WO2021204757A1 - Treatment of ards and other parameters related to covid-19 - Google Patents

Abstract

The present invention relates to pharmaceutical products that can be used to treat or ameliorate COVID-19 or symptoms thereof, and to methods wherein such products can be used.

Description

Treatment of ARDS and other parameters related to COVID-19

Field of the invention

The present invention relates to pharmaceutical products that can be used to treat or ameliorate COVID-19 and symptoms thereof, and to methods wherein such products can be used.

Background art

Coronavirus disease 2019 (COVID-19) infects mainly elderly and people with cardiovascular risk, such as hypertension (Guan W-J, Ni Z-Y, Hu Y, et al., N Engl J Med 2020; : 1- 13). It is caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), a positive- strand RNA virus. The genome of SARS-CoV-2 shares about 80% identity with that of SARS-CoV and is about 96% identical to the bat coronavirus BatCoV RaTG13. The clinical spectrum and imaging are so specific that MDs recognize this disease in an instant especially now that it is widespread. Many clinicians recognize that the virus does not cause disease similar to influenza, which carries the risk that designing targeted therapies based on the pathogenesis of influenza can fail in COVID-19. Research on Sars-CoV pathogenesis, which is sometimes thought to be very similar to Sars-CoV-2 pathogenesis, has focused on ACE inhibitors, on recombinant ACE2, and on ARBs. Because these pathways were extensively studied in SARS, it is a subject of intense investigation how they could fit in the pathogenesis of COVID-19 (Fang L, Karakiulakis G, Roth M., Lancet Respir2020; 2600: 30116; Batlle D, Wysocki J, Satchell K., Clin Sci (Lond) 2020; 134: 543- 5). For recombinant ACE2 this would be straight forward, and would at least be an attempt to bind and try to scavenge the virus (Battlle et al.). However, for ACE inhibitors and ARBs it is a much more complicated story. Since most of the attention has been focused on the RAS system and its interaction with modulating the vascular system and inflammation, the other major role of ACE and ACE2 for the regulation of the kinin-kallikrein system was lacking attention (Marceau F, Bawolak MT, Fortin JP, et al. Peptides 2018; 105: 37-50; Jurado-Palomo J, Caballero T. A Compr Rev Urticaria Angioedema 2017. DOI:10.5772/67704). Moreover, notable clinical deterioration seems to be associated with increased inflammatory status, especially around day 9 of the disease’s progress, which is associated with an increased inflammatory status. For SARS-CoV, the spike glycoprotein (S-protein) on the virion surface mediates receptor recognition and membrane fusion (Yan et al., Science, 2020 Vol. 367, Issue 6485, pp. 1444-1448 DOI: 10.1126/science. abb2762). During viral infection, the trimeric S protein is cleaved into S1 and S2 subunits and S1 subunits are released in the transition to the postfusion conformation. S1 contains the receptor binding domain (RBD), which directly binds to the peptidase domain (PD) of angiotensin-converting enzyme 2 (ACE2), whereas S2 is responsible for membrane fusion. When S1 binds to the host receptor ACE2, another cleavage site on S2 is exposed and is cleaved by host proteases, a process that is critical for viral infection. The S protein of SARS-CoV-2 can also exploit ACE2 for host infection. The structure of the S protein of SARS-CoV-2 shows that the ectodomain of the SARS-CoV-2 S protein binds to the PD of ACE2 with a dissociation constant (Kd) of ~15 nM. ACE2 can be hijacked by various strains of coronavirus. Its primary physiological role is in the maturation of angiotensin (Ang), a peptide hormone which controls blood pressure and vasoconstriction. ACE2 is a type I membrane protein and is expressed in lungs, heart, kidneys, and intestine. Decreased expression of ACE2 is associated with cardiovascular diseases. Full-length ACE2 comprises an N-terminal PD and a C-terminal collectrin-like domain (CLD) that ends with a single transmembrane helix and a intracellular segment of about 40 residues. The PD of ACE2 cleaves Ang I to produce Ang-(1-9), which is then processed by other enzymes to become Ang-(1- 7). ACE2 can also directly process Ang II to yield Ang-(1-7).

Cleavage of the S protein of SARS-CoV occurs in endosomes and is facilitated by cathepsin L, pointing to a mechanism of receptor-mediated endocytosis (Simmons et al., Proc. Natl. Acad. Sci. U.S.A. 102, 11876-11881 (2005). doi:10.1073/pnas.0505577102pmid:16081529). Further characterization is required to examine The interactions between ACE2 and the viral particle as well as the effect of cofactors on this process are under investigation. It is known that cleavage of the ACE2 C-terminal segment, particularly residues 697 to 716, by proteases such as transmembrane protease serine 2 (TMPRSS2), enhances viral entry as driven by the S protein. Residues 697 to 716 form the third and fourth helices in the neck domain and map to the dimeric interface of ACE2. Because ACE2 is expressed in the lungs, there is a hypothesis that ACE2 ligands such as B°AT1 can suppress SARS-CoV-2 infection by blocking ACE2 cleavage.

The high-resolution structure of full-length ACE2 in a dimeric assembly is available (Yan et al.). Docking the S protein trimer onto the structure of the ACE2 dimer with the RBD of the S protein bound suggests simultaneous binding of two S protein trimers to an ACE2 dimer. Structure-based rational design of binders with enhanced affinities to either ACE2 or the S protein of the coronaviruses is thought to facilitate development of decoy ligands or neutralizing antibodies for suppression of viral infection.

There is no definitive treatment for COVID-19. Mechanical ventilation using high positive end-expiratory pressure (PEEP) is applied in an attempt to alleviate symptoms, but such ventilation may damage the patients' lungs. There is anecdotal evidence that chloroquine can have beneficial effects. There is an urgent need for improved treatment of COVID-19. There is a articular need for means to reduce the severity of the most intense cases, or to reduce the duration of intense symptoms in affected patients, to ensure that medical facilities retain sufficient capacity for treatment. Severe COVID-19 patients can suffer acute respiratory distress syndrome (ARDS) or may be suffering cytokine release storms. Prevention or reduction in severity of these life- threatening situations are much desired. Improved ventilation techniques are much desired.

Description of embodiments

The inventors have found that for treatment of COVID-19 it is effective to target the bradykinin receptor family, particularly to inhibit or antagonize it. Disease progress all starts with ACE2 and its role in the kallikrein-kinin system, which to date has not been investigated in the pathogenesis of SARS or COVID-19. The kinin-kallikrein system is a zymogen system that after activation leads to the release of the nona-petide bradykin that after binding to the B2-receptor on endothelial cells leads to capillary leakage and thus angio-edema. The prototype diseases of local peripheral transient increased bradykinin release are hereditary or acquired angio-edema (Jurado- Palomo et al.) The clinical picture of COVID-19 is in line with a single-organ failure of the lung that is due to edema at the site of inflammation. Moreover, the presence of an elevated D-dimer without thrombosis or microangiopathy is in line with the high D-dimers in angioedema. This reflects the leakage of plasma substances involved in the coagulation cascade leading to fibrin, which due to kallikrein activity is processed into D-dimer that subsequently leaks back into the circulation, reflecting subendothelial activation and kallikrein activity.

For more than ten years now, ACE2 and its role in the RAS system has been suggested to play a role in the pulmonary edema of ARDS and SARS (Imai Y, et al., Nature 2005; 436: 112-6). Pulmonary edema by ACE2 dysfunction was thought to be due to increased hydrostatic pressure as a result of vasoconstriction of the pulmonary vasculature due to high angiotensin II (a vasoconstrictor). However, further experiments showed no difference in hydrostatic pressure (Kuba K, et al. A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury Nat Med 2005; 11 : 875-9). The inventors found that increased bradykinin explains this observation without increased hydrostatic pressure. Notably, the RAS system controls vasoconstriction and vasodilatation, and the bradykinin system controls permeability and vasodilatation, whereas ACE2 regulates both.

