CN1331081A - Tetraphosphine quatorphosphonium salt, its preparing process, and two-step tetraphosphine medicine kit and its application - Google Patents

Abstract

The invention relates to a tetrofosmin quaternary phosphonium salt and a preparation method thereof. The tetrofosmin quaternary phosphonium salt has the formula [(C ₂ H ₅ OC ₂ H ₄ ) ₂ P ⁺ (H)C ₂ H ₄ P ⁺ (H)(C ₂ H ₄ OC ₂ H ₅ ) ₂ ]· 2X ^- , the definition of X in the formula is as stated in the description. The present invention also relates to a tetrofosmin kit in which the tetrofosmin raw material is made into a tetrofosmin quaternary phosphonium salt and a preparation method thereof. The tetrofosmin kit is obtained by preparing bottle A and bottle B respectively through a two-step method. The present invention also relates to the use of the tetrofosmin kit in the preparation of radioactive technetium-99m tetrofosmin injection with improved biological performance as an imaging agent in human or animal organs or tissues.

Description

A kind of tetrofosmin quaternary phosphonium salt and its preparation method, two-step method tetrofosmin kit and its application

The present invention relates to a ^99m Tc-labeled radiopharmaceutical, especially a bidentate organic phosphine compound, tetrofosmin quaternary phosphonium salt, and a preparation method thereof; Two-step tetrofosmin kit and preparation method thereof; in addition, the present invention further relates to radioactive technetium-99m tetrofosmin injection prepared by said two-step tetrofosmin kit and preparation method thereof; meanwhile, The present invention also relates to the use of the tetrofosmin kit in the preparation of radioactive technetium-99m tetrofosmin injection as an imaging agent in human or animal organs or tissues, especially in myocardium or tumors Use as imaging agent.

At present, cardiovascular disease is one of the main diseases that seriously endanger the life and health of our citizens. Early diagnosis of cardiovascular diseases is of great significance to the prevention and treatment of diseases. In nuclear cardiology, myocardial perfusion imaging using radionuclides can realize early diagnosis of cardiovascular diseases, and this diagnosis has become an important part of cardiology diagnostic examinations.

Since the 1980s, due to the use of Mo-Tc generators and the emergence of sterile, non-pyrogenic freeze-dried kits, ^99m Tc-labeled myocardial perfusion imaging agents have developed rapidly. Among them, ^99m Tc-MIBI, the structure is shown in formula a (Wackers FJT, Berman DS, Maddahi J et a1. Technetium-99mhexakis-2-methoxy-isobutyl-isonitrile: human biodistribution, dosimetry, safety and preliminary comparison to thallium-201 formyocardial perfusion imaging.J NuclMed, 1989, 30:301) ^99m Tc-teboroxime, whose structure is shown in formula b (Nunn AD et al., J Nucl Med 1986, 27:893) and ^99m Tc-tetrofosmin (structure shown in formula c) The development of imaging agents is relatively mature, and has been approved by the US FDA and widely used in clinical practice.

Several ^99m Tc-labeled Myocardial Perfusion Imaging Agents

Among the above three ^99m Tc-labeled myocardial perfusion imaging agents, ^99m Tc-tetrofosmin (tetrofosmin=tetrofosmin) is the latest type of organophosphine myocardial perfusion imaging agent released by British Amersham Company in the early 1990s. Its crude drug tetrofosmin (tetrofosmin, P53) was developed and synthesized for the first time by James D.Kelly et al. in 1991 by Amersham Company (J DKelly et al. USP5045302), and its labeled compound: [TcO ₂ (tetrofosmin) ₂ ] ⁺ shows Good myocardial uptake rate and blood, liver clearance performance, is considered to be an ideal myocardial perfusion imaging agent.

Subsequently, JDKelly et al. conducted further research on the labeled compound. In addition to preparing through liquid formulations, Kelly et al. also developed a sterile freeze-dried product preparation kit (Myoview ^TM ) of the compound, and carried out a test on the labeled compound. Further studies on structure determination and biological properties. (People such as JDKelly, Technetium-99m-tetrofosmin as NewRadiopharmaceutical for Myocardial Perfusion Imaging J Nucl Med1993, 34:222-227). Myoview ^TM , a sterile pyrogen-free freeze-dried product kit, was approved by the U.S. FDA in February 1996 and was launched on the market (Myoview ^TM kit for the preparation of Tc-99m-tetrofosmin), which can be directly labeled at room temperature to obtain ^99m Tc-tetrofosmin injection with a radiochemical purity greater than 90%, making it widely used in clinical practice abroad.

^99m Tc-tetrofosmin is not only used as a myocardial perfusion imaging agent, a large number of clinical studies have shown that it can also be used in the diagnosis of breast cancer, parathyroid cancer and other malignant tumors and the study of tumor multidrug resistance (MDR). ([1] Jain D, et al., J Nucl Med 1993, 34: 1354; [2] Tark Basoglu, et al., Eur J Nucl Med 1995, 22: 687; [3] Talahashi Norio, Reinhardt, Christopher P, et al., Circulation 1996, 94: 2605; [4] Tjeld Jan G, Erichsen Kunt, et al., J Nucl Med 1997, 38: 831; [5] Batista J F, Solano M E, et al., Nucl Med Commun 1997, 18:338)

