RU2718052C2 - Preparation for magnetic resonance diagnostics of oncological diseases containing deuterated 3-o-methylglucose, and diagnostic method using said preparation - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: group of inventions refers to medicine, namely to means for magnetic resonance diagnostics of oncological diseases. For this purpose, a diagnostic preparation and diagnostic method based on using a diagnostic preparation in form of 3-O-methylglucose containing deuterium bound with carbon in an amount greater than natural content or mixture of 3-O-methylglucose with different deuterium content. Method according to the invention involves magnetic resonance tomography and/or magnetic resonance spectroscopy on deuterium nuclei after the introduction of the diagnostic preparation after a time sufficient for accumulation of the diagnostic preparation in the tumor tissue of the subject, to obtain, respectively, MR-image and/or MR-spectrum.EFFECT: group of inventions enables diagnosing oncologic diseases with high information value.22 cl, 10 dwg, 4 ex

Description

Technical field

The invention relates to medicine, namely to means for magnetic resonance diagnosis of cancer.

State of the art

Diagnosis of cancer, including early diagnosis, is a priority in healthcare. One of the informative methods for diagnosing such diseases is magnetic resonance imaging (MRI).

Most types of MRI used in clinical practice are based on the registration of the magnetic resonance signal of protons ( ¹ H nuclei), which are part of the water in the human body. ¹ N MRI provides a high degree of anatomical detail and in many cases allows you to detect areas with an abnormal signal that correspond to neoplasms. At the same time, it is known from clinical practice that MRI is not always able to distinguish malignant neoplasms from benign ones that do not require urgent treatment (low specificity of the method). In this regard, early diagnosis of oncological diseases is also difficult, as there is a high risk of a false positive result.

The main method for increasing the information content of ¹ H MRI is the use of contrast agents that change the parameters of the signal in their environment [Topics in Current Chemistry, Contrast Agents I, Magnetic Resonance Imaging, Editors: Krause, Werner, 2002]. A wide range of contrast agents are known that are used in MRI diagnostics, including the commercially available Omniscan ^® , Magnevist ^® , ProHance ^® and Clariscan ^® , which are gadolinium complexes, as well as Feridex ^® and Resovist ^® , which are aqueous suspensions of stabilized magnetic nanoparticles. These substances are introduced into the patient’s bloodstream and make it possible to assess the degree of blood supply to areas with suspected malignancy.

An alternative to performing ¹ H MRI with contrast agents is to record the signal of other nuclei, in particular, ³¹ P, ¹³ C, ¹⁹ F, ² H, ²³ Na. One such nucleus is deuterium ( ² N). This is a non-radioactive isotope of hydrogen, the natural content of which in biological objects is 0.0156%, and the sensitivity is several times lower than that of a proton.

To date, several cases of ² H NMR and / or ² H MRI in vivo have been described. US20030211036 A1 has proposed a method for measuring perfusion of tumor tissues using isotopically labeled compounds, including deuterated compounds.

In the document US5042488 was shown the possibility of detecting the background signal of deuterium, as well as the signal of deuterium after injection of D ₂ O and 1-deuteroglucose in vivo (in rat liver). It is noted that the invention can also be carried out using other deuterium-labeled blood flow indicators.

US20100322865 A1 describes the use of metabolic precursors of water to evaluate metabolic rate by conducting ² H MRI. An example of a metabolic precursor of HOD is 1,2,3,4,5,6,6-deuterated glucose. In the framework of the described invention, only NMR signals are recorded on deuterium of metabolic water and an aliphatic chain of fatty acids, and there are no NMR signals of deuterated glucose.

Rat brain glucose consumption was estimated based on the recorded NMR spectra after 6.6-deuterated glucose was introduced in vivo [Ming Lu et. al., Quantitative assessment of brain glucose metabolic rates using in vivo deuterium magnetic resonance spectroscopy, JCBFM, 2017, 1-13].

None of the above methods are used in practice for the diagnosis of cancer, including because of the need to use very large doses of marker compounds.

Despite the existing methods for diagnosing diseases using MRI, there is a need to develop new, more effective approaches for conducting MRI diagnostics of oncological diseases.

Disclosure of Invention

The objective of the invention is to develop a new effective diagnostic drug for the diagnosis of cancer through MRI and / or MR spectroscopy and a diagnostic method, including the use of the specified drug.

The technical result of this invention is to create a new and effective diagnostic drug that can be used in the diagnosis of cancer, in particular breast cancer. The technical result of the present invention is also the development of a new effective and informative method for the diagnosis of cancer by magnetic resonance imaging and / or magnetic resonance spectroscopy on deuterium nuclei, including the introduction of a diagnostic drug according to the invention, which is able to accumulate in tumor tissue in a concentration sufficient to register informative deuterium tomogram or ² H-NMR spectrum in vivo.

The diagnostic preparation according to the invention is characterized by the fact that it implements a combination of such factors as a high content of deuterium atoms in the preparation, its ability to accumulate in the tumor in an acceptable amount of time at a concentration sufficient to conduct diagnostics, which is characterized by low toxicity, and practically completely remove the drug from organism unchanged. This allows you to diagnose using harmless dosages of the drug.

The method according to the invention is also characterized in that it is carried out without the harmful effects of ionizing radiation (typical, for example, for CT, PET, SPECT), which in turn increases the safety of studies, makes it possible to conduct more frequent repeated studies, in particular, makes the method attractive pediatrics. The invention is also aimed at obtaining diagnostic information similar to the method of positron emission tomography, but, unlike the latter, eliminates the risks associated with the ionizing radiation of radiopharmaceuticals.

