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WO2022166885A1 - Recombinant super-compound interferon (rsifn-co) for treating covid-19 patients with or without symptoms - Google Patents

Recombinant super-compound interferon (rsifn-co) for treating covid-19 patients with or without symptoms Download PDF

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Publication number
WO2022166885A1
WO2022166885A1 PCT/CN2022/074977 CN2022074977W WO2022166885A1 WO 2022166885 A1 WO2022166885 A1 WO 2022166885A1 CN 2022074977 W CN2022074977 W CN 2022074977W WO 2022166885 A1 WO2022166885 A1 WO 2022166885A1
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rsifn
covid
subject
spray
days
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PCT/CN2022/074977
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French (fr)
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Guangwen Wei
Rongbing Guo
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Sichuan Huiyang Life Science & Technology Corp.
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00Drugs for disorders of the respiratory system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404Indoles, e.g. pindolol
    • A61K31/4045Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425Thiazoles
    • A61K31/427Thiazoles not condensed and containing further heterocyclic rings
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47Quinolines; Isoquinolines
    • A61K31/47064-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/56Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19Cytokines; Lymphokines; Interferons
    • A61K38/21Interferons [IFN]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • A61P31/14Antivirals for RNA viruses

Definitions

  • the present invention relates to recombinant super-compound interferon (rSIFN-co) and its use to treat COVID-19 patients with or without symptoms.
  • COVID-19 Common symptoms of COVID-19 include fever (83–99%) , cough (59–82%) , fatigue (44–70%) , anorexia (40–84%) , shortness of breath (31–40%) , and myalgias (11–35%) [3] . Other reported symptoms have included, but are not limited to, sore throat, nasal congestion, headache, diarrhea, nausea and vomiting, anosmia, and ageusia [3] . The mortality of COVID-19 varies by geographical area. According to a recent analysis conducted by the Centers for Disease Control and Prevention (CDC) of the United States, more than 1.3 million laboratory-confirmed cases were reported between January and May 2020.
  • CDC Centers for Disease Control and Prevention
  • COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) , a newly discovered virus in the family of Coronaviridae (beta) , which has 89%nucleotide identity with bat SARS-like-CoVZXC21 and 82%with human SARS-CoV [6] .
  • SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA virus. Virus entry is achieved through binding of viral spike (S) protein to its receptor, angiotensin-converting enzyme 2 (ACE2) , on the host cell [7] .
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • ACE2 angiotensin-converting enzyme 2
  • the present invention discloses a method that prevents and/or treats COVID-19 (SARS-Cov-2) coronavirus infection, including mild COVID-19 infection and asymptomatic infection, in a subject suffering from diseases in digestive system, liver, heart and/or kidney.
  • the method comprises administering to the subject therapeutically effective doses, e.g., from 5 to 100 ⁇ g per day, of recombinant super-compound interferon (rSIFN-co) for a period of at least 3 days and up to 30 days.
  • the administration can be by nasal spray or intravenous injection.
  • Figure 1 is an illustration of the study schema.
  • Figure 2 compares structure of rSIFN-co and IFN- ⁇ 2b
  • Figure 3 compares structure of rSIFN-co and Infergen of Amgen
  • Figure 4 shows vector diagram of rSIFN-co in Plasmid pHY-5.
  • Figure 5 shows a manufacturing flowchart for rSIFN-co drug product.
  • interferons have long been used as antiviral treatments.
  • the preclinical studies of rSIFN-co have demonstrated its superiority of anti-SARS-CoV-2 capacity than conventional IFNs.
  • dysregulated IFNs was found in COVID-19 patients, implicating the role of IFN in COVID-19 pathogenesis [18] .
  • the potential of rSIFN-co as a protective and therapeutic agent against SARS-CoV-2 infection is suggested.
  • Impairment of type I interferon (IFN) response was discovered in severe and critical COVID-19 patients. No IFN- ⁇ and low IFN- ⁇ production/activity were observed and was associated with a persistent blood viral load and an exacerbated inflammatory response [17] .
  • IFNs type I interferon
  • IFN ⁇ -1a the efficacy and safety of IFN ⁇ -1a were evaluated in patients with severe COVID-19.
  • the patients in the IFN group received subcutaneous injection of 12 million IU/mL of IFN ⁇ -1a three times per week for two weeks [22] .
  • time to reach clinical response was not significantly improved, the proportions of subjects discharged on Day 14 were significantly higher in the IFN group [22] .
  • Recombinant super-compound interferon is a product of patented technological research developed by Sichuan Huiyang Life Science and Technology Corporation. It is a recombinant form of the naturally occurring cytokine interferon-alpha (IFN- ⁇ ) which has a modified spatial configuration.
  • rSIFN-co has the same amino acid sequence as the Amgen product (Interferon Alfacon-1) consisting of 167 amino acids (MCDLPQTHSLGNRRALILLAQMRRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSAAWDESLLEKFYTELYQQLNDLEACVIQEVGVEETPLMNVDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSLSTNLQERLRRKE, SEQ ID NO: 1) .
  • the amino acid homology of and rSIFN-co are naturally occurring interferon ⁇ -2a and interferon ⁇ -2b, with only a difference of 18 and 19 amino acids, respectively.
  • rSIFN-co This amino acid difference results in rSIFN-co having 10 times greater affinity for IFN-specific cell surface receptors IFNAR1 compared to interferon ⁇ -2a and interferon ⁇ -2b [15] .
  • rSIFN-co binds to IFN-specific cell surface receptors, resulting in the transcription and translation of genes whose protein products have antiviral, antiproliferative, anticancer, and immune-modulating effects.
  • the 3-dimensional conformational change improves efficacy and causes fewer side effects compared to IFN-a.
  • the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO. 1 is as follows (SEQ ID NO: 2) .
  • the rSIFN-co can be produced by the method comprising the following steps: introducing a nucleotide sequence comprising SEQ ID NO: 2 that encodes the recombinant interferon into an isolated host cell; culturing the host cell under appropriate condition for expression of the recombinant interferon; and harvesting the recombinant interferon, wherein the recombinant interferon has an amino acid sequence of SEQ ID NO: 1, and the recombinant interferon inhibits secretion of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) of Hepatitis B Virus.
  • the host cell is Escherichia coli.
  • the nucleotide sequence comprising SEQ ID NO: 2 is under the control of the promoter PBAD.
  • the harvesting step comprises extraction of the interferon from the fermentation broth, collection of the inclusion bodies, denaturation and renaturation of the harvested interferon. Still further, the harvesting step also comprises separation and purification of the recombinant interferon. Met at the N-terminus of SEQ ID NO: 1 is resected in mature protein.
  • the study used reagents including DMEM (Gibco) , fetal calf serum (Gibco) , DMSO (sigma) , double-antibody, pancreatine, etc., kits including Cell Counting Kit-8 (CCK-8) (B34304, bimake) , QIAamp viral RNA mini kit (52906, Qiagen) , Novel Coronavirus (2019-nCoV) Real Time RT-PCR kit (by Wuhan Institute of Virology) , and main instruments such as Varioskan Flash (by Thermo Fisher Scientific) , QIAcube HT 9001793 (by Qiagen) and CFX96 Touch Real-Time PCR Detection System (by Bio-rad) .
  • DMEM Gibco
  • Gibco fetal calf serum
  • DMSO double-antibody
  • pancreatine pancreatine, etc.
  • kits including Cell Counting Kit-8 (CCK-8) (B34304, bimake)
  • the final set of concentrations of rSIFN-co was 6 ⁇ 10 8 , 3 ⁇ 10 8 , 1.5 ⁇ 10 8 , 7.5 ⁇ 10 7 , 3.75 ⁇ 10 7 , 1.875 ⁇ 10 7 and 0 pg/ml
  • the final set of concentrations of recombinant human Interferon ⁇ -2b injection was 2 ⁇ 10 7 , 1 ⁇ 10 7 , 5 ⁇ 10 6 , 2.5 ⁇ 10 6 , 1.25 ⁇ 10 6 , 6.25 ⁇ 10 5 and 0 pg/ml
  • the final set of concentrations of Remdesivir was 320, 160, 80, 40, 20, 10 and 0 ⁇ M;
  • a) Grow Vero-E6 cells in a 24-well plate, 8 ⁇ 10 4 cells per well. Put the plate into 37 °Cincubator with 5%CO 2 . When the confluence reaches 70%-80%, using DMEN with 2%FBS for 2 times gradient dilution of rSIFN-co, recombinant human Interferon ⁇ -2b injection (pseudomonas) and Remdesivir, respectively.
  • qRT-PCR real time RT-PCR
  • RNA quantitative detection on collected virus. Take 20 ⁇ L collected supernatant after infection and extract RNA following instructions of QIAamp viral RNA mini kit. Conduct the qRT-PCR by using Novel Coronavirus (2019-nCoV) Real Time RT-PCR kit (TaqMan probe method) ;
  • Inhibition rate (%) 1 -Viral RNA Copies in test groups /Viral RNA Copies in drug free group ⁇ 100%. Analyze the half effective concentration of drugs (EC 50 ) by GraphPad PrisM6.0; and
  • Therapeutic Index (TI) half cytotoxicity concentration (CC 50 ) /half effective concentration (EC 50 ) .
  • the study showed that the EC 50 and CC 50 of rSIFN-co were 14.6 pg/mL and 7.12 ⁇ 10 8 pg/mL, respectively, whereas the EC 50 and CC 50 of IFN ⁇ -2b were 162 pg/mL and 1.37 ⁇ 10 7 pg/mL, respectively.
  • the results suggested that rSIFN-co exhibited a higher efficacy against SARS-CoV-2 and a lower cytotoxicity as compared with IFN ⁇ -2b.
  • PK pharmacokinetic
  • rSIFN-co induces more 2′-5′-oligoadenylate synthetase (2′, 5′-OAS; a specific marker triggered by interferon) over a longer time period, when compared to 2′, 5′-OAS induced by
  • the incidence of adverse events in the rSIFN-co group was lower than that in the group.
  • Type I interferons are known to play an important role in first-line defense against viruses by stimulating the expression of proteins such as ribonucleic acid (RNA) -dependent protein kinase, 2′, 5′-OAS, RNase L, and RNA-specific adenosine deaminase [16] .
  • RNA ribonucleic acid
  • 2′, 5′-OAS ribonucleic acid
  • RNase L RNA-specific adenosine deaminase
  • rSIFN-co severe acute respiratory syndrome
  • Sichuan aprovince in China
  • rSIFN-co spray was allocated to 3,000 users, including doctors and nurses in hospitals, populated areas with a high risk for SARS, and the National research group.
  • the highest dose was 16 million international unit (IU) and the longest treatment duration was over two months. No side effects connected to the use of the spray was reported, and none of users administered with rSIFN-co spray has been infected by SARS.
  • the present invention provides a method of preventing or treating Covid-19 (SARS-Cov-2) coronavirus infection in a subject.
  • the method comprises administering to the subject a therapeutically effective dose of recombinant super-compound interferon (rSIFN-co) for a period of at least 3 days.
  • rSIFN-co recombinant super-compound interferon
  • the rSIFN-co is administered to said subject by nasal spray, pharynx spray or intravenous injection.
  • the therapeutically effective dose ranges from 5 ⁇ g to 100 ⁇ g per day.
