Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins

Definitions

• the present application relates to new compounds including a VCAM-l binding peptide, which can be used in methods for detecting early atherosclerotic lesions in the blood vessels, in particular the arteries, of human subjects.
• such compounds are biotin derivatives or DOTA derivatives incorporating the above-mentioned peptide moiety.
• Atherosclerosis is the most common form of vascular disease and constitutes the major cause of death, with 17.5 million related deaths annually (31% of global mortality) [1] Atherosclerosis begins in childhood, as an accumulation of fatty streaks-lipid-engorged macrophages and T lymphocytes in the intima of the arteries.
• Atherosclerotic plaques remain asymptomatic (sub-clinical disease), some become obstructive and might cause symptoms because of impaired maximal blood flow (stable angina, intermittent claudication, or mesenteric ischemia are examples), and a small percentage, in some individuals, become thrombosis-prone (vulnerable) and lead to atherothrombotic events such as acute coronary syndromes (ACS), stroke, critical limb ischemia and sudden death [2,3]
• ACS acute coronary syndromes
• stroke critical limb ischemia
• sudden death [2,3]
• CAD coronary artery disease
• coronary angiography is still the gold standard for the assessment of CAD, it simply shows the coronary lumen, and is unable to address plaque vulnerability or provide information on the extent and severity of atherosclerosis.
• CVD cardiovascular disease
• Non-invasive ultrasound imaging represents a safe, fast, and comparatively cheap method of assessing atherosclerosis, although its use is largely confined to the carotid and peripheral vasculature.
• Non-invasive imaging techniques are: ultrasounds (US), electron beam computed tomography (EBCT), multi- detector CT (MDCT), magnetic resonance imaging (MRI) and nuclear imaging, including single photon emission computed tomography (SPECT) and positron emission tomography (PET).
• Radiopharmaceuticals are“Any medicinal product which, when ready for use, contains one or more radionuclides (radioactive isotopes) included for a medicinal purpose”, for diagnosis or therapy of various diseases.
• a receptor ligand often termed as“targeting biomolecule” (BM) serves as the“vehicle” to carry the radionuclide to the diseased tissue, which is known to contain a substantial concentration of the target receptor.
• BM targeting biomolecule
• Radiotracer imaging (often called“molecular imaging”) advantageous over traditional scintigraphic imaging using, for example, simple technetium complexing radiopharmaceuticals or other imaging modalities such as X-ray computed tomography (CT), ultrasound (US), and nuclear magnetic resonance imaging (MRI) [4]
• CT computed tomography
• US ultrasound
• MRI nuclear magnetic resonance imaging
• the high specificity of receptor binding results in selective uptake and distribution of the radiolabeled receptor ligand at diseased tissues.
• the BMs can be small molecules, peptides, monoclonal antibodies (mAbs), nanobodies or mAh fragments, and, ideally, their affinity for the biological target should be unchanged after radiometal labeling [5]
• BFC bifunctional chelate
• DOTA analogues as BFCs
• the advantage of using DOTA analogues as BFCs is the extremely high kinetic inertness of their metal chelates; conversely, the kinetic lability of acyclic metal chelates often results in dissociation of the radiometal from the metal chelate, and leads to radiation toxicity to non-target organs, such as bone marrow [4]
• VCAM-l vascular cell adhesion molecule- 1
• IAM-l intercellular adhesion molecule- 1
• P- and E-selectin promoting monocyte recruitment to the vascular wall and subsequent lesion development.
• VCAM-l is an immunoglobulin (Ig)-like transmembrane adhesion molecule, highly conserved in evolution, and participates in a variety of cellular functions in health and disease.
• Human VCAM-l has 2 isoforms, the predominant 7 Ig-domain isoform and a minor, alternatively spliced isoform with 6 Ig domains, whereas in mice the second isoform consists of the first 3 domains attached to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor.
• Ig immunoglobulin
• VCAM-l is minimally expressed on most resting vascular endothelial cells and is inducible in many tissue vascular beds following injury or stress. Because of this activation, VCAM-l has been implicated in the pathophysiology of certain autoimmune diseases, atherosclerosis, and allograft rejection.
• VCAM-l is constitutively expressed in bone marrow stromal/endothelial cells and certain classes of hematopoietic cells (B cells, follicular dendritic cells, and macrophages).
• VCAM-l is a promising marker for molecular imaging of vascular inflammation in atherosclerosis, since it is not constitutively expressed in normal vessels but is rapidly up-regulated on vascular endothelial cells in both early and advanced lesions and is readily accessible to blood-borne, targeted contrast agents. VCAM-l is also up-regulated in macrophages and smooth muscle cells in atherosclerotic plaques [7]
• ligand Its major ligand is the integrin VLA-4, with binding sites located in the first and fourth Ig domains, whereas other ligands bind with less affinity and include a4b7, a9b1 and a ⁇ b2 [8]
• CLIO dextran- coated iron oxide
• the VCAM-l -targeted nanoparticles could detect VCAM-l expression on the endothelial cells by magnetic resonance and optical imaging.
• VHSPNKK- modified magneto fluorescent nanoparticles had l2-fold higher binding affinity to VCAM-l than VCAM-l monoclonal antibodies and, importantly, had low binding affinity to macrophages [10]
• VHPKQHR another peptide, VHPKQHR, that had a sequence homology to VLA-4, the natural ligand of VCAM-l, and a binding affinity of 33.7 ⁇ 8 nM [11,12] It was conjugated to magnetofluorescent nanoparticles through a GGSK(FITC)C linker (VINP-28).
• VINP-28 had high binding affinity to endothelial cells, but low binding affinity to macrophages and smooth muscle cells, and was also able to detect endothelial cells and other VCAM-l expressing cells in resected human carotid artery lesions ex vivo [11]
• a SPECT radiotracer has been also developed, in which the residue 75-84 (B2702-p) of the major histocompatibility complex- 1 (MHC-l) molecule B2702 was radiolabeled with "mTc, for in vivo molecular imaging of VCAM-l expression in atherosclerotic plaques [14]
• Nanobodies recognising both human and mouse VCAM-l have been investigated as potential targeting ligands for SPECT.
• Nanobodies are single-domain antibody fragments that occur naturally in sharks and camelids.
• SPECT-CT imaging "mTc- radiolabeled VCAM-l nanobodies enabled in vivo detection of VCAM-l expression in aortic arch atherosclerosis in apolipoprotein E deficient (apoE-/-) mice [15].
• the problem underlying the present invention was that of providing new radiopharmaceuticals suitable for application in diagnostic nuclear imaging techniques aimed at detecting early atherosclerotic lesions in the blood vessels of human subjects.
• R is a peptide with the sequence VHPKQHRGGSKGC, linked to the succinimidyl ring through the -SH group of the C-terminal cysteine.
• This biotin derivative can be used in a method for detecting early atherosclerotic lesions in the blood vessels of a human subject, wherein the method includes the steps of
• parenterally administering including a chelating moiety, a biotin moiety and a metal radionuclide, and
• the chelating moiety of said chelating agent is preferably selected from the group comprising DOTA (l,4,7,l0-tetraazacyclododecane-l,4,7,l0-tetraacetic acid) derivatives, NOTA (1 ,4,7- triazacyclononane-N,N',N"-triacetic acid) derivatives, and TETA (1,4,8,11- tetraazacyclotetradecane-l,4,8,l 1 -tetraacetic acid) derivatives.
• DOTA l,4,7,l0-tetraazacyclododecane-l,4,7,l0-tetraacetic acid
• NOTA (1 ,4,7- triazacyclononane-N,N',N"-triacetic acid
• TETA 1,4,8,11- tetraazacyclotetradecane-l,4,8,l 1 -tetraacetic acid
• the chelating moiety is DOTA, which is obtained from a DOTA derivative selected from the group comprising DOTA, maleimido-DOTA (l,4,7,l0-tetraazacyclododecane-l,4,7- tris-acetic acid-lO-maleimidoethylacetamide), DOTA-NHS-ester (l,4,7,l0-tetraazacyclodecane- 1 ,4,7, 10-tetraacetic acid mono-N-hydroxysuccinimide ester), p-SCN-Bn-DOTA (S-2-(4- isothiocyanatobenzyl)- 1 ,4,7, 10-tetraazacyclododecane tetraacetic acid).
• DOTA maleimido-DOTA
• DOTA-NHS-ester l,4,7,l0-tetraazacyclodecane- 1 ,4,7, 10-tetraacetic acid mono-N-hydroxysuccinimide ester
• the chelating agent includes a DOTA derivative containing a biotin moiety, selected from the group consisting of BisDOTA-C3 (bis[(9H-fluoren-9-yl)methyl]-3,3-[2-oxo-2-[[6-[[5- [(3aS,4S,6aR)-exahydro-2-oxo-lH-thieno-[3,4-d]imidazol-
• a DOTA derivative containing a biotin moiety selected from the group consisting of BisDOTA-C3 (bis[(9H-fluoren-9-yl)methyl]-3,3-[2-oxo-2-[[6-[[5- [(3aS,4S,6aR)-exahydro-2-oxo-lH-thieno-[3,4-d]imidazol-
• the metal radionuclide is preferably a radioactive isotope of an element selected among the group consisting of Sr, Rh, Pd, Sm, Er, Au, Bi, In, Lu, Y, Ce, Pr, Nd, Pm, Sa, Eu, Gd, Tb, Dy, Ho, Tm, Yb, Ga, Ni, Co, Fe, Cu, Re, Th and Zr. 68 Ga is particularly preferred.
• PET Positron Emission Tomography
• the molar ratio between said biotin derivative and avidin, neutravidin or streptavidin is preferably 1 :1.
• biotin derivative of formula (I) can also be used in a method for detecting early atherosclerotic lesions in the blood vessels of a human subject, wherein the method includes the consecutive steps of:
• a chelating agent including a chelating moiety, a biotin moiety and a metal radionuclide
• biotin derivative of formula (I) can also be used in a method for treating a disease of a human patient characterized by an overexpression of VCAM-l, which method comprises the steps of:
• biotinylated nanodispersed or microdispersed system such as nanoparticles, liposomes and micelles.
• biotin derivative of formula (I) can also be used in a method for treating a disease of a human patient characterized by an overexpression of VCAM-l, which method comprises the steps of:
• biotinylated nanodispersed or microdispersed system selected among nanoparticles, liposomes and micelles, and
• biotinylated nanodispersed or microdispersed system exploiting the high affinity of biotin moieties to avidin, neutravidin or streptavidin, forms a non covalent complex with at least one of the three residual bindig site of the complex of said biotin derivative with avidin, neutravidin or streptavidin.
• the biotin derivative of formula (I) can also be used in a method for treating a disease of a human patient characterized by an overexpression of VCAM- 1 , wherein the method includes the consecutive steps of:
• VCAM-l a biotinylated nanodispersed or microdispersed system, selected among nanoparticles, liposomes and micelles.
• the disease characterized by an over expression of VCAM-l is preferably selected from the group consisting of inflammatory diseases, atherosclerosis, multiple sclerosis, neurodegenerative diseases, neoplastic diseases and neuroinflammatory diseases.
• the above-mentioned nanoparticles, liposomes and micelles can be loaded with at least one drug suitable for treating said disease characterized by an overexpression of VCAM-l .
• the disease is preferably selected from the group consisting of inflammatory diseases, atherosclerosis, tumors, multiple sclerosis, Parkinson’s disease, neurodegenerative diseases and neuroinflammatory diseases.
• biotin derivative of formula (I) can also be used in a method for treating tumors in a human patient, which method includes the steps of
• a chelating agent including a chelating moiety, a biotin moiety and a metal radionuclide.
• the metal radionuclide is an energy- emitting radioisotope and is preferably selected from the group consisting of 177 Lu, 90 Y, m In, 186 Re, 188 Re, 153 Sm, 89 Sr, 169 Er and 223 Ra.
• the biotin derivative of formula (I) can be prepared by a method which comprises reacting a compound of formula (II)
• the compound of formula (II) is prepared by reacting norbiotinamine of formula (III)
• X is an anion, in particular TFA (CF3COO ),
• R is a peptide with the sequence VHPKQHRGGSKGC, linked to the succinimidyl ring through the -SH group of the C-terminal cysteine.
• the compound of formula (V) can be used in a method for detecting early atherosclerotic lesions in the blood vessels of a human subject, wherein the method includes the steps of
• the metal radionuclide is preferably a radioactive isotope of an element selected among the group consisting of Sr, Rh, Pd, Sm, Er, Au, Bi, In, Lu, Y, Ce, Pr, Nd, Pm, Sa, Eu, Gd, Tb, Dy, Ho, Tm, Yb, Ga, Ni, Co, Fe, Cu, Re, Th e Zr. 68 Ga is particularly preferred.
• PET Positron Emission Tomography
• the compound of formula (V) can be prepared by a method that comprises reacting a compound of formula (VI)
• parenterally administering or“parenteral administration” an intravascular administration, preferably an intravenous or intraarterial injection or infusion, is meant.
• Fig. 1 is a DSC spectrum of ASAM (N-hydroxysuccinimidyl-3-maleimido propionic acid) heated from 30°C to l 85°C at lO°C/min.
• ASAM N-hydroxysuccinimidyl-3-maleimido propionic acid
• Fig. 2 is the 1R spectrum of ASAM.
• Fig. 3 is a HPLC purity assessment of isolated N-Boc-norbiotinamine.
• Fig. 4 is the DSC profile of N-Boc-norbiotinamine heated from 40°C to 200°C at lO°C/min.
• Fig. 5 is the 1R spectrum of N-Boc-norbiotinamine.
• Fig. 6 is a RP-HPLC chromatogram of NAM reaction mixture.
• Fig. 7 is the comparison between RP-HPLC chromatograms of NAM reaction mixtures at room temperature (solid line) and at 40°C (dashed line).
• Fig. 8 is the comparison between RP-HPLC chromatograms of NAM reaction mixture with (dashed line) and without (solid line) NBA substrate.
• Fig. 9 is an RP-HPLC chromatogram (method H-NAM2) of NAM reaction mixture.
• Fig. 10 is the IR spectrum of NAM.
• Fig. 11 is the H-NMR spectrum of NAM.
• Fig. 12 is an RP-HPLC chromatogram of NAMP reaction mixture in phosphate buffer.
• Fig. 13 is a comparison between RP-HPLC chromatograms of NAMP reaction mixture (solid line) and of a solution of the peptide in phosphate buffer (dashed line).
• Fig. 14 shows the comparison between RP-HPLC chromatograms of NAMP reaction mixture (solid line) and a solution including NAM and TCEP without the peptide (dashed line).
• Fig. 15 shows the comparison between RP-HPLC chromatograms of NAMP reaction mixture (dashed line) and of a NAM solution in phosphate buffer (solid line).
• Fig. 16 shows the comparison between RP-HPLC chromatograms of peptide solubilised in water (dashed line) and in phosphate buffer (solid line), after 24h.
• Fig. 17 shows a comparison between RP-HPLC chromatograms of the peptide in phosphate buffer, before (dashed line) and after TCEP addition (solid line).
• Fig. 18 is an RP-HPLC chromatogram of NAMP reaction mixture in phosphate buffer without TCEP.
• Fig. 19 is an RP-HPLC chromatogram of NAMP reaction mixture in ultrapure water.
• Fig. 20 is the mass spectrum of NAMP.
• Fig. 21 is a MS 3 spectrum of the peak with m/z of 507.58 in Fig. 20.
• Fig. 22 is the mass spectrum of NAMP derivative.
• Fig. 23 shows the comparison between RP-HPLC chromatograms of MMA-DOTA (dashed line) and peptide (dotted line) standard solutions and the reaction mixture in phosphate buffer (solid line).
• Fig. 24 shows the comparison between RP-HPLC chromatograms of MMA-DOTA in phosphate buffer (dashed line) and in water (solid line).
• Fig. 25 shows the comparison between RP-HPLC chromatograms of MacroP reaction mixtures in water (A) and in phosphate buffer (B).
• Fig. 26 is an RP-HPLC chromatogram of MacroP reaction mixture in water after 48h.
• Fig. 27 is the mass spectrum of MacroP.
• Fig. 28 is a MS 2 fragmentation spectrum of the peak with m/z 384.39 in Fig. 27.
• Fig. 29 shows the comparison between HPLC chromatograms of ultrafiltrates in the presence (A) and absence (B) of avidin.
• Fig. 30 shows the comparison of CE electropherograms of the ultrafiltrate retentate (dotted line) of the product of avidin and NAMP incubation with NAMP (dashed line) and avidin (solid line) solutions.
• Fig. 31 shows the comparison of CE electropherograms of NAMP-avidin + BisDOTA (dashed line) and a standard BisDOTA solution (solid line).
• Fig. 32 shows the comparison of CE electropherograms of BisDOTA (A) and NAMP (B) standard solutions and of (C) the ultrafiltrate of NAMP incubated with avidin and BisDOTA.
• Fig. 33 shows the comparison between ITLC of 68 Ga-BisDOTA (left) and of NAMP-avidin- 68 Ga- BisDOTA complex (right).
• Fig. 34 shows the comparison between CE electropherogram of MacroP standard solution (A) and MacroP-VCAM-l complex (B).
• Fig. 35 is an ITLC of raw 68 Ga-MacroP in 1 M ammonium acetate, using 0.1 M sodium citrate buffer as mobile phase.
• Fig. 36 is a ITLC of raw 68 Ga-MacroP in 1 M ammonium acetate in water/methanol 1 :1.
• Fig. 37 is an RP-HPLC of raw 68 Ga-MacroP.
• Fig. 38 is an RP-HPLC of pure 68 Ga-MacroP, radiolabeled with MacroP2 method.
• Fig. 39 is a calibration curve for the spectrophotometric determination of MacroP chemical purity, by arsenazo-Pb method.
• Fig. 40 is a diagram showing VCAM-l mRNA expression on HUVECs before (CTR) and after TNF-a stimulation for 4, 18 and 24 hours.
• Fig. 41 is a flow cytometric histogram for control cells (A), 4h (B), l 8h (C) and 24h (D) TNF-a stimulated HUVECs.
• Fig. 42 is a graph showing 68 Ga-BisDOTA-avidin-NAMP uptake of activated and unstimulated
• Fig. 43 is a graph showing 68 Ga-MacroP uptake of activated and unstimulated HUVECs.
• Figure 44 is a confocal fluorescence micrograph of TNF- stimulated HUVECs incubated with
• NAMP neutravidin and liposomes and control HUVECs.
• the present invention envisages the use of a specific VCAM-l binding peptide, namely a peptide with the sequence VHPKQHRGGSKGC, containing the sequence VHPKQHR, homologous to the natural ligand of VCAM-l (VLA-4), whose C-terminal arginine is linked by the spacer GGSKG, to C terminal cysteine.
• VCAM-l binding peptide is commercially available from Innovagen AB (Sweden).
• BM targeting biomolecule
• two classes of radiopharmaceuticals namely one (i.e. the one based on the compound of formula (V)) in which the VCAM-l ligand is directly linked to a chelating agent and the other (i.e. the one based on the compound of formula (I)) in which the VCAM-l ligand is separated from the chelating agent and only in vivo the molecule containing the BM will be associated to a chelating agent moiety through a biotin-avidin system.
• radiopharmaceuticals will have also different uptake properties, depending on ligand-receptor and biotin-avidin affinities, and on biodistribution and pharmacokinetic behaviour, which are determined by the physical and chemical properties of the structure linked to the peptide.
• the two classes are different for the aspects involved in the radiolabeling procedure.
• the peptide is linked to chelating agent during the radiolabeling while in the second class, the prototype of which is named NAMP, the radiolabeling procedure does not involve the peptide.
• Both classes of molecules can be used in SPECT and PET nuclear imaging, and also in magnetic resonance imaging (MRI).
• NAMP compound of formula (I)
• Radiolabeled derivatives of biotin are being developed for application to“pretargeting”, usually employed in tumor therapy with radiolabeled monoclonal antibodies (MoAbs) [16-19]
• the strategy is based on the separate administration of a BM, i.e. a monoclonal antibody (MoAb), that binds, subsequently, a second radiolabeled component [17].
• a BM i.e. a monoclonal antibody (MoAb)
• MoAb monoclonal antibody
• the modified BM is injected first and allowed to distribute throughout the body, to bind the cells expressing the receptor and to clear substantially from the other tissues. Then, the radiolabeled second component is administered and, ideally, it localises at the sites where the modified BM has accumulated. If the second component has higher permeation, clearance and diffusion rates than BM, more rapid radionuclide localisation to the tumour and higher tumour selectivity are possible, thus resulting in higher tumour to non-tumour ratios [16].
• the Applicant thought to exploit the biotin-avidin system for diagnostic and therapeutical purpose in atherosclerosis and all other pathologies overexpressing the VCAM-l protein.
• the VCAM-l ligand is biotinylated to bind avidin, neutravidin or streptavidin,
• a second component or chelating agent suitably biotinylated and radiolabeled.
• Avidin, neutravidin and streptoavidin are capable of binding biotin with high affinity.
• avidin is a 66-kDa glycoprotein found in egg white, made of four identical subunits, each bearing a single binding site for biotin. One mole of protein can therefore bind up to four moles of biotin [16].
• Neutravidin is a deglycosylated version of avidin, with a mass of approximately 60,000 daltons. As a result of carbohydrate removal, lectin binding is reduced to undetectable levels, yet biotin binding affinity is retained because the carbohydrate is not necessary for this activity.
• Avidin has a high pi but neutravidin has a near-neutral pi (pH 6.3), minimizing non-specific interactions with the negatively-charged cell surface or with DNA/RNA.
• Neutravidin yields the lowest nonspecific binding among the known biotin-binding proteins.
• the specific activity for biotin binding is approximately 14 pg/mg of protein, which is near the theoretical maximum activity. It is available e.g. from Thermo ScientificTM as“Neutr Avidin Protein” (catalog number 31000).
• Streptavidin is a 52.8 kDa protein purified from the bacterium Streptomyces avidinii. With respect to avidin, streptavidin lacks any carbohydrate modification and has a near-neutral pi. Avidin shows only 30% of sequence identity to streptavidin, but almost identical secondary, tertiary and quaternary structure.
• biotin The strong hydrogen bonding interactions within the binding site of biotin is due to the presence of the ureido group and the nonoxidized thioether, that results in remarkable avidity in the femtomolar range.
• the high affinity of biotin for avidin, neutravidin and streptavidin and its fast blood clearance gives the advantage of better tissue-blood ratio over conventionally used radiolabeled drugs. This makes the avidin-biotin, neutravidin-biotin or streptavidin-biotin systems an ideal method for therapeutic and diagnostic purposes [20]
• biotin involved in binding leave the carboxylic group available for modification.
• radiolabeled biotin derivatives generally have been prepared by direct functionalization, generally with an amide, of the carboxyl group of the vitamin [21]
• Norbiotinamine is an interesting compound because it is exploitable for conjugation reactions with the carboxylic group of aminoacids or other molecules used for targeting and the resulting reversed amide bond is stable to biotinidase cleavage [21]
• the molecules containing biotin moieties and VCAM-l ligand should be injected in vivo first, in order to target the VCAM-l expressed on the surface of endothelial cells in correspondence of the atherosclerotic lesion, followed by avidin, neutravidin or streptavidin injection, and finally by the injection of a radiolabeled biotin.
• a chasing step must be also considered, for example with biotinylated albumin.
• the molecules containing biotin moieties and VCAM-l ligand, before being injected, should be bound to avidin, neutravidin or streptavidin in a 1 :1 molar ratio and the injection should be followed by the parenteral administration of a molecule containing a biotin moiety linked with a chelating agent, e.g. a suitable bifunctional chelating agent like DOT A. Since only a small percentage of radioactivity really localizes on the lesion, the DOTA-biotin labeled conjugate should have high specific activity.
• a chelating agent e.g. a suitable bifunctional chelating agent like DOT A. Since only a small percentage of radioactivity really localizes on the lesion, the DOTA-biotin labeled conjugate should have high specific activity.
• the maximum allowed stoichiometry of a DOTA-conjugated molecule is one metallic radionuclide per each DOTA macrocyclic ring, and this may limit the imaging detection of VCAM-l on the tissue.
• the Applicant thought to increase the potentiality of this diagnostic approach using biotin derivatives carrying at least two DOTA groups, that can be labeled with at least two radionuclides to increase the radionuclide signal localized on the receptor.
• biotin derivatives carrying two DOTA groups per molecule (BisDOTA), in order to deliver a higher radiation dose to the tumors, for therapeutic purposes.
• BisDOTA DOTA groups per molecule
• biotin has been modified through bifunctional spacers of different lengths and chemical structures and conjugated with two molecules of DOTA.
• the BisDOTA-Lys derivatives showed avidin binding capacity higher than B1SDOTA-C3 and very similar to native biotin affinity [27] For this reason, BisDOTA-Lys-C3 was considered the best binding molecule to be exploited with NAMP and other analogous molecules.
• Both classes of molecules can be used to drag radionuclides (i.e. 177 Lu, 90 Y, m In, 186 Re, 188 Re, 153 Sm, 89 Sr and 169 Er) to neoplastic cell expressing the VCAM-l protein for antitumoral treatment.
• radionuclides i.e. 177 Lu, 90 Y, m In, 186 Re, 188 Re, 153 Sm, 89 Sr and 169 Er
• NAMP class molecules can be linked, through avidin, neutravidin or streptavidin to a biotinylated nanodispersed system (liposomes, nanoparticles, micelles), either drug-loaded or not, to exploit other therapeutic effects.
• NAMP i.e. a norbiotinamine conjugated with a VCAM-l binding peptide, through a linker, as it is clear from the following formula:
• R VHPKQHRGGSKGC, where the thiol group bonded to R is that of the C- terminal cysteine of VCAM-l peptide ligand.
• biotin moiety in NAMP allows to exploit the avidin-biotin binding system for 3 -step or 2-step pretargeting.
• an activated linker N-hydroxysuccinimidyl-3-maleimido propionic acid (ASAM)
• SAM N-hydroxysuccinimidyl-3-maleimido propionic acid
• the reaction occurs between norbiotinamine and the thiol group of the ligand through the linker named ASAM.
• the thiol group of R-SH is that of C-terminal cysteine of the VCAM-l ligand.
• N-hydroxysuccinimide 28 mmol
• dicyclohexylcarbodiimide DCC 47.7 mmol
• the filtrate was poured on ice to obtain the product as a white precipitate.
• the product was characterized by elemental analysis, thermal analysis, FT-IR, ⁇ -NMR, 13 C- NMR and TLC. Purity was evaluated by RP-HPLC.
• Table 1 shows the comparison between the theoretical percentage composition of AS AM and the measured one. The deviation of the elemental analysis from the theoretical composition is lower than 0.3%.
• Fig. 2 shows the infrared spectrum of ASAM.
• maleimide C-H stretching vibrations (3108 cm 1 ) and asymmetrical and symmetrical methylene stretching vibrations (2929 cm 1 , 2851 cm 1 ).
• NP-TLC with ethyl acetate/methanol 85:15 (v/v) mobile phase ASAM R f is 0.85.
• norbiotinamine syntheses reported in the literature refer to a work published by Szalecki [26] It consists in a one-pot reaction wherein biotin is added in equimolar amounts to TEA and DPPA, in t-BuOH at reflux, to form the derivate N-Boc- norbiotinamine. Norbiotinamine is then obtained by acidic hydrolysis, as reported in the scheme below.
• the reaction was carried out by solubilizing biotin in DMF and then adding TEA and, after 10 minutes, DPPA at room temperature. After 30 min t-BuOH was added at room temperature. After lh the mixture was heated gradually (20°C/h) up to 90°C and refluxed under stirring for 24h.
• N-Boc-norbiotinamine was isolated from the reaction mixture by a semipreparative RP-HPLC. N-Boc-norbiotinamine was obtained highly pure, with a yield of 50% (Fig. 3).
• NBA-BOC N-Boc-norbiotinamine
• NBA-BOC was hydrolyzed to NBA with TFA:dichloromethane 1 :l, at 0°C for 2h. Solvents were removed with Rotavapor and the obtained product was utilized for the next reaction without further purification.
• the product was characterized by elemental analysis, thermal analysis, FT-IR, H-NMR and 13 C- NMR.
• Table 2 shows the comparison between the theoretical percentage composition of N-Boc- norbiotinamine and the experimental one. The deviation of the elemental analysis from the theoretical composition is lower than 0.3%.
• Table 2 Theoretical and actual percentage composition of N-Boc-norbiotinamine.
• the thermal profile of the substance shows a single endotherm at l68°C (extrapolated onset), which is the melting point of N-Boc-norbiotinamine, in accordance with the hot stage microscopy observation (Fig. 4).
• the N-Boc-norbiotinamine infrared spectrum is shown in Fig. 5.
• the N-H stretching 3535 cm 1 , 3296 cm 1
• stretching of methyl and methylene groups (2978 cm 1 , 2930 cm 1 and 2865 cm 1 ).
• NBA-BOC was hydrolyzed to NBA with TFA:dichloromethane 1 :l, at 0°C for 2h. Solvents were removed with Rotavapor and the obtained product was utilized for the next reaction without further purification.
• NAM N-norbiotinyl- -maleimidopropionylamide
• reaction was performed applying the aforementioned conditions, adding TEA in molar ratio 1 : 1 with norbiotinamine, in order to favour the nucleophilic attack of the amine on the activated carboxyl group of ASAM, according to the following scheme.
• reaction mixture dried and dissolved in the mobile phase was analysed by RP-HPLC with H-NAM1 method (Fig. 6).