This technical result is achieved through the development and creation of a diagnostic drug, including a deuterated derivative of 3-O-methylglucose or a mixture of at least two different deuterated derivatives of 3-O-methylglucose, for the diagnosis of cancer by magnetic resonance imaging and / or magnetic -resonance spectroscopy on deuterium nuclei.

In particular embodiments of the invention, the diagnostic preparation further includes at least one pharmaceutically acceptable excipient. In particular embodiments, the pharmaceutically acceptable excipient is a carrier, excipient and / or solvent.

In particular embodiments, the deuterated 3-O-methyl glucose derivative is 3-O-CD ₃ -glucose, 3-O-CD ₂ H-glucose, 3-O-CDH _2- glucose, 6,6-dididero-3- O-CD ₃ -glucose, 6,6-didytero-3-O-CD ₂ H-glucose, 6,6-didytero-3-O-CDH ₂ -glucose, 6,6-didytero-3-O-CH ₃ glucose.

In other particular embodiments, the diagnostic preparation is a mixture of at least two different deuterated derivatives of 3-O-methylglucose selected from 3-O-CD ₃ -glucose and / or 3-O-CD ₂ H-glucose and / or 3-O-CDH ₂ glucose and / or 6,6-diditero-3-O-CD ₃ glucose and / or 6,6-diditero-3-O-CD ₂ H-glucose and / or 6.6 -deutero-3-O-CDH ₂ -glucose and / or 6,6-dididero-3-O-CH ₃ -glucose and / or any other deuterated derivatives of 3-O-methylglucose. In particular embodiments of the invention, the diagnostic preparation optionally comprises non-deuterated 3-O-methylglucose.

In particular embodiments of the invention, the components of the diagnostic preparation along with the deuterium atoms bonded to the carbon atoms contain deuterium atoms that partially or completely replace the mobile hydrogen atoms bonded to the oxygen atoms.

The invention also includes the preparation of a diagnostic preparation according to the invention.

The achievement of the specified technical result is also ensured by the implementation of a method for diagnosing an oncological disease in a subject, comprising the following steps:

administering to the subject a diagnostic preparation of the invention;

conduct magnetic resonance imaging and / or magnetic resonance spectroscopy on the nucleus of deuterium after the introduction of the diagnostic drug after a time sufficient to accumulate the diagnostic drug in the tumor tissue, to obtain, respectively, tomograms and / or NMR spectrum;

diagnose the presence or absence of cancer based on the observed signal intensity of deuterium nuclei, reflecting the level of accumulation of the diagnostic drug.

In private embodiments of the invention, in the absence of areas of accumulation of a diagnostic drug, the subject is diagnosed with the absence of an oncological disease.

In particular embodiments of the invention, at least one additional medical examination is performed, selected from magnetic resonance imaging on nuclei other than deuterium nuclei, and / or ultrasound and / or computed tomography and / or radiography and / or palpation and / or biopsies and / or analysis of biological fluids for tumor markers and / or radionuclide diagnostics and / or visual observation.

In particular embodiments of the invention, the presence or absence of an oncological disease is diagnosed based on a comparison of the signal intensity of deuterium nuclei with the typical signal intensity observed in healthy subjects in the corresponding tissue or corresponding organ.

In particular embodiments of the invention, the presence or absence of cancer is diagnosed based on a comparison of the signal strength of deuterium nuclei in areas corresponding to normal and abnormal tissue according to additional medical research.

In particular embodiments of the invention, the presence or absence of an oncological disease is diagnosed based on a comparison of a deuterium tomogram with an image obtained by MRI on protium nuclei.

In particular embodiments of the invention, the presence or absence of an oncological disease is diagnosed based on a comparison of the signal intensity of deuterium nuclei in neighboring voxels located on opposite sides of the normal tissue and tissue boundary with suspected neoplasm according to ¹ H MRI.

In particular embodiments of the invention, based on the observed signal strength of the deuterium nuclei, a conclusion is made about the malignancy or aggressiveness or degree of differentiation of the tumor.

In a particular embodiment, the diagnosed cancer is breast cancer.

In particular embodiments of the invention, tomograms are recorded using selective excitation of the deuterium nuclei of the diagnostic drug.

In particular embodiments of the invention, tomograms are recorded using broadband excitation of the deuterium nuclei of a diagnostic drug.

In particular embodiments of the invention, the diagnostic drug is administered orally to the subject.

In other particular embodiments of the invention, the diagnostic drug is administered parenterally.

In particular embodiments of the invention, magnetic resonance imaging and / or magnetic resonance spectroscopy on deuterium nuclei is performed 20-210 minutes after administration of the diagnostic drug.

In particular embodiments, a diagnostic preparation is administered to a subject in an amount corresponding to 1-3.5 g of a deuterated derivative of 3-O-methylglucose or a mixture of deuterated derivatives of 3-O-methylglucose per 1 kg of body weight of a subject.

The invention also includes the use of a diagnostic preparation according to the invention for the diagnosis of cancer through magnetic resonance imaging and / or magnetic resonance spectroscopy on deuterium nuclei.

Brief Description of the Drawings

Figure 1. ² H NMR spectrum of a sample simulating the accumulation of 3-OCD ₃ -glucose in a tumor in an animal body.

Figure 2. Deuterium tomogram of a sample containing a dilute solution of a deuterated diagnostic preparation (a) broadband excitation, b) selective excitation at a frequency of 3-O-methylglucose).

Figure 3. Distribution of 3-OCCD ₃ -glucose in the mouse body with a tumor in the abdominal region (a) and the control mouse without tumor (b) 70 minutes after drug administration.

Figure 4. Tomograms of a control mouse without tumor 40 min after administration of 72 mg of 3-OCCD ₃ -glucose:

a) ² N tomogram;

b) ¹ N tomogram.

