WO2012113733A1 - Nanoparticules en tant qu'agent de contraste d'irm pour le diagnostic du carcinome hépatocellulaire - Google Patents

Nanoparticules en tant qu'agent de contraste d'irm pour le diagnostic du carcinome hépatocellulaire Download PDF

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WO2012113733A1
WO2012113733A1 PCT/EP2012/052811 EP2012052811W WO2012113733A1 WO 2012113733 A1 WO2012113733 A1 WO 2012113733A1 EP 2012052811 W EP2012052811 W EP 2012052811W WO 2012113733 A1 WO2012113733 A1 WO 2012113733A1
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nanoparticles
nanoparticle
dtpa
contrast agent
hcc
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PCT/EP2012/052811
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German (de)
English (en)
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Jörg KREUTER
Karsten ULBRICH
Klaus Langer
Thomas Knobloch
Albrecht Piiper
Verena KÖBERLE
Hüdayi KORKUSUZ
Thomas J. VOGEL
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Johann Wolfgang Goethe-Universität, Frankfurt Am Main
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
    • A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
    • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
    • A61K49/1878—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles the nanoparticle having a magnetically inert core and a (super)(para)magnetic coating
    • A61K49/1881—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles the nanoparticle having a magnetically inert core and a (super)(para)magnetic coating wherein the coating consists of chelates, i.e. chelating group complexing a (super)(para)magnetic ion, bound to the surface

Definitions

  • the present invention relates to nanoparticles comprising a gadolinium compound and / or incorporated perfluorooctylbromide for use as contrast agents in a magnetic resonance imaging (MRI) based diagnosis of liver diseases, in particular hepatocellular carcinoma (HCC).
  • MRI magnetic resonance imaging
  • HCC hepatocellular carcinoma
  • the PLGA and HSA nanoparticles according to the invention were loaded with a gadolinium compound (Gd-DTPA) and could be used successfully for improved contrasting in the MRI of HCC.
  • the invention describes novel contrast agents comprising the nanoparticles according to the invention, in particular for use in the diagnosis, monitoring and / or early detection of HCC.
  • a method for diagnosis, monitoring and / or early detection of HCC is provided.
  • HCC hepatocellular carcinoma
  • HCC liver transplantation or resection. While the transplantation can be performed in BCLC stages 0, A and possibly B, tumor resection only makes sense in stage 0. The remaining, higher grade tumor stages can only be treated by local ablation procedures (LITT, TACE) or systemic therapy palliatively (chemoradiation with sorafenib). The early diagnosis is therefore crucial for a successful therapy.
  • HCC Surveillance the semi-annual ultrasound examination and the determination of the tumor marker ⁇ -fetoprotein has been established (Greten and Manns, 2008) .This usually results but only from a tumor diameter of about 1.5 cm to the diagnosis. Serum levels of ⁇ -fetoprotein, however, are inappropriate for diagnosis because of their low sensitivity and specificity (Daniele et al, 2004).
  • HCC High-risk patients for the development of HCC
  • HCC Patients with liver cirrhosis, as well as the diagnosis of suspicious nodules are central to a significant improvement in the prognosis of HCC patients.
  • the most reliable HCC diagnostic imaging enables dynamic Contrast Magnetic Resonance Imaging (MRI) and Contrast Computed Tomography.
  • MRI Magnetic Resonance Imaging
  • Contrast Computed Tomography In these studies, HCCs show a characteristic arterial venous and venous flow behavior due to the supply via the A. hepatica, which is utilized by the low-molecular contrast media (Magnevist®, Primovist®).
  • Tumor vessels have an increased permeability, which extends in particular to macromolecules such as nanoparticles. Since lymphatic drainage in the tumor tissue is also reduced, macromolecular substances injected into the blood circulation, such as nanoparticles but not low molecular weight substances, can deposit selectively in the tumor tissue.
  • Magnetic resonance imaging is an imaging technique that is used primarily in medical diagnostics to represent the structure and function of tissues and organs in the body. Magnetic resonance imaging is based on very strong magnetic Fields and electromagnetic alternating fields in the radio frequency range, with which certain nuclei (usually the hydrogen nuclei / protons) are excited resonantly in the body, which then induce electrical signals in the receiver circuit. Contrast agents serve to improve the representation of structures and functions of tissues / organs in MRI. In magnetic resonance imaging, gadolinium chelates are primarily used as contrast agents, which due to the paramagnetic property of the gadolinium atom lead to a shortening of the relaxation times in the vicinity of the contrast agent and thus to a brighter (more signal rich) representation of structures.
  • Nanoparticles made from the polymers human serum albumin (HSA) or poly (D, L-lactic-co-glycolic) acid (PLGA) are well tolerated. There is no unwanted long-term accumulation of these carrier systems in the body, since the nanoparticles are broken down by the body.
  • the functional groups present on the particle surface allow conjugation with different ligands for specific targeting.
