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WO2021245541A1 - A composition of pegylated interferon alpha-2b for the treatment of sars-cov-2 infection and related manifestations - Google Patents

A composition of pegylated interferon alpha-2b for the treatment of sars-cov-2 infection and related manifestations Download PDF

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Publication number
WO2021245541A1
WO2021245541A1 PCT/IB2021/054776 IB2021054776W WO2021245541A1 WO 2021245541 A1 WO2021245541 A1 WO 2021245541A1 IB 2021054776 W IB2021054776 W IB 2021054776W WO 2021245541 A1 WO2021245541 A1 WO 2021245541A1
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Prior art keywords
composition
buffer
interferon alpha
pegylated interferon
acid
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Application number
PCT/IB2021/054776
Other languages
French (fr)
Inventor
Sanjeev Kumar Mendiratta
Pankaj Kalita
Kevinkumar KANSAGRA
Deven V PARMAR
Sanjay Bandyopadhyay
Original Assignee
Cadila Healthcare Limited
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Publication of WO2021245541A1 publication Critical patent/WO2021245541A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic 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
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic 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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4706—4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic 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
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

Definitions

  • the present invention relates to a composition of PEGylated interferon alpha- 2b for use in treatment of SARS-CoV-2 infection and its related clinical manifestations.
  • the said composition according to the present invention is a pharmaceutical composition.
  • Current invention also provides dose and dose regimen of PEGylated interferon alpha-2b for use in treatment of severe acute respiratory syndrome related coronavims, preferably SARS-CoV-2 and its related clinical manifestations.
  • the invention also discloses a method of treatment of severe acute respiratory syndrome related coronavims, preferably SARS-CoV-2 and its related clinical manifestations using the composition of PEGylated interferon alpha-2b as disclosed herein. Further disclosed is the use of the composition of PEGylated interferon alpha-2b as disclosed herein below for the treatment of severe acute respiratory syndrome related coronavims, preferably SARS-CoV-2 and its related clinical manifestations.
  • MERS-CoV Middle East respiratory syndrome coronavims
  • SARS-CoV severe acute respiratory syndrome coronavims
  • 2019-nCoV 2019 novel coronavims
  • WHO World Health Organization
  • SARS-CoV was first reported in Guangdong, China in 2002.
  • SARS-CoV caused human-to-human transmission and resulted in the 2003 outbreak with about 10% case fatality rate (CFR), while MERS-CoV was reported in Saudi Arabia in June 2012. Even though with its limited human-to human transmission, MERS-CoV showed a CFR of about 34.4%.
  • 2019-nCoV was first reported in Wuhan, China in December 2019 from patients with pneumonia, and it has exceeded both SARS-CoV and MERS- CoV in its rate of transmission among humans.
  • 2019-nCoV was renamed SARS- CoV-2 by Coronaviridae Study Group (CSG) of the International Committee on Taxonomy of Viruses (ICTV), while it was renamed HCoV-19, as a common virus name, by a group of virologists in China.
  • the disease and the virus causing it were named Coronavirus Disease 2019 (COVID-19) and the COVID-19 virus, respectively, by WHO.
  • the COVID-19 virus outbreak represents a pandemic threat that has been declared a public health emergency of international concern (PHEIC) (1).
  • the present invention provides a pharmaceutical composition of
  • PEGylated interferon alpha-2b for use in treatment of SARS-CoV-2 infection.
  • prophylactic and therapeutic interventions such as lopinavir/ritonavir, chloroquine, remdesivir, alisporivir, thymosin-a, ribavirin, type I interferons (IFN-a/b), arbidol (ARB) and combinations of these have been tested to strengthen COVID-19 prevention and control measures with no dramatically successful outcomes.
  • type I IFNs-a/b are broad spectrum antivirals, exhibiting both direct antiviral effects on viral replication and supporting an immune response to clear vims infection (3).
  • IFN-a and IFN-b have been demonstrated for suppressing SARS-CoV-2 infection in vitro (4).
  • PEGylated interferon alpha-2a was evaluated for anti-SARS-CoV-2 activity and inhibition of coronavirus replication in vitro (5).
  • type I interferon in the treatment of both MERS and SARS-CoV-1, its role in therapeutic intervention in humans is less clear with reports of protective effects for MERS and pathogenic effects for SARS-CoV-1 (6).
  • SARS-CoV-2 virus infection leads to a suppression of production of type I interferons in direct correlation with disease intensity (7).
  • type I interferon production in response to SARS-CoV-2 is significantly lesser than with other respiratory viruses (8).
  • type I interferons have been used for infection by SARS- CoV-2 vims, is a human interferon-stimulated gene (ISG) which may get overexpressed with the use of type I interferons leading to enhancement of infection (9). Yet, IFN beta is being used in combination with remdesivir (10). Similarly, IFN alpha has been used prophylactically and therapeutically in China with limited success.
  • ISG human interferon-stimulated gene
  • the current invention provides a composition of PEGylated interferon alpha-2b (PEG-IFN alpha 2b) for use in treatment of SARS-CoV-2 infection and its related clinical manifestations.
  • the said pharmaceutical composition is administered subcutaneously at an effective dose.
  • PEGylated interferon alpha-2b that can be used in treatment of SARS-CoV-2 infection and its related clinical manifestations. It also provides safe and effective dose and dose regimen of PEG-IFN alpha 2b that can be used in treatment of SARS-CoV-2 infection and its related clinical manifestations.
  • the invention also discloses a method of treatment of severe acute respiratory syndrome related coronavims, preferably SARS-CoV-2 and its related clinical manifestations using the composition of PEGylated interferon alpha-2b as disclosed herein. Further disclosed is the use of the composition of PEGylated interferon alpha-2b as disclosed herein below for the treatment of severe acute respiratory syndrome related coronavims, preferably SARS-CoV-2 and its related clinical manifestations.
  • Figure 1 depicts percentage (%) inhibiton of SARS-CoV-2 virus by PEGylated interferon alpha-2b composition of the current invention in in-vitro model.
  • SARS-CoV-2 refers to coronavirus which had an outbreak in December 2019 and was first reported in Wuhan, China.
  • SARS-CoV-2 infection refers to an infection caused by or otherwise associated with growth of coronavirus in a subject, in the family Coronaviridae (subfamily Coronavirinae).
  • pharmaceutical formulation refers to preparations, which are in such form as to permit the biological activity of the active ingredients to be unequivocally effective.
  • pharmaceutical formulation pharmaceutical composition
  • composition can be used here interchangeably.
  • the “lyophilized formulation” is a dosage form, which is prepared by lyophilization or freeze-drying process.
  • the lyophilization was performed with conventional lyophilization technique known in the literatures.
  • Lyophilization process is a process whereby water is sublimed from a composition after it is frozen- which can provide a stable form to the biological over a desired period of time. It may involves steps such as freezing, primary drying, secondary drying and optionally annealing.
  • excipient refers to an agent that may be added to a formulation to stabilize the active drug substance in the formulated form to adjust and maintain ionic strength, osmolality and pH of the pharmaceutical preparations.
  • excipients include, but are not limited to, buffers, sugars, polyols, amino acids, surfactants and polymers.
  • “Pharmaceutically acceptable” excipients are those which can reasonably be administered to a subject to provide an effective dose of the active ingredient employed.
  • buffer or “buffer solution” or “buffer system” refers to generally aqueous solution comprising a mixture of an acid (usually a weak acid) and conjugate base.
  • a buffered solution prevents change of pH of the solution due to the “buffering capacity” imparted by the “buffering agent(s)”.
  • buffering agent refers to an acid or base component (usually a weak acid or weak base) of a buffer or buffer solution.
  • a buffering agent maintain the pH of a given solution at or near to a pre-determined value, and the buffering agents are generally chosen to complement the pre-determined value.
  • a buffering agent is suitably a single compound which gives rise to a desired buffering effect, especially when said buffering agent is mixed with (and suitably capable of proton exchange with) an appropriate amount (depending on the pre-determined pH desired) of “acid/base conjugate”, or if the required amount of “acid/base conjugate” is formed in situ - this may be achieved by adding strong acid or base until the required pH is reached.
  • buffering agent and “buffers” can be used here interchangeably.
  • salt refers to the conjugate acid or conjugate base (whichever is relevant at a particular pH) of a particular “buffering agent”.
  • salt according to the present invention is a component which is not a part of a buffer system.
  • Salt according to the present invention includes organic salt and inorganic salt.
  • Preferred salts are inorganic salts which is not a part of buffer system.
  • inorganic salt refers to any salt that does not contain C- H bonds as opposed to organic salt that contains C-H bonds.
  • Inorganic salts such as sodium chloride, potassium chloride etc. are widely used as stabilizers or tonicity agents in addition to their use in a buffer system in the pharmaceutical formulations. Examples of inorganic salts include, but are not limited to, sodium chloride (NaCl), potassium chloride (KC1), calcium chloride (CaCE), barium chloride (BaCE), ferric chloride (FcCE,) etc.
  • stabilizers generally includes agents, which provide stability to the protein from freezing -induced stresses.
  • stabilizers include salt(s) such as sodium chloride or potassium chloride polyol(s) such as mannitol or sorbitol, saccharide(s) such as sucrose, surfactant(s) such as polysorbate, poloxamer or polyethylene glycol, amino acid(s) such as arginine and the like.
  • a “stable” formulation is one in which the protein therein essentially retains its physical stability and/or chemical stability and/or biological activity upon storage. Preferably, the formulation essentially retains its physical and chemical stability, as well as its biological activity upon storage. The storage period is generally selected based on the intended shelf-life of the formulation.
  • Various analytical techniques for measuring protein stability are available in the art and are reviewed in Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature for a selected time period. The formulation is stable between +2 °C and +8 °C for at least one month.
  • the formulation is preferably stable following freezing (to, e.g., - 70 °C) and thawing of the formulation, for example following 1, 2, 3 or 10 cycles of freezing and thawing.
  • Stability can be evaluated qualitatively and/or quantitatively in a variety of different ways, including evaluation of aggregate formation (for example using size exclusion chromatography, by measuring turbidity, and/or by visual inspection); by assessing charge heterogeneity using cation exchange chromatography or capillary zone electrophoresis; amino-terminal or carboxy- terminal sequence analysis; mass spectrometric analysis; SDS-PAGE analysis to compare reduced and intact antibody; peptide map (for example tryptic or LYS-C) analysis; evaluating biological activity or antigen binding function of the antibody; etc.
  • aggregate formation for example using size exclusion chromatography, by measuring turbidity, and/or by visual inspection
  • charge heterogeneity using cation exchange chromatography or capillary zone electrophoresis
  • Instability may involve any one or more of: aggregation, deamidation (e.g. Asn deamidation), oxidation (e.g. Met oxidation), isomerization (e.g. Asp isomerization), clipping/hydrolysis/fragmentation (e.g. hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, glycosylation differences, etc.
  • deamidation e.g. Asn deamidation
  • oxidation e.g. Met oxidation
  • isomerization e.g. Asp isomerization
  • clipping/hydrolysis/fragmentation e.g. hinge region fragmentation
  • succinimide formation unpaired cysteine(s)
  • N-terminal extension e.g. Asp isomerization
  • C-terminal processing e.g., glycosylation differences, etc.
  • prevention refers to the reduction in risk of coronavirus infection in patients. Such prevention may not be absolute, i.e. it may not prevent all such patients developing a coronavirus infection, or may only partially prevent an infection.
  • prevention and “prophylaxis” may be used interchangeably.
  • treatment refers to any treatment of a disease in a mammal, particularly in a human. It includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
  • a “therapeutically effective amount” or “effective amount” of IFN refers to an amount effective for use in the treatment of SARS-CoV-2 infection and its related clinical manifestations.
  • the term “about” refers to variation in the numerical quantity that can occur, for example, through typical measuring and handling procedures used for making compounds, compositions, concentrates or formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods, and other similar considerations.
  • the term “about” also encompasses amounts that differ due to aging of a formulation with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a formulation with a particular initial concentration or mixture.
  • patient and “subject” are used interchangeably and are used in their conventional sense to refer to a living organism suffering from or prone to a condition that can be prevented or treated by administration of a composition of the present invention, and includes both humans and non-human animals.
  • subjects include, but are not limited to, humans, chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like.
  • the term does not denote a particular age. Thus, adult, juvenile and new bom individuals are of interest.
  • cryoprotectant(s) or “lyoprotectant(s)” as used herein includes agent(s) that provide stability to the protein during water removal from the system during the drying process, presumably by maintaining the proper conformation of the protein through hydrogen bonding. Cryoprotectant(s) can also have lyoprotectant effects and therefore, the term “cryoprotectant” or “lyoprotectant” can be used interchangeably herein. It includes agent(s) which provides stability to the protein from freezing-induced stresses. For example, sugar(s) or sugar alcohol, such as sucrose, lactose, trehalose, mannitol and the like. The cryoprotectant may be used alone or in suitable combinations thereof. One such preferred cryoprotectant is lactose according to the present invention. ABBREVIATIONS USED IN THE CURRENT APPLICATION %: percentage
  • ACE2 Angiotensin-converting enzyme 2
  • CMC Carboxymethyl cellulose
  • DNA Deoxyribonucleic acid
  • EGTA Ethylene g 1 y c o 1 - b i s ( b - a m i n o cl h y 1 ether)-N,N,N’,N’-tetraacetic acid
  • fM Femtomolar
  • HP-IEC High-performance ion-exchange chromatography
  • Hrs Hours
  • IFN-a Interferon alpha
  • IFN-b Interferon beta
  • IFN-co Interferon omega
  • ISG Interferon-stimulated gene kDa: kilodalton kg: kilogram meg: microgram
  • MERS-CoV Middle East respiratory syndrome coronavims mg: milligram mITT: modified intent-to -treat mlTT: Modified intent-to-treat mL: milliliter mM: millimolar ng: nano gram PEG: Polyethylene glycol
  • PEG-IFN alpha 2b PEGylated interferon alpha 2b
  • PEG IFN-a2b PEGylated interferon alpha 2b
  • PEG-IFN PEGylated interferon PEI: Polyethyleneimine PP: Per protocol PT : Prefered term q.s.: Quantity sufficient r-DNA: Recombinant DNA RR: Respiratory rate (RR) RT-PCR: Reverse transcription polymerase chain reaction
  • SARS Severe acute respiratory syndrome
  • SARS-CoV Severe acute respiratory syndrome (SARS) coronavirus
  • SEC Size exclusion chromatography
  • SC Subcutaneous SDS: Sodium dodecyl sulphate
  • SOC Standard of care
  • SOP Standard operating procedure
  • SpO Saturation of peripheral oxygen
  • TEAE Treatment emergent adverse event
  • TNF Tumor necrosis factor
  • WFI Water for injection WHO: World Health Organization
  • b-HPCD Hydroxypropyl-beta-cyclodextrin pg: microgram EMBODIMENTS OF THE INVENTION
  • the current invention provides a composition of PEGylated interferon alpha-2b (PEG-IFN alpha 2b) for use in treatment of SARS- CoV-2 infection and its related clinical manifestations.
  • PEG-IFN alpha 2b PEGylated interferon alpha-2b
  • it provides a pharmaceutical composition of PEGylated interferon alpha-2b comprising therapeutically effective amount of PEGylated interferon alpha-2b.
  • the composition of PEGylated interferon alpha- 2b according to the current invention can be used for treatment of SARS-CoV-2 infection.
  • PEGylated interferon alpha-2b according to the current invention is in the concentration range of 0.01 mg / mL to 5.0 mg / mL, preferably 0.03 mg / mL to 2.5 mg / mL, more preferably 0.1 mg / mL to 0.5 mg / mL.
  • the pharmaceutical composition comprises therapeutically effective amount of PEGylated interferon alpha-2b, either alone or in combination with other suitable excipient(s).
  • suitable excipient(s) are selected from buffer(s), salt(s), carbohydrate(s), surfactant(s), amino acid(s), anti- oxidant(s), chelating agent(s), tonicity modifier(s), acid(s), base(s), glycerine, water for injection (WFI) and suitable combination thereof and the like.
  • WFI water for injection
  • buffer(s) and other suitable excipient(s) where in buffer(s) is selected from phosphate buffer (e.g., sodium phosphate buffer, etc.), arginine-phosphate buffer, histidine buffer, arginine-histidine buffer, histidine chloride buffer, glycine buffer, sodium glycinate buffer, arginine buffer, citrate buffer (e.g., sodium citrate buffer, etc.), arginine-citrate buffer, succinate buffer (e.g.
  • succinic acid buffer sodium succinate buffer, etc.
  • potassium succinate buffer arginine- succinate buffer, acetate buffer, arginine-acetate buffer, arginine- glutamate buffer, arginine-aspartate buffer, tromethamine buffer and suitable combination thereof.
  • the suitable excipient(s) is selected from salt(s), carbohydrate(s), surfactant(s), amino acid(s), anti-oxidant(s), chelating agent(s), tonicity modifier(s), acid(s), base(s), glycerine, water for injection (WFI) and suitable combination thereof and the like.
  • the pharmaceutical composition comprising PEGylated interferon alpha-2b, buffer(s), carbohydrate(s), surfactant(s) and other suitable excipient(s).
  • the suitable excipient(s) is selected from salt(s), amino acid(s), anti-oxidant(s), chelating agent(s), tonicity modifier(s), acid(s), base(s), glycerine, water for injection (WFI) and suitable combination thereof and the like.
  • Carbohydrate(s) may act as a cryoprotectant(s) or as a lyoprotectant(s) according to one of the aspects of the present invention.
  • the pharmaceutical composition of PEGylated interferon alpha-2b according to the present invention comprising stabilizer(s).
  • the stabilizer(s) of present invention is selected from surfactant(s), sugar(s), amino acid(s), polyol(s) and the like or suitable derivative or mixtures thereof.
  • the pharmaceutical composition of PEGylated interferon alpha- 2b according to the present invention comprising buffer(s).
  • the buffer(s) of present invention is selected from phosphate buffer (e.g., sodium phosphate buffer, etc.), arginine -phosphate buffer, histidine buffer, arginine-histidine buffer, histidine chloride buffer, glycine buffer, sodium glycinate buffer, arginine buffer, citrate buffer (e.g., sodium citrate buffer, etc.), arginine-citrate buffer, succinate buffer (e.g.
  • succinic acid buffer sodium succinate buffer, etc.
  • potassium succinate buffer arginine- succinate buffer
  • acetate buffer arginine-acetate buffer
  • arginine-glutamate buffer arginine-aspartate buffer
  • tromethamine buffer tromethamine buffer and suitable combination thereof and the like.
  • the pharmaceutical composition of PEGylated interferon alpha-2b comprising carbohydrate(s).
  • the carbohydrate(s) of present invention is sugar(s) and / or polyol(s) selected from dextran, trehalose, raffinose, sucrose, polyvinylpyrrolidone, lactose, inositol, sorbitol, mannitol, dimethyl sulfoxide, glycerol, albumin, calcium lactobionate, fructose, maltose, galactose, glucose, D- mannose, sorbose, cellobiose, melezitose, maltodextrins, starches, lactitol, xylitol, pyranosyl sorbitol, myoinositol, hydroxypropyl-beta-cyclodextrin (b-HPCD) and suitable combination thereof and the like.
  • Carbohydrate(s) may act as a cryoprotectant(s) or as a lyoprotectant(s) according to the present invention.
  • Cryoprotectant(s) or lyoprotectant(s) include agent(s) that provides stability to the protein during water removal from the system during the drying process, presumably by maintaining the proper conformation of the protein through hydrogen bonding.
  • the pharmaceutical composition of PEGylated interferon alpha-2b comprising surfactant(s).
  • the surfactant(s) of present invention is selected from polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (e.g. Brij), alkylphenylpolyoxyethy lene ethers (e.g. Triton-X), polyoxyethylene - polyoxypropylene copolymer (e.g. Poloxamer, Pluronic), polyethylene glycol (PEG), polyethyleneimine, sodium dodecyl sulphate (SDS) and suitable combination thereof and the like.
  • Tween polyoxyethylensorbitan fatty acid esters
  • polyoxyethylene alkyl ethers e.g. Brij
  • alkylphenylpolyoxyethy lene ethers e.g. Triton-X
  • polyoxyethylene - polyoxypropylene copolymer e.g
  • the pharmaceutical composition of PEGylated interferon alpha-2b according to the present invention comprising acid(s).
  • the acid(s) according to the present invention is selected from hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, succinic acid, sulfuric acid, fumaric acid, and suitable combinations thereof.
  • the pharmaceutical composition of PEGylated interferon alpha-2b according to the present invention comprising base(s).
  • the base(s) according to the present invention is selected from sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium succinate, sodium format, sodium sulfate, potassium sulfate, potassium fumarate, and suitable combinations thereof.
  • the pharmaceutical composition of PEGylated interferon alpha- 2b according to the present invention comprising amino acid(s).
  • the amino acid(s) of present invention is selected from arginine, glycine, asparagine, glutamine, lysine, threonine, histidine, glutamic acid, aspartic acid, isoleucine, valine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, cysteine, their suitable salt form and combination thereof and the like.
  • the pharmaceutical composition of PEGylated interferon alpha-2b according to the present invention comprising chelating agent(s) selected from ethylenediaminetetraacetic acid (EDTA), pentetic acid and ethylene g 1 yco 1 -b i s(P - a m i noct h y 1 ether)-N,N,N’,N’-tetraacetic acid (EGTA).
