EA046784B1 - TOOLS FOR DIAGNOSTIC IMAGING OF CANCER - Google Patents

Description

The present invention relates to a ligand-GEA-chelator conjugate containing in one molecule: (a) a ligand capable of binding to PSMA, (b) a silicofluoride acceptor moiety (SIFA) containing a covalent bond between silicon and a fluorine atom labeled F, and ( c) a chelating group containing a chelated non-radioactive cation.

Documents cited in this application include patent applications and manufacturers' manuals. The descriptions of these documents are incorporated into this application by reference in their entirety, but do not prejudice the patentability of the present invention. More specifically, all documents referred to herein are incorporated herein by reference to the same extent as if each individual document were specifically and individually identified as being incorporated by reference.

Prostate cancer.

Prostate cancer (PCa) has remained the most common malignancy in men over the past decades, with low survival rates. Prostate specific membrane antigen (PSMA) or glutamate carboxypeptidase II (GCP II), due to its overexpression in prostate cancer, has proven to be an excellent target for the development of highly sensitive radiolabeled agents for endoradiotherapy and PCa imaging. Prostate-specific membrane antigen is an extracellular hydrolase whose catalytic center contains two zinc(II) ions bridged by a hydroxy ligand. It is highly upregulated in metastatic and hormone-resistant prostate carcinomas, but has also been reported to be physiologically expressed in the kidney, salivary glands, small intestine, brain and, to a lesser extent, also in healthy prostate tissue. In the intestine, PSMA facilitates the absorption of folate by converting pteroylpoly-γglutamate to pteroylglutamate (folate). In the brain, it hydrolyzes Na-acetyl-L-aspartyl-L-glutamate (NAAG) to Na-acetyl-L-aspartate and glutamate.

Prostate specific membrane antigen (PSMA).

Prostate-specific membrane antigen (PSMA) is a type II transmembrane glycoprotein overexpressed on prostate cancer epithelial cells. Despite the name, PSMA expression is also found to varying degrees in the neovascular network of a large number of cancer types outside of prostate cancer. The most common types of non-prostate cancers that demonstrate PSMA expression include breast, lung, colorectal, and renal cell carcinoma.

The general required structures of PSMA-targeting molecules include a binding group containing a zinc-binding group (such as urea, phosphinate or phosphoramidate) linked to a glutamate ^P1 ' moiety, which ensures high affinity and specificity for PSMA and is usually additionally linked to an effector functionality. The effector part is more flexible and to some extent tolerant of structural modifications. The entry funnel contains two other structures important for ligand binding. The first is the arginine epitope, a positively charged region on the wall of the inlet funnel, and preferentially negatively charged functional groups at the P1 position of PSMA based on a mechanistic explanation. This appears to be the reason for the preferential incorporation of negatively charged residues into the scaffold ligand. To our knowledge, an in-depth analysis of the effect of positive charges on PSMA ligands has not yet been carried out. Following binding, the concerted repositioning of arginine side chains can result in the opening of the hydrophobic accessory pocket S1, a second important structure that has been shown to accommodate the iodine-benzyl group of several urea-based inhibitors, contributing to their high affinity for PSMA.

Zhang et al. discovered a remote PSMA binding site that can be used for the bidentate binding mode (Zhang et al., Journal of the American Chemical Society 132, 12711-12716 (2010)). The so-called arene binding site is a simple structural motif formed by the side chains of Arg463, Arg511 and Trp541 and is part of the GCPII entry cap. Engagement of the arene binding site by the distal fragment of the inhibitor can lead to a significant increase in the affinity of the inhibitor for PSMA due to avidity effects. Thus, PSMA I&T was designed to interact with PSMA, although crystal structure analysis of the binding mode is not available. The required structure according to Zhang et al. is a linker group (suberic acid in the case of PSMA I&T) that promotes the open conformation of the GCPII entrance cap and thereby allows accessibility of the arene binding site. In addition, the structural composition of the linker has been shown to have a significant impact on tumor targeting and biological activity, as well as imaging contrast and pharmacokinetics, properties critical to both high-quality imaging and effective targeted endoradiotherapy.

There are currently two categories of PSMA-targeting inhibitors used in clinical settings. On the one hand, there are labeled substances with chelating groups for complexation of radionuclides, for example, PSMA I&T or related compounds. On the other hand, there are small molecules that include targeting group and effector molecules.

Introduction of the ¹⁸ F mark.

Recently, several groups have focused on the development of novel urea-based inhibitors labeled with ¹⁸ F for the diagnosis of PCa. The ¹⁸ F-labeled urea-based PSMA inhibitor ¹⁸ F-DCFPyl has shown promising results in the detection of primary and metastatic diseases. Based on the structure of PSMA-617, an ¹⁸ F-labeled analogue PSMA1007 was recently developed, showing a comparable tumor/organ ratio.

An attractive approach to introducing ^18F tags is the use of silicofluoride acceptors (SIFAs). Silicofluoride acceptors are described, for example, in Lindner et al., Bioconjugate Chemistry 25, 738-749 (2014). To maintain the silicofluoride bond, the use of silicofluoride acceptors necessitates the creation of sterically complex groups around the silicon atom. This, in turn, makes the silicofluoride acceptors highly hydrophobic. With respect to binding to a target molecule, in particular a PSMA target molecule, the hydrophobic moiety provided by the silicofluoride acceptor can be used to interact a radiodiagnostic or therapeutic compound with the hydrophobic pocket, as described in Zhang et al., Journal of the American Chemical Society 132, 12711-12716 (2010). However, prior to binding, the higher degree of lipophilicity introduced into the molecule poses a major challenge for the development of radiopharmaceuticals with suitable in vivo biodistribution, i.e., low nonspecific binding to non-target tissue.

Failure to solve hydrophobicity problem.

Despite numerous attempts, the problem of hydrophobicity caused by silicofluoride acceptors has not been satisfactorily solved in the prior art.

For example, Schirrmacher E. et al. (Bioconjugate Chem. 2007, 18, 2085-2089) synthesized various ¹⁸ G-labeled peptides using the highly efficient labeled synthon p-(di-tert-butylfluorosilyl)benzaldehyde ([ ¹⁸ F]SIFA-A), which is one example of a silicofluoride acceptor. The SIFA method resulted in unexpectedly efficient ^19F - ^18F isotopic exchange yielding ^{the 18F} synthon in near quantitative yield with high specific activity ranging from 225 to 680 GBq/μmol (6081-18378 Ci/mmol) without the use of HPLC purification. Finally, [ ¹⁸ F]SIFA-benzaldehyde was used to label the N-terminal aminooxy (N-AO) derivatized peptides AO-TyroZoctreotate (AO-TATE), cyclo(fK(AO-N)RGD) and N-AO-PEG ₂ -[D-Tyr-Gln-Trp-Ala-Val-Ala-His-Thi-Nle-NH ₂ ] (AO-BZH3, bombesin derivative) with high radiochemical yields. However, labeled peptides are highly lipophilic (as determined by HPLC retention time using the conditions described in this publication) and are therefore not suitable for further evaluation in animal models or humans.

In the publication Wangler S. et al. (Bioconjugate Chem., 2009, 20 (2), pp. 317-321) describes the first Kit-like radiofluorination of a protein (rat serum albumin, RSA) based on SIFA. As a labeling agent, 4-(di-tert-butyl[ ^18F ]fluorosilyl)benzenethiol (Si[ ^18F ]FA-SH) was prepared by simple isotope exchange with a radiochemical yield of 40 to 60% (RCY), and the product was coupled directly to maleimide-derivatized serum albumin with total RCY 12% for 20 to 30 min. The technically simple labeling procedure does not require any complex purification procedures and is a direct example of the successful application of Si- ^18F chemistry for in vivo PET imaging. Activity-time curves and pPET images of mice showed that most of the active substance was localized in the liver, thereby demonstrating that the labeling agent is too lipophilic and targets the in vivo probe for hepatobiliary excretion and extensive hepatic metabolism.

Wangler S. et al. (see Bioconjug Chem. 2010 Dec 15, 21(12):2289-96) subsequently tried to overcome the main disadvantage of the SIFA method, the high lipophilicity of the resulting radiopharmaceuticals, by synthesizing and evaluating new analogues of SIFA-octreotate (SIFA-Tyr3-octreotate, SIFA- Asn(AcNH-e-Glc)Tyr3-octreotate and SIFA-Asn(AcNH-β-Glc)-PEG-Tyr3-octreotate). In these compounds, hydrophilic linkers and pharmacokinetic modifiers, ie carbohydrate and PEG linker plus carbohydrate, were introduced between the peptide and the SIFA moiety. As a measure of the lipophilicity of the conjugates, the log P(ow) value was determined, which was 0.96 for SIFA-Asn(AcNH-β-Glc)-PEG-Tyr3-octreotate and 1.23 for SIFA-Asn(AcNH-β-Glc) -Tyr3-octreotate. These results indicate that the high lipophilicity of the SIFA moiety can only be partially compensated for by the use of hydrophilic moieties. The first imaging study demonstrated excessive hepatic clearance/uptake by the liver and thus was not carried over to the first human study.

Bernard-Gauthier et al. (Biomed Res Int.2014, 2014: 454503) a large number of different types of SIFA have been reported in the literature, ranging from small prosthetic groups and other low molecular weight compounds to labeled peptides and more recently described affibody molecules. Based on these data, the problem of lipophilicity of prosthetic groups on

- 2 046784 The basis of SIFA has not yet been resolved, that is, a method for reducing the overall lipophilicity of a SIFA-conjugated peptide to a log D below approximately -2.0 has not been described.

In the publication Lindner S. et al. (Bioconjug Chem. 2014 Apr 16, 25 (4): 738-49) describe pegylated bombesin derivatives (PESIN) as specific GRP receptor ligands and RGD peptides (single letter codes for arginine-glycine-aspartic acid) as specific binders ave3 substances were prepared and labeled with a silicofluoride acceptor moiety (SIFA). To compensate for the high lipophilicity of the SIFA fragment, various modifications to the hydrophilic structure were introduced, resulting in lower logD values.

SIFA-Asn(AcNH-3-Glc)-PESIN, SIFASer(3-Lac)-PESIN, SIFA-Cya-PESIN, SIFA-LysMe3-PESIN, SIFA-y-KapOoKCH-d-Glu-PESIN,

S IF A-Cya2-PESIN, SIFA-LysMe3-Y-Kap6oKCH-d-Glu-PESIN, SIFA-(Y-Kap6oKCH-d-Glu)2PESIN, SIFA-RGD, SIFA-Y- _K ap6oKCH-d-Glu- RGD, SIFA-(Y _- Kap6oKCH-d-Glu)2-RGD, SIFALysMe3-Y-Kap6oKcn-d-Glu-RGD.

All of these peptides, already improved and modified to reduce lipophilicity, showed logD values ranging from +2 to -1.22.

In view of the above, the technical problem underlying the present invention relates to the provision of radiodiagnostic agents containing a silicofluoride scavenger and at the same time characterized by favorable properties in vivo.

As will be apparent from the following, the present invention provides a test and proof of principle for the use of specific conjugates that bind with high affinity to prostate specific membrane antigen (PSMA) as a target. Accordingly, a further technical problem underlying the present invention is to provide improved diagnostics for the medical indication of cancer, preferably prostate cancer.

The object of the present invention solves these technical problems. Accordingly, in a first aspect, the present invention provides a ligand-SIFA-chelator conjugate having the formula

where M ³⁺ is a chelated non-radioactive cation containing in one molecule: (a) a ligand capable of binding to PSMA, (b) a silicofluoride acceptor fragment (SIFA) containing a covalent bond between a silicon atom and a fluorine atom, and which is labeled ¹⁸ F , and (c) a chelating group containing a chelated non-radioactive cation.

The ligand-SIFA-chelator conjugate contains three separate moieties. The three separate moieties are: (a) a ligand capable of binding to PSMA, (b) a silicofluoride acceptor moiety (SIFA) containing a covalent bond between silicon and a fluorine atom, and (c) a chelating group containing a chelated non-radioactive cation.

For diagnostic imaging, the fluorine atom in the SIFA fragment is ¹⁸ F. ¹⁸ F can be introduced by isotopic exchange with ¹⁹ F.

Although certain ligands capable of binding to a disease-related target molecule may be cyclic peptides, such cyclic peptides are not chelating groups as contemplated herein, since the problem of the hydrophobic moiety of SIFA is not addressed in the absence of an additional chelating moiety. Thus, the compounds of the present invention require a hydrophilic chelating group in addition to a ligand that is capable of binding to PSMA. A hydrophilic chelating group is required to reduce the hydrophobic nature of the compounds caused by the presence of the SIFA moiety.

The ligand in relation to the first aspect of the present invention is defined in functional terms. This is so because the present invention is not structurally dependent on the specific nature of the ligand. Rather, the key aspect of the present invention is the combination within a single molecule of a silicofluoride acceptor and a chelator or chelate. These two structural elements

- 3 046784 ment, SIFA and chelator, exhibit spatial proximity. Preferably, the shortest distance between two atoms of two elements is less than or equal to 25 A, more preferably less than 20 A, and even more preferably less than 15 A. Alternatively or additionally, it is preferred that no more than 25 covalent bonds separate a SIFA moiety atom from a chelator atom, preferably no more 20 chemical bonds and even more preferably no more than 15 chemical bonds.

The cation according to paragraph (c) of the first aspect is a non-radioactive cation. Preferably a non-radioactive metal cation. Examples are given below.

We have surprisingly discovered that placing a silicofluoride acceptor in proximity to a hydrophilic chelator, such as, but not limited to, DOTAGA or DOTA, screens or effectively compensates for the lipophilicity of the SIFA moiety to the extent that it shifts the overall hydrophobicity of the compound to a range where the compound suitable for in vivo administration.

An additional advantage of the PSMA-targeting compounds of the present invention is their unexpectedly low accumulation in mouse kidneys compared to other PSMA-targeting radiopharmaceuticals such as PSMA I&T. Without wishing to be bound by any particular theory, it is the combination of the SIFA building block with a chelator that provides a surprising reduction in renal accumulation.

With regard to lipophilicity/hydrophilicity, the logP value (sometimes also called the logD value) is a recognized measure in the art.

The term lipophilicity refers to the ability to dissolve or be absorbed in lipid solutions, or adsorb to a lipid-like surface or matrix. This means a preference for lipids (literal meaning) or organic or non-polar liquids, or liquids, solutions or surfaces with a small dipole moment compared to water. The term hydrophobicity is used herein in an equivalent meaning. The adjectives lipophilic and hydrophobic are used in the same sense as the nouns described above.

The mass flux of a molecule at the interface between two immiscible or essentially immiscible solvents is determined by its lipophilicity. The more lipophilic a molecule, the more soluble it is in the lipophilic organic phase.

The partition coefficient of the molecule, which is observed between water and n-octanol, has been adopted as a standard measure of lipophilicity. The distribution coefficient P of type A is determined as the ratio P = [A] _n-octanol / [A] _water . Typically the value given is logP, which is the logarithm of the distribution coefficient. In case the molecule is ionizable, many different microspecies (ionized and non-ionized forms of the molecule) will in principle be present in both phases. The value describing the overall lipophilicity of the ionizable species is the distribution coefficient D, defined as the ratio D = [sum of concentrations of all microspecies] _n-octanol / [sum of concentrations of all microspecies] _water . As with logP, the logarithm of the distribution coefficient, logD, is often reported. Often a buffer system, such as phosphate-buffered saline, is used as an alternative to water in the above-described logP determination.

If the lipophilic nature of a substituent on a first molecule is to be assessed and/or quantified, then a second molecule corresponding to such substituent can be assessed, where said second molecule is obtained, for example, by breaking the bond connecting said substituent to the remainder of the first molecule and connecting the free(s) valence(s) thus obtained with hydrogen(s).

Alternatively, the contribution of the substituent to the logP of the molecule can be determined. The contribution of ^{the πχ x} substituent X to the logP of the RX molecule is determined as πχχ = logPR-χ - logPR-н, where RH represents the unsubstituted parent compound.

P and D values greater than one, and logP, logD, and πχ x values greater than zero indicate lipophilic/hydrophobic character, while P and D values less than one, and logP, logD, and ^{πχ χ} values less than zero indicate hydrophilic character corresponding molecules or substituents.

The above-described parameters characterizing the lipophilicity of the lipophilic group or the entire molecule of the present invention can be determined by experimental means and/or predicted by computational methods known in the art (see, for example, Sangster, Octanol-water Partition Coefficients: fundamentals and physical chemistry, John Wiley & Sons, Chichester (1997)).

In a preferred embodiment, the logP value of the compounds of the present invention is from -5 to -1.5. Particularly preferably, the logP value is between -3.5 and -2.0.

The ligand of the present invention contains amino acids and binds with high affinity to prostate specific membrane antigen (PSMA).

According to the present invention, the silicofluoride acceptor moiety (SIFA) has the structure

- 4 046784 represented by formula (Ia)

where t-Bu represents a tert-butyl group, and F represents both ¹⁹ F and ¹⁸ F.

According to the present invention, the chelating group is the residue of the chelating agent, which is 1,4,7,10-tetracyclododecane-H\NO,NO',NO-tetraacetic acid (DOTA), the remainder of which is provided by covalent binding of the carboxyl group contained in the chelating agent , with the rest of the conjugate through an ester or amide bond.

