WO2022180344A1

WO2022180344A1 - Device for intra-tissue nmr detection and administering of molecules

Info

Publication number: WO2022180344A1
Application number: PCT/FR2022/050344
Authority: WO
Inventors: Yannick Cremillieux; Noël PINAUD; Justine DEBORNE
Original assignee: Universite de Bordeaux; Centre National De La Recherche Scientifique
Priority date: 2021-02-25
Filing date: 2022-02-24
Publication date: 2022-09-01
Also published as: FR3119980A1

Abstract

The invention relates to a micro-probe for imaging or for magnetic resonance spectroscopy, comprising means for administering a compound of interest and detection means comprising an implantable NMR micro-antenna, wherein the administering means and the micro-antenna are adjacent and extend along a longitudinal axis X of the implantable device, a distal end of the administering means and of the detection means forming an implantable distal end of the device. The invention thus also relates to a device for imaging or for magnetic resonance spectroscopy comprising a micro-probe, as well as to a method for metabolic analysis of or for imaging a biological sample using the micro-probe.

Description

DESCRIPTION

TITLE: Device coupling intra-tissue NMR detection and administration of molecules TECHNICAL FIELD

The present invention relates to the field of nuclear magnetic resonance (NMR). The invention relates, more specifically, to the field of microprobes that can be implemented in a device for spectroscopic analysis by NMR (SRM) or imaging by NMR (MRI), in particular for the analysis of a biological sample. TECHNOLOGICAL BACKGROUND

NMR is a spectroscopic method for analyzing matter, based on the magnetic properties of certain atomic nuclei. Despite its widespread use and its varied applications, particularly in the medical field, the techniques that derive from NMR such as magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) are inherently insensitive techniques compared to techniques analysis by fluorescence, mass spectrometry or nuclear imaging. The sensitivity of NMR is assessed by measuring the signal-to-noise ratio (SNR) of acquisitions in the time or frequency domain. This ratio depends, among other things, on the size of the sample, the concentration of the nuclei, the Larmor frequency and the Bi/i efficiency of the NMR probe, where B1 is the oscillating magnetic field generated by the probe NMR and i is the intensity of the current applied in the probe. The Bi/i ratio is inversely proportional to the size of the NMR probe. For this reason, the size of the NMR probes is, almost systematically, adapted to the volume of the sample, of the tissue or of the organ analyzed or imaged by NMR. The sensitivity of the NMR analysis is thus partly linked to the size of the NMR probe. Currently, implantable NMR probes are centimeter or millimeter in size and come in the form of rigid planar or solenoidal structures. The rigidity of their structure allows the implantation of the NMR probe in soft tissues (such as the brain or the peritoneal cavity) and the planar geometry of the probe ensures detection sensitivity on the tissues located opposite. of the probe. One of the problems posed by this type of structure is linked to their invasive and destructive aspect with respect to the tissues. Also, the dimensions of this type of NMR probe are constrained by the need to preserve the tissues at the time of insertion, along the path and at the final position of implantation of the NMR probe. It is for this reason that current NMR probes are generally implanted in the lumen of organs (stomach, esophagus, etc.) in order to avoid the destruction of the surrounding tissues. Thus, the NMR probes are not implanted directly in the tissue to be analyzed, but only in close proximity.

The current compromise between safety and performance (sensitivity) of the NMR probe does not allow the use of NMR probes directly in the tissues or organs to be imaged or analyzed, since their implantation within the tissue of interest would lead to the destruction of the surrounding tissues.

Another problem with current NMR probes is that they do not allow real-time evaluation of the effect of compounds of interest, such as therapeutic molecules, at the implantation site. The evaluation of such effects requires many adjustments and manipulations that require dedicated qualified personnel. In particular, the administration of the compounds and the analysis of their effects by NMR includes several successive steps of placement and removal of the probe, which are incompatible with real-time analysis. These repeated manipulations also present a risk for the patient.

There is therefore a need for probes that can be used for the analysis or imaging of biological samples, which allow the administration of a compound and an accurate NMR analysis of its effect in real time, without detrimental destruction of the surrounding tissues. . There is more particularly a need for probes allowing the administration of a therapeutic molecule such as a drug or a drug candidate, and the monitoring, advantageously in real time, of the effect of said therapeutic molecule by NMR analysis.

SUMMARY OF THE INVENTION The present invention therefore aims to remedy at least partially the drawbacks of current NMR probes, by proposing an NMR microprobe of millimeter or submillimeter size coupled to means for administering compounds. The invention allows minimally invasive implantation of microprobes, while ensuring biocompatibility and NMR detection sensitivity suitable for in vivo SRM/MRI studies. These probes allow the administration of compounds of interest, in particular therapeutic molecules, at the implantation site and the analysis of their effect in real time. The implantable microprobes according to the invention can thus advantageously allow the localized administration of therapeutic molecules, and the monitoring of their effect in real time. These implantable microprobes according to the invention can advantageously be used to carry out MRS (magnetic resonance spectroscopy) or MRI (magnetic resonance imaging) analyzes with high detection sensitivity, in particular on low-volume biological samples. The sensitivity of the probes according to the invention makes it possible to envisage the profiling (detection, quantification) of biological chemical species (for example metabolites, neurotransmitters or small molecules), of nuclei (for example 23Na, 31P or 13C) present in weak quantities in the samples analyzed, in particular in quantities less than one nanomole, or even to produce high spatial resolution images of biological samples.

The invention thus relates to a device combining means for administering a compound of interest, such as a therapeutic molecule, and NMR detection means, allowing in particular, according to a particular embodiment, the analysis of the effects of the compound administered to the tissue, the organ or any other biological sample in which the device according to the invention is implanted. The detection means make it possible in particular to produce images and NMR analysis spectra with increased spatial resolution and detection sensitivity.

In particular, the subject of the invention is an implantable magnetic resonance imaging or spectroscopy device comprising means for administering a compound of interest, and detection means comprising an implantable NMR micro-antenna, in which the administration means and the implantable NMR micro-antenna are adjacent and extend parallel along a longitudinal axis of the implantable device, a distal end of said administration means and of said detection means forming an implantable end of said device.

Advantageously, the administration means are chosen from the group consisting of a needle, a syringe, a cannula and a microdialysis probe, preferably a microdialysis probe.