In China, since the literature reports, Beijing, Shanghai and other places have successively started the synthesis of tetrofosmin raw material (tetrofosmin), the development of freeze-dried product kits, the preparation of the labeled compound ^99m Tc-tetrofosmin and the research on its biological characteristics . Such as: Beijing Jiangzhong Institute of Materia Medica (Zhang Guofeng et al., Synthesis of Tetrofosmin and Development of a Kit, Summary Collection of the Sixth National Academic Conference on Radiopharmaceuticals and Labeled Compounds, P17, 1996), School of Pharmacy, Shanghai Medical University (Zhu Gengen et al., Research on the new myocardial perfusion imaging agent ^99m Tc-P53, summary compilation of the Sixth National Conference on Radiopharmaceuticals and Labeled Compounds, P24, 1996), Department of Nuclear Medicine, Peking Union Medical College Hospital, Peking Union Medical College, China (Chen Fang, Wang Shizhen, Myocardial Perfusion Preparation and experimental research of imaging agent ^99m Tc-tetrofosmin, Chinese Nuclear Medicine 17(1): 13, 1997; Chen Fang, Zhou Qian, etc., preclinical research on myocardial perfusion imaging agent ^99m Tc-tetrofosmin, Chinese Nuclear Medicine 17( 1): 16, 1997), Beijing Shihong Drug Development Center, etc., including Beijing Jiangzhong Institute of Materia Medica and Beijing Division 1997), Beijing Shihong Drug Research Center, etc., including Beijing Jiangzhong Institute of Materia Medica and Beijing Shihong Drug Development Center The center has applied for the new drug clinical research of the drug.

Since the bulk drug tetrofosmin itself is a trivalent organic phosphine compound, this type of substance is easily oxidized in the presence of air, thus causing the bulk drug tetrofosmin and stannous tetrofosmin kits to be stored and transported ( Especially under high temperature and high humidity conditions in summer) quality problems occur, and the quality problems of the kit will directly affect the preparation of technetium-99m tetrofosmin injection and the image effect of myocardial imaging. In order to solve the above problems, prolong the shelf life of raw materials and kits, and provide reliable technetium-99m tetrofosmin injection for clinical use, we have made tetrofosmin raw materials into phosphonium salts, and further developed a two-step replacement method. Trifosmin kit and obtained technetium-99m tetrofosmin injection with improved biological properties.

Therefore, the object of the present invention is to provide a kind of new tetrofosmin quaternary phosphonium salt and preparation method thereof;

Another object of the present invention is to provide a tetrofosmin kit in which the tetrofosmin original drug is made into a tetrofosmin quaternary phosphonium salt and a preparation method thereof;

Another object of the present invention is to use the tetrofosmin kit to prepare radioactive technetium-99m tetrofosmin injection and its preparation method;

A further object of the present invention is to use the tetrofosmin kit as an imaging agent in the preparation of radioactive technetium-99m tetrofosmin injection in human or animal organs or tissues, especially in the myocardium. Or use as an imaging agent in tumors.

Tetrofosmin is a known compound, its chemical formula is (C ₂ H ₅ OC ₂ H ₄ ) ₂ PC ₂ H ₄ P(C ₂ H ₄ OC ₂ H ₅ ) ₂ , it is a bidentate organic phosphine compound, which is The tetrofosmin quaternary phosphonium salt shown in the following formula (I) obtained from the former medicine is a novel series of compounds:

[(C ₂ H ₅ OC ₂ H ₄ ) ₂ P ⁺ (H)C ₂ H ₄ P ⁺ (H)(C ₂ H ₄ OC ₂ H ₅ ) ₂ ]·2X ^- ...(I)

where X is a halogen such as Cl or Br,

Or the sulfonate ion of the following formula (II):

R ₂ is -H, -OH or -COOH.

The phosphonium salt reduces the activity of P(III) reacting with oxygen and can be stored for a long time, and the phosphonium salt can be easily converted into tetrofosmin in the original free state under neutral or alkaline conditions.

The present invention relates to a tetrofosmin quaternary phosphonium salt, characterized in that it has the following formula (I), [(C ₂ H ₅ OC ₂ H ₄ ) ₂ P ⁺ (H)C ₂ H ₄ P ⁺ (H )(C ₂ H ₄ OC ₂ H ₅ ) ₂ ]·2X ^- ...(I)

In the formula: X can be a halogen, such as chlorine or bromine;

Or the sulfonate group of the following formula (II),

In the formula, R ₁ is -H or -COOH, preferably -COOH;

R ₂ is -H, -OH or -COOH, preferably -OH.

The present invention also relates to the preparation method of the tetrofosmin quaternary phosphonium salt with following formula (I),

[(C ₂ H ₅ OC ₂ H ₄ ) ₂ P ⁺ (H)C ₂ H ₄ P ⁺ (H)(C ₂ H ₄ OC ₂ H ₅ ) ₂ ]·2X ^- ...(I)

In the formula: X is a halogen,

Or the sulfonate group of the following formula (II),

In the formula: R ₁ is -H, -COOH,

R ₂ is -H, -OH or -COOH,

The method includes:

Tetrofosmin (C ₂ H ₅ OC ₂ H ₄ ) ₂ PC ₂ H ₄ P(C ₂ H ₄ OC ₂ H ₅ ) ₂ and the reactant of formula HX

The definition of X in the formula is identical with the definition of formula (I),

Under the protection of an inert gas, the quaternary phosphonium salt of tetrofosmin, which is easily soluble in water, is formed in a non-polar medium at -10°C-25°C.

In the above preparation method, the inert gas can be nitrogen; the non-polar medium can be benzene, toluene, ether or petroleum ether, etc.

The composition of the two-step tetrofosmin kits A and B prepared by using the above-mentioned tetrofosmin quaternary phosphonium salt cannot be accurately expressed at present, so it can only be characterized by its preparation method.

