CN113769121A

CN113769121A - Radiotherapeutic medicine for diseases caused by coronavirus or influenza virus and preparation method thereof

Info

Publication number: CN113769121A
Application number: CN202111091713.XA
Authority: CN
Inventors: 罗志福; 李凤林; 樊彩云; 陈宝军; 刘子华; 解清华
Original assignee: China Institute of Atomic of Energy
Current assignee: China Institute of Atomic of Energy
Priority date: 2020-09-18
Filing date: 2021-09-17
Publication date: 2021-12-10
Anticipated expiration: 2041-09-17
Also published as: CN113769121B

Abstract

The invention provides a radioactive therapeutic medicine for diseases caused by coronavirus or influenza virus and a preparation method thereof.

Description

Radiotherapeutic medicine for diseases caused by coronavirus or influenza virus and preparation method thereof

Technical Field

The invention belongs to the field of radiopharmaceuticals in nuclear technology application, and particularly relates to a radiotherapeutic medicament for diseases caused by coronavirus or influenza virus.

Background

Coronaviruses are a large family of viruses known to cause the common cold and more serious diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). The novel coronavirus "2019-nCoV" is a new strain of coronavirus which has never been found in human bodies before, the disease caused by 2019-nCoV is named as "COVID-19" by WHO, the international committee for virus classification announces that the formal classification of the novel coronavirus is SARS-CoV-2, namely "Severe acute respiratory syndrome coronavirus 2".

Coronaviruses were first discovered in the 60's of the 20 th century and are the longest sequence RNA viruses known in nature with single strand positive gene sequences as long as 26-32kb capable of causing infections of the respiratory, digestive and nervous systems of humans and animals. Coronavirus infection is very common in the world, about 10% -30% of people in winter are caused by coronavirus, and the coronavirus is the 2 nd cause of common cold, and the infection rate of children is high. Coronavirus is also an important pathogen for acute exacerbation of adult chronic tracheitis patients. Coronaviruses occur mainly in winter and early spring, and coronaviruses can cause stealth infections in the population, which further promote latent transmission. Part of coronaviruses can cause wide spread and high mortality, researches show that the mortality rate of pneumonia caused by SARS virus in mainland China in 2003 is 7%, the mortality rate caused by MERS virus in 2012 is higher than that of SARS, and the current 2019-nCoV causes millions of people to be infected globally, and the mortality rate is about 2%.

Influenza viruses belong to the family orthomyxoviridae in the taxonomic group of viruses, the genus influenza, single-stranded negative-strand RNA viruses. Influenza virions are generally spherical or filamentous, with spherical virus diameters of 80-120 nm. The viral genome consists of single-stranded RNA fragments of 8 negative strands. Influenza viruses are classified into 3 types in total according to the difference in antigenicity between the viral Nucleoprotein (NP) and the Matrix Protein (MP): influenza A virus is also called A type, influenza B virus is also called B type, influenza C virus is also called C type. Among them, influenza a virus has the strongest antigenic variability, is susceptible to infection of human, pig, horse, poultry and other animals, is easy to cause moderate and severe diseases, can attack all age groups of people, and often causes a worldwide pandemic. The influenza A virus can be divided into 15 subtypes according to the difference of hemagglutinin antigenicity (H) on the surface of the influenza A virus, namely H1-H15; the virus can be divided into 9 subtypes according to the Neuraminidase Antigenicity (NA) of the virus surface glycoprotein, namely N1-N9; the two can be combined freely, 135 subtypes of influenza A virus can be combined at most, the main influenza A viruses which are spread in human and have caused the global pandemic are H1N1 subtype, H2N2 subtype and H3N2 subtype, and the prevalence of H9N2 subtype and H5N1 subtype in human is found in recent years. Influenza a viruses undergo major variation approximately every ten years. Influenza viruses that once led to a pandemic worldwide were all influenza a viruses 3 times.

Influenza causes a far greater number of deaths per year in humans than other viruses. According to the World Health Organization (WHO) estimates that seasonal influenza can cause 300-. In 2009, severe influenza a H1N1 occurred in the united states and mexico, with 5900 million americans infected, 26.5 million hospitalized, and 1.2 million deaths, when WHO characterized it as a global emergency public health emergency 1 st time. The overall mortality rate for this event is about two ten-thousandths, with hospitalized patients having a mortality rate of 4.5%. Recently, according to the latest estimation published by the american centers for disease prevention and control in recent years, in 9 months from 2019 to the present, at least 3400 million people in the united states are infected with influenza, 35 million people are hospitalized, the number of deaths reaches 2 million, and the mortality of inpatients reaches 5.5%. Data have shown that an average of 2.7 to 7 million deaths due to influenza occur annually in the united states.

Coronavirus and influenza viruses share many similarities in viral structure, variability, transmission pathways, post-infection clinical symptoms, and the like. Structurally, the coronavirus particle is coated with a fat membrane, and the surface of the membrane has three glycoproteins: spike glycoprotein (S protein), a receptor binding site, cytolytic effect and a major antigenic site; the small envelope glycoprotein (E protein) is small and is a protein that binds to the envelope; membrane glycoproteins (M proteins) responsible for transmembrane transport of nutrients, budding release of nascent viruses and formation of viral envelope; a few classes also have hemagglutinin esterase glycoproteins (HE proteins). The envelope of influenza virus contains two important glycoproteins: hemagglutinin (HA) and Neuraminidase (NA), which highlight viral in vitro, are called spike proteins; the hemagglutinin is a receptor binding site, can assist the mutual fusion of the virus envelope and the host cell membrane, and has immunogenicity; neuraminidase can hydrolyze sialic acid, cut off the connection between virus and host cells to enable the virus to be released smoothly, and continuously infect next host cell, and the protein is an action target of influenza therapeutic drugs. Therefore, the coronavirus and the influenza virus both contain spike proteins which are receptor binding sites, can assist mutual fusion of virus envelopes and host cell membranes, and have immunogenicity, so that the coronavirus and the influenza virus are important targets for designing therapeutic drugs, and the coronavirus and the influenza virus have certain targeting property. Secondly, studies have demonstrated that all coronaviruses and influenza viruses have a high rate of genetic variation, and that viral genes can recombine when 2 different genotypes of virus strain infect the same cell. In theory, common coronaviruses currently circulating worldwide or coronaviruses infecting only some animals, as well as various types of influenza viruses, there may be recombinant variation of viral genes to produce new strains, leading to unpredictable epidemic host range and pathogenicity, and outbreak and wide spread of new coronaviruses in the present year.

After infection with influenza virus, most of the cases are mild, and influenza vaccine inoculation is still the most effective means for preventing influenza. The treatment of patients who have not been vaccinated or have not acquired immunity after vaccination can be divided into general treatment, antiviral treatment, traditional Chinese medicine treatment and the like. The high risk group of influenza is easy to cause severe influenza, and antiviral treatment should be carried out as early as possible to relieve symptoms and reduce the fatality rate. The existing commonly used anti-influenza virus medicines mainly comprise neuraminidase inhibitors such as oseltamivir, zanamivir and peramivir, are effective to influenza A and B, and can obviously reduce severe influenza and mortality when being taken in early influenza, especially within 48 hours of morbidity. However, influenza virus is very easy to be mutated, no specific medicine effective to all types of influenza exists at present, and the development speed of the vaccine is far behind the virus mutation speed, so that the influenza vaccine cannot prevent the influenza virus by 100 percent.

