A drug for treating T-cell acute lymphoblastic leukemia and its application

Abstract

This invention relates to a drug for the prevention and / or treatment of T-cell acute lymphoblastic leukemia and its application. The active ingredient in the drug is anserin P-3, with the molecular formula C32H43ClN2O9. Anserin P-3 specifically targets the oncogene C-MYC, demonstrating significant efficacy against T-cell acute lymphoblastic leukemia and showing promising application prospects in the clinical treatment of this disease.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a drug for treating T-cell acute lymphoblastic leukemia. Background Technology

[0002] Anserin P-3 (AP-3, CAS: 66584-72-3) is a natural product extracted from actinomycetes and is an effective inhibitor of tubulin polymerization. Studies have shown that AP-3 has certain anti-tumor activity, inducing cell cycle arrest in tumor cells during the G2 / M phase. This process is mainly achieved by activating cell cycle checkpoint proteins and apoptosis-related proteins. Furthermore, AP-3 can also induce tumor cell apoptosis by activating both intrinsic and extrinsic apoptosis pathways. AP-3 can upregulate Bax protein expression and decrease Bcl-2 protein expression, thereby promoting the loss of mitochondrial membrane potential, releasing cytochrome c, activating the caspase cascade, and ultimately leading to apoptosis. These results indicate that AP-3 induces tumor cell apoptosis through multiple pathways, thereby effectively inhibiting tumor growth and spread. However, the anti-tumor spectrum of AP-3 is not clear, its effects on hematological malignancies are limited, and its anti-tumor targets are also unclear.

[0003] T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive, heterogeneous, and rapidly progressing hematologic malignancy, accounting for approximately 15%–30% of all acute lymphoblastic leukemia (ALL). Newly diagnosed adult T-ALL patients have a poor prognosis, with a 5-year overall survival rate of only 30%–40%. The prognosis is even worse for relapsed / refractory T-ALL patients, with a 3-year survival rate of only 10%–15%, and even transplantation does not significantly improve the prognosis. Currently, the standard treatment for T-ALL relies primarily on high-intensity chemotherapy. However, this treatment method faces numerous challenges, including chemotherapy resistance, high relapse rates, and significant treatment-related toxicities, all of which are key reasons for treatment failure. In stark contrast, B-cell acute lymphoblastic leukemia (B-ALL) and acute myeloid leukemia (AML) have entered a new phase of targeted therapy, achieving promising clinical results through targeted drugs, antibody drugs, or CAR-T cell therapy. Clearly, T-ALL is lagging behind and urgently needs in-depth exploration of new targets and drugs to develop more efficient and less toxic treatment strategies. This would improve the depth and effectiveness of initial treatment for T-ALL, reduce drug resistance and relapse, and is crucial for improving patient prognosis and overcoming current treatment bottlenecks.

[0004] C-MYC is the driver gene of T-ALL and a key molecule for T-ALL drug resistance and relapse. Discovering inhibitors targeting C-MYC is one of the current focuses of T-ALL new drug development, but so far no direct C-MYC inhibitor has been translated into clinical practice.

[0005] Therefore, it is necessary to find an effective targeted drug for T-ALL to meet clinical needs. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned technical problems. In a first aspect, this invention provides the use of anserin P-3 (AP-3) in the preparation of a medicament for the prevention and / or treatment of T-cell acute lymphoblastic leukemia.

[0007] This invention demonstrates through research that ansomniacin P-3 (AP-3) prevents and / or treats T-ALL by acting on the C-MYC target.

[0008] In some embodiments, the molecular formula of anserin P-3 (AP-3) is C32H43ClN2O9, and its structural formula is shown below:

[0009]

[0010] In some embodiments, the T-cell acute lymphoblastic leukemia is a T-cell acute lymphoblastic cell that highly expresses C-MYC.

[0011] In some embodiments, the anserin P-3 achieves prevention and / or treatment of T-cell acute lymphoblastic leukemia by inhibiting the expression of C-MYC.

[0012] In some embodiments, anserin P-3 (AP-3) is the sole active ingredient in the application.

