New PET Tracer for imaging of the functional hepatic reserve

Field of the Invention

The present invention relates to new and inventive compounds, especially compounds useful as PET (positron emission tomography) tracer. The present invention further relates to a pharmaceutical or diagnostic composition comprising such a compound. The present invention further relates to a method of synthesizing the compound. The present invention further relates to a method of imaging hepatocytes, comprising a) contacting the hepatocyte with such a compound and b) visualizing the compound that is in contact with the hepatocyte. Also, the present invention relates to a method of determining functional hepatic reserve, comprising a) contacting the liver with such a compound, and b) visualizing the compound that is in contact with the liver. The present invention further relates to the use of the compound for imaging hepatocytes as well as to the use for determining functional hepatic reserve.

Background of the Invention

The asialoglycoprotein receptor (ASGR) is a c-type lectin mainly expressed on the basolateral side of hepatocytes, where up to 500.000 receptors per cell can be found (D'Souza AA et al. J Control Release 2015;203:126-39) . In contrast, in the remainder of the body expression is low making it a promising target for e.g. drug delivery into hepatocytes. The main physiological function of the ASGR is to maintain serum glycoprotein homeostasis by clearing of de- sialylated glycoproteins bearing D-galactose (Gal) or N-acetylgalactosamine (GalNAc) as terminal carbohydrates. Therefore, these glycoproteins are internalized via receptor-mediated endocytosis. This is initiated by binding of the glycoprotein to the ASGR, followed by the migration of the receptor-ligand complex to active internalization sites (clathrin coated pits) where corresponding vesicles are formed leading to the internalization of the complex. Subsequently, in the cell, the glycoprotein is degraded in the lysosome and the ASGR is recycled (Stockert RJ. et al. Physiol Rev 1995;75:591-609; de Graaf W et al. J Nucl Med 2010;51 :742-52). The ASGR consists of two homologous subunits H1 and H2, which are responsible for Gal/GalNAc recognition (Lee YC et al. J Biol Chem 1983;258:199-202). Carbohydrate binding was elucidated by X-ray structure analysis of the carbohydrate recognition domain (CRD) of the H1 subunit (Meier M et al. J Mol Biol 2000;300:857-65).

Non-invasive methods allowing quantitative determination of the functional liver mass are of great interest for patient management in a diversity of clinical settings comprising liver surgery and liver transplantation (de Graaf W et al. J Nucl Med 2010;51 :742-52; Hoekstra LT et al. Ann Surg 2013;257:27-36; Kaibori M et al. Ann Nucl Med 2011 ;25:593-602) as well as diagnosis (Virgolini I et al.. Br J Cancer 1990;61 :937-41 ; Kurtaran A et al. J Nucl Med 1995;36:1875-81) and treatment monitoring (Virgolini I et al. Br J Cancer 1993;68:549-54) of cancer. Additionally, it has been revealed that the evaluation of remnant liver function can help to discriminate different stages of alcoholic liver cirrhosis (Virgolini I et al. Nucl Med Commun 1991 ;12:507- 17) and could be used to differentiate areas of steatosis, fibrosis and cholestasis (Bennink RJ et al. Semin Nucl Med 2012;42:124-37). Moreover, control of liver status before and during peptide receptor radionuclide therapy (PRRT) (Mansi L et al. Eur J Nucl Med Mol Imaging 2011 ;38:605-12) could lead to an optimized patient management. Further, patients who are potentially suitable for selective internal radiation therapy (SIRT) (D'Arienzo M et al. Nucl Med Commun 2012;33:633-40) may also benefit from such a diagnostic method, allowing stratifying patients according to their peri-interventional risk.

Due to the restricted expression, the ASGR is an optimal target structure for non-invasive monitoring of the liver function. [ ^99m Tc]Tc-diethylenetriarnine-pentaacetic acid galactosyl human serum albumin ([ ^99m Tc]Tc-GSA) (Kokudo N et al. Nucl Med Biol 2003;30:845-9) has been developed to image ASGR expression using single photon emission tomography (SPECT). It has been shown that [ ^99m Tc]Tc-GSA and dynamic SPECT allows estimation of regional hepatic function based on the determination of the ASGR density (Kudo M et al. Hepatology 1993;17:814-9; Vera DR et al. J Nucl Med 1996;37:160-4). To combine the superior performance of positron emission tomography (PET) compared with SPECT concerning imaging resolution and quantifying properties with the excellent properties of GSA in ASGR targeting, we developed a [ ⁶⁸ Ga]Ga -labelled analogue (Haubner R et al. Eur J Nucl Med Mol Imaging 2016;43:2005-13). [ ⁶⁸ Ga]Ga-DTPA-GSA showed comparable targeting properties as found for [ ^99m Tc]Tc-GSA but lacks high metabolic stability. By replacing DTPA with 2-S-(4-isothiocyanatobenzyl)-1 ,4,7-triazacyclononane-1 ,4,7-triacetic acid (p-NCS-Bn- NOTA), [ ⁶⁸ Ga]Ga-NOTA-GSA was developed which demonstrated the desired high metabolic stability, which was even higher as for the original ^99m Tc-labelled DTPA-GSA, allowing the use of pharmacokinetic modeling approaches for more detailed quantification (Haubner R et al. Mol Imaging Biol 2017;19:723-30). Moreover, it confirms the superiority of PET, which includes the possibility of time-resolved 3D volume determination allowing accurate quantification of the tracer dynamics throughout the entire liver and enabling the reader to delineate exactly the volumes of interest and move from a rough estimation of liver function to a spatially resolved quantification. Despite the good imaging performance of human GSA-based radiotracers the translation of this class of compounds into clinical practice is limited due to regulations of biological products isolated from human material. The requirements in the production of a GMP-compliant labelling precursor, which are essential for use in humans, increased during the last years. Thus, despite the good imaging performance, the translation of this human serum albumin based labelling precursor into clinical practice was not possible indicating the need of radiolabelled low molecular weight, synthetic derivatives as galactose carrier. Therefore, there is a particular research interest in the development of synthetic small molecule radiotracers based on galactose.

Summary of the Invention

It is the objective of the present invention to provide low molecular weight PET tracer for the non-invasive determination of the asialoglycoprotein receptor expression, and in particular for determining functional hepatic reserve.

This problem is solved according to the present invention by the following embodiments and aspects of the invention.

In a first aspect, the present invention relates to a compound according to formula (I) wherein D, R, L ¹ -L ³ , G and n are defined as disclosed below.

In a further aspect, the present invention relates to a pharmaceutical composition comprising the compound according to formula (I) and a pharmaceutically acceptable excipient.

In a further aspect, the present invention relates to a method of producing the compound according to formula (I), the method comprising synthesizing the compound.

In a further aspect, the present invention relates to a pharmaceutical or diagnostic composition comprising the compound according to formula (I) and at least one additive.

In a further aspect, the present invention relates to a method of imaging hepatocytes, comprising a) contacting the hepatocyte with a compound according to formula (I) or with a pharmaceutical or diagnostic composition comprising said compound, and b) visualizing the compound that is in contact with the hepatocyte.

In a further aspect, the present invention relates to a method of determining functional hepatic reserve, comprising a) contacting the liver with the compound according to formula (I) or with a pharmaceutical or diagnostic composition comprising said compound, and b) visualizing the compound that is in contact with the liver.

In a further aspect, the present invention relates to the use of the compound according to formula (I) or a pharmaceutical or diagnostic composition comprising said compound for imaging hepatocytes.

In a further aspect, the present invention relates to the use of the compound according to formula (I) or a pharmaceutical or diagnostic composition comprising said compound for determining functional hepatic reserve. Brief Description of the Drawings

The invention will be described in more detail in the following section and illustrated in accompanying figures.

Figure 1 shows a radio-HPLC of [ ⁶⁸ Ga]Ga-NODAGA-TriGalactan. Column: Reprosil Pur C AQ 150 x 4.6 mm, Gradient: 5-60 % B in 15 min. tp = 7.19 min.

Figure 2 shows biodistribution data (% ID/g) of [ ⁶⁸ Ga]Ga-NODAGA-TriGalactan (n = 3) and [ ^99m Tc] Tc-GSA (n = 3) in healthy BALB/c mice at 10, 30 and 60 min p.i.

Figure 3 shows the liver-to-organ ratios for [ ⁶⁸ Ga]Ga-NODAGA-TriGalactan and [ ^99m Tc] Tc- GSA 10, 30 and 60 min p.i.

Figure 4 shows biodistribution data (% ID/g) of [ ⁶⁸ Ga]Ga-NODAGA-TriGalactan (n = 3) in healthy BALB/c mice at 30 min p.i. and at 30 min blocked with 277 mM galactose.

Figure 5 shows a radio-HPLC of [ ¹⁸ F]AIF-NOTA-TriGalactan. Column: Phenomenex Kinetex C , 250 x 4.6 mm, Gradient: 5-60 % B in 15 min., 1.0 mL/min tp = 8.3 min.

Figure 6 shows a radio-HPLC of [ ⁶⁸ Ga]Ga-TRAP-Galactan. Column: Reprosil Pur C AQ 150 x 4.6 mm, Gradient: 5-15 % B in 15 min. tp = 12.7 and 13.3 min.

Figure 7 shows a radio-HPLC of [ ⁶⁸ Ga]Ga-Galacto-TRAP. Column: Reprosil Pur C AQ 150 x 4.6 mm, Gradient: 1-10 % B in 15 min. tp = 8.5 and 9.8 min.

Figure 8 shows biodistribution data (% ID/g) of [ ⁶⁸ Ga]Ga-TRAP-Galactan (n = 3) and [ ⁶⁸ Ga]Ga- Galacto-TRAP (n = 3) in healthy BALB/c mice at 10, 30 and 60 min p.i.

Figure 9 shows biodistribution data (% ID/g) of [ ⁶⁸ Ga]Ga-TRAP-Galactan (n = 3) and [ ⁶⁸ Ga]Ga- Galacto-TRAP in healthy BALB/c mice at 30 min p.i. and at 30 min blocked with 277 mM galactose.