Bradykinin (BK) is a linear nonapeptide that is formed by the proteolytic activity of kallikrein on kininogens (Bhoola KD, et al. Pharmacol. Rev. 1992; 44: 1-80.) Kallikreins are serine proteases and can be divided in plasma kallikrein and tissue kallikreins (Fig. 1). The plasma and tissue kallikreins release the vasoactive peptides known as kinins (all sorts of BKs) that cause relaxation of vascular smooth muscle and increased vascular permeability (Nhoola et al.). Plasma kallikrein processes high-molecular-weight kininogen (HMWK produced by the liver) into bradykinin, while tissue kallikrein processes low-molecular-weight kininogen (LMWK produced by the liver) and results in Lys-BK (Fig. 1). These are the ligands forthe constitutively expressed bradykinin receptor B2 on endothelial cells (Jurado-Palomo et al.). In addition, some enzymes (carboxypeptidase M (CPM) and carboxypeptidase N (CPN)) can further process BK and Lys-BK into des-Arg9-BK and Lys-des-Arg9-BK respectively, which are ligands for bradykinin receptor B1 , a receptor on endothelial cells that is upregulated under proinflammatory conditions. These kinins have strong vasopermeable and vasodilatory capacity and need to be tightly controlled to prevent excessive angioedema. ACE and ACE2 both have roles in inactivating the ligands forthe bradykinin receptors (Gralinski LE et al., MBio 2018; 9: 1-15.). ACE mainly inactivates bradykinin which is the major ligand for B2 receptors. ACE inhibition has been linked to systemic acquired angioedema since it can result in excessive presence of bradykinin that activates B2 receptor.

Interestingly, ACE2 does not inactivate bradykinin, but can inactivate des-Arg9-BK which is a potent ligand of the B1 receptor in the lung (Fig. 1) (Sodhi CP et a., Am J Physiol - Lung Cell Mol Physiol 2018; 314: L17—31). In this way it can be protective against pulmonary edema especially in the setting of inflammation, which is further supported by the role of ACE2 in acute pulmonary injury. ACE2 is almost undetectable in serum, but is expressed in the lung on bronchiolar exocrine cells and pneumocytes type II (Sodhi et al.). The Sars-CoV-2 Spike (S) antigen binds to ACE2 and internalizes (Walls AC et al., Cell, 2020, 1-12). It has been suggested that the expression of ACE2 and its capacity of enzyme activity is decreased in SARS-CoV and inflammatory conditions (Imai, Kuba, Sodhi). The inventors found that Sars-CoV-2 interaction with ACE2 at the surface can downregulate ACE2 expression and function, leading to a deficiency to inactivate the B1 ligand locally in the lung, which in this way directly links the virus to local pulmonary angioedema.

In 2005 it was proposed that the RAS system was responsible for complications due to Sars-CoV. RAS regulates vasodilatation and vasoconstriction, and it was thought that increased angiotensin II as a result of ACE2 deficiency would result in pulmonary edema due to increased hydrostatic pressure since angiotensin II would cause vasoconstriction. However, there was no effect observed on the hemodynamics of the pulmonary vasculature in ACE2 deficiency, while there was clear vascular leakage. AT1 R knockout mice and AT1 R blockade were protected from lung edema due to inflammation but this was not explained by a mechanism linking AT1 R to vascular leakage. The inventors found that bradykinin is the missing link, since AT1 R forms heterodimers with the B2 receotor and AT1 R can syergise with B1 receptor in the induction of reactive oxygen species (ROS) in endothelial cells (Quitterer U, Abdalla S., Biochem Pharmacol 2014; 88: 284-90; Ceravolo GS et al., PLoS One 2014; 9. DOI:10.1371 /journal. pone.0111117).

The inventors found that this dysregulated bradykinin pathway is present already early in COVID-19 disease. Patients can worsen clinically after days of illness (especially around day 9) which is accompanied by an increase in proinflammatory status often resulting in intensive care unit (ICU) admission and with necessity of supportive mechanical ventilation. This second hit is reminiscent to observations in SARS-CoV where 80% of patients with SARS-CoV that developed acute respiratory disease coincided with antiviral IgG seroconversion (Fu Y, Cheng Y, Wu Y., Virol Sin 2020; 12250. DOI:10.1007/s12250-020-00207-4). Moreover, patients who developed the anti- S-neutralizing antibody early in disease had a higher chance of dying from the disease. In a macaques model it was clear that the anti-S-neutralizing antibody lead to worsening of pulmonary disease (Liu L et al., JCI insight 2019; 4. DOI:10.1172/jci. insight.123158). The proposed mechanism was an induced hyperinflammation via FcG receptor. Prior administration of anti-S-antibodies lead to an overwhelming influx of monocytes and macrophages to the lung already at day 2 of SARS- CoV infection. This abundance of monocyte/macrophages has also been reported in autopsy reports in three patients with COVID-19 from China (PMID 32172546). In this context there might also be a role for an exaggerated complement activation (Gralinski et al). In addition, a phenomenon named antibody-dependent enhancement of viral infection might be responsible for persistent viral loads and subsequently cause a direct or indirect effect on ACE2 activity in the lung (Fu et al.).

Important to note is that anti-inflammatory strategies can thus have a role at the time of seroconversion and the development of anti-S antibodies (Fig. 2). This will not only result in less damage to the environment but it will also protect against further inflammation-induced B1 upregulation on endothelial cells. However, it must be kept in mind that it will not have any direct effect on the pulmonary edema that is driven by bradykinin, since kallikrein activity is not affected, kinins will still be present, and B1 and B2 receptors are still expressed on endothelial cells. This pathway is not responsive to corticosteroids or adrenaline, meaning as long as the virus persists ACE2 dysfunction is present and the bradykinin pathway is active and pulmonary edema at the site of infection will persist. On the other hand, clinicians know how fast patients with bradykinin-related angioedema can recover once this pathway is blocked or once the trigger is gone. Thus a very fast recovery of pulmonary edema and recovery of hypoxia and disease is foreseen once the bradykinin- related angioedema is adressed.

As long as the virus persists, the dysregulated kinin-kallikrein pathway is playing a role in disease via the absence of optimal ACE2 function in the lung. When disease progresses accompanied by increased proinflammatory status, which often results in critical illness, aggressive innate anti-inflammatory strategies should be pursued, however this must be done in the presence of blocking the bradykinin pathway. Several targets in the kallikrein-kinin pathway can be amendable to intervention, namely 1. at the level of blocking tissue kallikrein activity and thus reducing the production of kinins, 2. activating the degradation of kinins by treating with recombinant active enzymes such as ACE2, 3. at the level of B1 and B2 receptors, 4. by inhibiting the common downstream signaling of B1 and B2 receptors, and 5. by suppressing local NO which is largely responsible for the endothelal leakage.

The inventors found that the best approach is to block B1 and preferably also B2 receptor signaling. B2 receptor inhibitors exist in the clinics. Several B1 receptor drugs have been tested in pre-clinical and in phase I/ll trials as therapeutic target for inflammation related processes already since the 1970s. Dual inhibition of both the B1 and B2 receptor is the preferred way of treating COVID-19, particularly of treating ARDS associated with COVID-19.

In addition, the inventors found that combination with anti-inflammatory strategies can be advantageous. In particular, innate cytokines that upregulate B1 on endothelial cells at the site of inflammation should be blocked, preferably in combination with B1 and/or B2 receptor blockade. IL- 1 (comprising IL-1a and IL-1 b) and TNF are potent inducers of B1. Blockade of NF-KB translocation, TNF-a, or IL-1 prevents the functional and molecular up-regulation of B1 receptors by inducers such as lipopolysaccharide (LPS) (Passos GF et al., J Immunol 2004; 172: 1839-47). Therefore, a preferred combination therapy is with anakinra, which has an excellent safety profile and not only blocks I L- 1 b , but also IL-1 a. IL-1 a is likely to be extremely elevated locally due to its release from damaged cells. Blocking TNF is an option, but is not the first choice because it is associated with more infectious complications. Complement activation has been described as playing a role in this stage of disease, so another possible combination is C5 blockade such as with eculizimab. A randomized trial with eculizimab in COVID-19 is being performed (NCT04288713). Also corticosteroids are an option.

The inventors noticed that some patients have persistent disease and at some point develop a proinflammatory profile, especially a rise in CRP (reflecting IL-6 elevation), which often leads to ICU admission. This is a suitable timepoint to initiate the anti-inflammatory therapy. For most patients this timepoint will be identified before the need of ICU admission and thus an antiinflammatory drug can prevent ICU admission. This anti-inflammatory combination strategy must thus be initiated together with B1 and preferably also B2 blockade, and preferably also with available antivirals. The anti-inflammatory strategies will buy time, but will not resolve the disease by themselves as long as the virus is present, and/or as long as the bradykinin angioedema is not resolved. A summary of these targeted treatments and timing of treatment is depicted in Fig. 2.

In addition to the bradykinin-driven pulmonary edema and cytokine-related clinical detonation the inventors developed a strategy on how to ventilate in this context. “Preventive” mechanical ventilation is discouraged because the risk for ventilator induced lung injury is high. After intubation, patients should be turned prone to improve oxygenation and keep PEEP at the lowest possible level. Early assisted ventilation should be stimulated unless a high respiratory drive (Po i < -4 cm H2O or a predicted Pmus > 15 cm H2O or a predicted transpulmonary pressure > 17 cm H2O is present. In those cases, appropriate respiratory drive suppression can be attempted. Patients should be kept as dry as possible.

