Antimicrobial composition from copepods

The present invention relates to an antimicrobial composition, and to a process for the preparation of such a composition. The invention also relates to the use of such an antimicrobial composition. The present invention further relates to the use of the antimicrobial composition as a pharmaceutical.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Phase of PCT/EP2009/064229 filed Oct. 28, 2009, which claims priority of Norwegian Patent Application 20084555 filed Oct. 28, 2008.

All patent and non-patent references cited in the present application, are also hereby incorporated by reference in their entirety.

The norwegian patent application 20084555 and the references cited herein are hereby incorporated in the patent application in its entirety.

FIELD OF INVENTION

The present invention relates to an antimicrobial composition obtained from the marine copepodCalanus finmarchicus, and to a process for the preparation of such a composition. The invention also relates to the use of such an antimicrobial composition.

BACKGROUND OF INVENTION

Copepods are a group of small crustaceans found in the sea and nearly every freshwater habitat. According to the classification system of Martin and Davies (2001), the copepods form a subclass belonging to the subphylum Crustacea (crustaceans). Subphylum Crustacea is a large group of the phylum Arthropoda, comprising almost 52,000 described species. Six classes of the Crustaceans are usually recognized. Subclass Copepoda of the class Maxillopoda comprise ten orders, of which the order Calanoida include 43 families with about 2000 species. Many species are planktonic (drifting in sea waters), but more are benthic (living on the ocean floor), and some continental species may live in limno-terrestrial habitats and other wet terrestrial places, such as swamps, under leaf fall in wet forests, bogs, springs, ephemeral ponds and puddles, damp moss, or water-filled recesses (phytotelmata) of plants such as bromeliads and pitcher plants. Many live underground in marine and freshwater caves, sinkholes, or stream beds.

Two of the most abundant northern calanoid species isC. finmarchicuswhich is commonly regarded as a northern boreal species inhabiting North Atlantic Ocean, whileC. hyperboreusis an arctic species.

The evolution of antibiotic-resistant pathogenic bacteria has stimulated the search for alternative antimicrobial agents from natural sources. Antimicrobial activity has previously been detected in several decapod crustaceans, including lobster, crabs, shrimps and freshwater crayfish. The search for novel compounds displaying antimicrobial activity has led to the identification of several antimicrobial peptides and proteins in decapod crustaceans (Haug et al., 2002).

SUMMARY OF INVENTION

The present invention relates to an antimicrobial composition from a marine copepod, such asCalanus finmarchicus, and to a process for the preparation of such a composition. The invention also relates to the use of such an antimicrobial composition. The present invention further relates to a pharmaceutical composition obtained fromCalanus finmarchicusand to the use of a composition obtained fromCalanus finmarchicusin the treatment of microbial infections in an individual in need thereof.

DEFINITIONS

The term proteinaceous is defined as any molecule comprising amino acids connected by amide (peptide) bonds. Non-proteinaceous is any molecule, which does not comprise amino acids connected by amide (peptide) bonds.

A protein in the present context is an organic macromolecule made of amino acids. A protein is a biopolymer. Proteins consist of one or more polypeptide molecules.

A peptide in the present context is defined as a molecule consisting of 2 or more amino acids. Peptides are smaller than proteins. The dividing line between a peptide and a protein/polypeptide is at about 50 amino acids. Depending on the number of amino acids, peptides are called dipeptides, tripeptides, tetrapeptides, and so on.

A nucleotide is composed of a nucleobase (nitrogenous base), a five-carbon sugar (either ribose or 2′-deoxyribose), and one to three phosphate groups. Together, the nucleobase and sugar comprise a nucleoside. The phosphate groups form bonds with either the 2, 3, or 5-carbon of the sugar, with the 5-carbon site most common. Ribonucleotides are nucleotides where the sugar is ribose, and deoxyribonucleotides contain the sugar deoxyribose. Nucleotides can contain either a purine or pyrimidine base. Nucleic acids are polymeric macromolecules made from nucleotide monomers. In DNA, the purine bases are adenine and guanine, while the pyrimidines are thymine and cytosine. RNA uses uracil in place of thymine.

A nucleic acid is a macromolecule or a biopolymer composed of chains of monomeric nucleotides. A non-nucleic acid is a molecule which does not contain nucleotides.

An aerobic organism or aerobe is an organism that can survive and grow in an oxygenated environment. Obligate aerobes require oxygen for aerobic cellular respiration. Facultative anaerobes can use oxygen, but also have anaerobic methods of energy production. Microaerophiles are organisms that may use oxygen, but only at low concentrations. Aerotolerant organisms can survive in the presence of oxygen, but they are anaerobic because they do not use it as a terminal electron acceptor.

An anaerobic organism or anaerobe is any organism that does not require oxygen for growth and may even die in its presence. There are three types: obligate anaerobes, which cannot use oxygen for growth and are even harmed by it; aerotolerant organisms, which cannot use oxygen for growth, but tolerate the presence of it; and facultative anaerobes, which can grow without oxygen, but if present can utilize it.

A microorganism or microbe is an organism that is microscopic.

Microorganisms are very diverse; they include bacteria, fungi, archaea, viruses and protists; microscopic plants (called green algae); and animals such as plankton and the planarian. Pathogenic microorganisms cause infection.

Bacteria can be classified on the basis of cell structure, cellular metabolism or on differences in cell components such as DNA, fatty acids, pigments, antigens and quinones. By combining morphology and Gram-staining, most bacteria can be classified as belonging to one of four groups: Gram-positive cocci, Gram-positive bacilli, Gram-negative cocci and Gram-negative bacilli. Bacteria can be aerobic, anaerobic, or facultative anaerobic.

An antimicrobial is a substance that kills or inhibits the growth of microorganisms such as bacteria, fungi, or protozoans, as well as destroying viruses. Antimicrobial drugs either kill microbes (microbicidal) or prevent the growth of microbes (microbistatic). The main classes of antimicrobial agents are antibiotics (antibacterials), antivirals and antifungals targeting bacteria, viruses and fungi respectively.

The term broad-spectrum antibiotic refers to an antibiotic with activity against a wide range of disease-causing bacteria. It is also means that it acts against both Gram-positive and Gram-negative bacteria. This is in contrast to a narrow-spectrum antibiotic which is effective against only specific families of bacteria.

Antiseptics are antimicrobial substances that are applied to living tissue/skin to reduce the possibility of infection, sepsis, or putrefaction.

A preservative is a compound that is added to products such as foods, pharmaceuticals, paints, biological samples, wood, etc. to prevent decomposition by microbial growth or by undesirable chemical changes.

Disinfectants are antimicrobial agents that are applied to non-living objects to destroy microorganisms, the process of which is known as disinfection. Disinfection may be defined as: Cleaning an article of some or all of the pathogenic organisms which may cause infection.

Small molecules are low molecular weight organic compounds, which by definition are not polymers. The upper molecular weight limit for a small molecule is approximately 1000 Daltons (Da). Biopolymers such as nucleic acids, proteins, and polysaccharides (such as starch or cellulose) are not small molecules. Very small oligomers are also usually considered small molecules, such as dinucleotides, small peptides such as the antioxidant glutathione, and disaccharides such as sucrose. One group of small molecules are known as secondary metabolites.

Secondary metabolites are small organic compounds of metabolism that are not directly involved in the normal growth, development, or reproduction of organisms.

A biomolecule is any organic molecule that is produced by a living organism, including large polymeric molecules such as proteins, polysaccharides, and nucleic acids as well as small molecules such as primary metabolites, secondary metabolites, and natural products.

Organic compounds are molecules that contain carbon with the exception of a few types of compounds such as carbonates, simple oxides of carbon and cyanides, as well as the allotropes of carbon, which are considered inorganic. They can be either natural or synthetic organic compounds. Based upon the size of organic compounds, they can be classified as either small molecules or polymers.

A solvent is a liquid, solid, or gas that dissolves another solid, liquid, or gaseous solute, resulting in a solution. The most common solvent in everyday life is water.

