FIELD OF THE INVENTION

The present invention refers to a pharmaceutical preparation or feed supplement comprising anthrotainin, specifically from Metapochonia lutea, and the use or anthrotainin in the treatment of a disease condition in a subject which suffers from or is going to suffer from an infection caused by antibiotic resistant bacteria.

BACKGROUND OF THE INVENTION

The spread of antibiotic-resistant prokaryotes such as bacteria causes a great threat to public health and is getting worse as the development of new antibiotics is limited (Brown ED and Wright GD, 2016). Apart from the introduction of carbapenems in 1985, all new antibiotics between the early 1960s and 2000 were synthetic derivatives of existing scaffolds, which often allow resistant strains to arise rapidly (Fischbach MA and Walsh CT, 2009). In the U.S.A., the economic loss incurred by antibiotic resistance is estimated to be up to 55 billion USD per year with some data suggesting that the total cost may be even higher (Smith R. and Coast J., 2013).

As an example, Tetracycline-antibiotics target the protein synthesis by preventing the binding of aminoacyl-tRNA to the 30s ribosomal subunit, causing a disruption of protein synthesis and thereby prohibiting the growth of sensitive bacteria. Tetracyclines are broad-spectrum antibiotics that are widely used against gram-negative and grampositive bacteria. The use of tetracyclines for the treatment of animal and human infections has been increasing in recent years. However, this has led to the emergence of tetracycline-resistant bacteria limiting the use of tetracycline-antibiotics.

Also, archaea, such as methanogenic archaea, are highly resistant against antibiotics such as tetracycline, ampicillin, streptomycin, gentamicin, rifampicin, ofloxacin, and amphotericin B (Dridi B. et al., 2011).

Bacteria are exceptionally adaptable organisms and have repeatedly proven their ability to resist novel antibiotic agents. Many current antibiotics exhibit undesirable properties such as systemic toxicity, short half-life, and increased susceptibility to bacterial resistance. With the increasingly rapid appearance and global spread of antibiotic-resistant bacteria, prevention of infections with appropriately targeted drugs assumes greater urgency and importance. The rise of multidrug resistance in Gramnegative bacteria has become a particularly serious challenge. Gram-negative bacteria differ from gram-positive bacteria in the structure of the outer envelope, and as a result, in the penetration and retention of chemical agents. The gram-negative bacteria outer envelope consists of three principal layers: (1) the outer membrane, containing the lipopolysaccharide, (2) the peptidoglycan cell wall with partially crosslinked peptide chains, and (3) the cytoplasmic or inner membrane.

Gram-positive bacteria generally lack the outer membrane, which serves as a permeability barrier that excludes certain drugs and antibiotics from penetrating into the cell wall. This feature is one of the main factors contributing to the intrinsic antibiotic resistances observed in gram-negative bacteria.

In addition, the antibiotic resistance in gram-positive bacteria is a serious concern. Gram-positive bacteria (e.g., staphylococci, streptococci and enterococci) are among the most common bacterial causes of clinical infection and are associated with a diverse spectrum of pathologies, ranging from mild skin and soft tissue infections to lifethreatening systemic sepsis and meningitis.

As an example, Methicillin-resistant Staphylococcus aureus is a pathogen of concern due to its inherent resistance to almost all P-lactam antibiotics (penicillins, cephalosporins, carbapenems), apart from the novel cephalosporins, yet is almost entirely susceptible to vancomycin. Nevertheless, resistance of Methicillin-resistant S. aureus to such antibiotics is particularly problematic in the context of severe infections, where use of second-line agents confers a proven loss of survival-benefit. Similarly, glycopeptide-resistant enterococci are recognized as emerging pathogens, particularly in immunocompromised or hospitalized patients, and have been associated with outbreaks in healthcare facilities globally.

WO2016/014643A1 describes the preparation and evaluation of viridicatumtoxin analogs. Anthrotainin (TAN-1652, TAN-1612) is reported as being a tetracycline antibiotic.

JP H06 40995 A discloses tetracyclinic compounds as anti-inflammatory agents.

Bycroft B.W. et al., 2013 discloses a list of antibiotics.

Grossmann T.H., 2016, provides a general overview of tetracycline antibiotics. Roberts M.C., 2003, discloses the use of tetracyclines for treatment of infections.

The provision of novel compounds with anti-prokaryotic activity is therefore one of the most acute issues of modern chemical biology, biotechnology, and medicine. Despite a broad range of available antibiotics, there are significant problems of drug resistance and side effects which remain unsolved until now.

Therefore, despite the apparent availability of many new antibiotics, there is a pressing need for active antibiotic agents with novel spectra of activities and the potential to combat the continuing emergence of antibiotic resistance.

SUMMARY OF THE INVENTION

It is the objective of the present invention to provide a new antibiotic agent, specifically for targeting antibiotic resistant prokaryotes, archaea and bacteria, specifically gram-positive bacteria.

The objective is solved by the subject matter of the present invention.

The inventors of the present invention have surprisingly found that anthrotainin has antibacterial activity against a wide variety of prokaryotes, specifically bacteria. Thereby, although anthrotainin has been described as tetracyclic compound (Wong S. M. et al., 1993), the inventors of the present invention surprisingly found that anthrotainin has antibacterial activity especially against tetracycline-resistant prokaryotes.

Specifically, the anthrotainin had been successfully isolated by the inventors from filamentous fungus, Metapochonia lutea (Ascomycota, Hypocreales, Clavicipitaceae).

