Ark composition and use thereof in tumor diagnosis

Abstract

A composition and use thereof in tumor diagnosis. Specifically provided is a composition comprising a compound represented by formula I, a ligand for one or more radionuclides, a surfactant, a skeleton proppant, and a pH regulator. The composition has excellent stability, has a relatively high labeling rate for a radionuclide, and can be used for preparing a SPECT developing agent, enabling localization and diagnosis of HER2-positive tumors, lesion detection, patient screening for HER2-targeted tumor therapy, and evaluation of the efficacy of tumor therapy.

Description

Ark Compositions and Their Application in Tumor Diagnosis Technical Field

[0001] This invention relates to the field of medical imaging diagnosis, and more specifically to a composition and its application in tumor diagnosis. Background Technology

[0002] Breast cancer is one of the most common malignant tumors in women, seriously threatening their lives and health. Clinically, approximately 30% of breast cancer patients are human epidermal growth factor receptor 2 (HER2) positive. HER2 overexpression can lead to malignant transformation of cells and is closely related to breast cancer invasion, metastasis, and recurrence. Early-stage breast cancer patients have a high cure rate, but due to the lack of obvious early symptoms, they often miss the optimal treatment window. The advent of molecular imaging has made early diagnosis of tumors possible. Nuclear medicine imaging (PET, SPECT) is becoming increasingly important in clinical practice due to its high sensitivity, strong tissue penetration, and in vivo quantitative capabilities. Trastuzumab (Herceptin) is a humanized monoclonal antibody targeting HER2 and is a first-line treatment for HER2-positive breast cancer, significantly improving overall survival and showing good efficacy in both early and advanced (metastatic) breast cancer. However, not all patients are sensitive to it, and resistance may develop after long-term use. This makes assessing changes in HER2 expression levels before and during treatment crucial.

[0003] Currently, the most widely used technique in clinical tumor imaging is... 18 F-FDG PET imaging is a non-specific imaging technique used for tumor metabolism and cannot indicate the HER2 expression status of tumors. Additionally, radiolabeled trastuzumab has been used in clinical studies for imaging, but due to the slow metabolism of the antibody in vivo and its self-blocking effect, real-time monitoring of efficacy is difficult. Therefore, developing imaging agents that target the HER2 site has significant clinical application prospects, enabling precise assessment of patients' HER2 expression levels, optimization of treatment plans, and effective prognostic evaluation, ultimately achieving the goal of personalized treatment. Compared to PET technology, the nuclear medicine molecular imaging technique SPECT has lower equipment and drug costs, higher clinical penetration, and a better application foundation. However, it lacks a class of... 18 The limited availability of effective imaging agents for FDG restricts the role of SPECT in tumor diagnosis and efficacy evaluation. Therefore, developing tumor HER2 imaging agents for SPECT is of significant clinical importance.

[0004] Summary of the Invention

[0005] Polypeptide-containing compositions and their preparation

[0006] Based on existing technology, the present invention provides a stable polypeptide-containing composition, wherein the polypeptide can be labeled with radionuclides at a high labeling rate.

[0007] Specifically, the present invention provides a composition comprising a compound of formula I, a ligand for one or more radionuclides, a surfactant, a framework support agent, and a pH adjuster.

[0008] In some embodiments, the radionuclide is 99 mTc.

[0009] In some embodiments, the ligand for the radionuclide is selected from hydroxyl-containing ligands and phosphine ligands, such as TPPTS, Tricine, or combinations thereof.

[0010] In some embodiments, the surfactant is selected from polysorbate 80, poloxamer, etc. (polyoxyethylene (23) lauryl ether), Triton X-100 and sodium dodecyl sulfate.

[0011] In some embodiments, the skeletal support is selected from trehalose, sucrose, maltose, lactose, glycine, and mannitol.

[0012] In some implementations, the pH adjuster is selected from succinate buffer systems and HEPES buffer systems.

[0013] In some embodiments, the composition comprises the compound of formula I, TPPTS, Tricine, trehalose, polysorbate 80, and a succinate buffer system.

[0014] In some embodiments, the composition comprises 2-30 parts by weight of the compound of formula I, 50-500 parts by weight of TPPTS, 100-1000 parts by weight of Tricine, 1000-15000 parts by weight of a skeleton support agent, 1-100 parts by weight of a surfactant and a pH adjuster, wherein the amount of the pH adjuster is such that the pH of the composition is adjusted to 5.6-7.0.

[0015] In some embodiments, the composition comprises 3-15 parts by weight of the compound of formula I, 100-500 parts by weight of TPPTS, 250-650 parts by weight of Tricine, 2000-10000 parts by weight of trehalose, 1-100 parts by weight of polysorbate 80, and a succinate buffer system, wherein the amount of the succinate buffer system is such that the pH of the composition is 6.20-7.0.

[0016] In some embodiments, the composition comprises 1 part by weight of the compound of formula I, 60 parts by weight of TPPTS, 80 parts by weight of Tricine, 2000 parts by weight of trehalose, 10 parts by weight of polysorbate 80, and a succinate buffer system, wherein the amount of the succinate buffer system is such that the pH of the composition is adjusted to 6.20-7.0.

[0017] In this document, the succinate buffer system is a sodium succinate buffer solution. In some embodiments, the sodium succinate buffer solution is prepared by adding 0.1 mol / L sodium hydroxide aqueous solution to a 0.1 mol / L succinic acid aqueous solution to adjust the pH to 6.5.

[0018] In some embodiments, the composition is a solid dosage form.

[0019] In some embodiments, the composition is a lyophilized formulation, preferably a lyophilized powder for injection, such as a sterile powder for injection.

[0020] In another aspect, the present invention provides a method for preparing the composition described in any one of the preceding claims, comprising the following steps:

[0021] The ligands of one or more radionuclides and the framework support agent are added to an aqueous solution of the pH adjuster, the surfactant and the compound of formula I are added, the pH is optionally adjusted, and the mixture is prepared to obtain the composition.

[0022] In some implementations, the method is characterized by one or more of the following:

[0023] (1) Each step is carried out under inert gas protection;

[0024] (2) Each step shall be carried out under light-protected conditions;

[0025] (3) Perform each step using an aqueous solution of the ligand of the radionuclide, an aqueous solution of the skeleton support, an aqueous solution of the surfactant and / or an aqueous solution of the compound of Formula I;

[0026] (4) Each step is carried out at 2℃ to room temperature (e.g., 25℃), for example, 2℃ to 8℃;

[0027] (5) After the mixing step, a filtration step is also included. The filtration is preferably carried out using a filter membrane made of polyvinyl fluoride (PVDF), nylon, polyethersulfone (PES), or cellulose acetate (CA), preferably a PVDF filter membrane with a diameter of 0.2-0.3 μm.

[0028] In some embodiments, the composition comprises, by weight percentage: 0.002%-0.03% of the compound of formula I, 0.05%-0.5% of TPPTS, 0.1%-1% of Tricine, 1%-15% of a support matrix, 0.001%-0.1% of a surfactant, a pH adjuster, and the balance being water, wherein the amount of the pH adjuster is such that the pH of the composition is adjusted to 5.6-7.0.

[0029] In some embodiments, the composition contains 0.003%-0.015% of the compound of formula I, 0.1%-0.5% of TPPTS, 0.25%-0.65% of Tricine, 2%-10% of trehalose, 0.001%-0.1% of polysorbate 80, a succinate buffer system, and the balance being water, wherein the amount of the succinate buffer system is such that the pH of the composition is adjusted to 6.20-7.0;

[0030] Preferably, the composition contains 0.005% of the compound of formula I, 0.3% of TPPTS, 0.4% of Tricine, 10% of trehalose, 0.05% of polysorbate 80, a succinate buffer system, and the balance water, wherein the amount of the succinate buffer system is such that the pH of the composition is 6.20-7.0.