Chemical Polarity refers to a separation of electric charge leading to a molecule having an electric dipole. Polar molecules can bond together due to dipole-dipole intermolecular forces between one molecule (or part of a large molecule) with asymmetrical charge distribution and another molecule also with asymmetrical charge distribution. Molecular polarity is dependent on the difference in electronegativity between atoms in a compound and the asymmetry of the compound's structure. For example, a molecule of water is polar because of the unequal sharing of its electrons in a “bent” structure, whereas methane is considered non-polar because the carbon shares the electrons with the hydrogen atoms uniformly. A molecule may be polar either as a result of polar bonds due to differences in electronegativity as described above, or as a result of an asymmetric arrangement of non-polar covalent bonds and non-bonding pairs of electrons known as a full molecular orbital. Due to the polar nature of the water molecule itself, polar molecules are generally able to dissolve in water. A non-polar compound occurs when there is an equal sharing of electrons between different atoms. Examples of household non-polar compounds include fats, oil and petrol/gasoline. Therefore (per the “oil and water” rule of thumb), most non-polar molecules are water insoluble (hydrophobic) at room temperature. However many non-polar organic solvents, such as turpentine, are able to dissolve polar substances.

An infection is the colonization of a host organism by a foreign species, usually a microorganism. In an infection, the infecting organism seeks to utilize the host's resources to multiply, usually at the expense of the host. The infecting organism, or pathogen, interferes with the normal functioning of the host. Primary and secondary infection may either refer to succeeding infections or different stages of one and the same infection.

A fungus is any member of a large group of eukaryotic organisms that includes microorganisms such as yeasts and molds, as well as the more familiar mushrooms.

An antifungal is defined as any compound capable of killing or inhibiting the growth of a fungus.

Anti-fouling: The effect of controlling, reducing and/or eliminating over time the number of undesirable microorganisms in a bio-film.

Bio-film: Habitation of microbial organisms on a solid or semi-solid surface.

Resistance or drug resistance is the reduction in effectiveness of a drug in curing a disease or improving a patient's symptoms. When the drug is not intended to kill or inhibit a pathogen, then the term is equivalent to dosage failure or drug tolerance. More commonly, the term is used in the context of diseases caused by pathogens. Pathogens are said to be drug-resistant when drugs meant to neutralize them have reduced effect. When an organism is resistant to more than one drug, it is said to be multi-resistant. Drug resistance is an example of evolution in microorganisms. Individuals that are not susceptible to the drug effects are capable of surviving drug treatment, and therefore have greater fitness than susceptible individuals. By the process of natural selection, drug resistant traits are selected for in subsequent offspring, resulting in a population that is drug resistant.

A gel is a semirigid colloidal dispersion of a solid preferably with a liquid. A gel can further be defined as a solid, jelly-like material that can have properties ranging from soft and weak to hard and tough. Gels are defined as a substantially dilute crosslinked system, which exhibits no flow when in the steady-state.

A lotion is a low- to medium-viscosity, topical preparation intended for application to unbroken skin; creams and gels have a higher viscosity than lotions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention was discovered through the surprising observation that samples of the marine copepodCalanus finmarchicus(crustacea) could be stored for several days at ambient temperature without developing off-odors or other signs of spoilage. Furthermore, it was observed that the samples did not contain bacteria detectable by the standard methods for enumeration of aerobic micro-organisms, and that an extract fromCalanus finmarchicuspossesses antimicrobial activity directed against various bacterial strains and some fungi.

Another copepod species,C. hyperboreus, was also investigated for antimicrobial activity. Antimicrobial activity was not detected in theC. hyperboreus, suggesting that the antimicrobial activity discovered inC. finmarchicusextracts is specific for this species ofCalanuscopepods.

The present invention thus relates in one embodiment to an antimicrobial composition, wherein the composition is obtained from copepods, such asCalanusspecies, particularly the marine copepodCalanus finmarchicus.

In the present invention the antimicrobial composition comprises one or more identical or different antimicrobial compounds.

In one embodiment the antimicrobial composition comprises a single antimicrobial compound.

In other embodiments the antimicrobial composition comprises two or more different antimicrobial compounds, such as three, for example four, such as five, for example six, such as seven, for example eight, such as nine, for example ten different antimicrobial compounds.

Calanusis a genus of marine copepod in the family Calanidae (Order Calanoida). Calimidae is the largest taxonomic family of calanoid copepods. The genusCalanusmay be the most abundant animal genus on Earth. Copepods of the generaCalanusandNeocalanusare ecologically important in the Arctic and subarctic regions of the world's oceans.

Calanus finmarchicusis a zooplankton species, which is found in enormous amounts in the North Sea. The body length is up to 5.4 mm for females and 3.6 mm for males.C. finmarchicusis high in protein and contains valuable omega-3 fatty acids. It contains also high amounts of antioxidant.Calanus finmarchicusis the dominant copepod in the northern North Atlantic, it plays a vital role in economy of the oceans, forming middle link in food chain leading from phytoplankton up to commercially important fish species, many of which feed on this species either as larvae or as adults. Plays equally important role in global carbon cycle, since large proportion of fixed carbon dioxide passes through oceanic food web as phytoplankton consumed byC. finmarchicus.

Methods for Obtaining the Antimicrobial Composition fromC. finmarchicus

The antimicrobial composition of the present invention can be prepared formC. finmarchicusby any method suitable for obtaining a composition with antimicrobial activity fromC. finmarchicus.

In one embodiment the present invention relates to a process for producing a composition comprising one or more antimicrobial compounds, said process comprising the steps of:i) providing a sample comprisingC. finmarchicusor parts ofC. finmarchicus,ii) performing one or more purification steps and/or isolation steps and/or concentration steps resulting in purification and/or isolation and/or concentration from said sample of a composition comprising one or more antimicrobial compounds, wherein at least some of said antimicrobial compounds are preferably non-proteinaceous and non-nucleic acid antimicrobial compounds.

The purpose of the purification step is the removal of undesirable substances. Undesirable substances in the present context are any substances that do not contribute directly or indirectly to the antimicrobial effect of the composition. Undesirable substances can be, but are not limited to salts, natural environmental compounds, structural elements of the copepods not exhibiting antimicrobial activity, etc. In one embodiment purification can be performed by dividing the sample into two or more fractions or phases and discarding the fraction or phase not comprising the antimicrobial composition.

The purpose of the isolation step is to retain desirable substances. Desirable substances in the present context are any substances that directly or indirectly contribute to the antimicrobial effect of the composition. The isolation can be performed by isolating or separating the one or more antimicrobial compounds according to chemical and/or physical properties. Examples of chemical properties include affinity for one or more compounds and chemical stability. Examples of physical properties include mass or size, charge, solubility, polarity, distribution, melting point, boiling point and absorbance.

The purpose of the concentration step is to remove solvents fully or partly to obtain a composition with a higher antimicrobial activity than before said concentration step was performed. The solvent can be any liquid, wherein the one or more antimicrobial compounds are comprised. Concentration can in one embodiment be performed by evaporation of the solvent.

In one embodiment, an aqueous solution comprising the antimicrobial composition is obtained by performing at least one physical processing step, such as by centrifugation.

The invention in one embodiment relates to a process for producing an antimicrobial composition, wherein a sample comprisingC. finmarchicusis subjected to the following process steps;i) providing a sample comprisingC. finmarchicusor parts ofC. finmarchicus,ii) separating said sample into at least two, such as three phases by centrifugation, i.e. at least two of a sediment phase, an oil phase and an aqueous phase,iii) and isolation of said aqueous phase, wherein said aqueous phase comprises the antimicrobial composition.

The invention in one embodiment relates to a process for producing an antimicrobial composition, wherein a sample comprisingC. finmarchicusis subjected to the following process steps;i) providing a sample comprisingC. finmarchicusor parts ofC. finmarchicus,ii) separating said sample into at least two, such as three phases by centrifugation, i.e. at least two of a sediment phase, an oil phase and an aqueous phase,iii) and isolation of said sediment phase, wherein said sediment phase comprises the antimicrobial composition.