More specifically, the chelator is presented below.

Metal chelating or cationic macrocyclic compounds are well known in the art and are available from a number of manufacturers.

According to the present invention, the chelating group contains a chelated cation that is not radioactive.

Preferable examples of cations that can be chelated by the chelating group are the non-radioactive cations Sc, Cr, Mn, Co, Fe, Ni, Cu, Ga, Zr, Y, Tc, Ru, Rh, Pd, Ag, In, Sn, Te , Pr, Pm, Tb, Sm, Gd, Tb, Ho, Dy, Er, Yb, Tm, Lu, Re, Pt, Hg, Au, Pb At, Bi, Ra, Ac, Th, more preferably Sc, Cu cations , Ga, Y, In, Tb, Ho, Lu, Re, Pb, Bi, Ac, Th and Er. The cation may be Ga. The cation may be Lu.

According to the present invention, the ligand is capable of binding to prostate specific membrane antigen (PSMA) and has the structure represented by formula (IIa)

where n is 2, and where the ligand is attached to the rest of the conjugate through a bond designated λλλλ/vw.

For use in PET imaging, the compounds of the present invention require a positron active atom. Connections include ¹⁸ F for medical use. The most preferred compounds of the present invention are those wherein F includes ¹⁸ F and M ³⁺ refers to a non-radioactive metal cation.

In the compounds of the present invention, the acid groups with which the non-radioactive metal cation M is chelated are designated COO ^- .

The conjugate according to the second aspect of the present invention is

Preferred labeling schemes for the specified compound are disclosed herein.

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In a further aspect, the present invention relates to a pharmaceutical imaging composition containing one or more of the conjugates of the present invention disclosed hereinabove.

In an additional aspect, the present invention relates to a diagnostic composition containing one or more of the conjugates of the present invention disclosed hereinabove.

The pharmaceutical composition may further contain pharmaceutically acceptable carriers, excipients and/or diluents. Examples of suitable pharmaceutical carriers, excipients and/or diluents are well known in the art and include phosphate buffered saline, water, emulsions such as oil/water emulsions, various types of wetting agents, sterile solutions, etc. Compositions containing such carriers can be prepared by well-known conventional methods. Such pharmaceutical compositions can be administered to a subject at a suitable dose. Administration of suitable compositions can be accomplished in a variety of ways, for example, by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. Particularly preferably, said administration is carried out by injection and/or delivery, for example, to the pancreas, or to a cerebral artery, or directly to brain tissue. The compositions can also be administered directly to the target site, for example, by biolistic delivery to an external or internal target site, for example, in the pancreas or brain. The dosage regimen is determined by the attending physician and clinical factors. As is well known in the medical field, dosages for any patient depend on many factors, including patient parameters, body surface area, age, the specific compound administered, sex, time and route of administration, general health, and consideration of other drugs administered concomitantly. The amount of pharmaceutically active substance may be from 0.1 ng to 10 mg/kg body weight per dose, however, doses lower or higher than this exemplary range are contemplated, especially taking into account the above factors.

In a further aspect, the present invention relates to one or more of the compounds of the present invention disclosed hereinabove for use in diagnostic medicine.

A preferred medical application is a nuclear medicine application, for example nuclear diagnostic imaging, also referred to as nuclear molecular imaging, and/or targeted radiation therapy for diseases associated with overexpression of, preferably, PSMA on diseased tissue.

In a further aspect, the present invention provides a compound of the present invention disclosed hereinabove for use in a method for diagnosing and/or staging a cancer, preferably prostate cancer. Prostate cancer is not the only cancer in which PSMA expression is observed.

Non-prostate cancers in which PSMA expression is observed include breast, lung, colorectal and renal cell carcinoma. Thus, any compound disclosed herein containing a PSMA binding moiety can be used in the diagnosis, imaging, or treatment of cancer characterized by PSMA expression.

Preferred indications are the detection or staging of cancer, such as, but not limited to, high-grade glioma, lung cancer, and especially prostate cancer and metastatic prostate cancer, detection of metastatic disease in patients with primary prostate cancer from intermediate risk to high risk, and detection of metastatic sites even at low serum PSA values in patients with biochemically recurrent prostate cancer. Another preferred indication is imaging and imaging of neoangiogens.

In a further aspect, the present invention provides a conjugate or compound of the present invention disclosed hereinabove for use in a method for diagnosing and/or staging a cancer, preferably prostate cancer.

Brief description of graphic materials

Fig. 1: Approximate ratio of determination of nine reference substances in OriginPro 2016G.

Fig. 2a: quality control [ ¹⁹ F][ ^nat Ga]-rhPSMA7-rac ([ ¹⁹ F][ ^nat Ga]D/L-Dap-R/S-DOTAGA-rhPSMA-7rac), (batch 10, precursor for ( [ ¹⁸ F][ ^nat Ga]D/L-Dap-R/S-DOTAGA-rhPSMA-7 at the Department of Nuclear Medicine, Technical University of Munich. HPLC conditions: solvent A: H ₂ O + 0.1% TFA (trifluoroacetic). acid), solvent B: MeCN + 0.1% TFA. Gradient: from 25 to 35% B from 0 to 40 min, from 95 to 95% B from 40 to 45 min, from 35 to 35% B from 45 to 50 min, flow: 1 ml/min, column: Nucleosil 100-5 C ¹⁸ , 125x4.6 mm, sample: 1 mmol/l (DMSO (dimethyl sulfoxide)), 10 µl.

Fig. 2b: Peak assignment: D-Dap-R-DOTAGA-rhPSMA-7.1 rhPSM7-rac from FIG. 2a co-injected with enantiomerically pure D-Dap-R-DOTAGA-rhPSMA-7.1. HPLC conditions: solvent A:

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H2O+0.1% TFA, solvent B: MeCN+0.1% TFA. Gradient: 25 to 35% B from 0 to 40 min, 95 to 95% B from 40 to 45 min, 35 to 35% B from 45 to 50 min, flow: 1 ml/min, column: Nucleosil 100- 5 C ¹⁸ , 125x4.6 mm, sample: 1 mmol/l (DMSO), 10 µl.

Fig. 2c: HPLC profile of D-Dap-R-DOTAGA-rhPSMA-7.1. HPLC conditions: solvent A: H ₂ O + 0.1% TFA, solvent B: MeCN + 0.1% TFA. Gradient: 25 to 35% B from 0 to 40 min, 95 to 95% B from 40 to 45 min, 35 to 35% B from 45 to 50 min, flow: 1 ml/min, column: Nucleosil 100- 5 C ¹⁸ , 125x4.6 mm, sample: 1 mmol/l (DMSO), 10 µl.

Fig. 3a: Peak assignment: L-Dap-R-DOTAGA-rhPSMA-7.2 rhPSM7-rac from Fig. 2a co-injected with enantiomerically pure L-Dap-R-DOTAGA-rhPSMA-7.2. HPLC conditions: solvent A: H ₂ O + 0.1% TFA, solvent B: MeCN + 0.1% TFA. Gradient: 25 to 35% B from 0 to 40 min, 95 to 95% B from 40 to 45 min, 35 to 35% B from 45 to 50 min, flow: 1 ml/min, column: Nucleosil 100- 5 C ¹⁸ , 125x4.6 mm, sample: 1 mmol/l (DMSO), 10 µl.

Fig. 3b: HPLC profile L-Dap-R-DOTAGA-rhPSMA-7.2 HPLC conditions: solvent A: H ₂ O + 0.1% TFA, solvent B: MeCN + 0.1% TFA. Gradient: 25 to 35% B from 0 to 40 min, 95 to 95% B from 40 to 45 min, 35 to 35% B from 45 to 50 min, flow: 1 ml/min, column: Nucleosil 100- 5 C ¹⁸ , 125x4.6 mm, sample: 1 mmol/l (DMSO), 10 µl.

Fig. 4a: Peak assignment: D-Dap-S-DOTAGA-rhPSMA-7.3 rhPSM7-rac from Fig. 2a co-injected with enantiomerically pure D-Dap-S-DOTAGA-rhPSMA-7.3. HPLC conditions: solvent A: H ₂ O + 0.1% TFA, Solvent B: MeCN + 0.1% TFA. Gradient: 25 to 35% B from 0 to 40 min, 95 to 95% B from 40 to 45 min, 35 to 35% B from 45 to 50 min, flow: 1 ml/min, column: Nucleosil 100- 5 C ¹⁸ , 125x4.6 mm, sample: 1 mmol/l (DMSO), 10 µl.

Fig. 4b: HPLC profile D-Dap-D-DOTAGA-rhPSMA-7.3 HPLC conditions: solvent A: H ₂ O + 0.1% TFA, solvent B: MeCN + 0.1% TFA. Gradient: 25 to 35% B from 0 to 40 min, 95 to 95% B from 40 to 45 min, 35 to 35% B from 45 to 50 min, flow: 1 ml/min, column: Nucleosil 100- 5 C ¹⁸ , 125x4.6 mm, sample: 1 mmol/l (DMSO), 10 µl.

Fig. 5a: Peak assignment: L-Dap-S-DOTAGA-rhPSMA-7.4 rhPSM7-rac from Fig. 2a co-injected with enantiomerically pure D-Dap-S-DOTAGA-rhPSMA-7.3. HPLC conditions: solvent A: H ₂ O + 0.1% TFA, solvent B: MeCN + 0.1% TFA. Gradient: 25 to 35% B from 0 to 40 min, 95 to 95% B from 40 to 45 min, 35 to 35% B from 45 to 50 min, flow: 1 ml/min, column: Nucleosil 100- 5 C ¹⁸ , 125x4.6 mm, sample: 1 mmol/l (DMSO), 10 µl.

Fig. 5b: HPLC profile of L-Dap-D-DOTAGA-rhPSMA-7.4. HPLC conditions: solvent A: H ₂ O + 0.1% TFA, solvent B: MeCN + 0.1% TFA. Gradient: 25 to 35% B from 0 to 40 min, 95 to 95% B from 40 to 45 min, 35 to 35% B from 45 to 50 min, flow: 1 ml/min, column: Nucleosil 100- 5 C ¹⁸ , 125x4.6 mm, sample: 1 mmol/l (DMSO), 10 µl.

Fig. 6a: binding affinity (IC ₅₀ [nmol/l]) of rhPSMA7.1 and 7.2 to PSMA. Affinity was determined using LNCaP cells (150,000 cells/well) and ((4-[ ^125I ]iodobenzoyl)KuE ([ ^125I ]IB-KuE, c=0.2 nmol/L) as radioligand (1 h, 4 °C, HBSS+1% BSA) Data are expressed as mean ± standard deviation (SD) (n=3, from 3 different experiments).

Fig. 6b: determination of binding affinity [nmol/l] of rhPSMA7 isomers to PSMA. Each of the four columns shows individual affinity measurements for rhPSAM7.1 (left) to rhPSMA7.4 (right). The conditions are as described for FIG. 6a.

Fig. 7: Illustration of individual IC ₅₀ [nmol/l] measurements shown in FIG. 6a and 6b. Value number 5 rhPSMA7.1 has been removed. The conditions are as described for FIG. 6a.

Fig. 8: Illustration of individual internalization measurements [% of [ ¹²⁵ I]IB-KuE]. Internalized active substance (c=0.5 nmol/l) after 1 h as a % of reference ligand ([ ¹²⁵ I]I-BA)KuE (c=0.2 nmol/l) determined on LNCaP cells (37°C , DMEM F12+5% BSA, 125,000 cells/well). Data are corrected for nonspecific binding (10 μmol PMPA) and expressed as mean ± standard deviation (n=3).

Fig. 9: Illustration of individual logP measurements of rhPSMA isomers.

Fig. 10: Biodistribution (%ID/g) of ^18F -labeled rhPSMA at 1 h pi in SCID mice bearing LNCaP tumor. Data are expressed as mean ± standard deviation (n=4 for rhPSMA7.1, n=5 for 7.2, n=4 for 7.3, n=5 for 7.4 and n=3 for 7-rac).

Fig. 11: Biodistribution [% ID/g] ^{of 18} F-rhPSMA co-injected with PMPA (8 mg/kg) 1 h post-injection in SCID mice bearing LNCaP tumor. Data are expressed as mean ± standard deviation (n=3).

Fig. 12: possible species formed by metabolic cleavage of amide bonds. iL: when cleaved, a species with increased lipophilicity is formed, DF: defluorination, nd: not detected, since it is not radioactive.

Fig. 13: left: graphical analysis of overlapping peaks 1 (rhPSMA7.2) and 2 (rhPSMA7.3), right: deconvolution and integration of peak profiles using Systat PeakFit Software), top: aligned experimental data, bottom: individual peaks after deconvolution.

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Fig. 14a: Quantification of relative changes (% change in racemic mixture introduced) to assess the reproducibility of classical integration (using HPLC software) and deconvolution (using PeakFit) of peak 4 (rhPSMA7.1). Both methods show the same performance for this peak.

Fig. 14b: Quantification of relative changes (% change in racemic mixture introduced) to evaluate the reproducibility of classical integration (using HPLC software) and deconvolution (using PeakFit) of peak 3 (rhPSMA7.4). Both methods show the same performance for this peak.

Fig. 15: percentage change of each rhPSAM7.1-7.4 isomer in blood, liver, kidney, tumor and urine relative to its proportion in the administered solution ([ ¹⁸ F][ ^nat Ga]rhPSMA7-rac. Data are expressed as means ± standard deviation (n=4, see also Fig. 16).

Fig. 16: Percentage change of each rhPSAM7.1-7.4 isomer for each sample and experiment) in blood, liver, kidney, tumor and urine relative to its proportion in the administered solution ([ ¹⁸ F][natGa] rhPSMA7-rac). Analyzes were performed using Systat PeakFit.

Fig. 17: left: TLC scanner profile of a TLC plate with a liver sample (07/30/2018, total number of counts: 142 counts). Due to their poor statistics and limited reliability, data series with imp less than 200 were removed. Right: Photo image of a TLC plate with a liver sample: the long tail of the moving marker.

Fig. 18: left: TLC scanner profile of a TLC plate with a quality control (QC) sample (08/01/2018, total number of counts: 384). Right: A typical photographic image of a TLC plate containing urine, kidney, liver, tumor, blood, and QC samples.

Fig. 19: radio-TLC [F-18] rhPSMA7-rac (07/30/20 ¹⁸ ) as part of quality control in the nuclear medicine department before clinical use of the tag. Note that a trace of the labeled substance is observed even in the formulation buffer (and therefore in the absence of proteins).

Fig. 20: Quantification of free p-18]fluoride and intact [F-18]rhPSMA7-rac using radio-TLC of a urine sample (07/30/2018).

Fig. 21: Left: Radio-HPLC analysis of urine collected and pooled from 4 normal mice injected with the corresponding [F-18]rhPSAM-7.x tag. Right: radio-HPLC analysis of cold urine spiked with appropriate label [F-18] rhPSAM-7.x for 1 h (7.1, 7.2), 0.5 h (7.3) and 2 h (7.4). HPLC conditions: solvent A: H ₂ O + 0.1% TFA, solvent B: MeCN + 0.1% TFA, Gradient: 5% isocratic from 0 to 3 min, from 25 to 35% B from 3 to 43 min, from 95 to 95% B from 43 to 48 min, flow: 1 ml/min, column: Nucleosil 100-5 C18, 125x4.6 mm.

Fig. 22: separation of radioactive substances in urine by cartridge fixation and TLC. Top: Radio-HPLC urine analysis 30 min after [F-18]rhPSMA7.3 injection in mice showing a small fraction at 1.6 min and an intact label at approximately 34.5 min. Bottom (Left): Mouse urine 30 min after [F-18]rhPSMA7.3 injection was diluted and fixed with a STRATA-X cartridge. The cartridge was washed and eluted with MeCN/water (60/40 v/v + 1% TFA), and only intact label was detected. Bottom (right): Both the cartridge fixation breakthrough (components not retained) and the fraction finally eluted with MeCN/water from the cartridge were analyzed by TLC (bottom, right). While 96.1% [F-18]rhPSMA7.3 and only 3.9% [F18]fluoride were found in the cartridge eluate, the opposite ratio was found in the cartridge breakthrough (3.4% [F-18]rhPSMA7 .3 and only 96.6% p-18]fluoride).

Fig. 23: [F-18]rhPSMA7.3 was added to fresh and non-radioactive mouse urine, followed by 0.5 µmol of cold F-19-fluoride, incubated for 2 hours. The radioactive substance was completely (98.5%) converted into very hydrophilic fraction, representing p-18]fluoride (peak at 1.6 min). Note: The peak at 1.6 min was subsequently immobilized on a QMA cartridge and eluted with NaCl (1M) (=fluoride).

Fig. 24: Clinical biodistribution and accumulation in tumor lesions of ¹⁸ F-rhPSMA-7 (left) and ¹⁸ F-rhPSMA-7.3 (right), as demonstrated by SUV,,,^ (standardized uptake index). Data are expressed as mean ± standard deviation.

Fig. 25: Clinical biodistribution and accumulation in tumor lesions of ¹⁸ F-rhPSMA-7 (left) and ¹⁸ F-rhPSMA-7.3 (right) as demonstrated by SUV^^e Data are expressed as mean ± standard deviation.

Fig. 26: Clinical biodistribution and accumulation of ¹⁸ F-rhPSMA-7 (left) and ¹⁸ F-rhPSMA-7.3 (right) in tumor lesions as demonstrated by SUV,^ to background ratio. Data are expressed as mean ± standard deviation.