However, the research on the prevention and treatment drugs aiming at the new coronavirus mainly focuses on three directions: vaccine research, small molecule chemical drug research and traditional Chinese medicine research. The most studied is still focused on chemical drugs, and there are three major classes of targets currently designed and screened as chemical drugs against new coronaviruses: firstly, the virus enters the cell stage, key proteins mediating the invasion process such as virus receptor S protein and host cell receptor angiotensin converting enzyme 2(ACE2) can be used as target proteins, and antibodies and vaccines also act in the stage and the region; the second is the viral replication stage, involving the synthesis of viral nucleic acid RNA, translation and processing of proteins, etc., wherein RNA polymerase (RdRp), which is essential for RNA replication, and proteases, such as 3-chymotrypsin-like protease (3Clpro) and papain-like protease (Plpro), which process viral proteins, are effective antiviral drug targets; the virus release stage is related to the assembly and budding of virus particles, and few reports are reported in the literature about the molecular mechanism of the coronavirus release process. However, up to now, there is no specific therapeutic agent for this new coronavirus.

Considering that the nuclear technology has wide application in modern medicine, the nuclear technology has become an indispensable means for diagnosis and treatment of diseases and medical research in clinical work, and is an important method for solving three major diseases of heart, cerebral vessels and tumors at present. Under the situation that seasonal outbreak of influenza exists, the new coronavirus continuously spreads all over the world at this time and no special-effect medicine exists in 2019-nCOV, the invention provides a new nuclear technology method for treating the diseases caused by the new coronavirus such as the coronarism and the influenza or the coronavirus such as the influenza by using a radionuclide to mark molecules of a targeted virus spike protein and killing the influenza virus with the spike protein and the coronavirus such as the 2019-nCOV by means of the ray energy of the radionuclide.

So far, in the case that a novel coronavirus is continuously spread in the world, no specific therapeutic drug exists, a large number of death cases are caused by seasonal outbreak of influenza viruses every year, but no specific drug effective for various influenza viruses exists, further intensive research, development of new therapeutic drug research and development ideas, and development of all available resources into broad-spectrum antiviral specific drugs for the coronavirus or influenza virus such as 2019-nCOV are urgently needed, so that the spreading and prevention of the novel coronavirus and the influenza virus are met, and the challenge of possible variant coronavirus and influenza virus on the public health safety of human beings in the future is met.

Disclosure of Invention

It is well known that ultraviolet light can kill viruses, and studies have shown that 2019-nCOV and influenza viruses are also sensitive to ultraviolet light. Ultraviolet radiation is electromagnetic radiation with a wavelength shorter than visible light but longer than X-ray, and has an energy of 3eV-124eV, and ultraviolet radiation can destroy the RNA structure of viruses, so that the viruses lose the ability to produce proteins and the ability to reproduce, thereby killing the viruses. In addition, in order to kill all viruses in the irradiation range, it takes a certain time for ultraviolet rays to kill viruses, and people are not allowed to enter during killing to avoid damage. The radioactive nuclide has far higher ray energy than ultraviolet ray, and the energy generated by one photon of gamma ray or X ray generated by the decay of some radioactive nuclides has the capability of destroying chemical bonds between molecules, so that the radioactive nuclide has great potential for killing viruses.

Therefore, if a proper radionuclide is labeled on a proper molecule targeting coronavirus such as 2019-nCOV or influenza virus, the radiation generated by radionuclide decay can directly cause the breakage of a virus RNA chain, and in addition, free radicals generated by the radiation ionization effect can indirectly cause the breakage of the RNA chain by the action of groups on the virus RNA chain, so that the coronavirus such as 2019-nCOV and the influenza virus are killed in the range of virus particles (the diameter of the virus particles is about 100nm), and diseases caused by the coronavirus such as new coronary pneumonia and the influenza virus are treated. Meanwhile, the radionuclide is marked on the molecules targeting new coronavirus or influenza virus to kill the virus, which is similar to tumor targeting treatment, most of ray energy generated by radionuclide decay is deposited on viral protein or RNA in a targeting manner to destroy and inactivate the virus structure, so that the required dosage is low, and the human body cannot be damaged. At present, the method for treating the diseases caused by the coronavirus such as new coronary pneumonia, influenza and the like or the influenza virus by using the radioactive treatment medicine has no relevant report at home and abroad.

Firstly, a proper radionuclide needs to be selected, and nuclides suitable for radiotherapy of diseases caused by new coronavirus or influenza virus such as coronary pneumonia and influenza have the following characteristics: the ray energy is moderate, so that the target spot is ensured to be irradiated with enough dose, and meanwhile, the normal tissue is less damaged; ② half-life period (T)_1/2) The physical half-life of the therapeutic nuclide is appropriate, and is generally ideal from several days to dozens of days, so that the preparation of a subsequent marker is convenient; the production and preparation are convenient, the material is easy to obtain in time and the price is low; and fourthly, the chemical property is active so as to easily mark other substances.

Based on the full research, we found that Auger electron emission is obtained after comparing the properties of various treatable nuclides¹²⁵I substantially meets the above requirements.¹²⁵I is one of the first radionuclides used for disease treatment, the decay of which is divided into two steps, the first step is electron capture and the second step is internal conversion, in the course of the two steps, equal amounts of auger electrons are respectively emitted, 22 are provided, the energy of auger electrons is 20-500eV, the range in biological tissues is 1-10nm, LET is 10-25 keV/mum, compared with gamma ray, the biological effect in vivo is higher, and the toxic and side effects are smaller. In addition, the first and second substrates are,¹²⁵i also emits X-rays of 27.5keV energy and gamma-rays of 35.5keV energy, but within the organism¹²⁵I Immunoactive irradiation of most proteins, primarily by Auger electron-released energy to kill the targetThe damage influence is small, the stability of the marker is good, and meanwhile, the radiation damage to tissues and organs is small.¹²⁵Long half life I (T)_1/260.14 days), and can be produced at home at present, and is cheap and easy to obtain. Thus, it is possible to provide¹²⁵I is one of the best nuclides for labeling antibodies and small molecule compounds for radiotherapy. At the same time, the radionuclide with the most nuclear medicine application and the best clinical treatment effect is one of the radionuclides, and at present¹²⁵The I is applied in a large amount in tumor treatment in the form of a particle source, and achieves good effect.

Secondly, after the radiotherapeutic nuclides are determined, molecules with high targeting to 2019-nCOV and other coronavirus and influenza virus need to be screened out to serve as vectors, which is another key factor for successfully treating diseases caused by new coronavirus such as coronary pneumonia and influenza by utilizing the radionuclides. The targeting molecule suitable for treating diseases caused by coronavirus such as new coronary pneumonia and influenza radiotherapy has the following characteristics: targeting new coronavirus or influenza virus protein, and having strong binding property with protein molecules; the targeting property of the virus to new coronavirus or influenza virus is not changed or is slightly changed after the virus is marked; and molecules which have been shown to inhibit new coronavirus or influenza virus, preferably, to accelerate the development of radiopharmaceuticals.

After systematic research and analysis of many reported molecules with potential activity to coronavirus and influenza virus, we believe that biological macromolecules, such as monoclonal antibodies, small molecule polypeptides and chemical drugs, capable of targeting coronavirus and influenza virus are all protected by¹²⁵I, and the like. The monoclonal antibody and the polypeptide are bioactive molecules, have unique targeting property, can prevent diseases from happening and progressing, can accurately activate the microcosm of a human immune system to resist the diseases, is the direction researched and developed firstly after the new crown epidemic situation outbreak, and can protect human cells from being invaded by specifically combining with new crown viruses and inhibiting the activity of the viruses. The most studied small molecule chemical drugs can act on different stages of virus entering human body, and during this new crown epidemic situation, a plurality of research teams have screened out a plurality of small chemical drugsMolecular drugs, in part, have entered clinical trials.

Therefore, under the situation that the new coronavirus does not have specific therapeutic drugs and a great amount of death is caused by seasonal global outbreak of influenza, the drug molecules which are screened by various research teams and have an inhibiting effect on the coronavirus such as the new coronavirus or the influenza virus are used as target molecules, the target molecules are labeled with radionuclide, the coronavirus or the influenza virus is killed by utilizing rays generated by decay of the radionuclide, broad-spectrum special-effect radioactive therapeutic drugs aiming at the coronavirus such as the new coronavirus or the influenza virus are developed, and a nuclear technology new method is provided for treating diseases such as the new coronaviruses and the influenza and has important significance.