The invention in one embodiment relates to a process for producing an antimicrobial composition, wherein a sample comprisingC. finmarchicusis subjected to the following process steps;i) providing a sample comprisingC. finmarchicusor parts ofC. finmarchicus,ii) separating said sample into at least two, such as three phases by centrifugation, i.e. at least two of a sediment phase, an oil phase and an aqueous phase,iii) and isolation of said oil phase, wherein said oil phase comprises the antimicrobial composition.

In one embodiment of the present invention, a concentrated extract with increased antimicrobial activity as compared to the antimicrobial activity of the aqueous phase is obtained by performing an extraction of a sample comprisingC. finmarchicus, using an extraction agent such as methanol.

Accordingly, the invention in another embodiment relates to a process for the isolation or extraction of an antimicrobial composition, wherein a sample comprisingC. finmarchicusis subjected to the following method steps:i) providing a sample comprisingC. finmarchicusor parts ofC. finmarchicus,ii) extracting said sample using one or more extraction agents,iii) removing said one or more extraction agents to obtain a concentrated extract, and optionallyiv) dissolving the concentrated extract,
thereby obtaining a concentrated antimicrobial composition comprising one or more antimicrobial compounds.

The invention in another embodiment relates to a process for the isolation or extraction of an antimicrobial composition, wherein a sample comprisingC. finmarchicusis subjected to the following method steps:i) providing a sample comprisingC. finmarchicusor parts ofC. finmarchicus,ii) extracting said sample using one or more extraction agents,iii) removing said one or more extraction agents to obtain a concentrated extract, and optionallyiv) dissolving the concentrated extract,v) further subjecting said concentrated extract to one or more fractionation steps and/or one or more purification steps and/or one or more isolation steps, thereby obtaining a concentrated antimicrobial composition comprising one or more antimicrobial compounds. The one or more fractionation steps and/or one or more purification steps and/or one or more isolation steps comprises one or more of the following steps: HPLC, Wessel-Flügge extraction, size exclusion, anion exchange, cation exchange, reversed phase chromatography, semi-preparative reversed phase chromatography.

In one embodiment the sample comprisingC. finmarchicuspreferably comprises wholeC. finmarchicusand/or wholeC. finmarchicuscells and/or disrupted/degratedC. finmarchicuscells. In other embodiments the sample comprisingC. finmarchicusis a previously processedC. finmarchicussample, such as comprising crushedC. finmarchicus. In one embodiment theC. finmarchicussample is aC. finmarchicushomogenate obtained by state of the art methods. TheC. finmarchicushomogenate can in one embodiment comprise and/or consist of particles with an average diameter of less than 1 mm, 0.5 mm, 0.25 mm, 0.1 mm, 0.05 mm and/or 0.01 mm. In one embodiment theC. finmarchicushomogenate can be use as an antimicrobial composition. In one embodiment theC. finmarchicushomogenate is used for treatment of an individual in need thereof.

In one embodiment, the sample comprising wholeC. finmarchicusis dried prior to the extraction step. Drying may be performed in any suitable way. In a preferred embodiment the drying is performed by freeze-drying. Other drying methods may be used such as e.g. heat pump drying, hot air drying, indirect and direct steam drying.

The extraction in step ii) above may be performed in any suitable way. In a preferred embodiment the extraction agent or solvent used is methanol. In other embodiments acetone and/or ethanol may be used as the extraction agent.

The removal of the extraction agent or solvent may in one embodiment be performed by evaporation. The solvent may be removed fully or partly. Evaporation of the solvent can be performed in any suitable way known in the art. In one embodiment, evaporation of the solvent is performed by vacuum evaporation. In another embodiment atmospheric evaporation is used.

The concentrated extract obtained in step iv) above can be dissolved in any suitable solvent. In one embodiment, the solvent is an aqueous solvent. In a preferred embodiment of the present invention, the solvent is water, more preferred deionised water.

In one embodiment, the antimicrobial composition is obtained by a process comprising a step of deproteinisation, i.e. removing proteinaceous compounds from the composition. In one example the deproteinisation could be performed by a method comprising the step of acetone precipitation and/or salting out. Salting out is a method of separating proteins based on the principle that proteins are less soluble at high salt concentrations. The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein. Dialysis can be used to remove the salt if needed.

In one embodiment, the preparation of the antimicrobial composition according to the present invention further comprises the step of filtering the obtained solution containing the antimicrobial composition. Filtering can be performed on or more times using one or more filter types. Filtering methods are known in the art and can for example be performed by filtering through a glass fiber filter and/or a microporous cellulose acetate filter or any other suitable filtering procedure as determined by the skilled person. In a particular embodiment, a microporous cellulose acetate filter with pore size 0.2 μm is used.

According to the present invention, filtering can be performed using filters selected from the group consisting of purified cotton filters, glass fiber filters, paper filters and microporous cellulose acetate filters.

In one embodiment of the present invention, the one or more antimicrobial compounds comprised within the antimicrobial composition are further isolated by performing one or more further extraction steps.

In one embodiment, the one or more further extraction step comprises a Wessel-Flügge extraction.

In one embodiment, the one or more further extraction step comprises one or more chromatography steps, such as one further chromatography extraction, for example two further chromatography extractions, such as three further chromatography extractions.

In one embodiment, the further extraction step comprises a solid-phase extraction, such as QMA anion exchange or reverse-phase column chromatography.

The present invention is also directed at the further isolation of the one or more antimicrobial compounds comprised within the antimicrobial composition by performing one or more chromatography steps, such as by HPLC, and selecting the fractions wherein the antimicrobial activity is retained.

In one embodiment of the present invention, the antimicrobial composition is further analysed by mass spectroscopy.

In one embodiment the present invention is directed to an antimicrobial composition comprising one or moreC. finmarchicusantimicrobial compounds, wherein said composition is obtained or obtainable by a process comprising the steps of:i) providing a sample comprisingC. finmarchicusor parts thereof,ii) performing one or more purification steps and/or isolation steps and/or concentration steps resulting in purification and/or isolation and/or concentration from said sample of a composition comprising one or more antimicrobial compounds, wherein at least some of said antimicrobial compounds are preferably non-proteinaceous and non-nucleic acid antimicrobial compounds.

In another embodiment the present invention is directed to an antimicrobial composition comprising one or moreC. finmarchicusantimicrobial compounds, wherein said composition is obtained or obtainable by any process mentioned in this applications.

The invention further relates to a method for producing one or more anti-microbial compounds, said method comprising the steps of:i) providing an extract or a homogenate ofC. finmarchicus, andii) isolating from said extract or homogenate one or more anti-microbial compounds,wherein said one or more anti-microbial compounds are characterised by exhibiting m/z values in a mass spectroscopical analysis of a) from 235 to 255 and/or from b) 335 to 360, essentially as illustrated inFIG. 9.

The compounds are preferably isolated by one or more chromatographical purification steps; such as purification steps selected from, but not limited to, a Wessel-Flügge extraction step, a solid phase extraction (anion exchange) step, a solid phase extraction (reverse phase) step; and a high pressure liquid chromatography (HPLC) step (with or without polar end-capping); including reverse phase HPLC and normal phase HPLC.

The invention also relates to one or more anti-microbial agent(s) or a composition comprising same, said agent(s) or said composition being obtainable by a method comprising the steps of:1. providing an extract or a homogenate ofC. finmarchicus, and2. isolating from said extract or homogenate said one or more anti-microbial compounds,wherein said one or more anti-microbial compounds are characterised by exhibiting m/z values in a mass spectroscopical analysis of a) from 235 to 255 and/or from b) 335 to 360, essentially as illustrated inFIG. 9.wherein said one or more anti-microbial compounds are preferably isolated by one or more chromatographical purification steps; such as purification steps selected from, but not limited to, a Wessel-Flügge extraction step, a solid phase extraction (anion exchange) step, a solid phase extraction (reverse phase) step; and a high pressure liquid chromatography (HPLC) step (with or without polar end-capping); including reverse phase HPLC and normal phase HPLC.