In one embodiment, the NMR micro-antenna comprises at least one magnetic resonance circuit. The distal end of the magnetic resonance circuit can have, for example, a generally looped, U-shaped, twisted shape or a helical shape. In one embodiment, the administration means comprise or consist of a microdialysis probe, a microdialysis membrane of said microdialysis probe forming the distal end of the administration means. The distal end of the NMR micro-antenna can advantageously at least partially surround the microdialysis membrane of the microdialysis probe.

In one embodiment, the device further comprises an outer sleeve in which the administration means and the detection means extend at least partially.

In one embodiment, an external diameter of the implantable distal end of the device is less than or equal to 2 millimeters +/- 10%, preferably less than or equal to 1.5 millimeters +/- 10%, preferably between 0 .5 and 1.5 millimeters +/- 10%, or in which an external diameter of the implantable end of the device is less than or equal to 500 micrometers +/- 10%, preferably between 50 and 500 micrometers +/- 10 %.

The invention also relates to a kit comprising the implantable imaging or spectroscopy device according to the invention, a tuning-matching circuit capable of being placed at a distance from the magnetic resonance circuit of the NMR micro-antenna and optionally a compound of interest intended to be administered.

A subject of the invention is also the ex vivo or in vitro use of an implantable device or of a kit according to the invention, for measuring the biodistribution of a compound of interest in a biological sample and/or for evaluating the effect of a compound of interest on a biological sample, in particular on the cellular metabolism of the biological sample.

The invention also relates to a method for in vitro or ex vivo molecular analysis in a biological sample, said method comprising the introduction of a distal end of an implantable device according to the invention into a biological sample, G administration of a compound of interest by the means of administration of the implantable device, the detection of the compounds administered or of their by-products in the biological sample by the implantable NMR micro-antenna and optionally the molecular and structural analysis of the sample biological by spectroscopy or magnetic resonance imaging by the implantable NMR micro-antenna.

In one embodiment, the method further comprises the recovery and analysis of a sample taken from the biological sample, said sample being taken by the implantable device. Advantageously, the compound of interest is a therapeutic molecule or molecule for therapeutic purposes.

DETAILED DESCRIPTION OF THE INVENTION Implantable device

The subject of the invention is an implantable magnetic resonance imaging or spectrometry microprobe comprising:

- means for administering a compound of interest, and

- detection means comprising an implantable NMR micro-antenna, in which the administration means and the implantable NMR micro-antenna are adjacent and extend along a longitudinal axis X of the implantable device, a distal end of said administration means and said sensing means forming an implantable distal end of said device.

In particular, the subject of the invention is an implantable magnetic resonance imaging or spectrometry microprobe comprising:

- means for administering a compound of interest, and

- detection means comprising an implantable NMR micro-antenna, in which the means of administration and the implantable NMR micro-antenna are adjacent and extend parallel along a longitudinal axis X of the implantable device, a distal end of said means of administration and said detection means forming an implantable distal end of said device.

The term "magnetic resonance imaging or spectroscopy device" or "microprobe for nuclear magnetic resonance" means the part of an apparatus for analysis by nuclear magnetic resonance intended to generate a radiofrequency magnetic field to excite the nuclear spins of a sample and/or to detect a magnetic field. Such a microprobe generally comprises an LC-type resonant circuit (RLC circuit) which provides coupling with an external radio frequency magnetic field, as well as an impedance matching circuit.

By “distal end” is meant the portion of the element considered furthest from the manipulator and/or closest to the implantation zone. Conversely, “proximal end” means the portion of the element considered closest to the manipulator and/or furthest from the implantation zone. By “adjacent”, it is meant that the means of administration and the implantable NMR micro-antenna extend in the immediate vicinity of one another. According to the invention, said elements also extend along the same longitudinal axis, so that they extend parallel. In particular, the administration means and the implantable NMR micro-antenna can be placed side by side, separated by a short distance, be concentric, etc.

By “implantable device”, it is meant that the device can be inserted into a sample, in particular into a biological sample in vivo or ex vivo, such as a tissue, possibly within an organ.

By “biological sample” is meant a set of cells grouped together in a cluster, network or bundle. In one embodiment, the biological sample is an organ or an organ portion. In particular, the biological sample may be a tissue sample obtained for example by biopsy, a part of an organ, a cancerous tissue, etc. Alternatively, the biological sample can be a tissue or an organ in vivo, that is to say within a living individual. In a particular embodiment, the biological sample is a liquid or a fluid, preferably contained in a tissue or bone cavity, such as cerebrospinal fluid, pleural fluid, peritoneal fluid, cardiac fluid as well as physiological fluids such as plasma or blood. This term also covers any liquid surrounding biological tissue, such as organs and organ preparations, such as intercellular or interstitial fluid. Samples can be of animal, human or plant origin. The biological sample can be analyzed or imaged in vivo, in vitro or ex vivo.

By “compound of interest” is meant any molecule or substance inducing or likely to induce an effect at the implantation site where the compound is administered. This compound can be a chemical substance, a peptide, a hormone or a neurotransmitter for example.

Preferably, the compound of interest is a therapeutic molecule or molecule for therapeutic purposes. For example, the compound of interest may be a drug candidate. According to a preferred embodiment, the compound of interest is a molecule whose therapeutic effect is known, such as a drug, in particular a drug that has received marketing authorization.

Thus, the device according to the invention can for example allow monitoring in a tissue or organ ex vivo or in vivo of the therapeutic effect of a therapeutic compound, advantageously in real time and/or continuously over a period of time. . The term "therapeutic effect" is understood to mean a measurable effect, immediate or delayed, transient or definitive, reflecting an improvement in the state of health or well-being of a subject in relation to the use of a therapeutic compound and, explainable by one or more of its pharmacological properties, in particular by the effectiveness of the active ingredient contained therein. The state of health of the subject can in particular be analyzed on the basis of one or more known biomarkers for a given disease or condition.

A “biomarker” can be any measurable biological indicator. For example, the biomarkers can be molecular markers such as metabolites. For a given disease or condition, the expression or concentration of a biomarker is deregulated (increased or decreased) in the biological sample analyzed, in particular compared to a reference sample, such as a healthy sample. The healthy sample is preferably of the same histological origin as the sample tested. For example, when the biological sample is brain tissue, the reference sample is healthy brain tissue.

