Patent Application
20250144087
The present disclosure generally relates to viral infection, and more particularly to the prevention and/or treatment of coronavirus infections such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.
Coronaviruses are large, roughly spherical, RNA viruses with bulbous surface projections that cause diseases in mammals and birds. In humans, these viruses cause respiratory tract infections that can range from mild to lethal. Mild illnesses include some cases of the common cold (which is also caused by other viruses, predominantly rhinoviruses), while more lethal varieties can cause severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and Coronavirus disease 2019 (COVID-19). Coronaviruses have four structural proteins, namely the Spike (S), Envelope (E), and Membrane (M) proteins, forming the viral envelope, as well as the Nucleocapsid (N) protein, holding the viral RNA genome.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the strain of coronavirus that causes COVID-19, the respiratory illness responsible for the COVID-19 pandemic. The spike protein of SARS-CoV-2 is the glycoprotein responsible for allowing the virus to attach to and fuse with the membrane of a host cell; specifically, its S1 subunit contains the receptor-binding domain (RBD) that interacts with the cellular receptor angiotensin-converting enzyme 2 (ACE2) enabling virus attachment, and its S2 subunit possesses the fusion machinery, which can mediate host-viral membrane fusion after S1 shedding. The main receptor involved in SARS-CoV-2 entry into human cells is the angiotensin converting enzyme 2 (ACE2). After attachment of a SARS-CoV-2 virion to a target cell, the cell's protease transmembrane protease, serine 2 (TMPRSS2) cuts open the spike protein of the virus, exposing a fusion peptide in the S2 subunit, enabling viral fusion.
Multiple variants of SARS-CoV-2 are circulating globally and within the United States. New variants that have rapidly become dominant within their countries have aroused concerns: B.1.1.7 (also known as VOC-202012/01 or Alpha), 501Y.V2 (B.1.351 or Beta), P.1 (B.1.1.28.1 or Gamma), Delta (B.1.617.2) and B.1.1.529 (Omicron, which includes BA.1, BA.2 and BA.3 sublineages).
The B.1.1.7 variant (23 mutations with 17 amino acid changes) was first described in the United Kingdom in December 2020; the 501Y.V2 variant (23 mutations with 17 amino acid changes) was initially reported in South Africa in December 2020; and the P.1 variant (approximately 35 mutations with 17 amino acid changes) was reported in Brazil in January 2021. By February 2021, the B.1.1.7 variant had been reported in 93 countries, the 501Y.V2 variant in 45, and the P.1 variant in 21. All three variants have the N501Y mutation, which changes the amino acid asparagine (N) to tyrosine (Y) at position 501 in the receptor-binding domain of the spike protein. The 501Y.V2 and P.1 variants both have two additional receptor-binding-domain mutations, K417N/T and E484K. These mutations increase the binding affinity of the receptor-binding domain to the angiotensin-converting enzyme 2 (ACE2) receptor. Four key concerns stemming from the emergence of the new variants are their effects on viral transmissibility, disease severity, reinfection rates (i.e., escape from natural immunity), and vaccine effectiveness (i.e., escape from vaccine-induced immunity). Recently, two more SARS-CoV-2 variants, B.1.427 and B.1.429, which were first detected in California, have been shown to be approximately 20% more transmissible than pre-existing variants and have been classified by the CDC as variants of concern. The B.1.617.2 Delta variant comprises the following substitutions in the Spike protein that are known to affect transmissibility of the virus: D614G, T478K, P681R and L452R. The B.1.1.529 (Omicron) variant was reported to the WHO in November 2021 and comprises 32 mutations in the Spike protein. Studies on these variants have provided compelling evidence that they have the potential to escape naturally-induced immunity as well as the immunity induced by currently approved vaccines. Indeed, recent evidence suggests that dual-Ab cocktails from Regeneron (casirivimab and imdevimab, targeting adjacent, non-overlapping epitopes) and Eli Lilly (bamlanivimab and etesevimab, targeting overlapping epitopes) are much less effective at neutralizing Omicron (Wilhelm et al., eBioMedicine, vol. 82, 104158 (2022); Gruell et al., Nature Medicine volume 28, pages 477-480 (2022)), and that current vaccines confer a reduced protection against infections by the Omicron variant (Buchan et al., JAMA Netw Open. 2022; 5(9): e2232760).
Thus, there is a need for the development of therapies against SARS-CoV-2, including SARS-CoV-2 variants, and that minimize the risk of viral escape.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
The present disclosure provides the following items 1 to 36:
Other objects, advantages and features of the present disclosure will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
In the appended drawings:
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the technology (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.
The use of any and all examples, or exemplary language (“e.g.”, “such as”) provided herein, is intended merely to better illustrate embodiments of the claimed technology and does not pose a limitation on the scope unless otherwise claimed.
No language in the specification should be construed as indicating any non-claimed element as essential to the practice of embodiments of the claimed technology.
Herein, the term “about” has its ordinary meaning. The term “about” is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value, or encompass values close to the recited values, for example within 10% of the recited values (or range of values).
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into the specification as if they were individually recited herein.
Where features or aspects of the disclosure are described in terms of Markush groups or list of alternatives, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member, or subgroup of members, of the Markush group or list of alternatives.
Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in virology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).
Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).
In the studies reported herein, the present inventors have shown that VE607 has the ability to inhibit or neutralize SARS-CoV-2, including SARS-CoV-2 variants of concern (VOC) such as Delta and Omicron.
The present disclosure provides a method for preventing or treating a SARS-CoV-2 infection or a related disease in a subject in need thereof, the method comprising administering to the subject an effective amount of VE607 or a pharmaceutically acceptable salt thereof. The present disclosure provides the use of VE607 or a pharmaceutically acceptable salt thereof for preventing or treating a SARS-CoV-2 infection or a related disease in a subject. The present disclosure provides the use of VE607 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for preventing or treating a SARS-CoV-2 infection or a related disease in a subject. The present disclosure provides VE607 or a pharmaceutically acceptable salt thereof for use in preventing or treating a SARS-CoV-2 infection or a related disease in a subject.
The present disclosure also provides a method for reducing the risk of developing a SARS-CoV-2-related disease or the severity of a SARS-CoV-2-related disease in a subject, the method comprising administering to the subject an effective amount of VE607 or a pharmaceutically acceptable salt thereof. The present disclosure provides the use of VE607 or a pharmaceutically acceptable salt thereof for reducing the risk of developing a SARS-CoV-2-related disease or the severity of a SARS-CoV-2-related disease in a subject. The present disclosure provides the use of VE607 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for reducing the risk of developing a SARS-CoV-2-related disease or the severity of a SARS-CoV-2-related disease in a subject. The present disclosure provides VE607 or a pharmaceutically acceptable salt thereof for use in reducing the risk of developing a SARS-CoV-2-related disease or the severity of a SARS-CoV-2-related disease in a subject.
The present disclosure also provides a method (in vitro or in vivo) for blocking the entry of SARS-CoV-2 in an ACE2-expressing cell, the method comprising contacting the SARS-CoV-2 and/or cell with an effective amount of VE607 or a pharmaceutically acceptable salt thereof. The present disclosure provides the use of VE607 or a pharmaceutically acceptable salt thereof for blocking the entry of SARS-CoV-2 in an ACE2-expressing cell. The present disclosure provides the use of VE607 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for blocking the entry of SARS-CoV-2 in an ACE2-expressing cell. The present disclosure provides VE607 or a pharmaceutically acceptable salt thereof for use in blocking the entry of SARS-CoV-2 in an ACE2-expressing cell.
VE607 (3,3′-(1,3-phenylenebis(oxy))bis(1-(piperidin-1-yl)propan-2-ol) is a compound of the following structure:
The term VE607 as used herein encompasses mixtures (e.g., racemic mixtures) of any one of the enantiomers of VE607, as well as isolated (pure) enantiomers, such as the (S,S)-VE607, (R,R)-VE607, and (R,S)-VE607 (meso) enantiomers.
In an embodiment, a racemic mixture is used or administered. In another embodiment, the (R,R)-VE607 enantiomer is used or administered. In another embodiment, the (S,S)-VE607 enantiomer is used or administered. In another embodiment, the (R,S)-VE607 enantiomer is used or administered.
The term “pharmaceutically acceptable salt” refers to salts of VE607 that are pharmacologically acceptable and substantially non-toxic to the subject to which they are administered. More specifically, these salts retain the biological effectiveness and properties of VE607 and are formed from suitable non-toxic organic or inorganic acids or bases.
For example, these salts include acid addition salts of VE607. Such acid addition salts include acetates, adipates, alginates, lower alkanesulfonates such as a methanesulfonates, trifluoromethanesulfonatse or ethanesulfonates, arylsulfonates such as a benzenesulfonates, 2-naphthalenesulfonates, or toluenesulfonates (also known as tosylates), ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cinnamates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides, hydrobromides, hydroiodides, hydrogen sulphates, 2-hydroxyethanesulfonates, itaconates, lactates, maleates, mandelates, methanesulfonates, nicotinates, nitrates, oxalates, pamoates, pectinates, perchlorates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates, sulfonates, tartrates, thiocyanates, undecanoates and the like. In an embodiment, the pharmaceutically acceptable acid salt of VE607 is a hydrochloride salt, including a dihydrochloride salt (3,3′-(1,3-phenylenebis(oxy))bis(1-(piperidin-1-yl)propan-2-ol)dihydrochloride).
VE607 or the pharmaceutically acceptable salt thereof binds to the receptor binding domain (RBD) of SARS-CoV-2 Spike (S) protein and blocks S protein-ACE2-mediated SARS-CoV-2 entry.
The amino acid sequence of the full-length Spike protein from SARS-CoV-2 (Wuhan strain, NCBI Reference Sequence YP_009724390.1, SEQ ID NO: 1) is depicted below:
Residues 1-12 correspond to the signal peptide, residues 13-685 correspond to the Spike protein subunit S1 and residues 686-1273 correspond to the Spike protein subunit S2. The receptor-binding domain (RBD) is defined by residues 319-541 (receptor-binding motif=residues 437-508). Residues 816-837 define the fusion peptide 1, residues 835-855 define the fusion peptide 2, residues 920-970 define the heptad repeat 1 and residues 1163-1202 define the heptad repeat 2.
