Patent Application
20230190933
The disclosure belongs to the field of “photothermal immune” anti-tumor medicine, in particular relates to an immunologically active peptide-biliverdin conjugate, a preparation method therefor and an application thereof in cancer diagnosis, and/or tumor immunotherapy, and/or tumor “photothermal immunotherapy” (tumor photothermal therapy combined with immunotherapy).
Traditional tumor treatment methods, such as surgery, chemotherapy, radiotherapy, etc., are faced with different degrees of side effects, and “photothermal immunotherapy” is the product of the combination of tumor photothermal therapy and tumor immunotherapy, which is a safe, accurate and broad-spectrum new treatment for tumor, shows a good therapeutic effect for metastatic and multiple-focus advanced tumor. The basic principle is that photothermal immune drugs actively targeted at, or are passively targeted at the tumor site, under the excitation of laser with specific wavelength; the drugs gathered at the tumor site absorb photothermal energy and convert it into thermal energy, which can locally raise the temperature of tumor, thus effectively killing tumor cells and inhibiting the tumor development process or clearing tumor tissues; the tumor antigen produced by photothermal therapy and the immunologically active part of photothermal immune drugs activate or enhance the immune system function, enhance the activation of immune cells and the release of immune-related factors, thus further inhibiting tumor recurrence and metastasis.
At present, various photothermal preparations, immune peptide preparations, preparation methods therefor and applications thereof have been disclosed: for example, the application of a ferrite nanomaterial in the preparation of diagnosis and treatment drugs targeted at tumor (Publication No. 106310255A); a photothermal preparation based on graphene (Publication No. CN107080844A); a controllable preparation method of selenium compounds nanosheets of copper for tumor photothermal therapy (Publication No. 106902352B); an organic micromolecule (3,6-bis (2-thienyl)-2,5-dihydropyrrolo [3,4-c] pyrrole-1,4-dione (DPP) derivative) nano tumor photothermal therapeutic reagent and preparation method thereof (Publication No. 106008525B); a nano photothermal therapeutic agent formed by polymers covalently linked through mPEG-PLGA and/or PEG-PLGA with porphyrin compounds and preparation method thereof (Publication No. 105327348A), etc. These published photothermal preparations all show high photothermal conversion efficiency and can effectively inhibit the growth of tumor, but there are still some universal problems: 1) the long-term biological safety performance needs to be studied; 2) the metabolic mechanism is not yet clear; 3) tumor metastasis and recurrence after photothermal therapy. For example, anti-tumor related peptides and related anti-cancer vaccine compositions for inducing anti-tumor immune response of colorectal cancer have been disclosed (Publication No. 103360466A); a tumor-related peptide composition and related anti-cancer vaccine for treating gliomas and other cancers have been disclosed (Publication No. 102170901A); a pharmaceutical compound (FOXP3 SIRNA-protamine-anti-CD25 antibody compound) which can enhance the anti-tumor immune response has been disclosed (Publication No. 101455840A), etc. At the same time, some immunoregulatory peptide related drugs have been commercialized, such as thymopentin for injection, recombinant human interferon a-2b injection, coriolus versicolor glycopeptide capsule, etc. However, these published or commercialized immunoregulatory drugs have the following problems: 1) small molecules with short half-life and easy degradation; 2) weak immune effect; 3) complex ingredients and serious immune-related adverse events. Therefore, it is urgent to further develop new therapeutic preparation and methods for tumor on the basis of the currently disclosed photothermal preparation and immune preparation.
Combining phototherapy with immunotherapy is expected to further inhibit tumor metastasis and recurrence on the basis of tumor ablation, and bring better survival benefits to tumor patients. Rakuten Medical developed an Antibody-Drug Conjugates (ADC) composed of cetuximab and IRDye700DX, which is used for the combination of photodynamic therapy and immunotherapy, specifically, cetuximab-mediated targeted delivery is used to achieve high tumor specificity, and IRDye700DX photodynamic effect is used to achieve tumor ablation. At present, in the clinical trials for locally recurrent head and neck cancer, this technique has achieved a therapeutic effect of 50% remission rate, 16.7% complete remission rate and 86.7% disease control rate, and good biological safety. However, its complex structure has caused difficulties in synthesis and production to some extent. At the same time, photodynamic therapy is strongly dependent on oxygen which is therefore not suitable for hypoxic tumor. The corresponding tumor “photothermal immunotherapy” has shown remarkable advantages, especially its broad spectrum and accuracy. The success of this therapy mainly depends on the photo-thermal conversion efficiency of “photothermal immune” drugs and the immune effect. At the same time, the biological safety, immune-related adverse events (irAEs) and metabolic mechanism of “photothermal immune” preparations are also the keys to the successful implementation of tumor photothermal immunotherapy.
