CN113683653B

CN113683653B - Adenosine crystal form, preparation method and application thereof

Info

Publication number: CN113683653B
Application number: CN202110931201.3A
Authority: CN
Inventors: 陈阳; 张磊; 付永慧; 丁薇; 李佳杨
Original assignee: Yuanda Life Science Liaoning Co ltd
Current assignee: Yuanda Life Science Liaoning Co ltd
Priority date: 2021-06-28
Filing date: 2021-08-13
Publication date: 2024-03-19
Anticipated expiration: 2041-08-13
Also published as: CN113683653A

Abstract

The invention provides an adenosine crystal form, a preparation method and application thereof. The crystalline form has diffraction peaks at 5.5 ° ± 1, 7.1 ° ± 1, 13.5 ° ± 1, 14.2 ° ± 1, 14.8 ° ± 1, 16.3 ° ± 1, 18.3 ° ± 1 and 19.6 ° ± 1 using Cu-ka radiation, X-ray powder diffraction pattern expressed in terms of 2θ angle. The crystal form has the advantages of good stability, difficult degradation of products, capability of meeting the pharmaceutical requirements of production, transportation and storage, high dissolution speed and obviously shortened preparation process time.

Description

Adenosine crystal form, preparation method and application thereof

The present application claims the priority of the Chinese patent application with application number 202110719209.3, which is filed on 6/28 of 2021 and is entitled "an adenosine crystal form, preparation method and application thereof".

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and particularly relates to an adenosine crystal form, a preparation method and application thereof.

Background

Adenosine (Ado, adenosine) is known as adenine nucleoside, and is formed by combining adenine and pentose. The CAS number is 58-61-7, the chemical name is 6-amino-9-beta-D-ribofuranosyl-9H-purine, and the chemical structural formula is shown in formula I.

Adenosine, an endogenous active substance, has wide application in clinical aspects such as diagnosing coronary heart disease. Adenosine, which is a precursor of adenine nucleotides and a metabolite thereof, is a physiological regulator widely existing in organisms. Adenosine activates adenosine receptors (a receptors) to exert physiological and pharmacological actions. Up to now, 4 receptors for adenosine have been found, namely the A1, A2b, A3 receptors. Al receptors in the heart are mainly distributed on the surfaces of atrial muscles, ventricular muscles, sinus nodes and atrioventricular cells, adenosine and analogues thereof produce negative time-varying, force-varying and conduction-varying actions by exciting the Al receptors, and myocardial protection actions are produced by post-receptor signal transduction mechanisms. The A2 receptor is classified into a high affinity A2 receptor and a low affinity A2b receptor according to its affinity for adenosine. Adenosine binds to the A2 receptor and increases cAMP, thereby dilating vascular smooth muscle. Adenosine binds to A3 receptor, increases the activities of superoxide dismutase (SOD) and glutathione enzymes, activates the antioxidant system of cells, and reduces myocardial damage. The activated A3 receptor in turn increases the release of mast cell allergic mediators by promoting mast cell degranulation. The mechanism of action of adenosine on myocardial protection may be: (1) adenosine promotes the maintenance of microvascular blood flow; (2) adenosine inhibits neutrophil function; (3) adenosine may improve myocardial energy supply; (4) adenosine inhibits the production of oxygen radicals; (5) adenosine restores calcium balance; (6) adenosine promotes vascular repair and inhibits ventricular remodeling; (7) adenosine mediated ischemic preconditioning and post-ischemic conditioning; (8) adenosine inhibits apoptosis of cardiomyocytes. Cardioprotection by adenosine is associated with agonism of all of the A1, A2 and A3 receptors.

Adenosine can be prepared by a variety of routes including chemical synthesis, biological fermentation, and by isolation and purification of biological tissue. Patent CN1629178 discloses a process for the preparation of adenosine, which comprises obtaining needle-like, columnar or plate-like crystals by crystallization to increase the yield of adenosine preparation, but does not disclose specific information of the crystal form.

The preparation concentration of the adenosine injection was 3mg/mL, but the solubility of adenosine in water was 5.1mg/mL. Therefore, in the preparation concentration process, the solubility of the adenosine is close to the saturation concentration, so that the adenosine is dissolved for a long time, and the method is one of the longest procedures in the preparation process of the adenosine injection. And may leave a fine undissolved adenosine product during dissolution, resulting in a risk of an adenosine injection having a low level or unacceptable insoluble particles.

