阅读说明：本技术 包含氘代2-氨基-2-甲基丙酸和/或2-(n-甲基氨基)-2-甲基丙酸用于肿瘤疾病的磁共振诊断的制剂及使用其的诊断方法 (Preparation for magnetic resonance diagnosis of tumor diseases comprising deuterated 2-amino-2-methylpropanoic acid and/or 2- (n-methylamino) -2-methylpropanoic acid, and diagnostic method using same ) 是由 A·V·莱西夫 P·E·伊瓦希金 M·V·古利亚耶夫 E·O·多洛菲娃 A·V·科森科夫 于 2017-11-21 设计创作，主要内容包括：本发明涉及医学,尤其涉及用于肿瘤疾病的磁共振诊断的试剂。为此目的开发了诊断制剂和基于该诊断制剂的诊断方法,其中2-氨基-2-甲基丙酸或2-(N-甲基氨基)-2-甲基丙酸和/或其药学上可接受的盐的氘代衍生物,或至少两种2-氨基-2-甲基丙酸和/或2-(N-甲基氨基)-2-甲基丙酸和/或其药学上可接受的盐的氘代衍生物的混合物被用作所述试剂。根据本发明的方法,在施用所述诊断试剂后足以使诊断制剂在受试者的肿瘤组织中蓄积以产生磁共振图像和/或磁共振频谱的时间,对氘核进行磁共振成像和/或磁共振波谱。所提出的方法能够有效诊断肿瘤疾病。(The invention relates to medicine, in particular to reagents for magnetic resonance diagnosis of neoplastic diseases. For this purpose, diagnostic preparations and diagnostic methods based on the diagnostic preparations have been developed, wherein 2-amino-2-methylpropanoic acid or 2- (N-methylamino) -2-methylpropanoic acid and/or deuterated derivatives of pharmaceutically acceptable salts thereof, or mixtures of at least two 2-amino-2-methylpropanoic acids and/or 2- (N-methylamino) -2-methylpropanoic acids and/or deuterated derivatives of pharmaceutically acceptable salts thereof are used as the agent. According to the method of the invention, magnetic resonance imaging and/or magnetic resonance spectroscopy of the deuterons is performed for a time sufficient to allow accumulation of the diagnostic agent in the tumor tissue of the subject to produce a magnetic resonance image and/or a magnetic resonance spectrum after administration of the diagnostic agent. The proposed method enables an effective diagnosis of tumor diseases.)

1. A diagnostic agent comprising 2-amino-2-methylpropionic acid and/or 2- (N-methylamino) -2-methylpropionic acid and/or a deuterated derivative of a pharmaceutically acceptable salt thereof, or a mixture of at least two different 2-amino-2-methylpropionic acids and/or 2- (N-methylamino) -2-methylpropionic acid and/or deuterated derivatives of a pharmaceutically acceptable salt thereof, for use in the diagnosis of a neoplastic disease by magnetic resonance imaging and/or magnetic resonance spectroscopy at deuterium frequencies.

2. The diagnostic reagent of claim 1, further comprising at least one pharmaceutically acceptable excipient.

3. Diagnostic agent according to claim 2, characterized in that the pharmaceutically acceptable excipient is a carrier, a filler and/or a solvent.

4. The diagnostic reagent of claim 1, whereinIn that the deuterated derivative of 2-amino-2-methylpropionic acid is 2-amino-2- (CD)₃)-3,3,3-D₃-propionic acid or 2-amino-2- (CD)₂H)-3,3,3-D₃-propionic acid or 2-amino-2- (CD)₂H)-3,3,3-D₃-propionic acid or 2-amino-2-methyl-3, 3,3-D₃-propionic acid or 2-amino-2- (CD)₂H)-3,3-D₂-propionic acid or 2-amino-2- (CDH)₂)-3,3-D₂-propionic acid or 2-amino-2-methyl-3, 3-D₂-propionic acid or 2-amino-2- (CDH)₂) -3-D-propionic acid or 2-amino-2-methyl-3-D-propionic acid.

5. The diagnostic drug of claim 1, wherein the deuterated derivative of 2- (N-methylamino) -2-methylpropanoic acid is 2-methylamino-2- (CD)₃)-3,3,3-D₃-propionic acid or 2- (N- (CD)₃) Amino) -2-methylpropanoic acid or 2- (N- (CD)₃) Amino) -2- (CD₃)-3,3,3-D₃-propionic acid.

6. The diagnostic reagent of claim 1, comprising a mixture of at least two different deuterated derivatives of 2-amino-2-methylpropionic acid and/or 2-amino 2- (N-methylamino) -2-methylpropionic acid selected from the group consisting of: 2-amino-2- (CD)₃)-3,3,3-D₃-propionic acid and/or 2-amino-2- (CD)₂H)-3,3,3-D₃-propionic acid and/or 2-amino-2- (CD)₂H)-3,3,3-D₃-propionic acid and/or 2-amino-2-methyl-3, 3,3-D₃-propionic acid and/or 2-amino-2- (CD)₂H)-3,3-D₂-propionic acid and/or 2-amino-2- (CDH)₂)-3,3-D₂-propionic acid and/or 2-amino-2-methyl-3, 3-D₂-propionic acid and/or 2-amino-2- (CDH)₂) -3-D-propionic acid or 2-amino-2-methyl-3-D-propionic acid and/or 2-methylamino-2- (CD)₃)-3,3,3-D₃-propionic acid and/or 2- (N- (CD)₃) Amino-2-methylpropanoic acid and/or 2- (N- (CD)₃) Amino) -2- (CD₃)-3,3,3-D₃-propionic acid.