The compound of interest can in particular be chosen from a drug against cancer, an infectious disease such as a fungal, bacterial or viral disease, a metabolic disease such as hepatic steatosis or diabetes, an autoimmune disease, an neurodegenerative disease such as Parkinson's or Alzheimer's, a genetic disease or a mental illness such as depression. The compound of interest can thus be, according to a particular embodiment, an antiseptic, antibacterial, antifungal, antiviral, analgesic, antidepressant, anxiolytic, anti-inflammatory, immunotherapeutic, immunosuppressive or chemotherapeutic drug.

The compound of interest can take any form known to those skilled in the art, in particular a chemical molecule, a protein or a peptide, an antibody, DNA, cDNA, RNA or one of their fragments. The means of administration and detection are more particularly described below.

Means of administration

The device according to the invention comprises means for administering a compound of interest.

In particular, the administration means allow local or localized administration of a compound of interest. Thus, by "means of administration" is understood any means allowing the administration, injection or diffusion of a compound of interest at the site of implantation of the device according to the invention. Preferably, the distal end of the administration means forms an implantable distal end of said device.

According to one embodiment, the administration means are chosen from the group consisting of a needle, a syringe, a cannula, a catheter and a microdialysis probe.

Thus, the compound of interest is generally included in a solution or liquid, facilitating its administration. The solution comprising the compound of interest may in particular comprise an aqueous solution (perfusate) which closely resembles the ionic composition of the surrounding tissue fluid. Preferably, the solution or the liquid comprising the compound of interest is adapted to the implantation site, and is in particular isotonic with the biological sample in which the device is implanted. Alternatively, the perfusate has a higher concentration of molecules than that found in the biological sample.

Preferably, the means of administration are a microdialysis probe. In this embodiment, a microdialysis membrane of said microdialysis probe forms the distal end of the administration means, intended to be implanted in a biological sample. The microdialysis membrane is thus located at the implantation site and allows the release of the compound of interest through the microdialysis membrane.

A dialysis membrane is a semi-permeable film containing pores of different sizes. Dialysis works by differential diffusion across the membrane. As used herein, a "microdialysis membrane" is a semi-permeable membrane that has nanopores to exchange small molecules between a solution (or infusion fluid) included in the microdialysis probe and the fluid in a tissue in which the device according to the invention is implanted. The microdialysis site thus corresponds to the implantation site, i.e. the place of diffusion of the substances or compounds of interest through the membrane at the level of the implantation site (for example tissue or organ) .

Various designs of microdialysis probes have been developed for particular sites or tissue types. For example, microdialysis probes can be flexible or rigid, and the inlet and outlet flow paths can be looped, side-by-side, or concentric, for example as described in US Patent No. 5,191,900. Dialysis is preferably bidirectional in that there is exchange of molecules in both directions across the membrane. The difference in concentrations of a specific molecule across the dialysis membrane will determine the direction of the diffusion gradient. Due to this property, the microdialysis technique allows not only the measurement and quantification of endogenous molecules, but also the delivery of site-specific drugs into the extracellular space (Rongquist, G. et al. (1992) Acta Neurochir 114: 8-11; Muller, M. et al (1997) Clin Pharmacol Ther 62: 165-170).

Advantageously, the microdialysis probe has a concentric design. The flow or delivery of solution through the microdialysis probe at the implant site is generally referred to as perfusate, and the flow of fluid collected by the microdialysis probe from the implant site is generally referred to as dialysate.

According to one embodiment, the perfusion medium (perfusate) generally enters the probe through an inlet channel in which it is transported and it flows to the distal end of this channel which is surrounded by the membrane of microdialysis. The solution comprising the compound of interest is therefore diffused at the level of the dialysis site, that is to say at the level of the implantation site.

Substances from the interstitial fluid can in turn cross the semi-permeable membrane by means of passive diffusion. Preferably, the resulting dialysate exits the probe by flowing through an exit channel, where it can be collected for analysis. The extraction of the dialysate makes it possible to reduce the pressure at the level of the implantation site. The microdialysis probe can thus comprise an inlet channel for the injection of a compound of interest, a microdialysis membrane for the diffusion of the compound of interest at the level of the implantation site and an outlet channel for the extraction of the dialysate.

The inlet and outlet channels can typically be microdialysis catheters.

The inlet and outlet channels can be connected to external elements making it possible to circulate and monitor/analyze the dialysis solution, for example such as a pump or a syringe.

The device according to the invention can thus comprise a device allowing the active transport of fluid comprising a compound of interest, such as for example a pressure pump and/or a suction pump. This device can be configured to selectively transport and/or controllably the administration of the solution comprising the compound of interest and/or the withdrawal of the dialysate.

Preferably, the biological sample is generally perfused continuously at a relatively low rate, preferably about 0.1 to 5 µl per minute. Means of detection

According to one embodiment, the detection means comprise an implantable NMR micro-antenna operating in transmission-reception or in reception only and allowing the detection and acquisition of nuclear magnetic resonance signals. In particular, the detection means comprise at least one magnetic resonance circuit. The magnetic resonance circuit can be realized using micro-electro-mechanical systems (MEMS) fabrication procedures. Specifically, the magnetic resonance circuit is formed of a conductive metal. Preferably, the magnetic resonance circuit comprises at least one conductive metal microwire, such as a copper microwire. In one embodiment, said microwire has a diameter of less than 200 micrometers, less than 150 micrometers or less than 100 micrometers, preferably less than 70 micrometers, 60 micrometers, 50 micrometers, 40 micrometers, 30 micrometers or at 20 micrometers. Preferably, the microwire has a diameter less than or equal to 50 micrometers. Alternatively, the microwire has an outer diameter comprised between 10 and 70 micrometers, between 20 and 60 micrometers or between 30 and 50 micrometers. Preferably, the microwire has an outer diameter of between 30 and 50 micrometers.

Preferably, the distal end of the magnetic resonance circuit has the general shape of a loop, U, twist or a helical shape. This makes it possible to generate an alternating magnetic field for the detection of nuclear magnetic resonance signals. According to the invention, the means of administration and the at least one magnetic resonance circuit are adjacent and extend along a longitudinal axis X of the implantable device, a distal end of said means of administration and of said detection means forming an end implantable distal of said device.

Advantageously, the detection means partially surround the administration means. Preferably, the detection and administration means are in fluid communication. By “fluidic communication” is meant that the detection and administration means are positioned relative to each other so as to be able to allow a fluid or a solution comprising a compound of interest to circulate. Preferably, the compound of interest is free to diffuse means of administration towards the site of implantation.