  • the therapeutically effective dose is selected from the group consisting of 8 ⁇ g, 16 ⁇ g, 32 ⁇ g, 64 ⁇ g and 96 ⁇ g per day.
  • the period is 5 to 30 days.
  • the subject suffers from one or more diseases in one or more of the digestive system, liver, heart, and kidney.
  • the subject receives one or more additional treatments selected from the group consisting of Remdesivir, hydroxychloroquine, and dexamethasone.
  • the subject is healthy or has an asymptomatic infection of Covid-19 coronavirus, and said subject receives 2 sprays per nostril and 4 sprays through pharynx once daily, each spray comprising 0.2-5.0 ⁇ g of said rSIFN-co.
  • the subject shows mild covid-19 infection and receives 2 sprays per nostril and 4 sprays through pharynx twice daily, each spray comprising 0.2-5.0 ⁇ g of said rSIFN-co.
  • the rSIFN-co has a specific activity greater than 5.0 x 10 8 IU/mg protein.
  • the nasal spray, pharynx spray or intravenous injection comprises one or more of stabilizer, bacteriostat, surfactant, metal complex, isosmotic adjusting agent, buffering agent, and viscosity modifier.
  • the stabilizer is human serum albumin.
  • the metal complex is disodium edetate.
  • the surfactant is Tween 80.
  • the buffering agent is citric acid.
  • the viscosity modifier is glycerol.
  • the isosmotic adjusting agent is sodium chloride.
  • the bacteriostat is benzyl alcohol.
  • the nasal spray, pharynx spray or intravenous injection has a pH value of about 5.0.
  • the nasal spray or pharynx spray is stable under 4-8°C for 3 months, at 25°C for 2 months and at 37°C for 2 weeks.
  • the present provides a method of preventing or treating Covid-19 (SARS-Cov-2) coronavirus infection in a subject.
  • the method comprises administering to the subject a composition comprising a therapeutically effective dose of recombinant super-compound interferon (rSIFN-co) for a period of at least 3 days
  • the present provides a method of preventing or treating Covid-19 (SARS-Cov-2) coronavirus infection in a subject by administering a composition consisting of a therapeutically effective dose of rSIFN-co for a period of at least 3 days.
  • SARS-Cov-2 Covid-19 coronavirus
  • the primary objective is to assess the safety and tolerability of rSIFN-co in healthy subjects in close contact with confirmed COVID-19 case (s) and patients with mild COVID-19 or asymptomatic infection.
  • the secondary objective is to assess the efficacy of rSIFN-co in healthy subjects in close contact with confirmed COVID-19 case (s) and patients with mild COVID-19 or asymptomatic infection.
  • the primary endpoint is the safety and tolerability profiles of rSIFN-co.
  • the secondary endpoint for Healthy subjects in close contact with confirmed COVID-19 case (s) is:
  • the secondary endpoint for patients with mild COVID-19 or asymptomatic infection is:
  • the exploratory endpoint for Healthy subjects in close contact with confirmed COVID-19 case (s) is the subjects’ evaluation on ease of use, discomfort using the study product, and overall satisfaction.
  • the exploratory endpoint for patients with mild COVID-19 or asymptomatic infection is:
  • the study is designed as a randomized, double-blind, placebo-controlled study to minimize the potential for subjective bias.
  • the inclusion of placebo arms will provide a control for comparison of efficacy endpoints between the treatment groups.
  • the study will be conducted at multiple study centers to facilitate subject enrollment and to increase the generalizability of the study results.
  • the doses evaluated for this study 8 and 16 million IU, are based on the totality of the data from previous trials in adults.
  • An eligible subject must fulfill ALL of the following inclusion criteria:
  • RT-PCR Negative reverse transcriptase polymerase chain reaction
  • Healthy subjects in close contact with confirmed COVID-19 case (s) Subjects with exposure to confirmed COVID-19 case (s) within 96 hours and belong to one of the following exposure situations per health department directives:
  • ICU intensive care unit
  • First responders i.e., first aid personnel, paramedics
  • Level 2 protection refers to wearing a disposable cap, medical protective mask (N95 or higher level medical protective mask) , goggles (anti-fog type) or protective mask (anti-fog type) , medical protective suit or overalls (white gown) , disposable gloves and disposable shoe covers.
  • Asymptomatic or presymptomatic infection Individuals who test positive for SARS-CoV-2 by virologic testing using a molecular diagnostic (e.g., polymerase chain reaction) or antigen test, but have no symptoms.
  • a molecular diagnostic e.g., polymerase chain reaction
  • antigen test e.g., antigen test, but have no symptoms.
  • rSIFN-co or placebo, for a maximum of 10 to 28 days.
  • rSIFN-co will be administered with one or more other agents, wherein the control group will receive said one or more other agents only.
  • rSIFN-co The dose of rSIFN-co given by nebulization or injection is equivalent to 8 to 16 million IU (32 ⁇ g) per day. Other agents will be given in their usual dosages.
  • Randomize eligible subjects into one of the two treatment arms i.e., placebo or rSIFN-co
  • Vital sign measurements include blood pressures (systolic blood pressure [SBP] and diastolic blood pressure [DBP] ) , pulse rate, respiratory rate, body temperature, and SpO 2 . Vital signs will be assessed at each visit. Results will be documented in the CRF. Measurements of all vital signs shall be completed in the during the visit within a time frame of 2 hours. Measurements should be taken in a seated position after resting for approximately 10 minutes. The same thermometer should be used consistently throughout the study. Subjects in Group B will be provided with an oximeter to help monitor their oxygen level. The subjects will be instructed to document daily oxygen level on the diary card.
  • SBP systolic blood pressure
  • DBP diastolic blood pressure
  • Fever is defined as rectal, ear, or forehead temperature greater than 38 °C. Site personnel should measure ear temperature of subject if possible. If other methods are used, the same method should be used consistently throughout the study.
  • a complete physical examination will be conducted by the Investigator or designee at screening.
  • Complete physical examination items include general appearance, HEENT (head, eyes, ears, nose, and throat) , mouth, skin, neck (including thyroid) , lymph nodes, spine, cardiovascular system, respiratory system, gastro-intestinal system, nervous system, musculoskeletal system, blood and blood forming organs, mental status, and other body systems if applicable for describing the status of subject’s health.
  • HEENT head, eyes, ears, nose, and throat
  • mouth skin, neck (including thyroid)
  • lymph nodes spine
  • cardiovascular system respiratory system
  • gastro-intestinal system gastro-intestinal system
  • nervous system musculoskeletal system
  • blood and blood forming organs forming organs, mental status, and other body systems if applicable for describing the status of subject’s health.
  • an abbreviated physical examination comprising a brief medical history and targeted physical examination will be performed at the discretion of the Investigator to evaluate possible adverse events.
  • Electrocardiogram ECG
  • Single 12-lead ECG will be obtained at screening using an ECG machine that automatically calculates the heart rate and measures PR, QRS, QT, and QTc (either Bazette or Fridericia formulas) intervals.
  • the assessment will follow the current SOPs at each participating study center. Throughout the study, additional ECGs may be obtained if clinically indicated.
  • the Investigators will compare ECGs to the screening ECG and document overall interpretation of specific ECGs as either normal, abnormal with clinical significance, or abnormal without clinical significance. An AE will be recorded if clinically significant.
  • Laboratory tests (including hematology, biochemistry, and coagulation) will be performed at screening, Days 5, 10, 15, 21, 28, and 60. All samples will be analyzed by the study center’s local laboratory per the standard operating procedures (SOPs) of the laboratory.
  • the examination items will include analytes in the following table:
  • Additional tests may be conducted at any time during the study as determined necessary by the Investigator. A clinically significant change from baseline will be recorded as an AE.
  • nasopharyngeal swabs are preferred [26] , but if these are not obtainable or additional samples are required by local regulations, oropharyngeal or nasal swabs may be obtained if necessary.
  • Samples will be delivered to the laboratories selected by the Sponsor to quantitatively and qualitatively detect viral RNA via RT-PCR assays approved by local regulatory authorities. Preparation, handling, storage, shipment, and examinations of specimens will follow the lab SOPs.
  • Thoracic CT-scan or chest X-ray will be performed at Screening, Day 5 (only for mild COVID-19 patients or asymptomatic infection) , and End of Treatment (Day 10) .
  • a standardized framework will be used to assess the chest imaging at each study site.
  • Clinical improvement will be defined as a decrease of at least one point on the 11-point scale compared to the baseline value (e.g., from 2 to 1; from 2 to 0) .
  • COVID-19 disease severity and symptoms will be assessed and documented at screening and throughout the entire study. Typical symptoms for patients with mild disease include fever, cough, fatigue, anorexia, shortness of breath, myalgias, sore throat, nasal congestion, headache, gastrointestinal symptoms, loss of smell (anosmia) , loss of taste (ageusia) , and without evidence of viral pneumonia or hypoxia.
  • Asymptomatic or presymptomatic infection Individuals who test positive for SARS-CoV-2 by virologic testing using a molecular diagnostic (e.g., polymerase chain reaction) or antigen test, but have no symptoms;
  • Mild disease Individuals who have any of the various signs and symptoms of COVID-19 (e.g., fever, cough, sore throat, malaise, headache, muscle pain) and a SpO2 ⁇ 94%on room air at sea level, without shortness of breath, dyspnea, or abnormal chest imaging;
  • COVID-19 e.g., fever, cough, sore throat, malaise, headache, muscle pain
  • SpO2 ⁇ 94%on room air at sea level, without shortness of breath, dyspnea, or abnormal chest imaging
  • Moderate disease Individuals who have evidence of lower respiratory disease by clinical assessment or imaging and SpO2 ⁇ 94%on room air at sea level;
  • Severe disease Individuals who have respiratory frequency >30 breaths per minute, SpO2 ⁇ 94%on room air at sea level, ratio of arterial partial pressure of oxygen to fraction of inspired oxygen (PaO 2 /FiO 2 ) ⁇ 300 mmHg, or lung infiltrates >50%; and
  • ⁇ Critical disease Individuals who have respiratory failure, septic shock, and/or multiple organ dysfunction.
  • the Master Cell Bank (MCB) and Working Cell Bank (WCB) for rSIFN-co are manufactured under cGMP.
  • the DS manufacture process involves bioproduction and harvest then purification of rSIFN-co from the E. coli inclusion bodies using upstream and downstream manufacturing processing typical for this type of production.
  • the rSIFN-co cDNA was cloned into the E. coli high expression vector pHY-4 plasmid to yield the final recombinant plasmid pHY-5 ( Figure 4) .
  • Typical production and purification can be found in US Patent No. 8,415,151 and Patent No. 8,846,025.
  • ⁇ PBAD promoter which derived from bacterial arabinose operon (araBAD operon) and was mainly regulated by CAP-cAMP and L-arabinose binding protein AraC i.e. product of ara Cgene
  • ⁇ ⁇ -Lactamase coded by Bla gene could provide ampicillin resistance.
  • ⁇ ColE1 replicon determined the copy number and replication of plasmid in bacteria.
  • Animal-based ingredients, tryptone and acid hydrolyzed casein, are used in upstream manufacture and are of GMP quality and certified that they are sourced in a controlled and validated way as to reduce the risk of contamination or exposure to any diseases, especially Bovine Spongiform Encephalopathy.