Bradykinin and its receptors

The bradykinin receptor family is a group of G-protein coupled receptors (GPCRs) whose principal ligand is bradykinin. There are two Bradykinin receptors: the B1 receptor and the B2 receptor.

Bradykinin receptor B1 (B1) is a G-protein coupled receptor encoded by the BDKRB1 gene in humans. Its principal ligand is bradykinin, a 9 amino acid peptide generated in pathophysiologic conditions such as inflammation, trauma, burns, shock, and allergy. The B1 receptor is one of two G protein-coupled receptors that have been found which bind bradykinin and mediate responses to these pathophysiologic conditions. B1 is not normally expressed, and is synthesized de novo following tissue injury. Receptor binding leads to an increase in cytosolic calcium ion concentration, ultimately resulting in chronic and acute inflammatory responses. It has been shown that the kinin B1 receptor recruits neutrophil via chemokine CXCL5 production.

The B2 receptor is also a G protein-coupled receptor, coupled to Gq and Gi. The B2 receptor participates in bradykinin's vasodilatory role. Gq stimulates phospholipase C to increase intracellular free calcium and Gi inhibits adenylate cyclase. Furthermore, the receptor stimulates the mitogen-activated protein kinase pathways. It is ubiquitously and constitutively expressed in healthy tissues. The B2 receptor forms a complex with angiotensin converting enzyme (ACE), and this is thought to play a role in cross-talk between the renin-angiotensin system (RAS) and the kinin- kallikrein system (KKS). The heptapeptide angiotensin (1-7) also potentiates bradykinin action on B2 receptors.

Bradykinin (CAS 58-82-2) is an inflammatory mediator. It causes blood vessels to dilate via the release of prostacyclin, nitric oxide, and Endothelium-Derived Hyperpolarizing Factor (EDHF). It is a physiologically and pharmacologically active peptide of the kinin group, and comprises nine amino acids. Angiotensin converting enzyme (ACE) inactivates bradykinin. ACE inhibitors effectively increase bradykinin levels by inhibiting its degradation, thereby increasing its blood pressure lowering effect. ACE inhibitors are approved for the treatment of hypertension and heart failure. The kinin-kallikrein system generates bradykinin by proteolytic cleavage of its kininogen precursor, high-molecular-weight kininogen (HMWK or HK), via the enzyme kallikrein. In humans, bradykinin is broken down by three kininases: angiotensin-converting enzyme (ACE), aminopeptidase P (APP), and carboxypeptidase N (CPN), which cleave the 7-8, 1-2, and 8-9 positions, respectively.

Bradykinin is a potent endothelium-dependent vasodilator and mild diuretic, which may cause a lowering of the blood pressure. It also causes contraction of non-vascular smooth muscle in the bronchus and gut, and is also involved in the mechanism of pain. Importantly, it increases vascular permeability. During inflammation, it is released locally from mast cells and basophils during tissue damage. Accordingly, increased activity of bradykinin in the lungs may lead to increased influx of liquids into the lungs, leading to ARDS. When the S protein of Sars-Cov-2 binds ACE, the ACE loses its ability to reduce bradykinin levels, increasing B1 activity and thus promoting influx of liquids. Local inflammation can exacerbate this by promoting B1 expression.

Bradykinin is also thought to be the cause of the dry cough in some patients on widely prescribed angiotensin-converting enzyme (ACE) inhibitor drugs. It is thought that bradykinin is converted to inactive metabolites by ACE, therefore inhibition of this enzyme leads to increased levels of bradykinin; increased bradykinin sensitizes somatosensory fibers and thus causes hyperalgesia. Bradykinin may mediate this via pro-inflammatory peptides (e.g. substance P, neuropeptide Y) and a local release of histamine. In severe cases, the elevation of bradykinin can result in angioedema, a medical emergency. People of African descent have up to 5x increased risk of ACE inhibitor induced angioedema due to hereditary predisposing risk factors such as hereditary angioedema. This refractory cough is a common cause for stopping ACE inhibitor therapy. Overactivation of bradykinin is thought to play a role in a rare disease called hereditary angioedema, formerly known as hereditary angio-neurotic edema.

Compounds for use

The invention provides a bradykinin receptor antagonist for use as a medicament in the treatment of coronavirus disease 2019 (COVID-19). Features and definitions for COVID-19 have been provided above. This is referred to herein as the bradykinin receptoy agonist (for use) according to the invention. In preferred embodiments, the bradykinin receptor antagonist is an antagonist for bradykinin receptor B1 or for bradykinin receptor B2, preferably for bradykinin receptor B1. In more preferred embodiments both B1 and B2 are inhibited.

As used herein, antagonist and inhibitor are broadly used interchangeably. Also, a bradykinin receptor inhibitor is sometimes referred to as a bradykinin inhibitor. Bradykinin inhibitors (antagonists) are continuously being developed as potential therapies for hereditary angioedema. Bromelain, a substance obtained from the stems and leaves of the pineapple plant, suppresses trauma-induced swelling caused by the release of bradykinin into the bloodstream and tissues and can be considered a general bradykinin receptor inhibitor(Lotz-Winter H, 1990, Planta Medica. 56 (3): 249-53. doi:10.1055/s-2006-960949). Other substances that act as bradykinin inhibitors include aloe (Yagi A et al., 1982, J. Pharm. Sci. 71 (10): 1172-4. doi:10.1002/jps.2600711024), and polyphenols such as those found in red wine or green tea (Richard T et al., 2003, J. Biomol. Structure & Dynamics. 21 (3): 379-85. doi:10.1080/07391102.2003.10506933). Preferred polyphenols are green tea polyphenols and red wine polyphenols.

In preferred embodiments the invention provides the bradykinin receptor antagonist for use according to the invention, wherein the bradykinin receptor agonist is selected from the group consisting of safotibant, [Leu8]-bradykinin1-8, MK-0686, BI11382, ELN-441958, SSR 240612, NVP-SAA164, R-715, bromelain, a polyphenol, aloe, icatibant, [d-Phe7]-bradykinin, [Thi5,8,d- Phe7]-bradykinin, WIN 64338, and FR 173657, preferably selected from the group consisting of safotibant, [Leu8]-bradykinin1-8, MK-0686, BI11382, ELN-441958, SSR 240612, NVP-SAA164, R- 715, icatibant, [d-Phe7]-bradykinin, [Thi5,8,d-Phe7]-bradykinin, WIN 64338, and FR 173657; or wherein the bradykinin receptor agonist is a B1 receptor agonist such as safotibant, [Leu8]- bradykinin1-8, MK-0686, BI11382, ELN-441958, SSR 240612, NVP-SAA164, or R-715, preferably safotibant; or wherein the bradykinin receptoy agonist is a B2 receptor agonist such as icatibant, [d-Phe7]-bradykinin, [Thi5,8,d-Phe7]-bradykinin, WIN 64338, or FR 173657, preferably icatibant or FR 173657, more preferably icatibant. Safotibant is N-[[4-(4,5-Dihydro-1H-imidazol-2-yl)phenyl]methyl]-2-[2-[[(4-methoxy-2,6- dimethylphenyl)sulfonyl]methylamino]ethoxy]-N-methylacetamide and is also known as LF22-0542. It is preferably used as its fumarate. It is a non-peptide bradykinin B1 antagonist withbinding Ki values of 0.35 and 6.5 nM at cloned human and mouse B1 receptors, respectively, while having no affinity for either human, mouse, or rat B2 receptors at concentrations up to 10 pM. [Leu8]-bradykinin1-8 is a B1 receptor antagonist, which is bradykinin 1-8 where the phenylalanine at position 8 is replaced by leucine (Seabrook et al., doi: 10.1111/j.1476- 5381.1995.tb15887.x).

A series of biphenylaminocyclopropane carboxamide based bradykinin B1 receptor antagonists has been developed that possesses good pharmacokinetic properties, one of which is MK-0686 (Kuduk SD et al. J Med Chem 2007. DOI:10.1021/jm061094b)(CAS 578727-68-1).