In addition, the present invention relates to a kind of tetrofosmin medicine box that prepares tetrofosmin quaternary phosphonium salt from the tetrofosmin bulk drug, which is obtained by preparing bottle A and bottle B respectively according to the following two-step method:

Preparation of bottle A:

Reducing agent: intermediate ligand: auxiliary material: excipient: nitrogen-filled secondary water=(0.01-0.1): (1-5): (0.2-2.5): (1-10): (0.6-1) The weight ratio is mixed evenly, so that the above four materials are fully dissolved in the amount of nitrogen-filled secondary water, and the pH is adjusted to 5.0-8.5 with a pH regulator. After sterile filtration, it is divided into containers, and then freeze-dried , A bottle of medicine box A that is filled with nitrogen and sealed to be freeze-dried;

Wherein the reducing agent is Sn(II) halogen salt; the intermediate ligand is D-gluconic acid or its alkali metal or alkaline earth metal salt; the auxiliary materials are dihydrate sulfosalicylic acid, dihydrate sulfowater Sodium cylate or sodium sulfosalicylate, citric acid or its sodium salt, tartaric acid or its potassium or sodium salt; the excipient is sodium chloride or mannitol; the pH regulator is sodium hydroxide , sodium bicarbonate or disodium hydrogen phosphate;

Preparation of bottle B:

With the described tetrofosmin quaternary phosphonium salt as main bulk drug: auxiliary material: the weight ratio of solvent=(1-5): (1-10): (0.2-1.0), make described tetrofosmin quaternary phosphonium The salt and auxiliary materials are dissolved in the solvent, and after being fully dissolved, they are subpackaged in containers to obtain the medicine box B bottle; wherein the auxiliary materials are Tween emulsifiers or cyclodextrins; the solvents are water, Ethanol in water or absolute ethanol.

For the above-mentioned tetrofosmin kit, in the preparation of bottle A, the Sn(II) halogen salt is SnCL ₂ 2H ₂ O or SnF ₂ ; in the preparation of bottle B, the Tween emulsifier is Tween 20, Tween 40 or Tween 80, the cyclodextrin is γ-cyclodextrin or β-cyclodextrin.

Simultaneously, the present invention also relates to the preparation method of the above-mentioned tetrofosmin kit, which is prepared by preparing bottle A and bottle B respectively according to the above-mentioned two-step method.

In addition, the present invention relates to a radioactive technetium-99m tetrofosmin injection prepared by the two-step method tetrofosmin kit, which is prepared according to the following steps:

1) Preparation of bottle A:

Reducing agent: intermediate ligand: auxiliary material: excipient: nitrogen-filled secondary water=(0.01-0.1): (1-5): (0.2-2.5): (1-10): (0.6-1) The weight ratio is mixed evenly, so that the above four substances are fully dissolved in the amount of nitrogen-filled secondary water, and the pH is adjusted to 5.0-8.5 with a pH regulator. After sterile filtration, it is divided into containers, and then freeze-dried , A bottle of medicine box A that is filled with nitrogen and sealed to be freeze-dried;

Wherein the reducing agent is Sn(II) halogen salt; the intermediate ligand is D-gluconic acid or its alkali metal or alkaline earth metal salt; the auxiliary materials are dihydrate sulfosalicylic acid, dihydrate sulfowater Sodium cylate or disodium sulfosalicylate, citric acid or its sodium salt, tartaric acid or its potassium or sodium salt; the excipient is sodium chloride or mannitol; the pH regulator is hydrogen Sodium, sodium bicarbonate, or disodium hydrogen phosphate;

2) Preparation of bottle B:

With the described tetrofosmin quaternary phosphonium salt as main bulk drug: auxiliary material: the weight ratio of solvent=(1-5): (1-10): (0.2-1.0), make described tetrofosmin quaternary phosphonium The salt and auxiliary materials are dissolved in the solvent, and after being fully dissolved, they are subpackaged in containers to obtain the medicine box B bottle;

Wherein said adjuvant is Tween emulsifier or cyclodextrin; said solvent is water, ethanol aqueous solution or absolute ethanol;

3) Under aseptic operation, inject an appropriate amount of the radioactivity of ^99m TcO ₄ ^- (obtained from a medical ⁹⁹ Mo/ ^99m Tc generator) into the above prepared medicine box A bottle, shake well, and then The solution in the bottle B of the kit prepared above was taken out and poured into the above bottle A, shaken well, and left at room temperature for 15-20 minutes to obtain the radioactive technetium-99m tetrofosmin injection.

For the above-mentioned tetrofosmin injection, in the preparation of bottle A, the described Sn(II) halogen salt is Sncl ₂ 2H ₂ O or SnF ₂ ; in the preparation of bottle B, the Tween emulsifier Tween 20, Tween 40 or Tween 80, and the cyclodextrin is γ-cyclodextrin or β-cyclodextrin.

At the same time, the present invention also relates to the preparation method of the above-mentioned radioactive technetium-99m tetrofosmin injection, which is prepared according to the above-mentioned steps. The present invention further relates to the use of the tetrofosmin kit in the preparation of radioactive technetium-99m tetrofosmin injection as an imaging agent in organs or tissues of humans or animals, especially for use in myocardial or Use as an imaging agent in tumors.