  • the release testing specifications for the DS include protein content, biological activity, specific activity, purity, molecular weight, exogenous DNA, host protein, antibiotic residue, endotoxin and a number of characterization assays.
  • the biological activity of rSIFN-co was determined based on the effect of rSIFN-co to protect cells against a viral cytopathic effect. It is quantified using a CPE potency assay, referenced against IFN ⁇ -2a reference standard, which has been tested and calibrated in International Units against the WHO reference standard.
  • the three-dimensional structure of the present recombinant interferon (SEQ ID NO: 3, without N-terminal M) is different from the three-dimensional structure of IFN ⁇ -2b (SEQ ID NO: 4) published in the art (see Figure 2) and the three-dimensional structure of based on computational modeling. There are obvious differences between the AB loops of the two, and their BC loops also cannot overlap completely (see Figure 3) .
  • Stability was tested according to ICH guideline for biological products Q1A and Q5C.
  • the stability of the rSIFN-co composition is tested under 4-8°C for 6 months, -80°C for 24 months, and accelerated condition at 25°C for 1 month. Repeated freeze/thaw cycle tests are done for 7 cycles. Stability data for 2 representative batches of drug substance 201801003, and 201801004 is shown in the following Tables 7-10.
  • a composition of rSIFN-co was prepared by obtaining a 80%of the total volume of water for injection, adding a prescribed amount of disodium edetate, polysorbate 80, citric acid, glycerol, sodium chloride and benzyl alcohol in turn while stirring. Add the next component after the former component is fully dissolved. After each component is added, stir for more than 60 minutes until well combined. Cool the solution to below 20°C and adjust pH value to about 5.0 with diluted hydrochloric acid. Stir the solution fully, add the prescribed amount of human serum albumin and rSIFN-co protein stock solution and then test pH value. If pH deviates, adjust pH value again to 5.0 and stir slightly. Dilute with water for injection cooled to below 20°C to volume, homogenizing by gently stirring for more than 30 minutes before filter and sterilize with a 0.22 ⁇ m filter to obtain the semi-finished rSIFN-co spray.
  • a typical workflow is shown in Figure 5.
  • composition (alternatively “drug product” ) is stable under 4-8°C for 3 months and the real time study is on-going. It is also stable under accelerated condition at 25°C for 2 months and at 37°C for 2 weeks.
  • the main components of the drug product are shown in the Table 11.
  • the product is a homogeneous colorless or light yellow with slightly sticky liquid spray.
  • the product is filled in high-density polyethylene bottles for pharmaceuticals with a mechanical nasal spray pump. Each bottle contains 8 ml and each pump is 0.1 ml.
  • a solution is prepared according to the formula followed by sterilization and filtering by a 0.22 ⁇ m filter.
  • the filtered liquid was transferred into a sterile container to obtain a semi-finished product.
  • the sterile filtered solution is packaged into 10 ml medicinal high-density polyethylene bottle for external use by filling machine and equip with medicinal spray pump.
  • the final product is stored at a controlled temperature of between 2-8°C, and the latest stability data showed that the drug product is stable under 4-8°C for 3 months and at accelerated condition of 25°C for 2 months and at 37°C for 2 weeks. Based on these test results at this time, the data supports a shelf life of 12 months and the stability program is on-going to support longer shelf life.
  • the drug product is supplied in spray bottle with mechanical pump.
  • the components of the container closure system are in conformance to the compendial requirements for spray bottle containers for pharmaceutical use.
  • the drug product is produced in GMP qualified facilities under ISO5, ISO7, and ISO8 clean room classification.
  • Stability was established according to ICH guideline for biological products Q1A and Q5C. Sponsor plans to test the drug product stability under 4-8°C for 36 months, at accelerated conditions 25°C for 6 months and 37°C for 1 month. Stability data for 3 representative batches of drug product 20200209, 20200210, and 20200311 is shown in the following Tables 14-17.
  • Example 3 Treatment by rSIFN-co of COVID-19 patients with symptoms
  • COVID-19 patients with symptoms such as mild pneumonia are treated by rSIFN-co or placebo for up to 10 to 28 days.
  • rSIFN-co is administered by nebulization or injection twice a day, 8 to 12 million International Units (IU) each time.
  • Clinical improvement is determined by assessing radiological improvement and/or virus nucleic acid negative conversion at the end of treatment period. The median time to reach clinical improvement is approximately 8 to 16 days, and an overall rate of clinical improvement at the end of treatment is approximately 80%to 90%.
  • Example 4 Treatment by rSIFN-co of COVID-19 patients without symptoms
  • COVID-19 patients without symptoms are treated by rSIFN-co or placebo for up to 10 to 28 days.
  • rSIFN-co is administered by nebulization or injection twice a day, 8 to 12 million International Units (IU) each time. Any COVID-19 symptoms are assessed during the course of treatment.
  • Patients treated with rSIFN-co are expected to show no symptom throughout the study, or significantly less treated patients show symptoms compared to patients receiving placebo.
  • Example 5 Treatment by rSIFN-co in combination with other agents of COVID-19 patients with symptoms
  • COVID-19 patients with symptoms such as mild pneumonia are treated by rSIFN-co in the presence of one or more agents for up to 10 to 28 days.
  • rSIFN-co is administered by nebulization or injection twice a day, 8 to 12 million International Units (IU) each time.
  • one or more of the following agents are also administered: (1) lopinavir (twice a day, 200 mg each time) , (2) ritonavir (twice a day, 50 mg each time) , (3) arbidol or umifenovir (three times a day, 200 mg each time) , (4) Remdesivir (100 mg per day) , (5) hydroxychloroquine (200 mg per day) , and (6) dexamethasone (4 mg per day) .
  • Clinical improvement is determined by assessing radiological improvement and/or virus nucleic acid negative conversion at the end of treatment period. The median time to reach clinical improvement and virus nucleic acid negative conversion is expected to be significantly shorter in patients receiving rSIFN-co than patients not receiving rSIFN-co.
  • Example 6 Safety and tolerability in healthy subjects receiving rSIFN-co
  • subjects receive rSIFN-co by nebulization or injection, or placebo for up to 10 to 28 days. Any symptoms or adverse events are assessed and recorded during the course of the study.
  • Example 7 Treatment by rSIFN-co of COVID-19 patients on respirators
  • COVID-19 patients on respirators are treated by rSIFN-co or placebo for up to 10 to 28 days.
  • rSIFN-co is administered by nebulization or injection twice a day, 8 to 12 million International Units (IU) each time.
  • Clinical improvement is determined by assessing blood oxygen level during and at the end of treatment period. The median time to reach clinical improvement is approximately 8 to 16 days, and an overall rate of clinical improvement at the end of treatment is approximately 80%to 90%.
  • Example 8 Treatment by rSIFN-co Spray
  • healthy subjects or patients with an asymptomatic infection of Covid-19 receive 2 sprays per nostril and 4 sprays through pharynx once daily. Each spray comprises 2 ⁇ g of rSIFN-co.
  • the period of treatment is approximately 5 days to 20 days.
  • Subjects treated with rSIFN-co are expected to show no symptom throughout the study, or significantly less treated patients show symptoms compared to patients receiving placebo.
  • each spray comprises 2 ⁇ g of rSIFN-co.
  • the period of treatment is approximately 10 days to 30 days.
  • the median time to reach clinical improvement and virus nucleic acid negative conversion is expected to be significantly shorter in patients receiving rSIFN-co than patients not receiving rSIFN-co.

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Abstract

The present invention discloses a method for preventing and/or treating COVID-19 (SARS-Cov-2) coronavirus infection by administering to a subject in need thereof a therapeutically effective dose of recombinant super-compound interferon (rSIFN-co) for a period. In one embodiment, the dose is from 1.0 to 100 μg per day and the period is at least 3 days and up to 30 days. In one embodiment, the rSIFN-co is administered by nasal spray, pharynx spray or intravenous injection.

Description

RECOMBINANT SUPER-COMPOUND INTERFERON (RSIFN-CO) FOR TREATING COVID-19 PATIENTS WITH OR WITHOUT SYMPTOMS FIELD OF THE INVENTION
The present invention relates to recombinant super-compound interferon (rSIFN-co) and its use to treat COVID-19 patients with or without symptoms.
BACKGROUND OF THE INVENTION
Since the outbreak of coronavirus disease 2019 (COVID-19) in December 2019, the disease has exploded into a global pandemic. As of December 8, 2020, nearly 66 million COVID-19 cases and 1,500,000 deaths have been reported [1] . From a report of 72, 314 COVID-19 patients in China, 81%of the cases were categorized into mild diseases (defined as no pneumonia or mild pneumonia) , 14%were severe (defined as dyspnea, respiratory frequency ≥30 breaths/min, SpO 2 ≤93%, PaO 2/FiO 2 <300 mmHg, and/or lung infiltrates >50%within 24 to 48 hours) , and 5%were critical (defined as respiratory failure, septic shock, and/or multiple organ dysfunction or failure) [2] . Common symptoms of COVID-19 include fever (83–99%) , cough (59–82%) , fatigue (44–70%) , anorexia (40–84%) , shortness of breath (31–40%) , and myalgias (11–35%) [3] . Other reported symptoms have included, but are not limited to, sore throat, nasal congestion, headache, diarrhea, nausea and vomiting, anosmia, and ageusia [3] . The mortality of COVID-19 varies by geographical area. According to a recent analysis conducted by the Centers for Disease Control and Prevention (CDC) of the United States, more than 1.3 million laboratory-confirmed cases were reported between January and May 2020. Of these cases, 14%of patients required hospitalization, 2%were admitted to an intensive care unit, and 5%died [4] . Although individuals of all ages are at risk of infection and severe disease, known risk factors for rapid deterioration, severe disease, and/or increased mortality include older age (more than 60 years) , noncommunicable diseases (e.g., diabetes, hypertension, cardiac disease, chronic lung disease, chronic kidney disease, immunosuppression and cancer) , and smoking [5] .
COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) , a newly discovered virus in the family of Coronaviridae (beta) , which has 89%nucleotide identity with bat SARS-like-CoVZXC21 and 82%with human SARS-CoV [6] . SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA virus. Virus entry is achieved through binding of viral spike (S) protein to its receptor, angiotensin-converting enzyme 2 (ACE2) , on the host cell [7] .
Current studies suggest that transmission of SARS-CoV-2 is primarily occurs through direct, indirect, or close contact with infected people via secretions such as respiratory droplets or saliva containing SARS-CoV-2 [3, 8] . According to the China National Health Commission’s guideline for diagnosis and treatment of COVID-19, virus could be found in human respiratory epithelial cells using in vitro culture system in about 96 hours [9] . Investigations of COVID-19 case clusters showed that transmission can occur from presymptomatic patients, from 1 to 3 days prior to symptom onset [10, 11] , which causes difficulties to contact tracing and disease control. As reports have suggested that viral shedding is highest at the time of symptom onset and that decreased viral load was correlated with reduced infectivity [12, 13] , transmissibility of COVID-19 may peak on or before symptom onset [14] . Therapeutics targeting virus reproduction may not only present with potential of disease treatment but also restricting its spreading.
