WO2009087253A1 - Tubular nanostructured materials having anisotropic magnetic properties, method for obtaining same and use thereof - Google Patents

Abstract

The invention relates to a contrast agent comprising a tubularly arranged nanostructured material soluble in aqueous solutions, having anisotropic properties and containing paramagnetic metals or superparamagnetic or ferromagnetic structures, which can be used in the production of an NMR diagnostic pharmaceutical composition for determining the laminar or turbulent flow and/or the rheological properties of fluids flowing through tubular pipes.

Description

 NUBESTRUCTURED TUBULAR MATERIALS WITH

ANISOTROPIC MAGNETIC PROPERTIES, PROCEDURE

OF OBTAINING AND ITS APPLICATIONS

SECTOR OF THE TECHNIQUE

The present invention can be applied in many different fields such as biomedicine, in the imaging of Magnetic Resonance Imaging (MRI) methods for the clinical diagnosis of neurodegenerative and vascular diseases, as for example in the early diagnosis of atherosclerosis. Likewise, it can be applied in fields such as physics or engineering, determining laminar or turbulent flow and, in general, the Theological properties of fluids that circulate in tubular conduits.

STATE OF THE TECHNIQUE

Magnetic Resonance Imaging (MRI) is one of the most powerful tools in current clinical diagnosis due mainly to its non-invasive nature (V. M. Runge, K. L. Nelson. In

Magnetic Resonance Imaging; D. D. Stark, W. G. Bradley, Ed .; Mosby: St. Louis, 1999; VoI 1, pp. 257-275).

It is a technique that allows you to obtain Magnetic Resonance Imaging (MRI) of a certain object by introducing it into a static magnetic field. In this way the three coordinates of the space of each of the volume elements of said object are obtained, by means of the application of three orthogonal magnetic gradients and one or more radiofrequency selective pulses. Thus, each element of volume of the object is defined spatially by its three coordinates x, y, z, imposed by the intensity of the magnetic gradients applied in each direction. The result obtained represents the distribution of water molecules in the object. Each element of the image (pixel) has a characteristic intensity that is determined by the amount of water in the original volume element (voxel), its longitudinal (n) and transverse (r ₂ ) magnetic relaxation properties and the dynamics of water flow through the object.

Since the water content of the tissues is very similar, the main determinants of the contrast between two points of an image

RM are the longitudinal (Ti) and transverse (T ₂ ) relaxation times of water protons (C. Lauterbur, Nature, 1973, 242, 190; P. Mansfield, A.

A. Maudsley, British J. Radiol. 1977, 50, 188).

MRI is a technique in constant development; each time more efficient image sequences are obtained and new contrast agents (ACs) are used that allow to increase the quality, resolution and specificity of the images obtained.

ACs significantly reduce relaxation times in the tissues where they are distributed. In many cases, the use of these agents represents a great improvement in the clinical diagnosis, in terms of high specificity, better characterization of the tissues, reduction of artifacts in the image and increase of their functional information.

The ACs most used in biomedicine are paramagnetic chelates of gadolinium, Gd (III) (H. Gries, Top. Curr. Chem 2002, 221, 1; Tóth, E .; HeIm, L .; Merbach, AE In The Chemistry of Contrast Agents in Medical Magnetic Resonance Imaging; Merbach, AE, Toth, E., Ed .; John Wiley & Sons, Ltd .: Chichester, 2001, pp. 45-119). The effectiveness of this family of compounds to act as ACs is valued, in the first place, by their relajativity, ri ₍₂₎ , that is, by the net increase they induce in the relaxation of water protons. Of this family of compounds, the most commonly used in diagnostic imaging are gadolinium complexes derived from diethylenetriaminepentaacetic acid (DTPA) and 1, 4,7,10-tetraaza-1, 4,7,10-cyclododecanotetraacetic acid (DOTA) ( S. Aime, S. Geninati Crich, E. Gianolio, GB Giovenzana, L. Tei, E. Terrain,

Coord. Chem. Rev. 2006, 250, 1562; M. Woods, DE Woessner, AD Sherry, Chem. Soc. Rev. 2006, 35, 500; Caravan, P .; Ellison, JJ; McMurry, TJ; Lauffer, RB Chem. Rev. 1999, 99, 2293; Lauffer, RB Chem. Rev. 1987, 87, 901 (Figure 6).