  • HSA Human serum albumin
  • PLGA-based microparticles As a contrast substance, Gd-DTPA was included in the PLGA microparticles with a diameter of 900-1800 nm. To test the thus obtained contrast agent, a suspension of agarose and the microparticles were analyzed. No analyzes were performed on living tissue or even mouse models (Doiron AL et al., 2009).
  • nanoparticles based on an iron oxide core containing a specific tumor binding substance (chlorotoxin), a fluorophore and a biopolymer has been found to be advantageous.
  • chlorotoxin chlorotoxin
  • fluorophore a fluorophore
  • biopolymer a specific tumor binding substance
  • the object of the invention to provide new possibilities for the diagnosis, prognosis and in particular for the early detection of hepatocellular carcinoma (HCC) as well as for the monitoring of a therapy of such a disease.
  • the technique of magnetic resonance imaging in the diagnosis of HCC should be improved.
  • the present invention is intended to offer a novel MRI contrast agent, which exceeds the contrast agent currently available for imaging HCC in MRI in terms of specificity and sensitivity. This allows early detection of even small HCCs and also allows the differentiation of HCC and cirrhotic regenerate nodules and other benign changes.
  • nanoparticles which are composed of a matrix of human serum albumin (HSA) or PLGA are preferred.
  • paramagnetic species such as free radicals (eg, stable nitrooxides), as well as elements of the transition metals, lanthanides, and actinides, which, if desired, are covalently or noncovalently attached to complexing agents (chelators) or amino acids containing macromolecules can be bound.
  • elements selected from the group consisting of Gd (III), Mn (II), Cu (II), Cr (III), Fe (II), Fe (III), Co (II), Er (II), Ni (II), Eu (III) and Dy (III).
  • Particularly preferred elements are Gd (III), Mn (II), Cu (II), Fe (II), Fe (III), Eu (III) and Dy (III), especially Mn (II) and Gd (III).
  • the nanoparticle according to the invention comprises a paramagnetic substance as compound of an element selected from the group of lanthanides. It is particularly preferred that the lanthanoid compound is a gadolinium compound.
  • Preferred chelators of the present invention include: acetylacetone (acac), ethylenediamine (s), 2- (2-aminoethylamino) ethanol (AEEA), diethylenetriamine (diene), imino-diacetate (ida), triethylenetetramine (triene), triaminotriethylamine, nitrilotriacetate ( nta), ethylenediaminotriacetate (ted), ethylenediaminetetraacetate (edta), diethylenetriamine pentaacetate (DTPA) 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetate (DOTA), oxalate (ox), tartrate (tart ), Citrate (cit), dimethylglyoxime (dmg), 8-hydroxyquinoline, 2,2'-bipyridine (bpy) and
  • the present invention relates to a nanoparticle wherein the gadolinium compound is a complex of gadolinium and a chelator, in particular wherein the chelator is selected from the group consisting of diethylenetriamine pentaacetic acid (DTPA) and 1,4,7,10-tetraazacyclododecane - 1,4,7,10-tetraacetic acid (DOTA).
  • the gadolinium compound is Gd-DTPA, or derivatives of this compound, such as Gd-EOB-DTPA (Primovist®).
  • a "chelator” in the context of the present invention is to be understood as meaning a ligand having more than one free electron pair which can occupy at least two coordination sites (binding sites) of a central atom, in particular a gadolinium atom , the binding electron pair is provided solely by the ligand.
  • the entire complex of central atom and ligand is called a "chelate complex”.
  • the paramagnetic substance ie in particular the gadolinium compound
  • the paramagnetic substance can be incorporated both in the nanoparticle and / or bound to the surface of the nanoparticle covalently or noncovalently, for example adsorptively.
  • the binding of the gadolinium compound to an HSA particle matrix is accomplished, for example, by the use of n- (3-dimethylaminopropyl) -N-ethylcarbodiimide (EDC).
  • EDC n- (3-dimethylaminopropyl) -N-ethylcarbodiimide
  • the underlying idea is the introduction of a linker that "mediates" a covalent bond between the particle and the gadolinium compound, in which both molecules must react with the linker, and the gadolinium compound (eg, Gd-DTPA) binds to the surface of the PLGA
  • Gd-DTPA gadolinium compound
  • the nanoparticles of the invention further contain incorporated perfluorooctyl bromide and / or Gd-DTPA.
  • nanoparticle of the invention described herein may be further modified, preferably.
  • the nanoparticle is coated with surfactants, or conjugated to a tumor-specific ligand.
  • nanoparticles Modifications of nanoparticles have been shown to be beneficial, particularly in connection with the transport of drugs across the blood / brain barrier. In the context of the invention, it is therefore desirable that the nanoparticles and / or contrast agents according to the invention can be modified according to the disease to be diagnosed.