[0031] In some embodiments, a drying step is further included, such as freeze drying, preferably a two-stage freeze drying: first stage: -40±2℃, 30±5Pa, drying for 2 hours; second stage: 25±2℃, 30±5Pa, drying for 1 hour.

[0032] Radionuclide labeling

[0033] The compound of formula I, TPPTS, and Tricine in the composition of this invention can combine with radionuclides to form a triple metal complex, which can be used for in vivo diagnostic imaging, especially SPECT imaging.

[0034] In another aspect, the present invention provides a liquid formulation or reconstituted liquid formulation obtained by dissolving the composition described in any one of the preceding claims in a solution containing a radionuclide.

[0035] In some embodiments, the solution containing the radionuclide is added to the composition to dissolve it, and then heated at 90-100°C for 20-30 minutes to obtain the liquid formulation or reconstituted liquid formulation.

[0036] In some embodiments, the radionuclide is 99m Tc.

[0037] In some embodiments, the solution containing the radionuclide is technetium […]. 99m Sodium Tc (Na) 99m TcO4) solution. In some embodiments, the activity of the radionuclide in the liquid formulation or reconstituted liquid formulation is 20-50 mCi, for example 20-35 mCi. In some embodiments, the liquid formulation or reconstituted liquid formulation is an injectable.

[0038] In some embodiments, the unit dose of the liquid formulation or reconstituted liquid formulation is 0.5-1.5 mL.

[0039] In some embodiments, the compound of Formula I and the ligand of the radionuclide combine with the radionuclide to form a metal complex.

[0040] In some embodiments, the liquid formulation or reconstituted liquid formulation comprises, by weight percentage:

[0041] 0.002%-0.03% of Formula I compounds (e.g., 0.002%-0.003%, 0.002%-0.004%, 0.002%-0.005%, 0.002%-0.006%, 0.002%-0.007%, 0.002%-0.008%, 0.002%-0.009%, 0.002%-0.00%). 10%, 0.002%-0.015%, 0.002%-0.020%, 0.002%-0.025%, 0.002%-0.030%, 0.003%-0.004%, 0.003%-0.005%, 0.003%-0.006%, 0.003%-0.007%, 0.003%-0.008%, 0.003%-0.009%, 0.003%-0.010%, 0.003%-0.015%, 0.003%-0.020%, 0.003%-0.025%, 0.003%-0.030%, 0.004%-0.005%, 0.004%-0.006%, 0.004%-0.007%, 0.004%-0.008%, 0.004%-0.009%, 0.004%-0.010%, 0.004%-0.015%, 0. 0.004%-0.020%, 0.004%-0.025%, 0.004%-0.030%, 0.005%-0.006%, 0.005%-0.007%, 0.005%-0.008%, 0.005%-0.009%, 0.005%-0.010%, 0.005%-0.015%, 0.005%-0.020%, 0.005%-0.025%, 0.005%-0.030%, 0.006%-0.007%, 0.00 6%-0.008%, 0.006%-0.009%, 0.006%-0.010%, 0.006%-0.015%, 0.006%-0.020%, 0.006%-0.025%, 0.006%-0.030%, 0.007%-0.008%, 0.007%-0.009%, 0.007%-0.010%, 0.007%-0.015%, 0.007%-0.020%, 0.007%-0.025%, 0.007% -0.030%, 0.008%-0.009%, 0.008%-0.010%, 0.008%-0.015%, 0.008%-0.020%, 0.008%-0.025%, 0.008%-0.030%, 0.009%-0.010%, 0.009%-0.015%, 0.009%-0.020%, 0.009%-0.025%, 0.009%-0.030%, 0.010%-0.015%, 0.010%-0.(0.020%, 0.010%-0.025%, 0.010%-0.030%, 0.015%-0.020%, 0.015%-0.025%, 0.015%-0.030%, 0.020%-0.025%, 0.020%-0.030% or 0.025%-0.030%).

[0042] 0.05%-0.5% TPPTS (e.g., 0.05%-0.1%, 0.05%-0.15%, 0.05%-0.2%, 0.05%-0.25%, 0.05%-0.3%, 0.05%-0.35%, 0.05%-0.4%, 0.05%-0.45%, 0.05%-0.5%, 0.1%-0.5%) 0.15%, 0.1%-0.2%, 0.1%-0.25%, 0.1%-0.3%, 0.1%-0.35%, 0.1%-0.4%, 0.1%-0.45%, 0.1%-0.5%, 0.15%-0.2%, 0.15%-0.25%, 0.15%-0.3%, 0.15%-0.35%, 0.15% -0.4%, 0.15%-0.45%, 0.15%-0.5%, 0.2%-0.25%, 0.2%-0.3%, 0.2%-0.35%, 0.2%-0.4%, 0.2%-0.45%, 0.2%-0.5%, 0.25%-0.3%, 0.25%-0.35%, 0.25%-0.4%, 0.2 5%-0.45%, 0.25%-0.5%, 0.3%-0.35%, 0.3%-0.4%, 0.3%-0.45%, 0.3%-0.5%, 0.35%-0.4%, 0.35%-0.45%, 0.35%-0.5%, 0.4%-0.45%, 0.4%-0.5% or 0.45%-0.5%);

