IT201900007647A1 - NEW CONTRAST AGENTS FOR MAGNETIC RESONANCE FOR IMAGES - Google Patents

Description

DESCRIPTION OF THE INDUSTRIAL INVENTION TITLE:

"New contrast agents for magnetic resonance imaging"

TEXT OF THE DESCRIPTION

FIELD OF THE INVENTION

The present disclosure relates to novel magnetic resonance imaging contrast agents.

BACKGROUND OF THE INVENTION

In recent decades, magnetic resonance imaging (MRI) has become a leading modality in the clinical setting due to its superb spatial resolution and its exceptional ability to differentiate soft tissues. Contrast in an MR image mainly arises from differences in the relaxation times of tissue water protons as a consequence of their interaction with macromolecules and biological membranes. Variations in longitudinal (T1) and transverse (T2) relaxation times are the determinants of contrast.

It is known from in vitro studies that the spin-lattice relaxation time, T1, of a given tissue changes as a function of the applied magnetic field strength. This behavior (known as "T1-scattering") is a marker of disease, but is invisible to conventional fixed-field MRI scanners. Measurement of T1-dispersion is often referred to as "relaxometry" and the resulting graph of T1 (or R1 = 1 / T1) versus magnetic field strength (or equivalent Larmor frequency) is known as the magnetic resonance dispersion curve nuclear (NMRD) or NMRD profile. Rapid Field Cycle (FFC) is the only practical way to measure T1-dispersion in vivo; involves switching the magnetic field between different field strengths during the measurement procedure. The time to move from one level to another is usually less than the T1 of the sample, in which case the technique is known as a "fast" field cycle (Figure 1). The polarization of nuclear spins is created during the first period, at the field strength Bop- The spin-lattice relaxation occurs during the evolution period (duration tE) at the field strength BOE, so the NMR signal is detected during the final period, always at the same field strength BOD- The sequence is repeated, updating BOE each time, to measure Ti as a function of BOE- The fact that the NMR signal measurement always takes place with the same magnetic field strength (BOD) is fundamental, because it means that the radio frequency coils (transmitting Tx and receiving Rx or Tx / Rx in combination) do not need to be retuned when collecting data for a Ti-dispersion curve measurement. FFC relaxometry on small samples (~ 1 ml) has been studied 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, mostly for the work of the Lurie group at the University of Aberdeen, where two prototype full-body-sized FFC-MRI scanners were built.

FFC introduces a whole new dimension in MRI, namely the strength of the applied magnetic field. By making measurements as a function of magnetic field strength, totally new methods can be employed to obtain the contrast between normal and diseased tissues and organs.

An example of a phenomenon that is completely invisible to conventional (fixed-field) MRI is the existence of "quadrupole peaks" (QP): significant increases in the measured relaxation rate (R1) of samples containing immobile protein molecules, in fields where the proton NMR frequency and the quadrupole nuclear resonance frequency (NQR) of <14> N coincide (Sunde et al., 2010; Koening et al., 1998; Fries et al., 2015) (Figure 2) . Note that in the FFC field it is usual to display scatter plots as R1 vs Larmor proton frequency rather than T1, in which R1 = 1 / 'Ti and this format will be adopted from now on.

In the case of biological tissues, the <14> N-QPs are normally detected at the proton NMR frequencies of 0.7, 2.1 and 2.8 MHz, equivalent to field strengths of 16 mT, 49 mT and 65 mT. For such systems, it is well known that QP detection is associated with the presence of amide peptide bonds deriving from endogenous protons. Their amplitude is proportional to the amount of protein present in the sample considered and may possibly be associated with the presence of pathological alterations. There are currently no compounds available that can be used as contrast agents in FFC-MRI for In vivo diagnostic purposes.

Currently, paramagnetic species are commonly exploited as fixed-field MRI contrast agents. The systems currently used in the clinic are represented by complexes with gadolinium ion polyamine carboxylate (Gd <3+>) which contains seven unpaired electrons. The ligands are multidentates (seven or eight donor atoms) with high thermodynamic stability to strongly limit the release of free metal ions which are highly toxic since, for example, they interfere with the Ca <2+> pathways. About 40-50% of MRI exams use Gd-based contrast agents substantially to report physiological changes accompanying a specific disease, i.e. the loss of the blood brain barrier in the case of tumor lesions in the brain or in the presence of multiple sclerosis. hypervascularization and for the change in the "washing in / washing out" rate to evaluate the malignancy of other tumors, etc. There is currently much concern in MRI about the potential toxicity of conventional Gd-containing contrast agents in patients with impaired renal function. Therefore, much attention worldwide is devoted to the development of metal-free contrast agents that employ novel mechanisms.

PURPOSE AND SUMMARY OF THE INVENTION

One purpose of the present disclosure is the development of novel MRI contrast agents for use in the rapid field cycle magnetic resonance imaging (FFC-MRI) technique. The MRI contrast agents described here are capable of shortening the relaxation time and generating image contrast at certain frequencies without using any paramagnetic contrast agent containing gadolinium or other metal.

According to the invention, the aforesaid object is achieved thanks to the object referred to specifically in the following claims, intended as an integral part of the present description.

In one embodiment, the present disclosure describes a magnetic resonance imaging (MRI) contrast agent comprising a compound comprising imidazole, wherein the compound comprising imidazole comprises at least 5 imidazole moieties (groups), and wherein the imidazole moieties they are in an immobilized state, so that, in use, the longitudinal relaxation rate of the water proton at 1.38 0.3 MHz (Larmor proton frequency) increases by at least 101 of the pre-contrast value.