SARS-CoV2 variants comprise mutations in the Spike protein including L5F, S13I, L18F, T19R, T20N, P26S, A67V, del69-70, G75V, T76I, D80Y, D80A, T95I, S98F, R102I, D138Y, G142D, del142-144, del144, W152C, E154K, EFR156-158G, F157L, R190S, ins214EPE, D215G, A222V, del246-252, D253G, W258L, N354D, F342L, V367F, K417N, K417T, A435S, W436R, N439K, N440K, G446V, L452R, Y453F, K458R, G476S, S477N, S477G, T478K, V483A, E484K, E484Q, F490S, N501Y, N501S, N501T, A570D, Q613H, D614G, A626S, A653V, H655Y, Q677H, Q677P, P681H, P681R, A701V, T716I, D796H, D796Y, T859N, F888L, D950N, S982A, T1027I, Q1071H, E1092K, H1101Y, D1118H, V1176F, G1219V, and V1122L.
The Delta variant comprises the following Spike protein mutations: T19R, (V70F*), T95I, G142D, E156-, F157-, R158G, (A222V*), (W258L*), (K417N*), L452R, T478K, D614G, P681R, D950N.
The Omicron variant (sublineage BA.1) comprises the following Spike protein mutations: A67V, del69-70, T95I, del142-144, Y145D, del211, L2121, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F.
In an embodiment, the methods and uses described herein relates to the inhibition of a SARS-CoV-2 variant comprising one of more of the above-noted mutations, e.g., to the prevention or treatment of diseases caused by such SARS-CoV-2 variant. In an embodiment, the methods and uses described herein relates to the inhibition of a SARS-CoV-2 variant comprising at least 50%, 60%, 70%, 80%, 90% or 95% of the Spike protein mutations of variant Omicron (BA.1, BA.2 and/or BA.3), e.g., to the prevention or treatment of diseases caused by such SARS-CoV-2 variant. In an embodiment, the methods and uses described herein relates to the inhibition of a SARS-CoV-2 variant comprising all of the Spike protein mutations of variant Omicron, e.g., to the prevention or treatment of diseases caused by such SARS-CoV-2 variant.
In another embodiment, VE607 or the pharmaceutically acceptable salt thereof is present in a composition. In an embodiment, the composition further comprises VE607 or the pharmaceutically acceptable salt thereof, and a carrier or excipient, in a further embodiment a pharmaceutically acceptable carrier or excipient. Such compositions may be prepared in a manner well known in the pharmaceutical art by mixing VE607 or the pharmaceutically acceptable salt thereof having a suitable degree of purity with one or more optional pharmaceutically acceptable carriers or excipients (see Remington: The Science and Practice of Pharmacy, by Loyd V Allen, Jr, 2012, 22nd edition, Pharmaceutical Press; Handbook of Pharmaceutical Excipients, by Rowe et al., 2012, 7th edition, Pharmaceutical Press). The carrier/excipient can be suitable for administration VE607 or the pharmaceutically acceptable salt thereof by any conventional administration route, for example, for oral, intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracisternal, intraperitoneal, intranasal or pulmonary (e.g., aerosol) administration. In an embodiment, the carrier/excipient is adapted for administration of VE607 or the pharmaceutically acceptable salt thereof by the oral route.
An “excipient” as used herein has its normal meaning in the art and is any ingredient that is not an active ingredient (drug) itself. Excipients include for example binders, lubricants, diluents, fillers, thickening agents, disintegrants, plasticizers, coatings, barrier layer formulations, lubricants, stabilizing agent, release-delaying agents and other components. “Pharmaceutically acceptable excipient” as used herein refers to any excipient that does not interfere with effectiveness of the biological activity of the active ingredients (VE607 or the pharmaceutically acceptable salt thereof) and that is not toxic to the subject, i.e., is a type of excipient and/or is for use in an amount which is not toxic to the subject. Excipients are well known in the art, and the present system is not limited in these respects. In certain embodiments, the composition includes excipients, including for example and without limitation, one or more binders (binding agents), thickening agents, surfactants, diluents, release-delaying agents, colorants, flavoring agents, fillers, disintegrants/dissolution promoting agents, lubricants, plasticizers, silica flow conditioners, glidants, anti-caking agents, anti-tacking agents, stabilizing agents, anti-static agents, swelling agents and any combinations thereof. As those of skill would recognize, a single excipient can fulfill more than two functions at once, e.g., can act as both a binding agent and a thickening agent. As those of skill will also recognize, these terms are not necessarily mutually exclusive. Examples of commonly used excipient include water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or auxiliary substances, such as emulsifying agents, preservatives, or buffers, which increase the shelf life or effectiveness. In an embodiment, VE607 or the pharmaceutically acceptable salt thereof is/are encapsulated in a vesicle or vesicle-like particle, such as a lipid vesicle (e.g., liposome). The term “lipid vesicle” (or “lipid-based vesicle”) as used herein encompasses macromolecular structures which as the main constituent include lipid or lipid derivatives. Suitable examples hereof are liposomes and micelles including detergent micelles/lipid emulsion, liposomes prepared from palmitoyloleoylphosphatidylcholine, hydrogenated soy phosphatdylcholine, and solid lipid nanoparticles prepared from steric acid or tripalmitin. The term liposome is used herein in accordance with its usual meaning, referring to microscopic lipid vesicles composed of a bilayer of phospholipids or any similar amphipathic lipids encapsulating an internal aqueous medium. The liposomes may be unilamellar vesicles such as small unilamellar vesicles (SUVs), which typically have a diameter of less than 0.2 μm (e.g., between 0.02 and 0.2 μm), and large unilamellar vesicles (LUVs), and multilamellar vesicles (MLV), which typically have a diameter greater than 0.45 μm (in some cases greater than 1 μm). No particular limitation is imposed on the liposomal membrane structure in the present disclosure. The term liposomal membrane refers to the bilayer of phospholipids separating the internal aqueous medium from the external aqueous medium. The composition may also comprise one or more additional active agents for the treatment the targeted disease/condition or for the management of symptom(s) of the targeted disease/condition (e.g., pain killers, anti-nausea agents, anti-coagulants, anti-inflammatory agents, immunotherapeutic agents, etc.).
