WO2016133560A1 - Protéine cc10 humaine recombinante pour le traitement de la grippe et de l'ebola - Google Patents

Protéine cc10 humaine recombinante pour le traitement de la grippe et de l'ebola Download PDF

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WO2016133560A1
WO2016133560A1 PCT/US2015/057267 US2015057267W WO2016133560A1 WO 2016133560 A1 WO2016133560 A1 WO 2016133560A1 US 2015057267 W US2015057267 W US 2015057267W WO 2016133560 A1 WO2016133560 A1 WO 2016133560A1
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ccio
rhccio
infection
viral
influenza
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PCT/US2015/057267
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English (en)
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Aprille L. PILON
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Therabron Therapeutics, Inc.
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Priority claimed from US14/623,362 external-priority patent/US9168285B2/en
Application filed by Therabron Therapeutics, Inc. filed Critical Therabron Therapeutics, Inc.
Publication of WO2016133560A1 publication Critical patent/WO2016133560A1/fr

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    • 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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • 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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4721—Lipocortins

Definitions

  • Embodiments of the present invention relate to methods of reducing viral titers in vivo and treating a viral respiratory or hemorrhagic infection in a patient.
  • Embodiments of the present invention also relate to methods of treating influenza infection, including Type A influenza, particularly H1N1 influenza and ebola virus disease (EVD) also known as ebola hemorrhagic fever (EHF).
  • EHF ebola hemorrhagic fever
  • embodiments of the present invention also relate to methods of treating the above using intranasally-administered and/or intravenously- administered, and/or inhaled recombinant human CCIO.
  • CCIO 10 kDa protein
  • UG uteroglobin
  • CCIO consists of two identical subunits of 70 amino acid residues, each with the "four helical bundle” secondary structure motif, joined in antiparallel orientation by two disulfide bonds between Cys 3 and 69', 3' and 69 (Matthews, 1994; Morize, 1997).
  • CCIO is the first member of an emerging family of small globular proteins that share the same secondary, tertiary and quaternary structure and are thought to mediate similar functions.
  • CCIO is an anti-inflammatory and immunomodulatory protein that has been characterized with respect to various interactions with other proteins, receptors and cell types (reviewed in Mukherjee, 1999 and Pilon, 2000). Lower levels of CCIO protein or mRNA have been found in various tissue and fluid samples for a number of clinical conditions characterized by some degree of inflammation including pneumonia (Nomori, 1995).
  • CCIO knockout mouse The physiology of CCIO protein in different types of pulmonary infections has been studied in one strain of CCIO knockout mouse.
  • CCIO knockout and wild type mice were each infected with either Pseudomonas aeruginosa or adenovirus, two common human respiratory pathogens, the wild type mice experienced more rapid clearing of the pathogens, with greater killing of the pathogens by the innate immune system, suggesting a benefit to CCIO deficiency during viral and bacterial infection (Hayashida, 1999; Harrod, 1998).
  • CCIO recombinant human CCIO protein
  • RSV respiratory syncytial virus
  • rhCCIO can prevent the development of acquired immunity, specifically antigen-specific T cells, when present at the same time that dendritic cells are exposed to antigen (Johansson, 2007), which again indicates that administration of rhCCIO may not benefit a patient with an infection.
  • the current state of knowledge regarding the potential hazards or benefits of rhCCIO treatment during a respiratory infection is conflicting and allows no conclusions to be drawn regarding the safe and/or efficacious use of CCIO to treat different types of respiratory infections. No information regarding the effect of CCIO on influenza infection is available.
  • Influenza has caused four major outbreaks (1889, 1918, 1957, and 1968) in the past 120 years, causing the deaths of an estimated 50-100 million people worldwide.
  • Influenza is an orthomyxovirus, an R A virus that is transmitted by aerosols as well as by direct contact of contaminated surfaces with nasal mucosa and targets respiratory epithelial cells.
  • Influenza infection may cause severe symptoms, including fever, sore throat and muscle aches, malaise, weight loss, respiratory congestion, and sometimes respiratory failure and death.
  • Influenza elicits an acquired immune response (cytotoxic T cells and antibodies) that typically clears the infection in 1-2 weeks in normal healthy individuals.
  • H5N1 avian influenza
  • H3N2 seasonal influenza H3N2
  • swine flu H1N1
  • antiviral agents such as neuraminidase inhibitors.
  • the rapid rate of mutation in influenza has led to the development of drug-resistant strains (Moscona, 2009), such that widespread use of antiviral agents for prevention and or treatment will lead to acceleration of the development of resistance to these drugs.
  • New therapeutic agents are therefore needed to treat, cure and prevent influenza infection.
