WO2020240431A1 - Magnetic resonance imaging contrast agents including an imidazole-comprising compound - Google Patents

Abstract

Novel Magnetic Resonance Imaging (MRI) contrast agents including an imidazole-comprising compound,wherein the imidazole-comprising compound comprises at least 5 imidazole moieties, and wherein the imidazole moieties are in an immobilized state so that in use the longitudinal water proton relaxation rate at 1.38±0.3MHz (Proton Larmor Frequency) increases of at least 10%of the pre-contrast value.

Description

MAGNETIC RESONANCE IMAGING CONTRAST AGENTS INCLUDING AN IMIDAZOLE-COMPRISING COMPOUND

★★★★

FIELD OF THE INVENTION

The present disclosure concerns novel magnetic resonance imaging contrast agents.

BACKGROUND OF THE INVENTION

In the last few decades, Magnetic Resonance Imaging (MRI) has become one of the key modalities in clinical settings thanks to its superb spatial resolution and its outstanding ability to differentiate soft tissues. The contrast in a MR image arises mainly from differences in the relaxation times of tissue water protons as a consequence of their interaction with biological macromolecules and membranes. Variation in the longitudinal (Ti) and transverse (T₂) relaxation times are the determinants of the contrast.

It is known from in vitro studies that the spin- lattice relaxation time, Ti, of a given tissue changes as a function of the applied magnetic field strength. This behavior (known as "Ti-dispersion" ) is a marker of disease, but is invisible to conventional, fixed-field MRI scanners. The measurement of Ti-dispersion is often termed "relaxometry" and the resulting graph of Ti (or Ri=l/Ti) versus magnetic field strength (or the equivalent Larmor frequency) is known as a Nuclear Magnetic Resonance Dispersion (NMRD) curve or an NMRD profile. Fast Field-Cycling (FFC) is the only practical way of measuring Ti-dispersion in vivo; it involves switching the magnetic field between different field strengths during the measurement procedure. The time to switch between levels is usually less than the sample Ti, in which case the technique is known as "fast" field- cycling (Figure 1) . The nuclear spin polarization is built up during the first period, at field strength B_0p. Spin-lattice relaxation occurs during the Evolution period (duration t_E) at field strength B₀E then the NMR signal is detected during the final period, always at the same field strength B₀D · The sequence is repeated, updating B₀E each time, in order to measure Ti as a function of B₀E · The fact that measurement of the NMR signal always occurs at the same magnetic field strength (BOD) is key, because it means that the radiofrequency coils (transmit Tx and receive Rx, or Tx/Rx combined) do not have to be retuned during the collection of data for a Ti-dispersion curve measurement . FFC relaxometry on small samples (~1 ml) has been investigated and exploited for many years in laboratories around the world (Broche et al . , 2012; Broche et al . , 2012) . However, FFC has only recently been applied to MRI, largely due to the work of the Lurie group at Aberdeen University where two prototype human whole-body sized FFC-MRI scanners have been built .

FFC introduces an entirely new dimension into MRI, namely the strength of the applied magnetic field. By making measurements as a function of magnetic field strength, totally new methods for obtaining contrast between normal and diseased tissues and organs can be employed .

An example of a phenomenon that is completely invisible to conventional (fixed-field) MRI is the existence of "quadrupole peaks" (QPs) : significant increases in the measured relaxation rate (Ri) of samples containing immobile protein molecules, at magnetic fields where the proton NMR frequency and the ¹⁴N nuclear quadrupole resonance (NQR) frequency coincide (Sunde et al . , 2010; Koening et al . , 1998; Fries et al . , 2015) (Figure 2) . Note that it is usual within the FFC community to display dispersion plots as Ri vs. Proton Larmor Frequency rather than Ti, where Ri=l/Ti, and that format will be adopted henceforth.

In the case of biological tissues, the ¹⁴N-QPs are normally detected at the proton NMR frequencies of 0.7, 2.1 and 2.8 MHz, equivalent to field strengths of 16 ml, 49 mT and 65 mT . For such systems, it is well established that the detection of QP is associated with the occurrence of amidic peptide bonds arising from the endogenous protons. Their amplitude is proportional to the amount of protein present in the considered specimen and may eventually be associated with the occurrence of pathological changes. At the moment, no compounds able to be used as contrast agents in FFC-MRI for diagnostic purposes in vivo are available.

Nowadays, paramagnetic species are commonly exploited as contrast agents for fixed-field MRI . The systems currently used in clinic are represented by polyaminocarboxylate complexes of gadolinium ion (Gd³⁺) that contains seven unpaired electrons. The ligands are multidentate (seven or eight donor atoms) with high thermodynamic stabilities in order to strongly limit the release of free metal ions that are highly toxic as, for example, they interfere with Ca²⁺ pathways. About 40-50% of the MRI exams make use of Gd-based contrast agents essentially to report about physiological variations that accompany a specific disease, i.e. loss of blood brain barrier in the case of tumour lesions in the brain or in the presence of multiple sclerosis, hypervascularization and for variation in the "washing in/washing out" rate to assess the malignancy of other tumours, etc. Currently there is much concern in the MRI community about the potential toxicity of conventional Gd-containing contrast agents in patients with impaired renal function. Thus, much attention worldwide is devoted to the development of metal-free contrast agents employing novel mechanisms.

OBJECT AND SUMMARY OF THE INVENTION

An object of the present disclosure is to develop novel MRI contrast agents for use in the Fast Field- Cycling Magnetic Resonance Imaging (FFC-MRI) technique. The MRI contrast agents herein disclosed are able to shorten relaxation time and generate image contrast at given frequencies without using any paramagnetic gadolinium- or other metal-containing contrast agents.

According to the invention, the above object is achieved thanks to the subject matter recalled specifically in the ensuing claims, which are understood as forming an integral part of this disclosure.

In an embodiment, the present description discloses a Magnetic Resonance Imaging (MRI) contrast agent including an imidazole-comprising compound, wherein the imidazole-comprising compound comprises at least 5 imidazole moieties, and wherein the imidazole moieties are in an immobilized state, so that in use the longitudinal water proton relaxation rate at 1.38 ± 0.3 MHz (Proton Larmor Frequency) increases of at least 10% of the pre-contrast value, wherein the MRI contrast agent is free of paramagnetic metal (s) .

The MRI contrast agents herein disclosed, characterized by comprising in their structure at least five imidazole moieties, provide the generation of detectable ¹⁴N-QPs that fall at a frequency, namely at 1.38±0.3 MHz, well distinguishable from the frequencies associated with the amidic peptide bonds from endogenous proteins, that occur at 0.7, 2.1 and 2.8 MHz. Thus, the QPs arising from the imidazole-comprising compounds - when administered to a subject before the MRI scans - can be easily identified and their detection will not be affected by variations of the endogenous QPs.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in detail, purely by way of illustrative and non-limiting examples, with reference to the attached figures, wherein:

— FIGURE 1. Generic Fast Field-Cycling NMR pulse sequence for the measurement of Ti-dispersion . Note that in an imaging (FFC-MRI) pulse sequence, magnetic field gradients are applied during the Detection period.