  • nanoparticles This can be achieved, on the one hand, by coating the nanoparticles with specific surfactants (polysorbate 80, or poloxamer 188, Gelperina et al., 2002), or by attaching specific ligands, such as apolipoprotein A1 and E (Zensi et al., 2009), Transferrin, insulin or antibodies to the corresponding receptors (Ulbrich et al., 2009, Ulbrich et al., 2010), but not by PEGylation.
  • pegylation of the nanoparticles may be beneficial in prolonging the circulation time of the nanoparticles in the blood. Modifications to allow targeted delivery of the nanoparticles to the tumor are known.
  • a preferred embodiment of the invention is directed to a nanoparticle which is coupled to a tumor-specific ligand, wherein the tumor-specific ligand is selected from the group comprising transferrin, an antibody against the transferrin receptor, insulin, an antibody against the insulin receptor and folic acid.
  • the tumor-specific ligand is selected from the group comprising transferrin, an antibody against the transferrin receptor, insulin, an antibody against the insulin receptor and folic acid.
  • all such substances are preferred as ligands which allow targeted taring of the nanoparticles to the tumor, in particular ligands which bind the nanoparticles to HCC in a targeted manner.
  • the nanoparticles of the invention should preferably be used as contrast agents in a magnetic resonance imaging method.
  • a magnetic resonance tomography for the diagnosis, prognosis and / or monitoring of a disease.
  • the nanoparticles described herein have a particularly good contrast of HCC in contrast to the surrounding liver parenchyma and non-malignant liver tissue readings. Since tumor vessels have an increased permeability and furthermore the lymphatic outflow in the tumor tissue is reduced, macromolecular substances injected into the blood circulation, but not low-molecular substances, can deposit selectively in the tumor tissue - this is the EPR effect.
  • the nanoparticles of the present invention have particularly surprisingly advantageous properties, which favor a contrasting of the HCC caused by the EPR effect.
  • nanoparticles have reduced penetration in malignancies (Jain and Stylianopoulos, Delivering nanomedicine to solid tumors, Nat Rev Clin Oncol 2010; doi: 10.1038 / nrclinonc.2010.139).
  • the nanoparticle-based contrasting of the tumors could also be due to a decreased accumulation of the nanoparticle-based contrast agent in the tumor. Since the accumulation of nanoparticles takes several hours, it is to be expected that the contrasting of the tumors by the nanoparticles will initially be based on a reduced accumulation of nanoparticles in the tumor, which compensates or reverses under the influence of the EPR effect. This should be a characteristic of malignancies like HCCs.
  • the nanoparticles and / or contrast agents of the present invention in conjunction with a 3-tesla scanner in the MRI.
  • the nanoparticles of the invention can be used for contrasting, and thus for diagnosing, a variety of diseases. Diseases which are associated with a disturbance of the barrier function of the vessels and reduced lymphatic drainage in the pathologically altered region are preferred. This is the case for most solid malignancies, in particular mammary carcinoma, colon carcinoma, pancreatic carcinoma, gastric carcinoma, ovarian carcinoma, bile duct carcinoma, prostate carcinoma, vicinal carcinoma, glioblastoma or tumor metastases.
  • the disease to be diagnosed is a liver disease, in particular cirrhosis and / or hepatocellular carcinoma (HCC).
  • HCC hepatocellular carcinoma
  • the structure of the nanoparticles according to the invention and / or contrast agents makes it possible to use them in the early detection of a hepatocellular carcinoma, in particular for the detection of a hepatocellular carcinoma of a size smaller than 1.5 cm.
  • a representation of such small HCC has not been possible via the known MRI methods. Therefore, the nanoparticles of the invention can be used especially for the diagnosis of HCC at an early stage. Therapy is characterized by a significantly improved prognosis for a patient diagnosed with HCC at an early stage, and therefore particularly advantageous.
  • the nanoparticles according to the invention are smaller than 500 nm, preferably smaller than 300 nm and most preferably have a size of 100-300 nm.
  • a contrast agent for use in magnetic resonance imaging comprising a nanoparticle of the invention described above.
  • the formulation and composition of pharmaceutically acceptable contrast media, in particular with regard to possible allergic reactions, are known to the person skilled in the art and can be found in the relevant prior art.
  • the contrast agent according to the invention also contains a tumor homing peptide, in particular iRGD.
  • the iRGD can be administered in parallel with the nanoparticles, or else coupled to the nanoparticles and / or incorporated in the nanoparticles.
  • iRGD consists of an RGD motif which binds to integrin a v , an integrin preferentially expressed in the endothelium of tumor vasculature (Ruoslahti, 2002) and a CendR motif which binds to neuropilin-1.
  • the binding of CendR and neuropilin is required for the enhanced tumor-selective penetration effect. Remarkably, this increased penetrability persists for several days after iRGD administration (Sugahara et al, 2010).