  • EDTA ethylenediaminetetraacetic acid
  • pentetic acid ethylene g 1 yco 1 -b i s(P - a m i noct h y 1 ether)-N,N,N’,N’-tetraacetic acid (EGTA).
  • EGTA ethylenediaminetetraacetic acid
  • the pharmaceutical composition of PEGylated interferon alpha- 2b according to the present invention comprising salt(s) selected from sodium chloride, potassium chloride, potassium sulfate and sodium
  • the pharmaceutical composition of PEGylated interferon alpha- 2b is free from salt(s). In a preferred embodiment, the pharmaceutical composition of PEGylated interferon alpha-2b is free from inorganic salt(s).
  • the pharmaceutical composition according to the present invention has pH in the range of about 4.0 to about 7.1, preferably about 6.5 to about 7.1, more preferably about 6.8.
  • the pharmaceutical composition comprising PEGylated interferon alpha-2b, buffer(s), carbohydrate(s), and surfactant(s).
  • the pharmaceutical composition comprising PEGylated interferon alpha- 2b, buffer(s), carbohydrate(s) as a cryoprotectant(s) or as a lyoprotectant(s), and surfactant(s).
  • the specific pH of the composition can be achieved by using acid or base.
  • the pharmaceutical composition comprising PEGylated interferon alpha- 2b, succinate buffer, lactose, polysorbate 80 and other suitable excipient(s).
  • the pharmaceutical composition comprising PEGylated interferon alpha-2b, succinate buffer, lactose and polysorbate 80.
  • the pharmaceutical composition of PEGylated interferon alpha-2b comprising therapeutically effective amount of PEGylated interferon alpha-2b, succinate buffer, lactose and polysorbate 80.
  • the composition of PEGylated interferon alpha-2b according to the current invention can be used for treatment of SARS-CoV-2 infection.
  • the preferred therapeutically effective amount of PEGylated interferon alpha-2b is in the range of 0.1 mg / mL to 0.5 mg / mL.
  • the current invention provides a pharmaceutical composition of PEGylated interferon alpha-2b (PEG-IFN alpha 2b) along with succinate buffer, lactose and polysorbate 80 for use in treatment of SARS-CoV-2 infection and its related clinical manifestations.
  • PEG-IFN alpha 2b PEGylated interferon alpha-2b
  • succinate buffer, lactose and polysorbate 80 for use in treatment of SARS-CoV-2 infection and its related clinical manifestations.
  • the current invention provides a pharmaceutical composition of PEGylated interferon alpha-2b comprising about 0.1 mg / mL to about 0.5 mg / mL of PEG-IFN alpha 2b, about 1 mM to about
  • the current invention provides a pharmaceutical composition of PEGylated interferon alpha-2b comprising therapeutically effective amount of PEG-IFN alpha 2b, 10 mM of succinate buffer, 105 mg / mL of lactose monohydrate and 0.1 mg / mL polysorbate 80 and optionally with other suitable excipient(s), wherein amount of PEGylated interferon alpha-2b in the pharmaceutical composition is in range of 0.1 mg / mL to 0.5 mg / mL, preferably 0.16 mg / mL or 0.20 mg / mL.
  • the current invention provides a pharmaceutical composition of PEGylated interferon alpha-2b comprising therapeutically effective amount of PEG-IFN alpha 2b, 10 mM of succinate buffer, 105 mg / mL of lactose monohydrate and 0.1 mg / mL polysorbate 80 and optionally with other suitable excipient(s), wherein amount of PEGylated interferon alpha- 2b in the pharmaceutical composition is in range of 0.1 mg / mL to 0.5 mg / mL, preferably 0.16 mg / mL or 0.20 mg / mL for use in treatment of SARS-CoV-2 infection and its related clinical manifestations.
  • the current invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising PEG-IFN alpha 2b in the concentration range of 0.1 mg / mL to 0.5 mg / mL; preferably 0.16 mg / mL or 0.20 mg / mL, 1.18 mg / mL of succinic acid, 105 mg / mL lactose and 0.1 mg / mL polysorbate 80 optionally with sodium hydroxide.
  • the current invention provides a pharmaceutical composition comprising 0.10 mg / mL of PEG-IFN alpha 2b, 1.18 mg / mL succinic acid, 105 mg / mL lactose and 0.1 mg / mL polysorbate 80 optionally with sodium hydroxide.
  • the current invention provides a pharmaceutical composition comprising 0.16 mg / mL of PEG-IFN alpha 2b, 1.18 mg / mL succinic acid, 105 mg / mL lactose and 0.1 mg / mL polysorbate 80 optionally with sodium hydroxide.
  • the current invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising 0.20 mg / mL of PEG-ILN alpha 2b, 1.18 mg / mL succinic acid, 105 mg / mL lactose and 0.1 mg / mL polysorbate 80 optionally with sodium hydroxide.
  • the current invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising 0.24 mg / mL of PEG-IFN alpha 2b, 1.18 mg / mL succinic acid, 105 mg / mL lactose and 0.1 mg / mL polysorbate 80 optionally with sodium hydroxide.
  • the current invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising 0.30 mg / mL of PEG-IFN alpha 2b, 1.18 mg / mL succinic acid, 105 mg / mL lactose and 0.1 mg / mL polysorbate 80 optionally with sodium hydroxide.
  • the succinic acid described in above mentioned specific embodiments are equivalent to 10 mm of succinate buffer.
  • the lactose described in above mentioned specific embodiments and used in the current invention to prepare composition of PEGylated interferon alpha 2b is lactose monohydrate.
  • the current invention provides a pharmaceutical composition including PEGylated interferon alpha- 2b in a lyophilized form or liquid form.
  • the current invention provides a pharmaceutical composition including PEGylated interferon alpha-2b in a lyophilized form.
  • the lyophilization process is a process whereby water is sublimed from a composition after it is frozen- which can provide a stable form to the biological over a desired period of time.
  • the said composition can be stored as a dry powder and reconstituted before use.
  • the current invention provides a pharmaceutical composition including PEGylated interferon alpha- 2b in a liquid form.
  • the current invention provides lyophilized and stabilized composition of PEGylated interferon alpha-2b.
  • the composition of PEGylated interferon alpha-2b comprising therapeutically effective amount of PEGylated interferon alpha- 2b, succinate buffer, lactose and polysorbate 80.
  • the current invention provides process of preparing a composition of PEGylated interferon alpha-2b for use in treatment of SARS-CoV-2 infection and its related clinical manifestations.
  • the present invention provides a method of administration to the patient of a composition of IFN, preferably PEGylated interferon alpha-2b (PEG-IFN alpha 2b) for use in treatment of SARS-CoV-2 infection and its related clinical manifestations.
  • a composition of IFN preferably PEGylated interferon alpha-2b (PEG-IFN alpha 2b) for use in treatment of SARS-CoV-2 infection and its related clinical manifestations.
  • the present invention provides a method of treating SARS-CoV-2 infection by administering PEGylated interferon alpha-2b.
  • the present invention provides a method of treating SARS-CoV-2 infection by administering composition of PEGylated interferon alpha-2b comprising PEGylated interferon alpha-2b in suitable therapeutic amount optionally with other suitable excipient(s).
  • excipient(s) examples include, but are not limited to, buffer(s), salt(s), carbohydrate(s), surfactant(s), amino acid(s), anti-oxidant(s), chelating agent(s), tonicity modifier(s), cryoprotectant(s) or lyoprotectant(s), acid(s), base(s), glycerine, water for injection (WFI) and suitable combination thereof and the like.
  • the current invention provides use of PEGylated interferon alpha- 2b for the treatment of SARS-CoV-2 infection and its related clinical manifestations.
  • the current invention provides use of composition of PEGylated interferon alpha-2b comprising PEGylated interferon alpha-2b in suitable therapeutic amount optionally with other suitable excipient(s) for the treatment of SARS-CoV-2 infection and its related clinical manifestations.
  • excipient(s) examples include, but are not limited to, buffer(s), salt(s), carbohydrate(s), surfactant(s), amino acid(s), anti oxidants), chelating agent(s), tonicity modifier(s), cryoprotectant(s) or lyoprotectant(s), acid(s), base(s), glycerine, water for injection (WFI) and suitable combination thereof and the like.
  • the PEGylated interferon alpha-2b of the present invention has at least one of the following characteristic(s): a. shows not more than about 1 °C difference in thermal transition value as compared to the unmodified interferon alpha-2b; b. exhibits certain degree of depegylation, preferably 30 % or more upon hydroxylamine treatment; c. shows about 8 - 9 positional isomers of interferon alpha; d. exhibits low level of ( ⁇ 5 %) oxidation on one of the methionine residues of interferon at the position 148; e.
  • k. contains no more than 10 % of high molecular weight species variants and less than 5 % of unmodified interferon.
  • the degree of depegyltion when termed as ‘30 % or more’ herein is 30 % or maximally 60 % of depegylation.
  • the term ‘no more than 10 % high molecular weight species’ as used herein may include 0 - 10 % of high molecular weight species.
  • the term ‘less than 5 % unmodified interferon’ as used herein may include 0-5 % of unmodified interferon (free IFN).
  • the PEGylated interferon alpha- 2b of the present invention has all the characteristics as listed above (points a. to points k.)
  • the pharmaceutical composition of present invention includes PEGylated interferon alpha-2b, which can be used for parenteral administration.
  • Parenteral administration includes intravenous, subcutaneous, intra peritoneal, intramuscular administration or any other route of delivery generally considered to be falling under the scope of parenteral administration and as is well known to a skilled person.
  • the pharmaceutical composition of present invention includes PEGylated interferon alpha-2b used for subcutaneous administration.
  • the pharmaceutical composition of present invention includes PEGylated interferon alpha-2b, which can be delivered into lungs by atomization. In another embodiment, the pharmaceutical composition of present invention includes PEGylated interferon alpha-2b, which can be delivered intranasally in the form of nasal drops.
  • the current invention provides safe and effective dose and dose interval of PEGylated interferon alpha-2b for use in treatment of SARS-CoV-2 infection and its related clinical manifestations.
  • the dose range of the composition of the invention, including PEGylated interferon alpha- 2b is from about 0.1 mcg/kg to about 30 mcg/kg on a weekly, twice in a week, thrice in a week, daily basis, twice in a day or every other week for use in treatment of SARS-CoV-2 infection.
  • this dose range may include for example, about 0.1 mcg/kg/week, about 0.5 mcg/kg/week, about 1 mcg/kg/week, about 1.5 mcg/kg/week, about 2 mcg/kg/week, about 5 mcg/kg/week, about 10 mcg/kg/week, about 20 mcg/kg/week, and/or about 30 mcg/kg/week.
  • the dose of PEGylated interferon alpha-2b is 1 mcg/kg/week.
  • the current invention provides treatment of SARS-CoV-2 infection and its related clinical manifestations comprising administration of single dose of PEG-IFN alpha 2b or its composition to the subject.
  • the said single dose of PEG-IFN alpha 2b according to the current invention is selected from 0.1 mcg/kg, about 0.5 mcg/kg, about 1 mcg/kg, about 1.5 mcg/kg, about 2 mcg/kg, about 5 mcg/kg.
  • the current invention provides composition of PEGylated interferon alpha-2b comprising buffer(s), carbohydrate(s) and surfactant(s) to deliver 1 mcg/kg of PEG-IFN alpha 2b to neutralize SARS-CoV-2 virus. In one of the embodiments, the current invention provides composition of PEGylated interferon alpha- 2b that can neutralize 100% SARS-CoV-2 vims.
  • the current invention provides a composition of IFN, preferably PEGylated interferon alpha- 2b which can be administered to the patient in combination with standard of care.
  • Standard of care is either hydroxychlorquine or any suitable antiviral agents, e.g., remdesivir, and / or ritonavir, and / or liponavir and / or anti- viral monoclonal antibodies, and / or any anti-inflammatory therapy such as anti-IL-6 or anti-TNF alpha or such, and / or any other therapy being given to the COVID-19 patient as a part of SOC, etc.
  • SOC is as per local regulatory guideline/ institute SOP.
  • the current invention provides a composition of IFN, preferably PEGylated interferon alpha- 2b which can be administered to the patient in combination with TNF inhibitor.
  • TNF inhibitor as mentioned herein is adalimumab, infliximab, or etanercept.
  • the current invention provides a composition of PEGylated interferon alpha- 2b which can be administered in combination with adalimumab.
  • said adalimumab is co-administered at a dose in the range of about 20 mg to about 160 mg on a weekly, twice in a week, thrice in a week, daily basis or every other week with the said composition of PEG-IFN alpha 2b.
  • the pharmaceutical composition of present invention includes PEGylated interferon alpha-2b, which can be administered to the patient in combination with anti-IL-6 antibody.
  • anti-IL-6 antibody is tocilizumab.
  • a composition according to the present invention comprises therapeutically effective dose of PEGylated interferon alpha-2b (PEGIFN alpha-2b) for use in treatment of SARS-CoV-2 infection and its related clinical manifestations.
  • PEG-IFN alpha 2b PEGylated interferon alpha- 26
  • a composition according to the present invention comprises therapeutically effective dose of PEGylated interferon alpha-2b (PEGIFN alpha-2b) for use in treatment of SARS-CoV-2 infection and its related clinical manifestations.
  • the present invention provides a method of treating SARS-CoV-2 infection by administering PEGylated interferon alpha-2b.
  • the present invention provides a method of treating SARS-CoV-2 infection by administering composition of PEGylated interferon alpha-2b comprising PEGylated interferon alpha-2b in suitable therapeutic amount optionally with other suitable excipient(s).
  • suitable excipient(s) include, but are not limited to, salt(s), carbohydrate(s), surfactant(s), amino acid(s), anti-oxidant(s), chelating agent(s), tonicity modifier(s), cryoprotectant(s) or lyoprotectant(s), acid(s), base(s), glycerine, water for injection (WFI) and suitable combination thereof and the like.
  • the current invention provides use of PEGylated interferon alpha-2b for the treatment of SARS-CoV-2 infection and its related clinical manifestations.
  • the current invention provides use of composition of PEGylated interferon alpha-2b comprising PEGylated interferon alpha-2b in suitable therapeutic amount optionally with other suitable excipient(s) for the treatment of SARS-CoV-2 infection and its related clinical manifestations.
  • excipient(s) examples include, but are not limited to, salt(s), carbohydrate(s), surfactant(s), amino acid(s), anti oxidants), chelating agent(s), tonicity modifier(s), cryoprotectant(s) or lyoprotectant(s), acid(s), base(s), glycerine, water for injection (WFI) and suitable combination thereof and the like.
  • WFI water for injection
  • SARS-CoV-2 The third zoonotic human coronavims, SARS-CoV-2, is transmissible from person to person. SARS- CoV-2 vims could induce symptoms including fever, dry cough, dyspnea, fatigue, and lymphopenia in infected patients. In general, aged people are more susceptible to COVID-19 and more likely to be severe than people younger than 50 years; this may be due to more health issues and comorbidities in this population (11).
  • interferon alpha as used herein means the family of highly homologous species-specific proteins that inhibit viral replication and cellular proliferation and modulate immune response.
  • suitable interferon-alphas include, but are not limited to, recombinant IFN alpha 2b such as Intron-A interferon available from Schering Corporation, recombinant IFN alpha 2a such as Roferon interferon available from Hoffmann-La Roche, recombinant IFN alpha 2c such as Berofor alpha 2 interferon available from Boehringer Ingelheim Pharmaceutical, Inc., interferon alpha-nl, a purified blend of natural alpha interferons such as Sumiferon available from Sumitomo, Japan or as Wellferon IFN-alphanl (INS) available from the Glaxo -Wellcome Ltd., London, Great Britain, or a consensus alpha interferon such as those described in U.S.
  • IFN alpha 2b such as Intron-A interferon available from Schering Corporation
  • Patent Nos. 4,897,471 and 4,695,623 (especially Examples 7, 8 or 9 thereof) and the specific product available from Amgen, Inc., Newbury Park, CA, or interferon alpha-n3 a mixture of natural alpha interferons made by Interferon Sciences and available from the Purdue Frederick Co., Norwalk, CT., under the Alferon Tradename.
  • Type I interferon IFN-a/b
  • ISGs interferon-stimulated genes
  • Interferons inhibit viral infection by inducing innate and adaptive immune responses like altering the intracellular environment to restrict viral replication and inducing signaling events that activate immune cell populations and elicit an antiviral immune response.
  • Interferon alfas have been used in the treatment of hepatitis B and C; and to induce inhibition of respiratory coronavims infections, albeit unrelated to the Urbani strain of SARS-CoV (13).
  • the use of IFN alpha either IFN alpha 2a or IFN alpha 2b, is preferred according to the present invention.
  • the manufacture of interferon alpha 2b is described in U.S. Patent No. 4,530,901.
  • the interferon alpha administered is selected from interferon alpha-2a, interferon alpha-2b, a consensus interferon, a purified IFN alpha product or a PEG-IFN alpha 2a or PEG-IFN alpha 2b.
  • PEG-IFN are interferon molecules covalently attached to a PEG molecule.
  • the PEG-IFN is PEG-IFN alpha, preferably interferon alpha-2a
  • interferon alpha-2b PEG-Intron, Schering- Plough; Sylantron, Merck; ViraferonPeg, Merck
  • interferon alpha-2c Boehringer Ingelheim, Ingelheim, Germany
  • consensus interferon as defined by determination of a consensus sequence of naturally occurring interferon alphas (Infergen, Amgen, Thousand Oaks, Calif.). Pegylation of interferons has been reported in U.S. Pat. Nos.
  • the approximate molecular weight of the polymer is designated with a number following the name of the repeated chemical unit.
  • “PEGnooo” or “Polyethylene glycol (12000)” refers to a polymer of polyethylene glycol having an average molecular weight of approximately 12,000 daltons. Conjugation of polymers to proteins may result in a single polymer molecule conjugated to a protein or multiple such conjugations to a single protein. The degree of conjugation is dependent upon the reaction conditions and desired result.
  • the IFN alpha 2b in the compositions of the present invention comprises a single IFN alpha 2b conjugated to a single PEG12000 molecule.
  • the IFN alpha 2b molecule is linked to the PEG 12000 molecule with a urethane bond.
  • Molecular weight of the conjugated PEGylated interferon alpha- 2b molecule is approximately 31 kDa.
  • processes for preparing a PEG-IFN conjugates are known in the art and such processes and the products derived from such processes are considered to be encompassed within the scope of the present invention. Examples of such process (es) for producing this protein-polymer conjugate may be found in U.S. Pat. No. 5,612,460 (Zalipsky) and U.S. Pat. No. 5,711,944 (Gilbert et al).
  • the preferred concentration of PEG-IFN alpha 2b is 0.01 to 5.0 mg IFN alpha 2b per mL.
  • the resulting conjugated PEG-IFN alpha 2b may be in the form of a single or mixture of positional isomers.
  • One such mixture of positional isomers could mean, the PEG-IFN alpha 2b has PEG linked at a histidine residue of the IFN alpha 2b molecule, while another PEG-IFN alpha 2b where PEG is linked to another site of the IFN alpha 2b molecule, for example the lysine residue or cysteine residue.
  • the PEGylated interferon alpha 2b has about 8 -9 positional isomers of interferon alpha.
  • the different isomers of PEGylated interferon alpha 2b of the current invention was identified by high-performance ion-exchange chromatography (HP-IEC) technique.
  • the PEGylated interferon alpha 2b of the current invention has about 1 °C difference in thermal transition value as compared to the unmodified interferon alpha 2b.
  • the term ‘unmodified interferon alpha 2b’ as used herein means the interferon alpha 2b without PEGylation.
  • the said thermal transition value (Tm) was assessed by differential scanning calorimetry (DSC) analysis.
  • the PEGylated interferon alpha 2b produced according to the present invention has interferon alpha 2b to polyethylene glycol present in a ratio of 1:2.
  • PEGylated interferon alpha 2b produced according to the current invention exhibits certain degree of depegylation upon hydroxylamine treatment as assessed by size exclusion chromatography (SEC-HPLC) method.
  • certain degree of depegylation according to the current invention is 30 % or more maximum up to 60 % of depegylation.
  • the PEGylated interferon alpha-2b prepared according to the current invention contains no more than 10 % of high molecular weight species variants and less than 5 % of unmodified interferon.
  • the PEGylated interferon alpha- 2b prepared according to the current invention exhibits low level of ( ⁇ 5 %) oxidation on one of the methionine residues of interferon at the position 148.
  • the PEGylated interferon alpha- 2b prepared according to the current invention has less than 5 % of oxidized variant of interferon at Methionine residue at position 148.
  • the PEGylated interferon alpha- 2b prepared according to the current invention exhibits low level of ( ⁇ 5 %) deamidation on one of the asparagine residues of interferon at the position 93.
  • the PEGylated interferon alpha-2b prepared according to the current invention has less than 5 % of deamidated variant of interferon at asparagine residue at position 93. Said oxidized variant as well as deamidated variants were analysed by mass -spectrometry.
  • lyophilisation can be used. Lyophilization is a process of freeze-drying a composition wherein a frozen aqueous mixture is treated to remove water. Commonly, the process involves the sublimation of water from the aqueous solutions, usually under reduced pressure conditions. After lyophilization, the PEG-IFN alpha conjugate(s) can be stored for extended periods of time.
  • composition of PEG-IFN alpha 2b to treat SARS-CoV-2 infection and its related clinical manifestations.