Figure 10 shows liver-to-organ ratios for [ ⁶⁸ Ga]Ga-TRAP-Galactan (n = 3) and [ ⁶⁸ Ga]Ga- Galacto-TRAP (n = 3) 10, 30 and 60 min p.i.

Figure 11 shows biodistribution data (% ID/g) of [ ⁶⁸ Ga]Ga-TRAP-GalNAc (n = 3) and [ ⁶⁸ Ga]Ga- TRAP-Gluco (n = 3) in healthy BALB/c mice at 10, 30 and 60 min p.i.

Figure 12 shows a comparison of the biodistribution data (% ID/g) of [ ⁶⁸ Ga]Ga-TRAP-GalNAc (n = 3) and [ ^99m Tc]Tc-GSA (n = 3) in healthy BALB/c mice at 10, 30 and 60 min p.i.

Figure 13 shows a radio-HPLC of [ ⁶⁸ Ga]Ga-TRAP-GalNAc. Column: Reprosil Pur C AQ 150 x 4.6 mm, Gradient: 5-15 % B in 15 min. tp = 14.1 and 14.6 min.

Figure 14 shows a radio-HPLC of [ ⁶⁸ Ga]Ga-TRAP-Gluco. Column: Reprosil Pur C AQ 150 x 4.6 mm, Gradient: 5-15 % B in 15 min. tp = 13.9 and 14.5 min. Figure 15 shows PET/MR images, a-d PET/MR images of [ ⁶⁸ Ga]Ga-NODAGA-TriGalactan, image a shows a T2w-MRI signal, image b shows a representative frame of dynamic 60 min PET imaging, and image c is an overlay of images a and b. e-h PET/MR images of [ ¹⁸ F]AIF- NOTA-TriGalactan, image e shows a T2w-MRI signal, image f shows a representative frame of dynamic 60 min PET imaging, and image g is an overlay of images e and f.

Figure 16 shows PET/MR images of [ ⁶⁸ Ga]Ga-TRAP-GalNAc, image a shows a T2w-MRI signal, image b shows a representative frame of dynamic 60 min PET imaging, and image c is an overlay of images a and b.

Detailed Description of the Invention

All publications, including but not limited to patents, patent applications and scientific publications, cited in this description are herein incorporated by reference for all purposes as if each individual publication were specifically and individually indicated to be incorporated by reference.

The use of the term “comprising” as well as other grammatical forms such as “comprises” and “comprised” is not limiting. The terms “comprising”, “comprises” and “comprised” should be understood as referring to an open-ended description of an embodiment of the present invention that may, but does not have to, include additional technical features in addition to the explicitly stated technical features. In the same sense the term “involving” as well as other respective grammatical forms such as “involves” and “involved” is not limiting. The same applies for the term “including” and other grammatical forms such as “includes” and “included”. Section headings throughout the description are for organizational purposes only. In particular, they are not intended as limiting for various aspects and embodiments described therein, and it is to be understood that aspects and embodiments (and features therein) described under one subheading may be freely combined with aspects and embodiments (and features therein) described under another subheading. Further, the terms “comprising”, “involving” and “including”, and any grammatical forms thereof, are not to be interpreted to exclusively refer to embodiments that include additional features to those explicitly recited. These terms equally refer to embodiments that consist of only those features that are explicitly mentioned.

The skilled person understands that the present invention is not limited to the embodiments explicitly mentioned above and below, but also includes combinations of aspects, embodiments or features that are not explicitly discussed herein, but can be implicitly derived.

The present invention relates to a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein

D is a chelating group or a prosthetic group or a [ ¹⁸ F]F accepting group;

-L ¹ -R is absent or

L ¹ is a linker selected from the group consisting of *-N(H)-(CH2)x-C(O)-, aspartic acid, glutamic acid, asparagine, glutamine, lysine, *-N(H)-(CH2-CH2-O)y-(CH ₂ )z-C(O)-,

R is a multivalent group of the following formula

L ² is a linker selected from the group consisting of N~N ,*-CH2-C(O)-NH-, *-CH ₂ - wherein * designates i) the attachment point to D, if -L ¹ -R is absent, or ii) the attachment point to R, if -L ¹ -R is present;

L ³ is absent or a linker selected from the group consisting of ^# -(CH2-CH2-O) _m -CH2-CH2-,

^# -(CH ₂ )m-, and , wherein ^# designates the attachment point to L ² ;

G is Gal, GalNAc, or Lac; m is independently selected from 1-36; n is 3-4, with the proviso that n is 3 if — L ¹ -R is present; o is 3-6; q is independently selected from 1-16; r is independently selected from 1-36; s is 1-10; t is independently selected from 1-8; u is independently selected from 1-8; x is 1-16; y is 1-36; and z is 1-2.

In a preferred embodiment, the compound is excluded from formula (I).

In some embodiments, D is a chelating group selected from the group consisting of NODA GA, NOTA, DOTAGA, D03A, HBED, DPTA, optionally NCS-DTPA, DFO, optionally p-NCS-Bz- DFO, FusC, DafC, CNAAZTA, NOPO, TRAP, DOTPI, DOTAZA, sacrophagine-based chelators, TE2A derivatives such as CB-TE2A. Particularly preferred are NODAGA, NOTA and TRAP. More particularly, NODAGA and TRAP are preferred. In particular, NODAGA and NOTA are preferred if — L ¹ -R is present and TRAP is preferred if — L ¹ -R is absent. It is preferred that NODAGA and NOTA chelate [ ⁶⁸ Ga]Ga or [ ¹⁸ F]AIF. It is preferred that TRAP chelates [ ⁶⁸ Ga]Ga or [ ¹⁸ F]AIF. Coupling of NCS-DTPA or p-NCS-Bz-DFO to the linker (preferably L ¹ or L ² ) connects the DTPA or DFO moiety to the linker via a thiourea group.

In some embodiments, the moiety -L ¹ -R is absent. If — L ¹ -R is absent, L ² is directly bound to D.

In some embodiments, the moiety -L ¹ -R is present. Preferably, the linker L ¹ binds to the multivalent group R through the amino functionality

In some embodiments, -L ¹ -R is present and D is a chelating group selected from the group consisting of More preferably, -L ¹ -R is present and the chelator D is selected from

Most preferably, if — L ¹ -R is present in the compound according to formula (I), the chelator D is In some embodiments, L ¹ is a linker selected from the group consisting of *-N(H)-(CH2)x-C(O)- , aspartic acid, glutamic acid, asparagine, glutamine, lysine, *-N(H)-(CH2-CH2-O) _y -(CH2) _z -C(O)- In preferred embodiments, L ¹ is *-N(H)-(CH2)>rC(O)-, and * designates the attachment point to D. The index x in *-N(H)-(CH2)x-C(0)- represents an integer from 1-16. In preferred embodiments, x is 2-10, 2-8 or 2-6. Most preferably, x is 3.

In some embodiments, -L ¹ -R is present and L ² is selected from N N ,*-CH2-C(O)- NH-, and *-CH2-CH2-O-, wherein * designates the attachment point to R.

In a more preferred embodiment, -L ¹ -R is present and L ² is N N wherein * designates the attachment point to R.

In some embodiments, -L ¹ -R is present and L ³ is selected from ^# -(CH2-CH2-O) _m -CH2-CH2-, wherein ^# designates the attachment point to L ² . In more preferred embodiments, -L ¹ -R is present and L ³ is ^# -(CH2-CH2-O) _m -CH2-CH2-, and ^# designates the attachment point to L ² . The index m in ^# -(CH2-CH2-O) _m -CH2-CH2- represents an integer from 1-36, preferably from 2-24, 2-18, 2-12 or 2-6. Most preferably, m is 2. ^# -(CH2-CH2-O) _m - CH2-CH2- for m=2 is ^# -CH ₂ -CH2-O-CH2-CH2-O-CH2-CH ₂ -.

In some embodiments, -L ¹ -R is present and the linker unit -L ² -L ³ - connecting the sugar moiety G with the hydroxy functionalities of the multivalent group R has a length of about 10-30 A, preferably about 14-20 A. For instance, the length of -L ² -L ³ - can be about 11 A, 12 A, 13 A, 14 A, 15 A, 16 A, 17 A, 18 A, 19 A, 20 A, 21 A, 22 A, 23 A, 24 A, 25 A, 26 A, 27 A, 28 A or 29 A.

In a preferred embodiment, the compound is In a further preferred embodiment, the compound is

In some embodiments, the moiety -L ¹ -R is absent; the compounds of the present invention are then represented by structural formula (II)

Compounds comprised by structural formula (II) have a group D as the central functionality to which sugar moieties G are attached via the linker group L ² -L ³ . Preferably, D is a chelating group. In some embodiments, -L ¹ -R is absent and D is a chelating group selected from the group consisting of In more preferred embodiments, -L ¹ -R is absent and the chelator D is

In some embodiments, -L ¹ -R is absent and L ² is selected from N N , wherein * designates the attachment point to D.

In more preferred embodiments, -L ¹ -R is absent and L ² is wherein designates the attachment point to D.

The index q ranges from 1-16, preferably from 1-12, 1-6, 1-4 or 1-3. In a more preferred embodiment, the index q is 1 or 2. The index r ranges from 1-36, preferably from 1-30, 1-24, 1-18, 1-12, 1-10, 1-8, 1-6, 1-4 or 1-3. In a more preferred embodiment, the index r is 1 or 2. The index s ranges from 1-10, preferably from 1-8, 1-6, 1-4 or 1-3. In a more preferred embodiment, the index s is 1 or 2. In some embodiments, -L ¹ -R is absent and L ³ is selected from ^# -(CH2-CH2-O) _m -CH2-CH2-, wherein ^# designates the attachment point to L ² . In more preferred embodiments, -L ¹ -R is absent and L ³ is ^# -(CH2-CH2-O) _m -CH2-CH2-, wherein ^# designates the attachment point to L ² . The index m in ^# -(CH2-CH2-O) _m -CH2-CH2- represents an integer from 1-36, preferably from 2-24, 2-18, 2-12 or 2-6. Most preferably, m is 2.