Therefore, when used as a raw material of injection, the adenosine has good development stability and high dissolution speed, and can effectively prolong the effective period of the injection as a raw material of the adenosine, thereby shortening the working time of the preparation and improving the safety of the injection.

Disclosure of Invention

The inventor of the invention examines the crystal products of the adenosine under different crystallization conditions, and performs X-diffraction, infrared detection, thermogravimetric analysis and DSC detection on the obtained crystal products, and discovers that under certain specific crystallization conditions, the adenosine crystal form with good stability can be obtained.

The present invention provides an adenosine crystalline form having an X-ray powder diffraction pattern expressed in terms of 2θ with diffraction peaks at 5.5°±1, 7.1°±1, 13.5°±1, 14.2°±1, 14.8°±1, 16.3°±1, 18.3°±1 and 19.6°±1 using cu—kα radiation.

Preferably, the X-ray powder diffraction pattern expressed in terms of 2θ using Cu-Ka radiation of the adenosine crystalline form has diffraction peaks at 5.5 ° ± 1, 7.1 ° ± 1, 8.2 ° ± 1, 11.4 ° ± 1, 13.5 ° ± 1, 14.2 ° ± 1, 14.8 ° ± 1, 16.3 ° ± 1, 16.5 ° ± 1, 18.3 ° ± 1, 19.6 ° ± 1, 21.4 ° ± 1, 23.2 ° ± 1, 24.7 ° ± 1, 26.4 ° ± 1, 27.9 ° ± 1, 29.8 ° ± 1 and 36.2 ° ± 1.

More preferably, the X-ray powder diffraction pattern of the adenosine crystalline form, expressed in terms of 2 theta angles, using Cu-K alpha radiation is shown in figure 1.

The differential scanning calorimetry curve of the adenosine crystalline form has an endothermic peak at 54.2+ -5deg.C, 110.3+ -5deg.C, 235.7+ -5deg.C, and an exothermic peak at 151.7+ -5deg.C.

Preferably, the differential scanning calorimetric curve of the adenosine crystalline form is shown in fig. 2.

Preferably, the thermogravimetric analysis of the adenosine crystalline form is as shown in figure 3.

Preferably, the moisture content of the adenosine crystal form is 8.0% -15.0%.

In another aspect, the present invention also provides a preparation method of the above adenosine crystal form, which includes: dissolving adenosine in the mixed solution of water and organic solvent, standing for crystallization.

In the above preparation method, the ratio of the volume of the mixed solution to the weight of adenosine may be 10 to 200mL:1g, preferably 100 to 150mL:1g.

In the above preparation method, the volume ratio of the water and the organic solvent in the mixed solution may be 1:0.1 to 10, preferably 1:0.25 to 4.

In the above preparation method, the temperature of the dissolution may be 30 to 85 ℃.

In the above preparation method, the organic solvent may be selected from one or more of tetrahydrofuran, acetonitrile, methanol, ethanol, or acetone.

In the above preparation method, the crystallization is performed at a temperature of 0 to 30 ℃, preferably 15 to 30 ℃, under atmospheric pressure or vacuum.

Preferably, the method further comprises the step of filtering and drying the crystals after crystallization.

In another aspect, the invention also provides a pharmaceutical composition comprising the above-described adenosine crystalline form or comprising an adenosine crystalline form prepared by the above-described method of preparation, and one or more pharmaceutically acceptable carriers, diluents or excipients.

On the other hand, the invention also provides the application of the adenosine crystal form or the adenosine crystal form prepared by the preparation method in preparing medicaments for preventing and/or treating heart diseases.

Preferably, the heart disease is angina, myocardial infarction, myocardial ischemia or paroxysmal supraventricular tachycardia.

On the other hand, the invention also provides the application of the adenosine crystal form or the adenosine crystal form prepared by the preparation method in preparing medicaments for diagnosing cardiovascular diseases, evaluating sinus node functions, judging myocardial activity and prognosis evaluation of coronary heart disease; preferably, the cardiovascular disease is tachycardia, implicit pre-excitation syndrome, coronary artery disease and Chronic Coronary Syndrome (CCS).

