Artenin lectins and combination formulations for treatment or prevention of viral infections in humans or animals caused by coronavirus

Abstract

The present invention addresses the problem of the category of active ingredients that can be used to treat viral infections in humans or animals caused by coronavirus. Treatment includes emergency treatment and prevention of already existing viral diseases. The present invention relates to bromelain, bromelain, artenin-like lectin, pineapple plant stem extract and / or fruit extract, a combination preparation, a bromelain inhibitor, a protein / protease mixture and a saccharified bromelain protein produced by exogenous non-enzymatic saccharification, the compound is used for treating or preventing viral infection of human or animals caused by coronavirus.

Description

[0001] This application is a divisional application of the Chinese patent application with application number "202180056115.0", application date "June 10, 2021", and invention name "Bromelain, bromelain, japanese lectin, extracts from pineapple plant stems and / or fruits, combined preparations, bromelain inhibitors, protein-protease mixtures and saccharified bromelain proteins produced by exogenous non-enzymatic saccharification for treating or preventing viral infections in humans or animals caused by coronavirus". Technical Field

[0002] The object of the present invention is to provide active ingredient classes with which viral diseases in humans or animals caused by coronaviruses can be treated. Treatment in this regard includes acute treatment of already existing viral diseases and their prevention. Background Art

[0003] In light of the ongoing COVID-19 pandemic, there remains a pressing need to provide additional active ingredients or classes of active ingredients to treat viral diseases. Summary of the invention

[0004] According to a first aspect, the present invention relates to bromelain (synonymous "pineapple enzyme") for use in the treatment or prevention of viral infections in humans or animals caused by coronaviruses. The bromelain is particularly selected from stem bromelain (SBM) (EC 3.4.22.32, CAS No.: 37189-34-7), fruit bromelain (EC 3.4.22.33, CAS No.: 9001-00-7), ananain (EC 3.4.22.31) and mixtures and combinations thereof.

[0005] The preparation and isolation of bromelain from the stem or fruit of the pineapple plant (Ananas comosus), in particular stem bromelain (SBM) (EC 3.4.22.32) and fruit bromelain (EC 3.4.22.33), is known in the prior art, for example as described by Rowan, AD, Buttle, DJ & Barrett, AJ in Arch. Biochem. Biophys. 267, 262-270 (1988) or in ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, Vol. 267, No. 1, November 15, pp. 262-270 (1988).

[0006] In addition to direct antiviral effects, the bromelain also has systemic effects that are generally helpful in treating viral infections, in particular anti-inflammatory effects that inhibit excessive immune responses, anti-edema effects such as eliminating pulmonary edema, and anti-coagulant effects that counteract thrombosis caused by inflammation and / or an overactive coagulation system.

[0007] According to a second aspect, the present invention relates to japanese lectins for use in treating or preventing viral infections in humans or animals caused by coronaviruses.

[0008] Here, the japanese lectin is particularly selected from the group consisting of mannose-specific lectin and glucose-specific lectin.

[0009] The japanese lectin is particularly preferably selected from the group consisting of pineapple lectin (japanese lectin from pineapple (AcmJRL)), japanese lectin, cassia bark lectin, MPA lectin, Jerusalem artichoke lectin, red algae lectin, and mixtures and combinations thereof.

[0010] A third aspect of the present invention also relates to an extract of the stem and / or fruit of a pineapple plant for use in treating or preventing viral infections in humans or animals caused by coronaviruses.

[0011] The pineapple plant is in particular pineapple and pineapple. Such extracts contain one or more than one of the previously mentioned bromelains and / or ananains.

[0012] According to a fourth aspect, the present invention also relates to a combined preparation comprising at least one bromelain and at least one japanese lectin for use in the treatment or prevention of viral infections in humans or animals caused by coronaviruses.

[0013] In a preferred embodiment, the weight ratio of the total amount of at least one bromelain to the total amount of at least one japanese lectin is 50:50 to 0.1:99.9, preferably 40:60 to 1:99, more preferably 30:70 to 2:98, more preferably 20:80 to 3:97, particularly preferably 10:90 to 5:95.

[0014] More advantageously, the combined preparation according to the invention comprises at least one bromelain, the total concentration of all bromelains in the extract or combined preparation preferably being between 0.01% and 50.0% by weight, more preferably between 0.1% and 20.0% by weight, particularly preferably between 1.0% and 10.0% by weight.

[0015] Furthermore, advantageously, the combined preparation comprises at least one japanese lectin, the total concentration of the at least one japanese lectin in the extract being preferably 0.01 to 60.0% by weight, more preferably 1.5 to 50.0% by weight, particularly preferably 2.0 to 30% by weight.

[0016] In 1973, Perlstein & Kezdy discovered protease inhibitors in bromelain base powder (BBP), which is bromelain obtained from the stems of pineapple plants (Perlstein & Kezdy, Struct., Vol. 1, 249-254). These protease inhibitors can inhibit the enzymatic activity of bromelain in BBP.

[0017] Seven isoforms of these bromelain inhibitors were first described in 1973 (Perlstein & Kezdy, Struct., Vol. 1, 249-254). In 1975, Reddy et al. fully revealed the primary structure of isoform VII and pointed out the microscopic heterogeneity of the seven isolated isoforms (Reddy et al., J. Biol. Chem., Vol. 250, 1741-1750).

[0018] Lenarcic et al. revealed the primary structure of another bromelain inhibitor in 1992 and also revealed its inhibitory effect on cathepsin L (Lenarcic et al., Biol. Chem., Vol. 373, 459-464).

[0019] Hatano et al. published the primary and secondary structures of Bromelain inhibitor VI in 1995 (Hatano et al., Eur. J. Biochem., Vol. 232, 335-343). In the following year, the similarity of Bromelain inhibitor VI to the already described Bowman-Birk trypsin inhibitor was revealed (Hatano et al., Biochemistry, Vol. 35, 5379-5384). Hatano et al. finally published the amino acid sequences of all seven previously described Bromelain inhibitor isoforms in 1998 (Hatano et al., J. Biochem., Vol. 124, 457-461).

[0020] All bromelain inhibitor separation processes previously described in the literature use two-step chromatography. The basic principle of this method has been published by Perlstein & Kezdy in 1973 (Perlstein & Kezdy, Struct., Vol. 1, 249-254). In the first dimension, i.e. in the first purification step, the chromatography uses size exclusion chromatography (SEC). SEC can be used to separate the molecules contained in BBP from each other according to their different sizes. Bromelain (about 25 kDa) and bromelain inhibitor (about 6 kDa) can be separated in particular.

[0021] According to the method of Perlstein & Kezdy, anion exchange chromatography (e.g. weak anion exchange chromatography = WAX) is usually used for the second chromatographic separation. Using this method, Perlstein & Kezdy successfully separated seven bromelain inhibitors (seven different isoforms) from BBP.

[0022] It has been reported that about 3 mg of bromelain inhibitor isoform VII or 1 mg of bromelain inhibitor isoform VI was obtained from 1 g of BBP using this method (Reddy et al., Biol. Chem., Vol. 250, 1741-1750; Hatano et al., Eur. J. Biochem., Vol. 232, 335-343).

[0023] The purification method published by Perlstein & Kezzy, which may also be referred to as SEC / WAX, has the disadvantage that it was not successful in isolating the bromelain inhibitor isoforms in high purity or pure form.

[0024] It is generally agreed that all previously purified Bromelain inhibitor isoforms are actually mixtures of different Bromelain inhibitor isoforms (Hatano et al., Biol. Chem., Vol. 383, 1151-1156).

[0025] In addition, the SEC / WAX method is not cost-effective to scale up to industrial scale. What is decisive here is the use of the SEC method in the first dimension, i.e. in the first purification step. Compared with the adsorption chromatography protein purification method, the capacity and separation power of SEC are smaller. This is particularly evident in the fact that Hatano et al. repeated the first SEC step three times for separating 1 mg of bromelain protease inhibitor, which shows that even at this small scale (1 g BBP), a lot of time is needed (Hatano et al., 1995, Eur. J. Biochem., Vol. 232, 335-343).

[0026] Since three chromatography steps are considered to be the upper limit of economic efficiency, repeated SEC chromatography is not economical for industrial applications. For example, to obtain about 1 g of bromelain inhibitor using the SEC / WAX method, a SEC column with a volume of about 100 liters must be used. Therefore, the SEC / WAX method is not economical in terms of both technical materials and time economy, and has little value for commercial use.

[0027] For the purification of bromelain, different methods have been described in the literature (Rowan et al., Arch. Biochem. Biophys., Vol. 267, 262-270; Harrach et al., Protein Chem., Vol. 14, 41-52).