[0043] Tricine at concentrations of 0.1%-1% (e.g., 0.1%-0.15%, 0.1%-0.2%, 0.1%-0.25%, 0.1%-0.3%, 0.1%-0.35%, 0.1%-0.4%, 0.1%-0.45%, 0.1%-0.5%, 0.1%-0.55%, 0.1%-0.6%, 0.1%). -0.65%, 0.1%-0.7%, 0.1%-0.75%, 0.1%-0.8%, 0.1%-0.85%, 0.1%-0.9%, 0.1%-0.95%, 0.1%-1%, 0.15%-0.2%, 0.15%-0.25%, 0.15%-0.3%, 0.15%-0.35%, 0.15% -0.4%, 0.15%-0.45%, 0.15%-0.5%, 0.15%-0.55%, 0.15%-0.6%, 0.15%-0.65%, 0.15%-0.7%, 0.15%-0.75%, 0.15%-0.8%, 0.15%-0.85%, 0.15%-0.9%, 0.15%-0. 95%, 0.15%-1%, 0.2%-0.25%, 0.2%-0.3%, 0.2%-0.35%, 0.2%-0.4%, 0.2%-0.45%, 0.2%-0.5%, 0.2%-0.55%, 0.2%-0.6%, 0.2%-0.65%, 0.2%-0.7%, 0.2%-0.75%, 0.2%-0.8%, 0.2%-0.85%, 0.2%-0.9%, 0.2%-0.95%, 0.2%-1%, 0.25%-0.3%, 0.25%-0.35%, 0.25%-0.4%, 0.25%-0.45%, 0.25%-0.5%, 0.25%-0.55%, 0.25%-0.6%, 0.25%-0.65%, 0.25%-0.7%, 0.25%-0.75%, 0.25%-0.8%, 0.25%-0.85%, 0.25%-0.9%, 0.25%-0.95%, 0.25%-1%, 0.3%-0.35%, 0.3%-0.4%, 0.3%-0.45%, 0.3%-0.5%, 0.3%-0.55%, 0.3%-0.6%, 0.3%-0.65%, 0.3%-0.7%, 0.3%-0.75%, 0.3%-0.8%, 0.3%-0.85%, 0.3%-0.9%, 0.3%-0.95%, 0.3%-1%, 0.35%-0.4%, 0.35%-0.45%, 0.35%-0.5%, 0.35%-0.55%, 0.35%-0.6%, 0.35%-0.65%, 0.35%-0.7%, 0.35%-0.75%, 0.35%-0.8%, 0.35%-0.85%, 0.35%-0.9%, 0.35%-0.95%, 0.35%-1%, 0.4%-0.45%, 0.4%-0.5%, 0.4%-0.55%, 0.4%-0.6%, 0.4%-0.65%, 0.4%-0.7%, 0.4%-0.75%, 0.4%-0.8%, 0.4%-0.85%, 0.4%-0.9%, 0.4%-0.95%, 0.4%-1%, 0.45%-0.5%, 0.45%-0.55%, 0.45%-0.6%, 0.45%-0.65%, 0.45%-0.7%, 0.45% %-0.75%, 0.45%-0.8%, 0.45%-0.85%, 0.45%-0.9%, 0.45%-0.95%, 0.45%-1%, 0.5%-0.55%, 0.5%-0.6%, 0.5%-0.65%, 0.5%-0.7%, 0.5%-0.75%, 0.5%-0.8%, 0.5%-0.85%, 0.5%-0.9%, 0.5%-0.95%, 0.5%-1%, 0.55%-0.6%, 0.55%-0.65%, 0.55%-0.7%, 0.55%-0.75%, 0.5% 5%-0.8%, 0.55%-0.85%, 0.55%-0.9%, 0.55%-0.95%, 0.55%-1%, 0.6%-0.65%, 0.6%-0.7%, 0.6%-0.75%, 0.6%-0.8%, 0.6%-0.85%, 0.6%-0.9%, 0.6%-0.95%, 0.6%-1%, 0.7% (0.75%, 0.7%-0.8%, 0.7%-0.85%, 0.7%-0.9%, 0.7%-0.95%, 0.7%-1%, 0.75%-0.8%, 0.75%-0.85%, 0.75%-0.9%, 0.75%-0.95%, 0.75%-1%, 0.8%-0.85%, 0.8%-0.9%, 0.8%-0.95%, 0.8%-1%, 0.85%-0.95%, 0.85%-1%, 0.9%-0.95%, 0.9%-1% or 0.95%-1%).

[0044] 1%-15% of the matrix support (e.g., 1%-2%, 1%-4%, 1%-6%, 1%-8%, 1%-10%, 1%-12%, 1%-14%, 1%-15%, 2%-4%, 2%-6%, 2%-8%, 2%-10%, 2%-12%, 2%-14%, 2%-15%, 4%-6%, 4%-8%, 4%-10%) 4%-12%, 4%-14%, 4%-15%, 6%-8%, 6%-10%, 6%-12%, 6%-14%, 6%-15%, 8%-10%, 8%-12%, 8%-14%, 8%-15%, 10%-12%, 10%-14%, 10%-15%, 12%-14%, 12%-15%, or 14%-15%);

[0045] 0.001%-0.1% of surfactant (e.g., 0.001%-0.005%, 0.001%-0.01%, 0.001%-0.05%, 0.005%-0.01%, 0.005%-0.05%, 0.005%-0.10%, 0.01%-0.05%, 0.01%-0.10%, or 0.05%-0.10%); and,

[0046] A pH adjuster is used in an amount that brings the pH of the composition to 5.6-7.0 (e.g., 5.6-5.8, 5.6-6.0, 5.6-6.2, 5.6-6.4, 5.6-6.6, 5.6-6.8, 5.6-7.0, 5.8-6.0, 5.8-6.2, 5.8-6.4, 5.8-6.6, 5.8-6.8, 5...). 6.0-7.0, 6.0-6.2, 6.0-6.4, 6.0-6.6, 6.0-6.8, 6.0-7.0, 6.2-6.4, 6.2-6.6, 6.2-6.8, 6.2-7.0, 6.4-6.6, 6.4-6.8, 6.4-7.0, 6.6-6.8, 6.6-7.0, or 6.8-7.0);

[0047] And, radionuclides with an activity of 20-50 mCi, preferably 20-35 mCi.

[0048] In some embodiments, the chemical purity of the radionuclide is above 90%, for example above 90%, above 91%, above 92%, above 93%, above 94%, above 95%, above 96%, above 97%, above 98%, or above 99%.

[0049] In some embodiments, the liquid formulation or reconstituted liquid formulation is used for SPECT imaging.

[0050] In another aspect, the present invention provides a medicine box product comprising the composition described in any of the preceding claims, and optionally a radiopharmaceutical (e.g., a radiopharmaceutical for imaging) and / or instructions.

[0051] In some embodiments, the radiopharmaceutical is sodium pertechnetate [99mTc] injection.

[0052] In some embodiments, the activity of the radiopharmaceutical is 20-50 mCi, for example 20-35 mCi.

[0053] In some implementations, the unit dose of the radiopharmaceutical is 0.5-1.5 mL.

[0054] Diagnostic contrast imaging

[0055] The compositions, liquid formulations, or reconstructed liquid formulations or kits described in this invention can be used for in vivo diagnostic imaging of HER2-positive tumors, for auxiliary diagnosis of tumor localization, lesion detection, and neovascularization density, and for efficacy evaluation of anti-tumor angiogenesis therapy.

[0056] In another aspect, the present invention provides the use of any of the compositions, liquid formulations or reconstituted liquid formulations or kit products described in the foregoing in the preparation of a medicament or reagent, wherein the medicament or reagent is used for diagnostic contrast imaging of tumors (e.g., HER2-positive tumors).

[0057] In another aspect, the present invention provides a diagnostic imaging method for tumors (e.g., HER2-positive tumors), comprising the step of administering to a subject an effective amount of any of the compositions, liquid formulations, or reconstituted liquid formulations or cassette products described above.

[0058] The compositions, liquid formulations, or reconstituted liquid formulations or kit products described in this invention can be used for in vivo diagnostic imaging of HER2-positive tumors. These HER2-positive tumors include HER2-positive breast cancer, gastric cancer, colon cancer, bladder cancer, ovarian cancer, endometrial cancer, lung cancer, etc.

[0059] Terminology Definition

[0060] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to technical terms herein refer to techniques commonly understood in the art, including variations or equivalent substitutions of techniques that are obvious to one of ordinary skill in the art. While it is believed that the following terms will be well understood by one of ordinary skill in the art, the following definitions are set forth to better explain the invention.

[0061] As used herein, the term “tris(3-sulfonylphenyl)phosphine sodium salt hydrate (TPPTS)” has the following chemical structural formula:

[0062] Molecular formula: C 18 H 12 Na3O9PS3·4H2O; Molecular weight: 640.49; Appearance: White or off-white crystalline powder.

[0063] As used herein, the term "tris(hydroxymethyl)methylglycine" has the following chemical structural formula:

[0064] Molecular formula: C6H 13 NO5; Molecular weight: 179.17; Appearance: White crystals or crystalline powder.

[0065] As used herein, the term "labeling rate" refers to the percentage of stable radionuclides introduced into a labeled compound molecule relative to the total amount of nuclides used in the labeling reaction; it is a parameter reflecting labeling efficiency. The same radionuclide can have multiple different valence states, thus forming different compounds. To describe the relative quantitative relationships between the various chemical forms of the same radionuclide, the concept of radiochemical purity, also known as labeling rate, is defined. Commonly used methods for determining this include radiometric paper chromatography, thin-layer chromatography, column chromatography, and protein precipitation. Attached Figure Description

[0066] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0067] Figure 1 shows the effect of TPPTS dosage on the labeling rate.