The contrast agents for MRI described here, characterized by comprising in their structure at least five imidazole moieties, provide the generation of detectable <14> N-QPs that fall at a frequency, i.e. 1.38 0.3 MHz, well distinguishable from frequencies associated with amide peptide bonds from endogenous proteins, occurring at 0.7, 2.1, and 2.8 MHz. Therefore, QPs arising from compounds comprising imidazole - when administered to a subject prior to MRI scans - may be easily identified and their detection will not be affected by changes in endogenous QPs.

BRIEF DESCRIPTION OF THE DRAWINGS

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

- FIGURE 1. Sequence of generic fast field cycle NMR pulses for measurement of T1-dispersion. Note that in the Imaging Pulse Sequence (FFC-MRI), magnetic field gradients are applied during the detection period.

- FIGURE 2. Left: (a) NMRD profile of a BALB / c mouse paw, (b) Gaussian functions of QPs, after background subtraction, (c) Energy levels of nuclei of <14> N (S = 1 ) due to interactions of QP with the gradients of the local electric field. The two levels on the left are in axial symmetry. The top level splits when the axial symmetry breaks, as shown on the right. A local maximum of the R1 profile of protons occurs when the Zeeman transition energy of this spin I is equal to the difference in energy of two levels of the spin S.

FIGURE 3. Schematic representation of the polyamino acid polyhistidine.

- FIGURE 4. A) NMRD profile acquired from 0.01 to 20 MHz of poly-His (301 weight / weight) in water compared to the NMRD profile of fresh liver tissue ex vivo (T = 25 ° C) with expansion of the QP region. The asterisk indicates the imidazole peak. B) Gaussian functions of the QPs, after the 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 shown in panel A) in the range 0,8-4 MHz.

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

FIGURE 7. Schematic representation of an oligo-His-PLGA nanoparticle (NP). The oligo-His-PLGA NPs were prepared according to the solvent extraction procedure in oil / water (o / w) emulsion with PVA coating. Abbreviations: oligo-His-PLGA = Cys (Gly) 2His15βAla conjugated with PLGA-FEG2-Maleimide; PVA = Polyvinyl alcohol.

- FIGURE 8. NMRD profile of Oligo-His compared with that acquired on a commercially available poly-His solution, both dissolved in aqueous solution at 301 w / w. The insert shows the interpolation of the peak relative to the contribution of the imidazole, after the subtraction of the background.

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

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

- FIGURE 11. <1> H-NMR spectrum in dmso-d6 at 37 ° C of PLGA-PEG2-Mal.

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

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

DETAILED DESCRIPTION OF THE INVENTION

In the following description, numerous specific details are provided to allow a thorough understanding of the embodiments. The embodiments can be practiced without one or more specific details, or with other processes, components, materials, etc. In other cases, well-known structures, materials or operations are not shown or described in detail to avoid confusing aspects of the embodiments.

Reference throughout this description to "one embodiment only" or "one embodiment" means that a / a particular aspect, structure or feature described / described in connection with the embodiment is included / included in at least one embodiment of realization. Therefore, the forms of the expressions "in one embodiment" or "in one embodiment" at various points throughout the present disclosure are not necessarily all referred to the same embodiment. Furthermore, the particular aspects, structures or features can be combined / combined in any suitable way in one or more embodiments.

The titles 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 that has atoms held together by 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 one embodiment, the present disclosure describes a magnetic resonance imaging (MRI) contrast agent including a compound comprising imidazole, wherein the compound comprising imidazole comprises at least 5, preferably 5 to 150, more preferably 10 to 50 moìety imidazole, and in which the imidazole moieties are in an immobilized state, so that, in use (i.e. when the compound comprising imidazole is administered to a human or animal), the longitudinal relaxation rate of the water proton is 1, 38 0.3 MHz (Larmor proton frequency) increases by at least 101 of the pre-contrast value. In practice, the longitudinal relaxation rate of the water proton at 1.38 0.3 MHz increases by at least 101 of the pre-contrast value in the region of interest of the human or animal body in which the compound comprising imidazole is present and / or localized.

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

% boost = [(R1 (post-contrast) -R1 (pre-contrast)) / R1 (pre-contrast)] * 100

wherein R2 (post-contrast) is the relaxation rate measured after administration of the contrast agent for MRI; R1 (pre-contrast) is the relaxation rate measured prior to 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 reorientation time in the order of tens of nanoseconds (ns).

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

m which

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 chosen between a direct bond,

A is chosen from Cys and propargyl-Gly;

B is selected from Gly, β-Ala and Phe-Ala;

C is chosen between β-Ala and nothing;

r is an integer between 0 and 5;

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

n is an integer equal to or greater than 1.

In one or more embodiments, R1 is selected from polyglycolic acid (PGA), polylactic acid (PLA) and poly (lactic-co-glycolic) acid (PLGA); S is chosen from - (CH2) 2 is a linker

chosen from

A is chosen from Cys and propargyl -Gly; B is selected from Gly, β-Ala and Phe-Ala; C is chosen between β-Ala and nothing; r is an integer between 0 and 5; t is an integer equal to or greater than 5, preferably between 5 and 150, more preferably between 10 and 50; and n is an integer equal to 1.