7. The diagnostic reagent of claim 1, further comprising non-deuterated 2-amino-2-methylpropanoic acid or 2- (N-methylamino) -2-methylpropanoic acid.

8. Diagnostic reagent according to claim 1, characterized in that the deuterated derivative of 2-amino-2-methylpropanoic acid or 2- (N-methylamino) -2-methylpropanoic acid and/or of a pharmaceutically acceptable salt thereof comprises, in addition to the deuterium atom attached to the carbon atom, a deuterium atom partially or completely replacing a dissociable hydrogen atom attached to an oxygen and/or nitrogen atom.

9. A method of diagnosing a neoplastic disease comprising the steps of:

a) administering a diagnostic agent of claim 1 to a subject;

b) performing a magnetic resonance imaging study and/or magnetic resonance spectroscopy at the frequency of deuterons after a time sufficient for the diagnostic agent to accumulate in the tumor after administration of the diagnostic drug to obtain an MR image and/or an NMR spectrum, respectively;

c) the presence or absence of a tumor disease is diagnosed based on the observed signal intensity of deuterons, reflecting the level of accumulation of the diagnostic agent.

10. The method of claim 9, wherein the subject is diagnosed as not having cancer in the absence of the accumulation region of the diagnostic agent.

11. The method according to claim 9, characterized in that at least one further medical study is performed, selected from magnetic resonance imaging at a frequency of nuclei other than deuterium, or ultrasound, or computed tomography, or X-ray, or palpation, or biopsy, or tumor marker screening of biological material, or radionuclide diagnosis or physical examination.

12. The method of claim 8, wherein the presence or absence of a neoplastic disease is diagnosed based on a comparison of the signal intensity of deuterons in the examined subject with typical signal intensities observed in corresponding tissues or organs in healthy subjects.

13. The method of claim 11, wherein the presence or absence of a neoplastic disease is diagnosed based on a comparison of signal intensities of deuterons in a region corresponding to normal tissue and pathological tissue in accordance with additional medical studies.

14. The method of claim 11, wherein the method is based on¹H MRI, based on a comparison of the signal intensities of deuterons in neighboring voxels located on different sides of the interface between normal and suspect tissue, to diagnose the presence or absence of a tumor disease.

15. The method of claim 11, wherein the presence or absence of a tumor disease is diagnosed based on a comparison of a deuterium image to a 1H magnetic resonance image from the same subject.

16. The method according to claim 9, characterized in that the intensity of the deuterium signal and/or its variation over time allows conclusions to be drawn on the structure, malignancy grade, invasiveness or differentiation degree of the tumor.

17. The method of claim 9, wherein the neoplastic disease is breast cancer, glioma.

18. The method according to claim 9, characterized in that during the registration of the images, the selective excitation of deuterons contained in the mixture of 2-amino-2-methylpropanoic acid or 2- (N-methylamino) -2-methylpropanoic acid or deuterated derivatives of pharmaceutically acceptable salts thereof, or 2-amino-2-methylpropanoic acid and/or 2- (N-methylamino) -2-methylpropanoic acid and/or deuterated derivatives of pharmaceutically acceptable salts thereof is used.

19. The method according to claim 9, characterized in that during the registration of the images a broadband excitation of the deuterons contained in the mixture of 2-amino-2-methylpropanoic acid or 2- (N-methylamino) -2-methylpropanoic acid or a deuterated derivative of a pharmaceutically acceptable salt thereof, or 2-amino-2-methylpropanoic acid and/or 2- (N-methylamino) -2-methylpropanoic acid and/or a deuterated derivative of a pharmaceutically acceptable salt thereof is used.

20. The method of claim 9, wherein the diagnostic agent is administered orally to the subject.

21. The method of claim 9, wherein the diagnostic agent is administered parenterally to the subject.

22. The method of claim 9, wherein the magnetic resonance tomography and/or magnetic resonance spectroscopy on deuterons is performed within 20-360 minutes after administration of the diagnostic agent.

23. The method of claim 9, wherein the diagnostic agent is administered to the subject in an amount equivalent to 0.25-1g 2-amino-2-methylpropionic acid and/or 2- (N-methylamino) -2-methylpropionic acid and/or a deuterated derivative of a pharmaceutically acceptable salt thereof, or a mixture of 2-amino-2-methylpropionic acid and/or 2- (N-methylamino) -2-methylpropionic acid and/or a deuterated derivative of a pharmaceutically acceptable salt thereof per kilogram body weight of the subject.

Technical Field

The present invention relates to medicine, in particular to an apparatus for magnetic resonance imaging in oncology.

Background

Cancer diagnosis, including early detection, is a major focus of global healthcare. One of the powerful diagnostic methods for such diseases is Magnetic Resonance Imaging (MRI).

The majority of MRI nmr for clinical applications is based on proton magnetic resonance signals: (¹H nuclei) is detected, and the detection is carried out,¹the H nucleus is a portion of the water molecule in the human body.¹H MRI provides a high degree of anatomical detail and in many cases can detect regions of abnormal signal corresponding to tumors. At the same time, it is known from clinical practice that MRI is not always able to distinguish malignant tumors from benign tumors or tumors that do not require urgent treatment (method specificity is low). In this case, early diagnosis of tumor diseases is also hampered due to the high risk of false positive results.