The invention has the following beneficial effects:

1. the adenosine prepared by the invention has good stability, the product is not easy to degrade, and the pharmaceutical requirements of production, transportation and storage can be met;

2. the product has high dissolution speed, obviously shortens the preparation process time, can avoid the generation of impurities in the preparation process, can reduce the risk of pollution of samples, and improves the safety of the preparation;

3. the new crystal form has good stability under the conditions of illumination, high temperature, high humidity, heating and the like, and the product is not easy to degrade;

4. the product can effectively prolong the effective period of the product as a medicine raw material, and can meet the pharmaceutical requirements of production, transportation and storage;

5. the production process is stable, repeatable and controllable, and can meet the requirements of industrial production.

Drawings

Specific embodiments of the invention are described with reference to the following drawings:

FIG. 1 is an X-ray powder diffraction pattern of the crystalline form of adenosine prepared in example 1 of the present invention.

FIG. 2 is a DSC chart of the adenosine crystalline form prepared in example 1 of the present invention.

FIG. 3 is a TGA spectrum of the adenosine crystalline form prepared in example 1 of the present invention.

FIG. 4 is a DVS spectrum of the adenosine crystalline form prepared in example 1 of the present invention.

FIG. 5 is a graph of the X-ray powder diffraction contrast of the crystalline form of adenosine prepared in example 1 of the present invention before and after the DVS test.

FIG. 6 is an X-ray powder diffraction pattern of the crystalline form of adenosine prepared in example 1 of the present invention after 30 days of examination at 60 ℃.

FIG. 7 is an X-ray powder diffraction pattern of the crystalline form of adenosine prepared in example 1 of the present invention after 30 days of light.

FIG. 8 is an X-ray powder diffraction pattern of the crystalline form of adenosine prepared in example 1 of the present invention after 6 months of examination under accelerated conditions (40 ℃ C., RH 75%).

Fig. 9 is a TGA profile of an adenosine crystalline form prepared by the method disclosed in patent CN 1629178.

FIG. 10 is an HPLC chromatogram of a formulated solution with all of the adenosine crystalline forms prepared in example 1 of the present invention dissolved.

Fig. 11 is an HPLC chromatogram of a formulation solution of the crystalline form obtained in patent CN1629178A at 60min during the formulation process.

Fig. 12 is an HPLC chromatogram of the formulated solution obtained in patent CN1629178A after complete dissolution of the crystalline form (120 min).

Detailed Description

The present invention will be further described with reference to examples, which are not intended to limit the invention in any way, so that those skilled in the art will more fully understand the invention.

Powder X-ray diffraction (XRPD) data for the adenosine crystalline forms of the present invention were measured by a bruker X-ray diffractometer under the following conditions:

rays: monochromatic Cu-ka radiation (λ=1.5406);

scanning mode: θ/2θ, scan range: 5-40 degrees;

voltage: 45KV, current: 40mA;

DSC, TGA test conditions were:

parameters (parameters)	TAG	DSC
			Method	Linear temperature rise	Linear temperature rise
Sample tray	Aluminum plate, open	Aluminum plate, gland/not gland
			Temperature range	Room temperature to set end temperature	25 ℃ to set end point temperature
Scanning speed (. Degree. C./min)	10	10
			Protective gas	Nitrogen gas	Nitrogen gas

The DVS test conditions were:

EXAMPLE 1 preparation of adenosine Crystal form

10g of adenosine was dissolved in 1200mL of acetonitrile aqueous solution (acetonitrile/water=1/1) at 85℃and left to evaporate naturally under vacuum at 20℃to precipitate crystals, and the crystals were filtered and dried to give 8.9g of adenosine with a purity of 99.7%. FIG. 1 is an X-ray powder diffraction (XRPD) pattern of the crystalline form having diffraction peaks at 5.5, 7.1, 8.2, 11.4, 13.5, 14.2, 14.8, 16.3, 16.5, 18.3, 19.6, 21.4, 23.2, 24.7, 26.4, 27.9, 29.8 and 36.2. FIG. 2 shows a DSC profile of this crystalline form having an endothermic peak at 54.2.+ -. 5 ℃, 110.3.+ -. 5 ℃, 235.7.+ -. 5 ℃ and an exothermic peak at 151.7.+ -. 5 ℃. This form is defined as the new form of adenosine. Fig. 3 shows the TGA profile of this crystal form, and it can be seen that the moisture content of the adenosine crystals is 12.1%.