[0013] A second aspect of the present invention provides the use of a pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of T-cell acute lymphoblastic leukemia, said pharmaceutical composition comprising anserine P-3 (AP-3) and pharmaceutically acceptable excipients.

[0014] In some embodiments, the pharmaceutical composition is prepared into various pharmaceutically acceptable dosage forms, such as injections, tablets, capsules, granules, solutions, etc.

[0015] In some embodiments, anserin P-3 (AP-3) is the sole active ingredient in the composition.

[0016] In some embodiments, the composition further includes other therapeutic agents, said therapeutic agents being drugs for the prevention and / or treatment of T-cell acute lymphoblastic leukemia.

[0017] A third aspect of the invention provides the use of anserin P-3 (AP-3) in the preparation of medicaments for the prevention and / or treatment of diseases with high expression of C-MYC.

[0018] In some embodiments, the disease with high C-MYC expression is T-cell acute lymphoblastic leukemia.

[0019] In a fourth aspect, the invention provides the use of anserin P-3 (AP-3) in inhibiting the activity of T-cell acute lymphoblastic leukemia cell lines.

[0020] In some embodiments, T-cell acute lymphoblastic leukemia cell lines include one or more of Jurkat, ZYXY-T1, Molt-3, Molt-4, and Loucy.

[0021] The beneficial effects of this invention are:

[0022] This invention utilizes a natural product library containing 2541 natural compounds for high-throughput drug screening and drug sensitivity experiments, discovering that AP-3 is highly sensitive to T-ALL cells and is a highly effective anti-T-ALL natural compound. Furthermore, transcriptome sequencing following AP-3 treatment, combined with computed molecular docking, protein imprinting, and surface plasmon resonance, clarified the direct target of AP-3 in treating T-ALL.

[0023] This invention is the first to discover that AP-3 can treat T-cell acute lymphoblastic leukemia, and at the same time, it clarifies that its target is C-MYC, providing a new strategy for the clinical treatment of T-cell acute lymphoblastic leukemia. Attached Figure Description

[0024] Figure 1 Screening of natural compounds against T-ALL. A shows the screening process for natural compounds against T-ALL, and B is a heatmap of the inhibitory effects of 76 effective natural compounds obtained from the initial screening at a concentration of 1 μM on T-ALL cells at an action concentration of 10 nM.

[0025] Figure 2The cytotoxic effects of anserine P-3 (AP-3) on different T-ALL cell lines and normal blood cells. A shows the cytotoxic effects of 13 selected compounds on T-ALL cells; B shows the cytotoxic effects of different concentrations of AP-3 on AML cell lines (HL60, OCI2-AML, OCI3-AML, and U937) and T-ALL cell lines (Jurkat and ZYXY-T1); C shows the cytotoxic effects of different concentrations of AP-3 on T-ALL cell lines Jurkat, Molt-3, Molt-4, ZYXY-T1, Loucy, and T-ALL cells from a primary T-ALL patient; D shows the cytotoxic effects of different concentrations of AP-3 on Jurkat and normal blood cells.

[0026] Figure 3 The growth inhibitory effects of anthracycline P-3 on different T-ALL cell types. A shows the growth inhibitory effects of different concentrations of AP-3 on Jurkat cells. B shows the growth inhibitory effects of different concentrations of AP-3 on ZYXY-T1 cells. C shows the growth inhibitory effects of different concentrations of AP-3 on Molt-3 cells.

[0027] Figure 4 The therapeutic effect of anserine P-3 on T-ALL model mice. Figure A is the experimental flowchart. Figure B shows the in vivo tumor burden at different time points in T-ALL model mice that received or did not receive AP-3 treatment. Figure C shows the survival curves of T-ALL model mice that received or did not receive AP-3 treatment.