[0028] Therefore, the object of the present invention is also to provide an improved method for separating bromelain inhibitor and bromelain, and further to provide a bromelain inhibitor and bromelain mixture with high purity and rich biological activity.

[0029] In a fifth aspect, the present invention therefore relates to a bromelain inhibitor for use in the treatment or prevention of viral infections in humans or animals caused by coronaviruses, the bromelain inhibitor comprising or consisting of at least one peptide having an amino acid sequence that is at least 90%, preferably 95%, particularly preferably 100% identical to one of the sequences of SEQ ID NO: 1 to SEQ ID NO: 7, wherein the at least one peptide preferably has a purity of at least 80% by weight, particularly preferably at least 95% by weight.

[0030] According to the invention, there is also provided a process for purifying at least one bromelain inhibitor, characterized in that an aqueous solution containing a dissolved pineapple stem extract is purified by cation exchange chromatography, the aqueous solution having a pH value of 6.5 to 9.5, the cation exchange material binding bromelain within this pH value, and the chromatography pass comprising at least one bromelain inhibitor.

[0031] Aqueous solutions and / or other solutions (eg elution buffer) that can be used for cation exchange chromatography optionally contain a buffer substance having a buffering effect at a pH value of 6.5 to 9.5.

[0032] High capacity and separation efficiency can be achieved using strong cation exchange chromatography or weak cation exchange chromatography (SCX or WCX), ie cation exchange chromatography using strong ion exchange materials or weak ion exchange materials.

[0033] Preferably, the buffer conditions are selected so that in the cation exchange chromatography, the bromelain inhibitor from the stem extract of the pineapple plant does not initially bind to the cation exchange material (cation exchange column), but all the bromelain binds to the cation exchange material. The bromelain inhibitor is thus located in the flow-through of the cation exchange chromatography, which can be collected and subjected to further chromatography steps.

[0034] In a preferred embodiment, the buffer conditions are selected so that the cation exchange chromatography is carried out at a pH of 5.0 to 10, preferably 7.0 to 9.5. The aqueous solution in the method may contain a buffer substance, for example a buffer substance selected from TRIS, HEPES, sodium phosphate, and / or potassium phosphate. In a preferred embodiment, the aqueous solution has a weak electrical conductivity. The concentration of the buffer substance contained in the aqueous solution may be 5 mM to 100 mM, preferably 10 mM to 50 mM.

[0035] Bromelain bound to the cation exchange material can be released from the cation exchange material and isolated in a further step. This can be accomplished by eluting the bromelain using a suitable elution buffer.

[0036] The buffer conditions in the elution buffer are selected to decouple the bromelain from the cation exchange material. The buffer may have a salt (e.g., NaCl or KCl) concentration of 0.01 M to 1.0 M, preferably 0.10 M to 0.5 M. Alternatively, elution from the cationic buffer material may be performed by an elution buffer having a pH higher than the pH of the aqueous solution or higher than the pH of the wash buffer.

[0037] Elution can be performed using a step gradient or a linear gradient. If at least one further purification step of bromelain is provided, elution is preferably performed using a linear gradient. In a particularly preferred embodiment, the individual fractions of the eluate are separated during this process. Elution with a step gradient produces a bromelain mixture having a biological activity that is enriched compared to BBP.

[0038] The eluate of the cation exchange material comprises bromelain which is substantially free of inhibitors. In this regard, the bromelain may contain less than 5.0 wt. %, preferably less than 2.0 wt. %, further preferably less than 1.0 wt. %, more preferably less than 0.5 wt. %, more preferably less than 0.2 wt. %, particularly preferably less than 0.1 wt. % of bromelain inhibitors relative to the total peptide mass. The purity of the bromelain may be determined by SEC-HPLC or RP-HPLC.

[0039] If the Bromelain inhibitor is to be further purified, the cation exchange material passthrough containing the Bromelain inhibitor can be purified in a subsequent second step by anion exchange chromatography (AX), hydrophobic chromatography (HIC), reverse phase HPLC (RP-HPLC), or SEC.

[0040] The conditions of the buffer are selected so that at least one bromelain inhibitor binds to an anion exchange material, a material for hydrophobic chromatography or a material for RP-HPLC and then separated. In addition to SEC, the separation is preferably washed with one buffer and then eluted with another buffer. Since the bromelain inhibitor does not bind to the SEC chromatographic material at all, this step is unnecessary in SEC. Suitable buffer conditions for the corresponding materials (for binding, washing and / or elution of peptides or proteins) are known to those skilled in the art from the prior art.

[0041] The person skilled in the art is familiar with the term “washing”. “Washing” is preferably understood as contacting the chromatography material with a buffer which weakens the binding of undesired materials to the chromatography column, particularly preferably completely eliminates such binding, while maintaining the binding of the bromelain inhibitor to the chromatography material.

[0042] Elution can be performed using a step gradient or a linear gradient and the eluate can optionally be collected in separate fractions.

[0043] Depending on the desired quality of the Bromelain inhibitor, a third chromatography step may be performed after the second step. In this third step, further purification is performed, wherein at least one separated Bromelain inhibitor is purified by RP-HPLC, AX, or SEC, the buffer conditions being selected so that the at least one Bromelain inhibitor binds to the material of the RP-HPLC or the anion exchange material and then separated. In addition to SEC, the separation preferably uses one buffer for washing and another buffer for elution. Since the Bromelain inhibitor does not bind to the SEC material, no washing or elution is required (a running buffer is sufficient).

[0044] In all methods according to the present invention, the cation exchange material can be selected from strong cation exchange materials and weak cation exchange materials; the anion exchange material can be selected from strong anion exchange materials and weak anion exchange materials; the material for hydrophobic chromatography can be selected from linear, cyclic, non-aromatic and aromatic ligands with carbon numbers of C3 to C20; the material for RP-HPLC can be selected from linear, cyclic, non-aromatic and aromatic ligands with chain lengths of C4 to C18 (preferably linear ligands); the material for gel filtration chromatography can be Superdex material (e.g., Superdex30 Prepgrade from GE Healthcare).

[0045] In a preferred embodiment of the method according to the invention, the extract from pineapple plant stem is BBP.

[0046] An advantage of the process according to the invention is that a mixture of Bromelain inhibitor isoforms is obtained which is free of other components of the stem extract of the pineapple plant.

[0047] By means of the method, a Bromelain inhibitor mixture (i.e. the totality of all Bromelain inhibitor isoforms) can be provided, the yield of which is about 1% to 50% by weight, preferably 5% to 30% by weight, of the starting material of Bromelain base powder (BBP = commercially available stem extract of pineapple plant). Since the individual Bromelain inhibitor isoforms reported in the prior art are 0.1% or 0.3% by weight, this means that the method according to the invention is a great improvement over the prior art. Therefore, the method according to the invention for separating Bromelain inhibitor isoforms from BBP is more efficient and economical than the methods of the prior art.

[0048] It is also possible to provide individual ultrapure Bromelain inhibitor isoforms. This can be achieved by performing AX chromatography in the second dimension (= second step) and RP-HPLC chromatography in the third dimension (= third step). Thus, individual ultrapure Bromelain inhibitor isoforms can be isolated from a stem extract of a pineapple plant.

[0049] The present invention includes a bromelain inhibitor comprising or consisting of at least one peptide having an amino acid sequence that is at least 90%, preferably 95%, particularly preferably 100% identical to the sequence of one of SEQ ID NO. 1 to SEQ ID NO. 7 (sequences of known bromelain inhibitors I to VII).

[0050] According to the present invention, at least one peptide in the Bromelain inhibitor (e.g. at least one isoform of the Bromelain inhibitor) may have a purity of at least 80% by weight, preferably at least 90% by weight, particularly preferably at least 95% by weight relative to contaminating molecules and other Bromelain isoforms.

[0051] According to the present invention, a bromelain inhibitor is also provided, which is a mixture of at least two, preferably at least three, particularly preferably at least four bromelain inhibitor isoforms. The mixture may optionally include all seven known bromelain inhibitor isoforms (see SEQ ID NO. 1 to SEQ ID NO. 7). Compared to a single bromelain inhibitor isoform, a mixture of multiple bromelain inhibitor isoforms has the advantage that it can bind to a wider range of molecular targets and therefore has a better effect.

[0052] In other preferred embodiments, the Bromelain inhibitor according to the present invention comprises at least Bromelain inhibitor isoform IV (SEQ ID NO. 4) and / or Bromelain inhibitor isoform V (SEQ ID NO. 5).

[0053] The mixture may have a purity of at least 80% by weight, preferably at least 90% by weight, particularly preferably at least 95% by weight, in terms of the ratio of bromelain inhibitor to non-bromelain inhibitor contaminating molecules (eg bromelain).