For the prevention, treatment or reduction in the severity of a given disease or condition (viral disease such as COVID-19), the appropriate dosage of VE607 or the pharmaceutically acceptable salt thereof (or pharmaceutical composition described herein) will depend on the type of disease or condition to be treated, the severity and course of the disease or condition, whether VE607 or the pharmaceutically acceptable salt thereof, or pharmaceutical composition, is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to VE607 or the pharmaceutically acceptable salt thereof, and the discretion of the attending physician. The VE607 or the pharmaceutically acceptable salt thereof may be suitably administered to the patient at one time or over a series of treatments. Preferably, it is desirable to determine the dose-response curve in vitro, and then in useful animal models prior to testing in humans. The present disclosure provides dosages for VE607 or the pharmaceutically acceptable salt thereof, or pharmaceutical composition. For example, depending on the type and severity of the disease, about 1 μg/kg to 1000 mg per kg (mg/kg) of body weight per day. Further, the effective dose may be 0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg/25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, and may increase by 25 mg/kg increments up to 1000 mg/kg, or may range between any two of the foregoing values. A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
As used herein the term “treating” or “treatment” in reference to viral infection or disease is meant to refer to administration of the agent after infection that leads to a reduction/improvement in one or more symptoms or pathological features associated with said viral disease (e.g., COVID-19). Non-limiting examples include a decrease in viral load, reduction of cough, fever, fatigue, shortness of breath, reduction/prevention of acute respiratory distress syndrome (ARDS), reduction/prevention of multi-organ failure, septic shock, and blood clots, hospitalization, etc.
As used herein the term “preventing” or “prevention” in reference to viral infection or disease is meant to refer to administration of the agent prior to infection that leads to protection from being infected or from developing the viral disease (e.g., COVID-19), to a delay in the development of the disease, or to a reduction of one or more symptoms or pathological features associated with the viral disease.
VE607 or the pharmaceutically acceptable salt thereof, or pharmaceutical composition described herein may be used alone or in combination with other prophylactic agents such as antivirals, anti-inflammatory agents, anti-coagulants, vaccines, immunotherapies (antibody-based therapies), etc. The combination of active agents and/or compositions comprising same may be administered or co-administered (e.g., consecutively, simultaneously, at different times) in any conventional dosage form. Co-administration in the context of the present disclosure refers to the administration of more than one therapeutic in the course of a coordinated treatment to achieve an improved clinical outcome. Such co-administration may also be coextensive, that is, occurring during overlapping periods of time. For example, a first agent (e.g., VE607 or the pharmaceutically acceptable salt thereof) may be administered to a patient before, concomitantly, before and after, or after a second active agent (e.g., an antiviral or anti-inflammatory agent) is administered. The agents may in an embodiment be combined/formulated in a single composition and thus administered at the same time. In another embodiment, VE607 or the pharmaceutically acceptable salt thereof is used in combination with one or anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof (or nucleic acids encoding same), i.e., an antibody cocktail.
In an embodiment, VE607 or the pharmaceutically acceptable salt thereof is for administration prior to exposure to SARS-CoV-2. In another embodiment, VE607 or the pharmaceutically acceptable salt thereof is for administration after exposure to SARS-CoV-2. In another embodiment, VE607 or the pharmaceutically acceptable salt thereof is for administration prior to and after exposure to SARS-CoV-2.
In an embodiment, VE607 or the pharmaceutically acceptable salt thereof is for administration prior to development of the viral disease (e.g., COVID-19). In another embodiment, VE607 or the pharmaceutically acceptable salt thereof is for administration after development of the viral disease (e.g., COVID-19). In another embodiment, VE607 or the pharmaceutically acceptable salt thereof is for administration prior to and after development of the viral disease (e.g., COVID-19).
