One step 64Cu-BaBaSar-RGD2 production method

Abstract

A method of preparing a .sup.64Cu-BaBaSar-RGD.sub.2 solution is provided. The method includes lyophilizing a solution of BaBaSar-RGD.sub.2 and adding a .sup.64Cu solution to the lyophilized BaBaSar-RGD.sub.2.

Claims

1. A method of preparing a .sup.64Cu-BaBaSar-RGD.sub.2 solution comprising: lyophilizing a solution of BaBaSar-RGD.sub.2 comprising a buffer salt to obtain a powder of BaBaSar-RGD.sub.2 and the buffer salt; and adding a .sup.64Cu solution comprising .sup.64CuCl.sub.2 to the powder of BaBaSar-RGD.sub.2 and the buffer salt.

2. The method of claim 1, wherein the buffer salt is sodium acetate buffer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Kit production process of .sup.64Cu-BaBaSar-RGD.sub.2.

(2) FIG. 2: Structure of RGD peptide and the synthesis route for .sup.64Cu-BaBaSar-RGD.sub.2.

(3) FIG. 3: Analytical radio trace HPLC chromatogram for the purity of the .sup.64Cu-BaBaSar-RGD.sub.2.

(4) FIG. 4: Decay-corrected anterior maximum-intensity projections of PET/CT at 1, 5, 10, 20, 40, 60, 120, and 180 min after injection of .sup.64Cu-BaBaSar-RGD.sub.2 in macaque monkey.

(5) FIG. 5: Structures of AnAnSar, BaAnSar, BaMalSar, and MalMalSar.

(6) FIG. 6: Additional peptides to be used in present invention.

DETAILED DESCRIPTION OF THE INVENTION

(7) Unless otherwise indicated herein, all terms used herein have the meanings that the terms would have to those skilled in the art of the present invention. Practitioners are particularly directed to current textbooks for definitions and terms of the art. It is to be understood, however, that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.

(8) To promote the clinical application of .sup.64Cu-BaBaSar-RGD.sub.2 in humans, the present invention provides a straightforward, one-step synthesis of .sup.64Cu-BaBaSar-RGD.sub.2 radiopharmaceutical using a preloaded cold kit.

(9) The present invention provides a one-step production of radiopharmaceutical .sup.64Cu-BaBaSar-RGD.sub.2 with a kit preloaded with all the precursors. FIG. 1 discloses the process of production. Furthermore, this method is not limited to the production of .sup.64Cu-BaBaSar-RGD.sub.2. When biological ligands other than RGD peptides are conjugated with the BaBaSar chelator, the same kit method associated with BaBaSar chelator could be used too. For example, other peptides can be used in place of RGD. In addition, other chelators, preferably sarcophagine based chelators, such as AnAnSar, BaAnSar, BaMalSar, and MalMalSar can be used in place of BaBaSar. Therefore, this kit method provides a universal method for .sup.64Cu radiopharmaceutical production.

(10) General Method

(11) BaBaSar-RGD.sub.2 was synthesized as previously reported (23). All commercial chemicals were of analytic grade and used without further purification. .sup.64Cu in hydrochloric acid was obtained from Washington University (St. Louis, Mo.) or produced in the Molecular Imaging Center Cyclotron Facility. Analytic reversed-phase high-performance liquid chromatography (RP-HPLC) using a Phenomenex Luna column (5μ, C.sub.18, 250×4.6 mm) were performed on a Dionex U3000 chromatography system with a diode arrays detector and radioactivity flow-count (Eckert & Ziegler, Valencia, Calif.). The recorded data were processed using Chromeleon version 7.20 software. The flow rate of analytical HPLC was 1.0 mL/min. The mobile phase starts from 95% solvent A (0.1% trifluoroacetic acid [TFA] in water) and 5% solvent B (0.1% TFA in acetonitrile [MeCN]). From 2 to 32 min, the mobile phase ramped to 35% solvent A and 65% solvent B. The ultraviolet (UV) detector of HPLC was set at 254 nm. The endotoxin analysis was performed on a portable Endosafe®-PTS™ system consisting of LAL reagent and endotoxin controls applied to a single use, polystyrene cartridge.