[0054] At least one peptide in the Bromelain inhibitor according to the present invention may also:

[0055] a) contains a post-translational modification specific to pineapple plants, preferably glycosylation; and / or

[0056] b) does not contain any post-translational modifications.

[0057] The Bromelain inhibitor according to the present invention can preferably be prepared using the method according to the present invention.

[0058] The Bromelain inhibitors according to the invention can be used in medicine, preferably in the treatment and / or prevention of diseases characterized by an enhanced expression of at least one cellular protease, preferably at least one cysteine ​​protease.

[0059] The invention also includes a mixture of bromelains containing less than 0.5% by weight, preferably less than 0.2% by weight, particularly preferably less than 0.1% by weight of bromelain inhibitors relative to the total peptide mass. The person skilled in the art can determine the residual content of bromelain inhibitors in the bromelain mixture, for example, by analytical SEC or RP-HPLC.

[0060] The Bromelain mixture according to the invention can be prepared according to variants of the method according to the invention.

[0061] In a sixth aspect, the present invention relates to a bromelain mixture for use in treating or preventing viral infections in humans or animals caused by coronaviruses, wherein the bromelain mixture comprises less than 5.0% by weight, preferably less than 2.0% by weight, more preferably less than 1.0% by weight, more preferably less than 0.5% by weight, more preferably less than 0.2% by weight, and particularly preferably less than 0.1% by weight of bromelain inhibitor relative to the total peptide mass.

[0062] In a seventh aspect, the present invention also relates to a saccharified bromelain protein comprising at least one saccharide unit covalently bound to the bromelain protein. The saccharified bromelain protein is produced by exogenous non-enzymatic saccharification, in particular saccharified japanese lectin, saccharified bromelain, saccharified bromelain protease inhibitor, or a mixture thereof.

[0063] Preferably, saccharified bromelain is used for treating or preventing viral infections in humans or animals caused by coronaviruses.

[0064] In a more preferred embodiment herein, at least one sugar unit covalently bound to the bromelain protein is selected from monomeric hexoses or oligomeric hexoses, in particular glucose, galactose, and / or mixtures and combinations thereof.

[0065] 1 to 10, preferably 1 to 5, particularly preferably 1, 2 or 3 sugar units are covalently bound to the bromelain protein via the Maillard reaction.

[0066] The saccharified bromelain protein mentioned above can be prepared, in particular, by mixing at least one bromelain protein with at least one reducing sugar and performing a Maillard reaction.

[0067] The coronavirus may be selected, for example, from an orthocoronavirus, preferably an alphacoronavirus, a betacoronavirus, a gammacoronavirus or a deltacoronavirus, and a letovirus, preferably an alphaletovirus, in particular a milecovirus, such as microhylaletavirus1 (MLev-1).

[0068] Here, positive coronaviruses are particularly selected from:

[0069] Alphacoronavirus, selected from coronavirus, such as bat coronavirus CDPHE15; decacovirus, such as horseshoe bat alphacoronavirus HuB-2013; Duvinacovirus, such as human coronavirus 229E (HCov229E); luchacovirus, such as Lucheng brown rat coronavirus; minacovirus, such as ferret coronavirus or mink coronavirus 1; minunacovirus, such as long-winged bat coronavirus 1 or long-winged bat coronavirus HKU8; myotacovirus, such as big-footed myotacovirus alphacoronavirus Sax-2011 or Zhonghuashan Bat alphacoronavirus SC-2013; pedacovirus, such as porcine epidemic diarrhea virus (PEDV) or yellow bat coronavirus 512; Rhinacovirus, such as horseshoe bat coronavirus HKU2 or porcine acute diarrhea syndrome coronavirus (SADS-CoV); Setracovirus, such as human coronavirus NL63 (HCov-NL63) or NL63-related bat coronavirus strain BtKYNL63-9b; Tegacovirus, such as alphacoronavirus 1, in particular canine coronavirus (CCoV), feline coronavirus (FCoV) or transmissible gastroenteritis virus (TGEV);

[0070] Betacoronavirus is selected from sarbecovirus, such as severe acute respiratory syndrome-associated coronavirus ("SARS-associated coronavirus", in particular severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and synonymous or non-synonymous mutants thereof, including variants B.1.1.7, B.1.351, B.1.617, P.1, P.2, B.1.525, B.1.427, B.1.429, L452R, Fin-796H, B.1.526, S :H66D、S:G142V、S:D215G、S:V483A、S:D614G、S:H655Y、S:G669S、S:Q949R、S:N1187D、ORF6:K23-、ORF6:V24 -, ORF6:S25-, ORF6:I26-, ORF6:W27-, ORF6:N28-, ORF6:L29-, ORF6:D30-, ORF6:Y31-, S:Y144-, E484K, VOC 20I / 484Q, B.1, R.1, A.2.5, C.36, B.1.1.318, B.1.621, B.1.623, severe acute respiratory syndrome coronavirus (SARS-CoV, SARS coronavirus, also known as SARS-CoV-1), Embecovirus, such as betacoronavirus 1, in particular bovine coronavirus (BCoV), equine coronavirus (ECoV-NC99), human coronavirus OC43 (HCoV-OC43), porcine hemagglutinating encephalomyelitis virus (HEV), puffinosis coronavirus (PCoV), human enteric coronavirus (HECoV); Chinese rat coronavirus 24; human coronavirus HKU1 (HCoV-HKU1); murine coronavirus such as mouse hepatitis virus (MHV) or rat coronavirus (RtCoV); hibecovirus, such as horseshoe bat beta coronavirus Zhejiang 2013; merbecovirus (formerly MERS-related coronavirus, MERSr-CoV), such as hedgehog coronavirus 1, Middle East respiratory syndrome-related coronavirus (MERS-CoV), sphenops ...

[0071] The gammacoronavirus is selected from the group consisting of cegavirus, such as beluga coronavirus SW1; or igacovirus, such as avian coronavirus, in particular turkey coronavirus (TCov), pheasant coronavirus (PhCoV), infectious bronchitis virus (IBV); and

[0072] The delta coronavirus is selected from andecovirus, such as ruddy duck coronavirus HKU20; buldecovirus, such as BuCoV HKU11, coronavirus HKU15, Munia coronavirus HKU13 (MunCoV HKU23), white-eye coronavirus HKU16, or thrush coronavirus HKU12 (ThCoV HKU2); herdecovirus, such as night heron coronavirus HKU19; and moordecovirus, such as moorhen coronavirus HKU21.

[0073] According to the present invention, bromelain, japanese lectin, extract, combination preparation, bromelain inhibitor, bromelain mixture or glycosylated bromelain protein as described above are suitable for use according to the present invention in treating symptoms caused by coronavirus infection, in particular fever, cough, pneumonia, community-acquired pneumonia with lymphocytopenia (L-CAP), pleurisy, dyspnea, discomfort and / or fatigue, sputum, loss of smell and / or loss of taste, shortness of breath, myositis and / or pharyngitis, joint pain, chest pain, pharyngitis, headache, back pain, malaria, nausea and / or vomiting, chills, diarrhea, hemoptysis, lymphocytopenia, rash on the hands, feet or mouth, bilateral non-purulent conjunctivitis, hypotonia, shock, hypertyrosinemia, myocardial dysfunction, pericarditis and / or heart valve inflammation, coagulopathy, multisystem inflammatory syndrome in children (MIS-C) and conjunctival swelling.

[0074] Advantageously, in the aforementioned extract or combined preparation, the weight ratio of the total amount of at least one bromelain to the total amount of at least one japanese lectin is 1:99 to 99.9:0.1, preferably 50:50 to 99:1, more preferably 60:40 to 98:2, more preferably 70:30 to 97:3, more preferably 80:20 to 96:4, particularly preferably 90:10 to 95:5.

[0075] The weight ratios or amounts of JRL / bromelain are defined in claims 10 to 12, such as they are present in the natural product (extract) or used in their natural proportions. The extract or combined preparation preferably comprises bromelain, and the total concentration of all bromelains in the extract or combined preparation is preferably from 0.1% to 80.0% by weight, more preferably from 1.0% to 50.0% by weight, particularly preferably from 5.0% to 20.0% by weight.

[0076] More advantageously, the extract or combined preparation comprises at least one japanese lectin, the total concentration of the at least one japanese lectin in the extract being preferably 0.01 to 50.0% by weight, more preferably 0.1 to 10.0% by weight, particularly preferably 2.0 to 8.0% by weight.

[0077] Advantageously, the bromelain, japanese lectin, extract, combined preparation, bromelain inhibitor, bromelain mixture or saccharified bromelain protein in all the above embodiments is in the form of or as a component of a powder, granules, tablet, in particular a film tablet, a hard capsule, a soft capsule, an effervescent tablet, a solution, in particular an injection solution or an infusion solution, an emulsion, a suspension, an ointment, a cream, a paste, a gel, a tincture, an eye drop, a powder mist, a nasal spray, a suppository or a transdermal patch.

