WO2022024058A1

WO2022024058A1 - Use of heme arginate for the manufacture of a medicament for the treatment of beta coronavirus infection

Info

Publication number: WO2022024058A1
Application number: PCT/IB2021/056961
Authority: WO
Inventors: Zora MELKOVA; Pavel Martasek; Martina KOZIAR VASAKOVA; Lucie HOZNAUEROVA
Original assignee: Univerzita Karlova
Priority date: 2020-07-30
Filing date: 2021-07-30
Publication date: 2022-02-03
Also published as: CZ2020437A3; CZ308937B6

Abstract

Heme arginate, commonly used to treat acute porphyrias, has been found to inhibit the replication of beta coronaviruses, especially SARS-CoV-2, while protecting infected cells from damage. In addition, heme arginate acts as an immunomodulator. Heme arginate is usable for the treatment of patients infected with SARS-CoV-2 and related beta coronaviruses.

Description

Use of heme arginate for the manufacture of a medicament for the treatment of beta coronavirus infection

Field of Technology

The present invention relates to the treatment of SARS-CoV-2 virus infection by administration of heme arginate.

State of the Art

Introduction

SARS-CoV-2 appeared in China in December 2019, spread rapidly to other countries, and on March 11, 2020, a global pandemic was declared, affecting millions of people (17 million) and causing hundreds of thousands of deaths (over 669,000). SARS-CoV-2 belongs to beta coronaviruses, RNA viruses. The name is derived from the most severe symptom of the infection, Severe Acute Respiratory Syndrome, and its sequence is about 80% identical to the previously emerged SARS virus. In the absence of effective causal therapy, the treatment is only symptomatic. The most serious cases require artificial lung ventilation, progress to severe damage of the lung parenchyma and result in a multiorgan failure due to dysregulation of immune responses, the so-called cytokine storm. The disease mortality is around 6 % of reported cases worldwide, but numbers vary significantly from country to country depending on many factors - specific situation and quarantine measures, frequency of testing, health system readiness and overall health status of the population (0.5 to 14 %; tries; July 30, 2020).

Current solutions

The proposed or tested treatment of COVID-19 caused by the SARS-CoV-2 mainly includes antivirals developed for the treatment of other infections, other anti-infectives or immunomodulators and substances affecting the cellular receptor for this virus, ACE2 (Angiotensin-converting enzyme 2; [Costanzo, M, M.A.R. De Giglio, and G.N. Roviello, SARS- CoV-2: Recent Reports on Antiviral Therapies Based on Lopinavir /Ritonavir, Darunavir/Umifenovir, Hydroxychloroquine, Remdesivir, Favipiravir and Other Drugs for the Treatment of the New Coronavirus. Curr Med Chem, 2020; Saber-Ayad, M., M.A. Saleh, and E. Abu-Gharbieh, The Rationale for Potential Pharmacotherapy of COVID-19. Pharmaceuticals (Basel), 2020. 13(5)]. Among the antivirals developed against other viral infections, Remdesivir, an adenosine analogue originally developed against Ebola which is effective against a wide range of other viruses, including the coronaviruses SARS, MERS and now SARS-CoV-2, is particularly promising. It acts primarily as an inhibitor of virus-encoded RNA-dependent RNA polymerase (RdRP) and leads to cessation of viral RNA synthesis [Wang, M, et al, Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res, 2020. 30(3): p. 269-271; Ledford, H., Hopes rise for coronavirus drug remdesivir. Nature, 2020.]. The use of Favipiravir, a guanine analogue approved for the treatment of influenza A and B in some Asian countries that also inhibits RdRP, is also being tested [Furuta, Y., T. Komeno, and T. Nakamura, Favipiravir (T-705), a broad spectrum inhibitor of viral RNA polymerase. Proc Jpn Acad Ser B Rhys Biol Sci, 2017. 93(7): p. 449-463.]. The use of protease inhibitors used to treat HIV-1 (especially lopinavir and ritonavir) is also being tested, in addition in combination with other antiviral drugs - the combination of lopinavir-ritonavir, interferon beta-lb and ribavirin appears to be effective [Hung, I.F., et al, Triple combination of interferon beta-lb, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with COVID-19: an open-label, randomised, phase 2 trial. Lancet, 2020. 395(10238): p. 1695- 1704.]. Interferons are endogenously produced cytokines with a significant antiviral activity; ribavirin is an inhibitor of inosine monophosphate dehydrogenase, a key enzyme in de novo guanosine synthesis. Furthermore, the combination of umifenovir, a broad-spectrum anti -viral agent [Haviernik, ./., et al, Arbidol (Umifenovir): A Broad-Spectrum Antiviral Drug That Inhibits Medically Important Arthropod-Borne Flaviviruses. Viruses, 2018. 10(4)], with pegylated interferon b is being tested. Most recently, a clinical study has shown a positive effect of administering the inhaled form of interferon b alone (https:// www. sy nairgen . eomAvp- content/uplo¾ds/2020/07/200720-Synairgen-¾nnounces-posltive-results-from-trial-of- SNG00idn4iospitali^dTX¾VTD_: 19 j3atients_j?df; July 30, 2020).