In some embodiments, -L ¹ -R is absent and the linker unit -L ² -L ³ - connecting the sugar moieties G with the chelator D has a length of 10-30 A, preferably 14-20 A. For instance, the length of -L ² -L ³ - can be about 11 A, 12 A, 13 A, 14 A, 15 A, 16 A, 17 A, 18 A, 19 A, 20 A, 21 A, 22 A, 23 A, 24 A, 25 A, 26 A, 27 A, 28 A or 29 A. In a preferred embodiment, the compound is In a further preferred embodiment, the compound is

(TRAP-GalNAc)

In a preferred embodiment, -L ¹ -R and L ³ are both absent. In a more preferred embodiment, the compound is

D is a chelating group (chelator), a prosthetic group or a [ ¹⁸ F]F accepting group.

The term “chelator” or “chelating group” is known in the art and refers to organic compounds that are polydentate ligands that form two or more coordinate bonds with a radionuclide. The chelator may contain different donor groups for metal complexation such as oxygen, nitrogen, sulphur, (carboxyl, phosphonate, hydroxamate, amine, thiol, thiocarboxylate or derivatives thereof) and comprises acyclic and macrocyclic chelators such as polyaminopolycarboxylic ligands. The introduction of the radionuclide into the chelator is typically performed after conjugation of the chelator to the remainder of the molecule. The term “prosthetic group” is known in the art and refers to a bifunctional labelling agent that is a small organic molecule that can be easily radiolabelled, for example with a radionuclide, and conjugated to a second molecule, e.g., a biomolecule such as a sugar containing molecule. Generally, prosthetic groups are conjugated to amines, thiols or carboxylic acid functions present in the second molecule. Also conjugation via click chemistry is possible. The term “[ ¹⁸ F]F accepting group” refers to a group that can be selectively covalently modified with [ ¹⁸ F]F. In some embodiments, D is a chelating group and coordinates to (chelates) a radionuclide. The radionuclide is preferably selected from [ ⁶⁸ Ga]Ga, [ ¹⁸ F]AIF, [ ⁸⁹ Zr]Zr, [ ⁶⁴ Cu]Cu and [ ⁸⁶ Y]Y, more preferably [ ⁶⁸ Ga]Ga or [ ¹⁸ F]AIF, most preferably [ ⁶⁸ Ga]Ga.

In some embodiments, the chelator is selected from the group consisting of NODAGA (1 ,4,7- triazacyclononane-1 -glutaric acid-4, 7-diacetic acid), NOTA (1 ,4,7-triazacyclononane-1 ,4,7- triacetic acid), DOTAGA (1 ,4,7, 10-Tetraazacyclododecane-1 -glutaric acid-4,7, 10-triacetic acid), DO3A (1 ,4,7,10-Tetraazacyclododecane-1 ,4,7-triacetic acid), HBED (N,N'-di(2- hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid), NCS-DTPA (S-2-(4- lsothiocyanatobenzyl)-diethylene-triamine pentaacetic acid), p-NCS-Bz-DFO (N1-hydroxy-N1- (5-(4-(hydroxy(5-(3-(4-isothiocyanatophenyl)thioureido)penty l)amino)-4- oxobutanamido)pentyl)-N4-(5-(N-hydroxyacetamido)pentyl)succi namide), FusC (Fusarinine C), DafC (Diacetyl-Fusarinine C), CNAAZTA (1 ,4-bis(carboxymethyl)-6- [bis(carboxymethyl)]amino-6-(9-carboxynonyl)-perhydro-1 ,4-diazepine), NOPO (3-(((4,7- bis((hydroxy(hydroxymethyl)-phosphoryl)methyl)-1 ,4,7-triazonan-1-yl)methyl)(hydroxy)- phosphoryl)propanoic acid), TRAP (3,3',3”-(((1 ,4,7-triazonane-1 ,4,7- triyl)tris(methylene))tris(hydroxyphosphoryl))tripropanoic acid, DOTPI (1 ,4,7,10- tetraazacyclododecane-1 ,4,7, 10-tetrakis[methylene(2-carboxyethylphosphinic acid)), DOTAZA (1 ,4,7,10-Tetra-azacyclododecan-1 ,4,7,10-tetra-azidoethylacetic acid), sacrophagine-based chelators, and TE2A derivatives such as CB-TE2A (2,2'-(1 ,4,8, 11 - tetraazabicyclo[6.6.2]hexadecane-4,11-diyl)diacetic acid).

The radionuclide is preferably a positron-emitting isotope of a metal or halogen suitable for PET imaging. Suitable radionuclides to be coordinated to the chelating group include but are not limited to [ ⁸⁹ Zr]Zr, [ ⁴⁴ Sc]Sc, [ ⁴⁵ Ti]Ti, [ ⁵¹ Mn]Mn, [ ⁶⁴ Cu]Cu, [ ⁶¹ Cu]Cu, [ ⁶³ Zn]Zn, [ ⁶⁸ Ga]Ga, [ ¹¹ C]C, [ ¹²⁴ l]l, and [ ¹⁸ F]AIF. In some embodiments, the radionuclide is a metal ion such as [ ⁶⁸ Ga]Ga, [ ⁸⁹ Zr]Zr, [ ⁴⁴ Sc]Sc, [ ⁶⁴ Cu]Cu or [ ⁸⁶ Y]Y, more preferably [ ⁶⁸ Ga]Ga or [ ⁶⁴ Cu]Cu. Most preferably, the radionuclide is [ ⁶⁸ Ga]Ga. In some other embodiments, the radionuclide is a halide such as [ ¹²⁴ l]l or [ ¹⁸ F]AIF, most preferably [ ¹⁸ F]AIF. In some embodiments, the radionuclide is selected from [ ⁶⁸ Ga]Ga and [ ¹⁸ F]AIF.

It is to be understood that various factors may be included in the selection of a suitable positron-emitting isotope. Such factors include a sufficient half-life of the isotope to allow preparation of a diagnostic composition prior to administration to the patient, and a sufficient remaining half-life to yield sufficient activity to allow non-invasive measurement by PET. The isotope should have a sufficiently short half-life to limit patient exposure to unnecessary radiation. Suitable isotopes to be coordinated by the chelating groups of the present invention are ⁶⁸ Ga and [ ¹⁸ F]AIF. Preferred prosthetic groups of the present invention are labelled with radionuclides of halogens such as iodine, bromine or fluorine, preferably fluorine, more preferably [ ¹⁸ F]F. The introduction of the radionuclide into the prosthetic group in typically performed before conjugation of the prosthetic group to the remainder of the molecule (e.g. conjugating D to L ¹ -R-(L ² -L ³ -G) _n or (L ² - L ³ -G) _n ).

In some embodiments the radiolabelled prosthetic group is selected from a group comprising but not limited to 4-[ ¹⁸ F]fluorophenacyl bromide, N-succinimidyl-4-[ ¹⁸ F]fluorobenzoate ([ ¹⁸ F]SFB), N-succinimidyl-4-([ ¹⁸ F]fluoromethyl)benzoate, 4-[ ¹⁸ F]fluorobenzaldehyde, ¹⁸ F-6- fluoropyridine-3-carboxylic acid 2,3,5,6-tetrafluorophenyl ester ([ ¹⁸ F]F-Py-TFP), silicon- containing building blocks such as N-succinimidyl 3-(di-tert-butyl[ ¹⁸ F]fluorosilyl)benzoate ([ ¹⁸ F]SiFB), carbohydrate-based radiolabelled prosthetic groups, such as [ ¹⁸ F]fluoro- deoxyglucose, preferably 2-[ ¹⁸ F]fluoro-2-deoxyglucose ([ ¹⁸ F]FDG), and [ ¹⁸ F]fluoro- deoxymannose, preferably [ ¹⁸ F]2-fluoro-2-deoxymannose, or derivatives thereof, maleimide- based and heterocyclic methylsulfone-based ¹⁸ F-synthons, ¹⁸ F-labelled prosthetic groups such as ¹⁸ F-azides or ¹⁸ F-alkynes permitting labelling via click chemistry, ¹⁸ F-labelled organotrifluoroborates and [ ¹⁸ F]fluoropyridines. The preferred radiolabelled prosthetic group is [ ¹⁸ F]-6-fluoropyridine-3-carboxylic acid 2,3,5,6-tetrafluorophenyl ester (F-Py-TFP). The linkers are designed to have a N-terminal amino function allowing labelling via any prosthetic group, which reacts with an amine.

In some embodiments a chelator-based labelling approach using aluminium-fluoride ([ ¹⁸ F]AIF) is applied for radiofluorination.

In some embodiments, D is a [ ¹⁸ F]F accepting group. In some embodiments, direct labelling of the compound of the present invention is achieved by attaching a [ ¹⁸ F]F accepting group to the remainder of the molecule. For instance, N,N,N-trimethyl-5-[(2, 3,5,6- tetrafluorophenoxy)carbonyl]pyridin-2-aminium trifluoromethanesulfonate can be conjugated to the amino function of R group under release of tetrafluorophenol. The trimethylammonium group acts as a leaving group and can be replaced by, e.g. [ ¹⁸ F]F.

Also contemplated is the use of para-substituted di-tert-butylfluorosilylbenzene structural motif, which is known as the silicon-fluoride acceptor (Si FA) , for incorporating radioactive [ ¹⁸ F]F into the compound of the present invention. Labelling with [ ¹⁸ F]F is performed by isotopic exchange of [ ¹⁹ F]F from SiFA with [ ¹⁸ F]F.

In some embodiments, the compound has the following structure . In some embodiments, the fluorine of the tert-butylfluorosilylbenzene moiety is ¹⁸ F.

In some embodiments, the compound has the following structure . in some embodiments, the trimethylammonium group is replaced with ¹⁸ F.