[0078] Also advantageously, in the above-mentioned various aspects of the invention, the extract, bromelain, japanese lectin, bromelain inhibitor, bromelain mixture or saccharified bromelain protein is administered orally, by intravenous, subcutaneous or intramuscular injection, by infusion into the nasal mucosa, oral mucosa or pharyngeal mucosa, by inhalation, by application to the ocular surface, rectum, and / or by a transdermal patch.

[0079] According to an exemplary embodiment of various aspects of the present invention: administration is performed once a week to once an hour, preferably once a day to 10 times a day, particularly preferably 1 to 3 times a day, or continuously.

[0080] The subject matter according to the invention will be explained in more detail with reference to the following figures and the following examples, without being restricted to the specific embodiments shown here. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 The primary structure and molecular weight of previously known bromelain inhibitor isoforms are shown (according to Hatano et al., (2002) Biol. Chem., Vol. 383:1151-1156).

[0082] Figure 2 The strong cation exchange chromatography (SEC) elution profile of BBP is shown.

[0083] Figure 3 Shown is the size exclusion chromatography (SCX) purification profile of BBP.

[0084] Figure 4 A schematic diagram of the HCov229E test is shown.

[0085] Figure 5Lectins with anti-coronavirus activity in the HCoV229E reporter virus screening are shown; A) to F) Compounds (specified starting concentrations) were pre-incubated with HCoV229E virus at a ratio of 1:10 for 30 minutes (Table 2), then titrated continuously on target cells Huh-7.5-FLuc and incubated for 48 hours. The activities of RLuc and FLuc were measured as indicators of residual infectivity and cell viability. Data were normalized to TBS control. Black arrows mark Figure 6 Standardize at a 1:500 dilution or a 1:2500 dilution.

[0086] Figure 6 Lectins with anti-coronavirus activity in the HCoV229E reporter virus screening are shown. Compounds were pre-incubated with HCoV229E virus at a ratio of 1:10 for 30 minutes (Table 2) and then titrated along target cells. A) Values ​​for HCoV229E virus replication and cell viability are shown for ( Figure 5 A) Black arrow in the figure) Normalized values ​​relative to TBS control. B) Normalized representation of 1:2500 dilution.

[0087] Figure 7 Lectins with anti-coronavirus activity in the HCoV229E reporter virus screening are shown. The compounds were pre-incubated with the HCoV229E virus at a ratio of 1:2 for 30 minutes (Table 2) and then titrated along the target cells. Remdesivir was used as a positive control and DMSO as a negative control. A) Dose-effect titration of the lectins was performed starting from a 1:500 dilution. The infectivity of the HCoV229E virus (absolute values) is shown on the left, and the corresponding cell viability of Huh-7.5-FLuc cells (log values) is shown on the right. B) The values ​​of HCoV229E virus replication and cell viability at a dilution of 1:500 or at a dilution of 1:2500 (red rectangle in A) are shown as normalized values ​​relative to the BSA control. .

[0088] Figure 8 Exemplary UV / VIS spectra of various prepared pineapple lectin batches are shown.

[0089] Fig. 9 A detailed evaluation of an exemplary pineapple lectin UV / VIS spectrum by forming second derivatives is shown. The maximum of the UV / VIS spectrum is 280.0 nm; significant subbands are shown in red; they correspond to typical absorption layers of the aromatic amino acids Phe, Tyr, and Trp.

[0090] Fig.10SDS-PAGE analysis of a representative lectin batch is shown. Displayed as follows: Lanes 1 to 3: (5.6 / 11.1 / 22.2) μg HZI 2-09. Lane 4: 4.5 μl SERVAChrom protein standard III. Lanes 5 to 7: (8.9 / 17.8 / 35.6) μg HZI 2-10. (T=14%, C=5%; reduced with DTT and denatured by adding SDS at 95°C for 3 minutes before use of the samples).

[0091] Fig.11 RP-HPLC UV chromatogram of pineapple lectin batch HZI 2-09 is shown. The absorption was detected at λ=214 nm (blue) and λ=280 nm (red).

[0092] Figures 12A-12E Surface plasmon resonance spectroscopy (SPR) binding kinetics and affinity testing of SARS-CoV-2-spike protein and different lectin batches isolated from bromelain A) HZI 2-05, (B) HZI 02-06, C) HZI 2-09, D) HZI 2-10, and E) Acm JRL 2 are shown. Single cycle measurements were performed by injecting increasing concentrations of ligand (highest concentration: A) 100 μM, B) 80 μM, C+D) 50 μM, E) 20 μM, starting from a 51:1 dilution). The sensorgram shows that the binding of lectins to the spike protein is faster than the dissociation (binding and dissociation rates). The surface containing the spike protein was regenerated with 80% ethylene glycol between different.

[0093] Fig.13 Binding kinetics by surface plasmon resonance spectroscopy (SPR) and affinity testing for the ACE-2 receptor and the lectin HZI 2-09 are shown. Increasing concentrations of ligand were injected (highest concentration: 50 μM, starting from 5 1:1 dilutions) for single cycle measurements.

[0094] Fig.14 Deconvoluted mass spectra of recombinant SARS-CoV-2 spike (shown at the top) and recombinant SARS-CoV-spike digested with PNGase F (shown at the bottom) are shown to detect glycosylation.

[0095] Fig.15 SDS-PAGE for detecting the hydrolytic degradation of recombinant SARS-CoV-2 spike and the inhibitory function of HZI 2-07 is shown. The material ratio of SARS-CoV-2 spike and bromelain (HTI 2-08) and bromelain inhibitor (HZI 2-07) is 1:1:1. 1) PageRuler pre-staining. The rectangle marks the complete spike protein of about 150 kDa; the dashed rectangle shows the spike degradation product of about 24 kDa.

[0096] Fig.16 SDS-PAGE for detecting the hydrolytic degradation of recombinant SARS-CoV-2 spike and the inhibitory function of HZI 2-07 on the bromelain fraction HZI 2-08 is shown. The material ratios of SARS-CoV-2 spike, bromelain fraction HZI 2-08 and bromelain inhibitor HZI 2-07 are 1:1:1 and 1:1:10. 1) PageRuler pre-staining. The rectangle marks the complete spike protein of about 150 kDa.

[0097] Fig.17 SDS-PAGE for detecting the hydrolytic degradation of recombinant SARS-CoV-2 spike and the inhibitory function of HZI 2-07 on the bromelain separation part HZI 2-08 is shown. The material ratios of SARS-CoV-2 spike and bromelain (HZI 2-08) are 10:1 and 100:1, and the material ratios of SARS-CoV spike and bromelain HZI 2-08 and bromelain inhibitor HZI 2-07 are 1:1:0.5. 1) PageRuler pre-staining. The rectangle marks the complete spike protein of about 150kDa; the dotted rectangle shows the spike degradation product of about 24kDa.

[0098] Fig.18 Shown are the total ion chromatogram and deconvoluted mass spectra of the recombinant SARS-CoV-2 spike, which was incubated with the bromelain fraction HZI-2-08 at a 100:1 mass ratio at 37°C for 10 minutes. The arrow marks the intact spike protein of approximately 150 kDa.

[0099] Fig.19 Shown is the total ion chromatogram and deconvoluted mass spectrum of the recombinant SARS-CoV-2 spike, which was incubated with the bromelain-isolated fraction HZI-2-08 at a 100:1 mass ratio at 37°C for 2 hours. The right arrow marks the intact spike protein of approximately 150 kDa; the left arrow shows the 24 kDa spike degradation product.

[0100] Fig. 20 Shown is the total ion chromatogram and deconvoluted mass spectrum of the recombinant SARS-CoV-2 spike, which was incubated with the bromelain-isolated fraction HZI-2-08 at a 1:1 material ratio at 37°C for 2 hours. The right arrow marks the intact spike protein of approximately 150 kDa; the left arrow shows the 24 kDa spike degradation product.

[0101] Fig.21Shown are the total ion chromatogram and deconvoluted mass spectra of the recombinant SARS-CoV-2 spike, which was incubated with the bromelain fraction HZI 2-08 and the bromelain inhibitor HZI 2-07 at a 1:1:1 ratio for 2 hours at 37°C. The arrow marks the intact spike protein of approximately 150 kDa.

