WO2021198534A1

WO2021198534A1 - Plasma kallikrein inhibitors for use in the treatment of coronaviral disease

Info

Publication number: WO2021198534A1
Application number: PCT/EP2021/058970
Authority: WO
Inventors: Peter Verhamme; Marc VANHOVE
Original assignee: Oxurion NV; Katholieke Universiteit Leuven
Priority date: 2020-04-04
Filing date: 2021-04-06
Publication date: 2021-10-07

Abstract

The present invention relates to the treatment of coronaviral diseases and the respiratory diseases associated therewith. The present invention provides inhibitors of plasma kallikrein and the kallikrein-kinin pathway for such use, as well as pharmaceutical compositions comprising such inhibitors and methods for their use.

Description

PLASMA KALLIKREIN INHIBITORS FOR USE IN THE TREATMENT OF CORONAVIRAL DISEASE

FIELD OF INVENTION

BACKGROUND OF THE INVENTION In the last 20 years, several viral epidemics developed such as the severe acute respiratory syndrome coronavirus (SARS-CoV-1) in 2002 to 2003, the H1N1 influenza in 2009, and the Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012. Most recently, in December, 2019, a series of pneumonia cases of unknown etiology emerged in Wuhan, Hubei, China, with clinical presentations greatly resembling viral pneumonia. Subsequent investigations revealed a novel coronavirus. Initially, the new virus was called 2019-nCoV, but was later termed SARS-CoV-2 due to its similarity to the one that caused the SARS outbreak (SARS-CoV-1) in 2002.

Coronaviruses (CoVs) are a large family of single-stranded RNA viruses with a crown-like appearance under an electron microscope due to the presence of spike glycoproteins on the envelope and have become the major pathogens of emerging respiratory disease outbreaks. The virus can be transmitted from animal-to-human as was presumed to be the main mechanism for the first cases but can also be transmitted from human-to-human. The transmission is believed to occur through respiratory droplets from coughing and sneezing similar to other respiratory pathogens. The SARS-CoV-2 virus can trigger a potentially deadly respiratory disease called COVID-19. COVID-19 affects the most the elderly and the immunocompromised with chronic medical conditions. The entry of SARS-CoV-1 into human host cells is mediated mainly by a cellular receptor angiotensin-converting enzyme 2 (ACE2), which is expressed in human airway epithelia, pulmonary parenchyma, vascular endothelia, kidney cells, and small intestine cells. However, the presence of ACE2 solely is not sufficient to make host cells susceptible to infection. For example, some ACE2-expressing endothelial cells and human intestinal cell lines failed to be infected by SARS-CoV-1 (Chan et al., Med Virol. 2004;74:1-7.; Ding et al J Pathol. 2003;200:282-289), while some cells without a detectable expression level of ACE2, such as hepatocytes could also be infected by SARS-CoV-1 (To and Lo, J Pathol. 2004;203:740-743). In light of the high similarity between SARS-CoV-1 and SARS-CoV2, it is assumed that SARSCoV-2 also possesses a similar potential (Li et al., Med Virol. 2020:1-4, DOI: 10.1002/jmv25728). The clinical spectrum of COVID-19 varies from asymptomatic carrier stage to acute respiratory disease (such as mild fever, dry cough, sore throat, dyspnoe, myalgia, fatigue) and to pneumonia of varying degrees of severity. Chest CT scans of all 41 patients presented in one of the first reports by Huang et al showed pneumonia with abnormal findings. Approximately, one third of the patients (N=13; 32%) required ICU care and 6 patients (15%) died. (Huang et al., The Lancet, Vol 395 February 15, 2020: 497- 506).

Other complications included acute respiratory distress syndrome (12 [29%]), anemia (six [15%]), acute cardiac injury (five [12%]) and secondary infection (four [10%]). Compared with non-ICU patients, ICU patients had higher plasma levels of IL2, IL7, IL10, GSCF, IP10, MCP1, MIP1A, and TNFa.

Authors of clinical and epidemiological data from the Chinese CDC based on 72,314 cases (Wu and McGoogan, JAMA 2020 Feb) divided the clinical manifestation of the disease by severity:

• Mild disease: non-pneumonia and mild pneumonia (81% of the cases)

• Severe disease: dyspnea (14%)

• Critical disease: respiratory failure, septic shock, and/or multiple organ dysfunction (MOD) or failure (MOF) (5%) Since then, other reports and health policy agencies divide the clinical manifestations according to the severity such as mild, moderate or severe. Only the severe clinical manifestations include severe pneumonia, acute respiratory distress syndrome (ARDS), sepsis and septic shock.

Acute respiratory distress syndrome may develop in 3.4% [Guan NEJM 2020 (n=1099)] - 17% [Chen Lancet 2020 (n=99)] of patients based on different reports. The fatality rate is about 1.4-2% based on the report of the WHO of Feb 25, 2020 and Guan et al (Guan W, Ni Z, Hu Y et al. Clinical characteristics of coronavirus disease 2019 in China. NEJM :10.1056/NEJMMoa2002032)

A meta-analysis by Sun et al (Sun P et al., J Med Virol 2020. https://doi.org/10.1002/ jmv.25735. https://doi.Org/10.1016/j.ijid.2020.03.017),) based on ten studies, included a total number of 50466 patients. It confirmed that a vast majority of patients with SARS-CoV-2 infection (96.6%) have abnormal chest CT examination. In this analysis, the incidence of acute respiratory distress syndrome (ARDS) reached 14.8%.

Another meta-analysis by Rodriguez-Morales et al (Rodriguez-Morales et al., Travel Medicine and Infectious Disease, Mar 2020 https://doi.Org/10.1016/j.tmaid.2020.101623) based on 656 patients showed that 32.8% were presented with acute respiratory distress syndrome (ARDS) (95%CI 13.7-51.8).

Acute respiratory distress syndrome (ARDS) is a type of respiratory failure characterized by increased permeability of the edema, which is interpreted as being an accumulation of protein-rich edema fluid in the alveoli and is mediated by inflammation of various mechanisms. Symptoms include shortness of breath, rapid breathing, and bluish skin coloration. For those who survive, a decreased quality of life is common. Causes may include sepsis, pancreatitis, trauma, pneumonia, and aspiration. The underlying mechanism involves diffuse injury to cells which form the barrier of the microscopic air sacs of the lungs, surfactant dysfunction, activation of the immune system, and dysfunction of the body's regulation of blood clotting. In effect, ARDS impairs the lungs' ability to exchange oxygen and carbon dioxide. The primary treatment involves mechanical ventilation together with treatments directed at the underlying cause.

Tian et al (Tian et al., J. Thorac Oncol. 2020 Feb 28) reported histopathological data obtained on the lungs of two patients who underwent lung lobectomies for adenocarcinoma and retrospectively found to have had the infection at the time of surgery. Apart from the tumors, the lungs of both cases showed edema and important proteinaceous exudates as large protein globules. The authors also reported vascular congestion combined with inflammatory clusters of fibrinoid material and multinucleated giant cells ad hyperplasia of pneumocytes.

Without an effective cure for treating and preventing COVID-19 and other coronaviral diseases, present research focuses on ACE2, which is required for cellular entry (Zhou et al., Nature 2020; 579:270- 273 and Floffmann et al. Cell 2020 Mar 4). Strategies under consideration include anti-ACE2 antibodies as well as soluble ACE2 to prevent cellular entry. In addition, Transmembrane protease, serine 2 (TMPRSS2) receives significant attention as it has been shown to lead to SARS-CoV-2 S protein priming and as administration of theTMPRSS2 inhibitor camostat mesilate partically blocked SARS-CoV-2 entry into Caco- 2 and Vero-TMPRSS2 cells (Floffmann et al. Cell 2020 Mar 4). Interestingly, Milewska et al. (BioRxiv, 2 Mar 2020) postulated that the tissue kallikrein named human kalli krein-related peptidase 13 (KLK13) would be essential for S protein priming in human coronavirus HKU1 (HCoV-HKUl). They observed that inhibition of KLK13 inhibited HCoV-HKUl replication, while other tissue kallikrein inhibitors did not.