To evaluate the efficacy of safety of nebulized rSIFN-co for moderate-to-severe COVID-19 patients, a phase II, randomized, single-blind study was conducted in China. Ninety-four (94) subjects were randomized in a 1: 1 ratio to receive either 12 million IU of rSIFN-co (twice daily) or 5 million IU of IFN alfa (twice daily) for a maximum of 28 days in addition to treatment with standard antiviral agents. It was found that subjects in the rSIFN-co arm had a significantly shorter time to clinical improvement (11.5 days vs 14.0 days) , radiological improvement (8.0 days vs 10.0 days) , and viral nucleic acid negative conversion (7.0 vs 10.0 days) than the IFN alfa arm. On Day 14, a significant higher proportion of patients receiving rSIFN-co had clinical improvement (65.2%vs 39.6%) . The percentage of subjects reporting adverse event during the study was lower in the rSIFN-co than the IFN alfa arm (28.3%vs 37.5%) [23] . However, there is no study suggesting  whether rSIFN-co alone would treat COVID-19 infection, nor the effective dose of rSIFN-co for treating COVID-19 infection with no/limited side effects.
SUMMARY OF THE INVENTION
The present invention discloses a method that prevents and/or treats COVID-19 (SARS-Cov-2) coronavirus infection, including mild COVID-19 infection and asymptomatic infection, in a subject suffering from diseases in digestive system, liver, heart and/or kidney. The method comprises administering to the subject therapeutically effective doses, e.g., from 5 to 100 μg per day, of recombinant super-compound interferon (rSIFN-co) for a period of at least 3 days and up to 30 days. The administration can be by nasal spray or intravenous injection.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is an illustration of the study schema.
Figure 2 compares structure of rSIFN-co and IFN-α2b
Figure 3 compares structure of rSIFN-co and Infergen of Amgen
Figure 4 shows vector diagram of rSIFN-co in Plasmid pHY-5.
Figure 5 shows a manufacturing flowchart for rSIFN-co drug product.
DETAILED DESCRIPTION OF THE INVENTION
Currently, there are no specific treatment for COVID-19, and no known agent administered before or after exposure can prevent SARS-CoV-2 infection. Symptomatic and supportive treatment is still the mainstay for the disease. Given the highly contagious nature of COVID-19 and its devastating social and economic impacts, development of effective pharmacologic interventions for disease prevention and control is of urgent need.
With abilities of immunomodulation and interfering with viral replications, interferons (IFNs) have long been used as antiviral treatments. The preclinical studies of rSIFN-co have demonstrated its superiority of anti-SARS-CoV-2 capacity than conventional IFNs. Moreover, dysregulated IFNs was found in COVID-19 patients, implicating the role of IFN in COVID-19 pathogenesis [18] .  Hence, the potential of rSIFN-co as a protective and therapeutic agent against SARS-CoV-2 infection is suggested.
Type I interferon for COVID-19
Impairment of type I interferon (IFN) response was discovered in severe and critical COVID-19 patients. No IFN-β and low IFN-α production/activity were observed and was associated with a persistent blood viral load and an exacerbated inflammatory response [17] . By blocking viral infection and eliciting immune responses, administration of IFNs at early stage of disease in patients or as protective agents in healthy subject may help prevent disease exacerbation or development [18] .
In the China National Health Commission’s guideline for diagnosis and treatment of COVID-19, 5 million IU or equivalence of IFN α is recommended for the treatment of COVID-19 at a frequency of two times a day, with a duration of no more than 10 days [9] . The application of IFN α/β for COVID-19 has also been suggested by others [19, 20] .
In a recent open-label study conducted in Hubei, China, 2415 subjects were treated with recombinant human IFN α nasal drops (2-3 drops/nostril/time; 4 times/day) for 28 days. No new-onset COVID-19 was observed in these subjects. While no flu-like symptoms was reported, some subjects experienced transient irritation of nasal mucosa (e.g., itching) , which was subsequently resolved without discontinuing study intervention [21] .
In another study, the efficacy and safety of IFN β-1a were evaluated in patients with severe COVID-19. In addition to the standard of care, the patients in the IFN group received subcutaneous injection of 12 million IU/mL of IFN β-1a three times per week for two weeks [22] . Although time to reach clinical response was not significantly improved, the proportions of subjects discharged on Day 14 were significantly higher in the IFN group [22] .
rSIFN-co for COVID-19
Recombinant super-compound interferon (rSIFN-co) is a product of patented technological research developed by Sichuan Huiyang Life Science and Technology Corporation. It is a  recombinant form of the naturally occurring cytokine interferon-alpha (IFN-α) which has a modified spatial configuration. rSIFN-co has the same amino acid sequence as the Amgen product 
Figure PCTCN2022074977-appb-000001
(Interferon Alfacon-1) consisting of 167 amino acids (MCDLPQTHSLGNRRALILLAQMRRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSAAWDESLLEKFYTELYQQLNDLEACVIQEVGVEETPLMNVDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSLSTNLQERLRRKE, SEQ ID NO: 1) . The amino acid homology of
Figure PCTCN2022074977-appb-000002
and rSIFN-co are naturally occurring interferon α-2a and interferon α-2b, with only a difference of 18 and 19 amino acids, respectively. This amino acid difference results in rSIFN-co having 10 times greater affinity for IFN-specific cell surface receptors IFNAR1 compared to interferon α-2a and interferon α-2b [15] . rSIFN-co binds to IFN-specific cell surface receptors, resulting in the transcription and translation of genes whose protein products have antiviral, antiproliferative, anticancer, and immune-modulating effects. The 3-dimensional conformational change improves efficacy and causes fewer side effects compared to IFN-a. The nucleic acid sequence encoding the amino acid sequence of SEQ ID NO. 1 is as follows (SEQ ID NO: 2) .
Figure PCTCN2022074977-appb-000003
In one embodiment, the rSIFN-co can be produced by the method comprising the following steps: introducing a nucleotide sequence comprising SEQ ID NO: 2 that encodes the recombinant interferon into an isolated host cell; culturing the host cell under appropriate condition for expression of the recombinant interferon; and harvesting the recombinant interferon, wherein the  recombinant interferon has an amino acid sequence of SEQ ID NO: 1, and the recombinant interferon inhibits secretion of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) of Hepatitis B Virus. Further, the host cell is Escherichia coli. Further, the nucleotide sequence comprising SEQ ID NO: 2 is under the control of the promoter PBAD. Further, the harvesting step comprises extraction of the interferon from the fermentation broth, collection of the inclusion bodies, denaturation and renaturation of the harvested interferon. Still further, the harvesting step also comprises separation and purification of the recombinant interferon. Met at the N-terminus of SEQ ID NO: 1 is resected in mature protein.
In one embodiment, this invention provides a crystalline recombinant interferon comprising the amino acid sequence of SEQ ID NO: 1. Further, this crystal belongs to the trigonal system. In one embodiment, the space group of this crystal is P3 121. In some embodiments, the unit cell parameters of this crystal are
Figure PCTCN2022074977-appb-000004
α = β = 90°, γ = 120°, with a variability of at most 5%in all cell parameters. In some embodiments, said crystal contains two molecules in one asymmetric unit. In some embodiments, said crystal comprises covalently or non-covalently bound metal ions. Further, said metal ions can be magnesium ion, zinc ion and the like, these metal ions can mediate the formation of the interferon dimers in the crystal.
In an in vitro study, the anti-SARS-CoV-2 activities of rSIFN-co has been investigated in Vero-E6 cell to assess rSIFN-co’s inhibitory effect on 2019-nCoV’s replication at cell level. The Vero-E6 cell line was kept in the lab at a multiplicity of infection (MOI) of 0.005. The study used reagents including DMEM (Gibco) , fetal calf serum (Gibco) , DMSO (sigma) , double-antibody, pancreatine, etc., kits including Cell Counting Kit-8 (CCK-8) (B34304, bimake) , QIAamp viral RNA mini kit (52906, Qiagen) , Novel Coronavirus (2019-nCoV) Real Time RT-PCR kit (by Wuhan Institute of Virology) , and main instruments such as Varioskan Flash (by Thermo Fisher Scientific) , QIAcube HT 9001793 (by Qiagen) and CFX96 Touch Real-Time PCR Detection System (by Bio-rad) .
The following method was used to determine the cytotoxicity of rSIFN-co and recombinant human Interferon α-2b injection (pseudomonas) :
a) Grow Vero E6 cells in 96 micro well plate one day in advance, 1×10 4 cells per well (Attention: The peripheral wells on the plate shall not be used for experiment, and add in PBS to prevent the volatilization of medium in other wells; ) 
b) Observe the cells and when the confluence reaches 50%, use 2×DMEN with 4%FBS to dilute rSIFN-co 2.17 times to obtain the maximum concentration, i.e., 6×10 8 pg/ml, and dilute recombinant human Interferon α-2b injection (pseudomonas) 1.5 times to obtain the maximum concentration, i.e., 2×10 7 pg/ml. Then use DMEN with 2%FBS for 2 times gradient dilution of rSIFN-co, recombinant human Interferon α2b injection (pseudomonas) and Remdesivir respectively. The final set of concentrations of rSIFN-co was 6×10 8, 3×10 8, 1.5×10 8, 7.5×10 7, 3.75×10 7, 1.875×10 7 and 0 pg/ml, the final set of concentrations of recombinant human Interferon α-2b injection was 2×10 7, 1×10 7, 5×10 6, 2.5×10 6, 1.25×10 6, 6.25×10 5 and 0 pg/ml, and the final set of concentrations of Remdesivir was 320, 160, 80, 40, 20, 10 and 0 μM;
c) Add the above solutions to the plate at 100 μL/well, and 4 wells per concentration. Set control group (drug free) and blank group (cell free) . Put plates into an incubator with 5%CO 2 at 37 ℃; and
d) After adding drug 48 hours, add Cell Counting Kit-8 (CCK-8) at ratio of 1/10 cell culture medium and mix well, avoiding bubbles. Put plates into the incubator at 37 ℃ for about one hour until it turns orange. Zero setting microplate reader with blank group, then read OD 450 values of the whole plate. Calculate the survival rate by the formula: Survival rate = Drug group OD 450 /Control group OD 450 × 100%, and then calculate the half cytotoxicity concentration (CC 50) of drugs.
Results are summarized in Table 1.