The sugar moiety G is galactose (Gal, D-galacto-hexopyranose), galactose with an acetylated amino residue at position 2 (GalNAc, 2-(acetylamino)-2-deoxy-D-galactose), or lactose (Lac). More preferably, G is Gal or GalNAc. The sugar moiety may be connected to the linker L ³ via an O-glycosidic linkage, an N-glycosidic linkage, an S-glycosidic linkage or a C-glycosidic linkage. In a preferred embodiment, the linkage is O-glycosidic.

The present invention further relates to pharmaceutical compositions, preferably for diagnostic use, comprising the compound described herein and a pharmaceutically acceptable additive.

The present invention further provides a method for synthesizing the compound described herein. The method comprises the steps of (la) activating a group R to allow coupling of R with a modified sugar residue;

(2a) reacting the activated group of step (1a) with a modified sugar residue;

(3a) conjugation of L ¹ to R; and

(4a) attaching D to the resulting product of step (3a); or

(l b) activating D to allow coupling with a modified sugar residue, and

(2b) reacting the activated D with a modified sugar residue.

The present invention also relates to a method of imaging cells as described herein. The method of imaging cells described herein makes use of the compound described herein.

The present method of imaging cells makes us of established methods of imaging. Preferably, positron emission tomography (PET) is used for imaging the cells. PET is a nuclear medicine imaging technique that detects pairs of gamma rays emitted indirectly by a positron-producing radionuclide. Compared to other nuclear medicine imaging techniques such as Single Photon Emission Computed Tomography (SPECT), PET is known to show higher sensitivity, better spatial resolution, greater signal to noise ratio, and superior tracer quantification in both preclinical and clinical applications.

Combining the functional imaging obtained by PET, which depicts the spatial distribution of metabolic or biochemical activity in the body, with computer tomography (CT) allows to more precisely align or correlate the results with the anatomy of the body. Combination of PET with magnetic resonance imaging (MRI) combines the exquisite structural and functional characterization of tissue provided by MRI with the extreme sensitivity of PET imaging of metabolism and tracking of uniquely labelled cell types or cell receptors.

Radiopharmaceuticals such as the compounds of the present invention are of great interest for patient management in a diversity of clinical settings comprising liver surgery and liver transplantation, diagnosis, and treatment monitoring of cancer, as well as other liver diseases like alcoholic liver cirrhosis and liver fibrosis. The optimal liver function assessment will allow a precise evaluation of the different liver segments aimed at better predicting liver failure following major liver surgery.

The compounds of the present invention overcome the drawbacks of the human GSA-based radiotracers, e.g., [ ^99m Tc]Tc-GSA, in the art because they can be easily prepared and put to practice in clinical routine. Surprisingly, the present inventors have found that the compounds of the present invention show higher affinity for the target receptors compared to the GSA- based radiotracer. Without being bound by theory, it is thought that due to the lower molecular mass, elimination from the body is faster resulting, at least for later time points, in improved liver to organ ratios compared with GSA-based radiotracer such as [ ^99m Tc]Tc-GSA.

In one aspect, the present invention provides a method of imaging hepatocytes, comprising a) contacting the hepatocyte with a compound of the present invention and b) visualizing the compound that is in contact with the hepatocyte.

In a further aspect, the present invention provides a method of determining functional hepatic reserve, comprising a) contacting the liver with the compound of the present invention, and b) visualizing the compound that is in contact with the liver.

In some embodiments, the visualizing is performed via positron emitting computer tomography (PET).

The methods of the invention may be performed in vivo or in vitro.

The contacting of the hepatocyte or the contacting of the liver with the compound can be achieved, for example, by administering the compound to a patient. Administration to the patient can be by injection, in particular, by intravenous injection.

For administration to a patient, the compound is formulated in a suitable dosage form. One suitable dosage form is a pharmaceutical or diagnostic composition, comprising at least one additive, such as saline.

In some embodiments, the molar activity of the radiolabelled compound ranges from about 50 MBq/nmol up to about 33 GBq/nmol.

In some embodiments, if D is a chelator group and the radionuclide is [ ⁶⁸ Ga]Ga, the molar activity of the radiolabelled compound is about 50-500 MBq/nmol. In preferred embodiments, the molar activity of the radiolabelled compound is about 50-450 MBq/nmol, about 50-400 MBq/nmol, about 50-450 MBq/nmol, about 50-400 MBq/nmol, about 100-500 MBq/nmol, about 100-450 MBq/nmol, about 100-400 MBq/nmol, or about 120-400 MBq/nmol. In a more preferred embodiment, the molar activity of the radiolabelled compound is about 100-400 MBq/nmol.

In some embodiments, if D is a prosthetic group and the radionuclide is [ ¹⁸ F]F or if D is an [ ¹⁸ F]F acceptor group, the molar activity of the radiolabelled compound ranges from 1000 MBq/nmol up to 33 GBq/nmol. In preferred embodiments, the molar activity of the radiolabelled compound is about 3000 MBq/nmol - 20 GBq/nmol, about 3500 MBq/nmol - 10 GBq/nmol, about 3500-5000 MBq/nmol, or about 3500-4500 MBq/nmol. In a more preferred embodiment, the molar activity of the radiolabelled compound is about 3500-5000 MBq/nmol.

In some embodiments, if D is a chelator group and the radionuclide is [ ¹⁸ F]AIF, the molar activity of the radiolabelled compound ranges from 10 MBq/nmol up to 2 GBq/nmol. In some embodiments, if the radiolabelled compound is produced via isotopic exchange reaction, the molar activity is about 50-500 MBq/nmol, more preferably about 50-450 MBq/nmol.

Examples

The compounds of the present invention can be prepared by a combination of methods known in the art including the procedures described in schemes 1 , 2 and 3 below. The following reaction schemes are meant only to exemplify and do not limit the invention.

The labelling precursor NODAGA-TriGalactan was obtained in a seven-step synthesis (Scheme 1). For the first reaction step, Boc-protected Tris (1) was reacted with propargylbromide to give the respective ether conjugate (2). Next, Cu-catalyzed click chemistry was used to attach three galactose moieties to the backbone followed by removal of the Boc- protection group using trifluoroacetic acid (3). In a subsequent step y-amino butyric acid was introduced as a linker on the free amino function of Tris (4). Coupling of R-NODAGA and subsequent removal of all acetyl protection groups from the galactoses yielded NODAGA- TriGalactan (6).

Scheme 1

The labelling precursor TRAP-Galactan (4) was obtained in a four-step synthesis (Scheme 2).

For the first reaction step the chelator TRAP-Pr (1) was functionalized with propargylamine to give TRAP-Alkynes (2). Next, Cu-catalyzed click chemistry was applied to attach three galactose moieties to the chelator followed by demetallation of the resulting Cu-complex. (3).

Subsequent removal of all acetyl protection groups from the galactoses yielded TRAP-

Scheme 2

The labelling precursor Galacto-TRAP (6) was obtained in a four-step synthesis (Scheme 3). For the first reaction step the chelator TRAP-Pr (1) was functionalized with propargylamine to give TRAP-Alkynes (2). Next, Cu-catalyzed click chemistry was used to attach three galactose moieties to the chelator followed by demetallation of the resulting Cu-complex. (5). Subsequent removal of all acetyl protection groups from the galactoses yielded Galacto-TRAP (6).

Scheme 3

Example 1 : Synthesis of NODAG A-TriGalactan Step 1 : Synthesis of Boc-Tris(Proparqyl)3 (2)

Formula Weight: 335.3948

Monoisotopic Mass: 335.173273 Da

To a 100 mL round bottom flask 500 mg (2.3 mmol, 1 eq) of Boc-protected Tris ((1) in Scheme 1) were added as a solid and dissolved in 6 mL of dry DMF. The solution was cooled on ice followed by addition of 1.5 mL of propargylbromid in toluene (80%, 13.9 mmol, 6.2 eq) as well as 1 g (17.9 mmol, 7.9 eq) of fine powdered KOH. The reaction mixture was stirred on ice for 15 min and was allowed to warm up to room temperature afterwards. For workup, 20 mL of ethylacetate were added and the resulting solution was transferred into a separation funnel where the organic layer was washed three times with 20 mL of distilled water. The organic layer was separated, and the aqueous extracts were washed twice with 20 mL of ethylacetate. The combined organic fractions were dried over Na2SO4, filtered and all volatiles removed in vacuo yielding the raw product as a yellowish oil which was subsequently purified on column chromatography using a gradient of n-hexane/ethylacetate (90/10 - 40/60). The product containing fractions were pooled and all volatiles were removed in vacuo to yield the product as a yellowish oil that solidified upon storage in the refrigerator.

¹ H-NMR (CDCh): 6 (ppm) = 4.91 (s, 1 H, NH), 4.13 (m, 6H, CH ₂ -C C), 3.75 (s, 6 H, O-CH ₂ ), 2.48 (t, 3 H, ⁴ J = 1.8 Hz, C CH), 1.41 (s, 9H, CH ₃ ).

MALDI-MS (m/z) = 336.3 [M+H] ⁺ , 358.3 [M+Na] ⁺ .

Step 2: Synthesis of Tris(Ac4-Gal)3 (3)

Formula Weight: 361.33928

Monoisotopic Mass: 361.148509 Da

Compound 2 (24.9 , 74.2 pmol, 1 eq) was weighed into a 15 mL Falcon-Tube and a solution of 135 mg (267 pmol, 3.6 eq) 1-Azido-1-deoxy-p-D-Galactopyranoside in 600 pL of MeOH was added. The reaction was started by subsequent addition of an aqueous solution of CU(OAC) ₂ *H ₂ O (17.8 mg, 89.1 pmol, 1.2 eq) and sodium ascorbate (586 mg, 3 mmol, 40 eq) and the resulting mixture was heated on a water bath at 60°C for 1 h. For workup all volatiles were removed in vacuo and for Boc-deprotection the residue was resuspended in 4 mL of TFA/TIPS/H ₂ O (95/2.5/2.5) and incubated for 1 h at room temperature. After removal of TFA under a stream of Argon, the raw product was dissolved in 1.6 mL of H ₂ O/30% MeCN (vol/vol) and subjected to semipreparative RP-HPLC (30-50% B in 20 min). The product was obtained as a colorless oil (27 mg 14.5 pmol, 20%).