In the absence of efficacious prevention and treatment options of coronaviral diseases, there is an urgent need for alternative approaches and therapies. Additionally, patients that contract the disease should have access to therapies that improve their outcome and reduce, potentially long-term or chronic, effects on the respiratory tract.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that plasma kallikrein inhibitors are effective for such treatment. Therefore, in a particular embodiment, the present invention provides a plasma kallikrein inhibitor for use in the treatment of a coronaviral disease. Although not limited thereto, the inhibitors of the present invention are found to be particularly effective for reducing pulmonary edema associated with coronaviral diseases. Therefore, in another particular embodiment, the present invention provides a plasma kallikrein inhibitor for use in the treatment and/or prevention of pulmonary edema associated with a coronaviral disease.

In a particular embodiment, the coronaviral disease is caused by a Betacoronavirus, such as OC43, HKU1, SARS-CoV-1, SARS-CoV-2, and MERS-CoV. In a further embodiment, the coronaviral disease is caused by a Severe Acute Respiratory Syndrome-related coronavirus (SARS-CoV). In a preferred embodiment, the coronaviral disease is caused by SARS-CoV-2. The plasma kallikrein inhibitor for use in the invention is preferably a direct plasma kallikrein inhibitor.

It may for example be selected from the group consisting of an antibody (such as lanadelumab), a peptide (such as ecallantide or Cl inhibitor), a nucleotide (such as a silencing RNA), or a chemical molecule (such as berotralstat or a compound of Formula A)

Formula A.

Additionally, the plasma kallikrein inhibitor for use in the invention is preferably a selective inhibitor for plasma kallikrein that shows no or only weak inhibition of other serine proteases. In a particular embodiment, the plasma kallikrein inhibitor has an inhibition constant that is at least 100 times lower for plasma kallikrein than for tissue kallikrein and transmembrane protease, serine 2 (TMPRSS2).

Particular plasma kallikrein inhibitors that may be used in the invention are selected from the group consisting of ecallantide, lanadelumab, Cl inhibitor, a compound of formula A, or a compound comprising a polypeptide sequence comprising at least three cysteine residues separated by two peptide loops, wherein the at least three cysteine residues are covalently attached to a molecular scaffold and wherein each peptide loop independently comprises from 2 to 10 amino acids.

In a further embodiment, the plasma kallikrein inhibitor for use in the invention is a compound comprising a polypeptide sequence comprising at least three cysteine residues separated by two peptide loops, wherein the at least three cysteine residues are covalently attached to a molecular scaffold, wherein each peptide loop independently comprises from 2 to 10 amino acids, and wherein the first peptide loop comprises the sequence SFPYR (SEQ ID NO: 1), wherein optionally P is replaced with azetidine carboxylic acid and/or R is replaced with homoarginine or N-methylarginine. For example, the plasma kallikrein inhibitor may be a compound comprising [Cl]SF(Aze)Y(HArg)[C2]VYYPDI[C3] (SEQ ID NO: 2), wherein Aze is azetidine carboxylic acid, HArg is homo-arginine, and wherein the cysteine residues [Cl] to [C3] are attached via thioether linkages to the molecular scaffold. Alternatively, the plasma kallikrein inhibitor may be a compound comprising [Cl]SF(Aze)Y(HArg)[C2](Ala(i]jCH₂NH))HQDL[C3] (SEQ ID NO: 3) wherein Aze is azetidine carboxylic acid, HArg is homo-arginine, Ala(i]jCH₂NH) is alanine wherein the carbonyl is reduced to methylene, and wherein the cysteine residues [Cl] to [C3] are attached via thioether linkages to the molecular scaffold. The present invention further provides pharmaceutical compositions comprising a plasma kallikrein inhibitor as defined herein and a pharmaceutically acceptable carrier, for use in treating a coronaviral disease, in particular for treating and/or preventing pulmonary edema in a subject having a coronaviral disease.

The treatment preferably comprises intravenous or pulmonary administration of the plasma kallikrein inhibitor or the pharmaceutical composition according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Chemical structure of cpdl, referred to as Ac-(06-34-18) Phe2 Aze3 Tyr4 Harg5

Ala(i]jCH₂NH)6 in International Patent Application Publication WO 2014/167122 A1 FIG. 2 Chemical structure of cpd2, referred to as Ac-(06-550)-Sar₃-(DArg₂) Aze3HArg5 in International Patent Application Publication WO 2015/063465 A2.

FIG. 3 Plasma kallikrein activity in patients with COVID-19 and without COVID-19 (control). Individual sample levels are marked with circles, while the mean is shown with a line.

FIG. 4 Bradykinin and kinin levels in patients with COVID-19 and without COVID-19 (control). Individual sample levels are marked with circles, while the mean is shown with a bar.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have identified that the kallikrein-kinin pathway is important in the development of coronaviral diseases, especially in the second phase of these diseases wherein the infection leads to respiratory diseases. The kallikrein-kinin pathways, also referred to as the kallikrein- kinin system, is a pathway involving blood proteins best known for its effects in contact-activated coagulation. In the pathway, the zymogen plasma prekallikrein is converted to the active serine protease plasma kallikrein through the action of Factor XI la by the cleavage of an internal Arg-lle bond. Plasma kallikrein liberates bradykinin from high molecular weight (HMW) kininogens. In addition, plasma kallikrein is able to convert plasminogen into plasmin. The inventors have now identified that inhibition of the kallikrein-kinin pathway is able to treat coronaviral diseases and especially respiratory diseases associated with coronaviral diseases. Therefore, in a first aspect, the present invention provides an inhibitor of the kallikrein-kinin pathway for treating a coronaviral disease. As the skilled person will understand, inhibition of the pathway can be obtained through inhibition of one of its components, in particular through inhibition of Factor XI I, inhibition of the conversion of Factor XII to Factor XI la, inhibition of Factor XI la, inhibition of prekallikrein, inhibition of the conversion of prekallikrein to plasma kallikrein, inhibition of plasma kallikrein, antagonism of bradykinin, or inhibition of bradykinin receptor B1 or B2.

Therefore, in a particular embodiment, the present invention provides an inhibitor of Factor XII for use in the treatment of a coronaviral disease. In another particular embodiment, the present invention provides an inhibitor of Factor XI la for use in the treatment of a coronaviral disease. In another particular embodiment, the present invention provides an inhibitor of prekallikrein for use in the treatment of a coronaviral disease. In yet another particular embodiment, the present invention provides an inhibitor of plasma kallikrein for use in the treatment of a coronaviral disease. In yet another particular embodiment, the present invention provides a bradykinin antagonist for use in the treatment of a coronaviral disease. ln yet another particular embodiment, the present invention provides an inhibitor of bradykinin receptor B1 for use in the treatment of a coronaviral disease. In yet another particular embodiment, the present invention provides an inhibitor of bradykinin receptor B2 for use in the treatment of a coronaviral disease. Particularly preferred is the inhibition of plasma kallikrein. As used herein, the term "coronavirus" includes any member of the family Coronaviridae, including, but not limited to, the subfamilies Letovirinae and Orthocoronavirinae. They thus include the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus.

As used herein, the term "coronaviral disease" refers to a disease associated with an infection with a coronavirus, particularly a disease caused by the infection with a coronavirus. As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and 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.

The term "inhibition" or "inhibitor" refers to a reduction in the biological activity of a given molecule, such as a reduction of the enzymatic activity of the molecule, a reduction of the expression or amount of the molecule, or a reduction of the receptor signaling activity of the molecule. Inhibitors in the context of the invention thus also refer to antagonists. In a preferred embodiment, inhibitor refers to a molecule that binds to and inhibits the proteolytic activity of the target molecule.