— FIGURE 2. On the left: (a) NMRD profile of a BALB/c mouse leg, (b) Gaussian fits of the QPs, after background subtraction, (c) Energy levels of ¹⁴N nuclei (S=l) due to QP interactions with local electric field gradients. The two levels to the left occur for axial symmetry. The upper level splits when the axial symmetry is broken, as shown to the right. A local maximum of the Ri profile of protons occurs when the Zeeman transition energy of this I spin is equal to the energy difference of two levels of the S spin.

— FIGURE 3. Schematic representation of the poly amino acid poly-histidine.

— FIGURE 4. A) NMRD profile acquired from 0.01 to 20 MHz of poly-His (30% w/w) in water compared with the NMRD profile of an ex-vivo fresh liver tissue (T=25 °C) with the expansion of the QPs region. The asterisk indicates the imidazole peak. B) Gaussian fits of the QPs, after background subtraction.

— FIGURE 5. A) NMRD profiles of poly-His solutions at different concentrations (50 mg/ml, 25 mg/ml, 14 mg/ml, 5 mg/ml; pH = 7.5 and T = 25 °C) . B) Expansion of the NMRD profiles showed in panel A) in the 0.8-4 MHz range .

- FIGURE 6. QP peak intensity, measured at 1.38 MHz, for a 15 mg/ml poly-His solution as a function of the pH.

— FIGURE 7. Schematic representation of an oligo- His-PLGA nanoparticle (NP) . The oligo-His-PLGA NPs were prepared according to the oil/water (o/w) emulsion solvent extraction method with PVA coating. Abbreviations: oligo-His-PLGA = Cys (Gly) 2His;i^Ala conjugated to the PLGA-PEG2-Maleimide; PVA=Polyvinyl Alcohol .

— FIGURE 8. Oligo-His NMRD profile compared with that acquired on a solution of the commercially available poly-His, both dissolved in 30% w/w water solution. The insert reported the fitting of the peak related to the imidazole contribution, after the background subtraction .

- FIGURE 9. NMRD profiles of two oligo-His-PLGA NP preparations, compared to the one obtained from a commercial poly-His, at the same [His] . The insert is an amplification of the QPs region.

— FIGURE 10. ¹H-NMR spectrum in dmso-d₆ at 37 °C of oligo-His peptide.

— FIGURE 11. ¹H-NMR spectrum in dmso-d₆ at 37 °C of PLGA-PEG_2-Mal .

— FIGURE 12. ¹H-NMR spectrum in dmso-d₆ at 37 °C of the oligo-His-PLGA.

— FIGURE 13. Schematic representation of the oligo- His-PLGA-NP preparation.

DETAILED DESCRIPTION OF THE INVENTION

In the following description, numerous specific details are given to provide a thorough understanding of embodiments. The embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, etc. in other instances. Well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the embodiments.

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments .

The headings provided herein are for convenience only and do not interpret the scope or meaning of the embodiments .

As used herein, the term "compound" refers to a molecule having atoms held together via covalent and/or ionic bonds .

As used herein, the term "contrast agent" refers to a substance used to enhance the contrast of structures or fluids within a body in medical imaging.

As used herein, the term "MRI contrast agent" refers to a substance that can enhance the contrast of structures or fluids within a body during an MRI scan.

In an embodiment, the present description discloses a Magnetic Resonance Imaging (MRI) contrast agent including an imidazole-comprising compound, wherein the imidazole-comprising compound comprises at least 5, preferably 5 to 150, more preferably 10 to 50 imidazole moieties, and wherein the imidazole moieties are in an immobilized state, so that, in use (i.e. once the imidazole-comprising compound is administered to a human or animal) , the longitudinal water proton relaxation rate at 1.38 ± 0.3 MHz (Proton Larmor Frequency) increases of at least 10% of the pre-contrast value, wherein the MRI contrast agent is free of paramagnetic metal (s) . In practice, the longitudinal water proton relaxation rate at 1.38 ± 0.3 MHz increases of at least 10% of the pre-contrast value in the region of interest of the human or animal body in which the imidazole comprising compound is present and/or localized.

As used herein the expression "pre-contrast value" means the value of the longitudinal water proton relaxation rate measured in the region of interest before administration of the imidazole-comprising compound. In other words, the contrast in the MRI image obtained in using the MRI contrast agent is directly proportional to the relaxation rate enhancement defined as follows:

% enhancement = [ (Ri (post-contrast ) -Ri (pre-contrast )) /

Ri (pre-contrast) ] *100 wherein Ri (post-contrast ) is the relaxation rate measured after the administration of the MRI contrast agent; Ri (pre-contrast ) is the relaxation rate measured before the administration of the MRI contrast agent.

As used herein the expression "the imidazole moieties are in an immobilized state" means that the imidazole moieties are characterized by a tumbling time in the order of tens of nanoseconds (ns) .

In one or more embodiments, the imidazole comprising compound has formula (I) :

Ri— [S—L— (B)_r— (His)_t—C]_n (I) wherein

Ri is selected from polyglycolic acid (PGA) , polylactic acid (PLA) , poly (lactic-co-glycolic acid) (PLGA) , alginate, hyaluronic acid (HA) and chitosan;

S is a spacer selected from a direct bond, -(CH₂₎₂ 0

B is selected from Gly, Phe, Ala or b-Ala;

C is selected from b-Ala and none;

r is an integer number between 0 and 5;

t is an integer number equal to or greater than 5, preferably comprised between 5 and 150, more preferably comprised between 10 and 50;

n is an integer number equal to or greater than 1.

In one or more embodiments, Ri is selected from polyglycolic acid (PGA) , polylactic acid (PLA) and poly (lactic-co-glycolic acid) (PLGA) ; S is selected from - (CH₂) ₂-0- (CH₂) ₂-0- and - (CH₂) ₂-0-

a linker selected from

;B is selected from Gly, Phe, Ala or b

Ala ; C is selected from b-Ala and none; r is an integer number between 0 and 5; t is an integer number equal to or greater than 5, preferably comprised between 5 and 150, more preferably comprised between 10 and 50; and n is an integer number equal to 1.

In one or more embodiments, Ri is poly (lactic-co- glycolic acid) (PLGA); S is - (CH₂) ₂-0- (CH₂) ₂-0-; L is

; B is Gly; C is b-Ala; r is equal to 2; t is an integer comprised between 10 and 50 and n is equal to 1.

In one or more embodiments, Ri is poly (lactic-co- glycolic acid) (PLGA); S is - (CH₂) _2-0- (CH₂) _2-0-; L is

; B is Gly; C is b-Ala; r is equal to 2; t is equal to 15; and n is equal to 1.