• Peak 1 was identified as N-hydroxysuccinimide, which is cleaved from ASAM after the nucleophilic attack of the amine on the carboxylic group.
• Peak 2 and peak 4 correspond to unreacted ASAM and its impurity, respectively.
• Peak 3 was attributed to NAM, as confirmed by further RP-HPLC analyses.
• Fig. 9 shows the analysis of the reaction mixture with the new RP-HPLC method and the peak assignment.
• the aqueous phase containing N-hydroxysuccinimide and NAM was vacuum dried by Rotavapor and NAM was purified from the residual solid mixture by repeated washings with diethylether, obtaining a N-norbiotinyl- -maleimidopropionylamide yield of 23% w/w.
• the elemental analysis, 1R analysis, H-NMR spectroscopy confirmed NAM identity.
• Chloroform was replaced with acetonitrile, more able to solubilize norbiotinamine, and, after various attempts, the best yield was obtained by heating at 80°C for 24h.
• NAM N-norbiotinyl-fj-maleimidopropionylamide
• NBA about 0.15 mmol
• TEA 0.15 mmol
• ASAM 0.24 mmol
• the product was collected by precipitation at -20°C after 48h and obtained pure after four washings with diethylether. The yield obtained was 60 % w/w.
• the characterization was performed by elemental analysis, 1R analysis, H-NMR spectroscopy and RP-HPLC.
• Fig. 10 shows the IR spectrum of NAM.
• the IR spectrum of NAM shows peaks similar to the spectrum of ASAM, such as 828 cm 1 and 696 cm 1 signals, which may be related to the maleimidic CH.
• the spin-spin decoupling was used for simplifying the spectrum and determining the positions of some protons in the molecule.
• the decoupling of the signal of the amidic NH proton (7.91 ppm) permitted to convert the multiplet at 2.98 ppm in a triplet, confirming the formation of the amide binding between ASAM and norbiotinamine.
• the disulfide bonds of the peptide are reduced by a 10-fold molar excess of a reducing agent such as dithiothreitol (DTT) or tris-(2-carboxyethyl) phosphine hydrochloride (TCEP-HC1), for 2 hours, at room temperature.
• a reducing agent such as dithiothreitol (DTT) or tris-(2-carboxyethyl) phosphine hydrochloride (TCEP-HC1)
• TCEP-HC1 is a strong reducing agent that reduces even very stable alkyl disulfides, rapidly and cleanly in water, at room temperature and pH 5 [31].
• TCEP-HC1 has pKa of 7-8, which is a common pH range for performing bioconjugations, and, as such, the trialkylphosphines are more- effective nucleophiles than thiol-based reducing agents to effect reduction within this pH range.
• disulfide reductions utilizing alkylphosphines are irreversible and driven by phosphorus-oxygen bond formation, unlike the reversible mechanism of disulfide reduction observed with thiol-containing reducing agents [32]
• TCEP has been advertised as being less reactive than DTT with thiol-reactive compounds, thereby eliminating the need to remove it before labeling [33]
• Peaks 5 and 6 are related to NAM, in particular peak 5 forms also in a mixture including NAM and TCEP (Fig. 14), while peak 6 correspond to a NAM derivative that forms in phosphate buffer over time (Fig. 15).
• Peak 3 shows a behaviour similar to peak 6 and seems to be related to peak 4: it increases over time in phosphate buffer, while peak 4 decreases.
• peak 3 was supposed to be the hydrolysed maleimidic derivative of NAMP shown here below, in accordance with a work published by D. Fontaine et al. in 2015 [34]
• Peaks 4 and 3 were isolated by semi-preparative RP-HPLC and characterized by mass spectrometry (Figs. 20 and 22) which confirmed that they correspond to NAMP and to its hydrolysed derivatives respectively.
• the maleimide-thiol conjugates form through Michael addition of a thiolate (RS ) to the double bond of the maleimide to produce a succinimidyl thioether (SITE), but the succinimidyl moiety of a SITE undergoes irreversible hydrolysis to provide two isomeric succinamic acid thioethers (SATE). Since, in the presence of excess of other thiolate (R’S ), as in most biological environments, a new conjugate could form with this thiol and the original SITE considered to be irreversibly cleaved (see scheme here below).
• a strategy to stabilize maleimide-thiol conjugates is to intentionally hydrolyse the conjugate prior to its exposure to exogenous thiol.
• the two SATE derivatives of NAMP can be useful because they bind VCAM-l with a longer spacer between the biotin derivative and the peptide, which in some pathological tissue conditions could increase the affinity of the tracer to VCAM-l, with respect to the unhydrolysed molecule, or also stabilize the tracer in vivo.
• the first possible change was not to incubate the peptide with TCEP before the coupling reaction with the maleimide.
• the peptide, dissolved in phosphate buffer was analysed by RP-HPLC, applying method N-NAMP2 since with method N-NAMP1 the peptide co-elutes with the phosphate buffer.
• Fig. 16 shows a comparison between the peptide dissolved in water and in phosphate buffer, analysed after 24h.
• the reaction was repeated in ultrapure water, without using the phosphate buffer, at pH in the range 5-6, a pH value that allows to keep the selectivity of the nucleophilic attack of the thiol group on the maleimide. Moreover the reaction temperature was increased to 37°C and the reaction time prolonged from 2h to 24h.
• a stock solution of NAM was prepared by dissolving 0.6 mg of NAM in 1.5 mL of ultrapure water, in a 1.5 mL polypropylene tube.
• the peptide solution was prepared by solubilising 1 mg in 500 pL of ultrapure water.
• 1.05 mL of NAM stock solution (1.3 pmol) and 450 pL of peptide solution (0.65 pmol) were mixed in a 1.5 mL polypropylene tube, and heated at 37°C, under orbital shaking at 400 rpm for 24h, under nitrogen.
• the isolation or purification of NAMP was performed by semi-preparative RP-HPLC.
• NAMP characterization was performed by mass spectrometry, using the LTQ OrbitrapTM Velos Pro.
• the LTQ OrbitrapTM is a high-performance LC-MS and MSn system, combining rapid LTQ ion trap data acquisition with high accuracy Orbitrap mass analysis.
• Fig. 20 shows the mass spectrum of the purified molecule. The measured mass coincides with the calculated monoisotopic mass.
• cleavage of the backbone typically occurs at the peptide amide bond to produce b ions, if the amino terminal fragment retains the charge; y ions, if the carboxy-terminal fragment retains the charge.
• the y series is sometimes accompanied by peaks formally corresponding to loss of NH3 from the y ions if the fragment includes arginine, asparagine, lysine or glutamine as aminoacids, or loss of H2O from y ions if the fragment includes serine, threonine, glutamic acid or aspartic acid.
• ylO related to the loss of valine, histidine and proline
• y2 in which only cysteine and glycine remained.
• the y2 ion permitted to confirm that the peptide was conjugated to the maleimide through the thiol group of the cysteine.
• MMA-DOTA maleimido-monoamide DOTA
• VCAM-l binding peptide was initially performed in phosphate buffer at pH 7, at 37°C for 2h, in accordance with an analogous procedure described in the literature. To avoid the formation of byproducts in the reaction mixture, no reducing agent (e.g. TCEP or DTT) was added.
• TCEP reducing agent
• reaction mixture was analysed by analytical RP-HPLC and by the comparison with standard solutions of MMA-DOTA and peptide in phosphate buffer at pH 7, and the peaks in the chromatogram were identified (Fig. 23).
• the MMA-DOTA solution in phosphate buffer showed two peaks, attributed to the maleimidic portion of MMA-DOTA.
• an aqueous solution of MMA-DOTA was analysed at the same concentration of the phosphate buffer solution and, as shown in Fig. 24, it presented only the second peak; therefore, the first peak resulted to be a MMA-DOTA derivative forming exclusively in phosphate buffer at pH 7.
• the reaction was also repeated using ultrapure water as reaction medium.
• Fig. 25 shows the comparison between the reaction mixtures in water and in phosphate buffer, after stirring at 37°C for l9h.
• the amount of MMA-DOTA was increased from a molar ratio of 1.2 to 2 with respect to the amount of peptide;
• MMA-DOTA 1.4 pmol
• peptide 0.72 pmol
• the reaction was heated at 37°C, under orbital shaking at 400 rpm for 48h, in atmosphere of nitrogen.
• MacroP was purified by semi-preparative RP-HPLC.
• the purified MacroP was analysed by analytical RP-HPLC, resulting highly pure.
• the pure MacroP has been characterized by mass spectrometry (Fig. 27).
• the mass spectrometry analysis of MacroP was performed by a LTQ Orbitrap Velos Pro, in order to confirm the identity of the substance and to identify the thioether bond between the thiol group of the VCAM-l binding peptide and the maleimide of MMA-DOTA.
• the calculated monoisotopic mass is 1915.9, but the presence of peaks with a mass of 1916.9 is justified by the abundance of the different isotopes of the elements in the molecule.
• mass spectrometry cannot detect single molecules but is influenced by the millions of copies of a molecule that includes different isotope species.
• the monoisotopic mass is most often used: it is the sum of the masses of the atoms in a molecule using the principle (most abundant) isotope mass of each atom instead of the isotope averaged atomic mass (atomic weight).
• the mass spectral peak representing the monoisotopic mass is not always the most abundant isotopic peak in a spectrum, although it stems from the most abundant isotope of each atom type. In fact, as the number of atoms in a molecule increases, the probability for the entire molecule to contain at least one heavy isotope increases.
• NAMP capability of binding avidin was tested by RP-HPLC and CE.
• the ultrafiltrate was analysed by RP-HPLC in order to verify the presence of unbound NAMP (Fig. 29 A), while the retentate was examined by CE.
• the retentate was analysed by CE and compared with standard solutions of avidin and NAMP.
• the formation of NAMP-avidin complex was indicated in the CE electropherogram with a new peak with a migration time different from the avidin and NAMP standard solution peaks and with a different peak profile (Fig. 30).
• NAMP-avidin-BisDOTA complex was incubated with a BisDOTA ( B is DOT A- Lys-C ) solution, in order to verify the formation of NAMP-avidin-BisDOTA complex by CE.
• B is DOT A- Lys-C
• BisDOTA was radiolabeled with 68 Ga by using the BisDOTA 1 method reported in the below “materials and methods” section, in order to develop a preliminary test for evaluating the formation of NAMP-avidin-radiolabeled [ 68 Ga] BisDOTAcomplex.
• the method should be able to distinguish the free radiolabeled BisDOTA from the BisDOTA bound to the complex.
• MacroP ability of binding VCAM-l was tested by CE.
• MacroP and VCAM-l were mixed in equimolar amount and, after 400 rpm stirring at 37 °C for 2 h, the solution was analyzed by CE and compared with a MacroP standard solution at the same concentration (Fig. 34)
• the radiolabeling was performed using the Eckert & Ziegler Eurotope Modular Lab Standard ® automated synthesis system: briefly, after the elution of 68 Ge/ 68 Ga from the generator, 68 Ga is trapped on the SCX while 68 Ge and other metallic impurities are eluted in the waste.
• 68 Ga is eluted from the SCX with a 0.02 M HC1 solution in 98% acetone into the reaction vial containing MacroP.
• the compound was dissolved in 0.2 M ammonium acetate buffer (pH 4), 800 pL of 0.02 M HC1 acetone solution were added, and the mixture was heated at 95°C for 400 seconds; after cooling, a RP-HPLC was carried out using a standard MacroP solution at lower concentration as reference, and no differences were found.
• MacroP was radiolabeled and RCP tested by ITLC and RP-HPLC, before purification.
• the product RCP resulted to be higher than 97% with both methods.
• the product was also analysed by RP-HPLC using the RPH1 method and the obtained RCP resulted to be higher than 90% (Fig. 37).
• MacroP has been radiolabeled with method (MacroP2) deriving from the Mueller [38] one with slight modifications.
• the radioactivity yield was 57% with a RCP equal to 97%, when calculated by ITLC, and of 99%, when measured by RP-HPLC RPH2 method (Fig. 38).
• Table 4 Stability study of 68 Ga-MacroP in saline.
• the spectrophotometric assay is based on the decrease of the 656 nm absorption of the complex between Pb ++ and arsenazo (AA), for the concurrent formation of the Pb-DOTA complex.
• Fig. 39 shows the calibration curve for the spectrophotometric determination of MacroP chemical purity, by arsenazo-Pb method.
• VCAM-l The expression of VCAM-l on HUVECs was induced by TNF-a stimulation for different times and was evaluated by qRT-PCR and FACS analysis.
• VCAM-l mRNA level in HUVECs To assess the VCAM-l mRNA level in HUVECs, a quantitative assay utilizing reverse transcription-polymerase chain reaction was performed. As shown in Fig. 40, TNF-a incubation caused a stimulatory effect on VCAM-l mRNA levels in HUVECs as corrected by GAPDH and 28S transcripts. The maximum of the VCAM-l expression was observed within l8h stimulation, then the mRNA expression descended.
• Histograms in Fig. 41 plot the fluorescence intensity (on the x-axis) against the cell count (on the y-axis).
• the P3 gate identifies a region of the plot in which the cells are vital and show a high fluorescence intensity, which is related to an increased VCAM-l expression.
• Table 5 Comparison between the % of total vital cells and the mean fluorescence intensity for TNF-a stimulated HUVEC at different times.
• TNF-a stimulated and unstimulated HUVEC were incubated first with NAMP then with avidin, before adding 6 MBq of 68 Ga-BisDOTA in 4 mL of the culture medium.
• the culture medium was replaced with 3 mL of saline solution containing 5 MBq of radiolabeled MacroP. The results were analysed by using the Ligandtracer ® technology, based on repeated differential measurements of surface-associated proteins.
• An increase of the signal is related to the uptake of the radioactivity by the cells, that is not visible in the control cells.
• Fig. 42 and Fig. 43 show the plots of NAMP-avidin-BisDOTA and MacroP in vitro tests, respectively.
• MacroP has been successfully radiolabeled with 68 Ga and in vitro tests confirmed its ability to bind the VCAM-l .
• VCAM-l The expression of VCAM-l on HUVECs was evaluated by FACS analysis.
• TNF-a stimulated HUVECs were incubated at 4°C first with NAMP then with neutravidin, before adding fluorescently labeled biotinylated liposomes in 1 mL of the culture medium.
• Control TNF-a stimulated HUVECs were incubated at 4°C with neutravidin, before adding fluorescently labeled biotinylated liposomes in 1 mL of the culture medium.
• TNF-a stimulated and control HUVECs were finally fixed with 1% paraformaldehyde and stained with DAPI.
• Figure 44 shows that a diffused red fluorescent signal is detectable on HUVECs incubated with NAMP, neutravidin and liposomes; the same fluorescent signal is not visible in control cells. Control HUVECs are on the left, HUVECs incubated with NAMP, neutravidin and liposomes are on the right.
• Acetic acid glacial 100% (AcOH) (Merck); aluminium sheet silica gel 60 F254 plates (Merk); b- alanine 98% (Alfa Aesar); biotin (Amresco); diphenylphosphorylazide (DPPA) (Alfa Aesar); maleic anhydride 98+% (Alfa Aesar); N-hydroxysuccinimide 98+% (Alfa Aesar); N,N’- dicyclohexylcarbodiimide 99% (DCC) (Alfa Aesar); N,N’-dimethylformamide for peptide synthesis (DMF) (Merk); tert-butanol (t-BuOH) (Alfa Aesar); triethylamine (TEA) (Sigma Aldrich); trifluoroacetic acid (TFA) (Merck); tris-(2-carboxyethyl)phosphine
• the DSC thermal analyses were supported by hot stage microscopy analysis, performed at the same conditions.
• IR spectra were collected on a Perkin Elmer 2000 FT-IR spectrometer from samples prepared as KBr pellets.
• Analytical reversed-phase high-performance liquid chromatography Analytical RP-HPLC was performed on Hewlett Packard Series II 1090 with a UV-visible detector.
• a RP- HPLC method was performed using a gradient solvent system at a flow rate of 1 ml . /min.
• the gradient mixture was composed of 0.1% aqueous AcOH (solvent A) and methanol gradient grade (solvent B).
• the chromatograms were acquired at 200, 254 and 340 nm wavelengths.
• the injection volume was 10 pL.
• ASAM was dissolved in methanol; the other analytes were dissolved in the mobile phase.
• Method H-NAM1 it was performed using a gradient solvent system at a flow rate of 1 ml . /min.
• the gradient mixture was composed of 0.1% aqueous AcOH (solvent A) and methanol gradient grade (solvent B). Gradient elution: 0-2 min 40% B; 2-7 min linear gradient from 40% to 100% B; 7-11 min 100% B. Post run to 40% B: 3 min.
• the chromatograms were acquired at 200, 254 and 340 nm wavelengths.
• the injection volume was 10 pL and the solutions were prepared using the mobile phase as solvent.
• Method H-NAM2 it was performed using a gradient solvent system at a flow rate of 1 mL/min.
• the gradient mixture was composed of ultrapure water (solvent A) and acetonitrile gradient grade (solvent B). Linear gradient from 20% to 100% B in 10 min. Post run to 20% B: 3 min.
• the chromatograms were acquired at 200, 254 and 340 nm wavelengths.
• the injection volume was 10 pL and the mobile phase was used as solvent.
• Method H-NAMP1 10 minutes isocratic method at a flow rate of 1 mL/min.
• the mobile phase was composed of 85% A and 15% B.
• Method H-NAMP2 this method was suggested by the peptide manufacturer Innovagen. Gradient solvent system at a flow rate of 1 mL/min. Linear gradient from 6% to 31% B in 25 min. Post run to 6% B: 3 min.
• the chromatograms were acquired at 198, 210, 225 and 254 nm wavelengths.
• the injection volume was 10 pL and samples were dissolved in ultrapure water.
• a RP- HPLC method was applied using a gradient solvent system at a flow rate of 1 mL/min.
• the gradient mixture was composed of 0.1% aqueous TFA (solvent A) and acetonitrile gradient grade (solvent B). Linear gradient from 6% to 16% B in 10 min. Post run to 6% B: 3 min.
• the chromatograms were acquired at 198, 210, 225 and 254 nm wavelengths.
• the injection volume was 10 pL and the samples were dissolved in ultrapure water.
• the purification of NBA-BOC was performed by semi-preparative RP-HPLC, using a gradient solvent system at a flow rate of 5 ml . /min.
• the gradient mixture was composed of 0.1% aqueous AcOH (solvent A) and methanol gradient grade (solvent B). Gradient elution: 0-2 min 40% B; 2-12 min linear gradient from 40% to 100% B. Post run to 40% B: 2 min.
• the chromatograms were acquired at 200, 254 and 340 nm wavelengths.
• the injections were performed using a 1 mL loop.
• NAMP N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(trimethyl)-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
• the mobile phase was composed of 85% 0.1% aqueous TFA and 15% acetonitrile gradient grade.
• the chromatograms were acquired at 198, 210, 225 and 254 nm wavelengths.
• the injections were performed with a 500 pL loop, using the partial loop-fill injection method.
• the mass spectrometer LTQ-Orbitrap Velos Pro was operated in positive ionization mode. Single MS survey scans were performed in the Orbitrap, recording a mass window between 150 and 2000 m/z. The Full Scan resolution was set to 120000. Sample were diluted 1 :100 with a water/acetonitrile (50:50, v/v) solution containing 1% AcOH and introduced into the mass spectrometer by means of direct infusion at a flow rate of 5 pL/min with a syringe pump.
• the spectrophotometric assay is based on the decrease of the 656 ran absorption of the complex between Pb ++ and Arsenazo (AA), for the concurrent formation of the Pb-DOTA complex.
• absorbance at 656 ran was determined, 10 min after its preparation, at room temperature and protected from light.
• a stock solution of avidin was prepared by dissolving 0.5 mg of protein in 200 pL of ultrapure water. 100 pL of this solution (3.8 x 10 6 mmol) were mixed with 100 pL of approx. 0.17 mM NAMP aqueous solution (17 x 10 6 mmol). The reaction mixture was heated at 37°C, under 400 rpm orbital shaking for 2h.
• the reaction mixture containing NAMP and avidin was placed in an ultrafiltration system with a regenerated cellulose membrane with 30 kDa NMWL-nominal molecular weight limit, and washed with 250 pL of ultrapure water at 14000 g for 20 min at l5°C.
• the retentate was recovered by inverted spinning at 1000 g for 3 min.
• reaction mixtures containing NAMP, avidin and BisDOTA were transferred in an ultrafiltration system with a a regenerated cellulose membrane having a 30 kDa cut-off, and washed with 250 pL of ultrapure water at 14000 g for 20 min, at 15°C.
• the retentate was recovered by inverted spinning at 1000 g for 3 min.
• CE analyses were performed using an Agilent Technologies 7100 Capillary Electrophoresis. The separations were carried out in a bare fused-silica capillary 50 pm (i.d.) x 64.5 cm (L) x 50 cm (1) (G1600-61239), thermostated at 30°C.
• the capillary was activated with 1 M NaOH aqueous solution, 0.1 M NaOH aqueous solution and washed with ultrapure water; the conditioning and the run were performed with 50 mM sodium phosphate buffer pH 2.5. Before sample injection, a preconditioning with this buffer was performed for 120 s.
• Samples were injected hydrodynamically applying a 100 mbar pressure for 2 s, followed by injection of buffer by applying a 100 mbar pressure for 1 s.
• the run was carried out with a gradient voltage from 0 to 30 kV in 0.2 min (positive polarity). Electropherograms were acquired at 198, 205, 215, 254 and 300 nm wavelengths.
• BisDOTAl method the radiolabeling was performed with the Eckert & Ziegler Eurotope Modular Lab Standard ® automated synthesis system.
• 68 Ga was eluted from 68 Ge/ 68 Ga generator with 6 mL of 0.1 M HC1 solution and concentrated on a Strata-X-C ion exchange cartridge (SCX).
• SCX Strata-X-C ion exchange cartridge
• 68 GaCl3 was eluted into the reaction vial containing 2 mL of 0.2 M sodium acetate buffer (pH 4) and 7.6 nmol of BisDOTA.
• the reaction was carried out at 95°C for 400 seconds. Without purification, the radiolabeled product was analyzed by ITLC.
• BisDOTA2 method the radiolabeling and purification of BisDOTA were performed with the Eckert & Ziegler Eurotope Modular Lab Pharm-Tracer ® automated synthesis system.
• the 68 Ge/ 68 Ga generator was eluted with 6 mL of 0.1 M HC1 solution and 68 GaCl 3 was trapped on the SCX.
• the radioactive was released from the cartridge to the sample solution with 5.5 M HC1 in 5 M NaCl solution.
• Solution A was prepared by dissolving 0.29 g of sodium acetate in 2 mL of B.Braun water and adding 128 pL of 30% HC1 solution.
• the sample solution was prepared by mixing 400 pL of solution A with 2 mL of B.Braun water and 9.2 pL of a 1 mg/mL BisDOTA solution (7 nmol).
• the radiolabeling was performed at 95°C for 5 minutes and at the end saline at room temperature was added for cooling.
• the purification was performed with a Cl 8 ion exchange cartridge, preconditioned with ethanol/water 1 : 1 (v/v) and washed with water. The product was eluted from the cartridge with ethanol/water 1 : 1 (v/v) and diluted in saline.
• the radiochemical purity of BisDOTA was determined by RP-HPLC.
• Reverse-phase HPLC chromatography was carried out using a Pursuit C18, 3 pm 3.0 x 150 mm, 200 A column.
• a gradient method at a flow rate of 0.6 mL/min was applied.
• the gradient mixture was composed of 0.1% aqueous TFA (solvent A) and 0.1% TFA in acetonitrile gradient grade (solvent B). Linear gradient from 5% to 30% B in 20 min. Post run to 5% B: 3 min.
• the injection volume was 20 pL.
• the chromatograms were acquired at 220 nm wavelength.
• the sample application volumes were 5 pL and 0.9% NaCkacetonitrile 1 :1 (v/v) was used as mobile phase for the development by ascending chromatography.
• the radiochromatographic profile was determined by an autoradiographic system that uses a high performance storage phosphor screen.
• VCAM-l A stock solution of VCAM-l was prepared by dissolving 50 pg of protein in 100 pL of ultrapure water. 24 pL of this solution diluted 1 :1 with water (0.08 nmol) were mixed with 26 pL of approx. 3.2 pM MacroP aqueous solution (0.08 nmol). The reaction mixture was incubated at 37°C, under 400 rpm orbital shaking for 2h.
• MacroP 1 method the radiolabeling was performed with Eckert & Ziegler Eurotope Modular Lab Standard ® automated synthesis system.
• 68 Ga was eluted from 68 Ge/ 68 Ga generator with 6 mL of 0.1 M HC1 solution and concentrated on a SCX.
• 68 GaCl 3 was eluted with 800 pL of 0.02 M HC1 in acetone 98% into the reaction vial containing 2 mL of 0.2 M sodium acetate (pH 4) and 0.027 pmol of MacroP.
• the reaction was carried out at 95°C for 400 seconds and, after cooling, the solution was manually injected with a syringe in a Cl 8 light reverse phase silica cartridge, preconditioned with ethanol/water 1 :1 (v/v). The cartridge was washed with water for removing free 68 Ga and the product was eluted with ethanol 50% and diluted in saline.
• MacroP 2 method the radiolabeling and purification of MacroP were performed with the Eckert & Ziegler Eurotope Modular Lab Pharm-Tracer ® automated synthesis system.
• the 68 Ge/ 68 Ga generator was eluted with 6 mL of 0.1 M HC1 solution and 68 GaCl 3 was trapped on the SCX.
• the radioactive was released from the cartridge to the sample solution with 5.5 M HC1 in 5 M NaCl solution.
• Solution A was prepared by dissolving 0.29 g of sodium acetate in 2 mL of B.Braun water and adding 128 pL of 30% HC1 solution.
• the sample solution was prepared by mixing 400 pL of solution A with 2 mL of B.Braun water and 15 pL of 0.833 M MacroP solution.
• the radiolabeling was performed at 95°C for 5 minutes and at the end some saline, stored at 2-8°C overnight, was added for cooling.
• the purification was performed with a Cl 8 ion exchange cartridge, preconditioned with ethanol/water 1 : 1 (v/v) and washed with water. The product was eluted from the cartridge with ethanol/water 1 : 1 (v/v) and diluted in saline.
• the radiochemical purity of MacroP was determined by ITLC and by RP-HPLC.
• the sample application volumes were 5 pL.
• the radiochromatographic profile was determined by an autoradiographic system that uses a high performance storage phosphor screen.
• RPH1 method RP-HPLC was performed on a Merck Hitachi LaChrom L-7100/L7100 HPLC with a L-7400 UV-visible detector and a Radiomatic 150TR flow scintillation analyzer, with Vydac Everest C18, 5 pm, 4.6 x 150 mm, 300 A as stationary phase.
• a gradient method at a flow rate of 1 mL/min was applied. The gradient mixture was composed of 0.1% aqueous TFA (solvent A) and 0.1% TFA in acetonitrile gradient grade (solvent B). Linear gradient from 6% to 16% B in 10 min. Post run to 6% B: 3 min. The injection volume was 20 pL. The chromatograms were acquired at 220 nm wavelength.
• RPH2 method RP-HPLC was performed on a UltiMate 3000 UHPLC systems with a Dionex UltiMate 3000 variable wavelength detectors and GABI Star (Raytest).
• Reverse-phase HPLC chromatography was carried out using a Pursuit C18, 3 pm 3.0 x 150 mm, 200 A column.
• a gradient method at a flow rate of 0.6 mL/min was applied.
• the gradient mixture was composed of 0.1% aqueous TFA (solvent A) and 0.1% TFA in acetonitrile gradient grade (solvent B).
• Gradient elution 0-2 min 100% B; 2-7 min linear gradient from 100% to 40% B; 7-12 min 40% B.
• the injection volume was 20 pL.
• the chromatograms were acquired at 220 nm wavelength.
• HAVEC Human Umbilical Vein Endothelial Cells
• TNF-a TNF-alpha
• Single stranded cDNA products were analyzed by real-time PCR using the SsoFastTM EvaGreen mix on a CFX96 Touch real-time PCR.
• Cycling conditions were set at 94 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s, for 37 cycles.
• the specific primer pairs used for polymerase chain reaction amplification were designed on the mature transcripts and are shown in Table 6.
• Table 6 List of primer designed for the quantification of specific mRNAs by qRT-PCR in HUVECs
• GPDH glyceraldehyde-3- phosphate dehydrogenase
• Fluorescence active cells sorting /FACS ' analysis of cell adhesion molecule expression
• VCAM-l The surface expressions of VCAM-l on HUVECs were evaluated by a flow cytometry analysis using a fluorescence-activated cell sorter system.
• TNF-a After stimulation with 20 ng/ml TNF-a, the time-course experiment was performed by FACS analysis at time 0, 4h, 18h and 24h. Control and TNF-a stimulated HUVECs were detached with trypsin, washed in PBS and analyzed for VCAM-l expression using the antibody anti-CD 106 (VCAM-l) conjugated with the fluorescent dye phycoerythrin. Appropriate IgG isotype-matched antibodies and unstained cells were used as negative control. Data were acquired on BD FACSCanto II and analyzed by BD FACSDiva software.
• HUVECs were incubated at 37°C with 4 nmol of NAMP for 20 minutes and afterwards with 3.5 nmol of avidin for 10 minutes. After washing and replacing the culture medium, the binding assay of 68 Ga-BisDOTA with the NAMP-avidin complex, bound to the VCAM-l expressed on HUVECs, was monitored using LigandTracer ® White, according to the manufacturer’s instructions.
• Culture dish containing 3 mL cell culture medium, was placed on the cell dish holder of the instrument and before starting the assay, the culture medium was replaced with 3 mL of the saline solution containing 5 MBq of 68 Ga-MacroP. The binding of radioactivity to cell-containing areas and reference areas was recorded. The same procedures were also applied to a control dish seeded with unstimulated HUVECs.