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. The terms "peptide," "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

The term "antibody" as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e. an antigen-binding portion. Examples of immunologically active portions of immunoglobulin molecules include scFV and dcFV fragments, Fab and F (a b') 2 fragments which can be generated by treating the antibody with an enzyme such as papain or pepsin, respectively. The antibody can be a polyclonal, monoclonal, recombinant, e.g. a chimeric or humanized, fully human, non-human, e.g. murine, or single chain antibody.

The term "specifically binds," refers to an antibody or a ligand, which recognizes and binds with a cognate binding partner protein, but the antibody or ligand does not substantially recognize or bind other molecules in the sample.

The term "subject", refers to any living organisms which can be infected with a coronavirus (e.g., mammals, human). In an embodiment the subject is a human. A subject may be of any age. In an embodiment the subject is a human subject of 50 years or older, in particular 65 years or older. In an embodiment, a subject is an adult subject, e.g., older than 18 years of age. As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity.

As used herein, the terms "comprising" and "including" are inclusive or open-ended and do not exclude additional unrecited elements, compositional components, or method steps. Accordingly, the terms "comprising" and "including" encompass the more restrictive terms "consisting essentially of" and "consisting of".

Coronaviruses and coronaviral diseases

As described above, coronavirus refers to any member of the family Coronaviridae, including, but not limited to, the subfamilies Letovirinae and Orthocoronavirinae, preferably a member of the Orthocoronavirinae. Therefore, in a particular embodiment of the invention, the coronavirus is an Orthoconavirinae member selected from the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. More in particular a coronavirus selected from the group consisting of:

• Alphacoronavirus: Human coronavirus 229E, Human coronavirus NL63, Miniopterus bat coronavirus 1, Miniopterus bat coronavirus HKU8, Porcine epidemic diarrhea virus, Rhinolophus bat coronavirus HKU2, and Scotophilus bat coronavirus 512; in particular Human coronavirus 229E or Human coronavirus NL63;

• Betacoronavirus: Betacoronavirus 1 (Human coronavirus OC43), Human coronavirus HKU1, Murine coronavirus, Pipistrellus bat coronavirus HKU5, Rousettus bat coronavirus HKU9, Severe acute respiratory syndrome-related coronavirus (SARS-CoV-1, SARS-CoV-2), Tylonycteris bat coronavirus HKU4, Middle East respiratory syndrome-related coronavirus (MERS-CoV), Hedgehog coronavirus 1 (EriCoV); in particular Human coronavirus OC43, Human coronavirus HKU1, SARS-CoV-1, SARS-CoV-2, and MERS-CoV; more in particular SARS-CoV-1, SARS-CoV-2, and MERS-CoV;

• Gammacoronavirus: Beluga whale coronavirus SW1, and Infectious bronchitis virus;

• Deltacoronavirus: Bulbul coronavirus HKU11, and Porcine coronavirus HKU15.

In a particular embodiment, the coronavirus belongs to the genus of Alpha- or Betacoronavi ruses, more in particular to the genus of betacoronavi ruses. In a further embodiment, the coronavirus is selected from SARS-CoV-1, SARS-CoV-2, and MERS-CoV. In a preferred embodiment, the coronavirus is a SARS- related coronavirus (SARS-CoV), such as SARS-CoV-1 or SARS-CoV-2. In a further preferred embodiment, the coronavirus is SARS-CoV-2. As will be understood by the skilled person from the disclosures herein, in a preferred embodiment, the coronavirus is a coronavirus that causes a respiratory tract disease. In another preferred embodiment, the coronavirus is a human coronavirus, meaning that the coronavirus has the ability to infect a human.

As described above, a coronaviral disease is a disease associated with an infection with a coronavirus, particularly a disease caused by the infection with a coronavirus. In a further embodiment, the coronaviral disease is MERS, SARS or COVID-19. In another particular embodiment, the coronaviral disease is a respiratory tract disease associated with an infection with a coronavirus, particularly a respiratory tract disease caused by the infection with a coronavirus. In yet another particular embodiment, the coronaviral disease is a respiratory tract infection with a coronavirus. In another embodiment, the coronaviral disease is a respiratory syndrome caused by a coronavirus, particularly an acute respiratory syndrome caused by a coronavirus, more in particular a severe acute respiratory syndrome caused by a coronavirus. In a further particular embodiment, the coronaviral disease is acute respiratory distress syndrome (ARDS) caused by a coronavirus. In another embodiment, the coronaviral disease is pneumonia caused by the infection with a coronavirus.

It has been identified that the inhibitors of the invention are particularly effective in treating and preventing pulmonary edema associated with coronaviral infections. It has been found that these inhibitors, and especially plasma kallikrein inhibitors, are able to reduce pulmonary edema, thereby improving respiratory function, improving ease of breathing and reducing coughing, reducing respiratory failure and avoiding or reducing damage to the lungs. Therefore, in another aspect of the invention, the present invention provides an inhibitor or antagonist as disclosed herein for use in the treatment of pulmonary edema in a subject having a coronaviral disease. In a further, preferred embodiment, the present invention provides a plasma kallikrein inhibitor as disclosed herein for use in the treatment of pulmonary edema in a subject having a coronaviral disease.

Subjects

As mentioned above, a subject refers to any living organisms which can be infected with a coronavirus (e.g., mammals, human). In a preferred embodiment, the subject is a living organism that has been confirmed or is suspected of having been infected with a coronavirus. In a further embodiment, the subject has been confirmed (diagnosed) to have a coronaviral disease as detailed therein. Therefore, as an example, in another further embodiment, the subject has been confirmed (diagnosed) to have MERS, SARS, or COVID-19, preferably COVID-19. ln a preferred embodiment, the subject has been confirmed or is suspected of having been infected with a coronavirus and has or is at risk of developing a respiratory disease. In a particular embodiment, the subject has been diagnosed to have an infection with a coronavirus as detailed herein and has or is at risk of developing a respiratory disease. In a further embodiment, the subject has been diagnosed of having a respiratory disease suspected of being caused by a coronavirus. In an even further embodiment, the subject has been diagnosed of having a respiratory disease caused by a coronavirus as detailed herein.

Evident from the disclosures herein, the present treatments are particularly suitable to address pulmonary edema associated with a coronaviral infection. Therefore, in a particularly preferred embodiment, the subject the subject has been confirmed or is suspected of having been infected with a coronavirus and has or is at risk of developing pulmonary edema. In a particular embodiment, the subject has been diagnosed to have an infection with a coronavirus as detailed herein and has or is at risk of developing pulmonary edema. In a further embodiment, the subject has been diagnosed of having pulmonary edema suspected of being caused by a coronavirus. In an even further embodiment, the subject has been diagnosed of having pulmonary edema caused by a coronavirus as detailed herein, such as MERS-CoV, SARS-CoV-1, or SARS-CoV-2.

In a preferred embodiment, the subject is a human.

