WO2023121629A1

WO2023121629A1 - Combination of camostat mesilat and umifenovir hydrochloride with proton pump inhibitors

Info

Publication number: WO2023121629A1
Application number: PCT/TR2022/051566
Authority: WO
Inventors: Tambay Taskin; Ismail Tuncer DEGIM
Original assignee: Tambay Taskin; Degim Ismail Tuncer
Priority date: 2021-12-22
Filing date: 2022-12-22
Publication date: 2023-06-29
Also published as: TR2021020737A2

Abstract

The present invention is the combination of Camostat Mesylate, Umifenovir Hydrochloride and 40-80 mg Esomeprazole Magnesium Trihydrate, which is used for the prevention, delay or treatment of COVID- 19 disease. In the combination of the invention, Camostat Mesylate, Umifenovir Hydrochloride and Esomeprazole Magnesium Trihydrate may be present in separate pharmaceutical compositions or in the same pharmaceutical composition. The COVID-19 patients to whom the inventive combination will be administered will preferably be uncomplicated but probable or definitively diagnosed COVID-19 patients.

Description

COMBINATION OF CAMOSTAT MESILAT AND UMIFENOVIR HYDROCHLORIDE WITH PROTON PUMP INHIBITORS

Technical Field

This invention relates to the combination of camostat mesylate, umifenovir hydrochloride and proton pump inhibitors in uncomplicated probable/definite diagnosed COVID- 19 cases. In this invention, it is envisaged to prepare a combination of Umifenovir, Camostat and Protom pump inhibitors (preferably s -omeprazole) as a tablet or hard gelatin capsule formulation for an effective antiviral formulation. These uncomplicated patients are those with symptoms such as fever, muscle/joint pain, cough, sore throat, and nasal congestion, without respiratory distress, tachypnea, and SPO2 < 90%, and with normal chest X-ray and/or lung tomography.

State of the Art

While infections caused by severe acute respiratory syndrome coronavirus SARS-CoV and Middle East respiratory syndrome, or ODSS (Middle East respiratory syndrome) MERS-CoV remain fresh in our memories, a new deadly corona virus type was detected in Yuhan, China in 2019. SARS- CoV emerged and spread rapidly all over the world. Around 2020, the International Committee on Virus Taxonomy officially adopted the name "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2) for this virus. SARS-CoV-2, as the name suggests, is the new form of SARS-CoV. After a century of Spanish flu tragedy, humanity has been battling a brand new frightening threat, the coronavirus 2019 disease (COVID-19), for over a year now. (Wu Z, 2020; published online Feb 24. ) (Chen N, 2020; 395: 507-13.) (Zhou F, 2020; 395: 1054-62.). Every day, an incalculable mass of people is added to the number of infected people. Unfortunately, as of December 15, 2021, more than 270 million people in the world have been infected and there are over 5 million deaths.

At least half of patients with coronavirus disease 2019 (COVID-19) requiring invasive mechanical ventilation have died in hospital, overwhelmingly increasing the burden on healthcare systems, particularly intensive care units in many countries (Zhou F, 2020; 395: 1054-62.). Although most infections tend to resolve on their own, severe pneumonia requiring supplemental oxygen may develop in 15% of cases. Of these, 5% develop severe pneumonia leading to hypoxemic respiratory failure, acute respiratory distress syndrome, and multi-organ failure requiring ventilation support. This picture can usually persist for several weeks (Wu Z, 2020; published online Feb 24. ) (Chen N, 2020; 395: 507-13.) (Zhou F, 2020; 395: 1054-62.). At least half of patients with coronavirus disease 2019 (COVID-19) requiring invasive mechanical ventilation have died in hospital, overwhelmingly increasing the burden on healthcare systems, particularly intensive care units in many countries (Zhou F, 2020; 395: 1054-62.).

Although some licensed drugs and investigational agents have demonstrated antiviral activity against SARS-CoV-2 in vitro (Wang M, 2020; 30: 2-71.) (Liu J, 2020; 6: 16) there is currently no antiviral therapy with proven efficacy in the treatment of severely ill COVID-19 patients. In a multicenter, open-label, randomized controlled trial of hydroxychloroquine involving 150 adults hospitalized with a diagnosis of COVID- 19, it was reported that the drug had no significant effect on accelerating viral clearance (Tang W, 2020; published online April 14. DOI: 10.1101/2020.04.10.20060558 (preprint).).

In another study, favipiravir and umifenovir were compared in terms of clinical improvement. In the favipiravir group, 98 of 116 patients were moderately severe COVID- 19 and 18 were severe COVID-19 patients, while hypertension and diabetes were detected in 42 COVID-19 patients. There were 120 patients in the umifenovir group, of which 111 were moderate and 9 were severe COVID- 19 patients. It was determined that 35 of the patients in this group had comorbidities such as hypertension and diabetes. While clinical improvement rates were 51.67% (62/120) in patients using umifenovir for 7 days and 61.21% (71/116) in patients using favipiravir, no statistically significant difference was found between them (P=0.1396) (Chen C, 2020; published online April 15. DOI: 10.1101/2020.03.17.20037432 (preprint).)

