ Ac.

It should be understood that the complexing agent or linker or chelating moiety Z according to the present invention may also encompass derivatives of the above compounds (such as derivatives of EDTMP, DOTA, eg p-SCN-Bn-DOTA, and TCMC, eg p-SCN- Bn-TCMC). Of course, it should be understood that such derivatives must retain the ability to form a complex with ²¹² Pb with a higher stability constant than with ²²⁴ Ra.

The chelating moiety Z may be selected from the group consisting of acyclic chelators, cyclic chelators, cryptands, crown ethers, porphyrins, or cyclic or non-cyclic polyphosphonates, DOTMP, EDTMP and bisphosphonate derivatives, DOTA, a DOTA derivative such as p-SCN-Bn- DOTA, pamidronate conjugated to DOTA, TCMC, a derivative of TCMC such as p-SCN-Bz-TCMC, pamidronate conjugated to TCMC, DOTA conjugated to an antibody, TCMC conjugated to an antibody, HBED-CC, NOTA, NODAGA, TRAP , NOPO, PCTA, DFO, DTPA, CHX-DTPA, AAZTA, DEDPA and oxo-Do3A.

In one embodiment of the present invention, the linker is DOTA or a DOTA derivative such as p-SCN-Bn-DOTA.

In another embodiment of the present invention, the linker is TCMC or a TCMC derivative such as p-SCN-Bn-TCMC.

The complexing agent may be bound via a carbon backbone that allows all of the "bonding arms" of the chelator molecule to interact with the metal. Alternatively, one of the arms can be used as a linker.

Suitable chelators include DOTA derivatives such as p-isothiocyanatobenzyl-1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraacetic acid (p-SCN-Bz-DOTA) and DOTA-NHS ester.

Thus, in the case of p-SCN-Bn-DOTA or p-SCN-Bn-TCMC, the complexing agent can be linked to the rest of the compound via a carbon backbone (C-backbone).

In one embodiment, the linker is an octadentate hydroxypyridinone containing ligand, such as 3,2-HOPO. Such ligands will typically contain at least one chelating group of the following structure as a substituted pyridine (I):

-17-R, I,

where R is an optional N-substituent group, and thus it may be absent or may be selected from hydrocarbon, OH-, O-hydrocarbon, SH- and S-hydrocarbon groups, with any or each hydrocarbon moiety independently selected from short hydrocarbyl groups such as a C1-C8 hydrocarbon, including C1-C8 alkyl, -alkenyl or alkynyl groups, or may be an OH or O hydrocarbon. R may also include a linker moiety, as defined below, and/or may contain a linker moiety, as also defined below.

In Formula I, each of the R2-R6 groups can be independently selected from H, OH, ═O, short hydrocarbon (described herein), linker moiety (described herein), and/or linker moiety (described herein). Typically, at least one of the R-R6 groups will be OH. Typically, at least one of the groups R2 - R6 will be ═O.

Typically, at least one of the R-R6 groups will be a linker moiety. Preferably exactly one of the groups R2 - R6 will be ═O. Preferably exactly one of the R-R6 groups will be OH. Preferably, exactly one of the R-R6 groups will be a linker moiety (described herein). The remaining R-R6 groups may be any of the moieties specified herein, but are preferably H. If the linker moiety or any additional linker, template, or chelating groups attached to the linker moiety do not provide for a linker moiety, then one of the R- R6 is preferably a linker (described herein).

In a preferred embodiment, one of the R-R6 groups will be OH and one of the groups from R2 onwards will be ═O, and the OH and ═O groups will be located on adjacent ring atoms.

Thus, in a preferred embodiment, OH and ═O can be located at atoms 1,2; 2.3; 3.2; 3.4;

or 4.3, respectively (as expected, the numbering is from nitrogen).

Particularly preferred are octadentate ligands having at least one chelating moiety, where the OH and ═O groups are present at positions 3 and 2, respectively. Octadentate ligands may have 2, 3 or 4 such chelating groups, with 2 or 4 such groups being particularly preferred.

In a particular embodiment, the PSMA-targeting urea complex is labeled with ²¹² Pb or ²²⁷ Th while being mixed with osteotropic ²²⁴ Ra or ²²³ Ra, respectively, to provide dual targeting by (1) targeting cellular PSMA and (2) targeting bone synthesis associated with skeletal metastases using radium cations.

Complexing agents for ²²⁷ Th, including those described in WO2011098611, US20170319721, Ramdahl et al. (Bioorganic & Medicinal Chemistry Letters

Volume 26, Issue 17, 1 September 2016, Pages 4318-4321) and Hagemann et al. (Mol Cancer Ther. 2016 Oct;15(10):2422-2431. Epub 2016 Aug 17), conjugated to a urea derivative to target PSMA. The complexing agents mentioned herein are thus incorporated by reference.

Thus, one embodiment of the present invention relates to a PSMA-targeting urea derivative containing a TCMC moiety, such as p-SCN-Bn-TCMC, for ²¹² Pb chelation.

Another embodiment of the present invention relates to a PSMA-targeting urea derivative containing HOPO for ²²⁷ Th chelation.

A further embodiment of the present invention relates to a PSMA-targeting urea derivative containing DOTA, such as p-SCN-Bn-TCMC labeled with either ²¹² Pb or ²²⁷ Th.

In yet another embodiment, the complexing agent does not complex with ²²⁴ Ra or does not substantially complex with ²²⁴ Ra in the pharmaceutical composition.

In another further embodiment, the complexing agent forms a complex with ²¹² Pb with a higher stability constant than with ²²⁴ Ra.

In one embodiment, the stability constant for ²¹² Pb is at least twice the affinity for ²²⁴ Ra, such as at least four times higher, such as at least 8 times higher, or such as at least 10 times higher .

In one embodiment, said complexing agent is selected from the group consisting of ligand-conjugated DOTA, such as ligand-conjugated p-SCN-Bn-DOTA, or ligand-conjugated TCMC, such as ligand-conjugated p-SCN-Bn-TCMC.

The ligand may be an antibody or a polypeptide.

In an additional embodiment, the number of ²²⁴ Ra and ²¹² Pb is in radioactive equilibrium.

In yet another further embodiment, the ratio of activity (in MBq) of ²¹² Pb to ²²⁴ Ra is 0.5 to 2, such as 0.8 to 1.5, or such as 0.8 to 1.3, or preferably, such as 0.9-1.15.

In the present context, the term "radioactive equilibrium" refers to the fact that the ratio in MBq between two radionuclides remains the same or substantially the same over time. The term "activity ratio", for example ²¹² Pb to ²²⁴ Ra, refers to the ratio of ²¹² Pb to ²²⁴ Ra expressed in MBq. The figure 5 presents a table (table 2), which shows the dynamics of this ratio of activity over time. It can be seen that after two days a radioactive equilibrium of 1.1 was established for the ratio of ²¹² Pb to ²²⁴ Ra activity (7.3 divided by 6.8). Thus, in figure 5 it can also be seen that after approximately 2 days a radioactive equilibrium between ²¹² Pb and ²²⁴ Ra was reached.

In the context of the present invention, the terms "complexing agent", "acceptor", "linker", "chelating Z fragment" and "chelating agent" are used interchangeably. These terms refer to agents capable of complexing with ^212Pb , preferably by chelation, and with significant strength, based on the results of measurements in test systems, while the presence of such a complex does not significantly affect radium, based on the results of measurements in test systems .

The assays include in vivo biodistribution determination as well as an in vitro cation exchanger or size-dependent retention and centrifugal concentration cartridge to detect radionuclide binding to the chelate-antibody complex. Alternatively, thin layer chromatography can be used as a test system.

In the present context, the term "acceptance" (or complexation) is defined as at least 50% binding according to thin layer chromatography (TLC), centrifugal separation or biodistribution profiles.

This means, for example, at least 50% less accumulation ^{of 212} Pb in the blood with a low molecular weight chelator. In the case of an antibody-conjugated chelator, where blood accumulation is not a reliable indicator, at least 50% binding is predicted based on TLC assays.

In one embodiment, the present invention provides for at least 60% binding.

In another embodiment, the present invention provides for at least 70% binding.

In another embodiment, the present invention provides for at least 80% binding.

In another embodiment of the present invention, at least 85% binding is provided.

In another embodiment, the present invention provides for at least 90% binding.

The compound or compounds may also have the ability to complex with more radionuclides than ²¹² Pb alone.

In one embodiment of the present invention, the compound and/or complex is present at a concentration of 1 ng/mL to 1 g/mL.

In another embodiment of the present invention, the compound and/or complex is present at a concentration of 100 ng to 10 mg/mL.

The complex may contain one, two, three, four, five or more compounds.

In one embodiment, a solution is provided in a volume of 100 µl to 1000 ml, such as 500 µl to 100 ml, 1 ml to 10 ml.

In one embodiment of the present invention, the radioactivity of the solution is 1 kBq to 1 GBq, such as 10 kBq to 100 MBq, such as 100 kBq to 10 MBq.

In another embodiment of the present invention, the radioactivity of the solution is between 100 kBq and 100 MBq.

In another embodiment of the present invention, the complexing agent is conjugated to a compound selected from the group consisting of urea peptides and peptidomimetic derivatives having an affinity for PSMA.

In another embodiment of the present invention, the complexing agent is a chelator of TCMC, such as p-SCN-Bn-TCMC, or DOTA, such as p-SCN-Bn-DOTA, conjugated to a compound selected from the group consisting of a monoclonal antibody, a polyclonal antibody, antibody fragment, synthetic protein, peptide, hormone or hormone derivative, or vitamin or vitamin derivative.

For dosing purposes, a pure ²¹² Pb solution may be used. Alternatively, ²²⁴ Ra can be used in admixture with ²¹² Pb, using the latter to provide dual targeting, i.e. ²²⁴ Ra targets bone metastases and ²¹² Pb targets systemic cancer via its urea carrier. In the case of administration of solutions containing ²²⁴ Ra, they can be stored for some time, for example for 1 day or more preferably at least two days, for example 1-2 days or 1-3 days, to ensure that an equilibrium is reached between ²²⁴ Ra and ^212Pb / ^212Bi . This will provide ²¹² Pb to ²²⁴ Ra activity ratio values of 0.83 to 1.14. This can be achieved, for example, by the manufacturer by simply keeping the product for a day or so before shipping.

Alternatively, ²¹² Pb can be added to ²²⁴ Ra solutions to obtain a certain ratio of radionuclides. For example, if the soft tissue tumor burden is much greater than the skeletal tumor burden, a pure ²¹² Pb preparation or a solution with a high ratio of ²¹² Pb to ²²⁴ Ra can be used.