  • the pharmaceutical composition of present invention is stabilized composition of PEG-IFN alpha 2b, which includes one or more suitable pharmaceutically acceptable excipient(s).
  • suitable pharmaceutically acceptable excipient(s) include, but are not limited to, buffer(s), sugar(s), polyol(s), amino acid(s), surfactant(s), preservative(s), salt(s), anti-oxidant(s), tonicity modifier(s), acid(s), base(s), chelating agent(s), and polymer(s).
  • Interferon alpha-2b in the said composition is monopegylated interferon alpha-2b.
  • monopegylated interferon alpha-2b according to the present invention are interferon alpha-2b molecules covalently linked to one or more “PEG molecule/s” or “monomethoxy PEG molecule/s”.
  • Interferon alpha-2b is recombinant according to the present invention. Recombinant IFN alpha 2b was produced by r- DNA technology in Pichia pastoris (KM71) as a secretory protein and it is a non- glycosylated protein with a molecular weight of 19.268 kDa.
  • compositions of PEG-IFN alpha 2b are prepared using suitable combinations of a buffer, stabilizer, cryoprotectant(s) and/or lyoprotectant(s) and a solvent, optionally with other excipients and suitably lyophilized and stored as a dry powder to be reconstituted before use.
  • the composition of PEG- IFN alpha 2b are prepared using suitable combinations of succinate buffer, lactose and/or polysorbate 80, optionally with other excipients and suitably lyophilized and stored as a dry powder to be reconstituted before use.
  • the pharmaceutical composition comprising therapeutically effective amount of PEGylated interferon alpha-2b, either alone or in combination with other suitable excipient(s).
  • suitable excipient(s) is selected from salt(s), carbohydrate(s), surfactant(s), amino acid(s), anti-oxidant(s), chelating agent(s), tonicity modifier(s), acid(s), base(s), glycerine, water for injection (WFI) and suitable combination thereof and the like.
  • the PEGylated interferon alpha-2b used for the treatment of SARS-CoV-2 infection and its related clinical manifestations or for the administration as a part of method of treating SARS-CoV-2 infection is in the concentration range of 0.01 mg / mL to 5.0 mg / mL, preferably 0.03 mg / mL to 2.5 mg / mL, more preferably 0.1 mg / mL to 0.5 mg / mL.
  • the pharmaceutical composition according to the present invention comprising PEGylated interferon alpha- 2b in the concentration range of 0.01 mg / mL to 5.0 mg / mL, preferably 0.03 mg / mL to 2.5 mg / mL, more preferably 0.1 mg / mL to 0.5 mg / mL.
  • the concentration range of 0.1 mg / mL to 0.5 mg / mL includes 0.1 mg / mL, 0.16 mg / mL, 0.20 mg / mL, 0.24 mg / mL, 0.3 mg / mL.
  • the pharmaceutical composition according to the present invention comprising PEGylated interferon alpha-2b in the concentration range of 10 pg to 5000 pg per mL, preferably 30 pg to 2500 pg per mL, more preferably 100 pg to 500 pg per mL.
  • concentration range of 100 pg to 500 pg per mL includes 100 pg, 160 pg, 200 pg, 240 pg, 300 pg per mL.
  • the pharmaceutical composition comprising
  • buffer(s) and other suitable excipient(s) where in buffer(s) is selected from phosphate buffer (e.g., sodium phosphate buffer, etc.), arginine-phosphate buffer, histidine buffer, arginine-histidine buffer, histidine chloride buffer, glycine buffer, sodium glycinate buffer, arginine buffer, citrate buffer (e.g., sodium citrate buffer, etc.), arginine-citrate buffer, succinate buffer (e.g.
  • succinic acid buffer sodium succinate buffer, etc.
  • potassium succinate buffer arginine- succinate buffer, acetate buffer, arginine-acetate buffer, arginine- glutamate buffer, arginine-aspartate buffer, tromethamine buffer and suitable combination thereof and the like.
  • the suitable excipient(s) is selected from salt(s), carbohydrate(s), surfactant(s), amino acid(s), anti-oxidant(s), chelating agent(s), tonicity modifier(s), acid(s), base(s), glycerine, water for injection (WFI) and suitable combination thereof and the like.
  • the pharmaceutical composition comprising PEGylated interferon alpha-2b, buffer(s) and other suitable excipient(s), where in buffer(s) is selected from succinate buffer, phosphate buffer, sodium succinate buffer, potassium succinate buffer, histidine chloride buffer, sodium glycinate buffer and suitable combination thereof and the like.
  • the pharmaceutical composition comprising PEGylated interferon alpha- 2b, buffer and other suitable excipient(s), where in buffer is succinate buffer.
  • the suitable excipient(s) is selected from salt(s), carbohydrate(s), surfactant(s), amino acid(s), anti-oxidant(s), chelating agent(s), tonicity modifier(s), acid(s), base(s), glycerine, water for injection (WFI) and suitable combination thereof and the like.
  • the pharmaceutical composition comprising PEGylated interferon alpha-2b, buffer(s), carbohydrate(s), surfactant(s) and other suitable excipient(s).
  • the pharmaceutical composition comprising PEGylated interferon alpha-2b, buffer(s), carbohydrate(s) as a cryoprotectant(s) or as a lyoprotectant(s), surfactant(s) and other suitable excipient(s).
  • the suitable excipient(s) is selected from salt(s), amino acid(s), anti oxidants), chelating agent(s), tonicity modifier(s), acid(s), base(s), glycerine, water for injection (WFI) and suitable combination thereof and the like.
  • the current invention provides pharmaceutical composition comprising PEGylated interferon alpha-2b, buffer(s) and carbohydrate(s) as a cryoprotectant(s) or as a lyoprotectant(s), optionally with other suitable excipient(s). Furthermore, it provides pharmaceutical composition comprising PEGylated interferon alpha-2b, buffer(s) and surfactant(s), optionally with other suitable excipient(s).
  • compositions that are anticipated here utilize a suitable buffer(s), suitable carbohydrate(s) which may act as a cryoprotectant(s) and/or lyoprotectant(s), suitable surfactant(s), and solvent(s), alone or in suitable combination, optionally with other suitable excipients, in addition to the PEGylated interferon alpha conjugate.
  • a suitable buffer(s) suitable carbohydrate(s) which may act as a cryoprotectant(s) and/or lyoprotectant(s), suitable surfactant(s), and solvent(s), alone or in suitable combination, optionally with other suitable excipients, in addition to the PEGylated interferon alpha conjugate.
  • the pharmaceutical composition of PEGylated interferon alpha-2b according to the present invention comprising stabilizer(s).
  • the stabilizer(s) of present invention is selected from surfactant(s), sugar(s), amino acid(s), polyol(s) and the like or suitable derivative or mixtures thereof.
  • Stabilizer include an excipient capable of preventing aggregation or other physical and chemical decompositions (e.g., self-decomposition, deamidation, oxidation, etc.) in aqueous and solid states. In the present invention, it stabilizes the PEGylated interferon alpha conjugates to prevent depegylation during and after lyophilisation - a phenomenon commonly associated with the PEG- interferon alpha 2b product.
  • Stabilizers commonly used in pharmaceutical compositions include, but not limited to, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, glycine, arginine hydrochloride, sodium dodecyl sulphate (SDS), polysorbate; poly-hydroxy compounds including polysaccharides such as dextran, starch, hydroxyethyl starch, cyclodextrins, N- methyl pyrollidene, cellulose, hyaluronic acid, sodium chloride and suitable combination thereof.
  • One such preferred stabilizing agent is polyoxyethylensorbitan-fatty acid ester, polysorbate 80 (Tween 80), according to the present invention.
  • Polysorbate 20 can also be used as a stabilizing agent according to the present
  • Buffers suitable for use in each of the compositions of the present invention include buffers that are compatible with PEGylated interferon alpha-2b and suitable for administration to a subject. Buffers of each of the compositions of the present invention are selected from phosphate buffer (e.g., sodium phosphate buffer, etc.), arginine -phosphate buffer, histidine buffer, arginine-histidine buffer, histidine chloride buffer, glycine buffer, sodium glycinate buffer, arginine buffer, citrate buffer (e.g., sodium citrate buffer, etc.), arginine-citrate buffer, succinate buffer (e.g.
  • phosphate buffer e.g., sodium phosphate buffer, etc.
  • arginine -phosphate buffer arginine -phosphate buffer
  • histidine buffer arginine-histidine buffer
  • histidine chloride buffer histidine chloride buffer
  • glycine buffer sodium glycinate buffer
  • succinic acid buffer sodium succinate buffer, etc.
  • potassium succinate buffer arginine- succinate buffer
  • acetate buffer arginine-acetate buffer
  • arginine-glutamate buffer arginine-aspartate buffer
  • tromethamine buffer tromethamine buffer and suitable combination thereof and the like.
  • the buffer of each of the compositions according to the present invention is phosphate buffer (e.g., sodium phosphate buffer, etc.). In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is arginine-phosphate buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is histidine buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is arginine-histidine buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is histidine chloride buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is glycine buffer.
  • phosphate buffer e.g., sodium phosphate buffer, etc.
  • the buffer of each of the compositions according to the present invention is arginine-phosphate buffer.
  • the buffer of each of the compositions according to the present invention is histidine buffer.
  • the buffer of each of the compositions according to the present invention is sodium glycinate buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is arginine buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is citrate buffer (e.g., sodium citrate buffer, etc.). In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is arginine-citrate buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is succinate buffer (e.g. succinic acid buffer, sodium succinate buffer, etc.).
  • succinate buffer e.g. succinic acid buffer, sodium succinate buffer, etc.
  • the buffer of each of the compositions according to the present invention is potassium succinate buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is arginine- succinate buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is acetate buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is arginine- acetate buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is arginine-glutamate buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is arginine-aspartate buffer.
  • the buffer of each of the compositions according to the present invention is tromethamine buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is combinations of buffers as described in the present invention.
  • the buffers included in the pharmaceutical composition according to the present invention may be a buffer that maintains the pH of the composition at a pH, ranging from about pH 4.0 to about pH 8.0.
  • the pH range of about 4.0 to about 8.0 according to the present invention includes about pH 4.0, about pH 5.0, about pH 6.0, about pH 7.0 or about pH 8.0 or any non-integer number in between them (e.g., about pH 4.5 or about pH 5.3 or about pH 6.8 and the like).
  • the pharmaceutical composition of PEGylated interferon alpha-2b according to the present invention having a pH in the range of about pH 4.0 to about pH 7.1.
  • the pharmaceutical composition of PEGylated interferon alpha- 2b according to the present invention having a pH in the range of about pH 6.5 to about pH 7.1.
  • About pH 6.5 to about pH 7.1 according to the present invention includes about pH 6.5, about pH 7.0 or any non-integer or integer number in between them.
  • the pharmaceutical composition of PEGylated interferon alpha-2b according to the present invention having a pH of about 6.8.
  • the concentration of buffer according to the present invention is in the range of about 1 mM to about 500 mM.
  • the buffer concentration according to the present invention is in the range of about 1 mM to about 100 mM.
  • the concentration range according to present disclosure include each integer and non-integer number between a particular range.
  • the concentration range of about 1 mM to about 100 mM include about 5 mM, about 9 mM, about 10 mM, about 13 mM, about 15 mM, about 18 mM, about 20 mM, about 25 mM, about 30 mM, about 45 mM, about 60 mM, about 70 mM or any integer or non-integer number in between them.
  • the concentration of buffer when present in combination with other suitable excipient(s) according to the present invention is in the range of about 1 mM to about 500 mM.
  • the buffer concentration according to the present invention is in the range of about 1 mM to about 100 mM.
  • the concentration range according to present disclosure include each integer and non-integer number between a particular range.
  • the concentration range of about 1 mM to about 100 mM include about 5 mM, about 9 mM, about 10 mM, about 13 mM, about 15 mM, about 18 mM, about 20 mM, about 25 mM, about 30 mM, about 45 mM, about 60 mM, about 70 mM or any integer or non-integer number in between them.
  • composition according to the present invention does not contain buffer.
  • the present invention provides the pharmaceutical composition of PEGylated interferon alpha- 2b comprising buffer in the range of about 1 mM to about 500 mM, preferably about 1 mM to about 100 mM.
  • the present invention provides the pharmaceutical composition of PEGylated interferon alpha- 2b comprising succinate buffer at a concentration of about 10 mM.
  • the buffering agent(s) or buffering species or buffering component(s) according to the present invention is present in amount of about 0.01 mg / mL to about 100 mg / mL.
  • concentration range according to present disclosure include each integer and non-integer number between particular range.
  • the concentration range of about 0.01 mg / mL to about 100 mg / mL include about 0.1 mg / mL, about 0.7 mg / mL, about 1.18 mg / mL, about 1.50 mg / mL, about 2 mg / mL, about 2.3 mg / mL, about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 50 mg / mL or any integer or non-integer number in between them.
  • the pharmaceutical composition of PEGylated interferon alpha-2b according to the present invention comprising carbohydrate(s).
  • a carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may also be employed.
  • carbohydrate excipients of interest include, but are not limited to, for example, monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.
  • monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like
  • Carbohydrate(s) may act as a cryoprotectant(s) or as a lyoprotectant(s) according to the present invention.
  • the composition may include a sugar.
  • Preferred sugars suitable for use in the compositions of the present invention include sugars that are compatible with PEGylated interferon alpha- 2b and suitable for administration to a subject.
  • suitable sugar include, but not limited to, dextran, trehalose, raffinose, sucrose, polyvinylpyrrolidone, lactose, inositol, sorbitol, mannitol, dimethyl sulfoxide, glycerol, albumin, calcium lactobionate, fructose, maltose, galactose, glucose, D- mannose, sorbose, cellobiose, melezitose, maltodextrins, starches, lactitol, xylitol, pyranosyl sorbitol, myoinositol, hydroxypropyl-beta-cyclodextrin (b-HPCD) and suitable combination thereof and the like.
  • the pharmaceutical composition of PEGylated interferon alpha- 2b according to the present invention comprising lactose or sucrose or raffinose or trehalose.
  • the pharmaceutical composition of PEGylated interferon alpha-2b according to the present invention comprising carbohydrate(s), either as a cryoprotectant(s) or as a lyoprotectant(s), selected from sucrose, lactose and mannitol.
  • the carbohydrate according to the present invention is lactose.
  • the carbohydrate according to the present invention is sucrose.
  • the carbohydrate according to the present invention is raffinose.
  • the carbohydrate according to the present invention is trehalose.
  • the concentration of carbohydrate(s) according to the present invention is in the range of about 10 mg / mL to about 250 mg / mL, preferablylO mg / mL to about 200 mg / mL.
  • the carbohydrate(s) concentration according to the present invention is in the range of about 10 mg / mL to about 200 mg / mL.
  • the concentration range according to present disclosure include each integer and non-integer number between a particular range.
  • the concentration range of about 10 mg / mL to about 200 mg / mL include about 50 mg / mL, about 70 mg / mL, about 100 mg / mL, about 105 mg / mL, about 125 mg / mL, about 160 mg / mL, about 195 mg / mL or any integer or non-integer number in between them.
  • the present invention provides the pharmaceutical composition of PEGylated interferon alpha- 2b comprising carbohydrate in the range of about 10 mg / mL to about 250 mg / mL, preferably about 10 mg / mL to about 200 mg / mL.
  • the present invention provides the pharmaceutical composition of PEGylated interferon alpha-2b comprising lactose at a concentration of about 105 mg / mL.
  • compositions of the present disclosure in addition to the PEGylated interferon alpha- 2b, may include carbohydrate(s) as a cryoprotectant(s) or as a lyoprotectant(s).
  • concentration of cryoprotectant(s) according to the present invention is in the range of about 10 mg / mL to about 250 mg / mL, preferably about 10 mg / mL to about 200 mg / mL.
  • the compositions of the present disclosure, in addition to the PEGylated interferon alpha-2b, buffer(s) and carbohydrate(s), may include surfactant(s).
  • surfactants include, but not limited to polyoxyethylensorbitan fatty acid esters (Tweens), polyoxyethylene alkyl ethers (e.g. Brij), alkylphenylpolyoxyethylene ethers (e.g. Triton-X), polyoxyethylene - polyoxypropylene copolymer (e.g. Poloxamer, Pluronic), polyethylene glycol (PEG), polyethyleneimine (PEI), sodium dodecyl sulphate (SDS) and suitable combination thereof and the like.
  • Teweens polyoxyethylensorbitan fatty acid esters
  • polyoxyethylene alkyl ethers e.g. Brij
  • alkylphenylpolyoxyethylene ethers e.g. Triton-X
  • polyoxyethylene - polyoxypropylene copolymer e.g. Poloxamer, Pluronic
  • PEG polyethylene glycol
  • PEI polyethyleneimine
  • SDS sodium dodec
  • the pharmaceutical composition of PEGylated interferon alpha- 2b according to the present invention comprising surfactant, wherein the surfactant is polyoxyethylensorbitan-fatty acid esters (Tween).
  • the pharmaceutical composition of PEGylated interferon alpha-2b according to the present invention comprising polysorbate 20 or polysorbate 80.
  • the surfactant according to the present invention is polyoxyethylensorbitan fatty acid esters (Tween).
  • the surfactant according to the present invention is polyoxyethylene alkyl ethers (e.g. Brij).
  • the surfactant according to the present invention is alkylphenylpolyoxyethylene ethers (e.g. Triton-X).
  • the surfactant according to the present invention is polyoxyethylene-polyoxypropylene copolymer (e.g. Poloxamer, Pluronic).
  • the surfactant according to the present invention is polyethylene glycol (PEG).
  • the surfactant according to the present invention is polyethyleneimine.
  • the surfactant according to the present invention is sodium dodecyl sulphate (SDS).
  • the surfactant according to the present invention is in the range of about 0.001 mg / mL to about 1 mg / mL.
  • the surfactant concentration according to the present invention is in the range of about 0.01 mg / mL to about 2 mg / mL.
  • the concentration range according to present disclosure include each integer and non-integer number between particular range.
  • the concentration range of about 0.01 mg / mL to about 2 mg / mL include about 0.1 mg / mL, about 0.2 mg / mL, about 0.5 mg / mL, about 0.9 mg / mL or any integer or non-integer number in between them.
  • the present invention provides the pharmaceutical composition of PEGylated interferon alpha- 2b comprising surfactant in the range of about 0.001 mg / mL to about 1 mg / mL, preferably about 0.01 mg / mL to about 2 mg / mL.
  • the present invention provides the pharmaceutical composition of PEGylated interferon alpha-2b comprising polysorbate 80 at a concentration of about 0.1 mg / mL.
  • Surfactant(s) can act as stabilizer in the pharmaceutical compositions.
  • compositions of the present disclosure may include amino acid in addition to the PEGylated interferon alpha- 2b.
  • Amino acids suitable for use in the compositions of the present disclosure include amino acids that are compatible with the PEGylated interferon alpha-2b and suitable for administration to a subject.
  • suitable amino acids include, but are not limited to, arginine, glycine, asparagine, glutamine, lysine, threonine, histidine, glutamic acid, aspartic acid, isoleucine, valine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, cysteine, their suitable salt form and combination thereof and the like.
  • Amino acid can be a part of buffer as a potential counter ion.
  • compositions of the present disclosure may also include other component(s), such as additional pharmaceutically acceptable excipient(s) or a dosage delivery vehicle as part of the composition.
  • suitable excipient(s) according to the present invention may include, but are not limited to, salt(s), anti- oxidant(s), chelating agent(s), tonicity modifier(s), acid(s), base(s), vegetable oils, phospholipids, glycerine, water for injection (WFI) and suitable combination thereof and the like.
  • WFI water for injection
  • salts according to the present invention may include, but are not limited to, sodium chloride, potassium chloride, potassium sulfate and sodium sulfate.
  • the pharmaceutical composition of PEGylated interferon alpha- 2b according to the present invention comprising salt(s) selected from sodium chloride, potassium chloride, potassium sulfate and sodium sulfate.
  • the pharmaceutical composition of PEGylated interferon alpha-2b can be free from salt(s).
  • the pharmaceutical composition of PEGylated interferon alpha-2b can be free from inorganic salt(s).
  • Acids or bases may also be present in composition of the present disclosure. Acids or bases according to the present invention can be a part of buffering agent or acid / base conjugate.
  • Examples of acid according to the present invention may include, but are not limited to hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, succinic acid, sulfuric acid, fumaric acid, and any combination thereof.
  • Examples of base according to the present invention include, but are not limited to sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium succinate, sodium format, sodium sulfate, potassium sulfate, potassium fumarate, and any combination thereof.
  • chelating agent(s) of the present invention is selected from ethylenediaminetetraacetic acid (EDTA), pentetic acid and ethylene glycol -bi s(p- aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA).
  • EDTA ethylenediaminetetraacetic acid
  • EGTA ethylene glycol -bi s(p- aminoethyl ether)-N,N,N’,N’-tetraacetic acid
  • Suitable solvent for the present composition is water, preferably the solvent may be water for injection.
  • the pharmaceutical composition of the present invention comprising
  • PEGylated interferon alpha-2b buffer(s), carbohydrate(s) and surfactant(s).
  • the pharmaceutical composition of the present invention comprising PEGylated interferon alpha-2b, buffer(s), carbohydrate(s) as a cryoprotectant(s) or as a lyoprotectant(s), and surfactant(s).
  • the PEGylated interferon alpha- 2b composition of the present invention comprising PEGylated interferon alpha- 2b and buffer(s).