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Abstract

Description

Title: Compounds and methods for detecting early atherosclerotic lesions in blood vessels

DESCRIPTION

Field of application

The present application relates to new compounds including a VCAM-l binding peptide, which can be used in methods for detecting early atherosclerotic lesions in the blood vessels, in particular the arteries, of human subjects.

In particular, such compounds are biotin derivatives or DOTA derivatives incorporating the above-mentioned peptide moiety.

Prior art

Atherosclerosis is the most common form of vascular disease and constitutes the major cause of death, with 17.5 million related deaths annually (31% of global mortality) [1] Atherosclerosis begins in childhood, as an accumulation of fatty streaks-lipid-engorged macrophages and T lymphocytes in the intima of the arteries.

During a life-time, most atherosclerotic plaques remain asymptomatic (sub-clinical disease), some become obstructive and might cause symptoms because of impaired maximal blood flow (stable angina, intermittent claudication, or mesenteric ischemia are examples), and a small percentage, in some individuals, become thrombosis-prone (vulnerable) and lead to atherothrombotic events such as acute coronary syndromes (ACS), stroke, critical limb ischemia and sudden death [2,3]

Despite recent advances in medical and interventional percutaneous or surgical therapies, coronary artery disease (CAD) continues to be a major cause of morbidity and mortality throughout the world.

Although coronary angiography is still the gold standard for the assessment of CAD, it simply shows the coronary lumen, and is unable to address plaque vulnerability or provide information on the extent and severity of atherosclerosis.

The most effective way of preventing and intervening in cardiovascular disease (CVD) is to be able to diagnose the first clinical signs.

For diagnostic purpose, non- invasive ultrasound imaging represents a safe, fast, and comparatively cheap method of assessing atherosclerosis, although its use is largely confined to the carotid and peripheral vasculature. Non-invasive imaging techniques are: ultrasounds (US), electron beam computed tomography (EBCT), multi- detector CT (MDCT), magnetic resonance imaging (MRI) and nuclear imaging, including single photon emission computed tomography (SPECT) and positron emission tomography (PET).

Ultrasound remains limited in its ability to distinguish plaque constituents and morphology. Nuclear imaging uses radiopharmaceuticals.

According to the community code relating to medicinal products for human use (DIRECTIVE 2001/83/EC), Radiopharmaceuticals are“Any medicinal product which, when ready for use, contains one or more radionuclides (radioactive isotopes) included for a medicinal purpose”, for diagnosis or therapy of various diseases.

