RU2541090C1 - Gadopentetate-modified beta-cyclodextrin derivatives - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: gadopentetate-modified beta-cyclodextrin derivatives, characterised by that the contrast medium used is beta-cyclodextrin containing one, two or three gadopentetate residues or a mixture thereof with general formula (C₄₂H_70-nO₃₅)(C₁₄H₂₁N₃O₈)_nGd_n, where n=1-3.

EFFECT: improved properties.

2 cl, 1 tbl, 3 ex, 9 dwg

Description

The invention relates to medicine, and more specifically to means for magnetic resonance contrast diagnostics and binary radiation therapy of malignant tumors (neutron capture and photon capture therapy). The invention can be used in both experimental and clinical studies.

Magnetic resonance contrast media are widely used in clinical practice for the diagnosis of various pathological conditions. Magnetic resonance contrast medium increases the sensitivity, specificity and diagnostic reliability of magnetic resonance imaging, contributes not only to identifying a pathological focus, but also to more accurately determine the extent of its prevalence, making a differential diagnosis, shortens, ultimately, the study time and, therefore, makes the latter is more economical.

In neutron-capture and photon-capture radiation therapy, drugs containing gadolinium are used to generate additional energy in the irradiated target, which is formed as a result of nuclear-physical interactions of gadolinium with thermal neutrons or photons;

n + ¹⁵⁷ Gd → ¹⁵⁸ Gd → photons (7.88 meV) + conversion electrons (45-66 keV) + Auger and Kester-Kronig electrons (5-9 keV);

γ (~ 60 keV) + Gd, Bi ~ x-ray radiation (-30 keV) + electrons (~ 30 keV)

Currently, a number of commercial magnetic resonance contrast media are known that have been used in clinical practice as diagnostic drugs. All of them are aqueous solutions of gadolinium complex compounds with organic ligands. These are preparations Magnevist and Gadovist of the German company Bayer AG, Omniscan of the American company GE Healthcare, Dotareram of the French company Laboratore Gubert. ProHans and MultiHans of the Italian company Bracco.

The most widely used drugs are based on diethylenetriaminopentaacetic acid and, in particular, the drug Magnevist, which was chosen as an analogue [DE No. 3129906, A61, publ. 1981].

The drug Magnevist is a 0.5 M aqueous solution of dimeglumine salt of gadopentetate with a pH of ~ 7, a viscosity of 4.9 cP at 20 ° C and a density of not more than 1.2 g / cm ³ at 20 ° C.

The use of Magnevist and other magnetic resonance contrast media to form additional energy release in binary radiation technologies is limited by the high rate of elimination from the place of their interstitial injection. Systemic routes of administration of these drugs are not effective for this purpose, since they are all extracellular substances and are not able to accumulate inside normal and tumor cells. In addition, the drug Magnevist is not safe for medical use, as it is difficult to control its quality.

A known dosage form of gadopentetate is a 0.5 M solution of the disodium salt of a complex of gadolinium with diethylene triaminopentaacetic acid with a pH of 5.0-85, containing up to 5% polyvinylpyrrolidone with a molecular weight of 12,000 ± 1,500 daltons. This dosage form is taken by us as a prototype [RU No. 2150961, A61K 49/00, publ. 06/20/2000]. The presence in the dosage form of polyvinylpyrrolidone with a molecular weight of 12,000 ± 1,500 daltons provides a delayed elimination of the main substance of gadopentetate during the intratumoral route of administration. The disadvantage of the prototype dosage form is the presence of polyvinylpyrrolidone with a molecular weight of 12,000 ± 1,500 daltons, which is forbidden for medical use (Circular of the Federal Service for Supervision of Health and Social Development (No. 1100-Pr / 05 of 05.24.2005). elimination gadopentetata when intratumoral administration of the dosage form in neutron-capture technology and photon radiotherapy gripping characterized elimination half period T _{1 / 2E} equal to 50 ± 5 minutes, which is not the optimum value d I ray binary implementation tehnoloty.