Analytical HPLC (ReproSil Pur, 30-100% B in 15 min, 1.0 mL/min) tp = 8.5 min (70% B)

MALDI-MS (m/z) = 1750.2 [M+H] ⁺ , 1772.2 [M+Na] ⁺ .

Step 3: Synthesis of GABA-Tris(Ac4-Gal)3 (4)

Formula Weight: 446.44376

Monoisotopic Mass: 446.201273 Da

In a V-shaped vial HOAt (3.95 mg, 29.0 pmol, 2 eq), HATLI (11.0 mg, 29.0 pmol, 2 eq), and Boc-GABA (5.89 mg, 29.0 pmol, 2 eq) were dissolved in 100 pL of anhydrous DMF and DI PEA (15.2 pL, 87.0 pmol, 6 eq). After 10 minutes of pre-activation this solution was added dropwise to a solution of compound 3 (27.0 mg, 14.5 pmol, 1 eq) in 350 pL of dry DMF and 15.2 pL (87.0 pmol, 6 eq) DI PEA. The reaction mixture was allowed to stand at room temperature for 2 h before 500 pL of water were added to deactivate the remaining coupling agents. The solution was transferred to a 10 mL round bottom flask and all volatiles were removed in vacuo. For Boc-deprotection the residue was resuspended in 1 mL TFA/TIPS/H2O (95/2.5/2.5) and incubated for 1 h at room temperature. After removal of TFA under a stream of Argon the raw product was dissolved in 660 pL H2O/30% MeCN and subjected to semipreparative RP-HPLC (30-45% B in 20 min). The product was obtained as a colorless oil (15.9 mg, 8.16 pmol, 56%).

Analytical HPLC (ReproSil Pur, 20-80% B in 15 min, 1.0 mL/min) tp = 11.2 min (65% B).

MALDI-MS (m/z) = 1835.8 [M+H] ⁺ , 1857.2 [M+Na] ⁺ . Step 4: Synthesis of NODAGA-TriGalactan (6)

Formula Weight: 1689.72144

Monoisotopic Mass: 1688.772751 Da

(R)-NODAGA-NHS (7.17 mg, 9.79 pmol, 1.2 eq) was weighed into a 1.5 mL Eppendorf reaction vial and was dissolved in 100 pL of anhydrous DMF and DI PEA (4.27 pL, 24.5 pmol, 3 eq). This mixture was then added dropwise to a solution of compound 4 (15.9 mg, 8.16 pmol, 1 eq) in 300 pL of anhydrous DMF and DI PEA (8.53 pL, 49.0 pmol, 6 eq). The pH was adjusted by addition of another 3 eq DI PEA and the mixture was stirred over night at room temperature. For removal of the acetyl protection groups the reactions mixture was reduced to dryness and the residue was dissolved in 3 mL of NEt ₃ /MeOH/H ₂ O (1/6/2). After 12 h all volatiles were removed in vacuo and the raw product was subjected to purification via semi-preparative RP- HPLC (12% B isocratic). After lyophilisation the product was obtained as a white solid (4.48 mg, 2.34 pmol, 29%).

Analytical HPLC (ReproSil Pur, 5-60% B in 15 min, 1.0 mL/min) tp = 7.2 min (32% B).

MALDI-MS (m/z) = 1689.7 [M+H] ⁺ .

Example 2: Labelling of NODAGA-TriGalactan with ⁶⁸ Ga- and ^nat Ga-gallium ⁶⁸ Ga-NODAGA-TriGalactan For labelling with Gallium-68 a fractionated elution approach was applied. An overall activity of 200 MBq was eluted from the ⁶⁸ Ga/ ⁶⁸ Ge generator in 1.5 mL fractions and the activity content of each fraction was calculated using a dose calibrator. From the fraction containing the highest activity, 550 pL were added to a mixture of 5 nmol (1 mM, 5 pL) of the labelling precursor in 100 pL of a 1 M NaOAc/HOAc-buffer (pH = 5). The mixture was heated to 56°C for 10 min and completion of the labelling reaction was monitored using radio-TLC and radio- HPLC (ReproSil Pur, Gradient: 10-60 %B in 15 min). If required the labelled compound was further purified via fixation on a Waters (Milford, Massachusetts, USA) Sep Pak Ct 18 Light cartridge. For pre-conditioning the cartridge was flushed with 5 mL of EtOH followed by 10 mL of water and 1mL of air. The labelling solution was loaded onto the cartridge and 10 mL of water were used for washing. The final compound was eluted using 300 pL of EtOH/H2O (1 :1) and diluted with PBS for further usage. ^nat Ga-NODAGA-TriGalactan

Complexation with ^nat Ga was done accordingly to a published procedure (A. Wurzer et al. J Nucl Med, 2020). A 2 mM stock solution of NODAGA-TriGalactan (500 pL, 1 pmol, 1 eq) was mixed with 150 pL of a 20 mM ^nat GaBr3 solution (3 pmol, 3 eq) and incubated for 10 min at 56°C.

RP-HPLC (ReproSil Pur, 5-60% B in 15 min) tp = 7.2 min. Calculated monoisotopic mass (C ₆ 8Hii3GaNi ₄ O35): 1754.7 Da; found (m/z) = 1756.1 [M+H] ⁺ , 1778.0 [M+Na] ⁺ , 1794.0 [M+K] ⁺ . Radiolabelling of NODAGA-TriGalactan with ⁶⁸ Ga was achieved in high radiochemical yields and purity as determined by radio-HPLC (> 99%) (Fig. 1) and radio-TLC (> 98%). Molar activities were usually between 8 - 10 MBq/nmol. Labelling with ^nat Ga with a 3-fold molar excess over the labelling precursor occurred instantly and led to formation of [ ^nat Ga]Ga- NODAGA-TriGalactan in quantitative yields and high purity (> 97%) as determined by HPLC and MS analysis.

Example 3: Synthesis of NOTA-TriGalactan

Step 1 : Synthesis of GABA-Tris(PEG3-Gal)3 (7)

Formula Weight: 1332.36 g/mol Monoisotopic Mass: 1331 .62 Da Molecular Formula: C ₆₃ H ₉₃ N ₁₁ O ₂₈

Compound 4 of Scheme 1 (4.77 mg, 2.45 pmol, 1.0 eq) was treated over night with a mixture of NEts/MeOH/FW (1 :6:2). All volatiles were removed in vacuo and the deprotected trimer was purified via semipreparative HPLC (12-17% B in 25 min). Subsequent lyophilisation yielded 1.95 mg (1.47 pmol, 60 %) of a colorless oil.

MALDI-MS (m/z) = 1332.7 [M+H] ⁺ , 1394 [M+H+Na+K] ⁺ .

Step 2: Synthesis of NOTA-TriGalactan (8)

Formula Weight: 1617.66 g/mol Monoisotopic Mass: 1616.75 Da

Molecular Formula: C _Kq H ₁₁ ,N ₁ ,O„

Compound 7 (750 pg, 560 nmol, 1.0 eq) was dissolved in 50 pL of dry DMF and 1 pL of DI PEA. To this mixture a solution of NOTA-NHS (940 pg, 1.4 pmol, 2.5 eq) in 50 pL of dry DMF was added and the pH was adjusted to 9 by addition of another 0.5 pL of DIPEA. After 1.5 h at room temperature, the reaction mixture was diluted ten-fold with Millipore water and directly subjected to purification via HPLC (5-25% B in 15 min). Lyophilization yielded 600 pg (370 nmol, 66%) of a white solid.

Analytical HPLC (ReproSil Pur, 5-60% B in 15 min, 1.0 mL/min) tp = 7.4 min (28% B)

ESI-MS (m/z) = 1617.8 [M+H] ⁺ .

Example 4: Labelling of NOTA-TriGalactan with [ ¹⁸ F]AIF

For fluorination ¹⁸ F (=2 GBq) was trapped on a QMA cartridge (HCCh' form) and washed with 5 mL of Millipore water. The activity was eluted inversely from the cartridge in 200 pL fractions using an 0.1 M NaOAc/HOAc buffer (pH 4). To the fraction containing the highest activity (»1.6 GBq) 10 pL of a 2 mM AlCh solution in 0.1 M NaOAc/HOAc buffer (pH 4) were added and incubated for 5 min at RT. From that solution 600-800 MBq were transferred to a 1.5 mL protein-low bind tube, followed by the addition of 15 nmol NOTA-TriGalactan (15 pL, 1 mM) as well as 100 pL of an 0.1 M NaOAc/HOAc buffer (pH 4). The mixture was heated to 99°C for 15 min at 1300 rpm and passed through a preconditioned C SepPak Light Plus cartridge. Unchelated [ ¹⁸ F]AIF was removed by flushing the cartridge with 10 mL of Millipore water and the labelled compound was eluted using 500 pL of water/ethanol (1 :1). For injection into animals the tracer was further diluted 1 :5 with PBS.

Radio-HPLC (Pheneomenex Kinetex C , 5-60% B in 15 min, 1.0 mL/min) tp = 8.3 min (32% B).

The radio-HPLC of [ ¹⁸ F]AIF-NOTA-TriGalactan is shown in Figure 5.

Example 5: Synthesis of TRAP-Galactan

Step 1 : Synthesis of TRAP-Alkynes

Formula Weight: 690.602286 Monoisotopic Mass: 690.246085 Da

TRAP-Pr (200 mg, 346 pmol, 1.00 eq) was weighed into a 10 mL round bottom flask and got dissolved in 1 mL DMSO and 724 pL DIPEA (4.16 mmol, 12.0 eq). The solution stirred for about 5 min at room temperature before propargylamine hydrochloride (158 mg, 1.73 mmol, 5.00 eq) and HATLI (1.18 g, 3.12 mmol, 9.00 eq) were added as a solid. The mixture stirred for 1 h at room temperature, then all volatiles were removed in vacuo and the oily residue got transferred into a 15 mL Falcon tube followed by addition of 3 x 500 pL Millipore water to deactivate the remaining coupling agents. After centrifugation (10 min, 3000 rpm) the supernatant was syringe filtered, acidified by addition of 25 pL of TFA and subjected to semipreparative RP-HPLC purification (12% B isocratic). After lyophilization the product was obtained as a colorless oil (232 mg, 288 pmol, 83%).