Figure 5. Tomograms of a mouse with 4T1 breast carcinoma 20 min after administration of 72 mg of 3-OCCD ₃ -glucose:

a) ² N tomogram (the position of the surface coil is shown by a white outline);

b) ¹ N tomogram;

c) combined tomogram.

Figure 6. Tomograms of a mouse with 4T1 breast carcinoma 40 minutes after administration of 72 mg of 3-OCCD ₃ -glucose:

a) ² N tomogram;

b) ¹ N tomogram;

c) combined tomogram.

Figure 7. Tomograms of a mouse with 4T1 breast carcinoma 210 minutes after the administration of 72 mg of 3-OCCD ₃ -glucose:

a) ² N tomogram;

b) ¹ N tomogram;

c) combined tomogram.

Figure 8. Tomograms of a mouse with 4T1 breast carcinoma 320 min after administration of 72 mg of 3-OCCD ₃ -glucose:

a) ² N tomogram;

b) ¹ N tomogram;

Figure 9. Tomograms of a mouse with 4T1 breast carcinoma 50 minutes after administration of 20 mg of 3-OCCD ₃ -glucose:

a) ² N tomogram;

b) ¹ N tomogram;

c) combined tomogram.

Figure 10. Tomograms of a mouse with 4T1 breast carcinoma after oral administration of 70 mg of 3-OCCD ₃ -glucose:

(a) ² N MRI after 15 minutes;

(b) ¹ N MRI after 15 minutes;

(c) application of ² N MRI and ¹ N MRI after 15 minutes;

(g) ² N MRI after 100 minutes;

(d) ¹ N MRI after 100 minutes;

(e) overlay of ² N MRI and ¹ N MRI after 100 minutes.

Definitions and Terms

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

In the description of the present invention, the terms “includes” and “including” are interpreted as meaning “includes, among other things.” These terms are not intended to be construed as “consists of only”.

The term “deuterated” or “deuterated derivative” as used herein refers to a compound containing deuterium bound to carbon in an amount exceeding its natural content in at least one position. In particular cases of the embodiment of the invention, the deuterium content in at least one position exceeds 30%, in other particular cases - 90%. The symbol “D” in this document denotes a hydrogen atom represented by the isotope ² N in a fraction exceeding its natural content. By “mixture of at least two different deuterated derivatives” is meant a mixture of compounds containing deuterium at different positions of a 3-O-methylglucose molecule, or containing a different amount of deuterium at the same position.

The term "voxel" in this document refers to the sample volume arbitrarily selected by adjusting the magnetic field parameters in which the nuclear magnetic resonance signal is recorded.

The diagnostic preparation of the invention may include one or more of any pharmaceutically acceptable excipients suitable for the particular dosage form, in particular any carriers, solvents and / or excipients, such as can be administered to the patient along with the compound of the invention , and which do not destroy these compounds, and are non-toxic when administered.

Detailed disclosure of the invention.

Despite the fact that the accumulation of very small amounts of radiolabeled [ ¹⁸ F] fluorodeoxyglucose in tumors is a well-studied phenomenon and is used for diagnostics in positron emission tomography, the relatively low sensitivity of MRI and the background content of deuterium in water (HOD) and tissues require the creation of tumors of a much higher concentration of the diagnostic drug. So, [ ¹⁸ F] fluorodeoxyglucose is used in dosages of the order of 10 ^-11 mol / kg (taking into account the typical specific activity of about 10 ³ Ci / mol and a typical dose of about 10 mCi), however, such data do not allow to predict the efficiency of accumulation of the compounds of the invention in tumor cells in the concentration necessary for the implementation of this invention (according to the results of studies conducted by the authors, the practically applicable doses of the preparation according to the invention are of the order of 10 ^-3 mol / kg).

For the successful implementation of the diagnosis of cancer using ² N MRI or ² N NMR, it is necessary to create a sufficiently high concentration of deuterium in the tumor tissue. To satisfy this criterion, the diagnostic product must:

1) quickly and selectively accumulate in the tumor tissue (in particular, the presence of a sufficiently effective mechanism of membrane transport);

2) characterized by sufficiently slow excretion (provides sufficient time for the accumulation of large quantities of the drug in the tumor, as well as for the continuous registration of ² N tomograms);

3) not undergo significant metabolism (minimizes possible side effects, including from the inclusion of deuterium in biomolecules, and allows for repeated diagnostics several hours after drug administration, without changing the background signal of the tumor);

4) possess low toxicity in the required concentration (makes it possible to administer sufficiently large doses of the drug);

5) contain a large amount of deuterium (necessary to achieve sufficient signal intensity).

The authors of this invention conducted studies to study deuterated glucose derivatives as diagnostic drugs, and it was found that not all glucose derivatives can be used to diagnose cancer using ² H MRI or ² H NMR. So, in particular, preliminary experiments with 6,6-D ₂ -glucose indicate the impossibility of reliable tumor imaging using this compound (low contrast, low signal to noise ratio).

It was unexpectedly found that deuterated derivatives of 3-O-methylglucose are able to accumulate in the tumor tissue in a concentration sufficient to visualize tumors in vivo using ² H MRI or ² H NMR, which in turn allows for the effective diagnosis of cancer using magnetic resonance imaging on deuterium nuclei.

Due to the low deuterium content in the body (0.015% of hydrogen atoms), background signals in ² N MRI are several orders of magnitude lower than in ¹ N MRI. The development of similar methods using non-deuterated diagnostic drugs based on ¹ H MRI is difficult due to the existence of a large number of background signals of naturally occurring low molecular weight compounds with an intensity comparable to the maximum achievable signal intensity of an undeuterated diagnostic drug. At the same time, the presence of a HOD background signal limits the minimum acceptable concentration of a diagnostic drug in a tumor for ² N MRI. The feasibility of the practical implementation of the diagnostic method according to the invention depends on the pharmacokinetics and pharmacodynamics of a particular diagnostic drug.