Endogenous pigment biliverdin, a secondary metabolite of hemoglobin in animals, has a clear metabolic mechanism and multiple biological activities (antioxidant, anti-inflammatory, anti-tumor, etc.). Biliverdin is a bioactive pigment with linear tetrapyrrole structure, which has remarkable near-infrared absorption and can effectively convert near-infrared light into thermal energy (Patent Publication No. 109224073A), and has a broad application prospect in the development of tumor therapeutic drugs and photothermal anti-tumor fields. Studies have proved that tumor inflammatory microenvironment can further promote tumor metastasis and recurrence, but it has not been publicly reported that the anti-inflammatory activity of biliverdin molecule can be used to reverse tumor inflammatory microenvironment and achieve tumor immunotherapy. At the same time, small-molecule immunologically active peptides are substances with specific amino acid sequences and multiple biological functions (immunoregulation, anti-tumor, etc.), and gradually show unique advantages in the biomedical field.
There is no public report on the conjugate formed by biliverdin molecule and immunologically active peptide molecule by means of chemical synthesis, preparation method therefor and application thereof in tumor “photothermal immunotherapy”. An immunologically active peptide-biliverdin conjugate disclosed in this application has remarkable advantages in the field of cancer diagnosis, and/or tumor immunotherapy, and/or tumor “photothermal immunotherapy”: 1) an extended half-life and enhanced stability, compared with the disclosed immunologically active peptide and composition thereof; 2) high biological safety, single component and clear metabolism mechanism in vivo; 3) an immunologically active peptide end which can stimulate an organism to generate a tumor-immune response and enhance immune function; the biliverdin end can achieve cancer diagnosis and photothermal treatment, relieve and eliminate tumor inflammation, and remodel the tumor inflammatory microenvironment. In summary, the molecular conjugate of the present disclosure and related preparations, dosage forms thereof and preparation methods thereof are of great significance in promoting clinical application in the treatment of tumor, and have great application potential in eliminating primary tumor, inhibiting tumor metastasis and recurrence, etc.
The disclosure discloses an immunologically active peptide-biliverdin conjugate, a preparation method therefor and an application thereof in tumor imaging, tumor immunotherapy and tumor “photothermal immunotherapy”. The disclosed conjugate has the following advantages: 1) an extended half-life and enhanced stability, compared with the disclosed immunologically active peptide and composition thereof; 2) high biological safety, single component and clear metabolism mechanism in vivo; 3) An immunologically active peptide end which can stimulate an organism to generate a tumor-immune response and enhance immune function; the biliverdin end can realize tumor multi-mode imaging and tumor photothermal treatment, achieve tumor ablation, and also can relieve and eliminate tumor inflammation, remodel the tumor inflammatory microenvironment, and prevent tumor metastasis and recurrence.
To achieve the purpose of the disclosure, the disclosure adopts the following technical solution:
On the second aspect, the present disclosure provides the conjugate according to the first aspect, characterized in that R1 and R2 are the same or different.
On the third aspect, the present disclosure provides the conjugate according to the first to second aspects, characterized in that R1 and R2 are any sequences of the above, also can be peptides or protein comprising any sequences of the above, or derivatives of any sequences of the above, or amino acids, peptides or protein with similar functions;
On the fourth aspect, the present disclosure provides the conjugate according to the first to third aspects, characterized in that involving molecular conjugates and preparations or dosage forms derived from the molecular conjugates:
On the fifth aspect, the present disclosure provides the method for preparing the conjugate according to the first to fourth aspects, characterized in that comprising the following steps:
On the sixth aspect, the present disclosure provides the conjugate according to the first to fifth aspects and the preparation method thereof, which is characterized in that the conjugate has “photothermal immune” anti-tumor use.