On the basis of the research, the invention provides the following technical scheme:

a radiotherapeutic agent for a disease caused by a coronavirus or an influenza virus, comprising a targeting molecule for targeting a coronavirus or an influenza virus protein, the targeting molecule being labelled with a radionuclide.

In some embodiments, the targeting molecule is selected from the group consisting of small molecule compounds, polypeptides, and proteins.

In some embodiments, the radionuclide includes, but is not limited to¹²⁵I、¹²³I、¹³¹I、⁶⁷Ga、¹¹¹In、²⁰¹Tl、¹⁹¹Pt、¹¹⁹Sb、¹⁶¹Tb、¹⁷⁷Lu、⁸⁹Zr、⁷⁷Br、⁸⁹S、⁹⁰Y、³²P、¹⁵³Sm and¹⁸⁸any one of Re or a combination thereof.

In some embodiments, the radionuclide is selected from¹²⁵I、¹²³I、¹³¹I、⁶⁷Ga、¹¹¹In、²⁰¹Tl、¹⁹¹Pt、¹¹⁹Sb、¹⁶¹Tb、¹⁷⁷Lu、⁸⁹Zr、⁷⁷Br、⁸⁹S、⁹⁰Y、³²P、¹⁵³Sm and¹⁸⁸any one of Re or a combination thereof.

In some embodiments, the small molecule compounds include, but are not limited to, abidol, ridciclovir, favipiravir, chloroquine phosphate, hydroxychloroquine, and analogs thereof; the polypeptides include, but are not limited to HR2L, HR2P, P1, CP-1, LCB1, LCB3, AHB2, SBP1 and variants thereof, including, but not limited to, coronavirus and influenza glycoprotein antibodies SARS-CoV-2SpikeRBDNanobody, SAntibody, neocorona nano-neutralizing antibody R173C1, neocorona antibody k79d9, S309, B38, H4, LY-CoV555, STE90-C11, influenza virus antibodies HA antibodiy, NP antibiody, PA antibiody and variants thereof.

In some embodiments, the small molecule compound is selected from the group consisting of arbidol, ridiflovir, favipiravir, chloroquine phosphate, hydroxychloroquine, and analogs thereof, the polypeptide is selected from the group consisting of HR2L, HR2P, P1, CP-1, LCB1, LCB3, AHB2, SBP1, and variants thereof, and the protein is selected from the group consisting of coronavirus and influenza glycoprotein antibodies SARS-CoV-2SpikeRBDNanobody, SAntibody, neocorona nano-neutralizing antibody R173C1, neocorona antibody k79d9, S309, B38, H4, LY-CoV555, STE90-C11, influenza virus antibodies HA antibody, NP antibody, PA antibody, and variants thereof.

In some embodiments, the amino acid sequence of HR2L is SIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKELGNYTYYNKW (SEQ ID NO: 1);

in some embodiments, the amino acid sequence of HR2P is SLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKEL (SEQ ID NO: 2);

in some embodiments, the amino acid sequence of P1 is LTQINTTLLDLTYEMLSLQQVVKALNESYIDLKEL (SEQ ID NO: 3);

in some embodiments, the amino acid sequence of CP-1 is GINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYE (SEQ ID NO: 4);

in some embodiments, the amino acid sequence of LCB1 is DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAERLLEEVER (SEQ ID NO: 5);

in some embodiments, the amino acid sequence of LCB3 is NDDELHMLMTDLVYEALHFAKDEEIKKRVFQLFELADKAYKNNDRQKLEKVVEELKELLERLLS (SEQ ID NO: 6);

in some embodiments, the amino acid sequence of AHB2 is ELEEQVMHVLDQVSELAHELLHKLTGEELERAAYFNWWATEMMLELIKSDDEREIREIEEEARRILEHLEELARK (SEQ ID NO: 7);

in some embodiments, the amino acid sequence of SBP1 is IEEQAKTFLDKFNHEAEDLFYQS (SEQ ID NO: 8).

In some embodiments, the variant refers to a sequence as described above that contains one or more amino acid substitutions, deletions or insertions, and the variant is also capable of targeting coronavirus and influenza virus proteins.

In another aspect, the present invention provides a method for preparing the radiotherapeutic medicament, comprising labeling a radionuclide on a targeting molecule targeting a coronavirus or influenza virus protein.

In some embodiments, the labeling methods include the chloroglycoluril (Iodogen) method, the chloramine T method, the Lactoperoxidase (LPO) method, and the acylating reagent (Bolton and Hunter reagents) method.

In some embodiments, the chloroglycoluril process comprises: adding target molecules to be marked and a radionuclide solution into a reaction container with the inner wall coated with the chloroglycoluril, and reacting at room temperature for 1-60min to obtain the radionuclide-marked target molecules.

In some embodiments, a method of coating chloroglycoluril on the interior wall of a reaction vessel comprises: dissolving the chloroglycoluril in a volatile organic solvent to prepare a solution, transferring the prepared solution into a reaction container, drying the solution by nitrogen to form a uniform chloroglycoluril film on the inner wall of the reaction container, wherein the organic solvent is dichloromethane or chloroform, the concentration of the solution is 0.04-1mg/mL, and the content of the chloroglycoluril on the inner wall of the reaction container is 5-50 mu g, preferably 10-50 mu g, 10-40 mu g, 10-30 mu g, 10-20 mu g, 20-30 mu g, 30-40 mu g, such as 10 mu g, 20 mu g, 30 mu g or 40 mu g.

In some embodiments, the labeling method of the small molecule compound further comprises reacting the small molecule compound, the catalyst auxiliary, the reaction auxiliary and the reducing agent in an organic solvent at 30-60 ℃ overnight under an inert gas atmosphere, and removing the solvent after cooling the reaction mixture and purifying to obtain a precursor compound;

dissolving the precursor compound in methanol to prepare a solution, adding the prepared methanol solution and acetic acid into a reaction container coated with chloroglycoluril, uniformly mixing, adding a solution of radionuclide, shaking for 2-10min, and then removing the reactant to terminate the reaction; purifying the reaction product to obtain the final product of the radionuclide.

In some embodiments, the inert gas is nitrogen.

In some embodiments, the catalyst includes, but is not limited to, palladium acetate, palladium on carbon, bis (benzonitrile) palladium dichloride, bis (triphenylphosphine) palladium dichloride, tetrakis (triphenylphosphine) palladium, [1, 1-bis (diphenylphosphino) ferrocene ] palladium dichloride, and bis (tri-tert-butylphosphino) palladium.

In some embodiments, the catalyst is selected from the group consisting of palladium acetate, palladium on carbon, bis (benzonitrile) palladium dichloride, bis (triphenylphosphine) palladium dichloride, tetrakis (triphenylphosphine) palladium, [1, 1-bis (diphenylphosphino) ferrocene ] palladium dichloride, and bis (tri-tert-butylphosphino) palladium.

In some embodiments, the catalyst is palladium acetate.

In some embodiments, the catalytic adjunct includes, but is not limited to, triphenylphosphine, tricyclohexylphosphine, and 2,2 '-bis (diphenylphosphino) -1,1' -binaphthyl.

In some embodiments, the catalytic adjuvant is selected from triphenylphosphine, tricyclohexylphosphine, and 2,2 '-bis (diphenylphosphino) -1,1' -binaphthyl.

In some embodiments, the catalyst adjunct is 2,2 '-bis (diphenylphosphino) -1,1' -binaphthyl.

In some embodiments, the reaction promoter is preferably tetramethylethylenediamine.

In some embodiments, the reducing agent is preferably sodium borohydride.

In some embodiments, the organic solvent is selected from tetrahydrofuran, 1, 4-dioxane, and diethylene glycol dimethyl ether.

In some embodiments, the radionuclide-labeled targeting molecule is purified using a chromatography column.