Fig. 27: Clinical biodistribution and accumulation of ¹⁸ F-rhPSMA-7 (left) and ¹⁸ F-rhPSMA-7.3 (right) in tumor lesions as demonstrated by SUVcpe^ee to background ratio. Data are expressed as mean ± standard deviation.

Fig. 28: two clinical examples of PET imaging ¹⁸ F-rhPSMA-7.3.

The present invention is illustrated by suitable examples.

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Example 1: materials and methods.

Fmoc-(9^nyopeHu.nMeTOKCUKap6oHu.n-) and all other protected amino acid analogues were purchased from Bachem (Bubendorf, Switzerland) or Iris Biotech (Marktredwitz, Germany). Trityl chloride polystyrene resin (TCP) was purchased from PepChem (Tübingen, Germany). The chelators DOTAGA-anhydride, (R)-DOTA-GA(tBu)4 and (S)-DOTA-GA(tBu)4 were purchased from Chematech (Dijon, France). All necessary solvents and other organic reagents were purchased from Alfa Aesar (Karlsruhe, Germany), Sigma-Aldrich (Munich, Germany), or VWR (Darmstadt, Germany). Solid-phase peptide synthesis was performed manually using an IntelliMixer syringe shaker (Neolab, Heidelberg, Germany). Analytical and preparative reversed-phase high-pressure chromatography (RP-HPLC) was performed using Shimadzu gradient systems (Shimadzu Deutschland GmbH, Neufahrn, Germany), each equipped with an SPD-20A ultraviolet-visible detector (PDA) (220 nm, 254 nm). A Nucleosil 100 C18 column (125 x 4.6 mm, particle size 5 μm) (CS GmbH, Langerwee, Germany) was used for analytical measurements at a flow rate of 1 mL/min. The text provides both specific gradients and corresponding retention times t _R . Preparative HPLC purification was performed on a Multospher 100 RP18 column (250x10 mm, 5 μm particle size) (CS GmbH, Langerwee, Germany) at a constant flow rate of 5 mL/min. Analytical and preparative radio-RP-HPLC were performed using a Nucleosil 100 C18 column (5 μm, 125 x 4.0 mm) (CS GmbH, Langerwee, Germany). Water (solvent A) and acetonitrile (solvent B), both containing 0.1% trifluoroacetic acid, were used as eluents for all HPLC operations. Electrospray ionization mass spectra for substance characterization were obtained on an ExpressL CMS mass spectrometer (Advion Ltd., Harlow, UK). NMR spectra were recorded on Bruker AVHD-300 or AVHD-400 spectrometers at 300 K. pH values were measured using a SevenEasy pH meter (Mettler Toledo, Gieeen, Germany).

Reception protocols.

1) Solid-phase peptide synthesis according to the Fmoc strategy.

Loading TCP resin (GP1).

Loading of trityl chloride polystyrene (TCP) resin with Fmoc-protected amino acid (AA) was accomplished by mixing a solution of TCP resin (1.95 mmol/g) and Fmoc-AA-OH (1.5 eq.) in anhydrous DCM (dichloromethane ) with DIPEA (diisopropylethylamine) (4.5 eq.) at room temperature for 2 hours. The remaining trityl chloride was blocked by adding methanol (2 ml/g resin) for 15 minutes. The resin was then filtered and washed with DCM (2x5 ml/g resin), DMF (2x5 ml/g resin), methanol (5 ml/g resin) and dried in vacuum. The final loading of Fmoc-AA-OH was determined using the following equation:

[-mmol-ι (m ₂ ^{- m} i) ^x SOOO I- g -I (M _w - M _HC i) t ₂ where m ₂ = mass of loaded resin [g];

m ₁ = mass of unloaded resin [g];

M _W = molecular weight of AA [g/mol];

M _H cl=molecular weight of HCl [g/mol].

Formation of amide bond on resin (GP2).

To conjugate the building block to the resin-bound peptide, a mixture of TBTU (tributylthiourea) and HOBT (hydroxybenzotriazole) was used to pre-activate with DIPEA or 2,4,6-trimethylpyridine as a base in DMF (10 ml/g resin) for 5 min. The exact stoichiometry and reaction time for each conjugation step are specified in the preparation protocol. After the reaction, the resin was washed with DMF (6x5 ml/g resin).

Removing Fmoc protection on resin (GP3).

The resin-bound Fmoc peptide was treated with 20% piperidine in DMF (v/v, 8 ml/g resin) for 5 min and then for 15 min. After this, the resin was thoroughly washed with DMF (8x5 ml/g resin).

Removing Dde protection on resin (GP4).

Dde-protected peptide (1.0 eq.) was dissolved in a solution of 2% hydrazine monohydrate in DMF (v/v, 5 ml/g resin) and shaken for 20 min (GP4a). In the case of Fmoc groups present, Dde deprotection was carried out by adding a solution of imidazole (0.92 g/g resin), hydroxylamine hydrochloride (1.26 g/g) in n-methyl-2-pyrrolidone (5.0 ml) and DMF (1.0 ml) for 3 hours at room temperature (GP4b). After deprotection, the resin was washed with DMF (8x5 ml/g resin).

Cleavage of the peptide from the resin with simultaneous deprotection of acid-labile protecting groups (GP 5).

The fully protected resin bound peptide was dissolved in TFA/TIPS/water (v/v/v, 95/2.5/2.5) and shaken for 30 min. The solution was filtered and the resin was treated in the same way for another 30 minutes. Both filtrates were combined, stirred for an additional 5 hours and concentrated under a stream of nitrogen.

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After dissolving the residue in a mixture of tert-butanol and water and subsequent lyophilization, the crude peptide was obtained.

^l-complexation (GP6).

To form a complex with ^nat Ga, the peptide (1.0 eq.) was dissolved in a mixture of 3:1 (v/v) TBuOH in H2O and an aqueous solution of Ga(NO) ₃ (3.5 eq.) was added. After heating the resulting mixture for 30 min at 75°C, the peptide was purified using RP-HPLC.

2) Preparation of the PSMA binding motif.

Glu-urea-Glu((tBuO)EuE(OtBu) ₂ )

tBu-protected Glu-urea-Glu (EuE) binding motif was prepared according to a previously described method (Scheme 1) for tBu-protected Glu-urea-Lys (EuK) (Weineisen, M.; Simecek, J.; Schottelius, M. .; Schwaiger, M.; Wester, H. J., Synthesis and preclinical evaluation of DOTAGA-conjugated PSMA ligands for functional imaging and endoradiotherapy of prostate cancer. EJNMMI research 2014, 4 (1), 63).

Di-tert-butyl-(1H-imidazole-1-carbonyl)^-glutamate (i).

A solution of DCM containing 2.0 g (7.71 mmol, 1.0 eq.) Ndi-tert-butyl-G-glutamate-NS'! was cooled on ice for 30 min and then treated with 2.69 ml TEA ( 19.28 mmol, 2.5 eq.) and 3.3 mg (0.3 mmol, 0.04 eq.) DMAP. After addition with constant stirring for 5 minutes, 1.38 g (8.84 mmol, 1.1 eq.) of 1,1'-carbonyldiimidazole (CDI) dissolved in DCM was slowly added over 30 minutes. The reaction mixture was further stirred overnight and allowed to warm to room temperature. The reaction was stopped using 8 ml saturated NaHCO ₃ with accompanying water (2x) and brine (2x) washing steps and dried over Na ₂ SO ₄ . The remaining solvent was removed in vacuo and the crude product ^)-di-tert-butyl-2-(1H-imidazole-1-carboxamido)pentanedioate (i) was used without further purification.

-Benzyl-1 -(tert-butyl)-(((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)carbamoyl)^-glutamate (ii).

2.72 g (7.71 mmol, 1.0 eq.) of crude product ^)-di-tert-butyl-2-(1H-imidazole-1carboxamido)pentanedioate (i) was dissolved in 1,2-dichloroethane (DCE) and cooled on ice for 30 minutes. To this solution were added 2.15 ml (15.42 mmol, 2.0 eq.) TEA and 2.54 g (7.71 mmol, 1.0 eq.) H-L-Glu(OBzl)OtBu HCl and the solution was stirred in overnight at 40°C. The remaining solvent was evaporated and the crude product was purified by flash chromatography on silica gel with ethyl acetate/hexane/TEA (500:500:0.8, v/v/v). After removal of the solvent, 5-benzyl-1(tert-butyl)-((^)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)carbamoyl)^-glutamate (ii) was obtained as colorless oil.

(tBuO)EuE(OtBu) ₂ (iii).

To obtain (tBuO)EuE(OtBu) ₂ , 3.17 g (5.47 mmol, 1.0 eq.) 5-benzyl-1-(tert-butyl)-((^)-1,5-ditert- butoxy-1,5-dioxopentan-2-yl)carbamoyl)^-glutamate (ii) was dissolved in 75 ml of EtOH and to this solution was added 0.34 g (0.57 mmol, 0.1 eq.) palladium on activated carbon (10%). The flask containing the reaction mixture was first purged with H ₂ and the solution was stirred overnight at room temperature and gentle pressure with H ₂ (balloon). The crude product was purified through celite and the solvent was evaporated in vacuo. Product (iii) was obtained as a hygroscopic solid (84%). HPLC (from 10 to 90% B in 15 min): t _R =11.3 min. Calculated monoisotopic mass (C ₂₃ H ₄₉ N ₂ O ₉ ): 488.3, found: m/z=489.4 [M+H]+, 516.4 [M+Na]+.

Scheme 1. Preparation of (tBuO)EuE(OtBu) ₂ : a) DCI, TEA (triethanolamine), DMAP (DCM), b) H-LGlu(OBzl)-OtBu HCl, TEA (DCE), c) Pd/C ( 10%), H2 (EtOH).

3) Preparation of the silicofluoride acceptor. 4-(di-tert-butylfluorosilyl)benzoic acid (SiFA-BA)

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SiFA-BA was prepared according to a previously described method (Scheme 2) (lovkova, L.; Wangler, V.; Schirrmacher, E.; Schirrmacher, R.; Quandt, G.; Boening, G.; Schurmann, M.; Jurkschat, K., para-Functionalized aryl-di-tertbutylfluorosilanes as potential labeling synthons for (18)F radiopharmaceuticals. Chemistry (Weinheim an der Bergstrasse, Germany) 2009, 15 (9), 2140-7). All reactions were carried out in dried reaction vessels under an argon atmosphere using a vacuum gas manifold.

((4-Bromobenzyl)oxy)(tert-butyl)dimethylsilane (i).

Imidazole (2.04 g, 30.0 mmol, 1.2 eq) was added to a stirred solution of 4-bromobenzyl alcohol (4.68 g, 25.0 mmol, 1.0 eq) in anhydrous DMF (70 mL). and TBDMSCl (4.52 g, 30.0 mmol, 1.2 eq.), and the resulting mixture was stirred at room temperature for 16 hours. The mixture was then poured into ice-cold H ₂ O (250 ml) and extracted with Et ₂ O ( 5x50 ml). The combined organic fractions were washed with sat. aqueous NaHCO ₃ (2x100 ml) and brine (100 ml), dried, filtered and concentrated in vacuo to give the crude product, which was purified by flash column chromatography (silica, 5% EtOAc/gasoline) to give product i as a colorless oil ( 7.18 g, 95%).

¹ H NMR (400 MHz, CDCU): δ [ppm]=0.10 (6H, s, SiMe2t-Bu), 0.95 (9H, s, SiMe ₂ tBu), 4.69 (2H, s, CH ₂ OSi), 7.21 (2H, d), 7.46 (2H, d). HPLC (50 to 100% B in 15 min): tR=15 min.

Di-tert-butyl{4-[(tert-butyldimethylsilyloxy)methyl]phenyl}fluorosilane (ii).

At -78°C and with magnetic stirring, a solution of tBuLi in pentane (7.29 mL, 1.7 mol/L, 12.4 mmol, 2.4 eq.) was added to a solution of ((4-bromobenzyl)oxy )(tert-butyl)dimethylsilane (i) (1.56 g, 5.18 mmol, 1.0 eq.) in dry THF (15 ml). After stirring the reaction mixture for 30 min at -78°C, the resulting suspension was added dropwise over 30 min to a cooled (-78°C) solution of di-tert-butyldifluorosilane (1.12 g, 6.23 mmol, 1.2 eq.) in dry THF (10 ml). The reaction mixture was allowed to warm to room temperature over 12 hours and then hydrolyzed with saturated aqueous NaCl (100 ml). The organic layer was separated and the aqueous layer was extracted with diethyl ether (3x50 ml). The combined organic layers were dried over magnesium sulfate and filtered. The filtrate was concentrated in vacuo to give product ii as a yellowish oil (1.88 g, 95%). It was used for subsequent reactions without further purification. NMR spectra correspond to literature data (Iovkova, L.; Wangler, V.; Schirrmacher, E.; Schirrmacher, R.; Quandt, G.; Boening, G.; Schurmann, M.; Jurkschat, K., para-Functionalized aryl -di-tert-butylfluorosilanes as potential labeling synthons for (18)F radiopharmaceuticals (Weinheim an der Bergstrasse, Germany) 2009, 15 (9), 2140-7). HPLC (from 50 to 100% B in 20 min): t _R =19 min.

4-(Di-tert-butylfluorosilanyl)benzyl alcohol (iii).

A catalytic amount of concentrated aqueous HCl (0.5 ml) was added to a suspension of ii (1.88 g, 4.92 mmol, 1.0 eq.) in methanol (50 ml). The reaction mixture was stirred for 18 hours at room temperature, and then the solvent and volatiles were removed under reduced pressure. The residue was redissolved in diethyl ether (40 ml) and the solution was washed with saturated aqueous NaHCO ₃ solution. The aqueous layer was extracted with diethyl ether (3x50 ml). The combined organic layers were dried over magnesium sulfate and filtered. The filtrate was concentrated in vacuo to give product iii as a yellowish oil (1.29 g, 98%) which solidified. The product was used without further purification. NMR spectra correspond to literature data (Iovkova, L.; Wangler, V.; Schirrmacher, E.; Schirrmacher, R.; Quandt, G.; Boening, G.; Schumann, M.; Jurkschat, K., para-Functionalized aryl -di-tertbutylfluorosilanes as potential labeling synthons for (18)F radiopharmaceuticals. Chemistry (Weinheim an der Bergstrasse, Germany) 2009, 15 (9), 2140-7). HPLC (from 50 to 100% B in 15 min): t _R =8.2 min.

4-(Di-tert-butylfluorosilyl)benzaldehyde (iv).

A solution of alcohol iii (1.37 g, 5.10 mmol, 1.0 eq.) in dry dichloromethane (20 ml) was added dropwise to a stirred ice-cooled suspension of pyridinium chlorochromate (2.75 g, 12.8 mmol, 2. 5 eq.) in dry dichloromethane (60 ml). After stirring the reaction mixture for 30 minutes at 0°C and for 2.5 hours at room temperature, anhydrous diethyl ether (40 ml) was added and the supernatant solution was decanted from the black resinous material. The insoluble material was washed thoroughly with diethyl ether and the combined organic phases were passed through a short layer of silica gel (10 cm per g crude product) for filtration. The solvents were removed in vacuo to give aldehyde iv as a yellowish oil (1.31 g, 96%). NMR spectra correspond to literature data (Iovkova, L.; Wangler, V.; Schirrmacher, E.; Schirrmacher, R.; Quandt, G.; Boening, G.; Schurmann, M.; Jurkschat, K., para-Functionalized aryl -di-tert-butylfluorosilanes as potential labeling synthons for (18)F radiopharmaceuticals. (Weinheim an der Bergstrasse, Germany) 2009, 15 (9), 2140-7). HPLC (50 to 100%

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B in 15 min): t _R =10.5 min.

4-(Di-tert-butylfluorosilyl)benzoic acid (v).

At room temperature, 1 mol/L aqueous KMnO ₄ (30 ml) was added to a mixture of iv (1.31 g, 4.92 mmol, 1.0 eq.), tert-butanol (30 ml), dichloromethane (3.3 ml ) and 1.25 mol/L NaH ₂ PO ₄ -H ₂ O buffer (20 ml) at a pH value of 4.0 to 4.5. After stirring the mixture for 25 minutes, it was cooled to 5°C, after which excess KMnO ₄ (0.78 g, 4.92 mmol, 1.0 eq.) was added. The reaction was then quenched by adding saturated aqueous Na ₂ SO ₃ solution (50 ml). When 2 mol/L aqueous HCl was added, all MnO ₂ dissolved. The resulting solution was extracted with diethyl ether (3x100 ml). The combined organic layers were washed with saturated aqueous NaHCO ₃ , dried over MgSO ₄ , filtered and concentrated under reduced pressure to give a white solid, which was purified by recrystallization from Et ₂ O/n-hexane (1:3, for 12 hours) to give product v (0.84 g, 60%). NMR spectra correspond to literature data (Iovkova, L.; Wangler, V.; Schirrmacher, E.; Schirrmacher, R.; Quandt, G.; Boening, G.; Schumann, M.; Jurkschat, K., para-Functionalized aryl -di-tert-butylfluorosilanes as potential labeling synthons for (18)F radiopharmaceuticals (Weinheim an der Bergstrasse, Germany) 2009, 15 (9), 2140-7). HPLC (from 50 to 100% B in 15 min): t _R =8.5 min.