The ability to register a deuterium signal in vivo is also determined by a sufficient number of deuterium atoms in the structure of the compound. Thus, a diagnostic preparation according to the invention, including deuterated 3-O-methylglucose containing a CD ₃ group and / or a CD ₂ OH group, is a preferred embodiment of the present invention. The presence of such deuterated groups allows diagnosis using lower concentrations of the diagnostic drug, which minimizes side effects.

The method according to the invention allows to diagnose the presence or absence of cancer. The method according to the invention is based on the use of a deuterated diagnostic preparation and registration of tomograms and / or NMR spectra at the frequency of deuterium.

It is known that ¹ N MRI alone in many cases has insufficient diagnostic accuracy, while the method according to the invention provides data on the molecular transport of glucose, not available in traditional methods of embodiment of ¹ N MRI, and thus potentially provides a more accurate diagnostic information.

In one embodiment of the invention, the diagnostic process includes an MRI scan and is performed as follows:

a) in some embodiments, an MRI scan is performed on protium nuclei ( ¹ N). Registration of ¹ N MRI allows, firstly, anatomical binding of the deuterium signal, and secondly, to identify areas with suspected malignancy (in other embodiments of the invention, determining the area of ² N MRI can be carried out in other ways, in particular, by means of ultrasound research, computed tomography, radiography, palpation, biopsy, analysis of biological fluids for tumor markers, radionuclide diagnostics, visual observation);

b) a diagnostic drug is introduced;

c) after a time sufficient for the accumulation of the diagnostic drug in the tumor tissue of the subject, tomograms are recorded at the precession frequency of the deuterium nuclei of the diagnostic drug;

d) the obtained deuterium tomograms are analyzed in order to find areas with an abnormally high intensity and, therefore, corresponding to the accumulation of a diagnostic drug. In particular, it is possible to compare the tomograms obtained on ¹ N and ² N: if the abnormal areas on ¹ N and ² N coincide, we can talk about the greater likelihood of a malignant formation. Nevertheless, the presence of an anomaly on a ¹ N tomogram is not a prerequisite: there may be situations where the neoplasm does not appear on the tomogram obtained by ¹ N MRI, while there is an accumulation of the diagnostic drug on the tomogram obtained by ² N MRI. In the latter case, a ¹ N MRI is only used to anatomically attach a suspicious area.

In yet another particular embodiment of the invention, the diagnostic process includes NMR spectroscopy on deuterium nuclei and is carried out as follows:

a) ¹ H MRI is performed, as a result of which areas with suspected malignancy are identified (in other embodiments of the invention, the determination of the ² H NMR region can be carried out by other methods, in particular, by ultrasound, computed tomography, radiography, palpation, biopsy , analysis of biological fluids for tumor markers, radionuclide diagnostics, visual observation);

b) a diagnostic drug is introduced;

c) after a time sufficient for the accumulation of the diagnostic drug in the tumor tissue of the subject in voxels corresponding to the area with suspected malignancy (for example, according to the results of ¹ H MRI), the deuterium spectrum is recorded (in particular, using local spectroscopy); optionally, spectrum is recorded in neighboring voxels to compare signal intensity; in particular embodiments, spectroscopy can be carried out using transmitting, receiving, transmitting, bulk, implant, surface coils;

d) the signal intensity in voxels corresponding to the area with suspected malignancy is compared, in particular, with: (i) typical values for a given tissue (which must be determined previously in healthy subjects) and / or (ii) the intensity in neighboring voxels corresponding to the same organ or tissue and free from abnormalities according to ¹ H MRI. The increased signal intensity allows us to talk about the accumulation of a diagnostic drug and, as a consequence, the presence of a malignant neoplasm.

The order of steps a), b), c) in both of the above embodiments of the invention may be different, for example, you can enter a diagnostic drug, conduct ¹ N MRI, then conduct ² N MRI or ² N NMR spectroscopy; or conduct ¹ N MRI after ² N MRI or ² N NMR spectroscopy.

In particular cases of the invention, after identifying a region with a suspected malignancy, individual voxels are selected that lie both within and outside the suspicious region (in particular, a series of neighboring voxels lying on the same line crossing the border of the suspicious region can be selected). Registration of the integral signal of ² N or ² N spectra in the selected voxels with subsequent comparison of their intensity allows you to quickly and with greater sensitivity to detect the accumulation area of the diagnostic drug.

MRI images and MR spectra according to this invention can be obtained on any magnetic resonance imager equipped with equipment for detecting a deuterium signal.

In particular cases of the embodiment of the invention, the use of a diagnostic drug that gives a signal in an area free of the HOD background signal allows for MRI using a selective exciting pulse tuned to the frequency of the diagnostic drug. This allows you to get rid of the background HOD signal on the tomogram.

Due to the use of molecular mechanisms of transport and accumulation of the diagnostic preparation according to the invention in cells, the method according to the invention allows the metabolic activity of the test tissue to be evaluated and, as a consequence, to evaluate the malignancy or aggressiveness of the tumor tissue. Thus, the diagnostic potential of the method is increased in comparison with traditional ¹ H magnetic resonance imaging, as well as MRI methods based on perfusion assessment (including with contrast agents).