In the last 15 years, numerous articles have been published aimed at the study of the structure and dynamics of the complexes of Gd (III) (P. Caravan, Chem. Soc. Rev. 2006, 35, 512-523), providing a great advance in the understanding of the structural, dynamic and electronic parameters that determine the relaxivity of these complexes. Currently, research in this field is focused on the design and synthesis of new chelating agents that maintain high affinity for the corresponding paramagnetic metal, as well as improved longitudinal (ri) or transverse (r2) relaxing properties.

Recently, a new series of pyrazolylethyldiethylenetriaminetetraacetic acids have been described whose Gd (III) complexes have superior relaxivity properties than those previously used (P. Ballesteros García, E. Pérez Mayoral, Heterocyclic ligands and their gadolinium complexes ( III) with biomedical applications, PCT Int. Appl., (2006) WO 2006051142; P. Ballesteros García, E. Pérez Mayoral, Complexing agents derived from pyrazolylethyldiethylenetriaminetetraacetic acids. Gadolinium complexes (III) with applications in clinical magnetic resonance diagnosis , P20050245; P. Ballesteros García, E. Pérez Mayoral, 1-Pyrazolylethyl-1, 4,7,10-tetraazacyclododecane-4,7,10-triacetic acids. Application of its gadolinium (III) complexes in clinical diagnosis, PCT Int. Appl., (2006), WO 2006051142).

However, all ACs used so far have isotropic magnetic properties, so they do not allow to detect the physical characteristics of blood flow since they are not able to provide directional images and / or differentiate laminar or turbulent flow. Thus, the relaxivity observed in the MRI is independent of the spatial orientation of the agent, making it impossible to distinguish the orientation and the direction of the flow of the molecule that causes the change in observed relaxivity (Figure 1A), which makes it difficult to detect the neurodegenerative and / or vascular pathologies that modify it, such as, for example, atherosclerosis. As explained above, the ACs that are used so far in Magnetic Resonance (MR) do not allow to distinguish the orientation and direction of the flow of the molecule that causes the change in relaxivity that is observed in the MR image. This indicates that the relaxivity is independent of the direction of the magnetic field, the same intensity of the RM signal being observed in the three orthogonal planes of the image. This means that the ACs used so far do not serve to detect the physical characteristics of the flow. This is a great inconvenience when diagnosing some serious diseases early, such as neurodegenerative and / or vascular diseases, and more specifically, atherosclerosis.

Atherosclerosis is a systemic disease characterized by the accumulation of lipid plaques on the walls of blood vessels, constituting the main cause of mortality in developed countries worldwide. In order to be able to make an early diagnosis of this disease, together with the follow-up of its therapies, it is necessary to develop new ACs that allow distinguishing laminar flow from turbulence in the vascular system.

DESCRIPTION OF THE INVENTION

Brief description

An aspect of the invention constitutes a contrast agent useful for the preparation of a pharmaceutical NMR diagnostic composition, hereinafter referred to as a contrast agent of the invention, based on a nanostructured material organized in a tubular form, soluble in aqueous solutions, with properties anisotropic and with a content of paramagnetic metals or superparamagnetic or ferromagnetic structures, preferably Ti, Fe, Co and Ni.

A particular aspect of the invention constitutes the contrast agent of the invention in which the nanostructured material is a nanotube of a material belonging, by way of illustration and without limiting the scope of the invention, to the following group: carbon (NTCs) , silicon, or other materials of tubular nature and their derivatives.

Another object of the present invention constitutes a new method to obtain and purify, by osmotic exchange, the nanostructured contrast agent of the invention by using dialysis membranes submerged in deionized water, and periodically renewed until changes of pH, substantially simplifying the separation of nanostructured materials, by-products.

Thus, another aspect of the invention constitutes a pharmaceutical composition useful for the diagnosis of NMR, hereinafter pharmaceutical composition of the invention, constituted by a composition comprising at least one contrast agent of the invention and a pharmaceutically acceptable carrier, either an agent of a single type or a mixture of several different agents of the invention. Finally, another aspect of the present invention constitutes the use of the pharmaceutical diagnostic composition of the invention, hereinafter used of the invention, in a procedure by NMR for determining a laminar or turbulent flow and / or the Theological properties of fluids circulating in tubular conduits.