  • Ebola is a hemorrhagic fever virus, also known as a filovirus, originating in
  • embodiments of the invention by administering rhCCIO in a dosage range given at appropriate intervals, or in one dose, to reduce viral titer and treat, cure or prevent viral infection.
  • embodiments of the invention by administering CC10 in a dosage range given at appropriate intervals or in one dose where a patient is diagnosed with a viral infection by symptoms characteristic of the particular virus, and/or by detection of virus in patient samples through culturing of the virus, immunological detection of the virus, and/or detection of the viral nucleic acid, using standard methods.
  • CC10 in a dosage range given at appropriate intervals or in one dose where a patient is diagnosed with an influenza infection by one or more symptoms of fever, myalgia, and congestion, and/or by detection of influenza virus in patient samples (nasal lavages, blood or sputum samples) through culturing of the virus, immunological detection of the virus, and/or detection of the viral nucleic acid, using standard methods.
  • CC10 in a dosage range given at appropriate intervals or in one dose where a patient is diagnosed with an ebola viral infection by one or more symptoms of fever, bleeding from orifices, bloody cough, vomit, urine, or stool, sore throat, muscle pain, and headaches, vomiting, diarrhea, rash, decreased function of the liver and decreased function of the kidneys and/or by detection of ebola virus in patient samples (nasal lavages, blood or sputum samples) through culturing of the virus, immunological 125 detection of the virus, and/or detection of the viral nucleic acid, using standard methods.
  • CC10 is administered intranasally in a dose divided about equally between each nostril in a range of 1.5 micrograms to 1.5 milligrams per kilogram of body weight per day, or in multiple doses which taken together achieve this dosage range on a daily basis to reduce pulmonary viral titer and treat, cure or prevent 130 influenza or ebola infection.
  • CC10 is administered intravenously in a dose of up to 10 milligrams per kilogram of body weight per day, or in multiple doses which taken together achieve this dosage range on a daily basis to treat, cure or prevent influenza or ebola infection.
  • a non-human CC10 protein is administered in a dosage range given at appropriate intervals or in one dose where a patient is diagnosed with a viral infection by symptoms characteristic of the particular virus, and/or by detection of virus in patient samples through culturing of the virus, immunological detection of the virus, and/or detection of the viral nucleic acid, using standard methods.
  • secretoglobins is administered in a dosage range given at appropriate intervals or in one dose where a patient is diagnosed with a viral infection by symptoms characteristic of the particular virus, and/or by detection of virus in patient samples through culturing of the virus, immunological detection of the virus, and/or detection of the viral
  • Fig. 1 is a bar graph depicting H1N1 viral load at 2 days in lungs of infected cotton rats treated with intranasal rhCClO. Viral titer is expressed as (x 10 7 ) TCID50/gram of tissue.
  • Fig. 2 is a bar graph depicting H1N1 viral load at 2 days in lungs of cotton rats treated with intraperitoneal injection of rhCClO. Viral titer is expressed as (x 10 5 )
  • Fig. 3 is a bar graph depicting inhibition of viral replication in cultured cells by rhCClO. RhCClO was added to the culture medium of HEp2 cells at 100 micrograms/ml,
  • Fig. 4 is a bar graph comparison of rhCClO antiviral effects when given pre- infection and post-infection.
  • HEp2 cells were treated with 1 mg/ml rhCClO and infected with RSV as in figure 3.
  • 1 mg/ml of rhCClO was given at one hour after infection (treatment DO), 24 hours after infection (treatment Dl), and 48 hours after infection
  • treatment D2 Viral titers in culture media were measured on day 4 post-infection.
  • Fig. 5 is a summary schematic of rhCClO expression in bacteria and recovery.
  • Fig. 6 is a schematic overview of an rhCClO purification process.
  • Embodiments of the present invention relate to the use of CCIO to reduce pulmonary viral titer and treat, cure or prevent influenza infection.
  • the CCIO is preferably a
  • rhCClO 170 recombinant human CCIO protein obtained by the processes described in U.S. Patent App. Publication No.: 20030207795 and PCT/US09/43613 attached hereto at Exs. A and B respectively, all of which are incorporated by reference in their entirety, or via any other process which yields pharmaceutical grade rhCClO.
  • the rhCClO of the embodiments of the present invention may be administered with, without, before or after other intranasal,
  • human CC10 aka uteroglobin
  • the recombinant human CC10 that is active in suppressing viral replication in vitro and in vivo was synthesized and characterized as described in US Patent App. Publication No.: 20030207795.
  • PCT/US09/43613 represent further embodiments of the present invention that may be used to suppress viral replication in vivo, particularly in the nasal passages and sinuses.
  • rhCClO is
  • rhCClO administered intranasally, to each nostril 1-3 times per day, for 7-14 days, and every other day thereafter for another 14 days, and thereafter as needed. More preferably, rhCClO is administered as soon as the patient begins to experience fever, myalgia, and congestion or is diagnosed with influenza.