Among other anti-infectives, the use of antimalarials chloroquine and hydroxychloquine is particularly being clinically tested, as they stabilize cell membranes, block endocytosis, and inhibit the acidification of the endosome and its conversion into a lysosome, thereby inhibiting the early steps in cell infection caused by many viruses, including SARS-CoV-2 [Savarino, A., et al, Effects of chloroquine on viral infections: an old drug against today's diseases? Lancet Infect Dis, 2003. 3(11): p. 722-7.; Liu, .!., et al, Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro. Cell Discov, 2020. 6: p. 16.; die, Z.H., H.; Xi, H.; Zhi, Z., P. Multicenter collaboration group of Department of Science Technology of Guangdong, and p. Health Commission of Guangdong Province for chloroquine in the treatment of novel coronavirus, [Expert consensus on chloroquine phosphate for the treatment of novel coronavirus pneumonia] . Chin. ./. Tuberc. Respir. Dis., 2020. 43(0): p. 185- 188.]. They also block the production of various cytokines [Jang, C.H., et ah, Chloroquine inhibits production of TNF-alpha, IL-lbeta and IL-6 from lipopolysaccharide-stimulated human monocytes/macrophages by different modes. Rheumatology (Oxford), 2006. 45(6): p. 703-10.]. However, their serious side effects on the heart and kidneys limit their use to specialized clinical wards.

The use of immunomodulators, which would alleviate the excessive, dysregulated immune responses (development of a cytokine storm) occurring in severe cases of SARS-CoV-2 infection, is also being considered. Cytokine storm mainly consists in overproduction of interleukin-6 (IL-6) by macrophages, dendritic cells or monocytes, leading to overproduction of other cytokines, especially IL-17 and interferon gamma (IFNy), which stimulate macrophages to further production of IL-6 by a feedback mechanism [Moore, J.B. and C.H. June, Cytokine release syndrome in severe COVID-19. Science, 2020. 368(6490): p. 473-474.; Shimabukuro-Vornhagen, A., et al., Cytokine release syndrome. J Immunother Cancer, 2018. 6(1): p. 56.]. To alleviate or to prevent the cytokine storm, the use of Bazedoxifene, a selective estrogen receptor modulator (SERM) approved for the treatment of osteoporosis that inhibits the IL-6 signalling pathway, or use of various low molecular weight inhibitors and monoclonal antibodies that inhibit the effects of these and other cytokines may be considered [Tian, J., et ah, Bazedoxifene is a novel IL-6/GP 130 inhibitor for treating triple-negative breast cancer. Breast Cancer Res Treat, 2019. 175(3): p. 553-566.; Smetana, K., Jr. and J. Brabek, Role of Interleukin-6 in Lung Complications inPatients With COVID-19: Therapeutic Implications. In Vivo, 2020. 34(3 Suppl): p. 1589-1592.]. Further, administration of intravenous immunoglobulins or plasma of donors recovering from COVID-19 is also being tested [Saber- Ay ad, M., M.A. Saleh, andE. Abu-Gharbieh, The Rationale for Potential Pharmacotherapy of COVID-19. Pharmaceuticals (Basel), 2020. 13(5).].