The Boehringer Ingelheim drug BI113823 is another bradykinin B1 receptor antagonist (Nasseri et al., Crit Care Med. 2015 Nov;43(11):e499-507. doi: 10.1097/CCM.0000000000001268) (CAS 1439402-33-1). Other suitable B1 receptor antagonists are ELN-441958 (CAS 913064-47-8), also known as 7-chloro-2-[3-(9-pyridin-4-yl-3,9-diazaspiro[5.5]undecane-3-carbonyl)phenyl]-3H- isoindol-1-one, and SSR 240612 (CAS 465539-70-2), also known as (2R)-2-[[(3R)-3-(1 ,3- benzodioxol-5-yl)-3-[(6-methoxynaphthalen-2-yl)sulfonylamino]propanoyl]amino]-3-[4-[[(2S,6R)- 2,6-dimethylpiperidin-1 -yl]methyl]phenyl]-N-methyl-N-propan-2-ylpropanamide, and NVP-SAA164 which is also known as [1-[4-(2,2-diphenylethylamino)-3-(morpholine-4- carbonyl)phenyl]sulfonylpiperidin-4-yl]-(4-methylpiperazin-1-yl)methanone (Ritchie et al., J Med Chem. 2004 Sep 9;47(19):4642-4), and R-715, which is also known as BDBM50408804 and AKOS024457611 and has formula C57H81N13O12.

Icatibant is a second generation B2 receptor antagonist which has undergone limited clinical trials in pain and inflammation. Icatibant, trade name Firazyr, also known as Hoe 140, is medication that has been approved by the European Commission for the symptomatic treatment of acute attacks of hereditary angioedema (HAE) in adults with C1 -esterase-inhibitor deficiency. It is not effective in angioedema caused by medication from the ACE inhibitor class, as shown in a 2017 trial. It is a peptidomimetic consisting often amino acids, which is a selective and specific antagonist of bradykinin B2 receptors The licensed dose of icatibant for hereditary angioedema is 30 mg by subcutaneous injection as a single dose. Icatibant is a highly preferred B2 antagonist for use according to the invention.

[d-Phe7]-bradykinin and [Thi5,8,d-Phe7]-bradykinin are additional B2 antagonists (Vavrek RJ, Stewart JM. Competitive antagonists of bradykinin. Peptides.1985; 6:161-164), as is WIN 64338 (Salvino et al., J. Med. Chem. 1993, 36, DOI: 10.1021/jm00069a021). FR 173657 is another orally active non-peptide B2 antagonist that has undergone trials as analgesic and antiinflammatory drug; it is also known as (E)-3-(6-acetamido-3-pyridyl)-N-[N-[2-4-dichloro-3-[(2-methyl-8-quinolinyl) oxymethyl]phenyl]-N-methylaminocarbonyl-methyl]acrylamide).

Although blocking B1 is most effective for preventing, ameliorating, or reverting ARDS and pulmonary angioedema associated with COVID-19, a combined blockade of B2 is advantageous as it would prevent vasodilation. Therefore the invention provides a combination of a bradykinin receptor B1 antagonist and a bradykinin B2 antagonist, preferably for use as described elsewhere herein. The combination can be in separate containers or vials, or the inhibitors can be in a mixture or in a single composition. In some embodiments the combination of a B1 inhibitor and a B2 inhibitor relates to a single compound that inhibits both B1 and B2.

The invention also provides a composition, preferably a pharmaceutical composition, comprising one of: i) at least one bradykinin receptor B1 antagonist, preferably as defined above; ii) at least one bradykinin receptor B2 antagonist, preferably as defined above; or iii) at least one bradykinin receptor B1 antagonist and at least one bradykinin receptor

B2 antagonist, both preferably as defined above, this composition is preferably for use according to the invention. The composition can further comprise an additional pharmaceutical agent as defined elsewhere herein. The composition can further comprise a pharmaceutically acceptable excipient. Preferably, such a composition is formulated as a pharmaceutical composition. A preferred excipient is water, preferably purified water, more preferably ultrapure water. Further preferred excipients are adjuvants, binders, desiccants, and diluents. Further preferred compositions additionally comprise additional medicaments, preferably for treating viral infections and/or inflammation, more preferably for treating COVID-19 or inflammation. Suitable anti-inflammatory agents and anticiral agents are defined later herein Formulation of medicaments, ways of administration, and the use of pharmaceutically acceptable excipients are known and customary in the art and for instance described in Remington; The Science and Practice of Pharmacy, 21st Edition 2005, University of Sciences in Philadelphia.

Compositions and pharmaceutical compositions according to the invention may be manufactured by processes well known in the art; e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes, which may result in liposomal formulations, coacervates, oil-in-water emulsions, nanoparticulate/microparticulate powders, or any other shape or form. Compositions for use in accordance with the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent on the route of administration chosen.

For injection, the compounds according to the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. Subcutaneous injection is a preferred administration for products for use according to the invention, particularly 30 mg by subcutaneous injection.

For administration by inhalation, the compounds and compositions according to the invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and optionally a suitable powder base such as lactose or starch.

The compound or composition according to the invention may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. In this way it is also possible to target a particular organ, tissue, site of inflammation, etc. Formulations for infection may be presented in unit dosage form, e.g., in ampoules or in multi-dose container, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Treatment regimen

In preferred embodiments, the bradykinin receptor antagonist for use according to the invention, wherein treatment is continued as long as the viral load remains within 3 Iog10 of the viral load at the start of treatment. Preferably, it is continued as long as the viral load remains within 4 log 10, more preferably within 5 Iog10, even more preferably within 6 Iog10. Most preferably, treatment is continued until no viral load can be detected. Viral load, also known as viral burden, viral titre, or viral titer, is a numerical expression of the quantity of virus in a given volume. It is preferably expressed as viral particles, or infectious particles, or genome copies per ml_ depending on the type of assay. A higher viral load generally correlates with the severity of COVID-19. The quantity of virus / mL can be calculated by assaying the amount of virus in an involved body fluid. For example, it can be given in RNA copies per millilitre of blood plasma. A preferred method for determining viral load of Sars-CoV-2 is the N-gene-specific quantitative RT-PCR assay as described by Chu et al. (Clin Chem. 2020; published online Jan 31 , D0l:10.1093/clinchem/hvaa029). Samples used to determine viral load are preferably throat swab, sputum, respiratory, or stool samples, more preferably throat swab or sputum samples, most preferably throat swab samples.

The invention provides the bradykinin receptor antagonist for use according to the invention, wherein the subject has been affected with COVID-19 for at least 4 days, preferably for at least 6 days, most preferably for 9 days. The subject can be affected for 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 days or more. Fig. 2 provides an overview of recommended treatment at different points during disease progression. Because seroconversion generally occurs at day nine, the bradykinin receptor antagonist for use according to the invention is preferably administered around that time, preferably at that time. Many patients are known to worsen especially around day nine, although this can also occur much earlier. This worsening seems to be accompanied specifically with further increases in IL-6, CRP, ferritin, without elevated procalcitonin, indicative of a progressive inflammatory status, which is a clear different pattern of the first stage of the disease. Accordingly, in preferred embodiments, the bradykinin receptor antagonist for use according to the invention is for administration to a subject with at least one of increased IL-6, increased CRP, and increased ferritin. Preferably, the subject does not have increased procalcitonin. Most preferably, the subject has increased in IL-6, CRP, ferritin, without increased procalcitonin. All these factors are widely known in the art and standardized and validated assays for these parameters are in routine use in clinical laboratories throughout the world. A skilled person or a treating physician will know when such a factor is increased or not. In preferred embodiments, treatment is started in a subject who has seroconverted Sars-CoV-2. Seroconversion of Sars-CoV-2 can be detected using known methods such as described by Amanat et al. (DOI: 10.1101/2020.03.17.20037713).

The bradykinin receptor antagonist for use according to the invention is preferably administered n an effective amount. An “effective amount” of a bradykinin receptor antagonist is an amount which, when administered to a subject, is sufficient to reduce or eliminate either one or more symptoms of a disease, or to retard the progression of one or more symptoms of a disease, or to reduce the severity of one or more symptoms of a disease, or to suppress the manifestation of a disease, or to suppress the manifestation of adverse symptoms of a disease. An effective amount can be given in one or more administrations.

The “effective amount” of that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host to which the active ingredient is administered and the particular mode of administration. The unit dosage chosen is usually fabricated and administered to provide a desired final concentration of the compound in the blood.