According to one embodiment, the administration and detection means are integral. By “solidarity” is meant a close connection between the means of administration and the means of detection. Preferably, the distal end of the detection means is irreversibly attached to the distal end of the administration means.

In particular, when the administration means are a microdialysis probe and the detection means an NMR circuit, the distal end of the NMR circuit at least partially surrounds the membrane of the microdialysis probe.

The implantable device according to the invention may also comprise an outer sleeve in which the administration means and the detection means extend at least partially. Preferably, the device according to the invention can be mounted in translation in the outer sleeve.

In particular, the outer sleeve does not cover the NMR detection circuit or the microdialysis membrane.

Advantageously, the outer sleeve is retractable. Thus, during the insertion of the probe into the biological sample, the administration and detection means are protected by the outer sleeve, and are only deployed outside the outer sleeve at the level of the site of location of interest. The outer sleeve may include a beveled tip to facilitate its introduction into the biological sample.

According to one embodiment, the external diameter of the implantable distal end of the device is less than or equal to 5 millimeters +/- 10%, preferably less than or equal to 4 millimeters +/- 10%, less than or equal to 3 millimeters +/- 10%, less than or equal to 2 millimeters +/- 10%, less than or equal to 1.5 millimeters +/- 10%, preferably between 0.5 and 5 millimeters +/- 10%, between 0, 5 and 4 millimeters +/- 10%, between 0.5 and 3 millimeters +/- 10%, between 0.5 and 2 millimeters +/- 10%, between 0.5 and 1.5 millimeters +/- 10% . Alternatively, the outer diameter of the implantable end of the device is less than or equal to 500 micrometers +/- 10%, preferably between 50 and 500 micrometers +/- 10%.

The device according to the invention can also comprise a sensor or sensor allowing real-time analysis of the substances contained in the dialysate. The sensor module can be configured to directly detect at least one substance or compound of interest in the dialysate or to monitor the concentration of gas (including oxygen) present in the dialysate. The sensor can in particular allow the differential analysis of the composition of the perfusate and the dialysate. This sensor can in particular be located at the level of the proximal end of the device according to the invention. set

The invention also relates to a kit comprising the implantable imaging or spectroscopy device according to the invention, a tuning-matching circuit capable of being placed at a distance from the magnetic resonance circuit of the NMR micro-antenna and optionally a compound of interest intended to be administered. The kit may further comprise means for inserting and/or manipulating the probe in order to facilitate its manipulation and its introduction into a biological sample. The kit may further comprise a means for collecting a biological sample.

The invention thus also relates to a kit for inserting an implantable device comprising a hollow micro-needle and an implantable device according to the invention. Preferably, the microneedle has a beveled tip to facilitate its introduction into the biological sample. The positioning of NMR microprobes using a micro-needle included in an insertion kit, makes it possible, for example, to access and explore by SRM/MRI all the tissues of a biological sample in an invasive way at a minimum.

Preferably, a microprobe according to the invention can be mounted in translation in a microneedle. The kit can thus either comprise a ready-to-use microneedle, in which a microprobe is already housed in translation, or a microneedle and a microprobe ready to be mounted in translation inside the microneedle.

The insertion kit can also comprise a compound of interest, in particular therapeutic molecules or molecules for therapeutic purposes. In one embodiment, the insert kit includes therapeutic molecules for administration to a subject. In the context of the invention, the term “subject” designates a mammal, and preferentially a human, including an adult, a child or an infant. The term “subject” can also designate a non-human mammal, in particular a non-human primate.

In the context of the invention, such an administration of therapeutic molecules allows the analysis of the activity or of the effect of the therapeutic molecules administered by the device according to the invention. Thus, a subject of the invention is also the use of a kit for inserting a microprobe for the analysis of a sample or of a biological tissue. The biological sample can be analyzed in vivo, in vitro or ex vivo, in particular after taking a sample, tissue or organ from a subject. The kit according to the invention can also be used for analyzing and/or monitoring the therapeutic effect of a compound of interest in vivo, in particular in a living tissue or organ.

The invention also relates to a magnetic resonance imaging or spectroscopy device comprising the device according to the invention, said magnetic resonance imaging or spectroscopy device further comprising a tuning-matching circuit arranged at a distance of the magnetic resonance circuit of the microprobe.

The tuning and matching circuit is in particular composed of two variable capacitors. The so-called tuning capacitor makes it possible to tune the frequency of the resonant circuit of the NMR probe to the Larmor resonance frequency, which depends on the core and the magnetic field used. Preferably, the nucleus is a hydrogen nucleus, preferably composed of a single proton ('H). The capacity, called adaptation, makes it possible to optimize and maximize the power transfer between the NMR probe and the amplification, digitization and signal processing chain of the spectrometer of the magnetic resonance system, in particular for the production images and spectra.

In one embodiment, the microprobe is interfaced (via the amplification, digitization and signal processing chain) with the magnetic resonance imaging system for the production of magnetic resonance images and spectra.

Advantageously, the magnetic resonance imaging device comprising the microprobe according to the invention makes it possible to obtain spectra of a biological sample with a spectral resolution of less than 5 Hz. Method and use

The invention also relates to the in vivo, in vitro or ex vivo use of an implantable device or of the kit as described above, for measuring by MRI and/or MRS the biodistribution of a compound of interest in a biological sample and/or to evaluate the effect of a compound of interest on a biological sample, in particular on the cellular metabolism of the biological sample.

The invention also relates to the in vivo, in vitro or ex vivo use of an implantable device or of the kit as described above for the diagnosis of a disease or a condition, or for monitoring the progression of a disease or condition, in particular by identifying and/or monitoring biomarkers associated with this disease or condition. Preferably, these biomarkers are detected by SRM. In particular, the detection of one or more biomarkers associated with a disease in a biological sample reflects the state of health of the subject (for example sick or healthy), in particular in comparison with the analysis of a healthy tissue. .

For example, the detection by SRM in a cerebral biological sample of a decrease in the concentration of N-acetylaspartate (NAA) and an increase in the concentration of choline (Cho) and the Lipids/Lactate complex in comparison with a sample healthy brain, can translate the presence of a glioblastoma.