On the seventh aspect, the present disclosure provides the use according to the sixth aspect, which is characterized in that the conjugate molecules gathered in the tumor position can complete the conversion of light energy to thermal energy under the irradiation of laser with specific wavelength, and therefore realize tumor ablation, and at the same time, after illumination the tumor position can generate in-situ tumor specific antigen; the conjugate can further activate the immune response of the organism, eliminate tumor inflammatory microenvironment, enhance the specific immune response of the organism, thus achieving immuno-treatment of tumor and further preventing tumor metastasis and recurrence. That is, the combination of tumor photothermal therapy and tumor immunotherapy is achieved, which is characterized in that the combination of tumor ablation, immunoregulation and tumor inflammatory microenvironment regulation is achieved, and the treatment effect of tumor is obviously improved; wherein
On the eighth aspect, the present disclosure provides the use according to the sixth to seventh aspects, characterized in that the conjugate is used for tumor diagnosis and monitoring before, during and after “photothermal immunotherapy”, comprising nuclear magnetic resonance imaging, radionuclide imaging and photoacoustic imaging.
On the ninth aspect, the present disclosure provides the use according to the eighth aspect, characterized in that the conjugate is used for nuclear magnetic resonance imaging of tumor, and M is preferably selected from the following atoms or ions: Mn, Fe, Cu, Eu, Gd, Dy.
On the tenth aspect, the present disclosure provides the use according to the eighth aspect, characterized in that the conjugate is used for radionuclide imaging of tumor, and M is preferably selected from the following atoms or ions: 64,67Cu, 99mTc, 195Pt, 67,68Gd, 201Tl, 60Co, 111In and 51Cr.
On the eleventh aspect, the present disclosure provides the use according to the eighth aspect, characterized in that the conjugate is used for photoacoustic imaging of tumor, and M is preferably selected from the following atoms or ions: H and Zn.
On the twelfth aspect, the present disclosure provides the use according to the sixth to eleventh aspects, characterized in that the tumor is a primary tumor or a metastatic tumor, and is selected from single or multiple tumors such as brain cancer, head and neck cancer, esophageal cancer, breast cancer, lung cancer, stomach cancer, liver cancer, colon cancer, pancreatic cancer, lymphoma, melanoma, ovarian cancer, cervical cancer, prostate cancer and bladder cancer. Preferably, the tumor is a superficial tumor or a tumor with high surgical risk; for example, the superficial tumor or the tumor with high surgical risk comprising head and neck cancer, breast cancer, melanoma, cervical cancer, prostate cancer, pancreatic cancer and et al.
On the thirteenth aspect, the present disclosure provides the use according to the sixth to eleventh aspects, which is characterized in that it can be combined with tumor therapy strategies such as surgery, chemotherapy, radiotherapy and immunotherapy.
On the fourteenth aspect, the present disclosure provides the use according to the thirteenth aspect, which is characterized in that it is used for the treatment after postoperative of residual tumor lesions and/or metastatic tumor lesions.
On the fifteenth aspect, the present disclosure provides the use according to the thirteenth aspect, which is characterized in that it is used for the combination of chemotherapy and “photothermal immunotherapy”;
On the sixteenth aspect, the present disclosure provides the use according to the thirteenth aspect, which is characterized in that it is used for the combination of radiotherapy and “photothermal immunotherapy”;
preferably, the dose of radiation is 5%-40% of the conventional dose, and further preferably, 5%-20%.
On the seventeenth aspect, the present disclosure provides the use according to the thirteenth aspect, which is characterized in that it is used for the combination of immunotherapy and “photothermal immunotherapy”;
On the eighteenth aspect, the conjugate according to the first to seventeenth aspects, the preparation method therefor and application thereof in tumor therapy provided by the present disclosure have the advantages of high biological safety, good stability, difficulty to develop drug resistance, definite metabolic mechanism, extended half-life and the like, and can significantly enhance the anti-tumor effect.
1) At present, there are no reports of “photothermal immune” molecules, preparations and dosage forms based on biliverdin, and no related reports of its effects of tumor immunotherapy and/or “photothermal immunotherapy”, in particular, the conjugate of the disclosure can improve the photothermal effect of biliverdin;
2) An immunologically active peptide-biliverdin molecular conjugate, which is obtained by chemical synthesis from endogenous biliverdin and immunologically active peptide, has high biological safety, good stability and clear metabolic mechanism, and can effectively solve the problems of serious immune-related adverse events (irAEs) and poor biocompatibility.