Scheme 2. Preparation of SiFA-BA: a) TBDMSCl, imidazole (DMF), b) tBuLi, di-tert-butyldifluorosilane (THF), c) HCl (MeOH), d) pyridinium chlorochromate (DCM), f) KMnO ₄ (DCM, tert-butanol, NaH ₂ PO ₄ buffer).

4) Obtaining rhPSMA-7.1-7.4.

The first steps to prepare the four different isomers of rhPSMA-7 are identical and are performed together using the standard Fmoc-SPPS protocol described above, starting with Fmoc-D-Orn(Dde)OH bound to the resin. After cleavage of the Fmoc group with 20% piperidine in DMF (GP3), (tBuO)EuE(OtBu) ₂ (2.0 eq.) was conjugated with HOAt (2.0 eq.), TBTU (2.0 eq.) and DIPEA (6.0 eq.) in DMF for 4.5 hours. After removing the Dde group with a mixture of 2% hydrazine in DMF (GP4a), a solution of succinic anhydride (7.0 eq.) and DIPEA (7.0 eq.) in DMF and left to react for 2.5 hours. Conjugation of Fmoc-D-Lys(OtBu)-HCl (2.0 eq.) was achieved by adding a mixture of HOAt (2.0 eq.), TBTU (2 .0 eq.) and DIPEA (6.0 eq.) in DMF. After pre-activation for 5 min, Fmoc-D-Lys(OtBu)-HCl (2.0 eq) dissolved in DMF was added and allowed to react for 2.5 h (GP2). Subsequent removal of the Fmoc group was carried out by adding a mixture of 20% piperidine in DMF (GP3). Finally, the resin was cleaved to obtain rhPSMA-7.1-7.4 (Scheme 3).

Reference conjugate rhPSMA-7.1 (D-Dap-(R)-DOTA-GA)

Fmoc-D-Dap(Dde)-OH (2.0 eq.) was pre-activated in a mixture of HOAt (2.0 eq.), TBTU (2.0 eq.) and 2,4,6-trimethylpyridine (6.7 eq.) in DMF and added to the resin-bound peptide for 2.5 hours. Subsequent deprotection of orthogonal Dde was carried out using imidazole and hydroxylamine hydrochloride dissolved in a mixture of n-methyl-2-pyrrolidone and DMF for 3 hours. SiFA- BA (1.5 eq) was reacted with the free side chain amine with HOAt (1.5 eq), TBTU (1.5 eq) and DIPEA (4.5 eq) as activation reagents in DMF for 2 h. After deprotection of Fmoc with piperidine (GP3), conjugation of (R)-DOTA-GA(tBu) ₄ (2.0 eq.) with HOAT (2.0 eq.), TBTU (2.0 eq.) was carried out. and 2,4,6-trimethylpyridine (6.7 eq.) in DMF for 2.5 hours. Cleavage from the resin with simultaneous deprotection of acid-labile protecting groups was carried out in TFA according to GP5. ^nat Ga complexation of the peptide was performed as described in GP6.

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Reference conjugate rhPSMA-7.2 (L-Dap-(R)-DOTA-GA)

Fmoc-L-Dap(Dde)-OH (2.0 eq.) was pre-activated in a mixture of HOAt (2.0 eq.), TBTU (2.0 eq.) and 2,4,6-trimethylpyridine (6.7 eq.) in DMF for 2.5 h. After deprotection of the orthogonal Dde, SiFA-BA conjugation and Fmoc cleavage were performed as described for rhPSMA-7.1. Conjugation of (R)-DOTA-GA(tBu) ₄ (2.0 eq.) with HOAT (2.0 eq.), TBTU (2.0 eq.) and 2,4,6trimethylpyridine (6.7 eq.) was carried out. ) in DMF for 2.5 hours. Cleavage of acid-labile protecting groups from the resin was carried out in TFA; complexation of the peptide was carried out as described in GP6.

rhPSMA-7.3 (D-Dap-(S)-DOTA-GA) by simultaneous removal according to GP5. ^nat Ga-

Fmoc-D-Dap(Dde)-OH (2.0 eq.) was pre-activated in a mixture of HOAt (2.0 eq.), TBTU (2.0 eq.) and 2,4,6-trimethylpyridine (6.7 eq.) in DMF for 2.5 h. After deprotection of orthogonal Dde, SiFA-BA conjugation and Fmoc cleavage were performed as described for rhPSMA-7.1. Conjugation of (S)-DOTA-GA(tBu)4 (2.0 eq.) with HOAT (2.0 eq.), TBTU (2.0 eq.) and 2,4,6trimethylpyridine (6.7 eq.) was carried out. ) in DMF for 2.5 hours. Cleavage from the resin with simultaneous deprotection of acid-labile protecting groups was carried out in TFA according to GP5. Peptide complexation was performed as described in GP6.

Reference conjugate rhPSMA-7.4 (L-Dap-(S)-DOTA-GA)

Fmoc-L-Dap(Dde)-OH (2.0 eq.) was pre-activated in a mixture of HOAt (2.0 eq.), TBTU (2.0 eq.) and 2,4,6-trimethylpyridine (6.7 eq.) in DMF for 2.5 h. After deprotection of orthogonal Dde, SiFA-BA conjugation and Fmoc cleavage were performed as described for rhPSMA-7.1. Conjugation of (S)-DOTA-GA(tBu) ₄ (2.0 eq.) with HOAT (2.0 eq.), TBTU (2.0 eq.) and 2,4,6trimethylpyridine (6.7 eq.) was carried out. ) in DMF for 2.5 hours. Cleavage from the resin with simultaneous deprotection of acid-labile protecting groups was carried out in TFA according to GP5. Peptide complexation was performed as described in GP6.

Reference conjugate rhPSMA-7.1:

HPLC (from 10 to 70% B in 15 min): t _R =10.5 min.

HPLC (from 25 to 35% B in 40 min): t _R =31.4 min.

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Reference conjugate rhPSMA-7.2:

HPLC (from 10 to 70% B in 15 min): t _R =10.4 min.

HPLC (from 25 to 35% B in 40 min): t _R =27.9 min.

rhPSMA-7.3:

HPLC (from 10 to 70% B in 15 min): t _R =10.4 min.

HPLC (from 25 to 35% B in 40 min): t _R =28.1 min.

Reference conjugate rhPSMA-7.4:

HPLC (from 10 to 70% B in 15 min): t _R =10.5 min.

HPLC (from 25 to 35% B in 40 min): t _R =29.1 min. rhPSMA-7.1-7.4:

Calculated monoisotopic mass (C ₆₃ H ₉₆ FGaN ₁₂ O ₂₅ Si): 1536.6, found: m/z = 1539.4 [M+H]+, 770.3 [M+2H] ₂ +.

I rhPSMA-7.3] I fhPSMA-7.4]

Scheme 3. Preparation of rhPSMA-7.1-7.4: a) 20% piperidine (DMF), b) (tBuO)EuE(OtBu)2, NOLU TBTU, DIPEA, (DMF), c) 2% hydrazine (DMF), d) succinic anhydride, DIPEA (DMF), f) Fmoc-DLys(OtBu)-HCl, HOAt, TBTU, DIPEA, (DMF), f ¹ ) Fmoc-D-Dap(Dde)-OH, HOAt, TBTU, 2.4 ,6-collidine, (DMF), f ² ) Fmoc-L-Dap(Dde)-OH, HOAt, TBTU, 2,4,6-collidine, (DMF), g) imidazole, hydroxylamine hydrochloride (n-methyl- 2-pyrrolidone, DMF), h) SiFA-BA, HOAt, TBTU, DIPEA (DMF), i ¹ ) (R)DOTA-GA(tBu)4, HOAt, TBTU, 2,4,6-collidine (DMF) , i ² ) (S)-DOTA-GA(tBu)4, HOAt, TBTU, 2,4,6 collidine (DMF), j) elimination and deprotection: TFA, TIPS, H ₂ O, k) Ga(NO ₃ ) ₃ , (tBuOH, H ₂ O).

5) ^-labeling.

For ^18F labeling, a previously described method was used (Wangler, C; Niedermoser, S.; Chin, J.; Orchowski, K.; Schirrmacher, E.; Jurkschat, K.; Iovkova-Berends, L.; Kostikov, AP; Schirrmacher , R.; Wangler, V., One-step (18) F-labeling of peptides for positron emission tomography imaging using the SiFA methodology. Nat Protoc 2012, 7 (11), 1946-55), which was slightly modified. Briefly, aqueous ¹⁸ F ^was passed through a SAX cartridge (Sep-Pak Accell Plus QMA Carbonate light), which was pretreated with 10 ml of water. After drying 10 ml of air, the water was removed by washing the cartridge with 10 ml of anhydrous acetonitrile and then 20 ml of air. ¹⁸ F was eluted with 100 µmol [K ⁺ c2.2.2]OH ^- dissolved in 500 µl anhydrous acetonitrile. Before labeling, 30 μmol oxalic acid in anhydrous acetonitrile (1 mol/L, 30 μL) was added. This mixture was used as a whole or an aliquot to fluoridate with 10 to 25 nmol PSMA-SiFA (1 mmol/L in anhydrous DMSO). The resulting reaction mixture was incubated for 5 min at room temperature. To purify the labeled substance, a SepPak C18 light cartridge was used, pretreated with 10 ml of EtOH and then 10 ml of H2O. The labeled mixture was diluted with 9 ml PBS (pH 3) and passed through the cartridge, followed by 10 ml H2O. The peptide was eluted with 500 μl of a 4:1 mixture

- 14 046784 (v/v) EtOH in water. The radiochemical purity of the labeled compound was determined by radio-RP-HPLC and radio-TLC (silica gel 60 RP-18 F _254S , mobile phase: 3:2 (v/v) mixture of MeCN in H2O with the addition of 10% 2 mol/l aqueous solution NaOAc and 1% TFA).

6) ^|25 [-marking.

The reference ligand for in vitro studies ([ ¹²⁵ I]I-BA)KuE was prepared according to a previously described method (Weineisen, M.; Simecek, J.; Schottelius, M.; Schwaiger, M.; Wester, H. J., Synthesis and preclinical evaluation of DOTAGA-conjugated PSMA ligands for functional imaging and endoradiotherapy of prostate cancer. EJNMMI research 2014, 4(1), 63). Briefly, 0.1 mg of stannylated precursor (SnBu3-BA)(OtBu)KuE(OtBu)2 was dissolved in a solution containing 20 μL peracetic acid, 5.0 μL (21 MBq) [ ¹²⁵ I]NaI (74 TBq/mmol, 3.1 GBq/ml, 40 mmol/l NaOH, Hartmann Analytic, Braunschweig, Germany), 20 μl MeCN and 10 μl acetic acid. The reaction solution was incubated for 10 min at room temperature, loaded into a cartridge and washed with 10 ml of water (C18 Sep Pak Plus cartridge, pretreated with 10 ml of MeOH and 10 ml of water). After elution with 2.0 mL of 1:1 (v/v) EtOH/MeCN, the radioactive solution was evaporated to dryness under a gentle stream of nitrogen and treated with 200 μL of TFA for 30 min, followed by evaporation of the TFA. The crude product ([ ¹²⁵ I]I-BA)KuE was purified by RP-HPLC (20 to 40% B in 20 min): tR=13.0 min.

In vitro experiments.

1) Determination of IC ₅₀ .

PSMA-positive LNCaP cells were grown in Dulbecco's modified Eagle's medium/nutrient mixture F-12 with glutamax-I (1:1) (Invitrigon), supplemented with 10% fetal calf serum and maintained at 37°C in a humidified atmosphere at 5 %CO2. To determine the affinity of PSMA (IC ₅₀ ), cells were collected 24 ± 2 hours before the experiment and seeded in 24-well plates (1.5x105 cells in 1 ml/well). After removal of the culture medium, cells were treated once with 500 μl HBSS (Hank's balanced salt solution, Biochrom, Berlin, Germany, supplemented with 1% bovine serum albumin (BSA)) and left for 15 min on ice to equilibrate in 200 μl HBSS (1% BSA). Then 25 µl per well of solutions containing either HBSS (1% BSA, control) or the appropriate ligand in increasing concentrations (from 10 ^-10 to 10 ^-4 mol/l in HBSS, followed by the addition of 25 µl ([ ^125I ] I-BA)KuE (2.0 nmol/L) in HBSS (1% BSA). All experiments were performed at least three times for each concentration. After 60 min of incubation on ice, the experiment was completed by removing the medium and successively washing with 200 μl of HBSS. both steps were pooled into one fraction and the amount of free radioligand was reported. Cells were then lysed with 250 μL of 1 mol/L NaOH and combined with 200 μL of HBSS in the next washing step. Bound and free radioligand were quantified using a y-counter.

2) Internalization.

For internalization studies, LNCaP cells were collected 24 ± 2 h before the experiment and seeded in 24-well plates (1.25 x 105 cells in 1 ml/well). After removal of the culture medium, cells were washed once with 500 μl DMEM-F12 (5% BSA) and allowed to equilibrate for at least 15 min at 37°C in 200 μl DMEM-F12 (5% BSA). Each well was treated with either 25 μl DMEM-F12 (5% BSA) or 100 μM PMPA block solution. Then 25 μl of ⁶⁸ Ga/ ¹⁸ F labeled PSMA inhibitor (5.0 nmol/l) was added and the cells were incubated at 37°C for 60 min. The experiment was completed by placing the 24-well plate on ice for 3 min and sequentially removing the medium. Each well was washed with 250 μl of HBSS and fractions from these first two steps were pooled, representing the amount of free radioligand. Removal of surface bound active material was accomplished by incubating the cells with 250 μl of ice-cold PMPA solution (10 μmol in PBS) for 5 min and washing again with another 250 μl of ice-cold PBS. Internalized active substance was determined by incubating cells in 250 μl of 1 mol/l NaOH and combining with a subsequent wash step fraction of 250 μl of 1.0 mol/l NaOH. Each experiment (control and blocking) was performed in triplicate. Free, surface-bound and internalized active substance was quantified using a counter. All internalization studies were accompanied by reference studies using ([ ¹²⁵ I]I-BA)KuE (c=0.2 nmol/L), which were performed similarly. Data were corrected for nonspecific internalization and normalized to the specific internalization observed for the radioiodine-labeled reference compound.

3) Octanol-water separation coefficient.

Approximately 1 MBq of the labeled substance was dissolved in 1 ml of a 1:1 (v/v) mixture of phosphate-buffered saline (PBS, pH 7.4) and n-octanol in an Eppendorf tube. After vigorously stirring the suspension for 3 min at room temperature, the tube was centrifuged at 15,000 g for 3 min (Biofuge 15, Heraus Sepatech, Osterode, Germany) and 100 μL aliquots of both layers were measured on a gamma counter. The experiment was repeated at least six times.

4) HSA binding.

To determine HSA binding, a Chiralpak HSA column (50x3 mm, 5 μm, H13H-2433) was used at a constant flow rate of 0.5 ml/min. Mobile phase (A: NH ₄ OAc, 50 mmol/l in water, pH 7 and B:

- 15 046784 isopropanol) was prepared anew for each experiment and used only for one day. The column was stored at room temperature and each run was stopped after signal detection to reduce data acquisition time. All substances were dissolved at a concentration of 0.5 mg/ml in 50% 2propanol and 50% 50 mmol/L ammonium acetate buffer, pH value 6.9. The selected reference substances show a range of HSA binding from 13% to 99%, as a wide variety of albumin binding to peptides was expected. All nine reference substances were administered sequentially to establish a nonlinear regression with OriginPro 2016G.

Table 1

Reference substances (Yamazaki, K.; Kanaoka, M., Computational prediction of the plasma protein-binding percent of diverse pharmaceutical compounds. Journal of pharmaceutical sciences 2004, 93 (6), 1480-94) used to calibrate the HSA column

Reference substance tR LogtR Lit., HSA% Log To HSA p-benzyl alcohol 2.40 0.38 13.15 -0.82 Aniline 2.72 0.43 14.06 -0.79 Phenol 3.28 0.52 20.69 -0.59 Benzoic acid 4.08 0.61 34.27 -0.29 Carbamazepine 4.15 0.62 75.00 0.46 p-nitrophenol 5.62 0.75 77.65 0.52 Estradiol 8.15 0.91 94.81 1.19 Probenecid 8.84 0.95 95.00 1.20 Glibenclamide 29.18 1.47 99.00 1.69

The retention time is shown as an example for the experiment performed, t _R retention time, Lit. HSA human serum albumin binding value in [%] according to literature, Log K HSA logarithmic human serum albumin binding K value.

In vivo experiments.

All animal experiments were carried out in accordance with the general animal welfare regulations in Germany and the institutional guidelines for the care and use of animals. To generate tumor xenografts, LNCaP cells (10 ⁷ cells/200 μl) were suspended in a 1:1 (v/v) mixture of Dulbecco's modified Eagle's medium/nutrient mixture F-12 with glutamax-I (1:1) and Matrigel ( BD Biosciences, Germany) and injected subcutaneously into the right shoulder of 6- to 8-week-old CB17-SCID mice (Charles River, Sulzfeld, Germany). Mice were used when tumors reached 5 to 8 mm in diameter (3 to 4 weeks after inoculation).

1) Biodistribution.