[0028] Figure 5 Ansifen P-3 directly targets C-MYC in T-ALL. A shows the flowchart of RNA-seq sequencing and analysis of T-ALL cells before and after AP-3 treatment. B shows the differential pathway enrichment map of AP-3-treated T-ALL cells analyzed by Gene Enrichment Analysis (GSEA). C shows the effect of AP-3 treatment on the expression level of the oncogene C-MYC. D shows the binding ability of AP-3 to C-MYC analyzed by computer-aided molecular docking. E shows the direct binding ability of AP-3 to C-MYC analyzed by surface plasmon resonance (SPR). F shows the image of the effect of AP-3 treatment on the C-MYC protein expression level in ZYXY-T1 cells. G shows the image of the effect of AP-3 treatment on the C-MYC protein expression level in Molt-3 cells. Detailed Implementation

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; unless otherwise specified, the experimental materials used in the following embodiments are all purchased from commercial channels.

[0031] Related reagents, animals, and instruments:

[0032] NCG mice: purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., 5 weeks old.

[0033] Ansifen P-3: Purchased from Selleck, product number: S2447

[0034] Cell lines: ZYXY-T1 was kindly donated by Professor Jin Jie of the First Affiliated Hospital of Zhejiang University; HL60, OCI-AML2, OCI-AML3, U937, Jurkat, and Loucy cell lines were purchased from the American ATCC cell bank; molt-3 and molt-4 cell lines were purchased from Baidi Biotechnology; and primary T-ALL patient T-ALL cells were isolated from the peripheral blood of hospitalized T-ALL patients diagnosed at the People's Hospital Affiliated to Ningbo University.

[0035] Human peripheral blood lymphocyte separation medium (Solepro, Cat. No. P8610)

[0036] D-luciferin potassium salt (PerkinElmer, 122799)

[0037] Liposome Transfection Kit (GeneCopoeia, CAT.#EF001)

[0038] Small animal live imaging system (Caliper Life Sciences, IVIS Spectrum)

[0039] Isolation of normal blood cells:

[0040] Collect 5 ml of peripheral blood from healthy individuals and T-ALL patients with informed consent. Add 5 ml of human peripheral blood lymphocyte separation medium to a 15 ml sterile centrifuge tube. Use a pipette to add the healthy individual's peripheral blood to the centrifuge tube and centrifuge at 2000 rpm for 20 minutes. After centrifugation, collect the white membrane layer (the layer of normal blood cells) at the interface between the upper and lower plasma layers of the lymphocyte separation medium. Transfer the white membrane layer to a new 15 ml sterile centrifuge tube. Wash the normal peripheral blood cells twice with sterile 1×PBS and resuspend them in IMDM containing 10% FBS.

[0041] Preparation of Jurkat Luc cells

[0042] 293T cells at 1×10 6 Plating was performed on 10cm plates at a concentration of / ml, and virus packaging was carried out after 24 hours. The viral packaging plasmids VSVG, PMD, and plasmids containing the luciferase gene and blastcin gene were packaged at a mass ratio of 2:1:3, with a total amount of 10μg. Plasmids were added to a 1.5ml sterile EP tube, and the volume was increased to 200μl with sterile double-distilled water. Then, 200μl of liposome transfection reagent was added, and the mixture was thoroughly mixed and incubated at room temperature for 25min. 293T cells were then added, mixed, and incubated at 37℃ for 12 hours. The transfected 293T cells were then replaced with fresh DMEM medium. After 48 hours, the culture supernatant (viral supernatant) of the transfected 293T cells was collected, filtered through a 0.45μm filter, and stored at 4℃. The Jurkat cell line was prepared into 1×10⁶ cells / year. 6 For each ml of cell culture, mix 1 ml of cell culture, 3 ml of virus supernatant, and 2 ml of complete culture medium, then add the mixture to a culture flask for incubation. After 24 hours, transfer the cells to a new 15 ml sterile centrifuge tube, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, resuspend the cells in fresh complete culture medium, and continue incubation. After 48 hours, replace the culture medium with complete culture medium containing blast fungicide (5 μg / ml), and screen the cells until they grow normally in the medium containing blast fungicide. Then, take 200 μl of cultured cells + 20 μl of 15 mg / ml luciferase and verify the cells using a microplate reader with an autoluminescent program. After verification, expand the culture and freeze the cells.