The effective amount (i.e. the effective total daily dose), preferably for adults, is herein defined as a total daily dose of about 0.01 to 2000 mg, or about 0.01 to 1000 mg, or about 0.01 to 500 mg, or about 5 to 1000 mg, or about 20 to 800 mg, or about 30 to 800 mg or about 30 to 700 mg, or about 20 to 700 mg or about 20 to 600 mg, or about 30 to 600 mg, or about 30 to 500 mg, about 30 to 450 mg or about 30 to 400 mg, or about 30 to 350 mg or about 30 to 300 mg or about 50 to 600 mg, or about 50 to 500 mg, or about 50 to 450 mg, or about 50 to 400 mg or about 50 to 300 mg, or about 50 to 250 mg, or about 100 to 250 mg or about 150 to 250 mg. In the most preferred embodiment, the effective amount is about 200 mg. In preferred embodiments, the invention provides a bradykinin receptor antagonist for use according to the invention, or a composition for use according to the invention, characterized in that it is administered to a subject in an amount ranging from 0.1 to 400 mg/day, preferably from 0.25 to 150 mg/day, such as about 100 mg/day. Most preferably about 25 mg - 35 mg such as 30 mg per dose is administered, more preferably at one such dose per day.

In other highly preferred embodiments, 30 mg per dose is administered, for at least two doses, more preferably for three doses. Such doses are preferably administered at intervals of at least 2 hours, more preferably at intervals of at least 4 hours, most preferably at intervals of at least 6 hours. The intervals are preferably at most 24 hours, more preferably at most 12 hours, most preferably at most 8 hours. Preferred intervals are 6 hours. A preferred regimen is 3 doses at 6- hour intervals, particularly for icatibant, where a single such dose is preferably 30 mg.

Alternatively, the effective amount of the compound, preferably for adults, preferably is administered per kg body weight. The total daily dose, preferably for adults, is therefore about 0.05 to about 40 mg/kg, about 0.1 to about 20 mg/kg, about 0.2 mg/kg to about 15 mg/kg, or about 0.3 mg/kg to about 15 mg/kg or about 0.4 mg/kg to about 15 mg/kg or about 0.5 mg/kg to about 14 mg/kg or about 0.3 mg/kg to about 14 mg/kg or about 0.3 mg/kg to about 13 mg/kg or about 0.5 mg/kg to about 13 mg/kg or about 0.5 mg/kg to about 11 mg/kg.

The total daily dose for children is preferably at most 200 mg. More preferably the total daily dose is about 0.1 to 200 mg, about 1 to 200 mg, about 5 to 200 mg about 20 to 200 mg about 40 to 200 mg, or about 50 to 200 mg. Preferably, the total daily dose for children is about 0.1 to 150 mg, about 1 to 150 mg, about 5 to 150 mg about 10 to 150 mg about 40 to 150 mg, or about 50 to 150 mg. More preferably, the total daily dose is about 5 to 100 mg, about 10 to 100 mg, about 20 to 100 mg about 30 to 100 mg about 40 to 100 mg, or about 50 to 100 mg. Even more preferably, the total daily dose is about 5 to 75 mg, about 10 to 75 mg, about 20 to 75 mg about 30 to 75 mg about 40 to 75 mg, or about 50 to 75 mg.

Alternative examples of dosages which can be used are an effective amount of the compounds for use according to the invention within the dosage range of about 0.1 pg /kg to about 300 mg/kg, or within about 1 .0 pg /kg to about 40 mg/kg body weight, or within about 1 .0 pg/kg to about 20 mg/kg body weight, or within about 1.0 pg /kg to about 10 mg/kg body weight, or within about 10.0 pg /kg to about 10 mg/kg body weight, or within about 100 pg/kg to about 10 mg/kg body weight, or within about 1.0 mg/kg to about 10 mg/kg body weight, or within about 10 mg/kg to about 100 mg/kg body weight, or within about 50 mg/kg to about 150 mg/kg body weight, or within about 100 mg/kg to about 200 mg/kg body weight, or within about 150 mg/kg to about 250 mg/kg body weight, or within about 200 mg/kg to about 300 mg/kg body weight, or within about 250 mg/kg to about 300 mg/kg body weight. Other dosages which can be used are about 0.01 mg/kg body weight, about 0.1 mg/kg body weight, about 1 mg/kg body weight, about 10 mg/kg body weight, about 20 mg/kg body weight, about 30 mg/kg body weight, about 40 mg/kg body weight, about 50 mg/kg body weight, about 75 mg/kg body weight, about 100 mg/kg body weight, about 125 mg/kg body weight, about 150 mg/kg body weight, about 175 mg/kg body weight, about 200 mg/kg body weight, about 225 mg/kg body weight, about 250 mg/kg body weight, about 275 mg/kg body weight, or about 300 mg/kg body weight.

Compounds or compositions for use according to the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided dosage of two, three or four times daily. In a preferred embodiment of the invention, "subject", "individual", or "patient" is understood to be an individual organism, preferably a vertebrate, more preferably a mammal, even more preferably a primate and most preferably a human. In other embodiments the subject is not human. The subject can be a bat. Preferred subjects have confirmed SARS-CoV-2 infection, preferably as confirmed by a polymerase chain reaction (PCR) assay. The use according to the invention is particularly useful for subjects who have a computed tomography severity score of at least 5, more preferably of at least 7. Such scores are known in the art, for instance from Salaffi et al., doi: 10.1097/MD.0000000000022433. In brief, to establish such a score each lung is radiologically evaluated at 3 levels: the upper level (above the carina), the middle level (below the carina up to the upper limit of the inferior pulmonary vein), and the lower level (below the inferior pulmonary vein). The right and left lung are evaluated separately and the results are summed to give the final score for each level. The nature of the lung abnormalities in each area is defined on the basis of a 4-point scoring system (1 is normal parenchyma, 2 is at least 75% ground-cglass opacities or crazy-paving patter, 3 is combination thereof and consolidation provided that each accounts for less than 75% involvenement, 4 is at least 75% consolidation), and the extent of lung involvement at each level was categorized as 0 for normal lung; 1 for <25% lung abnormalities; 2 for 25% to 49% abnormalities; 3 for 50% to 74% abnormalities and 4 for >75% abnormalities. The 2 scores (the extent and nature of the abnormalities) are multiplied by each other and added to the scores of all six levels (3 levels on each side). A final radiological severity score ranging from 0 to 96 is thus attributed to parenchymal involvement. In a further preferred embodiment of the invention, the human is an adult, e.g. a person that is 18 years or older. In addition, it is herein understood that the average weight of an adult person is 62 kg, although the average weight is known to vary between countries. In another embodiment of the invention the average weight of an adult person is therefore between about 50 - 90 kg. It is herein understood that the effective dose as defined herein is not confined to subjects having an average weight. Preferably, the subject has a BMI (Body Mass Index) between 18.0 to 40.0 kg/m², and more preferably a BMI between 18.0 to 30.0 kg/m².

The use according to the invention is very suitable for increasing oxygen saturation. Hence a subject preferably has an oxygen saturation of less than 95%, more preferably of less than 93%, most preferably of less than 90%. This oxygen saturation is preferably the oxygen saturation without supplemental oxygen. Other preferred subjects are subjects in need of 1 L/min supplemental oxygen or more, more preferably in need of 2 L/min or more, most preferably in need of 3 L/min or more. It is preferred that a subject is not undergoing an acute ischemic event.

Alternatively, the subject to be treated is a child, e.g. a person that is 17 years or younger. In addition, the subject to be treated may be a person between birth and puberty or between puberty and adulthood. It is herein understood that puberty starts for females at the age of 10 -11 years and for males at the age of 11 - 12 year. Furthermore, the subject to be treated may be a neonate (first 28 days after birth), an infant (0-1 year), a toddler (1-3 years), a preschooler (3-5 years); a school- aged child (5-12 years) or an adolescent (13-18 years).

To maintain an effective range during treatment, the bradykinin receptor antagonist or composition may be administered once a day, or once every two, three, four, or five days. However preferably, the compound may be administered at least once a day. Hence in a preferred embodiment, the invention pertains to a bradykinin receptor antagonist for use according to the invention, or a composition for use according to the invention, characterized in that it is administered to a subject 4, 3, 2, or 1 times per day or less, preferably 1 time per day. The total daily dose may be administered as a single daily dose. Alternatively, the compound is administered at least twice daily. Hence, the compound as defined herein may be administered once, twice, three, four or five times a day. As such, the total daily dose may be divided over the several doses (units) resulting in the administration of the total daily dose as defined herein. In a preferred embodiment, the compound is administered twice daily.

In a preferred embodiment, the total daily dose is divided over several doses per day. These separate doses may differ in amount. For example for each total daily dose, the first dose may have a larger amount of the compound than the second dose or vice versa. However preferably, the compound is administered in similar or equal doses. Therefore in a most preferred embodiment, the compound is administered twice daily in two similar or equal doses.

In a further preferred embodiment of the invention, the total daily dose of the compound as defined herein above is administered in at least two separate doses. The interval between the administration of the at least two separate doses is at least about 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours, preferably the interval between the at least two separate doses is at least about 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours and more preferably the interval between the at least two separate doses is at least about 8, 9, 10, 11 or 12 hours.