  • the rhCClO may be produced in a process comprising the steps of: a) 190 providing a bacterial expression system capable of expressing rhCClO; b) inoculating a
  • fermenter with an inoculum comprising the bacterial expression system to form a
  • step d adding an induction agent to the fermentation culture to induce the expression of rhCClO by the bacterial expression system; d) harvesting the rhCClO expressed in step c; and e) purifying the rhCClO harvested in step d, wherein the purifying step
  • 195 comprises the use of at least one filter and at least one ion exchange column, as described in US Patent App. Publication No.: 20030207795.
  • the rhCCIO may also be expressed in alternative bacterial, fungal, insect, mammalian, or plant expression systems and purified to meet specifications for a pharmaceutical product suitable for administration to humans using standard methods.
  • the rhCCIO of the present invention that inhibits 205 viral replication also inhibits phospholipase A 2 (PLA 2 ) enzymes, as described in US Patent App. Publication No.: 20030207795.
  • PHA 2 phospholipase A 2
  • a dose or multiple doses of intranasal rhCCIO equaling a 210 dose ranging from about 1.5 micrograms to about 5 milligrams per kilogram of body weight per day may be administered.
  • rhCCIO may be administered in the dose range on a daily basis.
  • rhCCIO may be administered in the dose range on a daily basis for at least seven days consecutively.
  • rhCCIO may be administered in the dose range on a daily basis for at least 14
  • rhCCIO may be administered in the dose range every other day for 30 days consecutively.
  • rhCCIO may be administered in tapered dosages daily for ten consecutive days, said tapered dosages comprising a high dose at each administration for the first three days, an intermediate dose at each administration for the second three days, and a low dose at each administration for the
  • rhCCIO may be administered in the dose range or in tapered doses up to three times per day, approximately every eight hours.
  • the above doses of rhCCIO may be administered intranasally to the patient as an aerosol, by intranasal spray or lavage, or by deposition of a gel or cream, or other method of instillation in the nasal passages.
  • the above doses of rhCCIO may be administered by inhalation to the patient as an aerosol, by nebulizer or metered dose inhaler, or other method of direct application to the lungs and airways.
  • rhCCIO in treating or preventing influenza infection, is administered intravenously, in doses of 15 micrograms to 20 milligrams per kilogram of
  • rhCCIO may be administered in tapered dosages daily for ten consecutive days, said tapered dosages comprising a high dose at each administration for the first three days, an intermediate dose at each administration for the second three days, and a low dose at each administration for the last four days.
  • rhCCIO may be administered in the dose range or in tapered doses up to three times per day, approximately every eight hours.
  • the above doses of rhCCIO may be administered to the patient using a combination of intranasal, inhaled, and intravenous routes.
  • rhCCIO in accordance with the methods described above, may be administered
  • rhCCIO in accordance with the methods described above, may be administered to reduce pulmonary viral titer and treat, cure, or prevent influenza or ebola infection.
  • rhCCIO administered daily, more than once daily, three times daily, every other day or in a tapered fashion depending upon the severity of influenza infection being treated, the patient's overall health, and whether underlying conditions are present. For example, the more severe the infection, the higher the amount of rhCCIO would be required to effectively treat it. It is
  • rhCCIO Intranasal formulations, devices, and methods by which rhCCIO may be 255 administered intranasally have been described in PCT/US09/43613, which is incorporated herein by reference in its entirety.
  • the intravenous formulation of rhCCIO consists of a 5.5 mg/ml solution in 0.9% saline and has been described in U.S. Patent App. Publication No.: 20030207795, which is incorporated herein by reference in its entirety.
  • Influenza virus is propagated in MDCK cells (ATCC catalog# CCL-34) by infecting 60% confluent cell monolayer (150 cm flasks) with flu virus at a multiplicity of infection (MOI) of 0.01. Three to four days later, when
  • 265 cytopathic effect is generalized and most of the cells have detached from the culture vessel, the cells and supernatants are harvested. Cells are removed by centrifugation (800g) and the supernatant filtered (0.45 ⁇ ) and centrifuged (18000g) for 2 hours at 4°C to pellet viruses. The viral pellet is resuspended in DMEM medium, aliquoted and stored at -150°C. Influenza virus titer is determined by applying 0.1 mL of serially diluted viral stocks to MDCK cell
  • TCID 50 tissue culture infectious dose 50%
  • Sections of the left and right lobes from lungs of infected mice and cotton rats are aseptically removed, weighed and homogenized in
  • Viral Titers in amplified viral stocks and in lung
  • FFA foci forming assay
  • clarified media DMEM with 1% BS A
  • PFA plaque forming assay
  • FFA foci forming assay
  • Culture titers typically yield 10 7 - 109 pfu/ml for influenza.