[0043] auto-dock molecular docking

[0044] Download the 3D molecular structure file of AP-3 (compound CID: 5282049) from the Pubchem website (https: / / pubchem.ncbi.nlm.nih.gov / ). Download the crystal structure of C-MYC from the PDB website (https: / / www.rcsb.org / ), selecting the 6G6K crystal structure of C-MYC. Further molecular docking was performed using Auto Dock4 software; specific docking steps were performed according to the Auto Dock website (https: / / autodock.scripps.edu / ). Finally, PyMol software was used to display the docking results and output images.

[0045] Surface plasmon resonance (SPR) analysis of the direct binding ability of AP-3 and C-MYC

[0046] Coupling of target protein C-MYC: Using a CM5 chip, the sensor surface was activated by injection of a mixture of 50 mM N-hydroxysuccinimide (NHS) and 200 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) for 7 minutes. The target protein C-MYC was then diluted to 30 μg / mL with 10 mM acetate (pH 4.0) and immobilized on the CM5 chip surface at a flow rate of 10 μL / min for 1260 s. Finally, the surface was blocked with 1 M ethanolamine (pH 8.5).

[0047] Test conditions for the analyte: The binding characteristics of the target protein C-MYC to the small molecule compound AP-3 were initially determined and evaluated using manual mode. 50 μM was determined as the maximum analytical concentration of AP-3. Nine analytical concentrations were set using a 2-fold serial dilution: 0 μM, 0.39 μM, 0.78 μM, 1.56 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, and 50 μM. The flow rate was set at 30 μL / min, the binding time at 300 s, and the dissociation time at 300 s.

[0048] Solvent correction preparation: Inject a series of DMSO blank samples with varying concentrations. Plot the relative signal values ​​of the reference ligand channels (minus the reference channel signal values) against the reference channel signal values ​​to construct a correction curve, called the solvent correction curve. The solvent correction curve is used to calculate the bulk solvent effect on the final result under different relative response values ​​on the reference channel. In this way, the magnitude of the bulk solvent effect can be calculated for each sample using its relative response value to the reference channel, and the final result (minus the reference ligand channel response value) can be corrected accordingly.

[0049] Kinetic parameter determination: The experiment adopted a multi-cycle operation, with the analysis time as the abscissa and the response value as the ordinate. The obtained data were fitted using BIAcore S200 analysis software, and the fitting model used was the SteadyState Affinity binding model to determine the binding rate constant, dissociation rate constant, and binding-dissociation constant, among other kinetic constants.

[0050] Example 1: Screening of natural compounds against T-ALL

[0051] To screen for effective natural compounds against T-ALL, compounds against T-ALL were screened using the Selleck L1400 natural product library (L1400-Z716216-50 μL). The screening procedure is as follows: Figure 1 As shown in Figure A, specifically, T-ALL cell line ZYXY-T1 cells were divided into two groups of 2 × 10⁻⁶ cells. 6 The drug was seeded at a concentration of 90 μl / well in 96-well plates. Compounds from the L1400 library of 2541 natural products were diluted to 1 mM, and 10 μl was added to each well of the 96-well plate containing cell culture medium. The mixture was further stirred using a horizontal shaker to achieve a final drug concentration of 1 μM. Each 96-well plate included a positive control well (no drug, only cells) and a negative control well (no cells). The 96-well plates were incubated at 37°C and humidified for 48 hours. 20 μl of MTS was added to each well, and the plates were stained for another 4 hours. The OD value was then measured at 490 nm using a microplate reader. Cell viability in each well was determined using the formula (OD value of drug-treated well - absorbance of negative control) / (absorbance of positive control - absorbance of negative control). After screening with 1 μM, 76 compounds were found to have significant cytotoxic activity against T-ALL cells (activity ≤ 50%). Further, using the same method, the 76 compounds were diluted 100-fold to achieve a final concentration of 10 nM in the target cells for further screening. The results are as follows: Figure 1 As shown in B, a total of 13 compounds exhibited significant killing effects on T-ALL cells (activity ≤60%).