  • Cirrhotic liver tissue has elevated levels of neuropilin-1 compared to the normal liver (Cao et al, 2010); furthermore, the carbon tetrachloride-induced liver fibrosis in the mouse is enhanced by neuropilin-1 (Cao et al, 2010). Since iRGD improves the penetration of the tumor stroma into the HCC, increased extravasation of the nanoparticles and an increase in the EPR effect are to be expected, resulting in an improvement in the specificity and / or the sensitivity of the contrast enhancement in malignant HCC tissue.
  • the contrast agent according to the invention may additionally contain agents which cause an increase in the blood pressure of the subject to be diagnosed-a patient.
  • agents which cause an increase in blood pressure leads to an enhancement of the EPR effect.
  • the contrast agent of the invention contains angiotensin II.
  • Another possibility is the ac- Allowing cumulation of nanoparticles is that preferably nitric oxide (NO) donors, such as glyceryl trinitrate (GNT) is contained in the contrast agent of the invention.
  • NO nitric oxide
  • GNT glyceryl trinitrate
  • the contrast agent may contain a physiologically acceptable aqueous medium.
  • the contrast agent of the present invention is preferably provided in the form of an injection solution.
  • a third aspect of the invention provides a nanoparticle according to the invention and / or a contrast agent according to the invention which is suitable for enabling a differentiation between hepatocellular carcinoma and cirrhotic regenerate nodules in a magnetic resonance tomography method. Furthermore, a nanoparticle according to the invention and / or a contrast agent according to the invention can be used for the differentiation between HCC and liver cirrhosis.
  • the object of the present invention is achieved by a method for the diagnosis, monitoring and / or early detection of hepatocellular carcinoma, comprising administering a contrast agent according to the invention to a subject and a subsequent MRI examination of the subject.
  • the MRI examination is preferably carried out by means of a 3-Tesla tomograph.
  • a subject in the context of the invention is preferably a human (a patient), in particular a patient in whom cirrhosis and / or HCC is suspected, or in whom a preventive or follow-up examination for HCC is to be made.
  • the present invention may be used generally for imaging by means of an MRI in a patient.
  • the image recording process is carried out as follows: first, a sufficient amount of the contrast agent according to the invention is administered to a patient, in order then to perform a scan of the patient by means of magnetic resonance tomography. Thus, images of the internal structures of the patient of interest, particularly the diseased tissues, are taken.
  • the contrast agent according to the invention is particularly helpful for the visualization of liver tissues, but other regions can also be used to record images.
  • Administration of the contrast agent can be accomplished in any manner well known to those skilled in the art. This includes in particular intravenous, intracardiac (heart injection), oral, rectal, etc. administration of different formulations of the contrast agent.
  • the necessary dose of the contrast agent is varied according to the age, size and weight of the patient, as well as the area of the body to be examined.
  • contrast agents of the invention can be used both alone and in combination with other diagnostic, therapeutic or other substances.
  • other substances is meant in particular pharmaceutical excipients, flavorings or colorants, for example sucrose or a natural citrus flavor may be added to an orally administered formulation
  • the contrast agent of the invention be included in liposomes or other pharmaceutical carriers.
  • the use of the above-described nanoparticles and / or contrast agents in an MRI method is also provided.
  • a use of the nanoparticles according to the invention and / or contrast agents for the diagnosis, prognosis and / or early detection of an HCC, and / or the monitoring of a therapy of such a disease based on an MRI is preferred.
  • Figure 1 Physicochemical properties (particle size and polydispersity) of
  • PLGA 502H nanoparticles as a function of their loading with gadolinium.
  • PLGA 504H nanoparticles as a function of their loading with gadolinium.
  • Figure 3 MRI scan at a repetition time of 100 ms.
  • Figure 4 Application of contrast agents in the HCC mouse model. In all applications the same amount of Gd-DTPA was used. Red circles highlight the HCCs.
  • Mouse 1 MRI of the liver of a TGFalpha / c-myc mouse directly after injection with Primovist® (Oh), and after 24 h. Primovist® can penetrate into normal liver cells and tumor cells through transport proteins, thus first contrasting the vessels and then both the tumor and the surrounding liver parenchyma.
  • Mouse 2 MRI of the liver of a TGFalpha / c-myc mouse directly after injection with Magnevist® (Oh), and after 24 h.
  • Mouse 3 This mouse was treated with Primovist® and analyzed directly. Thereafter, the mouse received Gd-DTPA-PLGA nanoparticles 48 hours later and was measured immediately after injection, as well as 24 h and 64 h later.
  • Figure 7 Incorporation of fluorescently labeled nanoparticles in the liver.
  • A PLGA-NP rhodamine-labeled nanoparticles.
  • B HSA-NP-rhodamine-labeled nanoparticles.
  • Figure 8 MRI image natively and with NP-HSA-Gd-Rhodamine: HCC in the native image of the same tumor, which can not be detected without contrast medium.