Approximately 1 to 2 MBq (less than 0.2 nmol) of β-labeled PSMA inhibitor was injected into the tail vein of male CB-17 SCID mice bearing the LNCaP tumor and sacrificed 1 hour after injection (n = 4 to 5). Selected organs were removed, weighed and measured on a y-counter.

2) Metabolism studies.

a) Analytical setup.

Analytical reverse-phase high-pressure chromatography (RP-HPLC) was performed using Shimadzu gradient systems (Shimadzu Deutschland GmbH, Neufahrn, Germany) equipped with an SPD-20A ultraviolet-visible detector (220 nm, 254 nm). A Multospher 100 RP18 column (125 x 4.6 mm, 5 μm particle size) (CS GmbH, Langerwee, Germany) was used for analytical measurements at a flow rate of 1 mL/min. Water (solvent A) and acetonitrile (solvent B), both containing 0.1% trifluoroacetic acid, were used as eluents for all HPLC operations. Radioactivity was detected by connecting the output of the UV photometer to a HERM LB 500 detector (Berthold Technologies GmbH, Bad Wildbad, Germany). The gradient for all HPLC runs was: 5% isocratic from 0 to 3 min, 25 to 35% B from 3 to 43 min, 95 to 95% B from 43 to 48 min.

Radio thin layer chromatography was performed using aluminum sheets coated with silica gel 60 RP-18 F _254S with a mobile phase consisting of a 3:2 (v/v) mixture of MeCN in H2O with the addition of 10% 2 mol/L aqueous NaOAc and 1% TFA. Analysis was performed using a Scan-RAM radio-TLC detector (LabLogic Systems Ltd., Sheffield, UK) or a CR 35 BIO fluorescent phosphor imaging unit (Duerr Medical GmbH, Bietigheim-Bissingen, Germany).

b) Determination of metabolic stability of rhPSMA-7.1-7.4.

For in vivo micrometabolism studies, 8 to 12 MBq (less than 0.6 nmol) of the appropriate 18E-labeled ligand (rhPSMA-7.1-16 046784) was injected into the tail vein of healthy female CB17-SCID mice (n=4).

7.4) . Mice were left under anesthesia for 30 min and urine was collected using a bladder catheter. Urine samples were pooled and centrifuged for 5 min at 9000 rpm to remove suspended solids. The supernatant was directly used for radio-HPLC analysis under the above conditions. To demonstrate that isotopic exchange ^{of 19} F for peptide-bound ¹⁸ F occurs in urine, each compound was incubated for specified time intervals with urine samples from healthy female CB-17-SCID mice, which were analyzed by radio-HPLC and/or radio-LC. TLC. In addition, this experiment was carried out with the addition of excess Na ¹⁹ F (0.5 μmol) and incubation for 2 hours with ¹⁸ F labeled rhPSMA-7.3.

c) Determination of the in vivo distribution of rhPSMA-7.1-7.4.

To quantify the relative accumulation of each isomer (rhPSMA-7.1-7.4), a male mouse bearing a CB-17-SCID tumor was injected with a racemic mixture of rhPSMA-7 (180 to 280 MBq, S _a =ot 247 to 349 GBq/μmol, produced in Klinikum rechts der Isar in a fully automated way). The animal was left under anesthesia for 30 min and sacrificed. Urine, blood, liver, kidney, and tumor were collected and processed according to the methods described below. The urine sample was centrifuged for 5 min at 9000 rpm to obtain a clear solution and directly analyzed by radio-HPLC. The blood was diluted to 1 ml H ₂ O and centrifuged twice at 13,000 g for 5 minutes. The supernatant was collected and loaded into a Strata X cartridge (33 µm reverse phase polymer, 500 mg, pretreated with 5 ml MeOH followed by 5 ml H ₂ O). After washing with 5 mL H2O, the cartridge was eluted with 6:4 (v/v) MeCN in H2O supplemented with 1% TFA. The eluate was diluted with water and analyzed by radio-HPLC. The tumor, kidney, and liver were homogenized using a Potter-Elvehjem tissue homogenizer (Kontes Glass Co, Vineland, USA) or an MM-400 ball mill (Retsch GmbH, Haan, Germany).

I) Potter-Elvehjem fabric homogenizer.

The tumor and kidneys were separately homogenized in a tissue homogenizer with 1 mL of extraction buffer (850 μL of 1 mol/L HEPES pH 7.4, 100 μL of 20 mmol/L PMPA, and 100 μL of 1 mol/L NaCl) for 30 min. The resulting homogenate was collected and centrifuged at 13,000 g for 5 min. The supernatant was subsequently collected, centrifuged again (13,000 g, 5 min) and loaded into a Strata X cartridge (polymer reverse phase 33 μm, 500 mg, pretreated with 5 ml MeOH and then 5 ml H2O). After washing with 5 mL H2O, the cartridge was eluted with 6:4 (v/v) MeCN in H2O supplemented with 1% TFA. The eluate from each organ was diluted with water and analyzed by radio-HPLC.

II) Ball mill MM-400.

Organs (tumor, kidney, liver) were separately homogenized in a 2 ml tube along with 3 grinding balls (3 mm diameter) and 1 ml extraction buffer (850 μl 1 mol/l HEPES pH 7.4, 100 μl 20 mmol/l PMPA and 100 μl of 1 mol/l NaCl) for 10 min at 30 Hz. The homogenate was centrifuged at 13,000 g for 5 min and the supernatant was collected. The pellet was then suspended in 1 ml of extraction buffer and homogenized again using a ball mill for 10 min at 30 Hz. After centrifugation (13,000 g, 5 min), both supernatants were combined and loaded into a Strata X cartridge (33 μm polymer reverse phase, 500 mg, pretreated with 5 ml MeOH followed by 5 ml H2O). After washing with 5 mL H2O, the cartridge was eluted with 6:4 (v/v) MeCN in H2O supplemented with 1% TFA. The eluate from each organ was diluted in water and analyzed by radio-HPLC. To demonstrate that breakthrough during cartridge loading was not the result of unbound F-18, the supernatant was also examined by radio-TLC after centrifugation.

Finally, individual isomer ratios were determined from the HPLC profiles of the extracted samples and compared with isomer ratios from the rhPSMA-7 racemic mixture quality control. The extraction efficiency adjusted for decay and cartridge loading, as well as the overall extraction activity of the studied samples, are given in Table. 2. Cartridge elution efficiency was greater than 99% for all experiments.

Example 2: results.

Assignment of chromatographic peaks.

The assignment of chromatographic peaks was performed by comparing UV profiles.

a) racemic mixture rhPSMA7-rac;

b) a racemic rhPSMA7-rac mixture co-injected with each enantiomerically pure rhPSMA7 compound.

The following names were used for the various isomers:

rhPSMA-rac: [ ¹⁹ F] [ ^nat Ga]D/L-Dap-R/S-DOTAGA-rhPSMA7 reference rhPSMA-7-1: [ ¹⁹ F] [ ^nat Ga]D-Dap-R-DOTAGA-rhPSMA7 reference rhPSMA-7-2: [ ¹⁹ F] [ ^nat Ga]L-Dap-R-DOTAGA-rhPSMA7 rhPSMA-7-3: [ ¹⁹ F] [ ^nat Ga]D-Dap-S-DOTAGA-rhPSMA7 reference rhPSMA-7 -4: [ ¹⁹ F] [ ^nat Ga]L-Dap-S-DOTAGA-rhPSMA7.

- 17 046784

table 2

Assignment of different isomers, names, typical retention times (HPLC conditions are given in Fig. 2a-4^ and the percentage of each isomer in a typical rhPSAM7-rac mixture.

The exact amount may vary for each isomer

Ligand Name tR [min] typical percentage of total mixture [ ¹⁹ F][ ^nat Ga]D-Dap-R-DOTAGA-rhPSMA7 rhPSMA-7-1 31.6 21 [ ¹⁹ F][ ^nat Ga]L-Dap-R-DOTAGA-rhPSMA7 rhPSMA-7-2 28.3 22 [ ¹⁹ F][ ^nat Ga]D-Dap-S-DOTAGA-rhPSMA7 rhPSMA-7-3 28.9 37 [ ¹⁹ F][ ^nat Ga]L-Dap-S-DOTAGA-rhPSMA7 rhPSMA-7-4 30.1 20

Binding affinity.

The first set of values (rhPSMA-7.1 and rhPSMA-7.2, Fig. 6a) were determined using a series of dilutions of the solution immediately obtained after ^nat Ga complexation of the corresponding ligand. In the second data set (Fig. 6b), complexed ligands were purified using RP-HPLC to separate ^nat Ga salts not included in the complex. Since no significant differences were found, both series were combined and used to calculate the mean values (± standard deviation). Table 3

Illustration of individual IC50 [nmol/L] measurements (as shown in Figures 6a and 6b). The conditions are given in the ______________________ description of Fig. 6a_____________________

Number rhPSMA7.1 rhPSMA7.2 rhPSMA7.3 rhPSMA7.4 1 8.74 3.17 nd nd 2 6.91 2.97 nd nd 3 7.27 3.36 nd nd 4 5.04 2.64 3.17 3.4 5 1.21 (*) 2.76 2.91 3.56 6 7,I 3.94 5.35 2.79 7 8.31 5.8 5.74 4.57 8 4.97 4.31 4.59 3.78 9 6.44 4.32 4.45 nd Average 6.85 3.70 4.37 3.62 meaning Standard 1.36 1.01 1.14 0.65 deviation

* Value number 5 of the rhPSMA7.1 series has been removed (statistical outlier). Table 4

Binding affinity ( _IC50 [nmol/L]) of other selected PSMA inhibitors (*).

Number Inhibitor IC50 [nmol/l] 1 (I-BA)KuE 7.1 ±2.4 nM 2 DCFPyL 12.3 ±1.2 nmol/l 3 DKFZ1007 4.2 ±0.5 nmol/l

* Conducted in the inventors' laboratory using an identical binding assay. (Robu et al. EJNMMI Research 2018; 8:30).

- 18 046784

Research on internalization.

Table 5

Individual levels of internalization [% of [ ¹²⁵ I IB-KuE] rhPSMA7.1 rhPSMA7.2 rhPSMA7.3 rhPSMA7.4 1 61.8 188.7 156.6 209.6 1 70.6 182.7 156.0 202.6 1 68.0 169.5 171.7 209.8 1 67.9 205.3 - - 1 71.5 212.6 - - 1 77.5 192.3 - - Average value 69.55 191.83 161.41 207.33 Standard deviation 5.19 15.54 8.88 4.06

Table 6

Internalization values [% [ ¹²⁵ I]IB-KuE] from other selected PSMA inhibitors (*).

Number Inhibitor Internalization [%] 1 PSMA-1007 118±4 2 DCFPyL 118±5

* Conducted in the inventors' laboratory using an identical binding assay. (Robu et al. EJNMMI Research 2018; 8:30).

Lipophilicity (octanol-water partition coefficient).

Determination of logP values was carried out in phosphate-buffered saline (PBS, pH 7.4) and n-octanol (=logPoct/PBS).

Table 7

Individual log P measurements for rhPSMA7 isomers 7.1- to 7.4, determined in octanol/ _PBS7 mixtures. ₄ ______________

rhPSMA7.1 rhPSMA7.2 rhPSMA7.3 rhPSMA7.4 rhPSMA7rac 1 -2.79 -3.00 -3.03 -3.09 -3.23 2 -2.82 -3.01 -3.08 -3.12 -3.15 3 -2.80 -3.00 -3.07 -3.06 -3.17 4 -2.84 -3.00 -3.02 -3.07 -3.10 5 -2.88 -3.03 -3.06 -3.10 -3.17 6 -2.90 -2.96 -3.07 -3.04 -3.24 7 -2.85 -2.94 -3.06 -2.99 -3.80 8 -2.86 -2.89 -2.86 -2.97 -3.60 9 -3.14 -3.02 -3.39 -3.37 -3.87 10 -3.29 -3.02 -3.33 -3.43 -3.61 eleven -3.26 -3.06 -3.34 -3.29 -3.76 12 -3.02 -3.02 -3.34 -3.48 -3.65 13 -3.15 -2.99 -3.20 -3.52 -3.67 14 -3.57 -3.02 -3.39 -3.50 - 15 -3.40 -3.06 -3.40 -3.44 - 16 -3.32 -3.14 -3.41 -3.41 - 17 -3.64 -3.40 -3.48 -3.56 - 18 -3.92 -3.50 -3.49 -3.61 - 19 - -3.45 -3.32 -3.58 -

- 19 046784

20 - -3.45 -3.45 -3.54 - 21 - -3.53 -3.53 -3.42 - 22 - -3.48 -3.43 -3.57 - 23 - - -3.56 -3.67 - Average value -3.14 -3.13 -3.26 -3.33 -3.46 Standard deviation 0.34 0.22 0.19 0.22 0.29

Table 8

Log P values of isomers PSMA-1007, DCFPYL, rhPSMA7-rac and rhPSAM-7.1-7.4, (n=6), octanol/PBS7.4

Inhibitor logP PSMA-1007 -1.6 DCFPyL -3.4 ^nat Ga- ¹⁸ F-rhPSMA7-rac, -3.46 ± 0.29 ⁶⁸ Ga ^-nat F-rhPSMA7-rac ^nat Ga- ¹⁸ F-rhPSMA7.1 -3.14 ±0.34 ^nat Ga- ¹⁸ F-rhPSMA7.2 -3.13 ±0.22 ^nat Ga- ¹⁸ F-rhPSMA7.3 -3.26 ±0-19 ^nat Ga- ¹⁸ F-rhPSMA7.4 -3.33 ± 0.22

Binding of PSMA inhibitors to human plasma protein (HSA).

Table 9

Binding of isomers PSMA-1007, DCFPYL, rhPSMA7-rac and rhPSAM-7.1-7.4 to HSA, (n=6). Determined on a Chiralpak HSA column (50x3 mm, 5 µm, Н13Н-2433)

Inhibitor HSA binding [%] PSMA-1007 97.8 DCFPyL 14.3 ⁶⁸ Ga ^-nat F-rhPSMA7-rac 96.7 ^nat Ga- ¹⁸ F-rhPSMA7.1 97.7 ^nat Ga- ¹⁸ F-rhPSMA7.2 97.8 ^nat Ga- ¹⁸ F-rhPSMA.3 96.9 ^nat Ga- ¹⁸ F-rhPSMA7.4 96.6

Biodistribution of [F][ ^nat Ga]rhPSMA-7.1-7.4 1 hour after injection.

- 20 046784

Table 10

Biodistribution (% ID/g) of ¹⁸ F-rhPSMA 1 hour after injection in SCID mice bearing LNCaP tumor. Data are expressed as mean ± standard deviation (n=4 for rhPSMA7.1, n=5 for __________________7.2, n=4 for 7.3, n=5 for 7.4 and n=3 for 7-rac)_________________

[ ¹⁸ F][ ^nat Ga]rhPSMA-7-l [ ¹⁸ F][ ^nat Ga]- rhPSMA-7-2 [ ¹⁸ F][ ^nat Ga]rhPSMA-7-3 [ ¹⁸ F][ ^nat Ga]- rhPSMA-7-4 [ ¹⁸ F][ ^nat Ga]rhPSMA-rac Blood 0.53 ±0.13 0.56 ±0.20 0.96 ± 0.24 1.15 ±0.30 1.1 ±0.03 Heart 0.53 ± 0.03 0.32 ±0.13 0.87 ±0.17 0.71 ± 0.26 0.69 ± 0.07 Lung 1.1 ± 0.21 0.89 ±0.38 2.2 ±0.35 1.59 ±0.61 1.4 ±0.17 Liver 0.75 ± 0.62 0.35 ±0.08 0.69 ±0.13 0.69 ± 0.20 0.67 ± 0.07 Spleen 20.0 ± 4.2 10.1 ±6.3 16.6 ±2.6 18.4 ±9.77 11.1 ±2.3 Pancreas gland 0.45 ±0.12 0.21 ± 0.08 0.63 ± 0.44 0.50 ±0.30 0.60 ±0.10 Stomach 0.28 ±0.17 0.19 ±0.08 0.44 ± 0.23 0.25 ± 0.06 0.49 ± 0.07 Intestines 0.30 ±0.16 0.18 ±0.07 0.35 ± 0.07 0.37 ± 0.09 0.60 ± 0.27 Kidneys 220 ± 24.8 87.6 ±28.8 292 ±45.1 153 ±80.3 71.3 ± 13.3 Adrenal glands 2.0 ±0.25 1.3 ±0.8 2.2 ± 0.83 3.57 ±2.38 3.0 ± 0.45 Muscle 0.32 ±0.30 0.13 ±0.07 0.33 ±0.15 0.31 ±0.08 0.36 ± 0.06 Bone 0.50 ±0.31 0.31 ±0.24 0.38 ± 0.32 0.62 ± 0.30 0.91 ±0.11 Tumor 14.1 ±4.1 6.5 ±2.3 18.3 ±7.2 18.9 ±3.27 10.4 ±0.67

Biodistribution of [ ¹⁸ F][ ^nat Ga]rhPSMA-7.1-7.4 1 hour after injection with competition.