The m/z values in a mass spectroscopical analysis of a) from 235 to 255 mentioned above can in one embodiment be from 235 to 240, and/or from 240 to 245, and/or from 245 to 250, and/or 250 to 255.

The m/z values in a mass spectroscopical analysis of b) from 335 to 360 mentioned above can in one embodiment be from 335 to 340, and/or 340 to 345, and/or 345 to 350, and/or 350 to 355 and/or 355 to 360.

Chemical Polarity of Compounds

The antimicrobial composition according to the present invention preferably comprises one or more polar compounds exhibiting an antimicrobial activity.

The polarity of a compound is dependent on the difference in electronegativity between atoms in a compound and the asymmetry of the compound's structure. In a non-polar compound the electrons of the molecule are distributed uniformly, whereas in a polar compound, the electrons are distributed asymmetrically giving rise to a molecule with an asymmetrical charge distribution.

Polarity underlies a number of physical properties including surface tension, solubility, and melting- and boiling-points. Polarity of a compound can e.g. be assessed by its ability to dissolve in aqueous solvents, such as water. A water-soluble compound is polar, whereas a compound which is not soluble in water is non-polar.

The polarity of a particular compound or composition can be estimated by a range of different methods.

In organic chemistry and the pharmaceutical sciences, a partition—(P) or distribution coefficient (D) is the ratio of concentrations of a compound in the two phases of a mixture of two immiscible solvents at equilibrium. Hence these coefficients are a measure of differential solubility of the compound between these two solvents.

Normally one of the solvents chosen is water while the second is hydrophobic such as octanol. Hence both the partition and distribution coefficient are measures of how hydrophilic (“water loving”) or hydrophobic (“water fearing”) a chemical substance is, in other words how polar a compound is. Partition coefficients are useful for example in estimating distribution of drugs within the body. Hydrophobic drugs with high partition coefficients are preferentially distributed to hydrophobic compartments such as lipid bilayers of cells while hydrophilic drugs (low partition coefficients) preferentially are found in hydrophilic compartments such as blood serum.

The partition coefficient is a ratio of concentrations of un-ionized compound between the two solutions. To measure the partition coefficient of ionizable solutes, the pH of the aqueous phase is adjusted such that the predominant form of the compound is un-ionized. The logarithm of the ratio of the concentrations of the un-ionized solute in the solvents is called log P:

Reverse phase HPLC can be used to estimate log P of a compound and/or a composition.

In one embodiment of the present invention, the log P of the one or more antimicrobial compounds in the composition of the present invention is in the range of −3 to 6, more preferred −1.5 to 4, such as in the range of −1.5 to −1.4, for example −1.4 to −1.3, such as −1.3 to −1.2, for example −1.2 to −1.1, such as −1.1 to −1.0, for example −1.0 to −0.9, such as −0.9 to −0.8, for example −0.8 to −0.7, such as −0.7 to −0.6, for example −0.6 to −0.5, such as −0.5 to −0.4, for example −0.4 to −0.3, such as −0.3 to −0.2, for example −0.2 to −0.1, such as −0.1 to 0.0, for example 0.0 to 0.1, such as 0.1 to 0.2, for example 0.2 to 0.3, such as 0.3 to 0.4, for example 0.4 to 0.5, such as 0.5 to 0.6, for example 0.6 to 0.7, such as 0.7 to 0.8, for example 0.8 to 0.9, such as 0.9 to 1.0, for example 1.0 to 1.1, such as 1.1 to 1.2, for example 1.2 to 1.3, such as 1.3 to 1.4, for example 1.4 to 1.5, such as 1.5 to 1.6, for example 1.6 to 1.7, such as 1.7 to 1.8, for example 1.8 to 1.9, such as 1.9 to 2.0, for example 2.0 to 2.1, such as 2.1 to 2.2, for example 2.2 to 2.3, such as 2.3 to 2.4, for example 2.4 to 2.5, such as 2.5 to 2.6, for example 2.6 to 2.7, such as 2.7 to 2.8, for example 2.8 to 2.9, such as 2.9 to 3.0, for example 3.0 to 3.1, such as 3.1 to 3.2, for example 3.2 to 3.3, such as 3.3 to 3.4, for example 3.4 to 3.5, such as 3.5 to 3.6, for example 3.6 to 3.7, such as 3.7 to 3.8, for example 3.8 to 3.9, such as 3.9 to 4.0.

In another embodiment the log P value is less than 2, such as less than 1.9 for example less than 1.8, such as less than 1.7, for example less than 1.6, such as less than 1.5, for example less than 1.4, such as less than 1.3, for example less than 1.2, such as less than 1.1, for example less than 1.0, such as less than 0.9, for example less than 0.8, such as less than 0.7, for example less than 0.6, such as less than 0.5, for example less than 0.4, such as less than 0.3, for example less than 0.2, such as less than 0.1, for example less than 0.0, such as less than −0.1, for example less than −0.2, such as less than −0.3, for example less than −0.4, such as less than −0.5, for example less than −0.6, such as less than −0.7, for example less than −0.8, such as less than −0.9, for example less than −1.0, such as less than −1.1, for example less than −1.2, such as less than −1.3.

Type of Action

Overall, there are three types of action of antimicrobial agents; i) static action where growth is inhibited, ii) cidal action where organisms are killed and iii) lytic action where organisms are killed and lysed.

If a compound is static or biostatic, the growth of the microorganism is inhibited, but it is not killed by the treatment. If a population of microorganisms is treated with a static antimicrobial compound, the number of viable microorganisms is not decreased by the treatment compared to the total number of microorganisms.

If a compound is cidic or biocidic, the microorganism is killed by the treatment. If a population of microorganisms is treated with a cidic antimicrobial compound, the number of viable microorganisms is decreased compared to the total number of microorganisms.

If a compound is lytic or biolytic, the microorganism is lysed and killed by the treatment. If a population of microorganisms is treated with a lytic antimicrobial compound, both the number of viable and the total number of microorganisms is decreased to a similar extent.

In relation to bacteria, the terms used are bacteriostatic, bacteriocidic and bacteriolytic, respectively.

According to the present invention the antimicrobial composition can be static, cidic or lytic.

In one embodiment the antimicrobial composition is static.

In one embodiment the antimicrobial composition is cidic.

In one embodiment the antimicrobial composition is lytic.

In one embodiment the antimicrobial composition is both cidic and lytic.

In a preferred embodiment of the present invention, the antimicrobial composition of the present invention is bacteriocidic.

In another embodiment of the present invention, the antimicrobial composition of the present invention is not bacteriolytic.

The anti-fungal activity of the antimicrobial composition of the present invention is either static, cidic or lytic.

The type of antimicrobial action of a particular compound or composition can be determined by several different ways known in the art.

Characteristics of the Antimicrobial Composition

In one embodiment the antimicrobial composition comprises one or more compounds with the chemical characteristics disclosed in the examples and figures of the present invention.

In one embodiment the antimicrobial composition of the present invention comprises one or more antimicrobial compounds.

In one embodiment, the antimicrobial composition is water soluble.

In one embodiment the antimicrobial composition comprises one or more polar antimicrobial compounds.

In one embodiment, the one or more antimicrobial compounds of the present invention is/are heat stable. In one example the antimicrobial composition fully or partially retains its antimicrobial activity after heating. Heating of the antimicrobial composition can be performed at 60-130° C., such as in the range of 60-65° C., 65-70° C., 70-75° C., 75-80° C., 80-85° C., 85-90° C., 90-95° C., 95-100° C., 100-105° C., 105-110° C., 110-115° C., 115-120° C., 120-125° C., 125-130° C.

In one embodiment heating is performed at about 65-75° C., more preferred at about 70° C.

