Patent Application
20240016775
This application claims the priority of the Chinese patent application 2020101117554 which has the application date of Feb. 24, 2020. The above Chinese patent application is incorporated into the present application by reference in their entirety.
The present invention belongs to the field of biomedical technology and in particular relates to the application of a medication with a poly ADP ribose polymerase inhibitor as a main component and a pharmaceutically acceptable salt thereof, and a pharmaceutical composition and a kit containing the same in anti-coronavirus-induced diseases.
Coronavirus (CoV) is a class of enveloped single-stranded positive-stranded RNA viruses that can infect humans and a variety of animals. It has respiratory, gastrointestinal and nervous system tropisms, and causes serious illnesses in livestock and companion animals (such as pigs, cows, chickens, dogs, cats) and can lead to illnesses ranging from common cold to severe acute respiratory syndrome in humans. In the ninth report of the International Committee on Taxonomy of Viruses, coronaviruses are divided into four groups, i.e., α, β, γ and δ according to their evolutionary characteristics. Among them, the hosts of α and ρ groups are mainly mammals, and 7 and 6 groups are mainly found in birds and fowls.
As of February 2020, there are currently seven known coronaviruses that can infect humans, including human coronaviruses 229E (HCoV-229E), NL63 (HCoV-NL63), HKU1 (HCoV-HKU1), OC43 (HCoV-OC43) and middle east respiratory syndrome coronavirus (MERS-CoV) that cause common cold, a symptom of upper respiratory tract infection, and severe acute respiratory syndrome (SARS) related coronaviruses: coronavirus SARS-CoV (severe acute respiratory syndrome coronavirus) and SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). Coronaviruses are responsible for 15%-30% of human respiratory tract infections each year, and can cause more severe diseases in newborns, the elderly and other susceptible populations, who have even higher incidences of lower respiratory tract infections. Among them, the case fatality rates are as high as 10% and 36%, respectively, for the highly lethal coronaviruses SARS-CoV and MERS-CoV. From 2019 to the beginning of 2020, the novel coronavirus SARS-CoV-2 that mainly broke out in Hubei and other places in China, caused more than 70,000 people to be infected and could lead to severe COVID-19 pneumonia (novel coronavirus pneumonia), with the severe rate ranging from 15 to 30%, the fatality rate of about 2%, again drawing much attention of people to coronavirus. However, there is currently no approved specific drug for treatment of the infections caused by coronavirus. Even for patients with severe acute respiratory tract infections caused by SARS-CoV, SARS-CoV-2 and MERS-CoV infections, the clinical treatment is mainly symptomatic treatment to reduce the complications of the patients. Therefore, there is an urgent need to develop effective drugs to treat infections caused by coronaviruses.
Small molecule compounds are a hotspot in the research of antiviral drug candidates. Exploring new uses of existing drugs has become an important approach to drug research and development. Candidate drugs have great application prospects since there is already data of their pharmacological efficacy testing, functional targets and clinical safety that is helpful for further toxicological evaluation, pharmacokinetic evaluation and formulation development, and can greatly reduce the research and development risk, shorten the research time and cut the costs for research and development.
At present, the clinical first-line treatment for infections caused by coronaviruses mostly includes broad-spectrum antiviral drugs, such as anti-HIV drug lopinavir/ritonavir (Aluvia), Arbidol, anti-Ebola virus drug Remdesivir, etc. Arbidol is a broad-spectrum antiviral drug that mainly treats upper respiratory tract infections caused by influenza A and B viruses. In recent years, many studies have proven that it has a certain inhibitory activity against SARS-CoV and MERS-CoV coronaviruses. On Feb. 4, 2020, the latest research results of in vitro cell experiments performed by Li Lanjuan team have shown that Arbidol could effectively inhibit coronaviruses, and at a concentration of 10-30 micromoles, it was up to 60 times more effective than that in the untreated control group, and significantly inhibit the pathogenic effect of viruses on cells. However, from the current data, it is difficult to achieve the concentration of 10-30 micromoles in current clinical treatment. In the Diagnosis and Treatment Protocol for Novel Coronavirus Pneumonia (Trial Version 6), the recommended dose for Arbidol is 200 mg per time, 3 times a day. However, as described in the package insert of “Arbidol Tablets (Manuosu)”, after oral administration of single doses of Arbidol at 200 mg, 400 mg and 600 mg, the peak concentrations are 614.1±342.5 ng/mL, 904.2±355.6 ng/mL and 975.1±661.0 ng/mL, only equivalent to the level of 1-2 micromoles and far lower than the inhibitory concentration in vitro, which is speculated that it can only have limited antiviral activity in vivo. Meanwhile, it is stated in the package insert that adverse events occur in about 6.2% of subjects taking Arbidol, mainly manifested as nausea, diarrhea, dizziness and increased serum transaminases. Also, it should be cautious that in the human bioequivalence test of domestic Arbidol preparations, some healthy subjects have developed bradycardia (some of them having a heart rate of less than 60 beats/minute in some healthy subjects and a heart rate decrease of 2-24 beats/minute at 3 hours after taking the drug). The relationship between this event and the drug remains unknown. In addition, the drug should be used with caution or as directed by a doctor for pregnant and lactating women, and those with severe renal insufficiency. The drug has unclear significance in patients with nodal disease or insufficiency, so it is recommended that this product should be used with caution for this population. It is speculated that, based on the above data, the currently available drugs are not the best choice for treatment of coronaviruses due to their low antiviral activity, insufficient clinical evidence and safety concerns, even though they are included in the recommended treatments.
In addition to Arbidol, other antiviral drugs are also undergoing in vitro screening and clinical trials for anti-novel coronavirus effects. However, there is evidence that not all antiviral drugs are effective against the novel coronaviruses. Among them, Aluvia, an anti-AIDS drug, is not effective in treatment of pneumonia caused by novel coronavirus infections, and has toxic and side effects, so it is no longer recommended for continued use. Remdesivir, clinical research of which is ongoing, has also relatively serious risks of liver toxicity. Therefore, according to the existing evidence and experience, the treatment of coronaviruses has its uniqueness, and it is still necessary to develop proprietary drugs that have innovative mechanisms of action, and good efficacy and safety.