In one or more embodiments, Ri is selected from alginate and hyaluronic acid; S is selected from -(CH₂)_2-

0-(CH₂)_2-0- and- (CH₂) _2-0- (CH₂) _2-0- (CH₂) _2-0-; L is a linker

selected from

; B is selected from Gly, Phe, Ala or b-Ala; C is selected from b-Ala and none; r is an integer number between 0 and 5; t is an integer number equal to or greater than 5, preferably comprised between 5 and 150, more preferably comprised between 10 and 50; and n is an integer number greater than 1, preferably comprised between 2 and 200.

In one or more embodiments, Ri is chitosan; S is a

direct bond; L is a linker selected from

and ; B is selected from Gly, Phe, Ala or b-Ala; C is selected from b-Ala and none; r is an integer number between 0 and 5; t is an integer number equal to or greater than 5, preferably comprised between 5 and 150, more preferably comprised between 10 and 50; and n is an integer number greater than 1, preferably comprised between 2 and 200.

In one or more embodiments, PGA, PLA, PLGA have a molecular weight comprised between 10 and 75 kDa. Alginate and chitosan have a molecular weight comprised between 10 and 400 kDa. HA has a molecular weight comprised between 8 and 1000 kDa. Preferably, PGA, PLA, PLGA, alginate, chitosan, HA have a molecular weight comprised between 10 and 100 kDa.

The novel MRI contrast agents are specifically suitable in FFC-MRI technique thanks to the generation of detectable ¹⁴N-QPs that fall at a frequency (1.38±0.3 MHz) well distinguishable from the frequencies associated with the amidic peptide bonds from endogenous proteins (0.7, 2.1 and 2.8 MHz) .

The observation of ¹⁴N-QPs is strictly dependent on a substantial immobilization of the imidazole moieties present in the imidazole-comprising compounds at physiologic conditions. In fact, QPs are not seen for isotropically tumbling proteins or polymers, but require immobilization on the time scale of the inverse quadrupole coupling (ca. 50 ns) (Sunde et al . , 2010) . The ¹⁴N-⁴H coupling concerns not only the hydrogen atoms directly bound to the nitrogen atoms (i.e. the hydrogen atoms of the imidazole moieties of the imidazole comprising compounds), but also the hydration water protons or other labile protons (also named intermediate water protons) in proximity of the imidazole-comprising compounds, that directly or indirectly sense ¹⁴N-⁴H interaction. The protons of this intermediate water layer spread the ¹⁴N quadrupolar effect of the imidazole comprising compounds thanks to their exchange with both imidazole protons bound to the ¹⁴N and with bulk water protons. In order to be effective this intermediate water layer has to exchange on the microsecond time scale as occurs in a tissue or particle environment.

For these reasons, the imidazole-comprising compounds are in form of nanoparticles, wherein the nanoparticles are water permeable. In such a form, the imidazole moieties of the imidazole-comprising compounds, and - if present - the hydration water molecules of the imidazole-comprising compounds adjacent to/contained within the nanoparticles (coming from the water molecules surrounding the nanoparticles) are immobilized in a sort of solid-like state (namely, a gel state) , allowing thus the detection of the ¹⁴N-QPs generated by the imidazole-comprising compounds. The imidazole-comprising compounds of formula (I) in form of nanoparticle result the imidazole moieties to be in an immobilized state characterized by a tumbling time in the order of tens of ns. In presence of water molecules, the imidazole-comprising compounds of formula (I) in form of nanoparticle result the imidazole moieties and the water molecules permeated within the nanoparticles to be in a gel state, so that the imidazole moieties and the water molecules are in an immobilized state with a tumbling time in the order of tens of ns.

The imidazole-comprising compounds in form of nanoparticles are injected intravenously, intra-tumour or implanted subcutaneously. In fact, it is well known that neo-formed vessels of solid tumours show a significantly higher permeability to nanoparticles that leads to their enhanced accumulation with respect to healthy tissues (EPR effect) . The nanoparticles are preferably injected intravenously before the imaging. In order to improve the targeting efficiency, the external surface of the nanoparticles can be functionalized with specific ligands able to recognize specifically the tumour cell surface. Examples of such ligands are antibodies (monoclonal, polyclonal, chimeric and humanized antibodies and fragments thereof) able to specifically bind to protein (s) expressed on the tumour cell surface.

In another embodiment, the imidazole-comprising compounds of formula (I) are in form of scaffolds for cells, wherein these scaffolds are used for tissue engineering in the field of regenerative medicine. These scaffolds act as a temporary substitute for extracellular matrices, providing an initial mechanical support for transplanted cells until the regenerated tissue can stabilize the initial structure.

However, the successful use of scaffolds - whatever form they may take - depends strongly on their stability after insertion and it is vitally important that an implant's status can be assessed in vivo, may it be for monitoring in clinical trials on humans or to take early corrective actions when implanted. In this context, the imidazole-comprising compounds subject of the present invention act as a sensor for the detection of the scaffold stability after transplantation in the host organism. The imidazole-comprising compounds are in fact distributed homogenously either on the external surface and in the interior of the scaffold itself. The information provided by the QPs generated by the imidazole moieties in sensing the scaffold stability comes from their pH-dependent solubility in water. A pH decrease (pH<7), due to cell death and/or to scaffold matrix degradation, leads to changes in the QP intensity (see the results described below) dependent on an increase in the mobility of the imidazole moieties of the imidazole-comprising compounds. In fact, as it was described above, the immobilization of the imidazole moieties is mandatory for the generation of the ¹⁴N-QPs. Accordingly, the appearance of positive charges on imidazole moieties (pKa ca. 6.8) due to their protonation increases water solubility of the imidazole-comprising compound, with a proportional increase in mobility and disappearance of the detectable QPs. Therefore, the pH of the microenvironment in which the imidazole- comprising compound is located can be assessed by the changes in the intensity of the imidazole QP .

The pH sensing ability of the imidazole-comprising compounds object of the present application is also very useful in oncological applications. In fact, the measurement of tumour tissue pH has a marked prognostic relevance as the presence of slightly acidic zones (e.g. pH = 6.6-6.8) surrounding hypoxic and necrotic zones promotes cell migration and metastasis. The pH sensing of nanoparticles formed by imidazole-comprising compounds is allowed by their water and H⁺ permeability and the consequent fast equilibration of the internal and external pH.

In an embodiment, the present description discloses novel imidazole-comprising compounds of formula (I) for use (i) as contrast agents and/or (ii) as pH sensing agent in FFC-MRI in mammals, preferably humans.

In an embodiment, the instant description discloses a composition comprising imidazole-comprising compounds of formula (I) and a pharmaceutically acceptable vehicle, preferably sterile water or a sterile buffer, such as phosphate buffered saline (PBS) . Preferably, the composition has a pH ranging from 7.2 to 7.4.