The present invention provides anthrotainin for use in the treatment of a disease condition in a subject which suffers from or is going to suffer from an infection caused by prokaryotes, specifically by bacteria.

Specifically, the infection is caused by by gram-positive or gram-negative bacteria.

According to a specific embodiment of the invention, the disease is caused by archaea, selected from Haloarchaea, methanogenic archaea, such as Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanobrevibacter oralis, Methanomassiliicoccus luminyensis, Methanomassiliicoccales, including Methano- methylophilaceae, Haloferax, such as Haloferax massiliensis and Halorubrum lipolyticum, Sulfolobales and Nitrososphaerales.

According to a further specific embodiment of the invention, the infection is caused by bacteria selected from the group consisting of Caryophanales such as Alicyclobacillaceae, Bacillaceae, Caryophanaceae, Listeriaceae, Paenibacillaceae, Pasteuriaceae, Sporolactobacillaceae, Staphylococcaceae, and Thermoactino- mycetaceae; Lactobacillales, such as Aerococcaceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leconostocaceae, and Streptococcaceae; Pseudomonadales, such as Acinetobacter, Methanomonadaceae, Moraxellaceae, Pseudomonadaceae, Thiorhodaceae, and Ventosimonadaceae; Neisseriales, such as Chromobacteriaceae and Neisseriaceae, Enterobacterales such as Budviciaceae, Enterobacteriaceae, Erwiniaceae, Hafniaceae, Morganellaceae, Pectobacteriaceae, Thorselliaceae, and Yersiniaceae; Actinobacteria; such as Corynebacteriaceae, Dietziaceae, Mycobacteriaceae, Nocardiaceae, Segniliparaceae, and Tsuka- murellaceae; Chlamydiae; Coccobacillus, Pasteurellales, such as Pasteurellaceae and Psittacicellaceae; Hyphomicrobiales such as Aestuariivirgaceae, Afifellaceae, Ahrensi- aceae, Alsobacteraceae, Amorphaceae, Ancalomicrobiacae, Aurantimonadaceae, Bartonellaceae, Beijerinckiaceae, Blastochloridaceae, Boseaceae, Breoghaniaceae, Brucellaceae, Chelatococcaceae, Cohaesibacteraceae, Devosiaceae, Hyphomicro- biaceae, Kaistiaceae, Lichenibacteriaceae, Lichenihabitantaceae, Methylo- bacteriaceae, Methylocystaceae, Nitrobacteraceae, Notoacmeibacteraceae, Parvi- baculaceae, Phreatobacteraceae, Phyllobacteriaceae, Pleomorphomonadaceae, Pseudoxanthobacteraceae, Rhabdaerophilaceae, Rhizobiaceae, Rhodobiaceae, Rosei- arcaceae, Salinarimonadaceae, Egnochrobactraceae, Stappiaceae, Tepida- morphaceae, Xanthobacteraceae; and Proteobacteria.

Specifically, the infection is caused by bacteria selected from Acinetobacter baumannii, Bacillus anthracis, Brucella abortus, Burkholderia cepacia, Burkholderia mallei, Burkholderia pseudomallei, Burkholderia thailandensis, Citrobacter freundii, Corynebacterium jeikeium, Enterobacter sp, Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Escherichia coll, Francisella tularensis, Haemophilus influenza, Klebsiella aerogenes, Klebsiella pneumoniae, Listeria monocytogenes, Moraxella catarrhalis, Morganella morganii, Neisseria meningitides, Neisseria gonorrhoea, Proteus mirabilis, Providencia stuartii, Pseudomonas aeruginosa, Serratia marcescens, Shigella sp, Staphylococcus aureus, Staphylococcus epidermis, Staphylococcus haemolyticus, Staphylococcus saprophyticus, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus bovis, Streptococcus constellatus, Streptococcus mitis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus oralis, Streptococcus sanguinis, Group C Streptococcus, Yersinia pestis, Salmonella sp., Salmonella enteritidis, Salmonella typhi, Pseudomonas sp., Moraxella sp., Helicobacter sp., Helicobacter pylori, Bdellovibrio sp., Legionella sp., and Legionella pneumophila.

According to an embodiment of the invention, the infection is caused by antibioticresistant prokaryotes, specifically by archaea or bacteria.

Specifically, the infection is caused by tetracycline-resistant bacteria.

According to the invention, the subject is a human or an animal.

Specifically, the anthrotainin is administered by topical, systemic, parenteral, subcutaneous, transdermal, rectal, oral, intravaginal, intranasal, intrabronchial, intraocular, intra-aural, intravenous, intramuscular, or intraperitoneal administration.

According to a specific embodiment, the anthrotainin is combined with one or more further active substances, preferably selected from the group consisting of antiviral, anti-cancer, anti-inflammatory, and antibiotic substances.

Also provided herein is a pharmaceutical composition comprising anthrotainin and a pharmaceutically acceptable excipient.

Specifically, the excipient is selected from the group consisting of a carrier, a diluent, and an adjuvant.

More specifically, the pharmaceutical composition described herein further comprises one or more further active substances, preferably selected from the group consisting of antiviral, anti-cancer, anti-inflammatory, and antibiotic substances.

The pharmaceutical composition described herein can be used as a medicament, specifically in the treatment of a disease condition in a subject which suffers from or is going to suffer from an infection caused by bacteria.