Increase of¹H MRI diagnosisThe main method of outliers is the use of contrast agents that change the signal parameters in the vicinity [ subject of current chemistry, contrast agent I, magnetic resonance imaging, editing: krause, Werner,2002 (Topics in Current Chemistry, Contrast Agents I, Magnetic Resonance Imaging, Editors: Krause, Werner,2002)]. Various contrast agents are used in MRI diagnosis, including those commercially available

Andthey are gadolinium complexes, andandthey are stable aqueous suspensions of magnetic nanoparticles. These substances are injected into the blood of a patient and the degree of blood supply in the region suspected of having a malignant tumor is evaluated.

With contrast agents¹Alternative methods for H MRI are the recording of other nuclear signals, in particular³¹P、¹³C、¹⁹F、²H、²³And (4) Na. One of these nuclei is deuterium (²H) In that respect This nonradioactive isotope of hydrogen, which has a natural content of 0.0156% in biological objects, has a sensitivity several times lower than that of protons.

So far, in vivo use has been described²H NMR and/or²Several cases of H MRI. Document US20030211036a1 proposes a method for measuring the perfusion of tumor tissue using isotopically labeled compounds, including deuterated compounds.

Document US5042488 demonstrates the detection of background deuterium signal and the injection of D in vivo (in rat liver)₂Signals generated by O or 1-deuterated glucose are possible. Note that the invention may also be practiced using other blood flow indicators labeled with deuterium.

Document US20100322865a1 describes a method for the production of a shoe by means of a shoeLine of²H-MRI to assess metabolic rate using metabolic precursors of water. 1,2,3,4,5,6, 6-deuterated glucose is an example of a precursor of HOD metabolism. Within the described invention, only the deuterium nuclear magnetic resonance signals of the aliphatic chains of metabolic water and fatty acids are recorded, but not the deuterium glucose.

None of the above methods has been used in practice for the diagnosis of neoplastic diseases, largely due to the need to use very large doses of deuterated compounds.

Despite the excellent performance of the current forms of MRI, there is still a need to develop new, more effective methods for MRI diagnosis of neoplastic diseases.

Disclosure of Invention

The object of the present invention is to develop new effective diagnostic agents for the diagnosis of tumor diseases by MRI and/or MR spectroscopy, as well as diagnostic methods comprising the use of specific agents.

The present invention relates to the development of novel effective diagnostic reagents useful in the diagnosis of neoplastic diseases, particularly breast cancer. Another technical result of the present invention is the development of a new oncologically efficient and informative diagnostic method by means of magnetic resonance imaging and/or magnetic resonance spectroscopy at the frequency of deuterons, which method comprises the administration of a diagnostic agent according to the invention, said agent being capable of accumulating in the tumor in sufficient concentration to record an in vivo deuterium MR image or to record an in vivo deuterium MR image²H-NMR spectrum.

The diagnostic reagents according to the invention are characterized by the following combination of properties: high content of deuterium atoms; accumulate in the tumor within a reasonable time to a concentration sufficient for diagnosis; the low toxicity and intact agent is almost completely excreted from the body. This allows effective diagnosis using a dose that is not harmful to the human body.

The method of the invention is independent of any detrimental effect of ionizing radiation (typically, for example, for CT, PET, SPECT methods), which in turn increases the safety of the study and allows studies to be repeated more frequently and to be adapted to pediatric patients. The present invention aims to obtain diagnostic information similar to Positron Emission Tomography (PET), but unlike the latter, it can eliminate the risks associated with ionizing radiation of radiopharmaceuticals.

The invention relates to the development of diagnostic reagents comprising at least two different mixtures of 2-amino-2-methylpropanoic acid and/or 2- (N-methylamino) -2-methylpropanoic acid and/or deuterated derivatives of pharmaceutically acceptable salts thereof or 2-amino-2-methylpropanoic acid and/or 2- (N-methylamino) -2-methylpropanoic acid and/or deuterated derivatives of pharmaceutically acceptable salts thereof for the diagnosis of tumor diseases by means of magnetic resonance imaging and/or magnetic resonance spectroscopy at the frequency of deuterons.

In a particular embodiment of the invention, the diagnostic reagent additionally comprises at least one additional pharmaceutically acceptable component. In a particular embodiment of the invention, the pharmaceutically acceptable component is a carrier, filler and/or solvent.

In a particular embodiment of the invention, the deuterated derivative of 2-amino-2-methylpropionic acid is 2-amino-2- (CD)₃)-3,3,3-D₃-propionic acid or 2-amino-2- (CD)₂H)-3,3,3-D₃-propionic acid or 2-amino-2- (CDH2) -3,3, 3-D3-propionic acid or 2-amino-2-methyl-3, 3, 3-D3-propionic acid or 2-amino-2- (CD)₂H)-3,3-D₂-propionic acid or 2-amino-2- (CDH)₂)-3,3-D₂-propionic acid or 2-amino-2-methyl-3, 3-D₂-propionic acid or 2-amino-2- (CDH)₂) -3-D-propionic acid or₂-amino-2-methyl-₃-D-propionic acid.