The non-overlapping side effect profile of radium cations and alpha particle emitters conjugated to PSMA-binding urea derivatives makes the mixture of ²²⁴ Ra cation and ²¹² Pb-PSMA targeting agent particularly attractive for dual targeting. This is because a lower dose of each compound can be used to obtain antitumor activity because, at least in the case of a skeletal moiety, two different compounds will target lesions independently of each other.

It is important to store the radium primarily as an uncomplexed or weakly complexed cation, as this ensures maximum accumulation in bone and in bone metastases, as well as favorable excretion of the eliminated product, mainly through the intestines.

By adding a complexing agent to the radium solution, the daughter product can be endowed with affinity for bone or tumor, as well as increasing the therapeutic potential of the radium solution instead of its health hazard. However, it is the complexing agent that should not have a negative effect on the osteotropic properties of radium. For example, the ability of a TCMC-labeled urea derivative to accept ²¹² Pb generated in radium solution during transport and storage between the place of manufacture and hospitals where the product is to be administered.

Although this susceptibility can be reduced by the addition of radiolysis inhibitors, tumor-targeting peptides and peptidomimetics are often more susceptible to radiolysis and should probably be supplied in a kit format that adds hours to minutes before administration of the solutions. ²²⁴ Ra with a relatively long shelf life.

It is known in the art that calixarenes and EDTA can complex to some extent with radium and can also complex with lead and bismuth. However, in the present work, the present inventors have found chelators that leave radium largely uncomplexed or weakly complexed, as determined by in vivo biodistribution measurements, and yet are capable of complexing rapidly and with appropriate stability with the longest-lived ^212Pb daughter. Selective complexation can be used to produce at least bone or tumor tropic lead while maintaining the favorable properties of radium in the treatment of sclerotic diseases such as skeletal metastases. A complex with ^212Pb that targets bone or tumor cells forms a ^212Bi alpha particle emitter due to ^212Pb decay. Thus, a ²¹² Pb beta emitter is used as an indirect alpha emitter to irradiate target cells or tissue. Other potential chelates that may be suitable for accepting the ²²⁴ Ra derived daughter nuclide, in addition to TCMC and DOTA, include, but are not limited to, porphyrins, DTPA and DTPA derivatives, and carboxyl-bound DOTA.

Lead-212 is the longest lived of the ^224Ra decay progeny, and it is the most important for complexing as it generates the short lived ^212Bi alpha particle emitter in vivo. If ^{the 212} Pb chelate accumulates in bone or tumor cells, ²¹² Bi is also likely to be retained in the target. In a solution ^{of 224} Ra in equilibrium with the daughter products of the decay of ²¹² Pb atoms, there will be more than 10 times more than ²¹² Bi atoms. Thus, the amount of radiation generated by ²¹² Bi atoms in these solutions is small and probably has no toxicological significance compared to the decay chain ^{of 224} Ra and ²¹² Bi. The amount of ²¹² Bi is comparable to that of ²¹¹ Pb, which indirectly produces an alpha particle in the ²²³ Ra chain, and this was not a significant problem for the registration and clinical use ^{of 223} Ra in equilibrium with progeny decay products.

However, if a high degree of chelation of ^{the 212} Bi in the injection solution is also required, at least in some cases the addition of a stabilizing agent such as NaI or HI may be required, since bismuth tends to be in aqueous solutions in a state that is less suitable for chelation.

Compared to the currently approved alpha particle generating pharmaceutical for the treatment ^of skeletal metastases, i. targeting the daughter nuclide to circulating cancer cells and, to some extent, also to soft tissue metastases. This may prevent recurrence due to CTC-mediated skeletal recolonization by cancer cells.

Another aspect is that the shorter half-life of ²²⁴ Ra compared to ²²³ Ra may indeed be of some benefit as radium is incorporated into the bone matrix. Due to the high density of the mineral component of bone, the range of alpha particles is greatly reduced in bone compared to soft tissues. When using an alpha particle generating pharmaceutical with a bulk affinity, the insertion process can be important, especially in areas of rapid mineralization such as bone cancer metastases.

Therefore, ²²⁴ Ra can provide an improvement in the antitumor dose, since on average its introduced amount at the time of decay will be less.

Diseases for which the novel ²¹² Pb solutions with or without ²²⁴ Ra may be used include, but are not limited to, primary and metastatic cancers, autoimmune diseases, and atherosclerosis. This drug can be administered intravenously or topically, including intraperitoneally or under conditions of perfusion of the limb.

The chelators used in the new solutions can be acyclic as well as cyclic chelators and cryptands, crown ethers, porphyrins, or cyclic or non-cyclic polyphosphonates, including DOTMP and EDTMP. A bisphosphonate, such as pamidronate conjugated with DOTA, TCMC or the like, can also be used as an acceptor in the ²²⁴ Ra solution.

It can be argued that the amount of ²¹² Pb in the therapeutic solution ^{of 224} Ra can be moderate to negligible (ie, approximately 1.1 times ^{the 224} Ra at equilibrium). If a dose of ²²⁴ Ra is assumed to be similar to that given to ²²³ Ra in patients, but adjusted for differences in half-life, then the dose administered would be approximately 150 kBq per kg of body weight.

At equilibrium, this can be translated into a dose of ²¹² Pb-antibody conjugate equal to 11.5 MBq per 5 liters of blood in a 70 kg patient (if ²¹² Pb is quantitatively chelated). The number of tumor cells circulating in the bloodstream is usually less than 10 cells per ml, so there are less than 50,000 tumor cells in total in 5 liters of blood. If only 1 in 100,000 administered ^212Pb -antibody conjugate molecules binds to tumor cells, this would mean at least 0.0023 Bq per cell, which is equivalent to about 127 bound ^212Pb atoms per cell, which would be highly damaging because it was previously reported that an average of 25 cell-bound ²¹² Pb per cell led to the destruction of 90% of the cell population.

Connections

One aspect of the present invention relates to a compound X associated with a chelating moiety Z, which is defined by formula I:

formula I

or a pharmaceutically acceptable salt thereof,

Where

W is a PSMA-targeting ligand;

A ⁴ is a bond or divalent linking fragment containing 1-10 carbon atoms in the chain, ring, or combinations thereof, where at least one carbon atom is optionally replaced by O, -NR ³ - or -C(O)-;

G is C=O, C=S, C-NH ₂ or C-NR ³ ;

R ¹ represents hydrogen or a protecting group for a carboxylic acid group;

R ³ is selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl and heteroaryl;

each of R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ and R ¹⁶ independently represents hydrogen, alkyl, alkoxy, or

each of R ¹⁷ and R ¹⁸ independently represents hydrogen, alkyl, aryl or alkylaryl;

R ¹⁹ is selected from the group consisting of alkyl, alkoxy, halide, haloalkyl and

CN;

m is an integer from 1 to 6; And

o is an integer from 0 to 4, and if o is greater than 1, then each R ¹⁹ is the same or different.

One aspect of the present invention relates to a complex according to the present invention, or a pharmaceutically acceptable salt thereof, wherein

A ⁴ is a bond, (CH ₂ ) _n , -HC(O)-, -(OCH ₂ CH ₂ ) _n -, -(HCH ₂ CH ₂ ) _n -, -H(CO)CH ₂ -, -HC (O)CH ₂ (OCH ₂ CH ₂ ) _n - or -HC(O)CH ₂ (HCH ₂ CH ₂ ) _n -; And

L is a bond, (CH ₂ ) _n , -(OCH ₂ CH ₂ ) _n -, -(HCH ₂ CH ₂ ) _n - or -C(O)(CH ₂ ) _n -;

wherein n is independently 1, 2, or 3.

One aspect of the present invention relates to the complex in accordance with the present invention or its pharmaceutically acceptable salt, where A ⁴ represents a bond, -(OCH ₂ CH ₂ ) _n - or -HC(O)CH ₂ (OCH ₂ CH ₂ ) _n - ; And

L is a bond or -(OCH ₂ CH ₂ ) _n -;

wherein n is independently 1 or 2.

One aspect of the present invention relates to a complex according to the present invention, or a pharmaceutically acceptable salt thereof, wherein

W is characterized by the structure:

where each of R ²⁰ and R ²¹ independently represents an amino acid residue associated through its amino group with the neighboring group -C(O)-.

One aspect of the present invention relates to a complex according to the present invention, or a pharmaceutically acceptable salt thereof, wherein

W is characterized by the structure:

where R is hydrogen or a protecting group for a carboxylic acid group.

One aspect of the present invention relates to a complex according to the present invention, or a pharmaceutically acceptable salt thereof, having the structure:

or a pharmaceutically acceptable salt thereof,

where R ¹⁷ represents aryl.

One aspect of the present invention relates to a complex according to the present invention, or a pharmaceutically acceptable salt thereof, wherein the complex is PSMA-617:

wherein the radionuclide, such as ²¹² Pb, is bonded/chelated to four N.

One aspect of the present invention relates to the complex in accordance with the present invention, in which the DOTA unit, such as p-SCN-Bz-DOTA, is replaced by a TCMC unit, such as p-SCN-Bz-TCMC.

The linker in PSMA-617 can also be covalently bonded to the C atom in the backbone instead of bonding to N, as can be seen in the figure above.

Thus, the urea derivative can be conjugated to DOTA or TCMC via bonding to the C atom of the backbone or bonding to the N atom.

In the case of conjugation of p-SCN-Bn-DOTA or p-SCN-Bn-TCMC via C-backbone bonding, the compound is:

where Z is:

and where X represents -OH or NH ₂ .

X is -OH for p-SCN-Bn-DOTA and X is NH ₂ for p-SCN-Bn-TCMC.

This means that the formula for C-linked p-SCN-Bn-DOTA or p-SCN-Bn-TCMC would be:

,

,

where X represents -OH or NH ₂ .

C-linked p-SCN-Bn-DOTA is:

,

,

p-SCN-Bn-DOTA-PSMA ligand 2.

Thus, one aspect of the present invention relates to a compound that is p-SCN-Bn-DOTA-PSMA Ligand 2.

C-linked p-SCN-Bn-TCMC is:

,

,

p-SCN-Bn-TCMC-PSMA ligand 1.

Thus, one aspect of the present invention relates to a compound that is p-SCN-Bn-TCMA-PSMA Ligand 1 or p-SCN-Bn-DOTA-PSMA Ligand 2 as disclosed above and in the examples.