In certain forms of epilepsy, the proton MRS makes it possible to highlight a reduction in the concentration of NAA and an increase in the concentrations of creatine and choline at the level of the epileptogenic focus. This is of great interest, especially during a preoperative assessment of resection surgery of the cerebral area where the epileptogenic focus has been precisely identified by MRS.

The analysis by MRS, in a prostatic biological sample, of a decrease in the concentrations of citrate and polyamines and an increase in the concentration of choline, compared to a healthy prostate tissue, is an indicator of the presence of a tumor. . The evolution of the ratio between the concentration of citrate and the concentration of choline can also be a marker of tumor severity.

The invention also relates to the in vivo, in vitro or ex vivo use of an implantable device or of the kit as described above for monitoring a therapeutic treatment by monitoring biomarkers associated with a disease or condition. . Preferably, the biomarker(s) are detected by SRM. For example, the disappearance of biomarkers indicating the presence of a disease following the administration of a compound of interest can be associated with a therapeutic effect of said compound. The return to a level of expression or concentration of the biomarker at a level similar to the level observed in a healthy sample can also be associated with a therapeutic effect of said compound. The device can be used, for example, in the diagnosis of a medical condition, the monitoring of the progression of a progressive pathology (eg traumatic brain injury) or surgical procedures, or for the evaluation of appropriate treatment regimens . Typically, the device or kit according to the invention can be used to evaluate the effectiveness of a known drug or a candidate drug for the treatment of a disease or condition.

The disease diagnosed or monitored by means of the device according to the invention can in particular be chosen from cancer, an infectious disease such as a fungal, bacterial or viral disease, a metabolic disease such as hepatic steatosis or diabetes, an auto immune system, a neurodegenerative disease such as Parkinson's or Alzheimer's, a genetic disease or a mental illness such as depression.

Specifically, the pathologies affected by the use of the device according to the invention are:

- cancer, for example for the development of drugs, the evaluation of the grade of the tumor, the monitoring of the tumor, the evaluation of therapeutic treatments. The type of cancer targeted, which may be glioma, breast cancer, liver cancer, lung cancer, or sarcomas for example,

- neurodegenerative diseases, for example for the quantification of neurotransmitters in Parkinson's disease, the development of drugs in Alzheimer's disease and multiple sclerosis,

- metabolic pathologies such as hepatic steatosis or diabetes. The invention particularly relates to a method for the molecular analysis of a biological sample, said method comprising:

- the introduction of a distal end of an implantable device according to the invention into a biological sample; - the administration of a compound of interest by the means of administration of the implantable device;

- the detection of the administered compounds or their by-products in the biological sample by the implantable NMR micro-antenna; - optionally, molecular and structural analysis of the biological sample by spectroscopy or magnetic resonance imaging using the implantable NMR micro-antenna.

The invention also relates to a method for the molecular analysis of a biological sample, said method comprising:

- the introduction of a distal end of an implantable device according to the invention into a biological sample;

- optionally the administration of a compound of interest by the means of administration of the implantable device;

- detection and/or monitoring of one or more biomarkers associated with a disease, preferably by magnetic resonance spectroscopy. The invention also relates to a method for diagnosing a disease in a biological sample from a subject, said method comprising:

- the introduction of a distal end of an implantable device according to the invention into a biological sample; and

- detection and/or monitoring of one or more biomarkers associated with this disease, preferably by magnetic resonance spectroscopy.

Preferably, a comparison with a reference spectrum is carried out, the reference spectrum preferably being that of a healthy biological sample.

In particular, the deregulation of one or more biomarkers associated with this disease is indicative of the diseased state of the subject. The invention also relates to a method for the molecular analysis of a biological sample, said method comprising: - the introduction of a distal end of an implantable device according to the invention into a biological sample;

- the administration of a therapeutic molecule or molecule for therapeutic purposes by the means of administration of the implantable device; and - the evaluation and/or monitoring of the therapeutic effect of said molecule by the detection of one or more biomarkers associated with a disease, preferably by magnetic resonance spectroscopy.

The invention also relates to a method for the therapeutic treatment of a sick subject, said method comprising: - the introduction of a distal end of an implantable device according to the invention into a biological sample in vivo, in particular into a tissue or organ of a living subject;

- the administration of a therapeutic molecule by the means of administration of the implantable device; and

- the evaluation and/or monitoring of the therapeutic effect of said molecule by the detection of one or more biomarkers associated with a disease, preferably by magnetic resonance spectroscopy.

Advantageously, the follow-up is carried out until the subject's state of health improves. For example, the disappearance of the biomarker(s) associated with a disease in the biological sample is an indicator of an improvement in the state of health of the subject. The return to a level of expression or concentration of the biomarker at a similar level observed in a healthy sample is also an indicator of an improvement in the state of health of the subject.

The invention also relates to a method for selecting a therapeutic molecule, said method comprising:

Preferably, the analysis of the therapeutic effect is carried out by SRM. In particular, a comparison with a reference spectrum is carried out, the reference spectrum preferably being that of a healthy biological sample. The process can be repeated in order to administer different therapeutic molecules, thus making it possible to select the therapeutic molecule(s) most suited to the treatment of the patient.

The process can be carried out in vitro, in vivo or ex vivo. Preferably, the biological sample is a tissue and/or an organ of a subject and the microprobe is inserted at an anatomical location of interest in a tissue and/or an organ of a subject. The use of such a probe is thus similar to an in situ and non-destructive biopsy method on living tissue.

According to one embodiment, the method further comprises the recovery and analysis of a sample taken from the biological sample, said sample being taken by the implantable device.

The detection of the administered compounds or its by-products in the biological sample by the implantable NMR micro-antenna makes it possible to evaluate the molecular and structural profile of the biological sample. This detection can be carried out several times over time to obtain trends in the chemical changes of the biological sample.

According to one embodiment, the effect of a compound of interest, administered by the administration means according to the invention within a biological sample, is evaluated by magnetic resonance experiments carried out by the micro-antenna NMR, in particular, by magnetic resonance spectroscopy (MRS) and/or magnetic resonance imaging (MRI) experiments.

According to one embodiment, the effect of a compound of interest is evaluated by SRM, in particular by NMR spectrum analysis. An NMR spectrum includes peaks or series of peaks called signals which correspond to the resonance of the different protons present in a biological sample. In particular, the NMR spectrum indicates a quantity called "chemical shift" denoted d and expressed in parts per million (ppm), reflecting the offset between the resonance frequency of the protons of the biological sample and a reference resonance frequency. Preferably, the reference frequency (zero chemical shift) corresponds to the resonance frequency of the protons of the tetramethylsilane (TMS) molecule.

