Patent Application
20200129646
The invention relates to a radioactive labeled long-acting targeted peptide pharmaceutical and a production method thereof, in particular to a targeted peptide pharmaceutical produced with improved labeling efficiency and minimum dosage required that reduces preparation time and cost.
According to the Global Cancer Report 2014 published by the World Health Organization (WHO), global cancer cases are growing rapidly from 14 million in 2012 to 19 million in 2025, and will reach 24 million by 2035. That is an increase of more than 70% in 23 years. At present, the development of targeted peptide pharmaceuticals has been a trend in decade. Tumor cells grow to 1-2 mm while migrate and grow due to their new blood vessels, and integrin acts as an influence on cell attachment, migration, growth and differentiation, and angiogenesis are highly expressed on inflammation and tumor endothelial cells, also, αvβ3 or αvβ5 plays an important role in breast cancer angiogenesis.
According to the Global Cancer Report 2014 published by the World Health Organization (WHO), global cancer cases are growing rapidly, from 14 million in 2012 to 19 million in 2025, and will reach 24 million by 2035. That is, an increase of more than 70% in 23 years. At present, the development of targeted peptide pharmaceuticals has been a trend in the past decade.
Tumor cells grow to 1-2 mm and migrate and grow due to their new blood vessels. Integrin acts as an influence on cell attachment, migration, growth and differentiation and angiogenesis is highly expressed in inflammation and tumor endothelial cells, in which, αvβ3 or αvβ5 also play an important role in breast cancer angiogenesis.
Therefore, the RGD peptide is used as an integrin-ligand interaction inhibitor, and the circular RGD peptide preferentially binds to integrin αvβ3 as a tumor cell targeting probe. When the peptide is labeled with a long half-life and a short half-life of radioisotopes, the molecular imaging can be used as a diagnostic and therapeutic agent for tumors.
Since members of the integrin family play an important role in the regulation of cell activation, migration, proliferation, survival, and differentiation, integrin αvβ3 has been found in osteoclasts and invasive tumors, such as advanced glioblastoma, breast and prostate tumors, malignant melanoma, and ovarian cancer, can be highly expressed, and clinical trials have also begun using radiolabeled RGD-peptides for integrin αvβ3, such as F-18-labeled RGD, for non-invasive breast cancer testing and staging. RGD peptides and labeling radionuclides, such as I-131, In-111, Tc-99m, Ga-68, F-18, etc. are used for visualizing tumor sites, such as some F-18-galactose RGD, F-18-RGD-K5, Ga-68-NOTA-RGD, Ga-68-NOTA-(RGD)2, F-18-(RGD)2 and F-18-Alfatide. At present, the development of clinical pharmaceutical RGD pharmaceuticals is mainly RGD or c(RGDfV) or c(RGDfK), and most of them are diagnostic agents.
Since members of the integrin family play an important role in the regulation of cell activation, migration, proliferation, survival and differentiation, integrin αvβ3 has been found highly expressed in osteoclasts and invasive tumors, such as advanced glioblastoma, mammary gland and prostate tumors, malignant melanoma, and ovarian cancer; clinical use of radiolabeled RGD-peptides for integrin αvβ3, such as F-18-labeled RGD, has also begun to be used for non-invasive breast cancer detection and staging. Contains RGD peptides and uses labeled radionuclides, such as I-131, In-111, Tc-99m, Ga-68, F-18, etc. for imaging tumor sites, such as some F-18-galactose, RGD, F-18-RGD-K5, Ga-68-NOTA-RGD, Ga-68-NOTA-(RGD)2, F-18-(RGD)2 and F-18-Alfatide. At present, the development of clinical pharmaceutical RGD pharmaceuticals is mainly RGD. c(RGDfV), or c(RGDfK), and most of them are diagnostic agents.
However, the peptide pharmaceutical is good in water solubility, and the amount of tumor accumulation in the actual application is not as high as that of the protein pharmaceutical, and is rapidly metabolized in the body. In the process of commercialization, it is still necessary to overcome the T/M ratio during radiography, the decrease of tumor accumulation amount with time, and the rapid elimination of small molecule peptide pharmaceuticals. In the prior arts WO2016209795A1 and CN 104650217A, National Institutes of Health revealed the modified Evans Blue (EB) as the technology for the development of RGD-related pharmaceuticals, but it is also difficult to effectively overcome problem of radiation decay in storage and transport.
The main object of the present invention is to provide a radioactive long-acting targeted peptide pharmaceutical DOTA-EB-cRGDfK (1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic Acid, DOTA), which is based on the highly specific tumor and the angiogenesis-identifying cyclic RGD peptide pharmaceutical cRGDfK, and develop long-acting target nuclear medicine for systemic transfer of integrin αvβ3 tumors, such as breast cancer etc., for diagnosis and treatment; and the main mechanism and design are as follows:
1) cyclic RGD derivatives: The cyclic peptide cRGDfK is highly specifically integrated with neovascularization and highly expressed integrin αvβ3 on some tumors.
2) the cyclic RGD is connected with a molecule which can be highly affined with albumin in the blood, which can prolong the circulation time of the peptide pharmaceutical in the body, and finally achieve goal of the specific binding amount of the tumor and reduce the non-specificity of the normal tissue in accumulation.
3) bonding a metal chelating agent (DOTA) and labeling radioisotopes with In-111, Lu-177, or Ga-68.
The advantages of the above-mentioned radioactive peptide pharmaceuticals. for example, breast cancer, can accurately assess the distribution and size of tumors in the body, mainly for systemic metastatic breast cancer that cannot be accurately diagnosed by surgery, ultrasound, tomography and reduce the probability of misdiagnosis.