TABLE 1. The Half Cytotoxicity Concentration (CC 50) of rSIFN-co and Recombinant Human Interferon α2b Injection (Pseudomonas) to Vero E6 Cells
Figure PCTCN2022074977-appb-000005
The following method was used to evaluate the inhibitory effect of rSIFN-co and recombinant human Interferon α-2b injection (pseudomonas) on 2019-n-CoV:
a) Grow Vero-E6 cells in a 24-well plate, 8×10 4 cells per well. Put the plate into 37 ℃incubator with 5%CO 2. When the confluence reaches 70%-80%, using DMEN with 2%FBS for 2 times gradient dilution of rSIFN-co, recombinant human Interferon α-2b injection (pseudomonas) and Remdesivir, respectively. Discard the culture medium in each well and add in 1 ml 2019-nCoV (COVID-19) solution (MOI as 0.005) and the DMEM with drugs (set the concentrations of Remdesivir as 10, 5, 2.5, 1.25, 0.625, 0.3 and 0 μM, concentration of rSIFN-co as 60, 30, 15, 7.5, 3.75 and 0 pg/ml, and concentration of recombinant human Interferon α-2b injection (pseudomonas) as 300, 150, 75, 37.5, 18.75 and 0 pg/ml) . Set a control group at the same time. Collect supernatant 24 hours after infection;
b) Use real time RT-PCR (qRT-PCR) for RNA quantitative detection on collected virus. Take 20 μL collected supernatant after infection and extract RNA following instructions of QIAamp viral RNA mini kit. Conduct the qRT-PCR by using Novel Coronavirus (2019-nCoV) Real Time RT-PCR kit (TaqMan probe method) ;
c) Calculate the drug inhibition rate at each concentration. Inhibition rate (%) = 1 -Viral RNA Copies in test groups /Viral RNA Copies in drug free group × 100%. Analyze the half effective concentration of drugs (EC 50) by GraphPad PrisM6.0; and
d) Calculate the therapeutic index of drugs by formula: Therapeutic Index (TI) = half cytotoxicity concentration (CC 50) /half effective concentration (EC 50) .
Results are summarized in Tables 2 and 3.
Table 2. Half Effective Concentration (EC 50) of rSIFN-co and Recombinant Human Interferon α2b Injection (Pseudomonas) for Inhibiting 2019-nCoV (COVID-19) Replication in Vero-E6 Cell Model
Figure PCTCN2022074977-appb-000006
Table 3. Therapeutic Index (TI) of rSIFN-co and Recombinant Human Interferon α2b Injection (Pseudomonas)
Figure PCTCN2022074977-appb-000007
In summary, the study showed that the EC 50 and CC 50 of rSIFN-co were 14.6 pg/mL and 7.12×10 8 pg/mL, respectively, whereas the EC 50 and CC 50 of IFN α-2b were 162 pg/mL and  1.37×10 7 pg/mL, respectively. The therapeutic index (TI=CC 50/EC 50) was 4.88×10 7 for rSIFN-co and 8.46×10 4 for IFN α-2b. The results suggested that rSIFN-co exhibited a higher efficacy against SARS-CoV-2 and a lower cytotoxicity as compared with IFN α-2b. In a separate study, rSIFN-co further exhibited a lower EC 50 at a MOI of 0.05 and 0.5 than IFN α-2b or IFN β-1b (EC 50 when the MOI=0.05 for rSIFN-co: 133.75 pg/mL, IFN α-2b: 575.65 pg/mL, IFN β-1b: 194.60 pg/mL; EC 50 when the MOI=0.5 for rSIFN-co: 164.15 pg/mL, IFN α-2b: 2432.50 pg/mL, IFN β-1b: 1240.00 pg/mL) .
In a pharmacokinetic (PK) study comparing rSIFN-co and
Figure PCTCN2022074977-appb-000008
21 healthy male volunteers were enrolled at the Drug Clinical Research Base, West China Hospital of Sichuan University. The subjects were given a single dose of 9 μg rSIFN-co (Group A) or 9 μg
Figure PCTCN2022074977-appb-000009
 (Group B) , and later switched over, and their blood taken at specified intervals. The PK results suggest that rSIFN-co induces more 2′-5′-oligoadenylate synthetase (2′, 5′-OAS; a specific marker triggered by interferon) over a longer time period, when compared to 2′, 5′-OAS induced by 
Figure PCTCN2022074977-appb-000010
In addition, the incidence of adverse events in the rSIFN-co group was lower than that in the
Figure PCTCN2022074977-appb-000011
group. The majority of the adverse effects experienced after the treatment of both rSIFN-co and
Figure PCTCN2022074977-appb-000012
included fever, headache, myalgia, fatigue and chills.
Type I interferons are known to play an important role in first-line defense against viruses by stimulating the expression of proteins such as ribonucleic acid (RNA) -dependent protein kinase, 2′, 5′-OAS, RNase L, and RNA-specific adenosine deaminase [16] . In the phase II clinical study of rSIFN-co in patients with chronic hepatitis B, rSIFN-co at 9 μg exhibited similar efficacy as 50 μg interferon α-1b, which was demonstrated by the suppression of hepatitis B virus replication, HBeAg/HBeAb seroconversion, and ALT reversion to normal levels. The rSIFN-co treatment was also better tolerated compared to interferon α-1b.
A set of experiments was also conducted to compare rSIFN-co, IFN α2b, β-1b, and hydrochloroquine at a high Multiplicity of Infection (MOI) of 0.5. The TI of rSIFN-co was is 4.34×10 6 which is 770 times higher than that of IFN α2b; EC50 for rSIFN-co is 164.15 pg/mL  which is 7 times lower in concentration as compare to β-1b which requires 1240.00 pg/mL. The result is summarized in Table 4.
Table 4. Comparison of rSIFN-co, INFα2b, IFN β-1b, and Hydrochloroquine in MOI of 0.5
Figure PCTCN2022074977-appb-000013
An in vitro study was conducted to compare rSIFN-co protection in viral infection to various forms of commercially available IFN products and Remdesivir. This includes IFN-α2b, IFNα8, IFNα14, IFNβ-1b, IFNω. As shown in Table 5, this study demonstrated that rSIFN-co at a concentration of 50U/mL is effective in protecting viral infection of up to 48 hours. This study further demonstrated that rSIFN-co could induce 2’-5’-oligoadenylate synthetase (2’, 5’-OAS) and under the same dose could have a better protective mechanism.
Table 5. Infection Protection comparing rSIFN-co to various commercial IFNs
Figure PCTCN2022074977-appb-000014
Furthermore, during the outbreak of severe acute respiratory syndrome (SARS) , rSIFN-co was distributed with the consent of the Sichuan (aprovince in China) working group on SARS prevention and control. rSIFN-co spray was allocated to 3,000 users, including doctors and nurses in hospitals, populated areas with a high risk for SARS, and the National research group. Among all users, the highest dose was 16 million international unit (IU) and the longest treatment duration was over two months. No side effects connected to the use of the spray was reported, and none of users administered with rSIFN-co spray has been infected by SARS.
In one embodiment, the present invention provides a method of preventing or treating Covid-19 (SARS-Cov-2) coronavirus infection in a subject. In one embodiment, the method comprises administering to the subject a therapeutically effective dose of recombinant super-compound interferon (rSIFN-co) for a period of at least 3 days.
In one embodiment, the rSIFN-co is administered to said subject by nasal spray, pharynx spray or intravenous injection.
In one embodiment, the therapeutically effective dose ranges from 5 μg to 100 μg per day.
In one embodiment, the therapeutically effective dose is selected from the group consisting of 8 μg, 16 μg, 32 μg, 64 μg and 96 μg per day.
In one embodiment, the period is 5 to 30 days.
In one embodiment, the subject suffers from one or more diseases in one or more of the digestive system, liver, heart, and kidney.
In one embodiment, the subject receives one or more additional treatments selected from the group consisting of Remdesivir, hydroxychloroquine, and dexamethasone.
In one embodiment, the rSIFN-co forms a crystal of the space group of P3 121, and the unit cell parameters of the crystal are
Figure PCTCN2022074977-appb-000015
α=β=90°, and γ=120°.
In one embodiment, the subject is healthy or has an asymptomatic infection of Covid-19 coronavirus, and said subject receives 2 sprays per nostril and 4 sprays through pharynx once daily, each spray comprising 0.2-5.0 μg of said rSIFN-co.
In one embodiment, the subject shows mild covid-19 infection and receives 2 sprays per nostril and 4 sprays through pharynx twice daily, each spray comprising 0.2-5.0 μg of said rSIFN-co.
In one embodiment, the rSIFN-co has a specific activity greater than 5.0 x 10 8 IU/mg protein.
In one embodiment, the nasal spray, pharynx spray or intravenous injection comprises one or more of stabilizer, bacteriostat, surfactant, metal complex, isosmotic adjusting agent, buffering agent, and viscosity modifier.
In one embodiment, the stabilizer is human serum albumin. In one embodiment, the metal complex is disodium edetate. In one embodiment, the surfactant is Tween 80. In one embodiment, the buffering agent is citric acid. In one embodiment, the viscosity modifier is glycerol. In one embodiment, the isosmotic adjusting agent is sodium chloride. In one embodiment, the bacteriostat is benzyl alcohol.
In one embodiment, the nasal spray, pharynx spray or intravenous injection has a pH value of about 5.0.
In one embodiment, the nasal spray or pharynx spray is stable under 4-8℃ for 3 months, at 25℃ for 2 months and at 37℃ for 2 weeks.
In one embodiment, the present provides a method of preventing or treating Covid-19 (SARS-Cov-2) coronavirus infection in a subject. In one embodiment, the method comprises administering to the subject a composition comprising a therapeutically effective dose of recombinant super-compound interferon (rSIFN-co) for a period of at least 3 days
In one embodiment, the present provides a method of preventing or treating Covid-19 (SARS-Cov-2) coronavirus infection in a subject by administering a composition consisting of a therapeutically effective dose of rSIFN-co for a period of at least 3 days.
Objectives
The primary objective is to assess the safety and tolerability of rSIFN-co in healthy subjects in close contact with confirmed COVID-19 case (s) and patients with mild COVID-19 or asymptomatic infection.
The secondary objective is to assess the efficacy of rSIFN-co in healthy subjects in close contact with confirmed COVID-19 case (s) and patients with mild COVID-19 or asymptomatic infection.
The primary endpoint is the safety and tolerability profiles of rSIFN-co.
The secondary endpoint for Healthy subjects in close contact with confirmed COVID-19 case (s) is:
1. Incidence of laboratory-confirmed SARS-CoV-2 infection over 28 days (Day 1 to Day 28)
2. Severity and clinical status of COVID-19 over the study period, and
3. Incidence of COVID-19-related complications.
The secondary endpoint for patients with mild COVID-19 or asymptomatic infection is:
1. Proportion of subjects with COVID-19-related hospitalization by Day 28,
2. Proportion of subjects who require supplemental oxygen or mechanical ventilation by Day 28,
3. Clinical improvement rates on Days 5 and 10,
4. Change from baseline per FDA definition ordinal scale on Days 5 and 10,
5. Time to clinical improvement from the lowest outcome scores,
6. Change in disease severity from baseline to Days 5 and 10,
7. Qualitative and quantitative polymerase chain reaction (PCR) for SARS-CoV-2 in respiratory tract on  Days  1, 2, 3, 5, 7, 10, 15, and 21,
8. Time to clearance of SARS-CoV-2, defined as 2 consecutive negative swabs (sampling interval ≥ 24 hours) , and
9. Time to resolution of all symptoms present at baseline.
The exploratory endpoint for Healthy subjects in close contact with confirmed COVID-19 case (s) is the subjects’ evaluation on ease of use, discomfort using the study product, and overall satisfaction.
The exploratory endpoint for patients with mild COVID-19 or asymptomatic infection is:
1. Incidence of COVID-19 pneumonia, cough, and fever over the study period,
2. Proportion of subjects without fever on  Days  1, 5, 10, and 15,
3. Subjects’ evaluation on ease of use, discomfort using the study product, and overall satisfaction,
4. Incidence of admission to a critical care unit, and
5. COVID-19 seroconversion rate for IgG and IgM on Days 5, 10, 15, and 21.
STUDY DESIGNS
General Design
This is a phase II, multi-site, randomized, double-blind, placebo-controlled pilot study that aims to evaluate the safety and preliminary efficacy of rSIFN-co in healthy subjects in close contact with confirmed COVID-19 case (s) as well as patients with mild COVID-19 or asymptomatic infection. Approximately 120 subjects are planned to be enrolled, including 60 healthy subjects and 60 patients.