In one embodiment, the NMR spectrum of the biological sample is compared to the NMR spectrum of a reference sample.

Thus, obtaining and/or analyzing NMR spectra can be performed to compare two conditions. This makes it possible, for example, to evaluate the effect of the administration of a compound of interest compared to a reference condition (absence of the compound of interest or other compound whose effect is known), to evaluate the effect of a compound of interest over time or to detect the presence of a disease. In particular, the comparison of the profiles of the NMR spectra, in particular the presence and amplitude of the peaks of the metabolites of the sample and, possibly, of the compound of interest, testifies to the modifications of the molecular metabolism of the biological sample.

The comparison of spectra can thus be carried out before and after administration of a compound of interest or between a sample A and a sample B. Preferably, sample A is from a subject to be tested and sample B is a reference sample, such as a sample from a healthy tissue or organ.

Preferably, the obtaining and/or the analysis of the NMR spectra is carried out in real time.

In particular, the obtaining and/or the analysis of the NMR spectra is carried out continuously over a given period of time.

According to one embodiment, the effect of a compound of interest is evaluated by MRI. The presence of a compound of interest can in fact lead to functional and morphological changes in the biological sample (necrosis, oedema, etc.), visible on MRI.

The device according to the invention thus allows precise evaluations by SRM and/or MRI thanks to the positioning of the NMR micro-antenna in the immediate vicinity of the zone of administration of the compound of interest. The evaluation of the effect of the compound of interest can thus be carried out in real time, without requiring analysis on the dialysate recovered at the outlet of the means of administration, in particular such as the microdialysis membrane. Alternatively, the information from the MRI or SRM analysis can be compared or correlated with the dialysate analysis. In particular, the dialysate can also be collected and analyzed repeatedly to obtain trends of chemical changes in the biological sample. This analysis can be carried out by means of biomolecular analysis known to those skilled in the art or via the sensor described above.

In this context, it is thus possible to analyze the molecular and structural profile of the biological sample over time or over a given period of time not only thanks to the detection means of the device according to the invention, but also thanks to dialysate analysis. This makes it possible to reinforce the acuity of the experimental data measured.

According to one embodiment, when the device comprises a microdialysis probe allowing both the administration of a compound of interest via an inlet channel, and the sampling of a fluid via an outlet channel as detailed above, the invention may also relate to a method for identifying or sampling molecules in a biological sample, which consists of inserting the distal end of the device according to the invention into the sample and then causing a solution from an inlet channel of the microdialysis probe through the microdialysis membrane and a return of a fluid through an outlet channel, allowing its collection at an outlet orifice. One or more fractions of the fluid are collected as it emerges from the exit orifice, in particular over a period and at a frequency of interest. A collected fraction can then be analyzed for the presence of the molecule or for the search for other compounds, in parallel with detection by MRI or SRM.

The information from the MRI or SRM analysis can be compared or correlated with an earlier and/or later analysis of the tissue or organ in which the device according to the invention is implanted by any means known to those skilled in the art. In particular, all or part of the tissue or organ can be collected and analyzed. Preferably, only a fragment of tissue or organ is removed. The sample can be taken by means known to those skilled in the art, in particular by biopsy. The analysis of the sample can be carried out by any technique of microscopy and molecular biology known to those skilled in the art.

In this context, it is thus possible to analyze the molecular and structural profile of the biological sample over time or over a given period of time not only thanks to the detection means of the device according to the invention, but also thanks to the analysis of the sample tissue, organ or fragment thereof. This analysis can also be combined with the dialysate analysis mentioned above.

The MRI or SRM analysis of the sample allows the precise detection of the occurrence of a biological event, such as the production or secretion of a compound or substance of interest. In this context, the method according to the invention may comprise the collection of the dialysate at the time of the occurrence of such an event, in particular for the cellular production of molecules of interest.

In one embodiment, the insertion of the microprobe into a biological sample and the metabolic analysis of this sample are carried out in vivo or ex vivo, preferably on a sample previously taken from the subject. The analysis can also be carried out in vitro, in particular on cell cultures or organoids.

Alternatively, the insertion of the microprobe into a biological sample and the metabolic analysis of this sample are carried out in vivo, i.e. in the organ or tissue of a living subject. In particular, the microprobe according to the invention has a detection volume of the biological sample of between 0.1 and 1 microliter, particularly between 100 and 400 nanoliters, between 100 and 300 nanoliters or between 100 and 200 nanoliters, preferably between 150 and 300 nanoliters, between 150 and 250 nanoliters, between 150 and 200 nanoliters or between 200 and 250 nanoliters. Preferably, the microprobe has a biological sample detection volume of about 200 nanoliters. By “detection volume” is meant the volume of the region of the biological sample in which the microprobe has the capacity to collect and transmit the NMR signal.

For example, the sensitivity of the microprobe according to the invention was evaluated in vitro on lactate solutions as well as in an implantation situation in vivo in animals and in situ in grapes. Detection thresholds of 1 mM on lactate were obtained with acquisition times of the order of a second. These detection thresholds are compatible with the concentrations of numerous metabolites and molecules of interest present in vivo (eg: NAA, choline, lactate, creatine, glutamate, etc.). FIGURES

The invention will be better understood on reading the following description and on examining the accompanying figures. These are presented for information only and in no way limit the invention.

FIG. 1 represents an implantable magnetic resonance imaging or spectroscopy device, according to a first embodiment;

Figure 2 is an enlargement seen from the front (A) and seen from the side (B) of the implantable end of the device according to Figure 1;

Figure 3 schematically shows the molecular exchanges between the implantable device according to Figure 1 and the implantation zone of said device; FIG. 4 represents an implantable magnetic resonance imaging or spectroscopy device, according to a second embodiment;

Figure 5 is an enlargement seen from the front of the implantable end of the device according to Figure 4.