General synthesis of imidazole-comprising compounds and preparation of nanoparticles thereof

In the following the synthesis methods for preparing the imidazole-comprising compounds as well as nanoparticles and scaffolds thereof subject of the present invention are disclosed. Those skilled in the art will appreciate that other synthetic routes may be used to synthesize the imidazole-comprising compounds as well as nanoparticles and scaffolds thereof. Although specific starting materials and reagents are depicted in the Schemes and discussed below, other starting materials and reagents can be easily substituted to provide a variety of derivatives and/or reaction conditions. In addition, many of the imidazole comprising compounds as well as nanoparticles and scaffolds thereof prepared by the methods described below can be further modified in light of this disclosure using conventional chemistry procedures well known to those skilled in the art .

The following abbreviations are used below:

BOP : (Benzotriazol-l-yloxy) tris (dimethylamino) phosphonium hexafluorophosphate

DCC : N, N ' -Dicyclohexylcarbodiimide

HATU : 1- [Bis (dimethylamino) methylene] -lH-1, 2, 3-triazole

[ 4 , 5-b] pyridinium 3-oxid hexafluorophosphate

HBTU : 0- (Benzotriazol-l-yl) -N,N,N',N'- tetramethyluronium hexafluorophosphate

PyBOP : Benzotriazole-l-yl-oxy-tris-pyrrolidino- phosphonium hexafluorophosphate

TBTU : 0- (Benzotriazol-l-yl) -N,N,N',N'- tetramethyluronium tetrafluoroborate

NHS : N-Hydroxysuccinimide

DIPEA: N, N-Diisopropylethylamine

TIS: Triisopropylsilane

TFA: Trifluoroacetic acid

Boc: tert-butyloxycarbonyl

Fmoc: 9-fluorenylmethoxycarbonyl

Trt : Trityl

NMP : N-Methyl-2-pyrrolidone

CH₃CN : Acetonitrile

CH2CI2 : dichloromethane

Et20: diethyl ether

pGly: propargyl-glycine

PGA: Poly (glycolic acid)

PLA: Poly (lactic acid)

PLGA: Poly (lactic acid-co-glycolic acid) PEG₂ : - (CH₂) 2 0 (CH₂) 2-0

PEG₃ : - (CH₂) ₂-0- (CH₂) ₂-0 (CH₂) 2 0

PVA: Polyvinyl Alcohol

Example 1

Scheme 1 shows the diagram representing the synthesis of the compound of formula (I) :

Ri— [S-L-(B)_r-(His)t—C]_n (I) wherein

Ri is selected from polyglycolic acid (PGA) , polylactic acid (PLA) , poly ( lactic-co-glycolic acid) (PLGA) , alginate, hyaluronic acid (HA) ;

S is a spacer selected from - (CH₂) ₂ ^~0- (CH₂) ₂ ^~0- and

B is selected from Gly, Phe, Ala or b-Ala;

C is selected from b-Ala and none;

r is an integer number between 0 and 5;

t is an integer number equal to or greater than 5, preferably comprised between 5 and 150, more preferably comprised between 10 and 50;

n is an integer number equal to or greater than 1.

Scheme 1

R iS-HSHhish-Q,

The single steps will now be described in details.

Such linker reagents are either commercially available (i.e. Sigma Aldrich or BroadPharm®) , or can be prepared from commercially available starting materials using methodology known in the art. The compounds Maleimide- (PEG) 2_or3^_amine and Azide- (PEG) 2_or3^_amine are prepared via a coupling reaction between 3- (Maleimido) propionic acid NHS ester or Azidoacetic acid NHS ester and t-Boc-A/-amido-PEG2_, 3-amine (BroadPharm®) . The coupling reaction to form the amide bond is generally carried out in a dry organic solvent, preferably under an inert atmosphere such as nitrogen or argon. Acetonitrile, chloroform, dichloromethane and tetrahydrofuran are used as solvent in the presence of a base, preferably trialkylamines such as triethyl amine, pyridine, 4- (dimethylamino) pyridine . The desired products are isolated by silica gel column chromatography. Then, to generate the desired compounds the t-butylcarbonyl (Boc) amine protecting group is removed according to standard methods known to those skilled in the art, such as by treatment with TFA:CH₂Cl2 (1:1 v/v) or ( 4M) HCl/dioxane (1:1 v/v) at room temperature. Reaction times ranging from 2-6 h.

Synthesis of R - [S-LJ_n

Ri is linked to Maleimide-PEG2, 3-amine or Azide- PEG2, 3-amine after activation of carboxylate carried out following standard procedures reported in literature. Briefly, activating agents such as N-hydroxysuccinimide in the presence of a carbodiimide (N- ( 3- Dimethylaminopropyl ) -N ' -ethylcarbodiimide

hydrochloride, EDC) have been used. The procedures are described in details in references (Cheng J, et al 2007; Dong X, et al . 2010) .

The reaction can also be carried out by using coupling reagents such as BOP, PyBOP, TBTU, HBTU, HATU originally developed for peptide synthesis, in the presence of a base using DMF, DMSO or CH₃CN as solvent, which depends on the solubility of polymer Ri .

Synthesis of (B) _r- (His) _t ^~C

The compounds of formula (B) _r- (His ) _t ^_C functionalized with thiol or azide terminated groups are prepared by means of standard solid-phase peptide synthesis techniques and preferably an automated peptide synthesizer, using Fmoc chemistry.

Typically, using such technique, an N-a-Fmoc protected amino acid and the amino acid attached to the growing peptide chain on the resin are coupled, in an inert solvent (i.e. dimethylformamide, N- methylpyrrolidinone ) , in the presence of coupling agents (i.e. DCC, HBTU, HATU or PyBOP) in the presence of a base such as DIPEA. Here amino acids are Fmoc-His (Trt ) - OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc^Ala-OH or Fmoc-Ala- OH . Fmoc-Cys (Trt ) -OH or Fmoc-propargyl-Gly-OH can be introduced in the amino-terminus of the peptide in order to link the sulfhydryl or the alkyne functionalized peptide with Ri^~ [S-L]_n. Fmoc group is cleaved by bases, preferably a solution of piperidine 20% v/v in DMF . After the completion of the entire sequence, the terminal Fmoc is removed and the resin is dried in vacuo. A cleavage cocktail solution (TFA, H₂0, Phenol and TIS in a ratio 88:5:5:2 v/v/wt/v) is used to cleave the peptide from the resin and to obtain the side-chain deprotection. After 4 h, the cleavage solution is collected and concentrated to dryness. Et₂0 is added to the residue to precipitate the crude peptide, which is collected. A C4, C8 or C18 preparative column is used to isolate peptides, and purity is determined using a C4, C8 or C18 analytical column. Solvents (A=0.1% TFA/water and B=0.1% TFA/CH₃CN) are delivered to the analytical column at a flow rate of 1.0 ml/min and to the preparative column at 20 ml/min.