In one embodiment heating is performed at about 90-110° C., more preferred at about 100° C.

In yet another embodiment heating is performed at about 120-125° C., more preferred at about 121° C.

According to the present invention, heating can be performed for shorter or longer periods of time, such as from a minute to several hours. Heating can for example be performed for a few minutes such as in the range of about 1-5 minutes, 5-10 minutes, 10-15 minutes, 15-20 minutes, 20-25 minutes, 25-30 minutes, 30 minutes-1 hour.

In a preferred embodiment, heating is performed for about 5-20 minutes, more preferred for about 10-15 minutes.

In one embodiment heating is performed at about 65-75° C., more preferred at about 70° C. such as for about 10-15 minutes.

In one embodiment heating is performed at about 90-110° C., more preferred at about 100° C. such as for about 10-15 minutes.

In yet another embodiment heating is performed at about 120-125° C., more preferred at about 121° C. such as for about 10-15 minutes.

In one embodiment, the one or more antimicrobial compounds are not proteinaceous compounds, i.e. they are not peptides or proteins. In one example, the antimicrobial composition is resistant to proteolytic enzymes, thereby indicating that the compound does not contain amide or peptide bonds.

In one example, the antimicrobial composition is resistant to pepsin treatment, meaning that the antimicrobial composition fully retains its antimicrobial activity following pepsin treatment.

In another example, the antimicrobial composition is resistant to alcalase treatment, meaning that the antimicrobial composition fully retains its antimicrobial activity following alcalase treatment.

In another example, the antimicrobial composition is resistant to Proteinase K treatment, meaning that the antimicrobial composition fully retains its antimicrobial activity following Proteinase K treatment.

In one embodiment the antimicrobial composition does not contain an antimicrobial compound in the form of a nucleotide or nucleic acids, i.e. it is a non-nucleic acid.

In one embodiment the antimicrobial composition contains one or more antimicrobial nucleotides or nucleic acids.

In one embodiment the one or more antimicrobial compounds are proteinaceous compounds, such as one or more cyclic peptides.

Carbohydrates are simple organic compounds that are aldehydes or ketones with many hydroxyl groups added, usually one on each carbon atom that is not part of the aldehyde or ketone functional group. The basic carbohydrate units are called monosaccharides.

In one embodiment at least one of the one or more antimicrobial compounds of the present invention comprises a carbohydrate.

In another embodiment at least one of the one or more antimicrobial compounds of the present invention does not comprise a carbohydrate.

In one embodiment, the one or more antimicrobial compounds comprised within the antimicrobial composition is not a strong anionic compound.

In one embodiment the one or more antimicrobial compounds comprised within the antimicrobial composition is a small molecule.

In one embodiment the size of the one or more antimicrobial compounds is less than 1 kDa. In other embodiments the size of the antimicrobial compound is less than 900 Da, for example less than 800 Da, such as less than 700 Da, for example less than 600 Da, such as less than 500 Da, for example less than 400 Da, such as less than 300 Da, for example less than 200 Da, such as less than 100 Da.

In other embodiments, the size of the one or more antimicrobial compounds comprised within the antimicrobial composition is in the range of 100 Da-1 kDa, such as 100-200 Da, for example 200-300 Da, such as 300-400 Da, for example 400-500 Da, such as 500-600 Da, for example 700-800 Da, such as 800-900 Da, for example 900-1000 Da (1 kDa).

In other embodiments the size of the one or more antimicrobial compounds is close to 1 kDa, but not necessarily less than 1 kDa, for example less than 1.1 kDa, such as less than 1.2 kDa, for example less than 1.3 kDa, such as less than 1.4 kDa, for example less than 1.5 kDa, such as less than 1.6 kDa, for example less than 1.7 kDa, such as less than 1.8 kDa.

In one embodiment the antimicrobial composition comprises less that 20% (weight/weight %) protein, for example less than 10%, such as less than 8%, for example less than 6%, such as less 4%, for example less than 1%.

In one embodiment the antimicrobial composition comprises less that 20% (weight/weight %) ash, for example less than 10%, such as less than 8%, for example less than 6%, such as less 4%, for example less than 1%.

In one embodiment the antimicrobial composition comprises less that 20% (weight/weight %) lipid, for example less than 10%, such as less than 8%, for example less than 6%, such as less 4%, for example less than 1%.

In one embodiment the antimicrobial composition comprises less that 20% (weight/weight %) carbohydrate, for example less than 10%, such as less than 8%, for example less than 6%, such as less 4%, for example less than 1%.

In one embodiment the antimicrobial composition comprises a compound with mass 346 and/or a m/z of 347 as depicted inFIG. 12.

In one embodiment the antimicrobial composition comprises a compound with UV-spectra similar or identical to the UV spectra depicted inFIG. 8.

In one embodiment the antimicrobial composition comprises penostatin or a similar compound.

Secondary Metabolites

Secondary metabolites, also known as natural products, are those products (chemical compounds) of metabolism that are not essential for normal growth, development or reproduction of an organism. In this sense they are “secondary”.

Secondary metabolites, including antibiotics, are produced in nature and serve survival functions for the organisms producing them. Secondary metabolites serve: (i) as competitive weapons used against bacteria, fungi, amoebae, plants, insects, and large animals; (ii) as metal transporting agents; (iii) as agents of symbiosis between microbes and plants, nematodes, insects, and higher animals; (iv) as sexual hormones; and (v) as differentiation effectors.

The function or importance of these compounds to the organism's development is usually of ecological nature as they are used as defence against predators (herbivores, pathogens etc.), for interspecies competition, and to facilitate the reproductive processes.

Contrary to primary metabolites these compounds are not ubiquitous in the living organisms who produce them nor are they necessarily expressed continuously. Although plants are better known as a source of secondary metabolites, bacteria, fungi and many marine organisms (sponges, tunicates, corals, snails) are very interesting sources, too.

Secondary metabolites can be classified by their chemical structure or physical properties into one or more of the following groups: alkaloids, terpenoids, polyketides, aliphatic, aromatic, and heteroaromatic organic acids, phenols, iridoids, steroids, saponins, peptides, ethereal oils, resins and balsams.

In one embodiment of the present invention at least one of the one or more antimicrobial compounds comprises or consists of a secondary metabolite such as one or more compounds selected from the group consisting of alkaloids, terpenoids, polyketides, aliphatic, aromatic, and heteroaromatic organic acids, phenols, iridoids, steroids, saponins, peptides, ethereal oils, resins and balsams.

Industrial Uses of the Antimicrobial Composition fromC. finmarchicus

In one embodiment the antimicrobial composition is an antiseptic. Antiseptics are antimicrobial substances that are applied to living tissue/skin to reduce the possibility of infection and/or sepsis, and/or putrefaction. Antiseptics are generally distinguished from antibiotics by their ability to be transported through the lymphatic system to destroy bacteria within the body, and from disinfectants, which destroy microorganisms found on non-living objects.

The microbial composition according to the present invention can be a true germicides, capable of destroying microbes (bacteriocidal), or bacteriostatic and only prevent or inhibit their growth. Antibacterials are antiseptics that have the proven ability to act against bacteria especially if they target systems which kill only bacteria. Microbicides which kill virus particles are called viricides or antivirals. The antimicrobial composition of the present invention can be a true germicide, an antibacterial, a microbicide, a viricide and/or an antiviral.

In one embodiment the antimicrobial composition is a disinfectant. In one embodiment the antimicrobial composition can be used in cleaning of hospitals such as in cleaning of an operating room and/or surgery equipment.