EXAMPLE 2 preparation of adenosine Crystal form

10g of adenosine was dissolved in 2000mL of tetrahydrofuran aqueous solution (tetrahydrofuran/water=10/1) at 30℃and left to evaporate naturally under vacuum at 30℃to precipitate crystals, and the crystals were filtered and dried to give 8.8g of adenosine with a purity of 99.8%. The X-ray diffraction pattern of the obtained crystal sample was confirmed to be identical to the crystal form obtained in example 1 by research and comparison. The moisture content of the adenosine crystals was 8.0%.

EXAMPLE 3 preparation of adenosine Crystal form

10g of adenosine was dissolved in 100mL of aqueous solution of methanol and acetone (methanol/acetone/water=4/1/50) at 50℃and left to evaporate naturally at 15℃to precipitate crystals, and 9.0g of adenosine was obtained after filtration and drying with a purity of 99.9%. The X-ray diffraction pattern of the obtained crystal sample was confirmed to be identical to the crystal form obtained in example 1 by research and comparison. The moisture content of the adenosine crystals was 15.0%.

EXAMPLE 4 preparation of adenosine Crystal form

10g of adenosine was dissolved in 1000mL of ethanol aqueous solution (ethanol/water=1/4) at 40℃and left to evaporate naturally under vacuum at 25℃to precipitate crystals, and the crystals were filtered and dried to give 8.5g of adenosine with a purity of 99.9%. The X-ray diffraction pattern of the obtained crystal sample was confirmed to be identical to the crystal form obtained in example 1 by research and comparison. The moisture content of the adenosine crystals was 10.0%.

EXAMPLE 5 preparation of adenosine Crystal form

10g of adenosine is dissolved in 1500mL of aqueous solution of acetone and ethanol (acetone/ethanol/water=2/2/1) at 30 ℃ and placed at 0 ℃ for natural volatilization, crystals are separated out, 7.8g of adenosine is obtained after filtration and drying, and the purity is 99.8%. The X-ray diffraction pattern of the obtained crystal sample was confirmed to be identical to the crystal form obtained in example 1 by research and comparison. The moisture content of the adenosine crystals was 14.0%.

EXAMPLE 6 adenosine Crystal TGA experiment

The TGA test conditions were:

parameters (parameters)	TAG
		Method	Linear temperature rise
Sample tray	Aluminum plate, open
		Temperature range	Room temperature to set end temperature
Scanning speed (. Degree. C./min)	10
		Protective gas	Nitrogen gas

TGA measurements were performed using the sample of example 1 and the adenosine powder obtained by the method of patent CN1629178A, respectively. The results are shown in fig. 3 and 9.

The results showed that the sample of example 1 of the present invention was a hydrate with a moisture content of 12.1% and the sample prepared in patent CN1629178A did not lose weight before 200 ℃ and did not begin to lose weight until about 260 ℃ and the temperature was the degradation temperature of adenosine, indicating that the loss of weight was due to degradation of adenosine rather than loss of water, thus judging that the adenosine powder was anhydrous.

EXAMPLE 7 adenosine Crystal DVS experiment

The samples of example 1 were selected for hygroscopicity assessment, and as shown in fig. 4, the adenosine crystal form showed little weight change between 20% rh and 100% rh, and only showed significant weight loss below 10% rh, but with increasing humidity, the lost water molecules could be recombined with adenosine, thus maintaining the stability of the crystal form. And as shown in fig. 5, XRPD patterns before and after DVS experiments were compared, with no change in adenosine crystalline form. The crystal form is a stable crystal form and has good stability.

Example 8 experiment of influence factor of adenosine Crystal

The adenosine crystalline form prepared in example 1 was uniformly distributed to an open dish with a thickness of about 5mm, and was subjected to sampling test at high temperature (60 ℃) and under light (4500 lx.+ -. 500 lx) for 5 days, 10 days and 30 days, respectively, and compared with the results of 0 days, and the results are shown in tables 1 and 2. Wherein, the X-ray powder diffraction diagram of the adenosine crystal form after 30 days at 60 ℃ is shown in figure 6, and the X-ray powder diffraction diagram of the adenosine crystal form after 30 days of illumination is shown in figure 7.