Poly(ADP-ribose) polymerases (PARPs) play an important role in DNA damage repair and apoptosis, and are also involved in a series of cellular processes including infections and immune regulation. This protein family consists of 17 members, including PARP1, PARP2 and the like. PARP inhibitors were originally developed in the industry to block DNA damage repair in highly mutated cancer cells, thereby producing anti-cancer effects through “toxic damage” accumulation and “synergistic lethal” effect. At present, there are 4 PARP inhibitors that have been marketed in the world, namely, Olaparib, Niraparib, Rucaparib and Talazoparib, respectively. At the same time, several PARP inhibitors have been at the clinical study stage at home and abroad, including fluzoparib, mefuparib, simmiparib, IMP4297, BGB-290, ABT-888, etc.
It is reported in literatures that PARP may be closely related to invasion, integration and replication of viruses and the formation of capsid proteins. In 2001, Hyo Chol Ha et al. reported that PARP-1 enzyme played a key role in the replication, integration and transcription of HIV-1 during the replication of HIV virus, and Proc Natl Acad Sci USA. 2001; 98(6): 3364-8 mentioned that PARP inhibitors helped inhibit HIV infection. It was also reported that PARP-1 could inhibit the nuclear localization of PARP-1 through participating in the process of stimulating gene transcription and expression by inflammatory pathway NF-KB, thereby inhibiting the NFKB transcription pathway and achieving the effect of inhibiting virus replication. Finally, PARP-1 can induce apoptosis of immune cells including T cells, so inhibition of PARP-1 may block the integration of HIV in host cells, thereby achieving the effect of treating AIDS. Similar findings have also been demonstrated in HPV, EBV, HSV and the like. In 2010, Tempera et al. confirmed that PARP1 inhibitors had inhibitory effects on EBV (J Virol. 2010; 84(10): 4988-97). In 2012, Grady et al. confirmed that PARP1 inhibitors had inhibitory effects on HSV (J Virol. 2012; 86(15): 8259-68). In 2017, Matthew E. Grunewald et al. proposed that the capsid protein of coronavirus was modified by ADP ribosylation and speculated that some subtypes of PARP were involved in the formation of coronavirus capsid protein (Virology. 2018, 517: 62-68), but they have not further clarified which PARP subtype selectivity is extremely important, nor do they suggest whether PARP inhibitors can inhibit viral replication or capsid protein formation. On the other hand, in 2016, Chad. V. Kuny et al. published an article discussing the role of PARP enzymes in virus-host interactions. Among them, PARP13 can degrade the reverse-transcribed RNA of viruses to block viral replication, and PARPs 10, 12, 13, 14, etc., can inhibit viral replication through the production of interferons (PLoS pathogens, 2016, 12(3): e1005453). In 2019, Matthew E. Grunewald mentioned in an article that PARP enzymes had both antiviral and immunomodulatory functions, confirming that the presence of PARP12 and PARP14 could inhibit viral invasion and replication (PLoS pathogens, 2019, 15(5): e1007756). It can be seen that some subtypes of PARP enzymes can inhibit viral infections. At present, the functions of PARP are not conclusive at home and abroad, and the inhibitory effect of PARP inhibitors on various viruses remains unclear.
In summary, it is urgent to look for a drug with a lower effective concentration and higher antiviral activity when inhibiting the viruses while ensuring high safety and fewer side effects, so that it can effectively inhibit the viruses when being used in the clinical treatment of diseases caused by coronaviruses.
The technical problems to be solved by the present invention are to overcome the problems in the prior art, such as too high effective concentration and insufficient antiviral activity of drugs for treatment of diseases caused by coronavirus, resulting in limited antiviral activity in vivo when being clinically used, so the present invention provides a poly ADP ribose polymerase (PARP) inhibitor or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition and a kit comprising the same, for use against viruses e.g., coronaviruses, wherein the antiviral treatment can be either the PARP inhibitor of the present invention as an antiviral agent, or the PARP inhibitor for treatment of diseases caused by viruses. The poly ADP ribose polymerase inhibitor of the present invention or a pharmaceutically acceptable salt thereof, as well as a pharmaceutical composition and a kit containing the same, can achieve a lower effective concentration for inhibiting viruses and a higher antiviral activity while ensuring low toxicity and high safety when being used in humans, so that when used in the clinical treatment of diseases caused by coronaviruses, the inhibitor of the present invention can effectively inhibit the viruses.
Through extensive experiments, it was unexpectedly found that a poly ADP ribose polymerase inhibitor or a pharmaceutically acceptable salt thereof of the present invention can significantly inhibit the replication of coronavirus in vitro. The present invention proves for the first time that the poly ADP ribose polymerase inhibitor or the pharmaceutically acceptable salt thereof can inhibit the infection of host cells by a coronavirus and the replication of the coronavirus and can be used for treatment of diseases caused by the coronavirus infection, and is of great significance for prevention, control and treatment of infections with coronaviruses, especially coronavirus SARS-CoV-2.
To solve the above-mentioned technical problems, the first aspect of the present invention provides application of a poly ADP ribose polymerase inhibitor or a pharmaceutically acceptable salt thereof in the preparation of antiviral agents or in the preparation of medicines for the treatment of diseases caused by viruses, wherein the viruses are β-coronavirus viruses.
In the present invention, the SARS-related coronaviruses are the coronaviruses causing severe acute respiratory syndrome (SARS). The diseases caused by SARS-related coronaviruses are symptoms or diseases of viral infections, and their early stage is mainly reflected in respiratory diseases, and the clinical manifestations include fever, fatigue, dry cough, cough, rapid respiratory rate or acute respiratory distress syndrome, shortness of breath, etc., and symptoms such as nasal congestion, runny nose and sore throat in a small number of patients. The imaging manifestations include different degrees of changes in the lungs, such as multiple spot-like and ground-glass-like shadows. The viruses are mainly transmitted by close-range droplets or contact with patients' respiratory secretions. There may also be gastrointestinal symptoms such as diarrhea, acute gastroenteritis in infants and neonates, and in rare cases, neurological syndromes. At a later stage, many complications may occur, including metabolic acidosis and coagulation dysfunctions difficult to remedy, respiratory failure, fulminant myocarditis to multiple organ failures such as heart failure, liver and kidney failure, and septic shock. Critically ill patients often develop dyspnea and/or hypoxemia one week after onset, and in severe cases, rapidly progress to acute respiratory distress syndrome, septic shock, metabolic acidosis and coagulation dysfunctions difficult to remedy, and multiple organ failures. It is expected that all of these diseases will be treated with the PARP inhibitor of the present invention. In addition, some virus carriers do not have the above symptoms, and may also receive the PARP inhibitor of the present invention to inhibit viral replication and spread, so as to prevent the virus carriers from getting sick or spreading the viruses to generate new patient cases. In an embodiment, the viruses of genus β-coronavirus are viruses that cause acute respiratory syndrome, such as SARS-related coronaviruses; preferably, the SARS-related coronaviruses are SARS-CoV (severe acute respiratory syndrome coronavirus) and/or SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2).