RP-HPLC (5-15% B in 15 min) t _R = 11.7 min. ESI-MS (m/z) = 691.2 [M +H] ⁺ .

Step 2: Synthesis of TRAP-Galactan

Formula Weight: 1702.581246

Monoisotopic Mass: 1701.691629 Da

TRAP-Alkynes (17.7 mg, 22.0 pmol, 1.00 eq) was dissolved in 35 pL Millipore water and was added to a solution of 40.0 mg (79.1 pmol, 3.60 eq) Ac4-Gal-PEG3-Azid in 200 pL of MeOH. Furthermore aqueous solutions of 5.27 mg (26.4 pmol, 1.20 eq) Cu(OAc)2*H2O and 174 mg (880 pmol, 40.0 eq) of sodium ascorbate were added and the resulting mixture was stirred for 1 h at 60°C. After that the reaction mixture was centrifuged (14000 rpm, 5 min) and the supernatant was added to a solution of 330 mg (662 pmol, 30.0 eq) DTPA in 300 pL of Millipore water. The pH was adjusted to 2.2 using concentrated HCI and the resulting solution was heated to 60°C for 1 h. Completion of the demetallation reaction was monitored by MS and the acetyl protected intermediate was isolated by semi-preparative RP-HPLC (38% B isocratic). Fractions of the intermediate were collected and reduced to dryness before 3 mL of NEt3/MeOH/H2O (1/6/3) were added for deacetylation. After 24 h at room temperature all volatiles were removed in vacuo and the residue was dissolved in 1.5 mL of H2O/10% MeCN. Purification via semi-preparative RP-HPLC (5-10 % B in 25 min) followed by lyophilization yielded 21.8 mg (11.3 pmol, 51 %) of a waxy solid.

RP-HPLC (5-15% B in 15 min) t _R = 13.5 min.

MALDI-MS (m/z) = 1748.3 [M+2Na] ⁺ , 1762.3 [M+Na+K] ⁺ .

Example 6: Synthesis of Galacto-TRAP

Formula Weight: 1306.108206

Monoisotopic Mass: 1305.455696 Da

TRAP-Alkynes (10.0 mg, 12.4 pmol, 1.00 eq) were dissolved in 20 pL of Millipore water and was added to a solution of 1-Azido-1-deoxy-p-D-Galactose (16.7 mg, 44.6 pmol, 3.60 eq) in 150 pL hot MeOH. Furthermore aqueous solutions of 2.95 mg (14.8 pmol, 1.20 eq) CU(OAC)2*H2O and 73.7 mg (372 pmol, 30.0 eq) of sodium ascorbate were added and the resulting mixture was stirred for 1 h at 60°C. After that the reaction mixture was centrifuged (14 000 rpm, 5 min) and the supernatant was added to a solution of 185 mg (372 pmol, 30.0 eq) DTPA in 500 pL of Millipore water. The pH was adjusted to 2.2 using concentrated HCI and the resulting solution was heated to 60°C for 1 h. Completion of the demetallation reaction was monitored by MS and the acetyl protected intermediate was isolated by semipreparative RP-HPLC (38% B isocratic). Fractions of the intermediate were collected and reduced to dryness before 3 mL of NEt ₃ /MeOH/H ₂ O (1/6/3) were added for deacetylation. After 24 h at room temperature all volatiles were removed in vacuo and the residue was dissolved in 300 pL of H2O/5% MeCN. Purification via semi-preparative RP-HPLC (1-10% B in 30 min) followed by lyophilization yielded 4.83 mg (3.15 pmol, 25.4%) of a white solid.

RP-HPLC (1-10% B in 15 min) t _R = 8.9 min. MALDI-MS (m/z) = 926.2 [2(M+2Na+K)] ³⁺ , 1305.8 [M+H] ⁺ , 1421.5 [M+H+3K] ⁺ .

Example 7: Labelling of TRAP-Galactan and Galacto-TRAP with ⁶⁸ Ga- and ^nat Ga-gallium

Radiolabelling of TRAP-Galactan and Galacto-TRAP with ⁶⁸ Ga was achieved in high radiochemical yields and purity as determined by radio-HPLC (> 98%) (Fig. 6 and 7) and radio- TLC (> 98%). Molar activities were usually between 10 - 15 MBq/nmol. Labelling with ^nat Ga with a 1.5-fold molar excess over the labelling precursor occurred instantly and led to formation of the respective ^nat Ga-TRAP complexes in quantitative yields and high purity (> 97%) as determined by HPLC and MS analysis. MS analysis also shows that both peaks correspond to the same mass and thus, might be different conformers of the desired products. Of interest is also that the ratio of both peaks is similar for both compounds.

Example 8: Synthesis of TRAP-GalNAc

Formula Weight: 1825.74 g/mol Monoisotopic Mass: 1824.77 Da

Molecular Formula: C ₆₀ H ₁₂₃ N ₁₈ O ₃₃ P ₃ TRAP-Alkynes (Compound 2 of Scheme 2) (3.5 mg, 4.4 pmol, 1.0 eq) was dissolved in 100 pL of Millipore water and mixed with a methanolic solution of 2-[2-(2-azidoethoxy)ethoxy]ethyl 2- acetamido-2-deoxy-p-D-galactopyranoside (6 mg, 15.8 pmol, 3.6 eq). Addition of Cu(OAc)2 (1.0 mg, 5.3 pmol, 1.2 eq), as well as an aqueous solution of sodium ascorbate (34.8 mg, 176 mmol, 40.0 eq) started the reaction. After 1 hour at 60 °C, a solution of DTPA (65.6 mg, 30.0 eq) was added and the pH was adjusted to 2.2 with concentrated HCI. After another hour at 60°C the resulting mixture was injected into semipreparative HPLC (8-12% B in 25 min). Lyophilization of the product fraction yielded 880 pg (428 nmol, 10%) of a white solid.

Analytical HPLC (ReproSil Pur, 5-15% B in 15 min, 1.0 mL/min) t _R = 14.4 min (14% B).

MALDI-MS (m/z) = 1823.8 [M-H]’, 1885.5 [M-H+Na+K]'.

Example 9: Labelling of TRAP-GalNAc with ⁶⁸ Ga-gallium

Following the procedure described above for TRAP-Galactan and Galacto-TRAP in Example 7 yielded [ ⁶⁸ Ga]Ga-TRAP-GalNAc. (Fig. 13).

Example 10: Synthesis of reference compound [ ^99m Tc]Tc-GSA

Preparation of the clinically established reference ligand [ ^99m Tc]Tc-GSA was done according to reference (Kudo M et al. Methods Enzymol, 1994). In brief, 130 pg of GSA-Kit formulation were dissolved in 110 pL of Millipore water and 100 pL of ^99m Tc-pertechnetate solution (approx. 200 MBq) were added as well as 88 nmol (22 mM, 4 pL) of a freshly prepared SnCh solution in 0.1 M HCI. The mixture was gently shaken (300 rpm) and incubated for 10 min at 37°C. Completion of the labelling reaction was monitored with Radio-HPLC (Jupiter, Gradient: 10- 60% B in 15 min). The labelled compound was then diluted with PBS without further purification. For in vivo studies the ^99m Tc- pertechnetate solution was diluted with 0.9% saline and an activity of approx. 15 MBq/100 pL was added to reach the same molar activity as for the gallium labelled tracer.

Radio-HPLC (Jupiter, 10-60% B in 15 min, 1.0 mL/min) t _R = 9.0 min (40% B).

Example 11 : Synthesis and labelling protocol for reference compound TRAP-Gluco

Formula Weight: 1702.58 g/mol Monoisotopic Mass: 1701.69 Da

Molecular Formula: C ₆₃ H ₁₁₄ N ₁₆ O ₃₃ P ₃

TRAP-Alkynes (Compound 2 of Scheme 2) (7.8 mg, 9.8 pmol, 1.0 eq) was dissolved in 100 pL of Millipore water and mixed with a methanolic solution of 2-[2-(2-azidoethoxy)ethoxy]ethyl 2- acetamido-2-deoxy-p-D-glucopyranoside (11.8 mg, 35.0 pmol, 3.6 eq) as well as 2.3 mg (11.8 pmol, 1.2 eq) of Cu(OAc)2. The reaction was started by addition of an aqueous solution of sodium ascorbate (66 mg, 333 pmol, 34.0 eq). After 1 hour at 60°C, DTPA (149 mg, 300 pmol, 30.0 eq) was dissolved in 185 pL of water and was added to the solution. The pH was adjusted to 2.2 with concentrated HCI and the resulting mixture was heated to 60°C for another hour. Purification via semipreparative HPLC (8-12% B in 25 min) and subsequent lyophilization yielded 2.42 mg (1.42 pmol, 14%) of a white solid.

Analytical HPLC (ReproSil Pur, 5-15% B in 15 min, 1.0 mL/min) tp = 13.6 min (13% B).

MALDI-MS (m/z) = 678.8 [M-3H] ⁵ ', 1190.8 [2(M-H+2Na+K)] ³ ’, 1703.2 [M+H]’.

Following the procedure described above for TRAP-Galactan and Galacto-TRAP in Example 7 yielded [ ⁶⁸ Ga]Ga-TRAP-Gluco. (Fig. 14). Example 12: In vitro evaluation

Partition coefficient and protein binding

For determination of logarithmic partition coefficients (JogD) n-Octanol (500 pL) and PBS (500 pL) were pipetted into eight 1.5 mL Protein Low-Bind Eppendorf tubes followed by addition of 300 kBq of labelled compound. The vials were vortexed for 5 min at 1 ,500 rpm and then centrifuged for 5 min at 20,000 ref (Eppendorf Centrifuge 5424). Aliquots of 100 pL were taken out of each phase and the amount of activity was quantified in a y-counter (Perkin Elmer 2480 Wizard2 3”; Waltham, Massachusetts, USA).