A known contrast composition for magnetic resonance imaging and x-ray studies based on the gadolinium complex, according to the invention, containing from 0.1 to 1.5 mol / l of the gadolinium complex (Gd ³⁺ ) with disodium salt of diethylene triaminopentaacetic acid (DTPA) or dimethylamide diethylenetriamine penta acetic acid 6-8 and additionally containing from 0.0001 to 0.5% metalchrome indicator by weight of gadolinium. As a metalchromic indicator, chromazurol S or pure-blue BPD chromoxane is used, the solutions of which are stable for a long time. Chromazurol S and chromoxane pure blue BPD have a contrast transition (yellow - purple) in the working pH range from 6 to 8 units. The complexation constant of these indicators is 12 to 16 units. The color change of the solution of the composition is due to the formation of the gadolinium-indicator complex during the decomposition of the gadolinium-DTPA or gadolinium-DMADTPA complex (RU 2308290, A61K 49/06, A61K 49/04, publ. 20.10.2007). This source is accepted as a prototype.

A positive result obtained by the implementation of the proposed composition is the high safety of the drug as a magnetic resonance and x-ray contrast composition. But in the presence of such positive qualities, this contrast agent has the disadvantage of insufficient visualization of specific enzymatic activity and activation of transcription factors in living systems.

The present invention is aimed at achieving a technical result, which consists in increasing the efficiency of use in neutron capture and photon capture therapy by enhancing the visualization of specific enzymatic activity and activation of transcription factors in living systems.

The specified technical result is achieved by the fact that beta-cyclodextrin derivatives, in particular beta-cyclodextrin, containing one, two, three gadopentetate residues, or a mixture thereof with the general formula, are used as a contrast agent.

(C ₄₂ H _70-n O ₃₅ ) (C ₁₄ H ₂₁ N ₃ O ₈ ) _n Gd _n , where n = 1-7.

The indicated technical result is also achieved by the fact that beta-cyclodextrin derivatives, in particular beta-cyclodextrin, containing one, two, three residues of gadopentetate or a mixture thereof with the general formula are used as a contrast agent.

(C ₄₂ H _70-n O ₃₅ ) (C ₁₄ H ₂₁ N ₃ O ₈ ) _n Gd _n , where n = 1-7, wherein the mixture consists of 70-80% of a mono-derivative beta-cyclodextrin, 10-20%, a di-derivative of beta-cyclodextrin and 5-10% of a tri-derivative of beta-cyclodextrin.

These features are significant and are interconnected with the formation of a stable set of essential features sufficient to obtain the desired technical result.

The present invention is illustrated by illustrative material, where:

figure 1 - front MP tomogram of a mouse with transplantable tumor of melanoma B16, T2 - weighted image,

figure 2 is a frontal MP tomogram of a mouse with a transplantable tumor of melanoma B16, T1 weighted image before the introduction of the sample;

figure 3 - front MP tomogram of a mouse with transplantable tumor of melanoma B16, T1 weighted image after injection of the sample, the tumor growth area is highlighted;

4 is a frontal MP tomogram of the pelvic region of a mouse with transplantable epidermoid lung carcinoma Lewis LLC1, T2 - weighted image;

5 is a frontal MP tomogram of the pelvic region of a mouse with transplantable epidermoid lung carcinoma Lewis LLC1, T1 weighted image before the introduction of the sample;

6 is a frontal MP tomogram of the pelvic region of a mouse with transplantable epidermoid lung carcinoma Lewis LLC1, T1 weighted image after injection of the sample, the tumor growth area is highlighted.

7 is a frontal MP tomogram of the pelvic region of a mouse with transplantable mammary adenocarcinoma Ca755, T2 is a weighted image;

Fig. 8 is a frontal MP tomogram of the pelvic region of a mouse with transplantable mammary adenocarcinoma Ca755, T1 weighted image before sample administration;

Fig.9 is a frontal MP tomogram of the pelvic region of a mouse with transplantable adenocarcinoma of the mammary gland Ca755, T1 weighted image after injection of the sample, the tumor growth area is highlighted.

The use of contrast agents with paramagnetic properties is one of the most promising areas for improving the diagnostic capabilities of magnetic resonance imaging (MRI). Until now, contrast preparations are created on the basis of complexes with Gd + 3. Paramagnetic relaxation properties of Gd + 3 favorably show the possibility of creating paramagnetic contrast agents with relaxation. Contrast media (KB) is used for MRI in cases where it is necessary to identify a tumor, its size, cell composition and borders. Contrast allows a more detailed study of the structure of tissues. Gadolinium based components are non-toxic and highly soluble in water. In order for gadolinium to cease to be toxic, special chelate complexes (compositions, substances) are created, since its simple salts are poisonous. The use of contrast increases the return of the signal from the area of interest of the body. The speed of contrast depends primarily on how densely the area is covered with blood vessels.