In an embodiment, the poly ADP ribose polymerase inhibitor is an inhibitor against PARP 1 and/or PARP 2.
In an embodiment, the poly ADP ribose polymerase inhibitor is Substance A, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof;
The Substance A is selected from the group consisting of tarazoparib, fluzoparib, simmiparib, IMP4297, BGB-290, ABT-888, one or more of PARP inhibitors as described in CN1342161A, PARP inhibitors as described in CN1788000A, PARP inhibitors as described in CN103242273A, PARP inhibitors as described in CN101415686A, and PARP inhibitors as described in CN101578279A. They are only examples herein, and do not exclude the possibility that Substance A can be selected from other compounds not listed here.
In the present invention, the “one or more selected from . . . ” includes the situation where the listed compounds are used in combination. In some embodiments of the present invention, there will be better therapeutic effects when two or more compounds are used in combination.
In an embodiment, the PARP inhibitor as described in CN1342161A is the compound shown in formula I;
In the formula, R11 is H, halogen, cyano, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C4 cycloalkyl, 3-6-membered heterocycloalkyl “containing 1-2 heteroatoms selected from one or more of O, N and S”, C6-C10 aryl, 5-10-membered heteroaryl “containing 1-2 heteroatoms selected from one or more of O, N and S”, C1-C4 alkyl substituted by one or more R11-1, C2-C4 alkenyl substituted by one or more R11-1, C2-C4 alkynyl substituted by one or more R11-1, C3-C4 cycloalkyl substituted by one or more R11-1, 3-6-membered heterocycloalkyl “containing 1-2 heteroatoms selected from one or more of O, N and S”, C6-C10 aryl substituted by one or more R11-1 (the “C6-C10 aryl” is, for example
5-10-membered heteroaryl “containing 1-2 heteroatoms selected from one or more of O, N and S”, substituted by one or more R11-1, —C(═O)—R11-2, —C(═O)—O—R11-3 or —C(═O)—NR11-4R11-5;
In an embodiment, the PARP inhibitor as described in CN1788000A is the compound shown in formula II;
In the formula, X2, Y2 and the carbon atoms connected thereto together form a C6-C10 aryl (for example, phenyl), or a C6-C10 aryl substituted by one or more RX2-1;
When Z is —NRZ-1—, m will be 1 or 2; when Z is —CRZ-2RZ-3—, m will be 1;
—C(═O)ORZ-1-4, —C(═S)NRZ-1-5RZ-1-6, —S(═O)2RZ-1-7, C1-C20 alkyl substituted by one or more RZ-1-8, C6-C20 aryl substituted by one or more RZ-1-9, or 3-20-membered heterocyclyl “containing 1-6 heteroatoms selected from one or more of O, N and S”, substituted by one or more RZ-1-10;
In an embodiment, the PARP inhibitor as described in CN103242273A is the compound shown in formula III;
In an embodiment, the PARP inhibitor as described in CN101415686A is the compound shown in formula IV;
“5-membered heteroaromatic ring containing 1, 2, 3 or 4 heteroatoms independently selected from O, N and S but not more than one being O or S”, “6-membered heteroaromatic ring containing 1, 2 or 3 nitrogen atoms”, or “7- to 15-membered unsaturated, partially saturated or saturated heterocycle containing 1, 2, 3 or 4 heteroatoms independently selected from N, O and S”;
Ra-1 and Ra-3 are independently “4-membered saturated heterocycle containing one N atom”, or “5-, 6- or 7-membered saturated or partially saturated heterocycle containing 1, 2 or 3 N atoms and 0 or 1 O atom”;
In an embodiment, the compound shown in formula I has the following definitions:
In the formula, R11 is C6-C10 aryl substituted by one or more R11-1 (the “C6-C10 aryl” is for example, phenyl; the “C6-C10 aryl substituted by one or more R11-1” is for example
In an embodiment, the compound shown in formula II has the following definitions:
—C(═O)ORZ-1-4, —C(═S)NRZ-1-5RZ-1-6 or —S(═O)2RZ-1-7;
In an embodiment, the compound shown in formula III has the following definitions:
In an embodiment, the compound shown in formula III has the following definitions:
In an embodiment, the compound shown in formula IV has the following definitions:
or, “7-15-membered unsaturated, partially saturated or saturated heterocycle containing 1, 2, 3 or 4 heteroatoms independently selected from N, O and S”.
In an embodiment, the compound shown in formula IV has the following definitions:
In an embodiment, the compound shown in formula I is any of the following compounds:
In an embodiment, the compound shown in formula II is any of the following compounds.
In the formula, R is selected from
In an embodiment, the compound shown in formula III is any of the following compounds:
In an embodiment, the compound shown in formula IV is any of the following compounds:
And pharmaceutically acceptable salts or tautomers thereof.
Other particular compounds within the scope of the present invention are:
Trifluoroacetate
In an embodiment, the compound shown in formula I is
In an embodiment, the compound shown in formula II is
In an embodiment, the compound shown in formula III is
In an embodiment, the compound shown in formula IV is
In an embodiment, the Substance A is selected from one or more of niraparib, tarazoparib, fluzoparib, simmiparib, IMP4297, BGB-290, ABT-888, rucaparib, olaparib and mefuparib;
In an embodiment, the pharmaceutically acceptable salt is a hydrochloride salt.
In an embodiment, the pharmaceutically acceptable salt of substance A is
In an embodiment, the poly ADP ribose polymerase inhibitor or a pharmaceutically acceptable salt thereof is present in the form of a pharmaceutical composition comprising the same. Preferably, the pharmaceutical composition uses the poly ADP ribose polymerase inhibitor or a pharmaceutically acceptable salt thereof as the only active ingredient of the pharmaceutical composition; and/or, the pharmaceutical composition further comprises pharmaceutical acceptable carriers, such as pharmaceutically acceptable excipients.