3) Multi-functional synergy can be achieved in tumor therapy: the immunologically active peptide end can stimulate the organism to generate tumor immune response and enhance immune function; the biliverdin end can realize tumor imaging, tumor photothermal treatment and tumor ablation, and also can relieve and eliminate tumor inflammation, remodel the tumor inflammatory microenvironment, and reduce tumor metastasis rate and recurrence rate.
To further explain the technical solution and effects of the present disclosure, the technical solution of the present disclosure will be further explained below with reference to the preferred embodiments of the present disclosure, but the present disclosure is not limited to the scope of the embodiments.
The embodiments without specific technology or conditions are carried out according to the technology or conditions described in the literature in the field or according to the product description. The reagents or instruments used without indicating the manufacturer are conventional products that can be purchased through regular channels.
According to the following steps, the biliverdin-SIINFEKL conjugate is obtained through chemical synthesis: certain amounts of biliverdin, EDC-HCl, NHS and DMF were weighed, sequentially added into a reactor and mixed uniformly; the obtained mixture was stirred at room temperature in the dark for 24 h; water was added and stirred, and precipitate was collected; anhydrous DMF was added into the obtained precipitate, and the mixture was mixed uniformly, then SIINFEKL peptide and anhydrous triethylamine were added and stirred, and these substances reacted at room temperature in the dark for 24 h; the precipitate of the above reaction was collected, and purified by size exclusion chromatography; the obtained substance was recrystallized to obtain a pure molecular conjugate. Wherein, the concentration of biliverdin was 100 mM, the concentration of EDC. HCI was 100 mM, the concentration of NHS was 50 mM and the concentration of peptide was 200 mM. 1H NMR information of the prepared conjugate was as follows:
1H NMR (600 MHz) 6 = 11.95 - 13.00 (3H), 10.68 (s, 1H), 8.85 (s, 1H), 8.56 (s, 1H), 8.40 (s, 1H), 8.32 (s, 2H), 8.21 (s, 2H), 8.08 (s, 1H), 7.50 (s, 1H), 7.41 (s, 1H), 7.30 (m, 3H), 7.16 (m, 3H), 7.03 (q, 2H), 6.93 (s, 1H), 6.49 (t, 2H), 5.57 (s, 1H), 5.20 (m, 4H), 4.16 (m, 1H), 3.95 (m, 1H), 4.55 (m, 2H), 4.92 (m, 1H), 4.34 (m, 2H), 4.94 (m, 1H), 4.84 (m, 1H), 4.61 (1H), 4.44 (m, 2H), 3.44 (m, 1H), 3.18 (m, 1H), 2.81 (m, 1H), 2.69 (m, 4H), 2.49 - 2.42 (7H), 2.35 (m, 3H), 2.23 (m, 2H), 2.12 (m, 6H), 2.06 (m, 2H), 1.95 (m, 3H), 1.49 (m, 1H), 1.75 (m, 4H), 1.55 (m, 6H), 1.25 (m, 2H), 1.11 (m, 6H), 0.90 - 1.00 (12 H).
The biliverdin-SIINFEKL conjugate was prepared from biliverdin molecule and SIINFEKL according to the chemical synthesis method of Example 1. A certain amount of conjugate was weighed, after being pre-dissolved in trace DMSO solution, the conjugate was directly dissolve in PBS solution, filtration sterilization was conducted after stirring and dissolving, and the pH value was adjusted to neutral. The prepared conjugates with different concentration gradients were incubated with Human Umbilical Vein Endothelial Cells HUVEC in the dark, and the biological safety of the conjugates was evaluated by MTT colorimetric method.
Firstly, biliverdin was chemically synthesized with excess zinc acetate to obtain biliverdin-Zn metal complex, and the experimental method was as follows: biliverdin and excess zinc acetate were dissolved in methanol solution, stirred at 60° C. for 4 hours, solid was obtained by removing solvent from the obtained solution by rotary evaporation, the solid wad then purifed by reversed-phase chromatographic column to obtain biliverdin-Zn complex, wherein the mass concentration ratio of biliverdin and zinc acetate is 1:5. The biliverdin-Zn-NYSKPTDRQYHF conjugate was prepared from biliverdin-Zn metal complex and NYSKPTDRQYHF according to the above chemical synthesis method. A certain amount of conjugate was weighed, after being pre-dissolved in trace DMSO solution, the conjugate was directly dissolve in PBS solution, filtration sterilization was conducted after stirring and dissolving, and the pH value was adjust to neutral. The prepared conjugates with different concentration gradients were incubated with mouse skin melanoma cells B16-F10 in the dark, and the biological safety of the conjugates was evaluated by MTT colorimetric method.