The main purpose of using KB in MRI is to accelerate the relaxation of the spins of water protons in the surrounding tissue, i.e. shortening the spin-lattice T1 and spin-spin T2 relaxation times by applying the gadolinium salt Gd (III), since the cation Gd (III) has 7 unpaired f-electrons, and a symmetric S state, which determines, on the one hand, a large magnetic moment, and on the other hand, slow relaxation of electrons, which is necessary from the point of view of the paramagnetic quantum well efficiency. The general theory of relaxation in dilute solutions of paramagnetic substances was developed by Blumbergen, Solomon and Morgan in the 1950s. According to this theory, the relaxation frequency (relaxivity R _(1,2) = 1 / T _(1,2) , i.e., the reciprocal of the relaxation time) in the presence of the cation Gd (III) 1 / T _(1,2) = 1 / Td _(1,2) + 1 / Tp _(1,2) = 1 / Td _(1,2) + r _(1,2) [Gd], where 1 / Td _(1,2) is a diamagnetic term that describes relaxation in the absence of KB; and r - the so-called molar relaxivity, which is the coefficient of proportionality between relaxivity and the [Gd] concentration of the paramagnetic cation. Relaxation is usually understood as proton relaxation. Paramagnetic relaxation of water protons occurs due to dipole-dipole interactions between nuclear spins and a fluctuating local magnetic field KB, which is created due to the presence of spin in unpaired electrons. In order for the paramagnetic relaxation effect to affect a large number of protons of molecules, they must be in the immediate sphere of influence of the KB field, since this field rapidly decreases with distance from the paramagnetic atom. For complexes of paramagnetic metals, a similar specific interaction is the coordination binding of water molecules with KV, i.e. interaction in the inner coordination shell. In addition to the internal-shell effect, there is also an external-shell effect, which depends on the stochastic translational diffusion of protons near the QW. In addition, water molecules form hydrogen bonds with a chelating agent. These water molecules in the secondary coordination shell contribute to the externally shell effect. Thus, the total relaxivity of water protons in a simplified form is equal to the sum of relaxations, where R = R ^IS + R ^OS , IS is the inner shell, and OS is the outer shell. The reciprocal of the spin-lattice relaxation time in the inner coordination shell (ie, relaxivity) is written as: 1 / T1 = (([Gd] · q / 55.5) (T _1m = τ _m ) ^-1 , where q is the number water molecules bound to Gd (hydration number), T _1m is the relaxation time of bound water, and τ _m is the lifetime of the water molecule in the inner coordination shell.

Proton relaxation in the presence of chelated paramagnetic cations is exposed to a whole range of factors. If the chelating substance prevents the formation of coordination bonds between water and the cation in the inner shell, the number of hydration of the cation is q <1, and the relaxation of the chelated cation is much less than the relaxation of the hydrated cation. The most frequently used in practice Gd (III) chelates allow the formation of coordination bonds with q = 1. If the relation T _1m << τ _m holds, then the exchange rate of water molecules coordinated with KB is the only determining factor of relaxation.

These features made it possible to experimentally create a new HF. The essence of the invention lies in the fact that to isolate the lesion area during MRI, a new composition (substance) is used as a contrast agent, which is beta-cyclodextrin derivatives modified with gadopentetat, in particular beta-cyclodextrin containing one, two, three gadopentetat residues or their mixture.

The general formula of the substance is: (C ₄₂ H _70-n O ₃₅ ) (C ₁₄ H ₂₁ N ₃ O ₈ ) _n Gd _n where n = 1-7.

In particular, as an option, derivatives of beta-cyclodextrin modified with gadopentetat are considered, namely beta-cyclodextrin containing one, two, three residues of gadopentetate or a mixture thereof consisting of 70-80% of a mono-derivative of beta-cyclodextrin, 10-20% of a di-derivative beta-cyclodextrin and 5-10% of the tri-derivative of beta-cyclodextrin.