In one or more embodiments, R1 is acidic

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

In one or more embodiments, R1 is PLGA poly (lactic-co-glycolic) acid; S is L

And ; A is propargyl-Gly; B is Gly; C is β-Ala; r is equal to 2; t is equal to 15; and n is equal to 1.

In one or more embodiments, R1 is selected from alginate and hyaluronic acid; S is chosen from - (CH2) 2-O- I1 is a linker

chosen between A is chosen between Cys and propargyl-Gly; B is selected from Gly, β-Ala and Phe-Ala; C is chosen between β-Ala and nothing; r is an integer between 0 and 5; t is an integer equal to or greater than 5, preferably between 5 and 150, more preferably between 10 and 50; and n is an integer greater than 1, preferably between 2 and 200.

In one or more embodiments, R1 is chitosan; S is a direct link; L is a linker chosen from

; A is chosen from Cys and propargyl-Gly; B is selected from Gly, β-Ala and Phe-Ala; C is chosen between β-Ala and nothing; r is an integer between 0 and 5; t is an integer equal to or greater than 5, preferably between 5 and 150, more preferably between 10 and 50; and n is an integer greater than 1, preferably between 2 and 200.

In one or more embodiments, PGA, PLA, PLGA have a molecular weight between 10 and 75 kDa. Alginate and chitosan have a molecular weight between 10 and 400 kDa. HA has a molecular weight between G and 1,000 kDa. Preferably, PGA, PLA, PLGA, alginate, chitosan, HA have a molecular weight comprised between 10 and 100 kDa.

Novel MRI contrast agents are specifically suited for FFC-MRI due to the generation of detectable <14> N-QPs that fall at a frequency (1.38 0.3 MHz) that is well distinguishable from the frequencies associated with amide peptide bonds. endogenous proteins (0.7, 2.1 and 2.8 MHz).

The observation of <14> N-QP is strictly dependent on a substantial immobilization of the imidazole moieties present in the compounds comprising imidazole under physiological conditions. In fact, QFs are not observed for proteins or polymers in isotropic reorientation, but require the immobilization on a time scale of the inverse quadrupolar coupling (about 50 ns) (Sunde et al., 2010). The coupling <14> N- <1> H concerns not only the hydrogen atoms directly bound to the nitrogen atoms (i.e. the hydrogen atoms of the imidazole moieties of the compounds comprising imidazole), but also the protons of the water of hydration or other labile protons (also called intermediate water protons) in proximity to the compounds comprising imidazole, which directly or indirectly detect the <14> N- <1> H interaction. The protons of this intermediate water layer diffuse the quadrupolar effect of <14> N of the compounds comprising imidazole thanks to their exchange with the imidazole protons bound to <14> N and with water protons in mass. To be effective, this intermediate water layer must perform microsecond time-scale exchange as occurs in the environment of a tissue or particle.

For these reasons, the compounds comprising imidazole are in the form of nanoparticles, in which the nanoparticles are permeable to water. In this form, the imidazole moieties of the compounds comprising imidazole and - if present - the hydration water molecules of the compounds comprising imidazole adjacent to / contained in the nanoparticles (coming from the water molecules surrounding the nanoparticles) are immobilized in a sort of a solid-like state (i.e. a gel state), which thus allows the detection of the <14> N-QPs generated by the compounds comprising imidazole. The compounds comprising imidazole of formula (I) in the form of nanoparticle bring the imidazole moieties to be in an immobilized state characterized by a reorientation time in the order of tens of ns. In the presence of water molecules, the compounds comprising imidazole of formula (I) in the form of nanoparticle bring the imidazole moieties and the water molecules permeated inside the nanoparticles to be in a gel state, so that the imidazole moieties and the water molecules are in an immobilized state with a reorientation time in the order of tens of ns.

Compounds comprising imidazole in the form of nanoparticles are injected intravenously, intratumorally or implanted subcutaneously. In fact, it is known that newly formed vessels of solid tumors show a significantly greater permeability to nanoparticles which leads to their greater accumulation compared to healthy tissues (EPR effect). The nanoparticles are preferably injected intravenously prior to imaging. In order to improve targeting efficiency, the outer surface of the nanoparticles can be functionalized with specific ligands capable of specifically recognizing the surface of tumor cells. Examples of such ligands are antibodies (monoclonal, polyclonal, chimeric and humanized antibodies and their fragments) capable of binding specifically to a protein / proteins expressed / expressed on the surface of tumor cells.

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

However, the successful use of scaffolds - whatever form they may take - strongly depends on their stability after insertion and it is vital that the implant status can be assessed in vivo, can be monitored in studies. clinical on humans or corrective action can be taken early after implantation. In this context, the compounds comprising imidazole object of the present invention act as a sensor for detecting the stability of the scaffold after transplantation into the host organism. The compounds comprising imidazole are in fact homogeneously distributed on the external surface and inside the scaffold itself. The information provided by the QPs generated by the imidazole moieties in detecting the stability of the scaffold comes from their pH-dependent water solubility. A decrease in pH (pH <7), due to cell death and / or degradation of the scaffold matrix, leads to changes in the intensity of the QPs (see the results described below) depending on an increase in the mobility of the imidazole moieties of the compounds including imidazole. In fact, as described above, the immobilization of the imidazole moieties is mandatory for the generation of <14> N-QPs. Consequently, the appearance of positive charges on imidazole moieties (pKa about 6.8) for their protonation increases the water solubility of the compound comprising imidazole, with a proportional increase in mobility and disappearance of detectable QPs. Therefore, the pH of the microenvironment in which the imidazole-comprising compound is found can be assessed by changes in the intensity of the imidazole QPs.