Further encompassed herein is the use of anthrotainin for the manufacture of a medicament.

Specifically, Metapochonia lutea is used for the production of anthrotainin.

Further provided herein is a method of producing anthrotainin, said method comprising the sequential steps of: a) inoculating a production medium with Metapochonia lutea, preferably with spores of Metapochonia lutea', b) cultivating Metapochonia lutea in said production medium, preferably under conditions with reduced daylight; and c) isolating anthrotainin from the cultivated production medium. According to a further embodiment, herein provided is a feed or food additive comprising anthrotainin, specifically for use in animal feed.

Specifically, the anthrotainin of the food or feed additive is isolated from Metapochonia lutea.

FIGURES

Figure 1 : Bioactivity of M. lutea. A) Isolation plate from Danube river samples, arrows indicate M. lutea. B) Inhibition zones of the indicated bacterial lawns emanating from M. lutea agar plugs. Upper picture: Klebsiella pneumoniae, lower picture: Staphylococcus aureus.

Figure 2: Fractionation and purification of anthrotainin. A) Flash chromatogram showing a dominant signal (Rt=25-32 min) (arrow). B) Analytical HPLC chromatogram of the isolated fraction with detection at 420nm.

Figure 3: MS analysis of anthrotainin. A) Expected molecular mass of anthrotainin B) Full mass spectrum of anthrotainin, C) MS/MS spectra.

Figure 4: NMR Structure analysis of anthrotainin. A) 1 H-NMR, B) 13C NMR C

Figure 5: Structure analysis of anthrotainin. Structure model obtained from the diffraction pattern of anthrotainin isolated from M. lutea.

DETAILED DESCRIPTION

Unless indicated or defined otherwise, all terms used herein have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Sambrook et al, "Molecular Cloning: A Laboratory Manual" (4th Ed.), Vols. 1 -3, Cold Spring Harbor Laboratory Press (2012); Krebs et al., "Lewin's Genes XI", Jones & Bartlett Learning, (2017), Brock Mikrobiologie: Michael T. Madigan, Kelly S. Bender, et al. Pearson Studium - Biologie 2020; Tetracyclines in Biology, Chemistry and Medicine, Mark Nelson, Wolfgang Hillen, Robert A. Greenwald Birkhauser (2001).

The subject matter of the claims specifically refers to artificial products or methods employing or producing such artificial products, which may be variants of native (wild type) products, or products isolated by technical means. Though there can be a certain degree of sequence identity to the native structure, it is well understood that the materials, methods and uses of the invention, e.g., specifically referring to isolated nucleic acid sequences, amino acid sequences, fusion constructs, expression constructs, transformed host cells and modified proteins, are “man-made” or synthetic, and are therefore not considered as a result of “laws of nature”.

The terms “comprise”, “contain”, “have” and “include” as used herein can be used synonymously and shall be understood as an open definition, allowing further members or parts or elements. “Consisting” is considered as a closest definition without further elements of the consisting definition feature. Thus “comprising” is broader and contains the “consisting” definition.

The term “about” as used herein refers to the same value or a value differing by +/-5 % of the given value.

As used herein and in the claims, the singular form, for example “a”, “an” and “the” includes the plural, unless the context clearly dictates otherwise.

The term „antibiotic“ refers to drugs which may be used to treat a prokaryotic bacterial infection through either inhibiting the growth of archaea, bacteria or killing bacteria. Thereby, antibiotics can fall into a wide range of classes.

The term “antibiotic-resistance” or “antibiotic-resistant” refers to a loss or reduction in the effectiveness of an antibiotic (e.g., an antimicrobial agent, an antimicrobial peptide, a hydrocarbon-stapled and/or stitched peptide) to kill or inhibit the growth of a prokaryotic, specifically a bacterial strain that was once responsive to the antibiotic. In certain embodiments, an antibiotic has a cytostatic effect on a prokaryote instead of having a cytotoxic effect. In certain embodiments, a bacterium can develop antibiotic-resistance by genetically altering (e.g., mutating) the antibiotic target-binding site. In certain embodiments, a bacterium can develop antibiotic-resistance by preventing (e.g., inhibiting or reducing) penetrance of the antibiotic into the bacterium (e.g., genetically mutating the docking site of the antibiotic, genetically mutating the antibiotic transport pump (e.g., increasing the efflux of the antibiotic)). In certain embodiments, a bacterium can develop antibiotic-resistance by acquiring an antibioticresistance gene (e.g., by plasmid) by horizontal gene transfer (e.g., conjugation, transformation, or transduction) from another bacterial strain. In certain embodiments, a bacterium can develop antibiotic-resistance by expressing enzymes that inactivate the antibiotic. In a specific embodiment, the antibiotic is selected from the group consisting of: carbapenem, an aminoglycoside, vancomycin, methicillin, clarithromycin, cephalosporin, penicillin, ampicillin, fluoroquinolone, tetracycline, and a P-lactam, specifically the prokaryotic is resistant to tetracycline. In a specific embodiment, a gram-positive bacterium can have acquired resistance to an antibiotic (i.e. antibiotic-resistance). In some embodiments a grampositive bacterial infection can have acquired resistance to an antibiotic (i.e. an antibiotic-resistant gram-positive bacterial infection (e.g., a tetracycline-resistant grampositive bacterial infection. In some embodiments, a subject can have acquired resistance to an antibiotic (e.g., tetracycline-resistance).