In the last decades, the direction of research in this area has been shifted towards target-specific radiopharmaceuticals based on receptor binding of a radiolabeled receptor ligand in the diseased tissue [4]

A receptor ligand, often termed as“targeting biomolecule” (BM), serves as the“vehicle” to carry the radionuclide to the diseased tissue, which is known to contain a substantial concentration of the target receptor.

Accumulation of the radiotracer at diseased tissues relying on the localization of the radiolabeled receptor ligand, that binds to receptors with high affinity and specificity, makes receptor imaging (often called“molecular imaging”) advantageous over traditional scintigraphic imaging using, for example, simple technetium complexing radiopharmaceuticals or other imaging modalities such as X-ray computed tomography (CT), ultrasound (US), and nuclear magnetic resonance imaging (MRI) [4]

The high specificity of receptor binding results in selective uptake and distribution of the radiolabeled receptor ligand at diseased tissues.

The BMs can be small molecules, peptides, monoclonal antibodies (mAbs), nanobodies or mAh fragments, and, ideally, their affinity for the biological target should be unchanged after radiometal labeling [5]

The most convenient approach to bind a radionuclide to a BM durably is to use a suitable bifunctional chelate (BFC), which is simply a chelator with reactive functional groups that can be covalently coupled to the BM, forming a stable conjugate [5,6] Examples of such bifunctional chelating agents are reported here below.

The advantage of using DOTA analogues as BFCs is the extremely high kinetic inertness of their metal chelates; conversely, the kinetic lability of acyclic metal chelates often results in dissociation of the radiometal from the metal chelate, and leads to radiation toxicity to non-target organs, such as bone marrow [4]

To select a suitable targeting biomolecule BM for early diagnosis of atherosclerosis, the knowledge of the mechanisms involved in the evolution of the atherosclerotic plaque is important. Endothelial activation is a key event in early atherogenesis, characterised by up-regulation of adhesion molecules such as vascular cell adhesion molecule- 1 (VCAM-l), intercellular adhesion molecule- 1 (ICAM-l), P- and E-selectin, promoting monocyte recruitment to the vascular wall and subsequent lesion development.

Initial monocyte rolling along activated endothelium is mediated by P-selectin and its interaction with integrin P-selectin glycoprotein ligand- 1 (PSGL-l) expressed on monocytes, while firm adhesion of monocytes is mediated by VCAM-l (CD 106) and engagement of the integrin very late antigen-4 (VLA-4, also known as a4b1 integrin) expressed on monocytes [7]

VCAM-l is an immunoglobulin (Ig)-like transmembrane adhesion molecule, highly conserved in evolution, and participates in a variety of cellular functions in health and disease. Human VCAM-l has 2 isoforms, the predominant 7 Ig-domain isoform and a minor, alternatively spliced isoform with 6 Ig domains, whereas in mice the second isoform consists of the first 3 domains attached to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor.

VCAM-l is minimally expressed on most resting vascular endothelial cells and is inducible in many tissue vascular beds following injury or stress. Because of this activation, VCAM-l has been implicated in the pathophysiology of certain autoimmune diseases, atherosclerosis, and allograft rejection.

VCAM-l is constitutively expressed in bone marrow stromal/endothelial cells and certain classes of hematopoietic cells (B cells, follicular dendritic cells, and macrophages).

For these reasons VCAM-l is a promising marker for molecular imaging of vascular inflammation in atherosclerosis, since it is not constitutively expressed in normal vessels but is rapidly up-regulated on vascular endothelial cells in both early and advanced lesions and is readily accessible to blood-borne, targeted contrast agents. VCAM-l is also up-regulated in macrophages and smooth muscle cells in atherosclerotic plaques [7]

Its major ligand is the integrin VLA-4, with binding sites located in the first and fourth Ig domains, whereas other ligands bind with less affinity and include a4b7, a9b1 and aϋb2 [8]

For molecular imaging of vascular inflammation, Tsourkas et al. conjugated cross-linked dextran- coated iron oxide (CLIO) nanoparticles, labeled with the near-infrared fluorochrome Cy5.5, with an anti-VCAM-l antibody [9] The VCAM-l -targeted nanoparticles could detect VCAM-l expression on the endothelial cells by magnetic resonance and optical imaging.

Besides antibodies, different VCAM-l -targeting peptides have been selected using the phage display or other approaches [10,11] VHSPNKK- modified magneto fluorescent nanoparticles (VNP) had l2-fold higher binding affinity to VCAM-l than VCAM-l monoclonal antibodies and, importantly, had low binding affinity to macrophages [10] The same research group identified another peptide, VHPKQHR, that had a sequence homology to VLA-4, the natural ligand of VCAM-l, and a binding affinity of 33.7 ± 8 nM [11,12] It was conjugated to magnetofluorescent nanoparticles through a GGSK(FITC)C linker (VINP-28). In vitro experiments revealed a 20-fold higher cellular binding and internalization of VINP-28 by VCAM-l -expressing cells than the previous nanoparticles. VINP-28 had high binding affinity to endothelial cells, but low binding affinity to macrophages and smooth muscle cells, and was also able to detect endothelial cells and other VCAM-l expressing cells in resected human carotid artery lesions ex vivo [11]

The same peptide sequence has been utilized by Nahrendorf et al. to develop a VCAM-l targeting peptide-based radiotracer, called 18F-4V, which is internalised by endothelial cells and can detect VCAM-l expression in murine atherosclerotic plaques by in vivo hybrid PET-CT [13].

A SPECT radiotracer has been also developed, in which the residue 75-84 (B2702-p) of the major histocompatibility complex- 1 (MHC-l) molecule B2702 was radiolabeled with "mTc, for in vivo molecular imaging of VCAM-l expression in atherosclerotic plaques [14]

Recently, nanobodies recognising both human and mouse VCAM-l have been investigated as potential targeting ligands for SPECT. Nanobodies are single-domain antibody fragments that occur naturally in sharks and camelids. Using non-invasive SPECT-CT imaging, "mTc- radiolabeled VCAM-l nanobodies enabled in vivo detection of VCAM-l expression in aortic arch atherosclerosis in apolipoprotein E deficient (apoE-/-) mice [15].

SUMMARY OF THE INVENTION

The problem underlying the present invention was that of providing new radiopharmaceuticals suitable for application in diagnostic nuclear imaging techniques aimed at detecting early atherosclerotic lesions in the blood vessels of human subjects.

According to an aspect of the present invention, such a problem has been solved by the provision of a biotin derivative of formula (I)

in which R is a peptide with the sequence VHPKQHRGGSKGC, linked to the succinimidyl ring through the -SH group of the C-terminal cysteine.

This biotin derivative can be used in a method for detecting early atherosclerotic lesions in the blood vessels of a human subject, wherein the method includes the steps of

- forming a complex of said biotin derivative with avidin, neutravidin or streptavidin;

- parenterally administering the complex thus formed; - subsequently, parenterally administering a chelating agent including a chelating moiety, a biotin moiety and a metal radionuclide, and

- subjecting the human subject to a diagnostic nuclear imaging technique.

The chelating moiety of said chelating agent is preferably selected from the group comprising DOTA (l,4,7,l0-tetraazacyclododecane-l,4,7,l0-tetraacetic acid) derivatives, NOTA (1 ,4,7- triazacyclononane-N,N',N"-triacetic acid) derivatives, and TETA (1,4,8,11- tetraazacyclotetradecane-l,4,8,l 1 -tetraacetic acid) derivatives.

Preferably, the chelating moiety is DOTA, which is obtained from a DOTA derivative selected from the group comprising DOTA, maleimido-DOTA (l,4,7,l0-tetraazacyclododecane-l,4,7- tris-acetic acid-lO-maleimidoethylacetamide), DOTA-NHS-ester (l,4,7,l0-tetraazacyclodecane- 1 ,4,7, 10-tetraacetic acid mono-N-hydroxysuccinimide ester), p-SCN-Bn-DOTA (S-2-(4- isothiocyanatobenzyl)- 1 ,4,7, 10-tetraazacyclododecane tetraacetic acid).

Preferably, the chelating agent includes a DOTA derivative containing a biotin moiety, selected from the group consisting of BisDOTA-C3 (bis[(9H-fluoren-9-yl)methyl]-3,3-[2-oxo-2-[[6-[[5- [(3aS,4S,6aR)-exahydro-2-oxo-lH-thieno-[3,4-d]imidazol-

4yl]pentyl]amino]hexyl]amino]ethylazanodiyl]bis-(propane-l,3-diyl) Dicarbamate), BisDOTA- Lys-C3 (2,2,2-[l0-[l6-Carboxy-2,9-dioxo-22-[(3aS,4S,6aR)-hexahydro-2-oxo-lH-thieno[3,4- d]imidazol-4-yl]-7-[3-[2-[4,7, 10-tris-(carboxymethyl)- 1 ,4,7, 1 O-tetraazacyclododecan-N-l - yl]acetamido]propyl]-3,7,l 0, 17-tetraazadocosyl]-l ,4,7, 10-tetraazacyclo-dodecane-N,N,N-l ,4,7- triyljtriacetic acid), BisDOTA-Lys-(paB)-C3 (2,2,2-[l0-[2-[3-[[3-[2-[4,7,l0- tris(carboxymethyl)- 1 ,4,7, 10-tetraazacyclododecan-N- 1 -yljacetamidojpropyl] [2-[4- [5-carboxy- 5-[5-(2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanamido]- pentylcarbamoyl]phenylamino]-2-oxoethyl]amino]propylamino]-2-oxoethhyl]-l,4,7,l0- tetraazacyclododecane-N,N,N,-l,4,7-triyl]triacetic acid.

The metal radionuclide is preferably a radioactive isotope of an element selected among the group consisting of Sr, Rh, Pd, Sm, Er, Au, Bi, In, Lu, Y, Ce, Pr, Nd, Pm, Sa, Eu, Gd, Tb, Dy, Ho, Tm, Yb, Ga, Ni, Co, Fe, Cu, Re, Th and Zr. ⁶⁸Ga is particularly preferred.

Among the diagnostic nuclear imaging techniques, Positron Emission Tomography (PET) is particularly preferred.

In the complex of the biotin derivative of formula (1) with avidin, neutravidin or streptavidin, the molar ratio between said biotin derivative and avidin, neutravidin or streptavidin is preferably 1 :1.

The biotin derivative of formula (I) can also be used in a method for detecting early atherosclerotic lesions in the blood vessels of a human subject, wherein the method includes the consecutive steps of:

- parenterally administering the biotin derivative of formula (I);

- parenterally administering avidin, neutravidin or streptavidin;

- chasing any free avidin, neutravidin or streptavidin by parenterally administering biotinylated albumin or by any other chasing method suitable for avidin, neutravidin or streptavidin;

- parenterally administering a chelating agent including a chelating moiety, a biotin moiety and a metal radionuclide;

- subjecting the human subject to a diagnostic nuclear imaging technique.

The biotin derivative of formula (I) can also be used in a method for treating a disease of a human patient characterized by an overexpression of VCAM-l, which method comprises the steps of:

- forming a complex of said biotin derivative with avidin, neutravidin or streptavidin;

- parenterally administering to the patient the complex thus formed;

- subsequently, parenterally administering to the patient a biotinylated nanodispersed or microdispersed system, such as nanoparticles, liposomes and micelles.

The biotin derivative of formula (I) can also be used in a method for treating a disease of a human patient characterized by an overexpression of VCAM-l, which method comprises the steps of:

- forming a complex of said biotin derivative with avidin, neutravidin or streptavidin;

- adding said complex to a biotinylated nanodispersed or microdispersed system, selected among nanoparticles, liposomes and micelles, and

subsequently, parenterally administering to the patient said biotinylated nanodispersed or microdispersed system including said complex.

The above-mentioned biotinylated nanodispersed or microdispersed system, exploiting the high affinity of biotin moieties to avidin, neutravidin or streptavidin, forms a non covalent complex with at least one of the three residual bindig site of the complex of said biotin derivative with avidin, neutravidin or streptavidin. The biotin derivative of formula (I) can also be used in a method for treating a disease of a human patient characterized by an overexpression of VCAM- 1 , wherein the method includes the consecutive steps of:

- parenterally administering the biotin derivative of formula (I);

- parenterally administering avidin, neutravidin or streptavidin

- chasing any free avidin, neutravidin or streptavidin by parenterally administering biotinylated albumin or by any other chasing method suitable for avidin, neutravidin or streptavidin;

- subsequently, parenterally administering to the patient a biotinylated nanodispersed or microdispersed system, selected among nanoparticles, liposomes and micelles. The disease characterized by an over expression of VCAM-l is preferably selected from the group consisting of inflammatory diseases, atherosclerosis, multiple sclerosis, neurodegenerative diseases, neoplastic diseases and neuroinflammatory diseases.

The above-mentioned nanoparticles, liposomes and micelles can be loaded with at least one drug suitable for treating said disease characterized by an overexpression of VCAM-l . In such a case, the disease is preferably selected from the group consisting of inflammatory diseases, atherosclerosis, tumors, multiple sclerosis, Parkinson’s disease, neurodegenerative diseases and neuroinflammatory diseases.

The biotin derivative of formula (I) can also be used in a method for treating tumors in a human patient, which method includes the steps of

- forming a complex of said biotin derivative with avidin, neutravidin or streptavidin;

-parenterally administering said complex to the human patient.

-parenterally administering a chelating agent including a chelating moiety, a biotin moiety and a metal radionuclide.

The metal radionuclide is an energy- emitting radioisotope and is preferably selected from the group consisting of ¹⁷⁷Lu, ⁹⁰Y, ^mIn, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³ Sm, ⁸⁹ Sr, ¹⁶⁹Er and ²²³Ra.

The biotin derivative of formula (I) can be prepared by a method which comprises reacting a compound of formula (II)

with a peptide with sequence VHPKQHRGGSKGC.

The compound of formula (II) is prepared by reacting norbiotinamine of formula (III)

in which X is an anion, in particular TFA (CF3COO ),

with a compound of formula (IV)

(IV).

In another aspect of the present invention, the above-outlined technical problem has been solved by the provision of a compound of formula (V)

in which R is a peptide with the sequence VHPKQHRGGSKGC, linked to the succinimidyl ring through the -SH group of the C-terminal cysteine.

The compound of formula (V) can be used in a method for detecting early atherosclerotic lesions in the blood vessels of a human subject, wherein the method includes the steps of

- radiolabeling said compound with a metal radionuclide;

- parenterally administering the radiolabeled compound thus obtained; and - subjecting the human subject to a diagnostic nuclear imaging technique.

The metal radionuclide is preferably a radioactive isotope of an element selected among the group consisting of Sr, Rh, Pd, Sm, Er, Au, Bi, In, Lu, Y, Ce, Pr, Nd, Pm, Sa, Eu, Gd, Tb, Dy, Ho, Tm, Yb, Ga, Ni, Co, Fe, Cu, Re, Th e Zr. ⁶⁸Ga is particularly preferred.

Among the diagnostic nuclear imaging techniques, Positron Emission Tomography (PET) is particularly preferred.

The compound of formula (V) can be prepared by a method that comprises reacting a compound of formula (VI)

with a peptide with sequence VHPKQHRGGSKGC.

In the present specification, by“parenterally administering” or“parenteral administration” an intravascular administration, preferably an intravenous or intraarterial injection or infusion, is meant.

The present invention will be further explained in the following detailed description, in which reference is made to some figures, as described hereinafter.

Brief description of the drawings

Fig. 1 is a DSC spectrum of ASAM (N-hydroxysuccinimidyl-3-maleimido propionic acid) heated from 30°C to l 85°C at lO°C/min.

Fig. 2 is the 1R spectrum of ASAM.

Fig. 3 is a HPLC purity assessment of isolated N-Boc-norbiotinamine.

Fig. 4 is the DSC profile of N-Boc-norbiotinamine heated from 40°C to 200°C at lO°C/min.

Fig. 5 is the 1R spectrum of N-Boc-norbiotinamine.

Fig. 6 is a RP-HPLC chromatogram of NAM reaction mixture. Fig. 7 is the comparison between RP-HPLC chromatograms of NAM reaction mixtures at room temperature (solid line) and at 40°C (dashed line).

Fig. 8 is the comparison between RP-HPLC chromatograms of NAM reaction mixture with (dashed line) and without (solid line) NBA substrate.

Fig. 9 is an RP-HPLC chromatogram (method H-NAM2) of NAM reaction mixture.

Fig. 10 is the IR spectrum of NAM.

Fig. 11 is the H-NMR spectrum of NAM.

Fig. 12 is an RP-HPLC chromatogram of NAMP reaction mixture in phosphate buffer.

Fig. 13 is a comparison between RP-HPLC chromatograms of NAMP reaction mixture (solid line) and of a solution of the peptide in phosphate buffer (dashed line).

Fig. 14 shows the comparison between RP-HPLC chromatograms of NAMP reaction mixture (solid line) and a solution including NAM and TCEP without the peptide (dashed line).

Fig. 15 shows the comparison between RP-HPLC chromatograms of NAMP reaction mixture (dashed line) and of a NAM solution in phosphate buffer (solid line).

Fig. 16 shows the comparison between RP-HPLC chromatograms of peptide solubilised in water (dashed line) and in phosphate buffer (solid line), after 24h.

Fig. 17 shows a comparison between RP-HPLC chromatograms of the peptide in phosphate buffer, before (dashed line) and after TCEP addition (solid line).

Fig. 18 is an RP-HPLC chromatogram of NAMP reaction mixture in phosphate buffer without TCEP.

Fig. 19 is an RP-HPLC chromatogram of NAMP reaction mixture in ultrapure water.

Fig. 20 is the mass spectrum of NAMP.

Fig. 21 is a MS³ spectrum of the peak with m/z of 507.58 in Fig. 20.

Fig. 22 is the mass spectrum of NAMP derivative.

Fig. 23 shows the comparison between RP-HPLC chromatograms of MMA-DOTA (dashed line) and peptide (dotted line) standard solutions and the reaction mixture in phosphate buffer (solid line).

Fig. 24 shows the comparison between RP-HPLC chromatograms of MMA-DOTA in phosphate buffer (dashed line) and in water (solid line).

Fig. 25 shows the comparison between RP-HPLC chromatograms of MacroP reaction mixtures in water (A) and in phosphate buffer (B).

Fig. 26 is an RP-HPLC chromatogram of MacroP reaction mixture in water after 48h.

Fig. 27 is the mass spectrum of MacroP.

Fig. 28 is a MS² fragmentation spectrum of the peak with m/z 384.39 in Fig. 27.

Fig. 29 shows the comparison between HPLC chromatograms of ultrafiltrates in the presence (A) and absence (B) of avidin.

Fig. 30 shows the comparison of CE electropherograms of the ultrafiltrate retentate (dotted line) of the product of avidin and NAMP incubation with NAMP (dashed line) and avidin (solid line) solutions.

Fig. 31 shows the comparison of CE electropherograms of NAMP-avidin + BisDOTA (dashed line) and a standard BisDOTA solution (solid line).

Fig. 32 shows the comparison of CE electropherograms of BisDOTA (A) and NAMP (B) standard solutions and of (C) the ultrafiltrate of NAMP incubated with avidin and BisDOTA.

Fig. 33 shows the comparison between ITLC of ⁶⁸Ga-BisDOTA (left) and of NAMP-avidin-⁶⁸Ga- BisDOTA complex (right).

Fig. 34 shows the comparison between CE electropherogram of MacroP standard solution (A) and MacroP-VCAM-l complex (B).

Fig. 35 is an ITLC of raw ⁶⁸Ga-MacroP in 1 M ammonium acetate, using 0.1 M sodium citrate buffer as mobile phase.

Fig. 36 is a ITLC of raw ⁶⁸Ga-MacroP in 1 M ammonium acetate in water/methanol 1 :1.

Fig. 37 is an RP-HPLC of raw ⁶⁸Ga-MacroP.

Fig. 38 is an RP-HPLC of pure ⁶⁸Ga-MacroP, radiolabeled with MacroP2 method.

Fig. 39 is a calibration curve for the spectrophotometric determination of MacroP chemical purity, by arsenazo-Pb method.

Fig. 40 is a diagram showing VCAM-l mRNA expression on HUVECs before (CTR) and after TNF-a stimulation for 4, 18 and 24 hours.

Fig. 41 is a flow cytometric histogram for control cells (A), 4h (B), l 8h (C) and 24h (D) TNF-a stimulated HUVECs.

Fig. 42 is a graph showing ⁶⁸Ga-BisDOTA-avidin-NAMP uptake of activated and unstimulated

HUVECs. Fig. 43 is a graph showing ⁶⁸Ga-MacroP uptake of activated and unstimulated HUVECs.