Synthesis of Ri^~ [S-L- (B) _r- (His) _t ^~C]_n

Ri-[S-L]_n conjugated with maleimide functional group is linked to Thiol- (B) _r (His ) _t ^_C via maleimide-thiol chemistry following the procedures already described in the following references: Vasconcelos A, et al 2015; Holloway J. et al, 2014. Ri-[S-L]_n conjugated with azide functional group is linked to Alkyne- (B) _{r (}His ) _t ^_C via copper (I) catalyzed azide-alkyne click chemistry following the procedures described in the references: Yu Y, et al . , 2011; Zhou Z, et al . , 2015.

Example 2

Scheme 2 shows the diagram representing the synthesis of the compound of Formula (I) :

Ri— [S—L— (B)_{r— (}His)_t—C]_n (I) wherein

Ri is chitosan;

S is a direct bond;

L is selected from

B is selected from Gly, Phe, Ala or b-Ala;

C is selected from b-Ala and none;

r is an integer number between 0 and 5;

t is an integer number equal to or greater than 5, preferably comprised between 5 and 150, more preferably comprised between 10 and 50;

n is an integer number greater than 1, preferably comprised between 2 and 200, more preferably between 2 and 50.

Scheme 2

Synthesis of -R - [S-L]_n

Chitosan is linked to Maleimide moieties to form Ri— [S—L]_n (chitosan a) or Azide moieties to form Ri-[S- L] _n (chitosan b) via a coupling reaction between 3- (Maleimido) propionic acid NHS ester or Azidoacetic acid NHS ester in PBS or MES buffer, in water/dimethyl sulfoxide (DMSO) (1:1 v/v) . The products are isolated by dialysis against distilled water, according to the procedures described in Matsumoto M et al . , 2016; Kim LY et al . , 2008/ Wang H., 2010.

Synthesis of (B) _r- (His) _t ^~C

See example 1. Synthesis of Ri [S-L- (B) _r- (His) _t~C]_n

Chitosan a is conjugated to Thiol- (B) _r (His ) _t ^_C via maleimide-thiol chemistry by simply mixing chitosan a and Thiol- (B) _r (His ) _t ^_C in 1% (v/v) acetic acid solution according to the procedures described by Chan P et al . , 2007 and Malhotra M et al . , 2013.

Chitosan b is conjugated to Alkyne- (B) _r (His ) _S-C via copper (I) catalyzed azide-alkyne click chemistry according to the procedures described by Jung S. et al . , 2013.

Example 3

Nanoparticles preparation

The following sections (A-D) describe briefly some non-limiting examples of nanoparticle preparation found in the literature. They are not exhaustive due to the large amount of studies published on this topic. The nanoparticles thus obtained have diameters comprised between 100 and 500 nm.

(A) PLGA, PLA, PGA nanopartides preparation

PLGA, PLA, PGA nanoparticles are obtained using an oil-in-water emulsion solvent extraction method (Mariano N. et al . , 2014) The emulsion is prepared by dissolving the Ri- [S-L- (B) _r- (His) t-C] n (where R1 is PLGA or PLA or PGA) alone or in combination with a commercial PLGA or PLA or PGA (MW varying in the range 15-50 KDa) , in chloroform : methanol (3:1); this solution is called phase 1. PLGA is, for example, the Resomer® RG 502 H, molecular weight 7000-17000 Da, product number 719897, Sigma- Aldrich .

Phase 2 consists of a Poly (Vinyl Alcohol) aqueous solution (PVA, Mw 31000-50000 Da, 98-99% hydrolyzed, product number 363138, Sigma-Aldrich) . Phase 1 is added into phase 2 drop by drop and sonicated, keeping the mixture in an ice bath.

Phase 1 and phase 2 concentrations, sonication power, and sonication time are varied to control the nanoparticles sizes.

The emulsion is transferred to a round-bottom flask and put into a rotary evaporator (at 740 mmHg and 30 rpm) for 120 min to remove the organic solvent. Then the sample is subjected to dialysis (molecular weight cutoff of 14 000 Da) overnight at 4 °C against aqueous buffer.

When necessary, nanoparticles are concentrated by centrifugation with vivaspin filters (Sartorius) .

(B) Chitosan nanoparticles preparation

Chitosan nanoparticles are obtained by ionotropic gelation, using the sol-gel transition of chitosan polymers in the presence of a poly-anionic cross-linking agent, typically sodium tripolyphosphate (TPP, purchased from Sigma-Aldrich) .

Different chitosan (MW varying in the range 15-120 KDa) and salts concentration ratio are used to control the characteristics of chitosan particles ranging in size from nano- to micro-meters (Sreekumar S et al . , 2018) .

As an example, to obtain chitosan nanoparticles in the range of 100-200 nm the protocol reported by De la Fuente M. et al . , 2008 can be applied.

In brief, 0.2 ml of the cross-linker TPP (0.625 mg/ml or 0.42 mg/ml) is added dropwise to 1 ml of the aqueous solution containing the chitosan- [L- (B) _r- (His) _t ^_ C] _n compound 1-5 mg/ml under magnetic stirring for 10 min at room temperature.

The size of the particles formed are determined using dynamic light scattering (DLS) . (C) Alginate nanoparticles preparation

Alginate nanoparticles are synthesized by the ionic gelation method (Lopes M. et al, 2016) . Briefly, calcium chloride solution was added dropwise to the Alginate solution (low molecular weight alginate, 1-8% w/v) under a constant homogenization rate at 25 °C.

(D) Hyaluronic acid (HA) nanoparticles preparation

To obtain nanoparticles containing HA by the ionic gelification technique, it is necessary to couple it with a positively charged polymer, such as chitosan (de la Fuente M. et al, 2008) .

Briefly, chitosan (MW 10-12 KDa) is dissolved in water (2.5 mg ml^-1, solution A) . The solution of the Ri- [S-L- (B) _r- (His) _t-C] _n compound, wherein Ri is HA, is prepared in ultrapure water (0.5-5 mg ml^-1) and TPP is incorporated (0.25-1 mg ml^-1, solution B) . Nanoparticles are instantaneously obtained upon the addition of 1.5 ml of the solution B (pH=8-9) to a 3 ml solution A (pH = 3), under magnetic stirring at room temperature.

Example 4

Scaffold preparation

Porous scaffold based on the chitosan and alginate derivatives are prepared by freeze-gelation method (H- Ming-Hua H et al . , 2004) .

The Ri- [S-L- (B) _r- (His) _t-C] n compound wherein Ri is chitosan is dissolved in acetic-acid aqueous solution (1 M) to form a 2 wt% polymer solution. The polymer solution is placed in a mold and frozen at -20°C. The frozen solution is immersed in a NaOH/ethanol aqueous solution (pre-cooled to -20°C) to adjust its pH to allow for the gelation of chitosan.