In a particular embodiment of the invention, the deuterated derivative of 2- (N-methylamino) -2-methylpropionic acid is 2- (N-methylamino) -2- (CD)₃)-3,3,3-D₃-propionic acid or 2- (N- (CD)₃) Amino) -2-methylpropanoic acid or 2- (N- (CD)₃) Amino) -2- (CD₃)-3,3,3-D₃-propionic acid.

In other particular embodiments of the invention, the diagnostic agent is a mixture of at least two different deuterated derivatives of 2-amino-2-methylpropionic acid and/or 2- (N-methylamino) -2-methylpropionic acid selected from the group consisting of: 2-amino-2- (CD)₃)-3,3,3-D₃-propionic acid and/or 2-amino-2- (CD)₂H)-3,3,3-D₃-propionic acid and/or 2-amino-2- (CD)₂H)-3,3,3-D₃-propionic acid and/or 2-amino-2-methyl-3, 3,3-D₃-propionic acid and/or 2-amino-2- (CD)₂H)-3,3-D₂-propionic acid and/or 2-amino-2- (CDH)₂)-3,3-D₂-propionic acid and/or 2-amino-2-methyl-3, 3-D₂-propionic acid and/or 2-amino-2- (CDH)₂) -3-D-propionic acid or 2-amino-2-methyl-3-D-propionic acid and/or 2-methylamino-2- (CD)₃)-3,3,3-D₃-propionic acid and/or 2- (N- (CD)₃) Amino-2-methylpropanoic acid and/or 2- (N- (CD)₃) Amino) -2- (CD₃)-3,3,3-D₃-propionic acid.

In a particular embodiment of the invention, the diagnostic reagent of the invention optionally comprises non-deuterated 2-amino-2-methylpropionic acid or 2- (N-methylamino) -2-methylpropionic acid.

In a particular embodiment of the invention, the deuterated derivative of 2-amino-2-methylpropionic acid or 2- (N-methylamino) -2-methylpropionic acid comprises, in addition to the deuterium atom bound to a carbon atom, a deuterium atom partially or completely replacing a mobile hydrogen atom associated with an oxygen atom and/or a nitrogen atom.

The invention also includes a method of obtaining a diagnostic agent according to the invention.

The invention comprises the implementation of a method for diagnosing a tumor disease, comprising the following steps:

a) administering a diagnostic agent according to the invention to a subject;

b) deuterium tomography and/or NMR spectroscopy, magnetic resonance imaging and/or magnetic resonance spectroscopy to produce the respective deuterium ions at the frequency of the deuterium ions after a time sufficient to allow accumulation of 2-amino-2-methylpropionic acid and/or a deuterated derivative of 2- (N-methylamino) -2-methylpropionic acid and/or a pharmaceutically acceptable salt thereof or a mixture of at least two different 2-amino-2-methylpropionic acids and/or a deuterated derivative of 2- (N-methylamino) -2-methylpropionic acid and/or a pharmaceutically acceptable salt thereof in the tumour tissue²H-MR image and/or²H-NMR spectrum;

c) diagnosing the presence or absence of a neoplastic disease based on the observed intensity of the deuteron signal, reflecting the level of accumulation of 2-amino-2-methylpropanoic acid and/or 2- (N-methylamino) -2-methylpropanoic acid and/or a deuterated derivative of a pharmaceutically acceptable salt thereof or a mixture of at least two different 2-amino-2-methylpropanoic acids and/or 2- (N-methylamino) -2-methylpropanoic acids and/or deuterated derivatives of a pharmaceutically acceptable salt thereof.

In a particular embodiment of the invention, the subject is diagnosed as having no cancer in the absence of a diagnostic agent accumulation region.

In a particular embodiment of the invention, at least one further medical study is performed, selected from magnetic resonance imaging and/or ultrasound and/or computed tomography and/or X-ray and/or palpation and/or biopsy and/or tumour marker analysis of biological fluids and/or radionuclide diagnostics and/or physical examinations using nuclei other than deuterium.

In a particular embodiment of the invention, the presence or absence of a neoplastic disease is diagnosed based on a comparison of the signal intensity of deuterons with typical signal intensities observed in corresponding tissues or organs in healthy subjects.

In a particular embodiment of the invention, the presence or absence of a neoplastic disease is diagnosed based on a comparison of the signal intensity of deuterons in the region corresponding to normal and abnormal tissue, according to additional medical studies.

In a particular embodiment of the invention, the MR image and the MR image are based on¹Comparison of images obtained by H-MRI to diagnose the presence or absence of a neoplastic disease.

In a particular embodiment of the invention, conclusions are drawn about the malignancy or aggressiveness or the degree of differentiation of the tumor based on the observed signal intensity of the deuterons.

In a particular embodiment of the invention, the tumor disease diagnosed is breast cancer, glioma.

In a particular embodiment of the invention, during the registration of the deuterium MR images, a selective excitation of deuterium comprised in a mixture of 2-amino-2-methylpropionic acid and/or 2- (N-methylamino) -2-methylpropionic acid and/or a deuterated derivative of a pharmaceutically acceptable salt thereof or of 2-amino-2-methylpropionic acid and/or 2- (N-methylamino) -2-methylpropionic acid and/or a deuterated derivative of a pharmaceutically acceptable salt thereof is used.