[0102] Fig. 22 An SDS PAGE gel is shown, in which a recombinant SARS-CoV-2 spike protein incubated with bromelain (original fraction) was applied; a SARS-CoV-2 spike protein incubated without bromelain is also shown for comparison. Details of SDS-PAGE analysis: Lane 1: 2 μg lectin batch HZI 2-09. Lane 2: 2 μg lectin batch HZI 2-10. Lane 4: 40 μg bromelain (Ursapharm). Lanes 5 to 6: 1 μg recombinant SARS-CoV-2 spike and 0.4 μg bromelain after incubation at 37°C for 1 hour (substance ratio protein: protease about 1:1). Lanes 7 / 8: 1 / 1.5 μg recombinant SARS-CoV-2 spike. Lane 9: 2 μg horseradish peroxidase Lane 3 / 10: 3.5 μl SERVA tricolor protein standard III. (T=14%, C=5%; before use, the sample was reduced with DTT and denatured by adding SDS, heated at 95°C and centrifuged for 3 minutes). DETAILED DESCRIPTION

[0103] Figure 1 The primary structures (amino acid sequences) of seven known Bromelain inhibitors I, II, III, IV, V, VI, and VII are shown in tabular form. In addition, the average molecular mass and monoisotopic molecular mass of each isoform are given in Daltons.

[0104] Figure 2 The elution curve of BBP applied to preparative SEC is shown. The y-axis represents the absorption intensity at 214nm, and the x-axis reflects the elution volume. The first signal (1) of the elution curve is a BBP component with a molecular weight of about 24kDa, which is determined to be bromelain. The second signal (2) of the elution curve is a BBP component with a molecular weight of about 6kDa, which is determined to be a mixture of bromelain inhibitors. The two signals at (3) are the results of the first and second rechromatograms of the combined separated parts of the second signal (2).

[0105] Figure 3(a) shows a curve of BPP purification on a strong cation exchange column (SCX). The y-axis represents the absorption signal at 280nm, while the x-axis represents the volume of elution buffer. (a) A strong signal (signal I) appears in the SCX column pass, i.e., the molecule is obviously not bound to the SCX column. These molecules can be determined to be bromelain inhibitors by SDS-PAGE and mass spectrometry. At a higher volume of elution buffer and a higher ion concentration (linear gradient), another signal (signal II) appears. SDS-PAGE and mass spectrometry confirm that the molecule at this signal is bromelain. (b) Under the same conditions as the first run, the combined separated portion of the first signal (signal I) (i.e., the pass of the first chromatography) is chromatographed again using the same SCX column, and the results show that the second signal (peak II) no longer appears. Therefore, the bromelain representing the second signal can be separated from the bromelain inhibitor.

[0106] Example 1

[0107] Detection of the effectiveness of bromelain against alpha coronavirus HCoV229E

[0108] 1. Sample Preparation

[0109] The pineapple enzyme was dissolved in a TRIS buffered saline solution (TBS) at pH 7.3 (10 mg / ml). The dried material (HZI 2-01 to -03) was dissolved in TBS buffer. The suspension was briefly centrifuged to pellet the insoluble components. Samples HZI 2-01 and HZI 2-02 were insoluble and were not used for further testing. HZI 2-07 was suspended in a solution of 200 mM NaCl, 20 mM TRIS, pH 8.0.

[0110]

[0111]

[0112] Table 1 Almost all the samples used.

[0113] Table 1

[0114]

[0115]

[0116] MMTS: methyl methylthiosulfonate; RP-HPLC: reverse phase high performance liquid chromatography;

[0117] TBS: TRIS buffered saline solution (pH 7.3)

[0118] 2. Implementation of in vitro antiviral activity test of bromelain, isolated fractions, and lectins against HCoV229E

[0119] Bromelain and lectin were dissolved in TBS. The suspension was centrifuged briefly to pellet the insoluble components. Huh 7.5FLuc cells expressing firefly luciferase were cultured in DMEM medium (Gbico#41965-039) + 10% fetal calf serum (FCS) + 1% penicillin / streptomycin + 1% L-glutamine + 2% non-essential amino acids (= DMEM complete medium) + 5 mg / ml blasticidin. 2x10 4 Cells / well (96-well plate) were seeded in 100 μl of DEME complete medium.

[0120] Alphacoronavirus HCoV229E containing Renilla luciferase (RLuc) was pre-incubated with the compounds at room temperature in a 10:1 ratio (90 μl virus + 10 μl compound) for 30 minutes. The titer of the virus stock solution used for all experiments was 3.41x10 6 TCID50 / ml (For all experiments, the concentration of virus per ml at which 50% of the cells are infected. This corresponds to the number of infectious particles per ml). The mixture was diluted in culture medium and titrated on target cells in steps of 1:5 after preincubation. Figure 4 A schematic diagram of the implementation of the alpha-coronavirus CoV229E experiment is shown.

[0121] Pre-incubation was performed at a ratio of 1:2 (10 μl virus + 10 μl compound) for further experiments. Firefly luciferase-expressing Huh 7.5 FLuc cells were infected with HCoV229E one day after seeding in the presence of the indicated concentrations of compounds [serial titration of cells (1:5)]. Based on the starting concentration in the original sample, the virus starting dilution was 1:500 and is shown in Table 2. The specifications of the pineapple enzyme sample with a stock concentration of 10 mg / ml were similar to those of the HZI 2-03 sample and are not listed separately.

[0122]

[0123]

[0124] After inoculation and incubation of cells at 33°C and 5% CO2 for 48 hours, the virus inoculum was removed, the cells were washed twice with phosphate buffered saline (PBS), and the cells were lysed in 50 μl PBS / 0.5% Triton X-100. The lysis of the cells was further amplified by freezing the plates at -20°C. 20 μl of the lysate was used to measure cell viability by firefly luciferase signal, and another 20 μl of the lysate was used to analyze virus replication / infection efficiency by Renilla luciferase signal.

[0125] 3. Results of in vitro antiviral activity tests of bromelain, isolated fractions, and lectins against HCoV229E

[0126] Bromelain and lectin batches were first tested using Huh-7.5-FLuc cells that are highly permeable to HCoV229E infection with the HCoV229E Renilla luciferase reporter virus (alpha coronavirus). These cells were engineered to express the firefly luciferase reporter gene, allowing the assessment of cell viability in a dual luciferase reporter assay. All lectin samples were composed of different lectin isolates from bromelain. Samples HZI 2-01 and 2-02 were not used due to their insolubility in water. Bromelain at 2mg / ml or 10mg / ml was used as a control.

[0127] We were able to demonstrate that bromelain and lectin showed promising antiviral activity against the HCoV229E reporter virus ( Figure 5 ).

[0128] High concentration of bromelain ( Figure 5 E and Figure 5 F) leads to low cell viability. At 100% cell viability (indicated by the dotted line), it can be observed that the viral replication of the alpha coronavirus HCoV229E with bromelain is reduced by about 50% (dots). The black arrow marks the Figure 6 A standardized 1:500 dilution or a 1:2500 dilution was used for administration; the 1:2500 dilution showed less cytotoxicity due to the reduced concentration of compound, thus better demonstrating any antiviral effects that occurred, thereby also improving cell viability.

[0129] According to the results of about 100% cell viability in samples Acm-JR 1 and HZI 2-05, a reduction of about 25% in viral replication can also be identified at a dilution of 1:500. HZI 2-03 showed a reduction of about 20% in viral replication, while HZI2-04 was inactive. Pineapple enzyme was accompanied by a significant reduction in cell viability at both concentrations (2 mg / ml and 10 mg / ml), so no valid conclusion can be drawn about the reduction in viral replication. The reduction in cell viability is mainly caused by the activity of pineapple enzyme, which causes the separation of cells from the well matrix. The subsequent luciferase analysis only includes adhered cells, and cannot distinguish between dead cells and live cells floating in the culture medium, so erroneous results of cell viability appear.

[0130] In a 1:2500 dilution of all lectins, at approximately 100% cell viability, a reduction in viral replication of approximately 20% to 25% could be confirmed. Bromelain even showed a reduction of approximately 45% to 50% at a stock concentration of 2 mg / ml with cell viability >100%. Only the high concentration of bromelain at a stock concentration of 10 mg / ml showed reduced cell viability, so no valid conclusions about a reduction in viral replication could be drawn. However, a decrease in viral replication could also be recorded here.

[0131] These results should be confirmed by lectins in further experiments. In addition, the use of a higher substance to virus ratio in the preincubation (1:2 instead of 1:10) should amplify the antiviral effect. The starting concentration of the titration 1:500 was further set to correspond to the first pilot experiments. Samples Acm-JRL 1, HZI 2-03, and HZI 2-05 were used in the experiments. Remdesivir (1 μM in TBS), DMSO (1:40 in TBS), and BSA (5 mg / ml in TBS) were used as controls ( Figure 7 A).

