TWI229187B - A novel method for determination of radiochemical purity of technetium-99m-TRODAT-1 - Google Patents

Abstract

Technetium-99m-TRODAT-1 has been reported as the first 99mTc radiopharmaceutical for SPECT imaging of the dopamine transporters in human central nerve system. For clinical use of Tc-99m-TRODAT-1 in diagnosis of Parkinson's disease, it is necessary to confirm the radiochemical purity for good imaging quality. The HPLC method has been reported previously to determine the radiochemical purity for the tracer. Currently, we developed a new simple two-strip method with silica gel base for fast routine determination of radiochemical purity (RCP) for the reconstitution of TRODAT-1 kit. This convenient and facile method has been validated with HPLC method. The correlation coefficient between results from HPLC and that of ITLC was 0.99 if the main impurity was 99mTc-glucoheptonate, while it was found to be 0.98 if the main impurity was 99mTc pertechnetate. We conclude that this new two-strip method is a simple, fast and accurate one for routine RCP determination for 99mTc TRODAT-1.

Description

1229187 V. Description of invention α)

Parkinson's disease is a progressive dopamine neurological degenerative disease. The medical community has long recognized that the movement disorder caused by Parkinson's disease originates from the destruction of specific substantia nigra cells (Substantia N igra) located in the brain stem; these black Plasma cell axons extend to the striatum (Striatum) and are connected to the cortical areas of the brain that control body movements through nerve fibers. The substantia nigra cells release the neurotransmitting substance dopamine into the striatum, which stimulates the striatum to continue to release dopamine, and transmits the control information of coordinated action to the cerebral cortex via the advanced motion control center in the brain. Once the dopamine-producing cells die, the mechanism responsible for coordinating the movement of the cerebral cortex to control movement is lost, which is why the motor symptoms occur. (1) According to statistics, about 15% of the population over 65 years of age have Parkinson's syndrome ; At the end of August, the population over 65 years old in Taiwan, accounting for 8.44% of the total population, was about 18.86 million people (2), estimated at 15% incidence, about 28 million people are Parkinson's syndrome Among them, about 40,000 people suffer from true Parkinson's disease. Due to the high incidence of the elderly population and the problem of patients' inability to move, Parkinson's disease has gradually become a major healthcare issue in a global aging society.

At present, the most common methods used by neurologists to diagnose Parkinson's disease are observation of symptoms and assessment of severity. Although computed tomography (CT) or magnetic resonance imaging (MRI) can help clarify other diseases similar to Parkinson's disease, it still cannot be used to directly diagnose Parkinson's disease. To develop a high degree of specificity and directly diagnose Parkinson's disease Imaging technology, it must rely on the functional and biochemical imaging technology of nuclear medicine. Due to the differences in the radioactive species used to identify nuclear medicines, two types of nuclear medical imaging technologies have been developed: Positron Emission

Page 6

1229187, V. Description of the invention (2)

Tomography (PET) and Single-Photon Emission Computed Tomograpy (SPECT). PET Because PET must rely on accelerators to produce short half-life isotope I substances' to limit the popularity of PET, SPECT is still the main medical diagnostic imaging diagnosis for a small number of PET radiography equipment; considering medical resources & benefits and huge market demand, develop capabilities There is a need for SPECT contrast diagnosis and drugs. In SPECT diagnostic nuclear medicine, although I medicine has the advantages of easy development and diversified applications, one must rely on the # 素 produced by cyclotron (C yc 1 〇tr ο η), and then immediately deliver it Compared with the most important nuclear medicine application development chromium-9 9m (technetium 9 9m, Tc-9 9ffl), the finished pharmaceutical products that have been marked and manufactured by nuclear medicine plants obviously have many market restrictions. The main advantage of W-99m in nuclear medicine is its low cost and convenience, which makes chromium-9 9m nuclear medicines competitive in the nuclear medicine contrast agent market. In 1996, Professor Fanfan Yuan and others from the University of Pennsylvania School of Medicine in the United States and others established a N2S2 structure bound to a tropane structure to form TR0DAT-1 molecule-9. 9m can form a stable complex with the N, S2 structure of TR0DAT-1 molecule At the same time, without sacrificing the specific binding ability of trop ane structure and dopamine transporter, there is a major breakthrough in the development of chromium-9 9 m-labeled dopamine transporter in the diagnosis of nuclear medicine for sclerosis 6). Animal tests (7, 8) and initial clinical studies (9, 1ϋ) at home and abroad have confirmed the application of angiography and diagnosis of amine transporters, including Parkinson's disease and Sage drugs, nuclear investment) nuclear medicine- 1 2 3 After all, the nuclear is the isotope field of the isotope. The most competitive one is chromium. Hepakinson has completed dopamine.