In a particular embodiment of the invention, a broadband excitation of deuterium comprised in a mixture of 2-amino-2-methylpropionic acid and/or 2- (N-methylamino) -2-methylpropionic acid and/or deuterated derivatives of pharmaceutically acceptable salts thereof or 2-amino-2-methylpropionic acid and/or 2- (N-methylamino) -2-methylpropionic acid and/or deuterated derivatives of pharmaceutically acceptable salts thereof is used during the registration of the deuterium MR images.

In a particular embodiment of the invention, the diagnostic agent is administered orally to the subject.

In other particular embodiments of the invention, the diagnostic agent is administered parenterally to the subject.

In a particular embodiment of the invention, magnetic resonance imaging and/or magnetic resonance spectroscopy on deuterons is performed 20-360 minutes after administration of the diagnostic agent.

In a particular embodiment of the invention, the diagnostic agent is administered to the subject in an amount equivalent to 0.25-1g of 2-amino-2-methylpropionic acid and/or 2- (N-methylamino) -2-methylpropionic acid and/or a deuterated derivative of a pharmaceutically acceptable salt thereof, or a mixture of 2-amino-2-methylpropionic acid and/or 2- (N-methylamino) -2-methylpropionic acid and/or a deuterated derivative of a pharmaceutically acceptable salt thereof per kilogram of body weight of the subject.

The invention also comprises the use of the diagnostic agent according to the invention for the diagnosis of tumor diseases by magnetic resonance imaging and/or magnetic resonance spectroscopy at deuterium frequencies.

Brief description of the drawings

FIG. 1 shows a cross-sectional view of a CD having 2-amino-2- (CD)₃)-3,3,3-D₃Of samples of propionic acid²Optical spectrum

Figure 2. deuterium MR images of samples containing diluted solutions of deuterated diagnostic agents. (a) Broadband excitation; (b) with 2-amino-2- (CD)₃)-3,3,3-D₃-selective excitation of the frequency of propionic acid;

FIG. 3 administration of 20mg 2-amino-2- (CD)₃)-3,3,3-D₃MR images of mouse No. 1 with breast cancer of 4t1 after 40min of propionic acid:

a)²h Mr (the position of the surface coil is shown in white outline);

b)¹H MRI；

c) was performed.

FIG. 4. after administration of 20mg of 2-amino-2- (CD)₃)-3,3,3-D₃MR images of no tumor 2 mice after 40min of propionic acid:

a)²H MRI；

b)¹H MRI；

FIG. 5 administration of 20mg 2-amino-2- (CD)₃)-3,3,3-D₃MR images of mouse No. 3 with breast cancer of 4t1 after 115min of propionic acid:

a)²H MRI；

b)¹H MRI；

c) was performed.

FIG. 6 injection of 20mg 2-amino-2- (CD)₃)-3,3,3-D₃Tumor recovery from mouse No. 3 within 150 minutes after propionic acid²H MR image (a) and photograph (b).

FIG. 7 injection of 20mg 2-amino-2- (CD)₃)-3,3,3-D₃MR images of mouse No. 4 with breast cancer of 4t1 after 20min of propionic acid:

a)²H MRI；

b)¹H MRI；

c) was performed.

FIG. 8 is a schematic representation of the administration of 20mg of 2-amino-2- (CD)₃)-3,3,3-D₃MR images of mouse No. 4 with breast cancer of 4t1 after 360min of propionic acid.

a)²H MRI；

b)¹H MRI；

C) was performed.

FIG. 9 administration of 10mg 2-amino-2- (CD)₃)-3,3,3-D₃MR images of mouse No. 5 with breast cancer of 4t1 after 30min of propionic acid:

a)²H MRI；

b)¹H MRI；

c) was performed.

FIG. 10 administration of 5mg 2-amino-2- (CD)₃)-3,3,3-D₃-propionic acid 30miAfter n, MR images of mouse No. 6 with 4-t 1 breast cancer:

a)²H MRI；

b)¹H MRI；

c) was performed.

FIG. 11 administration of 150mg 2-amino-2- (CD)₃)-3,3,3-D₃MR images of C6 glioma-bearing rats 3 hours after propionic acid:

a)²H MRI；

b)¹H MRI；

c) was performed.

FIG. 12 is a schematic representation of the administration of 150mg of 2-amino-2- (CD)₃)-3,3,3-D₃MR images of tumor-free control rats 3 hours after propionic acid:

a)²H MRI；

b)¹H MRI。

definitions and terms

For a better understanding of the present invention, the following are some terms used in the description of the present invention.

In the description of the present invention, the terms "comprise" and "comprise" are to be interpreted as meaning "including, among others". These terms should not be construed as "consisting only of.

As used herein, the term "deuterated derivative" refers to a compound containing deuterium bound to carbon at least at one position, the deuterium content exceeding its natural content. In particular embodiments of the invention, the deuterium content in at least one position is more than 30%, in other particular embodiments 90%. A mixture of at least two different deuterated derivatives refers to a compound comprising deuterium at different positions of the molecule or a mixture of compounds comprising different amounts of deuterium at the same position. As used herein, the symbol "D" means enriched relative to its natural content²Hydrogen atom of the H isotope.