Finally, the effect of ACE inhibitors and blockers, which affect the function of the renin- angiotensin system and simultaneously increase ACE2 expression, needs to be elucidated [Kuba, K., et al, Trilogy of ACE2: a peptidase in the renin-angiotensin system, a SARS receptor, and a partner for amino acid transporters. Pharmacol Ther, 2010. 128(1): p. 119- 28.; Deshotels, M.R., et al, Angiotensin II mediates angiotensin converting enzyme type 2 internalization and degradation through an angiotensin II type I receptor-dependent mechanism. Hypertension, 2014. 64(6): p. 1368-1375.]. Their increased expression seems to reveal a protective effect on the lung parenchyma and to positively affect the consequences of coronavirus infection in the lungs due to reduced activation of the angiotensin type 1 receptor \Saber-Ayad, M, M.A. Saleh, and E. Abu-Gharbieh, The Rationale for Potential Pharmacotherapy of COVID-19. Pharmaceuticals (Basel), 2020. 13(5).\.

These and other therapeutic approaches are subject of ongoing clinical trials.

Use of heme compounds in connection with viral infections

Heme arginate (Normosang), or a similar compound Panhematin, are preparations containing human hemin used to treat acute porphyrias. Their administration replenishes the depleted heme pool in the body and affects the expression of enzymes in the heme biosynthetic pathway. Heme and probably heme arginate also act as a powerful ‘danger signal’ for the immune system and as an efficient immunomodulator [Igarashi, K. and M. Watanabe-Matsui, Wearing red for signaling: the heme-bach axis in heme metabolism, oxidative stress response and iron immunology. Tohoku J Exp Med, 2014. 232(4): p. 229-53.]. Heme or heme arginate further stimulate the expression of various genes \Mense, S.M. and L. Zhang, Heme: a versatile signalling molecule controlling the activities of diverse regulators ranging from transcription factors to MAP kinases. Cell Res, 2006. 16(8): p. 681-92.].

Normosang, as well as other preparations of heme or hemin, induces ubiquitous expression of heme oxygenase- 1 (HO-1), one of the most important antioxidant enzymes. HO-1 decomposes heme to Fe²⁺, carbon monoxide and biliverin, which is further converted by biliverdin reductase to bilirubin \Maines, M.D., G.M. Trakshel, andPK. Kutty, Characterization of two constitutive forms of rat liver microsomal heme oxygenase. Only one molecular species of the enzyme is inducible. J Biol Chem, 1986. 261(1): p. 411-9.; Shankaran, P., et ah, Heme arginate potentiates latent HIV-1 reactivation while inhibiting the acute infection. Antiviral Res, 2011. 92(3): p. 434-46.]. Bilirubin is a potent antioxidant; by the action of oxygen radicals it is converted into biliverdin and regenerated by the action of biliverdin reductase. This creates an effective antioxidant cycle that modulates excessive inflammatory responses by reducing redox stress [Baranano, D.E., et al, Biliverdin reductase: a major physiologic cytoprotectant. Proc Natl Acad Sci USA, 2002. 99(25): p. 16093-8.].