[0068] Figure 2 shows the effect of Tricine dosage on the labeling rate.

[0069] Figure 3 shows the composition of the present invention being... 99m Results of radioactive TLC detection after Tc labeling.

[0070] Figure 4 shows the effect of labeling temperature and time on the labeling rate.

[0071] Figure 5 shows tail vein administration in the SKBR3 breast cancer tumor model. 99m SPECT / CT images at different time points after Tc-HP-Ark2 (50mCi labeled injection) (white arrows indicate tumor locations).

[0072] Figure 6 shows tail vein administration in the SKBR3 breast cancer tumor model. 99mSPECT / CT images of unlabeled precursor HP-Ark2 (50mCi labeled injection) 0.5h after Tc-HP-Ark2 (50mCi labeled injection) blockade (10mg / mL) (white arrows indicate tumor location).

[0073] Figure 7 shows tail vein administration in the SKBR3 breast cancer tumor model. 99m Whole-body SPECT / CT images at different time points after Tc-HP-Ark2 (20mCi labeled injection) (white arrows indicate tumor locations).

[0074] Figure 8 shows tail vein administration in the SKBR3 breast cancer tumor model. 99m Distribution of radioactivity in tissues at different time points after Tc-HP-Ark2 (n=5 for each group).

[0075] Figure 9 shows tail vein administration in the SKBR3 breast cancer tumor model. 99m Distribution of radioactivity in tissues blocked by the unlabeled precursor HP-Ark2 (5 mg / mL) 0.5 h after Tc-HP-Ark2 administration (n = 5 per group). Note: *** indicates radioactivity distribution in tissues blocked by the unlabeled precursor HP-Ark2 (5 mg / mL). 99m Compared to the Tc-HP-Ark2 group, the tumor %ID / g decreased after 0.5 h following administration, the tumor %ID / g decreased after nerve block (p<0.001). Detailed Implementation

[0076] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0077] Labeling rate detection method

[0078] Example 1. Prescription Screening

[0079] I. Selection of Surfactant Types and Dosage

[0080] Using polysorbate 80 as a surfactant, the formulations were screened within the range of 0.001% to 0.1% of polysorbate 80. The screening results are shown in Tables 1 and 2.

[0081] Table 1. Formulation composition for polysorbate 80 dosage screening (single dose)

[0082] Note: A brief description of the complete formulation preparation process: Prepare a 0.1 mol / L succinic acid solution, and adjust the pH to 6.5 with 0.1 mol / L sodium hydroxide solution. Take 80% of the formulation amount of buffer salt solution, add the formulation amounts of TPPTS, Tricine, and Polysorbate 80, dissolve them, add the formulation amount of compound I solution, add water to make up to the total volume and stir well.

[0083] Distilled water was used instead of water for injection during the experiment.

[0084] Table 2. Results of the experiment on the selection of polysorbate 80 dosage:

[0085] Note: Prescriptions 1-7 are over-filled; the agreed volume should be increased to 102% of the total volume.

[0086] The content of each Formula I compound is compared by peak area, with the content of Formula I compound 1-1 before filtration being 100%.

[0087] Experimental conclusion:

[0088] (1) Compound I has high activity and certain surface activity, and can be adsorbed on the surface. After adding polysorbate 80, the content of compound I in the sample increased significantly. Polysorbate 80 helps to reduce the adsorption of compound I.

[0089] (2) The amount of polysorbate 80 used has no significant effect on the labeling rate of this product.

[0090] (3) As the content of polysorbate 80 increased from 0.001% to 0.05%, the content of compound I in the sample increased progressively. The 0.05% polysorbate 80 sample had the highest content of compound I and a better labeling rate.

[0091] II. Selection of Buffer Salts

[0092] This experiment used succinate and HEPES buffer systems for research. The experimental results are shown in Tables 3 and 4.

[0093] Table 3. Selection of buffer salts and formulation composition

[0094] [注1] Prepare a 0.1 mol / L succinic acid solution and adjust the pH to 6.5 by adding 0.1 mol / L sodium hydroxide solution.

[0095] [注2] Preparation of 10 mmol / L HEPEs buffer: Weigh 2.383 g of HEPES, add water to a final volume of 1 L, and adjust the pH to 6.5 with 0.5 M sodium hydroxide solution.

[0096] [注3]Distilled water was used instead of water for injection during the experiment.

[0097] Table 4. Experimental Results of Buffer Salt Selection

[0098] [注1] The same injection system was used before and after filtration, and the study was conducted based on the ratio of the main peak area.

[0099] Formulas 2-3 exhibited strong opalescence after reconstitution of the product following freeze-drying (reconstitution solvent: water).

[0100] Experimental conclusion:

[0101] (1) Samples prepared with HEPES buffer have a high content of compound I in the solution before lyophilization, which may help reduce the adsorption of compound I on the surface of the instrument.

[0102] (2) Samples prepared with 10 mmol / L HEPES buffer showed opalescence after lyophilization and reconstitution.

[0103] III. Selection of Excipient Types and Dosage

[0104] This experiment selected sugars (sucrose, trehalose), amino acids (glycine), and polyhydroxy compounds (mannitol) to study the types of excipients.

[0105] 1. Selection of Excipient Types and Dosage (Part 1)

[0106] Lactose, mannitol, and glycine were selected as excipients and added to the formulation at a dosage of 100 mg / vial. The effects of these three excipients on the samples were investigated.

[0107] Table 5. Selection of Excipient Types and Dosages (Part 1)

[0108] [注1] Prepare a 0.1 mol / L succinic acid solution and adjust the pH to 6.5 by adding 0.1 mol / L sodium hydroxide solution.

[0109] [注2] Use Watson's distilled water instead of water for injection.

[0110] Brief description of the complete prescription preparation process: Prepare a 0.1 mol / L succinic acid solution, and adjust the pH to 6.5 with 0.1 mol / L sodium hydroxide solution. Take 80% of the prescription volume of buffer salt solution, add the prescription volume of TPPTS, Tricine, lactose / mannitol / glycine, dissolve, add the prescription volume of compound I solution, add water to make up to the total volume and stir well.

[0111] Analysis of Results of Experiment 1 on Screening of Excipient Types and Dosage

[0112] (1) A flocculent precipitate appeared in the drug solution after glycine was added;

[0113] (2) When mannitol was added to the solution, a white precipitate formed;

[0114] (3) Lactose, as a reducing sugar, may undergo Maillard reaction with protein during the later stabilization process, and it is a bovine-derived feed additive.

[0115] 2. Screening of Excipient Types and Dosages (Part Two)

[0116] Sucrose and trehalose were selected as excipients to investigate their effects on the samples.

[0117] Table 6-1 Experiment (II) on the Selection of Excipient Types and Dosages: Formulation Composition

[0118] Table 6-1 Experiment (II) on the Selection of Excipient Types and Dosages: Formulation Composition

[0119] [注1] Prepare a 0.1 mol / L succinic acid solution and adjust the pH to 6.5 by adding 0.1 mol / L sodium hydroxide solution.

[0120] [注2] Brief description of the complete prescription preparation process: Take 80% of the prescription amount of buffer salt solution, add the prescription amount of TPPTS, Tricine, Trehalose / Sucrose, and Polysorbate 80, dissolve, add the prescription amount of Compound I solution, add water to make up to the total volume and stir evenly.

[0121] Table 7 Screening of Excipient Types and Dosages (II) Intermediate and Finished Product Properties, Product Marking Rate

[0122] Experimental conclusion:

[0123] (1) The formulation with added sucrose and trehalose has a clear and transparent intermediate solution, which is better than the previous formulation with mannitol and glycine.