In an embodiment, the subject or patient has a weakened immune system and a reduced ability to fight viral infections such as SARS-CoV-2 infection. In another embodiment, the subject or patient is an immunosuppressed or immunocompromised subject or patient. Immunosuppression may be caused by certain diseases or conditions, such as AIDS, cancer, diabetes, malnutrition, and certain genetic disorders, or certain drugs or treatments such as anticancer drugs, radiation therapy, and stem cell or organ transplant. In an embodiment, the subject or patient is an elderly subject or patient, for example a subject or patient having 60 years old or more, 65 years old or more, 70 years old or more, 75 years old or more, or 80 years old or more, who typically develop a weaker immune response to vaccines and infections.
In another aspect, the disclosure provides kits for preventing or treating a SARS-CoV-2 infection or a related disease in a subject, or for reducing the risk of developing a SARS-CoV-2-related disease or the severity of a SARS-CoV-2-related disease in a subject, for blocking the entry of SARS-CoV-2 in an ACE2-expressing cell, the kits comprising VE607 or the pharmaceutically acceptable salt thereof. Kits include one or more containers comprising by way of example, and not limitation, VE607 or the pharmaceutically acceptable salt thereof and instructions for use in accordance with any of the methods of the disclosure. The containers can be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
The kits are provided in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, syringes, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. A kit can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container can also have a sterile access port (e.g., the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Kits can optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container.
The present disclosure is illustrated in further details by the following non-limiting examples.
The plasmids expressing the human coronavirus Spike D614G of SARS-CoV-2 was kindly provided by Stefan Pöhlmann and was previously reported (Hoffmann et al., 2020b). The pNL4.3 R-E-Luc was obtained from NIH AIDS Reagent Program. The codon-optimized RBD sequence (encoding residues 319-541) fused to a C-terminal hexahistidine tag was cloned into the pcDNA3.1(+) expression vector and was reported elsewhere (Beaudoin-Bussieres et al., 2020). The mutants encoding variants Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1) were codon-optimized and synthesized by Genscript. Plasmids encoding the Delta and Omicron Spikes were generated by overlapping PCR using a codon-optimized wild-type SARS-CoV-2 Spike gene (GeneArt, ThermoFisher) that was synthesized (Biobasic) and cloned in pCAGGS as a template (Chatterjee et al., 2021; Tauzin et al., 2022). The vesicular stomatitis virus G (VSV-G)-encoding plasmid (pSVCMV-IN-VSV-G) was previously described (Emi et al., 1991).
293T human embryonic kidney cells (obtained from ATCC) and Vero E6 cells (ATCC CRL-1586™) were maintained at 37° C. under 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) (Wisent) containing 5% fetal bovine serum (VWR), 100 UI/ml of penicillin and 100 μg/ml of streptomycin (Wisent). The 293T-ACE2 cell line was previously reported (Prevost et al., 2020).
FreeStyle 293 F cells (Invitrogen) were grown in FreeStyle 293F medium (Invitrogen) to a density of 1×106 cells/mL at 37° C. with 8% CO2 with regular agitation (150 rpm). Cells were transfected with a plasmid coding for SARS-CoV-2 S RBD using ExpiFectamine 293 transfection reagent, as directed by the manufacturer (Invitrogen). One week later, cells were pelleted and discarded. Supernatants were filtered using a 0.22 μm filter (Thermo Fisher Scientific). The recombinant RBD proteins were purified by nickel affinity columns, as directed by the manufacturer (Invitrogen). The RBD preparations were dialyzed against phosphate-buffered saline (PBS) and stored in aliquots at −80° C. until further use. To assess purity, recombinant proteins were loaded on SDS-PAGE gels and stained with Coomassie Blue.
DSF experiments were essentially performed as described previously (Sharifahmadian et al., 2017). DSF was conducted using 5 μM of purified RBD, 10× concentration of SYPRO Orange (from 5000× stock solution, ThermoFisher) in 50 mM HEPES, 100 mM NaCl, pH 7.5 and 5% final concentration of DMSO. The small molecules were added to final concentrations of 5 mM. SYPRO Orange fluorescence was monitored over 20-95° C. with a LightCycler® 480 instrument (Roche, USA). The LightCycler® 480 Software (Roche) was used to calculate the first derivate of the resulting melting curve, with the steepest point of the slope being the Tm.
Neutralization assay using pseudoviral particles. Target cells were infected with single-round luciferase-expressing lentiviral particles as described previously (Prevost et al., 2020). Briefly, 293T cells were transfected by the calcium phosphate method with the lentiviral vector pNL4.3 R-E-Luc (NIH AIDS Reagent Program) and a plasmid encoding for SARS-CoV-2 Spike at a ratio of 5:4. Two days post-transfection, cell supernatants were harvested and stored at −80° C. until use. 293T-ACE2 target cells were seeded at a density of 1×104 cells/well in 96-well luminometer-compatible tissue culture plates (Perkin Elmer) 24 h before infection. Recombinant viruses in a final volume of 100 μl were incubated with the indicated concentrations of small molecules (VE607 and SSAA009E2) up to concentrations of 100 μM for 1 h at 37° C. and were then added to the target cells followed by incubation for 48 h at 37° C.; cells were lysed by the addition of 30 μl of passive lysis buffer (Promega) followed by one freeze-thaw cycle. An LB941 TriStar luminometer (Berthold Technologies) was used to measure the luciferase activity of each well after the addition of 100 μl of luciferin buffer (15 mM MgSO4, 15 mM KPO4 [pH 7.8], 1 mM ATP, and 1 mM dithiothreitol) and 50 μl of 1 mM d-luciferin potassium salt (Prolume). The neutralization half-maximal inhibitory dilution (ID50) represents the sera dilution to inhibit 50% of the infection of 293T-ACE2 cells by recombinant viruses.