1229187, V. Description of the invention (3) Market potential of diseases related to the nervous system. In order to facilitate the diagnosis and clinical use, we have completed a single bottle set formula that can immediately mark chromium-9 9 in-TR 0 DAT-1 (in the Republic of China new invention patent application). Contains: TRODAT-1 hydrochloride 126 Microgram, 320 micrograms of sodium glucoheptonate, 930 micrograms of Na2 · EDTA · 2H20, 32 micrograms of stannous chloride, manni to 1 20 milligrams, anhydrous Na2HP04 4.1 micrograms, NaH2P04 · 2Η20 460 micrograms. Radiochemical purity is an indicator of the labeling efficiency of nuclear medicines. The presence of impurities in the labeling process will affect the clinical imaging results due to the different biological distribution of the impurities and the desired product; the single bottle set is used in clinical cases. When it is used, it must be measured whether the radiochemical purity reaches the standard value after the flag reaction with the radionuclide and before the injection to the patient to ensure the quality of the drug. In the flagging process of Co-99m-TR0DAT-1, in the presence of glucoheptonate, 'Cr-9 9m sodium perchromate was first reduced from stannous gas to a pentavalent saw, and immediately formed the wrong compound with g 1 uc 〇hept ο nate Then, through ligand exchange reaction, TR0DAT-1 is substituted for glucoheptonate to generate -99m-TR0DAT-1. The reaction formula is as follows:

TRODAT-1

Tc-99m-TRODAT-l) 1229187 ............... ....... 5. Description of the invention (4)

Sn2 + " mTc04 ·-^ — ► reduced " mTc " mTc02

reduced 99mTc + glucoheptonate (GH) ► 99mTc- GH 99mTc-GH + TROD AT-1 ~ " ► 99mTc- TROD AT-1 + GH

According to the above reaction formula, it is known that the possible impurities in the process of the standard-99m-TRODAT-1 include: -99m-glucoheptonate, 鐯 -9 9m-sodium perchromate and reduced chromium-99m-hydrated dioxide chromium. According to the literature, the radiochemical purity of chromium-99m-TR0DAT-1 was measured using reversed-phase high-performance liquid chromatography (reversed phase HPLC), and the analytical column was Hami 1 ton PRP-1 (2 5 0, 4 · 1 mm). The eluent is a mixed solution of acetonitrile-5mM DMGA (acetonitrile-5mM dimethylglutaric acid) (pH 7 · 0) 8: 2 at a flow rate of 1 liter per minute (7). A typical map of chromium-99m-TRODAT-l under this condition is shown in Figure 1. The retention times of chromium-99111-but 1 ^ 01 ^ 1'-1, wrong-99 m-glucoheptonate and chromium-99m-sodium perchromate are shown in the table

Page 9 1229187 V. Description of the invention (5) Table 1 'Chromium-99m-TR0DAT-1 and other errors — 99m-Impurity retention time of HPLC species retention time (minutes) Chromium-99m-TRODAT-1 16-32!荅 -99m-glucoheptonate 2 ~ 5! 荅 -99m-gluconic acid sodium 2 ~ 5 HPLC has the advantages of the South Resolution, but in order to further improve the analysis efficiency for clinical use, the radiochemistry of 鍀 -99m-TRODAT-l is simplified The purity analysis step is necessary; due to the advantages of TLC analysis: (1) short analysis time 'about 10 minutes; (2) simple operation procedures; (3) equipment and analysis materials are cheap and easy to obtain; (4) The use space is small. Therefore, the thin layer chromatographic analysis (TLC) provided by the present invention can meet the needs of clinical use. 'Provide a fast and simple method for measuring the purity of 鐯 -99m-^ ⑽ 丁丁 丨 radionon purity, there are Helps improve the efficiency of drug quality control before clinical use. 2. The analysis method in this case mainly includes a combination of two systems. Systems (丨) and (2) both use ITLC-SG sheets as the stationary phase. A small amount of radioactive samples—ggm—trqdaT-1—are dropped on two ITLC-SG sheets. After the sample was air-dried, the sample was placed in a developing tank, and the mobile phase was developed in the system (1) using a 0 · g% sodium chloride aqueous solution. The system (2) was first developed with acetone, air-dried, and then ^ 9 % Gas-sodium hydroxide aqueous solution is unfolded, and after taking out, analyze the spectrum with a radiological thin-layer scanner or cut it into multiple segments and measure the activity of each segment with an appropriate radiation detector.