The Ri- [S-L- (B) _r- (His) _t-C] n compound wherein Ri is alginate is dissolved in deionized water to form a 2 wt% solution, which is then frozen at -20°C and then immersed in aqueous ethanol solution of CaCl2 at -20°C to induce gelation of alginate. Drying at room temperature is performed after gelation to obtain scaffolds.

Porous PLGA-based scaffolds are prepared by the porogen leaching technique (Holy CE et al . , 1999) . The Ri- [S-L- (B) _r- (His) _t-C] _n compound wherein Ri is PLGA, alone or in combination with a commercial PLGA (75 mg/ml), is dissolved in DMSO anhydrous and added to a mold containing glucose as porogens. The mold is cooled to - 20°C, then the frozen sample is immersed in distilled water, which is changed several times, and dried at room temperature .

RESULTS

To provide a general demonstration that the novel imidazole-comprising compounds subject of the present invention are suitable for use as contrast agents and pH sensing agents in FFC-MRI technique, the present Inventors collected some experimental data using (i) a commercial poly-Histidine (poly-His) and (ii) a compound of formula (I) as QPs generating compounds.

Figure 3 shows a schematic representation of poly-

His .

Poly-His shows a characteristic relaxation peak at 1.38±0.3 MHz due to the ¹⁴N nuclear quadrupole resonance frequency of the imidazole groups present in the polymeric chains. There are two further resonance frequencies from the imidazole group that generate broad peaks at 0.721 MHz and 0.647 MHz, respectively, but these cannot be used because they overlap with the QP at 0.7 MHz arising from the tissue protein amidic groups.

Figure 4 shows the 1/Ti NMRD profile of a commercially available (Sigma Aldrich catalogue number P9386) poly-His (MW = 15600 Da, dissolved in water, 30% w/w), showing its characteristic QPs compared with a 1/Ti NMRD profile acquired on a liver tissue sample. The QP at 1.38±0.3 MHz arising from imidazole is well detectable and distinguishable from the QPs generated by the background tissue. The relaxivity enhancement calculated at the maximum of the peak with respect the background profile is of ca . 60% corresponding to a relaxivity increase of about 5 s^_1.

Figure 5 shows 1/Ti NMRD profiles acquired on poly- His at different concentrations. Although the QP at 1.38±0.3 MHz still remains detectable at the lowest tested concentration (5 mg/ml), 14 mg/ml represents the minimum concentration giving a significant relaxation enhancement (20%) to be well detectable also in the presence of a consistent tissue background.

As pH controls the solid/liquid physical state of the poly-His and given that the ¹⁴N-QPs are only observed for immobilized systems, the pH of the microenvironment in which the imidazole-comprising compound is present can be assessed by changes in the intensity of the imidazole QP . There is increasing interest in the development of pH sensing agents able to measure in vivo tissue pH non-invasively, in particular in oncological applications. In fact, the measurement of tumour tissue pH has a marked prognostic relevance as the presence of slightly acidic zones (e.g. pH = 6.6-6.8) surrounding hypoxic and necrotic zones promotes cell migration and metastasis .

As described above, Poly-His QPs intensity is pH- dependent and its observation is possible only when poly- His is immobilized in a solid-like form, i.e. at pH > 6.6. At lower pHs the protonation of the imidazole groups (pKa~6.8) increases the solubility in water of the polymer with a consequent increase of its mobility and a proportional decrease of the QPs intensity (Figure 6) . Since the nanoparticle interior is water accessible the imidazole-comprising compounds subject of the present invention can be exploited as pH reporters in the range of interest for tumour tissue after the set-up of a proper calibration curve where the QP intensity is detected as a function of the pH measured using a standard pH Meter.

Imidazole-comprising compounds containing imidazole moieties, such as poly-His, may be useful for the design of contrast agents reporting on local pathological and physiological changes. Such a task may be tackled by confining the poly-His chains responsible for QPs inside a biocompatible particle while maintaining the suitable conditions for the generation of the peculiar imidazole QP at 1.38±0.3 MHz.

As a non-exhaustive example, the poly-His chain can be confined into poly (lactic-co-glycolic acid) (PLGA) nanoparticles. PLGA is approved by the US Food and Drug Administration (FDA) and European Medicine Agency (EMA) in several drug delivery systems for human use. The polymers are commercially available at different molecular weights and copolymer compositions. Furthermore, PLGA-NPs interior is accessible to bulk water to an extent that is inversely proportional to the NP size. A schematic representation of an oligo-His-PLGA NP is shown in Figure 7. To prepare this imidazole comprising compound, an oligo-histidine (His x 15) peptide containing a free thiol group as end group was synthesized and conjugated to a maleimide functionalized PLGA, as described in materials and methods. The peptide used in the present example is Thiol- (B) _r (His ) _t ^_C wherein aminoacid N-terminus is Cys, B is Gly, r is equal to 2, t is equal to 15, C is b-Ala, thus obtaining Cys (Gly) 2 (His ) _ΐ5bAΐ3 (named in the following as oligo- His) . Before its conjugation to PLGA the 1/Ti NMRD profile of oligo-His suspension was compared with the one obtained from the commercially available poly-His (at least 38 residues) . As shown in Figure 8, the QPs still remained well detectable in the synthesized oligo- His and were only slightly lower than those obtained for the commercial poly-His (about 10% less) .

In the following, a discussion about a preferred embodiment of the instant description is provided, wherein the imidazole comprising compound of the instant description presents the following chemical structure:

wherein x is equal to 77 and y is equal to 77.

The experimental data collected with the compound containing an imidazole moiety identified above are not limiting with respect to the actual scope of the present invention as claimed in the set of claims appended.

The 1/Ti NMRD profiles acquired on suspensions containing two different batches of oligo-His-PLGA nanoparticles (at a 5 mg/ml oligo-His concentration, the lower concentration tested with the commercial poly-His) are reported in Figure 9. In both samples the imidazole QPs are more pronounced (+75%) than the one obtained in the case of the commercial poly-His at the same concentration .

MATERIALS AND METHODS

Preparation of oligo-His-PLGA

In order to incorporate an oligo-His into the PLGA nanoparticles, the oligo-His-PLGA was synthesized according to a conjugation scheme 3. Scheme 3

The steps of the synthesis represented in scheme 3 will now be disclosed in details.

Oligo-His synthesis

Preferably, the peptide fragments of the present invention are synthesized by solid phase peptide synthesis (SPPS) techniques using standard FMOC protocols. (See Carpino et al . , 1970; Carpino et al . ,

1972) . In a preferred embodiment, peptide with specific amino acid sequences was synthesized, having the above formula Thiol- (B) _{r (}His ) _t ^_C, wherein aminoacid -terminus is Cys, B is Gly, r is equal to 2, t is equal to 15, C is b-Ala as a C-terminal amide. The Cys (Gly) 2 (His ) i^Ala peptide (SEQ ID No.: 1) was assembled on H-Rink amide ChemMatrixDresin (Sigma-Aldrich) (150 mg, resin loading

0.4-0.6 mmol/g) using HBTU/HOBt activation (CEM modello Liberty, s/n DP7026DU8276 Synthesizer) .