  • the PEGylated interferon alpha-2b composition of the present invention comprising PEGylated interferon alpha-2b, buffer(s) and carbohydrate(s).
  • the PEGylated interferon alpha- 2b composition of the present invention comprising PEGylated interferon alpha-2b, buffer(s) and carbohydrate(s) as a cryoprotectant(s) or as a lyoprotectant(s).
  • the PEGylated interferon alpha-2b composition of the present invention comprising PEGylated interferon alpha-2b, buffer(s) and surfactant(s).
  • the PEGylated interferon alpha- 2b composition of the present invention comprising PEGylated interferon alpha- 2b, surfactant(s) and carbohydrate(s).
  • the PEGylated interferon alpha-2b composition of the present invention comprising PEGylated interferon alpha- 2b, surfactant(s) and carbohydrate(s) as a cryoprotectant(s) or as a lyoprotectant(s).
  • the PEGylated interferon alpha-2b composition of the present invention comprising PEGylated interferon alpha- 2b, buffer(s), carbohydrate(s), surfactant(s) and other suitable excipient(s).
  • the PEGylated interferon alpha-2b composition of the present invention comprising PEGylated interferon alpha-2b, buffer(s), carbohydrate(s) as a cryoprotectant(s) or as a lyoprotectant(s), surfactant(s) and other suitable excipient(s).
  • Buffer(s), carbohydrate(s), surfactant(s) and other suitable excipient(s) for the PEGylated interferon alpha-2b composition of the present invention are as described herein above.
  • the present invention provides pharmaceutical composition comprising PEGylated interferon alpha- 2b, succinate buffer, lactose, polysorbate 80 and other suitable excipient(s).
  • the pharmaceutical composition of PEGylated interferon alpha-2b comprising therapeutically effective amount of PEGylated interferon alpha-2b, succinate buffer, lactose and polysorbate 80.
  • the composition of PEGylated interferon alpha-2b according to the current invention can be used for treatment of SARS-CoV-2 infection.
  • the composition of the invention has improved stability either in frozen, lyophilized, or liquid form.
  • the current invention provides a pharmaceutical composition including PEGylated interferon alpha-2b in a lyophilized form.
  • the current invention provides a pharmaceutical composition including PEGylated interferon alpha- 2b in a liquid form.
  • the present invention provides composition (e.g., frozen or lyophilized or liquid) and methods of making composition.
  • a process of lyophilizing the composition according to the current invention, to get a lyophilized powder comprising: a) preparing a composition comprising PEG-IFN alpha 2b, buffer, stabilizer and cryoprotectant; b) lyophilizing said formulation under suitable conditions and obtaining lyophilized powder.
  • the lyophilization process is a process whereby water is sublimed from a composition after it is frozen- which can provide a stable form to the biological over a desired period of time.
  • the lyophilization process to prepare composition of the present invention can be performed by a skilled person using the techniques available in the art.
  • the lyophilizing steps may include freezing, primary drying, secondary drying and optionally with the step of annealing.
  • the said lyophilized composition can be stored as a dry powder and reconstituted before use.
  • the pharmaceutical composition of PEG-IFN alpha 2b can be stored under +2 °C to +8 °C storage condition.
  • the composition (e.g., frozen or lyophilized or liquid) is a sterile composition.
  • sterile is meant that there are substantially no microbial contaminants (e.g., fungi, bacteria, viruses, spore forms, etc.).
  • the composition e.g., frozen or lyophilized or liquid
  • the present invention provides pharmaceutical composition of PEG-IFN alpha 2b that is substantially free of particles and / or aggregates and prevents the formation of particles and / or aggregates during storage and / or transport.
  • the current invention provides lyophilized and stabilized composition of PEGylated interferon alpha-2b comprising therapeutically effective amount of PEGylated interferon alpha-2b, succinate buffer, lactose and polysorbate 80 and optionally with other suitable excipient(s).
  • suitable excipient(s) for PEG-IFN alpha 2b are as described herein above.
  • the interferon alpha administered at safe and effective dose according to the current invention is a PEGylated interferon alpha- 2b and the amount of PEG-IFN alpha 2b administered is about 0.1 mcg/kg to about 30 mcg/kg on a weekly, twice in a week, thrice in a week, daily basis, twice in a day or every other week for use in treatment of SARS-CoV-2 infection.
  • the composition of PEG-IFN alpha 2b can be used for subcutaneous administration at variety of different dosage levels, for example, about 0.1 mcg/kg/week, about 0.5 mcg/kg/week, about 1 mcg/kg/week, about 1.5 mcg/kg/week, about 2 mcg/kg/week, about 5 mcg/kg/week, about 10 mcg/kg/week, about 20 mcg/kg/week, and/or 30 mcg/kg/week as per further described embodiment.
  • the current invention further provides treatment of SARS-CoV-2 infection and its related clinical manifestations comprising administration of single dose of PEG-IFN alpha 2b or its composition to the subject.
  • the said single dose of PEG- IFN alpha 2b according to the current invention is selected from 0.1 mcg/kg, about 0.5 mcg/kg, about 1 mcg/kg, about 1.5 mcg/kg, about 2 mcg/kg, about 5 mcg/kg.
  • the current invention provides composition of PEGylated interferon alpha-2b comprising buffer(s), sugar(s) and surfactant(s) to deliver 1 mcg/kg of PEG-IFN alpha 2b to neutralize SARS-CoV-2 virus.
  • the current invention provides composition of PEGylated interferon alpha- 2b that can neutralize 100% SARS-CoV-2 virus.
  • the compositions of the present invention may be suitable for any use, including both in vitro and in vivo uses.
  • the pharmaceutical composition of present invention includes PEGylated interferon alpha- 2b, which can be used for parenteral administration.
  • Parenteral administration includes intravenous, subcutaneous, intra peritoneal, intramuscular administration or any other route of delivery generally considered to be falling under the scope of parenteral administration and as is well known to a skilled person.
  • the pharmaceutical composition of present invention includes PEGylated interferon alpha-2b used for subcutaneous administration.
  • the pharmaceutical composition of present invention includes PEGylated interferon alpha- 2b, which can be delivered into lungs by atomization.
  • the pharmaceutical composition of present invention includes PEGylated interferon alpha-2b, which can be delivered intranasally in the form of nasal drops.
  • the current invention provides a composition of IFN, preferably
  • Standard of care is either hydroxychlorquine or any suitable antiviral agents, e.g., remdesivir, and / or ritonavir, and / or liponavir and / or anti- viral monoclonal antibodies, and / or any anti-inflammatory therapy such as anti-IL-6 or anti-TNF alpha or such, and / or any other therapy being given to the COVID-19 patient as a part of SOC, etc.
  • SOC is as per local regulatory guideline/ institute SOP.
  • the current invention provides a composition of IFN, preferably PEGylated interferon alpha-2b which can be administered to the patient in combination with TNF inhibitor.
  • TNF inhibitor as mentioned herein is adalimumab, infliximab, or etanercept.
  • the current invention provides a composition of PEGylated interferon alpha-2b which can be administered in combination with adalimumab.
  • said adalimumab is co-administered at a dose in the range of about 20 mg to about 160 mg on a weekly, twice in a week, thrice in a week, daily basis or every other week with the said composition of PEG-IFN alpha 2b.
  • the pharmaceutical composition of present invention includes PEGylated interferon alpha-2b, which can be administered to the patient in combination with anti-IL-6 antibody.
  • anti-IL-6 antibody is tocilizumab.
  • the compositions of the present invention can be used in the treatment of
  • SARS-CoV-2 infection and its related clinical manifestations in a subject are also included in the invention.
  • devices that may be used to deliver the formulation of the invention. Examples of such devices include, but are not limited to, a syringe, a pen, an implant, a needle-free injection device and a patch.
  • a syringe a pen
  • an implant a needle-free injection device
  • a patch a patch
  • the described pharmaceutical compositions of the present invention and the means of carrying out the invention to obtain a stable pharmaceutical dosage form of said composition of PEG-IFN alpha 2b. It will be appreciated that the examples are illustrative and such other suitable modifications/additions etc. as are well within the scope of the persons skilled in the art are meant to be encompassed within the scope of the present invention.
  • the efficacy of the PEG-IFN alpha 2b in the treatment of SARS-CoV- 2 infection or related clinical manifestations may be evaluated in vivo as described in following examples.
  • PEGylated interferon alpha 2b compositions as mentioned in table 1 were prepared following the process of making formulation as disclosed in patent document WO 2008/062481.
  • the composition as mentioned in table 1 were prepared in lyophilized form.
  • compositions of PEGylated interferon alpha 2b can be prepared with 50 pg / 0.5 mL of PEGylated interferon alpha 2b, 120 pg / 0.5 mL of PEGylated interferon alpha 2b and 150 pg / 0.5 mL of PEGylated interferon alpha 2b, respectively.
  • Example 2 Analysis of anti-viral effect of PEGylated interferon alpha-2b composition using plaque neutralization assay
  • plaque neutralization assay was performed using Vero E6 cells and SARS-CoV-2 vims (Strain: NIV-2020-770) sourced from ICMR-NIV Pune.
  • Log dilutions of PEGylated interferon alpha-2b (10 fold dilutions starting at a concentration of 10 pg / mL) were used to determine SARS-CoV-2 vims inhibition.
  • Vero cells were seeded in a 24 well cell culture plate and incubated for 24 hours with the dilutions of PEG-IFN alpha 2b.
  • the supernatant was discarded and the cells were infected with a known titer of SARS-CoV-2 vims. After vims infection for 1 hour, the supernatant was discarded and wells filled with medium containing CMC. The plates were incubated for further 72 hours in a CO2 humidified incubator at 37 °C. Post the incubation, the cells were fixed and the plaques enumerated by staining with crystal violet stain. The number of plaques were counted and percentage inhibition was calculated in comparison to the number of plaques obtained in the wells with virus (without PEG-IFN alpha 2b) as a positive control.
  • Post enrolment patients on test arm were provided the standard of care medication along with PEGylated interferon alpha-2b (1 mcg/kg) on day 1.
  • next dose (second dose) of 1 mcg/kg on day 8 could be optionally administered along with SOC.
  • the PEGylated interferon alpha- 2b was provided in a subcutaneous injection form.
  • patients could also be administered PEGylated interferon alpha-2b along with standard of care at a dose in the range of 0.5 mcg/kg/week to 2 mcg/kg/week for this study.
  • the PEGylated interferon alpha- 2b product was found to be safe and well tolerated. No serious adverse events and/or deaths were reported in the PEGylated interferon alpha- 2b treatment group and no persistent change in laboratory parameters were recorded. No clinically relevant findings from clinical examination, vital signs and ECG evaluations were attributed to PEGylated interferon alpha-2b. Overall, treatment with PEGylated interferon alpha-2b was found to be safe and well tolerated.
  • Example 4 250 patient clinical study of PEGylated interferon alpha-2b composition in treatment of SARS-CoV-2 infection
  • Eligible subjects were randomized in a 1:1 ratio to either PEG-IFN alpha 2b (1 pg/kg subcutaneous [SC] injection, single dose) plus SOC or SOC alone.
  • Standard of care treatments i.e., antipyretics, cough suppressants, antibiotics, steroids, vitamins, anticoagulants, hydroxychloroquine and antivirals e.g. remdesivir
  • Randomization was generated using SAS ® software (Version 9.4).
  • Each vial of PEG-IFN alpha 2b was reconstituted with 0.7 ml of water for injection for administration of up to 0.5 ml of solution.
  • Each 0.5 ml of solution for SC injection delivers 100 pg of PEG-IFN alpha 2b. All subjects were hospitalized, RT-PCR tests in pharyngeal swab were performed at screening, Day 7, Day 11, Day 15 and Day 29. The assessment was done on the basis of different evaluation criteria as described in example 3.
  • mITT modified intent- to-treat
  • Table 2 Analysis of proportion of subjects with clinical improvement in mITT population, measured using the WHO 7-point ordinal scale
  • Table 3 Analysis of proportion of subjects with negative qualitative RT- PCR in mITT population
  • PEG-IFN alpha 2b No clinically relevant findings from clinical examination, vital signs and ECG evaluations were attributed to PEG-IFN alpha 2b. Overall, single dose of PEG-IFN alpha 2b was safe and well tolerated in the study. Early treatment with PEG-IFN alpha 2b induced early viral clearance and improved the clinical status of patients with moderate COVID-19 disease. It also decreased the duration of supplemental oxygen. Treatment with PEG IFN-a2b provides a viable treatment option during the current pandemic situation. It can also limit the spread of virus in the community.
  • SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues, Cell, 2020, volume 181, issue 5, pages 1016-1035

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Abstract

The present invention relates to a composition of PEGylated interferon alpha-2b for use in treatment of SARS-CoV-2 infection and its related clinical manifestations. The said composition according to the present invention is a pharmaceutical composition. Current invention also provides dose and dose regimen of PEGylated interferon alpha-2b for use in treatment of severe acute respiratory syndrome related coronavirus, preferably SARS-CoV-2 and its related clinical manifestations. The invention also discloses a method of treatment of severe acute respiratory syndrome related coronavirus, preferably SARS-CoV-2 and its related clinical manifestations using the composition of PEGylated interferon alpha-2b as disclosed herein. Further disclosed is the use of the composition of PEGylated interferon alpha-2b as disclosed herein below for the treatment of severe acute respiratory syndrome related coronavirus, preferably SARS-CoV-2 and its related clinical manifestations.

Description

A COMPOSITION OF PEGYLATED INTERFERON ALPHA-2B FOR THE TREATMENT OF SARS-COV-2 INFECTION AND RELATED MANIFESTATIONS
FIELD OF THE INVENTION The present invention relates to a composition of PEGylated interferon alpha- 2b for use in treatment of SARS-CoV-2 infection and its related clinical manifestations. The said composition according to the present invention is a pharmaceutical composition. Current invention also provides dose and dose regimen of PEGylated interferon alpha-2b for use in treatment of severe acute respiratory syndrome related coronavims, preferably SARS-CoV-2 and its related clinical manifestations. The invention also discloses a method of treatment of severe acute respiratory syndrome related coronavims, preferably SARS-CoV-2 and its related clinical manifestations using the composition of PEGylated interferon alpha-2b as disclosed herein. Further disclosed is the use of the composition of PEGylated interferon alpha-2b as disclosed herein below for the treatment of severe acute respiratory syndrome related coronavims, preferably SARS-CoV-2 and its related clinical manifestations.
BACKGROUND OF THE INVENTION Three highly pathogenic human coronavimses (CoVs) have been identified so far, including Middle East respiratory syndrome coronavims (MERS-CoV), severe acute respiratory syndrome (SARS) coronavims (SARS- CoV), and a 2019 novel coronavims (2019-nCoV), as previously termed by the World Health Organization (WHO). Among them, SARS-CoV was first reported in Guangdong, China in 2002. SARS-CoV caused human-to-human transmission and resulted in the 2003 outbreak with about 10% case fatality rate (CFR), while MERS-CoV was reported in Saudi Arabia in June 2012. Even though with its limited human-to human transmission, MERS-CoV showed a CFR of about 34.4%. The 2019-nCoV was first reported in Wuhan, China in December 2019 from patients with pneumonia, and it has exceeded both SARS-CoV and MERS- CoV in its rate of transmission among humans. 2019-nCoV was renamed SARS- CoV-2 by Coronaviridae Study Group (CSG) of the International Committee on Taxonomy of Viruses (ICTV), while it was renamed HCoV-19, as a common virus name, by a group of virologists in China. The disease and the virus causing it were named Coronavirus Disease 2019 (COVID-19) and the COVID-19 virus, respectively, by WHO. The COVID-19 virus outbreak represents a pandemic threat that has been declared a public health emergency of international concern (PHEIC) (1). As of September 30, 2020, a total of 169 million confirmed cases of COVID-19 were reported, including more than 3.5 million deaths globally, in China, Europe, USA, India and at least 200 other countries and / or territories (2). Currently, the intermediate host of SARS-CoV-2 is still unknown, and no effective prophylactics or therapeutics are available. It shows an urgent need for the immediate development of vaccines and antiviral drugs for prevention and treatment of COVID-19 (1). More than 100 pre-clinical or clinical trials are going on, which include repurposing of drugs already approved for different indications such as antimalarial, anti-viral, anti-parasitic drugs, cytokines or complement targeting antibodies, etc. Still, there is a growing need of antivirals against novel coronavirus SARS-CoV-2. While a number of vaccines are in development and several of them have already reached the market, therapeutics would still be needed as vaccines even if highly successful, would need a long time to be vaccinate the entire population of 7.5 billion people. The present invention provides a pharmaceutical composition of
PEGylated interferon alpha-2b for use in treatment of SARS-CoV-2 infection. As per the various publications, several prophylactic and therapeutic interventions, such as lopinavir/ritonavir, chloroquine, remdesivir, alisporivir, thymosin-a, ribavirin, type I interferons (IFN-a/b), arbidol (ARB) and combinations of these have been tested to strengthen COVID-19 prevention and control measures with no dramatically successful outcomes. Of the several options used, type I IFNs-a/b are broad spectrum antivirals, exhibiting both direct antiviral effects on viral replication and supporting an immune response to clear vims infection (3). The potential efficacy of IFN-a and IFN-b have been demonstrated for suppressing SARS-CoV-2 infection in vitro (4). In addition, PEGylated interferon alpha-2a was evaluated for anti-SARS-CoV-2 activity and inhibition of coronavirus replication in vitro (5). Despite the use of type I interferon in the treatment of both MERS and SARS-CoV-1, its role in therapeutic intervention in humans is less clear with reports of protective effects for MERS and pathogenic effects for SARS-CoV-1 (6). SARS-CoV-2 virus infection leads to a suppression of production of type I interferons in direct correlation with disease intensity (7). In general type I interferon production in response to SARS-CoV-2 is significantly lesser than with other respiratory viruses (8). One concern with the use of type I interferons has been that the ACE2 receptor which is used for infection by SARS- CoV-2 vims, is a human interferon-stimulated gene (ISG) which may get overexpressed with the use of type I interferons leading to enhancement of infection (9). Yet, IFN beta is being used in combination with remdesivir (10). Similarly, IFN alpha has been used prophylactically and therapeutically in China with limited success.
Still, there is no prior publication which suggests or discloses PEGylated interferon alpha-2b for the treatment of SARS-CoV-2 infection. The current invention provides a composition of PEGylated interferon alpha-2b (PEG-IFN alpha 2b) for use in treatment of SARS-CoV-2 infection and its related clinical manifestations. The said pharmaceutical composition is administered subcutaneously at an effective dose.
SUMMARY OF THE INVENTION The present invention provides a pharmaceutical composition of
PEGylated interferon alpha-2b that can be used in treatment of SARS-CoV-2 infection and its related clinical manifestations. It also provides safe and effective dose and dose regimen of PEG-IFN alpha 2b that can be used in treatment of SARS-CoV-2 infection and its related clinical manifestations. The invention also discloses a method of treatment of severe acute respiratory syndrome related coronavims, preferably SARS-CoV-2 and its related clinical manifestations using the composition of PEGylated interferon alpha-2b as disclosed herein. Further disclosed is the use of the composition of PEGylated interferon alpha-2b as disclosed herein below for the treatment of severe acute respiratory syndrome related coronavims, preferably SARS-CoV-2 and its related clinical manifestations. BRIEF DESCRIPTION OF FIGURE
Figure 1 depicts percentage (%) inhibiton of SARS-CoV-2 virus by PEGylated interferon alpha-2b composition of the current invention in in-vitro model.
DEFINITIONS
The term “SARS-CoV-2”, “2019-nCoV” and “HCoV-19” as used herein refers to coronavirus which had an outbreak in December 2019 and was first reported in Wuhan, China.
The term “SARS-CoV-2 infection” or “coronavirus infection” as used herein, refers to an infection caused by or otherwise associated with growth of coronavirus in a subject, in the family Coronaviridae (subfamily Coronavirinae).
The term “pharmaceutical formulation” refers to preparations, which are in such form as to permit the biological activity of the active ingredients to be unequivocally effective. The term “pharmaceutical formulation”, “pharmaceutical composition” and “composition” can be used here interchangeably.
The “lyophilized formulation” is a dosage form, which is prepared by lyophilization or freeze-drying process. The lyophilization was performed with conventional lyophilization technique known in the literatures. Lyophilization process is a process whereby water is sublimed from a composition after it is frozen- which can provide a stable form to the biological over a desired period of time. It may involves steps such as freezing, primary drying, secondary drying and optionally annealing.
The term “excipient” refers to an agent that may be added to a formulation to stabilize the active drug substance in the formulated form to adjust and maintain ionic strength, osmolality and pH of the pharmaceutical preparations. Examples of commonly used excipients include, but are not limited to, buffers, sugars, polyols, amino acids, surfactants and polymers.
“Pharmaceutically acceptable” excipients are those which can reasonably be administered to a subject to provide an effective dose of the active ingredient employed.
The term “buffer” or “buffer solution” or “buffer system” refers to generally aqueous solution comprising a mixture of an acid (usually a weak acid) and conjugate base. A buffered solution prevents change of pH of the solution due to the “buffering capacity” imparted by the “buffering agent(s)”.