Figure 44 is a confocal fluorescence micrograph of TNF- stimulated HUVECs incubated with

NAMP, neutravidin and liposomes and control HUVECs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention envisages the use of a specific VCAM-l binding peptide, namely a peptide with the sequence VHPKQHRGGSKGC, containing the sequence VHPKQHR, homologous to the natural ligand of VCAM-l (VLA-4), whose C-terminal arginine is linked by the spacer GGSKG, to C terminal cysteine.

This VCAM-l binding peptide is commercially available from Innovagen AB (Sweden).

With this targeting biomolecule (BM) two classes of radiopharmaceuticals have been realized, namely one (i.e. the one based on the compound of formula (V)) in which the VCAM-l ligand is directly linked to a chelating agent and the other (i.e. the one based on the compound of formula (I)) in which the VCAM-l ligand is separated from the chelating agent and only in vivo the molecule containing the BM will be associated to a chelating agent moiety through a biotin-avidin system.

The two classes of radiopharmaceuticals will have also different uptake properties, depending on ligand-receptor and biotin-avidin affinities, and on biodistribution and pharmacokinetic behaviour, which are determined by the physical and chemical properties of the structure linked to the peptide.

Furthermore, the two classes are different for the aspects involved in the radiolabeling procedure. In fact, for the first class, the prototype of which is named MacroP (compound of formula (V)), the peptide is linked to chelating agent during the radiolabeling while in the second class, the prototype of which is named NAMP, the radiolabeling procedure does not involve the peptide. Both classes of molecules can be used in SPECT and PET nuclear imaging, and also in magnetic resonance imaging (MRI).

The class of molecules, the prototype of which is NAMP (compound of formula (I)) exploits the principle of the pretargeting using the strong affinity of the biotin-avidin system.

Radiolabeled derivatives of biotin are being developed for application to“pretargeting”, usually employed in tumor therapy with radiolabeled monoclonal antibodies (MoAbs) [16-19]

The concept of pretargeting has been exploited in an attempt to overcome the low uptake of MoAbs by the tumor and improve the tumor-to-blood ratio [16].

The strategy is based on the separate administration of a BM, i.e. a monoclonal antibody (MoAb), that binds, subsequently, a second radiolabeled component [17].

Conceptually, the modified BM is injected first and allowed to distribute throughout the body, to bind the cells expressing the receptor and to clear substantially from the other tissues. Then, the radiolabeled second component is administered and, ideally, it localises at the sites where the modified BM has accumulated. If the second component has higher permeation, clearance and diffusion rates than BM, more rapid radionuclide localisation to the tumour and higher tumour selectivity are possible, thus resulting in higher tumour to non-tumour ratios [16]. The Applicant thought to exploit the biotin-avidin system for diagnostic and therapeutical purpose in atherosclerosis and all other pathologies overexpressing the VCAM-l protein.

In the class of molecules object of this patent application, the prototype of which is NAMP, the VCAM-l ligand is biotinylated to bind avidin, neutravidin or streptavidin,

which in turn bind a second component or chelating agent, suitably biotinylated and radiolabeled. Avidin, neutravidin and streptoavidin are capable of binding biotin with high affinity.

Briefly, avidin is a 66-kDa glycoprotein found in egg white, made of four identical subunits, each bearing a single binding site for biotin. One mole of protein can therefore bind up to four moles of biotin [16].

Neutravidin is a deglycosylated version of avidin, with a mass of approximately 60,000 daltons. As a result of carbohydrate removal, lectin binding is reduced to undetectable levels, yet biotin binding affinity is retained because the carbohydrate is not necessary for this activity. Avidin has a high pi but neutravidin has a near-neutral pi (pH 6.3), minimizing non-specific interactions with the negatively-charged cell surface or with DNA/RNA. Neutravidin is a tetramer with a strong affinity for biotin (/% = 10 ¹ M).

Neutravidin yields the lowest nonspecific binding among the known biotin-binding proteins. The specific activity for biotin binding is approximately 14 pg/mg of protein, which is near the theoretical maximum activity. It is available e.g. from Thermo Scientific™ as“Neutr Avidin Protein” (catalog number 31000). Streptavidin is a 52.8 kDa protein purified from the bacterium Streptomyces avidinii. With respect to avidin, streptavidin lacks any carbohydrate modification and has a near-neutral pi. Avidin shows only 30% of sequence identity to streptavidin, but almost identical secondary, tertiary and quaternary structure.

The strong hydrogen bonding interactions within the binding site of biotin is due to the presence of the ureido group and the nonoxidized thioether, that results in remarkable avidity in the femtomolar range. The high affinity of biotin for avidin, neutravidin and streptavidin and its fast blood clearance gives the advantage of better tissue-blood ratio over conventionally used radiolabeled drugs. This makes the avidin-biotin, neutravidin-biotin or streptavidin-biotin systems an ideal method for therapeutic and diagnostic purposes [20]

In the context of this patent application, the use of avidin is to be considered interchangeable with that of neutravidin and streptavidin.

The structural elements of biotin involved in binding leave the carboxylic group available for modification.

For this reason, radiolabeled biotin derivatives generally have been prepared by direct functionalization, generally with an amide, of the carboxyl group of the vitamin [21]

Wilbur et al. [22-25] published a series of articles describing modifications of this amide bond by introducing close steric hindrance to stabilize it.

Szalecki [26] synthesized norbiotinamine, a biotin derivative with a terminal amino group in place of the carboxylic acid and developed its derivatives.

Norbiotinamine is an interesting compound because it is exploitable for conjugation reactions with the carboxylic group of aminoacids or other molecules used for targeting and the resulting reversed amide bond is stable to biotinidase cleavage [21]

In 3 -steps pretargeting, the molecules containing biotin moieties and VCAM-l ligand should be injected in vivo first, in order to target the VCAM-l expressed on the surface of endothelial cells in correspondence of the atherosclerotic lesion, followed by avidin, neutravidin or streptavidin injection, and finally by the injection of a radiolabeled biotin. Generally, in this kind of pretargeting a chasing step must be also considered, for example with biotinylated albumin.

In 2-steps pretargeting, the molecules containing biotin moieties and VCAM-l ligand, before being injected, should be bound to avidin, neutravidin or streptavidin in a 1 :1 molar ratio and the injection should be followed by the parenteral administration of a molecule containing a biotin moiety linked with a chelating agent, e.g. a suitable bifunctional chelating agent like DOT A. Since only a small percentage of radioactivity really localizes on the lesion, the DOTA-biotin labeled conjugate should have high specific activity. Generally, the maximum allowed stoichiometry of a DOTA-conjugated molecule is one metallic radionuclide per each DOTA macrocyclic ring, and this may limit the imaging detection of VCAM-l on the tissue. The Applicant thought to increase the potentiality of this diagnostic approach using biotin derivatives carrying at least two DOTA groups, that can be labeled with at least two radionuclides to increase the radionuclide signal localized on the receptor.

For example, Pratesi et al. [27] synthesized several biotin derivatives carrying two DOTA groups per molecule (BisDOTA), in order to deliver a higher radiation dose to the tumors, for therapeutic purposes. Substantially, biotin has been modified through bifunctional spacers of different lengths and chemical structures and conjugated with two molecules of DOTA. Some of these derivatives are reported here below.

The BisDOTA-Lys derivatives showed avidin binding capacity higher than B1SDOTA-C3 and very similar to native biotin affinity [27] For this reason, BisDOTA-Lys-C3 was considered the best binding molecule to be exploited with NAMP and other analogous molecules.

Both classes of molecules (i.e. those having NAMP and MacroP as the respective prototypes) can be used to drag radionuclides (i.e. ¹⁷⁷Lu, ⁹⁰Y, ^mIn, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³ Sm, ⁸⁹ Sr and ¹⁶⁹Er) to neoplastic cell expressing the VCAM-l protein for antitumoral treatment.

Furthermore, the NAMP class molecules can be linked, through avidin, neutravidin or streptavidin to a biotinylated nanodispersed system (liposomes, nanoparticles, micelles), either drug-loaded or not, to exploit other therapeutic effects.

Synthesis of NAMP

A compound according to this patent application is NAMP, i.e. a norbiotinamine conjugated with a VCAM-l binding peptide, through a linker, as it is clear from the following formula:

in which R = VHPKQHRGGSKGC, where the thiol group bonded to R is that of the C- terminal cysteine of VCAM-l peptide ligand.

The presence of the biotin moiety in NAMP allows to exploit the avidin-biotin binding system for 3 -step or 2-step pretargeting.

In order to link norbiotinamine with the thiol group of the cysteine of the VCAM-l binding peptide, an activated linker, N-hydroxysuccinimidyl-3-maleimido propionic acid (ASAM), has been synthesized. This linker has a carboxylic group, activated by N-hydroxysuccinimide, able to bind the amino group of norbiotinamine, and a maleimide group that can react selectively with the thiol group of C-terminal cysteine in the peptide, according to the following scheme:

The reaction occurs between norbiotinamine and the thiol group of the ligand through the linker named ASAM. The thiol group of R-SH is that of C-terminal cysteine of the VCAM-l ligand. Here after are described the synthesis and characterization of ASAM; the conversion of biotin into norbiotinamine and the characterization of the synthetic intermediate, N-Boc- norbiotinamine; the reaction between ASAM and norbiotinamine, to form N-norbiotinyl-b- maleimidopropionylamide (NAM) and its characterization; finally, the reaction between NAM and the VCAM-l binding peptide.

The main reaction steps are depicted here below:

Synthesis of ASAM or N-hvdroxysuccinimidyl-3-maleimido propionic acid

AS AM was prepared as described by Hong Y. Song et al [28]

b- Alanine (22.4 mmol) and a solution of maleic anhydride (22.4 mmol) in 25 mL of DMF were mixed and stirred at room temperature up to complete dissolution.

To the resulting solution, cooled to 0°C in an ice bath, N-hydroxysuccinimide (28 mmol) was added, followed by dicyclohexylcarbodiimide DCC (47.7 mmol), and the mixture was stirred o/n at room temperature.

The precipitated N,N’-dicyclohexylurea (DCU), formed during the reaction, was removed by centrifugation at 2170 rpm for 10 minutes, followed by paper filtration.

The filtrate was poured on ice to obtain the product as a white precipitate.

After centrifugation at 2170 rpm for 10 minutes and Buchner filtration, the solid was washed with water and dried under vacuum. The reaction yield was 54% w/w.

The product was characterized by elemental analysis, thermal analysis, FT-IR, ^-NMR, ¹³C- NMR and TLC. Purity was evaluated by RP-HPLC.

Elemental analysis

Table 1 shows the comparison between the theoretical percentage composition of AS AM and the measured one. The deviation of the elemental analysis from the theoretical composition is lower than 0.3%.

Table 1 Theoretical and actual percent composition ofASAM

Thermal analysis

A single endotherm at l63°C (extrapolated onset) was observed (see Fig. 1), corresponding to the melting point of ASAM in accordance to the hot stage microscopy analysis.

IR analysis

Fig. 2 shows the infrared spectrum of ASAM. At the high frequency portion of the spectrum, it is possible to observe maleimide C-H stretching vibrations (3108 cm ¹) and asymmetrical and symmetrical methylene stretching vibrations (2929 cm ¹, 2851 cm ¹). The most intense band occurs at 1720 cm ¹, corresponding to the C=0 stretching vibration; the asymmetric and symmetric bands due to the ester C-O-C stretching vibrations occur at 1213 and 1072 cm ¹ respectively.

¹ H-NMR and ¹³ C-NMR spectroscopy

¾-NMR (300 MHz, CDC1₃): 2.83 (4H, s, -CH₂CH₂-), 3.02 (2H, t, J = 7 Hz, -CH₂-N-), 3.93 (2H, t, J = 7 Hz, -CH₂-COO-), 6.74 (2H, s, -CH=CH-).

¹³C-NMR (300 MHz, CDCI3): 25.6 (-CH₂CH₂-), 29.7 (CH₂-COO-), 33.0 (-CH₂-N-), 134.3 (- CH=CH-), 166.0, 168.8, 170.1 (3 C=0).

Chromatographic purity assessment

NP-TLC: with ethyl acetate/methanol 85:15 (v/v) mobile phase ASAM R_f is 0.85.

RP-HPLC: ASAM retention time (t_R> is 2.4 minutes.

With both methods ASAM, so produced, showed maximum chromatographic purity, under the method detection limits.

Synthesis of norbiotinamine

Most norbiotinamine syntheses reported in the literature refer to a work published by Szalecki [26] It consists in a one-pot reaction wherein biotin is added in equimolar amounts to TEA and DPPA, in t-BuOH at reflux, to form the derivate N-Boc- norbiotinamine. Norbiotinamine is then obtained by acidic hydrolysis, as reported in the scheme below.

Initially, the reaction was attempted under the reported conditions, but it gave low reaction yield and purity. Moreover, the purification steps appeared more complicated in terms of cost, time and purity results.

For this reason, solvent and reaction conditions were modified.

Since biotin is very slightly soluble in t-BuOH, even in the presence of TEA, t-BuOH was replaced with a high dielectric constant and aprotic solvent, DMF, which had shown to dissolve biotin better than t-BuOH.

The reaction was carried out by solubilizing biotin in DMF and then adding TEA and, after 10 minutes, DPPA at room temperature. After 30 min t-BuOH was added at room temperature. After lh the mixture was heated gradually (20°C/h) up to 90°C and refluxed under stirring for 24h.

The modification introduced in this synthesis involved:

a different reaction solvent

a different sequence of addition of the reactants or catalyzers

a postponed and controlled addition of tertbutanol

an accurate control of thermal conditions with a gradual heating up to 90° C

an optimization of reaction time.

N-Boc-norbiotinamine was isolated from the reaction mixture by a semipreparative RP-HPLC. N-Boc-norbiotinamine was obtained highly pure, with a yield of 50% (Fig. 3).

Detailed synthesis of N-Boc-norbiotinamine (NBA-BOC)

In a flask, 500 mg of biotin (2.05 mmol) were dissolved in 37.5 mL of DMF, at room temperature. After complete dissolution, 620 pL of TEA (4.4 mmol) were added and stirred at room temperature for 10 minutes, followed by the addition of 620 pL of DPPA (2.9 mmol). After stirring at room temperature for 30 minutes, 90 mL of t-BuOH were added. Gradual heating (20°C/h) was carried out up to 90°C and the reaction was refluxed for 24h. After purification in semi-preparative RP-HPLC and drying by Rotavapor, NBA-BOC was obtained as a flocculated solid with a yield of 50% w/w.

NBA-BOC was hydrolyzed to NBA with TFA:dichloromethane 1 :l, at 0°C for 2h. Solvents were removed with Rotavapor and the obtained product was utilized for the next reaction without further purification.

Characterization of N-Boc-norbiotinamine

The product was characterized by elemental analysis, thermal analysis, FT-IR, H-NMR and ¹³C- NMR.

Elemental analysis

Table 2 shows the comparison between the theoretical percentage composition of N-Boc- norbiotinamine and the experimental one. The deviation of the elemental analysis from the theoretical composition is lower than 0.3%.

Table 2: Theoretical and actual percentage composition of N-Boc-norbiotinamine.

Thermal analysis

The thermal profile of the substance shows a single endotherm at l68°C (extrapolated onset), which is the melting point of N-Boc-norbiotinamine, in accordance with the hot stage microscopy observation (Fig. 4).

IR analysis

The N-Boc-norbiotinamine infrared spectrum is shown in Fig. 5. In the high frequency portion of the spectrum, it is possible to observe the N-H stretching (3535 cm ¹, 3296 cm ¹), followed by stretching of methyl and methylene groups (2978 cm ¹, 2930 cm ¹ and 2865 cm ¹). The most intense signal corresponds to the stretching C=0 of the carbamate group (1693 cm ¹), which has a shoulder probably related to the stretching C=0 of the ureic group (1675 cm ¹).

¹ H -NMR and ¹³ C-NMR spectroscopy

H-NMR and ¹³C-NMR spectra were acquired, obtaining results comparable with those found in the literature [29^].

‘H-NMR (300 MHz, CDC1₃): 4.50 (1H, dd, ³H), 4.34 (1H, dd, J= 4.5, 7.5 Hz, ⁴H), 3.13 (1H, m, ^SH), 2.99 (2H, m, ⁷H), 2.90 (1H, dd, J=5, 12.7 Hz, ⁶H), 2.73 (1H, d, J= 12.7 Hz, ⁶H), 1.70-1.52 (6H, m, ⁸H,⁹H,¹⁰H), 1.45 (9H, s, ⁿH).

¹³C-NMR (300 MHz, CDCI3): d 164.9, 157.6, 79.1, 61.6, 60.5, 55.3, 40.7, 40.4, 29.7, 28.5, 28.3, 25.6.

Synthesis of norbiotinamine

After identifying and characterizing N-Boc-norbiotinamine, the hydrolysis reaction was performed. The reaction was carried out at the condition reported in the literature and the obtained product was used for the following reaction without purification.

Detailed synthesis of norbiotinamine (NBA)

NBA-BOC was hydrolyzed to NBA with TFA:dichloromethane 1 :l, at 0°C for 2h. Solvents were removed with Rotavapor and the obtained product was utilized for the next reaction without further purification.

Synthesis of N-norbiotinyl- -maleimidopropionylamide (NAM)

One reaction is reported in the literature for the synthesis of N-norbiotinyl-P-malcimido propionylamide [26] Chloroform was used as solvent and the reaction conditions were room temperature for 18 hours, using a molar ratio of 1.5 between b-maleimidopropionate and norbiotinamine.

Initially, the reaction was performed applying the aforementioned conditions, adding TEA in molar ratio 1 : 1 with norbiotinamine, in order to favour the nucleophilic attack of the amine on the activated carboxyl group of ASAM, according to the following scheme.

At the end, the obtained reaction mixture dried and dissolved in the mobile phase was analysed by RP-HPLC with H-NAM1 method (Fig. 6).

The peak assignment of the chromatogram in Fig. 6 was performed against injections of standard solutions.

Peak 1 was identified as N-hydroxysuccinimide, which is cleaved from ASAM after the nucleophilic attack of the amine on the carboxylic group.

Peak 2 and peak 4 correspond to unreacted ASAM and its impurity, respectively.

Peak 3 was attributed to NAM, as confirmed by further RP-HPLC analyses.

First, the reaction was carried out at room temperature and at 40°C and the comparison of the two reaction mixtures is shown in Fig. 7.

The decrease of ASAM and the increases of N-hydroxysuccinimide and peak 3 compound at 40°C were considered indicative of the progression of the reaction. The possible atribution of peak 3 compound as NAM was indicated since a chloroform solution of TEA and ASAM, without norbiotinamine, heated at 40°C for l 8h did not show peak 3 formation (Fig. 8), and by UV spectrum comparison with that of ASAM.

For further evaluation, another RP-HPLC method, H-NAM2, was applied in order to improve peak resolution. Fig. 9 shows the analysis of the reaction mixture with the new RP-HPLC method and the peak assignment.

For NAM purification, due to its solubility in water, 3 extractions with water/chloroform 1 :1 were carried out to separate NAM from ASAM.

The aqueous phase containing N-hydroxysuccinimide and NAM was vacuum dried by Rotavapor and NAM was purified from the residual solid mixture by repeated washings with diethylether, obtaining a N-norbiotinyl- -maleimidopropionylamide yield of 23% w/w. The elemental analysis, 1R analysis, H-NMR spectroscopy confirmed NAM identity.

The original reaction conditions [26] were modified to improve the yield.

Chloroform was replaced with acetonitrile, more able to solubilize norbiotinamine, and, after various attempts, the best yield was obtained by heating at 80°C for 24h.

With these new conditions, the product precipitated by cooling the reaction mixture and maintaining it at -20°C for 48h; moreover it was possible to purify the product from N- hydroxysuccinimide by washings with diethylether, obtaining a final yield of 60% w/w.

Detailed synthesis ofN-norbiotinyl-fj-maleimidopropionylamide (NAM)

NBA (about 0.15 mmol) and TEA (0.15 mmol) were dissolved in 25 mL of acetonitrile, by sonication. 0.24 mmol of ASAM were added to the dispersion, and the reaction mixture was stirred in glycerine bath at 80°C for 24h, under reflux. The product was collected by precipitation at -20°C after 48h and obtained pure after four washings with diethylether. The yield obtained was 60 % w/w.

Characterization of NAM

The characterization was performed by elemental analysis, 1R analysis, H-NMR spectroscopy and RP-HPLC.

Elemental analysis

Elemental analysis on C, H, N and S was carried out in order to confirm the composition of the product. Table 3: Elemental analysis of NAM: theoretical and actual percentage composition.

IR analysis

Fig. 10 shows the IR spectrum of NAM.

In the high frequency portion of the spectrum, it is possible to observe the N-H stretching of the secondary amides (3286 cm ¹ and 3090 cm ¹), followed by methylene groups stretching (2928 cm ‘and 2856 cm ¹).