Cyclohadopentetate (CGP) is obtained in two stages. The degree of modification is determined by the ratio of components - cyclodextrin and DADTPA.

At the first stage, cyclodextrin is modified with diethylenetriaminopentaacetic acid dianhydride (DADTPA). To a solution of 30 g (0.0264 M) beta-cyclodextrin in dimethyl sulfoxide, 23.6 g of diethylene triaminopentaacetic acid dianhydride was added and stirring was continued at 60 ° C. for twenty hours. The solution is cooled to room temperature, poured into a glass with a capacity of 2 l, containing 600 ml of acetone. A white precipitate forms. The mixture is thoroughly mixed and incubated for 30 minutes to complete the formation of a precipitate. The reaction mixture was filtered in a vacuum of a water-jet pump on a funnel with a porous bottom and treated with acetone in five portions of 300 ml each and dried in vacuum (15 mm) with gentle heating (500 ° C) to constant weight. The yield of cyclopentetate (CP) (97%).

In the second stage, CHP is obtained by the interaction of CP with gadolinium nitrate. 45 g cyclopentetate is placed in a 750 ml flat-bottomed heat-resistant flask equipped with a reflux ball cooler and an armature for a magnetic stirrer, 450 ml of distilled water are poured and stirred for 15 minutes. To a solution of cyclopentetate in distilled water, 22 g of gadolinium hexahydrate are added and stirred at room temperature for one hour. The resulting solution was poured into a glass with a capacity of 2 l, containing 1000 ml of acetone. A white precipitate forms. The mixture is thoroughly mixed and incubated for 30 minutes to form a precipitate. The precipitate is filtered and dried. The output of CHP 98%.

To purify CHP, it is reprecipitated from an aqueous solution. For this, the dried intermediate is transferred into a glass with a capacity of 1 liter and dissolved in 400 ml of distilled water. The solution is poured into a 2 liter beaker containing 1000 ml of acetone. The precipitate formed is filtered off and dried.

The content of components with varying degrees of modification in the resulting product is determined by high performance chromatography. The characteristics of the mixtures obtained are presented in table 1.

Table 1 The content of mono-, di- and tri-modified DADTPA CGP and the relaxivity of mixtures and Magnevista ^® Sample, No. or Title % content of mono-, di-, tri-CHP Relaxivity, (mmol / L) ^-1 · s ^-1 one 20/15/25 5.13 2 40/15/15 5.54 3 80/0/20 5.81 four 60/30/10 6.29 5 80/10/10 6.08 6 35/0/0 5.39 Magnevist ^® 0/0/0 4.22

Relaxivity was determined on a BrukerBioSpinc ClinScan MP scanner (magnetic induction 7 T, resonant frequency 300 MHz for ¹ H).

MRI studies using the resulting mixtures were performed on a Bruker BioSpin ClinScan MRI scanner with a field strength of 7T (300 MHz for 1H) using a two-segment surface receiving coil. After obtaining fast orthogonal T1 weighted images (TI), T2 weighted images (TI) with frequency suppression of the fat signal in the frontal plane were obtained using the pulse sequence Turbo Spin Echo with parameters: TR = 2220 ms, TE = 49 ms, cut thickness 1 mm, 16 slices, 320 × 320 matrix, FOV read = 50 mm, K-space filling path - BLADE, BLADE coverage 100%. We also obtained T2-VI with frequency suppression of the fat signal in axial projection: Turbo Spin Echo, TR = 3310 ms, TE = 51 ms, slice thickness 1 mm, 26 slices, matrix 256 × 256, FOV read = 40 mm. T2 VIs were used to verify the presence of the tumor process, as well as more accurate positioning of subsequent sections. Then, with a similar geometry, we obtained two-dimensional gradient T1-VI with frequency suppression of fat, in axial: TR = 529 ms, TE = 4.3 ms, angle = 70 degrees, slice thickness 1 mm, 26 slices, matrix 259 × 384 and FOV read = 50 mm; and frontal planes: TR = 419 ms, TE = 4.03 ms, angle = 70 degrees, slice thickness 1 mm, 18 slices, matrix 259 × 384, FOV read = 50 mm, data acquisition time - 2:16 min. After the studied component was introduced into the animals, the dynamics of its distribution and excretion were monitored using frontal T1-VI images with a two-minute step (data acquisition time 2:16).