In an embodiment, the poly ADP ribose polymerase inhibitor or a pharmaceutically acceptable salt thereof is present in the form of a kit composition comprising the same, and the kit also contains drugs to treat coronaviruses-related diseases and/or drugs to treat diseases caused by other viruses.
In order to solve the above technical problems, the second aspect of the present invention provides a compound represented by formula III and/or a compound represented by formula IV or a pharmaceutically acceptable salt thereof in the preparation of antiviral agents or in the preparation of medicines for treatment of diseases caused by viruses according to any one of claims 3-6, the viruses being HIV, HPV, EBV, IFV and/or coronaviruses, preferably the subfamily Orthocoronavirinae viruses.
In an embodiment, the pharmaceutically acceptable salt is a hydrochloride salt. In a preferred example of the present invention, it is preferred that the pharmaceutically acceptable salt is mefuparib hydrochloride.
In an embodiment, the compound represented by formula III and/or a compound represented by formula IV or a pharmaceutically acceptable salt thereof is present in the form of a pharmaceutical composition comprising the same; preferably, the pharmaceutical composition uses the compound represented by formula III and/or a compound represented by formula IV or a pharmaceutically acceptable salt thereof as the only active ingredient of the pharmaceutical composition; and/or, the pharmaceutical composition further comprises pharmaceutical acceptable carriers, such as pharmaceutically acceptable excipients.
In an embodiment, the compound represented by formula III and/or a compound represented by formula IV or a pharmaceutically acceptable salt thereof is present in the form of a kit composition comprising the same, and the kit also contains drugs for other anti-coronavirus-induced diseases.
In an embodiment, viruses of the subfamily Orthocoronavirinae are α coronavirus, β coronavirus, γ coronavirus and/or δ coronavirus, preferably coronaviruses that cause upper respiratory tract infections, and viruses that cause acute respiratory syndrome such as SARS-related coronavirus and/or Middle East respiratory syndrome coronavirus (MERS-CoV).
Preferably, the coronaviruses that cause upper respiratory tract infections are human coronavirus 229E, human coronavirus HKU1 (HCoV-HKU1), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV-NL63) and/or mouse hepatitis virus A59 (MHV-A59).
Preferably, the SARS-related coronavirus is SARS-CoV (severe acute respiratory syndrome coronavirus) or SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2).
In a preferred embodiment of the present invention, the coronaviruses include severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).
In a preferred embodiment of the present invention, the coronavirus is severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).
In addition, in order to solve the above technical problems, the present invention also provides the application of a poly ADP ribose polymerase inhibitor or a pharmaceutically acceptable salt thereof in the preparation of a medicine for virus-related diseases.
Preferably, the poly ADP ribose polymerase inhibitor or a pharmaceutically acceptable salt thereof is as described in the first aspect of the present invention.
Preferably, the medicine for virus-related diseases is as described in the second aspect of the present invention.
To solve the above technical problems, the present invention also provides a medicine for treatment of virus-related diseases as described in the second aspect of the present invention, the medicine comprising the poly ADP ribose polymerase inhibitor or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention.
To solve the above technical problems, the present invention also provides a viral inhibitor, which comprises the poly ADP ribose polymerase inhibitor or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention. The viruses are those as described in the second aspect of the present invention.
To solve the above technical problems, the present invention also provides use of a poly ADP ribose polymerase inhibitor or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention for the treatment of the virus-related diseases as described in the second aspect of the present invention.
The term “pharmaceutically acceptable” refers to salts, solvents, excipients and the like that are generally non-toxic, safe, and suitable for use in patients. The “patient” is preferably a mammal, more preferably a human.
The term “pharmaceutically acceptable salts” refers to salts obtained by preparing the compounds of the present invention with relatively non-toxic, pharmaceutically acceptable acids or bases. When compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting neutral forms of such compounds with a sufficient amount of a pharmaceutically acceptable base in pure solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to, lithium, sodium, potassium, calcium, aluminum, magnesium, zinc, bismuth, ammonium, diethanolamine salts. When compounds of the present invention contain relatively basic functional groups, acid addition salt can be obtained by contacting neutral forms of such compounds with a sufficient amount of a pharmaceutically acceptable acid in pure solution or in a suitable inert solvent. The pharmaceutically acceptable acids include inorganic acids, including but not limited to: hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, phosphoric acid, phosphorous acid, sulfuric acid, and the like. The pharmaceutically acceptable acids include organic acids, including but not limited to: acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acid citrate, oleic acid, tannic acid, pantothenic acid, hydrogen tartrate, ascorbic acid, gentisic acid, fumaric acid, gluconic acid, sugar acid, formic acid, ethanesulfonic acid, pamoic acid (i.e. 4,4′-methylene-bis(3-hydroxy-2-naphthoic acid), amino acids (for example, glutamic acid, arginine), and the like. When the compounds of the present invention contain relatively acidic and relatively basic functional groups, they can be converted into base addition salts or acid addition salts. For details, see Berge et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science 66: 1-19 (1977) or Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl and Camille G. Wermuth, ed., Wiley-VCH, 2002).
The term “solvate” refers to a substance formed by combining a compound of the present invention with a stoichiometric or non-stoichiometric amount of a solvent. Solvent molecules in solvates can exist in the form of ordered or non-ordered arrangement. The solvent includes, but is not limited to, water, methanol, ethanol, and the like.
“Pharmaceutically acceptable salts” and “solvates” in the term “solvates of pharmaceutically acceptable salts” are as described above and refer to the substances formed by combining the compounds of the present invention with 1, with 2 obtained by preparing a relatively non-toxic, pharmaceutically acceptable compound, and with a stoichiometric or non-stoichiometric solvent. The “solvates of pharmaceutically acceptable salts” include, but are not limited to, hydrochloric acid monohydrates of the compounds of the present invention.
The term “plurality” refers to 2, 3, 4 or 5.
When any variable (for example, R11-1) appears multiple times in the definition of a compound, the definition that appears at each position of the variable is independent of the definitions that appear at other positions, and their meanings are independent of each other and do not affect each other. Therefore, if a group is substituted by 1, 2 or 3 R11-1 groups, that is, the group may be substituted by up to 3 R11-1 groups, the definition of R11-1 at this position will be independent of the definition of R11-1 at the remaining positions. In addition, combinations of substituents and/or variables are allowed only if such combinations produce stable compounds.