P-SCN-Bn-TCMC-PSMA Ligand (Ligand 1) and p-SCN-Bn-DOTA-PSMA Ligand (Ligand 2) (as shown in the examples) are bonded via a carbon backbone (C-backbone) to the rest of the compound and has an extended linker region including an isothiocyanatobenzyl linker and also uses a carbon substituted chelator with all 4 chelating arms being free unlike PSMA-617 which has a shorter linker region and uses one of the chelating arms as the attachment point of the linker. The following examples in this document show that these differences result in a significantly different biodistribution of the radionuclide labeled product, making it more suitable for targeting ^212Pb to PSMA-expressing tumors as it provides reduced renal exposure compared to PSMA-617. .

In one embodiment of the present invention, the compounds of the present invention, such as p-SCN-Bn-TCMC-PSMA Ligand 1 and p-SCN-Bn-DOTA-PSMA Ligand 2, may be in the form of a trifluoroacetic acid salt.

CTT1401 and CTT1403 and their derivatives (Choy et al, 2017) with TCMC or DOTA variants can also be used together with ^212Pb in the complexes of the present invention.

When preparing a PSMA-binding urea derivative according to the present invention with a readily available amino group or other group suitable for conjugation, p-SCN-Bn-TCMC can be conjugated to the PSMA-binding compound. Post-purification may be required before incorporation of the ²¹² Pb radioactive label with or without ²²⁴ Ra in solution.

Thus, in one embodiment of the present invention, the linker is p-SCN-Bn-TCMC. One embodiment of the present invention relates to a complex according to the present invention wherein the chelating linker is p-SCN-Bn-TCMC. One embodiment of the present invention relates to a complex in accordance with the present invention, where the linker is p-SCN-Bn-DOTA. In other words, -p-SCN-Bn- is the end of a linker region that is attached to the TCMC or DOTA chelator group via a carbon backbone.

Human serum albumin can be used to prolong the half-life of drugs. Thus, in one embodiment, ^{the 212} Pb-labeled PSMA-binding urea derivative of the present invention further comprises a moiety that can bind to albumin to increase the circulating half-life of the radionuclide labeled product.

A further embodiment of the present invention relates to a complex according to the present invention, wherein ^{the 212} Pb-labeled PSMA-binding urea derivative according to the present invention further comprises human serum albumin that has been directly conjugated to the complex or associated with the complex, e.g. via liposomes. .

Pharmaceutical compositions

One embodiment of the present invention relates to a solution containing a compound and/or complex of the present invention. The solution may also be a pharmaceutical composition.

Typically, an important element of a pharmaceutical composition is a buffer solution that substantially maintains the chemical integrity of the radioligand and is physiologically acceptable for infusion into patients.

In one embodiment of the present invention, the pharmaceutical composition contains one or more pharmaceutically acceptable carriers and/or excipients.

Acceptable pharmaceutical carriers include, without limitation, non-toxic buffers, excipients, isotonic solutions, and the like. More specifically, the pharmaceutical carrier may be, without limitation, normal saline (0.9%), semi-normal saline, Ringer's lactate solution, 5% dextrose, 3.3% dextrose/0.3% saline. The physiologically acceptable carrier may contain an anti-radiolytic stabilizer, such as ascorbic acid, which protects the integrity of the radiopharmaceutical during storage and transport.

One aspect of the present invention relates to a pharmaceutical composition containing a complex in accordance with the present invention and a diluent, carrier, surfactant and/or excipient.

One aspect of the present invention relates to ²¹² Pb-labeled ligand as a single agent. This will be the compound of the present invention complexed with ²¹² Pb and without the presence of any additional radionuclides such as ²²⁴ Ra.

One aspect of the present invention relates to providing dual targeting solution containing ²¹² Pb-labeled ligand and cationic or weakly complexed ²²⁴ Ra. This will be a compound of the present invention complexed with ²¹² Pb, together with ²²⁴ Ra present as a cationic or weakly complexed ²²⁴ Ra.

One aspect of the present invention relates to a pharmaceutical composition containing a compound and/or complex in accordance with the present invention, which additionally contains ²²⁴ Ra. ²²⁴ Ra may be cationic. The addition of ²²⁴ Ra enables dual targeting, for example, when it is present together with ²¹² Pb.

One aspect of the present invention relates to a pharmaceutical composition containing a complex in accordance with the present invention, where the radioactivity is from 100 kBq to 100 MBq per dose.

One aspect of the present invention relates to a pharmaceutical composition containing a complex in accordance with the present invention, where the amount of ²²⁴ Ra and ²¹² Pb is in radioactive equilibrium.

One aspect of the present invention relates to a pharmaceutical composition comprising a complex according to the present invention, wherein the activity ratio (MBq) ^{of 212} Pb to ²²⁴ Ra is from 0.5 to 2, such as 0.8 to 1.5, or as for example, 0.8-1.3, or preferably, such as 0.9-1.15.

Sets

The solution should be made physiologically injectable either at a centralized point of manufacture or by being obtained from a kit system, typically 2-4 vials, while still being physiologically injectable after pooling the contents of the kit vials.

One aspect of the present invention relates to a kit containing a first vial containing a radiopharmaceutical composition in accordance with the present invention and a second vial containing a neutralizing solution to adjust the pH and/or isotonicity of the radiopharmaceutical composition prior to administration to a patient.

In the case of, for example, a monoclonal antibody, it is generally recommended to keep the alpha particle generating radiopharmaceutical solution's own dose below 0.5 kGy to avoid reduction in binding properties due to radiolysis. Thus, in the case of concentrated solutions intended for shipment to remote locations, depending on the resistance to radiolysis of the generated radioligand, it is recommended to use a recruitment system that involves adding a ligand conjugated with a chelator to a ²²⁴ Ra solution (including daughter decay products) at a distance of several hours to 10 minutes time point prior to administration.

One aspect of the present invention relates to a kit containing a first vial containing a solution of ²²⁴ Ra; a second vial containing a complexing agent selected from the group consisting of p-SCN-Bn-DOTA-PSMA Ligand, p-SCN-Bn-TCMC-PSMA Ligand, acyclic chelators, cyclic chelators, cryptands, crown ethers, porphyrins or cyclic or non-cyclic polyphosphonates, DOTMP, EDTMP, bisphosphonate derivatives, DOTA, DOTA derivative, DOTA conjugated pamidronate, TCMC, TCMC derivative, TCMC conjugated pamidronate, DOTA antibody conjugated, TCMC antibody conjugated, HBED-CC , NOTA NODAGA, TRAP, NOPO, PCTA, DFO, DTPA, CHX-DTPA, AAZTA, DEDPA, and oxo-Do3A, wherein the complexing agent is capable of complexing with a ²²⁴ Ra-derived daughter nuclide such as ²¹² Pb while also complexing the agent does not form a complex with ²²⁴ Ra in a pharmaceutical solution; and optionally instructions for mixing the contents of the first vial and the second vial, thereby obtaining a pharmaceutical composition ready for administration to a patient 1 minute to 12 hours after mixing.

In one embodiment, the present invention provides a kit for use as a drug.

In a specific embodiment, the term "solution ²²⁴ Ra" should be understood as ²²⁴ Ra, which is present in solution in free form and is not associated, for example, with a surface such as resin.

In one embodiment, the kit contains a third vial containing a neutralizing solution to adjust the pH and/or isotonicity of the radiopharmaceutical solution prior to administration to a patient.

In yet another preferred embodiment, the amount of ²²⁴ Ra and ²¹² Pb is in radioactive equilibrium in the first vial.

In yet another preferred embodiment, the activity ratio (MBq) ^{of 212} Pb to ²²⁴ Ra in the first vial is between 0.5 and 2, such as 0.8-1.5, or such as 0.8-1.3 , or, for example, 0.9–1.15.

In yet another embodiment, the first vial has a radioactivity in the range of 100 kBq to 100 MBq.

In one embodiment of the present invention, the chelator-conjugated ligand is added to the ²²⁴ Ra solution (including daughter degradation products) over 30 minutes. – 5 hours before injection, such as 1-3 hours before injection.

In one embodiment of the present invention, the chelator-conjugated ligand is added to the ²²⁴ Ra solution (including the daughter degradation products) over 1 minute. - 20 minutes. before injection.

In one embodiment of the present invention, the chelator-conjugated ligand is added to the ²²⁴ Ra solution (including the daughter degradation products) over 1 minute. - 10 min. before injection.

Also part of the present invention is a kit with a chelated protein or peptide in one vial and a ²²⁴ Ra solution in another vial, which involves mixing the contents of these two vials 12 hours - 1 minute before administration. In one embodiment, mixing occurs several hours (such as 5) to 30 minutes prior to administration to the patient in order to bind ²¹² Pb and/or ²¹² Bi to the chelate.

In one embodiment of the present invention, the contents of both vials are mixed for 30 minutes. – 1 hour before injection.

In one embodiment of the present invention, the contents of both vials are mixed for 1 minute. - 20 minutes. before injection.

In an embodiment of the present invention, the contents of both vials are mixed for 1 minute. - 10 min. before injection.

Optionally, a third vial may be used containing a liquid used to dilute and adjust isotonicity prior to administration of the radiopharmaceutical solution. This third vial may contain EDTMP, which can chelate ²¹² Bi if necessary.

Medical applications

One aspect of the present invention relates to a radiopharmaceutical composition according to the present invention for use as a drug.

In one embodiment of the present invention, the disease is cancer.

One aspect of the present invention relates to a radiopharmaceutical composition according to the present invention for use in the treatment of soft tissue and/or skeletal disease. Treatment is directed to PSMA-expressing disease, including soft tissue and skeletal disease.

In one embodiment of the present invention, the skeletal disease is selected from the group consisting of soft tissue or skeletal metastases in breast, prostate, kidney, lung, bone, or multiple myeloma forms of cancer.

In one embodiment of the present invention, the cancer is prostate cancer. The cancer may also be breast cancer. The cancer may be kidney cancer. The cancer may also be lung cancer. The cancer may also be bone cancer. The cancer may also be multiple myeloma. The cancer may be metastases caused by these types of cancer.

In one embodiment of the present invention, the solution is administered at a dose in the range of 50-150 kBq per kg of body weight, such as 50-100 kBq per kg of body weight.

One aspect of the present invention relates to a method of treating a malignant or non-malignant disease by administering a radiopharmaceutical composition in accordance with the present invention to an individual in need thereof.

It can be used with purified ^212Pb -labeled ligand or in a dual-targeting solution where ^224Ra will act as a skeletal treatment and ^212Pb -labeled urea will act as a systemic therapy against cells expressing PSMA antigen, which is associated with advanced metastatic prostate cancer.

Thus, the complexes and solutions of the present invention can be used in the treatment of metastatic prostate cancer.

Another embodiment of the present invention relates to a pharmaceutical solution with dual-targeting properties, where ²¹² Pb is complexed with a urea-based PSMA targeting agent as described herein, and cationic ²²⁴ Ra targets bone metastases via calcium-mimic incorporation into bone.