(12) Radiopharmaceutical Preparation

(13) Preparation of .sup.64Cu-BaBaSar-RGD.sub.2 Production Kit

(14) The 18.2 MΩ.Math.cm water from in-house GenPure™ station was treated with chelex 100 resin 48 hours before use. All the solution hereafter was prepared with this treated water. The lyophilized BaBaSar-RGD.sub.2 (1.0 mg) was dissolved in 1.0 mL sodium acetate buffer (NaOAc, 0.1 M, pH 5.5). The pH of BaBaSar-RGD.sub.2 solution was adjusted to pH 5.5 using 0.1 M sodium hydroxide (NaOH). Then, the BaBaSar-RGD.sub.2 solution was equally aliquoted to 20 Eppendorf vials (1.5 mL). The filled vials were frozen using dry ice and then transferred to the bottles of the Labconco Freeze Dry System (pressure <100 mTorr). After the solvent was removed, the vials containing BaBaSar-RGD.sub.2 powder were then sealed and stored at −18° C. for .sup.64Cu labeling.

(15) .sup.64Cu-Labeling Chemistry

(16) 64CuCl2 (5-30 mCi) purchased from Washington University at St. Louis was reconstituted using 200-300 μL NaOAc buffer (0.1 M, pH 5.5) and added to a vial prepared in above section. The vial was gently shaking at room temperature for 5 min. After the reaction was quenched with 5.0 mL saline, the activity passed through a 0.22 μm sterile filter (Pall Corp.) into a 10 mL Allergy vial for quality control test and animal/human injection.

(17) Kit Preparation

(18) A cold kit can contain 50 μg BaBaSar-RGD.sub.2 ligand to which .sup.64CuCl.sub.2 is to be complexed, and buffer salts to adjust the pH suitable for the labelling conditions. The kits are prepared in a lyophilized form and have a long shelf life of over 3 months at room temperature. When the cold kits are stored in a refrigerator at 2-8° C., the shelf life is over a year.

(19) Radiochemistry

(20) The labeling chemistry for .sup.64Cu-BaBaSar-RGD.sub.2 is disclosed in FIG. 2. The .sup.64Cu-labeling yield for .sup.64Cu-BaBaSar-RGD.sub.2 was >99% based on HPLC analysis (FIG. 3). However, after passing through 0.22 μm Pall filter to remove pyrogen, approximately 15-20% .sup.64Cu-BaBaSar-RGD.sub.2 was trapped onto the filter and the overall recovered yield for .sup.64Cu-BaBaSar-RGD.sub.2 is about 80% calculated from the loaded .sup.64Cu. The radiochemical purity of .sup.64Cu-BaBaSar-RGD.sub.2 was >99% based on radiotrace analytical HPLC (FIG. 3). The retention times for free .sup.64CuCl.sub.2 and .sup.64Cu-BaBaSar-RGD.sub.2 on HPLC were 2.5 and 13.9 min, respectively. The reaction crude without purifications did not show free .sup.64Cu in HPLC chromatograms. Therefore, no further purification is needed for the final product.

(21) Quality Control

(22) All the quality control results met the pre-specified limits for 3 validation runs. These included half-life, appearance, pH value, identity, endotoxin amount, etc. (Table 1). The specific activity determined by HPLC analysis was between 389.2 and 605.4 mCi/μmol (average±SD, 473.0±116.2 mCi/μmol). Therefore, a human dose (<25 mCi) of .sup.64Cu-BaBaSar-RGD.sub.2 contained less than 125 μg of RGD peptide.

(23) TABLE-US-00001 TABLE 1 Quality control data from 3 synthesis runs. QC Test Release Criteria Run 1 Run 2 Run 3 Product (mCi) none 5.5 6.2 4.5 Visual Inspection Clear, colorless Yes Yes Yes Radiochemical Identity RRT = 0.9-1.1 1.0 1.0 1.0 Radiochemical Purity >90% 99% 100% 99% Specific Activity >100  15.7 14.4 22.4 (mCi/μmol) Dose pH 4.5-7.5 5.5 6.0 6.0 Sterile Filter >45 64 64 62 Integrity Test (psi) Radionuclidic Identity 12.6-12.8 h 12.7 12.7 12.7 (t.sub.1/2) Endotoxin Analysis  ≤17.5 <5 <5 <5 (EU/mL)
Absorbed Dose Estimates from Macaque Imaging

(24) The injection of 13.1-19.7 MBq/kg of .sup.64Cu-BaBaSar-RGD.sub.2 in macaque monkey produced no observable effects on vital signs (blood pressure, pulse, and electrocardiogram) during and 24-h after PET scan. The PET images at 1, 5, 10, 20, 40, 60, 120, and 180 min after injection are disclosed in FIG. 4. At 1 min, rapid uptake of .sup.64Cu-BaBaSar-RGD.sub.2 was observed in the heart, and liver. The bladder content was visualized at 10 min after injection and more and more activity was accumulated in urine bladder content. The bladder did not void because the macaque monkey was under anesthesia. Gallbladder uptake was not observed during the whole scan. Rapid clearance of activity in the liver was observed in the images at time points after 1 min. The urinary bladder had the highest uptake, with 51.37%±8.73% of injected activity at 1 h post injection. The maximum uptake for the liver, and kidneys were 37.40±6.63% ID (9 min) and 26.79±4.35% ID (0.5 min) respectively. At 3 h of post injection, 8.62%±1.41% of injected activity was found in the gallbladder, small intestine, and upper and lower portions of the large intestine.