However, the dose of umifenovir (Arbidol) used here was administered as 200 mg tid for 7-10 days. This dose is not sufficient for SARS-CoV-2 inhibition. The maximum umifenovir plasma concentration (Cmax) increased with increasing dose and peaked at 1.23 pg/ml at 1.6 - 1.8 hours after administration for a 200 mg dose (Metz R, Muth P, Ferger M, Kukes VG, Vergin H.). Ge et al. (2020) showed that arbidol alone at a concentration of 30 pM inhibited SARS-CoV-2 by 98% in cell cultures in vitro (Ge Y et al., 2020). At low concentrations, the antiviral effect is reduced. The dose used by Chen et al. is 600 mg per day. No superior effect was obtained than favipiravir as monotherapy. The recommended dose of umifenovir in this clinical trial protocol is 800 mg per day, which is the most compatible with the IC50 values obtained in in vitro cell cultures.

An open-label study showed that the lopinavir/ritonavir combination did not affect the primary outcome in terms of clinical improvement and did not result in reductions in viral RNA titers compared to control. The study by Zhu et al., published on April 14, 2020 and showing the superiority of umifenovir (Arbidol) monotherapy over lopinavir/ritonavir, took umifenovir one step further in this regard (Zhu Z, 2020). Chest CT scan was performed by taking throat swab from 50 patients diagnosed with COVID- 19, and all patients received conventional therapy, including oxygen inhalation (2L/min for half an hour and 3 times a day) and atomized inhalation of recombinant human interferon-a2b injection. These patients were divided into two groups: lopinavir/ritonavir (34 cases) and umifenovir (arbidol) (16 cases). 400mg/100mg b.i.d. for one week to the lopinavir/ritonavir group, umifenovir was administered at a dose of 3x200 mg. There was no difference between the baseline Ct values (cycle threshold) of both groups in terms of ORF lab and N genes (P>0.05). While viral load could not be detected in half of the patients given umifenovir on the seventh day of hospitalization, this rate was 23.5% in the lopinavir/ritonavir group. Interestingly, viral clearance was achieved in all patients receiving umifenovir on day 14. This rate was 44.1% in the lopinavir/ritonavir group. Patients in the umifenovir group had a shorter positive RNA duration compared to the lopinavir/ritonavir group (P<0.01) (Zhu Z, 2020).

Remdesivir is a monopho sphoramidate prodrug of an adenosine analog with a broad antiviral spectrum, including filoviruses, paramyxoviruses, pneumoviruses and coronaviruses (Wang Y. et al., 2020 Published online April 29, https://doi.org/10.1016/80140-6736(20)31022-9). In vitro, remdesivir inhibited all human and animal coronaviruses tested to date, including SARS (CoV-2, and SARS-CoV-1 and Middle East respiratory syndrome [MERS]). It has shown antiviral and clinical effects against coronaviruses in animal models. In the lethal murine model of MERS, remdesivir was superior to the combined interferon beta and lopinavir-ritonavir regimen. Remdesivir is a potent inhibitor of SARS-CoV-2 replication in human nasal and bronchial airway epithelial cells (Wang Y. et al., 2020 Published online April 29, https://doi.org/10.1016/80140-6736(20)31022-9). In the SARS-rhesus macaque model of CoV-2 infection, early administration of remdesivir has been shown to produce significant antiviral and clinical effects (reduced pulmonary infiltrates and virus titers in bronchoalveolar lavages). Intravenous remdesivir has been used on an individual compasionate basis over the past few months in patients with COVID-19 in Ebola virus disease, where it is adequately tolerated but less effective than several monoclonal antibody therapies, and in some countries. It has been reported to be beneficial in severely ill patients in COVID- 19 case studies. However, the clinical and antiviral efficacy of remdesivir in COVID-19 has not yet been determined. The study summarized below includes Wang et al.'s results of the first placebo -controlled randomized trial of remdesivir in patients with severe COVID-19, published April 29 in The Lancet.

Wang et al. found that the time to clinical improvement in the remdesivir group in the ITT population was not significantly different versus placebo in a multicenter, randomized, placebo-controlled study. This time was median 21.0 days in the remdesivir group [IQR 13 0-28 0] and 23 0 days [15 0-28 0] in the placebo group; It is recorded as HR 1 -23 [95% CI 0-87-1 -75] (Wang Y. et al., 2020 Published online April 29, https://doi.org/10.1016/80140-6736(20)31022-9).

The 28-day mortality was also similar between the two groups (22 [14%] in the remdesivir group and 10 (13%) in the placebo group; the difference was 1.1% [95% CI -8.1 to 10.3] ). In patients using remdesivir within 10 days of symptom onset, 28-day mortality was not significantly different between groups, although it was numerically higher in the placebo group; on the contrary, in the late-use patient group, remdesivir patients were found to have a numerically higher 28-day mortality, although there was no significant difference. While the clinical improvement rates on days 14 and 28 were not significantly different between the groups, a numerically higher figure was obtained in the remdesivir group compared to the placebo group. For patients assigned to the remdesivir group, the duration of invasive mechanical ventilation was not significantly different. No significant difference was observed between the two groups in terms of duration of oxygen support, length of hospital stay, days from randomization to discharge, days from randomization to death, and distribution of the six-category scales at days 7, 14, and 28.