The pH detection capability of the imidazole-comprising compounds of the present application is also very useful in oncological applications. In fact, the measurement of the pH of the tumor tissue has a marked prognostic importance as the presence of slightly acidic areas (for example pH = 6.6-6.8) surrounding hypoxic and necrotic areas promotes cell migration and metastasis. The detection of the pH of nanoparticles formed by compounds including imidazole is allowed by their permeability to water and to H <+> and the consequent rapid balancing of the internal and external pH.

In one embodiment, the present disclosure describes novel compounds comprising imidazole of formula (I) for use (i) as contrast agents and / or (ii) as pH sensing agent in FFC-MRI in mammals, preferably human beings.

In one embodiment, the present disclosure describes a composition comprising compounds comprising imidazole of formula (I) and a pharmaceutically acceptable carrier, preferably sterile water or a sterile buffer, such as isotonic phosphate buffer (PBS). Preferably, the composition has a pH ranging from 7.2 to 7.4.

General synthesis of compounds comprising imidazole and preparation of related nanoparticles

The synthesis processes for preparing the compounds comprising imidazole as well as the relative scaffolds and nanoparticles object of the present invention are described below. Those skilled in the art will recognize that other synthesis pathways can be used to synthesize compounds comprising imidazole as well as related scaffolds and nanoparticles. Although specific starting materials and reactants are described in the Schemes and discussed below, other starting materials and reactants can be easily substituted to provide a variety of derivatives and / or reaction conditions. Furthermore, many of the compounds comprising imidazole as well as related scaffolds and nanoparticles prepared by the methods described below can be further modified in the light of this disclosure using conventional chemistry procedures well known to those skilled in the art.

The following abbreviations are used below:

BOP: (benzotriazol-1-yloxy) tris (dimethylamino) phosphonium hexafluorophosphate

DCC: N, N'-Dicyclohexylcarbodiimide

HATU: 1- [Bis (dimethylamino) methylenes-1H-1,2,3-triazole [4,5-b] pyridinium 3-oxide hexafluorophosphate

HBTIJ: O- (benzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate

PyBOP: Benzotriazol-1-il-os sitris-pyrrolidino-phosphonium hexafluorophosphate

TBTU: O- (benzotriazol-1-yl) -N, N, N <1>, N'-tetramethyluronium tetrafluoroborate

NHS: N-Hydroxyisuccinimide

DIPEA: N, N-Diisopropylethylamine

TIS: Triisopropylsilane

TFA: trifluoroacetic acid

Boc: tert-butyloxycarbonyl

Fmoc: 9-fluorenylmethoxycarbonyl

Trt: Tritile

NMP: N-Methyl-2-pyrrolidone

CH3CN: Acetonitrile

CH2Cl2: dichloromethane

Et2O: diethyl ether

pGly: propargyl-glycine

PGA: Polyglycolic acid

PLA: Polylactic acid

PLGA: Poly (lactic-co-glycolic acid)

PEG2: - (CH2) 2-O- (CH2) 2-O-PEG3: - (CH2) 2-O- (CH2) 2-O- (CH2) 2-O-PVA: Polyvinyl alcohol

Example 1

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

in which

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

S is a spacer chosen from

L is a linker chosen from

A is chosen from Cys and propargyl-Gly;

B is selected from Gly, β-Ala and Phe-Ala;

C is chosen between β-Ala and nothing;

r is an integer between 0 and 5;

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

n is an integer equal to or greater than 1.

Scheme 1

The individual steps will now be described in detail.

Synthesis of S-L

Such linker reagents are commercially available (namely, Sigma Aldrich or BroadPharm®), or they can be prepared from commercially available starting materials using the methodology known in the art. The Maleimide- (PEG) 2 or 3-amine and Azide- (PEG) 2 or 3-amine compounds are prepared by a coupling reaction between NHS ester of 3- (maleimido) propionic acid or NHS ester of azidoacetic acid and t- Boc-N-amido-PEG2, 3-amine (BroadPharm®). The coupling reaction to form the amide bond is generally carried out in a dry organic solvent, preferably in an inert atmosphere such as nitrogen or argon. Acetonitrile, chloroform, dichloromethane and tetrahydrofuran are used as a solvent in the presence of a base, preferably trialkylamines such as triethylamine, pyridine, 4- (dimethylamino) pyridine. The desired products are isolated by silica gel column chromatography. Then, to generate the desired compounds, the protecting group of the amino group t-butylcarbonyl (Boc) is removed according to standard procedures known to those skilled in the art, for example by treatment with TFA: CH2Cl2 (1: 1 volume / volume) or ( 4M) HCl / dioxane (1: 1 volume / volume) at room temperature. Reaction times between 2 and 6 hours.

Synthesis of R1- [S-L] n

R1 is bound to maleimide-PEG2,3-amine or azide-PEG2, 3-amine after the activation of the carboxylate performed following the standard procedures reported in the literature. Briefly, activating agents such as N-hydroxysuccinimide were used in the presence of a carbodiimide (N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride, EDC). The procedures are detailed in the references (Cheng J, et al 2007; Dong X, et al., 2010).

The reaction can also be performed 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 C3CN as a solvent, which depends on the solubility of the Ri polymer.