Protein binding was determined for different time points (2 min, 30 min, 60 min, 120 min) in triplicates using Sephadex MicroSpin G-50 columns (GE-Healthcare, Chicago, Illinois, USA). The columns were spun initially at 20,000 ref for 1 min to remove the storage buffer. Then approx. 5 MBq of radioligand was added to 1 mL of fresh human serum and incubated at 37°C. At each given timepoint 25 pL of the mixture was loaded on to the column, which was then spun at 20,000 ref for 1 min. The activity content on the column (free radioligand) and in the filtrate (protein bound fraction) was quantified in a y-counter.

Stability studies

Ligand stability in human blood serum (n = 2) and PBS (n = 1) was determined for different time points (2 min, 30 min, 60 min, 120 min). Therefore, approx. 5 MBq of radioligand was added to 1 mL of fresh human serum or PBS and incubated at 37°C. At each given time point 100 pL of the mixture were transferred to a 1.5 mL Eppendorf tube containing 100 pL of acetonitrile. The vial was vortexed and centrifuged for 2 min at 14,000 rpm before 100 pL of the supernatant were diluted with 200 pL of water and injected into analytical radio-HPLC. The PBS solution was directly injected into the radio-HPLC. Ligand stability was determined by integrating the areas of the signals in the radio chromatogram in relation to the signal of the intact ligand.

In vitro binding studies

Inhibitory constant (IC50) values were determined in triplicates using an isolated receptorbased assay as described in literature (Eggink LL et al. J Immunother Cancer 2018; 6:28). Recombinant human ASGR1 was seeded out into Ni-coated 96 well plates (Pierce, Thermo Fisher) in PBS at a concentration of 2 pg/mL for at least 2 h at room temperature under mild agitation (200 1/min). Unbound receptor was removed by washing the wells twice with binding buffer (25 mM Tris x HCI, 150 mM NaCI, 0.05% TWEEN 20, pH = 7.4). Next 140 pL of binding buffer as well as 20 pL of a 19 mM CaCh-solution were added to the wells followed by addition of either 20 pL of PBS (control) or 20 pL of [ ^nat Ga]Ga-NODAGA-TriGalactan in PBS. Final ligand concentrations were ranging from 10' ⁵ - 10' ¹¹ M. Lastly 20 pL of ¹²⁵ l-Asialoorosomucoid (50 nM, 2.4 kBq per well) were added and the plate was incubated for 1 hour at room temperature under mild agitation (200 rpm). Supernatants were removed and the wells washed twice with 200 pL of PBS before receptor bound activity was released by addition of 200 pL of hot (60°C) 1 M NaOH. Wells were incubated with NaOH for 10 min. Lysates were removed and the wells washed again twice with 200 pL of hot NaOH. The activity content in the lysates was quantified in a y-counter and the values plotted in excel. Fitting of the sigmoidal binding curve and the calculation of the IC50 value was done with Excel’s solver plugin.

[ ⁶⁸ Ga]Ga-NODAGA-TriGalactan showed high stability in PBS (> 99 % intact tracer) and human blood serum (> 93%) over the time course of 2 h as determined by radio-HPLC. Furthermore, low protein binding could be observed, and rise from 7.6 ±3.9% (n = 3) after 2 min to 13.6 ± 3.2% (n=3) after 120 min incubation (Table 1). Determination of /ogD-values for [ ⁶⁸ Ga[Ga-NODAGA-TriGalactan (- 4.33 ± 0.09, n = 6) and the reference ligand [ ^99m Tc]Tc-GSA (- 1 .88 ± 0.02, n = 8) revealed high hydrophilicity of the tracer.

Table 1. Stability in human blood serum (n = 2), protein binding (n=3), IC50 (n=3) and logD (n=6) values of [ ⁶⁸ Ga]Ga-NODAGA-TriGalactan & [ ^99m Tc]Tc-GSA.

[ ⁶⁸ Ga]Ga-TRAP-Galactan showed high stability in PBS (> 99% intact tracer) and human blood serum (> 98%) over the time course of 2 hours as determined by radio-HPLC. Furthermore, low protein binding could be observed, and rise from 8.2 ± 3.4% (n = 3) after 2 min to 11.6 ± 2.2% (n=3) after 120 min incubation (Table 2). Determination of /ogD-values for [ ⁶⁸ Ga]Ga-TRAP-Galactan (- 4.26 ± 0.08, n = 6) and [ ⁶⁸ Ga]Ga-Galacto-TRAP (- 4.16 ± 0.09, n = 6) revealed high hydrophilicity of both tracers.

Table 2. Stability in human blood serum (n = 2) and protein binding (n=3) of [ ⁶⁸ Ga]Ga-TRAP- Galactan.

Serum stability (% of intact ligand) Protein binding (%)

2 min 30 min 60 min 120 min 2 min 30 min 60 min 120 min

97.7 + 0.3 97.6 + 0.2 97.6 + 0.1 97.9 + 0.1 8.2 + 3.4 6.4 + 1.8 12.0 + 3.1 11.6 + 2.2

[ ⁶⁸ Ga]Ga-TRAP-GalNAc and [ ⁶⁸ Ga]Ga-TRAP-Gluco show high serum stability after 120 min (Table 3). [ ⁶⁸ Ga]Ga-TRAP-GalNAc further shows highly hydrophilic properties (JogD = -4.2 ± 0.08). Protein binding tendency is low to moderate. Despite higher protein binding of [ ⁶⁸ Ga]Ga- TRAP-GalNAc, this conjugate showed best imaging performance (Fig. 12) indicating low influence of this parameter to the desired imaging properties.

Table 3. Stability in human blood serum and protein binding of [ ⁶⁸ Ga]Ga-TRAP-GalNAc and [ ⁶⁸ Ga]Ga-TRAP-Gluco

Example 13: In vivo evaluation

Biodistribution studies

All animal experiments were conducted in accordance with Austrian animal experiments law (BGBI. I Nr. 114/2012) and according to the institution’s animal welfare standards as approved by the government of Austria (2022-0.311.708).

For biodistribution studies 6 week old female BALB/c mice (n=3) were injected with 0.1 nmol (100 pL, approx. 1 MBq) of labelled compound via the lateral tail vein and sacrificed after 10, 30, or 60 min by cervical dislocation. The mice were dissected, blood and organs (spleen, pancreas, stomach, intestine, liver, kidney, heart, lung, muscle, and bone) were weighed, and their activity measured in a y-counter. For blocking experiments, mice were co-injected with 100 pL of a 277 mM Galactose/PBS solution. In case of [ ⁶⁸ Ga]Ga-TRAP-GalNAc a 277 mM N- Acetylgalactose solution was used. Biodistribution studies for [ ⁶⁸ Ga]Ga-NODAGA-TriGalactan showed high liver uptake reaching its maximum already at 10 min p.i. (33.4 ± 0.9 % I D/g) and staying at a constant high level up to 60 min p.i. (27.7 ± 3.1 % I D/g) (Fig. 2, Table 4). Activity elimination from blood is rapid with a blood activity concentration of 0.3 ± 0.04%ID/g 60 min p.i. This results in low non-liver organ uptake. Highest uptake in non-target organs could be found in the kidneys 10 min p.i. (8.2 ± 0.5% ID/g). However, even there the liver to organ ratio is 3.5 at 10 min p.i. increasing to 11.4 at 60 min p.i. Most favourable liver-to-organ ratios were reached after 60 min p.i. in almost all organs including blood, spleen, pancreas, heart, lung, and muscle demonstrating the described lack of ASGR1 expression in non-liver tissue and the good pharmacokinetic properties of this new compound (Fig. 3, Table 5). The rapid reduction of activity uptake in kidneys indicate fast renal excretion of the tracer. The only organ with increasing activity concentration over time is the intestine reaching 3.0± 0,4% ID/g 60 min p.i.

Blocking experiments were carried out by co-injecting a high excess of D-galactose and examining the mice 30 min p.i. (Fig. 4). A significant reduction in activity accumulation in the liver can be found (32.2 ± 3.7%ID/g vs. 13.9 ± 1.0%ID/g) whereas in most non-target organs comparable activity accumulation is observed. The only other organs with an activity reduction are stomach and intestine, where uptake might be affected due to elimination of the high galactose amount and not due to specific interaction with the tracer.

The reference compound [ ^99m Tc]Tc-GSA showed higher liver uptake 10 min p.i. (58.1 ± 6.7% ID/g,) but declines more significantly over the observation period of 60 min. (46.3 ± 5.9 % ID/g) (Fig. 2, Table 4 ). In contrast to [ ⁶⁸ Ga]Ga-NODAGA-TriGalactan elimination from the blood pool is much slower and activity concentration in kidneys remains almost stable over the observation period of 60 min. Again, highest non-target uptake was found in the kidneys. The increase of activity in the intestine is also higher compared to the new tracer resulting in 9.8 ± 1.6 %l D/g vs. 3.0 ± 0.4 %ID/g 60 min p.i. In general, activity concentration in observed organs and tissue is higher as found for [ ⁶⁸ Ga]Ga-NODAGA-TriGalactan resulting for the later time points in inferior liver-to-organ ratios (Table 5).

Table 4. Biodistribution of [ ⁶⁸ Ga]Ga-NODAGA-TriGalactan (n=3) and [ ^99m Tc]Tc-GSA (n=3) in healthy BALB/c mice. Data are expressed as a percentage of the injected dose per gram (% ID/g), mean value ± standard deviation.