The above results show that there is currently not an adequate antiviral agent against COVID-19 all over the world. Besides the availability of vaccines, the frightening and even more dramatic aspect of the issue is that no fully proven and effective treatment option has yet emerged. It is not possible to talk about a real treatment option to treat the coronavirus infection. Researchers in academia and the pharmaceutical industry are now focused on repositioning existing drugs and/or developing new drugs against COVID-19. It is observed that the authorities in the world have positive views on emergency use authorizations for potential drug candidates. To save time in the race to find effective treatment, repositioning still remains promising and is the main theme of this patent application.

It is very important to understand the mechanisms of virus entry and post-entry viral fusion in order to develop treatment strategies. The preparation of the coronavirus 2 (SARS-CoV- 2) spike protein for cell entry by host proteases upon contact with human mucosal cells is one of the most important initiation mechanisms (Benton DJ, Wrobel AG, Xu P, Roustan C, Martin SR, Rosenthal PB, Skehel JJ, Gamblin SJ., 2020). The preparation of the spike protein takes place by proteolysis on the surface of human respiratory mucosal cells. Furin and transmembrane serine protease-2 (TMPRSS2) are proteases abundant in airway mucosal membranes. Entry of SARS-CoV-2 into host cells is possible by preparation of the S protein by both furin and TMPRSS2. After the virus binds to the human angiotensin converting enzyme 2 (ACE2) receptor by upregulation of one of the S 1 trimer of the spike protein, the SI and S2 subunits of the SARS-CoV-2 S protein are cleaved by furin- mediated cleavage in the multibasic region, thus removing the barrier on the fusion peptide. The approach of TMPRSS2 to the S2' region of the spike protein becomes possible. Heptad repeat 1 (HR1, amino acids 987-1062) and heptad repeat 2 (HR2, amino acids 1263-1279) are activated in the S2 subunit to which the fusion peptide is attached by S2' proteolysis, and are ready to initiate fusion. For fusion, this process is a critical start. The HR forms then interact with each other to form a six -helix bundle (6-HB) for the fusion core. Formation of 6-HB fusion nuclei brings viral particles close to host cell membranes. At this critical stage, endosomal acidification is necessary for conformational change to bring the viral and host cell membranes together. Eventually, membrane fusion occurs and viral RNA is released into the cytoplasm (Zhang Q, Xiang R, Huo S, Zhou Y, Jiang S, Wang Q, Yu F. , 2021 )

In SARS-CoV-2, S protein pre-priming has a critical role in binding to ACE2 receptors, viral entry and expression of fusion peptides. Therefore, the host cell serine protease TMPRSS2 is one of the most important drug targets. Camostat mesylate, a clinically proven TMPRSS2 inhibitor, has an approved indication since 1994 under the brand name Foipan® for the treatment of major symptoms of chronic pancreatitis (Ono Pharmaceutical Co., Etd., 2009). Molecular docking analysis revealed that Camostat mesylate and its structural analogue Nafamostat strongly interact with residues His296 and Ser441 found in the catalytic triad of TMPRSS2 (Sonawane KD, Barale SS, Dhanavade MJ, Waghmare SR, Nadaf NH, Kamble SA, Mohammed AA, Makandar AM, Fandilolu PM, Dound AS, Naik NM, More VB., 2021). Camostat mesylate has been shown to significantly inhibit SARS CoV-2 in Calu-3 cells with EC50 ~lpM and CC50>500 pM (Hoffmann M, Kleine- Weber H, Schroeder S, Kruger N, Herrler T, Erichsen S, Schiergens TS, Herrler G, Wu NH, Nitsche A, Muller MA, Drosten C, Pbhlmann S., 2020). By the same investigators, camostat mesylate infects SARS -CoV-2 pseudovirus in human lung cells (Hoffmann M, Kleine-Weber H, Schroeder S, Kruger N, Herrler T, Erichsen S, Schiergens TS, Herrler G, Wu NH, Nitsche A, Muller MA, Drosten C, Pohlmann S., 2020) and Wu et al. It has also been demonstrated that it inhibits SARS-CoV-2 proliferation in Calu-3 cells (Wu Z, 2020; published online Feb 24. ). Although there are many incomplete clinical studies on the efficacy and safety of camostat mesylate in patients with COVID- 19, recently published clinical trial results are also increasingly entering the COVID-19 literature (Sakr Y, Bensasi H, Taha A, Bauer M, Ismail K; the UAE-Jena Research Group., 2021) (Hofmann- Winkler, H., Moerer, O., Alt-Epping, S., Brauer, A., Biittner, B., Muller, M., Fricke, T., Grundmann, J., Harnisch, L. O., Heise, D., Kernchen, A., Pressler, M., Stephani, C., Tampe, B., Kaul, A., Gartner, S., Kramer, S., Pohlmann, S., & Winkler, M, 2020).