Synthesis of A- (B) r- (Hi.s) t-C

The compounds of formula A- (B) r- (His) t-C, in which A is selected between Cys (a sulfhydryl-functionalized peptide) and propargyl-Gly (an alkyne-functionalized peptide), are prepared by peptide synthesis techniques in standard solid phase and preferably an automatic peptide synthesizer, using Fmoc chemistry.

Typically, using this technique, an amino acid protected with N-a-Fmoc and the amino acid attached to the peptide chain growing 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 diisopropylethylamine. Here the amino acids are Fmoc-His (Trt) -OH, Fmoc-Gly-OH, Fmoc-βAla-OH, Fmoc-Phe-OH. Fmoc-Cys (Trt) -OH or Fmoc-propargyl-Gly-OH are introduced into the amino terminus of the peptide. The Fmoc group is cleaved from the bases, preferably a 20% volume / volume piperidine solution in DMF. After the completion of the entire sequence, the terminal Fmoc is removed and the resin is dried under vacuum. A solution formed from a cleavage cocktail (TFA, H2O, phenol and TIS in an 88: 5: 5: 2 volume / volume / weight / volume ratio) is used to cleave the peptide from the resin and achieve side chain deprotection. After 4 h, the cleavage solution is collected and concentrated to dryness. Et2O is added to the residue to precipitate the crude peptide, which is collected. A C4, C8 or C18 preparative column is used to isolate the peptides, and purity is determined using a C4, C8 or C18 analytical column. The solvents (A = 0.11 TFA / water and B = 0.11 TFA / CH3CN) are introduced into the analytical column at a flow rate of 1.0 ml / min and in the preparative column at 20 ml / min.

Ri- [S-L] n conjugated with a maleimide functional group is linked to Cys- (B) r (His) t-C by 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 an azide functional group is bound to pGly- (B) r (His) t-C by azide-alkyne "click chemistry" with a copper (I) catalyst 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):

rn cur

Rx is chitosan;

S is a direct link;

L is chosen from

A is chosen from Cys and propargyl-Gly;

B is selected from Gly, β-Ala and Phe-Ala;

C is chosen between β-Ala and nothing;

r is an integer between 0 and 5;

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

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

Scheme 2

Chitosan is bound to maleimide parts to form R1- [S-L] n (chitosan a) or azide parts to form R1- [S-L] n (chitosan b) by a coupling reaction between NHS ester of 3 (maleimido) propionic acid or NHS ester of azidoacetic acid in PBS or MES buffer, in water / dimethyl sulfoxide (DMSO) (1: 1 volume / volume). 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.

See example 1.

Chi shear a is conjugated with

by maleimide-thiol chemistry simply by mixing chitosan a and Cys- (B) r (His) t-C in 1% (volume / volume) acetic acid solution according to the procedures described by Chan P et al., 2007 and Malhotra M et al., 2013.

Chitosan b is conjugated with pGly- (B) r (His) S-C by azide-alkyne "click chemistry" with copper (I) catalyst according to the procedures described by Jung S. et al., 2013.

Example 3

Preparation of nanoparticles

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

(A) Preparation of PLGA, PLA, PGA nanoparticles Nanoparticles of PLGA, PLA, PGA are obtained using an extraction procedure with an oil-in-water emulsion solvent (Mariano N. et al., 2014) The emulsion is prepared dissolving R1- [S-L-A- (B) r- (His) t-C] n (where Ri is PLGA or PLA or PGA) alone or in combination with a commercial PLGA or PLA or PGA (MW variable in the range 15-50 KDa ), in chloroform: methanol (3: 1); this solution is called phase 1. PLGA is, for example, Resomer® RG 502 H, molecular weight 7,000-17,000 Da, product number 71S8S7, Sigma-Aldrich.

Phase 2 consists of an aqueous solution of polyvinyl alcohol (PVA, Mw 31,000-50,000 Da, hydrolyzed to 98-991, product number 363138, Sigma-Aldrich). Phase 1 is added dropwise to phase 2 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 size of the nanoparticles.

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

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

(B) Preparation of chitosan nanoparticles

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

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

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

Briefly, 0.2 ml of the TPP crosslinker (0.625 mg / ml or 0.42 mg / ml) is added dropwise to 1 ml of the aqueous solution containing the chitosan compound- [L-A- (B) r- (His ) t-C] n 1-5 mg / ml under magnetic stirring for 10 min at room temperature.

The size of the particles formed is determined using dynamic light scattering (DLS).

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

(D) Preparation of hyaluronic acid (HA) nanoparticles

To obtain HA-containing nanoparticles by the ionic gelation technique, it is necessary to couple them 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 compound R1- [S-L-A- (B) r- (His) t-C] n, in which R1 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). The nanoparticles are obtained instantly by adding 1.5 ml of solution B (pH = 8-9) to a 3 ml solution A (pH = 3), under magnetic stirring at room temperature.

Example 4

Preparation of scaffolds

Porous scaffolds based on alginate and chitosan derivatives are prepared by a freeze-gelation process (H-Ming-Hua H et al., 2004).

The compound Ri- [S-L-A- (B) r- (His) t-C] n in which Ri is chitosan is dissolved in aqueous solution of acetic acid (1 M) to form a 21 by weight polymer solution. The polymer solution is placed in a mold and frozen at -20 ° C. The frozen solution is dipped in an aqueous solution of NaOH / ethanol (pre-cooled to -20 ° C) to adjust its pH to allow the chitosan to gel.