  • the cotton rat (S. hispidus), a type of vole, is an animal model in which influenza replicates 290 and generates a mild respiratory infection (Ottolini, 2005). The animals are infected by
  • intranasal inoculation with influenza virus and pulmonary viral titers peak two days (about 48 hours) after inoculation.
  • This model is used to screen for compounds that inhibit influenza replication in vivo.
  • Pathogen free cotton rats were purchased from Virion Systems, Inc. (Rockville, MD). A total 295 of eighteen cotton rats (S. hispidus, 6-8 weeks old) were infected with Type A influenza
  • Fig.1 illustrates the reductions in viral titer in lung tissue that were observed in the both rhCCIO dose groups. Viral titer in lung is expressed as (x 10 7 ) TCID 50 /gram of tissue.
  • IP intraperitoneal injection
  • FIG. 2 illustrates the statistically significant reduction (p ⁇ 0.01) in viral titer in lung tissue that was observed in the 5 mg/kg rhCCIO dose group, and the trend towards a lower viral titer in the 0.5 mg/kg dose group.
  • Viral titer in lung is expressed as (x 10 7 )
  • rhCCIO has been found to reduce viral titer in a
  • embodiments of the present invention provide an intranasal, and intravenous, or a combination rhCCIO based therapy effective at treating, curing or preventing influenza infection.
  • HEp2 cells ATCC, Manassas, VA were used to propagate RSV, strain A-2 (Advanced Biotechnologies, Inc., Columbia, MD) and generate viral stocks.
  • Cells were plated at 50,000 cells/well in 48 well plates and grown in MEM with 10% FBS to -80% 330 confluence. Cells were pre-treated with CCIO in 0.5 mL MEM for 4 hours. Medium was then changed and RSV infections were performed using 1 x 10 6 TCID 5 o per 100 mm TC dish for 1 hour. Non-adsorbed virus was removed by washing and 0.5 ml of MEM with 2% FBS, 4 mM L-glutamine, and rhCCIO was added. Supernatants were collected on day 4 post infection and the virus titrated.
  • Figure 3 shows that a concentration of 1 mg/ml CCIO 335 virtually eliminated RSV production, while 100 and 300 micrograms/ml showed a ⁇ 3-fold decrease.
  • CCIO also inhibited viral replication in cells when given at 1, 24, and 48 hours after infection.
  • Fig. 4 shows that rhCCIO is effective at reducing viral titer not only when added before infection, but also when added after infection. This is the first report of a direct
  • CCIO cerebral spastic organelles
  • this phenotype means that CCIO plays an active role in transport of secretory vesicles from the Golgi apparatus to the plasma membrane of the cell.
  • CCIO also modulates the uptake and processing of antigens in antigen-
  • CCIO is an important factor in the transport of materials both out of and in to many types of cells. We therefore infer that CCIO inhibits viral replication by interfering with viral transport in the cell. Since all viruses rely upon cellular transport processes to invade the cell and replicate, CCIO can be expected to inhibit the replication of all viruses. Likewise, other secretoglobins, which share similar
  • 355 structure to CCIO can also be expected to inhibit viral replication at the cellular level.
  • Vero cells were seeded in 96-well plates and cultured to 90-100% confluence. One hour prior to infection, culture media (EMEM/NEAA, 5% FBS) was replaced with fresh media with or without rhCCIO (1.5 or 0.5 mg/ml). After one hour of EMEM/NEAA, 5% FBS) was replaced with fresh media with or without rhCCIO (1.5 or 0.5 mg/ml). After one hour of EMEM/NEAA, 5% FBS). After one hour of
  • EBOV multiplicity of infection
  • MOI multiplicity of infection
  • Ebola glycoprotein (EBOV GP) for 20 minutes on a platform shaker. The plates were then washed in PBS and secondary antibody (goat anti-human AlexaFluor488) was added. After 20 minutes on a platform shaker, the plates were washed and placed in PBS containing the plasma membrane dye (CellMask Deep Red) and left overnight at 4°C. The plates were imaged the next day with an Operetta system. GP -positive cells were counted and
  • the percentage of cells infected with EBOV is the ratio of the number of EBOV GP -positive cells to the total number of Hoechst stained nuclei. At 24 hours postinfection, very few cells were infected at either MOI. At 48 hours post-infection, over 20% 385 of the cells were infected at the lowest MOI in the absence of rhCClO. At 48 hours postinfection at the higher MOI, there was a clear dose-dependent and statistically significant (ie. p value ⁇ 0.05) reduction in the number of infected cells at both concentrations of rhCClO. At 72 hours post-infection, the reduction in the number of infected cells was significant at both MOI at the highest concentration of rhCClO (1.5 mg/ml). The results of the experiment
  • FIG. 5 A summary schematic of the bacterial fermentation, expression of rhCClO, and harvest of cell paste containing rhCClO is shown in Figure 5.
  • a vial of the Production seed cell bank was thawed at room temperature.
  • One hundred microliters of the production seed was then used to inoculate each of the six, fernbach flasks containing one liter each of sterile Super Broth medium (Becton-Dickinson Select APS Super Broth, glycerol and WFI).
  • the cultures in the six flasks were then incubated at 32°C in a New Brunswick shaker-incubator with agitation (300 rpm) for approximately 20 hours.
  • the cultures in the six flasks were then used to inoculate 300 liters of Superbroth in a 400 liter New Brunswick Scientific Fermenter System (Model IF-400).