Detailed description of COVID-19 treatment

In some embodiments, the invention provides the bradykinin receptor antagonist for use according to the invention, wherein the treatment is for COVID-19-associated acute respiratory distress syndrome (ARDS), preferably wherein the ARDS is associated with at most slightly decreased pulmonary compliance. Preferably, pulmonary compliance is reduced by at most 40, 35, 30, 25, 20, 15, 10, or 5% as compared to a healthy subject. Inspiratory compliance is the preferred compliance to assess. In the setting of severe ARDS, where a patient is ventilated with lower tidal volumes and driving pressures, hypercapnia is inevitable. This permissive hypercapnia is made possible with deep sedation and pharmacological paralysis when severe. Hypercapnia is usually well tolerated but is known to have adverse physiologic effects.

Acute respiratory distress syndrome (ARDS) is a type of respiratory failure associated with fast onset of widespread inflammation in the lungs. Symptoms can include shortness of breath, rapid breathing, and blue skin coloration. Survivors often experience a decreased quality of life. COVID-19 may cause ARDS. For ARDS in general, 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. Diagnosis is based on a Pa0₂/FiC>₂ ratio (ratio of partial pressure arterial oxygen and fraction of inspired oxygen) of less than 300 mm Hg despite a positive end-expiratory pressure (PEEP) above 5 cm H2O.

The inventors have found that excessive activity of bradykinin receptors, particularly of B1 , is at the root of ARDS in COVID-19 patients. Treatment preferably further involves mechanical ventilation, together with treatments directed at the underlying cause such as administration of the bradykinin receptor inhibitor as definedherein. Ventilation strategies include using low volumes and low pressures. If oxygenation remains insufficient, lung recruitment maneuvers and neuromuscular blockers may be used. If this is insufficient, extracorporeal membrane oxygenation (ECMO) can be used.

The invention also provides the bradykinin receptor antagonist for use according to the invention, wherein the subject has at least one of the following conditions: i) pulmonary edema, preferably pulmonary edema that is resistant to corticosteroids, to andrenaline, or to both, preferably to both; ii) angioedema outside of the lungs, preferably in the gut; iii) unilateral or bilateral ground-glass opacities or clear consolidations as visible on CT scans of the subject; iv) antibodies against Sars-CoV-2 spike antigen; v) inflammation, preferably inflammation in the lungs; vi) an age of at least 40, more preferably at least 50, even more preferably at least 60, still more preferably at least 65, most preferably at least 70; vii) fever; viii) dry cough; ix) dyspnea; x) tachypnea; xi) increased D-fimers, preferably without evidence of thromboembolic events; xii) having suffered COVID-19 for at least nine days; xiii) elevated IL-6; xiv) elevated CRP; xv) elevated ferritin; xvi) any of xiii-xv without elevated procalcitonin, preferably all of xiii-xv without elevated procalcitonin; xvii) cytokine release syndrome.

Preferrable a subject to be treated has at least one of conditions ii, iii, iv, v, xvi, or xvii.

Cytokine release syndrome is also known as cytokine storm. It is a form of systemic inflammatory response syndrome that can be triggered by a variety of factors such as infections and is known to occur in some subjects suffering COVID-19. It occurs when large numbers of white blood cells are activated and release inflammatory cytokines, which in turn activate yet more white blood cells. The invention prevents deterioration via ARDS while a cytokine storm occurs.

Combined strategies

COVID-19 shows several symptoms that can each be individually addressed. Fig. 2 shows recommended treatment options.

Preferred embodiments provide the bradykinin receptor antagonist for use according to the invention, characterized in that it is administered together with a further pharmaceutical agent selected from an antiviral agent, an anti-inflammatory agent, an anti-fibrotic agent, and a neuromuscular blocker. Most preferably use of a bradykinin receptor antagonist and an antiviral agent are combined. In some embodiments, the bradykinin receptor antagonist is combined with a further pharmaceutical agent selected from an antiviral agent, an anti-inflammatory agent, and an anti-fibrotic agent. In some embodiments, the bradykinin receptor antagonist is combined with an antiviral agent and an anti-inflammatory agent. In some embodiments, the bradykinin receptor antagonist is combined with an antiviral agent, an anti-inflammatory agent, and an anti-fibrotic agent. In some embodiments, the bradykinin receptor antagonist is combined with an antiinflammatory agent.

Anti-inflammatory agents are widely known and are suitable to reduce or delay expression of additional B1 receptors, which will reduce their effect, making their antagonists more effective, or allowing a lower dose of the antagonists. Preferred anti-inflammatory agents in this context are anakinra, toclizumab, and eculizumab. Anakinra is most preferred.

Antivirals are widely known and help reduce the viral load, ultimately contributing to curing COVID-19. Examples of antiviral agents are oseltamivir, zanamivir, peramivir, and niclosamide, the latter of which is preferred. Additional preferred examples are favipiravir, remdesivir, chloroquine, ribavirin, arbidol, lopinavir, hydroxychloroquine, and ritonavir.

Anti-fibrotic agents are widely known and mitigate the onset of fibrosis. Examples of anti- fibrotic agents are nintedanib, angiotensin receptor blockers, and pirfenidone. A preferred anti- fibrotic strategy is to use corticosteroids, preferably at high doses.

Neuromuscular blockers are widely known and can improve mechanical ventilation. Examples of neuromuscular blockers are aminosteroids (pancuronium, vecuronium, rocuronium, rapacuronium, dacuronium, malouetine, duador, dipyrandium, pipecuronium, chandonium), acetylcholine, suxamethonium, decamethonium, compounds based on the tetrahydroisoquinoline moiety such as atracurium, mivacurium, and doxacurium; and gallamine. In case neuromuscular blockers do not achieve an effect, extracorporeal membrane oxygenation (ECMO) is preferably combined with administration of the bradykinin receptor inhibitor.

A subject to be treated is preferably a fluid restricted subject, or a subject on a fluid restriction diet. This is to prevent fluid overload, which can exacerbate ARDS.

In some embodiments, the bradykinin receptor antagonist for use according to the invention is combined with at least one of the following: an agent that blocks tissue kallikrein activity and/or which reduces the production of kinins, an example of such an agent is ecallantide; an agent that degrades the activity of kinis or that degrades kinins, such as recombinant ACE2 does; an agent that inhibits the common downstream signaling of B1 and B2 receptors; a treatment that suppresses local NO.

Subjects suffering COVID-19 are often mechanically ventilated. The inventors found that subjects generally have only slightly decreased pulmonary compliance. Driving pressure is usually low. Using high PEEP may therefore substantially increase functional residual capacity resulting in hyperinflation, high strain and considerable hypercapnia through an increase in dead space ventilation, but high PEEP can nonetheless be needed to prevent hypoxia. Furthermore, PEEP shows poor recruitability in most patients. Hereby mechanical ventilation may further contribute to lung damage. Pulmonary hypertension is not an important clinical component. When a subject is ventilated, PEEP is preferably at most 5 cm H2O, more preferably about 4 to 5 cm H2O.

In addition to the bradykinin-driven pulmonary edema and cytokine-related clinical detonation the inventors developed a strategy on how to ventilate in this context. “Preventive” mechanical ventilation is discouraged because the risk for ventilator induced lung injury is high. Accordingly, in preferred embodiments the subject is not ventilated, more preferably not undergoing preventive mechanical ventilation. After intubation, patients should be turned prone to improve oxygenation and keep PEEP at the lowest possible level. Accordingly, in preferred embodiments the subject is prone. Early assisted ventilation should be stimulated unless a high respiratory drive (P0 1 < -4 cm H2O, where P0 1 is airway occlusion pressure, the pressure generated at the airways during the first 100 msec of an inspiratory effort against an occluded airway) or a predicted Pmus > 15 cm H2O or a predicted transpulmonary pressure > 17 cm H2O is present. In preferred embodiments is provided the bradykinin receptor antagonist for use according to the invention, wherein the subject is undergoing assisted ventilation with an airway occlusion pressure (P0 1) below 5 cm H2O. Preferably P0 1 is below 4 cm H2O. For subjects with P0 1 above 4 cm H2O, appropriate respiratory drive suppression can be attempted. Patients should be kept as dry as possible. Accordingly, a preferred subject is a subject who is under a restricted fluid intake regimen.