Heme and HO-1 induction have been shown in the past to inhibit the growth and replication of various RNA and DNA viruses, such as Zika, Dengue, Ebola, HCV, influenza, RSV, HIV-1,2, SIV, HBV, HSV-1,2 [Singh, N, et al, Host heme oxygenase-1 : Friend or foe in tackling pathogens? IUBMB Life, 2018. 70(9): p. 869-880.]. For most of these viruses, the expression of interferons and cytokines is modulated. In addition, inhibition of the viral protease by heme degradation product, biliverdin, has been shown in Dengue, HCV, HIV-1,2, and SIV viruses [Tseng, C.K., et ah, Human heme oxygenase 1 is a potential host cell factor against dengue virus replication. Sci Rep, 2016. 6: p. 32176.; McPhee, F., et ah, Bile pigments as HIV-1 protease inhibitors and their effects on HIV-1 viral maturation and infectivity in vitro. Biochem J, 1996. 320 ( Pt 2): p. 681-6.]. Heme further inhibits DNA polymerases, including HIV-1 reverse transcriptase [Shankaran, P., etal, Heme arginate potentiates latent HIV- 1 reactivation while inhibiting the acute infection. Antiviral Res, 2011. 92(3): p. 434-46.; Levere, R.D., etal, Heme inhibits human immunodeficiency virus 1 replication in cell cultures and enhances the antiviral effect of zidovudine. Proc Natl Acad Sci USA, 1991. 88(5): p. 1756-9.].

Normosang inhibits HIV-1 replication at the level of HIV-1 reverse transcription [Shankaran, P., et al, Heme arginate potentiates latent HIV-1 reactivation while inhibiting the acute infection. Antiviral Res, 2011. 92(3): p. 434-46.], while heme degradation products carbon monoxide and bilirubin inhibit the reactivation of latent HIV-1. In contrast, iron, another degradation product of heme, temporarily induces latency reversal in HIV-1. Stimulatory effects of heme arginate on HIV expression have been reproduced in human peripheral mononuclear cells ex vivo [Shankaran, P., et al, Effects of heme degradation products on reactivation of latent HIV- 1. Acta Virol, 2017. 61(1): p. 86-96]

Description of the Invention

The present invention relates to the use of heme arginate for the treatment of SARS-CoV-2 virus infection or infections with related beta coronaviruses such as SARS or MERS.

In severe cases of Covid-19, which is caused by the SARS-CoV-2, complications of the oxygenation of patients’ blood occur. Therefore, extracorporeal circulation is used to oxygenate the blood in these severe cases. Unexpectedly, however, it turned out that there was no improvement or sufficient oxygen saturation of the blood in these patients. This has led the inventors to hypothesize that the virus interacts with heme structures and damages them, or that it causes structural and functional changes in the proteins responsible for binding and transport of oxygen, preventing efficient oxygenation of the blood during extracorporeal circulation. Therefore, the inventors decided to carry out an experiment to demonstrate changes or degradation of heme structures by the action of SARS-CoV-2. Heme arginate (HA) was chosen as the model heme structure. The effect of the virus on the concentration and structure of heme characterized by the absorption spectra of heme arginate was tested in vitro , but it was surprisingly observed that HA had an effect on the replication of the virus in tissue cultures. As expected, it was shown that the virus caused concentration and structural changes of heme arginate, as characterized by the absorbance; but contrary to the expectations and despite the observed changes, heme arginate was found to affect virus replication in host cells, and quite unexpectedly, it even protected the cells from virus-induced cytopathic effects. Cells infected with SARS-CoV-2 in the presence of HA produce 1000-times lower quantities of viral particles than in the absence of HA. Likewise, virus-induced cell apoptosis is reduced to about 7 to 17 % in the presence of HA. The cytoprotective effect of heme arginate is absolutely essential from the point of view of preserving the function of lung parenchyma in blood oxygenation as well as the function of other tissues and organs. Preserving the function of these organs can be a decisive factor for the survival and recovery of the diseased organism.

Administration of heme arginate inhibits SARS-CoV-2 replication and protects infected cells from damage and cell death in vitro. In vivo heme arginate reduces the production of inflammatory mediators. Such a use of heme arginate is usable in the treatment of patients infected with SARS-CoV-2 or related beta coronaviruses, or in infections with other agents causing cytokine storm. Experiments have shown that each Vero cell can release about 1 x 10⁶ copies of viral RNA into the culture medium. Once the cellular capacity is depleted, no more virus is produced. When the viral inoculum is diluted, by one, two or three orders of magnitude, the maximum possible production of the virus by the available number of cells is achieved after a certain time of culture, again at the limit of about 1 x 10⁶ copies of viral RNA per cell. However, when heme arginate is administered to the cells, virus replication is inhibited, with the production of the virus decreasing to about 1 x 10³ per cell as the virus is unable to make maximum use of the cell pool for its replication due to HA. Its replication rate is reduced 1000-times.