In some embodiments, methods of labeling polypeptides and proteins comprise: and sequentially adding polypeptide or protein, PBS buffer solution and radionuclide solution into a reaction container with the inner wall coated with chloroglycoluril, and reacting at room temperature for 1-60min to obtain polypeptide or protein labeled by the radionuclide, wherein the polypeptide and the protein contain tyrosine groups.

In some embodiments, the small molecule compound is preferably abidol or an analog thereof.

In some embodiments, the radionuclide is preferred¹²⁵I。

In some embodiments, of abidol or analogs thereof¹²⁵The preparation route of the I marker is as follows:

in particular, in some embodiments, of abidol or an analog thereof¹²⁵I or¹³¹The I marking method comprises the following steps: under nitrogen atmosphere, Arbidol or its analogue, catalyst palladium acetate (Pd (OAc)₂) And 2,2 '-bis (diphenylphosphino) -1,1' -Binaphthyl (BINAP), Tetramethylethylenediamine (TMEDA), and sodium borohydride (NaBH)₄) Reacting in an organic solvent at 30-60 ℃ overnight, cooling the reaction mixture, removing the solvent and purifying to obtain a precursor compound;

dissolving the precursor compound in methanol to obtain a solution, adding the solution and acetic acid into a reaction vessel coated with chloroglycoluril (Iodogen), mixing, and adding a solution containing chloroglycoluril¹²⁵I^-Or¹³¹I^-Shaking the solution for 2-10min, and removing the reactant to terminate the reaction; purifying the reaction product to obtain¹²⁵I or¹³¹I labeled final product.

In some embodiments of the present invention, the substrate is,¹²⁵the preparation route of the I-labeled polypeptide or protein is shown as follows:

in particular, in some embodiments, of the polypeptide or protein¹²⁵I or¹³¹The I marking method comprises the following steps:

adding polypeptide or protein, PBS buffer solution and¹²⁵I^-or¹³¹I^-Reacting the solution at room temperature for 1-60min, removing the reaction solution from the reaction tube or adding buffer solution for dilution to stop the reaction to obtain the product¹²⁵I or¹³¹I labeled polypeptide or protein.

In some embodiments, the chromatography column is selected from a Sep-pak C18 column and a PD-10 column.

A pharmaceutical composition comprising said radiotherapeutic agent and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a method for preparing a stable therapeutic agent corresponding to a radiotherapeutic agent, comprising binding a corresponding stable nuclide of a radionuclide, preferably iodine, to a targeting molecule targeting a coronavirus or influenza virus protein, characterized in that the small molecule precursor compound, the iodinating agent, and the reaction solvent are reacted at a reaction temperature of 10-50 ℃, preferably 20-25 ℃, for 12-96 hours, preferably 72 hours, and the reaction mixture is purified by a silica gel column to obtain the corresponding stable compound.

In some embodiments, the iodinating agent includes, but is not limited to, N-iodosuccinimide, iodine chloride, elemental iodine, sodium iodide, potassium iodide, and tert-butoxy iodide.

In some embodiments, the iodinating agent is selected from the group consisting of N-iodosuccinimide, iodine chloride, elemental iodine, sodium iodide, potassium iodide, and tert-butoxy iodide.

In some embodiments, the iodinating agent is N-iodosuccinimide.

In some embodiments, the reaction solvent includes, but is not limited to, trifluoroacetic acid, trifluoroethanol, hexafluoroisopropanol, dimethylformamide, dimethyl sulfoxide, and pyridine.

In some embodiments, the reaction solvent is selected from trifluoroacetic acid, trifluoroethanol, hexafluoroisopropanol, dimethylformamide, dimethyl sulfoxide, and pyridine.

In some embodiments, the reaction solvent is trifluoroacetic acid.

In some embodiments, the SARS-CoV-2Spike RBD Nanobody is an Antibody targeting the S protein RBD region of SARS-CoV-2, S Antibody is an Antibody targeting the S protein of SARS-CoV-2, R173C1 and R175a2 are neo-crown nano-neutralizing antibodies targeting the S protein RBD region of SARS-CoV-2, k79d9 is an Antibody targeting the S protein RBD region of SARS-CoV-2, S309 is an Antibody targeting the S protein RBD region of SARS-CoV-2 and the S protein RBD region of SARS-CoV, B38, H4, LY-CoV555, and STE90-C11 are antibodies targeting the S protein RBD region of SARS-CoV-2. HA Antibody is an Antibody targeting HA protein of influenza virus, NP Antibody is an Antibody targeting NP protein of influenza virus, and PA Antibody is an Antibody targeting PA protein of influenza virus.

Has the advantages that:

research shows that small molecular compounds, polypeptides, monoclonal antibodies and other targeting molecules screened by various research teams in vitro cell experiments can effectively inhibit new coronavirus and other coronavirus and influenza virus, and obviously inhibit the pathological effect of the viruses on cells. But the clinical application has the problems of overlarge administration dosage, stronger toxic and side effect on human bodies, limited curative effect on new coronavirus or influenza virus and the like. After the molecules of the targeted coronavirus or the influenza virus are labeled by the radioactive marker provided by the invention, the new coronavirus and other coronavirus and the influenza virus are killed by mainly utilizing the ray energy of the radioactive nuclide, so that the problems of overlarge administration dosage and strong toxic and side effects in the current clinical application can be solved.

Drawings

FIG. 1 shows nuclear magnetism of compound CIAE-001 in example 1 of the present invention¹HNMR) spectrum;

FIG. 2 is a liquid mass (LC-MS) spectrum of CIAE-001 of the compound in example 1 of the present invention;

FIG. 3 shows the affinity of Arbidol for RBD (sensorgram: left, fitted graph: right);

FIG. 4 shows the affinity of CIAE001-1 for RBD (sensorgram: left, fitted graph: right).

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments.

In the description of the present invention, reference to "one embodiment" means that a particular feature, structure, or parameter, step, or the like described in the embodiment is included in at least one embodiment according to the present invention. Thus, appearances of the phrases such as "in one embodiment," "in one embodiment," and the like in this specification are not necessarily all referring to the same embodiment, nor are other phrases such as "in another embodiment," "in a different embodiment," and the like. Those of skill in the art will understand that the particular features, structures or parameters, steps, etc., disclosed in one or more embodiments of the present description may be combined in any suitable manner.

1. Feasibility analysis of virus killing by radioactive rays

First, the radioactive nuclide has much higher energy than ultraviolet ray, and the gamma ray or X ray generated by the decay of some radionuclides has the ability to break intermolecular bonds, and research shows that the bond energy of RNA chain is generally 3-11eV, and that when the energy deposited on the particle is greater than 12.65eV, one chain is broken, so that the radionuclides have great potential to kill virus. Most of Auger electrons have energy of 20-500eV, range of 1-10nm in biological tissues and LET of 10-25 keV/mum, and compared with gamma rays or X rays, the Auger electrons have high LET, higher in vivo biological effect and smaller toxic and side effects, so that the Auger electrons have better effect of killing viruses.

Secondly, although the mechanism of virus inactivation by using the radiotherapy medicine is the same as that of radiation sterilization, the required radiation dose is different, and the radiation sterilization is to kill all bacteria and viruses in a certain space, because the farthest end even a blocked position is required to reach the killing dose to ensure complete killing, the apparent dose is very high. Meanwhile, because the targeting is not available, most of the radiation dose is not deposited on bacterial viruses, so that the required dose is further increased, and the dose is generally 15-50 kGy. Studies have shown that doses of 25-40kGy can inactivate many viruses and mycoplasma in animal sera. The silkworm grape spot virus can be completely eliminated when the silkworm grape spot virus is irradiated by gamma rays and reaches 10 kGy. The process of labeling radionuclide on the molecule targeting new coronavirus or influenza virus to kill virus is similar to tumor targeting therapy, and most of the radioactive ray energy generated by radionuclide decay is deposited on virus protein or RNA in a targeting mode to destroy and inactivate virus structures, so that the required dosage is low.