Inhibitors Compounds for use in the invention are inhibitors or antagonists of the kallikrein-kinin pathway, in particular inhibitors of plasma kallikrein. As mentioned herein before, an inhibitor refers to any compound that leads to a reduction in the biological activity of a given molecule, such as a reduction of the enzymatic activity of the molecule, a reduction of the expression or amount of the molecule, or a reduction of the receptor signaling activity of the molecule. For example, a plasma kallikrein inhibitor may be a molecule that reduces the expression of the prekallikrein protein, that reduces the conversion of the zymogen prekallikrein to the active molecule plasma kallikrein, that inhibits the enzymatic activity of plasma kallikrein, or that increases the degradation of plasma kallikrein. Examples of inhibitors include an antibody, a peptide, a nucleotide, or a chemical molecule (also referred to in the art as a small molecule or inorganic molecule) as well as synthetic derivatives thereof. Preferred inhibitors for use in the invention are specific inhibitors, referring to molecules that have a higher specificity for the target molecule than for another molecule outside of the kallikrein-kinin pathway, particularly than for another serine protease. For example, a specific plasma kallikrein inhibitor refers to a molecule having an inhibition constant that is lower for plasma kallikrein than for another serine protease, in particular than for tissue kallikrein and/or transmembrane protease, serine 2 (TMPRSS2). In a particular embodiment, a specific inhibitor refers to an inhibitor that has an inhibition constant that is at least 10 times, in particular at least 100 times, more in particular at least 1000 times lower for the target molecule in the kallikrein-kinin pathway than for a serine protease outside of said pathway. In a particular embodiment, a specific inhibitor refers to an inhibitor that has an inhibition constant that is at least 10 times, in particular at least 100 times, more in particular at least 1000 times lower for the target molecule in the kallikrein-kinin pathway than for tissue kallikrein. In another embodiment, a specific inhibitor refers to an inhibitor that has an inhibition constant that is at least 10 times, in particular at least 100 times, more in particular at least 1000 times lower for the target molecule in the kallikrein-kinin pathway than for transmembrane protease, serine 2 (TMPRSS2). In a preferred embodiment, the inhibitor has an inhibition constant that is at least 10 times, in particular at least 100 times, more in particular at least 1000 times lower for the target molecule in the kallikrein-kinin pathway than for tissue kallikrein and transmembrane protease, serine 2 (TMPRSS2). Assays to determine inhibition constants (K,) are well-known in the art. We determining inhibition constants for different targets in order to compare specificity, the assay features are kept as identical as possible and the assays are preferably ran simultaneously in parallel.

In a particular embodiment, the inhibitor for use in the invention has an inhibition constant for the target in the kallikrein-kinin pathway of 50 mM or less, in particular 10 mM or less, more in particular 1 mM or less. In preferred embodiment, the inhibitor has a inhibition constant for the target in the kallikrein-kinin pathway of 500 nM or less, in particular 100 nM or less, preferably 50 nM or less.

In another preferred embodiment, the inhibitor is a direct inhibitor, meaning that it directly binds to and interferes with the target protein, or the gene or mRNA from which it is derived. In a further preferred embodiment, the inhibitor binds to and interferes with the target protein. In an even further embodiment, the inhibitor binds to and reduces the enzymatic activity of its target protein.

Plasma kallikrein inhibitors As is evident from the above, preferred molecules (herein also referred to as compounds) for the methods of the invention are plasma kallikrein inhibitors. In the context of the invention, suitable plasma kallikrein inhibitors reduce the expression of prekallikrein, reduce the conversion of prekallikrein to kallikrein or inhibit the enzymatic activity of plasma kallikrein. Among these, direct plasma kallikrein inhibitors that bind to plasma kallikrein and reduce its enzymatic activity are preferred.

Suitable, preferred, plasma kallikrein inhibitors for the invention have, for example, been developed by Bicycle Therapeutics and are disclosed in W02013050616 Al, WO2014167122 Al, and WO2015063465 Al, which are hereby incorporated by reference. These plasma kallikrein inhibitors will also be referred to as bicyclic peptide or bicyclic plasma kallikrein inhibitors herein. These inhibitors comprise a polypeptide sequence comprising at least three cysteine residues separated by two peptide loops, wherein the at least three cysteine residues are covalently attached to a molecular scaffold and wherein each peptide loop independently comprises from 2 to 10 amino acids. Amino acids refer to naturally occurring as well as non-natural amino acids and peptide bonds may be chemically modified. The compounds can be denoted as comprising a peptide sequence [Cl] - Loopl - [C2] - Loop2 - [C3], wherein [Cl] to [C3] denote a cysteine which is covalently attached to the molecular scaffold and wherein Loop 1 and Loop2 denote the first and second loop. For full clarity, in the nomenclature used in the context of the invention, [Cl] to [C3] are not considered to be part of the first and second peptide loop in these bicyclic compounds. The chemical scaffold is preferably trisbromomethylbenzene (TBMB). Therefore, in a particular embodiment, the plasma kallikrein inhibitor for use in the invention is a compound comprising a polypeptide sequence comprising at least three cysteine residues separated by two peptide loops, wherein the at least three cysteine residues are covalently attached to a molecular scaffold and wherein each peptide loop independently comprises from 2 to 10 amino acids. In a further embodiment, the peptidic compound of the invention comprises two peptide loops, wherein each loop comprises 4, 5 or 6 amino acids; preferably 5 or 6 amino acids. In yet another particular embodiment, the peptide loops in the peptidic compounds of the invention comprise in total of at least 5, preferably at least 8, more preferably at least 10 amino acids, which are optionally chemically modified. In a further embodiment, the peptide loops comprise in total from 5 to 25 amino acids; in particular from 8 to 20 amino acids; more in particular from 10 to 15 amino acids; even more in particular 10 or 11 amino acids.

In a particular embodiment, the plasma kallikrein inhibitor is a bicyclic peptide disclosed in the tables of W02013050616 Al, WO2014167122 Al, or WO2015063465 Al, particularly those bicyclic peptides for which the tables show a Ki value towards human (plasma) kallikrein of 100 nM or less. In a further embodiment, the plasma kallikrein inhibitor is a bicyclic peptide disclosed in the tables of W02013050616 Al, WO2014167122 Al, or WO2015063465 A1 for which the table shows a Ki value towards human (plasma) kallikrein of 50 nM or less, preferably 25 nM or less.

In a further embodiment, the first peptide loop comprises the sequence SFPYR (SEQ ID NO: 1), wherein optionally P is replaced with azetidine carboxylic acid and/or R is replaced with homoarginine or N-methylarginine. The presence of this motif in the peptidic compounds described herein imparts plasma kallikrein inhibitory activity to the molecule. In a particularly preferred embodiment, the plasma kallikrein inhibitor is a bicyclic peptide disclosed in the tables of W02013050616 Al, WO2014167122 Al, or WO2015063465 Al wherein the first peptide loop comprises the sequence SFPYR (SEQ ID NO: 1), wherein optionally P is replaced with azetidine carboxylic acid and/or R is replaced with homoarginine or N- methylarginine. In particular, those bicyclic peptides for which the tables show a Ki value towards human (plasma) kallikrein of 100 nM or less. In a further embodiment, the plasma kallikrein inhibitor is a bicyclic peptide disclosed in the tables of W02013050616 Al, WO2014167122 Al, or WO2015063465 Al wherein the first peptide loop comprises the sequence SFPYR (SEQ ID NO: 1), wherein optionally P is replaced with azetidine carboxylic acid and/or R is replaced with homoarginine or N-methylarginine, and for which the table shows a Ki value towards human (plasma) kallikrein of 50 nM or less, preferably 25 nM or less.

In a further embodiment, the first peptide loop comprises the sequence SF(Aze)Y(FIArg) (SEQ ID NO: 4) wherein Aze is azetidine carboxylic acid, FIArg is homo-arginine. In a particularly preferred embodiment, the plasma kallikrein inhibitor is a bicyclic peptide disclosed in the tables of W02013050616 Al, WO2014167122 Al, or WO2015063465 Al wherein the first peptide loop comprises the sequence SF(Aze)Y(FIArg) (SEQ ID NO: 4) wherein Aze is azetidine carboxylic acid, FIArg is homo-arginine. In particular, those bicyclic peptides for which the tables show a Ki value towards human (plasma) kallikrein of 100 nM or less. In a further embodiment, the plasma kallikrein inhibitor is a bicyclic peptide disclosed in the tables of W02013050616 Al, WO2014167122 Al, or WO2015063465 Al wherein the first peptide loop comprises the sequence SF(Aze)Y(FIArg) (SEQ ID NO: 4) wherein Aze is azetidine carboxylic acid, FIArg is homo-arginine, and for which the table shows a Ki value towards human (plasma) kallikrein of 50 nM or less, preferably 25 nM or less.