Another object of the present invention is to provide a radiation-labeled long-acting targeted peptide pharmaceutical, which is designed in a kit to produce a long-acting targeted peptide pharmaceutical for the whole operation. It is extremely convenient, and it can be safely stored and transported without being affected by radiation half-time. It can be of more than 90% labeling efficiency being tested by Radio-HPLC and ITLC, and it does not need to be purified by column.
Still another object of the present invention is to provide a radioactive labeled long-acting targeted peptide pharmaceutical which can be combined with molecular imaging medical technology to develop diagnosis and treatment techniques, improving the diagnosis accuracy and cure rate of tumors which can be diagnosed and treated at earlier stage, and reducing cost.
The chemical structure of DOTA-EB-cRGDfK of the present invention is shown In
Please refer to
1) The content of DOTA-EB-cRGDfK is 40 to 60 μg, the In111 activity is 6mCi, and the reaction is carried out at 95° C. for 10 to 20 minutes at pH 4.5-6, and the labeling efficiency is greater than 90%.
2) The content of DOTA-EB-cRGDfK is 40 to 60 μg, the In111 activity is 6 to 13mCi, and the reaction is carried out at 95° C. for 15 minutes at pH6, and the labeling efficiency is greater than 90%.
Referring to
Referring to
In the practical application, the above-mentioned radioactive labeled DOTA-EB-cRGDfK of the present invention can be prepared into a DOTA-EB-cRGDfK cryostat bottle, which is convenient for long-term storage and transportation, and can improve the convenience of use; DOTA- The preparation method of EB-cRGDfK cryostat bottle is as follows:
adding 4-5 μL of DOTA-EB-cRGDfK/DMSO solution with concentration 20 μg/μL to 333 μL in volume of 3M sodium acetate solution, adding injection water to obtain a volume of 1000 μL with pH 7, and freeze-drying at −25° C. for 96 hours, refilling with nitrogen for about 1 minute, and sealing the cap with a plastic soft plug and aluminum cap.
In practical application, the DOTA-EB-cRGDfK cryostat bottle can be combined with a solution bottle, a filtering device and an adsorption device to form a kit suitable for production of the radioactive labeled DOTA-EB-cRGDfK of In111 and Ga68. The solution bottle has a content of 3 mL of hydrochloric acid or acetic acid of pH 3 for use as a conditioning agent and pH adjustment.
The components of the DOTA-EB-cRGDfK cryostat bottle include:
1) a content of 60-120 μg, preferably 100 μg of DOTA-EB-cRGDfK peptide;
2) dimethyl sulfoxide DMSO having a content of 4-10 μL;
3) sodium acetate in an amount of 80-100 mg.
The adsorption device has an adsorption column, the composition of which has two layers, and the upper layer is an enhanced anion exchange resin, and the composition thereof includes Dowex 1×8-200, 100-200 mesh, 8% cross-linking, the dosage is 0.5 g to 2 g, and the lower layer is titanium dioxide particles with a size 75-300 μm and the amount is zero or 0.1 g to 1 g, and its composition includes rutile or anatase filled in a Biorad column (731-1550). The filtration device is a 0.22 μm PVDF (polyvinylidene fluoride) filter.
Referring to
injecting 1 to 2 mL of pH 3 acidic solution from a solution bottle into a cryostat bottle of DOTA-EB-cRGDfK;
using 0.1 N HCl to wet an adsorption column of an adsorption device and placing the adsorption column on the cryostat bottle;
injecting radiation solution of 68GaCl3 or 111InCl3 or 177LuCl3 having 3 to 12mCi radiation activity into the adsorption column, and the radiation solution flows into the cryostat bottle after purification;
heating a chemical reaction bottle from 85 to 95° C., preferably 95° C., for 10 to 30 minutes for further mixing the radiation solution, and then filtering through a filtering device to obtain an initial product;
a final product of the radioactive labeled 111In-DOTA-EB-cRGDfK or 68Ga-DOTA-EB-cRGDfK or 177Lu-DOTA-EB-cRGDfK is produced after a Radio-ITLC labeling efficiency test.
The above DOTA-EB-cRGDfK cryostat was subjected to the In-111 labeling test, and the labeling efficiency and radiochemical purity test were carried out by ITLC and Radio-HPLC, and the 111In-DOTA-EB-cRGDfK labeling efficiency and radiochemical purity were greater than 90%, its cryostat bottle can still reach radiochemical purity and labeling efficiency of greater than 90% after maintaining at −25° C. for 1 month.
The above-mentioned radiation labeled long-acting targeted peptide pharmaceutical of the present invention has the following advantages:
1) Increasing the amount of pharmaceutical specific to the tumor, and reducing the amount of pharmaceutical accumulation in the non-tumor tissue.
2) Improving shortcomings of rapid elimination rate of peptide pharmaceuticals.
3) The amount of salt composition is small to litigate the burden on the body.
4) To be used for diagnosis and treatment.
5) The preparation process is short and personnel exposure is reduced.
6) Radiation species, such as, 68Ga, 111In, and 177Lu that can be used for diagnosis and treatment.
In addition, the combination of its pharmaceutical and albumin in the blood can prolong the circulation time of the peptide pharmaceutical in the body, increase the specific binding amount of the tumor, and reduce the non-specific accumulation of normal tissues to meet the development trend of precision medicine.
In practical applications, the 177Lu/90Y-DOTA-EB-cRGDfK for therapeutic use can be further developed in the future to achieve the goal of personalized medicine by diagnosis and screening first, and followed by precise treatment. The advantage is to accurately assess the distribution of breast cancer in the body and the size of the tumor, mainly for the surgical operation, ultrasound, tomography and those other methods, which can't be used accurately diagnose the systemic metastatic Integrin αvβ3 tumor and thus increasing the diagnostic accuracy.