The term “anthrotainin” as used herein refers to the chemical compound of the following structure and structural isomers (tautomers), derivatives, and salts thereof.

Anthrotainin is of following chemical structure:

Synonyms for anthrotainin are: 2-Naphthacenecarboxamide, 3,4,4a, 5, 12, 12a- hexahydro-1 ,4a,10,11 ,12a-pentahydroxy-8-methoxy-3,12-dioxo-, (cis)-. The following PubChem ID refers to anthrotainin: 54692192. The IUPAC name for anthrotainin is: (AaR, 12aR)-1 ,4a, 10,11 ,12a-pentahydroxy-8-methoxy-3, 12-dioxo-4,5-dihydrotetracene- 2-carboxamide.

According to a specific embodiment, anthrotainin is isolated from Metapochonia lutea.

According to a specific embodiment, a derivative of anthrotainin may be analogous to tetracycline derivatives such as chlortetracycline, oxytetracycline, demeclocycline, doxycycline, minocycline, lymecycline, meclocycline, methacycline, rolitetracycline, tigecycline, omadacycline, sarecycline, eravacycline, or other derivatives on the C7, and/or C9 positions of anthrotainin, and optionally other positions.

According to a specific embodiment, a derivative of anthrotainin may be 5- Hydroxyanthrotainin (IUPAC name: 4aS,5S,12aS)-1 ,4a,5,10,11 ,12a-hexahydroxy-8- methoxy-3,12-dioxo-4,5-dihydrotetracene-2-carboxamide), 8-O-desmethyl-anthrotainin (IUPAC name: 4aR, 12aS)-2-carbamoyl-4a,8, 10,11 ,12a-pentahydroxy-3, 12-dioxo-4,5- dihydrotetracen-1-olate), or 5-Hydroxy-Desmethylanthrotainin (IUPAC name: (4aS,5S, 12aS)-2-carbamoyl-4a,5,8, 10,11 ,12a-hexahydroxy-3, 12-dioxo-4,5- dihydrotetracen-1 -olate). According to a specific embodiment, a salt of anthrotainin may be a pharmaceutically acceptable salt.

A “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and nonhuman primates such as monkeys), and rodents (e.g., rabbits, mice and rats). In certain embodiments, the individual or subject is a human.

As used herein, “treatment”, “treat” or “treating” refers to clinical intervention in an attempt to alter the natural course of the subject being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the anthrotainin of the invention is used to delay development of a disease or to slow the progression of a disease.

The term “at risk of’ a certain disease condition, refers to a subject that potentially develops such a disease condition, e.g., by a certain predisposition, exposure to prokaryotes or prokaryotic-infected subjects, or that already suffers from such a disease condition at various stages, particularly associated with other causative disease conditions or else conditions or complications following as a consequence of prokaryotic, e.g. bacterial infection. The risk determination is particularly important in a subject, where a disease has not yet been diagnosed. This risk determination therefore includes early diagnosis to enable prophylactic therapy.

Specifically, the term “prophylaxis” refers to preventive measures which is intended to encompass prevention of the onset of pathogenesis or prophylactic measures to reduce the risk of pathogenesis.

“Prokaryotes” are organisms whose cells lack a nucleus and other organelles. Prokaryotes are divided into two groups, bacteria and archaea.

“Archaea” (archaebacteria) lack cell nuclei and are therefore prokaryotes. Current classification systems organize archaea into groups of organisms that share structural features and common ancestors. These classifications rely heavily on the use of the sequence of ribosomal RNA genes to reveal relationships among organisms. Archae include, but are not limited to Haloarchaea, methanogenic archaea, such as Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanobrevibacter oralis, Methanomassiliicoccus luminyensis, Methanomassiliicoccales, including Methano- methylophilaceae, Haloferax, such as Haloferax massiliensis and Halorubrum lipolyticum, Sulfolobales and Nitrososphaerales.

“Gram-positive bacteria” are bacteria that give a positive result in the Gram stain test. Gram-positive bacteria take up the crystal violet stain used in the test, and then appear to be purple-colored when seen through an optical microscope. This is because the thick peptidoglycan layer in the bacterial cell wall retains the stain after it is washed away from the rest of the sample, in the decolorization stage of the test. Gram-positive bacteria possess a thick cell wall containing many layers of peptidoglycan and teichoic acids. Gram-positive bacteria include cocci and bacilli, e.g. Corynebacterium, Clostridium, Listeria, Bacillus, Staphylococcus, and Streptococcus.

„Gram-negative bacteria" cannot retain the violet stain after the decolorization step; alcohol used in this stage degrades the outer membrane of gram-negative cells, making the cell wall more porous and incapable of retaining the crystal violet stain. Their peptidoglycan layer is much thinner and sandwiched between an inner cell membrane and a bacterial outer membrane, causing them to take up the counterstain (safranin or fuchsine) and appear red or pink. Gram-negative bacteria have a relatively thin cell wall consisting of a few layers of peptidoglycan surrounded by a second lipid membrane containing lipopolysaccharides and lipoproteins. Non-limiting example species are E. coll, Salmonella, Shigella, and other Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, acetic acid bacteria, Legionella. Other notable groups of gram-negative bacteria include the cyanobacteria, spirochaetes, green sulfur, and green non-sulfur bacteria. Medically relevant gram-negative cocci including Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Salmonella enteritidis, Salmonella typhi, Acinetobacter baumannii.

Anthrotainin is administered to a subject in an effective amount.