Microneutralization with authentic virus. One day prior to infection, 2×104 Vero E6 cells were seeded per well in the 96-well flat bottom plate and incubated overnight to permit Vero E6 cell adherence. Compounds dilutions ranged from 0, 0.316, 1, 3.16, 10, 31.6 and 100 μM were performed in a separate 96 well culture plate using DMEM supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), HEPES, 0.12% sodium bicarbonate, 2% FBS and 0.24% BSA. 104 TCID50/mL of SARS-CoV-2 virus was prepared in DMEM+2% FBS and combined with an equivalent volume of diluted compounds for one hour. After this incubation, all media was removed from the 96 well plate seeded with Vero E6 cells and virus: compounds mixture was added to each respective well at a volume corresponding to 600 TCID50 per well and incubated for one hour further at 37° C. Both virus only and media only (MEM+2% FBS) conditions were included in this assay. All virus-compounds supernatant was removed from wells without disrupting the Vero E6 monolayer. Each diluted compound (100 μL) was added to its respective Vero E6-seeded well in addition to an equivalent volume of MEM+2% FBS and was then incubated for 48 hours. Media was then discarded and replaced with 10% formaldehyde for 24 hours to cross-link Vero E6 monolayer. Formaldehyde was removed from wells and subsequently washed with PBS. Cell monolayers were permeabilized for 15 minutes at room temperature with PBS+0.1% Triton X-100, washed with PBS and then incubated for one hour at room temperature with PBS+3% non-fat milk. An anti-mouse SARS-CoV-2 nucleocapsid protein (Clone 1C7, Bioss Antibodies) primary antibody solution was prepared at 1 μg/mL in PBS+1% non-fat milk and added to all wells for one hour at room temperature. Following extensive washing (3 ×) with PBS, an anti-mouse IgG HRP secondary antibody solution was formulated in PBS+1% non-fat milk. One hour post-room temperature incubation, wells were washed with 3×PBS, substrate (ECL) was added and an LB941 TriStar luminometer (Berthold Technologies) was used to measure the signal each well.
Using the standard calcium phosphate method, 10 μg of Spike expressor and 2.5 μg of a green fluorescent protein (GFP) expressor (pIRES-GFP) were transfected into 2×106 293T cells. 48 h post-transfection, Spike-expressing cells were incubated with 100 μM of VE607 or equivalent volume of vehicle (DMSO) and incubated for 30 min at room temperature. CV3-25 (5 μg/ml) or with sACE2 (100 μg/ml) was added to the cells and incubated for 45 min at 37° C. and sACE2 binding was detected using a polyclonal Goat anti-human ACE2 (RND Systems) at 1/100 dilution at room temperature for 30 min. AlexaFluor-647-conjugated goat anti-human IgG (H+L) Ab (Invitrogen) and AlexaFluor-conjugated donkey anti-goat IgG (H+L) Ab (Invitrogen) was used as secondary antibodies. AF647 and analyzed by flow cytometry. The percentage of transfected cells (GFP+ cells) was determined by gating the living cell population based on viability dye staining (Aqua Vivid, Invitrogen). sACE2-AF594 Binding levels were normalized to signals obtained with the conformationally-independent, anti-S2 CV3-25 mAb (Li et al., 2022; Prevost et al., 2021; Tauzin et al., 2022). Samples were acquired on a LSRII cytometer (BD Biosciences, Mississauga, ON, Canada) and data analysis was performed using FlowJo vX.0.7 (Tree Star, Ashland, OR, USA).
General Procedures. All reactions were conducted in oven-dried glassware under an inert atmosphere of nitrogen, unless otherwise stated. All solvents were reagent or high-performance liquid chromatography (HPLC) grade. Anhydrous THF was obtained from the Pure Solve™ PS-400 system under an argon atmosphere. All reagents were purchased from commercially available sources and used as received. Reactions were magnetically stirred under a nitrogen atmosphere, unless otherwise noted and were monitored by thin layer chromatography (TLC) was performed on pre-coated silica gel 60 F-254 plates (40-55 micron, 230-400 mesh) and visualized by UV light or staining with KMnO4 and heating. Yields refer to chromatographically and spectroscopically pure compounds. Optical rotations were measured on a JASCO P-200 polarimeter. Proton (1H) and carbon (13C) NMR spectra were recorded on a Bruker Avance III 500-MHz spectrometer. Chemical shifts (6) are reported in parts per million (ppm) relative to chloroform (δ 7.26) or methanol (δ 3.31) for 1H NMR, and chloroform (δ 77.2) or methanol (δ 49.0). High resolution mass spectra (HRMS) were recorded at the University of Pennsylvania Mass Spectroscopy Service Center on either a VG Micromass 70/70H or VG ZAB-E spectrometer. Lyophilization was performed in a Labconco FreeZone 12 Plus lyophilizer (0.148 mbar). The purity of new compounds were judged by NMR and LCMS (>95%).