The most intense signal is the stretching C=0 at 1703 cm ¹, which is related to the carbonyl of the maleimide.

This signal has a shoulder at 1680 cm ¹, which could be the signal of the C=0 stretching of the biotin ureic cycle. The signal at 1638 cm ¹ might be both C=0 stretching and NH bending of the amide, since when these signals have similar frequencies they can turn out into one peak.

Finally, the IR spectrum of NAM shows peaks similar to the spectrum of ASAM, such as 828 cm ¹ and 696 cm ¹ signals, which may be related to the maleimidic CH.

¹ H-N R spectroscopy

H-NMR spectrum (Fig. 11) was acquired in DMSO-d₆ for the low solubility of NAM in chloroform, dichloromethane and methanol.

‘H-NMR (300 MHz, DMSO-d): d 7.91 (1H, t, NH⁹), 7.01 (2H, s, CH=CH¹²), 6.40 (H, d, J=2l .O Hz, NH¹), 4.31 (1H, dd, CH² ), 4.13 (1H, dd, CH² ), 3.60 (2H, t, J=7.3 Hz, CH₂ ⁿ), 3.07 (1H, m, CH⁴), 2.98 (2H, m, CH₂ ⁸), 2.93 (lH,dd, J=5, 12.4 Hz, CH₂ ³), 2.57 (1H, d, J=l 1.6 Hz, CH₂ ³ ), 2.31 (2H, t, J=7.l Hz, CH₂ ¹⁰), 1.23-1.58 (6H, m, CH₂ ⁵-CH₂ ⁶- CH₂ ⁷).

The spin-spin decoupling was used for simplifying the spectrum and determining the positions of some protons in the molecule. The decoupling of the signal of the amidic NH proton (7.91 ppm) permitted to convert the multiplet at 2.98 ppm in a triplet, confirming the formation of the amide binding between ASAM and norbiotinamine.

The decoupling of the signal at 3.60 ppm, related to the CH2^{1 1} protons, converted the triplet of CH2¹⁰ into a singlet, showing the integrity of the structure of ASAM in the molecule. Synthesis of NAMP

Most of the procedures reported in the literature for the conjugation of a maleimide with peptides by thioether linkage are carried out in a buffer at pH ranging from 7.0 to 7.5, in a 10-100 mM phosphate, Tris or HEPES buffer. In this pH range, the thiol group is sufficiently nucleophilic for reacting almost exclusively with the maleimide, even in the presence of a more prominent lysine amino group, which is protonated and relatively unreactive.

Usually, before coupling, the disulfide bonds of the peptide are reduced by a 10-fold molar excess of a reducing agent such as dithiothreitol (DTT) or tris-(2-carboxyethyl) phosphine hydrochloride (TCEP-HC1), for 2 hours, at room temperature.

The commercial availability of TCEP-HC1, which is odorless and water-soluble, makes this reagent safe and convenient to use [30]

TCEP-HC1 is a strong reducing agent that reduces even very stable alkyl disulfides, rapidly and cleanly in water, at room temperature and pH 5 [31].

TCEP-HC1 has pKa of 7-8, which is a common pH range for performing bioconjugations, and, as such, the trialkylphosphines are more- effective nucleophiles than thiol-based reducing agents to effect reduction within this pH range.

Furthermore, disulfide reductions utilizing alkylphosphines are irreversible and driven by phosphorus-oxygen bond formation, unlike the reversible mechanism of disulfide reduction observed with thiol-containing reducing agents [32]

In addition, TCEP has been advertised as being less reactive than DTT with thiol-reactive compounds, thereby eliminating the need to remove it before labeling [33]

For this reason, initially it was decided to perform the reaction between the maleimide of NAM and the thiol group of the VCAM-l binding peptide cysteine in phosphate buffer, at pH 7 and in the presence of TCEP, without removing it from the reaction mixture, according to the following scheme.

The reaction mixture was analysed by RP-HPLC, using the H-NAMP1 method (Fig. 12). Peaks 1-2 of the chromatogram in Fig.12 are related to the phosphate buffer, although traces of peptide could co-elute with peak 2, as shown in Fig. 13.

Peaks 5 and 6 (Fig. 12) are related to NAM, in particular peak 5 forms also in a mixture including NAM and TCEP (Fig. 14), while peak 6 correspond to a NAM derivative that forms in phosphate buffer over time (Fig. 15).

Peak 3 (Fig. 12) shows a behaviour similar to peak 6 and seems to be related to peak 4: it increases over time in phosphate buffer, while peak 4 decreases.

Assuming that peak 4 could be NAMP, peak 3 was supposed to be the hydrolysed maleimidic derivative of NAMP shown here below, in accordance with a work published by D. Fontaine et al. in 2015 [34]

Peaks 4 and 3 (Fig. 12) were isolated by semi-preparative RP-HPLC and characterized by mass spectrometry (Figs. 20 and 22) which confirmed that they correspond to NAMP and to its hydrolysed derivatives respectively.

In fact, the maleimide-thiol conjugates form through Michael addition of a thiolate (RS ) to the double bond of the maleimide to produce a succinimidyl thioether (SITE), but the succinimidyl moiety of a SITE undergoes irreversible hydrolysis to provide two isomeric succinamic acid thioethers (SATE). Since, in the presence of excess of other thiolate (R’S ), as in most biological environments, a new conjugate could form with this thiol and the original SITE considered to be irreversibly cleaved (see scheme here below).

Thus, a strategy to stabilize maleimide-thiol conjugates is to intentionally hydrolyse the conjugate prior to its exposure to exogenous thiol.

The two SATE derivatives of NAMP can be useful because they bind VCAM-l with a longer spacer between the biotin derivative and the peptide, which in some pathological tissue conditions could increase the affinity of the tracer to VCAM-l, with respect to the unhydrolysed molecule, or also stabilize the tracer in vivo.

The original reaction conditions were modified to improve NAMP yield.

The first possible change was not to incubate the peptide with TCEP before the coupling reaction with the maleimide. To verify the necessity of using a reducing agent to decompose the dipeptide possibly deriving from the formation of disulphide-bond between two cysteines, the peptide, dissolved in phosphate buffer, was analysed by RP-HPLC, applying method N-NAMP2 since with method N-NAMP1 the peptide co-elutes with the phosphate buffer.

No dipeptide was detected, but when the analysis was repeated after 24h a second peak, with higher t_¾ appeared; this phenomenon did not occur when the peptide was dissolved in water. Fig. 16 shows a comparison between the peptide dissolved in water and in phosphate buffer, analysed after 24h.

For establishing that the second peak, forming in phosphate buffer, was the dipeptide, TCEP was added to the solution ln this condition, the second peak disappeared and the first peak increased (Fig. 17). So the dipeptide forms only under specific conditions and is not detectable at the beginning of the reaction, so TCEP resulted to be not strictly necessary.

For these reasons, the NAMP synthesis was repeated without TCEP: as a result, the compound corresponding to peak 5 did not form and the area of NAMP peak increased (Fig. 18).

Finally, in order to further increase the NAMP yield, the reaction was repeated in ultrapure water, without using the phosphate buffer, at pH in the range 5-6, a pH value that allows to keep the selectivity of the nucleophilic attack of the thiol group on the maleimide. Moreover the reaction temperature was increased to 37°C and the reaction time prolonged from 2h to 24h.

Under these conditions, a reaction mixture including only NAMP and NAM, with complete peptide consumption, was obtained (Fig. 19).

Detailed synthesis of NAMP

A stock solution of NAM was prepared by dissolving 0.6 mg of NAM in 1.5 mL of ultrapure water, in a 1.5 mL polypropylene tube. The peptide solution was prepared by solubilising 1 mg in 500 pL of ultrapure water. 1.05 mL of NAM stock solution (1.3 pmol) and 450 pL of peptide solution (0.65 pmol) were mixed in a 1.5 mL polypropylene tube, and heated at 37°C, under orbital shaking at 400 rpm for 24h, under nitrogen. The isolation or purification of NAMP was performed by semi-preparative RP-HPLC.

Characterization of NAMP

NAMP characterization was performed by mass spectrometry, using the LTQ Orbitrap™ Velos Pro. The LTQ Orbitrap™ is a high-performance LC-MS and MSn system, combining rapid LTQ ion trap data acquisition with high accuracy Orbitrap mass analysis.

ft permitted to confirm the structure of the new radiopharmaceutical, identifying the formation of a thioether binding between the peptide and NAM.

Fig. 20 shows the mass spectrum of the purified molecule. The measured mass coincides with the calculated monoisotopic mass.

By the fragmentation in MS² of the peak with m/z of 439.97 and the fragmentation in MS³ of the peak with m/z of 507.58, it was possible to identify the fragment ion types that are produced by cleavage of different bonds along the peptide backbone.

The fragmentations are described following the specific nomenclature [35, 36]: cleavage of the backbone typically occurs at the peptide amide bond to produce b ions, if the amino terminal fragment retains the charge; y ions, if the carboxy-terminal fragment retains the charge. The y series is sometimes accompanied by peaks formally corresponding to loss of NH3 from the y ions if the fragment includes arginine, asparagine, lysine or glutamine as aminoacids, or loss of H2O from y ions if the fragment includes serine, threonine, glutamic acid or aspartic acid.

In Fig. 21 the fragmentation in MS³ shows the following ions:

ylO: related to the loss of valine, histidine and proline;

y9: for the cleavage of the 4 terminal aminoacids and the related ions that lost NH₃ and successively H2O;

y8: related to further loss of glutamine;

y7 : for the cleavage of the amidic bond between arginine and histidine;

y2: in which only cysteine and glycine remained.

The y2 ion permitted to confirm that the peptide was conjugated to the maleimide through the thiol group of the cysteine.

By mass spectrometry, the two NAMP isomeric derivatives got by hydrolysis of the maleimide have been identified. In fact, the calculated monoisotopic mass of this substance (M_mi = 1773.8515) coincides with the measured value (Fig. 22). By fragmentation in MS² of the peak with m/z 592.29, it was possible to detect the y ions, deriving from the cleavage of different bonds along the peptide backbone.

Synthesis of the compound of formula (V) (also called MacroP)

The conjugation reaction between maleimido-monoamide DOTA (MMA-DOTA) and the VCAM-l binding peptide was initially performed in phosphate buffer at pH 7, at 37°C for 2h, in accordance with an analogous procedure described in the literature. To avoid the formation of byproducts in the reaction mixture, no reducing agent (e.g. TCEP or DTT) was added.

The reaction mixture was analysed by analytical RP-HPLC and by the comparison with standard solutions of MMA-DOTA and peptide in phosphate buffer at pH 7, and the peaks in the chromatogram were identified (Fig. 23).

The MMA-DOTA solution in phosphate buffer showed two peaks, attributed to the maleimidic portion of MMA-DOTA. In fact, an aqueous solution of MMA-DOTA was analysed at the same concentration of the phosphate buffer solution and, as shown in Fig. 24, it presented only the second peak; therefore, the first peak resulted to be a MMA-DOTA derivative forming exclusively in phosphate buffer at pH 7. Hence, to reduce the formation of impurities and increase the amount of the supposed product, the reaction was also repeated using ultrapure water as reaction medium.

Fig. 25 shows the comparison between the reaction mixtures in water and in phosphate buffer, after stirring at 37°C for l9h.

Carrying out the reaction in water, a remarkable increase of the peak of interest was obtained, furthermore, within the detection limits of the analytical method applied, it was not possible to detect any residual MMA-DOTA or other peaks.

These experimental evidences permitted to set other reaction conditions as follows:

1. The amount of MMA-DOTA was increased from a molar ratio of 1.2 to 2 with respect to the amount of peptide;

2. The reaction time was extended to 48h.

The chromatogram of the reaction mixture obtained by applying the new condition is shown in Fig. 26. It showed increased MacroP formation and the presence of unreacted MMA-DOTA. Detailed synthesis of MacroP

In a 1.5 mL polypropylene tube, 1.13 mg of MMA-DOTA (1.4 pmol) were dissolved in 500 pL of ultrapure water. 1 mg of peptide (0.72 pmol) was solubilised in 500 pL of ultrapure water and added to the MMA-DOTA solution. The reaction was heated at 37°C, under orbital shaking at 400 rpm for 48h, in atmosphere of nitrogen.

MacroP was purified by semi-preparative RP-HPLC.

Characterization of MacroP

The purified MacroP was analysed by analytical RP-HPLC, resulting highly pure.

The pure MacroP has been characterized by mass spectrometry (Fig. 27). The mass spectrometry analysis of MacroP was performed by a LTQ Orbitrap Velos Pro, in order to confirm the identity of the substance and to identify the thioether bond between the thiol group of the VCAM-l binding peptide and the maleimide of MMA-DOTA.

The calculated monoisotopic mass is 1915.9, but the presence of peaks with a mass of 1916.9 is justified by the abundance of the different isotopes of the elements in the molecule.

In fact, mass spectrometry cannot detect single molecules but is influenced by the millions of copies of a molecule that includes different isotope species.

In mass spectrometry, the monoisotopic mass is most often used: it is the sum of the masses of the atoms in a molecule using the principle (most abundant) isotope mass of each atom instead of the isotope averaged atomic mass (atomic weight).

The mass spectral peak representing the monoisotopic mass is not always the most abundant isotopic peak in a spectrum, although it stems from the most abundant isotope of each atom type. In fact, as the number of atoms in a molecule increases, the probability for the entire molecule to contain at least one heavy isotope increases.

It is possible to note that the molecular ion with mass 1916.9 is more abundant than the one with mass 1915.9.

By the MS² fragmentation spectrum obtained from the peak with m/z of 384.39, it was possible to identify the fragments related to the cleavage of the aminoacids occurred at the peptide amide bond and the relative y ions (Fig. 28).

Since during the synthesis and purification a metallic contamination could occur and cause the incapacity of MacroP to chelate the radionuclide, the complexing capacity of the product was evaluated, before radiolabeling.

On this purpose, a method adapted by Caviglioli et al. [37] was used based on the decrease of the 656 nm absorbance of a complex between lead and arsenazo, for the concurrent formation of the complex between the metal and the DOTA derivative. The chelating efficiency of the macrocycle after preparative reaction resulted unchanged.

EXAMPLE 1

NAMP capability of binding avidin and biotinylated chelating agent.

First, NAMP capability of binding avidin was tested by RP-HPLC and CE.

Since avidin can theoretically bind up to four molecules of biotin, a NAMP/avidin molar ratio of 4:1 was incubated for 2h and the solution was ultrafiltered on a 30kDa cut-off ultrafilter, since the MW of NAMP-avidin complex is 67000 Da.

The ultrafiltrate was analysed by RP-HPLC in order to verify the presence of unbound NAMP (Fig. 29 A), while the retentate was examined by CE.

To assay the possible aspecific binding of NAMP to the filter, ultrafiltration in the absence of avidin was also carried out and the ultrafiltrate was analysed by RP-HPLC (Fig. 29 B).

In Fig. 29, RP-HPLC comparison between the chromatograms of the ultrafiltrates shows that when NAMP was incubated with avidin, no detectable unbound NAMP was present in the ultrafiltrate.

The retentate was analysed by CE and compared with standard solutions of avidin and NAMP. The formation of NAMP-avidin complex was indicated in the CE electropherogram with a new peak with a migration time different from the avidin and NAMP standard solution peaks and with a different peak profile (Fig. 30).

To demonstrate the NAMP-avidin ability to complex a biotinylated chelant agent, the NAMP- avidin complex was incubated with a BisDOTA ( B is DOT A- Lys-C ) solution, in order to verify the formation of NAMP-avidin-BisDOTA complex by CE.

From the comparison between the CE electropherograms of the reaction mixture and a BisDOTA solution at the same concentration, it was possible to notice that the BisDOTA peak disappears in the presence of the NAMP-avidin complex (Fig. 31).

A further test was performed by CE analysing the ultrafiltrate of the solution of NAMP incubated with avidin and BisDOTA for lh at 37°C.

In Fig. 32 the comparison between the electropherograms of the ultrafiltrate and of standard BisDOTA and NAMP solutions is shown: considering the limits of detection of the applied method, BisDOTA and NAMP were not found in the ultrafiltrate, as they were bound to avidin. EXAMPLE 2

BisDOTA radiolabeling

BisDOTA was radiolabeled with ⁶⁸Ga by using the BisDOTA 1 method reported in the below “materials and methods” section, in order to develop a preliminary test for evaluating the formation of NAMP-avidin-radiolabeled [⁶⁸Ga] BisDOTAcomplex.

After the radiolabeling, a 1TLC method using saline:acetonitrile 1 : 1 mobile phase has been used as product control.

The method should be able to distinguish the free radiolabeled BisDOTA from the BisDOTA bound to the complex.

Fig. 33 shows the 1TLC comparison between radiolabeled BisDOTA and NAMP-avidin- radiolabeled BisDOTA complex: while free radiolabeled BisDOTA migrates with Rf= 0.6, when radiolabeled BisDOTA is incubated with NAMP-avidin complex it does not migrate.

The BisDOTA2 method was applied and the radiochemical purity of the product was analyzed by RP-HPLC: RCP >90%. EXAMPLE 3

MacroP binding with VCAM-l

MacroP ability of binding VCAM-l was tested by CE. MacroP and VCAM-l were mixed in equimolar amount and, after 400 rpm stirring at 37 °C for 2 h, the solution was analyzed by CE and compared with a MacroP standard solution at the same concentration (Fig. 34)

ft is possible to note a decrease of the MacroP peak after the incubation with VCAM-l, which indicated the interaction between MacroP and VCAM-l .

The radiolabeling was performed using the Eckert & Ziegler Eurotope Modular Lab Standard^® automated synthesis system: briefly, after the elution of ⁶⁸Ge/⁶⁸Ga from the generator, ⁶⁸Ga is trapped on the SCX while ⁶⁸Ge and other metallic impurities are eluted in the waste.

By the acetone method, ⁶⁸Ga is eluted from the SCX with a 0.02 M HC1 solution in 98% acetone into the reaction vial containing MacroP.

Before radiolabeling, to assess MacroP stability to the reaction conditions, the compound was dissolved in 0.2 M ammonium acetate buffer (pH 4), 800 pL of 0.02 M HC1 acetone solution were added, and the mixture was heated at 95°C for 400 seconds; after cooling, a RP-HPLC was carried out using a standard MacroP solution at lower concentration as reference, and no differences were found.

Subsequently, MacroP was radiolabeled and RCP tested by ITLC and RP-HPLC, before purification.

For the ITLC analysis, two different mobile phases were used:

-0.1 M sodium citrate buffer (Fig. 35) in which the product does not migrate, while free gallium migrates with the solvent front;

-1 M ammonium acetate in water/methanol 1 :1 (Fig. 36) in which the hydrolysed gallium remains at the application line, while the product migrates.

The product RCP resulted to be higher than 97% with both methods.

The product was also analysed by RP-HPLC using the RPH1 method and the obtained RCP resulted to be higher than 90% (Fig. 37).

EXAMPLE 4

Stability study of ⁶⁸Ga-MacroP in saline

MacroP has been radiolabeled with method (MacroP2) deriving from the Mueller [38] one with slight modifications.

The radioactivity yield was 57% with a RCP equal to 97%, when calculated by ITLC, and of 99%, when measured by RP-HPLC RPH2 method (Fig. 38).

A preliminary stability study at room temperature for 4h of radiolabeled ⁶⁸Ga-MacroP in saline was performed by ITLC, as reported in Table 4.

Table 4: Stability study of⁶⁸Ga-MacroP in saline.

EXAMPLE 5

Determination of MacroP chelating efficiency

This determination has been used as a titrimetric method to evaluate the chemical purity of MacroP. The spectrophotometric assay is based on the decrease of the 656 nm absorption of the complex between Pb⁺⁺ and arsenazo (AA), for the concurrent formation of the Pb-DOTA complex.

Fig. 39 shows the calibration curve for the spectrophotometric determination of MacroP chemical purity, by arsenazo-Pb method.

EXAMPLE 6

In vitro cell VCAM-l tests

In vitro cell tests were performed on human umbilical vein endothelial cells (HUVEC) stimulated overnight with 20 ng/mL TNF-a, for the expression of VCAM-l .

The expression of VCAM-l on HUVECs was induced by TNF-a stimulation for different times and was evaluated by qRT-PCR and FACS analysis.

To assess the VCAM-l mRNA level in HUVECs, a quantitative assay utilizing reverse transcription-polymerase chain reaction was performed. As shown in Fig. 40, TNF-a incubation caused a stimulatory effect on VCAM-l mRNA levels in HUVECs as corrected by GAPDH and 28S transcripts. The maximum of the VCAM-l expression was observed within l8h stimulation, then the mRNA expression descended.