The compound in which Ri is alginate is dissolved in deionized water to form a 2% by weight solution, which is then frozen at -20 ° C and then dipped in an aqueous solution of CaCl2 and ethanol at -20 ° C to induce the gelling of alginate. Room temperature drying is performed after gelation to obtain scaffolds.

PLGA-based porous scaffolds are prepared with the porogenic leaching technique (Holy CE et al-, 1999). The compound in which Ri is PLGA, alone or in combination with a commercial PLGA (75 mg / ml), is dissolved in anhydrous DMSO and added to a template containing glucose as a porogen. 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 new imidazole-comprising compounds of the present invention are suitable for use as contrast agents and pH sensing agents in the FFC-MRI technique, the present inventors have collected some experimental data using (i) a commercial polyhistidine (poly-His) and (ii) a compound of formula (I) as QP-generating compounds.

Figure 3 shows a schematic representation of poly-His.

Poli-His shows a characteristic relaxation peak at 1.38 0.3 MHz due to the resonance frequency of the nuclear quadrupole <14> N of the imidazole groups present in the polymer chains. There are two other resonant frequencies from the imidazole group that generate wide peaks at 0.721 MHz and 0.647 MHz, respectively, but these cannot be used because they overlap with the 0.7 MHz QP resulting from tissue protein amide groups.

Figure 4 shows the commercially available 1 / Ti NMRD profile of poly-His (Sigma Aldrich catalog number P9386) (MW = 15,600 Da, dissolved in water, 301 w / w), showing its characteristic QPs with respect to a NMRD 1 / Ti profile acquired on a liver tissue sample. The QP at 1.38 ± 0.3 MHz deriving from imidazole is well detectable and distinguishable from the QPs generated by the background tissue. The increase in relaxation calculated at the maximum peak with respect to the background profile is about 601 which corresponds to an increase in relaxation of about 5 s-1

Figure 5 shows 1 / T1 NMRD profiles acquired on poly-His at different concentrations. Although the QP at 1.38 ± 0.3 MHz is still detectable at the minimum tested concentration (5 mg / ml), 14 mg / ml represents the minimum concentration that gives a significant increase in relaxation (201) to be well detectable even in presence of a consistent background on the part of the fabric.

Since pH controls the solid / liquid physical state of poly-His and since <14> N-QPs are observed only for immobilized systems, the pH of the microenvironment in which the compound comprising imidazole is present can be evaluated based on the changes in the intensity of the QP of the imidazole. There is growing interest in the development of pH sensing agents capable of non-invasively measuring the pH of tissues in vivo, particularly in oncology applications. In fact, the measurement of the pH of a tumor tissue has a marked prognostic importance as the presence of slightly acidic areas (for example pH = 6.6-6.8) surrounding hypoxic and necrotic areas promotes cell migration and metastases .

As described above, the QP intensity of poly-His depends on the pH and its observation is possible only when poly-His is immobilized in a solid-like form, i.e. at pH> 6.6. At lower pH the protonation of the imidazole groups (pKa ~ 6.8) increases the water solubility of the polymer with a consequent increase in its mobility and a proportional decrease in the intensity of the QPs (Figure 6). Since the inside of the nanoparticle is accessible to water, the compounds comprising imidazole object of the present invention can be exploited as a pH reporter in the range of interest for the tumor tissue after the construction of an appropriate calibration curve in which the intensity of QP is detected as a function of the measured pH using a standard pH meter.

Compounds comprising imidazole containing imidazole moieties, such as poly-His, may be useful for the design of contrast agents that report local physiological and pathological changes. This task can be tackled by confining the poly-His chains responsible for QPs inside a biocompatible particle while maintaining the conditions suitable 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 to poly (lactic-glycolic) acid (PLGA) nanoparticles. PLGA is approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) in various human drug delivery systems. The polymers are commercially available at different molecular weights and copolymer compositions. Furthermore, the NP interior of PLGA is accessible to water in bulk in an inversely proportional measure to the size of the NPs. A schematic representation of an oligo-His-PLGA NP is shown in Figure 7. To prepare this compound comprising imidazole, an oligo-histidine peptide (His x 15) containing a free thiol group as a terminal group was synthesized and conjugated with Maleimide functionalized PLGA, as described in Materials and Methods. The peptide used in the present example is A- (B) r (His) t-C where A (amino terminus) is Cys, B is Gly, r is equal to 2, t is equal to 15, C is β-Ala, obtaining Like this

(hereinafter referred to as oligo-His). Before its conjugation with PLGA, the NMRD profile of the oligo-His suspension was compared with that obtained from 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 commercial poly-His (approximately 101 fewer).

In the following, a discussion of a preferred embodiment of the present disclosure is provided, in which the compound comprising imidazole of the present disclosure has the following chemical structure:

where x equals 77 and y equals 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 group of appended claims.

NMRD profiles acquired on suspensions containing two different charges of oligo-His-PLGA nanoparticles (at an oligo-His concentration of 5 mg / ml, the lowest concentration tested with commercial poly-His) are shown in Figure 9. In both samples, the QPs of imidazole are more pronounced (+751) than that obtained in the case of commercial poly-His at the same concentration.

MATERIALS AND METHODS

Preparation of oligo-His-PLGA

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

Scheme 3

The phases of the synthesis represented in scheme 3 will now be described in detail.