Firstly, biliverdin was chemically synthesized with excess ferrous chloride to obtain biliverdin-Fe metal complex, and the biliverdin-Fe-YMDGTMSQV conjugate was prepared from biliverdin-Fe metal complex and YMDGTMSQV according to the aforementioned chemical synthesis method. A certain amount of conjugate was weighted, after being pre-dissolved in a small amount of organic solvent, the conjugate was completely dissolved in PBS solution, filtration sterilization was conducted, and the pH value was adjusted to neutral. Fluorescein was used to label the conjugate, and the labeled conjugate was incubated with mouse Bone Marrow-derived Dendritic Cells BMDCs, the uptake of the conjugate by BMDCs was detected by flow cytometry.
The biliverdin-KIFGSLAFL conjugate was prepared from biliverdin molecule and KIFGSLAFL according to the above chemical synthesis method. A certain amount of conjugate was weighed, after being pre-dissolving in a small amount of organic solvent, the conjugate was completely dissolve in PBS solution, filtration sterilization was conducted, and the pH value was adjusted to neutral. The prepared conjugate molecular solution was cocultured with dendritic cells from peripheral blood of non-small cell lung cancer model mice, after 24 hours, the dendritic cells were collected, washed and fluorescently labeled, and the CD80, CD83 and CD86 on the cell surface were detected by flow cytometry to evaluate the promoting effect of the conjugate on the maturation of dendritic cells.
The biliverdin-FLWGPRALV conjugate was prepared from biliverdin molecule and FLWGPRALV according to the above chemical synthesis method. A certain amount of conjugate was weighed, directly dissolved in PBS solution, filtration sterilization was conducted after stirring and dissolving, and the pH value was adjusted to neutral. The prepared conjugate was co-incubated with human prostate cancer DU-145 cell line and LNCaP cell line, and analyzed by flow cytometry. The data were statistically analyzed by SPSS 12.0.
Firstly, biliverdin-Mn metal complex was chemically synthesized by biliverdin and excess manganese acetate tetrahydrate, and the biliverdin-Mn-YLEPGPVTA conjugate was prepared from biliverdin-Mn metal complex and YLEPGPVTA according to the aforementioned chemical synthesis method. A certain amount of conjugate was weighed, after being pre-dissolved in a small amount of organic solvent, the conjugate was completely dissolved in PBS solution, filtration sterilization was conducted, and the pH value was adjusted to neutral. 1 mL of this conjugate (at a concentration of 0.2 mg mL-1) was irradiated at 730 nm laser (0.3 W/cm2) for 10 min, and the temperature rise of the conjugate solution was investigated.
The biliverdin-IMDQVPFSV conjugate was prepared from biliverdin molecule and IMDQVPFSV according to the above chemical synthesis method. A certain amount of conjugate was weighted , after being pre-dissolved in trace DMSO solution, the conjugate was directly dissolved in PBS solution, filtration sterilization was conducted after stirring and dissolving, and the pH value was adjusted to neutral. According to the standard tumor mouse modeling method, C57BL/6 mouse model was established, and the mouse breast cancer cells 4T1 were inoculated subcutaneously, and then these mice were fed in SPF environment, the tumor growth was observed at any time, and relevant experiments were carried out after the average tumor volume reached about 80-100 mm3. The mice were divided into two groups (10 mice in each group), on the 1st, 2nd, 4th and 8th day, the mice in experimental group was injected with 100 uL of the conjugate (at a concentration of 0.2 mg mL-1) intraperitoneally, while the mice in blank group was injected with the same quality of normal saline. The growth of tumor volume in mice was monitored within 28 days.