The term “halogen” refers to fluorine, chlorine, bromine or iodine.
The term “hydrocarbyl” refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having 1 to 20 carbon atoms (unless otherwise specified), which may be aliphatic or cycloaliphatic and may be saturated or unsaturated (e.g., partially unsaturated, fully unsaturated). Thus, the term “hydrocarbyl” includes the subclasses of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, and the like.
The term “alkyl” refers to a straight or branched chain alkyl group having the specified number of carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the similar alkyl groups thereof.
The term “alkenyl” refers to a straight or branched chain alkenyl group having the specified number of carbon atoms.
The term “alkynyl” refers to a straight or branched chain alkynyl group having the specified number of carbon atoms.
The term “alkoxy” refers to the group —O—RX, wherein RX is an alkyl group as defined above.
The term “cycloalkyl” refers to a monovalent saturated cyclic alkyl group, preferably a monovalent saturated cyclic alkyl group having 3-7 ring carbon atoms, more preferably 3-6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.
The term “heterocyclyl” or “heterocycle” refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, the moiety having 3 to 20 ring atoms (unless otherwise specified), wherein 1 to 10 are ring heteroatoms and can be aromatic or non-aromatic. Preferably, each ring has 3 to 7 ring atoms, wherein 1 to 4 are ring heteroatoms.
The term “heterocycloalkyl” refers to a saturated monocyclic group having a heteroatom, preferably a 3-7 membered saturated monocyclic group containing 1, 2 or 3 ring heteroatoms independently selected from N, O and S. Examples of heterocycloalkyl groups are: pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, tetrahydropyridyl, tetrahydropyrrolyl, azacyclobutanyl, thiazolidinyl, oxazolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, azepanyl, diazepanyl, oxazepanyl and the like. Preferred heterocyclyl groups are morpholin-4-yl, piperidin-1-yl, pyrrolidin-1-yl, thiomorpholin-4-yl and 1,1-dioxo-thiomorpholin-4-yl.
The term “heteroaryl” or “heteroaromatic ring” refers to an aromatic group comprising a heteroatom, preferably comprising 1, 2 or 3 aromatic 5-6 membered monocyclic or 9-10 membered bicyclic rings independently selected from nitrogen, oxygen and sulfur, for example, furanyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, isoxazolyl, oxazolyl, diazolyl, imidazolyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, benzimidazolyl, indolyl, indazolyl, benzothiazolyl, benziisothiazolyl, benzoxazolyl, benzisoxazolyl, quinolinyl, isoquinolyl, and the like.
The pharmaceutical compositions involved in the present invention using the compounds of the present invention as active ingredients can be prepared according to methods known in the art. The compounds of the present invention may be formulated in any dosage form suitable for use in humans or animals. The weight content of the compound of the present invention in the pharmaceutical composition thereof is usually 0.1-99.0%.
The pharmaceutically acceptable carrier can be a conventional carrier in the art, and the carrier can be any suitable physiologically or pharmaceutically acceptable excipients. The pharmaceutical excipients are conventional pharmaceutical excipients in the art, preferably including pharmaceutically acceptable vehicles, fillers or diluents and the like. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the above-mentioned protein and/or the above-mentioned antibody-drug conjugate, and 0.01-99.99% of the pharmaceutical carrier, and the percentage being mass percentage of the pharmaceutical composition.
The compound of the present invention or the pharmaceutical composition containing it may be administered in the form of unit dose, and in the route which may be enteral or parenteral, for example, oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosal, intraocular, pulmonary and respiratory, skin, vaginal, rectal administration and the like.
The dosage form for administration may be a liquid dosage form, a solid dosage form or a semi-solid dosage form. Liquid dosage forms can be solutions (including both true and colloidal solutions), emulsions (including o/w, w/o and multiple emulsion), suspension, injections (including aqueous, powdered and infusion), eye drops, nasal drops, lotion, liniment and the like; the solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric-coated capsules), granules, powders, pellets, dripping pills, suppositories, films, patches, gas (powder) aerosols, sprays and the like; semi-solid dosage forms can be ointments, gels, pastes and the like.
The compound of the present invention can be prepared into ordinary preparations, and also sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle delivery systems.
On the basis of conforming to common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain the better examples of the present invention.
The reagents and raw materials used in the present invention are all commercially available.
The effect of the present invention terms of positive progress is: the present invention has been confirmed for the first time that the poly ADP ribose polymerase inhibitor can inhibit the infection of host cells by coronaviruses and the replication of the coronaviruses, and the effect is dose-dependent with no significant cytopathic action, and that it can be used for treatment of diseases related to anti-coronavirus infections. The poly ADP ribose polymerase inhibitor of the present invention or a pharmaceutically acceptable salt thereof, as well as a pharmaceutical composition and a kit containing the same, can achieve a lower effective concentration for inhibiting viruses and a higher antiviral activity while ensuring low toxicity and high safety when being used in humans, so that, when used in the clinical treatment of diseases caused by coronaviruses, the present inhibitor can effectively inhibit the viruses. In a preferred example of the present invention, the inhibition rate of the poly ADP ribose polymerase inhibitor against the viruses can be up to 35% (under the same conditions, the inhibition rate of the arbidol against the viruses is only 21%).
The present invention will be further described in the following examples, nevertheless, without being limited thereto. The experimental methods, specific conditions of which are not indicated in the following examples, are usually performed in accordance with conventional conditions, for example, described in common reference books in the art, such as Molecular Cloning: A Laboratory Manual (Third Edition, Science Publishing House, 2005), or according to the conditions suggested by the reagent manufacturer.
(1) Main instruments (name, number) are shown in Table 1 below.
(2) Experimental reagents and virus strains
1) Reagents are shown in Table 2 below.
2) SARS-CoV-2 strain
As identified, the genomes of the above two virus strains are completely identical, so they are both SARS-CoV-2 virus strains. The virus strains used in subsequent experiments are mainly 2019-nCoV-1.
(3) Drugs
Zanamivir, oseltamivir, remdesivir, baricitinib, olaparib and arbidol were all provided by MCE (Medchem Express, China). The PARP1 inhibitor mefuparib hydrochloride (CVL218, see also patent application 201210028895.0) has a purity of over 99.0%, which was provided by Pukang (Shanghai) Health Technology Co., Ltd.