The present invention can be illustrated more clearly by the following preparations and examples:

One, preparation example:

Synthesis of tetrofosmin:

For the synthesis of tetrofosmin [(C ₂ H ₅ OC ₂ H ₄ ) ₂ PC ₂ H ₄ P(C ₂ H ₄ OC ₂ H ₅ ) ₂ ], please refer to the literature of Kelly et al. [Kelly J D, KW Forster, Ina A. Latham . USP5045302, 1991]. Using phosphorus trichloride and absolute ethanol as raw materials, it is prepared through esterification, halogenation, reduction and free radical addition. The reaction route is as follows:

The obtained tetrofosmin is a colorless or light yellow viscous liquid, which is easily oxidized in air. Since tetrofosmin is a trivalent organic phosphine compound, under the protection of an inert gas, it can be formed with some organic or inorganic acids (such as: sulfosalicylic acid, HCl, etc.) of phosphonium salts. The phosphonium salt reduces the activity of P(III) reacting with oxygen, can be stored for a long time, and the phosphonium salt can be easily converted into tetrofosmin in the original free state under neutral or alkaline conditions. Two, embodiment:

Embodiment 1: tetrofosmin hydrochloride phosphonium salt

Preparation of [(C ₂ H ₅ OC ₂ H ₄ ) ₂ P ⁺ (H)C ₂ H ₄ P ⁺ (H)(C ₂ H ₄ OC ₂ H ₅ ) ₂ ]·2Cl ^-

3g tetrofosmin was dissolved in 10ml of anhydrous diethyl ether under the protection of _N , then passed through dry HCl gas under ice-bath cooling until the solution was separated, and after a large amount of oily matter was completely separated out, the oily matter was separated and vacuum-dried to obtain About 3.2 g of colorless (or light yellow) viscous liquid, the yield is about 90%. ¹ H NMR (CDCl ₃ , TMS): δ1.10-1.17 (12H, OCH ₂ CH ₃ ); 2.93-2.96 (8H, PCH ₂ CH ₂ OE _t ); 3.12-3.20 (4H, PC ₂ H ₄ P) ; 3.45-3.56 (8H, OCH ₂ CH ₃ ); 3.79-3.90 (8H, PCH ₂ CH ₂ OE _t ) ppm. ³¹ P NMR (CDCl ₃ , H ₃ PO ₄ ): δ 14.80 ppm. IR: ν(-COC) 1157-1017cm ^-1 , ν(PH) 2240cm ^-1 (liquid film) Example 2: Preparation of tetrofosmin sulfosalicylate

(R ₁ =COOH, R ₂ =OH)

10ml of anhydrous ether solution with 3g of tetrofosmin was added under _N2 protection into a 50ml round bottom flask equipped with electromagnetic stirring and an isobaric dropping funnel, then cooled in an ice bath and stirred dropwise into a solution containing 3.4g of sulfur Ether solution of acid salicylic acid was 15ml, and the precipitate was separated and vacuum-dried to obtain about 4.7g of a colorless (or light yellow) solid, and the yield was about 73%. ³¹ P NMR (CDCl ₃ , H ₃ PO ₄ ): δ 12.75 ppm. IR: ν(-COC) 1157-1017 cm ^-1 , ν(PH) 2310 cm ^-1 (liquid film).

Embodiment 3: the preparation of two-step method tetrofosmin kit:

The two-step method kit is divided into two parts, A bottle and B bottle. Among them, bottle A is a sterile pyrogen-free freeze-dried kit, which mainly contains reducing agents, intermediate ligands, excipients and other auxiliary materials for buffering the pH value. The function of bottle A is to reduce ^99m TcO ₄ ^-injection to ^99m Tc(V) valence, so that it can form a ^99m Tc(V) valence intermediate complex that can undergo ligand exchange with the tetrofosmin ligand.

1), bottle A: the preparation of sodium D-gluconate frozen in the kit (take 1000 as an example)

Take 1000 mg of sodium D-gluconate, 30 mg of SnCl 2H ₂ O, 400 mg of sodium sulfosalicylate dihydrate and 10,000 mg of mannitol and dissolve them in nitrogen-filled secondary water, and use the nitrogen-filled secondary water to dilute the mixed solution to 1000ml, adjust the pH value to 7.5-8.5 with NaHCO ₃ . Sterile filter the mixed solution, take 1000 vials of 10ml each, fill each vial with 1.0ml, and then freeze-dry and seal with nitrogen to obtain bottle A.

2), bottle B: preparation of tetrofosmin ampoule (take 1000 as an example)

The bottle B of the two-step kit is a colorless, sterile and pyrogen-free solution packed in ampoules, which mainly provides the main raw material for preparing technetium-99m tetrofosmin injection.

Get tetrofosmin hydrochloride phosphonium salt 1000mg prepared by implementing 1 and Tween 80 5000mg and be dissolved in dehydrated alcohol 200ml, after fully dissolving, get 1000 ampoules of each 1mg, subpackage 0.2ml this solution in every bottle, Sealed bottle B.

Embodiment 4: Preparation of Radioactive Technetium-99m Tetrofosmin Injection:

Under aseptic conditions, inject 1-6 milliliters of radioactive technetium-99m injection into the two-step method kit A bottle (one D-sodium gluconate lyophilized kit vial) prepared in Example 3 with a syringe, Shake well, take another syringe, take out the solution in the two-step method kit B bottle (a tetrofosmin ampoule) prepared in Example 3 and inject it into the described A bottle, after fully shaking up, place it at room temperature In 15-20 minutes, the radioactive technetium-99m tetrofosmin injection was obtained.

The performance determination of the two-step method tetrofosmin kit is described as follows:

1. The labeling method of the kit and the determination of its radiochemical purity:

1) Labeling method: Under aseptic operation, according to the technetium radioactivity of technetium [99m-Tc] sodium injection, take 1-6 ml into the above two-step kit A bottle (D-sodium gluconate freeze-dried medicine box), shake well, and take another syringe to take out the solution in bottle B (tetrofosmin ampoule) and inject it into bottle A. After shaking well, let it stand at room temperature for 15-20 minutes to obtain technetium-99m-tetrofosne Phosphine Injection.