Herein, the term "voxel" refers to a volume element in a sample that can be freely selected by adjusting the magnetic field parameters and generates a nuclear magnetic resonance signal.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt that, according to medical conclusions, is suitable for contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, and the like, and indicates a reasonable risk and benefit ratio. Pharmaceutically acceptable salts of amines, carboxylic acids, phosphonates and other types of compounds are well known in medicine. Salts may be prepared in situ during isolation or purification of the compounds of the invention and may be obtained separately by reacting the free acid or free base compound of the invention with the appropriate base or acid, respectively. Examples of pharmaceutically acceptable non-toxic salts of acids include salts of the amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, succinic acid or malonic acid, or obtained by other methods used in the art, for example, using ion exchange. Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorcarboxylates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, formates, fumarates, glucoheptonates, glycerophosphates, gluconates, hemisulfates, heptanoates, hexanoates, hydroiodides, 2-hydroxy-ethanesulfonates, lactobionates, lactates, laurates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectinates, persulfates, 3-phenylpropionates, phosphate esters, Picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate and the like. Typical salts of alkali and alkaline earth metals include sodium, lithium, potassium, calcium, magnesium, and the like. In addition, pharmaceutically acceptable salts can contain, if desired, nontoxic cations of ammonium, quaternary ammonium, and amines, obtained using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkane sulfonates, and aromatic sulfonates.

The diagnostic agent according to the present invention may comprise one or more pharmaceutically acceptable excipients suitable for the particular dosage form, in particular any carrier, solvent and/or excipient, which may be introduced into the patient together with the compound constituting the essence of the invention, and which does not negatively affect the deuterated compound and is non-toxic when administered.

Detailed disclosure of the invention

For successful implementation and use²H MRI or²H NMR diagnostic methods for neoplastic diseases require that deuterium concentrations in the tumor tissue be sufficiently high. To satisfy this condition, the diagnostic reagent:

1) should rapidly and selectively accumulate in tumor tissue (especially should have a sufficiently efficient membrane transport mechanism);

2) the expulsion should be characterized as being rather slow (which is a massive accumulation of the drug in the tumour and²extended registration of H MR images provides sufficient time);

3) should not be significantly metabolized (minimize possible side effects, including incorporation of deuterium into the biomolecule, and allow repeated examination within hours after the previous dose without changing background signals from the tumor);

4) low toxicity at the desired concentration (a sufficiently large dose of the agent can be administered);

5) deuterium should be contained in large amounts (needed to achieve sufficient signal intensity).

The authors of the present invention have unexpectedly found that the deuterated derivatives of 2-amino-2-methylpropionic acid and/or 2- (N-methylamino) -2-methylpropionic acid according to the invention are capable of accumulating in tumor tissue in sufficient concentration to pass through²H MRI method or²H NMR in vivo visualization allows for efficient diagnosis of tumor disease by magnetic resonance imaging on deuterons.

Due to the low deuterium content (0.015% of hydrogen atoms) in vivo²Background signal ratio in H MRI¹The background signal in H MRI is several orders of magnitude lower. Thus, even at low concentrations of diagnostic reagent, the signal does not overlap with the natural background signal component. Based on¹Analogy to H MRI using non-deuterated diagnostic agentsThe development of the method is very complex because there is a large background signal of natural low molecular weight compounds, whose intensity is comparable to the maximum signal intensity achievable with non-deuterated diagnostic reagents. At the same time, the presence of background HOD signals places a limit on the minimum acceptable concentration of H MRI of tumor diagnostic agents. The practical applicability of the diagnostic method according to the invention depends on the pharmacokinetics and pharmacodynamics of the particular diagnostic agent.

The ability to register deuterium signals in vivo also depends on whether a sufficient number of deuterium atoms are present in the compound structure. Thus, the diagnostic reagent according to the present invention comprises one or more CDs₃Deuterated 2-amino-2-methylpropanoic acid and/or 2- (N-methylamino) -2-methylpropanoic acid of the group are preferred embodiments of the present invention. The presence of such deuterated groups allows MRI scans to be performed using lower concentrations of diagnostic agents, thereby minimizing side effects.

The method of the invention allows diagnosing the presence or absence of a tumor disease. The method according to the invention is based on the use of deuterated diagnostic agents and the registration of MRI and/or NMR spectra at deuterium frequencies.

As is well known in the art,¹h MRI by itself is not sufficiently accurate in diagnosis in many cases, and the method of the present invention provides information about the transport of molecules of 2-amino-2-methylpropanoic acid and/or 2- (N-methylamino) -2-methylpropanoic acid, which is not available in the traditional mode of 1H MRI, and therefore more accurate diagnostic information is possible.

In one of the embodiments of the invention, the diagnostic procedure involves MRI and is performed as follows:

a) in some embodiments of the invention, execution is performed¹H MRI。¹Registration of H MRI may firstly establish an anatomical frame of reference for the deuterium signal, and secondly identify regions with suspicious malignancy (in other embodiments of the invention,²the definition of the field of view of H MRI can be performed in other ways, in particular by ultrasound, computed tomography, radiography, palpation, biopsy, tumor marker analysis of biological fluids, radionuclide diagnostics, physical examination);

b) administering a diagnostic agent;

c) after a time sufficient for the diagnostic agent to accumulate in the tumor tissue of the subject,²the HMR images are registered at the precession frequency of the diagnostic agent deuterons;

d) the obtained deuterium MR images are analyzed to find areas of abnormally high intensity, which is therefore the cause of the accumulation of diagnostic agents. In particular, can be compared in¹H and²image obtained on H: if it is not¹H and²an abnormal region on H matches, meaning that the probability of developing a malignant tumor is higher. However, it is possible to use a single-layer,¹whether there is an anomaly on the H-image is not a prerequisite: in some cases, by¹Tumors were not visible on the images obtained by H MRI, but passed²Accumulation of the diagnostic agent was observed on the image obtained by H MRI. In the latter case, the first and second parts of the device,¹h MRI is used only for anatomical reference of suspicious regions.