Umifenovir (Arbidol®) is a fusion inhibitor approved in China and Russia (Kadam RU, Wilson IA. , 2017) for the prophylaxis and treatment of influenza and is effective in inhibiting SARS-CoV-2 entry and post-cell entry (Vankadari N., 2020). Umifenovir has broad-spectrum in vitro antiviral activity against a variety of viruses, including influenza virus, coronaviruses, and Ebola. (Hulseberg CE, Feneant L, Szymanska-de Wijs KM, Kessler NP, Nelson EA, Shoemaker CJ, Schmaljohn CS, Polyak SJ, White JM., 2019) (Kadam RU, Wilson IA. , 2017). It inhibits viral entry with an IC50 of 2-20 pgml-1 against SARS-CoV-2 (Wang M, Cao R, Zhang L, Yang X, Liu J, Xu M, Shi Z, Hu Z, Zhong W, Xiao G., 2020). Unfortunately, the Cmax of umifenovir is less than 2 pgml-1 after administration of usual therapeutic doses (Deng P, Zhong D, Yu K, Zhang Y, Wang T, Chen X. , 2013) (Sun Y, He X, Qiu F, Zhu X, Zhao M, Li-Ling J, Su X, Zhao L„ 2013).

However, endosomal acidification inhibitors have been shown to increase the activity of umifenovir against coronaviruses. The antiviral activity of umifenovir was synergistically enhanced 10-fold after the addition of endosomal acidification inhibitors in an in vitro setting (Zhao H, To KKW, Lam H, Zhou X, Chan JF, Peng Z, Lee ACY, Cai J, Chan WM, Ip JD, Chan CC, Yeung ML, Zhang AJ, Chu AWH, Jiang S, Yuen KY., 2021). A pH modification of umifenovir has been shown to have antiviral activity against SARSCoV-2 at low concentrations (0.2-0.4 pgml-1) in Vero-E6 cells.

Considering all these facts, we anticipate that alteration of endosomal pH as well as repositioning of clinically proven drugs targeting TMPRSS2 (pre-entry) and fusion peptides (post-entry) can greatly influence the course of viral infection. This will confine the virus endosomally and the flow of positive-sense RNA into the cytoplasm will be blocked and the virus particle will not be able to replicate itself. To summarize the points discussed above, considering previously published pharmacodynamic/pharmacokinetic findings, COVID- 19 can be effectively treated with host protease and fusogenic peptide inhibitors when combined with proton pump inhibitors. We predict that the combination of camostat mesylate, umifenovir hydrochloride and proton pump inhibitors has not been tried so far for patients with PCR-proven mild and moderate COVID-19, and as such it will be an effective and safe treatment option.

Skinner- Adams and Davis showed that inhibition of H+/K+ATPase in gastric parietal cells inhibits the acidification of endosomes by suppressing the same pump in the endosomal environment. Skinner-Adams and Davis showed that inhibition of H+/K+ATPase in gastric parietal cells inhibits the acidification of endosomes by suppressing the same pump in the endosomal environment. Omeprazole was reported by these authors to show antimalarial activity in vitro by blocking the proton pump in parasitic vacuoles (Skinner- Adams T, Davis TM., 43(5): 1304-6.). This finding is in line with the recently published findings for coronaviruses by Zhao et al. (Zhao H, To KKW, Lam H, Zhou X, Chan JF, Peng Z, Lee ACY, Cai J, Chan WM, Ip JD, Chan CC, Yeung ML, Zhang AJ, Chu AWH, Jiang S, Yuen KY., 2021).

The dose of umifenovir we recommend within the framework of this invention is 800 mg per day, which is the most compatible with the IC50 values obtained in in vitro cell cultures. The above results show that there is currently not an adequate antiviral agent against COVID-19 all over the world. The first studies on umifenovir were made on influenza hemagglutinin. It may be a sufficient example to look at the affinity of hemagglutinin on Hl, H2, H3, H4 and H5, the influenza strains in which umifenovir has been most studied as a fusion inhibitor. Fusion peptides cannot bind to the host membrane at the endosomal membrane and fusion cannot occur. The same is true for the new SARS CoV-2 spike protein. It has been shown that umifenovir acts on the spike protein with a similar mechanism (Khamitov RA, Loginova Sla, Shchukina VN, Borisevich SV, Maksimov VA, Shuster AM., 2008 ). Today, it has been shown that many S proteins are dissociated at the SI and S2 interface, and this is called the S1/S2 region. This region is close to the N-terminal region of the fusion peptide. Separation of this region is important in terms of forming the mature N- terminus of the fusion peptide. Entry to the target cell membrane is through this peptide. If this does not happen, there cannot be a successful membrane fusion reaction (Hoffmann M., Hofmann-Winkler H., Pohlmann S., 2018) (Belouzard S, Chu VC, Whittaker GR., 2009) (Xia S, Xu W, Wang Q, Wang C, Hua C, Li W, Lu L, Jiang S., 2018).

In studies conducted against ribavirin with umifenovir and umifenovir mesylate, it has been shown that the addition of these agents to a medium containing GMK-AH-1 (D) cells cultured after infection, at concentrations of 50, 25 and 100 pg/ml, respectively, suppresses viral replication in a dose-dependent manner (Khamitov RA, Loginova Sla, Shchukina VN, Borisevich SV, Maksimov VA, Shuster AM., 2008 ). Umifenovir and its mesylate salt have been shown to have a direct antiviral effect on early viral replication in SARS virus cultured cells. The mesylate salt has been shown to be approximately 5 times more effective than arbidol in reducing the proliferation of SARS virus in cultured cells.