Microscopic analysis of cellular morphological changes caused by virus-induced programmed cell death (apoptosis) revealed that more than 80 % of cells survived in the presence of HA compared to controls without HA, as detected by fluorescence of the apoptotic nuclei of infected cells. More specifically, when HA is administered to the infected cells, only about 7 to 17 % of apoptotic cells are detected compared to samples without HA. The number of apoptotic cells decreases 6 to 13-times.

HA reveals these abilities even though its concentration (absorbance) in the culture medium is decreased and the structure of some HA molecules is significantly changed after the interaction with the virus, as evidenced by HA absorption spectra determined in supernatants of infected cells compared to uninfected samples.

As demonstrated in preliminary experiments in mice, the infection titre in the organs of SARS- CoV-2 -infected mice was reduced down to 10 % after HA administration, while a trend of additional reduction in infection titre after repeated HA administration was shown.

In a study in human patients, administration of HA proved to be very advantageous, especially in high-risk patients with other concomitant diseases. Upon HA administration, patients displayed a reduction in oxygen consumption and need already during or shortly after the HA infusion compared to the control group. HA administration prevented these patients from progressing worse, while it led to the faster improvement of their overall health condition and shortened the hospitalisation. Summary of presented drawings

Fig.1 : A: Calibration curve for SARS-CoV-2 gene quantification

B: Data for calibration curve for SARS-CoV-2 gene quantification Fig.2: A: Inhibition of SARS-CoV-2 replication by heme arginate (RT-qPCR), experiment 1, administration of HA 2.5 pl/ml

B: Inhibition of SARS-CoV-2 replication by heme arginate (RT-qPCR), experiment 2, administration of HA 2 mΐ/ml

C: Time course of SARS-CoV-2 replication, characterization using RT-qPCR, experiment 3, administration of HA 1.25 and 2.5 mΐ/ml D: Time course of SARS-CoV-2 replication, characterization using RT-qPCR, experiment 3, administration of Remdesivir 10, 1, 0.1, 0.01 mM E: Time course of SARS-CoV-2 replication, characterization using RT-qPCR, experiment 3, administration of Nelfmavir 10, 1, 0.1, 0.01 mM F: Time course of SARS-CoV-2 replication, characterization using RT-qPCR, experiment 3, administration of Favipiravir 10, 1, 0.1, 0.01 mM Fig.3 : A: Cytoprotective effect of heme arginate, Vero cells, fluorescence microscopy

B: Cytoprotective effect of heme arginate, the number of fluorescent dots corresponds to the number of apoptotic cells

Fig.4: A: Change of absorption spectrum of heme arginate in the supernatant of

SARS-CoV-2 infected cells at 1-5 days after infection

B.l: Detail of the absorption spectrum of heme arginate without the effect of the virus at time 0 and after 1 day of interaction with the virus according to Figure 4 A

B.2: Detail of the absorption spectrum of heme arginate without the effect of the virus at time 0 and after 2 days of interaction with the virus according to Figure 4 A

B.3: Detail of the absorption spectrum of heme arginate without the effect of the virus at time 0 and after 3 days of interaction with the virus according to Figure 4 A

B.4: Detail of the absorption spectrum of heme arginate without the effect of the virus at time 0 and after 4 days of interaction with the virus according to Figure 4 A B.5: Detail of the absorption spectrum of heme arginate without the effect of the virus at time 0 and after 5 days of interaction with the virus according to Figure 4 A

C: Differential absorption spectra of heme arginate - absorbance of supernatant of infected cells minus absorbance of supernatant of uninfected cells for each wavelength in 1-5 days after infection

Fig.5: Inhibition of SARS-CoV-2 replication in mice after administration of HA

Fig.6: A: Improvement of the health condition of patients infected with SARS-CoV-

2 after administration of HA compared to the control group without administration of HA

B: Characteristics of the health condition of patients infected with SARS-CoV- 2 after administration of HA