Detailed description

The nanostructured contrast agent of the present invention solves the problems described above, since the intensity of the RM signal provided by the nanostructured contrast agents with tubular geometry of the present invention depends on the average orientation of the ACs with respect to the field magnetic applied (Figure 1 B). The agents of the present invention spontaneously align with the magnetic field, causing a maximum of relaxivity in the direction of the B ₀ field, and a minimum, in the perpendicular plane. This makes it possible to determine the laminar or turbulent nature of the flow through a capillary, by means of measures of magnetic relaxivity by MRI in nanotube solutions, as indicated in Figure 1. The magnetization of a solution of nanotubes circulating in laminar flow through a capillary is the result of the vector product of the magnetic field vector by that of the flow vector. Since both maintain their magnitude and direction over time, the resultant is a single constant magnetization vector during the RM acquisition time, which originates an RM signal of defined relaxivity. However, when the nanotube solution circulates in turbulent flow, the resulting magnetization vector is smaller. This is due to the fact that it is the vector product of a vector with constant direction and modulus (the magnetic field vector Bo), and a constant modulus vector (the magnetization of the nanotube) and variable direction, (the orientation of the nanotube with respect to the flow ). In this case, the resulting magnetization is lower than that of the laminar flow, due to the cancellation of the magnetic moments of those nanotubes that rotate and move in the opposite direction, due to the turbulence.

Thus, one aspect of the invention constitutes a contrast agent useful for the preparation of a pharmaceutical composition for NMR diagnosis, hereinafter referred to as a contrast agent of the invention, based on a nanostructured material organized in a tubular form, soluble in aqueous solutions, with anisotropic properties and in a content of paramagnetic metals or superparamagnetic or ferromagnetic structures, preferably Ti, Fe, Co and Ni.

A particular aspect of the invention constitutes the contrast agent of the invention in which the nanostructured material is a nanotube of a material belonging, by way of illustration and without limiting the scope of the invention, to the following group: carbon (NTCs), silicon, or other materials of tubular nature and their derivatives.

A particular embodiment of the invention constitutes a contrast agent of the invention in which the paramagnetic metals are in the range between 0-1.5% Ti, 0-1% Fe, 0-3% Co , and 0-1% Ni (% by weight). Thus, a more particular embodiment of the invention constitutes the contrast agent of the invention in which the paramagnetic metals are in a range between 0-1.5% Ti, 0-1% Fe, 0-3 % of Co, and 0-1% of Ni (% by weight). Another object of the present invention constitutes a new method to obtain and purify, by osmotic exchange, the nanostructured contrast agent of the invention by using dialysis membranes submerged in deionized water, and periodically renewed until changes of pH, substantially simplifying the separation of nanostructured materials, by-products.

The contrast agent of the invention, which has been shown to be soluble in aqueous solutions, can be used either in the pure form of one type of agent or as a mixture of several of them to make a pharmaceutical composition together with solutions, suspensions or pharmaceutical preparations, preferably of paramagnetic complexes, superparamagnetic or ferromagnetic structures, which act as pharmaceutically acceptable carriers.

Thus, another aspect of the invention constitutes a pharmaceutical composition useful for the diagnosis of NMR, hereinafter pharmaceutical composition of the invention, constituted by a composition comprising at least one contrast agent of the invention and a pharmaceutically acceptable carrier, either an agent of a single type or a mixture of several different agents of the invention. Finally, another aspect of the present invention constitutes the use of the pharmaceutical diagnostic composition of the invention, in The use of the invention, in an NMR procedure for determining a laminar or turbulent flow and / or the Theological properties of fluids circulating in tubular conduits.

Another particular aspect is the use of the invention in which the procedure consists in the determination of laminar flow and perfusion of biological fluids in order to perform a clinical diagnosis in normal and pathological macro- and microvasculature, and in particular in the characterization of the tumor neovasculature and the cerebral and cardiac microvasculatures in neurodegenerative and cardiovascular diseases, more specifically, in the detection of atherosclerotic plaques in the vascular system, by means of the differentiation of the laminar or turbulent blood flow, which characterize the microcirculation in the normal or atherogenic vasculature (Figure 2) and the detection of lesions in organs (brain, heart, liver, kidney, etc.) affected by changes in the nature of laminar or turbulent blood flow.