2) Determination of the radiochemical purity of the intermediate ^99m Tc-gluconate:

Under aseptic operation, according to the radioactivity of technetium [99m-Tc] sodium injection, take 1-6 ml and inject it into bottle A of the two-step method kit, shake well, and place at room temperature for 5-10 minutes. Use physiological saline/polyamide thin-layer chromatography to identify, observe the Rf value of TcO ₄ ^- and TcO ₂ .xH2O in this chromatography system, judge the generation of ^99m Tc-gluconic acid, and calculate the radiochemical purity of the marker.

3) Determination of the radiochemical purity of the final product ^99m Tc-tetrofosmin:

Use acetonitrile/polyamide chromatography system to identify, observe the RF value of ^99m Tc-gluconic acid, TcO ₄ ^- , TcO ₂ ·xH2O and ^99m Tc-tetrofosmin in this chromatography system to judge the formation of ^99m Tc-tetrofosmin, and calculate the radiochemical purity.

4) Labeling of two-step kits (A, B) and determination of radiochemical purity:

Get each of the D-sodium gluconate freeze-dried kit (bottle A) and tetrofosmin hydrochloride ampoule (bottle B) prepared in Example 3, and under aseptic operation, high technetium [99m-Tc ] Inject 2ml (about 555MBq) of sodium D-gluconate injection into the freeze-dried kit of D-sodium gluconate, shake well and place at room temperature for 5 minutes. It was identified by physiological saline/polyamide thin-layer chromatography (chromatographic results are shown in Table 3), and the calculated radiochemical purity of the intermediate product ^99m Tc-gluconic acid was greater than 95%.

Take another syringe and take out the solution in bottle B (tetrofosmin ampoule) and inject it into the marked D-sodium gluconate kit. Trifosin injection. The radiochemical purity of ^99m Tc-tetrofosmin was all greater than 90% through identification by acetonitrile/polyamide chromatography system (the chromatography results are shown in Table 3).

Table 3 Chromatographic identification results Chromatographic system ^99m TcO ₄ ^{- 99m} TcO ₂ xH ₂ O ^9m Tc-tetrofosmin ^99m Tc-gluconic acid saline/polyamide 0 0 0-0.2 0.7-1.0 acetonitrile/polyamide 0.3- 0.5 0 0.8-1.0 0-0.1

2. Stability study of the two-step method kit:

1) Stability investigation of bottle A medicine box:

Store the samples of bottle A of the kit in a refrigerator at 0°C to 4°C, at room temperature and in a sealed water bath at 38°C to 40°C for 5 months, 30 days and 10 days, and take them out regularly for character observation and detection of labeled radiochemical purity , to investigate the stability of the A bottle kit under the above three conditions.

Take a total of 30 D-sodium gluconate freeze-dried kits prepared in Example 3, divide them into three groups of 10 in each group, and store them in a sealed water bath at 0°C to 4°C, room temperature, and 38°C to 40°C for 5 months. , 30 days and 10 days, take out regularly to observe the character and detect the radiochemical purity of the marker, and investigate the stability of the D-sodium gluconate kit under the above three conditions. The stability inspection results are shown in Tables 4, 5, and 6 below:

Table 4: Stability investigation of the medicine box stored in the refrigerator (0℃～4℃) for more than 3 months

Storage time (days) radiochemical purity (%) traits

1                                                                      

20 98.8 No significant change

60 97.4 No significant change

90 96.9 No significant change

120 96.7 No significant change

160 96.9 No significant change

As can be seen from the above data, the sample has been placed for five months, and its properties have no obvious change, and the labeled radiochemical purity is still greater than 95%, which meets the requirements, indicating that the two-step method tetrofosmin A bottle kit is relatively stable under this condition.

Table 5: Stability of the kit at room temperature (23°C-25°C) for 30 days

Storage time (days) radiochemical purity (%) traits

1                                                                      

7 98.7 No significant change

15 98.1 No significant change

28 97.4 No significant change

35 97.1 No significant change

From the above data, it can be seen that the properties and radiochemical purity of the medicine box have no obvious change after being placed at room temperature for 30 days, indicating that the medicine box is relatively stable under this condition.

Table 6: Stability study of the kits placed in a sealed water bath at 38°C to 40°C for 10 days

Storage time (days) radiochemical purity (%) traits

1 99.5 White powder

2 98.7 No significant change

3 98.2 No significant change

5 97.4 No significant change

7 97.9 No significant change

10 97.1 No significant change

It can be seen from the above data that the kit was placed in a sealed water bath at 38°C to 40°C for 10 days, there was no significant change in the observation of properties, and the radiochemical purity was still greater than 95%, indicating that the stability of the kit under this condition can meet the requirements of the kit in High heat and humid transportation conditions and time requirements in summer.

2) Stability investigation of bottle B kit:

Store the samples of the B bottle of the kit in a refrigerator at 0°C to 4°C, at room temperature and in a sealed water bath at 38°C to 40°C for 3 months, 15 days and 10 days, and take them out regularly for character observation and detection of labeled radiochemical purity , to investigate the stability of the B bottle kit under the above three conditions.

Take a total of 30 tetrofosmin hydrochloride ampoules prepared in Example 1, divide them into 3 groups of 10 in each group, and store them in a refrigerator at 0°C to 4°C, at room temperature and in a sealed water bath at 38°C to 40°C At 3 months, 15 days and 10 days, it was taken out regularly for character observation and detection of the radiochemical purity of the marker, to investigate the stability of tetrofosmin hydrochloride phosphonium salt ampoule under the above three conditions. The investigation results are shown in Tables 7, 8, and 9 below.

Table 7: Study on the stability of tetrofosmin hydrochloride phosphonium salt ampoule stored under refrigerator conditions (0°C-4°C) for more than 3 months

Storage time (days) radiochemical purity (%) traits

1 96.5 96.5 Colorless clear solution

7 97.0 No significant change

15 96.0 No significant change

30 96.5 No significant change

60 95.8 No significant change

90 95.4 No significant change

As can be seen from the above data, the sample has been placed for 3 months, and its properties have no obvious change, and the marked radiochemical purity is still greater than 90%, which meets the requirements, indicating that the tetrofosmin hydrochloride phosphonium salt ampoule is relatively stable under this condition.