C: Improvement of the health condition of patients infected with SARS-CoV- 2 after administration of HA, distribution of patients according to the NLR values

Examples of the Invention

Example 1A

Infection of Vero cells by SARS-CoV-2

Vero cells were incubated in culture medium Dulbecco’s Modified Eagle’s Medium (DMEM) with the addition of 2% fetal bovine serum (FBS); 2% FBS-DMEM. At a concentration of Vero cells 1.5 x 10⁵ per well/sample, the culture medium was aspirated and the cells were infected with 200 mΐ of SARS-CoV-2 isolate provided by the Biological Defence Department at Techonin in dilution lx, 10 ¹, 10², 10³. Then, the sample volume was made up to 1 ml with culture medium, and Vero cells were subsequently incubated in the culture medium 2% FBS- DMEM only or in culture medium with addition of heme arginate (HA) at concentrations of 2.5, 2 and 1.25 mΐ/ml, which corresponds to the hemin concentration of 96, 77 and 48 mM, respectively. After 4, or 3 days of culture, the virus was quantified in the cell supernatant according to the Example IB and the cytopathic effect of the virus was documented microscopically according to Example 1C.

Example IB

RNA quantification of SARS-CoV-2

Viral RNA was detected by l-step RT-qPCR. Total RNA was isolated from 200 mΐ of supernatant of SARS-CoV-2-infected cells using magnetic beads. SARS-CoV-2 RNA was determined by amplification of SARS-CoV-2 E-gene (Generi Biotech) using SensiFast Probe One-Step Kit (BioLine) and Light Cycler 480 II (Roche). Primers and probe used:

Concentration

Name Sequence 5’-3’ in reaction

E Sarbeco F 1 AC AGGT ACGTT AAT AGTT AAT AGCGT 400 nM E Sarbeco R2 AT ATTGC AGC AGT ACGC AC AC A 400 nM

E Sarbeco PI FAM-ACACTAGCCATCCTTACTGCGCTTCG-BHQ1 200 nM

Viral RNA was quantified absolutely using a calibration curve, Figs. 1 A and IB. The control glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) mRNA was determined using Human GAPDH TaqMan® Gene Expression Assay Hs02758991_gl VIC-MGB (Life Technologies), not shown. Due to detection in the culture medium, the presence of cellular GAPDH released into the supernatant serves as a control for RNA isolation, but it cannot be used for viral RNA normalization. Figs. 2A and 2B present changes in replication of SARS-CoV-2 characterized using RT-qPCR of SARS-CoV-2 E-gene in the culture medium alone and in the presence of heme arginate 2.5 or 2 mΐ/ml, experiment 1 and 2. The results indicate that after 4 days of infection in the culture medium alone, there was an increase of SARS-CoV-2 up to maximal values in several consecutive dilutions of inoculum, i.e. the growth of the virus at this time interval reached a maximum possible in respect to the number of cells and is no more proportional to the initial dilution. In contrast, a gradual decrease in virus replication in several dilutions of inoculum can be observed after HA administration.

Figs. 2C, 2D, 2E and 2F show the time course of SARS-CoV-2 replication characterized using RT-qPCR of SARS-CoV-2 E-gene in the culture medium alone and in the presence of heme arginate 1.25 and 2.5 mΐ/ml, in the presence of Remdesivir 0.01 to 10 mM, Nelfmavir 0.01 to 10 mM, and Favipiravir 0.01 to 10 mM = experiment 3. For Remdesivir, Nelfmavir and Favipiravir, a dose of 10 mM is toxic not only to the virus but also to the cells. In addition, a dose of 10 mM is harder to achieve in vivo. In contrast, HA concentration of 2.5 mΐ/ml (96 mM) is achieved in vivo during several hours after 1 infusion.