In one preferred embodiment, the plasma kallikrein inhibitor comprises [Cl]SF(Aze)Y( H Arg)[C2] (Ala(i)j CH ₂N H))HQDL[C3] (SEQ ID NO: 3) wherein Aze is azetidine carboxylic acid, HArg is homo-arginine, Ala( jCH₂NH) is alanine wherein the carbonyl is reduced to methylene, and wherein the cysteine residues [Cl] to [C3] are attached via thioether linkages to a molecular scaffold. In particular, the molecular scaffold being TBMB. In a most preferred embodiment, the plasma kallikrein inhibitor for use in the invention is cpdl, presented in Fig. 1.

In another preferred embodiment, the plasma kallikrein inhibitor comprises [Cl]SF(Aze)Y(HArg)[C2]VYYPDI[C3] (SEQ ID NO: 2), wherein Aze is azetidine carboxylic acid, FIArg is homo arginine, and wherein the cysteine residues [Cl] to [C3] are attached via thioether linkages to the molecular scaffold. In particular, the molecular scaffold being TBMB. In a most preferred embodiment, the plasma kallikrein inhibitor for use in the invention is cpd2, presented in Fig. 2.

Other plasma kallikrein inhibitors suitable for use in the treatments disclosed herein include Kunitz domain plasma kallikrein inhibitors, such as those developed by Dyax and disclosed in US5786328, US6333402, US6010880, and US9107928, which references are incorporated by reference herein. These kallikrein inhibitors comprise the consensus sequence amino acid sequence Xaal Xaa2 Xaa3 Xaa4 Cys Xaa6 Xaa7 Xaa8 Xaa9 XaalO Xaall Gly Xaal3 Cys Xaal5 Xaal6 Xaal7 Xaal8 Xaal9 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly Gly Cys Xaa39 Xaa40 Xaa41 Xaa42 Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48 Xaa49 Xaa50 Cys Xaa52 Xaa53 Xaa54 Cys Xaa56 Xaa57 Xaa58 (SEQ ID NO: 5) with Xaa being each independently from one another any amino acid.

In a particular embodiment, the plasma kallikrein inhibitor for use in the invention comprises the amino acid sequence of SEQ ID NO: 5, wherein

Xaal, Xaa2, Xaa3, Xaa4, Xaa56, Xaa57 or Xaa58 are, independently from one another, any amino acid or absent;

XaalO is an amino acid selected from the group consisting of Asp and Glu;

Xaall is an amino acid selected from the group consisting of Asp, Gly, Ser, Val, Asn, lie, Ala and

Thr;

Xaal3 is an amino acid selected from the group consisting of Arg, His, Pro, Asn, Ser, Thr, Ala, Gly, Lys and Gin;

Xaal5 is an amino acid selected from the group consisting of Arg, Lys, Ala, Ser, Gly, Met, Asn and

Gin;

Xaal6 is an amino acid selected from the group consisting of Ala, Gly, Ser, Asp and Asn; Xaal7 is an amino acid selected from the group consisting of Ala, Asn, Ser, lie, Gly, Val, Gin and

Thr;

Xaal8 is an amino acid selected from the group consisting of His, Leu, Gin and Ala;

Xaal9 is an amino acid selected from the group consisting of Pro, Gin, Leu, Asn and lie; Xaa21 is an amino acid selected from the group consisting of Trp, Phe, Tyr, His and lie;

Xaa22 is an amino acid selected from the group consisting of Tyr and Phe;

Xaa23 is an amino acid selected from the group consisting of Tyr and Phe;

Xaa31 is an amino acid selected from the group consisting of Glu, Asp, Gin, Asn, Ser, Ala, Val, Leu, lie and Thr; Xaa32 is an amino acid selected from the group consisting of Glu, Gin, Asp Asn, Pro, Thr, Leu, Ser,

Ala, Gly and Val;

Xaa34 is an amino acid selected from the group consisting of Thr, lie, Ser, Val, Ala, Asn, Gly and

Leu;

Xaa35 is an amino acid selected from the group consisting of Tyr, Trp and Phe; Xaa39 is an amino acid selected from the group consisting of Glu, Gly, Ala, Ser and Asp;

Xaa40 is an amino acid selected from the group consisting of Gly and Ala;

Xaa43 is an amino acid selected from the group consisting of Asn and Gly;

Xaa45 is an amino acid selected from the group consisting of Phe and Tyr;

Xaa6, Xaa7, Xaa8, Xaa9, Xaa20, Xaa24, Xaa25, Xaa26, Xaa27, Xaa28, Xaa29, Xaa41, Xaa42, Xaa44, Xaa46, Xaa47, Xaa48, Xaa49, Xaa50, Xaa52, Xaa53 and Xaa54 are, independently from one another, any amino acid.

In another particular embodiment, the plasma kallikrein inhibitor for use in the present invention is a peptide having the amino acid sequence of any of SEQ ID NO: 2 to SEQ ID NO: 43 of US9107928 B2. In a further particular embodiment, the plasma kallikrein inhibitor for use in the present invention is a peptide comprising the amino acid sequence Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro ArgTrp Phe Phe Asn lie Phe Thr Arg Gin Cys Glu Glu Phe lie Tyr Gly Gly Cys Glu Gly Asn Gin Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp (SEQ ID NO: 6). In a further particular embodiment, the plasma kallikrein inhibitor for use in the present invention is ecallantide, commercially available under the trade name Kalbitor. In yet another particular embodiment, the plasma kallikrein inhibitor for use in the present invention is aprotinin. This kunitz domain polypeptide is also known under its commercial name Trasylol. Another suitable plasma kallikrein inhibitor for use in the present invention is Cl-inhibitor (also known as Cl-INH or Cl esterase inhibitor). Cl-inhibitor is a naturally occurring plasma kallikrein inhibitor and often considered to the most important physiological inhibitor of plasma kallikrein.

Other plasma kallikrein inhibitors for use in the invention are anti-kallikrein antibodies or anti- prekallikrein antibodies that prevent conversion of prekallikrein to kallikrein. As defined herein, antibodies include antigen-binding fragments of full-length antibodies. Anti-kallikrein antibodies may bind both prekallikrein and kallikrein. In another embodiment, the anti-kallikrein antibodies bind to kallikrein but do not bind to prekallikrein. Anti-prekallikrein and anti-kallikrein antibodies are available commercially or can easily be obtained using the general knowledge in the art. Suitable anti-plasma kallikrein antibodies for use in the present invention include the antibodies disclosed in W02011085103 A2 and WO2012094587 Al, both reference are herewith incorporated herein. When using an antibody as plasma kallikrein inhibitor, lanadelumab is preferred.

Other plasma kallikrein inhibitors for use in the invention are chemical molecules (also referred to in the art as a small molecules or inorganic molecules). Suitable chemical molecules with plasma kallikrein inhibitory activity are known in the art. For example, suitable plasma kallikrein inhibitors include those disclosed in W02017072020 Al, W02017072021 A1 and WO2018192866 A1 developed by Boehringer Ingelheim International, as well as those disclosed in WO2019028362 Al developed by Dyax, and those disclosed in WO03076458 A2 and W02013005045 Al developed by Kalvista Pharmaceuticals, all reference herewith being incorporated by reference. In a particular embodiment, the plasma kallikrein inhibitor for use in the present invention is a compound selected from the compounds listed in claim 26 of WO2019028362 Al, berotralstat, and a compound of Formula A Formula A.

In a further embodiment, the plasma kallikrein inhibitor for use in the present invention is berotralstat or a compound of Formula A. In another embodiment, the plasma kallikrein inhibitor is an inhibitory nucleic acid molecules that targets prekallikrein RNA, e.g., antisense, siRNA, ribozymes, and aptamers, are used. In some embodiments, the inhibitory nucleic acid targets prekallikrein mRNA.