1.1 Experimental Groups:
1.2 Drug Dilution:
1.3 Experimental Steps:
1.3.1 Cell Preparation and Drug Pretreatment (Cell Culture Room)
Vero-E6 cells (purchased from ATCC cell bank) were seeded into 96-well culture plates at 1×104 cells/well, and cultured in DMEM containing 10% (v/v) fetal bovine serum for 16 hours to become 80% pellets, then the cell culture medium was aspirated and discarded from each well, the cells were washed once with sterile PBS, and different drugs (50 l/well) diluted in the cell maintenance solution as described in section 1.2 above were added to each well according to the experimental groups, with 4 duplicate wells set up for each group, and then they were placed in a 37° C., 5% CO2 incubator for 1 h pretreatment. Only 50 μl of the cell maintenance solution was added to the virus control group and the cell control group.
1.3.2 Virus Infection and Culture (BSL-3 Laboratory):
After drug pretreatment for 1 h, except for the cell control group, 2 μl of the SARS-CoV-2 strain was added to each well to bring the virus multiplicity of infection to 0.05 (MOI=0.05), the wells were placed in a 37° C., 5% CO2 incubator for adsorption for 2 h. After the adsorption was completed, the medium with the viruses was discarded, and a new drug-containing medium was added according to the experimental group, and then cultured in a 37° C., 5% CO2 incubator for 48 h. After culture, the cytopathic conditions were microscopically observed for each experimental group and recorded. 120 μl of the culture supernatant was pipetted from each well, placed at 56° C., and inactivated for 30 min. After the inactivation was completed, 100 μl was taken from each well and added to the lysate in the reagent tank of the nucleic acid extraction reagent. After the outer surface of the reagent tank was disinfected, the tank was transferred to the BSL-2 Laboratory for viral nucleic acid extraction and genetic testing.
1.3.3 Nucleic Acid Extraction:
Refer to the operating instructions of the automatic nucleic acid extractor and extraction kit for viral nucleic acid RNA extraction.
1.3.4 Fluorescence Quantitative PCR Detection
The PCR reaction system was configured according to the package insert of the Shanghai Bio-germ 2019 Novel Coronavirus Nucleic Acid Detection Kit. The specific reaction systems are: 6 μl of qRT-PCR reaction solution, 2 μl of qRT-PCR enzyme mixture, 2 μl of primer probe, and 2.5 μl viral nucleic acid RNA template extracted as described above; the reaction parameters are: 50° C. for 10 min, 95° C. for 5 min, 40 cycles of: 95° C. for 10 s, 55° C. for 40 s (at this step, fluorescence signals of the FAM channel and VIC channel were collected); viral replication levels were reflected by detecting the SARS-CoV-2 virus genes (ORFlab and N) transcript levels. The 2ΔCT value was calculated according to the CT value given by the PCR instrument to represent the relative virus content of the experimental group relative to the control group. The virus replication inhibition rate (%)=(1−2ΔCT)×100%.
1.4 Experimental Results
Compounds such as mefuparib hydrochloride (CVL218) and olaparib and their inhibitory effects on the SARS-CoV-2 coronavirus are shown in
In conclusion, both PARP1 inhibitors olaparib and CVL218 have exhibited inhibitory effects on SARS-CoV-2 replication; and CVL218 has a higher inhibition rate than olaparib. It should be noted that the antiviral efficacy of CVL218 exceeds that of Arbidol which is one of the standard treatments for COVID-19 in the Diagnosis and Treatment Protocol for Novel Coronavirus Pneumonia (6th Edition) promulgated by Chinese government.
2.1 Experimental Groups:
2.2 Drug Dilution:
Mefuparib hydrochloride was obtained from Pukang (Shanghai) Health Technology Co., Ltd., with a purity of >99.0%. It was dissolved in DMSO and then diluted in gradients according to Table 4 below (the maintenance solution was DMEM medium). Serial dilutions were performed for DMSO according to the same dilution gradients.
2.3 Experimental Steps:
2.3.1 Cell Preparation and Dosing Culture
Vero-E6 cells were seeded into 96-well culture plates at 1×104 cells/well, and cultured in DMEM containing 10% fetal bovine serum for 16 hours to become 80% pellets, then the cell culture medium was aspirated and discarded from each well, the cells were washed once with sterile PBS, and different drugs (200 μl/well) diluted in the cell maintenance solution were added to each well according to the experimental groups, with 3 duplicate wells set up for each group, and then the wells were placed in a 37° C., 5% CO2 incubator for 48 h.
For the DMSO control group, DMSO diluted with the cell maintenance solution to the corresponding concentration was added, and for the cell control group, 200 μl of the cell maintenance solution was added. For the blank control group, only cell maintenance solution without cells was added.
2.3.2 Detection of toxicity of mefuparib hydrochloride and Olaparib on Vero-E6 The cell state was observed and recorded under an inverted microscope, and the toxicity of the drug to Vero-E6 cells was detected according to the operation instructions of the CCK-8 kit: 20 μl of CCK-8 solution was added to all experimental wells and control wells, and the cell culture plate was placed in a 37° C., 5% CO2 incubator for 1 hour, and then its absorbance value at the wavelength of 450 nm was measured using a multifunctional microplate reader. Calculations were conducted according to the formula: cell activity inhibition rate (%)=[1−(drug group−blank control group)/(cell control group−blank control group)]×100%.
Graphs were plotted with the drug concentrations of mefuparib hydrochloride and Olaparib as the abscissa, and the cell proliferation inhibition rate as the ordinate. The results are shown as solid circles in
3.1 Indirect Immunofluorescence Assay
Vero E6 cells were treated with 5 μM, 15 μM and 25 μM of CVL218, respectively, following the “whole process” treatment procedure. Infected cells were fixed with 80% acetone in PBS, permeabilized with 0.5% Triton X-100, and then blocked with 5% BSA in PBS buffer containing 0.05% Tween 20 for 30 minutes at room temperature. Further, with SARS-CoV nucleocapsid protein rabbit polyclonal antibody (Cambridgebio, USA) as the primary antibody, the cells were incubated at a dilution of 1:200 for 2 hours, and then Alexa 488 labeled goat anti-rabbit antibody (Beyotime, China) was used as the secondary antibody for incubation at a dilution of 1:500. Nuclei were stained with DAPI (Beyotime, China). Immunofluorescence was observed with a fluorescence microscope.