[0124] (2) The amount of trehalose used in each vial is 20mg to 100mg, and the labeling rate is above 99%. The excipients do not theoretically affect the labeling rate.

[0125] (3) When the amount of trehalose and sucrose is less than 60mg, the appearance of the product is poor after 0 days of freeze drying.

[0126] (4) After freeze-drying, when the dosage of sucrose and trehalose in the prescription is above 60mg / vial, the product has a complete freeze-dried cake shape and a better appearance.

[0127] (5) After the samples were placed at a high temperature of 60°C for 5 days, only the prescription containing more than 80mg of trehalose was in good condition, while the other prescriptions were in poor condition.

[0128] (6) After the samples were placed at a high temperature of 60°C for 5 days, only the prescription containing 100mg of trehalose was in good condition, while the other prescriptions were in poor condition.

[0129] From Tables 5-8, we can see that:

[0130] Formulas containing sucrose exhibit poor heat resistance and are prone to melting. Formulas containing trehalose, however, show relatively better heat resistance, with trehalose at 100mg / vial yielding the best quality. Therefore, trehalose was chosen as the excipient, at a dosage of 100mg / vial.

[0131] IV. Selection of dosage of core ligand type I compound

[0132] In the formulation, compound I is the key ligand, and its dosage is related to the labeling rate. Therefore, it is necessary to investigate the effect of the dosage of compound I on the product.

[0133] 1. Wide-range screening of compound dosage in Formula I

[0134] Brief description of experimental method: The amounts of TPPTS, Tricine, and succinate in the formulation remained unchanged. Compound of Formula I was added at concentrations of 50 μg / vial, 100 μg / vial, 150 μg / vial, 200 μg / vial, and 250 μg / vial to prepare the samples.

[0135] Table 9. Screening of Compound Dosage in Formula I (I) - Formulation Composition

[0136] [注1] Prepare a 0.1 mol / L succinic acid solution and adjust the pH to 6.5 by adding 0.1 mol / L sodium hydroxide solution.

[0137] [注2] Use Watson's distilled water instead of water for injection.

[0138] Brief description of the complete prescription preparation process: Prepare a 0.1 mol / L succinic acid solution, and adjust the pH to 6.5 with 0.1 mol / L sodium hydroxide solution. Take 80% of the prescription volume of buffer salt solution, add the prescription volume of TPPTS and Tricine, dissolve them, add the solution of compound I (because the amount of compound I is small, it is prepared as a concentrated solution before addition), add water to make up to the total volume and stir well.

[0139] Table 10 Summary of data from the screening of compound dosage in Formula I (Part 1)

[0140] Experimental Results and Discussion:

[0141] (1) The amount of compound I of this product is 50 μg / vial or more, which is sufficient to meet the labeling rate of this product (>90%).

[0142] (2) As the amount of compound I increased to 200 μg / vial, the solution became cloudy and precipitated.

[0143] 2. Small-scale screening using compounds of Formula I

[0144] According to the selection of compound dosage for Formula I (I), 50 μg / vial is sufficient to meet the labeling rate requirement (>90%) for this product. This experiment will involve fine-tuning the dosage above and below 50 μg / vial to examine its impact on the labeling rate. The experimental results are shown in Table 12.

[0145] Table 11. Screening of Compound Dosage in Formula I (II) - Formulation Composition

[0146] [注1] Prepare a 0.1 mol / L succinic acid solution and adjust the pH to 6.5 by adding 0.1 mol / L sodium hydroxide solution.

[0147] [注2] Watson's distilled water was used instead of water for injection in this test.

[0148] In addition, the complete prescription preparation process is briefly described as follows: Take 80% of the prescription amount of buffer salt solution, add the prescription amount of TPPTS, Tricine, and polysorbate 80, dissolve them, add the prescription amount of compound I solution (because the amount of compound I is small, it is prepared as a concentrated solution in the laboratory and then added), add water to make up to the total volume and stir evenly.

[0149] Table 12 Summary of Labeling Rate Data for Compound I Dosage Screening (II)

[0150] Experimental Results and Discussion:

[0151] (1) When the amount of compound I is higher than 30 μg / vial, the labeling rate requirement can be met.

[0152] Summary of experiments (I) and (II) for screening the dosage of compounds of Formula I:

[0153] In this product formulation, a concentration of Formula I compound exceeding 30 μg / vial already meets the labeling requirement of >90%. Considering the extremely small dosage in this product (at the microgram level), further screening is not conducted. Furthermore, Formula I compound is a highly active polypeptide and serves as the core ligand in the kit. Given its extremely low dosage, degradation impurities may occur during prolonged storage. To ensure normal use throughout the shelf life, the concentration of Formula I compound in this product formulation is set at 50 μg / vial.

[0154] V. Selection of TPPTS Dosage as the Core Ligand

[0155] Tris(3-sulfonylphenyl)phosphine sodium salt hydrate (TPPTS) is a key ligand, and its dosage is related to the labeling rate. Therefore, it is necessary to investigate the effect of TPPTS dosage on the product.

[0156] Brief description of experimental methods: This lyophilization did not contain TPPTS. In the subsequent radiolabeling, different amounts of TPPTS were added to the kit, and the kit was treated with sodium pertechnetate solution. The labeling rate was examined using radioactive HPLC.

[0157] The results of TPPTS dosage selection are shown in Table 14 and Figure 1.

[0158] Table 13 Prescriptions without TPPTS kits

[0159] [注1] Prepare a 0.1 mol / L succinic acid solution and adjust the pH to 6.5 by adding 0.1 mol / L sodium hydroxide solution.

[0160] [注2] Watson's distilled water was used instead of water for injection in this preparation.

[0161] Brief description of the complete formulation preparation process: Prepare a 0.1 mol / L succinic acid solution, and adjust the pH to 6.5 with 0.1 mol / L sodium hydroxide solution. Take 80% of the prescribed amount of buffer salt solution, add the prescribed amounts of Tricine and mannitol, dissolve them, add the prescribed amount of compound I solution, add water to make up to the total volume and stir evenly, filter, fill and lyophilize.

[0162] Different amounts of TPPTS were added to the prepared kits, and the kits were treated with sodium pertechnetate solution. The labeling rate was then examined using radioactive HPLC.

[0163] Table 14 TPPTS Dosage Screening

[0164] Experimental Results and Discussion:

[0165] (1) When the amount of compound I and Tricine remains unchanged, when the amount of TPPTS is between 0 mg and 4 mg, the radiolabeling rate of the kit increases with the increase of the amount of TPPTS.

[0166] (2) When the dosage of TPPTS is ≥2mg, the labeling rate reaches 98% or more.

[0167] Because TPPTS exhibits both ligand and reducing agent properties in this system, and considering that the total TPPTS content may decrease due to factors such as oxidation during long-term storage, the dosage of tris(3-sulfonylphenyl)phosphine sodium salt hydrate (TPPTS) is set at 3 mg / vial to ensure normal use throughout the product's shelf life.

[0168] VI. Selection of Tricine Dosage (Core Ligand)

[0169] Tricine is a key ligand in this product's packaging, and its dosage is related to the labeling rate. Therefore, it is necessary to investigate the effect of Tricine dosage on the product's labeling rate.

[0170] Experimental Methods Overview: This lyophilization experiment did not include the Tricine kit. In the subsequent radiolabeling, different amounts of Tricine were added to the kit, and the mixture was treated with sodium pertechnetate solution. The labeling rate was then assessed using radioactive HPLC.

[0171] The experimental results are shown in Table 16.