The signal of the diagnostic preparation according to the invention can be recorded up to 3 hours after administration, and the distribution of the drug in the tumor and other organs varies throughout this time. So, the signal of the diagnostic drug is primarily manifested in the kidneys, then in certain areas of the tumor, probably corresponding to the areas of the most active growth and with the best blood supply. The maximum signal of deuterium in the entire tumor volume with intraperitoneal administration is observed after about 1 hour, in the future, the distribution pattern of the diagnostic drug continues to change, and the areas of the tumor with the best blood supply primarily lose the diagnostic drug. Due to this behavior, repeated tomography for several hours after the introduction of the diagnostic drug according to the invention allows to obtain information about the rate of its accumulation and leaching for individual parts of the tumor, as well as to evaluate the total volume of tumor spread. Obtaining such information about the structure of the tumor and its characteristic accumulation dynamics using, for example, a short-lived ¹¹ C-labeled analog for PET is not possible due to the very short half-life (20 minutes) and the inability to increase the dose of the radiopharmaceutical due to the concomitant increase in radiation load.

The experiments performed indicate the selectivity of the accumulation of the drug according to the invention in tumor tissue compared with the liver, skeletal muscle and other organs and tissues in doses acceptable for ² N MRI. It was also found that with the introduction of equivalent doses of the diagnostic drug according to the invention to animals with various tumors, differences in the dynamics of accumulation and maximum signal strength of deuterium are observed, which correlate with the type of tumor. In combination with ¹ H MRI data or other additional diagnostic methods, the preparation and method of the invention can be used to identify a tumor in cases where its type is unknown.

Studies conducted by the inventors indicate good tolerance of the diagnostic drug by animals, the absence of visible side effects when used in the indicated doses and the complete elimination of deuterium-containing compounds from the body within a few hours. So, with the intraperitoneal administration of the preparations according to the invention to mice with a dosage of 8 g / kg, no death of animals was observed, and 6 hours after administration, the drug was not observed in the tumor according to ² H magnetic resonance imaging. The background concentration of deuterium in the tumor and other tissues remained the same. Since the use of a diagnostic preparation according to the invention is not associated with the inclusion of deuterated compounds in metabolic processes, the likelihood of developing long-term side effects is reduced. In addition, the complete removal of deuterium from the tumor tissue allows for repeated diagnosis, for example, after 6 hours and to monitor the dynamics of the development of the tumor in the course of certain treatment methods.

The experiments showed that the results of imaging of the tumor significantly depend on the dose of the diagnostic drug within the acceptable range. Smaller doses make it possible to selectively visualize the areas of the tumor characterized by the most intense absorption, while increasing the dose leads to a more complete filling of the tumor borders with a deuterium signal. Due to this property of the diagnostic preparation according to the invention, dynamic studies are possible (multiple registration of tomograms) with a gradually increasing concentration of the drug in the blood over time (for example, as a result of slow intravenous infusion or a series of consecutive injections of small doses of the drug). Such studies can provide information simultaneously on the metabolic activity of different parts of the tumor and on the degree of spread of the tumor.

The diagnostic method according to the invention can be used, in particular, for the early diagnosis of tumors of various localization, metastatic lesions, assessment of the tumor response to treatment and conclusions on the effectiveness of the therapy, to clarify the diagnosis based on the results of ¹ N MRI and / or other diagnostic methods.

The method according to the invention extends the existing methods for the diagnosis of cancer and allows for effective diagnosis.

The implementation of the invention.

The possibility of an objective manifestation of a technical result when using the invention is confirmed by reliable data given in the examples containing experimental information obtained in the process of conducting research using methods adopted in this field. The invention is illustrated by figures.

It should be understood that these and all examples cited in the application materials are not limiting and are provided merely to illustrate the present invention.

The examples given in this document illustrate the principle of action of the developed method, and do not limit the range of doses used, as well as the time interval between administration of a diagnostic drug and registration of a deuterium signal, because depending on the sensitivity and other parameters of the equipment used, the diagnosed disease and the nature of the subject (person or laboratory animal) the required doses and the time required for accumulation may vary. In addition, the above parameters for recording spectra and tomograms, including the signal accumulation time, are part of specific embodiments of the invention and may vary depending on the equipment used and specific diagnostic tasks.

Synthesis of 3-O-CD ₃ -glucose

Diacetone-D-glucose (5 g, 19.2 mmol) was dissolved in DMF (10 ml), while cooling to 0 ° C and NaH (60%) (1.1 g) was added with stirring. The reaction mixture was kept under stirring for 1 hour at room temperature, and then CD ₃ I (1.5 ml) was added dropwise. After 2 hours, the reaction mixture was poured into ice water and the organic phase was extracted with ethyl acetate, washed twice with water. The organic phase was evaporated, then dissolved in THF (15 ml), CF ₃ COOH (2 ml) and water (1 ml) were added. The reaction mixture is boiled for 5 hours. Then add 10 ml of toluene and evaporated. 3-O-CD ₃ -glucose (2.6 g, 70%) was isolated from the residue by silica gel column chromatography.

α-Isomer: ¹ H NMR (600 MHz, D ₂ O): δ 5.21 (d, 1H, J _1-2 = 3.7 Hz, H-1), 3.83 (m, 2H, H-4, H-6 ^A ), 3.74 (dd, 1H, J _5-6B = 5.4 Hz, J _6A-6B = 12.4 Hz, H-6 ^B ), 3.58 (m, 1H, H-2), 3.47 (m, 2H, H-3 , H-5). ¹³ C NMR (151 MHz, D ₂ O): δ 93.3 (C-1), 83.8 (C-3), 72.7 (C-4), 72.2 (C-2), 70.3 (C-5), 61.7 ( C-6). β-Isomer: ¹ H NMR (600 MHz, D ₂ O): δ 4.84 (d, 1H, J _1-2 = 7.3 Hz, H-1), 3.88 (dd, 1H, J _5-6A = 1.8 Hz, J _6A-6B = 12.4 Hz, H-6 ^A ), 3.70 (dd, 1H, J _5-6B = 5.4 Hz, J _6A-6B = 12.4 Hz, H-6 ^B ), 3.47 (m, 2H, H- 4, H-5), 3.28 (m, 2H, H-2, H-3). ¹³ C NMR (151 MHz, D ₂ O): δ 97.1 (C-1), 86.5 (C-3), 77.0 (C-4), 74.7 (C-2), 70.2 (C-5), 61.9 ( C-6).