How to get

One aspect of the present invention relates to a method for obtaining a radiopharmaceutical composition in accordance with the present invention, the method provides for obtaining a first solution, where the amount of ²²⁴ Ra and ²¹² Pb is in radioactive equilibrium; preparation of a second solution containing a complexing agent selected from the group consisting of p-SCN-Bn-DOTA-PSMA ligand, p-SCN-Bn-TCMC-PSMA ligand, acyclic chelators, cyclic chelators, cryptands, crown ethers , porphyrins or cyclic or non-cyclic polyphosphonates, DOTMP, EDTMP, bisphosphonate, DOTA, DOTA derivative, DOTA conjugated pamidronate, TCMC derivative, TCMC conjugated pamidronate, DOTA antibody conjugated, TCMC antibody conjugated, HBED- CC, NOTA NODAGA, TRAP, NOPO, PCTA, DFO, DTPA, CHX-DTPA, AAZTA, DEDPA, and oxo-Do3A, wherein the complexing agent is capable of complexing with a ²²⁴ Ra-derived daughter nuclide such as ²¹² Pb, and the complexing agent does not complex with ²²⁴ Ra; and mixing the first composition and the second composition, thereby obtaining a pharmaceutical composition in accordance with the present invention.

PSMA derivatives

PSMA (also called prostate-specific cancer antigen, PSM, FGCP, FOLH, GCP2, mGCP, GCPII, NAALAD1, NAALA-dase, FOLH1, glutamate carboxypeptidase 2, glutamate carboxypeptidase II, membrane glutamate carboxypeptidase, N-acetylated-alpha-linked acid dipeptidase I, petroylpoly-gamma-glutamate carboxypeptidase, folylpoly-gamma-glutamate carboxypeptidase, folate hydrolase 1, prostate-specific membrane antigen, cell growth inhibitory protein 27) is a membrane antigen of prostate epithelial cells in the form of a type II transmembrane protein and consists of a short NH2-terminal cytoplasmic domain , a hydrophobic transmembrane region and a large extracellular domain. PSMA is an enzyme that in humans is encoded by the FOLH1 (folate hydrolase 1) gene. Human GCPII contains 750 amino acids and has a weight of approximately 84 kDa.

PSMA expression is restricted to a few healthy tissues such as lacrimal and salivary glands, proximal renal tubules, epididymis, ovary, inner ileum and jejunum, and astrocytes in the central nervous system (CNS); relatively low levels of PSMA are expressed in the healthy prostate, with expression limited to the apical epithelium of the secretory ducts. In these non-malignant tissues, the accumulation of PSMA-targeted probes may be limited by an intact blood-brain barrier, healthy inner side of the proximal small intestine, and reduced cytoplasmic PSMA expression in the normal prostate. PSMA is most commonly associated with androgen-independent, metastatic, high-grade tumors, although PSMA is expressed in most primary prostate tumors regardless of androgen status.

Small molecule PSMA inhibitors are zinc binding compounds and can be classified into three types: 1) phosphonate, phosphate and phosphoramidate compounds, 2) thiols and 3) ureas. Urea derivatives appear to have particularly interesting properties as a carrier for radionuclides for diagnostic and therapeutic purposes.

The present invention relates to the use of ²¹² Pb-labeled urea derivatives. They can be combined with androgen deprivation therapy to enhance PSMA expression for improved radioligand uptake (Bakht et al, 2017).

Activity level

If ^{the 212} Pb-labeled urea is used alone, the activity level will often be between 1 and 500 MBq per patient, and even more often between 10 and 100 MBq per patient.

If ²²⁴ Ra is in equilibrium with ²¹² Pb complexed with a PSMA-binding urea derivative, the dosage will often be from 0.1 MBq to 100 MBq per patient, more often from 1 to 20 MBq per patient.

In a specific embodiment, the PSMA-targeting urea complex of the present invention is labeled with ²²⁷ Th, which generates osteotropic ²²³ Ra to provide dual targeting.

²²⁷ Th may be pure or contain varying amounts of ²²³ Ra, such as 10%, 50%, 100% or 250% ²²³ Ra versus ²²⁷ Th.

General information

It should be understood that any feature and/or aspect discussed above in connection with the compounds of the present invention is by analogy applicable to the methods described herein.

The following figures and examples are presented below to illustrate the present invention. They are intended to be illustrative and should not be construed as limiting in any way.

EXAMPLES

In the following examples, the xenograft model used is a tumor model with an intermediate level of PSMA ligand accumulation in mouse xenografts using ¹⁷⁷ Lu-PSMA-617, typically 10–15% of administered dose per gram (%ID/g), as opposed to the PC3 PIP model used by other investigators, which shows an accumulation rate in mouse xenografts typically 30–40% ID/g.

Example 1 Novel PSMA Binding Chelating Ligands Versus PSMA-617

Prerequisites. There are several carrier molecules that target the radioligand to prostate-specific membrane antigen (PSMA). Lutetium-177-labeled PSMA-617 ( ¹⁷⁷ Lu-PSMA-617) is a compound in the latest stage of clinical development for use in radionuclide therapy. This molecule functions well and provides appropriate values for the ratio of accumulation in tumor and normal tissues for longer-lived (i.e., with a half-life of several days) radionuclides, including ¹⁷⁷ Lu and ²²⁵ Ac, but at the same time at early time points (usually a few hours after injection) is characterized by high accumulation in the kidneys. In the case of shorter lived radionuclides such as ²¹² Pb (half life 10.6 hours), initial accumulation in the kidneys presents a potential toxicity problem. Therefore, it is preferable to use a PSMA ligand with less accumulation in the kidneys, and this should not interfere with the accumulation in the tumor. The PSMA ligand molecules consist of (1) a PSMA binding region, (2) a linker region, and (3) a chelator, with the linker region connecting (1) and (3). The linker region is also used to control molecular size and polarity in order to influence distribution properties in vivo. The PSMA-binding region (motif) used in PSMA-617 is a structure that can be found in several molecules of this class developed by several different inventors and researchers, including PSMA-11 and PSMA I&T, as well as in labeled with ¹³¹ I and ²¹¹ At PSMA binding ligands.

As can be seen, p-SCN-Bn-TCMC-PSMA Ligand 1 has an extended linker region including an isothiocyanatobenzyl linker and also uses a carbon-substituted chelator with all 4 chelating arms being free, unlike PSMA-617, which has a shorter linker region and uses one of the chelator arms as the linker attachment point. The following examples in this document show that these differences result in significantly different biodistribution of the radionuclide labeled product, making p-SCN-Bn-TCMC-PSMA Ligand 1 more suitable for targeting ^212Pb to PSMA-expressing tumors, as it provides reduced renal exposure compared to PSMA-617.

Materials and methods. The PSMA ligand precursors for radionuclide labeling were synthesized by a subcontracting laboratory that carried out commercial synthesis.

PSMA-617 was synthesized according to procedures described in the literature. The p-SCN-Bn-TCMC precursors were synthesized according to procedures described in the literature. In the final step of the synthesis, the TCMC-PSMA ligand was synthesized by conjugating p-SCN-Bn-TCMC to the amino group of the PSMA binding ligand intermediate. Both PSMA-617 and TCMC-Bn-PSMA Ligand 1 were purified by HPLC to >98% purity and dried and stored as trifluoroacetic acid salts. The structures and masses of the molecules were determined by 1 ^H -NMR and MS analysis.

The trifluoroacetic acid salt of p-SCN-Bn-TCMC-PSMA ligand 1 has the chemical formula C ₆₅ H ₈₆ F ₁₂ N ₁₄ O ₂₁ S and a molecular weight of 1659.52 g/mol.

Chemical name (according to IUPAC nomenclature): (((1S)-1-carboxy-5-((2S)-3-(naphthalene-2-yl)-2-((1R,4S)-4-((3- (4-((1,4,7,10-tetrakis(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido)methyl)cyclohexane-1- carboxamido)propanamido)pentyl)carbamoyl)-L-glutamic acid; trifluoroacetic acid (1:4).

The trifluoroacetic acid salt of PSMA-617 has the chemical formula C ₅₇ H ₇₅ F ₁₂ N ₉ O ₂₄ and a molecular weight of 1498.25 g/mol.

Salt of trifluoroacetic acid and p-SCN-Bn-TCMC-PSMA ligand 1.

Salt of trifluoroacetic acid and PSMA-617.

As can be seen, p-SCN-Bn-TCMC-PSMA Ligand 1 has an extended linker region including a benzyl linker and also uses a carbon-substituted chelator with all 4 chelating arms being free, unlike PSMA-617, which has a shorter linker region and uses one of the chelator arms as the linker attachment point. The following examples show that these chemical differences result in a significantly different biodistribution of the radionuclide labeled product, making it more suitable for targeting ^212Pb to PSMA-expressing tumors as it provides reduced renal exposure compared to PSMA-617.

In summary, a new molecule with an identical PSMA binding region but a different linker region and different chelating properties compared to PSMA-617 is described.

Example 2

Carbon-substituted p-SCN-Bn-DOTA-PSMA ligand 2 for labeling with radioactive isotopes ^212Pb , ^212Bi , ^213Bi , ^225Ac , ^227Th , ^177Lu , etc.

Compared to PSMA-617, the ligand has a larger size and p-SCN-Bn-DOTA group, that is, it has both a different linker region and a different version of the DOTA chelator, where all four chelator arms are free to provide radionuclide chelation compared to PSMA- 617.

p-SCN-Bn-DOTA-PSMA Ligand 2 is the DOTA analogue of p-SCN-Bn-TCMC-PSMA Ligand 1 described in Example 1 and has a carbon linker that provides binding to the chelator backbone, leaving the chelator groups free for interaction with the radiolabel, and therefore it is expected to improve the stability of the chelate after radionuclide labeling compared to radionuclide labeled PSMA-617. Due to the presence of an additional lipophilic benzyl unit in the linker region and the larger molecular size, less accumulation in the kidneys is expected compared to PSMA-617. This molecule can be synthesized in the same manner as p-SCN-Bn-TCMC-PSMA Ligand 1 using a DOTA-based precursor instead of a TCMC-based precursor in the last step of the synthesis.

Salt of trifluoroacetic acid and p-SCN-Bn-DOTA-PSMA ligand 2.

The trifluoroacetic acid salt of p-SCN-Bn-DOTA-PSMA ligand 2 has the chemical formula C ₆₅ H ₈₂ F ₁₂ N ₁₀ O ₂₅ S and a molecular weight of 1663.46 g/mol.