Resorcinol (1) (100 mg, 0.908 mmol, 1 equiv) was dissolved in enantiopure (R)-epichlorohydrin (0.569 mL, 7.27 mmol, 8.0 equiv) and heated to 100° C. A solution of NaOH (72.7 mg, 1.82 mmol, 2.0 equiv) in EtOH (0.75 mL) was then added dropwise. After stirring at 100° C. for three hours, the solution was cooled to r.t., diluted with acetone and filtered through a fritted funnel packed with celite. The filtrate was then concentrated in vacuo which was purified via flash column chromatography (5% to 10% EtOAc/hexanes) to yield the title compound as a yellow oil in 97% yield. 1H NMR (500 MHz, CDCl3): δ 7.17 (t, J=8.08 Hz, 1H), 6.55-6.51 (m, 3H), 4.20 (dd, J=7.84, 3.17 Hz, 2H), 3.95 (dd, J=5.66, 5.29 Hz, 2H), 3.36-3.33 (m, 2H), 2.90 (t, J=4.77 Hz, 2H). 2.75 (dd, J=2.63, 2.35 HZ, 2H); 13C NMR (125 MHz, CDCl3): δ 159.76, 130.06, 107.44, 101.94, 68.83, 50.13, 44.87; HRMS (ESI) m/z: [M+Na]+ calcd 245.0790, found 245.0791; [α]D23 +17.28 (c 7.4, MeOH).
Epoxide 2R (25 mg, 0.113 mmol, 1.0 equiv) was dissolved in neat piperdine (0.177 mL, 1.8 mmol, 16 equiv) at 0° C. and stirred overnight at room temperature. Upon completion, solvent was removed in vacuo, dissolved in DCM, washed with brine, dried with Na2SO4, filtered through a cotton plug, and concentrated in vacuo. The crude product mixture was then dissolved in water, frozen, and lyophilized to remove excess piperdine. Product was isolated as a yellow oil in 99% yield. 1H NMR (500 MHz, CD3OD): δ 7.17-7.13 (m, 1H), 6.55-6.52 (m, 3H), 4.15-4.10 (m, 2H), 3.96 (dd, J=5.59, 4.31 Hz, 2H), 3.89 (dd, J=5.93, 3.82, 2H), 2.57-2.46 (m, 12H), 1.64-1.59 (m, 8H), 1.51-1.44 (m, 4H); 13C NMR (125 MHz, CD3OD): δ 161.62, 130.96, 108.15, 102.78, 72.15, 68.36, 63.18, 56.28, 47.47, 27.07, 26.77, 25.55, 25.23; HRMS (ESI) m/z: [M+H]+ calcd 393.2753, found 393.276; [α]D23+3.2 (c 3.3, MeOH).
Resorcinol (1) (100 mg, 0.908 mmol, 1 equiv) was dissolved in enantiopure (S)-epichlorohydrin (0.569 mL, 7.27 mmol, 8.0 equiv) and heated to 100° C. A solution of NaOH (72.7 mg, 1.82 mmol, 2.0 equiv) in EtOH (0.75 mL) was then added dropwise. After stirring at 100° C. for three hours, the solution was cooled to r.t., diluted with acetone and filtered through a fritted funnel packed with celite. The filtrate was then concentrated in vacuo which was purified via flash column chromatography (5% to 10% EtOAc/hexanes) to yield the title compound as a yellow oil in 97% yield. 1H NMR (500 MHz, CDCl3): δ 7.17 (t, 7.70 Hz, 1H), 6.55-6.51 (m, 3H), 4.20 (dd, J=7.76, 3.13 Hz, 2H), 3.93 (dd, J=5.77, 5.34 Hz, 2H), 3.36-3.33 (m, 2H), 2.90 (t, J=4.87, 2H), 2.75 (dd, 2.68, 2.29, 2H); 13C NMR (500 MHz, CDCl3): δ 159.74, 130.04, 107.42, 101.92, 68.81, 50.11, 44.74; HRMS (ESI) m/z: [M+Na]+ calcd 245.0805, found 245.0805; [α]D23 −17.09 (c 7.4, MeOH).
Epoxide 2 (25 mg, 0.113 mmol, 1.0 equiv) was dissolved in neat piperdine (0.177 mL, 1.8 mmol, 16 equiv) at 0° C. and stirred overnight at room temperature. Upon completion, solvent was removed in vacuo, dissolved in DCM, washed with brine, dried with Na2SO4, filtered through a cotton plug, and concentrated in vacuo. The crude product mixture was then dissolved in water, frozen, and lyophilized to remove excess piperdine. Product was isolated as a yellow oil in 99% yield. 1H NMR (500 MHz, CD3OD): δ 7.18-7.14 (m, 1H), 6.56-6.52 (m, 3H), 4.18-4.14 (m, 2H), 3.96 (dd, J=4.51, 4.45 Hz, 2H), 3.89 (dd, J=5.79, 5.73 Hz), 2.73-2.62 (m, 12H), 1.66-1.57 (m, 8H), 1.56-1.48 (m, 4H); 13C NMR (125 MHz, CD3OD): δ 121.91, 130.96, 108.14, 102.78, 72.15, 68.33, 63.18, 56.27, 47.59, 27.33, 26.78, 25.74, 25.23; HRMS (ESI) m/z: [M+H]+ calcd 415.2567, found 415.2567; [α]D23 −2.96 (c 3.3, MeOH).