The interferon-inducing effect of umifenovir is reported in the literature (Chen Qi, Yi- Ping, Chen Si-Yan, Yang, 2006) (Arastoo M, Khorram HR, Khorshid HRK, Radmanesh R, Gharibdoust F., 2014). In this way, it has an immunomodulatory effect. It also increased CD4 counts (Arastoo M, Khorram HR, Khorshid HRK, Radmanesh R, Gharibdoust F., 2014). The antioxidant properties of umifenovir were also discussed in the same publication. The interferon-inducing effect of umifenovir was studied in trypsinized chicken embryo fibroblasts. In order to induce interferon, umifenovir was mixed in 2% diethylaminoethyl dextran at a ratio of 1:4 and added as 0.2 ml to each fibroblast cell culture tube. After the drug-treated cells were kept at 37°C for 1 hour, the cells were washed. Chicken embryo fibroblast cells were titrated for interferon by reincubation for 8, 24, and 48 hours (Xia S, Xu W, Wang Q, Wang C, Hua C, Li W, Lu L, Jiang S., 2018)(Xiao LiuQ.-G. Zhou H. Li et al.). Interferon induction of umifenovir was studied against double- stranded RF.sub.2-phage RNA. Double- stranded RF.sub.2- phage RNA is one of the highly active inducers of interferon. These results indicate that umifenovir is a highly active inducer of interferon. At a concentration of 20 mg/ml, the interferon titer reached 64 — 1280 U/ml after 8 hours, and a maximum of 2560 U/ml after 24 hours. The efficacy, which was previously demonstrated experimentally by Hoffman et al., was started to be tested in two of the European countries for the first time. One is Denmark and the other is Germany. The spike protein is structurally a trimer protrusion. How it is processed by host proteases that it needs to get in is a complex issue. The proteolytic separation of the SI and 52 units of the viral spike protein can be called "priming", that is, "preparation", and thus, the COVID-19 S protein is given a structural flexibility that it can use during membrane fusion (Hoffmann M., Hofmann-Winkler H., Pbhlmann S., 2018). Initial studies with human HIV envelope protein and hemagglutinins of highly pathogenic avian influenza A viruses (FLUAV) showed that this degradation occurs in the secretory pathway of infected cells and is carried out by furin and subtilisin-like proteases.

Two cleavage sites (S1/S2)

Early studies showed that proteolytic cleavage of coronavirus “S” proteins is at the border between the surface and transmembrane units of glycoproteins, but there is more than one cleavage site for S protein activation (Belouzard S, Chu VC, Whittaker GR., 2009). In the study of Belouzard et al., they showed how important the activation of the S1-S2 border region by trypsin is in SARS-CoV S membrane fusion.

That is, fusion of SARS-CoV cannot occur without trypsin activity. Today, it has been shown that many S proteins are dissociated at the SI and S2 interface, and this is called the S1/S2 region. This region is close to the N-terminal region of the fusion peptide. Separation of this region is important in terms of forming the mature N-terminus of the fusion peptide. Entry to the target cell membrane is through this peptide. If this does not happen, a successful membrane fusion reaction cannot occur (Hoffmann M., Hofmann- Winkler H., Pbhlmann S., 2018) (Belouzard S, Chu VC, Whittaker GR., 2009) (Xia S, Xu W, Wang Q, Wang C, Hua C, Li W, Lu L, Jiang S., 2018).

The fact that coronaviruses use spike protein to enter the cell has highlighted the processes related to this protein and its activation as a drug target. It has been shown that the 2019-nCoV-S “priming” process is mediated by the host “Transmembrane Serine Protease-2 (TMPRSS2). TMPRSS2 proteases located in the membranes of lung target cells rather than endosomal proteases (cathepsin B and L) have been shown to be critical in CoV propagation and proliferation (Hoffmann M, Kleine -Weber H, Schroeder S, Kruger N, Herrler T, Erichsen S, Schiergens TS, Herrler G, Wu NH, Nitsche A, Muller MA, Drosten C, Pbhlmann S., 2020). Indeed, camostat mesylate, a serine protease inhibitor, has been shown to be a potent inhibitor of TMPRSS2 and inhibit the entry of 2019 n-CoV into CaCo-2 cells (Hoffmann M, Kleine-Weber H, Schroeder S, Kruger N, Herrler T, Erichsen S, Schiergens TS, Herrler G, Wu NH, Nitsche A, Muller MA, Drosten C, Pbhlmann S., 2020). It has been shown that E64d, an inhibitor of cathepsin B/L, an endosomal protease, has the opposite effect (Hoffmann M, Kleine -Weber H, Schroeder S, Kruger N, Herrler T, Erichsen S, Schiergens TS, Herrler G, Wu NH, Nitsche A, Muller MA, Drosten C, Pbhlmann S., 2020).

Summary of the Invention

Although some licensed drugs and agents under investigation have demonstrated antiviral activity against coronavirus in vitro, there is currently no antiviral drug with proven efficacy in the treatment of severely ill COVID-19 patients.