The term “effective amount” with respect to an antibiotic effect as used herein, shall refer to an amount (in particular a predetermined amount) that has a proven antibiotic effect. The amount is typically a quantity or activity sufficient to, when administered to a subject effect beneficial of desired results, including antibacterial or clinical results, and, as such, an effective amount or synonym thereof depends upon the context in which it is being applied.

An effective amount of a pharmaceutical preparation or drug is intended to mean that amount of a compound that is sufficient to treat, prevent or inhibit a disease, disease condition or disorder. Such an effective dose specifically refers to that amount of the compound sufficient to result in healing, prevention or amelioration of conditions related to diseases or disorders described herein.

In the context of disease, effective amounts (in particular prophylactically or therapeutically effective amounts) of anthrotainin as described herein are specifically used to treat, modulate, attenuate, reverse, or affect a disease or condition that benefits from its anti-prokaryotic, specifically anti-bacterial effect. The amount of anthrotainin that will correspond to such an effective amount will vary depending on various factors, such as the formulation, the route of administration, the type of disease or disorder, the identity of the subject being treated, the assessment of the medical situations and other relevant factors, but can nevertheless be routinely determined by one skilled in the art.

A treatment or prevention regime of a subject with an effective amount of anthrotainin described herein may consist of a single application or administration, or alternatively comprise a series of applications and administrations, respectively. For example, anthrotainin may be used at least once a day. However, in certain cases of an acute phase, e.g. upon suspected or confirmed exposure to prokaryotes, such as bacteria or archaea, or after bacterial infection has been determined, anthrotainin may be used more frequently, e.g. 2, or 3 times a day.

The length of the treatment period depends on a variety of factors, such as the severity of the disease, the age of the subject, and the concentration of anthrotainin. It will also be appreciated that the effective dosage used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art.

Anthrotainin is administered by topical, systemic, parenteral, subcutaneous, transdermal, rectal, oral, intravaginal, intranasal, intrabronchial, intraocular, intra-aural, intravenous, intramuscular, or intraperitoneal administration. Anthrotainin can be prepared in a pharmaceutical composition or medicinal product.

The pharmaceutical preparation or medicinal product described herein is specifically provided as human or veterinary pharmaceutical composition or medicinal product. Medicinal products are understood as substances that are used to treat diseases, to relieve complaints, or to prevent such diseases or complaints in the first place. This definition applies regardless of whether the medicinal product is administered to humans or to animals.

The pharmaceutical composition comprising the anthrotainin of the invention comprises a pharmaceutically acceptable excipient.

A “pharmaceutically acceptable excipient” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. Some examples of pharmaceutically acceptable excipients are carriers, diluents, adjuvants such as water, saline, phosphate buffered saline, amino acids such as glycine or histidine, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the compound.

The pharmaceutical compositions of this invention may be in a variety of forms, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application.

Anthrotainin can be combined with one or more further active substances, preferably selected from the group consisting of antiviral, anti-cancer, anti-inflammatory, and antibiotic substances.

According to one embodiment of the invention, anthrotainin or a pharmaceutical composition comprising anthrotainin may be used in combination therapies. Such a combination therapy may comprise the administration of an additional antimicrobial agent such as an antibiotic, an anti-viral, anti-cancer, anti-inflammatory, or a compound which mitigates one or more of the side effects experienced by the subject. According to a specific embodiment, the compounds of the present invention may be used in conjunction with another antibiotic. In some embodiments, the compounds may be used in conjunction with a narrow spectrum antibiotic which targets a specific bacteria type. Non-limiting examples of bactericidal antibiotics are penicillin, cephalosporin, polymyxin, rifamycin, lipiarmycin, quinolones, and sulfonamides. Nonlimiting examples of bacteriostatic antibiotics include macrolides, lincosamides, or tetracyclines. In some embodiments, the antibiotic of the invention is combined with an antibiotic selected from an aminoglycoside such as kanamycin and streptomycin, an ansamycin such as rifaximin and geldanamycin, a carbacephem such as loracarbef, a carbapenem such as ertapenem, imipenem, a cephalosporin such as cephalexin, cefixime, cefepime, and ceftobiprole, a glycopeptide such as vancomycin or teicoplanin, a lincosamide such as lincomycin and clindamycin, a lipopeptide such as daptomycin, a macrolide such as clarithromycin, spiramycin, azithromycin, and telithromycin, a monobactam such as aztreonam, a nitrofuran such as furazolidone and nitrofurantoin, an oxazolidonones such as linezolid, a penicillin such as amoxicillin, azlocillin, flucioxacillin, and penicillin G, an antibiotic polypeptide such as bacitracin, polymyxin B, and colistin, a quinolone such as ciprofloxacin, levofloxacin, and gatifloxacin, a sulfonamide such as silver sulfadiazine, sulfadimethoxine, or sulfasalazine, or a tetracycline such as demeclocycline, doxycycline, minocycline, oxytetracycline, or tetracycline. Other antibiotics that may be used for combination therapies may include arsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin, dalfopristin, thiamphenicol, tigecycline, tinidazole, or trimethoprim. Specifically, B-lactam antibiotics can be used, such as, but not limited to penicillins, cephalosporins, carbapenems, and monobactams.

As an alternative, anthrotainin described herein can be prepared as feed or food additive or supplement or feed material.

Specifically, anthrotainin isolated from Metapochonia lutea is used herein.