TABLE 1 high temperature stability test results for adenosine crystalline forms

Time (Tian)	Appearance of	Purity%％)
			0	White powder	99.7％
5	White powder	99.7％
			10	White powder	99.7％
30	White powder	99.7％

TABLE 2 results of test for the illumination stability of adenosine crystals

Time (Tian)	Appearance of	Purity (%)
			0	White powder	99.7％
5	White powder	99.7％
			10	White powder	99.7％
30	White powder	99.7％

Conclusion of the test: the crystal of the invention has good stability under high temperature and illumination, and can keep stable appearance and purity within 30 days.

EXAMPLE 9 accelerated stability test of adenosine

The packaged adenosine crystalline form prepared in example 1 was placed in a stability test chamber at 40 ℃ and 75% RH for six months, sampled and tested at the end of 3 and 6 months, respectively, and compared to the results for 0 month. The results are shown in Table 3. The X-ray powder diffraction pattern of the 6 month sample is shown in FIG. 8.

TABLE 3 accelerated stability test results of adenosine crystals

Time (month)	Appearance of	Purity (%)
			0	White powder	99.7％
3	White powder	99.7％
			6	White powder	99.6％

Conclusion of the test: the crystal of the invention has good stability under 40 ℃ and RH75%, and can maintain stable appearance and purity within 6 months.

EXAMPLE 10 experiment of dissolution rate of adenosine Crystal form

Adenosine powders obtained by the inventive adenosine crystalline form (prepared in example 1) and the method of patent CN1629178A were dissolved in physiological saline, respectively, the concentration process was simulated, and the dissolution rates were compared, and the results are shown in table 4:

TABLE 4 Experimental results of dissolution rate of adenosine crystals

The experimental results show that: the adenosine crystals prepared in the embodiment 1 of the invention have a dissolution rate obviously faster than that of adenosine powder obtained by the method of the patent CN1629178A, so that the production time of the preparation can be obviously shortened.

EXAMPLE 11 experiment of dissolution Rate of adenosine Crystal form at different temperatures

Adenosine powders obtained by the inventive adenosine crystalline form (prepared in example 1) and the method of patent CN1629178A were dissolved in physiological saline at different temperatures, respectively, the concentration process was simulated, and the dissolution rates were compared, and the results are shown in table 5:

TABLE 5 Experimental results of dissolution rate of adenosine crystals

The experimental results show that: the adenosine crystals prepared in the embodiment 1 of the invention have a dissolution rate which is obviously faster than that of the adenosine powder obtained by the method of the patent CN1629178A at different temperatures, and particularly, compared with the adenosine powder obtained by the method of the patent CN1629178A at 20 ℃ close to room temperature, the adenosine crystals prepared in the embodiment of the invention have a better dissolution rate, so that the preparation production man-hour can be obviously shortened, the generation of impurities in the preparation process can be avoided, and the risk of sample pollution can be reduced.

EXAMPLE 12 stability test of adenosine Crystal form preparation solution

Adenosine powders obtained by the inventive adenosine crystalline form (prepared by example 1) and the method of patent CN1629178A were dissolved in physiological saline, respectively, to simulate a concentration process. The specific experimental parameters are shown in table 6.

The adenosine crystal form of the present invention was assayed for the relevant substances after complete dissolution (5 min); the adenosine powder obtained by the method of patent CN1629178A was measured at 60min and after complete dissolution (120 min) during the formulation process, respectively.

The method for measuring the related substances adopts the method for measuring the related substances of adenosine in the 2020 edition of Chinese pharmacopoeia. Fig. 10 shows HPLC chromatograms of the formulation solution after the adenosine crystals of the present invention were completely dissolved, and fig. 11 and 12 show HPLC chromatograms of the formulation solution after the crystals of the crystal form obtained in patent CN1629178A were completely dissolved (120 min) at 60min during the formulation process, respectively.

TABLE 6 simulation of specific Experimental parameters for the rich process

Conclusion: after all adenosine crystal forms are dissolved in physiological saline, related substances in a sample solution are detected, an HPLC chromatogram is shown in a figure 10, the content of other impurities except adenosine is extremely small, and the purity of the sample solution is high. The adenosine powder obtained by the method of patent CN1629178A is sampled at 60min in the preparation process, and related substances in the sample solution are detected, wherein the HPLC chromatogram is shown in FIG. 11, and the content of other impurities except adenosine is increased; after the adenosine powder was completely dissolved in physiological saline (120 min), the relevant substances in the sample solution were detected, and the HPLC chromatogram is shown in FIG. 12, and the content of other impurities such as adenine was significantly increased except for adenosine, and the purity of the sample solution was affected.