  • Example 1 Gadolinium-DTPA PLGA Nanoparticles 1. Synthesis of the Gd-DTPA-PLGA nanoparticles
  • PLGA nanoparticles with covalently coupled gadolinium DTPA uncharged PLGA-NP prepared by emulsion diffusion evaporation, amino groups introduced by EDC and 1,4-diaminobutane, Gd-DTPA bound by EDC:
  • Unplated PLGA nanoparticles were prepared by emulsion diffusion evaporation.
  • the organic phase was washed with 10 ml of an aqueous, 1% stabilized polyvinyl alcohol (PVA, Mr 30,000-70,000 Da, degree of hydrolysis 96.8-97.6%, ester number 30-40, Sigma-Aldrich, Steinheim, Germany) solution by means of an Ultra - Turrax (Ultra Turrax® T25, IKA Laboratory, Staufen, Germany) emulsified at 17,000 rpm for 5 minutes.
  • PVA stabilized polyvinyl alcohol
  • the emulsion was added to a stirring PVA solution (550 rpm) in a 100 ml wide-bore condenser flask. As a result, the ethyl acetate diffuses into the aqueous phase and the polymer precipitates. By stirring overnight (-12 h), the ethyl acetate was removed by evaporation from the solution.
  • the resulting nanoparticles were purified by centrifugation (16,100 g 8 min, centrifuge 5804R, Eppendorf, Hamburg, Germany), exchange of the supernatant with MilliQ water and resuspension by means of ultrasound (ultrasonic bath Transsonic Digital, Elina, Singen, Germany). The purification cycle was repeated three times. At the last purification cycle, the nanoparticles were concentrated by resuspending in half of the original suspension volume. The exact nanoparticle content was determined gravimetrically (see below). 15 mg / ml of PLGA nanoparticles were brought to a suitable pH of 5.5 to 6.5 with the MES buffer.
  • the nanoparticles were subsequently purified. This was done as described above by centrifugation, exchange of the supernatant with MilliQ water. After 2 purification cycles, the nanoparticle content was determined gravimetrically.
  • PLGA nanoparticles with incorporated perflourooctyl bromide was carried out by means of emulsion-diffusion evaporation.
  • the organic phase was washed with 10 ml of an aqueous, 2% stabilized polyvinyl alcohol (PVA, Mr 30,000-70,000 Da, degree of hydrolysis 96.8-97.6%, ester number 30-40, Sigma-Aldrich, Steinheim, Germany) solution by means of an Ultra - Turrax (Ultra Turrax® T25, IKA Laboratory, Staufen, Germany) emulsified at 17,000 rpm for 5 minutes.
  • PVA polyvinyl alcohol
  • Ultra Turrax® T25 IKA Laboratory, Staufen, Germany
  • the emulsion was added to a stirring PVA solution (550 rpm) in a 100 ml wide-bore condenser flask. As a result, the ethyl acetate diffuses into the aqueous phase and the polymer precipitates. By stirring overnight (-12 h), the ethyl acetate was removed by evaporation from the solution.
  • the resulting nanoparticles were purified by centrifugation (16,100 g 12 min, centrifuge 5804R, Eppendorf, Hamburg, Germany) Exchange of the supernatant with MilliQ water and resuspension by means of ultrasound (Ultrasonic Bath Transsonic Digital, Elina, Singen, Germany). The purification cycle was repeated three times.
  • the concentration of nanoparticles was determined gravimetrically by the loss of drying (see above).
  • the size of the nanoparticles was determined by the PCS Photon Correlation Spectroscopy (Malvern Zetasizer 3000 HSA, Malvern, UK) using PCS cuvettes (PCS cuvettes (10x10x48), Sarstedt, Numbrecht, Germany).
  • the size measurements of two nanoparticle preparations show that particles have formed in the lower nanometer range (100-300 nm) ( Figures 1 and 2).
  • a satisfactory polydispersity of less than 0.3 (502H) or 0, 1 (504H) results ( Figures 1 and 2).
  • Particles with incorporated perfluorooctyl bromide have a size of about 260 nm and a polydispersity of less than 0.1.
  • the concentration of the nanoparticles was determined gravimetrically (analytical balance Supermicro S4, Sartorius, Göttingen, Germany) on the loss of drying. For this purpose, 50 ⁇ l of the nanoparticle suspension were weighed into a weighing boat (aluminum weighing boat, VWR, Darmstadt, Germany) and dried to constant weight at 80 ° C. in a drying cabinet (Ehret, Emmendingen, Germany).
  • the determination of the Gd-DTPA concentrations in the suspended nanoparticles was carried out by means of MRT in the 3 Tesla nuclear spin tomograph.
  • Figure 3 shows an MRI scan with a repetition time of 100 ms.
  • the numbers represent the theoretical Gd-DTPA concentration in the unit mg / ml.
  • PLGA means PLGA nanoparticles
  • HSA means HSA nanoparticles
  • the "+" plus sign means covalently coupled with e.g. Gd-DTPA.
  • the first column in Figure 3 shows a dilution series of the pure Gd-DTPA solution.