Similarly, using a reagent of the form CH _x D _(3-x) I (0 <x <3), partially deuterated 3-O- (CH _x D _(3-x) ) -glucose is obtained. When using 6,6-dideutero-diacetone-D-glucose as a substrate in combination with a reagent of the form CH _y D _(3-y) I (0≤y≤3), 3-O- (CH _y D _{(3-y )} ) -6,6-dideuteroglucose (in particular 3-O-methyl-6, dideuteroglucose or 3-O-CD ₃ -6,6-dideuteroglucose).

Using CF ₃ COOD and / or D ₂ O to remove the protective groups, as well as keeping the final product in D ₂ O followed by evaporation, deuterated 3-O-methylglucose with partially or fully deuterated hydroxyl groups is obtained.

To demonstrate the fundamental possibility of NMR detection of a deuterium signal during the accumulation of a small amount of small amounts of a diagnostic drug, an experiment was conducted simulating the accumulation of 3-OCD ₃ -glucose in a tumor in the animal’s body (example 1, figure 1). It was shown that the sensitivity of the method is sufficient to detect, using deuterium tomography, such small amounts of 3-OCD ₃ -glucose as 5 μmol distributed in a volume of 1 ml.

The possibility of registering a deuterium tomogram of a sample containing a solution of 3-OCD ₃ -glucose using a broadband and selective radio frequency pulse (example 2) is shown.

In vivo experiments (Example 3, 4) demonstrate the ability to register deuterium tomograms and in vivo NMR spectra and the ability of 3-OCCD ₃ glucose to accumulate in tumors. The possibility of diagnosing a tumor, in particular a breast tumor, was shown based on the observed signal of a deuterated diagnostic drug.

The following examples used a Bruker BioSpec BC70 / 30 USR tomograph with a constant field of 7.05 T, equipped with a surface transceiver coil with a diameter of 3 cm and a scanning depth of about 1 cm.

To register a deuterium tomogram, the pulse sequence FLASH (Fast low angle shot) was used.

The following settings were used for experiments with broadband excitation: the excitation frequency was determined from the ² N NMR spectrum and amounted to sfo1 ≈ 46.1745 MHz, a rectangular exciting pulse with a width of 1300 Hz and a power of 36 dB, deflection angle FA = 30 °, repetition time TR = 11.8 ms, time echo TE = 4.4 ms, scanning area 10 cm x 10 cm, scanning matrix 50 x 50, cutoff thickness 3 cm, frequency transmission width 12500 Hz, total scanning time 10 minutes (1030 accumulations).

The following settings were used for experiments with selective excitation: excitation frequency sfo1 = 46.1745 MHz, rectangular excitation pulse with a width of 130 Hz and a power of 48 dB, deflection angle FA = 30 °, repetition time TR = 25 ms, echo time TE = 10 ms, scanning area 10 cm x 10 cm, scanning matrix 50 x 50, slice thickness 3 cm, transmission bandwidth 25000 Hz, total scan time 10 minutes.

Example 1. Registration ² N spectrum of a diagnostic drug, concentrated in a small sample volume (model of accumulation of 3-OCD ₃ -glucose in the tumor in the animal body)

To demonstrate the fundamental possibility of detecting a deuterium signal when small amounts of 3-OCD ₃ -glucose are accumulated in a limited volume, the following experiment was carried out.

A solution of 1 mg (5 μmol) of 3-OCD ₃ -glucose in 1 ml of distilled water (with a natural deuterium content) was placed in a 1.5 ml cylindrical plastic tube. The resulting test tube with the solution was placed in a cylindrical plastic container with a volume of 50 ml and a diameter of 25 mm, filled with distilled water (with a natural deuterium content). The resulting container system was located in the cavity of the tomograph. A surface transceiver coil with a diameter of 3 cm and a scanning depth of about 1 cm was located directly above the internal tube. The deuterium spectrum was recorded with a reference frequency of 46.1746780 MHz. A pulse sequence consisting of one 90 ° pulse was used (256 accumulations, delay between pulses 0.1 s).

The figure 1 shows the spectrum of deuterium obtained in the above experiment. The large peak corresponds to the HOD signal (natural content 15 mmol / L), the small peak corresponds to deuterated 3-O-methylglucose. This experiment shows that ² H NMR spectroscopy using a surface coil has a sensitivity sufficient to detect such small amounts of 3-O-methylglucose as 5 μmol in a volume of 1 ml surrounded by a large excess of water.

Example 2. Registration of a deuterium tomogram of a sample containing a dilute solution of a deuterated diagnostic drug.

The following experiment was carried out to demonstrate the possibility of registering a deuterium tomogram of a diluted solution of a diagnostic drug.

A glass vial containing 5 ml of a solution of 3-O-CD ₃ -glucose (20 mg) in distilled water was placed in the cavity of the tomograph. A surface transceiver coil with a diameter of 3 cm and a scanning depth of about 1 cm was located directly above the bottle.

The figure 2 shows the deuterium tomograms of a sample with 3-O-CD ₃ -glucose, obtained using broadband (left) and selective (right) excitation.

Example 3. The accumulation of 3-OCD ₃ -glucose in a mouse tumor.

In this experiment, we used: a nonlinear mouse with a spontaneous tumor in the abdominal region and a nonlinear control mouse without a tumor.