Isolation of anthrotainin is performed by inoculating a production medium with Metapochonia lutea, preferably with spores of Metapochonia lutea; cultivating Metapochonia lutea in said production medium, preferably under conditions with reduced daylight; isolating anthrotainin from the cultivated production medium, and optionally purifying anthrotainin via chromatography. Specifically, cultivation is performed under reduced daylight, or even at complete darkness for several days, specifically 5 to 7 days, more specifically for 6 days. Specifically, the cultivation medium is a yeast extract sucrose agar medium, specifically comprising about 1 % yeast extract and about 15% saccharose.

EXAMPLES

The examples described herein are illustrative of the present invention and are not intended to be limitations thereon. Many modifications and variations may be made to the techniques described and illustrated herein without departing from scope of the invention. Accordingly, it should be understood that the examples are illustrative only and are not limiting upon the scope of the invention.

Example 1:

Anthrotainin was purified from the filamentous fungus, Metapochonia lutea (Labuda R. et al., 2018). The genus Metapochonia (and related Pochonia) comprises species living mainly in soil (Zare R. et al., 2001 , Gams W. et al., 2001 , Domsch K.H. et al., 2003). These fungi are producing secondary bioactive metabolites (Nonaka K. et al., 2013, Stadler M. et al., 2003, Degenkolb T. et al., 2016).

Figure 1 shows the bioactivity of M. lutea. A) Isolation plate from Danube river samples, Arrows indicate M. lutea. B) Inhibition zones of the indicated bacterial lawns emanating from M. lutea agar plugs.

Methods

Fermentation and Extraction

The fungal spore suspension (5.0 x 10 ⁶ spores/mL) is obtained after 7 days cultivation of the fungus (M. lutea BiMM-F96/DF4) on a potato dextrose agar (PDA, VWR Chemicals, Austria). Yeast extract sucrose agar medium (YES, Samson et al., 2000) with 15% sucrose was used for production of secondary metabolites. The cultivation continued for 7 days at 25°C in the dark. At the end of the cultivation the material was extracted with 2 I Ethylacetate. The organic phase was concentrated under reduced pressure at 45°C (Buchi Rotavapor R-114, Germany). The whole extraction procedure was repeated twice and yielded 2 g of crude culture extract.

Isolation of anthrotainin

The crude extract was purified by reversed-phase silica gel vacuum flash chromatography (Interchim Inc., puriFlash®450), using three consecutive Interchim puriFlash® 32 g silica IR-50C18-F0025 flash columns (particle size: 50pm). The columns were eluted with a binary solvent gradient (solvent A: H2O, solvent B: ACN). The starting linear gradient from 10% B to 27% B during 25 min at a flow rate 15 mL/min was followed by an isocratic elution at 52% B for 10 min. Then a linear gradient from 52% to 66% B over 7min was applied at the same flow rate. The target compound was found in fraction F4 (25 - 32Rt, yield: 160 mg). The outcome of the flash chromatography is depicted in Figure 2 A.

Figure 2 A) shows the flash chromatogram showing a dominant signal (Rt=25- 32 min) (arrow).

For further purification, the preparative HPLC was employed, as follows: The fraction F4 (containing the target substance) was resolved in a solvent mix (1 :1 :1 ; ACN/CH3OH/H2O) and further purified by an Agilent 1260 Infinity preparative HPLC (USA) on a reversed phase column Gemini NX C-18 (21.20 x 150 mm, 5 pm, 110 A). Gradient starting with 30 % ACN and 70 % H2O up to 90 % ACN in 10min (total time 34 min) and a flow rate of 25 ml/min. Four fractions (pF1 -pF4, time slice each 1 min) were collected, of which pF4 contained the target. Yield of target - anthrotainin vial 11 (tR 11 - 12 min) after one stage of prep HPLC was 60 mg. For purity check an Agilent 1200 system was used with the same stationary phase and gradient program.

Figure 2 B) shows the analytical HPLC Chromatogram of the Flash fraction of concentrated, performed with 15% AcN 45min gradient method + additional 420nm wavelength detection for yellow chromophores

LC-MS and NMR

A diluted solution of the purified metabolite was measured with liquid chromatography-high resolution mass spectrometry. Chromatographic separation was carried out with a reverse phase C18 column (Gemini ®, NX-C18, 5 pm, 110A, 150x2mm, Phenomenex, Torrance, CA, USA) in an UHPLC-system (Vanquish - Thermo Fisher Scientific, Bremen, Germany). 5 pL of sample solution were injected and gradient elution was carried out using water and acetonitrile (ACN) each containing 0.1 % formic acid (FA) as eluent A and B, respectively. The flow rate was 0.3 mL/min at 25°C. After two minutes of linear elution with 15% B, a 30 minutes gradient to 95% B followed by three minutes constant 95% B and re-equilibration of the system with 15% B for ten minutes was applied resulting in a chromatographic method of 45 minutes. The UHPLC- system was coupled to a QExactive HF Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) via a heated ESI interface operating in fast-polarity switching mode (positive/negative ionization). Full MS / TopN MS/MS scan events using an inclusion list were carried out for the positive and negative ionization mode. Full scan mass spectra were recorded in profile mode with a scan range m/z 100-1000 and a resolution of 120,000 FWHM (at m/z 200). If ions listed in the inclusion list were present in the full scan mass spectra, MS/MS was triggered with an isolation window of m/z ±1 and stepped collision energy (25, 35, 45 eV) in the HCD collision cell. MS/MS fragment spectra were recorded with a resolution setting of 15,000 FWHM (at m/z 200). Manual data evaluation was carried out with XCalibur.