Attached to DOTA via a carbon substitution, this molecule has properties that are highly suitable for labeling with the radioactive isotopes ^212Pb , ^212Bi , ^213Bi , ^225Ac , ^227Th , as well as positron emission tomography (PET) compatible radionuclides such as ⁶⁸ Ga.

Thus, a new PSMA binding molecule is described, which is p-SCN-Bn-DOTA-PSMA Ligand 2, suitable for radioligand imaging and therapy.

In summary, carbon-substituted p-SCN-Bn-DOTA-PSMA ligand 2 is described as having different size and chelating properties than PSMA-617.

Example 3 Tested radionuclides

Lutetium-177 was purchased as ready-to-use ¹⁷⁷ LuCl ₃ dissolved in diluted HCl. Lead-212 was obtained from solutions based on ²²⁴ Ra. Radium-224 was obtained from ²²⁸ Th bound to actinide resin (Eichrom Technologies, LLC) by eluting a column containing actinide resin immobilized with ²²⁸ Th with 1 M HCl. The eluate was purified on a second Ac-resin column and the eluate was evaporated to dryness using an evaporation vial with gas inlet and outlet cap placed in a heater block at about 110 ^° C and a small flow of nitrogen gas to evaporate the solvent. When the evaporation vial was free of solvent, 0.1 M HCl was added to dissolve the residue, typically 200-400 µl.

Example 4 Radioactive Labeling of PSMA Binding Ligands

As a rule, ¹⁷⁷ Lu and ²²⁴ Ra/ ²¹² Pb solutions were adjusted with HCl with 10% 5 M ammonium acetate to the desired volume and pH 5–6. PSMA-binding ligands were dissolved in 0.1 M HCl with 0.5 M ammonium acetate to a concentration of 1 mg/ml. As a rule, a concentration of 20 μg per 1 ml of radioactive solution was used. The reaction mixture was incubated on a shaker for 15–30 minutes, with labeling usually assessed by thin layer chromatography. Labeling with lead-212 was carried out at room temperature or 37 ^o C using p-SCN-Bn-TCMC-PSMA-ligand 1, while ¹⁷⁷ Lu-labeling and ²¹² Pb-labeling of PSMA-617 were carried out at 90 ^o C Typical yields for labeling with radionuclides were in the 90-100% range using test compounds and radionuclides, providing concentrations of 1 µg per 20 µl or higher.

In summary, radionuclide labeling of both ligands was successful. The new p-SCN-Bn-TCMC-PSMA ligand 1 could be labeled with the ²¹² Pb radionuclide at room temperature, in contrast to ¹⁷⁷ Lu-PSMA-617, which required labeling at elevated temperature.

Example 5 TLC Analyzes

Thin layer chromatography (TLC) was performed using chromatographic strips (Model No. 150-772, Biodex Medical Systems Inc, Shirley, NY, USA). Formulation buffer (FB) consisting of 7.5% human serum albumin and 5 mM EDTA in DPBS and adjusted to approximately pH 7 with NaOH was mixed with the antibody conjugates in 2:1 ratios for at least 5 minutes prior to application on strips to determine the free radionuclide. A small beaker with approximately 0.5 ml of 0.9% NaCl was used to place the sample spot strips in it. 1–4 µl of sample was typically added to the strip approximately 10% above the bottom edge of the strip. After the solvent front had moved approximately 20% from the top of the strip, the strip was cut in half and each half was placed in a 5 ml tube for radiation recording. In this system, the radionuclide-labelled ligand remains in the bottom half, while the radionuclide complexed with EDTA migrates to the top half. Both ²¹² Pb and ¹⁷⁷ Lu free cations were confirmed to complex with EDTA and move to the top.

In summary, a TLC system was used to quickly determine the yield of radionuclide labeling, which effectively distinguished between radioligand and free radionuclide.

Example 6. Separation of ²¹² Pb from solutions of ²²⁴ Ra

The radionuclide labeled ligand can be used as a component in ²²⁴ Ra dual targeting solutions where ²²⁴ Ra targets skeletal disease and the ligand targets systemic metastatic disease. Alternatively, ²²⁴ Ra generating solutions can be used to produce a ²¹² Pb labeled ligand by in situ labeling, ie the ligand forms a complex with ²¹² Pb in the presence of ²²⁴ Ra. To 40 µl of solutions of radium-224/ ²¹² Pb in 0.5 M ammonium acetate pH 5–6 was added 2 µl (1 µg/µl) of either p-SCN-Bn-TCMC-PSMA ligand 1 or PSMA-617, and carried out the reaction as described to generate ²¹² Pb-labeled ligands. To purify ^{the 212} Pb-PSMA ligand, about 10 μl of formulation buffer consisting of 7% bovine serum albumin, 10 mM EDTA and 10 mg/ml ascorbic acid was added to the product. The reaction mixture was then added to a Sephadex G-10 PD MiniTrap G-10 column (GE Healthcare Life Sciences) and eluted with 0.9% NaCl. The eluate containing ²¹² Pb, typically fractions eluted after addition of 0.7-1.5 ml, was collected and analyzed on a gamma radiometer, and analyzed by TLC and radioligand binding analysis. The product purification procedure was characterized by high radiochemical yield (typically >80%) and high radiochemical purity, with ²²⁴ Ra typically less than 0.4% compared to ²¹² Pb-labeled ligand.

In summary, p-SCN-Bn-TCMC-PSMA Ligand 1 and PSMA-617 can be labeled with ²¹² Pb radionuclide in the presence of ²²⁴ Ra resulting in a dual targeting solution for combined targeting of bone metastases with ²²⁴ Ra and systemic targeting to tumor cells using ²¹² Pb-labeled PSMA ligand.

It was also found that using a Sephadex G-10 gel filtration column, both tested ²¹² Pb-labeled PSMA ligands could be separated from ²²⁴ Ra in solution with a recovery level of greater than 80% and a ²²⁴ Ra penetration of less than 0.4%, providing obtaining a highly purified ²¹² Pb-labeled PSMA radioligand for independent targeting of PSMA.

Example 7 In vitro stability assay of ²¹² Pb-labeled ligands.

The radionuclide labeled ligand in ²²⁴ Ra/ ²¹² Pb solution was mixed 1:1 with PBS or bovine serum albumin and incubated at 37 ^° C. for up to 48 hours. TLC assays were performed after 1 hour, 4 hours, 24 hours and 48 hours of incubation.

The data are shown in Table 1 and show that ²¹² Pb, after its generation from ²²⁴ Ra, continuously reacts with the ligand and maintains a high percentage of radiochemical purity even after 48 hours. Thus, PSMA ligands are compatible with the use of a ²²⁴ Ra solution for in situ production of a radioligand, which is suitable for centralized manufacture and transport to the end user.

Table 1. Radiochemical purity of ²¹² Pb-TCMC-PSMA ligand 1 and ²¹² Pb-PSMA-617 in PBS and FBS

Conclusion. The data show that p-SCN-Bn-TCMC-PSMA Ligand 1 and PSMA-617 labeled with ²¹² Pb are stable in ²²⁴ Ra solutions for a long period of time, indicating compatibility with centralized production and transportation to the end user. ready to use product. Such solutions can be used for anti-cancer treatment.

Example 8 Binding of prostate cancer cells with p-SCN-Bn-TCMC-PSMA-ligand-1 and PSMA-617 labeled with ²¹² Pb

Bound cell fractions were measured by adding approximately 1 ng of radioligand to 0.2 ml of C4-2 cells (5 x 10 ⁷ cells per ml) in a 5 ml tube and incubating for 1 hour before measuring the induced activity, washing the cells 3 times with 0.5 ml of 0.5% bovine serum albumin in DPBS, and then the radiation of the washed cell pellet was re-registered. From several experiments, % binding was found to be in the range of 40-53% after subtracting non-specific binding. Non-specific binding was measured by blocking cells with an excess of 10 μg/ml of unlabeled ligand prior to addition of radioligand. No significant difference was found in cell binding between radionuclide labeled p-SCN-Bn-TCMC-PSMA Ligand 1 and PSMA-617. In summary, the radioligands of radionuclide labeled p-SCN-Bn-TCMC-PSMA Ligand 1 and PSMA-617 had similar cell binding properties in vitro, indicating that p-SCN-Bn-TCMC-PSMA Ligand 1 has a corresponding antigen-binding ability.

Example 9 Radiolytic Stability of p-SCN-Bn-TCMC-PSMA Ligand 1 Evaluated by Prostate Cancer Binding Radioligand Assay and TLC Assay.

Table 2 shows bound cell fractions measured by adding approximately 1 ng of radioligand to 0.2 ml of C4-2 cells (5 x 10 ⁷ cells per ml) in a 5 ml tube and incubating for 1 hour before measuring induced activity, washing cells 3 times with 0.5 ml of 0.5% bovine serum albumin in DPBS, and then re-register the radiation of the washed cell pellet. The initial activity in solution was about 5 MBq/ml, providing an absorbed dose in solution of approximately 1.8 Gy at 24 hours and 3.3 Gy at 48 hours. The data (Table 2) indicate a slight decrease at the longest exposure time, but in general, a relatively strong resistance of the ligand to radiolysis was observed, which is compatible with centralized production and transport to the end user of the ²²⁴ Ra-based generating solution with or without the implementation of ²²⁴ Ra removal. before product introduction.

Table 2. Binding capacity of ²¹² Pb-p-SCN-Bn-TCMC-PSMA ligand 1 when exposed to ²²⁴ Ra solution

Conclusion. The data indicate that 212Pb-p-SCN-Bn-TCMC-PSMA ligand 1 is stable in ²²⁴ Ra solutions for a long period of time, and PSMA ligand is able to complex ²¹² Pb as it is generated from ²²⁴ Ra during storage, and provided that the absorbed dose of radiation is maintained below about 2 kGy, such solutions can be used for anticancer treatment.

Example 10 Biodistribution of radionuclide labeled ligands in nude mice with PSMA positive C4-2 xenografts.

The biodistribution of ^224Ra / ^212Pb solutions with p-SCN-Bn-TCMC-PSMA Ligand 1 and PSMA-617 labeled with ^212Pb and ^177Lu was compared after intravenous injection in nude mice with C4-2 xenografts at various time points after injections. Each group usually consisted of three mice. ²¹² Pb labeling exceeded 92% for the products. The molar concentration of the ligand was significantly lower for ¹⁷⁷ Lu-PSMA-617 because a much higher radionuclide level was used for ¹⁷⁷ Lu compared to ²¹² Pb. Approximately 16 kBq ²²⁴ Ra/ ²¹² Pb were injected into each animal, ie approximately 0.2 nmol ligand per mouse. Animals were anesthetized and euthanized by cervical dislocation, followed by dissection and collection of tissue, blood, and urine samples. The samples were weighed and the radiation was recorded using a gamma radiometer.