In another particular embodiment of the invention, the diagnostic procedure comprises NMR spectroscopy at the frequency of deuterium and is performed as follows:

a) to carry out¹H MRI to identify regions with suspected malignancy (in other embodiments of the invention, this may be done in other ways²Definition of the H NMR region, in particular by ultrasound, computed tomography, radiography, palpation, biopsy, tumor marker analysis of biological fluids, radionuclide diagnostics, physical examination);

b) administering a diagnostic agent;

c) after a period of time sufficient for the diagnostic agent to accumulate in the tumor tissue of the subject, the deuterium spectra (particularly using local spectroscopy) are registered (e.g., based on)¹Results of H MRI); optionally, registering the spectra in neighboring voxels or comparing signal intensities; in particular embodiments, spectroscopy may be performed using a transmit coil, a transmit and receive coil, a volume coil, an implant coil, or a surface coil;

d) the signal intensity in the voxels corresponding to the suspected malignant tumor region is compared to: (i) typical values for this tissue (should be in healthy subjects)First defined) and/or (ii) correspond to the adjacency of the same tissue or organ and are in accordance with¹HMRI has no abnormal voxel intensities. An increase in signal intensity suggests accumulation of the diagnostic agent and the presence of a malignant tumor.

In the two particular embodiments of the invention described above, the sequence of stages a), b), c) may be different, for example, by first administering a diagnostic agent¹H MRI, then²H MRI or NMR spectroscopy; or¹H MRI is that²H MRI or²H NMR spectroscopy was followed.

In a particular embodiment of the invention, after determining a region with a suspected malignancy, a single voxel is selected that is inside and outside the suspicious region (in particular, a series of neighboring voxels that lie on the same line that crosses the boundary of the suspicious region may be selected). Registration in selected voxels²H or²The integrated signals of the H spectrum are then compared²The intensity in the H channel allows for rapid and sensitive detection of the accumulation of diagnostic agents.

The MR images and MR spectra according to the invention can be acquired using any MRI scanner equipped for registering deuterium signals.

In a particular embodiment of the invention, the use of a diagnostic agent that gives a signal in the region where there is no background HOD signal allows MRI to be performed by using selective excitation pulses configured at the frequency of the diagnostic agent. This eliminates the background signal of the HOD on the image.

By using the molecular mechanism of transport and accumulation of the diagnostic agent of the invention in cells, the method according to the invention enables the assessment of the metabolic activity of the tissue under investigation and thus the malignancy or invasiveness of the tumor. Thus, it is compared with the conventional¹The diagnostic potential of the method is improved compared to MRI methods based on perfusion assessment (including typical contrast agents).

The signals of the diagnostic agent according to the invention can be registered up to 6 hours after administration and the distribution of the agent in the tumor and other organs changes during this time. Thus, the signal of the diagnostic agent is mainly manifested in the liver and kidney, and then in certain areas of the tumor, possibly corresponding to the areas of most active growth and best blood supply. Maximal signal of deuterium throughout the tumor was observed within two hours after intraperitoneal injection, and then the distribution pattern of the diagnostic agent was constantly changing. Due to this behavior, repeated imaging within hours after administration of the diagnostic agent according to the invention allows to obtain the membrane transport rate and the perfusion level of different parts of the tumor, which in turn gives information about the structure and type of the tumor.

The experiments carried out by the authors prove that²Accumulation of the agents of the invention in tumor tissue is selective compared to brain, skeletal muscle and other organs and tissues under conditions where H MRI doses are acceptable.

2-amino-2-methylpropanoic acid and 2- (N-methylamino) -2-methylpropanoic acid are known to be non-protein amino acids, so their use does not lead to long-term fixation of deuterium in proteins. 2-amino-2-methylpropionic acid and 2- (N-methylamino) -2-methylpropionic acid are not metabolized to form deuterated cofactors or other metabolites that participate in the basic biochemical process. It is known from the prior art that the presence of deuterium can significantly alter the rate of enzymatic reactions, which in turn can lead to accumulation of toxic intermediates and other metabolic changes. Therefore, insufficient metabolism of the diagnostic agent according to the present invention is a factor to reduce the possibility of side effects. The experiments carried out showed that, irrespective of the mode of administration, the diagnostic reagent according to the invention has no metabolic transformation (blood, urine and in vivo)²No new signal in H NMR spectrum). Thus, the observed kinetics of accumulation of the agent in the tumor and its subsequent elimination depend only on the transport rate between the various tissues and the blood and are not affected by metabolic processes.

Studies carried out by the authors have shown that animals have good tolerance to diagnostic agents, have no significant side effects when used at the indicated doses, and can be completely cleared of deuterium containing compounds from the body within hours. Therefore, no animal death was observed after intraperitoneal injection of the drug according to the present invention to mice at a dose of 8g/kg, and within 72 hours after administration according to²H magnetic resonance imaging in tumorsNot observed in (1). Background concentrations of deuterium in tumors and other tissues remained the same, indicating that there was no long-term accumulation of the inventive drug in vivo. After complete removal of deuterium from the tumor tissue, the diagnosis can be repeated after 72 hours and the dynamics of tumor development monitored during the course of treatment.