  • the concentration range extends from 3 mg / ml to 0.5 mg / ml of Gd.
  • mice Male TGF ⁇ / c-myc bi-transgenic mice were obtained by cross-breeding with homozygous MT / TGF ⁇ (Jhappan et al, 1990) and ALB / C-myc (Murakami et al, 1993) single transgenic mice in a CD13B6CBA background (as described in Murakami et al, 1993). After the weaning phase, the mice were induced with ZnCl 2 to induce expression of TGF ⁇ and thus accelerated hepatocarcinogenesis. 18 to 22 weeks after the start of the zinc treatment, the animals were subjected to a contrast-enhanced MRI examination. After the MRI examination, the livers of the animals were prepared and examined microscopically for HCC. The presence of HCC was histopathologically detected by tissue sections.
  • mice were anesthetized after zinc induction by intraperitoneal injection of ketamine (60 mg / kg body weight) and xylazine (12 mg / kg body weight). Thereafter, Primovist® (Schering) was injected intravenously and a mouse MRI was performed immediately. Each mouse was scanned both before and after administration of the contrast agent. A 3-tesla tomograph was used (Siemens Magnetom Trio, Siemens Medical Solutions, Er Weg, Germany).
  • the quantitative MRI analysis of the present invention is based on the measurement of signal intensities (SI).
  • SI signal intensities
  • circular geometrical regions, so-called operator-defined regions of interest (ROI) were drawn into the MRI images.
  • the SI values of the ROIs were presented as averages with one standard deviation.
  • hepatocarcinogenesis is induced by zinc in drinking water. After induction of hepatocarcinogenesis for 20 to 22 weeks, the animals were first examined for the presence of HCC by Primovist® / Magnevist® enhanced MRI. It showed that Primovist® enabled a more sensitive representation of HCC than Magnevist®.
  • the HCCs in the TGFalpha / c-myc mouse model could be contrasted with Primovist® and correspond to the 10% human HCCs.
  • mice were subjected to another MRI scan one day later. Here it was shown that the contrast of the HCC by Primovist® was largely lost.
  • Nanoparticle-based contrast agents of the present invention therefore offer the option of additionally using the EPR effect, a characteristic of malignancies, in differential diagnosis.
  • Gd-EOB-DTPA (Primovist®) is a gadolinium-containing, water-soluble contrast agent. It differs from the MRI contrast agent Magnevist® described above by a lipophilic ethyl-oxy-benzyl group, so that the substance is specifically taken up by the hepatocytes and excreted biliary. For uptake into the cell, the albumin-binding organic anion transporter, which has only a low specificity, is responsible, whereas biliary excretion occurs in an ATP-dependent process with the help of glutathione S-transferase.
  • FIGS. 5 and 6 show that the nanoparticles according to the invention have a SIR ("signal / intensity ratio” signal / intensity ratio) as well as CNR ("contrast / noise ratio” / background ratio).
  • SIR signal / intensity ratio
  • CNR contrast / noise ratio
  • Figure 7 shows the distribution of fluorescently labeled PLGA and HSA nanoparticles.
  • 200 ⁇ of the PLGA nanoparticle rhodamines or HSA nanoparticle rhodamines were intravenously injected into the HCC mice. Thirty minutes later, the animals were sacrificed and cuts made by the liver. Fluorescence-labeled nanoparticles are seen to a lesser extent in HCCs compared to adjacent normal liver tissue. Accordingly, nanoparticles of the invention allow a clear contrast between tumor tissue and the surrounding liver tissue.
  • Figure 8 shows In order to investigate the contrasting effect of HSA-based nanoparticles in HCCs in TGFa / c-myc mice, an MRI sequence was first performed prior to injection of the nanoparticles. Subsequently, the animals were injected with Gd-DTPA-loaded HSA nanoparticles (with about 10 ⁇ Gd-DTPA / kg body weight) and MRI sequences were carried out immediately after the injection. One recognizes the demarcation of a HCC, which was not visible in the Nativfact.
  • Example 2 Gadolinium-DTPA HSA nanoparticles
  • the embedding of Gd-DTPA in an HSA particle matrix is carried out by ethanolic desolvation.
  • the polymer and the drug are first dissolved in a solvent.
  • the non-solvent is added to ethanol.
  • the addition of this substance results in an "ultrafine coacervation", which leads to the inclusion of the drug molecules in the polymer.
  • a part of the drug can be dissolved in the polymer.
  • a cross-linking substance such as glutaraldehyde is added its two free aldehyde functions with the terminal amino groups in the protein and thus leads to a cross-linking of the amino acid strands in the protein and thus to a stabilization or curing of the nanoparticles.
  • the addition of the non-solvent takes place slowly and uniformly Desolvation of the HSA nanoparticles is then performed in the final purification step and the nanoparticles are redispersed in MES buffer pH 4.7.
  • Binding of Gd-DTPA to the HSA particle matrix occurs by transient binding of n- (3-dimethylaminopropyl) -N-ethylcarbodiimide (EDC).