Vero cells, 1.5 x 10⁵/sample, were infected with SARS-CoV-2, 1.01 x 10⁶/sample, i.e. 7 copies of E-gene/cell. The results show that in the culture medium alone there was a gradual increase in SARS-CoV-2 by 4 orders of magnitude. In contrast, in the presence of HA 1.25 mΐ/ml virus replication was inhibited by 2 orders of magnitude in 3 days and in the presence of HA 2.5 mΐ/ml the virus almost did not replicate. Commonly used drugs presented by Remdesivir, Nelfmavir and Favipiravir at physiologically achievable drug concentrations are not nearly as good as the administration of HA. Following administration of physiologically achievable concentrations of Remdesivir, there is virtually no inhibition of the virus, same as with Nelfmavir and Favipiravir.

Example 1C

Microscopy

After 4 days of incubation, infected cells cultured in culture medium alone and in the presence of heme arginate 2.5 mΐ/ml were fixed by adding 4% paraformaldehyde at room temperature. Subsequently, the cells were washed with phosphate-buffered saline (PBS) and stained with fluorochrome Hoechst 33342, final concentration 0.1 pg/ml, which binds to cellular DNA. Infected cells were visualized in situ in tissue culture plates using Olympus IX-70 epifluorescence inverted microscope and a Progres MF Cool CCD camera (Jenoptik) with NIS- Elements Imaging Software (Nikon Instruments). The cytopathic effect of the virus was observed in phase contrast and under UV light, where the bright fluorescence of the condensed heterochromatin is visible after the addition of the fluorochrome Hoechst 33342, which is a typical feature of apoptosis. Figure 3 A clearly shows a reduction in the incidence of apoptotic cells in the presence of heme arginate, up to 7.52 %. Figure 3B documents Figure 3 A numerically, and represents the number of fluorescent dots in the image corresponding to the number of apoptotic cells.

Example 2

Change of absorption spectrum of heme arginate in the supernatant of SARS-CoV-2- infected cells

Vero cells were infected with SARS-CoV-2 as in Example 1 A and incubated in the presence of heme arginate (1.25 mΐ/ml). Control cells were treated analogously without virus infection of the cells. The absorption spectra of the culture medium were measured on individual days after infection - 2% FBS-DMEM without phenol red, an acid-base indicator, in the range of 250 to 800 nm using a spectrophotometer BioSpectrometr Kinetik (Eppendorf), and the differential spectra were calculated - Figure 4C, i.e. the absorbance of the supernatant of the infected cells minus the absorbance of the supernatant of the uninfected cells for each wavelength; all in duplicates. The graphs presented in Fig. 4A and 4B clearly indicate that viral infection causes changes in the absorption spectrum of heme arginate compared to controls.

Example 3

Inhibition of SARS-CoV-2 replication in mice after administration of HA

Female mice for this experiment were 5 months old, hemizygous for K18-hACE2 (Tg(K18- ACE2)2Prlmn), expressing hACE2 under control of the human keratin 18 (KRT18) promoter from Jackson Laboratory. These mice were infected intranasally with SARS-CoV-2 in PBS, at an infectious dose of 25.6 infection units/mouse. The infection dose was determined in Vero cells and corresponded to 9.56 x 10⁷ copies of the E-gene, as determined by RT-qPCR as in Example IB. Mice were infected under general anaesthesia with Avertin (2,2,2-tribromoethanol in tertiary amyl alcohol 1 mg/ml) administered intraperitoneally.

One group of infected mice received HA in two doses on days 4 and 6 after infection. The second group of infected mice received HA in three doses on days 4, 5, and 6 after infection. HA was administered intraperitoneally at the dose of 3 mg/kg in a single dose. The control group of mice received only phosphate buffer (PBS). For the purpose of this experiment, mice were humanely sacrificed under general anaesthesia on day 8 after infection, and individual organs were removed and frozen at -80 °C for further analysis. Portions of each organ were then homogenized in 2% FBS-DMEM, the homogenates were centrifuged, and the supernatants were used for serial dilution and Vero cell infection. The table in Fig. 5 summarizes the infection titre in the lungs and livers of control mice - without HA administration and mice with 2 or 3 doses of HA. Values represent the average of 2 mice in each group.