Page 10 1229187 V. Description of the invention (6) Draw a map. According to the conditions shown in the spectrum, the radiochemical purity of chromium-99m-TRODAT-1 (%) = 100- [percentage of liquid peak of system (1) + percentage of origin of system (2)]. Example 1 Preparation of Chromium-99m-TRODAT-1 Take a bottle of frozen crystal set of TROD AT-1, inject 5 ml, 30 millicurie of fresh sodium perrhenate solution, shake, and put into high-pressure steam sterilization The device was heated at 121 ° C. for 30 minutes and cooled to room temperature to complete the preparation. Example 2: Preparation of G-99m-glucoheptonate g 1 uc 〇hept ο nate; Dongjing set vial 'contains s odi inn glucoheptonate 320 micrograms, Na2 · EDTA, 2H20 930 micrograms, stannous gas 32 Μg, mannitol 20 μg, anhydrous Na2HP04 4.1 μg, NaH2P04 2 μ20 460 μg. Take a bottle of glucoheptonate frozen crystal set vial, inject 5 ml, 30 millicuries of fresh sodium perchromate solution ’, shake, perform flag reaction, and complete the preparation. Example III. Radiological thin-layer analysis method This analysis method includes two systems: System (1) One stationary phase: ITLC-SG sheet (1 · 5 X 13 cm) Mobile phase ·· 0 · 9% sodium chloride aqueous solution system (2) — Stationary phase_ · ITLC-SG sheet (1 · 5 X 13 cm)

Page 11 1229187 V. Description of the invention (7) Mobile phase: firstly develop with acetone, blow dry with cold air, and then spread a small amount of radioactive sample with 0.9% sodium carbonated aqueous solution to inject a needle or a micro pipette onto the I TLC-SG sheet At the lower edge of 2 cm (origin, 0 rigi η), the air-dried sample was dropped into a spreading tank and spread to about 12 cm (liquid peak, So 1 vent fr0nt), and then air-dried. The slice is analyzed with a radiological thin-layer scanner, or cut into multiple sections, and the activity of each section is measured with an appropriate radiation detector, and the map is drawn.

Since chromium-99m-glucoheptonate and chromium-99m ~ acid sodium are polar compounds, they will move to the liquid front with 0.9% gasified sodium hydroxide solution in system (1); wrong-9 9 m-TR 0 DAT _ 1 is a fat-soluble, low-polarity compound. Reduced chromium-99m-hydrated chromium dioxide is prone to form colloidal substances and will remain at the origin of the system. Chromium-9 9m-TROD AT-1 is a typical system (}) The analysis map is shown in Figure 2. In the analysis by system (2), acetone first brought the fat-soluble silver-99in-TRODAT-1 and osmium-99m-sodium superlaptopate to the liquid peak, and after air-drying the ITLC-SG sheet, it was chlorinated with 0.9%. Sodium aqueous solution shifted the polarity error -9 9m-glucoheptonate to the liquid peak. At this time, the original point of the slice only left the reduced state -9 9 m-hydrated dioxide record. Figure 2 shows the analysis by system (2),-99 m -Typical map of TRO DAT-1. "

鍩 -99m-TROD AT-1 radiochemical purity (%) = 100 ～ [% of liquid peak of system () + percentage of origin of system (2)] '' For each species in the radiological thin layer analysis method of the present invention R f values are shown in Table 2.