Table 11

Biodistribution [% ID/g] of ¹⁸ F-labeled rhPSMA co-injected with PMPA (8 mg/kg) 1 h post-injection in SCID mice bearing LNCaP tumor. Data are expressed as mean ± standard deviation (n=3)

[ ¹⁸ F][ ^nat Ga]- rhPSMA-7-l [ ¹⁸ F][ ^nat Ga]- rhPSMA-7-2 [ ¹⁸ F][ ^nat Ga]rhPSMA-7-З [ ¹⁸ F][ ^nat Ga]- rhPSMA-7-4 Blood 0.86 ± 0.40 1.1 ±0.31 0.55 ±0.14 0.82 ±0.17 Heart 0.37 ±0.16 0.47 ± 0.09 0.26 ± 0.04 0.37 ±0.05 Lung 0.85 ± 0.29 1.1 ±0.32 0.69 ±0.10 0.74 ±0.14 Liver 0.43 ± 0.07 0.46 ± 0.07 0.46 ±0.14 0.48 ±0.14 Spleen 0.21 ± 0.08 0.26 ± 0.07 0.35 ± 0.02 0.28 ±0.15 Pancreas gland 0.16 ±0.10 0.12 ±0.05 0.11 ±0.02 0.18 ±0.09 Stomach 0.97 ±0.81 0.21 ± 0.06 0.76 ± 0.74 0.20 ± 0.07 Intestines 0.66 ± 0.32 0.33 ±0.10 0.94 ± 0.97 0.36 ±0.08 Kidneys 10.9 ±2.5 10.9 ± 1.0 15.5 ±2.2 7.2 ± 2.4 Adrenal glands 0.003 ± 0.004 0.07 ±0.10 0.07 ± 0.09 0.03 ± 0.04 Muscle 0.17 ±0.15 0.09 ± 0.03 0.09 ± 0.02 0.20 ± 0.05 Bone 0.33 ± 0.24 0.57 ±0.39 0.34 ± 0.22 1.0 ±0.8 Tumor 0.94 ± 0.22 1.0 ± 0.13 1.5 ±0.4 0.99 ±0.19

Quantification of relative changes in the amount of each rhPSMA7.x isomer in blood, kidney, liver, urine, and tumor following [ ¹⁸ F]rhPSMA7-rac administration.

To quantify the relative changes of each rhPSMA7 isomer in the blood, liver, kidney, urine and tumor 30 min after injection of [ ¹⁸ F]rhPSMA7-rac into LNCaP tumor-bearing mice, two different homogenization methods (homogenizing and ball milling) were used to extract tags from kidney, liver and tumor tissue (see materials and methods section).

In table 12 summarizes the observed effectiveness of both homogenization methods and effectively

- 21 046784 the subsequent solid-phase extraction procedure (to separate the label from the protein fraction).

Table 12

Determination of the extraction of the active substance, corrected for degradation, from the studied tissue samples using a Potter-Elvehjem tissue homogenizer (n=1) and an MM-400 ball mill (n=3)

Potter-Elvehjem fabric homogenizer (η = 1): Efficiency [%] Sample extraction Loading a cartridge SPE Total Blood 93 93 86 Kidneys 91 66 60 Tumor 90 59 53 Ball mill MM-400 (n = 3): Efficiency [%] Sample extraction Loading a cartridge SPE Total Blood 98 ±2 94 ±2 92 ±3 Liver 97 89 ±2 86 ±2 Kidneys 63 ± 5 68 ±8 43 ± 8 Tumor 64 ± 18 65 ±3 42 ± 14

While the extraction of active substance from samples using a homogenizer was quite effective, the use of a ball mill did not live up to expectations. However, even with a ball mill, extraction efficiencies of over 60% were achieved.

Considering the possible species that could be formed by metabolic cleavage of the amide bonds of rhPSMA7, only a) species with significantly increasing lipophilicity b) F ¹⁸ -fluoride seem likely. Thus, it seems possible in principle that the iL species depicted in FIG. 12 are not extracted from the tissue sample (aqueous extraction) and therefore do not appear in the final analysis.

However, it should be noted that such species may appear in vivo in the liver and intestines (hepatobiliary excretion of lipophilic compounds) or must be bound to plasma proteins (resulting in high levels of the active substance in the blood, which, on the other hand, show excellent extraction efficiency).

A graphical approximation was initially used to quantify each isomer in the racemic mixture and especially for the poorly separated first and second peaks (rhPSMA 7.2 and rhPSMA 7.3). This method was based on the assumption that a) each isomer eluted from the HPLC column with an identical peak shape and b) different peak heights could be used as a first approximation for calculation using linear factors minus individual peaks (i.e. rhPSMA 7.2 and rhPSMA 7.3).

Based on these assumptions, the first analysis was performed using LNCaP tumor-bearing mice co-treated with [ ¹⁸ F][ ^nat Ga]rhPSMA7-rac. The Systat PeakFit software package was used to confirm these experiments with three additional experiments and improve the graphical analysis with a more reliable procedure. PeakFit automates the nonlinear separation, analysis, and quantification of HPLC elution profiles using deconvolution routines that employ a Gaussian response function with a Fourier deconvolution/filtering algorithm (https://systatsoftware.com/products/peakfit/).

Comparison of the graphical analysis of the first experiments showed that the second peak is overestimated in the graphical analysis (rhPSMA7.3), while the first peak is underestimated. Consequently, all datasets were reanalyzed and quantified using PeakFit.

HPLC analyzes of 4 independent experiments on tumor-bearing mice 30 min after injection.

1. Evaluation of peaks 3 and 4 (rhPSMA 7.4 and rhPSMA 7.1) using radio-HPLC. First, we determined whether the deconvolution method shows similar data for the last two peaks (rhPSMA7.4 and 7.1), which have good separation (although they are not separated by the baseline).

2. Evaluation of all peaks (rhPSMA7.1, 7.2, 7.3 and 7.4) by radio-HPLC.

- 22 046784

In fig. 14a and 14b summarize the percentage change of each rhPSAM7.n isomer in a given sample relative to its percentage in the injected solution ([ ¹⁸ F][ ^nat Ga]rhPSMA7-rac), the results for a separate experiment are shown in FIG. 14. The proportion of each isomer was quantified by HPLC elution profile analysis using Systat PeakFit. The percentage change of each isomer in a given sample was then calculated relative to its percentage in the injected solution.

3. Discussion of HPLC data.

Radio-HPLC analyzes of radioactive substances extracted from homogenized (kidney, liver, tumor) or diluted (blood) tissues and subsequently immobilized on and eluted from a solid-phase extraction cartridge showed no evidence of metabolic instability. Thus, no lipophilic metabolic moieties were detected. It should be noted that F-18-fluoride cannot be accurately detected by HPLC under the conditions used for sample preparation (see TLC Analysis).

Although there is a clear trend toward the use of D-Dap-derived rhPSMA7.1 and 7.3, the overall changes are minor (maximum 15%). In this context, it is also important to emphasize that in FIG. 15 and 16 show relative changes without taking into account the absolute values of accumulation.

Although rhPSMA7.1 has the weakest affinity and internalization of all rhPSMA7 compounds, it shows the largest positive percentage change in liver, kidney, and tumor blood.

Although the reason for this result is unclear, it can be assumed that homogenization of tissue samples, even using a ball mill, did not lead to quantitative cell destruction. Thus, the rhPSMA7 tags with the highest internalization (rhPSMA7.2: 191.83% ±15.54%, rhPSMA7.4: 207.33 ±4.06% and rhPSMA7.3: 161.41% ±8.88%) could be extracted less efficiently, whereas rhPSMA7.1, with its low internalization of only 69.55% ± 5.29%, was extracted efficiently and was therefore overestimated in HPLC analysis.

In addition, rhPSMA 7.2 and 7.4 compounds appear to be eliminated more quickly (see urine values). These compounds show generally negative changes in solid tissues and blood, although both compounds show higher affinity and internalization rates compared to rhPSMA7.1. Whether this could be caused by metabolic degradation of 7.2 and 7.4 (both L-Dap derivatives) is unclear since no metabolites, i.e. lyophilic metabolites, were detected. However, it is possible that such metabolites (see Fig. 11) due to their high logP value are not extracted by aqueous buffer solutions. In this case, they should appear in the liver (see biodistribution) and possibly in blood samples (high probability of high binding to serum proteins). Since increased accumulation of the active substance was not observed in biodistribution studies in liver tissue, and extraction of the active substance from the blood was highly efficient (see Table 3), the inventors assumed that significant degradation of rhPSMA7.2 and 7.4 did not occur. This assumption is supported by suspicious SUV values for liver tissue (gallbladder, intestine) in the context of clinical use of [ ¹⁸ F]rhPSMA-rac in humans.

TLC analysis in tumor-bearing mice 30 min after injection.

Radio-TLC analysis was performed: a) on urine samples by directly applying a small volume to a TLC strip, b) by analyzing a small volume of non-immobilized active during the SPE process (breakthrough fraction), and c) analyzing a small volume of cartridge eluates.

- 23 046784

Table 13

Analysis of blood, organs and urine by TLC

date Sample TLC scanner Installation for A comment* formation images on luminescent phosphorus coating Intensity 1"F. 1"F. Intact Intact TLC signal [by them] fluoride fluoride label G%1 label G%1 (generally low- [%] [%] short) 30.07.2 QC 018 Blood 3^44 44^4 47 Liver W9 44^04 442 Methodological Kidneys 94.04 5.96 369 Problems Urine 82.51 17.49 726 Tumor Q/1 1 o s £1 “1.1“ J.O 1 -- 472 01.08.2 — QC 94.27 5.73 96.02 3.98 384 018 Blood 40D4 4D4 44 Liver 92 ^9 7 51 93.92 ^# 6.08 ^# 473- Kidneys 94.58 5.42 572 Urine 96.2 3.80 98.55 1.55 395 Tumor pp o η αί , 1 U 444 02.08.2 — QC 97.43 2.57 016 Active level Blood substances 96.80 3.20 Liver too low for 74.15 ^# 25.85 ^# Kidneys TLC 96.48 3.52 Urine 95.85 4.15 Tumor 96.47 4.15

(*) Due to the low level of the active substance, TLC measurements with signal intensity less than 200 were removed.

Discussion of TLC data.

Since it is very difficult to detect p.s.a. ^{* 18} G-fluoride, using an RP18 chromatography column (due to the free Si-OH groups of the matrix that react with PS fluoride), thin layer chromatography was performed to quantify F-18-fluoride in the extracted solutions.

Because none of the reagents and salts typically used for protein precipitation are tested against cold fluoride, and to avoid the possible release of F-18 fluoride from the label by isotope exchange, protein precipitation was not used during sample preparation, although such protein loading often results to limited peak separation, peak lag, and activity that remains at the starting line. Solutions obtained after tissue extraction (or blood centrifugation) were directly used for TLC analysis.

Although the active substance available for analysis was quite low in all samples, the TLC results indicate that the total F-18-fluoride content was below approximately 6% in the tissue tested, with the exception of:

urine sample dated July 30, 2018 (17.49% free fluoride);

liver sample obtained on August 2, 2018 (25.85% free fluoride).

While the urine analysis obtained by TLC is considered a reliable result (see Profile in Fig. 20), the result obtained on the liver sample is caused by a significant lag in the peak representing the intact label (see Fig. 18). Furthermore, it can be concluded that the above-mentioned maximum 6% free fluoride represents an overestimation since the tail of the peak, even obtained during QC and the release of [F-18]rhPSMA7-rac in PBS (Fig. 18) for clinical use, shows the tail of the product peak. As the fluorescent phosphor coating images demonstrate, this tail is observed in almost every TLC analysis and contributes to the integrated area of F-18-fluoride.

It should be noted that neither biodistribution studies nor clinical PET scans in humans (as of July 2018: approximately 1400 scans with [F-18]rhPSMA7-rac) have resulted in any suspicious or identifiable accumulation of F-18 in bone due to released F-18 fluoride.

To further study the release of F-18 fluoride from [F-18]rhPSMA7-rac (observed in one urine sample), we examined the presence of F-18 fluoride in other urine samples (normal mice) using an RP-18 HPLC column ( new closed end column RP-18) and TLC.

Radio-TLC analysis of F-18-fluoride production in normal mice 30 min after injection.

For this purpose, normal mice were used. Urine samples were collected using a catheter over 30 min. The urine was centrifuged and directly subjected to HPLC and TLC.

As shown in FIG. 21, left column, free F-18-fluoride was detected in urine samples for all isomers and is also formed when fresh urine is incubated with [F-18]rhPSMA7.4. (right column). Identification of F-18 fluoride was achieved by demonstrating that a) the species is retained on QMA cartridges (data not shown), b) elutes in the dead volume of RP-18 columns, and c) cannot be retained or mobilized on RP-18 columns or TLC plates RP-18, respectively, regardless of the mobile phases used.

Due to the fact that such high amounts of F-18 fluoride were not detected in HPLC analyzes of blood or organs such as kidney, tumor, liver, etc., no increased uptake of the active substance in bones and have not observed increased bone uptake during clinical PET scans with the compound [F18]rhPSMA7-rac since [F-18]rhPSMA7-rac was established for clinical scanning at the end of 2017 at the Technical University of Munich (status at the end of July 2018: approximately 1400 PET scans in patients with prostate cancer), the inventors concluded that [F-18]fluoride may be formed after passing through glomerular filtration, leading to the formation and subsequent release of [F-18]fluoride no detectable uptake of F18-fluoride into blood, organs or bones.

This assumption is supported by the literature on fluoride toxicology, which describes corresponding amounts of fluoride in the kidneys and urine. The mice had normal urinary fluoride levels of 0.3 ppm. (Bouaziz H et al., Fluoride 2005, 38(1):23-31). In another publication, the average fluoride concentration in the urine of normal mice ranged from 0.13 to 0.14 μg/ml (Poesina ND et al. Rom J Morphol Embryol 2014, 55(2):343-349) and Inkielewicz I. et al. found that serum fluoride in rats was approximately 5% of the renal fluoride concentration (serum: 0.051 μg/ml, kidney: 0.942 μg/ml) (Fluoride, 36(4), 263-266). Considering that most of the tracer is specifically absorbed by the kidneys and is also excreted physiologically, elevated renal fluoride levels in combination with a body temperature of 36.6°C may result in continued excretion of F-18-fluoride rhPSMA compounds in the kidneys.

Therefore, fresh and non-radioactive urine samples collected from normal mice were incubated with [F-18]rhPSMA7.x for various periods of time (see description of Fig. 21). In fig. 22, right column, it is clearly seen that incubation of urine with [F-18]rhPSMA7.x ex vivo leads to the formation of free [F-18] fluoride to varying degrees, which is facilitated by different concentrations of cold F-19 fluoride in urine samples, increases over time.

To further confirm the hypothesis, 500 nmol of cold F-19 fluoride was added to fresh and non-radioactive urine of mice, followed by the addition of [F-18]rhPSMA7.3 and incubation for 2 hours. According to the hypothesis, a high concentration of [F-19] fluoride should lead to to the formation of a significant amount of [F-18] fluoride. In fig. Figure 23 shows that under these conditions, 98.5% of the radioactive substance is exchanged and [F-18] fluoride is formed within 2 hours (Figure 23).

Since isotope exchange rates depend on the concentration of the four corresponding species at equilibrium (p-18]fluoride, p-19]fluoride, [F-18]rhPSMA7.3 and [F-19]rhPSMA7.3), it was also examined whether addition resulted in p-18]fluoride to fresh and radioactive urine (20.6% [F-18] fluoride, 79.4%

- 25 046784

[F-18]rhPSMA7.3) followed by the addition of cold labeled [F-19]rhPSMA7.3 also to the radiopharmaceutical [F-18]rhPSMA7-3 labeling. Surprisingly, even the small amount of 5 nmol [F-19]rhPSMA7-3 in urine noted above resulted in an increase in [F-18]rhPSMA7.3 from 79.4% to 85.8% ^-18]fluoride decreased from 20 .6% to 14.2%) at room temperature.

The results obtained by urinary isotope exchange are considered to be representative of all tracers conjugated to the 4-(di-tert-butyl[(18^]fluorosilyl)benzyl)oxy moiety and therefore of all rhPSAM7 isomers.

Preclinical dosimetry, biodistribution in humans and uptake in tumor lesions.

Note that in the following example, 18F-rhPSMA-7 refers to ^nat Ga- ¹⁸ F-rhPSMA7-rac, and ¹⁸ F-rhPSMA-7.3 to ^nat Ga- ¹⁸ F-rhPSMA7.3.

A) Preclinical dosimetry of ¹⁸ F-rhPSMA-7 and ¹⁸ F-rhPSMA-7.3 in mice.

The objective was to evaluate the distribution and excretion of ¹⁸ F-rhPSMA-7 and ¹⁸ F-rhPSMA-7.3 at various time points up to 300 min after a single intravenous administration in mice and perform calculations for internal dosimetry.

Ways.

Three to five mice were injected at each time point with an average of 25.6 ± 3.6 MBq ^{of 18} FrhPSMA-7 and 28.5 ± 4.8 MBq of ¹⁸ F-rhPSMA-7.3, respectively. Mice with severe combined immunodeficiency (SCID) were used for experiments. All animal experiments were carried out in accordance with the general animal welfare regulations in Germany and the institutional guidelines for the care and use of animals.

Mice were sacrificed at the following time points:

¹⁸ F-rhPSMA-7: 10, 20, 40, 60, 120 and 180 min after administration.

¹⁸ F-rhPSMA-7.3: 10, 60, 120, 180 and 300 minutes after administration.