  • the culture was grown at 25°C to 40°C until a minimum optical density at 600 nm of 2.0 was reached.
  • rhUG also known as rhCClO
  • IPTG isopropyl-P-Dthiogalactopyranoside
  • the fermentation was maintained for at least one hour, preferably two hours post induction.
  • the bacterial culture is harvested by centrifugation with a Sharpie's continuous feed centrifuge.
  • the cell paste is partitioned and stored frozen at - 80°C for later purification.
  • the lysate (supernatant) was then processed using a 100 K nominal molecular weight cut off (NMWCO) membrane in a tangential flow filtration (TFF) system.
  • NMWCO nominal molecular weight cut off
  • TFF tangential flow filtration
  • the permeate from the 100 K step was concentrated by TFF using a 5 K NMWCO membrane and loaded onto a Macro Q anion exchange column.
  • the eluate from the anion exchange column was concentrated and diafiltered by TFF using a 5 K NMWCO membrane before being loaded onto a Type I Hydroxyapatite (HA) column.
  • HA Type I Hydroxyapatite
  • the eluate from the HA column was then loaded directly onto a column packed with Chelating Sepharose Fast Flow (CSFF) resin with copper as the chelate.
  • CSFF Chelating Sepharose Fast Flow
  • the rhUG passed through the column while the host-derived proteins present in the HA eluate bound to the column.
  • a positively charged Sartobind Q TFF membrane was also placed into the flowstream after the copper CSFF column to ensure that the maximum amount of endotoxin was removed from the final bulk material.
  • the pass- through from the Sartobind Q was concentrated and then extensively diafiltered using a 5 K NMWCO membrane with saline for injection (SFI) as the replacement buffer, both to remove residual copper as well as to properly formulate the final bulk material.
  • SFI saline for injection
  • the rhUG preparations made by this process, and minor variations thereof, are comparable in all respects: apparent size, molecular weight, charge, N-terminal amino acid sequence, amount of free thiol indicating correct formation of cystine-cystine bonds, immunological recognition techniques such as ELISA and Western blotting, and biological activity.
  • Protein purified using the copper CSFF column was tested for the presence of copper by Inductively Coupled Plasma (by QTI Inc.). No copper was detected and the detection limit of the assay was 0.5 ppm.
  • the following assays were established as in process assays, characterization assays and release assays for the production process and for the drug substance and drug product.
  • the rhUG drug substances and drug products were compared to standard research lot rhUG/7 where appropriate.
  • Bacterial Nucleic Acids Bacterial DNA content per dose of the rhUG drug substance
  • the molecular weight was determined by Electrospray Ionization 460 spectrometry by M-Scan Inc. Theoretical molecular weight was determined by PAWS (a shareware program for the determination of average molecular mass, obtained through Swiss Pro). A value of 16110.6 Da was determined by the PAWS program. The same value was found for cGMP batches of rhUG and was confirmed by MS analysis of standard research lot rhUG/7 as a control (determined molecular weight 16110.6 Da).
  • the pi was determined by isoelectric focusing using gels with a pH range of 3 to 7. The gels were obtained from Novex and were run under conditions as described by the manufacturer. Samples were run versus a standard from Sigma and a rhUG
  • the drug product was analyzed for the presence of aggregates by chromatography on either a Superose 12 or a Sephadex 75 size exclusion chromatography 500 (SEC) column (Amersham/Pharmacia).
  • SEC Sephadex 75 size exclusion chromatography 500
  • the column was run according to the manufacturer's instructions using the BioRad Biologic system and peak area was determined using EZLogic Chromatography Analysis software, also from BioRad.
  • the percent aggregation was determined by comparing the total area of all peaks vs. the area of peaks eluting prior to the main UG peak.
  • Endotoxin 505 Endotoxin. Endotoxin levels were tested by the rabbit pyrogenicity assay as described in the USP No. 151. An amount of rhUG equivalent to a single human dose was administered intravenously over a 10 minute period. Body temperature increase relative to the baseline predose temperature was monitored over the course of three hours. Acceptable results consist of no temperature rise equal to or greater than 0.5 °C over the baseline results.
  • AE adverse events
  • SAE serious adverse events
  • An AE is any untoward medical occurrence in a subject or a clinical investigation temporally associated with the use of the investigational drug whether or not the event is considered to have a causal relationship with the drug.
  • a pre-existing condition i.e., a disorder present before the AE reporting period started and noted on the pre-treatment medical history/physical examination form
  • Serious adverse events were defined as any untoward medical occurrence that, at any dose; 1) results in death, 2) is life -threatening, 3) requires hospitalization or prolongation of an existing hospitalization, 4)
  • Table 4 List of adverse events for patient receiving rhCCIO
  • intranasal rhCCIO administration was found to be safe and well- tolerated in humans when given once daily as an aerosol in a divided dose of 1.1 milligrams, 0.56 milligrams per nostril, for seven consecutive days.