The biliverdin-QQKFQFQFEQQ conjugate was prepared from biliverdin molecule and QQKFQFQFEQQ according to the above chemical synthesis method. A certain amount of conjugate was weighd, after being pre-dissolved in trace DMSO solution, the conjugate was directly dissolved in PBS solution, filtration sterilization was conducted after stirring and dissolving, and the pH value was adjusted to neutral. According to the standard tumor mouse modeling method, C57BL/6 mouse model was established, and the mouse colon cancer cells ct-26 were inoculated subcutaneously, and then these mice were fed in SPF environment. The tumor growth was observed at any time, and relevant experiments were carried out after the average tumor volume reached about 80-100 mm3. The mice were divided into the following four groups: blank group (normal saline), conjugate group (no light group) and conjugate group (light group), with 10 mice in each group. These mice were administered once on the 1st, 3rd, 8th and 12th day with the administration concentration being 2 mg kg-1. Wherein, the mice of the conjugate (light group) were irradiated with laser once 4 hours after administration on the first day, and the parameters were as follows: the laser intensity was 0.5 W/cm2 and the laser wavelength was 808 nm. The tumor inhibition of mice during the whole treatment cycle (the cycle is 45 days) was monitored. On the 29th day after the conjugate (light group) treatment, all the mice tumors were cleared, and the recurrence behavior of mice tumors was monitored from the 30th day to the 45th day.
Firstly, biliverdin-Ga metal complex was chemically synthesized by biliverdin and excess gadolinium chloride hexahydrate, and the biliverdin-Ga-FKFEFKFE conjugate was prepared from biliverdin-Ga metal complex and FKFEFKFE according to the aforementioned chemical synthesis method. A certain amount of conjugate was weighed, after being pre-dissolved in trace DMSO solution, the conjugate was directly dissolved in PBS solution. Filtration sterilization was conducted after stirring and dissolving, and the pH value was adjusted to neutral. According to the standard tumor mouse modeling method, the in-situ model of pancreatic cancer Pan02 in C57BL/6 mouse was established, and then these mice were fed in SPF environment, and the tumor growth was observed at any time, and relevant experiments were carried out after the average tumor volume reached about 80-100 mm3. The mice were divided into the following four groups: blank group (normal saline), conjugate group (no light group) and conjugate group (light group), with 10 mice in each group. The mice were administered once on the 1st, 3rd, 8th and 12th day with the administration concentration of 4 mg kg-1. Wherein, the mice of the conjugate (light group) were irradiated with laser once 4 hours after the first day of administration, and the parameters were as follows: the laser intensity was 0.5 W/cm2 and the laser wavelength was 730 nm. On the 15th day, the mice were euthanized and the tumor tissues of each group were taken, and the contents of immune-related factors (comprising IFN-7 with immune promoting effect and IL-4 and IL-10 with immune suppressing effect) in the supernatants of each group were measured by ELISA.
The biliverdin-LVVTPW conjugate was prepared from biliverdin molecule and LVVTPW according to the above chemical synthesis method. A certain amount of conjugate was weighted, after being pre-dissolved in trace DMSO solution,water was added to form the fiber dosage form of conjugate. And the concentration of the conjugate was 5 mg mL-1.
Firstly, biliverdin-Mn metal complex was chemically synthesized by biliverdin and excessive manganese chloride, and the biliverdin-Mn-ALCNTDSPL conjugate was prepared from the biliverdin-Mn metal complex and ALCNTDSPL according to the aforementioned chemical synthesis method. The conjugate was loaded into PLGA particles to prepare the conjugate emulsifier. According to the standard tumor mouse modeling method, the in-situ model of bladder cancer MB49 and MBT-2 in C57BL/6 mouse were estabblished, then these mice were fed in SPF environment, and the tumor growth was observed at any time, and relevant experiments were carried out after the average tumor volume reached about 80-100 mm3. The conjugate emulsifier was intravenously injected, after 6 hours, mice were placed under photoacoustic imager and nuclear magnetic resonance imager, and the photoacoustic signal and nuclear magnetic resonance signal intensity of tumor location were detected.
The biliverdin-EQLESIINFEKLTE conjugate was prepared from biliverdin molecule and EQLESIINFEKLTE according to the above chemical synthesis method. A certain amount of conjugate was weighed, after being pre-dissolved in trace DMSO solution, the conjugate was directly dissolved in PBS solution, after stirring and dissolving, filtration sterilization was conducted, and the pH value was adjusted to neutral. BALB/C cervical cancer U14 mouse model was established, and these mice were administered intraperitoneally with a concentration of 5 mg kg-1. On the 2nd, 4th and 7th day after administration, the contents of CD4+T and CD8+T cells in spleen and draining lymph nodes of mice were detected by immunofluorescence staining and flow cytometry.