In preferred embodiments is provided the bradykinin receptor antagonist for use according to the invention, wherein the subject is not simultaneously administered ACE inhibitors, angiotensin II receptor blockers, or recombinant ACE2. Alternately, the subject has not recently been administered ACE inhibitors, angiotensin II receptor blockers, or recombinant ACE2, or should not be scheduled for administration of ACE inhibitors, angiotensin II receptor blockers, or recombinant ACE2.

General Definitions

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb “to consist” may be replaced by “to consist essentially of” meaning that a combination or a composition as defined herein may comprise additional components) than the ones specifically identified, said additional components) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

The use of a substance as a medicament as described in this document can also be interpreted as the use of said substance in the manufacture of a medicament. Similarly, whenever a substance is used for treatment or as a medicament, it can also be used for the manufacture of a medicament for treatment. Products for use as a medicament described herein can be used in methods of treatments, wherein such methods of treatment comprise the administration of the product for use. For instance, the invention provides a method of treating COVID-19, the method comprising administration of an effective dose of a bradykinin receptor agonist as defined elsewhere herein, or of a combination as defined elsewhere herein, or of a composition as defined elsewhere herein, to a subject in need thereof.

In the context of this invention, a decrease or increase of a parameter to be assessed means a change of at least 5% of the value corresponding to that parameter. More preferably, a decrease or increase of the value means a change of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, or 100%. In this latter case, it can be the case that there is no longer a detectable value associated with the parameter.

The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 5% of the value.

Each embodiment as identified herein may be combined together unless otherwise indicated. The invention has been described above with reference to a number of embodiments. A skilled person could envision trivial variations for some elements of the embodiments. These are included in the scope of protection as defined in the appended claims. All patent and literature references cited are hereby incorporated by reference in their entirety. Description of drawings

Fig. 1 - Pathways involving kallikrein and kinins including bradykinin, showing the role of B1 and B2 receptors and their effect on angioedema in the lung.

Fig. 2 - Treatment according to the invention. Fig. 3A - normal state of pulmonary alveolus.

Fig. 3B - state during mild inflammation - note the appearance of B1 receptors.

Fig. 3C - state during hyperinflammation.

Examples Example 1 - Clinical observations

Patients admitted with symptomatic COVID-19 most commonly exhibited infection fever, dry cough, and dyspnea. Importantly, we observed that dyspnea and tachypnea can differ from hour to hour and a feeling of drowning is described with sometimes sudden recovery by patients. CT scans revealsunilateral or bilateral ground-glass opacities, that in some cases progress to more clear consolidations throughout the disease. Fluid restriction was found to improve oxygenation and ameliorate the feeling of dyspnea. Notably, plasma concentrations of D-dimers at this stage were found to be increased without evidence of thromboembolic events.

There is a phase during clinical admission where many patients are getting better, but some will worsen especially around day nine, although this can also occur much earlier. It was found thattthis worsening is specifically accompanied with further increases in IL-6, CRP, and ferritin, and that procalcitonin was not elevated. This is indicative of a progressive inflammatory status, which is a pattern that is clearly different from the first stage of the disease.

In the ICU several striking further observations were made. In contrast to patients with conventional forms of ARDS, most patients with COVID-19 and particularly with severe COVID-19 show an only slightly decreased pulmonary compliance. Driving pressure is usually low. Using high positive end- expiratory pressure (PEEP) can therefore substantially increase functional residual capacity resulting in hyperinflation, high strain, and considerable hypercapnia through an increase in dead space ventilation. However high PEEP can be needed to prevent hypoxia. Furthermore, PEEP shows poor recruitability in most patients. Hereby, mechanical ventilation further contributes to lung damage while pulmonary hypertension is not an important clinical component.

Most striking observations in COVID-19 patients are the hints on pulmonary edema (also seen on CT scans as ground glass opacities), dry cough, fluid restrictions to prevent more severe hypoxia, the huge PEEP that is needed while lungs are compliant, and the fact that anti-inflammatory therapies are not powerful enough to counter the severity of the disease. In some patients, symptoms are followed by a clinical worsening of disease around day 9 due to the formation antibodies directed against the spike (S)-antigen of the corona-virus that binds to ACE2 that can contribute to disease by enhancement of local immune cell influx and proinflammatory cytokines, leading to damage. This inflammation induces more B1 expression, and, via antibody-dependent enhancement of viral infection, leads to continued ACE2 dysfunction in the lung because of persistence of the virus. Example 2 - Treatment approach

It was found that the severity of the disease and many resulting deaths are due to a local vascular problem resulting from activation of B1 receptors on endothelial cells in the lungs. SARS-CoV-2 enters the cell via ACE2, a cell membrane bound molecule with enzymatic activity that next to its role in RAS is needed to inactivate des-Arg⁹ bradykinin, the potent ligand of the bradykinin receptor type 1 (B1). In contrast to bradykinin receptor 2 (B2), the B1 receptor on endothelial cells is upregulated by proinflammatory cytokines. Without ACE2 acting as a guardian to inactivate the ligands of B1 , the lung environment is prone to local vascular leakage leading to angioedema. Angioedema is likely a feature already early in disease, and can explain the typical CT scans and the drowning feeling of patients.

The inventors identified that a bradykinin-dependent local lung angioedema via B1 and B2 receptors is an important feature of COVID-19, resulting in a very high number of ICU admissions. It was found that blocking the B1 and B2 receptors has an ameliorating effect on disease caused by COVID-19. This kinin-dependent pulmonary edema is resistant to corticosteroids or adrenaline and should be targeted as long as the virus is present. Administration of safotibant thus ameliorates ARDS in COVID-19 patients.

Example 3 - AT1R blocking is not effective treatment for COVID-19 Several reports exist that suggest AT1 R blocking might protect from ARDS induced by SARS-CoV (Imai Y, Kuba K, Rao S, et al. Nature 2005; 436: 112-6; Kuba K, Imai Y, Rao S, et al. Nat Med 2005; 11 : 875-9). This is not correct. In the setting of SARS-CoV infection there is not a single data set that has investigated the infection with an AT1 R blocker.

What the Imai and Kuba papers have demonstrated is that in the setting of acid induced ARDS the deficiency of ACE2 aggravates the ARDS. This aggravation by the setting of ACE2 deficiency is angiotensin 2 dependent (hence the ACE-/- background ameliorating the ACE2 deficient effect) and AT1 R dependent. When translating this into the clinics: yes, when a person that would have no ACE2 would get an aspiration pneumonia driven ARDS, one could administer AT1 R blocker to compensate for the ACE2 deficiency, but this would not resolve ARDS. It can have a place in the treatment: when a COVID-19 aspirates one can administer an AT1 R blocker since the acid-induced worsening can be responsive to AT1 R blockade.The virus can get in the gut, where there is ACE2. Accordingly there might be angioedema with swelling in the gut due to bradykinin (similar to the lung), eventually causing gastric passage problems and nausua which we do see in the clinics, and which causes aspiration (or microaspiration). This then induces an extra component, namely an acid-induced ARDS component. And here AT1 R blocker might work and subjects with ACE-inhibitors might be protected from this. But is not a systematic approach to actually treating COVID-19. SARS-CoV infection without acid have been reported in ACE2 deficient subjects, and here the cause of ARDS cannot follow from the above AT1 R model.

Kuba et al. investigated Fc-Spike induced ARDS in an acid environment. The Fc-Spike induced ARDS worsened in the acid setting and could be attenuated by administering recombinant ACE2 or by blocking AT1R. However, Fc-spike itself has no effect in the absence of acid. This can be explained by the fact that in such studies there is no actual inflammatory hit, and thus no ARDS.

In the art, it is known that ACE2 deficiency generally worsens ARDS when there is inflammation (for example induced by CLP). However angiotensin 2 and AT1 R blocking has not been studied in this setting. It is known from Sodhi CP et al. (Am J Physiol - Lung Cell Mol Physiol 2018; 314: L17- 31) that LPS induced edema (ARDS) is blocked by B1 receptor antagonist in the setting of ACE2 inhibition. It follows that the treatment of the invention, which reduces the effect of des-arg9- bradykinin by pursuing B1 blockade is suitable for treating ARDS associated with COVID-19, and that AT1 R blockers will only be effective in specific conditions, and will only be effective to address those specific conditions. AT1R blockers can even be harmful because they upregulate ACE2.

Example 4 - Bradykinin 2 receptor antagonist icatibant improves oxygenation in patients with coronavirus disease 2019 (COVID-19)

Pulmonary edema is a prominent feature in patients with severe coronavirus disease 2019 (COVID- 19). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the cell via angiotensin-converting enzyme 2 (ACE2). ACE2 is involved in degrading the kinin des-Arg⁹- bradykinin, a potent vasoactive peptide that can cause vascular leakage. Loss of ACE2 can lead to plasma leakage and further activation of the plasma kallikrein-kinin system with more bradykinin formation that can contribute to pulmonary angioedema via stimulation of bradykinin 2 receptors. We demonstrate here that treatment with the bradykinin 2 receptor antagonist icatibant in patients with COVID-19 can be used as a treatment strategy.