Fmoc deprotection was performed using 20% v/v piperidine in DMF plus 0,1 M HOBt in two stages with an initial 0.3 min followed by a longer 3 min treatment (7 ml, 40 W, 75°C) . Coupling reagents were as follows: Fmoc- Aa-OH (0.2 M in DMF), HBTU (0.5 M in DMF), DIPEA (2.0 M in NMP) . Each coupling was done twice using a 5 fold excess of Fmoc-Aa-OH and slightly less than 5 fold excess of HBTU, 10 fold excess of DIPEA (for 5 min, 35 W, 75°C) . Power pulsing sequences of 25 W for 4 min were used for Cys coupling steps. The peptidyl-resin was washed with DMF (3 x 5 ml), DCM (3 x 5 ml) and dried. The peptide- resin was cleaved using a mixture of TFA/H₂0/Phenol/TIS (88:5:5:2 v/v/wt/v) , at room temperature for 4h with gentle tumbling agitation. The material was then filtered, washed with a minimum amount of TFA and the cleavage solution was collected and concentrated to dryness. Cold Et₂0 was added to the residue to precipitate the peptide, which was collected and dried. The peptide was obtained in 50% yield with 98% purity despite the omission of HPLC purification. HPLC assay conditions: RP-C18, 5% CH₃CN in 0.1% TFA over 5 min, 5-20% CH₃CN in 0.1% TFA over 10 min, 20-35% CH₃CN in 0.1% TFA over 15 min, 35-100% CH₃CN in 0.1% TFA over 3 min .

Column: Waters Atlantis C18, 5 pm, 4.6x150 mm

Flow rate: 1 mL/min

Detection: Diode Array, Single quadrupole MS

Mobile phase:

A: 0.1% aqueous TFA

B: 0.1% TFA in acetonitrile

Retention time: ^"11.5 minutes

ESI ( + ) : Calc, for CiooHi₂₄N₅oOi9S m/z[M+3H] ³⁺ = 788.5; m/ z [M+4H] ⁴⁺ = 591.6; m/ z [M+5H] ⁵⁺ = 473.5, observed 788, 4 ; 591,6; 473, 4.

^XH NMR (dmso-de, 600Mhz) d 8.92-8.80 (15H, -N=CH-N-

), 8.52-8.16 (amide-protons), 7.27 (15H, , C=CH-N) , 4.6- 4.5 (15H, NH-CH- (CH₂) -IM) , 3.36, 3.25 (m, 4H) , 3.10-2.99 (m, ~30H, —CH—CH_2—IM) , 2.26 (t, 2H, -NH-CH_2-CH_2-CONH₂ b- Ala) . (Figure 10 )

Maleimide-PE62-amine synthesis

3- (Maleimido) propionic acid NHS ester (0.3 g, 1.15 mmol) and TEA (0.24 ml, 1.5 eq) were dissolved in CH₃CN (10 ml) , followed by addition of t-Boc-N-amido-PEG_2- amine (0.28 g, 1.15 mmol) and the resulting reaction mixture was stirred for 2 h at room temperature. Then it was diluted with ethyl acetate and washed three times with water and one time with brine. The extract was dried over anhydrous sodium sulfate, concentrated under reduced pressure. Flash chromatography on silica gel using ethyl acetate and hexane afforded 0.46 g of the compound. ESI (+ ) m/ z (M+Na)⁺ 422.2. (M+H-Boc) ⁺ 300.2.

Subsequent N-Boc deprotection was performed using TFA:CH₂C1₂ (1:1 v/v, 5mL) for 1 h at room temperature. The solvent was removed in vacuo to yield the product as a white solid (0.3 g) .

HPLC assay conditions: RP-C18, 5% CH₃CN in 0.1% TFA over 5 min, 5-20% CH₃CN in 0.1% TFA over 10 min, 20-35% CH₃CN in 0.1% TFA over 15 min, 35-100% CH₃CN in 0.1% TFA over 3 min.

Column: Waters Atlantis C18, 5 pm, 4.6x150 mm

Flow rate: 1 mL/min

Detection: Diode Array, Single quadrupole MS

Mobile phase:

A: 0.1% aqueous TFA

B: 0.1% TFA in acetonitrile

Retention time: ^~8.8 minutes

ESI (+ ): Calc . forCi₃H_2i0₃0₅ m/z [M+H] ⁺=300.1 ; observed

300.1

¹H NMR (dmso-d₆, 600Mhz) d 8.00 (t, 5.4 Hz, CO-

NH, 1H) , 7.80 (s br,-NH₂,2H), 7.05 (s, maleimide methine protons) 3.65-3.55 (m, 8H) , 3.41 (t, 5.9 Hz,2H), 3.20 (m, 2H) , 3.02 (m, 2H) , 2.37 (t, 7.26Hz, 2H) .

PLGA-PEG2-Maleimide synthesis

PLGA Resomer® RG 502 H with a 50:50 monomer ratio PM= 12800, 0.5 g, 0.04 mmol) was dissolved in 10 mL of dry CH₃CN in a 25 mL flask. 20 pL (0.12 mmol) of DIPEA and HATU (0.017 g, 0.044 mmol) were added to the flask. After 5 min Maleimide-PEG2-amine (0.013 g, 0.044 mmol) dissolved in CH₃CN (1 mL) is added dropwise and then the mixture was stirred at room temperature for 6 h under N₂ atmosphere. The solvent was then removed under vacuum and the residue was dissolved with chloroform and washed three times with brine. The crude compound was purified via column chromatography (eluent: CH₂Cl₂/CH30H 98:2) to give product as a white solid (0.42 g) .

¹H NMR (dmso-d6, 600Mhz) d 8.0 (amide proton), 7.03 (s, maleimide methine protons), 5.31-5.0 (m, CH lactide proton), 4.98-4.85 (m, CH₂ glycolide proton), 1.54-1.46 (m, CH₃ lactide proton) . (Figure 11)

Oligo-His-PLGA conjugation

To a solution of PLGA-PEG2^~Mal (0.20 g, 0.015 mmol) in 2 mL of anhydrous DMF, a solution of Cys (Gly) 2 (His ) i^Ala peptide (0.034 g, 0.016 mmol) in DMF (0.5 mL) and N, N-diisopropylethylamine (DIPEA, 5 pL, 0.03 mmol) were added. The resulting mixture was magnetically stirred at room temperature for 3 h under nitrogen atmosphere. The desired oligo-His-PLGA was obtained by precipitation from cold diethyl ether/methanol 1:1 (10 mL) that was collected, washed with diethyl ether/methanol 1:1 (10 mL) twice and dried under vacuum yield, 85%) . ¹H NMR (dmso-d6, 600Mhz) d 8.46 (br, 15 H,—N=CH—N—) , 8.4-8.0 (amide protons), 7.12 (br, 15H, C=CH-N) , 5.31-5.01 (m, 94H, CH lactide proton), 4.98-4.85 (m, 196H, CH₂ glycolide proton), 4.57 (m, 15 H, H_a protons His ) , 3.08-2.99 (m, ~30H, Hp protons His), 2.26 (t, 2H, -NH-CH2-CH2-CONH2 b-Ala) , 1.55-1.47 (m, 298H, CH₃ lactide proton) (Figure 12) .