The object of the invention is the combined use of Camostat Mesylate, Umifenovir Hydrochloride and Proton Pump Inhibitors for the treatment of COVID-19. The term "combination" within the scope of the invention; means, but is not limited to, the presence of compounds of Camostat mesylate and Umifenovir hydrochloride and one of the Proton Pump Inhibitors in the same pharmaceutical composition (fixed dose combination), separate pharmaceutical compositions (combined use), and in the same kit package of different pharmaceutical compositions. In this invention, it is envisaged to prepare a combination of Umifenovir, Camostat and Proton pump inhibitors (preferably s-omeprazole) as a tablet or hard gelatin capsule formulation for an effective antiviral formulation. Here, simple mixtures of active ingredients can be prepared and compressed directly into tablets. The filling material to be used in this case is microcrystalline cellulose (Avicel). Because the stability of the proton pump and other substances is adversely affected in a high humidity environment. As another option, the active ingredients are dissolved in PEG or another suitable oil and placed in a soft gelatin capsule in dissolved or suspended form. In order not to adversely affect their stability and to prevent pharmaceutical incompatibilities, it should be ensured that the best formula and preparation are as follows:

0.2% Aerosil and 0.5% magnesium stearate are added to 500 g of Camostat , mixed and 75 mg of microcrystalline cellulose is added, mixed.

0.2% Aerosil is added to 500 g of Umifenovir, mixed and 75 mg of microcrystalline cellulose is added, mixed.

This mixture is combined and mixed.

In a separate tablet, 40 mg of proton pump (preferably s-omeprazole) is mixed with 20 mg of microcrystalline cellulose and pressed directly as a tablet. This tablet is used as the inner core tablet of the folded tablet.

A mixture of camostat and umifenovir and a proton pump (preferably s- omeprazole) as core tablet is pressed as folded tablet.

Camostat Mesylate and Umifenovir Hydrochloride should be understood when the terms Camostat and Umifenovir are used alone without specifying their salt form.

Detailed Description of the Invention

Umifenovir is a product licensed in Russia and China (Arbidol®) and has been used for nearly 30 years as a prophylaxis and treatment for influenza A and B infections. Unfortunately, it is not well known in western countries. It has come to the fore again with the COVID- 19 epidemic and has been included in many study protocols. Recent studies have shown that fusion inhibition applies to a wide family of viruses, including rhinoviruses and coronaviruses (Brooks MJ, Sasadeusz JJ, Tannock GA, 2004 )). In February, umifenovir entered protocols for use against coronavirus COVID- 19 and was published in the 7th edition of clinical trial guidelines in China (Dong L, Hu S2, Gao J., 2020) (Dong L, Hu S2, Gao J., 2020) (Diagnosis and Treatment Protocol for Novel Coronavirus Pneumonia -Trial Version 7-, 2020).

Mechanism of Action

Viral fusion mechanisms

The action of umifenovir is by inhibiting viral fusion and has the capacity to induce serum interferon (Teissier El) (Leneva IA). Umifenovir hydrochloride inhibits virus replication by inhibiting the fusion of the lipid membrane of the virus with host cells. Compared with the control group, umifenovir has been shown to effectively inhibit coronavirus up to 60- fold at a concentration of 10-30 pmol/L, significantly reducing the pathological effects of the virus on cells. Results of the docking study of umifenovir in the context of possible drug targets of the novel coronavirus were associated with non- structural proteins (Nsp7_Nsp8 complex, Nspl4, Nspl5), E-channel protein, and Spike, respectively, with the following scores: -136,087, -118.253, -118.253, -117.879 and 145,125 (Wu C, Liu Y, Yang Y, Zhang P, Zhong W, Wang Y, Wang Q, Xu Y, Li M, Li X, Zheng M, Chen L, Li H., 2020). Low score values indicate low binding energies and are inversely proportional to affinity.

Spike protein, TMPRSS2, etc. It is cleaved into SI and S2 by host cell protease. TMPRSS2 (transmembrane serine protease 2) is a multi-domain type II transmembrane serine protease responsible for preparing virus surface glycoproteins such as hemagglutinin and spike for entry and fusion. Without this process, cell entry and fusion cannot occur. The main function of SI is to bind to host cell surface receptors, and the S2 subunit mediates virus cell and cell cell membrane fusion. Spike's structural integrity and activation play a key role in virus transmission and virulence. Therapeutic strategies to block the entry of coronavirus into host cells by targeting Spike proteins or specific receptors on the host surface are valuable targets for the development of anti-viral drugs.

Camostat mesylate has been licensed in Japan since 1986. Its main indication is that it is a narrow spectrum TMPRSS2 inhibitor. In this way, unlike aprotinin and famostat, it does not act like other protease inhibitors that have an effect on hemodynamic balances and thrombosis process. Camostat will be an effective antiviral if given in combination with umifenovir hydrochloride at a dose of 3x200 mg for 8-10 days, and in combination with a dose of 4x200 mg.

In previous studies, both umifenovir and camostat mesylate were given individually and especially at a daily dose of 600 mg umifenovir. This time, umifenovir will reach the optimum plasma level against SARS CoV-2 as 4x200 mg. In this study, viral clearance will be achieved by giving both a fusion inhibitor and a serine rotase inhibitor, and a synergistic antiviral effect far higher than the effects seen in previous studies when applied individually, will have been obtained. Perhaps the most outstanding feature of the present invention is that a proton pump inhibitor, preferably esomeprazole, is added to this dual combination with a 2 x 40 posology.