The term “buffering agent” refers to an acid or base component (usually a weak acid or weak base) of a buffer or buffer solution. A buffering agent maintain the pH of a given solution at or near to a pre-determined value, and the buffering agents are generally chosen to complement the pre-determined value. A buffering agent is suitably a single compound which gives rise to a desired buffering effect, especially when said buffering agent is mixed with (and suitably capable of proton exchange with) an appropriate amount (depending on the pre-determined pH desired) of “acid/base conjugate”, or if the required amount of “acid/base conjugate” is formed in situ - this may be achieved by adding strong acid or base until the required pH is reached. The term “buffering agent” and “buffers” can be used here interchangeably.
The term “acid / base conjugate” refers to the conjugate acid or conjugate base (whichever is relevant at a particular pH) of a particular “buffering agent”. The term “salt” according to the present invention is a component which is not a part of a buffer system. Salt according to the present invention includes organic salt and inorganic salt. Preferred salts are inorganic salts which is not a part of buffer system. The term “inorganic salt” refers to any salt that does not contain C- H bonds as opposed to organic salt that contains C-H bonds. Inorganic salts such as sodium chloride, potassium chloride etc. are widely used as stabilizers or tonicity agents in addition to their use in a buffer system in the pharmaceutical formulations. Examples of inorganic salts include, but are not limited to, sodium chloride (NaCl), potassium chloride (KC1), calcium chloride (CaCE), barium chloride (BaCE), ferric chloride (FcCE,) etc.
The term “stabilizers” as used herein generally includes agents, which provide stability to the protein from freezing -induced stresses. Examples of stabilizers include salt(s) such as sodium chloride or potassium chloride polyol(s) such as mannitol or sorbitol, saccharide(s) such as sucrose, surfactant(s) such as polysorbate, poloxamer or polyethylene glycol, amino acid(s) such as arginine and the like.
A “stable” formulation is one in which the protein therein essentially retains its physical stability and/or chemical stability and/or biological activity upon storage. Preferably, the formulation essentially retains its physical and chemical stability, as well as its biological activity upon storage. The storage period is generally selected based on the intended shelf-life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature for a selected time period. The formulation is stable between +2 °C and +8 °C for at least one month. Furthermore, the formulation is preferably stable following freezing (to, e.g., - 70 °C) and thawing of the formulation, for example following 1, 2, 3 or 10 cycles of freezing and thawing. Stability can be evaluated qualitatively and/or quantitatively in a variety of different ways, including evaluation of aggregate formation (for example using size exclusion chromatography, by measuring turbidity, and/or by visual inspection); by assessing charge heterogeneity using cation exchange chromatography or capillary zone electrophoresis; amino-terminal or carboxy- terminal sequence analysis; mass spectrometric analysis; SDS-PAGE analysis to compare reduced and intact antibody; peptide map (for example tryptic or LYS-C) analysis; evaluating biological activity or antigen binding function of the antibody; etc. Instability may involve any one or more of: aggregation, deamidation (e.g. Asn deamidation), oxidation (e.g. Met oxidation), isomerization (e.g. Asp isomerization), clipping/hydrolysis/fragmentation (e.g. hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, glycosylation differences, etc.
The term “prevention” as used herein refers to the reduction in risk of coronavirus infection in patients. Such prevention may not be absolute, i.e. it may not prevent all such patients developing a coronavirus infection, or may only partially prevent an infection. The terms “prevention” and “prophylaxis” may be used interchangeably.
The term “treatment” or “therapeutics” as used herein, refers to any treatment of a disease in a mammal, particularly in a human. It includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. In the context of the present invention, a “therapeutically effective amount” or “effective amount” of IFN refers to an amount effective for use in the treatment of SARS-CoV-2 infection and its related clinical manifestations.
The term “about” refers to variation in the numerical quantity that can occur, for example, through typical measuring and handling procedures used for making compounds, compositions, concentrates or formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods, and other similar considerations. The term “about” also encompasses amounts that differ due to aging of a formulation with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a formulation with a particular initial concentration or mixture.
The terms “patient” and “subject” are used interchangeably and are used in their conventional sense to refer to a living organism suffering from or prone to a condition that can be prevented or treated by administration of a composition of the present invention, and includes both humans and non-human animals. Examples of subjects include, but are not limited to, humans, chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age. Thus, adult, juvenile and new bom individuals are of interest.
The term “cryoprotectant(s)” or “lyoprotectant(s)” as used herein includes agent(s) that provide stability to the protein during water removal from the system during the drying process, presumably by maintaining the proper conformation of the protein through hydrogen bonding. Cryoprotectant(s) can also have lyoprotectant effects and therefore, the term “cryoprotectant” or “lyoprotectant” can be used interchangeably herein. It includes agent(s) which provides stability to the protein from freezing-induced stresses. For example, sugar(s) or sugar alcohol, such as sucrose, lactose, trehalose, mannitol and the like. The cryoprotectant may be used alone or in suitable combinations thereof. One such preferred cryoprotectant is lactose according to the present invention. ABBREVIATIONS USED IN THE CURRENT APPLICATION %: percentage
2019-nCoV: novel coronavims
ACE2: Angiotensin-converting enzyme 2
AE: Adverse event
CFR: Case fatality rate
CMC: Carboxymethyl cellulose
CoV: Coronavims
COVID-19: Coronavims Disease 2019
CSG: Coronaviridae Study Group
DNA: Deoxyribonucleic acid
DSC: Differential scanning calorimetry
ECG: Electrocardiography
ECMO: Extracorporeal membrane oxygenation
EDTA: Ethylenediaminetetraacetic acid
EGTA: Ethylene g 1 y c o 1 - b i s ( b - a m i n o cl h y 1 ether)-N,N,N’,N’-tetraacetic acid fM: Femtomolar
HCoV-19: Human Coronavims
HP-IEC: High-performance ion-exchange chromatography Hrs: Hours
I.C50: Half-maximal inhibitory concentration
IFN: Interferon
IFN-a: Interferon alpha
IFN-b: Interferon beta
IFN-co: Interferon omega
INS: IFN-alphanl
ISG: Interferon-stimulated gene kDa: kilodalton kg: kilogram meg: microgram
MERS-CoV: Middle East respiratory syndrome coronavims mg: milligram mITT: modified intent-to -treat mlTT: Modified intent-to-treat mL: milliliter mM: millimolar ng: nano gram PEG: Polyethylene glycol
PEG-IFN alpha 2b: PEGylated interferon alpha 2b PEG IFN-a2b: PEGylated interferon alpha 2b PEG-IFN: PEGylated interferon PEI: Polyethyleneimine PP: Per protocol PT : Prefered term q.s.: Quantity sufficient r-DNA: Recombinant DNA RR: Respiratory rate (RR) RT-PCR: Reverse transcription polymerase chain reaction SARS: Severe acute respiratory syndrome
SARS-CoV: Severe acute respiratory syndrome (SARS) coronavirus SEC: Size exclusion chromatography SC: Subcutaneous SDS: Sodium dodecyl sulphate SOC: Standard of care SOP: Standard operating procedure SpO: Saturation of peripheral oxygen TEAE: Treatment emergent adverse event TNF: Tumor necrosis factor WFI: Water for injection WHO: World Health Organization b-HPCD: Hydroxypropyl-beta-cyclodextrin pg: microgram EMBODIMENTS OF THE INVENTION
In one embodiment, the current invention provides a composition of PEGylated interferon alpha-2b (PEG-IFN alpha 2b) for use in treatment of SARS- CoV-2 infection and its related clinical manifestations. In a specific embodiment of this invention, it provides a pharmaceutical composition of PEGylated interferon alpha-2b comprising therapeutically effective amount of PEGylated interferon alpha-2b. The composition of PEGylated interferon alpha- 2b according to the current invention can be used for treatment of SARS-CoV-2 infection. In one of the embodiments, the therapeutically effective amount of
PEGylated interferon alpha-2b according to the current invention is in the concentration range of 0.01 mg / mL to 5.0 mg / mL, preferably 0.03 mg / mL to 2.5 mg / mL, more preferably 0.1 mg / mL to 0.5 mg / mL.
In certain embodiment, the pharmaceutical composition comprises therapeutically effective amount of PEGylated interferon alpha-2b, either alone or in combination with other suitable excipient(s). The suitable excipient(s) are selected from buffer(s), salt(s), carbohydrate(s), surfactant(s), amino acid(s), anti- oxidant(s), chelating agent(s), tonicity modifier(s), acid(s), base(s), glycerine, water for injection (WFI) and suitable combination thereof and the like. In one of the embodiments, the pharmaceutical composition comprising
PEGylated interferon alpha-2b, buffer(s) and other suitable excipient(s), where in buffer(s) is selected from phosphate buffer (e.g., sodium phosphate buffer, etc.), arginine-phosphate buffer, histidine buffer, arginine-histidine buffer, histidine chloride buffer, glycine buffer, sodium glycinate buffer, arginine buffer, citrate buffer (e.g., sodium citrate buffer, etc.), arginine-citrate buffer, succinate buffer (e.g. succinic acid buffer, sodium succinate buffer, etc.), potassium succinate buffer, arginine- succinate buffer, acetate buffer, arginine-acetate buffer, arginine- glutamate buffer, arginine-aspartate buffer, tromethamine buffer and suitable combination thereof. The suitable excipient(s) is selected from salt(s), carbohydrate(s), surfactant(s), amino acid(s), anti-oxidant(s), chelating agent(s), tonicity modifier(s), acid(s), base(s), glycerine, water for injection (WFI) and suitable combination thereof and the like. In one of the embodiments, the pharmaceutical composition comprising PEGylated interferon alpha-2b, buffer(s), carbohydrate(s), surfactant(s) and other suitable excipient(s). The suitable excipient(s) is selected from salt(s), amino acid(s), anti-oxidant(s), chelating agent(s), tonicity modifier(s), acid(s), base(s), glycerine, water for injection (WFI) and suitable combination thereof and the like. Carbohydrate(s) may act as a cryoprotectant(s) or as a lyoprotectant(s) according to one of the aspects of the present invention.
In another embodiment, the pharmaceutical composition of PEGylated interferon alpha-2b according to the present invention comprising stabilizer(s). The stabilizer(s) of present invention is selected from surfactant(s), sugar(s), amino acid(s), polyol(s) and the like or suitable derivative or mixtures thereof.
In another embodiment, the pharmaceutical composition of PEGylated interferon alpha- 2b according to the present invention comprising buffer(s). The buffer(s) of present invention is selected from phosphate buffer (e.g., sodium phosphate buffer, etc.), arginine -phosphate buffer, histidine buffer, arginine-histidine buffer, histidine chloride buffer, glycine buffer, sodium glycinate buffer, arginine buffer, citrate buffer (e.g., sodium citrate buffer, etc.), arginine-citrate buffer, succinate buffer (e.g. succinic acid buffer, sodium succinate buffer, etc.), potassium succinate buffer, arginine- succinate buffer, acetate buffer, arginine-acetate buffer, arginine-glutamate buffer, arginine-aspartate buffer, tromethamine buffer and suitable combination thereof and the like.
In another embodiment, the pharmaceutical composition of PEGylated interferon alpha-2b according to the present invention comprising carbohydrate(s). The carbohydrate(s) of present invention is sugar(s) and / or polyol(s) selected from dextran, trehalose, raffinose, sucrose, polyvinylpyrrolidone, lactose, inositol, sorbitol, mannitol, dimethyl sulfoxide, glycerol, albumin, calcium lactobionate, fructose, maltose, galactose, glucose, D- mannose, sorbose, cellobiose, melezitose, maltodextrins, starches, lactitol, xylitol, pyranosyl sorbitol, myoinositol, hydroxypropyl-beta-cyclodextrin (b-HPCD) and suitable combination thereof and the like. Carbohydrate(s) may act as a cryoprotectant(s) or as a lyoprotectant(s) according to the present invention. Cryoprotectant(s) or lyoprotectant(s) include agent(s) that provides stability to the protein during water removal from the system during the drying process, presumably by maintaining the proper conformation of the protein through hydrogen bonding.
In another embodiment, the pharmaceutical composition of PEGylated interferon alpha-2b according to the present invention comprising surfactant(s). The surfactant(s) of present invention is selected from polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (e.g. Brij), alkylphenylpolyoxyethy lene ethers (e.g. Triton-X), polyoxyethylene - polyoxypropylene copolymer (e.g. Poloxamer, Pluronic), polyethylene glycol (PEG), polyethyleneimine, sodium dodecyl sulphate (SDS) and suitable combination thereof and the like.
In one of the embodiments, the pharmaceutical composition of PEGylated interferon alpha-2b according to the present invention comprising acid(s). The acid(s) according to the present invention is selected from hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, succinic acid, sulfuric acid, fumaric acid, and suitable combinations thereof. In one of the embodiments, the pharmaceutical composition of PEGylated interferon alpha-2b according to the present invention comprising base(s). The base(s) according to the present invention is selected from sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium succinate, sodium format, sodium sulfate, potassium sulfate, potassium fumarate, and suitable combinations thereof.
In one more embodiment, the pharmaceutical composition of PEGylated interferon alpha- 2b according to the present invention comprising amino acid(s). The amino acid(s) of present invention is selected from arginine, glycine, asparagine, glutamine, lysine, threonine, histidine, glutamic acid, aspartic acid, isoleucine, valine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, cysteine, their suitable salt form and combination thereof and the like. In one more embodiment, the pharmaceutical composition of PEGylated interferon alpha-2b according to the present invention comprising chelating agent(s) selected from ethylenediaminetetraacetic acid (EDTA), pentetic acid and ethylene g 1 yco 1 -b i s(P - a m i noct h y 1 ether)-N,N,N’,N’-tetraacetic acid (EGTA). In one more embodiment, the pharmaceutical composition of PEGylated interferon alpha- 2b according to the present invention comprising salt(s) selected from sodium chloride, potassium chloride, potassium sulfate and sodium sulfate.
In one of the embodiments, the pharmaceutical composition of PEGylated interferon alpha- 2b is free from salt(s). In a preferred embodiment, the pharmaceutical composition of PEGylated interferon alpha-2b is free from inorganic salt(s).
In another embodiment, the pharmaceutical composition according to the present invention has pH in the range of about 4.0 to about 7.1, preferably about 6.5 to about 7.1, more preferably about 6.8.
In one of the preferred embodiments, the pharmaceutical composition comprising PEGylated interferon alpha-2b, buffer(s), carbohydrate(s), and surfactant(s).
In a certain embodiment, the pharmaceutical composition comprising PEGylated interferon alpha- 2b, buffer(s), carbohydrate(s) as a cryoprotectant(s) or as a lyoprotectant(s), and surfactant(s). The specific pH of the composition can be achieved by using acid or base.
In one of the embodiments, the pharmaceutical composition comprising PEGylated interferon alpha- 2b, succinate buffer, lactose, polysorbate 80 and other suitable excipient(s).
In one of the preferred embodiments, the pharmaceutical composition comprising PEGylated interferon alpha-2b, succinate buffer, lactose and polysorbate 80.
In one of the specific embodiments, the pharmaceutical composition of PEGylated interferon alpha-2b comprising therapeutically effective amount of PEGylated interferon alpha-2b, succinate buffer, lactose and polysorbate 80. The composition of PEGylated interferon alpha-2b according to the current invention can be used for treatment of SARS-CoV-2 infection. The preferred therapeutically effective amount of PEGylated interferon alpha-2b is in the range of 0.1 mg / mL to 0.5 mg / mL.
In one of the specific embodiments, the current invention provides a pharmaceutical composition of PEGylated interferon alpha-2b (PEG-IFN alpha 2b) along with succinate buffer, lactose and polysorbate 80 for use in treatment of SARS-CoV-2 infection and its related clinical manifestations.
In one of the embodiments, the current invention provides a pharmaceutical composition of PEGylated interferon alpha-2b comprising about 0.1 mg / mL to about 0.5 mg / mL of PEG-IFN alpha 2b, about 1 mM to about
500 mM of buffer, about 10 mg / mL to about 250 mg / mL of carbohydrate, about 0.001 mg / mL to about 1 mg / mL of surfactant and optionally with other suitable excipient(s).
In one of the preferred specific embodiments, the current invention provides a pharmaceutical composition of PEGylated interferon alpha-2b comprising therapeutically effective amount of PEG-IFN alpha 2b, 10 mM of succinate buffer, 105 mg / mL of lactose monohydrate and 0.1 mg / mL polysorbate 80 and optionally with other suitable excipient(s), wherein amount of PEGylated interferon alpha-2b in the pharmaceutical composition is in range of 0.1 mg / mL to 0.5 mg / mL, preferably 0.16 mg / mL or 0.20 mg / mL. In one of the preferred specific embodiments, the current invention provides a pharmaceutical composition of PEGylated interferon alpha-2b comprising therapeutically effective amount of PEG-IFN alpha 2b, 10 mM of succinate buffer, 105 mg / mL of lactose monohydrate and 0.1 mg / mL polysorbate 80 and optionally with other suitable excipient(s), wherein amount of PEGylated interferon alpha- 2b in the pharmaceutical composition is in range of 0.1 mg / mL to 0.5 mg / mL, preferably 0.16 mg / mL or 0.20 mg / mL for use in treatment of SARS-CoV-2 infection and its related clinical manifestations.
In a certain specific embodiment, the current invention provides a pharmaceutical composition comprising PEG-IFN alpha 2b in the concentration range of 0.1 mg / mL to 0.5 mg / mL; preferably 0.16 mg / mL or 0.20 mg / mL, 1.18 mg / mL of succinic acid, 105 mg / mL lactose and 0.1 mg / mL polysorbate 80 optionally with sodium hydroxide.
In a certain specific embodiment, the current invention provides a pharmaceutical composition comprising 0.10 mg / mL of PEG-IFN alpha 2b, 1.18 mg / mL succinic acid, 105 mg / mL lactose and 0.1 mg / mL polysorbate 80 optionally with sodium hydroxide. In a certain specific embodiment, the current invention provides a pharmaceutical composition comprising 0.16 mg / mL of PEG-IFN alpha 2b, 1.18 mg / mL succinic acid, 105 mg / mL lactose and 0.1 mg / mL polysorbate 80 optionally with sodium hydroxide. In a certain specific embodiment, the current invention provides a pharmaceutical composition comprising 0.20 mg / mL of PEG-ILN alpha 2b, 1.18 mg / mL succinic acid, 105 mg / mL lactose and 0.1 mg / mL polysorbate 80 optionally with sodium hydroxide.
In a certain specific embodiment, the current invention provides a pharmaceutical composition comprising 0.24 mg / mL of PEG-IFN alpha 2b, 1.18 mg / mL succinic acid, 105 mg / mL lactose and 0.1 mg / mL polysorbate 80 optionally with sodium hydroxide.
In a certain specific embodiment, the current invention provides a pharmaceutical composition comprising 0.30 mg / mL of PEG-IFN alpha 2b, 1.18 mg / mL succinic acid, 105 mg / mL lactose and 0.1 mg / mL polysorbate 80 optionally with sodium hydroxide.
The succinic acid described in above mentioned specific embodiments are equivalent to 10 mm of succinate buffer. The lactose described in above mentioned specific embodiments and used in the current invention to prepare composition of PEGylated interferon alpha 2b is lactose monohydrate.
In one embodiment, the current invention provides a pharmaceutical composition including PEGylated interferon alpha- 2b in a lyophilized form or liquid form. In a preferred embodiment, the current invention provides a pharmaceutical composition including PEGylated interferon alpha-2b in a lyophilized form. The lyophilization process is a process whereby water is sublimed from a composition after it is frozen- which can provide a stable form to the biological over a desired period of time. The said composition can be stored as a dry powder and reconstituted before use. In another embodiment, the current invention provides a pharmaceutical composition including PEGylated interferon alpha- 2b in a liquid form.
In one of the embodiments, the current invention provides lyophilized and stabilized composition of PEGylated interferon alpha-2b. The composition of PEGylated interferon alpha-2b comprising therapeutically effective amount of PEGylated interferon alpha- 2b, succinate buffer, lactose and polysorbate 80.
In one of the embodiments, the current invention provides process of preparing a composition of PEGylated interferon alpha-2b for use in treatment of SARS-CoV-2 infection and its related clinical manifestations.
In one more embodiment, the present invention provides a method of administration to the patient of a composition of IFN, preferably PEGylated interferon alpha-2b (PEG-IFN alpha 2b) for use in treatment of SARS-CoV-2 infection and its related clinical manifestations.
In one more embodiment, the present invention provides a method of treating SARS-CoV-2 infection by administering PEGylated interferon alpha-2b. In a preferred embodiment, the present invention provides a method of treating SARS-CoV-2 infection by administering composition of PEGylated interferon alpha-2b comprising PEGylated interferon alpha-2b in suitable therapeutic amount optionally with other suitable excipient(s). Examples of suitable excipient(s) include, but are not limited to, buffer(s), salt(s), carbohydrate(s), surfactant(s), amino acid(s), anti-oxidant(s), chelating agent(s), tonicity modifier(s), cryoprotectant(s) or lyoprotectant(s), acid(s), base(s), glycerine, water for injection (WFI) and suitable combination thereof and the like.
In another embodiment, the current invention provides use of PEGylated interferon alpha- 2b for the treatment of SARS-CoV-2 infection and its related clinical manifestations. In another preferred embodiment, the current invention provides use of composition of PEGylated interferon alpha-2b comprising PEGylated interferon alpha-2b in suitable therapeutic amount optionally with other suitable excipient(s) for the treatment of SARS-CoV-2 infection and its related clinical manifestations. Examples of suitable excipient(s) include, but are not limited to, buffer(s), salt(s), carbohydrate(s), surfactant(s), amino acid(s), anti oxidants), chelating agent(s), tonicity modifier(s), cryoprotectant(s) or lyoprotectant(s), acid(s), base(s), glycerine, water for injection (WFI) and suitable combination thereof and the like.