  • EDC n- (3-dimethylaminopropyl) -N-ethylcarbodiimide
  • the underlying idea is the introduction of a linker that "mediates" a covalent bond between the particle and the drug, in which both molecules (particles and drug) must react with the linker
  • the Gd-DTPA complex is expressed in dissolved in the above-mentioned ethanol / water mixture and 0.6 ml of it with 0.2 ml of a freshly prepared EDC solution (10 mg / ml) .
  • This solution becomes the particles which in 1 ml of MES buffer pH 4 , Dispersed and shaken for two hours on an Eppendorf mixer at 600 rpm with exclusion of light.
  • the preparations contain an effective concentration of 6 mg /
  • Example 3 Further manufacturing methods of the nanoparticles of the invention
  • HSA nanoparticles were prepared by a desolvation process (Weber et al, 2000). 200 mg HSA was dissolved in 2 ml 10 mM NaCl 2 solution, adjusted to pH 8.4 and filtered through a 0.22 ⁇ filter. In this solution, the nanoparticles were formed under constant stirring at room temperature with the continuous addition of 8 ml of the desolvation agent ethanol (addition rate 1 ml / min). To the protein desolvation, 115 ⁇ of an 8% aqueous glutaraldehyde solution was added to achieve crosslinking of the particles. The resulting nanoparticles were purified by three centrifugation steps (16100xg, 8 min) and redispersion in the original volume with water.
  • DTPAa 5 mg was added to 1 ml of HSA solution adjusted to pH 9.0-9.5 and incubated for 3 h at room temperature with constant shaking at 600 rpm.
  • the DTPA-conjugated HSA nanoparticles were purified by four washings by centrifugation (16100xg, 10 min) and redispersed in 1 ml of water.
  • EDC l-ethyl-3- (3-dimethylaminopropyl) carbodiimide
  • a freshly prepared EDC solution (100 ⁇ , 10 mg / ml) was added to an HSA suspension and incubated for 30 min with constant shaking at 600 rpm.
  • the Gd-DTPA complex was subsequently synthesized by the addition of DTPAa and complexation with GdCl 3 to the HSA nanoparticles.
  • the Solution was quenched with 1 ⁇ M of hydroxylamine (500 mg / ml) for 20 min to stop the reaction. Subsequently, for the purification of the nanoparticles, three centrifugation cycles (16100 ⁇ g, 8 min) with redispersion in the original volume of water were carried out.
  • the particle size and polydispersivity index (PDI) were determined by dynamic light scattering (DLS). DLS and zeta potential analyzes were performed using the Zetasic Nano ZS (Malvern Instruments, Malvern, UK). An aliquot (15 ⁇ ) of the samples was diluted in 2 ml ultrapure water and the light scattering measured at 25 ° C at a 90 degree angle. The nanoparticle content of the suspension was determined gravimetrically, the concentration of Gd by atomic absorption spectroscopy. The surface morphology of the nanoparticles was determined by means of the scanning electron microscope (Hitachi, S 4500 SEM), the Gd loading of the nanoparticles by MRI.
  • DLS and zeta potential analyzes were performed using the Zetasic Nano ZS (Malvern Instruments, Malvern, UK). An aliquot (15 ⁇ ) of the samples was diluted in 2 ml ultrapure water and the light scattering measured at 25 ° C at a 90 degree angle
  • PLGA nanoparticles were prepared by the double-emulsion method by evaporation of the solvent. 500 mg of PLGA (50:50) was dissolved in 5 ml of ethyl acetate to form a polymer phase. Subsequently, this was mixed with 10 ml of 1% polyvinyl alcohol solution (PVA) and homogenized using an Ultra-Turrax (17000 rpm, 5 min). It resulted in a water-in-oil (w / o) emulsion. This primary emulsion was added with 40 ml of a 1% PVA solution and stirred for 18 h (550 rpm) to evaporate the ethyl acetate. In the last step, the nanoparticles were washed three times by centrifugation (16100xg, 8 min) and lyophilized for long-term storage.
  • PVA polyvinyl alcohol solution
  • Gd-DTPA-loaded PLGA nanoparticles were prepared by a modified double-emulsion solvent evaporation method. 5 mg of diethylenetriaminepentaacetic acid anhydride (DTPAa) was added to 1 ml of PLGA nanoparticles in a concentration of 10-50 mg / ml, homogenized using an Ultra-Turrax (17000 rpm, 1 min) and stirred at 600 rpm for 3 h , Subsequently, the DTPA-conjugated PLGA nanoparticles were washed three times by centrifugation (16100xg, 8 min) and redispersion in 1 ml of water.