Synthesis of oligo-His

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, a peptide with specific amino acid sequences has been synthesized, having the above formula A- (B) r (His) t-C, in which A (amino terminus) is Cys, B is Gly, r is equal to 2, t is equal to 15, C is β-Ala and a C-terminal aroma. The peptide (SEQ ID No .: 1) was assembled on a ChemMatrix® (Sigma-Aldrich) H-Rink amide resin (150 mg, 0.4-0.6 mmol / g resin load) using HBTU / HOBt as activation (CEM model Liberty, Synthesizer s / n DP7026DU8276).

Deprotection of Fmoc was performed using 201 volume / volume piperidine in DMF plus 0.1 M HOBt in two stages with an initial treatment of 0.3 min followed by a longer treatment of 3 min (7 ml, 40 W, 75 ° C). The 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 pairing was performed twice using a 5-fold excess of Fmoc-Aa-OH and a slightly less than 5-fold excess of HBTU, a 10-fold excess of DIPEA (for 5 min, 35 W, 75 ° C). Power pulse sequences of 25 W for 4 min were used for the Cys coupling steps. The peptidyl-resin complex was washed with DMF (3 x 5 ml), DCM (3 x 5 ml) and dried. The peptide-resin complex was cleaved using a mixture of TFA / H2O / phenol / TIS (88: 5: 5: 2 volume / volume / weight / volume), at room temperature for 4 h with gentle stirring by rotation. The material was then filtered, washed with a minimal amount of TFA and the cleavage solution collected and concentrated to dryness. Cold Et2O was added to the residue to precipitate the peptide, which was collected and dried. The peptide was obtained in a yield of 501 with a purity of 981 despite the omission of purification by HPLC. HPLC assay conditions: RP-C18, 5% CH3CN in 0.11 TFA in 5 min, 5-201 CH3CN in 0.11 TFA in 10 min, 20-351 CH3CN in 0.11 TFA in 15 min, 35-1001 CH3CN in 0.11 TFA in 3 min.

Column: Waters Atlantis C18, 5 μm, 4.6x150 mm Flow rate: 1 ml / min

Detection: Group of diodes, single quadrupole MS Mobile phase:

A: 0.1% aqueous TFA

B: 0.11 TFA in acetonitrile

Retention time: ~ 11.5 minutes

observed 788, 4;

Maleimide-PEG2-amine synthesis

NHS ester of 3- (maleimido) propionic acid (0.3 g, 1.15 mmol) and TEA (0.24 ml, 1.5 eq) were dissolved in CH3CN (10 ml), followed by the addition of t-Boc-N-amido-PEG2-amine (0.28 g, 1.15 mmol) and the resulting reaction mixture was stirred for 2 h at room temperature. It was then diluted with ethyl acetate and washed three times with water and once with brine. The extract was dried over concentrated anhydrous sodium sulfate under reduced pressure. Flash chromatography on silica gel using ethyl acetate and hexane yielded 0.46 g of the compound. ESI (+) m / s (M + Na) <+> 422.2. (M + H-BQC) <+> 300.2. Subsequent deprotection of N-Boc was performed using TFA: CH2Cl2 (1: 1 volume / volume, 5 ml) for 1 h at room temperature. The solvent was removed under vacuum to obtain the product as a white solid (0.3 g).

HPLC assay conditions: RP-C18, 5% C3CN in 0.1% TFA in 5 min, 5-20% CH3CN in 0.11 TFA in 10 min, 20-351 CH3CN in 0.11 TFA in 15 min, 35 -100% C3⁄4CN in 0.11 TFA in 3 min.

Column: Waters Atlantis C18, 5 μm, 4.6x150 mm Flow rate: 1 mi / <'> min

Detection: Group of diodes, single quadrupole MS Mobile phase:

A: 0.1% aqueous TFA

B: 0.1% TFA in acetonitrile

Retention time: ~ 8.8 minutes

ESI (+): Calc, for C13H21O3O5 m / z [M + H] <+> = 300.1; observed 300.1

<2> H NMR (dmso-d6, 600Mhz) δ 8.00 (t, 5.4 Hz, CO-NH, 1H), 7.80 (s br, -NH2.2H), 7.05 (s, methino maleimide 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).

Synthesis of PLGA-PEG2-Maleimide

PLGA Resomer® RG 502 H with a monomer ratio 50:50 MW = 12,800, 0.5 g, 0.04 mmol) was dissolved in 10 ml of anhydrous CH3CN in a 25 ml flask. 20 μl (0.12 mmol) of DIPEA and HATU (0.017 g, 0.044 mmol) were added to the flask. After 5 min, the Maleimide-PEG2-amine compound (0.013 g, 0.044 mmol) dissolved in CH3CN (1 ml) was added dropwise, then the mixture was stirred at room temperature for 6 h in an N2 atmosphere. The solvent was then removed in vacuo and the residue dissolved with chloroform and washed three times with brine. The crude compound was purified by column chromatography (eluent: CH2Cl2 / CH3OH 98: 2) obtaining a product as a white solid (0.42 g).

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

Conjugation of Oligo-His-PLGA

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) 25βAla peptide (0.034 g, 0.016 mmol) in DMF was added (0.5 ml) and N, N-diisopropylethylamine (DIPEA, 5 μl, 0.03 mmol). The resulting mixture was magnetically stirred at room temperature for 3 h under a nitrogen atmosphere. The desired PLGA-oligoHis was obtained by precipitation from a cold solution of diethyl ether / methanol 1: 1 (10 ml), which was collected, washed with diethyl ether / methanol 1: 1 (10 ml) twice and dried under vacuum (yield, 851). H NMR (dmsod6, 600Mhz) 68.46 (br, 15H, -N = CH-N-), 8.4-8.0 (amide protons), 7.12 (br, 15H, C = CH-N), 5.31-5.01 (m, S4H, CH lactide proton), 4.98-4.85 (m, 156H, CH glycolide proton), 4.57 (m, 15H , Hα protons of His), 3.08-2.99

lactide) (Figure 12).