In one of the embodiments, the PEGylated interferon alpha-2b of the present invention has at least one of the following characteristic(s): a. shows not more than about 1 °C difference in thermal transition value as compared to the unmodified interferon alpha-2b; b. exhibits certain degree of depegylation, preferably 30 % or more upon hydroxylamine treatment; c. shows about 8 - 9 positional isomers of interferon alpha; d. exhibits low level of (< 5 %) oxidation on one of the methionine residues of interferon at the position 148; e. exhibits low level of (< 5 %) deamidation on one of the asparagine residues of interferon at the position 93; f. conjugated with a polyethylene glycol molecule having an average molecular weight of approximately 12,000 daltons; g. conjugated with a polyethylene glycol molecule that is essentially free of diols; h. conjugated with a polyethylene glycol that shows a polydispersity index of about 1.00; i. synthesized by performing a reaction between interferon alpha and polyethylene glycol wherein the terminal activity of polyethylene glycol is no less than 90 %; j. synthesized by performing a reaction between interferon alpha and polyethylene glycol at 1:1 or 1:2 or 1:3 or 1:4 or 1:5 or 1:6 or 1:10 ratio wherein the polyethylene glycol is used in stoichiometric or molar excess amount over the interferon; and k. contains no more than 10 % of high molecular weight species variants and less than 5 % of unmodified interferon. The degree of depegyltion when termed as ‘30 % or more’ herein is 30 % or maximally 60 % of depegylation. The term ‘no more than 10 % high molecular weight species’ as used herein may include 0 - 10 % of high molecular weight species. The term ‘less than 5 % unmodified interferon’ as used herein may include 0-5 % of unmodified interferon (free IFN). In one of the preferred embodiments, the PEGylated interferon alpha- 2b of the present invention has all the characteristics as listed above (points a. to points k.)
In another embodiment, the pharmaceutical composition of present invention includes PEGylated interferon alpha-2b, which can be used for parenteral administration. Parenteral administration includes intravenous, subcutaneous, intra peritoneal, intramuscular administration or any other route of delivery generally considered to be falling under the scope of parenteral administration and as is well known to a skilled person. Preferably, the pharmaceutical composition of present invention includes PEGylated interferon alpha-2b used for subcutaneous administration.
In one of the embodiments, the pharmaceutical composition of present invention includes PEGylated interferon alpha-2b, which can be delivered into lungs by atomization. In another embodiment, the pharmaceutical composition of present invention includes PEGylated interferon alpha-2b, which can be delivered intranasally in the form of nasal drops.
In a further embodiment, the current invention provides safe and effective dose and dose interval of PEGylated interferon alpha-2b for use in treatment of SARS-CoV-2 infection and its related clinical manifestations. In a more preferred embodiment, the dose range of the composition of the invention, including PEGylated interferon alpha- 2b, is from about 0.1 mcg/kg to about 30 mcg/kg on a weekly, twice in a week, thrice in a week, daily basis, twice in a day or every other week for use in treatment of SARS-CoV-2 infection. However, this dose range may include for example, about 0.1 mcg/kg/week, about 0.5 mcg/kg/week, about 1 mcg/kg/week, about 1.5 mcg/kg/week, about 2 mcg/kg/week, about 5 mcg/kg/week, about 10 mcg/kg/week, about 20 mcg/kg/week, and/or about 30 mcg/kg/week. In one of the preferred embodiments, the dose of PEGylated interferon alpha-2b is 1 mcg/kg/week.
In one of the further embodiment, the current invention provides treatment of SARS-CoV-2 infection and its related clinical manifestations comprising administration of single dose of PEG-IFN alpha 2b or its composition to the subject. The said single dose of PEG-IFN alpha 2b according to the current invention is selected from 0.1 mcg/kg, about 0.5 mcg/kg, about 1 mcg/kg, about 1.5 mcg/kg, about 2 mcg/kg, about 5 mcg/kg. In one of the preferred embodiments, the current invention provides composition of PEGylated interferon alpha-2b comprising buffer(s), carbohydrate(s) and surfactant(s) to deliver 1 mcg/kg of PEG-IFN alpha 2b to neutralize SARS-CoV-2 virus. In one of the embodiments, the current invention provides composition of PEGylated interferon alpha- 2b that can neutralize 100% SARS-CoV-2 vims.
In one of the embodiments, the current invention provides a composition of IFN, preferably PEGylated interferon alpha- 2b which can be administered to the patient in combination with standard of care. Standard of care (SOC) is either hydroxychlorquine or any suitable antiviral agents, e.g., remdesivir, and / or ritonavir, and / or liponavir and / or anti- viral monoclonal antibodies, and / or any anti-inflammatory therapy such as anti-IL-6 or anti-TNF alpha or such, and / or any other therapy being given to the COVID-19 patient as a part of SOC, etc. According to the present invention, SOC is as per local regulatory guideline/ institute SOP.
In a separate embodiment, the current invention provides a composition of IFN, preferably PEGylated interferon alpha- 2b which can be administered to the patient in combination with TNF inhibitor. TNF inhibitor as mentioned herein is adalimumab, infliximab, or etanercept. In a preferred embodiment, the current invention provides a composition of PEGylated interferon alpha- 2b which can be administered in combination with adalimumab. In one of the embodiments, said adalimumab is co-administered at a dose in the range of about 20 mg to about 160 mg on a weekly, twice in a week, thrice in a week, daily basis or every other week with the said composition of PEG-IFN alpha 2b.
In one of the embodiments, the pharmaceutical composition of present invention includes PEGylated interferon alpha-2b, which can be administered to the patient in combination with anti-IL-6 antibody. One of the anti-IL-6 antibody as mentioned herein is tocilizumab.
DETAILED DESCRIPTION OF THE INVENTION
Current invention describes a composition of PEGylated interferon alpha- 26 (PEG-IFN alpha 2b) for use in treatment of SARS-CoV-2 infection and its related clinical manifestations. A composition according to the present invention comprises therapeutically effective dose of PEGylated interferon alpha-2b (PEGIFN alpha-2b) for use in treatment of SARS-CoV-2 infection and its related clinical manifestations. The present invention provides a method of treating SARS-CoV-2 infection by administering PEGylated interferon alpha-2b. Preferably, the present invention provides a method of treating SARS-CoV-2 infection by administering composition of PEGylated interferon alpha-2b comprising PEGylated interferon alpha-2b in suitable therapeutic amount optionally with other suitable excipient(s). Examples of suitable excipient(s) include, but are not limited to, salt(s), carbohydrate(s), surfactant(s), amino acid(s), anti-oxidant(s), chelating agent(s), tonicity modifier(s), cryoprotectant(s) or lyoprotectant(s), acid(s), base(s), glycerine, water for injection (WFI) and suitable combination thereof and the like. Alternatively, the current invention provides use of PEGylated interferon alpha-2b for the treatment of SARS-CoV-2 infection and its related clinical manifestations. Preferably, the current invention provides use of composition of PEGylated interferon alpha-2b comprising PEGylated interferon alpha-2b in suitable therapeutic amount optionally with other suitable excipient(s) for the treatment of SARS-CoV-2 infection and its related clinical manifestations. Examples of suitable excipient(s) include, but are not limited to, salt(s), carbohydrate(s), surfactant(s), amino acid(s), anti oxidants), chelating agent(s), tonicity modifier(s), cryoprotectant(s) or lyoprotectant(s), acid(s), base(s), glycerine, water for injection (WFI) and suitable combination thereof and the like. The detail of SARS-CoV-2 infection is given in a review article of ‘ International Journal of Antimicrobial Agents, 2020. 105948 This article describes transmission, clinical manifestations and diagnosis of SARS-CoV-2 infection.
A variety of zoonotic viruses are evolving in the wild. Particularly, coronavimses (CoVs) with zoonotic potential are so diverse. The third zoonotic human coronavims, SARS-CoV-2, is transmissible from person to person. SARS- CoV-2 vims could induce symptoms including fever, dry cough, dyspnea, fatigue, and lymphopenia in infected patients. In general, aged people are more susceptible to COVID-19 and more likely to be severe than people younger than 50 years; this may be due to more health issues and comorbidities in this population (11).
The term “interferon alpha” as used herein means the family of highly homologous species-specific proteins that inhibit viral replication and cellular proliferation and modulate immune response. Typical suitable interferon-alphas include, but are not limited to, recombinant IFN alpha 2b such as Intron-A interferon available from Schering Corporation, recombinant IFN alpha 2a such as Roferon interferon available from Hoffmann-La Roche, recombinant IFN alpha 2c such as Berofor alpha 2 interferon available from Boehringer Ingelheim Pharmaceutical, Inc., interferon alpha-nl, a purified blend of natural alpha interferons such as Sumiferon available from Sumitomo, Japan or as Wellferon IFN-alphanl (INS) available from the Glaxo -Wellcome Ltd., London, Great Britain, or a consensus alpha interferon such as those described in U.S. Patent Nos. 4,897,471 and 4,695,623 (especially Examples 7, 8 or 9 thereof) and the specific product available from Amgen, Inc., Newbury Park, CA, or interferon alpha-n3 a mixture of natural alpha interferons made by Interferon Sciences and available from the Purdue Frederick Co., Norwalk, CT., under the Alferon Tradename. Type I interferon (IFN-a/b) induces triggered antiviral states and ultimately a number of interferon-stimulated genes (ISGs) which encode for a variety of antiviral effectors (12). Interferons inhibit viral infection by inducing innate and adaptive immune responses like altering the intracellular environment to restrict viral replication and inducing signaling events that activate immune cell populations and elicit an antiviral immune response. Interferon alfas have been used in the treatment of hepatitis B and C; and to induce inhibition of respiratory coronavims infections, albeit unrelated to the Urbani strain of SARS-CoV (13). The use of IFN alpha, either IFN alpha 2a or IFN alpha 2b, is preferred according to the present invention. The manufacture of interferon alpha 2b is described in U.S. Patent No. 4,530,901. The interferon alpha administered is selected from interferon alpha-2a, interferon alpha-2b, a consensus interferon, a purified IFN alpha product or a PEG-IFN alpha 2a or PEG-IFN alpha 2b.
“PEG-IFN” are interferon molecules covalently attached to a PEG molecule. The PEG-IFN is PEG-IFN alpha, preferably interferon alpha-2a
(Pegasys, Roche Pharmaceuticals), interferon alpha-2b (PEG-Intron, Schering- Plough; Sylantron, Merck; ViraferonPeg, Merck), interferon alpha-2c (Berofor Alpha, Boehringer Ingelheim, Ingelheim, Germany) or consensus interferon as defined by determination of a consensus sequence of naturally occurring interferon alphas (Infergen, Amgen, Thousand Oaks, Calif.). Pegylation of interferons has been reported in U.S. Pat. Nos. 4,766,106 and 4,917,888 which describe inter alia beta interferon conjugated with activated polymers including mPEG-2, 4, 6-trichloro-S-triazme, mPEG-N-succinimidyl glutarate or mPEG-N- succinimidyl succinate. One such disclosure in U.S. Pat. No. 5,951,974 describes the conjugation of interferon to a substantially non-antigenic polymer at a histidine site. Another such disclosure in U.S. Pat. No. 5,981,709 describes the alpha interferon- polymer conjugate with relatively long circulating half-life in- vivo.
Often, the approximate molecular weight of the polymer is designated with a number following the name of the repeated chemical unit. For example, “PEGnooo” or “Polyethylene glycol (12000)” refers to a polymer of polyethylene glycol having an average molecular weight of approximately 12,000 daltons. Conjugation of polymers to proteins may result in a single polymer molecule conjugated to a protein or multiple such conjugations to a single protein. The degree of conjugation is dependent upon the reaction conditions and desired result. Preferably, the IFN alpha 2b in the compositions of the present invention comprises a single IFN alpha 2b conjugated to a single PEG12000 molecule. Particularly, the IFN alpha 2b molecule is linked to the PEG 12000 molecule with a urethane bond. Molecular weight of the conjugated PEGylated interferon alpha- 2b molecule is approximately 31 kDa. Several processes for preparing a PEG-IFN conjugates are known in the art and such processes and the products derived from such processes are considered to be encompassed within the scope of the present invention. Examples of such process (es) for producing this protein-polymer conjugate may be found in U.S. Pat. No. 5,612,460 (Zalipsky) and U.S. Pat. No. 5,711,944 (Gilbert et al). Without limiting the scope of the present invention, when such a protein-polymer conjugate is utilized in the formulation solutions of the present invention, the preferred concentration of PEG-IFN alpha 2b is 0.01 to 5.0 mg IFN alpha 2b per mL.
When a single interferon alpha-2b molecule is linked to a single PEG noon molecule, the resulting conjugated PEG-IFN alpha 2b may be in the form of a single or mixture of positional isomers. One such mixture of positional isomers could mean, the PEG-IFN alpha 2b has PEG linked at a histidine residue of the IFN alpha 2b molecule, while another PEG-IFN alpha 2b where PEG is linked to another site of the IFN alpha 2b molecule, for example the lysine residue or cysteine residue. In one of the aspects of the present invention, the PEGylated interferon alpha 2b has about 8 -9 positional isomers of interferon alpha. The different isomers of PEGylated interferon alpha 2b of the current invention was identified by high-performance ion-exchange chromatography (HP-IEC) technique. In another aspects of the current invention, the PEGylated interferon alpha 2b of the current invention has about 1 °C difference in thermal transition value as compared to the unmodified interferon alpha 2b. The term ‘unmodified interferon alpha 2b’ as used herein means the interferon alpha 2b without PEGylation. The said thermal transition value (Tm) was assessed by differential scanning calorimetry (DSC) analysis. The PEGylated interferon alpha 2b produced according to the present invention has interferon alpha 2b to polyethylene glycol present in a ratio of 1:2. PEGylated interferon alpha 2b produced according to the current invention exhibits certain degree of depegylation upon hydroxylamine treatment as assessed by size exclusion chromatography (SEC-HPLC) method. Preferably, certain degree of depegylation according to the current invention is 30 % or more maximum up to 60 % of depegylation. In one of the aspects, the PEGylated interferon alpha-2b prepared according to the current invention contains no more than 10 % of high molecular weight species variants and less than 5 % of unmodified interferon. Both the species, high molecular weight species and unmodified interferon were analysed by HP-SEC analysis. The PEGylated interferon alpha- 2b prepared according to the current invention exhibits low level of (< 5 %) oxidation on one of the methionine residues of interferon at the position 148. The PEGylated interferon alpha- 2b prepared according to the current invention has less than 5 % of oxidized variant of interferon at Methionine residue at position 148. The PEGylated interferon alpha- 2b prepared according to the current invention exhibits low level of (< 5 %) deamidation on one of the asparagine residues of interferon at the position 93. The PEGylated interferon alpha-2b prepared according to the current invention has less than 5 % of deamidated variant of interferon at asparagine residue at position 93. Said oxidized variant as well as deamidated variants were analysed by mass -spectrometry. To preserve the PEG-IFN alpha 2b in the most stable and active form, lyophilisation can be used. Lyophilization is a process of freeze-drying a composition wherein a frozen aqueous mixture is treated to remove water. Commonly, the process involves the sublimation of water from the aqueous solutions, usually under reduced pressure conditions. After lyophilization, the PEG-IFN alpha conjugate(s) can be stored for extended periods of time.
Current invention provides a pharmaceutical composition comprising therapeutically effective amount of PEG-IFN alpha 2b to treat SARS-CoV-2 infection and its related clinical manifestations. The pharmaceutical composition of present invention is stabilized composition of PEG-IFN alpha 2b, which includes one or more suitable pharmaceutically acceptable excipient(s). Examples of commonly used excipient(s) include, but are not limited to, buffer(s), sugar(s), polyol(s), amino acid(s), surfactant(s), preservative(s), salt(s), anti-oxidant(s), tonicity modifier(s), acid(s), base(s), chelating agent(s), and polymer(s).
As per one of the embodiments describing the present disclosure, Interferon alpha-2b in the said composition is monopegylated interferon alpha-2b. Herein, monopegylated interferon alpha-2b according to the present invention are interferon alpha-2b molecules covalently linked to one or more “PEG molecule/s” or “monomethoxy PEG molecule/s”. Interferon alpha-2b is recombinant according to the present invention. Recombinant IFN alpha 2b was produced by r- DNA technology in Pichia pastoris (KM71) as a secretory protein and it is a non- glycosylated protein with a molecular weight of 19.268 kDa.
The compositions of PEG-IFN alpha 2b are prepared using suitable combinations of a buffer, stabilizer, cryoprotectant(s) and/or lyoprotectant(s) and a solvent, optionally with other excipients and suitably lyophilized and stored as a dry powder to be reconstituted before use. Preferably, the composition of PEG- IFN alpha 2b are prepared using suitable combinations of succinate buffer, lactose and/or polysorbate 80, optionally with other excipients and suitably lyophilized and stored as a dry powder to be reconstituted before use.
In certain embodiment, the pharmaceutical composition comprising therapeutically effective amount of PEGylated interferon alpha-2b, either alone or in combination with other suitable excipient(s). The suitable excipient(s) is selected from salt(s), carbohydrate(s), surfactant(s), amino acid(s), anti-oxidant(s), chelating agent(s), tonicity modifier(s), acid(s), base(s), glycerine, water for injection (WFI) and suitable combination thereof and the like.
In one embodiment, the PEGylated interferon alpha-2b used for the treatment of SARS-CoV-2 infection and its related clinical manifestations or for the administration as a part of method of treating SARS-CoV-2 infection is in the concentration range of 0.01 mg / mL to 5.0 mg / mL, preferably 0.03 mg / mL to 2.5 mg / mL, more preferably 0.1 mg / mL to 0.5 mg / mL.
In one embodiment, the pharmaceutical composition according to the present invention comprising PEGylated interferon alpha- 2b in the concentration range of 0.01 mg / mL to 5.0 mg / mL, preferably 0.03 mg / mL to 2.5 mg / mL, more preferably 0.1 mg / mL to 0.5 mg / mL. The concentration range of 0.1 mg / mL to 0.5 mg / mL includes 0.1 mg / mL, 0.16 mg / mL, 0.20 mg / mL, 0.24 mg / mL, 0.3 mg / mL. In one more embodiment, the pharmaceutical composition according to the present invention comprising PEGylated interferon alpha-2b in the concentration range of 10 pg to 5000 pg per mL, preferably 30 pg to 2500 pg per mL, more preferably 100 pg to 500 pg per mL. The concentration range of 100 pg to 500 pg per mL includes 100 pg, 160 pg, 200 pg, 240 pg, 300 pg per mL. In one of the embodiments, the pharmaceutical composition comprising
PEGylated interferon alpha-2b, buffer(s) and other suitable excipient(s), where in buffer(s) is selected from phosphate buffer (e.g., sodium phosphate buffer, etc.), arginine-phosphate buffer, histidine buffer, arginine-histidine buffer, histidine chloride buffer, glycine buffer, sodium glycinate buffer, arginine buffer, citrate buffer (e.g., sodium citrate buffer, etc.), arginine-citrate buffer, succinate buffer (e.g. succinic acid buffer, sodium succinate buffer, etc.), potassium succinate buffer, arginine- succinate buffer, acetate buffer, arginine-acetate buffer, arginine- glutamate buffer, arginine-aspartate buffer, tromethamine buffer and suitable combination thereof and the like. The suitable excipient(s) is selected from salt(s), carbohydrate(s), surfactant(s), amino acid(s), anti-oxidant(s), chelating agent(s), tonicity modifier(s), acid(s), base(s), glycerine, water for injection (WFI) and suitable combination thereof and the like.
In one of the preferred embodiments, the pharmaceutical composition comprising PEGylated interferon alpha-2b, buffer(s) and other suitable excipient(s), where in buffer(s) is selected from succinate buffer, phosphate buffer, sodium succinate buffer, potassium succinate buffer, histidine chloride buffer, sodium glycinate buffer and suitable combination thereof and the like. In one of the more preferred embodiments, the pharmaceutical composition comprising PEGylated interferon alpha- 2b, buffer and other suitable excipient(s), where in buffer is succinate buffer. The suitable excipient(s) is selected from salt(s), carbohydrate(s), surfactant(s), amino acid(s), anti-oxidant(s), chelating agent(s), tonicity modifier(s), acid(s), base(s), glycerine, water for injection (WFI) and suitable combination thereof and the like.
In one of the embodiments, the pharmaceutical composition comprising PEGylated interferon alpha-2b, buffer(s), carbohydrate(s), surfactant(s) and other suitable excipient(s). In a certain embodiment, the pharmaceutical composition comprising PEGylated interferon alpha-2b, buffer(s), carbohydrate(s) as a cryoprotectant(s) or as a lyoprotectant(s), surfactant(s) and other suitable excipient(s). The suitable excipient(s) is selected from salt(s), amino acid(s), anti oxidants), chelating agent(s), tonicity modifier(s), acid(s), base(s), glycerine, water for injection (WFI) and suitable combination thereof and the like. The current invention provides pharmaceutical composition comprising PEGylated interferon alpha-2b, buffer(s) and carbohydrate(s) as a cryoprotectant(s) or as a lyoprotectant(s), optionally with other suitable excipient(s). Furthermore, it provides pharmaceutical composition comprising PEGylated interferon alpha-2b, buffer(s) and surfactant(s), optionally with other suitable excipient(s).