RNAi is a process whereby double-stranded RNA (dsRNA, also referred to herein as si RNAs or ds siRNAs, for double-stranded small interfering RNAs,) induces the sequence-specific degradation of homologous mRNA in animals and plant cells (Flutvagner and Zamore, Curr. Opin. Genet. Dev. 12:225-232 (2002); Sharp, Genes Dev., 15:485-490 (2001)). In mammalian cells, RNAi can be triggered by 21- nucleotide (nt) duplexes of small interfering RNA (siRNA) (Chiu et al., Mol. Cell. 10:549-561 (2002); Elbashir et al., Nature 411 :494-498 (2001)), or by micro-RNAs (miRNA), functional small-hairpin RNA (shRNA), or other dsRNAs which are expressed in vivo using DNA templates with RNA polymerase III promoters (Zeng et al., Mol. Cell 9:1327-1333 (2002); Paddison et al., Genes Dev. 16:948-958 (2002); Lee et al., Nature Biotechnol. 20:500-505 (2002); Paul et al., Nature Biotechnol. 20:505-508 (2002); Tuschl, Nature Biotechnol. 20:440-448 (2002); Yu et al., Proc. Natl. Acad. Sci. USA 99(9): 6047-6052 (2002); McManus et al., RNA 8:842-850 (2002); Sui et al., Proc. Natl. Acad. Sci. USA 99(6):5515-5520 (2002)).

The nucleic acid molecules or constructs can include dsRNA molecules comprising 16-30, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is substantially identical, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%) identical, e.g., having 3, 2, 1, or 0 mismatched nucleotide(s), to a target region in the mRNA, and the other strand is complementary to the first strand. The dsRNA molecules can be chemically synthesized, or can transcribed be in vitro from a DNA template, or in vivo from, e.g., shRNA. The dsRNA molecules can be designed using any method known in the art; a number of algorithms are known, and are commercially available. Gene walk methods can be used to optimize the inhibitory activity of the siRNA. ln another embodiment, an antisense nucleic acid is used that is complementary to the sense nucleic acid encoding prekallikrein and plasma kallikrein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to a TEF mRNA sequence. The antisense nucleic acid can be complementary to the entire coding strand of the target sequence, or to only a portion thereof. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence (e.g., the 5' and 3' untranslated regions). An antisense nucleic acid can be designed such that it is complementary to the entire coding region of a target prekallikrein mRNA, but can also be an oligonucleotide that is antisense to only a portion of the coding or noncoding region of the target mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of the target mRNA, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.

Based upon the known sequences of prekallikrein and plasma kallikrein and the non-coding neighboring regions, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules for use in accordance with the present invention.

Prekallikrein gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region (e.g., promoters and/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells. See generally, Helene, Anticancer Drug Des. 6:569-84 (1991); Helene, Ann. N.Y. Acad. Sci. 660:27-36 (1992); and Maher, Bioassays 14:807-15 (1992).

Ribozymes are a type of RNA that can be engineered to enzymatically cleave and inactivate other RNA targets in a specific, sequence-dependent fashion. By cleaving the target RNA, ribozymes inhibit translation, thus preventing the expression of the target gene. Ribozymes can be chemically synthesized in the laboratory and structurally modified to increase their stability and catalytic activity using methods known in the art. Alternatively, ribozyme genes can be introduced into cells through gene-delivery mechanisms known in the art. A ribozyme having specificity for a prekallikrein nucleic acid can include one or more sequences complementary to the nucleotide sequence of prekallikrein cDNA, and a sequence having known catalytic sequence responsible for mRNA cleavage (see US5093246 or Haselhoff and Gerlach Nature 334:585-591 (1988)). Alternatively, a target mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and Szostak, Science 261:1411-1418 (1993). In a particular embodiment of the present invention, the plasma kallikrein inhibitor for use in the invention is selected from the group consisting of ecallantide, lanadelumab, berotralstat, Cl inhibitor, cpdl, cpd2, and a compound of formula A

In a further particular embodiment, the plasma kallikrein inhibitor is a specific plasma kallikrein inhibitor and is selected from the group consisting of ecallantide, lanadelumab, berotralstat, cpdl, cpd2, and a compound of formula A. In a preferred embodiment, the plasma kallikrein inhibitor is cpdl or cpd2. Other kallikrein-kinin pathway inhibitors

Factor XII and Factor XI la inhibitors are known in the art. These are for example disclosed in Kenne and Renne (Drug Discovery Today 2014, 19:1459-1464) and Larsson et al. (Sci Transl Med 2014, 222:222ral7).

Bradykinin receptor antagonists are also known in the art. In a particular embodiment, the bradykinin receptor antagonist is a B1 receptor antagonist. In another particular embodiment, the bradykinin receptor antagonist is a B2 receptor antagonist. If a bradykinin receptor antagonist is used for the invention, it is preferably a bradykinin B1 receptor antagonist, as the inventors have experimentally identified a significantly increased expression of the bradykinin B1 receptor in patients admitted to the hospital for SARS-CoV-2 infections. Suitable antagonists for use in the methods of the invention include, but are not limited to, the bradykinin B1 receptor antagonists disclosed in W02010097372 A, W02011104203 Al and WO2012022795 Al, which are herewith incorporated by reference. In a further particular embodiment, the small molecule bradykinin receptor antagonist for use in the invention is selected from the group consisting of:

ln another further particular embodiment, the bradykinin receptor antagonist is selected from the group consisting of:

Compositions

It is a further object of the invention to provide compositions comprising an inhibitor as disclosed herein for use in treatment in accordance with the invention. Therefore, the present invention also provides a pharmaceutical composition comprising an inhibitor as disclosed herein and a pharmaceutically acceptable carrier for the uses of the invention. Pharmaceutically acceptable carriers enhance or stabilize the composition or can be used to facilitate preparation of the composition. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible, as is known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289- 1329; Remington: The Science and Practice of Pharmacy, 21st Ed. Pharmaceutical Press 2011; and subsequent versions thereof). Non-limiting examples of said pharmaceutically acceptable carrier comprise any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents.

Pharmaceutical compositions containing the compounds of the invention may be in any form suitable for the intended method of administration, including, for example, a solution, a suspension, or an emulsion. Liquid carriers are typically used in preparing solutions, suspensions, and emulsions. Liquid carriers contemplated for use in the practice of the present invention include, for example, water, saline, pharmaceutically acceptable organic solvent(s), pharmaceutically acceptable oils or fats, and the like, as well as mixtures of two or more thereof. The liquid carrier may contain other suitable pharmaceutically acceptable additives such as solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, thickening agents, viscosity regulators, stabilizers, and the like. Suitable organic solvents include, for example, monohydric alcohols, such as ethanol, and polyhydric alcohols, such as glycols. Suitable oils include, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, and the like. For parenteral administration, the carrier can also be an oily ester such as ethyl oleate, isopropyl myristate, and the like. Compositions of the present invention may also be in the form of microparticles, microcapsules, liposomal encapsulates, and the like, as well as combinations of any two or more thereof.

The compounds of the invention may be administered enterally, orally, parenterally, sublingually, by inhalation spray, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. For example, suitable modes of administration include oral, subcutaneous, transdermal, transmucosal, iontophoretic, intravenous, intramuscular, intraperitoneal, intranasal, subdural, and the like. Topical administration may also involve the use of transdermal administration such as transdermal patches or ionophoresis devices. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection, or infusion techniques.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-propanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, cyclodextrins, and sweetening, flavoring, and perfuming agents.

Effective amounts of the compounds of the invention generally include any amount sufficient to detectably treat viral infections and the diseases associated therewith.

Successful treatment of a subject in accordance with the invention may result in the inducement of a reduction or alleviation of symptoms in a subject afflicted with a medical or biological disorder to, for example, halt the further progression of the disorder, or the prevention of the disorder.