From the above, it is known that the contact time between the adenosine raw material and the external environment increases during the long-term preparation, which may lead to degradation of the raw material and increase of the impurity content. Thus, the formulation time of the adenosine solution is an important factor affecting the quality of the subsequent formulation during the test and production process.

Although the present invention has been described to a certain extent, it is apparent that appropriate changes may be made in the individual conditions without departing from the spirit and scope of the invention. It is to be understood that the invention is not to be limited to the described embodiments, but is to be given the full breadth of the claims, including equivalents of each of the elements described.

Claims

1. An adenosine crystalline form, characterized by: an X-ray powder diffraction pattern expressed in terms of 2θ using Cu-ka radiation has diffraction peaks at 5.5 ° ± 1, 7.1 ° ± 1, 13.5 ° ± 1, 14.2 ° ± 1, 14.8 ° ± 1, 16.3 ° ± 1, 18.3 ° ± 1 and 19.6 ° ± 1; the moisture content of the adenosine crystal form is 8.0% -15.0%.

2. The crystalline form of claim 1, characterized by: which uses Cu-Ka radiation, an X-ray powder diffraction pattern expressed in terms of 2 theta has diffraction peaks at 5.5 DEG + -1, 7.1 DEG + -1, 8.2 DEG + -1, 11.4 DEG + -1, 13.5 DEG + -1, 14.2 DEG + -1, 14.8 DEG + -1, 16.3 DEG + -1, 16.5 DEG + -1, 18.3 DEG + -1, 19.6 DEG + -1, 21.4 DEG + -1, 23.2 DEG + -1, 24.7 DEG + -1, 26.4 DEG + -1, 27.9 DEG + -1, 29.8 DEG + -1 and 36.2 DEG + -1.

3. The crystalline form of claim 2, characterized by: the X-ray powder diffraction pattern expressed in terms of 2 theta angles using Cu-K alpha radiation is shown in FIG. 1.

4. A crystalline form according to any one of claims 1 to 3, characterized by a differential scanning calorimetry curve having an endotherm at 54.2±5 ℃, 110.3±5 ℃, 235.7±5 ℃ and an exotherm at 151.7±5 ℃.

5. The process for the preparation of a crystalline form according to any one of claims 1 to 4, characterized in that the process comprises: dissolving adenosine in a mixed solution of water and an organic solvent, and standing for crystallization; the weight ratio of the volume of the mixed solution to the adenosine is 100-200 mL/1 g; the organic solvent is selected from one or more of tetrahydrofuran, acetonitrile, methanol, ethanol or acetone;

wherein the volume ratio of the water to the organic solvent in the mixed solution is 1:0.1-10; the dissolution temperature is 30-85 ℃; the crystallization is carried out at the temperature of 0-30 ℃ under the atmospheric pressure or vacuum;

the method further comprises the steps of filtering and drying the crystals after crystallization.

6. The method according to claim 5, wherein the ratio of the volume of the mixed solution to the weight of adenosine is 100 to 150 mL/1 g.

7. The method according to claim 5, wherein the volume ratio of water to the organic solvent in the mixed solution is 1:0.25-4.

8. The production method according to any one of claims 5 to 7, wherein the crystallization is performed at a temperature of 15 to 30 ℃.

9. A pharmaceutical composition comprising an adenosine crystalline form according to any one of claims 1 to 4 or prepared by a process according to any one of claims 5 to 8, and one or more pharmaceutically acceptable carriers.

10. Use of an adenosine crystalline form according to any one of claims 1 to 4 or prepared by a process according to any one of claims 5 to 8 in the manufacture of a medicament for the prophylaxis and/or treatment of heart disease.

11. The use according to claim 10, wherein the heart disease is angina, myocardial infarction, myocardial ischemia or paroxysmal supraventricular tachycardia.

12. Use of the adenosine crystalline form of any one of claims 1 to 4 or the adenosine crystalline form prepared by the method of any one of claims 5 to 8 in the manufacture of a medicament for diagnosing cardiovascular disease, evaluating sinus node function, determining myocardial viability and prognosis of coronary heart disease.

13. The use according to claim 12, wherein the cardiovascular disease is tachycardia, implicit pre-excitation syndrome, coronary artery disease and Chronic Coronary Syndrome (CCS).