Example 1

A suspension of B-16 murine melanoma cells (Bank of tumor strains of the Russian N. Blokhin Russian Academy of Medical Sciences RAMS) is transplanted under the skin and into the muscles of the right posterior thigh to BDF1 mice weighing 19-22 grams (2-3 months of life) in 0.3 ml of a complete Hanks solution with dye. When injected, animals are injected with 3.5-4.0 million tumor cells. Tumor development is controlled by measuring the size of the tumor. Animals are selected for further studies after reaching a tumor size of about 1 cm ³ .

Mice of the BDF1 line with transplantable B16 melanoma on the 8th day after inoculation are injected intravenously with 0.04 ml of aqueous CHP with pH = 7 with a gadolinium content of 0.4 mg. On MRI tomograms, the accumulation of the drug in the tumor is clearly visible. Figure 1-3 shows the front MP tomograms of a mouse with a transplantable tumor of melanoma B16: T2 is a weighted image, T1 is a weighted image before sample administration and T1 is a weighted image after sample administration. The area of tumor growth is clearly marked.

Example 2

A cell suspension of LLC1 epidermoid lung carcinoma of the lungs (Bank of tumor strains of FSBI RONTs im.N.N. Blokhin RAMS) was transplanted under the skin and into the muscles of the right back femur to BDF1 mice weighing 19-22 grams (2-3 months of life) in 0.3 ml of a complete Hanks solution with dye. When injected, animals are injected with 3.5-4.0 million tumor cells. Tumor development is controlled by measuring the size of the tumor. Animals are selected for further studies after reaching a tumor size of about 1 cm ³ .

Mice of the BDF1 line with transplantable Lewis LLC lung carcinoma on the 8th day after inoculation were intravenously administered 0.04 ml of aqueous CHP with pH = 7 containing 0.4 mg of gadolinium. On MRI tomograms, contrasting of the tumor is clearly visible. Figures 4-6 show frontal MP tomograms of the pelvic region of a mouse with transplantable epidermoid lung carcinoma of Lewis LLC1: T2 is a weighted image, T1 is a weighted image before sample administration and T1 is a weighted image after sample administration. The area of tumor growth is clearly marked.

Example 3

A suspension of mammary adenocarcinoma cells Ca755 (Bank of tumor strains of the Russian N. Blokhin Russian Cancer Research Center of the Russian Academy of Medical Sciences) was transplanted under the skin and into the muscles of the right posterior thigh to BDF1 mice weighing 19-22 grams (2-3 months of life) in 0.3 ml of a complete Hanks solution with dye. When injected, animals are injected with 3.5-4.0 million tumor cells. Tumor development is controlled by measuring the size of the tumor. Animals are selected for further studies after reaching a tumor size of about 1 cm ³ .

Mice of the BDF1 line with transplantable Ca755 adenocarcinoma on the 8th day after transplantation were intravenously administered 0.04 ml of aqueous CHP with pH = 7 containing 0.4 mg of gadolinium. On MRI tomograms, the accumulation of the drug is clearly visible. Figures 7-9 show frontal MP tomograms of the pelvic region of a mouse with transplantable adenocarcinoma of the mammary gland Ca755: T2 is a weighted image, T1 is a weighted image before sample administration and T1 is a weighted image after sample administration. The area of tumor growth is clearly marked.

Claims (2)

1. A tool for magnetic resonance diagnostics, which is modified with gadopentetatom derivatives of beta-cyclodextrin, where beta-cyclodextrin contains one, two, three residues of gadopeptetate or a mixture thereof with the General formula (C ₄₂ H _70-n O ₃₅ ) (C ₁₄ H ₂₁ N ₃ O ₈ ) _n Gd _n , where n = 1-3. 2. A tool for magnetic resonance diagnostics, which is modified with gadopentetatom derivatives of beta-cyclodextrin, where beta-cyclodextrin, which contains one, two, three residues of gadopentetat or their mixture, consisting of 70-80% of a mono-derivative beta-cyclodextrin, 10-20 % beta-cyclodextrin di-derivative and 5-10% beta-cyclodextrin tri-derivative with the general formula (C ₄₂ H _70-n O ₃₅ ) (C ₁₄ H ₂₁ N ₃ O ₈ ) _n Gd _n , where n = 1- 3.

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