For the healthy subjects in close contact with confirmed COVID-19 case (s) (Group A) , approximately 60 subjects will be randomized in a 1: 1 ratio to one of the two arms: placebo arm or rSIFN-co arm.
For the patients with mild COVID-19 or asymptomatic infection (Group B) , approximately 60 subjects will be randomly assigned in a 1: 1 ratio to receive standard of care (SoC) plus placebo or SoC plus rSIFN-co.
All eligible subjects will receive the study medications for 10 days, around 8 million IU (16 μg) of rSIFN-co for healthy subjects and 16 million IU (32 μg) for COVID-19 patients daily (see Dosage, Preparation and Administration of Investigational Product for details) . After 10 days of treatment, a safety follow-up visit on Days 15, 21, 28, and 60 will be conducted. The safety and efficacy parameters will be monitored throughout the study period.
Scientific Rationale for Study Design
To evaluate the safety and preliminary efficacy of the investigational product in healthy subjects in close contact with confirmed COVID-19 case (s) and patients with mild COVID-19 or asymptomatic infection, the study is designed as a randomized, double-blind, placebo-controlled study to minimize the potential for subjective bias. The inclusion of placebo arms will provide a control for comparison of efficacy endpoints between the treatment groups. The study will be conducted at multiple study centers to facilitate subject enrollment and to increase the generalizability of the study results.
As therapeutic options for patients with mild COVID-19 are limited, and symptomatic and supportive treatment is currently the mainstay for COVID-19. For this reason, local SoC for COVID-19 is incorporated and permitted for patients with mild COVID-19 or asymptomatic infection (Group B) in order to satisfy subjects’ need.
Justification for Dose
The doses evaluated for this study, 8 and 16 million IU, are based on the totality of the data from previous trials in adults.
In the previous phase I study conducted at the National Cancer Centre in Singapore, there were no dose-limiting toxicity observed in subjects subcutaneously administered with rSIFN-co at doses of up to 30 μg (refer to the IB for details) . Additionally, the phase II study evaluating the efficacy and safety of rSIFN-co in moderate-to-severe COVID-19 patients also demonstrated that nebulized rSIFN-co (12 million IU, twice daily) was safe and well tolerated [23] . With a dose of 12 million IU twice per day, no SAEs were reported and all AEs were classified as either  grade  1 or 2 in patients treated with rSIFN-co. As the present study include only healthy subjects or mild/asymptomatic COVID-19 patients, the daily dose of rSIFN-co is reduced to 8 and 16 million IU, respectively. The selected doses are considered adequate with its known safety profile.
Inclusion Criteria
An eligible subject must fulfill  ALL of the following inclusion criteria:
1. Male or females ≥ 18 years of age at the time of informed consent;
2. Willing and able to provide written informed consent for the trial;
3. Healthy subjects in close contact with confirmed COVID-19 case (s) or patients with mild COVID-19 or asymptomatic infection;
a. Healthy subjects in close contact with confirmed COVID-19 case (s) ;
I. With exposure to confirmed COVID-19 case (s) within 96 hours (refer to the definition of healthy subjects in close contact with confirmed COVID-19 case [s] ) ,
II. Negative reverse transcriptase polymerase chain reaction (RT-PCR) test for SARS-CoV-2 infection, with the sample collected within 72 hours prior to screening,
III. Have no previous confirmed COVID-19 diagnosis,
IV. Without symptoms of respiratory infection, including fever, cough, fatigue, anorexia, shortness of breath, myalgias, sore throat, nasal congestion, headache, gastrointestinal symptoms, loss of smell (anosmia) , loss of taste (ageusia) , or other symptoms associated with COVID-19, AND
V. With stable health status, as judged by the investigator and determined by physical examination, vital signs, medical history, and laboratory tests at screening;
b. Patients with mild COVID-19 or asymptomatic infection;
I. Meeting the criteria of mild COVID-19 or asymptomatic infection (refer to the definition of COVID-19 disease severity) ,
II. Positive RT-PCR test for SARS-CoV-2 infection, with the sample collected within 72 hours prior to screening, AND
III. Without evidence of viral pneumonia or hypoxia;
4. Women of childbearing potential must have a negative pregnancy test result at screening; and
5. Males and females who are fertile must adhere to contraception requirements for the duration of the study.
Definitions
The following terms, including abbreviations listed in Table 6 below, shall be used to describe the present invention. In the absence of a specific definition set forth herein, the terms used to describe the present invention shall be given their common meanings as understood by those of ordinary skill in the art.
TABLE 6. LIST OF ABBREVIATIONS
Figure PCTCN2022074977-appb-000016
Figure PCTCN2022074977-appb-000017
Figure PCTCN2022074977-appb-000018
Healthy subjects in close contact with confirmed COVID-19 case (s) : Subjects with exposure to confirmed COVID-19 case (s) within 96 hours and belong to one of the following exposure situations per health department directives:
1. Staying together in the same room with confirmed COVID-19 case (s) , e.g., living,  studying, working, dining and hanging out with confirmed COVID-19 case (s) , etc.
2. Taking the same transport with confirmed COVID-19 case (s) , e.g., all personnel in the cabin of civil aircraft; all passengers in the same carriage of a fully enclosed air-conditioned train and the crew members serving for this area; all personnel of fully sealed air-conditioned vehicles; all personnel in the same cabin of the ship and crew members serving for that cabin.
3. Medical staffs below without any protection of level 2 or above.
(1) People mainly working in the emergency room (e.g., doctors, nurses, assistants, triage personnel) ;
(2) People mainly working in intensive care unit (ICU) (e.g., physicians, nurses, support staff, respiratory therapists) ;
(3) People performing aerosol generation procedures (i.e., anesthesiologist, cRNA) ;
(4) First responders (i.e., first aid personnel, paramedics) ; and
(5) People working in the Department of general medicine, pulmonary medicine, infectious diseases, and isolation wards.
Note: Level 2 protection refers to wearing a disposable cap, medical protective mask (N95 or higher level medical protective mask) , goggles (anti-fog type) or protective mask (anti-fog type) , medical protective suit or overalls (white gown) , disposable gloves and disposable shoe covers.
COVID-19 disease severity (CDC definitions) [25]
- Asymptomatic or presymptomatic infection: Individuals who test positive for SARS-CoV-2 by virologic testing using a molecular diagnostic (e.g., polymerase chain reaction) or antigen test, but have no symptoms.
- Mild disease: Individuals who have any of the various signs and symptoms of COVID-19 (e.g., fever, cough, sore throat, malaise, headache, muscle pain) and a SpO 2 ≥94%on romm air at sea level, without shortness of breath, dyspnea, or abnormal chest imaging.
- Moderate disease: Individuals who have evidence of lower respiratory disease by clinical assessment or imaging and SpO 2 ≥94%on room air at sea level.
- Severe disease: Individuals who have respiratory frequency >30 breaths per minute, SpO 2 <94%on room air at sea level, ratio of arterial partial pressure of oxygen to fraction of inspired oxygen (PaO 2/FiO 2) <300 mmHg, or lung infiltrates >50%.
- Critical disease: Individuals who have respiratory failure, septic shock, and/or multiple organ dysfunction.
Dosage, Preparation and Administration of Investigational Product
The subjects will receive rSIFN-co or placebo, for a maximum of 10 to 28 days. For combination therapy, rSIFN-co will be administered with one or more other agents, wherein the control group will receive said one or more other agents only.
The dose of rSIFN-co given by nebulization or injection is equivalent to 8 to 16 million IU (32 μg) per day. Other agents will be given in their usual dosages.
Treatment Period
Healthy subjects (without symptoms) in close contact with confirmed COVID-19 case (s) 
Day 1
● Perform physical examination;
● Assess vital signs (including SpO 2) ;
● Document COVID-19-related symptoms, complications, clinical status, and disease severity, if applicable;
● Randomize eligible subjects into one of the two treatment arms (i.e., placebo or rSIFN-co) ;
● Collect respiratory tract sample (s) for viral RNA quantification and qualification;
● Dispense study medications and diary card; and
● Record AEs and concomitant medications;
● Review how to complete diary with subject.
Day 5
● Perform physical examination;
● Assess vital signs (including SpO 2) ;
● Document COVID-19-related symptoms, complications, clinical status, and disease severity, if applicable;
● Perform ECG if clinically indicated;
● Collect blood samples for laboratory tests;
● Collect respiratory tract sample (s) for viral RNA quantification and qualification;
● Collect blood samples for serologic test;
● Review electronic diary entries with subject prior to leaving clinic, including any AE/SAE information and have subject correct any discrepancies; and
● Record AEs and concomitant medications.
Day 10
● Perform physical examination;
● Assess vital signs (including SpO 2) ;
● Document COVID-19-related symptoms, complications, clinical status, and disease severity, if applicable;
● Obtain chest imaging;
● Perform ECG if clinically indicated;
● Collect blood samples for laboratory tests;
● Collect blood, stool, and respiratory tract sample (s) for viral RNA quantification and qualification;
● Collect blood samples for serologic test;
● Review electronic diary entries with subject prior to leaving clinic, including any AE/SAE information and have subject correct any discrepancies;
● Complete questionnaire for ease of use and discomfort using the study product; and
● Record AEs and concomitant medications.
COVID-19 patients with mild symptoms or asymptomatic infection
Day 1
● Perform physical examination;
● Assess vital signs (including SpO 2) ;
● Document COVID-19-related symptoms, complications, clinical status, and disease severity;
● Randomize eligible subjects into one of the two treatment arms (i.e., SoC+placebo or SoC+rSIFN-co) ;
● Collect respiratory tract sample (s) for viral RNA quantification and qualification;
● Dispense study medications and diary card; and
● Record AEs and concomitant medications.
Days  2, 3, and 7
● Perform physical examination;
● Assess vital signs (including SpO 2) ;
● Document COVID-19-related symptoms, complications, clinical status, and disease severity;
● Perform ECG if clinically indicated;
● Collect respiratory tract sample (s) for viral RNA quantification and qualification; and
● Record AEs and concomitant medications.
Days 5 and 10
● Perform physical examination;
● Assess vital signs (including SpO 2) ;
● Document COVID-19-related symptoms, complications, clinical status, and disease severity;
● Obtain chest imaging;
● Perform ECG if clinically indicated;
● Collect blood samples for laboratory tests;
● Collect respiratory tract sample (s) for viral RNA quantification and qualification;
● Collect blood and stool samples for viral RNA quantification and qualification (only for Day 10) ;
● Collect blood samples for serologic test;
● Collect diary card and used/unused study medications (Day 10) ;
● Review electronic diary entries with subject prior to leaving clinic, including any AE/SAE information and have subject correct any discrepancies;
● Record AEs and concomitant medications; and
● Complete questionnaire for ease of use and discomfort using the study product (only for Day 10) .