Firstly, the biliverdin was incubated with excess 99mTc to obtain radiolabeled biliverdin, and the biliverdin-99mTc-ISQAVHAAHAEEINEAGR conjugate was prepared from biliverdin-99mTc and ISQAVHAAHAEEINEAGR according to the aforementioned chemical synthesis method. A certain amount of conjugate was weighed, after being pre-dissolved in trace DMSO solution, the conjugate was directly dissolve in PBS solution, after stirring and dissolving, filtration sterilization was conducted, and the pH value was adjusted to neutral. The conjugate was injected into tumor model mice (BALB/C, mouse breast tumor cell C127, the initial tumor volume was about 100 mm3) by intravenous injection, and its accumulation at the tumor site was monitored by single photon emission computed tomography. It was found that at the 4th hour after administration, the conjugate showed the clearest image at the tumor site, and the accumulated amount reached the highest value, which provided a window for tumor treatment. Under this time window, the tumor location was irradiated with laser (laser wavelength was 730 nm, power was 0.2 W/cm2), and the temperature change of the tumor location was monitored by near infrared imaging equipment. The expression of CD8+T cells and CD 107 molecules on the surface of CD8+T cells in mouse tumors were monitored by fluorescent immunostaining method, and the immune effect was evaluated. With regard to the conjugate described in Example 14,
The biliverdin-PDRAHYNI conjugate was prepared from biliverdin molecule and PDRAHYNI according to the above chemical synthesis method. A certain amount of conjugate was weighed, after being pre-dissolved in a trace amount of organic solution, the conjugate was directly dissolved in PBS solution, after stirring and dissolving, filtration sterilization was conducted, and the pH value was adjusted to neutral. The C57BL/6 mouse model of skin melanoma B 16-F 10 and the C57BL/6 mouse model of lung cancer Lewis were established, and the combination of “photothermal immunotherapy” and immunotherapy was carried out. The administration concentration of the conjugate was 3 mg kg-1, and the administration dosage of the immune drug interferon was 20U/ mouse. The administration window was that the tumor growth entered the logarithmic phase, and the initial volume was 300 mm3, and the inhibition of tumor was monitored.
The biliverdin-MI,LAVLYCL conjugate was prepared from biliverdin molecule and MLLAVLYCL according to the above chemical synthesis method. A certain amount of conjugate was weighed, after being pre-dissolved in a trace amount of organic solution, the conjugate was directly dissolved in PBS solution. After stirring and dissolving, filtration sterilization was conducted, and the pH value was adjusted to neutral. The C57BL/6 mouse model of skin melanoma B16-F10 and the C57BL/6 mouse model of lung cancer Lewis were established, and the combination of “photo-immunotherapy” and chemotherapy was carried out. The administration concentration of the conjugate was 3 mg kg-1, and the administration concentration of the chemotherapeutic drug doxorubicin was 1 mg kg-1. The administration window was that the tumor growth entered the logarithmic phase, and the initial volume was 400 mm3, and the inhibition of tumor was monitored.
Firstly, biliverdin-Tb metal complex was chemically synthesized by biliverdin and excess terbium trichloride hexahydrate, and then biliverdin-Tb-VHFFKNIVTPTP conjugate was prepared from the biliverdin-Tb metal complex and VHFFKNIVTPTP according to the aforementioned chemical synthesis method. A certain amount of conjugate was weighed and directly dissolved in PBS solution, after stirring and dissolving, filtration sterilization was conducted, and the pH value was adjusted to neutral. The C57BL/6 mouse model of cutaneous melanoma B 16-F 10 was established, these mice were administered intravenously every other day for 5 times, and the dosage was 2 mg kg-1. After 30 days, the tissues of a mouse were taken, the indexes of main organs were measured, and the biological safety of the conjugate was evaluated.
The applicant declares that the detailed method of the present disclosure is illustrated by the above-mentioned embodiments, but the present disclosure is not limited to the above-mentioned detailed method, that is, it does not mean that the present disclosure must be implemented by the above-mentioned detailed method. It should be clear to those skilled in the technical field that any improvement of the disclosure, equivalent substitution of raw materials of the product of the disclosure, addition of auxiliary ingredients, selection of specific methods, etc. all fall within the scope of protection and disclosure of the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/089886 | 5/12/2020 | WO |