Another particular aspect is the use of the invention in which the diagnostic procedure by NMR determines the directionality of the anisotropic magnetic relaxation, preferably by obtaining orthogonal MR images acquired in the direction parallel and perpendicular to the magnetic field (B ₀ ) .

DESCRIPTION OF THE FIGURES

Figure 1. Illustrative scheme of orthogonal MRI images derived from the use of conventional isotropic contrast agents (A) or of the new anisotropic contrast agents described in the present invention. (B).

Figure 2. Distinction between laminar flow, characteristic of normal capillaries (upper panel) and turbulent flow, present in capillaries with vulnerable atherogenic plates (lower panel), by MRI using nanotubes with anisotropic relaxivity described in the present invention. Figure 3. Mannequins containing tubes with aqueous solutions of NTCs and water on a plasticine platform, used for the acquisition of standardized orthogonal RM images of anisotropic relativity. Figure 4. TEM image of the NTCs used in the present invention. Figure 5. A) Normalized image (perpendicular to B ₀ ), B) Normalized image (parallel to Bo) and C) Histogram of intensities of images A) and B) normalized with respect to water. Figure 6. Gadolinium complexes derived from diethylenetriaminepentaacetic acid (DTPA) and 1, 4,7, 10-tetraaza-1, 4, 7,10-cyclododecanotetraacetic acid (DOTA).

EXAMPLES OF EMBODIMENT Example 1. Obtaining and characterizing the nanotubes Commercial NTCs, single wall, 0.7-1, 2 nm in diameter and 2-20 m in length, synthesized by the CVD method (deposition method) are used chemical in vapor phase) and with an enrichment in NTCs of 10-40%. Commercial samples contain the following percentage of metals (% by weight): 1,27% Ti; 0.07% Fe; 2.25% Co and 0.75% Ni, identified by R-X by Total Reflection (TXRF).

NTCs are oxidized by the procedure described by Bourlinos et al. (Bourlinos, AB, Georgakilas, V., Tzitzios, V., Boukos, N., Herrera, R., Giannelis, EP Small 2006, 2, 1188) with variations in their isolation and purification, which do not affect its properties as described below.

A suspension of commercial NTCs (200 mg) in HNO3 (25 mL) is heated at reflux for 24 h. The reaction mixture is allowed to cool and diluted with deionized water (100 mL). Then, the suspension is centrifuged (3000 rpm for 10 min), and the resulting precipitate is resuspended in deionized water (50 mL), and introduced into a dialysis membrane (Spectra / Per 45 mm wide x 10 cm long, 3500 Dalton) that is submerged in deionized water. Water is renewed periodically until it reaches a pH ~ 5.5. Then, the suspension is centrifuged under the aforementioned conditions and the NTCs are dried in a desiccator. The CNTs used have been characterized by:

- Transmission Electron Microscopy (TEM), using a JEOL JEM 1010 (100 KV) transmission electron microscope. (see Figure 4)

- Fourier transform infrared (FT-IR), using a FT-IR Bruker vector 22 device with ATR accessory. The IR of the nanotubes of the present invention reveals two characteristic bands at 3304 and 1715 cm ^"

one

- Thermogravimetry (TG), using a thermal analysis equipment (TG-DTA) TA-SDT Q-600. The thermogravimetric profile obtained confirms a weight loss of approximately 10% in the temperature range between 200-300 ⁰ C, attributable to the loss of CO ₂ .

- R-X Fluorescence by Total Reflection (TXRF), using a TXRF EXTRA-II, Rich & Seifert (Germany) spectrometer. The oxidized NTCs of the present invention contain the following percentage of metals (% by weight): 1, 8% Co and 0.7% Ni.

Example 2. Magnetic Resonance (MR) methods.