[0132] The positive control remdesivir brought about a complete reduction in viral replication. At a dilution of 1:500, the lectins Acm-JRL 1 and HZI 2-05 showed a modest antiviral effect against the HCoV229E reporter virus ( Figure 7 B), while HZI 2-03 had no antiviral effect. Samples Acm-JRL 1 and HZI 2-05 showed a reduction of about 25% in HCoV229E viral replication at >100% cell viability. If viewed at a dilution of 1:2500, sample HZI 2-05 showed a 50% to 60% reduction in viral replication at >100% cell viability.

[0133] The above experimental results were confirmed in this experiment. Higher concentrations of lectin can further reduce viral replication in HZI 2-05 samples [ratio 1:2 reduced by about 50% to 60%, while 1:10 reduced the replication of HCoV229E by about 20% (such as Figure 6 B)].

[0134] It can be demonstrated that infection with HCoV229E can be prevented in in vitro tests.

[0135] Example 2

[0136] Separation of samples HZI 2-09 and HZI 2-05

[0137] 1200 mg of pineapple enzyme (Merck, order number: 1.01651, batch number: K38171251719) was dissolved in 30 ml of buffer A (50 mM TRIS, 500 mM NaCl, pH 7.2) at a concentration of 40 mg / ml and centrifuged at 9000 g for 15 minutes. A 5 ml aliquot of this solution was loaded onto a D-mannose chromatography column (filling volume 4 ml) of dimensions 10 mm×50 mm previously equilibrated with buffer A at a flow rate of approximately 2 ml / min. The column was flushed with at least 25 CV ("column volumes") until the original baseline was reached again or a constant baseline was obtained. The pineapple lectin was now eluted by switching to buffer B (50 mM TRIS, 500 mM NaCl, 1 M D-mannose, pH 7.2) and the separated fractions were collected. This use of 5 ml aliquots was repeated multiple times, for example 5 times. The eluted and collected fractions are pooled and loaded into a dialysis tube with a MWCO of 3.5 kDa. The volume of one eluted fraction is about 7 ml. Dialysis is performed with steady stirring at room temperature or at 4°C against 2 liters of buffer C (50 mM TRIS, 150 mM NaCl, pH 7.2), changing the buffer several times, until the dialyzed mannose concentration reaches ≤ 1 mM. After dialysis, the floccules are separated by centrifugation or filtration. The excess is reduced to a smaller volume by ultrafiltration membranes with a MWCO of 5 kDA or less, so that the original protein concentration after dialysis is increased several times. The protein concentration can be calculated as a first approximation from the UV absorption at 280 nm, for example by the theoretically calculated molar extinction coefficient of pineapple lectin according to www.uniprot.org, as was done here. Figure 8 A typical UV / VIS spectrum of an example batch is shown, Fig. 9 A detailed evaluation by forming the second-order derivative is shown.

[0138] Fig.10 Shown are the results of SDS-PAGE electrophoresis analysis of exemplary pineapple lectin batches. The two batches of samples showed a major band of approximately 15 kDa and multiple sub-bands; these sub-bands can be identified in particular by the heavy gel overloading of lanes 3 and 7.

[0139] Fig.11 RP-HPLC analysis of a representative batch is shown; by LC-MS coupling and calculation by deconvolution, the main mass 1 was determined to be 15388Da (about 15.38kDa). In addition, a species of +162Da can also be measured, indicating a glycosylated protein, see Gross et al. (2020) J.Pharm.Biomed.Anal.181,113075.

[0140] Example 3

[0141] Binding test of bromelain to the spike protein of SARS CoV-2 virus

[0142] 1. Sample Preparation

[0143] 1.1 Cloning, expression, and purification of SARS-CoV-2 spike protein

[0144] The nucleotide sequence of the extracellular domain of the SARS-CoV-2 spike protein (1-1213) was a synthetic gene obtained from Eurofins MWG. The gene was amplified by PCR and provided with the restriction interface 5′-BamHI / XhoI-3′. The gene was subsequently ligated in the eukaryotic expression vector pCAGGS by sticky end cloning with T4 DNA ligase (BamHI ligated again). The expression of the spike protein was performed in HEK293 cells. For this purpose, HEK293 cells were cultured in ultrafilters (growth area 1720 cm2) and grown to 80 to 90% confluence in DMEM complete (37°C, 5% CO2). Subsequently, transfection was performed with a 1:2 ratio of pCAGGS spike protein:polyethyleneimine (linear, average molecular weight 25000Da). After 5 hours, the DNA-PEI solution was removed and replaced with DMEM complete. The culture was further incubated for 48 hours at 37°C and 5% CO2 before the culture supernatant was harvested.

[0145] The culture supernatant was applied to a 5 ml GHisTrap HP column to purify the SARS-CoV-2 spike protein. After thorough washing with lysis buffer (200 mM NaCl, 20 mM TRIS, pH 8.0, 20 mM imidazole), the protein was eluted with elution buffer (200 mM NaCl, 20 mM TRIS pH 8.0, 500 mM imidazole). The obtained protein was then further separated by HiLoad 16 / 600 Superdex 200PG, and the peak was collected with an elution volume of 60 ml to 70 ml.

[0146] 1.2 SPR binding and affinity testing

[0147] The binding kinetics and affinity tests of lectins were performed on a Biacore X100 system (GE Healthcare). The purified extracellular domain of the spike protein (1-1213) and the purified extracellular domain of the ACE-2 receptor (Sigmal Aldrich, #SAE0064) were covalently immobilized on a CM5 sensor chip by amino coupling in 10mM sodium acetate buffer (pH4.5), with a final spike protein of 9600 and an ACE-2 receptor of 4000. Surface plasmon resonance (SPR) was measured at a flow rate of 30 μl / min in 1x HBS-EP (150mM NaCl, 10mM HEPES pH 7.4, 3mM EDTA, 0.005% Tween 20). Injection of increasing concentrations of lectins (1.25 μM to 20 μM) was performed for single-cycle measurements (contact time 120s, dissociation time 180s).

[0148] 1.3 SARS-CoV-2 SDS-PAGE and LC-MS analysis

[0149] Glycosylation of recombinant spike was demonstrated enzymatically: 20 μg of SARS-CoV-2 spike was in 200 mM NaCl, 20 mM TRIS, pH 8.0. 2 μl (1000 IU) PNGAse F was used as buffer and the protein was purified according to the New England Journal of Medicine. Inc., and incubated at 37°C for 6 h. Lyophilized bromelain inhibitor (HZI 2-07) was buffered and suspended in a solution of 200 mM NaCl, 20 mM TRIS, pH 8.0. The suspension was centrifuged in an Eppendorf vessel to pellet the insoluble components, and the components of the solution were determined photometrically. To examine the proteolytic degradation of the recombinant SARS-CoV-2 spike and the resulting peptide product fragment sizes and the inhibitory function of HZI 2-07, the recombinantly produced SARS-CoV-2 spike was mixed with bromelain HZI 2-08, with or without the inhibitor HZI 2-07, in a stoichiometric ratio and incubated at 37°C. Sample extraction for SDS-PAGE or LC-MS analysis was performed as follows:

[0150] The sample was mixed with SDS dye (1.2 g SDS, 6 mg bromophenol blue, 4 ml glycerol, 0.6 ml 1 M TRIS, pH 8.0, 5.4 ml H2O heated to dissolve all components; then 930 mg dithiothreitol (DTT) was added to obtain 6x SDS dye buffer) and immediately boiled at 100°C for 2 to 3 minutes.

[0151] Sample extraction for LC-MS analysis: Samples were immediately frozen in liquid nitrogen, stored at −80 °C, and thawed on ice for a few minutes before LC-MS measurement.

[0152] 10 μl of the prepared sample was loaded on an SDS-PAGE containing a 12 vol% polyacrylamide fraction. 6 μl of “PageRuler Prestained Protein Ladder” (Thermo Fisher Scientific) was loaded to monitor the progress of the SDS-PAGE and to estimate the approximate amount of separated protein after gel staining. Electrophoresis was performed on a “Mini- Tetra system" (BIO RAD), using SDS Laemmli buffer at 140 V for 90 min. After electrophoresis, SDS-PAGE was heated in a microwave oven with a dye solution (0.05% mass / volume Coomassie blue, 50% volume methanol, 7% volume pure acetic acid, 43% volume water) and stored in water for 24 h before photographing.