The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease undergoing therapy. The therapeutically effective amount for a given situation can be readily determined by routine experimentation and is within the skill and judgment of the ordinary clinician.

For purposes of the present invention, a therapeutically effective dose will generally be from about 0.01 mg/kg/day to about 1000 mg/kg/day, preferably from about 0.1 mg/kg/day to about 20 mg/kg/day, which may be administered in one or multiple doses.

While the compounds of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more other agents used in the treatment of disorders. The compounds of the invention may, for example, be combined with antiviral drugs. When additional active agents are used in combination with the compounds of the present invention, the additional active agents may generally be employed in therapeutic amounts as indicated in the Physicians' Desk Reference (PDR) 57th Edition (2003), PDR/Medical Economics Company, which is incorporated herein by reference, or such therapeutically useful amounts as would be known to one of ordinary skill in the art. The compounds of the invention and the other therapeutically active agents can be administered at the recommended maximum clinical dosage or at lower doses. Dosage levels of the active compounds in the compositions of the invention may be varied so as to obtain a desired therapeutic response depending on the route of administration, severity of the disease and the response of the patient. The combination can be administered as separate compositions or as a single dosage form containing both agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.

In a particular embodiment, a pharmaceutical composition of the invention comprises an inhibitor of the kallikrein-kinin pathway as described herein and does not comprise a second compound that is an inhibitor of a serine protease. In a further embodiment, the pharmaceutical composition of the invention does not comprise a second compound that is an inhibitor of tissue kallikrein or transmembrane protease, serine 2 (TMPRSS2). In another embodiment, the pharmaceutical composition of the invention does not comprise an angiotensin-converting-enzyme inhibitor. In another embodiment, the pharmaceutical composition of the invention does not comprise an inhibitor of the renin-angiotensin system. In a further embodiment, the pharmaceutical composition of the invention comprises an inhibitor of the kallikrein- kinin pathway as the only therapeutically active ingredient in the composition.

In a particular embodiment, the present invention provides a recipient comprising a pharmaceutical composition of the invention. In a further embodiment, the recipient is designed for systemic or pulmonary administration. In a preferred embodiment, the recipient comprising the pharmaceutical composition of the invention is designed for intravenous administration. In another preferred embodiment, the recipient comprising the pharmaceutical composition of the invention is designed for pulmonary administration.

Administration

The compounds/inhibitors and compositions of the invention can be administered to animals, e.g., mammals (human and non-human), fowl, and the like according to conventional methods well known to those skilled in the art (e.g., orally, subcutaneously, nasally, topically). In a preferred embodiment, the treatment comprises systemic or pulmonary administration of the inhibitors and compositions of the invention. Systemic administration is preferably oral, subcutaneous or intravenous administration.

Pulmonary drug delivery can itself be achieved by different approaches that are known to the skilled person. This includes liquid nebulizers, aerosol-based metered dose inhalers (MDI's), and dry powder dispersion devices. Such pulmonary drug delivery compositions are designed to be delivered by inhalation by the patient of a drug dispersion so that the active drug within the dispersion can reach the lung. Pulmonary administration is available for the inhibitors and compounds disclosed herein.

Oral administration is particularly useful for the chemical molecules (small molecules) disclosed herein. In a particular embodiment, the treatment comprises oral administration of a plasma kallikrein inhibitor as described in the paragraphs on plasma kallikrein inhibitors that are chemical molecules. In one preferred embodiment, the treatment of the present invention comprises oral administration of berotralstat.

Subcutaneous or intravenous administration is especially preferred for the bicyclic molecules, peptides and antibodies disclosed herein. In a particular embodiment, the treatment of the present invention comprises subcutaneous administration of a bicyclic molecule, peptide, or antibody as disclosed herein. In a further particular embodiment, the treatment of the present invention comprises subcutaneous administration of a plasma kallikrein inhibitor that is a bicyclic molecule, peptide, or antibody as disclosed herein. In one preferred embodiment, the treatment of the present invention comprises subcutaneous administration of lanadelumab, ecallantide, or Cl inhibitor; more in particular lanadelumab or ecallantide.

In another further particular embodiment, the treatment of the present invention comprises intravenous administration of a plasma kallikrein inhibitor that is a bicyclic molecule, peptide, or antibody as disclosed herein; in particular a plasma kallikrein inhibitor that is selected from a bicyclic molecule as disclosed herein, a Kunitz domain peptide, or Cl inhibitor. In a particularly preferred embodiment, the treatment of the invention comprises intravenous administration of a bicyclic molecule being a plasma kallikrein inhibitor as disclosed herein. In a further embodiment, the plasma kallikrein inhibitor for intravenous administration is a compound comprising [Cl]SF(Aze)Y(HArg)[C2]VYYPDI[C3] (SEQ ID NO: 2), wherein Aze is azetidine carboxylic acid, HArg is homo-arginine, and wherein the cysteine residues [Cl] to [C3] are attached via thioether linkages to the molecular scaffold, more in particular cpd2. In another particularly preferred embodiment, the plasma kallikrein inhibitor for intravenous administration is a compound comprising [Cl]SF(Aze)Y(HArg)[C2](Ala( jCH₂NH))HQDL[C3] (SEQ ID NO: 3) wherein Aze is azetidine carboxylic acid, FIArg is homo-arginine, Ala(i]jCFl NFI) is alanine wherein the carbonyl is reduced to methylene, and wherein the cysteine residues [Cl] to [C3] are attached via thioether linkages to the molecular scaffold, more in particular cpdl.

In another particular embodiment, the present invention provides a method for the treatment of a coronaviral disease as disclosed herein, the method comprising administering a subject in need thereof an inhibitor of the kallikrein-kinin pathway. In a further embodiment, the present invention provides a method for the treatment of a coronaviral disease as disclosed herein, the method comprising administering a subject in need thereof a plasma kallikrein inhibitor. In another embodiment, the present invention provides a method for inhibiting a plasma kallikrein inhibitor in a subject in need thereof, wherein the method comprises administering a plasma kallikrein inhibitor as disclosed herein and wherein the subject has or is suspected of having a coronaviral infection. In a further embodiment, the present invention provides a method for inhibiting a plasma kallikrein inhibitor in a subject in need thereof, wherein the method comprises administering a plasma kallikrein inhibitor as disclosed herein and wherein the subject has or is suspected of having lung edema associated with a coronaviral infection. In another particular embodiment, the present invention provides a method for treating lung edema in a subject in need thereof, wherein the method comprises administering an inhibitor of the kallikrein-kinin pathway to said subject. In a further embodiment, the subject has or is suspected of having a coronaviral disease. In a further embodiment, the subject is diagnosed as having a coronaviral disease, in particular a disease caused by MERS-CoV or a SARS-CoV. In another further embodiment, the subject has a respiratory disease associated with a coronaviral disease, in particular an acute respiratory syndrome associated with a coronaviral disease. In a further particular embodiment, the subject has symptoms of acute respiratory syndrome associated with a coronaviral infection.

Examples

EXAMPLE 1 - Sample collection

21 Hospitalized patients with COVID-19 and 19 hospitalized patients without COVID-19 were enrolled. COVID-19 was confirmed with a positive SARS-CoV-2 quantitative real-time transcription polymerase chain reaction test (qRT-PCR) performed on nasopharyngeal swabs and/or bronchoalveolar lavage (BAL) fluid. Patients without COVID-19 comprised i) patients suspected for COVID-19 with BAL resulting in an alternative diagnosis ii) patients without COVID19 who underwent BAL to rule out opportunistic co-infection and/or to remove mucus plugs and who subsequently tested negative for SARS- CoV-2 qRT-PCR on BAL fluid, or iii) patients with pulmonary disease from whom BAL fluid samples were banked prior to the outbreak of the pandemic.