Example 11 Comparison of Tumor Binding and Renal Accumulation of p-SCN-Bn-TCMC-PSMA Ligand 1 and ^212Pb Labeled PSMA-617 in Mice

The biodistribution of ^224Ra / ^212Pb solutions with p-SCN-Bn-TCMC-PSMA Ligand 1 and ^212Pb labeled PSMA-617 was compared after intravenous injection in nude mice with C4-2 xenografts 4 hours after injection. Provided for three mice in each group. ²¹² Pb labeling exceeded 92% for both products. The molar concentration was the same for both products, i.e. 12.5 nmol per MBq. Approximately 16 kBq ²²⁴ Ra/ ²¹² Pb were injected into each animal, ie approximately 0.2 nmol ligand per mouse. Animals were anesthetized and euthanized by cervical dislocation, followed by dissection and collection of tissue, blood, and urine samples. The samples were weighed and the radiation was recorded using a gamma radiometer. Results. Four hours after injection, the ratios of ²¹² Pb-p-SCN-Bn-TCMC-PSMA Ligand 1 to PSMA-617 were as follows: 1.35 for tumor; 0.20 for kidney; 1.21 for blood; 2.67 for the liver; 0.71 for the spleen. By recording the radiation from the samples after 3 days of storage, it was confirmed that the ²²⁴ Ra biodistribution was not significantly altered by any of the PSMA-targeted ligands. Discussion. ^212Pb p-SCN-Bn-TCMC-PSMA Ligand 1 showed a significantly different biodistribution from ^212Pb -PSMA-617, and the extremely low and favorable ratio for accumulation in the kidneys is particularly noteworthy, since the kidneys are expected to be the main dose-limiting normal tissue associated with PSMA radioligand therapy using relatively short lived alpha particle emitters (FIG. 1). In summary, 212Pb-p-SCN-Bn-TCMC-PSMA Ligand 1 shows very promising biodistribution at early time points compared to PSMA-617, which is important when using short lived radionuclides such as ²¹² Pb.

Example 12 Comparison of Tumor Binding and Kidney Accumulation of ²¹² Pb-Labeled p-SCN-Bn-TCMC-PSMA Ligand 1 and ¹⁷⁷ Lu-Labeled PSMA-617 in Mice

Ways. Tumor and kidney accumulation of ²¹² Pb-labeled p-SCN-Bn-TCMC-PSMA Ligand 1 and ¹⁷⁷ Lu-labeled PSMA-617 were compared 1 hour and 4 hours after intravenous injection of the products in nude mice with C4-2 xenografts as described in example 9. Results. Tumors and kidneys were tissues that accumulate the largest amount of radioactivity. As examples, tumor and kidney accumulation of ²¹² Pb-p-SCN-Bn-TCMC-PSMA averaged 13.9 and 8.1 percent of administered dose per gram (% ID/g), respectively, 4 hours after injection. Tumor and kidney accumulation of ¹⁷⁷ Lu-PSMA-617 averaged 13.6 and 17.4% ID/g, respectively, 4 hours after injection. It should be noted that the molar amount of injected ligand was much lower for PSMA-617, which is known to reduce renal accumulation, however, the new ²¹² Pb-labeled compound showed less renal accumulation. Tumor versus kidney uptake ratios at 4 hours were: 1.7 for ²¹² Pb-p-SCN-Bn-TCMC-PSMA ligand 1; 0.8 for ¹⁷⁷ Lu-PSMA-617.

Mean tumor versus kidney accumulation ratios determined 1 hour after administration were 0.40 for ²¹² Pb-p-SCN-Bn-TCMC-PSMA ligand 1 and 0.17 for ¹⁷⁷ Lu-PSMA-617. In summary, despite a higher molar concentration of ligand for ²¹² Pb-p-SCN-Bn-TCMC-PSMA ligand 1, it showed a better tumor uptake ratio compared to kidney than ¹⁷⁷ Lu-PSMA-617, indicating that it may be well suited for radioligand therapy with an alpha particle emitter based on ²¹² Pb.

Example 13 Biodistribution of Single Targeting Solution Containing ²¹² Pb-p-SCN-Bn-TCMC-PSMA Ligand 1 in PSMA Positive Xenograft Mice

Using a ²¹² Pb/ ²²⁴ Ra solution to react with p-SCN-Bn-TCMC-PSMA Ligand 1 as described, purified ²¹² Pb-p-SCN-Bn-TCMC-PSMA Ligand 1 was purified using a Sephadex gel filtration column. G-10, and approximately 30 kBq and 300 ng of purified radioligand product were injected into each animal. The data are shown in Table 3. As can be seen, renal activity declines relatively quickly while tumor accumulation shows good retention. Tumor versus tissue accumulation ratios (Table 4) indicate suitability for radioligand targeting with ²¹² Pb.

In summary, ²¹² Pb-p-SCN-Bn-TCMC-PSMA Ligand 1 exhibits appropriate targeting properties for use in radioligand therapy against PSMA-expressing prostate cancer.

Table 3. Biodistribution for ²¹² Pb-p-SCN-Bn-TCMC-PSMA Ligand 1 at various time points after injection

Table 4. Tumor versus tissue accumulation ratios for ²¹² Pb-p-SCN-Bn-TCMC-PSMA ligand 1 at different time points

Example 14 Dosage

The radiant energy generated for the two nuclides mainly comes from alpha particles and therefore only alpha particles are considered in the following evaluation.

²¹² Pb and short-lived decay daughters produce an average of 7.8 MeV of alpha radiation per ²¹² Pb atom. The half-life of ²¹² Pb is 10.6 hours.

²¹³ Bi and short-lived daughter decay products produce an average of 8.4 MeV of alpha radiation per ²¹³ Bi atom. The half-life of ²¹³ Bi is 46 min.

The equivalent dose for alpha particles is assumed to be 5 Sv/Gy.

Thus, 1 Bq of ²¹² Pb produces an equivalent dose of alpha particles of 1 x (10.6 x 60/46) x 7.8 / 8.4 = 12.6 Bq of ²¹³ Bi, upon complete decay.

Salivary glands, kidneys, and red bone marrow have been reported to be dose-limiting tissues for ²¹³ Bi and ²²⁵ Ac in complex with PSMA-617 (Kratochwil, et al, 2018).

Based on the imaging of PSMA-617 labeled with radionuclides suitable for positron emission tomography detection, it is expected that the maximum percentage of accumulation will be reached at the time point of 30 minutes when 70% of ²¹³ Bi atoms compared to 90% ^{of 212} Pb are reached and subjected to decay in the tumor (i.e. relative fraction of decay).

It is assumed that the relative disintegration fraction for dose-limiting tissues is 70% for all tissues when using ²¹³ Bi-PSMA-617. It is assumed that 50% of the accumulated ²¹² Pb-PSMA-617 will be degraded in the salivary glands, 30% in the kidneys, and 20% in the bone marrow.

After correcting for energy per Bq and relative disintegration fraction for PSMA-617 labeled with ²¹² Pb and ²¹³ Bi, the calculated doses for ²¹² Pb-PSMA-617 will be as presented in Table 5 along with previously published data for PSMA-617, labeled with ²¹³ Bi and ²²⁵ Ac. In addition, by comparing mouse data for ²¹² Pb-p-SCN-Bn-TCMC-PSMA Ligand 1 and ²¹² Pb-PSMA-617, and assuming a similar tissue uptake ratio in humans, a dosimetric estimate for ²¹² Pb-p-SCN-Bn-TCMC-PSMA ligand 1 is shown in Table 5.

Table 5. Dose estimation assuming similar product stability and affinity regardless of radionuclide

^a From Kratochwil et al 2017. ^b From Kratochwil et al 2018. ^c Assuming the same accumulation as ²¹² Pb-PSMA-617, except for renal accumulation, which is assumed to be reduced by 50%.

It has been reported that PSMA-617 labeled with both ²²⁵ Ac and ²¹³ Bi has been used clinically and has shown significant antitumor activity. Based on the evaluations in this example, ^{the 212} Pb-labeled urea-based PSMA inhibitor is shown to be a highly promising therapeutic agent against PSMA-expressing cancer.

Example 15 Dosimetric evaluation for dual targeting with ²²⁴ Ra cation and ²¹² Pb-labeled urea targeting PSMA

Used ²²⁴ Ra in equilibrium with ²¹² Pb-labeled urea derivative binding PSMA.

For a dose of 150 kBq ^{of 224} Ra, the total administered activity would be 10.5 MBq per 70 kg person. Using published dosimetry data for cationic ²²³ Ra in patients with prostate cancer and adjusting for the difference in half-life between ²²⁴ Ra and ²²³ Ra, assuming the same biodistribution and considering different retention times in different tissues, found that ²²⁴ Ra per administered MBq will provide 0.006 Gy for the kidneys, 0.029 Gy for the salivary glands, 0.26 Gy for the red bone marrow, and an estimated accumulation in tumors 5 times that for the red bone marrow, i.e. 1.3 Gy.

For the dose of alpha particles, an equivalent dose of 5 Sv/Gy is assumed and the data are converted to Sv per dose unit administered (10.5 MBq/patient) in Table 6.

Table 6. Dose assessment for cation ^224Ra + ^212Pb -labeled urea derivative binding PSMA*

*Assuming a 1:1 ratio between ²²⁴ Ra and ²¹² Pb.

^a From Lassmann et al, 2002. ^b It should be noted that red bone marrow is mainly exposed to Ra deposits on the bone surface, and due to the short path length of alpha particles from bone surfaces, significant areas of red bone marrow are out of reach of alpha particles from the surface bones. For a dose of alpha particles, an equivalent dose of 5 is assumed.

Example 15 Evaluation Dosimetry for ²²⁷ Th Labeled Urea Derivative Binding PSMA

In this example, p-SCN-Bn-DOTA-PSMA Ligand 2, or the HOPO-derived version of this molecule, is assumed to be labeled with ¹⁷⁷ Lu. Current estimates are based on adaptation of data from studies of ²²³ Ra in patients and ²²⁵ Ac-PSMA-617 in patients with prostate cancer (Chittenden et al., 2015, Kratochwil et al, 2017, 2018).

For radiation of alpha particles, an equivalent dose of 5 Sv/Gy is assumed.

It is assumed that ²²³ Ra generated in tissues will undergo local decay. It is estimated that 40% ^{of the 223} Ra generated from ²²⁷ Th circulation throughout the body is retained in the skeleton. Only the dose of alpha particles is considered, since it is 95% or more of the total generated radiation energy.