[0153] Direct intact protein UPLC-ESI-MS analysis was performed on an UltiMate 3000UPLC system connected to a MaXis4G Q-ToF mass spectrometer with an Apollo II ESI source. The measurements were performed in the forward mode. The samples were separated by an Aeris Widepore XB-C8 column (3.6 μm, 150 x 2.1 mm; Phenomenex). The separation was performed at a flow rate of 0.3 ml / min (eluent A: deionized water plus 0.1% by volume acetic acid, eluent B: distilled acetonitrile plus 0.1% by volume acetic acid) at 45°C with a gradient of 2% B for 30 seconds, followed by a linear gradient to 75% B in 10 minutes and then maintained at 75% B for 3 minutes. The flow rate was adjusted to 75 μl / min before entering the ion source. Mass spectra were generated in centroid mode at 2 Hz, 150 m / z to 2500 m / z. The parameters of the mass spectrometer ion source were set as follows: endplate offset of 500 V, capillary voltage of 4000 V, nebulizer gas pressure of 1.1 bar, dry gas flow of 6 L / min, and drying temperature of 180°C. The masses of the proteins were summed by total ion chromatograph from retention time period 6.5 min to 9 min, bromelain fractions (HZI 2-08) with and without bromelain inhibitor (HZI 2-07) to demonstrate glycosylation from 7.0 min to 7.6 min, and the native masses were deconvoluted at high resolution with Compass Data Analysis using the maximum entropy deconvolution algorithm at an instrument resolution of 8000.

[0154] 2. Results

[0155] SARS-CoV-2 spike protein binding analysis

[0156] The binding analysis of the SARS-Co-2 spike protein was performed in vitro with the aid of SPR on lectins that were differently isolated from bromelain. To this end, the purified SARS-CoV-2 spike protein was immobilized on a CM5 chip by amine coupling. The binding to lectins of the japanese aralia class was subsequently tested; their dissociation constants and binding kinetics were determined. In this regard, different binding properties and association / dissociation rates were observed, which will be presented in more detail below.

[0157] The properties of lectins isolated from different batches of bromelain (HZI 2-05, HZI 2-06, HZI2-09, HZI 2-10, and Acm-JRL 2) in binding assays were studied by SPR technology. In this regard, it was possible to determine subtle differences between the batches, first of all the variations in the association and dissociation rates. It can be observed here that the bound lectins of HZI 2-09 and Acm-JRL 2 dissociate from the spike protein more slowly than the association rate can be measured, so that the measured values ​​on the chip increase at a constant rate. The measured values ​​of different batches of lectins are shown in Figure 12 as a function of time. Between each measurement, the surface containing the bound spike protein was regenerated with 80% ethylene glycol.

[0158] Sensorgrams of SPR analysis for binding kinetics and affinity testing showing increasing concentrations of SARS-CoV2 spike protein and different lectin batches isolated from bromelain (HZI 2-05, HZI 2-06, HZI 2-09, HZI 2-10, Acm-JRL 2). K was calculated using Biacore X100 software. D The binding constants and Rmax values ​​were obtained (Table 3). We were able to demonstrate that lectin batches HZI 2-09, HZI 2-10, and Acm-JRL 2 have a high affinity for the SARS-CoV-2 spike protein in the micromolar range. In addition, a rapid binding of the spike protein was observed (K a value 10311 / Ms) and slow dissociation (K d value 0.003 1 / s).

[0159] Table 3 Binding kinetics and affinity tests of SARS-CoV2 spike protein and different lectin batches isolated from bromelain using surface plasmon resonance spectroscopy (SPR). The values ​​calculated using Biacore X100 software are shown below.

[0160]

[0161] In further experiments, SPR analysis was performed to analyze the binding kinetics and affinity testing of lectins on the ACE-2 receptor ( Fig.13 ). For this purpose, the concentration of lectin used was 3.125 μM to 50 μM.

[0162] Fig.13 Sensorgrams of SPR analysis of binding kinetics and affinity testing of ACE-2 receptor and lectin batch HTI 2-9 at increasing concentrations are shown. From the sensorgrams it can be concluded that the dissociation constant is 100 μM or greater. In addition, different binding characteristics related to the association rate / dissociation rate are shown.

[0163] K D It cannot be calculated from the measured value because it is outside the measured concentration.

[0164] 2.2 SARS-CoV-2 Spike SDS-PAGE and LC-MS Analysis

[0165] Glycosylation of the spike protein was confirmed by LC-MS analysis. Fig.14 Deconvoluted mass spectra are shown. PNGase F removes N-bound oligosaccharides from glycoproteins. In this regard, the cleavage occurs between the innermost GlC-NAc and asparagine residues of the oligosaccharide. After digestion of the recombinant SARS-CoV-2 spike protein with PNGase F, mass shifts can be identified in the mass spectrum, which are triggered by the loss of oligosaccharides from the spike protein.

[0166] SDS-PAGE and liquid chromatography-mass spectrometry were used to analyze and prove the proteolytic activity of the bromelain-isolated fraction HZI 2-08 and the inhibitory function of the peptide HZI 2-07 obtained from bromelain. The fragmentation of the recombinant SARS-CoV2 spike and the inhibitory function of HZI 2-07 were demonstrated by SDS-PAGE ( Figures 15 to 17 The mass spectrum and the neutral mass deconvoluted therefrom also confirm this fragmentation ( Figures 18 to 21 ).

[0167] Fig.15 Lanes 2 to 7 in the Figure 2 show proteolytic degradation of the SARS-CoV-2 spike protein by a protease (HTI 2-08). It could not be demonstrated that the major molecular weight of the intact spike protein is approximately 150 kDa. However, the major degradation product can be identified at a molecular weight of approximately 24 kDa ( Fig.15 After adding HZI 2-07 (= inhibitor), the inhibitory function becomes apparent - the degradation of the spike protein is inhibited. This results in the appearance of a band of approximately 150 kDa in lanes 8 to 10 ( Fig.15 rectangle in the image).

[0168] The inhibitory effect of the inhibitor on the protease (HZI 2-08) was maintained over a longer experimental period; the assay was performed for up to 120 minutes. Fig.16 The intact spike protein is marked by a rectangle in the middle.

[0169] Fig.17 The kinetics of proteolytic degradation of the spike protein are shown. In the case of a spike to protein ratio of 10:1, after 10 minutes of incubation, a clear band of approximately 150 kDa can be found (lane 2), indicating a complete spike protein. After a long incubation of 120 minutes (lane 5), it can be seen that the band has basically disappeared and the degree of fragmentation has increased. This is not the case at a ratio of 100:1 (lanes 3 and 6). After 120 minutes of incubation, intact proteins can also be seen here, indicating that the protease HZI 2-08 is not effective enough. Therefore, the optimal ratio of spike to protease is 1:1, and the results are shown in Figure 2. Fig.15 and Fig.16 As shown. It can be further concluded that the ratio of 1:1:0.5 (spike: protease: inhibitor) is unfavorable because the degradation of spike cannot be inhibited. The ratio of 1:1:1 is the most suitable here ( Fig.16 ).

[0170] Fig.18 Total ion chromatogram (TIC) and mass spectrum of SARS-CoV-2 spike protein are shown. After addition of protease (HZI 2-08; spike to protease ratio 100:1), no mass peak can be seen at 150 kDa after 10 minutes (arrow). The fragments that appear indicate efficient degradation of the spike protein after addition of protease. This confirms the results already shown by means of SDS-PAGE ( Fig.15 ).

[0171] After incubation with protease (HZI 2-08; spike:protease ratio 100:1) for 120 min, there was no mass peak at 150 kDa (right arrow, Fig.19 ). This indicates that the proteolytic action of the protease remains unchanged over a period of at least 120 min. In addition, a mass peak is now visible at m / z 24392, which represents the main degradation product of the spike protein. This confirms the results already shown by means of SDS-PAGE ( Fig.15 ).

[0172] Fig.17 In the Figure 2, the proteolytic action of the protease (HZI 2-08) becomes clear again. Here the spike appears in a 1:1 ratio with the protease: After 120 min of incubation, the peak of the degradation product (left arrow) is very obvious.

[0173] After adding the inhibitor HZI 2-07, the intact spike protein became visible at m / z 145196 ( Fig.21 ). No 24 kDa degradation product could be detected. This confirms that Fig.15 In the results of the study, it was possible to prove that the inhibitor inhibits the protease (HZI 2-08). Therefore, the spike protein cannot be degraded.

[0174] Fig. 22 The SDS-PAGE analysis of the recombinant SARS-CoV-2 spike protein after digestion with bromelain powder for 1 hour at 37°C is shown (the material amount ratio is about 1:1 protein: protease, lanes 5 and 6). The SARS-CoV-2 spike protein that was not incubated with bromelain is also shown for comparison. (lanes 7 and 8). The results show that the bromelain powder is also able to digest the spike protein.

[0175] Therefore, it can be proved that bromelain can bind to the spike protein of the coronavirus, and the lectin contained in bromelain (accounting for 5% of the total amount of bromelain) can bind to both the spike protein and mannose, but not in a similar K D Value binds to the host's ACE-2 receptor.

Claims

1. Use of japonin lectin in the preparation of a medicament for treating or preventing viral infection in humans or animals caused by coronavirus.