Table 8: Stability investigation of tetrofosmin hydrochloride phosphonium salt ampoule at room temperature (23°C-25°C) for 10 days

Storage time (days) radiochemical purity (%) traits

  1                                                                                          

3 96.9 No significant change

5 96.5 No significant change

7 95.8 No significant change

10 96.0 No significant change

From the above data, it can be seen that the properties and radiochemical purity of the ampoule of tetrofosmin hydrochloride phosphonium salt have no obvious change after being placed at room temperature for 10 days, indicating that the kit is relatively stable under this condition.

Table 9: Stability study of tetrofosmin hydrochloride phosphonium salt ampoule kit placed in a sealed water bath at 38°C to 40°C for 10 days

Storage time (days) radiochemically pure (%) traits

  1                                                                                      

                                                                                             

5                                                                      

7 95.1 No significant change

10 95.1 No significant change

It can be seen from the above data that the ampoule of tetrofosmin hydrochloride phosphonium salt was placed in a sealed water bath at 38°C to 40°C for 10 days, and there was no obvious change in the observation of properties, and the radiochemical purity was still greater than 90%, indicating the stability of the kit under this condition It can meet the high heat and humidity transportation conditions and time requirements of the medicine box in summer.

3. Acute toxicity test of injection:

Referring to the relevant experimental methods in the Pharmacopoeia, take 0.5ml of technetium-99m tetrofosmin injection prepared by the two-step kit, and inject it into the tail vein of mice with a body weight of 18-20g at a uniform speed for 4-5 seconds, a total of 5 mice, according to the requirements Observe whether there is abnormal death within 48 hours, and dissect to observe whether the heart, liver, spleen, lung, kidney and other organs are abnormal after 72 hours.

1) Acute toxicity test of injection in mice:

Take 0.5ml of technetium-99m tetrofosmin injection (370MBq/3ml) prepared in Example 4, and inject it into the tail vein of mice with a body weight of 18-20g at a uniform speed for 4-5 seconds, a total of 5 mice, and observe for 48 hours as required All the mice survived, and after 72 hours, no abnormalities were found in the heart, liver, spleen, lung, kidney and other organs.

The experimental results show that the dose of Kunming mice used in this experiment is 500 times higher than the human dose per kilogram of body weight. All 5 mice survived the experiment without any adverse reactions, and no abnormalities were found in the anatomical examination. Performance. The experimental results show that the toxicity of the injection is low, and it can be used for clinical trial research after passing the aseptic and non-pyrogenic inspection.

4. Biological properties of injection:

Take Kunming mice with a body weight of 18-20 g, and inject about 0.74 MBq/0.1 ml of radioactive technetium-99m-tetrofosmin injection prepared by a two-step kit into the tail vein, and wean the mice 2 to 60 minutes after injection. They were killed by neck, and the hearts, livers, lungs, muscles and blood were taken out, weighed separately, and their radioactive counts were measured in a well-type r detector to calculate the intake dose per gram of tissue.

1) In vivo distribution experiment of technetium 99m-tetrofosmin injection in mice:

Take a Kunming mouse with a body weight of about 15-17 g, inject 0.74 MBq/0.1 mL of the radioactive technetium-99m-tetrofosmin injection prepared in Example 4 into the tail vein, and take 2 minutes, 15 minutes, and 30 minutes after the injection After 60 minutes, the mice were killed by neck dislocation, the heart, liver, lung, kidney and other major organs, blood and muscle were taken out, weighed respectively, and the radioactive count was measured in the well-type gamma detector to calculate the uptake per gram of tissue Dose (%ID/g), the calculation of one percent injection dose (1%ID) is to take 0.1ml (injection amount) complex solution, dilute to 100 times and then take 0.1ml respectively in three small test tubes, Measure the radioactive count of the tissue while measuring the radioactive count, and one percent of the dose is the average of the radioactive counts in three small test tubes; and take the heart as the target organ, and compare it with the lung, liver, blood and muscle %ID/ g ratio. The results are shown in Table 10 below.

Table 10: Biodistribution of labeled compounds in mice after injection (n=3) Administration time 2 minutes 5 minutes 15 minutes 30 minutes 60 minutes

Average Standard Average Standard Standard Average Standard Average Standard Organization

Deviation Value Deviation Value Deviation Value Deviation Value Deviation

每克组织的摄取剂量(％ID/g)心           19.2    1.25   18.5    0.23   17.7    1.07   16.0   0.76   13.4   0.44肝           9.78    0.76   11.5    0.42   9.47    0.10   7.67   0.25   5.15   0.41肺           9.77    0.78   8.08    0.34   4.59    0.86   3.29   0.11   1.73   0.06肾           44.0    3.17   32.0    1.51 28.3 1.30 20.6 4.52 12.8 0.96 muscle 3.46 0.72 3.52 0.72 3.44 0.26 2.56 0.02 3.21 0.17 Blood 4.21 0.47 2.57 0.41 1.38 0.04 0.72 0.03 0.03

                            心肌与其它脏器的每克组织计数比值心/肝        2.00    0.17   1.44    0.18   1.96    0.52   2.09   0.34   2.75   0.09心/肺        1.97    0.10   2.54    0.07   3.89    0.11   4.87   0.48   6.93   0.21心/血        4.62    1.00   7.07    0.51   12.6    1.19   17.4   1.22   18.7   1.01

It can be seen from the biodistribution data in mice that its uptake in the myocardium is relatively high, and its retention is good, with high heart/blood and heart/lung ratios, which meet the basic requirements of myocardial imaging agents. The initial liver uptake is low, and the clearance is fast, and the heart/liver ratio is high, which is conducive to obtaining clear imaging images.