While the present invention is not limited to a particular composition, the compositions that are anticipated here utilize a suitable buffer(s), suitable carbohydrate(s) which may act as a cryoprotectant(s) and/or lyoprotectant(s), suitable surfactant(s), and solvent(s), alone or in suitable combination, optionally with other suitable excipients, in addition to the PEGylated interferon alpha conjugate. In another embodiment, the pharmaceutical composition of PEGylated interferon alpha-2b according to the present invention comprising stabilizer(s). The stabilizer(s) of present invention is selected from surfactant(s), sugar(s), amino acid(s), polyol(s) and the like or suitable derivative or mixtures thereof. Stabilizer (s) include an excipient capable of preventing aggregation or other physical and chemical decompositions (e.g., self-decomposition, deamidation, oxidation, etc.) in aqueous and solid states. In the present invention, it stabilizes the PEGylated interferon alpha conjugates to prevent depegylation during and after lyophilisation - a phenomenon commonly associated with the PEG- interferon alpha 2b product. Stabilizers commonly used in pharmaceutical compositions include, but not limited to, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, glycine, arginine hydrochloride, sodium dodecyl sulphate (SDS), polysorbate; poly-hydroxy compounds including polysaccharides such as dextran, starch, hydroxyethyl starch, cyclodextrins, N- methyl pyrollidene, cellulose, hyaluronic acid, sodium chloride and suitable combination thereof. One such preferred stabilizing agent is polyoxyethylensorbitan-fatty acid ester, polysorbate 80 (Tween 80), according to the present invention. Polysorbate 20 can also be used as a stabilizing agent according to the present invention.
Buffers suitable for use in each of the compositions of the present invention include buffers that are compatible with PEGylated interferon alpha-2b and suitable for administration to a subject. Buffers of each of the compositions of the present invention are selected from phosphate buffer (e.g., sodium phosphate buffer, etc.), arginine -phosphate buffer, histidine buffer, arginine-histidine buffer, histidine chloride buffer, glycine buffer, sodium glycinate buffer, arginine buffer, citrate buffer (e.g., sodium citrate buffer, etc.), arginine-citrate buffer, succinate buffer (e.g. succinic acid buffer, sodium succinate buffer, etc.), potassium succinate buffer, arginine- succinate buffer, acetate buffer, arginine-acetate buffer, arginine-glutamate buffer, arginine-aspartate buffer, tromethamine buffer and suitable combination thereof and the like.
In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is phosphate buffer (e.g., sodium phosphate buffer, etc.). In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is arginine-phosphate buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is histidine buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is arginine-histidine buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is histidine chloride buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is glycine buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is sodium glycinate buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is arginine buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is citrate buffer (e.g., sodium citrate buffer, etc.). In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is arginine-citrate buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is succinate buffer (e.g. succinic acid buffer, sodium succinate buffer, etc.). In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is potassium succinate buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is arginine- succinate buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is acetate buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is arginine- acetate buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is arginine-glutamate buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is arginine-aspartate buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is tromethamine buffer. In one of the preferred embodiments, the buffer of each of the compositions according to the present invention is combinations of buffers as described in the present invention. The buffers included in the pharmaceutical composition according to the present invention may be a buffer that maintains the pH of the composition at a pH, ranging from about pH 4.0 to about pH 8.0. The pH range of about 4.0 to about 8.0 according to the present invention includes about pH 4.0, about pH 5.0, about pH 6.0, about pH 7.0 or about pH 8.0 or any non-integer number in between them (e.g., about pH 4.5 or about pH 5.3 or about pH 6.8 and the like). In one embodiment, the pharmaceutical composition of PEGylated interferon alpha-2b according to the present invention having a pH in the range of about pH 4.0 to about pH 7.1. In a preferred embodiment, the pharmaceutical composition of PEGylated interferon alpha- 2b according to the present invention having a pH in the range of about pH 6.5 to about pH 7.1. About pH 6.5 to about pH 7.1 according to the present invention includes about pH 6.5, about pH 7.0 or any non-integer or integer number in between them. In a more preferred embodiment, the pharmaceutical composition of PEGylated interferon alpha-2b according to the present invention having a pH of about 6.8.
In one embodiment, the concentration of buffer according to the present invention is in the range of about 1 mM to about 500 mM. Preferably, the buffer concentration according to the present invention is in the range of about 1 mM to about 100 mM. The concentration range according to present disclosure include each integer and non-integer number between a particular range. For Example, the concentration range of about 1 mM to about 100 mM include about 5 mM, about 9 mM, about 10 mM, about 13 mM, about 15 mM, about 18 mM, about 20 mM, about 25 mM, about 30 mM, about 45 mM, about 60 mM, about 70 mM or any integer or non-integer number in between them. In one of the embodiments, the concentration of buffer when present in combination with other suitable excipient(s) according to the present invention is in the range of about 1 mM to about 500 mM. Preferably, the buffer concentration according to the present invention is in the range of about 1 mM to about 100 mM. The concentration range according to present disclosure include each integer and non-integer number between a particular range. For Example, the concentration range of about 1 mM to about 100 mM include about 5 mM, about 9 mM, about 10 mM, about 13 mM, about 15 mM, about 18 mM, about 20 mM, about 25 mM, about 30 mM, about 45 mM, about 60 mM, about 70 mM or any integer or non-integer number in between them. In one of the embodiments, composition according to the present invention does not contain buffer. In one of the embodiments, the present invention provides the pharmaceutical composition of PEGylated interferon alpha- 2b comprising buffer in the range of about 1 mM to about 500 mM, preferably about 1 mM to about 100 mM. In a preferred embodiment, the present invention provides the pharmaceutical composition of PEGylated interferon alpha- 2b comprising succinate buffer at a concentration of about 10 mM.
In one embodiment, the buffering agent(s) or buffering species or buffering component(s) according to the present invention is present in amount of about 0.01 mg / mL to about 100 mg / mL. The concentration range according to present disclosure include each integer and non-integer number between particular range. For Example, the concentration range of about 0.01 mg / mL to about 100 mg / mL include about 0.1 mg / mL, about 0.7 mg / mL, about 1.18 mg / mL, about 1.50 mg / mL, about 2 mg / mL, about 2.3 mg / mL, about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 50 mg / mL or any integer or non-integer number in between them.
In another embodiment, the pharmaceutical composition of PEGylated interferon alpha-2b according to the present invention comprising carbohydrate(s). A carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may also be employed. Some carbohydrate excipients of interest include, but are not limited to, for example, monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like. Carbohydrate(s) may act as a cryoprotectant(s) or as a lyoprotectant(s) according to the present invention. For instance, the composition may include a sugar. Preferred sugars suitable for use in the compositions of the present invention include sugars that are compatible with PEGylated interferon alpha- 2b and suitable for administration to a subject. Examples of suitable sugar include, but not limited to, dextran, trehalose, raffinose, sucrose, polyvinylpyrrolidone, lactose, inositol, sorbitol, mannitol, dimethyl sulfoxide, glycerol, albumin, calcium lactobionate, fructose, maltose, galactose, glucose, D- mannose, sorbose, cellobiose, melezitose, maltodextrins, starches, lactitol, xylitol, pyranosyl sorbitol, myoinositol, hydroxypropyl-beta-cyclodextrin (b-HPCD) and suitable combination thereof and the like. In a preferred embodiment, the pharmaceutical composition of PEGylated interferon alpha- 2b according to the present invention comprising lactose or sucrose or raffinose or trehalose. The pharmaceutical composition of PEGylated interferon alpha-2b according to the present invention comprising carbohydrate(s), either as a cryoprotectant(s) or as a lyoprotectant(s), selected from sucrose, lactose and mannitol. In one of the preferred embodiments, the carbohydrate according to the present invention is lactose. In one of the preferred embodiments, the carbohydrate according to the present invention is sucrose. In one of the preferred embodiments, the carbohydrate according to the present invention is raffinose. In one of the preferred embodiments, the carbohydrate according to the present invention is trehalose.
In one embodiment, the concentration of carbohydrate(s) according to the present invention is in the range of about 10 mg / mL to about 250 mg / mL, preferablylO mg / mL to about 200 mg / mL. Preferably, the carbohydrate(s) concentration according to the present invention is in the range of about 10 mg / mL to about 200 mg / mL. The concentration range according to present disclosure include each integer and non-integer number between a particular range. For Example, the concentration range of about 10 mg / mL to about 200 mg / mL include about 50 mg / mL, about 70 mg / mL, about 100 mg / mL, about 105 mg / mL, about 125 mg / mL, about 160 mg / mL, about 195 mg / mL or any integer or non-integer number in between them. In one of the embodiments, the present invention provides the pharmaceutical composition of PEGylated interferon alpha- 2b comprising carbohydrate in the range of about 10 mg / mL to about 250 mg / mL, preferably about 10 mg / mL to about 200 mg / mL. In a more preferred embodiment, the present invention provides the pharmaceutical composition of PEGylated interferon alpha-2b comprising lactose at a concentration of about 105 mg / mL.
The compositions of the present disclosure, in addition to the PEGylated interferon alpha- 2b, may include carbohydrate(s) as a cryoprotectant(s) or as a lyoprotectant(s). The concentration of cryoprotectant(s) according to the present invention is in the range of about 10 mg / mL to about 250 mg / mL, preferably about 10 mg / mL to about 200 mg / mL. The compositions of the present disclosure, in addition to the PEGylated interferon alpha-2b, buffer(s) and carbohydrate(s), may include surfactant(s). Examples of surfactants according to the present invention include, but not limited to polyoxyethylensorbitan fatty acid esters (Tweens), polyoxyethylene alkyl ethers (e.g. Brij), alkylphenylpolyoxyethylene ethers (e.g. Triton-X), polyoxyethylene - polyoxypropylene copolymer (e.g. Poloxamer, Pluronic), polyethylene glycol (PEG), polyethyleneimine (PEI), sodium dodecyl sulphate (SDS) and suitable combination thereof and the like.
In one embodiment, the pharmaceutical composition of PEGylated interferon alpha- 2b according to the present invention comprising surfactant, wherein the surfactant is polyoxyethylensorbitan-fatty acid esters (Tween). In a preferred embodiment, the pharmaceutical composition of PEGylated interferon alpha-2b according to the present invention comprising polysorbate 20 or polysorbate 80.
In one of the preferred embodiments, the surfactant according to the present invention is polyoxyethylensorbitan fatty acid esters (Tween). In one of the preferred embodiments, the surfactant according to the present invention is polyoxyethylene alkyl ethers (e.g. Brij). In one of the preferred embodiments, the surfactant according to the present invention is alkylphenylpolyoxyethylene ethers (e.g. Triton-X). In one of the preferred embodiments, the surfactant according to the present invention is polyoxyethylene-polyoxypropylene copolymer (e.g. Poloxamer, Pluronic). In one of the preferred embodiments, the surfactant according to the present invention is polyethylene glycol (PEG). In one of the preferred embodiments, the surfactant according to the present invention is polyethyleneimine. In one of the preferred embodiments, the surfactant according to the present invention is sodium dodecyl sulphate (SDS).
In another embodiment, the surfactant according to the present invention is in the range of about 0.001 mg / mL to about 1 mg / mL. Preferably, the surfactant concentration according to the present invention is in the range of about 0.01 mg / mL to about 2 mg / mL. The concentration range according to present disclosure include each integer and non-integer number between particular range. For example, the concentration range of about 0.01 mg / mL to about 2 mg / mL include about 0.1 mg / mL, about 0.2 mg / mL, about 0.5 mg / mL, about 0.9 mg / mL or any integer or non-integer number in between them. In one of the embodiments, the present invention provides the pharmaceutical composition of PEGylated interferon alpha- 2b comprising surfactant in the range of about 0.001 mg / mL to about 1 mg / mL, preferably about 0.01 mg / mL to about 2 mg / mL.
In a preferred embodiment, the present invention provides the pharmaceutical composition of PEGylated interferon alpha-2b comprising polysorbate 80 at a concentration of about 0.1 mg / mL. Surfactant(s) can act as stabilizer in the pharmaceutical compositions.
In another aspect, the compositions of the present disclosure may include amino acid in addition to the PEGylated interferon alpha- 2b. Amino acids suitable for use in the compositions of the present disclosure include amino acids that are compatible with the PEGylated interferon alpha-2b and suitable for administration to a subject. Examples of suitable amino acids include, but are not limited to, arginine, glycine, asparagine, glutamine, lysine, threonine, histidine, glutamic acid, aspartic acid, isoleucine, valine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, cysteine, their suitable salt form and combination thereof and the like. Amino acid can be a part of buffer as a potential counter ion.
Compositions of the present disclosure may also include other component(s), such as additional pharmaceutically acceptable excipient(s) or a dosage delivery vehicle as part of the composition. Other suitable excipient(s) according to the present invention may include, but are not limited to, salt(s), anti- oxidant(s), chelating agent(s), tonicity modifier(s), acid(s), base(s), vegetable oils, phospholipids, glycerine, water for injection (WFI) and suitable combination thereof and the like. Examples of salts according to the present invention may include, but are not limited to, sodium chloride, potassium chloride, potassium sulfate and sodium sulfate. Skilled artisan can use sugar(s) or polyol(s) or amino acid(s) as the tonicity modifiers instead of salts. In one embodiment, the pharmaceutical composition of PEGylated interferon alpha- 2b according to the present invention comprising salt(s) selected from sodium chloride, potassium chloride, potassium sulfate and sodium sulfate. The pharmaceutical composition of PEGylated interferon alpha-2b can be free from salt(s). In addition, the pharmaceutical composition of PEGylated interferon alpha-2b can be free from inorganic salt(s). Acids or bases may also be present in composition of the present disclosure. Acids or bases according to the present invention can be a part of buffering agent or acid / base conjugate. Examples of acid according to the present invention may include, but are not limited to hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, succinic acid, sulfuric acid, fumaric acid, and any combination thereof. Examples of base according to the present invention include, but are not limited to sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium succinate, sodium format, sodium sulfate, potassium sulfate, potassium fumarate, and any combination thereof. In one more embodiment, chelating agent(s) of the present invention is selected from ethylenediaminetetraacetic acid (EDTA), pentetic acid and ethylene glycol -bi s(p- aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA). Suitable solvent for the present composition is water, preferably the solvent may be water for injection. The pharmaceutical composition of the present invention comprising
PEGylated interferon alpha-2b, buffer(s), carbohydrate(s) and surfactant(s). The pharmaceutical composition of the present invention comprising PEGylated interferon alpha-2b, buffer(s), carbohydrate(s) as a cryoprotectant(s) or as a lyoprotectant(s), and surfactant(s). In one of the aspects, the PEGylated interferon alpha- 2b composition of the present invention comprising PEGylated interferon alpha- 2b and buffer(s). In one of the aspects, the PEGylated interferon alpha-2b composition of the present invention comprising PEGylated interferon alpha-2b, buffer(s) and carbohydrate(s). In one of the aspects, the PEGylated interferon alpha- 2b composition of the present invention comprising PEGylated interferon alpha-2b, buffer(s) and carbohydrate(s) as a cryoprotectant(s) or as a lyoprotectant(s). In one of the aspects, the PEGylated interferon alpha-2b composition of the present invention comprising PEGylated interferon alpha-2b, buffer(s) and surfactant(s). In one of the aspects, the PEGylated interferon alpha- 2b composition of the present invention comprising PEGylated interferon alpha- 2b, surfactant(s) and carbohydrate(s). In one of the aspects, the PEGylated interferon alpha-2b composition of the present invention comprising PEGylated interferon alpha- 2b, surfactant(s) and carbohydrate(s) as a cryoprotectant(s) or as a lyoprotectant(s). In one preferred aspect, the PEGylated interferon alpha-2b composition of the present invention comprising PEGylated interferon alpha- 2b, buffer(s), carbohydrate(s), surfactant(s) and other suitable excipient(s). In one preferred aspect, the PEGylated interferon alpha-2b composition of the present invention comprising PEGylated interferon alpha-2b, buffer(s), carbohydrate(s) as a cryoprotectant(s) or as a lyoprotectant(s), surfactant(s) and other suitable excipient(s). Buffer(s), carbohydrate(s), surfactant(s) and other suitable excipient(s) for the PEGylated interferon alpha-2b composition of the present invention are as described herein above.
The present invention provides pharmaceutical composition comprising PEGylated interferon alpha- 2b, succinate buffer, lactose, polysorbate 80 and other suitable excipient(s). In one preferred aspect, the pharmaceutical composition of PEGylated interferon alpha-2b comprising therapeutically effective amount of PEGylated interferon alpha-2b, succinate buffer, lactose and polysorbate 80. The composition of PEGylated interferon alpha-2b according to the current invention can be used for treatment of SARS-CoV-2 infection.
In one of the aspects, the composition of the invention has improved stability either in frozen, lyophilized, or liquid form. In one of the preferred embodiment, the current invention provides a pharmaceutical composition including PEGylated interferon alpha-2b in a lyophilized form. In another embodiment, the current invention provides a pharmaceutical composition including PEGylated interferon alpha- 2b in a liquid form.
In further embodiment, the present invention provides composition (e.g., frozen or lyophilized or liquid) and methods of making composition. A process of lyophilizing the composition according to the current invention, to get a lyophilized powder comprising: a) preparing a composition comprising PEG-IFN alpha 2b, buffer, stabilizer and cryoprotectant; b) lyophilizing said formulation under suitable conditions and obtaining lyophilized powder. The lyophilization process is a process whereby water is sublimed from a composition after it is frozen- which can provide a stable form to the biological over a desired period of time. The lyophilization process to prepare composition of the present invention can be performed by a skilled person using the techniques available in the art. The lyophilizing steps may include freezing, primary drying, secondary drying and optionally with the step of annealing. The said lyophilized composition can be stored as a dry powder and reconstituted before use. The pharmaceutical composition of PEG-IFN alpha 2b can be stored under +2 °C to +8 °C storage condition.
In certain instances, the composition (e.g., frozen or lyophilized or liquid) is a sterile composition. By “sterile” is meant that there are substantially no microbial contaminants (e.g., fungi, bacteria, viruses, spore forms, etc.). In one instance, the composition (e.g., frozen or lyophilized or liquid) contains low level of bacterial endotoxins. The present invention provides pharmaceutical composition of PEG-IFN alpha 2b that is substantially free of particles and / or aggregates and prevents the formation of particles and / or aggregates during storage and / or transport.
In the treatment of SARS-CoV-2 infection and its related clinical manifestations, the current invention provides lyophilized and stabilized composition of PEGylated interferon alpha-2b comprising therapeutically effective amount of PEGylated interferon alpha-2b, succinate buffer, lactose and polysorbate 80 and optionally with other suitable excipient(s). Other suitable excipient(s) for PEG-IFN alpha 2b are as described herein above.
When the interferon alpha administered at safe and effective dose according to the current invention is a PEGylated interferon alpha- 2b and the amount of PEG-IFN alpha 2b administered is about 0.1 mcg/kg to about 30 mcg/kg on a weekly, twice in a week, thrice in a week, daily basis, twice in a day or every other week for use in treatment of SARS-CoV-2 infection. In the current invention, the composition of PEG-IFN alpha 2b can be used for subcutaneous administration at variety of different dosage levels, for example, about 0.1 mcg/kg/week, about 0.5 mcg/kg/week, about 1 mcg/kg/week, about 1.5 mcg/kg/week, about 2 mcg/kg/week, about 5 mcg/kg/week, about 10 mcg/kg/week, about 20 mcg/kg/week, and/or 30 mcg/kg/week as per further described embodiment.
The current invention further provides treatment of SARS-CoV-2 infection and its related clinical manifestations comprising administration of single dose of PEG-IFN alpha 2b or its composition to the subject. The said single dose of PEG- IFN alpha 2b according to the current invention is selected from 0.1 mcg/kg, about 0.5 mcg/kg, about 1 mcg/kg, about 1.5 mcg/kg, about 2 mcg/kg, about 5 mcg/kg. In one of the preferred embodiments, the current invention provides composition of PEGylated interferon alpha-2b comprising buffer(s), sugar(s) and surfactant(s) to deliver 1 mcg/kg of PEG-IFN alpha 2b to neutralize SARS-CoV-2 virus. The current invention provides composition of PEGylated interferon alpha- 2b that can neutralize 100% SARS-CoV-2 virus. The compositions of the present invention may be suitable for any use, including both in vitro and in vivo uses. The pharmaceutical composition of present invention includes PEGylated interferon alpha- 2b, which can be used for parenteral administration. Parenteral administration includes intravenous, subcutaneous, intra peritoneal, intramuscular administration or any other route of delivery generally considered to be falling under the scope of parenteral administration and as is well known to a skilled person. Preferably, the pharmaceutical composition of present invention includes PEGylated interferon alpha-2b used for subcutaneous administration. In another aspect, the pharmaceutical composition of present invention includes PEGylated interferon alpha- 2b, which can be delivered into lungs by atomization. In one more aspect, the pharmaceutical composition of present invention includes PEGylated interferon alpha-2b, which can be delivered intranasally in the form of nasal drops. The current invention provides a composition of IFN, preferably
PEGylated interferon alpha-2b which can be administered to the patient in combination with standard of care. Standard of care (SOC) is either hydroxychlorquine or any suitable antiviral agents, e.g., remdesivir, and / or ritonavir, and / or liponavir and / or anti- viral monoclonal antibodies, and / or any anti-inflammatory therapy such as anti-IL-6 or anti-TNF alpha or such, and / or any other therapy being given to the COVID-19 patient as a part of SOC, etc. According to the present invention, SOC is as per local regulatory guideline/ institute SOP.