For tissue with a longer retention time of radionuclide-labeled PSMA-binding urea (salivary gland, kidney and tumors), it is assumed that the total activity of ²²³ Ra produced from ²²⁷ Th is 20% of ²²⁷ Th, and for red bone marrow is 5 %.

It is assumed that 20% and 5% of the radium generated from ²²⁷ Th in various tissues is in equilibrium with alpha emitting daughter radionuclides, so each radium decay essentially generates 26.4 MeV of alpha radiation, while ²²⁷ Th separately generates 5.9 MeV of alpha radiation.

Since the retention times for ²²⁵ Ac and ²²⁷ Th are lower than the half-lives, it is assumed that the total activity for ²²⁷ Th is only 10% higher than for ²²⁵ Ac (excluding ²²⁵ Ac decay progeny) in all organs. A total of 27.7 MeV of alpha radiation per ²²⁵ Ac atom is assumed to result from the decay of the parent nuclide and alpha emitting daughter decay products.

It is also assumed that in relation to the dose for red bone marrow and the dose for skeletal tumor, the latter increases by 2 times compared to the dose for red bone marrow due to the accumulation of ²²³ Ra in the skeleton generated during systemic circulation, etc. by the product with ²²⁷ Th.

The data are presented in Table 7. The data indicate a favorable tumor versus tissue accumulation ratio for the ²²⁷ Th-labeled PSMA-binding urea derivative.

Table 7. Estimated dosimetry of critical organ and tumor equivalent doses for ²²⁷ Th-labeled PSMA-binding urea derivative (MBq/patient)

*Includes added skeletal dose of ²²³ Ra generated from ²²⁷ Th during the circulating phase.

The data in Table 7 represent ²²⁷ Th-labeled PSMA-binding urea (eg, PSMA-617) that is purified from ²²³ Ra prior to injection. Alternatively, a ²²⁷ Th product solution containing varying amounts of ²²³ Ra can be used to increase the dose for bone metastases, for example if bone disease is highly dominant compared to soft tissue disease.

It is contemplated that the products described herein may be used as a single treatment or as repeated treatments.

In summary, dosimetric evaluations for ²¹² Pb or ²²⁷ Th labeled urea derivatives targeting PSMA indicate promising tumor versus tissue uptake ratios, indicating possible clinical efficacy when applied.

Example 16 Comparative experiment on therapy with ¹⁷⁷ Lu-PSMA-617 and ²¹² Pb-p-SCN-Bn-TCMC-PSMA Ligand 1 in nude mice with C4-2 xenografts

Male nude mice were inoculated with PSMA-positive human prostate cells of the C4-2 line in the lateral areas and 2 weeks later, when the tumors had a diameter of 5–7 mm. Groups of 8 animals each received saline, 52 MBq ¹⁷⁷ Lu-PSMA-617, or 320 kBq ²¹² Pb-p-SCN-Bn-TCMC-PSMA ligand 1. Animals were sacrificed when tumor size reached 20 mm, due to animal welfare requirements. Tumor dosimetry values were calculated based on the following assumptions: for ¹⁷⁷ Lu-PSMA-617, the effective half-life in the tumor was 3 days, 10% of the injected dose per gram is degraded in the tumor, and 80% of the radiation from decay in the tumors is absorbed by the tumors, and the radiation energy is 0.15 MeV per decay. It was assumed that the effective half-life for ²¹² Pb was 10.6 hours, 10% of the administered dose per gram is degraded in the tumor, and 100% of the radiation in the tumor is absorbed by the tumor, the retention of ²¹² Pb and daughter decay products in tumors is 100%, and the radiation energy is 8 MeV per decay. Tumor dosimetry of the activity of the injected substances showed an average tumor absorbed dose of 35.9 Gy for ^{the 177} Lu-PSMA-617 treated group and a tumor absorbed dose of 2.06 Gy for the ²¹² Pb-p-SCN-Bn-TCMC treated group. -PSMA-ligand 1 group, respectively.

Results. At day 30 post-treatment, the following data showed survival of 0%, 12.5%, and 75% for saline, ¹⁷⁷ Lu-PSMA-617, and ²¹² Pb-p-SCN-Bn-TCMC-PSMA Ligand 1 groups respectively (figure 2). Median survival was 15 days, 20 days, and >30 days (not achieved) for saline, ¹⁷⁷ Lu-PSMA-617, and ²¹² Pb-p-SCN-Bn-TCMC-PSMA-ligand 1 groups, respectively.

In summary, the data indicate a strong delay in tumor growth with ²¹² Pb-p-SCN-Bn-TCMC-PSMA Ligand 1 compared with ¹⁷⁷ Lu-PSMA-617 even though radiation dose estimates indicate that the radiation dose in terms of Gy, which delivers ¹⁷⁷ Lu-PSMA-617, is 17 times higher. Thus, the radiobiological efficacy (RBE) ^{of 212} Pb-p-SCN-Bn-TCMC-PSMA Ligand 1 relative to ¹⁷⁷ Lu-PSMA-617 was at least 17. This high radiobiological efficacy for therapeutic levels of the alpha particle emitter was quite unexpected , since the RBE for alpha particle emitters relative to beta particle emitters is generally expected to be 2–5.

Links

An efficient chelator for complexation of thorium-227.

Ramdahl T, Bonge-Hansen HT, Ryan OB, Larsen S, Herstad G, Sandberg M, Bjerke RM, Grant D, Brevik EM, Cuthbertson AS

Bioorg Med Chem Lett. 2016 Sep 1; 26(17):4318-21. In Vitro and In Vivo Efficacy of a Novel CD33-Targeted Thorium-227 Conjugate for the Treatment of Acute Myeloid Leukemia.

Hagemann UB, Wickstroem K, Wang E, Shea AO, Sponheim K, Karlsson J, Bjerke RM, Ryan OB, Cuthbertson AS

Mol Cancer Ther. 2016 Oct; 15(10):2422-2431.

Chittenden SJ., A phase 1, open-label study of the biodistribution, pharmacokinetics, and dosimetry of 223Ra-dicloride in patinets with hormone-refractory prostate cancer and skeletal metastases. J Nucl Med 56: 1304-1309 (2015).

Kratochwil C et al., Targeted α-therapy of metastatic castration resistant prostate cancer with 225Ac-PSMA-617: Dosimetry estimate and dose empirical finding J Nucl Med 58: 1624-1631 (2017).

Kratochwil C et al., Targeted α-therapy of mCRPC: Dosimetry estimate of 213Bismuth-PSMA-617. Eur J Nucl Med Mol Imaging 45:31-37 (2018)

Huang SS, Heston WDW. Should Low Molecular Weight PSMA Targeted Ligands Get Bigger and Use Albumin Ligands for PSMA Targeting? Theranostics 7:(7) 1940-1941 (2017)

Choy CJ et al., 177 Lu-Labeled Phosphoramidate-Based PSMA Inhibitors: The Effect of an Albumin Binder on Biodistribution and Therapeutic Efficacy in Prostate Tumor-Bearing Mice. Theranostics. 2017;7(7):1928–1939. doi:10.7150/thno.18719.

Lassmann, M et al., Therapy of ankylosing spondylitis with 224Ra-radium chloride: Dosimetry and risk considerations. Radiat Environ Biophys 41: 173-178 (2002).

ITEMS

1. Compound X, wherein Compound X is a urea derivative suitable for targeting cells and tissues expressing PSMA.

2. A complex containing compound X associated with ²¹² Pb, ¹⁷⁷ Lu, ²¹³ Bi, ²²⁵ Ac or ²²⁷ Th, where compound X is a urea derivative suitable for targeting cells and tissues expressing PSMA.

3. A compound according to claim 1 or a complex according to claim 2, wherein compound X is linked to ²¹² Pb or ²²⁷ Th via a Z chelating moiety.

4. Connection or complex according to any one of paragraphs. 1-3, wherein the chelating moiety Z is selected from the group consisting of acyclic chelators, cyclic chelators, cryptands, crown ethers, porphyrins, or cyclic or non-cyclic polyphosphonates, DOTMP, EDTMP, bisphosphonate, DOTA, a DOTA derivative such as p-SCN- Bn-DOTA, DOTA conjugated pamidronate, TCMC, TCMC derivative such as p-SCN-Bn-TCMC, TCMC conjugated pamidronate, DOTA antibody conjugated, TCMC antibody conjugated, HBED-CC, NOTA, NODAGA , TRAP, NOPO, PCTA, DFO, DTPA, CHX-DTPA, AAZTA, DEDPA, and oxo-Do3A.

4. Connection or complex according to any one of paragraphs. 1-4, where the linker is DOTA or a derivative of DOTA.

5. Connection or complex according to any one of paragraphs. 1-4, where the linker is a DOTA derivative such as p-SCN-Bn-DOTA.

6. Connection or complex according to any one of paragraphs. 1-4, where the linker is TCMC or a derivative of TCMC.

7. Connection or complex according to any one of paragraphs. 1-4 or claim 6 wherein the linker is a TCMC derivative such as p-SCN-Bn-TCMC.

8. Connection or complex according to any one of paragraphs. 1-7, where the linker is an octadentate hydroxypyridinone containing ligand such as 3,2-HOPO.

9. Connection or complex according to any one of paragraphs. 1-8, where the compound X associated with the chelating moiety Z is defined by formula I:

formula I

or a pharmaceutically acceptable salt thereof,

Where

W is a PSMA-targeting ligand;

A ⁴ is a bond or divalent linking fragment containing 1-10 carbon atoms in the chain, ring, or combinations thereof, where at least one carbon atom is optionally replaced by O, -NR ³ - or -C(O)-;

G is C=O, C=S, C-NH ₂ or C-NR ³ ;

R ¹ represents hydrogen or a protecting group for a carboxylic acid group;

R ³ is selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl and heteroaryl;

each of R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ and R ¹⁶ independently represents hydrogen, alkyl, alkoxy, or

each of R ¹⁷ and R ¹⁸ independently represents hydrogen, alkyl, aryl or alkylaryl;

R ¹⁹ is selected from the group consisting of alkyl, alkoxy, halide, haloalkyl and

CN;

m is an integer from 1 to 6; And

o is an integer from 0 to 4, and if o is greater than 1, then each R ¹⁹ is the same or different.

10. Connection or complex according to any one of paragraphs. 1-9 or a pharmaceutically acceptable salt thereof, where

A ⁴ is a bond, (CH ₂ ) _n , -HC(O)-, -(OCH ₂ CH ₂ ) _n -, -(HCH ₂ CH ₂ ) _n -, -H(CO)CH ₂ -, -HC (O)CH ₂ (OCH ₂ CH ₂ ) _n - or -HC(O)CH ₂ (HCH ₂ CH ₂ ) _n -; And

L is a bond, (CH ₂ ) _n , -(OCH ₂ CH ₂ ) _n -, -(HCH ₂ CH ₂ ) _n - or -C(O)(CH ₂ ) _n -;

wherein n is independently 1, 2, or 3.