This result was confirmed by flow cytometry analysis of the surface expression of VCAM-l on HUVECs at 0, 4, 18 and 24 h TNF-a stimulation, using the antibody anti-CDl06 (VCAM-l) conjugated with the fluorescent dye phycoerythrin.

Histograms in Fig. 41 plot the fluorescence intensity (on the x-axis) against the cell count (on the y-axis). The P3 gate identifies a region of the plot in which the cells are vital and show a high fluorescence intensity, which is related to an increased VCAM-l expression.

An increase of the fluorescence intensity is visible at 4h and mostly at l8h, while it decreases at 24h.

This is confirmed by the comparison between the % total of vital cells and the mean fluorescence intensity at 0, 4, 18 and 24 h TNF- a stimulation of HUVEC (Table 5).

While the % of total vital cells is approximately similar for 4, 18 and 24h stimulation, the mean fluorescence intensity is higher at 18h and decreases drastically at 24h.

Table 5: Comparison between the % of total vital cells and the mean fluorescence intensity for TNF-a stimulated HUVEC at different times.

TNF-a

% Mean fluorescence stilmulation

total cells intensity

time (h)

0 (CTR) 2.8 4.348

_

71 31.338

18 63 69.394

24 73.2 10.778

For NAMP in vitro test, TNF-a stimulated and unstimulated HUVEC were incubated first with NAMP then with avidin, before adding 6 MBq of ⁶⁸Ga-BisDOTA in 4 mL of the culture medium. Regarding MacroP, since the ⁶⁸Ga-MacroP specific radioactivity was lower than the one of ⁶⁸Ga- BisDOTA’s, the culture medium was replaced with 3 mL of saline solution containing 5 MBq of radiolabeled MacroP. The results were analysed by using the Ligandtracer^® technology, based on repeated differential measurements of surface-associated proteins.

By a reprocessing of the instrument data, it is possible to obtain a comparison plot between the radioactivity signal, subtracted by the background signal, of the TNF-a stimulated and unstimulated HUVEC (control cells).

An increase of the signal is related to the uptake of the radioactivity by the cells, that is not visible in the control cells.

Fig. 42 and Fig. 43 show the plots of NAMP-avidin-BisDOTA and MacroP in vitro tests, respectively.

Both synthesized molecules showed the ability to bind VCAM-l expressed on TNF-a stimulated HUVECs.

Furthermore, these tests demonstrated that the affinity of peptide“VHPKQHRGGSKGC” is preserved when it is conjugated with the ⁶⁸Ga-labeled macrocycle.

The other new radiopharmaceutical, MacroP has been successfully radiolabeled with ⁶⁸Ga and in vitro tests confirmed its ability to bind the VCAM-l .

EXAMPLE 7

In vitro cell VCAM-l tests

In vitro cell tests were performed on human umbilical vein endothelial cells (HUVEC) stimulated overnight with 20 ng/mL TNF-a, for the expression of VCAM-l .

The expression of VCAM-l on HUVECs was evaluated by FACS analysis.

For NAMP in vitro test, TNF-a stimulated HUVECs were incubated at 4°C first with NAMP then with neutravidin, before adding fluorescently labeled biotinylated liposomes in 1 mL of the culture medium.

Control TNF-a stimulated HUVECs were incubated at 4°C with neutravidin, before adding fluorescently labeled biotinylated liposomes in 1 mL of the culture medium.

TNF-a stimulated and control HUVECs were finally fixed with 1% paraformaldehyde and stained with DAPI.

The results were evaluated by live confocal fluorescence microscopy.

Figure 44 shows that a diffused red fluorescent signal is detectable on HUVECs incubated with NAMP, neutravidin and liposomes; the same fluorescent signal is not visible in control cells. Control HUVECs are on the left, HUVECs incubated with NAMP, neutravidin and liposomes are on the right.

The materials, instruments and methods used for the above-described experiments are reported here below.

Materials

Acetic acid glacial 100% (AcOH) (Merck); aluminium sheet silica gel 60 F254 plates (Merk); b- alanine 98% (Alfa Aesar); biotin (Amresco); diphenylphosphorylazide (DPPA) (Alfa Aesar); maleic anhydride 98+% (Alfa Aesar); N-hydroxysuccinimide 98+% (Alfa Aesar); N,N’- dicyclohexylcarbodiimide 99% (DCC) (Alfa Aesar); N,N’-dimethylformamide for peptide synthesis (DMF) (Merk); tert-butanol (t-BuOH) (Alfa Aesar); triethylamine (TEA) (Sigma Aldrich); trifluoroacetic acid (TFA) (Merck); tris-(2-carboxyethyl)phosphine hydrochloride (TCEP-HC1) (Sigma-Aldrich); ammonium acetate (AcONFM) (Merck); arsenazo III (Sigma- Aldrich); maleimido-monoamide-DOTA (MMA-DOTA) (Macrocyclics); phosphate buffer pH 2.5 and pH 7 for HPCE; TraceCERT®, Lead standard for AAS (Pb) (Sigma-Aldrich); trifluoroacetic acid (TFA) (Merck); VCAM-l binding peptide (Innovagen); Antibody CD 106 (VCAM-l) conjugated with the fluorescent dye phycoerythrin (PE) (Milteniy Biotec); attachment factor-coated plates (Thermo Fisher Scientific); avidin (Sigma Aldrich); culture medium EndoGRO-LS Complete Culture Media Kit (Millipore); high pure RNA isolation kit (Roche); human recombinant VCAM-l (Sigma Aldrich); human TNF-a (Milteniy Biotec); iScript cDNA synthesis kit (Bio-Rad); ITLC SG (Varian); Microcon®-30 centrifugal filters (Merck); MKC18F silica gel TLC plates (Whatman); ITLC-SG paper strips; phosphate buffer pH 2.5 e pH 7 for HPCE; sodium acetate trihydrate (Merck); saline for human use; trypsin (Gibco); 30% HC1 ultra-pure (Merck); iScript cDNA synthesis kit (Bio-Rad).

All chemicals were used as received, without further purification. The water used was purified with systems from Millipore.

Instruments

Bruker Avance DPX 300 NMR spectrometer; Buchi Rotavapor RE 128; Camag Linomat 5 and TLC scanner 3; Mettler Toledo Quattro MP220 Basic pHmeter; Nikon Alphaphot-2 YS2 microscope, provided with a Mettler Toledo FP82HT electrical furnace, controlled by a Mettler Toledo FP90 central processor; Perkin Elmer DSC 7; Perkin Elmer Sample Pan Aluminium 6.7 mm 900786.901/0219-0041; Perkin Elmer 2000 FT-IR System; Scaltec SBC 21, electronic analytical balance; Thermo Fisher Scientific FLASH 2000 OEA CHNS/O + MAS200R- SN 2015.F0227.

ALC PK 120 centrifuge; BD FACSCanto II (BD Biosciences); CFX96 touch real-time PCR (Bio- Rad); LigandTracer® White (Ridgeview Instruments AB, Uppsala, Sweden); Packard BioScience Cyclone CT; Hewlett Packard Series II 1090 liquid chromatograph; Merck Hitachi LaChrom L- 7100/L7100 HPLC; Mettler Toledo XS3DU electronic microbalance; Eckert & Ziegler Eurotope Modular Lab-Pharm Tracer®; Eckert & Ziegler Eurotope Modular Lab Standard®; SsoFastTM EvaGreen mix (Bio-Rad); Torrey-Pines Scientific EchoTherm™ SC25XT orbital mixing dry bath; LTQ Orbitrap Velos Pro; Perkin Elmer Radiomatic 150TR flow scintillation analyzer; Scaltec SBC 21 electronic analytical balance; Thermo Scientific UltiMate 3000 UHPLC systems. De Lama SteriPlus bench steam sterilizer; Hewlett Packard Series II 1090 liquid chromatograph; LTQ Orbitrap Velos Pro; Mettler Toledo Quattro MP220 Basic pHmeter; Mettler Toledo XS3DU electronic microbalance; EchoTherm™ SC25XT Torrey-Pines Scientific orbital mixing dry bath; Scaltec SBC 21 electronic analytical balance; Hewlett-Packard HP 8453 UV-Vis spectrophotometer; Waters 1525 binary pump HPLC; Waters 2992 photodiode array detector; Waters fraction collector III. LigandTracer®yellow - Ridgeview instruments AB.

Methods

Thermal analysis

The thermal analyses of ASAM and NBA-BOC were performed by differential scanning calorimetry (DSC).

For ASAM, carefully weighted samples of approx. 3.5 mg were subjected to a thermal gradient of 10°C/min under nitrogen flow, in a covered but not crimped aluminium pan. The scanning was carried out from 30°C to 185 °C.

For NBA-BOC, carefully weighed samples of approx. 2 mg in a covered but not crimped aluminium pan were heated under nitrogen flow from 40°C to 200°C at l0°C/min.

The DSC thermal analyses were supported by hot stage microscopy analysis, performed at the same conditions.

Infrared analysis (IR analysis)

IR spectra were collected on a Perkin Elmer 2000 FT-IR spectrometer from samples prepared as KBr pellets.

NMR analysis

NMR spectra were recorded on a Bruker Avance DPX 300 spectrometer at 300 MHz. Coupling constants (J) are expressed in Hertz; chemical shifts (d) are reported in parts per million (ppm) relative to tetramethylsilane (TMS). The following abbreviations are used to explain the multiplicities: s = singlet, d = doublet, dd = doublet of doublets, t = triplet, m = multiple!

¾- and ¹³C-NMR spectra of ASAM and NBA-BOC were recorded in CDCT; H-NMR spectrum of NAM in DMSO-de.

Analytical reversed-phase high-performance liquid chromatography (Analytical RP-HPLC) Analytical RP-HPLC was performed on Hewlett Packard Series II 1090 with a UV-visible detector.

The analysis of HPLC data was conducted on Hewlett-Packard HPLC ChemStation software. Reverse-phase HPLC chromatography was carried out using a RP 18, Waters X-Bridge Cl 8, 3.5 pm, 4.6 x 150 mm and a Waters X-Bridge BEH C18 Sentry Guard Cartridge 3.5 pm, 4.6 x 20 mm.

Analysis of ASAM, NBA-BOC and NBA

For the determination of ASAM, NBA-BOC and NBA purity, a RP- HPLC method was performed using a gradient solvent system at a flow rate of 1 ml . /min. The gradient mixture was composed of 0.1% aqueous AcOH (solvent A) and methanol gradient grade (solvent B). Gradient elution: 0-2 min 40% B; 2-12 min linear gradient from 40% to 100% B; 12-16 min 100% B. Post run to 40% B: 3 min.

The chromatograms were acquired at 200, 254 and 340 nm wavelengths.

The injection volume was 10 pL. ASAM was dissolved in methanol; the other analytes were dissolved in the mobile phase.

Analysis of NAM

The analysis of the reaction mixture and the determination of NAM purity were carried out by performing two different RP-HPLC methods: Method H-NAM1 : it was performed using a gradient solvent system at a flow rate of 1 ml . /min.

The gradient mixture was composed of 0.1% aqueous AcOH (solvent A) and methanol gradient grade (solvent B). Gradient elution: 0-2 min 40% B; 2-7 min linear gradient from 40% to 100% B; 7-11 min 100% B. Post run to 40% B: 3 min. The chromatograms were acquired at 200, 254 and 340 nm wavelengths. The injection volume was 10 pL and the solutions were prepared using the mobile phase as solvent. Method H-NAM2: it was performed using a gradient solvent system at a flow rate of 1 mL/min. The gradient mixture was composed of ultrapure water (solvent A) and acetonitrile gradient grade (solvent B). Linear gradient from 20% to 100% B in 10 min. Post run to 20% B: 3 min. The chromatograms were acquired at 200, 254 and 340 nm wavelengths. The injection volume was 10 pL and the mobile phase was used as solvent.

Analysis of NAMP

For the analysis of the reaction mixture and the determination of NAMP purity, two RP-HPLC methods were performed, using a mobile phase composed by 0.1% aqueous TFA (solvent A) and acetonitrile gradient grade (solvent B).

Method H-NAMP1 : 10 minutes isocratic method at a flow rate of 1 mL/min. The mobile phase was composed of 85% A and 15% B.

Method H-NAMP2 : this method was suggested by the peptide manufacturer Innovagen. Gradient solvent system at a flow rate of 1 mL/min. Linear gradient from 6% to 31% B in 25 min. Post run to 6% B: 3 min.

The chromatograms were acquired at 198, 210, 225 and 254 nm wavelengths. The injection volume was 10 pL and samples were dissolved in ultrapure water.

Analysis of MacroP

For the analysis of the reaction mixture and the determination of MacroP purity, a RP- HPLC method was applied using a gradient solvent system at a flow rate of 1 mL/min. The gradient mixture was composed of 0.1% aqueous TFA (solvent A) and acetonitrile gradient grade (solvent B). Linear gradient from 6% to 16% B in 10 min. Post run to 6% B: 3 min. The chromatograms were acquired at 198, 210, 225 and 254 nm wavelengths. The injection volume was 10 pL and the samples were dissolved in ultrapure water.

Semi-preparative reversed-phase high-performance liquid chromatography (Semi-preparative RP-HPLC)

Semi-preparative RP-HPLC was performed on a Waters 1525 binary pump HPLC with a Waters 2992 photodiode array detector. The analysis of HPLC data was conducted on Waters Empower™ 3 software. Reversed-phase HPLC chromatography was carried out using a RP18, Waters X- Bridge C18, 5 pm, 10 x 150 mm and a Waters X Bridge Prep C18, 5 pm, 10 x 10 mm Guard Cartridge. The pure product collections were performed using a Waters fraction collector III. Purification of NBA-BOC.

The purification of NBA-BOC was performed by semi-preparative RP-HPLC, using a gradient solvent system at a flow rate of 5 ml . /min.

The gradient mixture was composed of 0.1% aqueous AcOH (solvent A) and methanol gradient grade (solvent B). Gradient elution: 0-2 min 40% B; 2-12 min linear gradient from 40% to 100% B. Post run to 40% B: 2 min.

The chromatograms were acquired at 200, 254 and 340 nm wavelengths. The injections were performed using a 1 mL loop.

Purification of NAMP

The purification of NAMP was performed by semi-preparative RP-HPLC, using a 10 minutes isocratic method, at a flow rate of 3 mi . /min

The mobile phase was composed of 85% 0.1% aqueous TFA and 15% acetonitrile gradient grade. The chromatograms were acquired at 198, 210, 225 and 254 nm wavelengths. The injections were performed with a 500 pL loop, using the partial loop-fill injection method.

Purification of MacroP: purification was performed by semi-preparative RP-HPLC, using a gradient solvent system at a flow rate of 3 mi . /min. The gradient mixture was composed of 0.1% aqueous TFA (solvent A) and acetonitrile gradient grade (solvent B).

Linear gradient from 6% to 16% B in 10 min. Post run to 6% B: 3 min. The chromatograms were acquired at 198, 210, 225 and 254 nm wavelengths.

The injections were performed using a 500 pL loop, using the partial loop-fill injection method. Characterization of NAMP and MacroP by Mass spectrometry

The mass spectrometer LTQ-Orbitrap Velos Pro was operated in positive ionization mode. Single MS survey scans were performed in the Orbitrap, recording a mass window between 150 and 2000 m/z. The Full Scan resolution was set to 120000. Sample were diluted 1 :100 with a water/acetonitrile (50:50, v/v) solution containing 1% AcOH and introduced into the mass spectrometer by means of direct infusion at a flow rate of 5 pL/min with a syringe pump.

Determination of MacroP chelating efficiency

This determination has been exploited as a titrimetric method to evaluate the chemical purity of MacroP. The spectrophotometric assay is based on the decrease of the 656 ran absorption of the complex between Pb⁺⁺ and Arsenazo (AA), for the concurrent formation of the Pb-DOTA complex.

A stock solution of Pb-(II)-AA(III) in 0.15 M AcONFL buffer, pH=7.00 was prepared, containing 67.62 pmol/L of Pb(II) and 140 pmol/L of AA(III). Such stock solution must be stored protected from light, at a temperature of 2-6°C and used at room temperature within one day from its preparation. A stock solution of 0.26 mM DOTA in AcONFL buffer (0.15 M, pH=7.00) was also prepared.

For the calibration curve (Fig. 39), six standard solutions were used, containing increasing concentrations of DOTA from 0 to 0.024 mM: for their preparation 3.4 mL of Pb(II)-AA(III) stock solution, 200 pL of 1 M NaCl (in ammonium acetate buffer) and a variable volume of DOTA stock solution (0-500 pL), depending on the desired final concentration, were mixed. The final 4.2 mL volume of each standard solution was reached by adding a suitable volume of AcONFL buffer (0.15 M, pH=7.00).

The sample solution was prepared adding 40 pL of a 1.6 mM MacroP solution to a solution containing 3.4 mL of Pb(II)-AA(III) stock solution, 200 pL of 1 M NaCl, 560 pL of AcONFL buffer (0.15 M, pH=7.00).

For each solution, absorbance at 656 ran was determined, 10 min after its preparation, at room temperature and protected from light.

The reading was corrected subtracting the absorbance of a mixture made of 4.0 mL of AcONFL buffer (0.15 M, pH=7.00) and 200 pL of 1 M NaCl.

NAMP-avidin binding

A stock solution of avidin was prepared by dissolving 0.5 mg of protein in 200 pL of ultrapure water. 100 pL of this solution (3.8 x 10 ⁶ mmol) were mixed with 100 pL of approx. 0.17 mM NAMP aqueous solution (17 x 10 ⁶ mmol). The reaction mixture was heated at 37°C, under 400 rpm orbital shaking for 2h.

NAMP-avidin binding analysis by ultrafiltration

The reaction mixture containing NAMP and avidin was placed in an ultrafiltration system with a regenerated cellulose membrane with 30 kDa NMWL-nominal molecular weight limit, and washed with 250 pL of ultrapure water at 14000 g for 20 min at l5°C. The retentate was recovered by inverted spinning at 1000 g for 3 min.

NAMP-avidin-BisDOTA binding

To 36 pL of the retentate derived from NAMP-avidin ultrafiltration (1.13 x 10 ⁶ mmol) 15 pL of a 1 mg/mL BisDOTA solution (11.3 x 10 ⁶ mmol) were added. The reaction mixture was heated at 25°C, under orbital shaking at 400 rpm for lh.

Another similar test was performed, in which NAMP, avidin and BisDOTA were mixed in a molar ratio of 2: 1 :2 respectively and incubated at 25°C under orbital shaking at 400 rpm for lh. NAMP-avidin-BisDOTA analysis by ultrafiltration

The reaction mixtures containing NAMP, avidin and BisDOTA were transferred in an ultrafiltration system with a a regenerated cellulose membrane having a 30 kDa cut-off, and washed with 250 pL of ultrapure water at 14000 g for 20 min, at 15°C.

The retentate was recovered by inverted spinning at 1000 g for 3 min.

Capillary Electrophoresis (CE) analysis

CE analyses were performed using an Agilent Technologies 7100 Capillary Electrophoresis. The separations were carried out in a bare fused-silica capillary 50 pm (i.d.) x 64.5 cm (L) x 50 cm (1) (G1600-61239), thermostated at 30°C.

Before use, the capillary was activated with 1 M NaOH aqueous solution, 0.1 M NaOH aqueous solution and washed with ultrapure water; the conditioning and the run were performed with 50 mM sodium phosphate buffer pH 2.5. Before sample injection, a preconditioning with this buffer was performed for 120 s.

Samples were injected hydrodynamically applying a 100 mbar pressure for 2 s, followed by injection of buffer by applying a 100 mbar pressure for 1 s. The run was carried out with a gradient voltage from 0 to 30 kV in 0.2 min (positive polarity). Electropherograms were acquired at 198, 205, 215, 254 and 300 nm wavelengths.

Radiolabeling of BisDOTA with Gallium-68

Two different methods were used for BisDOTA radiolabeling.

BisDOTAl method: the radiolabeling was performed with the Eckert & Ziegler Eurotope Modular Lab Standard^® automated synthesis system.

⁶⁸Ga was eluted from ⁶⁸Ge/⁶⁸Ga generator with 6 mL of 0.1 M HC1 solution and concentrated on a Strata-X-C ion exchange cartridge (SCX). By rinsing the cation exchange column with 800 pL of 0.02 M HC1 in acetone 98%, ⁶⁸GaCl3 was eluted into the reaction vial containing 2 mL of 0.2 M sodium acetate buffer (pH 4) and 7.6 nmol of BisDOTA. The reaction was carried out at 95°C for 400 seconds. Without purification, the radiolabeled product was analyzed by ITLC.