2. The use according to the preceding claims, characterized in that The japanese lectin is selected from mannose-specific lectin and glucose-specific lectin.

3. The method according to claim 1, characterized in that The japanese lectin is selected from the group consisting of pineapple lectin (japanese lectin from pineapple (AcmJRL)), japanese lectin, cassia bark lectin, MPA lectin, Jerusalem artichoke lectin, red algae lectin, and mixtures and combinations thereof.

4. Use of a combined preparation containing at least one japanese lectin according to any of the preceding claims and at least one bromelain for the preparation of a medicament for the treatment or prevention of viral infections in humans or animals caused by coronaviruses.

5. Use according to the preceding claims, characterised in that The bromelain is selected from the group consisting of stem bromelain (SBM) (EC 3.4.22.32), fruit bromelain (EC 3.4.22.33), ananain (EC 3.4.22.31), and mixtures and combinations thereof.

6. The use according to any one of claims 4 to 5, characterized in that The weight ratio of the total amount of the at least one bromelain to the total amount of the at least one japanese lectin is 50:50 to 0.1:99.9, preferably 40:60 to 1:99, more preferably 30:70 to 2:98, more preferably 20:80 to 3:97, particularly preferably 10:90 to 5:

95.

7. The use according to any one of claims 4 to 6, characterized in that The total concentration of all bromelains in the combined preparation is 0.01% to 50.0% by weight, more preferably 0.1% to 20.0% by weight, particularly preferably 1.0% to 10.0% by weight.

8. The use according to any one of claims 4 to 7, characterized in that The total concentration of the at least one japanese lectin is 0.01% to 60.0% by weight, more preferably 1.5% to 50.0% by weight, particularly preferably 2.0% to 30% by weight.

9. Use according to any one of the preceding claims, characterised in that The coronavirus is selected from a betacoronavirus, an orthoconavirus, preferably an alphacoronavirus, a gammacoronavirus, or a deltacoronavirus, and a letovirus, preferably an alphaletovirus, in particular a milecovirus, such as microhyla letavirus1 (MLev-1).

10. Use according to the preceding claims, characterised in that The alpha coronavirus is selected from colacovirus, such as bat coronavirus CDPHE15; decacovirus, such as horseshoe bat alpha coronavirus HuB-2013; Duvinacovirus, such as human coronavirus 229E (HCov229E); luchacovirus, such as Lucheng brown rat coronavirus; minacovirus, such as ferret coronavirus or mink coronavirus 1; minunacovirus, such as long-winged bat coronavirus 1 or long-winged bat coronavirus HKU8; myotacovirus, such as big-footed myotacovirus alpha coronavirus Sax-2011 or middle Huashan bat alphacoronavirus SC-2013; pedacovirus, such as porcine epidemic diarrhea virus (PEDV) or yellow bat coronavirus 512; Rhinacovirus, such as horseshoe bat coronavirus HKU2 or porcine acute diarrhea syndrome coronavirus (SADS-CoV); Setracovirus, such as human coronavirus NL63 (HCov-NL63) or NL63-related bat coronavirus strain BtKYNL63-9b; Tegacovirus, such as alphacoronavirus 1, in particular canine coronavirus (CCoV), feline coronavirus (FCoV) or transmissible gastroenteritis virus (TGEV); The betacoronavirus is selected from sarbecovirus, such as severe acute respiratory syndrome-related coronavirus ("SARS-related coronavirus"), in particular severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and synonymous or non-synonymous mutants thereof, including variants B.1.1.7, B.1.351, B.1.617, P.1, B.1.525, P.2, B.1.427, B.1.429, L452R, Fin-796H, B.1.526, S:H66D, S:G142V, S:D215G, S:V483A, S:D614G, S:H655Y, S:G669S, S:Q949R, S:N1187D, ORF6:K23-, ORF6:V2 4-, ORF6:S25-, ORF6:I26-, ORF6:W27-, ORF6:N28-, ORF6:L29-, ORF6:D30-, ORF6:Y31-, S:Y144-, E484K, VOC 20I / 484Q, B.1, R.1, A.2.5, C.36, B.1.1.318, B.1.621, B.1.623, severe acute respiratory syndrome coronavirus (SARS-CoV, SARS coronavirus, also known as SARS-CoV-1), Embecovirus, such as beta coronavirus 1, in particular bovine coronavirus (BCoV), equine coronavirus (ECoV-NC99), human coronavirus OC43 (HCoV-OC43), porcine hemagglutinating encephalomyelitis virus (HEV), puffinosis coronavirus (PCoV), human enteric coronavirus (HECoV); Chinese rat coronavirus HKU24; human coronavirus HKU1 (HCoV-HKU1), mouse coronavirus, such as mouse hepatitis virus (MHV) or rat coronavirus (RtCoV ); hibecovirus, such as the horseshoe bat betacoronavirus Zhejiang 2013; merbecovirus (formerly MERS-related coronavirus, MERSr-CoV), such as hedgehog coronavirus 1, Middle East respiratory syndrome-related coronavirus (MERS-CoV), scutellaria avium coronavirus HKU5, flat-headed bat coronavirus HKU4; nobecovirus, such as fruit bat coronavirus GCCDC1 or fruit bat coronavirus HKU9; sarbecovirus, such as severe acute respiratory syndrome-related coronavirus (SARS-related coronavirus), in particular severe acute respiratory syndrome coronavirus (SARS-CoV, SARS coronavirus, also known as SARS-CoV-1), severe acute respiratory coronavirus 2 (SARS-CoV-2), or BatCoV RaTG13, Manis-CoV SRR10168377 and SRR10168378; The gammacoronavirus is selected from the group consisting of cegavirus, such as beluga coronavirus SW1; or igacovirus, such as avian coronavirus, in particular turkey coronavirus (TCov), pheasant coronavirus (PhCoV), infectious bronchitis virus (IBV); and The delta coronavirus is selected from andecovirus, such as ruddy duck coronavirus HKU20; buldecovirus, such as BuCoV HKU11, coronavirus HKU15, Munia coronavirus HKU13 (MunCoV HKU23), white-eye coronavirus HKU16 or thrush coronavirus HKU12 (ThCoV HKU2); herdecovirus, such as night heron coronavirus HKU19; and moordecovirus, such as moorhen coronavirus HKU21.

11. Use according to any one of the preceding claims, characterized in that The drug is used to treat symptoms caused by beta coronavirus infection, in particular fever, cough, pneumonia, lymphocytopenic community-acquired pneumonia (L-CAP), pleurisy, dyspnea, malaise and / or fatigue, sputum, loss of smell and / or loss of taste, shortness of breath, myositis and / or pharyngitis, joint pain, chest pain, pharyngitis, headache, back pain, malaria, nausea and / or vomiting, chills, diarrhea, hemoptysis, lymphocytopenia, rash on the hands, feet or mouth, bilateral non-purulent conjunctivitis, hypotonia, shock, hypertyrosinemia, myocardial dysfunction, pericarditis and / or heart valve inflammation, coagulopathy, multisystem inflammatory syndrome in children (MIS-c) and conjunctival swelling.

12. The use according to any one of claims 4 to 8, characterized in that The weight ratio of the total amount of the at least one bromelain to the total amount of the at least one japanese lectin is 1:99 to 99.9:0.1, preferably 50:50 to 99:1, more preferably 60:40 to 98:2, more preferably 70:30 to 97:3, more preferably 80:20 to 96:4, particularly preferably 90:10 to 95:

5.

13. The use according to any one of claims 4 to 8, characterized in that The total concentration of all bromelains in the combined preparation is 0.1% to 80.0% by weight, more preferably 1.0% to 50.0% by weight, particularly preferably 5.0% to 20.0% by weight.

14. The use according to any one of claims 4 to 8, characterized in that The total concentration of the at least one japanese lectin is 0.01% to 50.0% by weight, more preferably 0.1% to 10.0% by weight, particularly preferably 2.0% to 8.0% by weight.

15. Use according to any one of the preceding claims, characterized in that The japanese lectin or combined preparation is in the following forms or components of the following forms: powder, granules, tablets, especially film tablets, hard capsules, soft capsules, effervescent tablets, solutions, especially injection solutions or infusion solutions, emulsions, suspensions, ointments, creams, pastes, gels, tinctures, eye drops, powder mists, nasal sprays, suppositories or transdermal patches.

16. Use according to any one of the preceding claims, characterised in that The drug is administered orally, by intravenous, subcutaneous or intramuscular injection, by infusion into the nasal, oral or pharyngeal mucosa, by inhalation, by application to the eye surface, rectum, and / or by a transdermal patch.

17. Use according to any one of the preceding claims, characterised in that Administration is performed once a week to once an hour, preferably once a day to 10 times a day, particularly preferably 1 to 3 times a day, or continuously.

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