In order to further illustrate the stability of the two-step tetrofosmin kit, the following comparative examples are used to illustrate.

Comparative example 1: the stability comparison of the two-step method medicine box and the original one-step formula medicine box: Table 11 is the two-step method medicine box (A, B bottle) prepared by embodiment 3 and the one-step formula developed with reference to Amersham company The comparison of the stability of the two-step medicine kit under the condition of 38 ℃ ~ 40 ℃ can be seen from the comparison of the stability inspection results in the table. The other components are separated so that they are stored in a relatively favorable medium environment for their stability, thereby improving the stability of the entire medicine box (bottles A and B). The improvement of the quality stability of the medicine box is that the two-step method medicine box is better than the original separation, so that it is stored in a medium environment that is relatively conducive to its stability, thereby improving the stability of the entire medicine box (A, B bottles) . The improvement of the quality stability of the medicine box is an important aspect that the two-step medicine box is superior to the original Amersham company's formula one-step medicine box. Table 11: Comparison of the stability of the two-step formula kit and the original one-step formula kit at 38°C to 40°C Storage time (days) Labeled radiochemical purity (%) Two-step pill kit One-step pill kit* 13510 96.0 99.095.8 96.095.4 88.095.1 80.0

*The one-step kit data is taken from Zhu Gengen, Zhu Weijing. Determination of the Stability of ^99m Tc-tetrofosmin Kit, Compilation of Abstracts of the Seventh National Conference on Radiopharmaceuticals and Labeled Compounds, 1998.173p.

Due to the effect of the auxiliary agent added in the two-step method kit, the biological performance of ^99m Tc-tetrofosmin (tetrofosmin) injection is further improved. It is mainly manifested in the obvious reduction of the initial uptake of the liver and the significant increase of the early heart/liver ratio, which is more conducive to obtaining clear and high-quality imaging images and realizing early imaging of the myocardium in clinical practice.

Comparative Example 2: Comparison of the biodistribution in mice of the injection prepared by the two-step kit and the injection prepared by the original one-step kit

Table 12 is a comparison of the biological data in mice of the injection prepared in Example 4 and the injection prepared with reference to the original Amersham company kit formula kit.

Table 12: Comparison of biodistribution of two injections in mice (n=3) Administration time 5 minutes 30 minutes 60 minutes injection Mean Standard Deviation Mean Standard Deviation Mean Standard Deviation

The intake dose of each gram ( % ID/g) two -step method 18.5 0.23 16.0 0.76 13.5 0.44 Remarks liver 11.5 0.42 7.67 0.25 5.15 0.41 The ratio of the tissue count of the tissue of the other or other organs

Heart/Liver 1.44 0.18 2.09 0.34 2.75 0.09

Ingested dose per gram of tissue (%ID/g) one-step method Heart 15.1 0.77 13.7 0.30 10.9 0.93 Prepared injection Liver 15.0 0.67 10.7 0.66 7.40 0.85 Injection ^* The ratio of counts per gram of tissue between myocardium and other organs

Heart/Liver 1.01 0.27 1.28 0.21 1.47 0.08 ^* The biological data of the original one-step formula injection were obtained from the literature: Lu Jie, Wang Xuebin, Lu Gongxu. Preparation of a new ¹⁸⁸ Re-tetrofosmn complex and study on its biodistribution. Nuclear Technology, 1999, 22:695

As can be seen from Table 12, compared with ^{the 99m} Tc-tetrofosmin (tetrofosmin) injection prepared by the original one-step medicine box formula, the myocardial uptake of the injection made by the two-step medicine box in mice was significantly increased, while The initial liver uptake is significantly reduced, which greatly increases the heart/liver ratio, which is more conducive to obtaining clear, high-quality imaging images and realizing early imaging of the myocardium.

The beneficial effects that the present invention has are:

1. The main raw material tetrofosmin (tetrofosmin) is further phosphonium-salted to obtain a relatively stable tetrofosmin phosphonium salt in the air, so that it can be stored for a long time, which not only solves the storage of the raw material drug, but also The stability of its quality is guaranteed.

2. The two-step method kit separates the relatively unstable main drug substance from other components, so that it is stored in a relatively stable medium environment for tetrofosmin, which not only preserves the stability of the one-step method tetrofosmin kit in the injection solution. The convenience in preparation and the quality of the injection solution prolong the shelf life of the medicine box, especially for transportation and storage under high temperature conditions in summer.

Claims (10)

1. A tetrofosmin quaternary phosphonium salt is characterized in that it has the following formula (I), [(C ₂ H ₅ OC ₂ H ₄ ) ₂ P ⁺ (H)C ₂ H ₄ P ⁺ (H)( C ₂ H ₄ OC ₂ H ₅ ) ₂ ]·2X ^- ... (I) In the formula: X can be a halogen, Or the sulfonate group of the following formula (II),

In the formula: R1: for -H, -COOH, R ₂ is -H, -OH or -COOH. 2. The tetrofosmin quaternary phosphonium salt according to claim 1, characterized in that X in formula (I) is chlorine or bromine; in formula (II), _R1 is -COOH, and R2 is -OH. 3. A preparation method of tetrofosmin quaternary phosphonium salt with the following formula (I), [(C ₂ H ₅ OC ₂ H ₄ ) ₂ P ⁺ (H)C ₂ H ₄ P ⁺ (H)(C ₂ H ₄ OC ₂ H ₅ ) ₂ ]·2X ^- ...(I) In the formula: X is a halogen, Or the sulfonate group of the following formula (II),