Host cell serine proteases, TMPRSS2, ensure viral entry by cleavage of the S1/S2 anchor of the viral spike protein, also called "priming", and binding of the SI receptor-binding portion of the virus (RBD) to the ACE 2 receptor. Camostat mesylate is a potent TMPRSS2 inhibitor, preventing the virus from entering from the outside. The fusion of the virus, which managed to escape this effect and enter the cell, through the endosomal membrane is provided by the S2 fusion peptides of the spike protein. Umifenovir inhibits this process by binding to fusion peptides at this stage. In this way, even if the virus crosses the first barrier, the combination of camostat and umifenovir will create a synergistic pressure on the new coronavirus (SARS-CoV-2), since the virus will be attached to the second barrier. Based on this mechanism, the effect of the combined use of these two drugs on COVID-19 infection will be demonstrated for the first time in the world with this study. In addition, when this dual combination is potentiated with any proton pump inhibitor that inhibits endosomal acidification, the synergistic effect will be reflected in 10-fold antiviral potency, especially for umifenovir.

The secondary aim will be to reveal how strong the correlation between the individual mechanistic setup of the drugs used in this study and the clinical outcome is. For example, an important starting point is to prove that it is rational to target serine proteases such as TMRPSS2, which has no risk of mutation, rather than targeting viral proteases that are constantly mutating. The importance of host proteases in priming the spike protein of the virus is well known. It is true, then, that one of the targets against the virus is these critical enzymes. Without preliminary preparation, the SI part of the spike protein cannot be stripped from S2 and cannot bind to angiotensin converting enzyme 2 (ACE 2) and cannot enter the cell. Targeting the mechanisms that will release it from its confinement in the endosomal membrane to the cytoplasm in case of leakage explains why the drug that will join our synergy setup is a broad spectrum antiviral and fusion inhibitor. Therefore, testing the use of camostat mesylate, umifenovir hydrochloride and proton pump inhibitors, preferably omeprazole or esomeprazole, or lansoprazole or pantoprazole, in anticipation of such synergism, stands as an expected treatment option for this deadly disease. Camostat is a TMRPPS2 protease inhibitor and umifenovir is a fusion peptide inhibitor. Proton Pump Inhibitors are included here as endosomal H+/K+ ATP'ase inhibitors. In other words, if the virus enters through the main gate of the host cell, it can be considered as a second security barrier. The large number of preclinical mechanistic antiviral mechanisms made in this study may offer the chance to obtain data comparable to the standard treatment of human COVID-19 infection and even clinical outcomes when these drugs are used individually. All this is important in terms of playing a leading role in the selection of drug targets for all viral diseases using spike and ACE-2, as well as treatment, because this triple therapy scheme has never been applied in the world until now. The dose of umifenovir in the present invention is preferably 800 mg per day, which is most consistent with the IC50 values obtained in in vitro cell cultures. The present invention is the combination of Camostat Mesylate, Umifenovir Hydrochloride and Esomeprazole magnesium trihydrate, which is used for the prevention, delay or treatment of COVID-19 disease.

In one embodiment of the invention, Camostat Mesylate, Umifenovir Hydrochloride, and Esomeprazole Magnesium Trihydrate can be found in different pharmaceutical compositions. The combination of Camostat Mesylate, Umifenovir Hydrochloride and Esomeprazole Magnesium Trihydrate can preferably be administered simultaneously, separately or sequentially. In combination, the unit dose of Camostat Mesylate is preferably 200 mg, and the unit dose of Umifenovir Hydrochloride is preferably 200 mg. Camostat Mesylate can preferably be administered 3 to 4 times a day, Umifenovir Hydrochloride preferably 4 times a day. Omeprazole Magnesium Trihydrate can be administered with a posology of 40 mg once a day or 40 mg twice a day.

In another embodiment of the invention, the combination of Camostat Mesylate, Umifenovir Hydrochloride and Esomeprazole Magnesium Trihydrate, which is the subject of the invention, is used to prevent, delay or treat the COVID-19 disease; 200 mg Camostat Mesylate 3 times a day, 200 mg Umifenovir Hydrochloride 4 times a day and Esomeprazole Magnesium Trihydrate equivalent to 40 mg esomeprazole twice a day can be given in combination.

In another embodiment of the invention, said combination of Camostat Mesylate, Umifenovir Hydrochloride and Esomeprazole Magnesium Trihydrate is used to prevent, delay or treat COVID-19 disease; 200 mg of Camostat Mesylate 4 times a day, Umifenovir Hydrochloride 200 mg 4 times a day, and Esomeprazole Magnesium Trihydrate equivalent to 40 mg of esomeprazole twice a day can be given in combination.