Note that based on initial experiments demonstrating prolonged renal uptake of ¹⁸ F-rhPSMA-7.3, a late time point (300 min) was used for final experiments.

The following tissues/liquids were taken.

Urine, blood, heart, lungs, spleen, pancreas, liver, stomach (empty), small intestine (empty), large intestine (empty), kidney, bladder, testicle, fat, muscle (partial, femoral), femur , tail and brain. Urine was collected by pipette into a CO2 gas chamber. If there was no urination in the chamber, the bladder was aspirated with an insulin syringe. Blood was collected immediately after sacrifice using an insulin syringe from the heart. All other tissues and organs were excised and transferred directly to plastic containers.

The weight of samples in plastic containers was measured using an electronic balance. The weight of empty and pre-labeled plastic containers for isolated samples was measured in advance. The dead weight of the plastic containers was subtracted from the weight of the measured sample with the plastic container. The mass calculated in this way was designated as the mass of the measured sample.

Plastic containers containing the samples to be measured were placed in special racks of an automatic gamma counter (PerkinElmer-Wallac, Waltham, USA) to measure the counting rate for 60 seconds (cpm=cpm). In addition, a 1% (v/v) standard (n=5) with a known amount of radioactive material was measured along with the samples to convert the organ sample count rate to activity.

Data analysis.

The count rates of the measured samples were automatically corrected for decay. The distribution coefficients of radioactivity (unit: percentage of dose administered (% ID)) in the measured samples were determined using the equation below. The sum of the count rates for all measured samples obtained from one mouse was designated as the count rate for the administered radioactive substance.

counting rate of measured samples

Percentage of dose administered (%ID) the sum of the counting rate of all measured samples from one mouse * iqq

The distribution coefficient of radioactive substance per unit mass of measured sample (unit: % ID/g), excluding urine and feces samples, was determined using the equation below. The mass of the sample being measured was determined by subtracting the mass of the empty measurement container from the mass of the container containing the sample.

Percentage of dose administered (%ID)

Percentage of dose administered (%ID/r) = ------------------------— mass of sample measured (g)

Dosimetric analysis.

For consistency of statistical calculations, the same number of time points for ¹⁸ F-rhPSMA-7 and ¹⁸ F-rhPSMA-7.3 were used for each RFID tag. Therefore, for ¹⁸ F-rhPSMA-7, the 10 min and 20 min time points were combined to create the 15 min endpoint.

The time integral of activity during accumulation in important source organs (AUC) was obtained as

- 26 046784 by numerical integration and physical decomposition according to J Juan et al., Journal of Pharmaceutical Sciences, 1993, 82:762-763.

Kirshner et al. A method has been developed in which a linear scale of the percentage of the administered dose in an animal is used in relation to the mass of organs to the total body mass of the model animal for both species.

Kirschner AS, Ice RD, Beierwaltes WH. Radiation Dosimetry of 1311-19-Iodocholesterol. J Nucl Med. 1973 Sep 1; 14(9):713-7.

Kirschner A, Ice R, Beierwaltes W. Letters to the editor. J Nucl Med. (1975):248-9.

Briefly, calculation of human dosimetry from biodistribution in mice required extrapolation to account for differences between animals and humans. Normal organ doses were estimated for an anatomical model of a standard 70 kg adult using organ concentrations of the active agent over time (as a percentage of administered dose per gram, % ID/g) and whole body active agent concentrations measured in biodistribution studies in mice.

Tissue active concentrations in mice were converted to fractionated tissue actives from a standard 70 kg adult human using relative fractional organ masses from a standard adult human and a standard 25 g mouse. The whole body active versus time curve was exponential. function, and it was assumed that the difference between the active substance administered and the active substance in the whole body was excreted in the urine, since concentrations of the active substance in the liver and traces in the gastrointestinal tract were low at all time points studied.

The residence time in the organ was calculated by numerical integration using the trapezoidal rule, and the residence time in the rest of the body of ¹⁸ F was calculated as the difference between the residence time in the body as a whole and the sum of the residence times in the organ and in the urine. Bladder retention time was assessed using a dynamic voiding model in the OLINDA/EXM 1.0 dosimetry program. Finally, the standard mean organ dose equivalents for an adult (in mSv/MBq) and the effective dose (also in mSv/MBq) were calculated using OLINDA/EXM 1.0.

Final calculation of absorbed radiation dose and biodistribution dosimetry in mice: the tissues or organs in which significant accumulation of radioactive material occurs (i.e., the source organ) were the kidneys, spleen, lungs, liver, and heart. With regard to accumulation of the active substance and excretion, rapid clearance from the blood and excretion in urine, but relatively slow accumulation in the kidneys, was found.

Results.

Table 14

Dosimetry results for ¹⁸ F-rhPSMA-7 using a voiding interval of 3.5 hours

Target organ Alpha Beta Photon Total EED ED Adrenal glands Ο,ΟΟΕΟΟΟ 1.95Ε-03 5.85Ε-03 7.80Ε-03 Ο,ΟΟΕΟΟΟ 3.90Ε- Brain Ο,ΟΟΕΟΟΟ 1.95Ε-03 2.54Ε-03 4.49Ε-03 Ο,ΟΟΕΟΟΟ 2.24Ε- Breast Ο,ΟΟΕΟΟΟ 1.95Ε-03 2.29Ε-03 4.24Ε-03 6.36Ε-04 2D2E- Gall wall bubble Ο,ΟΟΕΟΟΟ 1.95Ε-03 5.54Ε-03 7.49Ε-03 Ο,ΟΟΕΟΟΟ

- 27 046784

Wall of the lower colon Ο,ΟΟΕΟΟΟ 1.95Ε-03 1.41Ε-02 1.61Ε-02 9.66Ε-04 1.93Ε- Small intestine Ο,ΟΟΕΟΟΟ 1.95Ε-03 8.40Ε-03 1D4E-02 Ο,ΟΟΕΟΟΟ 5D8E- Stomach wall Ο,ΟΟΕΟΟΟ 1.95Ε-03 5D5E-03 7D0E-03 Ο,ΟΟΕΟΟΟ 8.04Ε- Wall of the upper colon Ο,ΟΟΕΟΟΟ 1.95Ε-03 7.31Ε-03 9.26Ε-03 Ο,ΟΟΕΟΟΟ 4.63Ε- Heart wall Ο,ΟΟΕΟΟΟ 8.82Ε-04 ZD4E-OZ 4.42Ε-03 Ο,ΟΟΕΟΟΟ Kidneys Ο,ΟΟΕΟΟΟ 4.70Ε-02 1.80Ε-02 6.51Ε-02 3.91Ε-03 3.25Ε- Liver Ο,ΟΟΕΟΟΟ 6.35Ε-04 3.63Ε-03 4.27Ε-03 Ο,ΟΟΕΟΟΟ 2DZE- Lungs Ο,ΟΟΕΟΟΟ 1.25Ε-03 ZD4E-OZ 4DZE-OZ 5.16Ε-04 5D6E- Muscle Ο,ΟΟΕΟΟΟ 1.95Ε-03 5.73Ε-03 7.68Ε-03 Ο,ΟΟΕΟΟΟ 3.84Ε- Ovaries Ο,ΟΟΕΟΟΟ 1.95Ε-03 1D5E-02 1.55Ε-02 3.87Ε-03 3.09Ε- Pancreas gland Ο,ΟΟΕΟΟΟ 1.95Ε-03 6DZE-OZ 8D8E-03 Ο,ΟΟΕΟΟΟ 4.04Ε- Red bone marrow Ο,ΟΟΕΟΟΟ 1D9E-03 5.41Ε-03 6.08Ε-03 8.16Ε-04 8D6E- Osteogenic cells Ο,ΟΟΕΟΟΟ 4D8E-03 4.75Ε-03 8.93Ε-03 2.68Ε-04 8.93Ε- Leather Ο,ΟΟΕΟΟΟ 1.95Ε-03 2.98Ε-03 4.93Ε-03 Ο,ΟΟΕΟΟΟ 4.93Ε- Spleen Ο,ΟΟΕΟΟΟ 1D9E-02 Ι,ΟΙΕ-02 2.61Ε-02 1.56Ε-03 1.30Ε- Testes Ο,ΟΟΕΟΟΟ 1.95Ε-03 9.69Ε-03 1D6E-02 Ο,ΟΟΕΟΟΟ Thymus Ο,ΟΟΕΟΟΟ 1.95Ε-03 3.24Ε-03 5.18Ε-03 Ο,ΟΟΕΟΟΟ 2.59Ε- Thyroid Ο,ΟΟΕΟΟΟ 1.95Ε-03 3.23Ε-03 5.18Ε-03 1.55Ε-04 2.59Ε- Urinary wall bubble Ο,ΟΟΕΟΟΟ 2.45Ε-01 1.08Ε-01 ZD4E-01 2.12Ε-02 1.77Ε- Uterus Ο,ΟΟΕΟΟΟ 1.95Ε-03 2.61Ε-02 2.80Ε-02 1.68Ε-03 1.40Ε- Whole body Ο,ΟΟΕΟΟΟ 2.37Ε-03 5.39Ε-03 7.77Ε-03 Ο,ΟΟΕΟΟΟ EED - effective equivalent dose (mSv/MBq) 3.56E-02 ED - effective dose (mSv/MBq) 2.66E-02

Table 15

Dosimetry results for ¹⁸ F-rhPSMA-7 s. using a bladder emptying interval of 1.0 hours

Target organ Alpha Beta Photon Total ed ed

- 28 046784

Adrenal glands Ο,ΟΟΕΟΟΟ 1.95Ε-03 5.64Ε-03 7.59Ε-03 Ο,ΟΟΕΟΟΟ 3.79Ε- Brain Ο,ΟΟΕΟΟΟ 1.95Ε-03 2.54Ε-03 4.49Ε-03 Ο,ΟΟΕΟΟΟ 2.24Ε- Breast Ο,ΟΟΕΟΟΟ 1.95Ε-03 2.25Ε-03 4.20Ε-03 6.30Ε-04 2D0E- Gall wall Ο,ΟΟΕΟΟΟ 1.95Ε-03 4.96Ε-03 6.91Ε-03 Ο,ΟΟΕΟΟΟ bubble Wall of the lower department of the thick Ο,ΟΟΕΟΟΟ 1.95Ε-03 7.25Ε-03 9.20Ε-03 5.52Ε-04 Ι,ΙΟΕ- intestines Small intestine Ο,ΟΟΕΟΟΟ 1.95Ε-03 5.79Ε-03 7.73Ε-03 Ο,ΟΟΕΟΟΟ 3.87Ε- Stomach wall The wall of the upper Ο,ΟΟΕΟΟΟ 1.95Ε-03 4.70Ε-03 6.65Ε-03 Ο,ΟΟΕΟΟΟ 7.98Ε- department of the thick Ο,ΟΟΕΟΟΟ 1.95Ε-03 5.33Ε-03 7.27Ε-03 Ο,ΟΟΕΟΟΟ 3.64Ε- intestines Heart wall Ο,ΟΟΕΟΟΟ 8.82Ε-04 3.48Ε-03 4.36Ε-03 Ο,ΟΟΕΟΟΟ Kidneys Ο,ΟΟΕΟΟΟ 4.70Ε-02 1.76Ε-02 6.47Ε-02 3.88Ε-03 3.23Ε- Liver Ο,ΟΟΕΟΟΟ 6.35Ε-04 3.39Ε-03 4.02Ε-03 Ο,ΟΟΕΟΟΟ 2.01Ε- Lungs Ο,ΟΟΕΟΟΟ 1.25Ε-03 3.01Ε-03 4.26Ε-03 5D1E-04 5.1ΙΕ- Muscle Ο,ΟΟΕΟΟΟ 1.95Ε-03 4.01Ε-03 5.96Ε-03 Ο,ΟΟΕΟΟΟ 2.98Ε- Ovaries Ο,ΟΟΕΟΟΟ 1.95Ε-03 7.21Ε-03 9D6E-03 2.29Ε-03 1.83Ε- Pancreas Ο,ΟΟΕΟΟΟ 1.95Ε-03 5.86Ε-03 7.80Ε-03 Ο,ΟΟΕΟΟΟ 3.90Ε- gland red bone Ο,ΟΟΕΟΟΟ 1.39Ε-03 4.29Ε-03 5.68Ε-03 6.82Ε-04 6.82Ε- brain Osteogenic cells Ο,ΟΟΕΟΟΟ 4D8E-03 4.09Ε-03 8.27Ε-03 2.48Ε-04 8.27Ε- Leather Ο,ΟΟΕΟΟΟ 1.95Ε-03 2.38Ε-03 4.32Ε-03 Ο,ΟΟΕΟΟΟ 4.32Ε- Spleen Ο,ΟΟΕΟΟΟ 1.59Ε-02 9.09Ε-03 2.58Ε-02 1.55Ε-03 1.29Ε- Testes Ο,ΟΟΕΟΟΟ 1.95Ε-03 5.09Ε-03 7.03Ε-03 Ο,ΟΟΕΟΟΟ Thymus Ο,ΟΟΕΟΟΟ 1.95Ε-03 3.20Ε-03 5D5E-OZ Ο,ΟΟΕΟΟΟ 2.57Ε- Thyroid Ο,ΟΟΕΟΟΟ 1.95Ε-03 3.23Ε-03 5D7E-03 1.55Ε-04 2.59Ε- Urinary wall Ο,ΟΟΕΟΟΟ 7.87Ε-02 3.66Ε-02 1D5E-01 6.92Ε-03 5.76Ε- bubble Uterus Ο,ΟΟΕΟΟΟ 1.95Ε-03 1D2E-02 1.31Ε-02 7.87Ε-04 6.56Ε- Whole body Ο,ΟΟΕΟΟΟ 2.27Ε-03 3.93Ε-03 6D9E-03 Ο,ΟΟΕΟΟΟ EED - effective equivalent dose (mSv/MBq) 1.82Ε-02 ED - effective dose (mSv/MBq) 1.22E-02

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Table 16

Dosimetry results for ¹⁸ F-rhPSMA-7.3 using a bladder emptying interval of 3.5 hours_________________________________

Target organ Alpha Beta Photon Total EED ED Adrenal glands Ο,ΟΟΕΟΟΟ ZD2E-OZ 7.93Ε-03 ί,ΙΟΕ-02 Ο,ΟΟΕΟΟΟ 5.52Ε- Brain Ο,ΟΟΕΟΟΟ ZD2E-OZ 4.07Ε-03 7D9E-03 Ο,ΟΟΕΟΟΟ 3.59Ε- Breast Ο,ΟΟΕΟΟΟ ZD2E-OZ 3.55Ε-03 6.67Ε-03 Ι,ΟΟΕ-03 3.34Ε- Gall wall bubble Ο,ΟΟΕΟΟΟ ZD2E-OZ 7.46Ε-03 1.06Ε-02 Ο,ΟΟΕΟΟΟ Wall of the lower colon Ο,ΟΟΕΟΟΟ ZD2E-OZ 1.28Ε-02 1.59Ε-02 9.53Ε-04 1.91Ε- Small intestine Ο,ΟΟΕΟΟΟ ZD2E-OZ 9.42Ε-03 1.25Ε-02 Ο,ΟΟΕΟΟΟ 6.27Ε- Stomach wall Ο,ΟΟΕΟΟΟ ZD2E-OZ 7.02Ε-03 Ι,ΟΙΕ-02 Ο,ΟΟΕΟΟΟ 1.22Ε- Wall of the upper colon Ο,ΟΟΕΟΟΟ ZD2E-OZ 8.57Ε-03 1D7E-02 Ο,ΟΟΕΟΟΟ 5.85Ε- Heart wall Ο,ΟΟΕΟΟΟ 1.32Ε-03 5.39Ε-03 6.71Ε-03 Ο,ΟΟΕΟΟΟ Kidneys Ο,ΟΟΕΟΟΟ 5D1E-02 2.07Ε-02 7D8E-02 4.31Ε-03 3.59Ε- Liver Ο,ΟΟΕΟΟΟ 9.70Ε-04 5.02Ε-03 5.99Ε-03 Ο,ΟΟΕΟΟΟ 3.00Ε- Lungs Ο,ΟΟΕΟΟΟ 1.95Ε-03 4.66Ε-03 6.61Ε-03 7.93Ε-04 7.93Ε- Muscle Ο,ΟΟΕΟΟΟ ZD2E-OZ 6.55Ε-03 9.67Ε-03 Ο,ΟΟΕΟΟΟ 4.83Ε- Ovaries Ο,ΟΟΕΟΟΟ ZD2E-OZ 1.26Ε-02 1.57Ε-02 3.92Ε-03 ZD4E- Pancreas gland Ο,ΟΟΕΟΟΟ ZD2E-OZ 8.29Ε-03 1D4E-02 Ο,ΟΟΕΟΟΟ 5.70Ε- Red bone marrow Ο,ΟΟΕΟΟΟ 2.22Ε-03 6.79Ε-03 9.01Ε-03 1.08Ε-03 1.08Ε- Osteogenic cells Ο,ΟΟΕΟΟΟ 6.70Ε-03 6.52Ε-03 1.32Ε-02 3.97Ε-04 1.32Ε- Leather Ο,ΟΟΕΟΟΟ ZD2E-OZ 3.83Ε-03 6.95Ε-03 Ο,ΟΟΕΟΟΟ 6.95Ε- Spleen Ο,ΟΟΕΟΟΟ 1.52Ε-02 1D5E-02 2.67Ε-02 1.60Ε-03 1.34Ε- Testes Ο,ΟΟΕΟΟΟ ZD2E-OZ 8.96Ε-03 1.21Ε-02 Ο,ΟΟΕΟΟΟ Thymus Ο,ΟΟΕΟΟΟ ZD2E-OZ 5.08Ε-03 8.20Ε-03 Ο,ΟΟΕΟΟΟ 4D0E- Thyroid Ο,ΟΟΕΟΟΟ ZD2E-OZ 5D5E-03 8.27Ε-03 2.48Ε-04 4D4E- Urinary wall bubble Ο,ΟΟΕΟΟΟ 1.56Ε-01 7D4E-02 2.27Ε-01 1.36Ε-02 1D4E- Uterus Ο,ΟΟΕΟΟΟ ZD2E-OZ 2.04Ε-02 2.36Ε-02 1.41Ε-03 1D8E- Whole body Ο,ΟΟΕΟΟΟ 3.52Ε-03 6.33Ε-03 9.86Ε-03 Ο,ΟΟΕΟΟΟ EED - effective equivalent dose (mSv/MBq) 2.94E-02 ED - effective dose (mSv/MBq) 2D7E-02