Immunofluorescence microscopy showed that at 14 h after the SARS-CoV-2 infection, the expression of the viral nucleoprotein (NP) in the CVL218-treated cells had a dose-response relationship with the concentration of the treatment drug, and the expression in the CVL218-treated cells was significantly lower than that in the DSMO treated cells (
3.2 Study on Duration of Action
In order to systematically assess the inhibitory activity of compounds against SARS-CoV-2, we also performed a time-of-addition assay (method) to determine at which stage the compounds inhibited the viral infection. The duration of action was measured using relatively high doses of the test drugs (20 μM for CVL218 and 10 μM for remdesivir). Vero E6 cells at a density of 5×104 cells per well were treated with test drugs at different stages of the viral infection, or with DMSO as a control. The MOI was 0.05 for infecting cells with the virus. The “whole process” treatment was designed to evaluate the maximum antiviral effect, and the test drug in the cell culture medium was the same as described in the viral infection assay throughout the experiment. In the “when the virus enters” treatment, test drug was added to cells 1 hour prior to viral infection, then the cells were maintained in the drug-virus mixture for 2 hours during viral infection. Afterwards, the medium containing virus and test drug was replaced with fresh medium until the end of the experiment. In the “after the virus enters” experiment, the virus was first added to the cells and allowed to infect for 2 hours, and then the virus-containing supernatant was replaced with a drug-containing medium until the end of the experiment. At 14 hours after infection, the inhibitory effect of the drug on the virus in the cell supernatant was quantitatively detected by qRT-PCR and calculated with the DMSO group as the reference.
The results show that, compared with the DMSO control group, both CVL218 and remdesivir showed stronger antiviral activity during the whole process of SARS-CoV-2 infection of Vero E6 cells (
Interleukin-6 (IL-6) has recently been found to be one of the most important cytokines during viral infection (L. Velazquez-Salinas, A. Verdugo-Rodriguez, L. L. Rodriguez, M. V. Borca, The role of interleukin 6 during viral infections, Frontiers in microbiology 10 (2019) 1057). The new human and animal clinical studies suggest that IL-6 oversynthesis is associated with the persistence of many viruses, such as human immunodeficiency virus (HIV) (M. M. McFarland-Mancini, H. M. Funk, A. M. Paluch, M. Zhou, P. V. Giridhar, C. A. Mercer, S. C. Kozma, A. F. Drew, Differences in wound healing in mice with deficiency of IL-6 versus IL-6 receptor, The journal of immunology 184 (12) (2010) 7219-7228), foot-and-mouth disease virus (G. A. Palumbo, C. Scisciani, N. Pediconi, L. Lupacchini, D. Alfalate, F. Guerrieri, L. Calvo, D. Salerno, S. Di Cocco, M. Levrero, et al., IL6 inhibits HBV transcription by targeting the epigenetic control of the nuclear cccdna minichromosome, PLoS one 10 (11)) and vesicular stomatitis virus (VSV) (L. Velazquez-Salinas, S. J. Pauszek, C. Stenfeldt, E. S. OHearn, J. M. Pacheco, M. V. Borca, A. Verdugo-Rodriguez, J. Arzt, L. L. Rodriguez, Increased virulence of an epidemic strain of vesicular stomatitis virus is associated with interference of the innate response in pigs, Frontiers in microbiology 9 (2018) 1891). Furthermore, in vivo studies using the Friend retrovirus (FV) mouse model have shown that IL-6 blockade can reduce viral load and enhance virus-specific CD8+ T cell immunity (W. Wu, K. K. Dietze, K. Gibbert, K. S. Lang, M. Trilling, H. Yan, J. Wu, D. Yang, M. Lu, M. Roggendorf, et al., TLR ligand induced IL-6 counter-regulates the anti-viral CD8+ T cell response during an acute retrovirus infection, Scientific reports 5 (2015) 10501). These findings support a hypothesis that the rapid production of IL-6 may be a possible mechanism leading to deleterious clinical manifestations in viral pathogenesis (J. Zheng, Y. Shi, L. Xiong, W. Zhang, Y. Li, P. G. Gibson, J. L. Simpson, C. Zhang, J. Lu, J. Sai, et al., The expression of IL-6, tNF-α, and MCP-1 in respiratory viral infection in acute exacerbations of chronic obstructive pulmonary disease, Journal of immunology research 2017). A recently published study on the clinical characteristics of critically ill patients with SARS-CoV-2 infections has shown that IL-6 is significantly elevated, especially in those patients treated at ICU, causing an overactivated immune response (Y. Zhou, B. Fu, X. Zheng, D. Wang, C. Zhao, Y. Qi, R. Sun, Z. Tian, X. Xu, H. Wei, Aberrant pathogenic GM-CSF+ T cells and inflammatory CD14+ CD16+ monocytes in severe pulmonary syndrome patients of a new coronavirus, bioRxiv; J.-J. Zhang, X. Dong, Y.-Y. Cao, Y.-d. Yuan, Y.-b. Yang, Y.-q. Yan, C. A. Akdis, Y.-d. Gao, Clinical characteristics of 140 patients infected by SARS-CoV-2 in Wuhan, China, Allergy; B. Diao, C. Wang, Y. Tan, X. Chen, Y. Liu, L. Ning, L. Chen, M. Li, Y. Liu, G. Wang, et al., Reduction and functional exhaustion of T cells in patients with coronavirus disease 2019 (COVID-19), medRxiv; X. C. Huang, Y. Wang, X. Li, L. Ren, J. Zhao, Y. Hu, L. Zhang, G. Fan, J. Xu, Gu, et al., Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China, The lancet 395 (10223) (2020) 497-506; Y. B. Li, F. Feng, G. Yang, A. Liu, N. Yang, Q. Jiang, H. Zhang, T. Wang, P. Li, Mao, et al., Immunoglobulin G/M and cytokines detections in continuous sera from patients with novel coronaviruses (2019-nCoV) infection, Available at SSRN 3543609). The pathological role of IL-6 in SARS-CoV-2 infection suggests that IL-6 blockade may provide a feasible therapy for treatment of COVID-19.