[0172] Table 15 Prescriptions without Tricine Kit

[0173] [注1] Prepare a 0.1 mol / L succinic acid solution and adjust the pH to 6.5 by adding 0.1 mol / L sodium hydroxide solution.

[0174] [注2] Brief description of the complete prescription preparation process: Prepare a 0.1 mol / L succinic acid solution, and adjust the pH to 6.5 with 0.1 mol / L sodium hydroxide solution. Take 80% of the prescription volume of buffer salt solution, add the prescription volume of TPPTS and mannitol, dissolve them, add the prescription volume of compound I solution, add water to make up to the total volume and stir well, filter and freeze dry.

[0175] Different amounts of Tricine were added to the prepared kits, treated with sodium pertechnetate solution, and the labeling rate was examined using radioactive HPLC.

[0176] Table 16 Tricine Dosage Screening

[0177] Experimental Results and Discussion:

[0178] (1) With the amount of compound I and TPPTS remaining constant, the radiolabeling rate of the kit increases with the increase of the amount of Tricine.

[0179] (2) When the dosage of Tricine is ≥4mg, the labeling rate reaches ≥98%.

[0180] VII. pH Screening

[0181] In the preparation of injectable solutions, pH plays a decisive role in product stability. Changes in its quality, such as hydrolysis, oxidation, discoloration, decomposition, and clarity, are all related to pH. Therefore, it is necessary to study the suitable pH range for the product and investigate the required pH range for this drug solution.

[0182] The experimental results are shown in Tables 19 and 20.

[0183] Brief description of experimental method: The pH range above and below the initial prescription of pH 6.5 was studied to investigate the effect of pH on the sample.

[0184] Table 17 Initial prescription for pH studies

[0185] Table 18-1 pH Screening Experiment Recipe Information

[0186] Table 18 -2 pH Screening Experiment Recipe Information

[0187] [注1] Prepare a 0.1 mol / L succinic acid solution and adjust the pH value to the required level by adding a 0.1 mol / L sodium hydroxide solution.

[0188] [注2] Watson's distilled water was used instead of water for injection in this experiment.

[0189] Brief description of the complete prescription preparation process: Prepare a 0.1 mol / L succinic acid solution, and adjust the pH value to the required value (pH 5.8, pH 6.0, pH 6.2, pH 6.4, pH 6.6, pH 6.8, pH 7.0) by adding 0.1 mol / L sodium hydroxide solution. Take 80% of the prescription volume of buffer salt solution, add the prescription volume of TPPTS, Tricine, and Trehalose, dissolve them, add the prescription volume of polysorbate 80, add the prescription volume of compound I solution, retest the pH of the solution and adjust it to the required value, add water to make up to the total volume and stir well.

[0190] Table 19 Relevant indicators after preparation of drug solutions at different pH levels

[0191] [注1] When this study was conducted, the formulation-related substances method was under development, and the raw material purity method was used for detection.

[0192] Table 20 Data on clarity, pH, and labeling rate of drug solutions after freeze-drying at different pH values.

[0193] Experimental conclusion:

[0194] (1) After the medicine solution was prepared, there was no significant change in pH before and after filtration.

[0195] (2) After the drug solution was prepared, the clarity of the samples at pH 5.8 and pH 6.0 was slightly poor, while the clarity of the samples at pH 6.2 to pH 7.0 was good.

[0196] (3) There were no significant differences in the relevant substances in the drug solutions with different pH values.

[0197] (4) All pH solutions were clear and transparent after lyophilization. The pH after reconstitution was not significantly different from that before lyophilization. The labeling rate of samples with pH 5.8 to pH 7.0 was over 99%.

[0198] In summary, the pH range is 6.2 to 7.0.

[0199] Based on previous preparation processes, it is known that the ratio of 0.1 mol / L succinic acid to 0.1 mol / L sodium hydroxide solution used to prepare a pH 6.5 buffer solution is approximately 1:2. Therefore, the dosage of succinic acid and sodium hydroxide for a single dose is as follows:

[0200] The required amount of succinic acid per 1 ml is 1 ml * 80% * (1 / 3) * (1180 mg / 100 ml) = 3.1467 mg.

[0201] The amount of sodium hydroxide needed to fill 1 ml of water is 1 ml * 80% * (2 / 3) * (400 mg / 100 ml) = 2.1333 mg.

[0202] VIII. Prescription Confirmation

[0203] The updated prescription is:

[0204] Table 21 Updated Formulation of Arktide for Injection

[0205] Prescriptions selected through prescription screening were confirmed, and three small-scale trials were conducted (batch numbers: ARK-1, ARK-2, ARK-3).

[0206] Based on the established formula and process, three batches of samples were scaled up. The indicators of the three small-scale batches and the three pilot-scale batches were compared and studied. The results showed that the quality of the small-scale and pilot-scale products was basically the same.

[0207] Example 2. Stability Study

[0208] This experiment investigated the stability of the formulation under the conditions of 2–8℃, 25℃±2℃ / 60%±5%RH, and 40℃±2℃ / 75%±5%RH. The results are shown in Tables 22-24.

[0209] Example 3 and 99m Tc combination and testing

[0210] Experimental instruments

[0211] 3.1 Preparation of coordination compounds

[0212] Take 1 mL of sodium pertechnetate (Na) using a syringe. 99m Inject the TcO4 diluent into the drug solution of the prescription in Table 18. Shake for half a minute to completely dissolve the drug, heat upright in a boiling water bath for 20 minutes, remove and let cool naturally to obtain the final product.

[0213] Structural characterization:

[0214] Retention time: 21.574 min, peak area: 39860.8, labeling rate: 99.9%.

[0215] ESI+ve(m / z): 1260.88 [M+3H] 3+ .

[0216] 3.2 Trait Detection

[0217] The product prepared in 3.1 was extracted and placed in a 10 mL colorless glass bottle. The properties of the solution were observed, and no turbidity was observed when compared with physiological saline.

[0218] 3.3 pH

[0219] The pH of the product was tested using calibrated wide-range pH test paper (pH=1-14) and precision test paper (pH=5.5-9.0), and the result showed that the pH was 6.0.

[0220] 3.4 Specificity Detection

[0221] Take two TLC strips and spot the product prepared in 3.1 at the origin (2 μL). Develop the sample in phase A (Citrate, 0.1 M, pH = 5.0; preparation method: 2.88 g citric acid and 10.3 g sodium citrate dissolved in 500 mL pure water) and phase B (30% CAN-citrate, 0.1 M, pH = 5.0; preparation method: 90 mL CAN, added to 210 mL citrate buffer), respectively. After drying, perform radioactive TLC detection. The detection results are shown in Figure 3, where the main peak in phase A has an Rf value of 0–0.1, and the main peak in phase B has an Rf value of 0.9–1.

[0222] 3.5 Radiochemical purity

[0223] Following the method in 3.4, the TLC strips developed in phases A and B are divided into two equal segments (0-5cm and 5-10cm) from the middle section, and the radiochemical purity of the complex is calculated based on the radioactivity count.

[0224] Calculation formula;

[0225] Radiochemical purity = 100% - colloidal technetium content (%) - free technetium content (%)

[0226] Free technetium content = Count at the front of development system A / (Count at the origin of development system A + Count at the front of development system A)

[0227] Colloidal technetium content = Count at the origin of developed system B / (Count at the origin of developed system B + Count at the front of developed system B)

[0228] The test results are as follows:

[0229] Radiochemical purity >90%.

[0230] 3.6 Half-life

[0231] Take a TLC strip and spot the product prepared in 3.1 at the origin (2 μL), without developing. Repeat for three samples. Using a gamma counter, measure the radioactivity every 0.5–2 hours, recording the measurement time and radioactivity count, for a total of five times.