As summarized in Figure 5, in the lungs of HA-treated mice, the infection titre decreased up to 10 to 14% compared to the control group of mice infected without HA treatment. In the liver, the infection titre decreased to 67 to 88% compared to the control group. It can also be observed that the infection titre is lower in mice treated with three doses of HA compared to mice treated with only two doses of HA. From this result, it can be concluded that with additional doses of HA, the infectious titre would decrease even further.

Example 4

Improvement of the health condition of patients infected with the SARS-CoV-2 after administration of HA

The group of 6 patients with bilateral pneumonia and hypoxemic respiratory insufficiency who were included in the study after signing the informed consent consisted of 3 women and 3 men. In all patients, positivity for COVID-19 was proven in the laboratory and bilateral pneumonia by X-ray. Selected patients suffered from comorbidities, namely bronchial asthma, arterial hypertension, dyslipidemia, type 2 diabetes mellitus and overweight, which were a risk factor for the subsequent course of the disease. The overall health condition was assessed by the examining physician in accordance with the recommendations and contraindications of Normosang, i.e. heme arginate as the active substance.

All patients were treated according to SOC (Standard of Care) including oxygen therapy, administration of Remdesivir and systemic corticosteroids, prevention of thromboembolism, hydration, or antibiotics. HA in the form of Normosang was administered to the selected patients in addition to SOC. The infusion solution of HA was prepared from 10 ml of hem arginate (Normosang 25 mg/ml, 10 ml containing 250 mg of human hemin) and 100 ml 0.9% NaCl solution. The infusion solution contained 2273 pg of human hemin in 1ml solution. HA was administered to five patients in a single dose of 3 mg of hemin per 1 kg of body weight intravenously; patient no. 3 received HA in two doses, each dose representing 3 mg of hemin per 1 kg of body weight.

The parameters examined were mainly the neutrophil-to-lymphocyte ratio (NLR), C-reactive protein (CRP), length of hospitalization and the need for long-term oxygen therapy after discharge home (Home LTOT). The monitored averaged parameters are summarized in the table in Fig. 6A. Data from all HA-treated patients are shown in Fig. 6B. Fig. 6C shows the distribution of patients by NLR, where an NLR of 3.1 can be considered a criterion for assessing disease severity according to Hammad 2021 \HammadR, Eldosoky MAER, Fouad SH, Elgendy A, Tawfeik AM, Alboraie M, Abdelmaksoud MF. Circulating cell-free DNA, peripheral lymphocyte subsets alterations and neutrophil lymphocyte ratio in assessment of COVID-19 severity. Innate Immun. 2021 Apr; 27(3): 240-250]; the condition of patients with NLR lower than 3.1 is considered less severe and, conversely, the condition of patients with NLR higher than 3.1 is considered more severe, with a higher inflammatory component and a worse prognosis.

In all patients, administration of Normosang was considered beneficial by a pulmonologist for the following reasons:

1/ Oxygen consumption and need started to reduce during the infusion of Normosang or shortly after it;

2/ There was no progression to a worse condition, such as transfer to the ICU (intensive care unit), ECMO (ExtraCorporal Membrane Oxygenation), APV (Artificial Pulmonary Ventilation) or death;

3/ Overall condition improved faster than in similar patients, e.g. improved x-ray image;

4/ The length of hospitalization was shortened.

Applicability in Industry

Application of heme arginate inhibits SARS-CoV-2 replication and protects infected cells from damage and cell death in vitro. In vivo heme arginate reduces the production of inflammatory mediators. Such a use of heme arginate is usable in the treatment of patients infected with SARS-CoV-2 or related beta coronaviruses, or in infections with other agents causing cytokine storm.

Claims

1. Use of heme arginate for a manufacture of a medicament for a treatment of beta coronavirus infection.

2. Use of heme arginate for the manufacture of a medicament for a treatment of beta coronavirus infection according to Claim 1, wherein the beta coronavirus is SARS- CoV-2, SARS or MERS.

3. Use of heme arginate for the manufacture of a medicament for a treatment of beta coronavirus infection according to Claim 1, wherein the beta coronavirus is SARS- CoV-2.