To obtain MR images, dummies (eppendorf Safe-Lock microtubes of 1.5 mL, 38 mm high and 10 mm internal diameter) are used, inserted in a plasticine block (60 x 30 mm ² and 25 mm high ), containing tubes with water and with aqueous solutions of paramagnetic CNTs (2 mg in 1 mL) (Figure 3). RM images are acquired on a Bruker Pharmascan spectrometer (7.0 Tesla horizontal magnet / 16 cm in diameter) connected to a Hewlett-Packard console (Linux; Bruker Medical Gmbh, Ettlingen, Germany). The images are acquired with weighting in Ti, using an image sequence SPIN-ECO (MTX = 256 x 256, TR = 500 ms, TE = 10.6 ms; section width = 1 mm, AV = 3, FOV = 3.8 x 3.8 cm), making cuts of the mannequins in a parallel direction and perpendicular to the magnetic field (B ₀ ). Image processing and quantification are carried out using software developed in the inventors' laboratory with the MATLAB 7.4.0 (R2007a) program (The MathWorks, Inc., copyright 1984-2007; http: // www.mathworks .com). Figure 5 shows the normalized orthogonal images of the dummies studied and their corresponding histograms of signal intensity versus the repetition frequency of the signal intensity.

Figure 5 illustrates in its upper panels, how the intensity of the RM image, normalized with respect to water, obtained in the direction parallel to the magnetic field (upper right panel) is much higher than the intensity of the image obtained in the perpendicular direction (upper left panel). In fact, the intensity histograms show that all the pixels of the upper right image (in red) have an intensity greater than the pixels of the upper left image (in blue).

In short, these results show that the intensity of the RM image, using the anisotropic paramagnetic materials of the present invention, depends on the orientation of the cut in the image, being superior in the cuts parallel to the static magnetic field, than in the perpendicular cuts the same. These data indicate that the magnetically labeled nanotubes are oriented with the magnetic field as if they were the needle of a compass, marking the direction of the field with greater intensity.

Claims

1.- Contrast agent useful for the preparation of a pharmaceutical NMR diagnostic composition characterized in that it is a nanostructured material organized in a tubular form, soluble in aqueous solutions, with anisotropic properties and because it contains paramagnetic metals or superparamagnetic or ferromagnetic structures, preferably Ti , Faith, Co and Ni 2. Contrast agent according to claim 1 characterized in that the nanostructured material is a nanotube of a material belonging to the following group: carbon (NTCs), silicon, or other materials of tubular nature and its derivatives. 3. Contrast agent according to claim 1 characterized in that the paramagnetic metals are in the range between 0-1, 5% of Ti, 0-1% of Fe, 0-3% of Co, and 0-1% of Ni (% by weight). 4. Contrast agent according to claim 1 characterized in that it is a carbon nanotube with a percentage, by weight, of metals comprised in the range 0-3% for Co and which is preferably 1, 8%, and in the range 0-1% for Ni, preferably 0.7%. 5. Method for obtaining and purifying a contrast agent according to claims 1 to 4, characterized in that it is carried out by osmotic exchange, by using dialysis membranes immersed in deionized water, and periodically renewed until changes are not observed. pH 6. Pharmaceutical composition useful for the diagnosis of NMR characterized in that it comprises at least one contrast agent of the invention and a pharmaceutically acceptable carrier, either an agent of a single type or a mixture of several different agents according to claims 1 to Ia Four. 7.- Use of the pharmaceutical diagnostic composition of the invention in an NMR procedure for determining a laminar or turbulent flow and / or the Theological properties of fluids circulating in tubular conduits. 8. Use according to claim 7, characterized in that the procedure consists in determining the laminar flow and perfusion of biological fluids in order to perform a clinical diagnosis in normal and pathological macro- and microvasculature, and in particular in the characterization of the neovasculature tumor and cerebral and cardiac microvasculatures in neurodegenerative and cardiovascular diseases. 9. Use according to claim 7 characterized in that the cardiovascular disease consists in the detection of atherosclerotic plaques in the vascular system, by means of the differentiation of laminar or turbulent blood flow, which characterize the microcirculation in the normal or atherogenic vasculature and the detection of lesions in organs (brain, heart, liver, kidney, etc.) affected by changes in the nature of laminar or turbulent blood flow. 10. Use according to claim 7 characterized in that the diagnostic procedure by NMR determines the directionality of the anisotropic magnetic relaxation, preferably by obtaining orthogonal MR images acquired in the direction parallel and perpendicular to the magnetic field (B ₀ ).