Figure 6 represents the use of an implantable device according to the invention for the infusion of gadolinium (Gd)

Figure 7 represents the use of an implantable device according to the invention for the infusion of DMSO. Abbreviations: Cr: Creatine, Cho: Choline, DMSO: Dimethylsulfoxide, Glu: Glutamate, Gin: Glutamine, Lac: Lactate, MM: Macromolecules, NAA: N-acetylaspartate, Tau: Taurine Figure 8 represents the 'H spectra acquired by the implantable device according to the invention in a healthy rat and in a rat bearing a brain tumor (PRESS sequence, TR/TE = 2000/15.266 ms, voxel size = 2 x 2 x 4 mm3, 256 accumulations, acquisition: 8 min 32 s). Abbreviations: Cr: Creatine, Cho: Choline, Glu: Glutamate, Gin: Glutamine, Lac: Lactate, ml: myo-Inositol, NAA: N-acetylaspartate, Tau: Taurine. Figures 1 to 3 illustrate a first embodiment of a magnetic resonance imaging or spectroscopy device according to the invention.

In this embodiment of a device 1 according to the invention, the detection means 2 and the administration means 3 extend parallel to each other along a longitudinal axis X, partially inside a sleeve 4. An implantable distal end 5 of said device 1 comprises a distal end 6 of the detection means (magnetic resonance circuit) and a distal end 7 of the administration means (FIG. 2). The administration means consist of a microdialysis probe comprising an inlet channel 8, an outlet channel 9, extending into the sleeve 4, and a microdialysis membrane 10, extending out of the sleeve 4. In this embodiment, the distal end 6 of the magnetic resonance circuit has a general U-shape, surrounding the microdialysis membrane 10 of the microdialysis probe.

FIG. 3 schematically illustrates the molecular exchanges between the microdialysis membrane 10 and the implantation site, such as a biological sample. A perfusate is brought through the inlet channel 8 and diffuses into the biological sample at the level of the microdialysis membrane 10, while a dialysate rises through the outlet channel 9, towards a proximal end of the device, so that it can be collected and analyzed. The implantable end 6 of the NMR micro-antenna allows the continuous and simultaneous detection of the administered compounds or their by-products in the biological sample.

FIGS. 4 and 5 illustrate a second embodiment of a device 100 for magnetic resonance imaging or spectroscopy according to the invention.

In this embodiment, the device 100 comprises detection means 101 and administration means 102, which extend parallel to each other along a longitudinal axis X, partially inside a sleeve 103. An implantable distal end 104 of said device 100 comprises a distal end 105 of the detection means (magnetic resonance circuit) and a distal end 106 of the administration means. Figure 5 shows the implantable distal end 104 in more detail. The administration means 102 consist of a microdialysis probe comprising an inlet channel 107, an outlet channel 108, extending into the sleeve 103, and microdialysis membrane 109, extending out of the sleeve 4. In this embodiment, the distal end 105 of the magnetic resonance circuit comprises a metallic wire 110, such as a copper wire, twisted around the membrane of microdialysis 109 of the microdialysis probe. Molecular exchanges similar to those described in FIG. 3 for the first embodiment, can take place at the level of the implantable end 104 of the device 100. FIG. 6 shows the effective perfusion of the implantable device according to the invention, for the administration of a compound of interest and for its detection in MRI by the micro-antenna of the device. A solution of artificial cerebrospinal fluid (CSF) and gadolinium (0.5 mM), a contrast agent commonly used in MRI, was infused at a rate of 0.2 pL/min, through the microdialysis membrane of the device, previously implanted in the right cortex of healthy rats.

During the infusion, Tl-weighted MRI images were acquired with the NMR micro-antenna of the device according to the invention (detection volume=850 nL) using a FLASH (Fast Low Angle Shoot) sequence every 45 minutes with the following parameters: repetition time (TR)=200 ms, echo time (TE)=3.9 ms, tilt angle=10°, image matrix=256×256, spatial resolution=0.039×0.039 mm ² .

Over time, the increase in signal intensity (hypersignal), generated by the predominant Tl effect of gadolinium, is easily visualized on the Tl-weighted images acquired by the NMR micro-antenna.

Gadolinium is defined as being a contrast agent with a predominant Tl effect (or as a so-called "positive" contrast agent) because, by reducing the relaxation time Tl of the tissues with which it is in contact, it leads to an increase in signal strength. Indeed, on Tl-weighted sequences, tissues with short Tl appear in hypersignal.

Figure 7 shows the effective infusion of compound of interest by the implantable device according to the invention, in particular by a microdialysis membrane and its detection by the micro-antenna by MRS of the different resonance frequencies of the protons of the compound of interest and brain metabolites. A solution of artificial cerebrospinal fluid (CSF) and dimethylsulfoxide (DMSO - C2H60S) (30 mM) was infused at a flow rate of 1 pL/min, through the microdialysis membrane of the device, previously implanted in the right cortex of a rat.

A succession of NMR spectra was acquired by the NMR micro-antenna and using a single-voxel localized spectroscopy sequence called PRESS sequence (Point-RESolved Spectroscopy) with the following parameters: TR = 2000 ms, TE = 15 ms, 256 averages, total acquisition time = 8 min 32 s).

During the infusion, the increase in the amplitude, and therefore the concentration, of the DMSO resonance peak, at 2.55 ppm, is easily observable and quantifiable. The main cerebral metabolites (Cr, NAA, Cho, Tau, Glu, Gin, Lac) are also identifiable. This result shows the effective diffusion of a compound of interest (DMSO) by the implantable device according to the invention and the possibility of monitoring the concentration of this compound over time.

Figure 8 shows the spectra acquired by the implantable device according to the invention, in a healthy rat and a rat with a brain tumor. The two spectra presented in Figure 8 were obtained with a so-called PRESS spectroscopy sequence, repetition time = 2000 ms, echo time = 15.3 ms, voxel size = 2 x 2 x 4 mm3, 256 accumulations, d acquisition: 8 min 32 s.

For the spectrum acquired by the NMR micro-antenna, in the healthy rat, the device was implanted in the right cortex of the rat.

For the spectrum acquired by the NMR micro-antenna, in rats bearing a brain tumour, the device was implanted in the tumour. Before placement, by stereotactic surgery, of the cannula allowing the introduction of the device into the region of interest (here, the brain tumor), MRI anatomical images of the brain of the rat were taken in order to precisely determine the location of the the latter and therefore the future positioning of the device.