In another embodiment of the invention, Camostat Mesylate, Umifenovir Hydrochloride, and Esomeprazole Magnesium Trihydrate are combined in the same pharmaceutical composition. Said pharmaceutical composition may preferably contain 120-200 mg of Camostat Mesylate, 200 mg of Umifenovir Hydrochloride and Esomeprazole Magnesium Trihydrate equivalent to 20-80 mg of esomeprazole. More specifically, the pharmaceutical composition may contain Esomeprazole Magnesium Trihydrate equivalent to 150 mg of Camostat Mesylate, 200 mg of Umifenovir Hydrochloride, and 20 mg of someprazole. Or it may contain Esomeprazole Magnesium Trihydrate equivalent to 200mg of Camostat Mesylate, 200mg of Umifenovir Hydrochloride and 40mg of esomeprazole. Or it may contain 200mg of Camostat Mesylate, 200mg of Umifenovir Hydrochloride and Esomeprazole Magnesium Trihydrate equivalent to 80mg of esomeprazole.

Said pharmaceutical composition in which Camostat Mesylate and Umifenovir Hydrochloride are in the same pharmaceutical composition may preferably contain at least one pharmaceutically acceptable excipient.

Said pharmaceutical composition may preferably contain at least one pharmaceutically acceptable excipient, Esomeprazole Magnesium Trihydrate equivalent to 120-200mg of Camostat Mesylate, 200mg of Umifenovir Hydrochloride and 40-80mg of esomeprazole.

Said pharmaceutical composition may preferably contain between 150 mg of Camostat Mesylate, 200 mg of Umifenovir Hydrochloride and at least one pharmaceutically acceptable excipient. Said pharmaceutical composition may preferably contain 200 mg of Camostat Mesylate, 200 mg of Umifenovir Hydrochloride, Esomeprazole Magnesium Trihydrate equivalent to 40-80 mg of esomeprazole, and at least one pharmaceutically acceptable excipient.

The pharmaceutical composition may preferably be one of the tablets, capsules or other oral solid dosage forms and may be prepared by methods known in the art.

In another embodiment of the invention, Camostat Mesylate and Umifenovir Hydrochloride may be in the same kit package in different pharmaceutical compositions.

In all embodiments of the invention, the combination may be administered to said patient, preferably for 8-10 days.

In all embodiments of the invention, the COVID-19 patients to whom the combination will be administered will preferably be uncomplicated but probable or definitively diagnosed COVID- 19 patients, wherein uncomplicated patients; a) with symptoms such as fever, muscle and joint pain, cough, sore throat and nasal congestion; b) without respiratory distress, tachypnea and SPO2 < 90%, c) patients with normal chest X-ray and/or lung tomography.

With the developed invention, clinical studies will be conducted and the effectiveness of the combination of Camostat Mesylate and Umifenovir Hydrochloride and their combined use will be demonstrated.

Claims

CLAIMS Combination comprising Camostat Mesylate, Umifenovir Hydrochloride and Esomeprazole Magnesium Trihydrate for use in the prevention, delay or treatment of Covid- 19 disease. A combination according to claim 1, characterized in that Camostat Mesylate, Umifenovir Hydrochloride and Esomeprazole Magnesium Trihydrate are present in different pharmaceutical compositions. A combination according to the preceding claims, characterized in that said combination is applied simultaneously, separately or sequentially. A combination according to one of the preceding claims, characterized in that the unit dose of Camostat Mesylate is 200 mg, the unit dose of Umifenovir Hydrochloride is 200 mg and the unit dose of Esomeprazole Magnesium Trihydrate is equivalent to 40 mg of esomeprazole. A combination according to one of the preceding claims, characterized in that Camostat Mesylate is applied 3 to 4 times a day, Umifenovir Hydrochloride 4 times a day, and Esomeprazole Magnesium Trihydrate 2 times a day. A combination according to claim 1, characterized in that Camostat Mesylate, Umifenovir Hydrochloride and Esomeprazole Magnesium Trihydrate are present in the same pharmaceutical composition. A combination according to claim 6, characterized in that said pharmaceutical composition contains 120-200 mg of Camostat Mesylate, 200 mg of Umifenovir Hydrochloride and Esomeprazole Magnesium Trihydrate equivalent to 40-80 mg of esomeprazole. A combination according to claim 7, characterized in that said pharmaceutical composition contains 150mg of Camostat Mesylate, 200mg of Umifenovir Hydrochloride Esomeprazole Magnesium Trihydrate equivalent to 40-80mg of esomeprazole. A combination according to claim 7, characterized in that said pharmaceutical composition contains 200mg of Camostat Mesylate, 200mg of Umifenovir Hydrochloride and Esomeprazole Magnesium Trihydrate equivalent to 40-80mg of esomeprazole.

10. A combination according to any one of claims 6 to 9, characterized in that said pharmaceutical composition contains at least one pharmaceutically acceptable excipient. 11. A combination according to claim 1, characterized in that Camostat Mesylate,

Umifenovir Hydrochloride and Esomeprazole Magnesium Trihydrate are in the same kit package in different pharmaceutical compositions.

12. A combination according to any of the preceding claims, characterized in that said combination is administered to the patient for 8-10 days. 13. A combination according to any preceding claim, characterized in that said

COVID-19 patients are uncomplicated but probable or definitively diagnosed COVID- 19 patients, wherein uncomplicated patients; a. with symptoms such as fever, muscle and joint pain, cough, sore throat and nasal congestion; b. without respiratory distress, tachypnea and SP02 < 90%, c. patients with normal chest X-ray and/or lung tomography.