The <1> H-NMR analysis revealed the disappearance of the peak corresponding to the maleimide protons at 7.03 ppm. We used <1> H-NMR to determine the resulting molar ratio of FLGA and peptide by integration of PLGA

CH) and His (Hα protons). The calculated functionalization confirmed a 1: 1 molar ratio between PLGA and oligo-His. The preparation of oligo-His-PLGA nanoparticles (NPs)

NPs containing oligo-His were obtained by applying the solvent extraction procedure in oil / water (o / w) emulsion (Figure 13). The organic phase was prepared by dissolving oligo-His-PLGA and PLGA Resomer® RG 502 H (1: 1) in chloroform: methanol (3: 1). The aqueous phase was an aqueous solution of polyvinyl alcohol (PVA). PVA is the most commonly used emulsifier for the preparation of NP of PLGA because it allows to obtain particles that are relatively uniform, small in size and easy to redisperse in water. The organic phase was added to the aqueous phase, under sonication. To check the size of the NPs, different PVA concentrations and sonication conditions were tested. The solidification of the NPs was carried out by evaporation of the organic solvent 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 mean hydrodynamic diameters of the oligo-His-PLGA NPs were determined by dynamic light scattering (DLS) measurements and were equal to 150 nm.

Dynamic Light Diffusion (DLS)

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

Acquisition of the NMRD profile

Samples (usually 1 mL volume) were added to a glass tube (10 m diameter x 220 mm length).

The data was measured at 25 ° C on a continuum of magnetic field strength from 0.00024 to 0.47 T (corresponding to the Larmor proton frequency of 0.01-20 MHz) on a stelar fast field cycle relaxometer.

The temperature was controlled by a VTC-91 Stelar 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 of 1 / T1 values of 2%. Measurements Τ1 were performed using Non-Polarized and Pre-Polarized sequences as described by Ferrante and collaborators (Ferrante G et al., 2005) (Figure 1). T1 was determined by the saturation recovery procedure. 16 delay values (τ) between pulses were used. The number of experiments averaged was 2.

<1> H NMR spectra

The <1> H NMR spectra were acquired in deuterated solvent (dmso-d6) at 14 T on a Bruker Avance 600 spectrometer equipped with a 5 mm BBI or TXI probe with inverse Z gradient.

Claims (10)

CLAIMS 1. Magnetic resonance imaging (MRI) contrast agent including a compound comprising imidazole, in which the compound comprising imidazole comprises at least 5 imidazole moieties, and in which the imidazole moieties are in an immobilized state, corresponding to a reorientation time in the 'order of tens of nanoseconds (ns), so that, in use, the longitudinal relaxation rate of the water proton at 1.38 ± 0.3 MHz (Larmor proton frequency) increases by at least 10% of the pre -contrast. MRI contrast agent according to claim 1, wherein the compound comprising imidazole has formula (I): in which R1 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-O- (CH2) 2-O- and - (CH2) 2-O- (CH2) 2-O- (CH2) 2-O-; L is a linker chosen from A is chosen from Cys and propargyl-Gly; B is selected from Gly, β-Ala and Phe-Ala; C is chosen between β-Ala and nothing; r is an integer between 0 and 5; t is an integer equal to or greater than 5, preferably between 5 and 150, more preferably between 10 and 50; n is an integer equal to or greater than 1. Contrast agent for MRI according to claim 2, wherein R1 is selected from polyglycolic acid (PGA), polylactic acid (PLA) and poly (lactic-co-glycolic) acid (PLGA); S is selected from - (CH2) 2-O- (CH2) 2-O- and - (CH2) 2-O- (CH2) 2-O- (CH2) 2-O-; and n is an integer equal to 1. MRI contrast agent according to claim 2 or 3, wherein R1 is poly (lactic-co-glycolic) PLGA acid; S is - (CH2) 2-O- (CH2) 2-O-; L is A is Cys; B is Gly; C is β-Ala; r is equal to 2; t is an integer between 10 and 50. n is equal to 1. MRI contrast agent according to any one of claims 2 to 4, wherein R1 is poly (lactic-co-glycolic) PLGA acid; S is - (CH2) 2-O- (CH2) 2-O-; L is A is propargyl-Gly; B is Gly; C is β-Ala; r is equal to 2; t is equal to 15; n is equal to 1. 6. MRI contrast agent according to claim 2, wherein R1 is selected from alginate and hyaluronic acid; S is selected from - (CH2) 2-O- (CH2) 2-O- and - (CH2) 2-O- (CH2) 2-O- (CH2) 2-O-; and n is an integer greater than 1, preferably between 2 and 100. 7. MRI contrast agent according to claim 2, wherein R1 is chitosan; S is a direct link; n is an integer greater than 1, preferably between 2 and 200. MRI contrast agent according to any one of the preceding claims, wherein the compound comprising imidazole is in the form of a nanoparticle or scaffold. A pharmaceutical composition comprising an MRI contrast agent according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier. 10. Pharmaceutical composition according to claim 9 for use as a contrast agent and / or as a pH detection agent in the rapid field cycle magnetic resonance imaging (MRI) technique (FFC).