In the presence of a brain tumor, a significant decrease in the concentration of NAA and a significant increase in the concentration of choline (Cho) and the Lipid/Lactate complex are observed compared to healthy tissue. These three molecules are biomarkers of glioblastoma. These results show the ability of the device to differentiate healthy tissue from pathological tissue, especially on low-volume biological samples. In addition, it is possible to follow these biomarkers over time. During the administration of a therapeutic molecule, it is thus possible to evaluate the therapeutic effect of the molecule according to the evolution of these biomarkers over time. The high detection sensitivity obtained by the device also makes it possible to assess the grade and malignancy of the tumour. The acquisition of MRS and MRI data over time makes it possible to follow the evolution of the tumor.

The device according to the invention is implanted in a cerebral tumor of a rat and an antitumor drug is administered within the tumor. An exchange of molecules takes place, by passive diffusion, between two liquid media: the extracellular tumor medium and the perfusion medium containing the antitumor drug. MRS and/or MRI analyzes with high detection sensitivity antitumor drug effects are realized in real time. The detection volume, estimated by the MRI images, is 850 nL.

In particular, a molecule such as oxamate can be administered locally within the tumor by the implantable device according to the invention. An MRI image is obtained to verify device implantation and oxamate delivery efficiency. SRM analyzes are performed to determine the effects of oxamate on the tumor spectrum. Oxamate inhibits the process of conversion, by the enzyme LDH-A, of pyruvate into lactate. This leads to a decrease in the concentration of lactate and to the modification of resonance peaks of the main metabolites (eg creatine and choline) in the tumor spectrum. A decrease in these biomarkers indicates a therapeutic effect of oxamate and possibly an improvement in the subject's state of health.

Thus, the device according to the invention can be used for the development and evaluation of the efficacy of therapeutic compounds, such as candidate therapeutic compounds.

Moreover, thanks to an increasingly precise profiling of the tumor (morphological/functional characteristics and molecular profile obtained by MRI-SRM with the device), the implementation of personalized therapy/precision medicine may be facilitated.

The inventors also carried out two other studies, the main objective of which was to evaluate and highlight the potential of implantable NMR micro-antennas in the acquisition of NMR spectra and MRI images on regions of interest of low volume (less than one microlitre) such as those concerned by the device according to the invention, in healthy rats and in rats bearing a cerebral tumour.

A first study concerns the design and implementation of an implantable NMR micro-antenna, with a detection volume of 850 nL, in healthy rats. NMR experiments, at a magnetic field of 7 T, were carried out in vitro and in vivo on the brains of healthy rats. The results obtained with the implantable NMR micro-antenna were compared with those obtained with a conventional external NMR antenna and showed a gain in sensitivity greater than 50 compared to the detection offered by the external antenna. NMR spectra were acquired with the NMR micro-antenna implanted in the right cortex of the healthy rat. Concentrations of major brain metabolites (Cho, Gin, Glu, Cr, ml, Tau, NAA, Lac) were quantified and compared reference values available in the literature. The results obtained show a significant similarity with respect to the reference values and thus testify to the efficiency and the minimally invasive character of the NMR micro-antenna.

A second study focused on the use of an implantable NMR micro-antenna, with a detection volume of 450 nL, for the study of tumor metabolism. A comparative study of the spectroscopic results obtained by the NMR micro-antenna implanted in the right cortex of the healthy rat on the one hand and in the brain tumor on the other hand for the pathological rat, was carried out, at a magnetic field of 7T. A decrease in the concentration of NAA and an increase in the amplitude of the resonance peak of the Lipid/Lactate complex were observed. The gain in sensitivity obtained by the use of implantable NMR micro-antennas allows the detection of metabolic variations characteristic of tumor metabolism on small volumes equivalent to those concerned in the device according to the invention.

Claims

1. Implantable magnetic resonance imaging or spectroscopy device (1, 100) comprising:

- means (3, 102) for administering a compound of interest, and - detection means (2, 101) comprising an implantable NMR micro-antenna; wherein the delivery means and the implantable NMR micro-antenna are adjacent and extend parallel along a longitudinal axis (X) of the implantable device, a distal end of said delivery means (7, 106) and said detection means (6, 105) forming an implantable end (5, 104) of said device.

2. Device according to claim 1, in which the administration means are chosen from the group consisting of a needle, a syringe, a cannula and a microdialysis probe, preferably a microdialysis probe.

3. Device according to claim 1 or 2, in which the NMR micro-antenna comprises at least one magnetic resonance circuit, and in which the distal end of the magnetic resonance circuit preferably has the general shape of a loop, U, twist or a helical shape.

4. Device according to one of claims 1 to 3, wherein the administration means comprise or consist of a microdialysis probe, a microdialysis membrane (10, 109) of said microdialysis probe forming the distal end of the means administration, the distal end of the NMR micro-antenna preferentially at least partially surrounding the microdialysis membrane of the microdialysis probe.

5. Device according to one of claims 1 to 4, further comprising an outer sleeve (4, 103) in which extend at least partially the administration means and the detection means.

6. Device according to one of the preceding claims, in which an external diameter of the implantable distal end of the device is less than or equal to 2 millimeters +/- 10%, preferably less than or equal to 1.5 millimeters +/- 10%, preferably between 0.5 and 1.5 millimeters +/- 10%, or in which an external diameter of the implantable end of the device is less than or equal to 500 micrometers +/- 10%, preferably between 50 and 500 micrometers +/- 10%.

7. Kit comprising the implantable imaging or spectroscopy device according to any one of claims 1-6, a matching-matching circuit capable of being placed at a distance from the magnetic resonance circuit of the NMR micro-antenna and optionally a compound of interest intended to be administered.

8. Ex vivo or in vitro use of an implantable device according to one of claims 1-6 or of the kit according to claim 7, to measure the biodistribution of a compound of interest in a biological sample and/or to evaluate the effect of a compound of interest on a biological sample, in particular on the cellular metabolism of the biological sample.

9. Process for in vitro or ex vivo molecular analysis in a biological sample, said process comprising:

- the introduction of a distal end of an implantable device according to any one of claims 1 to 6 into a biological sample; - the administration of a compound of interest by the means of administration of the implantable device;

- the detection of the administered compounds or their by-products in the biological sample by the implantable NMR micro-antenna;

- optionally molecular and structural analysis of the biological sample by spectroscopy or magnetic resonance imaging by the implantable NMR micro-antenna;

- optionally the recovery and analysis of a sample taken from the biological sample, said sample being taken by the implantable device.

10. Kit according to claim 7, use according to claim 8, or analysis method according to claim 9, in which the compound of interest is a therapeutic molecule or molecule for therapeutic purposes.