¹²⁵I is one of the earliest radionuclides used in disease treatment,¹²⁵the decay is divided into two steps, the first step is electron capture, the second step is internal conversion, and the two steps respectively emit equal amount of auger electrons with 22, total energy of auger electrons is about 3.1keV, range is less than 10nm, and diameter is far less than that of new corona virus (about 100 nm). Therefore if will¹²⁵I is marked on a molecule targeting the surface spike protein (with the diameter of about a few nanometers) of the new coronavirus or influenza virus¹²⁵The I decay directly occurs on the virus spike protein, and almost all the energy of auger electrons is deposited on the protein, which can cause the breakage of hundreds of chemical bonds (when the energy is more than 12.65eV, one chain is broken), thus causing the serious damage of the structure of the virus spike protein, and finally causing the virus to lose the capacity of combining with the cell membrane of the human body and not to invade the human body.

The literature indicates that it is not possible to identify,¹²⁵i Auger electron absorption dose of each decay is 0.74-100Gy, and 1Gy absorption dose can cause 1000 DNA single strand breaks and 40 DNA double strand breaks, calculated¹²⁵Auger electron of I decay 7.4 × 10²-10⁵The single DNA strands are broken. Based on this calculation, theoryOn the upper part¹²⁵Auger electrons of each decay of I can be 7.4 × 10²-10⁵Breaking of viral RNA single strands, i.e.¹²⁵The I Auger electron can decay 7.4 × 10 per time²-10⁵Death of individual virus, 10¹¹The virus of copy number only needs 10⁶-1.35×10⁸Can be completely killed by secondary decay¹²⁵I the rate of decay occurring per day is about 1%, i.e. 10⁸-10¹⁰Is/are as follows¹²⁵Number of molecules of I, about 1 nCi-0.1. mu. Ci¹²⁵The I marker drug is reached, and therefore, the micro-population effective dose is inferred to break the RNA link of the virus, so that the virus loses activity.

According to the anatomical report data of SARS virus infected patient, the load capacity of SARS virus in human lung is averagely 10⁸Copy/g, average adult lung weight of 1kg, so that the total amount of SARS virus in lung tissue is 10¹¹The amount of the virus in other organs and tissues is much lower than that in lung, so the total amount of SARS virus in human is estimated to be 10¹¹Copy left and right. We therefore hypothesized that the total peak amount of new coronavirus or influenza virus in humans is also 10¹¹Copies were made, and by calculation we obtained a radioactivity of 1. mu. Ci¹²⁵The number of molecules of I is 10¹¹The number of the main components is one,¹²⁵i half-life of 60.14 days, a decay rate of about 1% per day, and a radioactivity of 0.1mCi¹²⁵I all viruses in the body can be killed within one day (assuming¹²⁵I each decay may result in inactivation of one virus). Microscopically decaying daily if the dose administered is set to 1-10mCi¹²⁵The number of I molecules can reach 10¹²-10¹³And the number of the new coronavirus or influenza virus is far more than that of the new coronavirus or influenza virus, even if only one percent (only 1 percent of the dose in the radioimmunotherapy can reach the target site) of the administration dose can reach the target organ after the medicine enters the body and is subjected to distributed metabolism, the new coronavirus or influenza virus can be killed theoretically. Meanwhile, in combination with the theory of recent research, it is thought that the virus binds to hemoglobin in blood, and thus the administration by intravenous injection further reduces the administration dose. Comprehensive analysis, application of milliCure level¹²⁵I radiopharmaceuticals to kill new coronavirusesOr influenza virus, is theoretically possible.

In addition, the first and second substrates are,¹²⁵i also emits X-rays of 27.5keV energy and gamma rays of 35.5keV energy while decaying to release auger electrons. During the transmission process of the gamma rays and the X rays, partial energy is transferred through reactions such as ionization, excitation and the like with molecules of a surrounding medium, and partial energy is deposited on the RNA chain of the virus, so that the RNA chain of the virus can be directly broken. In addition, the gamma rays interact with water molecules in the surrounding medium, so that the water molecules are ionized to generate free radicals, and the free radicals interact with bases or sugar phosphates on the RNA chain to indirectly cause the breakage of the RNA. In summary, it is feasible to use the energy of the radioactive nuclide to kill new coronavirus or influenza virus.

2. Radiation dose analysis of normal tissue for antiviral treatment

Since there is no current¹²⁵The effective dose of the I-labeled small molecular compound in a human body can be used as a reference, so that the effective dose is selected¹³¹The effective dose of the I-labeled small molecular compound o-iodohippuric acid in human body is used as a reference¹²⁵I effective dose of labeled small molecule compound produced in vivo is estimated. According to the data of the clinical nuclear medicine patient protection requirement of the national health industry standard WS 533 plus 2017,¹³¹the effective dose of I-o-iodohippuric acid in vivo is 5.2 multiplied by 10^-2mSv/MBq, at a dose of 0.1mCi¹³¹An effective in vivo dose of 0.19mSv for I-o-iodohippuric acid, whereby an estimated dose of 0.1mCi¹²⁵The effective dose of I-abidol produced in vivo in humans will be less than 0.19 mSv. The effective dose is far lower than the annual dose limit value of residents by 1mSv, so that radiation damage to human bodies is avoided.

Example 1 preparation of CIAE-001

1. Experimental reagents and instruments

Arbidol hydrochloride salt: shijiazhuang the fourth pharmaceutical factory; palladium acetate (Pd (O)Ac)₂) Diethylene glycol dimethyl ether (Diglyme), Tetramethylethylenediamine (TMEDA), 2 '-bis (diphenylphosphino) -1,1' -Binaphthyl (BINAP): analytically pure, Bailingwei science and technology Limited; chloroglycoluril (Iodogen): Sigma-Aldrich; na (Na)¹²⁵I: PerkinElmer; other reagents are analytically pure, national drug group chemical reagents, ltd.

2. Experimental methods

A250 mL three-necked flask was charged with nitrogen, to which were added 8.22g (17.2mmol) of Compound 1 (Abidol), 193mg (0.86mmol) of catalytic Palladium acetate (Pd (OAc))₂) 588mg (0.946mmol) of 2,2 '-bis (diphenylphosphino) -1,1' -Binaphthyl (BINAP) and 30mL of dry tetrahydrofuran are stirred at room temperature for 20 min. Then 4mL of Tetramethylethylenediamine (TMEDA) was added, and after stirring at room temperature for 20min, 30mL of dry tetrahydrofuran was added. Then, 52mL of sodium borohydride (NaBH) was slowly added dropwise to the reaction via a constant pressure dropping funnel₄) The solution (0.5M diethylene glycol dimethyl ether (Diglyme)) was left to react overnight at 30-60 ℃. The reaction mixture was cooled, and 40mL of water was added thereto to terminate the reaction. Extraction was performed with ethyl acetate (40 mL. times.3), and the organic phases were combined and dried over anhydrous magnesium sulfate. The drying agent was removed by suction filtration, the solvent was removed by rotary evaporation, and the product, compound 2(622mg, 9% yield) was obtained after silica gel column purification. The pretreatment is to remove bromine atoms in a small molecular structure so as to introduce radioactive iodine and stable iodine atoms into the small molecular structure in the next step, and the reaction has special requirements on the molecular structure, and some small molecules cannot be directly combined with the iodine atoms, so the pretreatment is needed to ensure that the small molecules can be combined with the iodine atoms under certain conditions.

The labeled precursor compound 2 was dissolved in methanol to prepare a solution of 30 mg/mL. To a reaction tube coated with chloroglycoluril (Iodogen) (20. mu.g, 0.5mg/mL), a mild, weak oxidizing agent, in high yield and with little effect on product activity, 5. mu.L of this solution (30mg/mL) was added. Then adding Na thereto¹²⁵Solution I was 4. mu.L (34MBq), and the reaction was stopped by shaking for 4min and removing the reaction. Using a polyamide film as a support, ethyl acetate: TLC analysis using 5:2(v/v) methanol as the developing solvent was calculated to have a labeling rate of 97%.