[0052] Example 2: Killing effect of anthracycline P-3 on different leukemia cell lines

[0053] To identify the compound with the strongest killing effect against T-ALL, T-ALL drug sensitivity experiments were conducted using different concentration gradients of the 13 compounds screened in Example 1. The concentration gradients for AP-3 were set as follows: 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 10 nM; and the concentration gradients for the other 12 compounds were set as follows: 1, 2.5, 5, 10, 25, 50 nM. The plating density of T-ALL cells was set to 2 x 10⁻⁶ cells / cm². 6 / ml, set up positive control wells with only cells and negative control wells with neither drug nor cells. Plated in 24-well plates, after 48 hours, mix the liquid in the 24-well plates with a pipette, transfer 100μl to each well of a 96-well plate, add 20μl of MTS, and after 4 hours measure the absorbance using a microplate reader. Calculate the IC50 of the 13 compounds against the T-ALL cell line ZYXY-T1 using Graphpad software. 50 The result is as follows Figure 2 As shown in Figure A: All 13 compounds exhibited cytotoxic effects against T-ALL cells, with AP-3 showing the strongest cytotoxic effect against T-ALL cells, and IC50... 50 It is 0.197 nM.

[0054] AP-3 drug sensitivity experiments were further conducted using four AML cell lines (HL60, OCI-AML2, OCI-AML3, and U937) and two T-ALL cell lines (Jurkat and ZYXY-T1). The results are as follows: Figure 2 As shown in B, AP-3 has cytotoxic effects on both AML and T-ALL cells, and on Jurkat cells with IC50. 50 The most sensitive value was 0.074 nM. Further drug sensitivity assays were used to analyze the IC50 of AP-3 against five different T-ALL cell lines (Jurkat, molt-3, molt-4, and loucy) and cells from one primary T-ALL patient. 50 The result is as follows Figure 2 As shown in C, AP-3 has an IC50 effect on T-ALL cells. 50 The range is between 0.069 and 1.958 nM.

[0055] To explore the medicinal value of AP-3, drug sensitivity experiments were subsequently used to analyze the cytotoxic effect of AP-3 on normal blood cells. The results are as follows: Figure 2 As shown in D. For normal blood cells, AP-3 has only a weak inhibitory effect even at a concentration of 1000 nM, IC50... 50 Unable to calculate the relative IC50 of AP-3 against T-ALL cells 50 It has exceeded 16,000 times.

[0056] The above results indicate that AP-3 has a significant inhibitory and killing effect on T-ALL cells, but no killing effect on normal blood cells, thus exhibiting a superior therapeutic window.

[0057] Example 3: Growth Inhibition Effect of Ansifen AP-3 on T-ALL Cells

[0058] To verify the inhibitory effect of anthrombin AP-3 on the growth of T-ALL cells, logarithmic growth phase T-ALL cell lines Jurkat, ZYXY-T1, and Molt-3 cells were cultured at a ratio of 2 x 10⁻⁶. 5 The plating concentration was calculated based on the concentration per ml, and the plates were plated in 96-well plates. The IC50 of Jurkat, ZYXY-T1, and Molt-3 cells obtained in Example 2 was used for detection. 50 Different AP-3 concentrations were used to inhibit the growth of different T-ALL cell lines. Jurkat cell lines were treated with AP-3 at concentrations of 0, 0.02, 0.04, and 0.06 nM; ZYXY-T1 cell lines at concentrations of 0, 0.12, 0.14, and 0.16 nM; and Molt-3 cell lines at concentrations of 0, 0.250, and 0.500 nM. Five monitoring points were set at hours 0 (on the day of plating), 24, 48, 72, and 96. The absorbance of different wells was measured using the MTS assay. Each concentration was plated in triplicate. Absorbance values ​​at different time points were collected for different T-ALL cell lines at different AP-3 concentrations. The absorbance value at hour 0 was used as a control. The absorbance values ​​at hours 24, 48, 72, and 96 were divided by the absorbance value at hour 0, and the multiples of the absorbance values ​​at different monitoring points were calculated. Growth curves for different T-ALL cell lines at different AP-3 concentrations were plotted using Graphpad software.