[ ⁶⁸ Ga]Ga-NODAGA-TriGalactan

10 min 30 min 60 min 30 min

Blood 3.3 ± 0.2 0.89 + 0.09 0.34 ± 0.04 1.19 ± 0.33

Spleen 0.78 ± 0.05 0.27 ± 0.02 0.16 ± 0.02 0.32 ± 0.03

Pancreas 0.87 ± 0.12 0.31 ± 0.05 0.19 ± 0.09 0.45 ± 0.01 Stomach 1.42 + 0.23 1.05 + 0.08 0.92 + 0.04 0.68 + 0.11

Intestine 1.13 ±0.07 1.95 + 0.17 2.99 ±0.43 0.77 ±0.19

Kidneys 8.15 0.54 2.8310.18 1.51 ±0.10 4.19±0.29

Stomach 0.91 ±0.01 1.61 ±0.20 2.43 ±1.45

Intestine 1.10 ±0.05 6.4111.05

Kidneys 7.35 ± 0.51 10.5 ±2.00 9.21 ± 0.21

Table 5. Liver-to-organ ratios of [ ⁶⁸ Ga]Ga-NODAGA-TriGalactan & [ ^99m Tc]Tc-GSA in healthy

BALB/c mice.

Biodistribution studies for [ ⁶⁸ Ga]Ga-TRAP-Galactan showed high liver uptake starting from (24.6 ± 1.6% I D/g) at 10 min p.i. and reaching its maximum 30 min p.i. (29.7 ± 1.7% ID/g) (Fig. 8, Table 6). Activity elimination from blood is rapid with a blood activity concentration of 0.3 ± 0.09%ID/g 60 min p.i. This results in low non-liver organ uptake. Highest uptake in nontarget organs could be found in the kidneys 10 min p.i. (7.4 ± 2.1 % ID/g). However, even there the liver to organ ratio is 3.7 at 10 min p.i. increasing to 15 at 60 min p.i. Most favourable liver- to-organ ratios were reached after 60 min p.i. in almost all organs including blood, spleen, pancreas, heart, lung and muscle demonstrating the described lack of ASGR1 expression in non-liver tissue and the good pharmacokinetic properties of this new compound (Fig. 10, Table 8). The rapid reduction of activity uptake in kidneys indicate fast renal excretion of the tracer. The only organ with increasing activity concentration over time is the intestine reaching 2.8 ± 0.2% ID/g 60 min p.i.

Biodistribution studies for [ ⁶⁸ Ga]Ga-Galacto-TRAP revealed moderate liver uptake peaking at 10 min p.i. (6.1 ± 0.5% ID/g) and declining to a value of 5.0 ± 0.6% ID/g at 60 min p.i. Highest non-target uptake was found in the kidneys (12.2 ± 1.4%ID/g) and blood pool (5.6 ± 0.2) 10 min p.i. Liver-to-organ ratios were generally lower than that for TRAP-Galactan, but still reached an optimum 60 min p.i. in spleen, pancreas, stomach, heart, lung, muscle, and femur.

Exchange of the sugar derivatives from galactose to N-acetyl-galactose surprisingly resulted in a 2-times higher liver uptake. On the other hand, the introduction of glucose dramatically reduced liver uptake to background activity demonstrating the selective receptor uptake based on galactose or /V-acetyl galactose (Table 7, Fig. 11).

Table 6. Biodistribution of [ ⁶⁸ Ga]Ga-TRAP-Galactan (n=3) & [ ⁶⁸ Ga]Ga-Galacto-TRAP (n = 3) in healthy BALB/c mice. Data are expressed as a percentage of the injected dose per gram (% I D/g), mean value ± standard deviation.

[ ⁶⁸ Ga]Ga-TRAP-Galactan

10 min 30 min 60 min 30 min

Blood 3.66 ± 0.2 1.6 ± 0.11 0.30 ± 0.09 1.35 ± 0.52

Spleen 0.84 ± 0.08 0.42 ± 0.07 0.17 ± 0.07 0.29 ± 0.03

Pancreas 0.87 ± 0.01 0.53 ± 0.01 0.16 ± 0.02 0.37 ± 0.04

Stomach 1.22 ± 0.20 1.00 ± 0.27 0.31 ± 0.21 0.49 ± 0.07

Intestine 1.24 ± 0.05 2.18 ± 0.10 2.80 ± 0.24 1.02 ± 0.18

Kidneys 7.39 ± 2.1 4.33 ± 0.35 1.72 ± 0.17 3.70 ± 0.53

Liver 24.6 ± 1.6 29.7 ± 1.70 25.0 ± 1.1 10.6 ± 1.4

Heart 1.28 ± 0.06 0.65 ± 0.08 0.16 ± 0.02 0.44 ± 0.09

Lung 2.31 ± 0.17 1.09 ± 0.03 0.32 ± 0.05 0.86 ± 0.14

Muscle 0.91 ± 0.08 0.37 ± 0.02 0.15 ± 0.03 0.30 ± 0.04

Femur 0.85 ± 0.33 0.32 ± 0.05 0.13 ± 0.03 0.27 ± 0.09 [ ⁶⁸ Ga]Ga-Galacto-TRAP

Blood 10 min 30 min 60 min 30 min

Spleen 5.58 ±0.17 0.52 + 0.23 1.84 + 0.14

Pancreas 1.20 + 0.03 0.49 + 0.07 0.12 + 0.02 0.43 + 0.02

Stomach 1.17 ±0.05 0.53 ±0.05

Intestine 1.8010.16 0.7910.15 0.1410.02 0.6710.15

Table 7. Biodistribution of [ ⁶⁸ Ga]Ga-TRAP-GalNAc (n=3) & [ ⁶⁸ Ga]Ga- TRAP-Gluco (n = 3) in healthy BALB/c mice. Data are expressed as a percentage of the injected dose per gram (% I D/g), mean value ± standard deviation.

Table 8. Liver-to-organ ratios of of [ ⁶⁸ Ga]Ga-TRAP-Galactan (n=3) & [ ⁶⁸ Ga]Ga-Galacto- TRAP (n = 3) in healthy BALB/c mice.

[ ⁶⁸ Ga]Ga-TRAP-Galactan

Stomach 20.814.0 31.817.8 117153 21.710.9

Intestine 19.912.1 13.710.8 9.010.4 10.712.0 Kidneys 3.65 + 1.2 6.9 + 0.97 14.6 + 0.9 2.90 + 0.4

Heart 19.4 + 2.1 46.8 ±7.1 159 +22 25.2 + 6.3

Lung 10.7 + 1.3 27.3 + 2.0 80.0 + 9.3 12.5 + 2.3

Muscle 27.1 ± 1.8 80.1 ±0.6 167 ±25 35.7 ±4.3

Femur 33.3 ± 11.4 94.9 ± 18 201 ± 50 41.7 ±8.4

[ ⁶⁸ Ga]Ga-Galacto-TRAP

10 min 30 min 60 min 30 min

Blood 1.09 ±0.1 1.97 ±1.3 10.8 ±3.5 1.76 ±0.05

Spleen 5.08 ±0.53 7.69 ±4.8 41.7 ± 1.7 7.54 ± 0.54

Pancreas 5.21 ±0.6 7.12 ±4.5 42.7 ±0.7 6.88 ± 0.76

Stomach 3.38 ±0.06 5.16 ±3.3 35.6 ±0.2 5.04 ± 0.78

Intestine 3.20 ±0.93 4.31 ±2.8 8.1 ±0.3 4.40 ± 0.38

Kidneys 0.50 ±0.02 0.83 ± 0.53 2.4 ±0.2 0.64 ± 0.06

Heart 3.09 ±0.23 5.57 ±3.6 41.3 ±0.8 4.79 ±0.5

Lung 1.87 ±0.10 2.75 ±1.7 13.8 ±0.0 2.42 ±0.18

Muscle 5.57 ±0.81 8.45 ±5.5 51.9 ± 12 8.70 ±1.6

Femur 5.29 ± 1.3 9.86 ±6.6 44.6 ±7.8 8.30 ±2.3

Blocking experiments for TRAP-Galactan and Galacto-TRAP were carried out by co-injecting a high excess of D-galactose and examining the mice 30 min p.i. (Fig. 9, Tables 6 and 8). A significant reduction in activity accumulation in the liver can be found for both tracers (10.6 ± 1.4%ID/g and 3.24 ± 0.2%ID/g), whereas in most non-target organs comparable activity accumulation is observed.

Comparison of the best performing TRAP-based radiopharmaceutical [ ⁶⁸ Ga]Ga-TRAP-GalNAc with the lead compound [ ^99m Tc]Tc-GSA shows that the liver uptake of this small molecular weight derivative is comparable to the liver uptake of the human serum albumin-based, approx. 84 kD large [ ^99m Tc]Tc-GSA. Moreover, the activity concentration in almost all other organs is already after 10 min clearly lower as found for the macromolecule. Thus, most target-to- background ratios are even better as found for [ ^99m Tc]Tc-GSA (Fig. 12).

PET/MR imaging

Three healthy C57BI/6 male mice were injected with 1.0 +- 0.1 MBg of the PET Tracer for a dynamic, 60 min PET acguisition, followed by a transmission scan for attenuation correction and a T2w-MRI for anatomical co-registration. PET images were reconstructed using a 3D- OSEM algorithm. For analysis, PET and MRI images were fused, regions of interest (ROIs) covering the liver, muscle, heart and kidney, were drawn on the basis of the MRI scan. Time- activity-curves (TACs) were generated for each organ. Representative frames of dynamic PET/MR imaging over 60 min after injection of [ ⁶⁸ Ga]Ga- NODAGA-TriGalactan and [ ¹⁸ F]AIF-NOTA-TriGalactan demonstrated an intensive liver signal and low background activity in the rest of the body (Fig. 15). Besides the liver, most activity is found in the kidneys indicating renal elimination. Comparison of the images with [ ⁶⁸ Ga]Ga- NODAGA-TriGalactan and [ ¹⁸ F]AIF-NOTA-TriGalactan indicates some increased activity accumulation in the bone for the latter. Whether this is due to incomplete separation of the unbound ¹⁸ F-fluoride during production or release of some ¹⁸ F-species from the injected radiopharmaceutical remains to be determined.

Representative frames of dynamic PET/MR imaging over 60 min after [ ⁶⁸ Ga]Ga-TRAP-GalNAc injection also demonstrated an intensive liver signal and low background activity in the rest of the body (Fig. 16).