All NMR spectra were recorded on a Bruker Avance II 400 (resonance frequencies 400.13 MHz for ¹ H and 100.63 MHz for ¹³ C) equipped with a 5 mm N2- cooled cryo probe head (Prodigy) with z-gradients at room temperature with standard Bruker pulse programmes. The sample was dissolved in 0.6 ml of MeOD (99.8 % D) and a few drops of DMSO-de (99.8 % D). Chemical shifts are given in ppm, referenced to residual solvent signals (3.31 ppm for ¹ H, 49.0 ppm for ¹³ C). ¹ H NMR data were collected with 32k complex data points and apodized with a Gaussian window function (lb = -0.3 Hz and gb = 0.3 Hz) prior to Fourier transformation. ¹³ C spectrum with WALTZ16 ¹ H decoupling was acquired using 64k data points. Signal-to-noise enhancement was achieved by multiplication of the FID with an exponential window function (lb = 1 Hz). All two-dimensional experiments were performed with 1 k x 256 data points, while the number of transients (2-16 scans) and the sweep widths were optimized individually. HSQC experiment was acquired using adiabatic pulse for inversion of ¹³ C and GARP- sequence for broadband ¹³ C-decoupling, optimized for ¹ (CH) = 145 Hz. For the NOESY spectrum a mixing time of 0.8 s was used.

Results and Discussion

Metapochonia lutea was isolated from the Danube River and showed visible inhibiting activity against bacteria by revealing inhibition zones (Figure 1). Upscaling of the fungus in liquid culture and extraction of the supernatant lead to the purification of an antibacterial active substance (see material section). The purified substance, designated as BiMM-00DF4, was isolated as yellow solid amorphous powder. Its purity was confirmed by analytical HPLC. Further analysis of the material by Mass spectrometry, NMR, and crystallography confirmed identity with anthrotainin (PubChem CID 54692192). In the analytical HPLC chromatogram BiMM-00DF4 and anthrotainin eluted with identical retention time (Rt=17.0 min) (Figure 2). Mass spectrometric analysis revealed a compound with an exact mass of 415.0903 (Figure 3) with Sum Formula: C20H17N109. H-NMR analysis and ¹³ C-NMR data (Figure 4) support the structure of the substance being anthrotainin. Structure analysis confirmed the identity and structure of the molecule as anthrotainin. (Figure 5).

Bioactivity of Anthrotainin:

A previous study reported the inhibition by anthrotainin of substance P binding to rat fore brain preparations (Wong S.M. et al., 1993). A MIC50 of 2.5 to 20 pg/ml was found for S. aureus isolates but higher MIC50s for P. aeruginosa and E. coli. The antibiotic activity against gram positive bacteria (MIC50 around 10pg/ml) is significantly stronger (at least 25-fold) than the cytotoxic effect of anthrotainin against HEC-293, CACO-2, and KB3-1.

Tetracycline resistance is not correlated with Anthrotainin resistance

Tetracycline resistant Staphylococcus aureus strains were tested on their sensitivity to anthrotainin (Table 1). The S. aureus strains had tetracycline MIC50 values (mean of four repetitions) reaching from 6 to above 50 pg/ml and anthrotainin MIC50 values from below 3,4 to 11 pg/ml. The wild type S. aureus has a MIC50 of 0.3 pg/ml against tetracycline in the test setup. The MIC50 values showed no correlation thereby demonstrating the anthrotainin sensitivity of tetracycline resistant S. aureus. It can be concluded that anthrotainin shows effectiveness as an antibiotic substance to tetracycline-resistant bacteria such as S. aureus. There was no linkage between tetracycline and anthrotainin resistance.

Table 1 S. aureus tet ^R isolates: MIC50 values of anthrotainin and tetracycline

Example 2: Anthrotainin effect on Escherichia coli

Anthrotainin was tested against 12 selected E. coli, three E. mamortae and one E. fergusonii strains obtained from natural sources. The mean MIC50 values were in the range of 30 pg/ml.

Example 3: Anthrotainin resistance formation

No resistant colonies were observed by plating 1*10 ¹⁰ cfu (colony forming units) of a S. aureus wild type strain on Muller Hinton Agar containing 100 pg/ml anthrotainin after 48hours. Anthrotainin was tested against a broad spectrum of problematic gram-negative pathogens of which some were carrying multiple antibiotic resistances (Table 2). The pathogens tested were clinical isolates obtained from the University Hospital St. Pblten. Anthrotainin showed good effects in combating a broad range of Klebsiella pneumoniae with MIC50s ranging from 3 to 15 pg/ml. Also highly problematic pathogens like Acinetobacter baumanii (MIC50 16 pg/ml) and Salmonella enteritidis (MIC50 4,7 pg/ml) have shown to respond to anthrotainin treatments.