The ¹H-NMR analysis revealed the disappearance of the peak corresponding to the maleimide protons at 7.03 ppm. We used ¹H-NMR to determine the resulting molar ratio of PLGA to peptide by integration of the PLGA (CH₃, CH₂, CH) and of the His (H_a protons) . The calculated functionalization confirmed a 1:1 molar ratio of PLGA to oligo-His .

The oligo-His-PLGA Nanoparticles (NP) preparation

The oligo-His containing NPs were obtained by applying the oil/water (o/w) emulsion solvent extraction method (Figure 13) . The organic phase was prepared by dissolving the oligo-His-PLGA and the PLGA Resomer® RG 502 H (1:1) in chloroform:methanol (3:1) . The water phase was a PolyVinyl Alcohol (PVA) aqueous solution. PVA is the most commonly used emulsifier for the preparation of PLGA-NPs because it yields particles that are relatively uniform, small sized, and easy to be re-dispersed in water. The organic phase was added to the aqueous phase, under sonication. To control the size of the NPs, different PVA concentrations and sonication conditions were tested. The solidification of NPs was carried out by organic solvent evaporation from the o/w emulsion in a rotary evaporator under vacuum (2.5 h) . The NPs were stored at 4 °C before their use. The average hydrodynamic diameters of the oligo-His-PLGA-NPs were determined by dynamic light scattering (DLS) measurements, and were equal to 150 nm.

Dynamic Light Scattering (DLS)

The hydrated mean diameter of nanoparticles is determined using a Malvern Zetasizer 3000HS (Malvern, U.K.) . All samples were analyzed at 25 °C in filtered (cutoff, 200 nm) water/buffer solution.

NMRD profile acquisition

Samples (usually 1 ml volume) were added in a glass tube (10 mm diameter x 220 mm length) .

Data were measured at 25°C over a continuum of magnetic field strengths from 0.00024 to 0.47 T (corresponding to 0.01-20 MHz proton Larmor Frequency) on a Stelar fast field-cycling relaxometer (Stelar, Mede, Italy) .

The temperature was controlled by a Stelar VTC-91 airflow heater, equipped with a copper-constantan thermocouple. The temperature in the probe head was measured with a Fluke 52 k/ j digital thermometer (Bassersdorf, Switzerland) . The relaxometer operated under complete computer control with an absolute uncertainty in the 1/Ti values of ±2%. Ti measurements were performed by using the Not Polarized and Pre- Polarized sequences as described by Ferrante and coworkers (Ferrante G et al . , 2005) (Figure 1) . Ti was determined by the saturation recovery method. 16 values of delay (t) between pulses were used. The number of averaged experiments was 2.

¹H NMR spectra

¹H NMR spectra were acquired in deuterated solvent (dmso-de) at 14 T on a Bruker Avance 600 spectrometer equipped with an inverse Z-gradient 5 mm BBI or TXI probe . REFERENCES

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Claims

1. A Magnetic Resonance Imaging (MRI) contrast agent including an imidazole-comprising compound, wherein the imidazole-comprising compound comprises at least 5 imidazole moieties, and wherein the imidazole moieties are in an immobilized state, corresponding to a tumbling time in the order of tens of nanoseconds (ns) so that in use the longitudinal water proton relaxation rate at 1.38±0.3 MHz (Proton Larmor Frequency) increases of at least 10% of the pre-contrast value, wherein the MRI contrast agent is free of paramagnetic metal (s) .

2 . The MRI contrast agent according to claim 1, wherein the imidazole-comprising compound has formula

(I) :

Ri- [S-L- (B) _r- (His)_t-C] n (I) wherein

Ri is selected from polyglycolic acid (PGA) , polylactic acid (PLA) , poly ( lactic-co-glycolic acid) (PLGA) , alginate, hyaluronic acid (HA) and chitosan;

S is a spacer selected from a direct bond, -(CH₂)₂- 0

B is selected from Gly, Phe, Ala or b-Ala;

C is selected from b-Ala and none;

r is an integer number between 0 and 5;

t is an integer number equal to or greater than 5, preferably comprised between 5 and 150, more preferably comprised between 10 and 50;

n is an integer number equal to or greater than 1.

3. The MRI contrast agent according to claim 2, wherein Ri is selected from polyglycolic acid (PGA) , polylactic acid (PLA) and poly ( lactic-co-glycolic acid) (PLGA) ; S is selected from- (CH₂) ₂-0- (CH₂) ₂-0- and -(CH₂)₂ ^_ 0- (CH₂) ₂ ^_0- (CH₂) ₂ ^_0-; and n is an integer number equal to 1.

4. The MRI contrast agent according to claim 2 or claim 3, wherein

Ri is poly ( lactic-co-glycolic acid) (PLGA) ;

B is Gly;

C is b-Ala;

r is equal to 2;

t is an integer comprised between 10 and 50.

n is equal to 1.

5. The MRI contrast agent according to any one of claims 2 to 3, wherein

Ri is poly ( lactic-co-glycolic acid) (PLGA);

S is - (CH₂) ₂-0- (CH₂) ₂-0-;

L is

B is Gly;

C is b-Ala;

r is equal to 2;

t is equal to 15;

n is equal to 1.

6. The MRI contrast agent according to claim 2, wherein Ri is selected from alginate and hyaluronic acid; S is selected from - (CH₂) ₂-0- (CH₂) ₂-0- and- (CH₂) ₂-0- (CH₂) ₂- 0-(CH₂)₂-0-; and n is an integer number greater than 1, preferably comprised 2 and 100.

7. The MRI contrast agent according to claim 2, wherein Ri is chitosan; S is a direct bond; n is an integer number greater than 1, preferably comprised 2 and 200.

8. The MRI contrast agent according to any one of the preceding claims, wherein the imidazole-comprising compound is in form of nanoparticle or scaffold.

9. A pharmaceutical composition comprising a MRI contrast agent according to any one of claims 1 to 8 and a pharmaceutically acceptable vehicle.

10. A pharmaceutical composition according to claim 9 for use as a contrast agent and/or as a pH sensing agent in Fast Field-Cycling (FFC) Magnetic Resonance Imaging (MRI) technique.