In the formula: R1 is -H, -COOH, R ₂ is -H, -OH or -COOH, The method includes: Tetrofosmin (C ₂ H ₅ OC ₂ H ₄ ) ₂ PC ₂ H ₄ P(C ₂ H ₄ OC ₂ H ₅ ) ₂ and the reactant of formula HX The definition of X in the formula is identical with the definition of formula (I), Under the protection of an inert gas, the quaternary phosphonium salt of tetrofosmin, which is easily soluble in water, is formed in a non-polar medium at -10°C-25°C. 4. The preparation method of tetrofosmin quaternary phosphonium salt according to claim 3, wherein the inert gas is nitrogen; the non-polar medium is benzene, toluene, ether or petroleum ether. 5, a kind of tetrofosmin medicine box that tetrofosmin former medicine is made into tetrofosmin quaternary phosphonium salt, it is characterized in that, it prepares (A) bottle and (B) bottle respectively according to following two-step method get: (A) Preparation of bottle: Reducing agent: intermediate ligand: auxiliary material: excipient: nitrogen-filled secondary water=(0.01-0.1): (1-5): (0.2-2.5): (1-10): (0.6-1) The weight ratio is mixed evenly, so that the above four substances are fully dissolved in the amount of nitrogen-filled secondary water, and the pH is adjusted to 5.0-8.5 with a pH regulator. After sterile filtration, it is divided into containers, and then freeze-dried , the medicine box (4) bottles that are filled with nitrogen and sealed to be freeze-dried; Wherein the reducing agent is Sn(II) halogen salt; the intermediate ligand is D-gluconic acid or its alkali metal or alkaline earth metal salt; the auxiliary materials are dihydrate sulfosalicylic acid, dihydrate sulfowater Sodium cylate or disodium sulfosalicylate, citric acid or its sodium salt, tartaric acid or its potassium or sodium salt; the excipient is sodium chloride or mannitol; the pH regulator is hydrogen Sodium, sodium bicarbonate, or disodium hydrogen phosphate; (B) Preparation of vials: With the tetrofosmin phosphonium salt described in claim 1 as main bulk drug: adjuvant: the weight ratio of solvent=(1-5): (1-10): (0.2-1.0), make described tetrofosmin The quaternary phosphonium salt and the adjuvant are dissolved in the solvent, and after being fully dissolved, they are subpackaged in containers to obtain the medicine box (B) bottle; Wherein the auxiliary material is Tween emulsifier or cyclodextrin; the solvent is water, ethanol aqueous solution or absolute ethanol. 6. The tetrofosmin kit according to claim 5, characterized in that, in (A) bottle preparation, the Sn(II) halogen salt is SnCL ₂ ·2H ₂ O or SnF ₂ ; in (B ) bottle preparation, the Tween emulsifier is Tween 20, Tween 40 or Tween 80, and the cyclodextrin is γ-cyclodextrin or β-cyclodextrin. 7. A radioactive technetium-99m tetrofosmin injection, characterized in that it is prepared according to the following steps: 1), the preparation of (A) bottle: Reducing agent: intermediate ligand: auxiliary material: excipient: nitrogen-filled secondary water=(0.01-0.1): (1-5): (0.2-2.5): (1-10): (0.6-1) The weight ratio is mixed evenly, so that the above four substances are fully dissolved in the amount of nitrogen-filled secondary water, and the pH is adjusted to 5.0-8.5 with a pH regulator. After sterile filtration, it is divided into containers, and then freeze-dried , the bottle of the lyophilized medicine box (A) filled with nitrogen and sealed; Wherein the reducing agent is Sn(II) halogen salt; the intermediate ligand is D-gluconic acid or its alkali metal or alkaline earth metal salt; the auxiliary materials are dihydrate sulfosalicylic acid, dihydrate sulfowater Sodium cylate or disodium sulfosalicylate, citric acid or its sodium salt, tartaric acid or its potassium or sodium salt; the excipient is sodium chloride or mannitol; the pH regulator is hydrogen Sodium, sodium bicarbonate, or disodium hydrogen phosphate; 2), (B) the preparation of bottle: With the tetrofosmin quaternary phosphonium salt as the main bulk drug described in claim 1: auxiliary material: the weight ratio of solvent=(1-5): (1-10): (0.2-1.0), make described tetrofosmin The phosphine phosphonium salt and auxiliary materials are dissolved in the solvent, and after being fully dissolved, they are subpackaged in containers to obtain the medicine box (B) bottle; Wherein said adjuvant is Tween emulsifier or cyclodextrin; said solvent is water, ethanol aqueous solution or absolute ethanol. 3) Under aseptic operation, according to the radioactivity of technetate ^99m T _c O ₄ ^- , take an appropriate amount into the bottle of the above-prepared medicine box (A), shake well, and then put the above-prepared medicine box (B) ) bottle solution into the above-mentioned (A) bottle, shake well, and place it at room temperature for 15-20 minutes to obtain the radioactive technetium-99m tetrofosmin injection. 8. Tetrofosmin injection according to claim 7, characterized in that, in the preparation of (A) bottle, said Sn(II) halogen salt is SnCL ₂ .2H ₂ O or SnF ₂ ; in (B) ) bottle preparation, the Tween emulsifier is Tween 20, Tween 40 or Tween 80, and the cyclodextrin is γ-cyclodextrin or β-cyclodextrin. 9. The use of the tetrofosmin kit according to claim 5 or 6 in the preparation of radioactive technetium-99m tetrofosmin injection for use as an imaging agent in human or animal organs or tissues. 10. The use according to claim 9, the radioactive technetium-99m tetrofosmin injection is used as an imaging agent in myocardium or tumor.