[0232] Gamma counter period detection results:

[0233] Half-life calculation results:

[0234] Half-life (h) = LN(2) / 0.0019 / 60

[0235] Half-life = 6.08h.

[0236] 3.7 Room temperature stability

[0237] Take 500 μL of the product prepared in 3.1 and place it at room temperature. At different time points, take samples (2 μL) onto paper strips and develop them in developing system A and developing system B, respectively. After development, the middle section is divided into two equal segments, and gamma counts are performed on each segment to calculate the radiochemical purity and room temperature stability of the complex.

[0238] Sampling time point:

[0239] 0h:

[0240] 3h:

[0241] 6h:

[0242] 9h:

[0243] 3.8 Accelerated stability (40℃)

[0244] Take 500 μL of the product prepared in 3.1 and place it in an air bath heater at 40 °C. At different time points, take samples (2 μL) onto paper strips and develop them in developing system A and developing system B, respectively. After development, the middle section is divided into two equal segments, and gamma counts are performed on each segment to calculate the radiochemical purity and room temperature stability of the complex.

[0245] Sampling time point

[0246] 0h:

[0247] 3h:

[0248] 6h:

[0249] 9h:

[0250] Example 4: Effect of Labeling Rate on Experiment

[0251] 4.1 99m Tc activity

[0252] Using a 1mL sterile syringe, add 1mL of sodium pertechnetate dilution solution with different activities to each kit. Mix well until the sample dissolves, then heat upright in a boiling water bath for 20 minutes. After labeling, remove the kit and allow it to cool naturally to room temperature. Inject the labeled sample from the kit and perform gradient elution using HPLC. Record the radioactivity HPLC chromatogram. The labeling rate results are as follows:

[0253] Tests showed that different activities of sodium pertechnetate injection solution affect the labeling rate of the tosylate nicotinamide hydrazone double-chain Ark peptide kit for injection.

[0254] The recommended activity of sodium pertechnetate injection for labeling nicotinamide hydrazone double-chain Ark peptide with tosylate for injection is 20-50 mCi (6:00 a.m.).

[0255] 4.2 99m Tc volume

[0256] Using a 1 mL sterile syringe, add 0.5 or 1.0 mL of sodium pertechnetate injection solution to each kit, along with different volumes of physiological saline. Mix well until the sample dissolves, then heat upright in a boiling water bath for 20 minutes. After labeling the kit, remove it and allow it to cool naturally to room temperature. Inject the labeled sample from the kit and perform gradient elution using HPLC. Record the radioactivity HPLC chromatogram. The labeling rate results are as follows:

[0257] Tests showed that different volumes of sodium pertechnetate injection solution affected the labeling rate of the Ark peptide tosylate nicotinamide hydrazone double-chain kit for injection. The recommended volume of sodium pertechnetate injection solution for labeling Ark peptide tosylate nicotinamide hydrazone double-chain kit for injection is 0.5–1.5 mL.

[0258] 4.2 Marking temperature and time

[0259] 1 mL of sodium pertechnetate diluent was drawn into each kit using a 1 mL sterile syringe and added to the kit. After mixing and dissolving the sample, the kits were labeled at different temperatures and times. After labeling, the kits were removed and allowed to cool naturally to room temperature. Gradient elution was performed according to the HPLC method, and the radioactive HPLC chromatogram was recorded. The labeling rate results are shown in the table below and Figure 4.

[0260] The results showed that different labeling temperatures and times affected the labeling of compound I. A labeling rate of 90-100℃ for 20-30 minutes was observed.

[0261] Example 5 In vivo biological evaluation

[0262] Imaging experiment: Using a syringe, take 0.5 mL (20 mCi) and 1.25 mL (50 mCi) of diluted technetium respectively. 99m [Tc] sodium nicotinamide hydrazone injection solution was injected into two injection kits containing nicotinamide hydrazone double-chain Ark peptide, and the mixture was shaken thoroughly to dissolve the lyophilized material. Under sealed conditions, the kits were heated upright in a boiling water bath for 20–30 minutes, then removed and cooled to room temperature. Five female SKBR3 human breast cancer cell-bearing mice were selected. The mice were injected with the solution. 99m Tc labeled with different activities (50 mCi and 20 mCi) 99mTc-HP-Ark2 was used to investigate drug distribution at 0.5h, 1h, 2h, and 4h post-administration imaging, and a closed group was used to investigate drug targeting at 0.5h post-administration imaging. Each mouse was anesthetized with 2% isoflurane-oxygen at the corresponding imaging time points after administration. NanoScan SPECT / CT was used to perform SPECT and CT imaging on the mice. SPECT scan parameters: four-probe parallel-aperture collimator, peak energy 140.5keV, 30s / frame, total acquisition time 27.5min; CT scan parameters: voltage 50kVp, energy 0.67mA, rotation 210°, exposure time 300ms. After image acquisition, the SPECT and CT images were reconstructed, fused, and processed using the instrument's Fusion software to obtain whole-body MIP imaging images (displayed using a posterior view).

[0263] Biodistribution experiment: Take 0.5 mL (20 mCi) of technetium [ 99m Sodium tcoate injection solution was diluted with 0.5 mL of physiological saline and injected into the injection kit for nicotinamide hydrazone double-chain Ark peptide. The kit was shaken thoroughly to dissolve the lyophilized product. Under sealed conditions, the kit was heated upright in a boiling water bath for 20–30 min, then removed and cooled to room temperature. Twenty-five female SKBR3 human breast cancer cell-bearing mice were randomly divided into five groups of five each. Four groups received the injection via tail vein. 99m Tc-HP-Ark2 injection was administered, and tissue samples were taken at 0.5h, 1h, 2h, and 4h after administration. A blocking group was also included (the blocking dose of HP-Ark2 was injected first, followed by the Tc-HP-Ark2 injection). 99m Tc-HP-Ark2 injection was administered, and tissue samples were collected 0.5 hours later. Animals were lightly anesthetized, blood was drawn, and tissue samples were collected from the heart, liver, spleen, kidneys, lungs, stomach (emptied contents), intestines (emptied contents), femur, muscles, brain, tumors, bladder, uterus, and ovaries. The weight of the organs was weighed and recorded, and gamma counters were used to calculate the %ID / g of the samples.

[0264] Experimental results:

[0265] Imaging experiment results: 99m Following injection of Tc-HP-Ark2 (50mCi labeled injection solution), radioactivity primarily accumulated in the tumor, kidney, and bladder. Over time, the radioactivity accumulation in the tumor and liver decreased, but significant accumulation was still observed at 4 hours. The large accumulation in the kidney and bladder indicated… 99m Tc-HP-Ark2 is mainly metabolized by the kidneys and excreted in urine, as shown in Figure 5.

[0266] Following a closed-dose injection of HP-Ark2, then administer... 99mTc-HP-Ark2 injection showed no radioactive enrichment at the tumor site (see Figure 6); this demonstrates... 99m The enrichment of Tc-HP-Ark2 in tumors can be specifically blocked by a large number of unlabeled HP-Ark2 molecules, and its tumor uptake is specifically mediated by HP-Ark2 target molecules and receptors.

[0267] 99m Following injection of Tc-HP-Ark2 (20mCi labeled injection), radioactivity primarily accumulated in the tumor, kidney, and bladder. Over time, the radioactivity accumulation in the tumor decreased, but significant accumulation was still observed at 4 hours. Large accumulations in the kidney and bladder indicated… 99m Tc-HP-Ark2 is primarily metabolized by the kidneys and excreted in the urine, as shown in Figure 7. 99m There was no significant difference in imaging results between 20mCi labeled injection and 50mCi labeled injection in Tc-HP-Ark2 injection.