Experiments have shown that the results of tumor visualization depend to a large extent on the dosage of the diagnostic agent. A smaller dose allows selective visualization of the most strongly absorbed tumor fraction, while an increased dose allows the deuterium signal to fill the tumor boundary more completely. Due to this property of the diagnostic agent according to the invention, it is possible to perform dynamic studies (multiple registration of images) while the concentration of the agent in the blood gradually increases over time (e.g. slow intravenous infusion or a series of consecutive injections of small doses of the agent). These studies can provide information about the metabolic activity at different sites of the tumor and the extent of the tumor disease.

The method of the invention is free of any deleterious effects of ionizing radiation (typically, for example, for CT, PET, SPECT methods), which in turn increases the safety of the study and allows repeated studies to be performed more frequently and makes the method suitable for pediatric patients.

The diagnostic method according to the invention can be applied, in particular, to the early diagnosis of tumors in different regions, to metastatic lesions, to the assessment of the response of tumors to therapy and to conclusions about the efficacy of therapy, in order to confirm the basis¹H MRI results and/or other diagnostic methods.

The method according to the invention can be used for the diagnosis of various tumors, in particular breast tumors and gliomas.

The method of the present invention extends existing oncology diagnostic methods and enables an effective diagnostic modality.

Practice of the invention

The reliable data presented in the examples demonstrate the possibility of objectively embodying the technical result when using the invention, including experimental information obtained during the study of the methods employed in this field. The invention is illustrated by the accompanying drawings.

It should be understood that these and all examples given in the application material are not limiting and are provided only to illustrate the present invention.

The examples given herein illustrate the principles of the developed method and do not limit the range of doses used and the time interval between administration of the diagnostic reagent and detection of deuterium, as this may vary depending on the sensitivity and other parameters of the equipment used, the disease diagnosed and the nature of the subject (human or laboratory animal). Furthermore, the registration parameters of the spectra and images, including the signal accumulation times, are part of a particular embodiment of the invention and may vary depending on the equipment used and the particular diagnostic task.

2-amino-2- (CD)₃)-3,3,3-D₃Synthesis of propionic acid

1.9g of acetone-d at 0-5 deg.C₆A solution in 5ml of diethyl ether was added to 2.0g of ammonium chloride in 5ml of D₂In solution in O. Then 1.6 g NaCN was slowly added to 3.5ml D₂And (4) in O. The reaction mixture was stirred for 1 hour and then left overnight. The ether layer was separated and the aqueous layer was extracted with six 3ml portions of ether. The combined ether extracts were evaporated and the residue was dissolved in 8ml of methanol. The resulting solution was saturated with gaseous ammonia and allowed to stand for 48 hours. The reaction mixture was evaporated, 6ml of water and 10ml of 48% hydrobromic acid were added to the residue, after which the mixture was boiled for 2 hours and then evaporated in vacuo, after which 5ml of water were added to the dried residue and evaporated again. The residue after evaporation was dissolved in 15ml of methanol and filtered. To the resulting solution was added 3ml of pyridine. Crystallizing 2-amino-2- (CD) within 10 hours₃)-3,3,3-D₃The propionic acid is filtered off, washed with methanol and dried in vacuo. Yield 1.1 g (35%)

¹H NMR(D₂O): 1.30 (residual signal of methyl protons).

¹³C NMR(D₂O)：178.1,23.5.

If D is to be₂O is used for hydrolysis and the final product is retained at D₂In O, followed by evaporation, the amino and carboxyl moieties are obtainedDeuterated 2-amino-2- (CD) with partial or complete deuteration₃)-3,3,3-D₃-propionic acid.

(example 1) shows the possibility of detecting a deuteron magnetic resonance image of a sample containing a deuterated 2-amino-2-methylpropionic acid solution using a broadband and selective radio frequency pulse.

In vivo experiments (examples 2-3) demonstrated the possibility of recording deuterium MR images and NMR spectra in vivo, as well as the ability of deuterated derivatives of 2-amino-2-methylpropionic acid to accumulate in tumors. Based on the observed signals of deuterated derivatives of 2-amino-2-methylpropionic acid, the possibility of diagnosing tumors, in particular breast tumor 4T1 and glioma C6, was shown.

In the following example, the MRI scanner Bruker BioSpec BC70/30USR has a constant magnetic field of 7.05T, equipped with surface transmit and receive coils of 3cm diameter and a scan depth of about 1 cm.

FLASH (fast low angle shot) pulse sequences were used to register the deuterium images.

For the broadband excitation experiments, the following settings were used: excitation frequency of²H NMR spectroscopy determined sfo1 ≈ 46.1745MHz, rectangular excitation pulse with a width of 1300Hz and a power of 36dB, flip angle FA of 30 °, time TR of 11.8MS, echo time TE of 4.4 MS, scan region of 10cmx10cm, scan matrix of 50x50, slice thickness of 3cm, bandwidth of 12500Hz, and total scan time of 10 minutes (average 1030).

For the selective excitation experiments, the following settings were used: excitation frequency sfo1 is 46.1745MHz, width is 130Hz, power is 48dB rectangular excitation pulse, flip angle FA is 30 °, repetition time TR is 25MS, echo time TE is 10MS, scan region 10cmx10cm, scan matrix 50x50, slice thickness 3cm, bandwidth 25,000Hz, total scan time 10 minutes.