The reaction mixture was diluted with 1mL of water and then purified using a solid phase extraction column to give Compound 3 (CIAE-001). The radiochemical purity of the purified product, Compound 3(CIAE-001), was calculated to be 98% by analysis by radioactive TLC or HPLC.

In this example, arbidol is the reaction substrate, palladium acetate (Pd (OAc)₂) Is used as catalyst, 2,2 '-bis (diphenylphosphino) -1,1' -Binaphthyl (BINAP) is used as catalytic adjuvant, tetrahydrofuran is used as solvent, Tetramethylethylenediamine (TMEDA) is used as reaction assistant, and sodium borohydride (NaBH)₄) As a reducing agent, diethylene glycol dimethyl ether (Diglyme) as a solvent; chloroglycoluril (Iodogen) is used as an oxidant; na (Na)¹²⁵I is an iodinating reagent used to introduce an iodine atom into the reaction substrate.

Nuclear magnetism of CIAE-001 in FIG. 1 and FIG. 2, respectively¹HNMR) spectrum and liquid mass (LC-MS) spectrum.

Example 2:¹²⁵preparation of I-targeting polypeptide (CIAE-002)

1. Process for preparing polypeptides¹²⁵I marking

Dissolving chloroglycoluril (Iodogen) in dichloromethane or other volatile organic solvent to obtain 0.4mg/mL solution, transferring 100 μ L of the solution into a reaction tube, blowing dry with nitrogen gas to volatilize the solvent and leave a uniformly coated chloroglycoluril film on the wall of the reaction tube, wherein the content of the chloroglycoluril is 40 μ g. The polypeptide structure contains a plurality of amino acids, and the invention selects the polypeptide containing tyrosine, wherein the tyrosine can be directly combined with iodine atoms under certain conditions, so that pretreatment is not needed.

290 mu L of polypeptide HR2L10 mu L, PBS buffer solution and radioactive Na are added into the reaction tube coated with the chloroglycoluril in sequence¹²⁵Solution I (1 μ L, 0.28mCi) is reacted at room temperature for 20min, and the reaction solution is removed from the reaction tube or diluted by adding a buffer solution to terminate the reaction, so as to obtain the product (CIAE-002). Samples were taken and the labeling rate was 96% by radioactive thin layer scanner or HPLC.

2. Separating and purifying

2.1 chromatographic column pretreatment

And taking a Sep-pak C18 solid phase extraction chromatographic column, sequentially leaching with 10mL of absolute ethyl alcohol and 10mL of pure water, and draining the liquid in the chromatographic column for later use.

2.2 separation and purification of CIAE-002

Adding the marked sample solution into the pretreated chromatographic column, adding pure water to rinse and remove free substances¹²⁵I, and discharging the liquid in the dry chromatographic column. Then adding 80% ethanol solution for leaching, draining liquid in the chromatographic column, and collecting eluent to obtain the final product¹²⁵I-targeting polypeptide (CIAE-002), samples were taken and the radiochemical purity of the marker was determined to be 98% by radioactive thin layer scanner or HPLC.

Example 3:¹²⁵preparation of I-glycoprotein antibody (CIAE-003)

Dissolving the chloroglycoluril in dichloromethane or other volatile organic solvent to prepare 0.4mg/mL solution, transferring 50 mu L of the solution into a reaction tube, drying the solution by blowing nitrogen to volatilize the solvent and leave a uniformly coated chloroglycoluril film on the wall of the reaction tube, wherein the content of the chloroglycoluril is 20 mu g. The glycoprotein antibody SARS-CoV-2Spike RBDNanobody, PBS buffer solution and radioactive Na are added into the reaction tube coated with the chloroglycoluril in sequence¹²⁵I solution (Na)¹²⁵I concentration in the reaction solution is not lower than 0.1mCi/mL), reacting at room temperature for 20min, removing the reaction solution from the reaction tube or adding buffer solution for dilution to stop the reaction to obtain¹²⁵I-labeled glycoprotein antibody (CIAE-003). Samples were taken and the labeling rate was 65% by radioactive thin layer scanner or HPLC.

2. Separating and purifying

2.1 chromatographic column pretreatment

Taking 1 PD-10 column (gel filtration chromatographic column), cutting off the seal at the lower end of the column to enable liquid in the column to naturally drip, then adding 25mL of 0.01mol/L PBS buffer solution (pH 7.4) for sufficient leaching, adding 0.3mL of 10% BSA after no liquid is accumulated on the column surface, and continuing adding 5mL of 0.01mol/L PBS buffer solution (pH 7.4) for leaching to balance for later use after no liquid is accumulated on the column surface.

2.2 isolation and purification of CIAE-003

Adding the marked sample solution into a pretreated PD-10 column, adding 0.01mol/L PBS solution for leaching after no liquid is accumulated on the column surface, and collecting eluent to obtain the target product¹²⁵The radiochemical purity of the I-glycoprotein antibody (CIAE-003), as determined by radioactive thin layer scanner or HPLC, was 98%.

Example 4 preparation of CIAE-001-1

A25 mL single-necked flask was charged with 750mg (1.88mmol) of compound 2(4- (dimethylaminomethyl) -5-hydroxy-1-methyl-2- (phenylthiomethyl) -1H-indole-3-carboxylic acid ethyl ester) and 6mL of trifluoroacetic acid (TFA), and 528mg (2.07mmol) of N-iodosuccinimide (NIS) as a solid were added in portions. The reaction is stirred for 48 to 72 hours at room temperature. The reaction solution was transferred to a separatory funnel containing 10mL of cold water, extracted with dichloromethane (5 mL. times.3), and the organic phases were combined and treated with 10% sodium metabisulfite (Na)₂S₂O₅) The aqueous solution was washed (15 mL. times.2), and dried over anhydrous magnesium sulfate overnight. The desiccant anhydrous magnesium sulfate was removed by suction filtration, the solvent was removed by rotary evaporation, and the residue was purified by silica gel column to give CIAE-001-1 (stable iodo-CIAE-001) (6.5mg, yield 0.66%).

Trifluoroacetic acid (TFA) as a solvent; n-iodosuccinimide (NIS) is an iodo reagent, the structure of which contains an iodine atom and is a source for introducing the iodine atom into the structure of the compound 2; na (Na)₂S₂O₅Is a reducing agent used to remove excess unreacted N-iodosuccinimide (NIS).

In the present invention, CIAE-001 is a compound containing a radionuclide in its structure, and CIAE-001-1 is a corresponding stable compound of CIAE-001. Since radioactive compounds and their corresponding stable compounds are essentially identical in many chemical properties, some of the chemical properties of radioactive compounds (cell binding, protein binding, etc.) are commonly studied using the corresponding stable compounds in the early stages of radiopharmaceutical development for ease of measurement and manipulation. In examples 5 and 6, protein binding and pseudovirus binding experiments were performed using the stable compound CIAE-001-1 to determine the binding ability of the radioactive compound CIAE-001 to protein and pseudovirus (ref.: Nuclear Medicine and Biology,2014,41: 355-

Example 5 affinity assay of Compound CIAE-001-1 with the Receptor Binding Domain (RBD) of the New coronavirus spike protein

1. Laboratory instruments, conditions and reagents

An experimental instrument: biacore 8K; experiment temperature: 25 ℃; reaction chip: CM5 type S series sensor chip (Cat 29149603, manufacturer: Situfang); an amino coupling kit (cargo number: BR100633, manufacturer: Situofen); the target protein: new coronavirus spike protein Receptor Binding Domain (RBD) (Cat No. 40592-V08B, manufacturer: Yi Qiao Shenzhou); running buffer: 1 XHBS-EP containing 5% DMSO (cat # D8418, manufactured by Sigma Aldrich)⁺(diluted from HBS-EP ⁺10 × concentrated buffer, cat No.: BR100669, manufacturer: cituofan), the specific components are as follows: 10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% (v/v) P20 (Tween 20), and 5% DMSO, pH 7.4.