Disinfectants should generally be distinguished from antibiotics that destroy microorganisms within the body, and from antiseptics, which destroy microorganisms on living tissue. Sanitizers are substances that reduce the number of microorganisms to a safe level. One official and legal definition states that a sanitizer must be capable of killing 99.999%, known as a 5 log reduction, of a specific bacterial test population, and to do so within 30 seconds. The main difference between a sanitizer and a disinfectant is that at a specified use dilution, the disinfectant must have a higher kill capability for pathogenic bacteria compared to that of a sanitizer. Very few disinfectants and sanitizers can sterilize (the complete elimination of all microorganisms), and those that can depend entirely on their mode of application. Bacterial endospores are most resistant to disinfectants, however some viruses and bacteria also possess some tolerance. The present invention relates in one embodiment to use of the antimicrobial composition as a sanitizer and/or a disinfectant.

Preservative

The invention also relates to the use of the antimicrobial composition as a preservative, such as in nutritional and/or pharmaceutical composition. A preservative is a natural or synthetic chemical that is added to products such as foods, pharmaceuticals, paints, biological samples, wood, etc. to prevent decomposition by microbial growth or by undesirable chemical changes.

An embodiment relates to the use of the antimicrobial composition as a preservative in a feed composition/food conservation.

In one embodiment the antimicrobial composition is used for anti-fouling. Anti-fouling is the process of removing or inhibiting the accumulation of biofouling. Biofouling or biological fouling is the undesirable accumulation of microorganisms, plants, algae, and animals on surfaces such as submerged structures like ships' hulls.

The antimicrobial composition can be used for controlling, reducing and/or eliminating over time the number of undesirable microorganisms in a bio-film.

Biofouling is also found in membrane systems, such as membrane bioreactors and reverse osmosis spiral wound membranes. In the same manner it is found as fouling in cooling water cycles of large industrial equipments and power stations. Biofouling is divided into microfouling—biofilm formation and bacterial adhesion—and macrofouling—attachment of larger organisms, of which the main culprits are barnacles, mussels, polychaete worms, bryozoans, and seaweed. Together, these organisms form a fouling community.

Biofouling can occur on any surface submerged in water such as for example on ships. Other examples of surfaces that can be exposed to biofouling are any installations, membranes, nets, measuring equipment or other equipment in aquaculture.

Biofouling can also occur in groundwater wells where buildup can limit recovery flow rates, and in the exterior and interior of ocean-laying pipes. In the latter case it has been shown to retard the seawater flow through the pipe and has to be removed with the tube cleaning process.

In one preferred embodiment the surface for application of the anti-fouling composition is a surface that is at least occasionally immersed in water, wherein said water includes fresh, salt or brackish water. The surface can be selected from the group consisting of the surfaces of vessels including boats and ships, ship hulls, off-shore equipment, pipes, substructures of bridges, piers and aquacultural apparatuses including fish farming nets.

The methods and compositions disclosed herein may be used on a variety of surfaces, including but not limited to boat hulls, marine markers, bulkheads, pilings, water inlets, floors, roofs, and shingles. For example, the methods and compositions may be used to minimize fouling of marine markers. Such markers constitute a large category of floating objects and are greatly impaired by the accumulation of marine growth. Similarly, the methods and compositions may be used on marine bulkheads. The accumulation of marine growth on bulkhead structures is detrimental to the bulkhead structure over the long term. Furthermore, the growth causes significant short term effects that are aesthetically displeasing and dangerous. Moreover, the harsh abrasive characteristics of the hard growth can result in major damage to vessels. Similarly, the present invention can be used to minimize blockages due to fouling by marine growth of heat exchangers, evaporators, condensers and fire and flushing systems, thus resulting in significant decreases in maintenance costs for all categories of marine structures.

The antimicrobial composition can in one embodiment be included in a paint such as a paint for marine vessels. Paints according to the invention include the antimicrobial composition in an amount effective to reduce the growth of unwanted or undesirable microorganisms. Such compositions and/or paints may be in a variety of forms, including paints, lacquers, pastes, laminates, epoxies, resins, waxes, gels, and glues in addition to other forms known to one of skill in the art.

The antimicrobial composition according to the present invention can be used for prevention and/or inhibition of any type of fouling including the types mentioned above.

The antimicrobial composition can also be used for conservation of e.g. food/feed, drinks/beverages, pharmaceuticals and cosmetics.

In another preferred embodiment the antimicrobial composition has an anti-bacterial effect. The antibacterial effect can in one embodiment be employed in food production such as in the dairy industry. In another embodiment the antimicrobial composition can be used in hospitals such as in an operating room.

In one embodiment the antimicrobial composition according to the present invention is aC. finmarchicushomogenate for use as a medicament.

Antiviral Effects

In one embodiment the antimicrobial composition according to the present invention is used to inhibit, kill, and/or prevent the growth of one or more fungi, such as those described elsewhere in the application.

The antimicrobial composition can also be used as an antimicrobial detergent, an antimicrobial hand wash, an antimicrobial toothpasta, an antimicrobial mouthwash, an antimicrobial textile treatment etc.

Use of the Composition fromC. finmarchicusas a Pharmaceutical

In one embodiment, the present invention relates to a pharmaceutical composition obtained fromCalanus finmarchicus.

The composition obtained fromCalanus finmarchicuscan in one embodiment of the present invention be used as a medicament.

In one embodiment of the present invention a composition obtained fromCalanus finmarchicusis used in the treatment of microbial infections in an individual in need thereof.

In another embodiment the composition obtained fromCalanus finmarchicusis used for the manufacture of a medicament for the treatment microbial infections.

The treatment can be ameliorating, curative or prophylactic treatment of one or more infectious diseases.

An infectious disease is in one embodiment a clinically evident disease resulting from the presence of pathogenic agents, including pathogenic viruses, pathogenic bacteria, fungi, protozoa, multicellular parasites, and aberrant proteins known as prions. These pathogens are able to cause disease in human beings, animals and/or plants. Microbial infections include infections caused by bacteria, fungi, protozoans, and viruses.

Infectious pathologies are usually qualified as contagious diseases (also called communicable diseases) due to their potentiality of transmission from one person or species to another. Transmission of an infectious disease may occur through one or more of diverse pathways including physical contact with infected individuals. These infecting agents may also be transmitted through liquids, food, body fluids, contaminated objects, airborne inhalation, or through vector-borne spread.

The term infectivity describes the ability of an organism to enter, survive and multiply in the host, while the infectiousness of a disease indicates the comparative ease with which the disease is transmitted to other hosts. An infection is not synonymous with an infectious disease, as an infection may not cause important clinical symptoms or impair host function.

In a preferred embodiment the present invention relates to treatment of bacterial infections.

In another preferred embodiment, the present invention relates to treatment of fungal infections.

In another embodiment, the present invention relates to treatment of viral infections.

Bacteria

In one embodiment, the composition of the present invention is directed against a bacteria, i.e. it is antibacterial or an antibiotic.

In one embodiment the antimicrobial composition of the present invention can be used as a broad-spectrum antibiotic.

In one embodiment, the antibacterial activity is directed against one or more bacteria selected from the group consisting of Gram positive bacteria, Gram negative bacteria, aerobic bacteria, anaerobic bacteria.

In a preferred embodiment, the antibacterial activity is directed against one or more pathogenic bacteria. More preferred, the antibacterial activity is directed against the bacteria selected from the group comprisingPseudomonas, Enterococcus, Listeria, Bacillus, Staphylococcus, Escherichia, SalmonellaandClostridium. In an even more preferred embodiment the bacteria are selected from the group comprisingPseudomonas aeruginosa, Enterococcus faecalis, Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, Escherichia coli, Salmonella berta, Clostridium perfringensandClostridium bifermentans.

In another embodiment the antibacterial activity is directed against on or more bacteria resistant to one or more conventional antibiotics. Examples of resistant or multiresistant bacteria include but are not limited to meticillin-resistantS. aureus, and multiresistantP. aeruginosa, Klebsiella pneumoniaeandAcinetobacter.

The strain is resistant to all β-lactam antibiotics. The treatment of MRSA is therefore difficult. The strain can cause both local and systemic infections.