An animal weighing 30 g was injected intraperitoneally with a solution of 60 mg of 3-O-CD ₃ -glucose in 0.3 ml of saline. After the injection, the animal was kept in a separate cage with free access to food and water for 70 minutes. To record the spectra, the animal was immobilized with the isoflurane preparation. The surface transceiver coil was fixed on the mouse body sequentially in four positions (head, chest, pelvis, near the tumor). Deuterium spectra were recorded on a Bruker BioSpec BC70 / 30 USR tomograph with a constant field of 7.05 T with a reference frequency of 46.1746780 MHz. A sequence consisting of one 90 ° pulse was used (64 accumulations, the delay between pulses was 2 s).

Figure 3 shows the distribution of deuterated 3-methylglucose in the body of a mouse with a tumor (Fig. 3, a) and a control mouse without a tumor (Fig. 3, b) 70 minutes after drug administration (from left to right: head, chest, pelvis, near tumors). As can be seen from the figure, in the case of a tumor-free mouse, 3-OCD ₃ -glucose is completely excreted, while a mouse with a tumor retains a deuterium signal near the tumor, which indicates the presence of a tumor disease. The experiment shows the possibility of accumulation of 3-O-CD ₃ -glucose in the tumor at a concentration sufficient for detection by deuterium spectroscopy.

Example 4. The use of deuterium tomography to visualize carcinoma of the mammary gland of mouse 4T1 in vivo using 3-OCD ₃ -glucose as a diagnostic drug.

In this example, experiments were performed on Balb / c mice inoculated with 4T1 breast carcinoma (injection of 5 × 10 ⁵ cells / 60 μl under the left front paw 10 days before the experiment) and on healthy Balb / c mice.

An animal weighing 20 g was injected intraperitoneally, intravenously or orally with a solution of 20-72 mg of 3-O-CD ₃ -glucose (intraperitoneally: 0.5 ml of saline; intravenously: 0.2 ml of water; oral: 0.25 ml of saline). After the injection, the animal was kept in a separate cage with free access to food and water for a specified time. To record the spectra, the animal was immobilized with the isoflurane preparation. The surface transceiver coil was attached to the mouse body from the dorsal side in the thoracic region. In figures 4-10 shows ² N tomograms obtained using broadband excitation of deuterium nuclei.

The figure 4 shows the tomograms obtained on a control mouse No. 1 without a tumor 40 minutes after intraperitoneal administration of 3-O-CD ₃ -glucose (a) ² N MRI; b) ¹ N MRI). There is no region with a strong deuterium signal in this figure.

The figure 5 shows the tomograms obtained on a mouse No. 2 with a tumor 20 minutes after intraperitoneal injection of 3-O-CD ₃ -glucose: (a) ² N MRI (the position of the surface coil is shown by a white outline); b) ¹ N MRI; c) the imposition of ² N MRI and ¹ N MRI).

The figure 6 shows the tomograms obtained on mouse No. 2 with a tumor 40 minutes after intraperitoneal injection of 3-O-CD ₃ -glucose: (a) ² N MRI; b) ¹ N MRI; c) the imposition of ² N MRI and ¹ N MRI).

The figure 7 shows the tomograms obtained on mouse No. 2 with a tumor 210 minutes after intraperitoneal administration of 3-O-CD ₃ -glucose: (a) ² N MRI; b) ¹ N MRI; c) the imposition of ² N MRI and ¹ N MRI).

The figure 8 shows the tomograms obtained on mouse No. 2 with a tumor 320 minutes after intraperitoneal administration of 3-O-CD ₃ -glucose: (a) ² N MRI; b) ¹ N MRI.

In figures 5-8, it can be seen that the deuterium signal is predominantly localized in the region corresponding to the tumor according to ¹ H MRI.

Based on the results illustrated by figures 5-8, we can conclude that:

1) the diagnostic preparations of the invention, in particular 3-O-CD ₃ -glucose, accumulate in the tumor at a concentration sufficient for imaging using ² N MRI;

2) the diagnostic preparations according to the invention, in particular, 3-O-CD ₃ -glucose, can be used as a diagnostic drug for the detection of malignant tumors using ² N MRI.

Based on the results illustrated by figures 5-8, it can be concluded that the allowable time range between administration of the diagnostic drug of the invention and registration of a deuterium signal can be for a mouse, in particular, 20-210 minutes. It should be noted that for other animals and for humans, this range can be significantly wider due to the difference in pharmacokinetics. In addition, based on these results, it can be concluded that the diagnostic drug is excreted from the tumor within a few hours.

The figure 9 shows the tomograms obtained on mouse No. 3 with a tumor 50 minutes after intravenous administration of 20 mg of 3-O-CD ₃ -glucose: (a) ² N MRI; (b) ¹ N MRI; (c) overlay of ² N MRI and ¹ N MRI. In this figure, there is an accumulation of 3-O-CD ₃ -glucose in the tumor. The tumor volume measured by 1H MRI was 180 mm ³ .

Based on the results illustrated by figures 5-9, it can be concluded that the permissible dose range of the diagnostic drug may be at least 1-3.5 g of 3-O-CD ₃ -glucose per 1 kg of animal weight. It should be noted that for other animals and for humans, as well as with a different method of administration (for example, oral or intravenous) and / or with other malignant diseases, this range may be different. In particular, taking into account the allometric equations describing the pharmacokinetics of drugs in organisms of various sizes, we can expect a reduction in the dose required for humans by several times compared with the doses described here.