Bronchoalveolar lavage (BAL) was performed according to routine clinical procedures by instilling approximately 20 ml of sterile saline with a retrieval of approximately 10 ml. If possible, this procedure was performed twice, in which case 2 ml of the second fraction was aliquoted for research purposes. BAL fluid was immediately placed on ice, transported to a Biosafety Level 3 (BSL-3) facility (REGA institute, KU Leuven) and centrifuged. The supernatant was frozen at -80°C for batch analyses. Plasma kallikrein activity was measured in non-virally inactivated BAL fluid samples because the viral inactivation procedure might affect enzyme activity. Before released from the BSL3 laboratory for batch analyses of kinin levels under BSL2 laboratory conditions, the virus in BAL fluid was inactivated by ultraviolet light treatment or by heating at 65°C for 30 minutes, respectively. The study was conducted according to the principles expressed in the Declaration of Helsinki. Ethical approval was obtained from the Research Ethics Committee of UZ Leuven (S63881). Informed consent was obtained from all individuals or their legal guardians. EXAMPLE 2 - Plasma kallikrein activity in COVID-19 BAL fluid

Plasma kallikrein activityxas measured in BAL fluid samples without prior viral neutralization using the synthetic fluorogenic substrate H-Pro-Phe-Arg-AMC (Bachem, cat. 1-1295.0050). BAL fluid samples were diluted (typically 3/10 or 1/10 (v/v)) in a reaction mixture composed of 20 mM Tris-HCI, 150 mM NaCI, 1 mM EDTA, 0-1 % (v/v) PEG-6000-8000, 0-1 % (v/v) Triton X-100 (pH 7-5) with or without the specific plasma kallikrein inhibitor THR-149 (100 nM final). Substrate hydrolysis was monitored by recording the increase in fluorescence at 480 nm with excitation at 360 nm in 96-well plate format using either a Spectramax M2e plate reader (Molecular Devices) or a Spark multimode microplate reader (Tecan) in such a way that no more than 10% of the substrate was hydrolyzed. All hydrolytic data were normalized with a well containing a fixed concentration of human plasma kallikrein (Molecular Innovations, cat. HPKA- 3900, typically 0-4 nM final) and expressed as human plasma kallikrein equivalent concentration.

Total hydrolytic activity of the kallikrein substrate was higher in BAL from patients with COVID-19 than for those without COVID-19 (median of 41-9 pM with interquartile range (IQR) [10-8 - 985-3] vs median of 14-4 pM with IQR [5-1-27-9], respectively, p = 0-038). Plasma kallikrein activity in BAL was at a median of 2-1 pM with IQR [0-0-4-5] in patients with COVID-19 vs 0-3 pM with IQR [0-0 - 0-8] in patients without COVID-19 (Figure 3).

EXAMPLE 3 - Detection of bradykinin and kinin levels

Levels of bradykinin and its metabolites (Lys-bradykinin-(l-8), bradykinin-(l-8), bradykinin-(l-7), and bradykinin-(l-5)) were measured by LC-MS/MS. Levels of bradykinin measured with an ELISA fell below the lower detection limit. Levels of bradykinin, and bradykinin-(l-8) were generally low with no detected significant difference in BAL fluid from patients with COVID-19 compared to those without COVID-19. In contrast, the levels of the more stable kinin degradation peptides were higher in BAL fluid from patients with COVID-19 than in those without COVID-19 for bradykinin-(l-8) (median 17-7 pg/mL with IQR [3-6-56-8] vs median 0-0 pg/ml with IQR [0-0-36-4], respectively, p = 0-062), bradykinin-(l-7) (median 97-2 pg/ml with IQR [30-0-196-0] vs median 28-4 pg/ml with IQR [11-3-91-9], respectively, p = 0-056), and bradykinin-(l-5) (median 89-6 pg/ml with IQR [32-8-211-0] vs median 0-0 pg/ml with IQR [0-0- 50-8], respectively, p = 0-001) (Figure 4).

In summary, higher plasma kallikrein activity was observed in BAL fluid from COVID-19 patients compared to patients without COVID-19. Additionally, metabolite levels of bradykinin were markedly increased in COVID-19 patients, supporting treatment of coronaviral disease by administration of plasma kallikrein inhibitors.

Claims

1. A plasma kallikrein inhibitor for use in the treatment of a coronaviral disease.

2. A plasma kallikrein inhibitor for use in treating pulmonary edema in a subject having a coronaviral disease.

3. The plasma kallikrein inhibitor for use according to claim 1 or 2, wherein the coronaviral disease is caused by a Betacoronavirus.

4. The plasma kallikrein inhibitor for use according to any one of the previous claims, wherein the coronaviral disease is caused by a Severe Acute Respiratory Syndrome-related coronavirus (SARS-CoV), in particular SARS-CoV-2.

5. The plasma kallikrein inhibitor for use according to any one of the previous claims, wherein the plasma kallikrein inhibitor is a direct plasma kallikrein inhibitor that is selected from the group consisting of an antibody, a peptide, a nucleotide, or a chemical molecule.

6. The plasma kallikrein inhibitor for use according to any one of the previous claims, wherein the plasma kallikrein inhibitor has an inhibition constant that is at least 100 times lower for plasma kallikrein than for tissue kallikrein and transmembrane protease, serine 2 (TMPRSS2).

7. The plasma kallikrein inhibitor for use according to any one of the previous claims, wherein the plasma kallikrein inhibitor is selected from the group consisting of ecallantide, lanadelumab, berotralstat, Cl inhibitor, a compound of formula A

Formula A, or a compound comprising a polypeptide sequence comprising at least three cysteine residues separated by two peptide loops, wherein the at least three cysteine residues are covalently attached to a molecular scaffold and wherein each peptide loop independently comprises from 2 to 10 amino acids.

8. The plasma kallikrein inhibitor for use according to any one of the previous claims, wherein the plasma kallikrein inhibitor is a compound comprising a polypeptide sequence comprising at least three cysteine residues separated by two peptide loops, wherein the at least three cysteine residues are covalently attached to a molecular scaffold, wherein each peptide loop independently comprises from 2 to 10 amino acids, and wherein the first peptide loop comprises the sequence SFPYR (SEQ ID NO: 1), wherein optionally P is replaced with azetidine carboxylic acid and/or R is replaced with homoarginine or N-methylarginine.

9. The plasma kallikrein inhibitor for use according to any one of the previous claims, wherein the plasma kallikrein inhibitor is a compound comprising [Cl]SF(Aze)Y(HArg)[C2]VYYPDI[C3] (SEQ ID NO: 2), wherein Aze is azetidine carboxylic acid, HArg is homo-arginine, and wherein the cysteine residues [Cl] to [C3] are attached via thioether linkages to the molecular scaffold.

10. The plasma kallikrein inhibitor for use according to any one of the previous claims, wherein the plasma kallikrein inhibitor is a compound comprising [Cl]SF(Aze)Y(HArg)[C2](Ala(i]jCH₂NH))HQDL[C3] (SEQ ID NO: 3) wherein Aze is azetidine carboxylic acid, FIArg is homo-arginine, Ala (I4JCH₂N H ) is alanine wherein the carbonyl is reduced to methylene, and wherein the cysteine residues [Cl] to [C3] are attached via thioether linkages to the molecular scaffold.

11. The plasma kallikrein inhibitor as defined in claim 10, for use in treating pulmonary edema in a subject having a coronaviral disease caused by SARS-CoV-2.

12. A pharmaceutical composition comprising a plasma kallikrein inhibitor as defined in any one of claims 1 to 10 and a pharmaceutically acceptable carrier, for use in treating lung edema in a subject having a coronaviral disease.

13. The plasma kallikrein inhibitor for use according to any one of claims 1 to 11, or the pharmaceutical composition according to claim 12, wherein the treatment comprises systemic or pulmonary administration of the plasma kallikrein inhibitor.