Follow-up Visits (Days 15, 21, 28 and 60)
● Perform physical examination;
● Perform pregnancy test Day15;
● Assess vital signs (including SpO 2) ;
● Document COVID-19-related symptoms, complications, clinical status, and disease severity, if applicable;
● Obtain chest imaging, if clinically indicated;
● Perform ECG, if clinically indicated;
● Collect blood samples for laboratory tests on Day 15, thereafter only if clinically indicated;
● Collect respiratory tract sample (s) for viral RNA quantification and qualification;
● Collect blood and stool samples for viral RNA quantification and qualification (only for Day 28) ;
● Collect blood samples for serologic test (for Group A, the samples will only be collected on Day 15 and/or Day 28, if a positive RT-PCR result for SARS-CoV-2 is obtained in the preceding test) ;
● Review electronic diary entries with subject prior to leaving clinic, including any AE/SAE information and have subject correct any discrepancies;
● Collect electronic diary on Day 60
● Record AEs and concomitant medications.
After Day 60
Subjects with positive RT-PCR results on Day 60 will be continuously followed every two (2) weeks until a negative result is obtained.
Vital Signs
Vital sign measurements include blood pressures (systolic blood pressure [SBP] and diastolic blood pressure [DBP] ) , pulse rate, respiratory rate, body temperature, and SpO 2. Vital signs will be assessed at each visit. Results will be documented in the CRF. Measurements of all vital signs shall be completed in the during the visit within a time frame of 2 hours. Measurements should be taken in a seated position after resting for approximately 10 minutes. The same thermometer should be used consistently throughout the study. Subjects in Group B will be provided with an oximeter to help monitor their oxygen level. The subjects will be instructed to document daily oxygen level on the diary card.
Fever is defined as rectal, ear, or forehead temperature greater than 38 ℃. Site personnel should measure ear temperature of subject if possible. If other methods are used, the same method should be used consistently throughout the study.
Physical Examinations
A complete physical examination will be conducted by the Investigator or designee at screening. Complete physical examination items include general appearance, HEENT (head, eyes, ears, nose, and throat) , mouth, skin, neck (including thyroid) , lymph nodes, spine, cardiovascular system,  respiratory system, gastro-intestinal system, nervous system, musculoskeletal system, blood and blood forming organs, mental status, and other body systems if applicable for describing the status of subject’s health. At subsequent visits, an abbreviated physical examination comprising a brief medical history and targeted physical examination will be performed at the discretion of the Investigator to evaluate possible adverse events.
The physical examination performed at screening will serve as the baseline. Any significant physical examination findings after dosing will be regarded as adverse events.
Electrocardiogram (ECG)
Single 12-lead ECG will be obtained at screening using an ECG machine that automatically calculates the heart rate and measures PR, QRS, QT, and QTc (either Bazette or Fridericia formulas) intervals. The assessment will follow the current SOPs at each participating study center. Throughout the study, additional ECGs may be obtained if clinically indicated. The Investigators will compare ECGs to the screening ECG and document overall interpretation of specific ECGs as either normal, abnormal with clinical significance, or abnormal without clinical significance. An AE will be recorded if clinically significant.
Laboratory Evaluations
Laboratory tests (including hematology, biochemistry, and coagulation) will be performed at screening, Days 5, 10, 15, 21, 28, and 60. All samples will be analyzed by the study center’s local laboratory per the standard operating procedures (SOPs) of the laboratory. The examination items will include analytes in the following table:
Figure PCTCN2022074977-appb-000019
Figure PCTCN2022074977-appb-000020
Additional tests may be conducted at any time during the study as determined necessary by the Investigator. A clinically significant change from baseline will be recorded as an AE.
SARS-CoV-2 Viral Load
Blood, stool and respiratory tract samples will be collected at scheduled visits. For respiratory tract samples, nasopharyngeal swabs are preferred [26] , but if these are not obtainable or additional samples are required by local regulations, oropharyngeal or nasal swabs may be obtained if necessary.
Samples will be delivered to the laboratories selected by the Sponsor to quantitatively and qualitatively detect viral RNA via RT-PCR assays approved by local regulatory authorities. Preparation, handling, storage, shipment, and examinations of specimens will follow the lab SOPs.
SARS-CoV-2 Serologic Test
For detection of immunoglobulin (Ig) M and IgG to SARS-CoV-2, blood samples will be collected at screening and on Days 5, 10, 15, and 28. Validated serologic tests for SARS-CoV-2 that are approved by local regulatory authorities will be applied and will be performed by the laboratories selected by the Sponsor. Preparation, handling, storage, shipment, and examinations of specimens will follow the lab SOPs.
Chest Imaging
Thoracic CT-scan or chest X-ray will be performed at Screening, Day 5 (only for mild COVID-19 patients or asymptomatic infection) , and End of Treatment (Day 10) . A standardized framework will be used to assess the chest imaging at each study site.
WHO Ordinal Scale
COVID-19-related clinical status will be assessed at each study visits. The WHO ordinal scale as enumerated in the below table will be applied to evaluate improvement in clinical status [27] .
Figure PCTCN2022074977-appb-000021
*If hospitalized for isolation only, record status as for ambulatory patient.
Clinical improvement will be defined as a decrease of at least one point on the 11-point scale compared to the baseline value (e.g., from 2 to 1; from 2 to 0) .
COVID-19 Disease Severity and Symptoms
COVID-19 disease severity and symptoms will be assessed and documented at screening and throughout the entire study. Typical symptoms for patients with mild disease include fever, cough, fatigue, anorexia, shortness of breath, myalgias, sore throat, nasal congestion, headache, gastrointestinal symptoms, loss of smell (anosmia) , loss of taste (ageusia) , and without evidence of viral pneumonia or hypoxia.
The following COVID-19 disease severity will be applied:
● Asymptomatic or presymptomatic infection: Individuals who test positive for SARS-CoV-2 by virologic testing using a molecular diagnostic (e.g., polymerase chain reaction) or antigen test, but have no symptoms;
● Mild disease: Individuals who have any of the various signs and symptoms of COVID-19 (e.g., fever, cough, sore throat, malaise, headache, muscle pain) and a SpO2 ≥94%on room air at sea level, without shortness of breath, dyspnea, or abnormal chest imaging;
● Moderate disease: Individuals who have evidence of lower respiratory disease by clinical assessment or imaging and SpO2 ≥94%on room air at sea level;
● Severe disease: Individuals who have respiratory frequency >30 breaths per minute, SpO2 <94%on room air at sea level, ratio of arterial partial pressure of oxygen to fraction of inspired oxygen (PaO 2/FiO 2) <300 mmHg, or lung infiltrates >50%; and
● Critical disease: Individuals who have respiratory failure, septic shock, and/or multiple organ dysfunction.
The present invention will be better understood by reference to the examples as follows, but those skilled in the art will readily appreciate that the specific examples detailed are only for illustrative purpose, and are not meant to limit the present invention as described herein, which is defined by the claims following thereafter.
Throughout this application, various references or publications are cited. Disclosures of these references or publications in their entireties are hereby incorporated by reference into the  application in order to more fully describe the state of the art to which this invention pertains. It is to be noted that the transitional term “comprising” , which is synonymous with “including” , “containing” or “characterized by” , is inclusive or open-ended, and does not exclude additional, un-recited elements or method steps.
EXAMPLES
Example 1: Preparation of rSIFN-co
The Master Cell Bank (MCB) and Working Cell Bank (WCB) for rSIFN-co are manufactured under cGMP. The DS manufacture process involves bioproduction and harvest then purification of rSIFN-co from the E. coli inclusion bodies using upstream and downstream manufacturing processing typical for this type of production.
To create rSIFN-co, the rSIFN-co cDNA was cloned into the E. coli high expression vector pHY-4 plasmid to yield the final recombinant plasmid pHY-5 (Figure 4) . Typical production and purification can be found in US Patent No. 8,415,151 and Patent No. 8,846,025.
Vector components in Figure 4 are:
● PBAD promoter, which derived from bacterial arabinose operon (araBAD operon) and was mainly regulated by CAP-cAMP and L-arabinose binding protein AraC i.e. product of ara Cgene
● Regulatory sequences araI, araO1 and araO2 of araBAD operon.
● araC gene coded PBAD promoter modulin –AraC.
● Ribosome binding site in the downstream of transcription start site, where PBAD promoter acted, followed by NdeI restriction enzyme cutting site.
● Transcription termination signal (rrnBT1T2)
● β-Lactamase coded by Bla gene could provide ampicillin resistance.
● ColE1 replicon determined the copy number and replication of plasmid in bacteria.
Fifty vials of MCB were manufactured and tested to ensure clone purity, stability, absence of adventitious agents and stable protein expression. The WCB was tested for mycoplasma, viruses, and non-host cell contamination. Additional tests were performed to confirm rSIFN-co identity via peptide map analysis and testing of the protein potency.
Animal-based ingredients, tryptone and acid hydrolyzed casein, are used in upstream manufacture and are of GMP quality and certified that they are sourced in a controlled and validated way as to reduce the risk of contamination or exposure to any diseases, especially Bovine Spongiform Encephalopathy.
The release testing specifications for the DS include protein content, biological activity, specific activity, purity, molecular weight, exogenous DNA, host protein, antibiotic residue, endotoxin and a number of characterization assays.
The identity of the primary sequence of rSIFN-co was confirmed using MS analysis. Mass-Spectrometry analysis was performed using MALDI-TOF-MS, and the results were consistent with the theoretical value of rSIFN-co minus methionine (19434.28Da) and reported in references (19434Da) . Traditional N-terminal sequencing by Edman degradation confirmed the identity of the first 15 amino acids. The identity was also confirmed by isoelectric focusing. In this gel method, the overall charge state of rSIFN-co (pI) was determined which is partly a measure of the number of charged amino acids in the molecule (primary structure) and partly the influence of structural considerations (secondary structure) around those charged residues. Secondary structure analysis was accomplished by CD Spectroscopy. This analysis also showed identical thermal structural stability.
The biological activity of rSIFN-co was determined based on the effect of rSIFN-co to protect cells against a viral cytopathic effect. It is quantified using a CPE potency assay, referenced against IFN α-2a reference standard, which has been tested and calibrated in International Units against the WHO reference standard. The three-dimensional structure of the present recombinant interferon (SEQ ID NO: 3, without N-terminal M) is different from the three-dimensional structure of IFN α-2b (SEQ ID NO: 4) published in the art (see Figure 2) and the three-dimensional structure of 
Figure PCTCN2022074977-appb-000022
based on computational modeling. There are obvious differences between the AB loops of the two, and their BC loops also cannot overlap completely (see Figure 3) .
Stability was tested according to ICH guideline for biological products Q1A and Q5C. The stability of the rSIFN-co composition is tested under 4-8℃ for 6 months, -80℃ for 24 months, and accelerated condition at 25℃ for 1 month. Repeated freeze/thaw cycle tests are done for 7 cycles. Stability data for 2 representative batches of drug substance 201801003, and 201801004 is shown in the following Tables 7-10.