The figure 10 shows the tomograms obtained on mouse No. 4 with a tumor after 15 and 100 minutes after oral administration of 70 mg of 3-O-CD ₃ -glucose: (a) ² N MRI after 15 minutes; (b) ¹ N MRI after 15 minutes; (c) application of ² N MRI and ¹ N MRI after 15 minutes; (g) ² N MRI after 100 minutes; (d) ¹ N MRI after 100 minutes; (e) overlay of ² N MRI and ¹ N MRI after 100 minutes. This figure shows how 3-O-CD ₃ glucose, originally located in the stomach (15 min.), Accumulates over time in the tumor (115 min.).

Based on the results illustrated in figure 10, it can be concluded that the diagnostic preparation according to the invention can be successfully used when administered orally.

Although the invention has been described with reference to the disclosed embodiments, it will be apparent to those skilled in the art that the specific experiments described in detail are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be understood that various modifications are possible without departing from the gist of the present invention.

Claims (25)

1. Diagnostic preparation, including 3-O-methylglucose containing deuterium bound to carbon in an amount exceeding the natural content, or a mixture of 3-O-methylglucose with different deuterium contents, for the diagnosis of cancer by magnetic resonance imaging and / or magnetic resonance spectroscopy on deuterium nuclei. 2. The diagnostic preparation according to claim 1, which further comprises at least one pharmaceutically acceptable excipient. 3. The diagnostic preparation according to claim 2, characterized in that the pharmaceutically acceptable excipient is a carrier, excipient and / or solvent. 4. The diagnostic preparation according to claim 1, wherein the 3-O-methylglucose compound containing deuterium bound to carbon in an amount exceeding the natural content is 3-O-CD ₃ -glucose, 3-O-CD ₂ H -glucose, 3-O-CDH ₂ -glucose, 6,6-didytero-3-O-CD ₃ -glucose, 6,6-didytero-3-O-CD ₂ H-glucose, 6,6-didytero-3 -O-CDH ₂ -glucose or 6,6-diditero-3-O-CH ₃ -glucose. 5. The diagnostic preparation according to claim 1, which comprises a mixture of at least two different 3-O-methylglucose compounds containing carbon deuterium in an amount exceeding the natural content selected from 3-O-CD ₃ -glucose, and / or 3-O-CD ₂ H-glucose, and / or 3-O-CDH ₂ -glucose, and / or 6,6-didytero-3-O-CD ₃ -glucose, and / or 6,6-didytero -3-O-CD ₂ H-glucose, and / or 6,6-dideutero-3-O-CDH ₂ -glucose, and / or 6,6-dideutero-3-O-CH ₃ -glucose. 6. The diagnostic preparation according to claim 1, which further includes non-deuterated 3-O-methylglucose. 7. The diagnostic preparation according to claim 1, wherein the 3-O-methylglucose compound containing deuterium bound to carbon in an amount exceeding the natural content, along with deuterium atoms bound to carbon atoms in the molecule, contains deuterium atoms, partially or completely replacing mobile hydrogen atoms bonded to oxygen atoms. 8. A method for diagnosing an oncological disease in a subject, comprising the following steps: a) administer to the subject a diagnostic preparation according to claim 1; b) conduct magnetic resonance imaging and / or magnetic resonance spectroscopy on the nucleus of deuterium after the introduction of the diagnostic drug after a time sufficient for its accumulation in the tumor tissue, to obtain, respectively, a tomogram and / or NMR spectrum; c) diagnose the presence or absence of cancer based on the observed signal intensity of deuterium nuclei, reflecting the level of accumulation of the diagnostic drug. 9. The method according to p. 8, in which in the absence of areas of accumulation of a diagnostic drug in a subject, the absence of an oncological disease is diagnosed. 10. The method according to p. 8, in which at least one additional medical study is selected, selected from magnetic resonance imaging on nuclei other than nuclei of deuterium, ultrasound, computed tomography, radiography, palpation, biopsy, analysis of biological fluids for tumor markers , radionuclide diagnostics and / or visual observation. 11. The method according to p. 8, in which the presence or absence of a cancer is diagnosed based on a comparison of the signal intensity of deuterium nuclei in the subject under study with the typical signal intensity observed in healthy subjects in the corresponding tissue or corresponding organ. 12. The method according to p. 10, in which they diagnose the presence or absence of cancer based on a comparison of the signal intensity of deuterium nuclei in areas corresponding to normal and abnormal tissues according to an additional medical study. 13. The method according to p. 10, in which the presence or absence of cancer is diagnosed based on a comparison of the signal intensity of deuterium nuclei in neighboring voxels located on opposite sides of the normal tissue and tissue boundary with suspected neoplasm according to ¹ H MRI. 14. The method according to p. 10, in which the presence or absence of an oncological disease is diagnosed based on a comparison of a deuterium tomogram of a subject under study with an image obtained by magnetic resonance imaging of a subject on protium nuclei. 15. The method according to p. 8, in which the intensity of the signal of the deuterium nuclei and / or its change in time concludes that the structure, malignancy, aggressiveness or degree of differentiation of the tumor. 16. The method of claim 8, wherein the oncological disease is breast cancer. 17. The method according to p. 8, in which, when registering a tomogram, selective excitation of the deuterium nuclei of the diagnostic drug is used. 18. The method of claim 8, wherein the tomogram is recorded using broadband excitation of deuterium nuclei. 19. The method of claim 8, wherein the diagnostic drug is administered orally to the subject. 20. The method of claim 8, wherein the diagnostic drug is administered parenterally. 21. The method according to p. 8, in which magnetic resonance imaging and / or magnetic resonance spectroscopy on deuterium nuclei is carried out 20-210 minutes after the introduction of the diagnostic drug. 22. The method according to p. 8, in which the diagnostic drug is administered to the subject in an amount corresponding to 1-3.5 g of deuterated 3-O-methylglucose or a mixture of deuterated derivatives of 3-O-methylglucose per 1 kg of body weight of the subject.

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