[0268] Results of tissue distribution experiment: 99m Over time, the radioactive uptake of Tc-HP-Ark2 injection in the blood significantly decreased. Within 0.5–4 hours after administration, varying degrees of radioactive enrichment were observed in tissues and organs. The kidneys, being the primary drug-metabolizing organ, showed the highest uptake, followed by tumors. The liver, lungs, bladder, and uterus also exhibited relatively high distribution. Other organs showed relatively low distribution. The brain showed the lowest uptake. The biodistribution results were consistent with the imaging results (see Figure 8). A closed dose of HP-Ark2 was administered followed by... 99m Tc-HP-Ark2 injection significantly reduced radioactive enrichment at the tumor site, and biodistribution experiments also proved that its tumor uptake was specifically mediated by HP-Ark2 target molecules and receptors (see Figure 9 for details).

[0269] The results showed that, within the range of 20 mCi to 50 mCi, different activities did not affect the labeling of Formula I compounds, nor their in vivo efficacy and pharmacokinetic properties.

[0270] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A composition comprising a compound of Formula I, one or more ligands of radionuclides, a surfactant, a matrix proppant, and a pH adjuster: ###0001### Formula I 2. The composition of claim 1, said radionuclide is 99m Tc; Preferably, the ligand of the radionuclide is selected from hydroxyl-containing ligands and phosphine ligands, such as TPPTS, Tricine or a combination thereof.

3. The composition of claim 1 or 2, the surfactant is selected from the group consisting of polysorbate 80, poloxamer, TWEEN® 20 (polyoxyethylene (23) lauryl ether), TRITON® X-100, and sodium dodecyl sulfate.

4. The composition of any one of claims 1-3, wherein the matrix support agent is selected from trehalose, sucrose, maltose, lactose, glycine and mannitol.

5. The composition of any one of claims 1-4, wherein the pH adjusting agent is selected from succinate buffer system, HEPES buffer system.

6. The composition of any one of claims 1-5, comprising the compound of Formula I, TPPTS, Tricine, trehalose, polysorbate 80 and succinate buffer system.

7. The composition of any one of claims 1-6, comprising 2-30 parts by weight of the compound of Formula I, 50-500 parts by weight of TPPTS, 100-1000 parts by weight of Tricine, 1000-15000 parts by weight of matrix support agent, 1-100 parts by weight of surfactant and pH adjusting agent, wherein the amount of the pH adjusting agent is such that the pH of the composition is to 5.6-7.0; Preferably, the composition contains 3-15 parts by weight of the compound of formula I, 100-500 parts by weight of TPPTS, 250-650 parts by weight of Tricine, 2000-10000 parts by weight of trehalose, 1-100 parts by weight of polysorbate 80 and a succinate buffer system, wherein, the amount of the succinate buffer system is such that the pH of the composition is to 6.20-7.0; Preferably, the composition comprises 1 part by weight of the compound of Formula I, 60 parts by weight of TPPTS, 80 parts by weight of Tricine, 2000 parts by weight of trehalose, 10 parts by weight of polysorbate 80 and succinate buffer system, wherein the amount of the succinate buffer system is such that the pH of the composition is to 6.20-7.

0.

8. The composition of any one of claims 1-7, which is a solid formulation; Preferably, it is a lyophilized formulation, preferably a lyophilized powder for injection, such as a sterile powder for injection.

9. A liquid formulation or reconstituted liquid formulation, which is obtained by dissolving the composition of any one of claims 1-8 in a solution containing a radionuclide; Preferably, the radionuclide is 99m Tc; Preferably, the solution containing the radionuclide is technetium […]. 99m [Tc] sodium acid solution; more preferably, the activity of the radionuclide in the liquid formulation or reconstituted liquid formulation is 20-50 mCi; more preferably, the unit dose of the liquid formulation or reconstituted liquid formulation is 0.5-1.5 mL; Preferably, the compound of Formula I and the ligand of the radionuclide form a metal complex with the radionuclide; Preferably, the liquid formulation or reconstituted liquid formulation is an injection; Preferably, the liquid formulation or reconstituted liquid formulation is used for SPECT imaging.

10. A method for preparing the composition of any one of claims 1-8, comprising the following steps: adding the one or more ligands of radionuclide and the matrix support agent to an aqueous solution of the pH adjusting agent, adding the surfactant and the compound of Formula I, optionally adjusting the pH, mixing, and the composition is obtained.

11. The method of claim 10, wherein one or more of the following: (1) each step is carried out under inert gas protection; (2) each step is carried out under light protection; (3) each step is carried out using an aqueous solution of the ligand of radionuclide, an aqueous solution of the matrix support agent, an aqueous solution of the surfactant and / or an aqueous solution of the compound of Formula I; (4) each step is carried out at 2°C to room temperature (e.g. 25°C), such as 2°C to 8°C; (5) after the mixing step, further comprising a filtration step, preferably using a filter membrane of polyvinylidene fluoride (PVDF), nylon, polyethersulfone (PES) or cellulose acetate (CA), preferably a 0.2-0.3 pm PVDF filter membrane.

12. The method of claim 10 or 11, wherein the composition comprises, by weight percentage, 0.002%-0.03% of the compound of Formula I, 0.05%-0.5% of TPPTS, 0.1%-1% of Tricine, 1%-15% of a skeletal support agent, 0.001%-0.1% of a surfactant, a pH adjusting agent, and the balance of water, wherein the pH adjusting agent is used in an amount such that the pH of the composition is 5.6-7.

0. Preferably, the composition contains 0.003% to 0.015% of the compound of formula I, 0.1% to 0.5% of TPPTS, 0.25% to 0.65% of Tricine, 2% to 10% of trehalose, 0.001% to 0.1% of polysorbate 80, a succinate buffer system and the balance water, wherein, the succinate buffer is used in an amount such that the pH of the composition is 6.20-7.

0. Preferably, the composition comprises 0.005% of the compound of Formula I, 0.3% of TPPTS, 0.4% of Tricine, 10% of trehalose, 0.05% of polysorbate 80, a succinate buffer, and the balance of water, wherein the succinate buffer is used in an amount such that the pH of the composition is 6.20-7.

0.

13. The method of any one of claims 10-12, further comprising a drying step, such as freeze-drying, preferably two-stage freeze-drying: first stage: -40±2°C, 30±5 Pa, drying for 2 h; second stage: 25±2°C, 30±5 Pa, drying for 1 h.

14. A kit product comprising the composition of any one of claims 1-8, and optionally a radiopharmaceutical (e.g., an imaging radiopharmaceutical) and / or an instruction; Preferably, the radiopharmaceutical is technetium […]. 99m Sodium Tcate Injection.

15. Use of the composition of any one of claims 1-8, the liquid preparation of claim 9, the reconstituted liquid preparation, or the kit product of claim 14 in the preparation of a medicament or an agent, wherein the medicament or the agent is used for the diagnosis of a tumor (e.g., a HER2-positive tumor), such as localization diagnosis, lesion detection, patient selection for HER2-targeted therapy of a tumor, or evaluation of the therapeutic effect of a tumor treatment.

16. A method of diagnosing a tumor (e.g., a HER2-positive tumor) comprising the step of administering to a subject an effective amount of the composition of any one of claims 1-8, the liquid formulation of claim 9, or a reconstituted liquid formulation, or the kit product of claim 14, wherein, The tumor diagnosis comprises tumor localization, lesion detection, patient selection for HER2-targeted therapy of a tumor, and evaluation of the therapeutic effect of a tumor treatment.

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