1229187 V. Description of the invention (8) Table 2. Chromium-99in_ TROD AT-1 and other impurities in the Rf value species analysis system (1) System (2) Chromium-99ίϋ-T_AT-1 丨 0 ~ 0 · 1 0 · 7 ~ 1 · 0 鍩 -99m-glucoheptonate 0, 9 ~ 1 · 0 0 · 9 ~ 1. 0 食 -99m-Sodium perchromate 0.9-1.0 0 · 9 ~ 1. 0 Original chromium -99m-Hydrochromium dioxide 0 ~ 0 · 1 0-0.1

Example 4: Error -99m-glucoheptonate-99m-TRODAT-1 Preparation and radiochemical purity analysis of the test sample The chromium-99m-TRODAT-1 prepared in Example 1 and the error prepared in Example 2-99m- Two kinds of radioactive samples of glucoheptonate were mixed according to different proportions to obtain different purity chromium-99m ~ TRODAT_l mixed liquid test samples containing impurities of -9-9 m-g 1 uc 〇hept ο nate. Table 3 shows the radiochemical purity of chromium-99m ~ TRODAT-1 measured by chromatographic analysis method and the radiation thin-layer analysis method in Example 3.

Page 13 1229187 I V. Explanation of the invention (9) Table III. Analysis of the radiochemical purity of chromium-99m-TRODAT-1 samples containing chromium-99m-glucoheptonate by HPLC and the TLC method of the present invention Sample No. Chromium-99m-TRODAT-1 radiation Chemical purity (HPLC method TLC method of the present invention ^^ 1 37. 76 37. 18 " ^^^ 2 51.15 43. 3 8 ^^ ~ 3 66.62 6 7. 6 8-4 74. 30 76.2 9 ~ 5 78 44 81 · 25 ^^ · 6 80. 72 84. 16 7 82.24 81.13 — ^ ^ 8 82.85 8 6.8 7 9 87.18 86 · 85 `` ^^^ 10 8 9.62 9 0.3 5 '11 92. 38 94,34 ^-12 96. 74 9 8. 6 8 ^ —— 13 96.88 9 7.16 —— 14 9 7.21 9 6,5 8 ^^ ~---- Example 5 Containing Error -99m-Sodium Fault -99 m-TR0DAT-1 Test Sample Preparation and Radiochemical Purity Analysis

Two types of chromium-99m-TROD AT-1 prepared in Example 1 and chromium-99m-sodium perchromate solution directly washed by a ~ 由 -99 / chrome-99m generator &

Page 14 1229187 V. Description of the invention (10) The radioactive samples were mixed in different proportions to obtain different purity 物 -99m-TRODAT-1 mixed liquid test samples containing 鐯 -g 9m-acid sodium, respectively. Reverse-phase high-performance liquid chromatography and the radiochemical thin layer analysis of Example 3 were used to measure the chromium-99m-TROD AT-1 of each sample. The radiochemical purity is shown in Table 40. Table 4 'Analysis by HPLC and the TLC method of the present invention Radiochemical Purity of Chromium-99111-Per # Sodium Chromium-9 9ra-TRODAT-1 Sample

Page 15 1229187 V. Explanation of the invention (11) Example 6. Linear regression analysis of TLC and HPLC Table 3 shows the results of HPLC and the TLC method of the present invention for the analysis of the errors of chromium-99m-glucoheptonate-99m-TR0DAT-1 The linear regression analysis of the radiochemical purity data is shown in Figure 4, and the correlation coefficient (r) was calculated to be 0.9908. The linear chemical analysis of the radiochemical purity data obtained from the analysis of the chromium-99m-sodium perchromate-containing chromium-9 9m-TROD AT-1 samples by HPLC and the TLC method of the present invention is shown in Table 5, and the r value is calculated. Is 0 · 9 8 7 5. Linear regression analysis shows that whether the main impurity is chromium-99m-glucoheptonate or chromium-99m-sodium acid, the radiochemical purity of chromium-99m-TRODAT-1 determined by the TLC method of the present invention is very good with the HPLC method used in the literature Linear relationship.