[0059] The results are as follows Figure 3 As shown in AC, anserin AP-3 can significantly inhibit the growth of Jurkat, ZYXY-T1, and Molt-3 cells.

[0060] Example 4: Validation of the efficacy of Ansifen AP-3 in T-ALL

[0061] To clarify the therapeutic effect of AP-3 on T-ALL mouse models, a live-in imaging T-ALL mouse CDX model was constructed using Jurkat cells (Jurkat Luc) carrying the luciferase reporter gene. The flowchart is shown below. Figure 4 As shown in Figure A. Five-week-old NCG mice were acclimatized within the SPF barrier for one week, and then fed 1x10... 6 / 100μl of Jurkat Luc cells were injected into NCG mice via tail vein. On day 5 post-injection, 50mg / ml of D-fluorescein potassium, a small animal in vivo imaging substrate, was injected intraperitoneally into the mice. In vivo imaging was performed using a small animal in vivo imaging system to detect tumor invasion and burden in the NCG mice. The model mice were further divided into a control group and a treatment group. The control group received saline via tail vein injection, while the treatment group received anserine AP-31.8mg / kg via tail vein injection once. Subsequently, in vivo imaging was performed three times on days 15, 25, and 35 post-injection of Jurkat Luc cells, and AP-31.8mg / kg was administered again via tail vein injection after imaging on day 25. Tumor invasion and survival time in the mice were observed.

[0062] The results showed that anserin AP-3 significantly reduced tumor invasion in T-ALL model mice. Figure 4 B), and can prolong the survival time of T-ALL model mice ( Figure 4 C).

[0063] Example 5: Ansifen P-3 inhibits T-ALL by targeting C-MYC.

[0064] 1×10T-ALL ZYXY-T1 cells were used. 6 Cells were seeded at 3 ml / well in 6-well plates, with 3 replicates per well, and divided into two groups: a control group without AP-3 and an experimental group with 0.35 nMAP-3. After 24 hours of incubation, cells were collected, centrifuged, and the supernatant was discarded. Cells were washed with 1×PBS and centrifuged again, retaining cell clumps. RNA-seq sequencing and data analysis were performed on both groups of cells (conducted by Shanghai Ouyi Biotechnology Co., Ltd.). The detailed flowchart is shown below. Figure 5 As shown in Figure A. The results indicate that the MYC pathway was significantly inhibited after treatment with anserine AP-3 ( Figure 5 B), the C-MYC gene is a significantly repressed gene ( Figure 5 C).

[0065] Auto-dock molecular docking revealed a binding site between AP-3 and C-MYC, with a binding energy of -3.58 ( ). Figure 5 D). The direct binding capability between AP-3 and C-MYC was clarified through SPR technology. Figure 5 E), protein imprinting confirmed that AP-3 treatment significantly reduced C-MYC expression levels in T-ALL cells. Figure 5 (FG). In summary, this experiment clarified, through different techniques, that the direct binding target of anserin AP-3 in killing T-ALL cells is C-MYC.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

Claims

1. Application of anserin P-3 (AP-3) in the preparation of drugs for the prevention and / or treatment of T-cell acute lymphoblastic leukemia.

2. Use according to claim 1, wherein The molecular formula of the anserin P-3 (AP-3) is C32H43ClN2O9, and its structural formula is as follows: 。 3. The use according to claim 1, wherein The T-cell acute lymphoblastic leukemia described is a T-cell acute lymphoblastic leukemia that highly expresses C-MYC.

4. The application as described in claim 3, characterized in that, The aforementioned anserin P-3 (AP-3) achieves prevention and / or treatment of T-cell acute lymphoblastic leukemia by inhibiting the expression of C-MYC.

5. The application as described in claim 3, characterized in that, In this application, anserin P-3 (AP-3) is the only active ingredient.

6. The use of a pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of T-cell acute lymphoblastic leukemia, said pharmaceutical composition comprising anserine P-3 (AP-3) and pharmaceutically acceptable excipients.

7. The application as described in claim 6, characterized in that, Ansifen P-3 (AP-3) is the sole active ingredient in the composition; or the composition may also include other therapeutic agents.

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