The current invention provides a composition of IFN, preferably PEGylated interferon alpha-2b which can be administered to the patient in combination with TNF inhibitor. TNF inhibitor as mentioned herein is adalimumab, infliximab, or etanercept. In one of the preferred embodiment, the current invention provides a composition of PEGylated interferon alpha-2b which can be administered in combination with adalimumab. In one of the embodiments, said adalimumab is co-administered at a dose in the range of about 20 mg to about 160 mg on a weekly, twice in a week, thrice in a week, daily basis or every other week with the said composition of PEG-IFN alpha 2b. In one of the embodiments, the pharmaceutical composition of present invention includes PEGylated interferon alpha-2b, which can be administered to the patient in combination with anti-IL-6 antibody. One of the anti-IL-6 antibody as mentioned herein is tocilizumab. The compositions of the present invention can be used in the treatment of
SARS-CoV-2 infection and its related clinical manifestations in a subject. Also included in the invention are devices that may be used to deliver the formulation of the invention. Examples of such devices include, but are not limited to, a syringe, a pen, an implant, a needle-free injection device and a patch. Following non-limiting examples illustrate the described pharmaceutical compositions of the present invention and the means of carrying out the invention to obtain a stable pharmaceutical dosage form of said composition of PEG-IFN alpha 2b. It will be appreciated that the examples are illustrative and such other suitable modifications/additions etc. as are well within the scope of the persons skilled in the art are meant to be encompassed within the scope of the present invention. The efficacy of the PEG-IFN alpha 2b in the treatment of SARS-CoV- 2 infection or related clinical manifestations may be evaluated in vivo as described in following examples. EXAMPLES
Example 1: Preparation of PEGylated interferon alpha-2b composition
PEGylated interferon alpha 2b compositions as mentioned in table 1 were prepared following the process of making formulation as disclosed in patent document WO 2008/062481. The composition as mentioned in table 1 were prepared in lyophilized form.
Table 1: Detail of PEGylated interferon alpha-2b composition
Figure imgf000039_0001
Figure imgf000040_0001
In the similar manner, other compositions of PEGylated interferon alpha 2b can be prepared with 50 pg / 0.5 mL of PEGylated interferon alpha 2b, 120 pg / 0.5 mL of PEGylated interferon alpha 2b and 150 pg / 0.5 mL of PEGylated interferon alpha 2b, respectively.
Example 2: Analysis of anti-viral effect of PEGylated interferon alpha-2b composition using plaque neutralization assay
To test the efficiency of PEGylated interferon alpha-2b composition in prevention of SARS-CoV-2 virus infection, plaque neutralization assay was performed using Vero E6 cells and SARS-CoV-2 vims (Strain: NIV-2020-770) sourced from ICMR-NIV Pune. Log dilutions of PEGylated interferon alpha-2b (10 fold dilutions starting at a concentration of 10 pg / mL) were used to determine SARS-CoV-2 vims inhibition. Briefly, Vero cells were seeded in a 24 well cell culture plate and incubated for 24 hours with the dilutions of PEG-IFN alpha 2b. Post the dilution, the supernatant was discarded and the cells were infected with a known titer of SARS-CoV-2 vims. After vims infection for 1 hour, the supernatant was discarded and wells filled with medium containing CMC. The plates were incubated for further 72 hours in a CO2 humidified incubator at 37 °C. Post the incubation, the cells were fixed and the plaques enumerated by staining with crystal violet stain. The number of plaques were counted and percentage inhibition was calculated in comparison to the number of plaques obtained in the wells with virus (without PEG-IFN alpha 2b) as a positive control. The data was plotted using GraphPad Prism software as shown in figure 1 and the I.C50 value (i.e., concentration of PEG-IFN alpha 2b at which 50% inhibition of plaques was observed) was calculated. The I.C50 obtained for PEG- IFN alpha 2b was found to be 0.3 ng/mL (15.57 fM). Example 3: Forty patient clinical study of PEGylated interferon alpha-2b composition in treatment of SARS-CoV-2 infection
The study evaluated whether an administration of PEGylated interferon alpha- 2b of the current invention along with the designated standard of care (SOC) for COVID-19, can provide benefits in terms of improvement in clinical status as well as early clearance of viral load. This was a randomized, controlled and open-label study to assess efficacy and safety of PEGylated interferon alpha- 2b in the treatment of SARS-CoV-2 infection.
In this study, the patients (population) with laboratory-confirmed SARS- CoV-2 infection as determined by Real-time reverse-transcription polymerase chain reaction (RT-PCR), and with Sp02 < 94% on room air or any other specific test showing illness like radiographic infiltrates by imaging (chest x-ray, CT scan, etc.) upon clinical assessment (evidence of rales/crackles on exam) were enrolled. The population included men and women aged >18 years. Overall, a total of 40 adult patients were randomly assigned to receive either Test (n = 20) or Reference (n = 20). Patients in the Test arm were given PEG-IFN 2b along with SOC (recommended as per SOP of local regulatory guideline/institute at the time of conduct of trial) while those in the Reference arm were given SOC only.
Post enrolment patients on test arm were provided the standard of care medication along with PEGylated interferon alpha-2b (1 mcg/kg) on day 1. After safety evaluation of first dose, next dose (second dose) of 1 mcg/kg on day 8 could be optionally administered along with SOC. The PEGylated interferon alpha- 2b was provided in a subcutaneous injection form. Moreover, patients could also be administered PEGylated interferon alpha-2b along with standard of care at a dose in the range of 0.5 mcg/kg/week to 2 mcg/kg/week for this study.
This was an open-label study in which patients were randomized in a 1:1 ratio between the two arms.
Criteria for Evaluation The affirmation of the benefit of PEGylated interferon alpha-2b for the treatment of COVID-19 was based on the following evaluations- • Evaluation of efficacy by analysing improvement on a 7-point ordinal scale: To evaluate the clinical efficacy of PEG-IFN alpha 2b at week 2 on the basis of change on a 7-point ordinal scale below, where the highest score of 7 meant death of the patient:
1. Not hospitalized, no limitations on activities. 2. Not hospitalized, limitation on activities.
3. Hospitalized, not requiring supplemental oxygen.
4. Hospitalized, requiring supplemental oxygen.
5. Hospitalized, on non-invasive ventilation or high flow oxygen devices.
6. Hospitalized, on invasive mechanical ventilation or extracorporeal membrane oxygenation (ECMO).
7. Death.
• Evaluation of efficacy by analysing RT-PCR :
This involved assessment by RT-PCR for the presence of SARS-CoV-2 virus in pharyngeal swab on screening day, day 7 and day 14 (while hospitalized). · Evaluation of safety:
The following safety assessments were performed during the study period:
1. Adverse events and serious adverse events
2. Physical examination
3. Vital signs 4. Clinical laboratory tests
5. PCR for SARS-CoV-2 in pharyngeal swab
Results and Conclusion
• Efficacy Conclusion: In the Test and Reference population, 19 (95.00%) and 13 (68.42%) patients respectively, achieved clinical improvement as assessed by the 7-point ordinal scale at the checkout visit. There was a statistical significant (p <0.05) difference observed in clinical improvement in the population given PEGylated interferon alpha- 2b along with SOC group when compared to the population given SOC alone, from day 0 to day 15.
In terms of viral reduction on Day 7 as assessed by RTPCR, of the 20 patients in the Test arm, 16 (80.00%) patients were observed to be RTPCR negative in comparison with the Reference arm, where out of 19, only 12 (63.16%) were observed to be RTPCR negative.
In terms of viral reduction on Day 14 as assessed by RTPCR, of the 20 patients in the Test arm, 19 (95.00%) patients were observed to be RTPCR negative in comparison with the Reference arm, where out of 19, only 13 (68.42%) were observed to be RTPCR negative. This difference was found to be statistically significant (p<0.05).
Patients in the Test arm given PEG-IFN alpha 2b along with SOC required supplemental oxygen for a shorter duration (median, 33.96 hours) than those in the Reference arm (median, 49.75 hours) (SOC alone).
None of the patients required mechanical ventilation during the study.
• Safety Conclusion:
The PEGylated interferon alpha- 2b product was found to be safe and well tolerated. No serious adverse events and/or deaths were reported in the PEGylated interferon alpha- 2b treatment group and no persistent change in laboratory parameters were recorded. No clinically relevant findings from clinical examination, vital signs and ECG evaluations were attributed to PEGylated interferon alpha-2b. Overall, treatment with PEGylated interferon alpha-2b was found to be safe and well tolerated.
Example 4: 250 patient clinical study of PEGylated interferon alpha-2b composition in treatment of SARS-CoV-2 infection
Study design
The multi-centre and open-label clinical study was undertaken at 20 study centres in India. Key inclusion criteria were age >18 years, RT-PCR confirmed SARS-CoV-2 infection, pneumonia with no signs of severe disease, respiratory rate (RR) >24 breaths/minute, Sp02 90% to 94%, and a negative pregnancy test prior to treatment for female patients of child-bearing potential. Interventions
Eligible subjects were randomized in a 1:1 ratio to either PEG-IFN alpha 2b (1 pg/kg subcutaneous [SC] injection, single dose) plus SOC or SOC alone. Standard of care treatments (i.e., antipyretics, cough suppressants, antibiotics, steroids, vitamins, anticoagulants, hydroxychloroquine and antivirals e.g. remdesivir) were administered as per the COVID-19 clinical management guidelines of the ministry of health, Government of India and the individual institutional practice. Randomization was generated using SAS® software (Version 9.4). Each vial of PEG-IFN alpha 2b was reconstituted with 0.7 ml of water for injection for administration of up to 0.5 ml of solution. Each 0.5 ml of solution for SC injection delivers 100 pg of PEG-IFN alpha 2b. All subjects were hospitalized, RT-PCR tests in pharyngeal swab were performed at screening, Day 7, Day 11, Day 15 and Day 29. The assessment was done on the basis of different evaluation criteria as described in example 3.
Results and Conclusion
Two-hundred and fifty subjects were randomized to the PEG-IFN alpha 2b plus SOC and the SOC arms. Efficacy analysis were performed according to the modified intent- to-treat (mITT) population with a 2-point improvement on the WHO 7-point ordinal scale. The mITT population included all randomized subjects who received either of the study medication and appeared for efficacy assessment.
Table 2: Analysis of proportion of subjects with clinical improvement in mITT population, measured using the WHO 7-point ordinal scale
Figure imgf000044_0001
In mITT population, the subjects of PEG-IFN alpha 2b plus SOC group had achieved more clinical improvement in comparison with SOC alone by day 8. On day 8 there was a statistically significant difference observed in clinical improvement in the population given PEG-IFN alpha 2b plus SOC group compared to the SOC alone (Table 2).
Table 3: Analysis of proportion of subjects with negative qualitative RT- PCR in mITT population
Figure imgf000045_0001
In mITT population, a greater percentage of subjects in the PEG-IFN alpha 2b plus SOC group had achieved RTPCR negative status in comparison with SOC alone by day 8. This difference was statistically significant (Table 3). Further, the proportion of adverse events (AE) were similar between the two groups. Of the 250 subjects, 21 (8.47%) subjects had at least one TEAE during the treatment period: 8 (6.67%) subjects in PEG-IFN alpha 2b plus SOC group and 13 (10.00%) subjects in SOC group alone. None of the subjects were discontinued from the study due to AEs in any of the treatment group. All AEs were followed up until the subject was ‘recovered’ or ‘recovered with sequelae’ or until the end of post treatment follow-up, whichever came first.
The most frequently reported PT of TEAEs in PEG-IFN alpha 2b along with SOC treatment group were: chest pain: 1 (0.77%), asthenia: 1 (0.77%), myalgia: 1 (0.77%), headache: 3 (2.31%), respiratory distress: 1 (0.77%) and pruritus: 1 (0.77%). While in SOC treatment group alone were: chest pain: 1 (0.83%), constipation: 2 (1.67%), diarrhoea: 1 (0.83%), gastritis: 1 (0.83%), nausea: 1 (0.83%), asthenia: 1 (0.83%), back pain: 1 (0.83%), cough: 1 (0.83%), respiratory distress: 2 (1.67%) and pruritus: 1 (0.83%). No clinically relevant findings from clinical examination, vital signs and ECG evaluations were attributed to PEG-IFN alpha 2b. Overall, single dose of PEG-IFN alpha 2b was safe and well tolerated in the study. Early treatment with PEG-IFN alpha 2b induced early viral clearance and improved the clinical status of patients with moderate COVID-19 disease. It also decreased the duration of supplemental oxygen. Treatment with PEG IFN-a2b provides a viable treatment option during the current pandemic situation. It can also limit the spread of virus in the community.
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2. http s ://w w w . worldometer s . info/coronaviru s/ 3. Wang et al., Global virus outbreaks: interferons as 1st responders, Seminars in
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11. Zhang et al., Clinical characteristics of 140 patients infected with SARS-CoV- 2 in Wuhan, China; Allergy, 2020, volume 75, pages 1730-1741 12. Schoggins et al., Interferon- stimulated genes and their antiviral effector functions, Current Opinion in Virology 2011, 1:519-525
13. Loutfy et al., Interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study, JAMA, 2003, volume 290, no. 24, pages 3222-3228 Incorporation by reference
The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes. Equivalents
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

We claim:
1. A composition of PEGylated interferon alpha-2b for use in treatment of SARS-CoV-2 infection and its related clinical manifestations.
2. The composition as claimed in claim 1 is pharmaceutical composition wherein the composition comprises therapeutically effective amount of PEGylated interferon alpha-2b optionally with other suitable excipient(s).
3. The PEGylated interferon alpha- 2b as claimed in claim 2 is in the concentration range of 0.01 mg / mL to 5.0 mg / mL, preferably 0.03 mg / mL to 2.5 mg / mL, more preferably 0.1 mg / mL to 0.5 mg / mL.
4. The composition as claimed in claim 2, wherein suitable excipient(s) is selected from buffer(s), salt(s), carbohydrate(s), surfactant(s), amino acid(s), anti-oxidant(s), chelating agent(s), tonicity modifier(s), acid(s), base(s), glycerine, water for injection (WFI) and suitable combination thereof.
5. The composition as claimed in claim 4, wherein buffer(s) is selected from phosphate buffer, arginine-phosphate buffer, histidine buffer, arginine -histidine buffer, histidine chloride buffer, glycine buffer, sodium glycinate buffer, arginine buffer, citrate buffer, arginine-citrate buffer, succinate buffer, potassium succinate buffer, arginine- succinate buffer, acetate buffer, arginine-acetate buffer, arginine-glutamate buffer, arginine- aspartate buffer, tromethamine buffer and suitable combination thereof.
6. The composition as claimed in claim 4, wherein carbohydrate(s) is selected from dextran, trehalose, raffinose, sucrose, polyvinylpyrrolidone, lactose, inositol, sorbitol, mannitol, dimethyl sulfoxide, glycerol, albumin, calcium lactobionate, fructose, maltose, galactose, glucose, D-mannose, sorbose, cellobiose, melezitose, maltodextrins, starches, lactitol, xylitol, pyranosyl sorbitol, myoinositol, hydroxypropyl-beta-cyclodextrin and suitable combination thereof.
7. The composition as claimed in claim 4, wherein surfactant(s) is selected from polyoxyethylensorbitan fatty acid esters, polyoxyethylene alkyl ethers, alkylphenylpolyoxyethylene ethers, polyoxyethylene -polyoxypropylene copolymer, polyethylene glycol, polyethyleneimine, sodium dodecyl sulphate and suitable combination thereof.
8. The composition as claimed in claim 4, wherein acid(s) is selected from hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, succinic acid, sulfuric acid, fumaric acid, and suitable combinations thereof.
9. The composition as claimed in claim 4, wherein base(s) is selected from sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium succinate, sodium format, sodium sulfate, potassium sulfate, potassium fumarate, and suitable combinations thereof.
10. The composition as claimed in claim 4, wherein salt(s) is selected from sodium chloride, potassium chloride, potassium sulfate and sodium sulfate.
11. The composition as claimed in claim 4, wherein amino acid(s) is selected from arginine, glycine, asparagine, glutamine, lysine, threonine, histidine, glutamic acid, aspartic acid, isoleucine, valine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, cysteine, their suitable salt form and combination thereof.
12. The composition as claimed in claim 4, wherein chelating agent(s) is selected from ethylenediaminetetraacetic acid, pentetic acid and ethylene g 1 yco 1 -b i s(P- a m i noct h y 1 ether)-N,N,N’ ,N’ -tetraacetic acid.
13. The composition as claimed in claim 5, wherein the concentration of buffer is in the range of about 1 mM to about 500 mM, preferably about 1 mM to about 100 mM.
14. The composition as claimed in claim 6, wherein the concentration of carbohydrate(s) s in the range of about 10 mg / mL to about 250 mg / mL, preferably about 10 mg / mL to about 200 mg / mL.
15. The composition as claimed in claim 7, wherein the concentration of the surfactant is in the range of about 0.001 mg / mL to about 1 mg / mL, preferably about 0.01 mg / mL to about 2 mg / mL.
16. The composition as claimed in claim 1, wherein the said composition has pH in the range of about pH 4.0 to about pH 7.1, preferably about pH 6.5 to about pH 7.1, more preferably pH 6.8.
17. The composition as claimed in any of preceding claims wherein the said composition is in a lyophilized form or liquid form, preferably in lyophilized form.
18. A pharmaceutical composition of PEGylated interferon alpha-2b comprising therapeutically effective amount of PEG-IFN alpha 2b, 10 mM of succinate buffer, 105 mg / mL of lactose monohydrate and 0.1 mg / mL polysorbate 80 and optionally with other suitable excipient(s), wherein amount of PEGylated interferon alpha-2b in the pharmaceutical composition is in range of 0.1 mg / mL to 0.5 mg / mL, preferably 0.16 mg / mL or 0.20 mg / mL.
19. The PEGylated interferon alpha 2b as claimed in claim 1 has at least one of the following characteristic(s): a. shows not more than about 1 °C difference in thermal transition value as compared to the unmodified interferon alpha-2b; b. exhibits certain degree of depegylation, preferably 30 % or more upon hydroxylamine treatment; c. shows about 8 - 9 positional isomers of interferon alpha; d. exhibits low level of (< 5 %) oxidation on one of the methionine residues of interferon at the position 148; e. exhibits low level of (< 5 %) deamidation on one of the asparagine residues of interferon at the position 93; f. conjugated with a polyethylene glycol molecule having an average molecular weight of approximately 12,000 daltons; g. conjugated with a polyethylene glycol molecule that is essentially free of diols; h. conjugated with a polyethylene glycol that shows a polydispersity index of about
1.00; i. synthesized by performing a reaction between interferon alpha and polyethylene glycol wherein the terminal activity of polyethylene glycol is no less than 90 %; j. synthesized by performing a reaction between interferon alpha and polyethylene glycol at 1:1 or 1:2 or 1:3 or 1:4 or 1:5 or 1:6 or 1:10 ratio wherein the polyethylene glycol is used in stoichiometric or molar excess amount over the interferon; and k. contains no more than 10 % of high molecular weight species variants and less than 5 % of unmodified interferon.
20. The composition as claimed in any of preceding claims wherein the said composition is administered to a subject by intravenous administration, subcutaneous administration, intra peritoneal, intramuscular administration or intranasal administration or into lungs by atomization.
21. The composition as claimed in any of preceding claims wherein the said composition is administered to a subject at a dose of about 0.1 mcg/kg to about 30 mcg/kg on a weekly, twice in a week, thrice in a week, daily basis, twice in a day or every other week, preferably at a dose of 1 mcg/kg/week.
22. The composition as claimed in any of preceding claims wherein the said composition is administered to the patient in combination with standard of care.
23. The composition as claimed in claim 22 wherein standard of care is either hydroxychlorquine or any suitable antiviral agent(s) or any anti-inflammatory therapy.
24. The composition as claimed in claim 23 wherein antiviral agent(s) is selected from remdesivir, ritonavir liponavir and anti-inflammatory therapy is selected from anti-IL- 6 and anti-TNF alpha antibody.
25. Use of PEGylated interferon alpha-2b or its composition for the treatment of SARS- CoV-2 infection and its related clinical manifestations.
26. The use of composition of PEGylated interferon alpha-2b as claimed in claim 25, wherein composition is as claimed in claims 1-24.
27. A method of treating SARS-CoV-2 infection by administering PEGylated interferon alpha-2b or its composition.
28. The method as claimed in claim 27 comprising administration of single dose of PEGylated interferon alpha 2b wherein single dose is selected from 0.1 mcg/kg, about 0.5 mcg/kg, about 1 mcg/kg, about 1.5 mcg/kg, about 2 mcg/kg, about 5 mcg/kg.
29. The method of treating SARS-CoV-2 infection by administering PEGylated interferon alpha-2b composition as claimed in claim 27, wherein composition is as claimed in claims 1-24.
PCT/IB2021/054776 2020-06-01 2021-06-01 A composition of pegylated interferon alpha-2b for the treatment of sars-cov-2 infection and related manifestations WO2021245541A1 (en)

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Citations (1)

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WO2006076014A2 (en) * 2004-04-30 2006-07-20 Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Interferon-alpha constructs for use in the treatment of sars

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WO2006076014A2 (en) * 2004-04-30 2006-07-20 Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Interferon-alpha constructs for use in the treatment of sars

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