2. Measurement method

The affinity of CIAE001-1 and Arbidol (control) for the Receptor Binding Domain (RBD) of the novel coronavirus spike protein was determined by Surface Plasmon Resonance (SPR) technique. The neocoronavirus spike-protein Receptor Binding Domain (RBD) was amino-coupled on CM5 chip (coupling amounts up to about 6-8kRU) according to the methods described in the amino-coupling kit instructions. A series of concentrations of test compound were flowed across the chip surface to test their affinity for the Receptor Binding Domain (RBD) of the new coronavirus spike-protein (dilution factor: 2, different concentration points (5, 10, 25, 50, 100. mu.M): at least 5 (including repeat concentrations), flow rate: 30. mu.L/min). All data were processed by double subtraction and solvent correction prior to fitting. Fitting software: biacore Instrument Evaluation Software v3.0, manufacturer: think and use the fan. Fitting a model: steady-state affinity model.

3. Results of the experiment

Within the tested concentration range, the binding signals of samples CIAE001-1 and Abidol (control) to the amino-coupled new coronavirus spike protein Receptor Binding Domain (RBD) are not saturated (out-fitting), and the fitting affinities are weaker than the highest concentration of the test, 100. mu.M. Probably, the binding site of the CIAE-001-1 of the Abidol and the derivative thereof and the new coronavirus is the three-dimensional configuration of the virus, and the binding domain (RBD) of the spike protein receptor of the new coronavirus is only a protein fragment and cannot meet the requirement of the binding site of the CIAE-001-1 of the Abidol and the derivative thereof, so that other methods are required to determine the affinity of the CIAE-001-1 and the new coronavirus.

Example 6 binding assay of Compound CIAE-001-1 with SARS-COV-2 pseudovirions

1. Experimental reagents, instruments and conditions

1x10⁷TU/mL pseudovirus containing S protein and 40. mu.L of RBD antibody to ACE2-Fc protein: baonosangya; 50% protein a magnetic bead resuspension in PBS: suzhou nanotechnology GmbH.

LC/MS/MS instrument model: SCIEX Triple quad 7500; LC/MS/MS quantitative software version: SCIEX OS 2.0.0.45330; ion mode: electrospray ion source, positive ion mode; scanning mode: multiple Reaction Monitoring (MRM); ion pair of target analyte: 525.4/480.1(EP:10, CE:27, CXP:15) _ CIAE-001, 477.5/432.1(EP:10, CE:25, CXP:13) _ Arbidol; ion pair of internal standard analyte: 472.7/436.4(EP:10, CE:38, CXP:14) _ Terfenadine. The liquid chromatography method comprises the following steps: a pump: a SCIEX AD pump; a chromatographic column: ACQUITY

Peptide BEH C181.7 μm 300A (50 mm. times.2.10 mm) Column; mobile phase: mobile phase A: water (1% formic acid), mobile phase B: acetonitrile: methanol: water: formic acid (85: 15: 5: 1). Column temperature (. degree. C.): 40. sample volume (μ L): abidol is 10 μ L; CIAE-001-1 is 0.5. mu.L. The liquid chromatography gradients are shown in the following table:

TABLE 1 liquid chromatography gradient

2. Binding and enrichment of compounds with SARS-COV-2 pseudovirions

The SARS-COV-pseudovirion is produced by using HEK293 cells, and the virus is dispensed into 2mL Eppendorf tubes (EP tubes for short) in a volume of 1.8 mL/tube. Is divided into four groups, which are respectively: (1) abidol (control compound) group, (2) CIAE-001-1 group, (3) DMSO blank control group, (4) medium was used instead of virus, and a virus-free control group was set.

Compound concentrations were diluted to 1mM with DMSO solutions, and 9 μ L of each diluted group of compounds was added to a viral EP tube, with the DMSO group serving as a blank control. Final compound concentration 5. mu.M, 0.5% DMSO. After gentle mixing, the samples of each experimental group were spun on a spinner at 37 ℃ for 30 min. To an EP tube was added 2. mu.g ACE2-Fc and 20. mu.L of 50% protein A magnetic beads. Mix gently and the sample is spun at 4 ℃ for 3 hours.

8000rpm/min, 4 ℃ for 1 min, gently sucking out the supernatant, retaining the precipitate, then adding 1mL of precooled PBS, gently mixing for 10 seconds with a vortex apparatus. This was repeated twice. Centrifuging at 8000rpm/min at 4 deg.C for 1 min, discarding the remaining supernatant, adding 50 μ L/tube of ethanol, mixing, and storing at-20 deg.C.

3. The analysis method comprises the following steps:

(1) sample volume: sample, working solution, blank matrix and quality control solution of 50 mu L;

(2) sample operation steps:

1) aspirate 5. mu.L of sample and add 495. mu.L of ethanol (dilute 100 times the sample with ethanol).

2) From the first step, 50. mu.L of sample was aspirated, and 450. mu.L of ethanol was added (the sample was diluted 10-fold with ethanol).

3) To all samples, 100. mu.L of water was added.

4) To all samples except the two blank solutions without DMSO and without arbidol was added 20. mu.L of internal standard Terfenadine (Terfenadine) at 5 ng/mL.

5) To two blank solutions without DMSO, without arbidol, 20 μ L of methanol/acetonitrile 1: 1.

6) Vortex for one minute and centrifuge for 15 minutes for injection.

(3) Sample matrix: ethanol

(4) Blank matrix: ethanol

(5) Standard curve range: 0.5-50nmol/L

(6) The regression equation: linearity

(7) And (3) weighting: 1/(x)

(9) Lower limit of quantitation: 0.5nmol

(10) Internal standard: terfenadine (5ng/mL) in methanol: acetonitrile (1:1, volume: volume)

(11) And calculating the concentration of the sample according to the established standard working curve.

3. Summary of the results

The concentrations of the samples tested were obtained from the established standard working curves, as shown in tables 2 and 3.

TABLE 2 measured concentration of Abidol

Sample name	Detection concentration (nmol/L)	Remarks for note
			DMSO blank control sample	BLOQ*	Non-pseudovirion particles
Pseudovirion control sample 1	BLOQ*	DMSO + pseudovirions
			Control sample 2 of pseudovirion	BLOQ*	DMSO + pseudovirions
Abidol negative control sample	16801	Non-pseudovirion particles
			Abidol pseudovirion binding sample 1	28389	Abidol + pseudovirions
Abidol pseudovirion binding sample 2	40029	Abidol + pseudovirions

BLOQ ═ below the lower limit of quantitation (< LLOQ), LLOQ ═ 0.5 (nmol/L);

sample 1 and sample 2 are parallel samples.

TABLE 3 detection concentrations of CIAE001-1

Sample name	Detection concentration (nmol/L)	Remarks for note
			DMSO blank control sample	BLOQ	Non-pseudovirion particles
Pseudovirion control sample 1	BLOQ	DMSO + pseudovirions
			Control sample 2 of pseudovirion	BLOQ	DMSO + pseudovirions
CIAE001-1 negative control sample	1604	Non-pseudovirion particles
			CIAE001-1 pseudovirion binding sample 1	7877	CIAE001+ pseudovirions
CIAE001-1 pseudovirion binding sample 2	9741	CIAE001+ pseudovirions

Lower than lower limit of quantitation (< LLOQ), LLOQ ═ 0.5(nmol/L)

Sample 1 and sample 2 are parallel samples.

In the experiment that the compounds of Abidol and CIAE-001-1 are respectively combined with SARS-COV-2 pseudovirion, the results of the experiment are compared with the results of two groups of negative control groups to prove that the Abidol and the CIAE-001-1 are combined with the SARS-COV-2 pseudovirion. The actual detection value of Abidol is 2.04 times of that of the negative control, and the actual detection value of CIAE001 is 5.49 times of that of the negative control. The results indicate that under the current experimental conditions, SARS-COV-2 pseudovirions will bind relatively more CIAE-001-1, and thus it is presumed that their corresponding radiolabeled compound CIAE001 will bind to SARS-COV-2 pseudovirions, indicating that CIAE-001 can bind to SARS-COV-2 virus and be useful in the treatment of SARS-COV-2 patients.