1229187 V. Description of the invention (12) References: 1'Moussa BHY, Peter R '(1 9 9) Understanding park i ηεοηφ s disease. Sc i Amer J, January: 39-45 2. Statistical Manual of the Accounting Office of the Executive Yuan , 1989. 3. Carroll FI, Scheffel U, Dannals RF, Boja JW, Kuhar MJ. (1 9 9 5) Development of imaging agents for the dopamine transporter. Med Res Rew, 1 5: 419-444. 4 . Meegalla SK, Plossl K, Kung MP, Stevenson DA, Liable-Sands LM, Rheingold AL, Kung HF. (1995) First example of a 9 9mTc complex as a dopamine transporter imaging agent. J Am Chem Soc, 117: 11037- 8. 5. Meega11 a SK, Plossl K, Kung MP, Chumpradit S, Stevenson A, Frederick D, Kung HF. (1 9 9 6)

Tc-99 in-labeled tropanes as dopamine transporter imaging agents. Bioconj Chem, 7: 421-429. 6. Meega11 a SK, Plossl K, Kung MP, Chumpradit S,

Stevenson DA, Kushner SA, McElgin WT, Mozley PD, Kung HF. (1 9 9 7) Synthesis and characterization of technetium-99m-1abe1ed tropanes as dopamine transporter-imaging agents. J Med Chem, 40: 9-17. 7 ' Kung MP, Stevenson A, Plossl K, Meegalla SK,

Page 17 1229187 V. Description of the invention (13)

Beckwith A, Essman WD, Mu M, Luck i I, Kung HF. (1997) [99mTc] TRODAT-1: a novel technetium-99m complex as a dopamine transporter imaging agent. Eur J Nuc 1 Med, 24: 372-380 8. Zeng Kaiyuan (1999) Research on Chromium-99 m-labeled TRO DAT-1 in Parkinson's disease-from basic to clinical.

9. Kung HF, Kim Η-J, Kung Μ-P, Meegalla SK, Plossl K, Lee HK. (1 9 9 6) Imaging of dopamine transporters in humans with technetium-99m TRODAT-1. Eur J Nucl Med, 23 : 1527-1530. 10. Mozley PD, Stubbs JB, Plossl K, Dresel SH,

Barrac 1 ough ED, Alavi A, Araujo L, Kung HF. (1 9 9 8) Biodistribution and dosimetry of TRODAT-1: a technet i um-9 9m tropane for imaging dopamine transporters. J Nucl Med, 3 9: 2 0 6 9-2 0 7 6.

Page 18 1229187 Brief description of the figure Figure 1 shows a typical spectrum of chromium-99m-TR0DAT-1 by PRP-1 reverse phase HPLC analysis. Figure 2 is a typical system (1) analysis spectrum of chromium-99m-TROD AT-1. Figure 3 is a typical system (2) analysis spectrum of chromium-99m-TRODAT-1. Figure 4 shows the radiochemical purity results of the error-99m-glucoheptonate-error-99m-TRODAT-l sample. The linear regression comparison chart of Η P L C and T L C is shown. Figure 5 shows the results of radiochemical purity of QOm-TRODAT-l samples containing the error of sodium-99m-permanganate. The linear regression comparison between HPLC and TLC

Claims (1)

A method for determining the chromium of the dopamine transporter contrast drug-VI. Patent application scope I. A method for the radiochemical purity, which is mainly used to analyze and determine the contrast drug by thin layer chromatography (TLC) method; The chromium-Mm-TRADOT-i is a radioactive compound sample containing at least one impurity of chromium-99m-glucoheptonate, chromium-99m-sodium perchromate and reduced state -9-9m-hydrated oxidized dioxide, and its measuring steps Including: a • Prepare two TLC sheets as a stationary phase, and use an injection needle or a micro pipette to drop a small amount of the above-mentioned radioactive compound sample onto the two sheets respectively; b. After air-drying, air-dry the two sheets 9。 Drug droplets of the thin slices were placed in the deployment tank, one system (丨) is using 0.9. / 〇 The gasification sodium aqueous solution is the mobile phase, and the sample on one of the sheets is unrolled. The other system (2) is to use acetone as the mobile phase to unfold the sample on the other sheet. After cold air drying, It was then re-expanded with a 0.9% sodium chloride aqueous solution until the two sheets were unfolded from the origin of the drip until a liquid peak appeared; c. After the two sheets were taken out of the expansion tank and air-dried, they were irradiated with thin films. The layer scanning instrument is used to analyze the spectrum, or it is cut into multiple segments to detect the radiation ^ Measure the activity of each segment and draw the spectrum; d · Finally, obtain the radiochemical purity of the analyzed sample according to the following formula: The analyzed sample The radiochemical purity (%) = 100- [percentage of liquid peak of system (1) + percentage of origin of system (2)]. Page 21