  • DTPAa diethylenetriaminepentaacetic acid anhydride
  • Rhodamine B / Rhodamine 123 was dissolved in 1 ml of ethyl acetate (0.5 mg / ml), added to 2.5 ml of a PLGA solution (in 100 mg / ml ethyl acetate) and 5 ml of a 1% PVA solution mixed. The homogenization was then carried out with the help of the Ultra-Turraxes (17000 rpm, 5 min). This primary emulsion was placed in 20 ml of a 1% PVA solution and stirred for 18 h (550 rpm) to allow the solvent to evaporate. Subsequently, the nanoparticles were washed three times by centrifugation (16100xg, 8 min) with ultrapure water and freeze-dried for long-term storage.
  • Particle size and polydispersity index (PDI) were determined by dynamic light scattering (DLS). DLS and zeta potential analyzes were performed using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). An aliquot (15 ⁇ ) of the samples was mixed with 2 ml ultrapure water and the light scattering at 25 ° C and a scattering angle of 90 degrees determined. The nanoparticle content of the suspension was determined gravimetrically. The concentration of Gd was measured by atomic absorption spectroscopy. The morphology of the nanoparticle surface was determined by scanning electron microscopy (Hitachi, S 4500 SEM). The loading of nanoparticles with Gd was determined by MRI.
  • DLS and zeta potential analyzes were performed using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). An aliquot (15 ⁇ ) of the samples was mixed with 2 ml ultrapure water and the light scattering at 25 ° C and

Abstract

L'invention concerne des nanoparticules comprenant un composé du gadolinium et/ou un bromure de perfluorooctyle incorporé, pour une utilisation en tant qu'agents de contraste dans un diagnostic assisté par imagerie par résonance magnétique (IRM) de maladies du foie, en particulier du carcinome hépatocellulaire (CHC). Les nanoparticules de PLGA et de HSA selon la présente invention ont été chargées en un composé du gadolinium (Gd-DTPA) et ont pu être utilisées avec succès pour un meilleur contraste dans l'IRM du CHC. La présente invention concerne en outre de nouveaux agents de contraste contenant les nanoparticules selon la présente invention, en particulier pour une utilisation dans le diagnostic, la surveillance et/ou la détection précoce du CHC. La présente invention concerne en outre un procédé pour le diagnostic, la surveillance et/ou la détection précoce du CHC.
PCT/EP2012/052811 2011-02-21 2012-02-17 Nanoparticules en tant qu'agent de contraste d'irm pour le diagnostic du carcinome hépatocellulaire WO2012113733A1 (fr)

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EP3216464A1 (fr) 2016-03-11 2017-09-13 Stichting Katholieke Universiteit Nijmegen Procédé de préparation de perles pour imagerie
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EP2708244A1 (fr) * 2012-09-14 2014-03-19 Stichting Katholieke Universiteit Agent de contraste et son utilisation pour l'imagerie
WO2014041150A1 (fr) 2012-09-14 2014-03-20 Stichting Katholieke Universiteit Agent de contraste et son utilisation pour l'imagerie
JP2015534549A (ja) * 2012-09-14 2015-12-03 スティヒティング カトリーケ ユニフェルシテイトＳｔｉｃｈｔｉｎｇ Ｋａｔｈｏｌｉｅｋｅ Ｕｎｉｖｅｒｓｉｔｅｉｔ 造影剤およびイメージングのためのその使用
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CN105051016A (zh) * 2012-11-12 2015-11-11 易格尼塔公司 苯达莫司汀衍生物及其使用方法
WO2016097963A1 (fr) * 2014-12-16 2016-06-23 Fondazione Istituto Italiano Di Tecnologia Nanoparticules d'albumine encapsulant du gadolinium et leur procédé de synthèse
CN107847614A (zh) * 2015-07-30 2018-03-27 阿尔布雷希特·皮佩尔 借助于iRGD和磁共振成像（MRT）用于诊断癌症的方法
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EP3124052A1 (fr) 2015-07-30 2017-02-01 Albrecht Piiper Procede de diagnostic de carcinomes a l'aide de irgd et tomographie par resonance magnetique
EP3216464A1 (fr) 2016-03-11 2017-09-13 Stichting Katholieke Universiteit Nijmegen Procédé de préparation de perles pour imagerie
US11286352B2 (en) 2016-03-11 2022-03-29 Stichting Radboud Universitair Medisch Centrum Process for preparation of beads for imaging
CN109486827A (zh) * 2018-12-04 2019-03-19 南京林业大学 一种肿瘤归巢穿膜肽tLyP-1修饰的去铁蛋白纳米笼及其制备方法
CN112587677A (zh) * 2020-12-23 2021-04-02 广东省第二人民医院（广东省卫生应急医院） 一种iRGD磁性靶向微泡造影剂及其应用
CN112587677B (zh) * 2020-12-23 2022-11-11 广东省第二人民医院（广东省卫生应急医院） 一种iRGD磁性靶向微泡造影剂及其应用
CN114767885A (zh) * 2022-03-15 2022-07-22 华中科技大学同济医学院附属协和医院 一种新型高效白蛋白钆基对比剂用于活体多功能磁共振成像
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