Sequence listing

Amino acid sequence of SEQ ID NO 1 HR2L

SIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKELGNYTYYNKW；

Amino acid sequence of SEQ ID NO 2 HR2P

SLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKEL；

3P1 amino acid sequence

LTQINTTLLDLTYEMLSLQQVVKALNESYIDLKEL；

Amino acid sequence of SEQ ID NO 4 CP-1

GINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYE；

Amino acid sequence of SEQ ID NO 5LCB1

DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAERLLEEVER；

Amino acid sequence of SEQ ID NO 6LCB3

NDDELHMLMTDLVYEALHFAKDEEIKKRVFQLFELADKAYKNNDRQKLEKVVEELKELLERLLS；

Amino acid sequence of SEQ ID NO 7 AHB2

ELEEQVMHVLDQVSELAHELLHKLTGEELERAAYFNWWATEMMLELIKSDDEREIREIEEEARRILEHLEELARK；

Amino acid sequence of SEQ ID NO 8SBP1

IEEQAKTFLDKFNHEAEDLFYQS。

Reference to the literature

1.Desiree Schütz et al.,Peptide and peptide-based inhibitors of SARS-CoV-2 entry,Advanced Drug Delivery Reviews,167(2020),47-65.

2.Dora Pinto et al.,Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody,https://doi.org/10.1038/s41586-020-2349-y.

3.Bryan E.Jones et al.,LY-CoV555,a rapidly isolated potent neutralizing antibody,provides protection in a non-human primate model of SARS-CoV-2infection,https://doi.org/10.1101/2020.09.30.318972.

4.Federico Bertoglio et al.,A SARS-CoV-2neutralizing antibody selected from COVID-19patients binds to the ACE2-RBD interface and is tolerant to most known RBD mutations,Cell Reports 36,109433,https://doi.org/ 10.1016/j.celrep.2021.109433.

5.Y.Wu et al.,A noncompeting pair of human neutralizing antibodies block COVID-19virus binding to its receptor ACE2,Science10.1126/science.abc2241(2020).

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A radiotherapeutic agent against a disease caused by a coronavirus or an influenza virus, comprising a targeting molecule targeting a coronavirus or an influenza virus protein, said targeting molecule being labelled with a radionuclide, optionally selected from the group consisting of¹²⁵I、¹²³I、¹³¹I、⁶⁷Ga、¹¹¹In、²⁰¹Tl、¹⁹¹Pt、¹¹⁹Sb、¹⁶¹Tb、¹⁷⁷Lu、⁸⁹Zr、⁷⁷Br、⁸⁹S、⁹⁰Y、³²P、¹⁵³Sm、¹⁸⁸Any one or combination of Re, preferably the radionuclide is¹²⁵I and/or¹³¹I。

2. The radiotherapeutic agent of claim 1, wherein the targeting molecule is selected from the group consisting of small molecule compounds, polypeptides and proteins.

3. The radiotherapeutic agent of claim 2 wherein the small molecule compound is selected from the group consisting of arbidol, ridciclovir, faviravir, chloroquine phosphate, hydroxychloroquine, and analogs thereof, the polypeptide is selected from HR2L, HR2P, P1, CP-1, LCB1, LCB3, AHB2, SBP1, and variants thereof, and the protein is selected from the group consisting of coronavirus and influenza glycoprotein antibodies SARS-CoV-2SpikeRBDNanobody, SAntibody, neocorona nano-neutralizing antibody R173C1, neocorona antibody k79d9, S309, B38, H4, LY-CoV555, STE90-C11, influenza virus antibody HA antibody, NP antibody, PA antibody and variants thereof.

4. A method of preparation of a radiotherapeutic according to any one of claims 1 to 3, comprising labelling a radionuclide on a targeting molecule targeting a coronavirus or influenza virus protein, the labelling method comprising the chloroglycoluril (Iodogen) method, the chloramine T method, the Lactoperoxidase (LPO) method and the acylating agent (Bolton and Hunter reagent) method, preferably the chloroglycoluril method comprising: adding target molecules to be marked and a radionuclide solution into a reaction container with the inner wall coated with the chloroglycoluril, and reacting at room temperature for 1-60min to obtain the radionuclide-marked target molecules.

5. The method of claim 4, wherein the step of coating the inner wall of the reaction vessel with chloroglycoluril comprises: dissolving the chloroglycoluril in a volatile organic solvent to prepare a solution, transferring the prepared solution into a reaction container, drying the solution by nitrogen to form a uniform chloroglycoluril film on the inner wall of the reaction container, wherein the organic solvent is dichloromethane or chloroform, the concentration of the solution is 0.04-1mg/mL, and the content of the chloroglycoluril on the inner wall of the reaction container is 5-50 mu g, preferably 10-50 mu g, 10-40 mu g, 10-30 mu g, 10-20 mu g, 20-30 mu g, 30-40 mu g, such as 10 mu g, 20 mu g, 30 mu g or 40 mu g.

6. The method according to claim 4 or 5, wherein the labeling method of the small molecule compound further comprises a step of subjecting the small molecule compound, a catalyst selected from palladium acetate, palladium/carbon, bis (benzonitrile) palladium dichloride, bis (triphenylphosphine) palladium dichloride, tetrakis (triphenylphosphine) palladium, [1, 1-bis (diphenylphosphino) ferrocene ] palladium dichloride and bis (tri-tert-butylphosphino) palladium, preferably palladium acetate, a catalytic adjuvant selected from triphenylphosphine, tricyclohexylphosphine and 2,2 '-bis (diphenylphosphino) -1,1' -binaphthyl, preferably 2,2 '-bis (diphenylphosphino) -1,1' -binaphthyl, a reaction adjuvant preferably tetramethylethylenediamine and a reducing agent preferably sodium borohydride to an organic solvent selected from tetrahydrofuran and 1, reacting 4-dioxane and diethylene glycol dimethyl ether at 30-60 ℃ overnight, cooling the reaction mixture, removing the solvent, and purifying to obtain a precursor compound;

7. The method according to claim 4 or 5, wherein the labeling method of the polypeptide or protein comprises: and sequentially adding polypeptide or protein, PBS buffer solution and radionuclide solution into a reaction container with the inner wall coated with chloroglycoluril, and reacting at room temperature for 1-60min to obtain polypeptide or protein labeled by the radionuclide, wherein the polypeptide and the protein contain tyrosine groups.

8. The method of any one of claims 5-8, wherein the radionuclide-labeled targeting molecule is purified using a chromatography column.

9. A pharmaceutical composition comprising the radiotherapeutic agent of any one of claims 1-3 and a pharmaceutically acceptable carrier.

10. A method for the preparation of a corresponding stable therapeutic agent of a radiotherapeutic agent according to any one of claims 1-3, comprising binding a corresponding stable nuclide of a radionuclide, preferably iodine, to a targeting molecule targeting a coronavirus or influenza virus protein, the method is characterized in that the micromolecule precursor compound, an iodination reagent (the iodination reagent is selected from N-iodosuccinimide, iodine chloride, iodine simple substance, sodium iodide, potassium iodide and tert-butoxy iodine, preferably N-iodosuccinimide), a reaction solvent (the reaction solvent is selected from trifluoroacetic acid, trifluoroethanol, hexafluoroisopropanol, dimethylformamide, dimethyl sulfoxide and pyridine, preferably trifluoroacetic acid) are reacted for 12-96 hours and 72 hours at the reaction temperature of 10-50 ℃ and preferably 20-25 ℃, and a corresponding stable compound is obtained after the reaction mixture is purified by a silica gel column.