System preparation, modeling, and docking calculation were performed using the Schrödinger Suite package (version 2020-4) [Maestro, Schrödinger, LLC, New York, NY, 2020], using default parameters unless otherwise noted. The target structures were taken from SARS-CoV-1 RBD (PDB ID: 6waq [doi: 10.2210/pdb6WAQ/pdb]) and SARS-CoV-2 RBD (PDB ID: 6w41 [doi: 10.2210/pdb6W41/pdb]) prepared using the Protein Preparation Wizard [doi: 10.1007/s10822-013-9644-8]. To prepare the structures, force field atom types and bond orders were assigned, missing atoms and side-chains were added, protonation states of ionizable amino acid side-chains were determined using PROPKA [doi: 10.1021/ct100578z], water orientations were sampled, and hydrogen bond networks were subsequently optimized by flipping Asn/Gln/His residues and sampling hydroxyl/thiol hydrogen. Constrained energy minimization was then performed using the imperf module from impact [Impact, Schrödinger, LLC, New York, NY, 2020] to generate the structure to be used in the subsequent modeling calculations. Potential binding sites were explored and characterized using the SiteMap tool [doi:10.1021/ci800324m; doi:10.1111/j.1747-0285.2007.00483.x]. VE607 compound was structurally preprocessed using LigPrep [LigPrep, Schrödinger, LLC, New York, NY, 2020] to generate multiple states for stereoisomers, tautomers, ring conformations, and protonation states at a selected pH range. Then, energy minimization was performed with the OPLS3e force field [doi: 10.1021/acs.jctc.8b01026]. The prepared molecular structures were docked into the putative binding sites using Glide [doi: 10.1021/jm030644s; doi: 10.1021/jm0306430] with the standard precision (SP) scoring function to evaluate enrichment of the calculated docked models.
Statistics were analyzed using GraphPad Prism version 8.0.2 (GraphPad, San Diego, CA, (USA). Every data set was tested for statistical normality and this information was used to apply the appropriate (parametric or nonparametric) statistical test. P values<0.05 were considered significant; significance values are indicated as * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.
It was initially tested whether the previously described SARS-CoV-1 inhibitors VE607 (Kao et al., 2004) and SSAA09E2 (Adedeji et al., 2013) bind the SARS-CoV-2 RBD (
To assess the effects of VE607 and SSAA09E2 using an in vitro model, the SARS-CoV-1 and SARS-CoV-2 S glycoproteins as well as the VSV-G glycoprotein as negative control were expressed on the surface of pseudoviral particles carrying the luciferase gene in their genomes. These particles were used for infection of ACE2-expressing 293T cells (Prevost et al., 2020) in the presence of increasing concentrations of VE607 and SSAA09E2. VE607 specifically inhibited pseudoviral particles bearing the SARS-CoV-1 Spike (IC50=1.47 μM,
Commercially available VE607 is a mixture of three stereochemical isomers, comprised of the (S,S)-VE607, (R,R)-VE607, and the meso (R,S)-VE607, mixed at a ratio of 1:1:2. Enantiomers was obtained by chemical synthesis and tested for their ability to neutralize pseudoviral particles bearing the SARS-CoV-2 S protein. No substantial differences in the SARS-CoV-2 pseudoviral inhibition among these enantiomers obtained by synthesis, with the meso (R,S)-VE607 enantiomer being slightly less potent (about 2-fold) than the two other enantiomers (
The in silico docking identified RBD residues Y505 and Q498 as potential contact sites for VE607 (
It was next assessed whether VE607 affects the RBD-ACE2 interaction. Briefly, the capacity of VE607 to compete with soluble ACE2 conjugated to Alexa-Fluor 594 for interaction with the full SARS-CoV-2 Spike, expressed at the cell surface, was measured by flow cytometry as described previously (Anand et al., 2020; Anand et al., 2021). Surprisingly, no competition between VE607 (100 μM) and sACE2-AF647 was observed (
SARS CoV-2 is in constant evolution as variants of concern keep emerging. It was tested whether VE607 inhibits pseudoviral particles bearing the Spike glycoproteins from the major VOCs (Alpha, Beta, Gamma, Delta and Omicron (BA.1, BA.4/5, BA.4.6, XBB or BQ.1.1 sublineage)). VE607 inhibited all VOCs with similar potency with IC50 values in the low micromolar range (
Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the word “comprising” is used as an open-ended term, substantially equivalent to the phrase “including, but not limited to”. The singular forms “a”, “an” and “the” include corresponding plural references unless the context clearly dictates otherwise.
The present application claims the benefit of U.S. provisional patent application No. 63/267,409, filed on Feb. 1, 2022, which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2023/050130 | 1/31/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63267409 | Feb 2022 | US |