Table 7. Stability Data of Long-term Test for Drug Substance of 2018 Batches (Y201801003 and Y201801004) at 4-8℃
Figure PCTCN2022074977-appb-000023
Table 8. Stability Data of Long-term Test for Drug Substance of 2018 Batches (Y201801003 and Y201801004) at -80℃
Figure PCTCN2022074977-appb-000024
Table 9. Stability Data of Accelerated Test for Drug Substance of 2018 Batches (Y201801003 and Y201801004) at 25±2℃
Figure PCTCN2022074977-appb-000025
Figure PCTCN2022074977-appb-000026
Table 10. Stability Data of Repeated Freezing and Thawing Test for Drug Substance of 2018 Batches (Y201801003 and Y201801004)
Figure PCTCN2022074977-appb-000027
Example 2: Composition of Drug Product and Preparation
A composition of rSIFN-co was prepared by obtaining a 80%of the total volume of water for injection, adding a prescribed amount of disodium edetate, polysorbate 80, citric acid, glycerol, sodium chloride and benzyl alcohol in turn while stirring. Add the next component after the former component is fully dissolved. After each component is added, stir for more than 60 minutes until well combined. Cool the solution to below 20℃ and adjust pH value to about 5.0 with diluted hydrochloric acid. Stir the solution fully, add the prescribed amount of human serum albumin and rSIFN-co protein stock solution and then test pH value. If pH deviates, adjust pH value again to 5.0 and stir slightly. Dilute with water for injection cooled to below 20℃ to volume, homogenizing by gently stirring for more than 30 minutes before filter and sterilize with a 0.22μm filter to obtain the semi-finished rSIFN-co spray. A typical workflow is shown in Figure 5.
The current stability program and data showed that the composition (alternatively “drug product” ) is stable under 4-8℃ for 3 months and the real time study is on-going. It is also stable under accelerated condition at 25℃ for 2 months and at 37℃ for 2 weeks.
In one embodiment, the main components of the drug product are shown in the Table 11.
Table 11. Representative Formulation
Figure PCTCN2022074977-appb-000028
In one embodiment, the product is a homogeneous colorless or light yellow with slightly sticky liquid spray. In one embodiment, the product is filled in high-density polyethylene bottles for  pharmaceuticals with a mechanical nasal spray pump. Each bottle contains 8 ml and each pump is 0.1 ml.
In one embodiment, a solution is prepared according to the formula followed by sterilization and filtering by a 0.22μm filter. The filtered liquid was transferred into a sterile container to obtain a semi-finished product. After the sterility of the filtered liquid sample and the integrity of the used filter are tested, the sterile filtered solution is packaged into 10 ml medicinal high-density polyethylene bottle for external use by filling machine and equip with medicinal spray pump.
The final product is stored at a controlled temperature of between 2-8℃, and the latest stability data showed that the drug product is stable under 4-8℃ for 3 months and at accelerated condition of 25℃ for 2 months and at 37℃ for 2 weeks. Based on these test results at this time, the data supports a shelf life of 12 months and the stability program is on-going to support longer shelf life.
The drug product is supplied in spray bottle with mechanical pump. The components of the container closure system are in conformance to the compendial requirements for spray bottle containers for pharmaceutical use. The drug product is produced in GMP qualified facilities under ISO5, ISO7, and ISO8 clean room classification.
The detailed specification of the drug product is in Table 12.
Table 12. Drug Product Specification
Figure PCTCN2022074977-appb-000029
Figure PCTCN2022074977-appb-000030
Three batches of drug product have been manufactured to-date with batch number 20200209, 20200210 and 20200311. The test results of these three batches are shown as follows.
Table 13. Test Results for 3 Batches of Drug Product
Figure PCTCN2022074977-appb-000031
Figure PCTCN2022074977-appb-000032
Stability was established according to ICH guideline for biological products Q1A and Q5C. Sponsor plans to test the drug product stability under 4-8℃ for 36 months, at accelerated conditions 25℃ for 6 months and 37℃ for 1 month. Stability data for 3 representative batches of drug product 20200209, 20200210, and 20200311 is shown in the following Tables 14-17.
Table 14. Stability Data of Long-term Test for Spray Drug Product of 2020 Batches (20200209, 20200210 and 20200311) at 4~8℃
Figure PCTCN2022074977-appb-000033
Figure PCTCN2022074977-appb-000034
Table 15. Stability Data of Accelerated Test for Spray Drug Product of 2020 Batches (20200209, 20200210 and 20200311) at 25±2℃
Figure PCTCN2022074977-appb-000035
Table 16. Stability Data of Accelerated Test for Spray Drug Product of 2020 Batches (20200209, 20200210 and 20200311) at 37±2℃
Figure PCTCN2022074977-appb-000036
Figure PCTCN2022074977-appb-000037
Example 3: Treatment by rSIFN-co of COVID-19 patients with symptoms
In this example, COVID-19 patients with symptoms such as mild pneumonia are treated by rSIFN-co or placebo for up to 10 to 28 days. rSIFN-co is administered by nebulization or injection twice a day, 8 to 12 million International Units (IU) each time. Clinical improvement is determined by assessing radiological improvement and/or virus nucleic acid negative conversion at the end of treatment period. The median time to reach clinical improvement is approximately 8 to 16 days, and an overall rate of clinical improvement at the end of treatment is approximately 80%to 90%.
Example 4: Treatment by rSIFN-co of COVID-19 patients without symptoms
In this example, COVID-19 patients without symptoms are treated by rSIFN-co or placebo for up to 10 to 28 days. rSIFN-co is administered by nebulization or injection twice a day, 8 to 12 million International Units (IU) each time. Any COVID-19 symptoms are assessed during the course of treatment. Patients treated with rSIFN-co are expected to show no symptom throughout the study, or significantly less treated patients show symptoms compared to patients receiving placebo.
Example 5: Treatment by rSIFN-co in combination with other agents of COVID-19 patients with symptoms
In this example, COVID-19 patients with symptoms such as mild pneumonia are treated by rSIFN-co in the presence of one or more agents for up to 10 to 28 days. rSIFN-co is administered by nebulization or injection twice a day, 8 to 12 million International Units (IU) each time. During the treatment period, one or more of the following agents are also administered: (1) lopinavir (twice a day, 200 mg each time) , (2) ritonavir (twice a day, 50 mg each time) , (3) arbidol or umifenovir (three times a day, 200 mg each time) , (4) Remdesivir (100 mg per day) , (5) hydroxychloroquine (200 mg per day) , and (6) dexamethasone (4 mg per day) . Clinical improvement is determined by assessing radiological improvement and/or virus nucleic acid negative conversion at the end of treatment period. The median time to reach clinical improvement and virus nucleic acid negative conversion is expected to be significantly shorter in patients receiving rSIFN-co than patients not receiving rSIFN-co.
Example 6: Safety and tolerability in healthy subjects receiving rSIFN-co
In this example, subjects receive rSIFN-co by nebulization or injection, or placebo for up to 10 to 28 days. Any symptoms or adverse events are assessed and recorded during the course of the study.
Example 7: Treatment by rSIFN-co of COVID-19 patients on respirators
In this example, COVID-19 patients on respirators are treated by rSIFN-co or placebo for up to 10 to 28 days. rSIFN-co is administered by nebulization or injection twice a day, 8 to 12 million International Units (IU) each time. Clinical improvement is determined by assessing blood oxygen level during and at the end of treatment period. The median time to reach clinical improvement is approximately 8 to 16 days, and an overall rate of clinical improvement at the end of treatment is approximately 80%to 90%.
Example 8: Treatment by rSIFN-co Spray
In this example, healthy subjects or patients with an asymptomatic infection of Covid-19 receive 2 sprays per nostril and 4 sprays through pharynx once daily. Each spray comprises 2 μg of rSIFN-co. The period of treatment is approximately 5 days to 20 days. Subjects treated with rSIFN-co are expected to show no symptom throughout the study, or significantly less treated patients show symptoms compared to patients receiving placebo.
As for patients with mild or severe infection of Covid-19 receive, they get 2 sprays per nostril and 4 sprays through pharynx twice daily. Each spray comprises 2 μg of rSIFN-co. The period of treatment is approximately 10 days to 30 days. The median time to reach clinical improvement and virus nucleic acid negative conversion is expected to be significantly shorter in patients receiving rSIFN-co than patients not receiving rSIFN-co.
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.

Claims (17)

  1. A method of preventing or treating Covid-19 (SARS-Cov-2) coronavirus infection in a subject, said method comprising administering to the subject a therapeutically effective dose of recombinant super-compound interferon (rSIFN-co) for a period of at least 3 days.
  2. The method of claim 1, wherein said rSIFN-co is administered to said subject by nasal spray, pharynx spray or intravenous injection.
  3. The method of claim 1, wherein said therapeutically effective dose ranges from 5 μg to 100 μg per day.
  4. The method of claim 1, wherein said therapeutically effective dose is selected from the group consisting of 8 μg, 16 μg, 32 μg, 64 μg and 96 μg per day.
  5. The method of claim 1, wherein said period is 5 to 30 days.
  6. The method of claim 1, wherein said subject suffers from one or more diseases in one or more of the digestive system, liver, heart, and kidney.
  7. The method of claim 1, wherein said subject receives one or more additional treatments selected from the group consisting of Remdesivir, hydroxychloroquine, and dexamethasone.
  8. The method of claim 1, wherein said rSIFN-co forms a crystal of the space group of P3 121, and the unit cell parameters of the crystal are
    Figure PCTCN2022074977-appb-100001
    α=β=90°, and γ=120°.
  9. The method of claim 2, wherein said subject is healthy or has an asymptomatic infection of Covid-19 coronavirus, and said subject receives 2 sprays per nostril and 4 sprays through pharynx once daily, each spray comprising 0.2-5.0 μg of said rSIFN-co.
  10. The method of claim 2, wherein said subject shows mild covid-19 infection and receives 2 sprays per nostril and 4 sprays through pharynx twice daily, each spray comprising 0.2-5.0 μg of said rSIFN-co.
  11. The method of claim 1, wherein the rSIFN-co has a specific activity greater than 5.0 x 10 8 IU/mg protein.
  12. The method of claim 2, wherein the nasal spray, pharynx spray or intravenous injection comprises one or more of stabilizer, bacteriostat, surfactant, metal complex, isosmotic adjusting agent, buffering agent, and viscosity modifier.
  13. The method of claim 12, wherein said stabilizer is human serum albumin, the metal complex is disodium edetate, the surfactant is Tween 80, the buffering agent is citric acid, the viscosity modifier is glycerol, the isosmotic adjusting agent is sodium chloride, and the bacteriostat is benzyl alcohol.
  14. The method of claim 2, wherein the nasal spray, pharynx spray or intravenous injection has a pH value of about 5.0.
  15. The method of claim 2, wherein the nasal spray or pharynx spray is stable under 4-8℃ for 3 months, at 25℃ for 2 months and at 37℃ for 2 weeks.
  16. A method of preventing or treating Covid-19 (SARS-Cov-2) coronavirus infection in a subject, said method comprising administering to the subject a composition comprising a therapeutically effective dose of rSIFN-co for a period of at least 3 days.
  17. A method of preventing or treating Covid-19 (SARS-Cov-2) coronavirus infection in a subject, said method comprising administering to the subject a composition consisting of a therapeutically effective dose of rSIFN-co for a period of at least 3 days.
PCT/CN2022/074977 2021-02-04 2022-01-29 Recombinant super-compound interferon (rsifn-co) for treating covid-19 patients with or without symptoms WO2022166885A1 (en)

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