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Abstract

L'invention porte sur des méthodes d'utilisation de CC10 humaine recombinante (rhCC10), connue également sous le nom d'utéroglobine humaine recombinante, pour réduire des titres viraux dans les tissus de patients, en particulier des titres de grippe et de filovirus dans les tissus pulmonaires. La rhCC10 peut être utilisée comme agent thérapeutique dans le traitement, le soin ou la prévention d'une infection virale, en particulier de l'infection par la grippe et l'Ebola. Plus particulièrement, l'invention concerne des méthodes, comprenant de manière large les gammes posologiques critiques de rhCC10, d'administration par voie intranasale et intraveineuse, qui peuvent être administrées pour traiter, soigner ou prévenir une infection par la grippe et par l'Ebola. L'invention porte également sur des compositions utiles dans les procédés ci-dessus et dans l'administration de rhCC10 à des êtres humains.
PCT/US2015/057267 2015-02-16 2015-10-25 Protéine cc10 humaine recombinante pour le traitement de la grippe et de l'ebola WO2016133560A1 (fr)

Applications Claiming Priority (2)

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US14/623,362 US9168285B2 (en) 2009-10-15 2015-02-16 Recombinant human CC10 protein for treatment of influenza and ebola
US14/623,362 2015-02-16

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WO2016133560A1 true WO2016133560A1 (fr) 2016-08-25

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US20110183887A1 (en) * 1997-05-28 2011-07-28 Clarassance, Inc. Methods and compositions for the reduction of neutrophil influx and the treatment of bronchopulmonary displasia, respiratory distress syndrome, chronic lung disease, pulmonary fibrosis, asthma and chronic obstructive pulmonary disease
US8470767B2 (en) * 1997-05-28 2013-06-25 Clarassance, Inc. Methods and compositions for the reduction of neutrophil influx and the treatment of bronchopulmonary displasia, respiratory distress syndrome, chronic lung disease, pulmonary fibrosis, asthma and chronic obstructive pulmonary disease
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