The strain has a plasmid-mediated, broad spectrum β-lactamase (Klebsiella PneumoniaeCarbapenemase (KPC) and is resistant to all clinical relevant β-lactam antibiotics. Furthermore, it is in vitro resistant to many other non β-lactam antibiotics. Infections with such microbes are difficult to treat with available antibiotics.K. pneumoniaeis a common cause of urinary tract infections, but can also cause systemic infections.

P. aeruginosais naturally resistant to a range of different antibiotics, and the spectre of efficient agents is narrow. Additionally, it has a high ability to develop resistance to new antibiotics. The actual strain is in vitro resistant to, or has reduced susceptibility to antimicrobial agents commonly used in the treatment ofP. aeruginosainfections. Severe infections withP. aeruginosaoccur with immune deficient patients and with weak, hospitalized patients.

Acinetobacteris naturally resistant to a range of different antibiotics, and the spectre of efficient agents is narrow. With one exception, the actual isolate in vitro resistant to, or have reduced susceptibility to all antimicrobial agents used for treatment ofAcinetobacterinfections. Severe infections withAcinetobacteroccur with immune deficient patients and with weak, hospitalized patients.

Fungi

In one embodiment the antimicrobial activity of the composition of the present invention is directed against a fungus, i.e. it is an antifungal. Some examples of fungi include yeasts, molds and mushrooms.

In one embodiment the antifungal activity is directed against one or more pathogenic fungi selected from the group consisting of, but not limited toCandida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis, Stachybotrys.

In one embodiment the antifungal activity is directed against one or more non-pathogenic fungi.

In a preferred embodiment, the antifungal activity is directed against a yeast, more preferred againstSaccharomyces cerevisiae.

In another preferred embodiment the antifungal activity is directed against a mold, more preferred againstAspergillus niger.

In one embodiment, the antifungal activity is not directed againstCandida albicans.

Virus

Microbial Infections

In one embodiment the present invention relates to treatment of one or more microbial infectious diseases by administration of the composition according to the present invention to an individual in need thereof. The infectious disease can be any type of infection including the types of infections mentioned elsewhere herein and those listed in Tables 1, 2, 3, or 4 herein below.

The individual treated can be a human being or an animal. The animal can be a dog, cat, horse, rabbit, hamster, mouse, rat, monkey, cow, pig, donkey, fish, bird, reptile or any other animal in need of treatment. In one embodiment the animal is a laboratory/test animal. In another embodiment the animal in need of treatment is a pet or livestock such as domesticated cows, pigs, sheep, poultry or farmed fish.

The human being can be a man, a woman, a post-menopausal women, a pregnant woman, a lactating woman, an infant, a child, or an adult. The individual such as a human being can be of any age such as from newborn to 120 years old, for example from 0 to 6 months, such as from 6 to 12 months, for example from 1 to 5 years, such as from 5 to 10 years, for example from 10 to 15 years, such as from 15 to 20 years, for example from 20 to 25 years, such as from 25 to 30 years, for example from 30 to 35 years, such as from 35 to 40 years, for example from 40 to 45 years, such as from 45 to 50 years, for example from 50 to 60 years, such as from 60 to 70 years, for example from 70 to 80 years, such as from 80 to 90 years, for example from 90 to 100 years, such as from 100 to 110 years, for example from 110 to 120 years.

The individual can be of any race such as a Caucasian, a black person, an East Asian person, a person of Mongoloid race, a person of Ethiopian race, a person of Negroid race, a person of American Indian race, a person of Australoid race, or a person of Malayan race.

Said human being or animal can be healthy or have one or more diseases. Said human being or animal can be diagnosed and/or treated for one or more diseases. In one embodiment said individual is genetically disposed for one or more diseases.

In one embodiment, the individual in need of treatment is a individual infected with one or more pathogenic or disease-causing bacteria. Disease-causing bacteria include, but are not limited to the group comprisingPseudomonas, Enterococcus, Listeria, Bacillus, Staphylococcus, Escherichia, SalmonellaandClostridium. In a preferred embodiment the bacteria are selected from the group comprisingPseudomonas aeruginosa, Enterococcus faecalis, Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, Escherichia coli, Salmonella berta, Clostridium perfringensandClostridium bifermentans.

In one embodiment, the individual in need of treatment is a individual infected with one or more bacteria resistant to one or more antibiotics. Examples of resistant or multiresistant bacteria include but are not limited to meticillin-resistantS. aureus, and multiresistantP. aeruginosa, Klebsiella pneumoniaeandAcinetobacter.

In another embodiment, the individual in need of treatment is a individual infected with one or more pathogenic fungi.

Administration Route and Dosage

The composition may be administered using one or more of the following routes of administration.

Routes of administration can broadly be divided into:Topical: local effect, substance is applied directly where its action is desired.Enteral: desired effect is systemic (non-local), substance is given via the digestive tract.

Parenteral: desired effect is systemic, substance is given by routes other than the digestive tract.

In one embodiment the antimicrobial composition of the present invention is administered topically.

In one embodiment the antimicrobial composition of the present invention is administered enterally.

In one embodiment the antimicrobial composition of the present invention is administered parentally.

Enteral administration is any form of administration that involves any part of the gastrointestinal tract and include:By mouth (peroral)By gastric feeding tube, duodenal feeding tube, or gastrostomyRectally

Parenteral by injection or infusion include:Intravenous (into a vein)Intraarterial (into an artery)Intramuscular (into a muscle),Intracerebral (into the cerebrum) direct injection into the brain.Intracerebroventricular (into the cerebral ventricles) administration into the ventricular system of the brainIntracardiac (into the heart)Subcutaneous (under the skin),Intraosseous infusion (into the bone marrow) is, in effect, an indirect intravenous access because the bone marrow drains directly into the venous system.Intradermal, (into the skin itself)Intrathecal (into the spinal canal)Intraperitoneal, (infusion or injection into the peritoneum)Intravesical infusion is into the urinary bladder.Intracavernosal injection is into the base of the penis.

Other parenteral administration modes include:Transdermal (diffusion through the intact skin),Transmucosal (diffusion through a mucous membrane), e.g. insufflation, sublingual, i.e. under the tongue, vaginal suppositoriesInhalational,Intracisternal: given between the first and second cervical vertebraeOther epidural (synonym: peridural) (injection or infusion into the epidural space)Intravitreal, through the eye

Peroral intake may be in the form ofTabletsCapsulesMixturesLiquidPowder

Injections may be either systemic or local injections

Other administration modes of the present invention include:Jet-infusion (micro-drops, micro-spheres, micro-beads) through skinDrinking solution, suspension or gelInhalationNose-dropsEye-dropsEar-dropsSkin application as ointment, gel, lotion, cream or through a patchVaginal application as ointment, gel, crème or washingGastro-Intestinal flushingRectal washings or by use of suppositories

Administration can be performed asSingle administration such as single intake, injection, application, washingMultiple administrations such as multiple intakes, injections, applications, washingsOn single day basisOver prolonged time as days, month, years

Drug dose and regimen can be modified during the course.

A dose or dosage of the composition according to the present invention may be given as a single dose or in divided doses. A single dose occurs only once, with the drug administered either as a bolus or by continuous infusion. Alternatively, the dose may be divided into multiple doses and given recurrently, such as twice (two times), for example three times, such as four times, for example five times, such as six times, for example seven times, such as eight times, for example nine times, such as ten divided doses. Furthermore, the dose may be given repeatedly, i.e. more than once, such as twice (two times), for example three times, such as four times, for example five times, such as six times, for example seven times, such as eight times, for example nine times, such as ten times a day. Alternatively, the dose may be in sustained release form. A bolus is in theory regarded as given immediately, and should be administered in less than 5 minutes.

It follows that the composition according to the present invention may be given once or more daily, or alternatively may be given with intervals of 1 day, such as 2 days, for example 3 days, such as 4 days, such as 5 days, for example 6 days, such as 7 days (1 week), for example 8 days, such as 9 days, such as 10 days, for example 11 days, such as 12 days, for example 13 days, such as 14 days (2 weeks), such as 3 weeks, for example 4 weeks, such as 5 weeks, for example 6 weeks, such as 7 weeks, such as 8 weeks, for example 12 weeks.