(25) The mean organ doses for the male human phantom were calculated with Olinda/EXM using .sup.64Cu-BaBaSar-RGD.sub.2 biodistribution in monkey (Table 2). The kidneys had the highest radiation-absorbed doses (108.43 μGy/MBq), followed by the bladder wall (87.07 μGy/MBq). The mean effective dose of .sup.64Cu-BaBaSar-RGD.sub.2 was 15.30±2.21 μSv/MBq. When 925-MBq of .sup.64Cu-BaBaSar-RGD.sub.2 is administrated into human subject, the effective dose for the non-voiding model is estimated to be 14.2 mSv, which is comparable to the estimated 6.23 mSv dose in a whole-body PET scan with 2-deoxy-2-[.sup.18F]fluoro-D-glucose (.sup.18F-FDG) (24). The estimated doses for the female human were higher by 18% because body and organ sizes of women are smaller than those men (data not shown).

(26) Venous blood samples were withdrawn from monkey during the PET scan. Based on the decay corrected activity per unit of blood sample, it was found that .sup.64Cu-BaBaSar-RGD.sub.2 was cleared rapidly from the blood. By 3 h after injection, 2.88±0.88% ID remained (range, 2.07-3.82% ID). At 22 h after injection, the activity in the blood decreased to 0.79±0.52% ID. Based on the percentage of injected dose in blood sample, the half life of .sup.64Cu-BaBaSar-RGD.sub.2 in blood pool was calculated as 12.1±4.0 min (n=3).

(27) TABLE-US-00002 TABLE 2 Estimated Human Absorbed Doses of .sup.64Cu-BaBaSar-RGD.sub.2 to Normal Organs Using Biodistribution Data from Macaque monkey. Organs Mean ± SD (μGy/MBq) Adrenals 3.34 ± 0.52 Brain 1.27 ± 0.22 Breasts 1.34 ± 0.23 Gall bladder Wall 3.07 ± 0.49 LLI Wall 2.86 ± 0.44 Small Intestine 4.53 ± 0.68 Stomach Wall 2.11 ± 0.34 ULI Wall 2.47 ± 0.39 Heart Wall 4.39 ± 0.62 Kidneys 108.43 ± 16.41  Liver 7.54 ± 1.15 Lungs 1.67 ± 0.28 Muscle 1.88 ± 0.31 Ovaries 2.88 ± 0.44 Pancreas 2.86 ± 0.45 Red Marrow 9.29 ± 1.02 Osteogenic Cells 7.01 ± 0.91 Skin 1.38 ± 0.24 Spleen 6.78 ± 0.88 Testes 2.03 ± 0.33 Thymus 1.56 ± 0.26 Thyroid 1.39 ± 0.24 Urinary Bladder Wall 87.07 ± 12.38 Uterus 4.16 ± 0.63 Total Body 2.76 ± 0.42 Effective Dose* 15.30 ± 2.21  *In unit of μSv/MBq

(28) Integrin αvβ3-targeted radiopharmaceutical .sup.64Cu-BaBaSar-RGD.sub.2 has been successfully synthesized with the one-step kit method. The straightforward method greatly simplifies the production process and benefits the clinical application of .sup.64Cu-BaBaSar-RGD.sub.2. Human radiation dosimetry of .sup.64Cu-BaBaSar-RGD.sub.2 was estimated after intravenous administration in macaque monkey, by PET imaging and OLINDA/EXM calculations. The critical organs were kidneys and urinary bladder wall. The mean effective dose, determined with the male adult model, was 15.30±2.21 Sv/MBq. This PET probe demonstrates an acceptable radiation dose comparable to other reported RGD-derived radiopharmaceuticals. These demonstrate great promise of .sup.64Cu-BaBaSar-RGD.sub.2 as an integrin marker, with a desirable biodistribution and safety characteristics in monkey. Therefore, .sup.64Cu-BaBaSar-RGD.sub.2 can safely be used in human scan for further evaluation of its performance as an integrin-targeting probe.

REFERENCES

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