Patients with COVID-19 were admitted from March to May 2020. We included 10 patients for treatment with 3 doses of 30 mg of icatibant (Firazyr; Shire Pharmaceuticals Ireland Limited) by subcutaneous injection at 6-hour intervals. Patients were eligible for icatibant treatment if they had confirmed SARS-CoV-2 by polymerase chain reaction assay, an oxygen saturation of less than 90% without supplemental oxygen, needed 3 L/min supplemental oxygen or more, and had a computed tomography severity score of 7 or greater. Patients with acute ischemic events at time of eligibility were excluded. For 9 patients who received icatibant on the ward, 2 matched control patients admitted prior to approval of this treatment were selected. Control patients with COVID-19 were matched on the factors sex, age, body mass index, and day of illness. One patient started receiving icatibant in the intensive care unit and was transferred to the ward with high-flow oxygen supplementation within 24 hours and discharged on day 7. We did not identify a matched control for this patient, so we were not able to evaluate the association of icatibant with outcomes for this individual. A change in oxygen need and oxygenation expressed as absolute number of liters per hour served as the primary outcome variable. Secondary outcomes included changes in D-dimer (dimerized plasmin fragment D), fever, and safety.

Nine cases were matched to 18 controls. The mean (SD) age was 55 (12.8) years for cases and 58 (10.5) years for controls. Most cases (9 of 10 [90%]) and controls (16 of 18 [90%]) were men. Patient and matched control characteristics are shown in table 4.1. Nine patients were prescribed icatibant on the ward. In all 9 patients, there was a marked decrease in oxygen supplementation (Table 4.2). After 3 injections of icatibant, 4 patients (44%) were no longer oxygen dependent within 10 to 35 hours. In 5 patients (56%), there was a substantial decrease of oxygen supplementation after treatment with icatibant (Table 4.2). Overall, in 8 of 9 patients (89%) treated with icatibant, a reduction of 3 L/min in oxygen supplementation or greater after 24 hours was observed (Table 4.2). Of 18 matched controls, only 3 (17%) showed a spontaneous reduction in oxygen supplementation of 3 L/min or greater within 24 hours. We noted that in 3 patients treated with icatibant there was a resurgence in the need for oxygen supplementation. This may be due to icatibant’s short half life of about 2 hours. Icatibant treatment was well tolerated in all 10 patients who received the drug. There were no severe adverse events. There was no clear association with D-dimer concentrations and fever.

Thus this study found evidence of an association between receipt of bradykinin receptor antagonist (here icatibant) and improved outcome of treatment of coronavirus disease 2019 (improved oxygenation). This demonstrates that targeting the kallikrein-kinin system in patients with COVID-19, especially in the early stages of disease when patients are hypoxic and are admitted to the hospital, is beneficial.

Table 4.1 - Characteristics of COVID-19 Patients With lcatibant Treatment and Matched Controls

Abbreviations: D-dimer, dimerized plasmin fragment D; F, female; M, male; NA, not applicable. SI conversion: to convert D-dimer to nmol/L, multiply by 5.476. ^a Days of illness at start icatibant or 24 hours after admission (controls). ^bD-dimer 24 hours after icatibant or 48 hours after admission (controls). Table 4.2 - Changes in Oxygen Supplementation at Baseline and 24 Hours in Individual

Patients and Controls

Claims

Claims

1 . A bradykinin receptor antagonist for use as a medicament in the treatment of coronavirus disease 2019 (COVID-19).

2. The bradykinin receptor antagonist for use according to claim 1 , wherein the treatment is for COVID-19-associated acute respiratory distress syndrome (ARDS), preferably wherein the ARDS is associated with at most slightly decreased pulmonary compliance.

3. The bradykinin receptor antagonist for use according to claim 1 or 2, wherein the antagonist is for bradykinin receptor B1 or for bradykinin receptor B2, preferably for bradykinin receptor B1 .

4. The bradykinin receptor antagonist for use according to any one of claims 1 -3, wherein the bradykinin receptor agonist is selected from the group consisting of safotibant, [Leu8]- bradykinin1-8, MK-0686, BI11382, ELN-441958, SSR 240612, NVP-SAA164, R-715, bromelain, a polyphenol, aloe, icatibant, [d-Phe7]-bradykinin, [Thi5,8,d-Phe7]-bradykinin, WIN 64338, and FR 173657, preferably selected from the group consisting of safotibant, [Leu8]-bradykinin1-8, MK-0686, BI11382, ELN-441958, SSR 240612, NVP-SAA164, R-715, icatibant, [d-Phe7]- bradykinin, [Thi5,8,d-Phe7]-bradykinin, WIN 64338, and FR 173657, or wherein the bradykinin receptor agonist is a B1 receptor agonist such as safotibant, [Leu8]-bradykinin1-8, MK-0686, BI11382, ELN-441958, SSR 240612, NVP- SAA164, or R-715, preferably safotibant, or wherein the bradykinin receptoy agonist is a B2 receptor agonist such as icatibant, [d-Phe7]-bradykinin, [Thi5,8,d-Phe7]-bradykinin, WIN 64338, or FR 173657, preferably icatibant or FR 173657, more preferably icatibant.

5. The bradykinin receptor antagonist for use according to any one of claims 1-4, characterized in that it is administered to a subject in an amount ranging from 0.1 to 400 mg/day, preferably from 0.25 to 150 mg/day, such as about 100 mg/day

6. The bradykinin receptor antagonist for use according to any one of claims 1-5, characterized in that it is administered together with a further pharmaceutical agent selected from an antiviral agent, an anti-inflammatory agent, an anti-fibrotic agent, and a neuromuscular blocker.

7. The bradykinin receptor antagonist for use according to any one of claims 1-6, wherein the subject is undergoing assisted ventilation with an airway occlusion pressure (Po i) below 5 cm H2O. Preferably P0 1 is below 4 cm H2O.

8. The bradykinin receptor antagonist for use according to any one of claims 1 -7, wherein the subject is not simultaneously administered ACE inhibitors, angiotensin II receptor blockers, or recombinant ACE2.

9. The bradykinin receptor antagonist for use according to any one of claims 1-8, wherein the subject has at least one of the following conditions: i) pulmonary edema, preferably pulmonary edema that is resistant to corticosteroids, to andrenaline, or to both, preferably to both; ii) angioedema outside of the lungs, preferably in the gut; iii) unilateral or bilateral ground-glass opacities or clear consolidations as visible on CT scans of the subject; iv) antibodies against Sars-CoV-2 spike antigen; v) inflammation, preferably inflammation in the lungs; vi) an age of at least 40, more preferably at least 50, even more preferably at least 60, still more preferably at least 65, most preferably at least 70; vii) fever; viii) dry cough; ix) dyspnea; x) tachypnea; xi) increased D-fimers, preferably without evidence of thromboembolic events; xii) having suffered COVID-19 for at least nine days; xiii) elevated IL-6; xiv) elevated CRP; xv) elevated ferritin; xvi) any of xiii-xv without elevated procalcitonin, preferably all of xiii-xv without elevated procalcitonin; xvii) cytokine release syndrome.

10. The bradykinin receptor antagonist for use according to the invention, wherein the subject has been affected with COVID-19 for at least 4 days, preferably for at least 6 days, most preferably for 9 days.

11. The bradykinin receptor antagonist for use according to any one of claims 1-10, wherein treatment is continued as long as the viral load remains within 3 log 10 of the viral load at the start of treatment.

12. A combination of a bradykinin receptor B1 antagonist and a bradykinin B2 antagonist, preferably for use as described in any one of claims 1-11.

13. A composition, preferably a pharmaceutical composition, comprising one of: i) at least one bradykinin receptor B1 antagonist, preferably as defined in claim 4; ii) at least one bradykinin receptor B2 antagonist, preferably as defined in claim 4; or iii) at least one bradykinin receptor B1 antagonist and at least one bradykinin receptor

B2 antagonist, both preferably as defined in claim 4, for use as described in any one of claims 1-11.

14. The composition according to claim 13, further comprising an additional pharmaceutical agent as defined in claim 6.

15. Method of treating COVID-19, the method comprising administration of an effective dose of a bradykinin receptor agonist as defined in any one of claims 1-11 , or of a combination as defined in claim 12, or of a composition as defined in claim 13 or 14, to a subject in need thereof.