11. The compound or complex according to any one of paragraphs. 1-10 or a pharmaceutically acceptable salt thereof, where A ⁴ is a bond, -(OCH ₂ CH ₂ ) _n - or -HC(O)CH ₂ (OCH ₂ CH ₂ ) _n -; And

L is a bond or -(OCH ₂ CH ₂ ) _n -;

wherein n is independently 1 or 2.

12. The compound or complex according to any one of paragraphs. 1-11 or a pharmaceutically acceptable salt thereof, where

W is characterized by the structure:

where each of R ²⁰ and R ²¹ independently represents an amino acid residue associated through its amino group with the neighboring group -C(O)-.

13. Connection or complex according to any one of paragraphs. 1-12 or a pharmaceutically acceptable salt thereof, where

W is characterized by the structure:

where R is hydrogen or a protecting group for a carboxylic acid group.

14. The compound or complex according to any one of paragraphs. 1-13 or a pharmaceutically acceptable salt thereof, characterized by the structure:

or a pharmaceutically acceptable salt thereof,

where R ¹⁷ represents aryl.

15. Connection or complex according to any one of paragraphs. 1-14 or a pharmaceutically acceptable salt thereof, where the complex is PSMA-617:

however, a radionuclide such as ²¹² Pb can be bonded/chelated to four N.

16. The compound or complex of claim 15, where the DOTA link is replaced by a TCMC link.

17. Connection or complex according to paragraphs. 15-16 where DOTA is p-SCN-Bn-DOTA and TCMC is p-SCN-Bn-TCMC.

18. Connection or complex according to paragraphs. 15–17, where p-SCN-Bn-DOTA or p-SCN-Bn-TCMC are linked to the C-atom of the backbone of the urea derivative (PSMA).

19. Connection or complex according to paragraphs. 15-18 where the compound is C-linked p-SCN-Bn-DOTA or p-SCN-Bn-TCMC:

where Z is:

and where X represents -OH or NH ₂ .

20. Connection or complex according to paragraphs. 15-19 where the compound is C-linked p-SCN-Bn-DOTA or p-SCN-Bn-TCMC:

,

,

where X represents -OH or NH ₂ .

21. Connection or complex according to paragraphs. 15-20, where the compound is C-linked p-SCN-Bn-DOTA, i.e. p-SCN-Bn-DOTA-PSMA ligand 2:

.

.

22. Connection or complex according to paragraphs. 15-20, where the compound is C-linked p-SCN-Bn-TCMC, i.e. p-SCN-Bn-TCMC-PSMA ligand 1:

.

.

23. A PSMA-targeting urea derivative containing a TCMC moiety for ²¹² Pb chelation.

24. PSMA-targeting urea derivative containing HOPO for ²²⁷ Th chelation.

25. PSMA-targeting urea derivative containing DOTA labeled with either ²¹² Pb or ²²⁷ Th.

26. Pharmaceutical composition containing a compound or complex according to paragraphs. 1-21 and/or PSMA-targeting urea derivative according to paragraphs. 15-17 and diluent, carrier, surfactant and/or excipient.

27. Radiopharmaceutical composition according to claim 26, additionally containing ²²⁴ Ra.

28. Radiopharmaceutical composition according to any one of paragraphs. 26–27 where the radioactivity is between 100 kBq and 100 MBq per dose.

29. Radiopharmaceutical composition according to any one of paragraphs. 26–28, where the amounts of ²²⁴ Ra and ²¹² Pb are in radioactive equilibrium.

30. Radiopharmaceutical composition according to any one of paragraphs. 26-29, where the activity ratio (MBq) ^{of 212} Pb to ²²⁴ Ra is from 0.5 to 2, such as 0.8-1.5, or, for example, 0.8-1.3, or preferably, such as 0.9–1.15.

31. A set containing:

- the first bottle containing the radiopharmaceutical composition according to any one of paragraphs. 26–30, and

- a second vial containing a neutralizing solution to adjust the pH and/or isotonicity of the radiopharmaceutical composition prior to administration to the patient.

32. A set containing:

- the first vial containing a solution containing ²²⁴ Ra, ²¹² Pb and/or ²²⁷ Th;

- a second vial containing a complexing agent selected from the group consisting of acyclic chelators, cyclic chelators, cryptands, crown ethers, porphyrins or cyclic or non-cyclic polyphosphonates, DOTMP, EDTMP, bisphosphonate derivatives, DOTA derivative of DOTA, pamidronate conjugated to DOTA, TCMC, TCMC derivative, pamidronate conjugated to TCMC, DOTA conjugated to antibody, TCMC conjugated to antibody, HBED-CC, NOTA, NODAGA, TRAP, NOPO, PCTA, DFO, DTPA, CHX-DTPA, AAZTA, DEDPA and oxo -Do3A or compounds according to any one of paragraphs. 1–21

wherein the complexing agent is capable of complexing with a ²²⁴ Ra-derived daughter nuclide such as ²¹² Pb, and the complexing agent does not complex with ²²⁴ Ra in the pharmaceutical solution; And

- optionally instructions for mixing the contents of the first vial and the second vial, thereby obtaining a pharmaceutical composition ready for administration to the patient after a period of 1 minute to 12 hours after mixing.

33. Set according to any one of paragraphs. 31-32, where the kit is intended for use as a medicinal product.

34. Radiopharmaceutical composition according to any one of paragraphs. 18-22 for use as a drug.

35. Radiopharmaceutical composition according to any one of paragraphs. 18-22 for use in the treatment of skeletal disease.

36. A radiopharmaceutical composition for use according to claim 35, wherein the skeletal disease is selected from the group consisting of skeletal metastases in forms of breast, prostate, kidney, lung, bone, or multiple myeloma or non-cancer diseases causing unwanted calcification, including ankylosing spondylitis.

37. Radiopharmaceutical composition for use according to any one of paragraphs. 34-36, where the solution is administered at a dose in the range of 50-150 kBq per kg of body weight, such as 50-100 kBq per kg of body weight.

38. A method of treating a malignant or non-malignant disease by introducing a radiopharmaceutical composition described in paragraphs. 26-30 to the individual in need.

39. The method of obtaining a radiopharmaceutical composition according to any one of paragraphs. 26–30, including:

a) obtaining the first solution, where the amount of ²²⁴ Ra and ²¹² Pb is in radioactive equilibrium;

b) obtaining a second solution containing a complexing agent, which is selected from the group consisting of acyclic chelators, cyclic chelators, cryptands, crown ethers, porphyrins or cyclic or non-cyclic polyphosphonates, DOTMP, EDTMP, bisphosphonate, DOTA derivative of DOTA, pamidronate conjugated with DOTA, TCMC, TCMC derivative, pamidronate conjugated to TCMC, DOTA conjugated to antibody, TCMC conjugated to antibody, HBED-CC, NOTA, NODAGA, TRAP, NOPO, PCTA, DFO, DTPA, CHX-DTPA, AAZTA, DEDPA and oxo-Do3A, wherein the complexing agent is capable of complexing with a ²²⁴ Ra-derived daughter nuclide, such as ²¹² Pb, while the complexing agent does not complex with ²²⁴ Ra; And

c) mixing the first composition and the second composition, thereby obtaining a pharmaceutical composition according to any one of paragraphs. 26-30.

Claims (28)

1. A compound comprising a PSMA-directed unit and a chelating unit, wherein the PSMA-directed unit is bonded via a carbon backbone to the chelating unit, and where the compound is characterized by the formula

where X represents -NH ₂ or -OH. 2. The compound according to claim 1, where the compound is

p-SCN-Bn-TCMC-PSMA ligand 1. 3. The compound according to claim 1, where the compound is

p-SCN-Bn-DOTA-PSMA ligand 2. 4. Complex for use in radioligand therapy, containing a compound according to any one of paragraphs. 1-3 complexed with a radionuclide selected from the group consisting of ²¹² Pb, ²¹² Bi, ²¹³ Bi, ²²⁵ Ac or ²²⁷ Th. 5. The complex of claim 4, wherein the compound of claim 2 (p-SCN-Bn-TCMC-PSMA ligand 1) is complexed with ²¹² Pb. 6. The complex of claim 4, wherein the compound of claim 3 (p-SCN-Bn-DOTA-PSMA ligand 2) is complexed with ²¹² Pb, ²¹² Bi, ²¹³ Bi, ²²⁵ Ac or ²²⁷ Th. 7. Pharmaceutical composition for use in radioligand therapy against PSMA-expressing prostate cancer, containing a complex according to any one of paragraphs. 4-6 and a diluent, carrier, surfactant and/or excipient. 8. Pharmaceutical composition according to claim 7, additionally containing ²²⁴ Ra. 9. Pharmaceutical composition according to claim 8, wherein the amounts of ²²⁴ Ra and ²¹² Pb are in radioactive equilibrium. 10. Pharmaceutical composition according to any one of paragraphs. 7-9, where the ratio of activity (MBq) ²¹² Pb to ²²⁴ Ra is from 0.5 to 2, such as 0.8-1.5, or, for example, 0.8-1.3, or preferably, as for example, 0.9-1.15. 11. Pharmaceutical composition according to any one of paragraphs. 7-10, where the composition is administered at a dose providing radioactivity from 100 kBq to 100 MBq per dose. 12. The use of a pharmaceutical composition according to any one of paragraphs. 7-11 as a drug for use in radioligand therapy against PSMA-expressing prostate cancer. 13. The use of a pharmaceutical composition according to any one of paragraphs. 7-11 in the treatment of PSMA-expressing disease, including soft tissue and skeletal disease. 14. Use according to claim 13 wherein the skeletal disease is selected from the group consisting of skeletal metastases in forms of breast, prostate, kidney, lung, bone or multiple myeloma cancer. 15. Application according to any one of paragraphs. 12-14 where the solution is administered at a dose in the range of 50-150 kBq/kg body weight. 16. A method of treating a disease characterized by the expression of PSMA, by introducing a pharmaceutical composition according to any one of paragraphs. 7-11 to the individual in need. 17. A set containing: - the first vial containing the pharmaceutical composition according to any one of paragraphs. 7-11, and - a second vial containing a neutralizing solution to adjust the pH and/or isotonicity of the pharmaceutical composition prior to administration to the patient. 18. A set for obtaining a complex, containing: - the first vial containing a solution containing ²²⁴ Ra, ²¹² Pb and/or ²²⁷ Th; - a second vial containing a compound according to any one of paragraphs. 1-3.

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