To test whether CVL218 can regulate IL-6 production in vitro, we stimulated IL-6 production by peripheral blood mononuclear cells (PMBCs) using CpG-ODN 1826, a potent stimulator of cytokines and chemokines. Incubation of PBMCs with 1 μM CpG-ODN 1826 for 6 hours (method) induced 40% IL-6 production compared to untreated cells (
Among them, the experimental steps of CpG-PDN1826-induced PBMCs to produce IL-6 are as follows: peripheral blood mononuclear cells (Beijing Yicon) were cultured in 96-well plates in RPMI1640 cell growth medium (Corning, Cat. 10-040-CVR) at 37° C. in a 5% CO2 atmosphere. For stimulation, PBMC cells were incubated with 1 μM CpG-ODN1826 (InvivoGen, Cat.tlrl-1826). To test whether CVL218 could inhibit IL-6 production, CVL218 was added to cell culture media at concentrations of 1 μM and 3 μM for 6 and 12 hours, respectively. Concentrations of IL-6 were determined by ELISA using a commercial kit (Dakewe Biotech, Cat. 1110602).
(I) Experimental Steps
5.1. Pharmacokinetic Studies
Sprague-Dawley rats were purchased from Animal Experiment Center of Shanghai, China. The experimental animals were housed in groups in wire cages, with no more than 6 animals per cage. The experimental conditions were good (temperature 25±2° C.; relative humidity 50±20%) and light-dark cycle (12 hours/12 hours). 144 Sprague-Dawley rats were randomized into 4 groups (18 animals/gender/group). CVL218 was administered at doses of 20, 40, 60 and 160 mg/kg. For all groups, 20 rats (10 animals/gender/group) were randomly selected, euthanized on day 28, and sections of various tissues and organs were obtained and frozen. Ten (5/gender/group) animals were euthanized after a 28-day drug-free period, and sections of their tissues and organs were taken and frozen. Six animals (3/gender/group) were euthanized after blood samples were collected. For pharmacokinetics and safety evaluations, the blood concentrations, clinical symptoms, mortality and body weight of animals were examined.
5.2. Tissue Distribution Study
Sprague-Dawley rats were randomized into 3 time-point groups (3 animals/gender/group). At 3, 6 and 8 hours after CVL218 administration, animals were sacrificed, and the brain, heart, lung, liver, spleen, stomach and kidney tissues were collected. The tissue samples were washed with ice-cold physiological saline and weighed after excess liquid was removed with paper towels. After tissue sample solutions were weighed, they were stored at −20±2° C. until the drug concentration was determined by LC-MS-MS.
5.3. Safety Study in Cynomolgus Monkeys.
Healthy male and female monkeys, aged 3-4 years, were purchased from Landao Biotechnology, Guangdong, China. Animals were fed in accordance with the Guide for Care and Use of Laboratory Animals. The animals were randomized (5 animals/gender/group), and were fed with CVL218 via nasogastric at dose levels of 0 (control), 5, 20 and 80 mg/kg. Monkeys were observed twice daily for any changes in viability/mortality and behavior, response to treatment or health status.
5.4. Statistical Analysis
All data represent the mean±standard deviations (SDs) of n values, where n corresponds to the number of data points used. Numbers were compiled using GraphPad Prism (GraphPad Software, USA). Statistical significance was calculated using SPSS (ver. 12), and two values were considered to be significantly different if p-value<0.05.
(II) Experimental Results and Conclusion
5.5. CVL218 Most Distributed in Rat Lung Tissues
The concentrations of CVL218 in different tissues at different time points after oral administration of different doses in rats are shown in
In addition, the results have shown that the pharmacokinetic parameters of CVL218 and arbidol are comparable, with similar plasma concentrations and drug exposures (Table 7). After administration to rats, arbidol is mainly distributed in the stomach and plasma. In contrast, CVL218 has a higher distribution in tissues, especially in the lung rather than in the plasma, which is higher than that of arbidol, indicating that CVL218 has a good pharmacokinetic profile, which may make it a potential antiviral therapeutic drug for SARS-CoV-2 lung infection.
After oral administration of 20/60/160 mg/kg CVL218 to rats for 28 days and then no administration for 28 days (method), we observed no significant difference in body weight between rats and cynomolgus monkeys at different doses and in the control group (
The in vivo data above suggest that CVL218 has favorable pharmacokinetic and safety profiles in rats and monkeys, and its high-level distribution in therapeutic target tissues (i.e., lungs) would be beneficial for treatment of SARS-CoV-2 infections.
Molecular docking was performed in the example to investigate the potential mode of interactions between two drugs, olaparib and CVL218, and the N-NTD of SARS-CoV-2.
A molecular interaction model of PARP1 inhibitors CVL218 and olaparib with the N protein N-terminal domain of SARS-CoV-2 (SARS-CoV-2-N-NTD) was generated using the docking program AutoDock4.2. The structure of SARS-CoV-2-N-NTD for molecular docking was established by homology modeling. The AutoGrid program was employed to generate grid maps with the spacing of 60×60×60 points of 0.375 Å, which were used to evaluate the binding energy between proteins and ligands.
The results showed (
In summary, it can be known that PARP1 inhibitors may have therapeutic potential in treatment of diseases caused by viruses such as COVID-19. First, during the life cycle of coronaviruses, PARP1 inhibitors may inhibit viral growth by inhibiting viral replication and preventing the binding of nucleocapsidin to viral RNAs, which can also be supported by our molecular docking results. Second, PARP1 inhibitors play a key role in controlling the inflammatory response by regulating pro-inflammatory factors such as IL-6, thereby providing clinical potential for alleviating cytokine storm and inflammatory response induced by SARS-CoV-2 infection. In the Diagnosis and Treatment Protocol for Novel Coronavirus Pneumonia (Trial Version 7) promulgated by the Chinese government, the monoclonal antibody drug tocilizumab against IL-6 is recommended for treatment of COVID-19, which also highlights the important role of inflammatory response in current SARS-CoV-2 treatment. CVL218 can effectively inhibit CpG-induced IL-6 production in PBMCs. This finding suggests that CVL218 may also have IL-6-specific anti-inflammatory effects in those patients with severe SARS-CoV-2 infection.
Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that these are only examples, and various changes or revisions may be made to these embodiments without departing from the principle and essence of the present invention. Therefore, the scope of the present invention shall be defined by the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010111755.4 | Feb 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/077610 | 2/24/2021 | WO |