Table 2: Anthrotainin effect against gram negative pathogens - MIC50 values Table 3: Resistance profiles of gram-negative pathogens A) B97 Salmonella enteritidis, B) B107 Pantoea ananatis, C) B36 Proteus mirabilis

A) Strain: B97 Salmonella enteritidis Microbiological-culture analysis: Fecal sample Gastroenteritis Bact. PCT; Aerobic conditions (+ = growth)

B) Strain: B107 Pantoea ananatis

Microbiological-culture analysis: smear Culture+resistance; Aerobic conditions

C) Strain: B36 Proteus mirabilis

Microbiological-cultural analysis: smear left MRGN/ESBL-screening; Aerobic conditions

Table 4: Resistance profiles of gram-negative pathogens A) B89 Acinetobacter baumanii, B) B11 Enterobacter cloacae, C) B54 Klebsiella pneumoniae

A) Strain B89 Acinetobacter baumanii Microbiological-culture analysis: smear MRGN/ESBL-Screening; Aerobic conditions

Determination of the minimum inhibitory concentration (MHK) for Colistin

Acinetobacter baumanii (MRE) +++ aerobic

Antibiotic Abs. rating (pg/ml) Rating M

Colistin 0.5 sensitive B) Strain: B11 Enterobacter cloacae

Microbiological-cultural analysis: smear Colonisation screening; Aerobic conditions

C) Strain: B54 Klebsiella pneumoniae Microbiological-culture analysis: permanent catheter urine UA: culture+resistance;

Aerobic conditions

Anthrotainin was tested on a set of E. coli, carrying different resistance plasmids (Table 5). As none of the plasmids resulted in a significant difference in vulnerability to anthrotainin compared to the empty-vector control one can conclude that these resistance mechanisms do not work for anthrotainin. Table 5: Anthrotainin resistance is not affected by different common resistance plasmids

Example 4: Cytotoxicity

In an EZ4U-Assay (Biomedica) HEK-293, KB-3-1 and Caco-2 cells anthrotainin is not cytotoxic in a concentration range of 5-80 pM (corresponding to 2,075 - 33,2 pg/ml) (Table 2). At higher concentrations in the range from 160 - 640 pM (corresponding to 66,4 - 256,6 pg/ml) there is a weak, concentration-dependent cytotoxic effect. However, IC50 was not reached, and thus IC50 is higher than 640 pM. Thus, the antibiotic activity against Gram-pos. bacteria (MIC50 around 10pg/ml) is significantly at least 25-fold higher than the cytotoxic effect of anthrotainin. Table 6: Cytotoxicity of anthrotainin

PUBLICATIONS

Labuda, R.; Bernreiter, A.; Schuller, C.; Kubatova, A.; Hellinger, R.; Strauss, J. Metapochonia lutea, a new species isolated from the Danube river in Austria. Nova Hedwig. 2018, 107, 487-500.

Zare, R.; Gams, W.; Evans, W.H.C. A revision of Verticillium section Prostrata. V. The genus Pochonia, with notes on Rotiferophthora. Nova Hedwig. 2001 , 73, 51-86.

Gams, W.; Zare, R. A taxonomic review of the clavicipitaceous anamorphs parasitizing nematodes and other microinvertebrates. In Clavicipitalean Fungi: Evolutionary Biology, Chemistry, Biocontrol and Cultural Impacts; White, J.F., Bacon, C.W., Hywel-Jones, N.L., Spatafora, J.W., Eds.; Marcel Dekker Inc.: New York, NY, USA, 2003.

Domsch, K.H.; Gams, W.; Anderson, T.H. Compendium of Soil Fungi, 2nd ed.; IHW-Verlag: Eching, Germany, 2007.

Nonaka, K.; Omura, S.; Masuma, R.; Kaifuchi, S.; Masuma, R. Three new Pochonia taxa (Clavicipitaceae) from soils in Japan. Mycologia 2013, 105, 1202-1218

Stadler, M.; Tichy, H.-V.; Katsiou, E.; Hellwig, V. Chemotaxonomy of Pochonia and other conidial fungi with Verticillium-like anamorphs. Mycol. Prog. 2003, 2, 95-122.

Degenkolb, T.; Vilcinskas, A. Metabolites from nematophagous fungi and nematicidal natural products from fungi as an alternative for biological control. Part I: Metabolites from nematophagous ascomycetes. Appl. Microbiol. Biotechnol. 2016, 100, 3799-3812.

S M Wong, R Kullnig, J Dedinas, K C Appell, G C Kydd, A M Gillum, R Cooper, R Moore Anthrotainin, an inhibitor of substance P binding produced by Gliocladium catenulatum PMID 7682212; DOI 10.7164/antibiotics.46.214; The Journal of antibiotics 1993 Feb; 46(2) :214-21. Dridi B. et al., The antimicrobial resistance pattern of cultured human methanogens reflects the unique phylogenetic position of archaea, J Antimicrob Chemother. 2011 Sep;66(9):2038-44. doi: 10.1093/jac/dkr251. Epub 2011 Jun 16.

Smith R. and Coast J., The true cost of antimicrobial resistance, BMJ. 2013, 346,:f1493, doi:10.1136/bmj.f1493.

Brown E.D. and Wright G.D., Antibacterial drug discovery in the resistance era, 2016, Nature, Vol. 529, p336-343.

Fischbach M.A. and Walsh C.T., Antibiotics for emerging pathogens, 2009, Science, Vol. 325, p1089-1093. Bycroft B.W. et al., Dictionary of antibiotics and related substrates, CRC Press,

2013

Grossmann T.H., Tetracycline antibiotics and resistance, Cold Spring Harbor Perspectives in Medicine, 2016, vol. 6(4), pp. 1-24

Roberts M.C., Tetracycline therapy: update, Clinical Infectious Diseases, 2003, vol. 36(4), pp. 462-467