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Table 17

Dosimetry results for ¹⁸ F-rhPSMA-7.3 using a 1.0 hour voiding interval

Target organ Alpha Beta Photon Total EED ED Adrenal glands Ο,ΟΟΕΟΟΟ ZD2E-OZ 7.79Ε-03 1D9E-02 Ο,ΟΟΕΟΟΟ 5.46Ε- Brain Ο,ΟΟΕΟΟΟ ZD2E-OZ 4.06Ε-03 7D8E-03 Ο,ΟΟΕΟΟΟ 3.59Ε- Breast Ο,ΟΟΕΟΟΟ ZD2E-OZ 3.53Ε-03 6.65Ε-03 9.97Ε-04 3.32Ε- Gall wall Ο,ΟΟΕΟΟΟ ZD2E-OZ 7D1E-03 1D2E-02 Ο,ΟΟΕΟΟΟ bubble Wall of the lower department of the thick Ο,ΟΟΕΟΟΟ ZD2E-OZ 8.45Ε-03 1D6E-02 6.94Ε-04 1.39Ε- intestines Small intestine Ο,ΟΟΕΟΟΟ ZD2E-OZ 7.78Ε-03 1D9E-02 Ο,ΟΟΕΟΟΟ 5.45Ε- Stomach wall The wall of the upper Ο,ΟΟΕΟΟΟ ZD2E-OZ 6.80Ε-03 9.92Ε-03 Ο,ΟΟΕΟΟΟ 1D9E- department of the thick Ο,ΟΟΕΟΟΟ ZD2E-OZ 7.33Ε-03 1.05Ε-02 Ο,ΟΟΕΟΟΟ 5.23Ε- intestines Heart wall Ο,ΟΟΕΟΟΟ 1.32Ε-03 5.36Ε-03 6.68Ε-03 Ο,ΟΟΕΟΟΟ Kidneys Ο,ΟΟΕΟΟΟ 5D1E-02 2.04Ε-02 7D6E-02 4.29Ε-03 3.58Ε- Liver Ο,ΟΟΕΟΟΟ 9.70Ε-04 4.87Ε-03 5.84Ε-03 Ο,ΟΟΕΟΟΟ 2.92Ε- Lungs Ο,ΟΟΕΟΟΟ 1.95Ε-03 4.63Ε-03 6.58Ε-03 7.90Ε-04 7.90Ε- Muscle Ο,ΟΟΕΟΟΟ ZD2E-OZ 5.47Ε-03 8.59Ε-03 Ο,ΟΟΕΟΟΟ 4,ZOE- Ovaries Pancreas Ο,ΟΟΕΟΟΟ ZD2E-OZ 8.61Ε-03 1D7E-02 2.93Ε-03 2.3 5Ε- Ο,ΟΟΕΟΟΟ ZD2E-OZ 8D2E-03 1D2E-02 Ο,ΟΟΕΟΟΟ 5.62Ε- gland red bone Ο,ΟΟΕΟΟΟ 2.22Ε-03 6.09Ε-03 8.31Ε-03 9.97Ε-04 9.97Ε- brain Osteogenic cells Ο,ΟΟΕΟΟΟ 6.70Ε-03 6D1E-03 1.28Ε-02 3.84Ε-04 1.28Ε- Leather Ο,ΟΟΕΟΟΟ ZD2E-OZ 3.45Ε-03 6.57Ε-03 Ο,ΟΟΕΟΟΟ 6.57Ε- Spleen Ο,ΟΟΕΟΟΟ 1.52Ε-02 1D4E-02 2.66Ε-02 1.59Ε-03 1.33Ε- Testes Ο,ΟΟΕΟΟΟ ZD2E-OZ 6.08Ε-03 9.20Ε-03 Ο,ΟΟΕΟΟΟ Thymus Ο,ΟΟΕΟΟΟ ZD2E-OZ 5.06Ε-03 8D8E-03 Ο,ΟΟΕΟΟΟ 4.09Ε- Thyroid Ο,ΟΟΕΟΟΟ ZD2E-OZ 5D5E-03 8.27Ε-03 2.48Ε-04 4DZE- Urinary wall Ο,ΟΟΕΟΟΟ 5D8E-02 2.66Ε-02 7.84Ε-02 4.70Ε-03 3.92Ε- bubble Uterus Ο,ΟΟΕΟΟΟ ZD2E-OZ Ι,ΙΙΕ-02 1.43Ε-02 8.55Ε-04 7DZE- Whole body Ο,ΟΟΕΟΟΟ 3.45Ε-03 5.42Ε-03 8.87Ε-03 Ο,ΟΟΕΟΟΟ EED - effective equivalent dose (mSv/MBq) 1.85Ε-02 ED - effective dose (mSv/MBq) 1.28E-02

Conclusion.

The highest radioactive distribution coefficients were observed in the kidneys after injection of ¹⁸ F-rhPSMA-7 and ¹⁸ F-rhPSMA-7.3 at all time points studied in mice. Moreover, the ratio was high in the spleen and bladder for both radiolabels compared with all other tissues evaluated where active ratios were less than 8% ID/g.

Since most of the active substances of ¹⁸ F-rhPSMA-7/ ¹⁸ F-rhPSMA-7.3 accumulate in the kidneys and excretion through the bladder shows high levels of active substances, the main route of elimination is determined through the kidneys and the urinary system.

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At voiding intervals of 3.5 hours and 1.0 hours, the extrapolated total effective doses were 2.66E-02 and 1.22E-02 mSv/MBq for ¹⁸ F-rhPSMA-7 and 2.17E-02 and 1.28E -02 mSv/MBq for ¹⁸ F-rhPSMA-7.3, respectively. Injecting up to 370 MBq (10 mCi) for a clinical scan will result in a beneficial radiation exposure of less than 5 mSv for both agents, assuming a 1-hour void interval.

The differences worth mentioning between both radioactive tracers are only apparent with respect to renal uptake, as ¹⁸ F-rhPSMA-7.3 tends to accumulate more gradually with longer retention. However, the exposure of both agents is comparable.

B) Biodistribution and uptake of ¹⁸ F-rhPSMA-7 and ¹⁸ F-rhPSMA-7.3 in human tumor lesions.

The following sections describe the biodistribution of ¹⁸ F-rhPSMA-7 and ¹⁸ F-rhPSMA-7.3.

A proof-of-concept study was conducted when used for humane reasons. The agent was administered in accordance with the German Medicines Act, AMG §13 2b, and in accordance with the authority of the regulatory authority (Government of Oberbayern).

All patients were examined on a Biograph mCT scanner (Siemens Medical Solutions, Erlangen, Germany). All PET scans were acquired in 3D mode with acquisition times ranging from 2 to 4 min in the supine position. The outlier data were corrected for randomness, dead time, scatter, and attenuation and were reconstructed iteratively using an ordered subset expectation maximization algorithm (four iterations, eight subsets) followed by a Gaussian smoothing filter after reconstruction (5 mm full width at half maximum).

Ways.

Biodistribution in humans was assessed by analyzing clinical studies of ¹⁸ F-rhPSMA-7- and ¹⁸ F-rhPSMA-7.3-n3T/KT in 47 and 32 patients, respectively. The mean potency of the administered active substance was 324 (range, 236 to 424) MBq versus 345 (range, 235 to 420) MBq, and absorption times were 84 (range, 42 to 166) minutes versus 76 (range, 59 to 122). ) min for ¹⁸ F-rhPSMA-7 compared to ¹⁸ F-rhPSMA-7.3, respectively.

The average and maximum standardized accumulation rates (SUVcp^e/SUV^c) were determined for the background (gluteus muscle), normal organs (salivary glands, blood pool, lungs, liver, spleen, pancreas, duodenum, kidney, bladder, bone ) and three typical tumor lesions. Tumor uptake was analyzed in 89 lesions (26 primary tumors/local recurrences, 23 bone, 38 lymph nodes and 2 visceral metastases) and 63 lesions (14 primary tumors/local recurrences, 30 bones, 18 lymph nodes and 1 visceral metastasis ) for ¹⁸ F-rhPSMA-7 and ¹⁸ F-rhPSMA-7.3, respectively.

To calculate SUV, circular ROIs were drawn around areas of focal increased attenuation in transaxial slices and automatically adapted to a three-dimensional volume of interest (VOI) at 50% isocontour. Organ-to-background and tumor-to-background ratios were calculated.

Results.

The biodistribution of ¹⁸ F-rhPSMA-7 and ¹⁸ F-rhPSMA-7.3 in humans showed a typical pattern known for other PSMA ligands. The uptake parameters of ¹⁸ F-rhPSMA-7 and ¹⁸ F-rhPSMA-7.3 were very similar with lower uptake in the bladder and higher uptake in tumor lesions for ¹⁸ F-rhPSMA-7.3: SUV _c| . _ie;uiee for ¹⁸ F-rhPSMA-7 compared to ¹⁸ F-rhPSMA-7.3 was 16.9 compared to 16.0 (parotid gland), 19.6 compared to 19.6 (submandibular gland), 2. 0 vs. 1.9 (blood pool), 0.7 vs. 0.7 (lungs), 7.0 vs. 7.3 (liver), 9.1 vs. 8.5 (spleen) , 32.4 compared to 35.5 (kidney), 2.5 compared to 2.8 (pancreas), 10.9 compared to 11.0 (duodenum), 1.1 compared to 1, 3 (healthy bone) and 10.2 versus 2.0 (bladder) for ¹⁸ F-rhPSMA-7 versus ¹⁸ F-rhPSMA7.3, respectively. Specifically, the uptake values of ¹⁸ F-rhPSMA-7.3 versus ¹⁸ F-rhPSMA-7 were significantly lower for bladder retention (2.0 ± 0.8 versus 6.3 ± 21.2, p < 0 .05) and significantly higher for tumor lesions (32.5±42.7 compared with 20.0±20.2, p<0.05).

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Table 18

SUV _MaKC and SUV _cpegHee of normal organs and tumor lesions using ¹⁸ F-rhPSMA-7. Data_____are shown as mean, minimum and maximum values_____

SUYmbkc ZiAverage average minimum maximum average minimum maximum Background 1.0 0.6 1.8 0.6 0.4 1.2 Parotid gland 23.8 8.2 42.3 16.9 5.5 32.7 Submandibular gland 27.0 Yu, 1 43.8 19.6 7.0 29.7 Blood pool 2.4 1.6 3.9 2.0 1.1 17.0 Lungs 1.1 0.5 3.1 0.7 0.3 2.0 Liver 9.5 4.5 25.2 7.0 3.2 17.7 Spleen 11.8 4.7 21.0 9.1 3.4 17.1 Bud 44.8 19.1 75.2 32.4 13.2 54.7 Pancreas 3.7 1.8 7.9 2.5 1.3 5.5 duodenum intestine 14.8 2.8 32.7 10.9 1.9 23.9 Bone 1.7 0.8 3.1 1.1 0.6 2.1 Bladder 8.5 0.5 112.0 6.3 o, s 85.7 Tumor 27.6 3.1 167.2 20.0 2.1 115.7

Table 19

SUV^ and SUVcpe^ee of normal organs and tumor lesions using ¹⁸ F-rhPSMA-7.3. Data_____are shown as mean, minimum and maximum values_____

SUYmbkc SUVcpeanee average minimum maximum average minimum maximum Background 1.0 0.6 1.7 0.7 0.4 1.1 Parotid gland 24.6 AND 2 38.3 16.0 8.2 25.0 Submandibular gland 28.4 14.6 47.4 19.6 10.4 33.4 Blood pool 2.8 1.9 3.9 1.8 1.3 2.5 Lungs 1.1 0.7 1.9 0.7 0.4 1.1 Liver 9.7 4.6 15.4 7.3 3.2 12.3 Spleen I,4 5.0 22.5 8.5 3.7 17.9 Bud 51.9 30.9 99.9 35.5 20.7 70.6 Pancreas 4.2 2.4 7.8 2.8 1.6 5.2 duodenum intestine 16.4 6.1 32.2 and about 3.0 23.0 Bone 2.1 1.1 3.4 1.3 0.7 2.2 Bladder 3.1 1.1 6.0 2.0 0.7 4.1 Tumor 44.0 2.4 316.0 32.5 1.6 224.1

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Table 20

Relation of SUV _MaKC and SUV _cpegHe e to the background of normal organs and tumor lesions using ¹⁸ F-rhPSMA-7. Data shown as average, minimum and maximum values

SUYmbkc relation ZPU ratio average average minimum maximum average minimum maximum Parotid gland 25.2 8.2 45.3 28.3 9.2 54.5 Submandibular gland 28.7 Yu, 1 54.7 33.3 I,7 61.8 Blood pool 2.5 1.3 4.8 3.2 1.6 21.3 Lungs 1.1 0.4 z, z 1.1 0.4 4.0 Liver 10.4 4.7 42.0 11.9 4.6 44.3 Spleen 12.5 4.7 35.0 15.1 5.7 39.5 Bud 48.1 18.2 98.7 55.2 19.8 109.3 Pancreas 3.9 1.5 from 4.3 1.9 10.8 duodenum intestine 15.7 2.8 31.3 18.4 3.2 35.3 Bone 1.7 0.9 2.9 1.8 1.0 3.2 Bladder 8.7 0.6 112.0 10.2 0.5 142.8 Tumor 32.0 3.1 278.6 36.0 3.5 289.3

Table 21

Ratio of SUV^ and SUV _cp ^ _ee to the background of normal organs and tumor lesions using ¹⁸ F-rhPSMA-7.3. Data shown as average, minimum and maximum values

ZSUmax ratio Ratio ZCUaverage average minimum maximum average minimum maximum Parotid gland 24.7 11.9 46.2 25.2 12.4 44.6 Submandibular gland 28.2 14.0 62.1 30.6 15.7 62.3 Blood pool 2.8 1.5 5.2 2.9 1.7 4.9 Lungs 1.0 0.6 1.8 1.0 0.6 1.8 Liver 9.7 4.0 19.0 I,4 4.1 20.7 Spleen I,4 3.6 22.4 13.3 3.9 28.6 Bud 51.8 25.7 93.0 55.6 27.6 95.5 Pancreas 4.1 2.2 6.9 4.4 2.3 7.8 duodenum intestine 16.2 6.9 34.3 17.1 4.7 39.4 Bone 2.0 1.1 3.2 2.1 1.0 3.6 Bladder 3.1 0.9 5.5 3.1 0.9 6.8 Tumor 43.6 1.7 321.2 50.8 1.8 356.4

Conclusion.

Biodistribution in humans is similar between ¹⁸ F-rhPSMA-7 and ¹⁸ F-rhPSMA-7.3 for most normal organs. However, the accumulation of tracer in the bladder is significantly lower, and the accumulation in tumor foci is significantly higher for ¹⁸ F-rhPSMA-7.3, which provides a clear advantage for clinical imaging. Examples of imaging with favorable human biodistribution and high tumor lesion accumulation ^{of 18} F-rhPSMA-7.3 are shown in FIG. 28.

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Claims (7)

CLAIM 1. A ligand-SIFA (silicofluoride acceptor fragment)-chelator conjugate, which is a compound of the formula where M ³⁺ is a chelated non-radioactive cation; or a pharmaceutically acceptable salt thereof. 2. The conjugate according to claim 1, where the chelator contains a chelated non-radioactive cation selected from the cations Sc, Cu, Ga, Y, In, Tb, Ho, Lu, Re, Pb, Bi, Ac, Th or Er. 3. The conjugate according to claim 2, where the chelator contains a chelated non-radioactive cation selected from Ga or Lu cations. 4. The conjugate according to claim 3, where the chelator contains a chelated non-radioactive cation selected from Ga ³⁺ cations. 5. The conjugate according to claim 1, which is a compound of the formula pharmaceutically acceptable salt. or him 6. A method for producing a ligand^[RL-chelator conjugate according to any one of claims 1-5, including isotopic exchange ^{of 19} F for ¹⁸ F in the ligand^[RL-chelator conjugate of the chemical formula is a chelated non-radioactive cation as defined in any of wherein M ³⁺ claims. 15. 7. Conjugate ligand^[RL-chelator chemical formula -