BisDOTA2 method the radiolabeling and purification of BisDOTA were performed with the Eckert & Ziegler Eurotope Modular Lab Pharm-Tracer^® automated synthesis system. The ⁶⁸Ge/⁶⁸Ga generator was eluted with 6 mL of 0.1 M HC1 solution and ⁶⁸GaCl₃ was trapped on the SCX. The radioactive was released from the cartridge to the sample solution with 5.5 M HC1 in 5 M NaCl solution.

Solution A was prepared by dissolving 0.29 g of sodium acetate in 2 mL of B.Braun water and adding 128 pL of 30% HC1 solution. The sample solution was prepared by mixing 400 pL of solution A with 2 mL of B.Braun water and 9.2 pL of a 1 mg/mL BisDOTA solution (7 nmol). The radiolabeling was performed at 95°C for 5 minutes and at the end saline at room temperature was added for cooling. The purification was performed with a Cl 8 ion exchange cartridge, preconditioned with ethanol/water 1 : 1 (v/v) and washed with water. The product was eluted from the cartridge with ethanol/water 1 : 1 (v/v) and diluted in saline.

Determination of BisDOTA radiochemical purity (RCP)

The radiochemical purity of BisDOTA was determined by RP-HPLC.

The analysis was performed on a UltiMate 3000 UHPLC systems with a Dionex UltiMate 3000 variable wavelength detectors and GABI Star (Raytest).

Reverse-phase HPLC chromatography was carried out using a Pursuit C18, 3 pm 3.0 x 150 mm, 200 A column. For BisDOTA analysis, a gradient method at a flow rate of 0.6 mL/min was applied. The gradient mixture was composed of 0.1% aqueous TFA (solvent A) and 0.1% TFA in acetonitrile gradient grade (solvent B). Linear gradient from 5% to 30% B in 20 min. Post run to 5% B: 3 min. The injection volume was 20 pL. The chromatograms were acquired at 220 nm wavelength.

NAMP-avidin-radiolabeled BisDOTA binding

12 pL of 0.031 mM NAMP-avidin solution (about 0.37 nmol) were added to 500 pL of radiolabeled BisDOTA solution (36 MBq). The reaction was performed at room temperature for 20 minutes.

Analysis of NAMP-avidin-radiolabeled BisDOTA complex by ITLC Instant thin-layer chromatography was carried out on Whatman MKC18F silica gel plate (60 A, size 2.5 x 7.5 cm, layer thickness 200 pm) without activation.

The sample application volumes were 5 pL and 0.9% NaCkacetonitrile 1 :1 (v/v) was used as mobile phase for the development by ascending chromatography. The radiochromatographic profile was determined by an autoradiographic system that uses a high performance storage phosphor screen.

MacroP-YCAM-l binding

A stock solution of VCAM-l was prepared by dissolving 50 pg of protein in 100 pL of ultrapure water. 24 pL of this solution diluted 1 :1 with water (0.08 nmol) were mixed with 26 pL of approx. 3.2 pM MacroP aqueous solution (0.08 nmol). The reaction mixture was incubated at 37°C, under 400 rpm orbital shaking for 2h.

Radiolabeling of MacroP with Gallium-68

Two different methods were used for MacroP radiolabeling.

MacroP 1 method: the radiolabeling was performed with Eckert & Ziegler Eurotope Modular Lab Standard^® automated synthesis system.

⁶⁸Ga was eluted from ⁶⁸Ge/⁶⁸Ga generator with 6 mL of 0.1 M HC1 solution and concentrated on a SCX. ⁶⁸GaCl₃ was eluted with 800 pL of 0.02 M HC1 in acetone 98% into the reaction vial containing 2 mL of 0.2 M sodium acetate (pH 4) and 0.027 pmol of MacroP.

The reaction was carried out at 95°C for 400 seconds and, after cooling, the solution was manually injected with a syringe in a Cl 8 light reverse phase silica cartridge, preconditioned with ethanol/water 1 :1 (v/v). The cartridge was washed with water for removing free ⁶⁸Ga and the product was eluted with ethanol 50% and diluted in saline.

MacroP 2 method : the radiolabeling and purification of MacroP were performed with the Eckert & Ziegler Eurotope Modular Lab Pharm-Tracer^® automated synthesis system. The ⁶⁸Ge/⁶⁸Ga generator was eluted with 6 mL of 0.1 M HC1 solution and ⁶⁸GaCl₃ was trapped on the SCX. The radioactive was released from the cartridge to the sample solution with 5.5 M HC1 in 5 M NaCl solution.

Solution A was prepared by dissolving 0.29 g of sodium acetate in 2 mL of B.Braun water and adding 128 pL of 30% HC1 solution. The sample solution was prepared by mixing 400 pL of solution A with 2 mL of B.Braun water and 15 pL of 0.833 M MacroP solution. The radiolabeling was performed at 95°C for 5 minutes and at the end some saline, stored at 2-8°C overnight, was added for cooling. The purification was performed with a Cl 8 ion exchange cartridge, preconditioned with ethanol/water 1 : 1 (v/v) and washed with water. The product was eluted from the cartridge with ethanol/water 1 : 1 (v/v) and diluted in saline.

Determination of MacroP radiochemical purity (RCP)

The radiochemical purity of MacroP was determined by ITLC and by RP-HPLC.

The ITLC was performed on ITLC-SG paper strips and using 2 different mobile phases: 0.1 M sodium citrate buffer (pH = 5) and 1 M NPLOAc in water/methanol 1 :1 (v/v). The sample application volumes were 5 pL.

The radiochromatographic profile was determined by an autoradiographic system that uses a high performance storage phosphor screen.

Two RP-HPLC methods were used for the ⁶⁸Ga-MacroP analysis.

RPH1 method : RP-HPLC was performed on a Merck Hitachi LaChrom L-7100/L7100 HPLC with a L-7400 UV-visible detector and a Radiomatic 150TR flow scintillation analyzer, with Vydac Everest C18, 5 pm, 4.6 x 150 mm, 300 A as stationary phase. For MacroP analysis, a gradient method at a flow rate of 1 mL/min was applied. The gradient mixture was composed of 0.1% aqueous TFA (solvent A) and 0.1% TFA in acetonitrile gradient grade (solvent B). Linear gradient from 6% to 16% B in 10 min. Post run to 6% B: 3 min. The injection volume was 20 pL. The chromatograms were acquired at 220 nm wavelength.

RPH2 method : RP-HPLC was performed on a UltiMate 3000 UHPLC systems with a Dionex UltiMate 3000 variable wavelength detectors and GABI Star (Raytest).

Reverse-phase HPLC chromatography was carried out using a Pursuit C18, 3 pm 3.0 x 150 mm, 200 A column. For MacroP analysis, a gradient method at a flow rate of 0.6 mL/min was applied. The gradient mixture was composed of 0.1% aqueous TFA (solvent A) and 0.1% TFA in acetonitrile gradient grade (solvent B). Gradient elution: 0-2 min 100% B; 2-7 min linear gradient from 100% to 40% B; 7-12 min 40% B. Post run to 100% B: 1 min, and 2 min waiting time. The injection volume was 20 pL. The chromatograms were acquired at 220 nm wavelength.

Cell culture

Human Umbilical Vein Endothelial Cells (HUVEC) were obtained from human umbilical cords collected from full-term women immediately after cesarean section at the Gynecology and Obstetrics department of International Evangelic Hospital in Genoa, with informed consent using the guidelines approved by the Institutional Committee. Each cord vein was individually processed for obtaining endothelial cells through mechanical dissociation of the tissue and collagenase digestion.

Cells were maintained in culture medium EndoGRO-LS Complete Culture Media Kit seeded on attachment factor-coated plates.

For labeling experiments 8x10⁵ cells were plated in Petri dishes in EndoGRO Basal Medium (SCME-BM) and allowed to attach overnight prior stimulation with 20 ng/mL of TNF-alpha (TNF-a). Cells without TNF-a treatment served as control.

RNA extraction and quantitative real-time PCR (qBT-

Total RNA was extracted using the high pure RNA isolation kit, according to the manufacturer’s instruction, and reverse transcribed into cDNA using the iScript cDNA synthesis kit.

Single stranded cDNA products were analyzed by real-time PCR using the SsoFast™ EvaGreen mix on a CFX96 Touch real-time PCR.

Cycling conditions were set at 94 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s, for 37 cycles.

The specific primer pairs used for polymerase chain reaction amplification were designed on the mature transcripts and are shown in Table 6.

Table 6: List of primer designed for the quantification of specific mRNAs by qRT-PCR in HUVECs

mRNA species Sense Sequence 5 '-3'

Forward GACC AC AT CT ACGCT G

Reverse AC

Human VCAM-1

GCAACTGAACACTTG

ACTG Forward ACCCACTCCTCCACCT

Reverse TTGA

GAPDH

CTGTTGCTGTAGCCAA

ATTCGT

Forward CCCAGTGCTCTGAATG

Reverse TCAA

28S

AGT GGGAAT CTCGTT C ATCC

Levels of target genes in each sample were normalized on the basis of glyceraldehyde-3- phosphate dehydrogenase (GAPDH) and 28S amplification and reported as relative values. All qRT-PCR runs included negative controls without mRNA templates and cDNA transcription to check reagents for contaminations.

Fluorescence active cells sorting /FACS^') analysis of cell adhesion molecule expression

The surface expressions of VCAM-l on HUVECs were evaluated by a flow cytometry analysis using a fluorescence-activated cell sorter system.

After stimulation with 20 ng/ml TNF-a, the time-course experiment was performed by FACS analysis at time 0, 4h, 18h and 24h. Control and TNF-a stimulated HUVECs were detached with trypsin, washed in PBS and analyzed for VCAM-l expression using the antibody anti-CD 106 (VCAM-l) conjugated with the fluorescent dye phycoerythrin. Appropriate IgG isotype-matched antibodies and unstained cells were used as negative control. Data were acquired on BD FACSCanto II and analyzed by BD FACSDiva software.

NAMP-avidin-⁶⁸Ga-BisDOTA in vitro test on TNF-q stimulated HUVEC

After removing TNF- a from the culture medium, HUVECs were incubated at 37°C with 4 nmol of NAMP for 20 minutes and afterwards with 3.5 nmol of avidin for 10 minutes. After washing and replacing the culture medium, the binding assay of ⁶⁸Ga-BisDOTA with the NAMP-avidin complex, bound to the VCAM-l expressed on HUVECs, was monitored using LigandTracer^® White, according to the manufacturer’s instructions.

Culture dish, containing 4 mL cell culture medium, was placed on the cell dish holder of the instrument and before starting the assay, 6 MBq of ⁶⁸Ga-BisDOTA were added to the culture medium. The binding of radioactivity to cell-containing areas and reference areas was recorded. The same procedures were also applied to a control dish seeded with unstimulated HUVECs. ⁶⁸Ga-MacroP in vitro test on TNF- a stimulated HUVECs

The binding assay of ⁶⁸Ga-MacroP with VCAM-l expressed on HUVEC was monitored using LigandTracer^® White, according to the manufacturer’s instructions.

Culture dish, containing 3 mL cell culture medium, was placed on the cell dish holder of the instrument and before starting the assay, the culture medium was replaced with 3 mL of the saline solution containing 5 MBq of ⁶⁸Ga-MacroP. The binding of radioactivity to cell-containing areas and reference areas was recorded. The same procedures were also applied to a control dish seeded with unstimulated HUVECs.

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Claims

1. A biotin derivative of formula (I)

in which R is a peptide with the sequence VHPKQHRGGSKGC, linked to the succinimidyl ring through the -SH group of the C-terminal cysteine.

2. The biotin derivative of formula (I) according to claim 1 for use in a method for detecting early atherosclerotic lesions in the blood vessels of a human subject, wherein the method includes the steps of

- forming a complex of said biotin derivative with avidin, neutravidin or streptavidin;

- parenterally administering the complex thus formed;

- subsequently, parenterally administering a chelating agent including a chelating moiety, a biotin moiety and a metal radionuclide, and

- subjecting the human subject to a diagnostic nuclear imaging technique.

3. The biotin derivative of formula (I) according to claim 1 for the use according to claim 2, wherein in said complex of the biotin derivative of formula (1) with avidin, neutravidin or streptavidin, the molar ratio between said biotin derivative and avidin, neutravidin or streptavidin is 1 :1.

4. The biotin derivative of formula (I) according to claim 1 for use in a method for detecting early atherosclerotic lesions in the blood vessels of a human subject, wherein the method includes the consecutive steps of:

- parenterally administering the biotin derivative of formula (I);

- parenterally administering avidin, neutravidin or streptavidin

- chasing any free avidin, neutravidin or streptavidin by parenterally administering biotinylated albumin or by any other chasing method suitable for avidin, neutravidin or streptavidin; - parenterally administering a chelating agent including a chelating moiety, a biotin moiety and a metal radionuclide;

- subjecting the human subject to a diagnostic nuclear imaging technique.

5. The biotin derivative for the use according to any one of claims 2 to 3, wherein the chelating moiety of said chelating agent is selected from the group comprising DOTA (1 ,4,7,10- tetraazacyclododecane-l,4,7,l0-tetraacetic acid) derivatives, NOTA (l ,4,7-triazacyclononane- N,N',N''-triacetic acid) derivatives, and TETA (1,4, 8,1 l-tetraazacyclotetradecane-l,4,8,l 1- tetraacetic acid) derivatives.

6. The biotin derivative for the use according to claim 5, wherein said chelating moiety is DOTA, which is obtained from a DOTA derivative selected from the group comprising maleimido-DOTA (1,4,7, 10-tetraazacyclododecane- 1 ,4,7-tris-acetic acid- 10-maleimidoethylacetamide), DOTA- NHS-ester (l,4,7,l0-tetraazacyclodecane-l,4,7,l0-tetraacetic acid mono-N-hydroxysuccinimide ester), p-SCN-Bn-DOTA (S-2-(4-isothiocyanatobenzyl)- 1 ,4,7, 10-tetraazacyclododecane tetraacetic acid).

7. The biotin derivative for the use according to any one of claims 2 to 6, wherein said chelating agent includes a DOTA derivative containing a biotin moiety selected from the group consisting of BisDOTA-C3 (bis[(9H-fluoren-9-yl)methyl]-3,3-[2-oxo-2-[[6-[[5-[(3aS,4S,6aR)-exahydro-2- oxo-lH-thieno-[3,4-d]imidazol-4yl]pentyl]amino]hexyl]amino]ethylazanodiyl]bis-(propane-l,3- diyl) dicarbamate), BisDOTA-Lys-C3 (2,2,2-[l0-[l6-carboxy-2,9-dioxo-22-[(3aS,4S,6aR)- hexahydro-2-oxo-lH-thieno[3,4-d]imidazol-4-yl]-7-[3-[2-[4,7,l0-tris-(carboxymethyl)-l,4,7,l0- tetraazacyclododecan-N- 1 -yljacetamidojpropyl] -3,7,10, 17-tetraazadocosyl]- 1 ,4,7, 10- tetraazacyclo-dodecane-N,N,N-l,4,7-triyl]triacetic acid), BisDOTA-Lys-(paB)-C3 (2,2,2-[l0-[2- [3-[[3-[2-[4,7, 10-tris(carboxymethyl)-l ,4,7, 1 O-tetraazacyclododecan-N-l- yl]acetamido]propyl][2-[4-[5-carboxy-5-[5-(2-oxohexahydro-lH-thieno[3,4-d]imidazol-4- yl)pentanamido]-pentylcarbamoyl]phenylamino]-2-oxoethyl]amino]propylamino]-2-oxoethhyl]- 1 ,4,7, 10-tetraazacyclododecane-N,N,N,- 1 ,4,7-triyl]triacetic acid.

8. The biotin derivative for the use according to any one of claims 2 to 7, wherein said metal radionuclide is a radioactive isotope of an element selected among the group consisting of Sr, Rh, Pd, Sm, Er, Au, Bi, In, Lu, Y, Ce, Pr, Nd, Pm, Sa, Eu, Gd, Tb, Dy, Ho, Tm, Yb, Ga, Ni, Co, Fe, Cu, Re, Th and Zr and is preferably ⁶⁸Ga.

9. The biotin derivative for the use according to any one of claims 2 to 8, wherein said diagnostic nuclear imaging technique is Positron Emission Tomography (PET).

10. The biotine derivative of formula (I) according to claim 1 for use in a method for treating a disease of a human patient characterized by an overexpression of VCAM-l, which method comprises the steps of:

- forming a complex of said biotin derivative with avidin, neutravidin or streptavidin;

- parenterally administering to the patient the complex thus formed;

- subsequently, parenterally administering to the patient a biotinylated nanodispersed or microdispersed system, selected among nanoparticles, liposomes and micelles.

11. The biotin derivative of formula (I) according to claim 1 for use in a method for treating a disease of a human patient characterized by an overexpression of VCAM-l, wherein the method includes the consecutive steps of:

- parenterally administering the biotin derivative of formula (I);

- parenterally administering avidin, neutravidin or streptavidin

- chasing any free avidin, neutravidin or streptavidin by parenterally administering biotinylated albumin or by any other chasing method suitable for avidin, neutravidin or streptavidin;

- subsequently, parenterally administering to the patient a biotinylated nanodispersed or microdispersed system, selected among nanoparticles, liposomes and micelles.

12. The biotine derivative of formula (I) according to claim 1 for use in a method for treating a disease of a human patient characterized by an overexpression of VCAM-l, which method comprises the steps of:

- forming a complex of said biotin derivative with avidin, neutravidin or streptavidin;

- adding said complex to a biotinylated nanodispersed or microdispersed system, selected among nanoparticles, liposomes and micelles, and

- subsequently, parenterally administering to the patient said biotinylated nanodispersed or microdispersed system including said complex.

13. The biotin derivative for the use according to any one of claims 10 to 12, wherein said nanoparticles, liposomes or micelles are loaded with at least one drug suitable for treating said disease.

14. The biotin derivative for the use according to any one of claims 10 to 12, wherein said disease characterized by an overexpression of VCAM-l is selected from the group consisting of inflammatory diseases, atherosclerosis, multiple sclerosis, neurodegenerative diseases, neoplastic diseases and neuroinflammatory diseases.

15. The biotin derivative for the use according to claim 13, wherein said disease characterized by an overexpression of VCAM-l is selected from the group consisting of inflammatory diseases, atherosclerosis, tumors, multiple sclerosis, Parkinson’s disease neurodegenerative diseases and neuroinflammatory diseases.

16. The biotin derivative of formula (I) according to claim 1 for use in a method for treating tumors in a human patient, which method includes the steps of

- forming a complex of said biotin derivative with avidin, neutravidin or streptavidin;

-parenterally administering said complex to the human patient.

-parenterally administering a chelating agent including a chelating moiety, a biotin moiety and a metal radionuclide.

17. The biotin derivative of formula (I) according to claim 1 for the use of claim 16, wherein said metal radionuclide is selected from the group consisting of ¹⁷⁷Lu, ⁹⁰Y, ^mIn, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³ Sm,

89 Sr, ¹⁶⁹Er and ²²³Ra.

18. The biotin derivative of formula (I) according to claim 1 for the use of claim 16 or 17, wherein said chelating agent is one of those defined in claim 6.

19. A method of preparing the biotin derivative according to claim 1, which comprises reacting a compound of formula (II)

with a peptide with sequence VHPKQHRGGSKGC.

20. The method according to claim 19, wherein said compound of formula (II) is prepared by reacting norbiotinamine of formula (III)

in which X is an anion, preferably TFA ,

with a compound of formula (IV)

21. A compound of formula (V)

in which R is a peptide with the sequence VHPKQHRGGSKGC, linked to the succinimidyl ring through the -SH group of the C-terminal cysteine.

22. The compound of formula (V) according to claim 21 for use in a method for detecting early atherosclerotic lesions in the blood vessels of a human subject, wherein the method includes the steps of

- radiolabeling said compound with a metal radionuclide;

- parenterally administering the radiolabeled compound thus obtained; and

- subjecting the human subject to a diagnostic nuclear imaging technique.

23. The compound for the use according to claim 22, wherein said metal radionuclide is a radioactive isotope of an element selected among the group consisting of Sr, Rh, Pd, Sm, Er, Au, Bi, In, Lu, Y, Ce, Pr, Nd, Pm, Sa, Eu, Gd, Tb, Dy, Ho, Tm, Yb, Ga, Ni, Co, Fe, Cu, Re, Th and Zr.

24. The compound of formula (V) according to claim 21 for use in a method for treating tumors in a human patient, which method includes the steps of

- radiolabeling said compound with a metal radionuclide selected from the group consisting of ¹⁷⁷LU, ⁹⁰Y, In, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³ Sm, ⁸⁹ Sr, ¹⁶⁹Er and ²²³Ra;

- parenterally administering the radiolabeled compound thus obtained.

25. A method of preparing the compound according to claim 21, which comprises reacting a compound of formula (VI)

with a peptide with sequence VHPKQHRGGSKGC.

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