In one embodiment the antimicrobial composition according to the present invention is administered to an individual in need thereof at a total daily dosage of from about 0.01 milligram to about 1000 milligram per kilogram of body weight. The dosage regimen may be adjusted within this range or even outside of this range to provide the optimal therapeutic response.

The composition according to the present invention is given in an effective amount to an individual in need thereof. The amount of composition according to the present invention in one preferred embodiment is in the range of from about 0.01 milligram per kg body weight per dose to about 1000 milligram per kg body weight per dose, such as from about 0.01 milligram per kg body weight per dose to about 0.025 milligram per kg body weight per dose, for example from about 0.025 milligram per kg body weight per dose to about 0.05 milligram per kg body weight per dose, such as from about 0.05 milligram per kg body weight per dose to about 0.075 milligram per kg body weight per dose, for example from about 0.075 milligram per kg body weight per dose to about 0.1 milligram per kg body weight per dose, such as from about 0.1 milligram per kg body weight per dose to about 0.25 milligram per kg body weight per dose, such as from about 0.25 milligram per kg body weight per dose to about 0.5 milligram per kg body weight per dose, for example from about 0.5 milligram per kg body weight per dose to about 0.75 milligram per kg body weight per dose, such as from about 0.75 milligram per kg body weight per dose to about 1.0 milligram per kg body weight per dose, for example from about 1.0 milligram per kg body weight per dose to about 2.5 milligram per kg body weight per dose, such as from about 2.5 milligram per kg body weight per dose to about 5 milligram per kg body weight per dose, for example from about 5 milligram per kg body weight per dose to about 7.5 milligram per kg body weight per dose, such as from about 7.5 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 10 milligram per kg body weight per dose to about 25 milligram per kg body weight per dose, such as from about 25 milligram per kg body weight per dose to about 50 milligram per kg body weight per dose, such as from about 50 milligram per kg body weight per dose to about 75 milligram per kg body weight per dose, for example from about 75 milligram per kg body weight per dose to about 100 milligram per kg body weight per dose, such as from about 100 milligram per kg body weight per dose to about 250 milligram per kg body weight per dose, for example from about 250 milligram per kg body weight per dose to about 500 milligram per kg body weight per dose, such as from about 500 milligram per kg body weight per dose to about 750 milligram per kg body weight per dose, for example from about 750 milligram per kg body weight per dose to about 1000 milligram per kg body weight per dose.

Co-Administration with One or More Drugs

The composition according to the present invention can be co-administered to an individual in need thereof in combination with one or more drugs such as one or more drugs with antimicrobial effect.

The composition according to the present invention can be co-administered to an individual in need thereof in combination with one or more antifungal drugs. The one or more antifungal drugs can be selected from the group consisting of polyene antimycotics such as Natamycin, Rimocidin, Filipin, Nystatin, Amphotericin B, and Candicin, imidazole and triazole antifungal drugs such as Imidazoles like Miconazole (Miconazole nitrate), Ketoconazole, Clotrimazole (marketed as Lotrimin, Canesten in the UK), Econazole, Bifonazole, Butoconazole, Fenticonazole, Isoconazole, Oxiconazole, Sertaconazole (marketed as Ertaczo), Sulconazole, Tioconazole, Fluconazole, Itraconazole, Isavuconazole, Ravuconazole, Posaconazole, Voriconazole, and Terconazole, Allylamines such as Terbinafine (marketed as Lamisil), Amorolfine, Naftifine (marketed as Naftin), and Butenafine (marketed as Lotrimin Ultra), Echinocandins such as Anidulafungin, Caspofungin, and Micafungin, Benzoic acid in combination with a keratolytic agent (such as in Whitfield's Ointment), Ciclopirox olamine, Flucytosine, or 5-fluorocytosine, Griseofulvin, and Gentian Violet Haloprogin Tolnaftate (marketed as Tinactin, Desenex, Aftate).

The composition according to the present invention can be co-administered to an individual in need thereof in combination with one or more antiparasitic drugs. The one or more antiparasitic drugs can be selected from the group consisting of Antinematodes such as Mebendazole (for most nematode infections), Pyrantel pamoate (for most nematode infections), Thiabendazole (for roundworm infections), and Diethycarbazine (for treatment of Lymphatic filariasis), The method according to item 197, wherein the one or more antiparasitic drugs comprises Anticestodes such as Niclosamide (for tapeworm infections), and Praziquantel (for tapeworm infections), Antitrematodes such as Praziquantel, Antiamoebics such as Rifampin, Amphotericin B, Clioquinol, Iodoquinol Metronidazole, Tinidazole, Ornidazole, Secnidazole Atovaquone, Emetine, Fumagillin, and Trimetrexate, Antiprotozoals such as Amphotericin, Antimony, Eflornithine, Furazolidone, Melarsoprol, Metronidazole, Miltefosine (Impavido), Ornidazole, Paromomycin sulfate, Pentamidine, Pyrimethamine, and Tinidazole.

Kit of Parts

In a further embodiment the present invention relates to a kit of parts comprising the antimicrobial composition according to the present invention. The kit of parts comprises at least one additional component, such as instructions for use, and/or one or more drugs for co-administration.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1shows the phylogeny of the Crustacea. The closest relatives to Copepoda are the Cephalocarida and the Ostracoda. It is clear from the phylogenetic tree of the Crustacea subphylum that copepods and decapods are not closely related. Decapods include many familiar groups, such as crayfish, crabs, lobsters, prawns and shrimp.

FIG. 2shows the Development of the number of viableSalmonella bertacells in Nutrient Broth and Nutrient Broth with added 10% aqueous phase fromCalanus finmarchicus(catch 2) during incubation at 37° C.

FIG. 3. HPLC-chromatogram of the anion exchange extract. The majority of the compounds including the active compound(s) elutes early during the run. This suggests that there are mostly polar or charged compounds in the extracts. Furthermore, C18-reversed phase chromatography is not the preferred method for separating the bioactive peak from contaminants.

FIG. 4. Comparison of analytic chromatography of early fractions re-chromatographed on a conventional C18 (left), or polar endcapped C18 (right). Fraction 2 is the lower, and fraction 7 the upper chromatogram. The right panel show better separation of the peaks making it possible to distinguish the different peaks. The active fraction (fraction 4, the third chromatogram counting from below) is separated into four different peaks with the Aquasil column (right panel), whereas the conventional C18 column gives no base-line separation of the same fraction (left panel).

FIG. 5. Chromatogram of the semipreparativ C18-purification. Similar to the analytical run, the bioactivity eluted early during the run (see also Table 22).

FIG. 6. Isocratic normal phase chromatography of the bioactive fraction from the semi-preparative reversed phase purification (FIG. 5). The lower panel is 40/60 and the upper panel is 1/99 EtOAc/MeOH. The chromatogram shows that normal phase gives a good separation of at least 4 different peaks that occur as one peak during the reversed phase chromatography. Normal phase chromatography is probably the best method for separation of the active compound.

FIG. 7. Isocratic normal phase chromatography of the active peak from the semi-preparative reversed phase fractionation (FIG. 5). See Table 22 for bioactivity of the fractions. The active fraction (4, approximately 3-4 min elution time) consists of several compounds, as seen by the shoulders of the peaks occurring at around 4 minutes. This could be due to the presence of several contaminants, but also several variants of the same compounds.

FIG. 8. UV-spectra of the various peaks from the normal phase chromatography shown inFIG. 7. The UV-spectra collected at different time-points in peak to varied little, suggesting that peak two consists of variants of the same compound.

FIG. 9. MS-analysis of fraction 4 fromFIG. 8. When the masses from the background (blank injection) was subtracted, the 245 and 347 peaks stood out as the most interesting.

FIG. 12. The structure of penostatin with a mass=346 Da.

EXAMPLES

Preparation of Extracts fromC. finmarchicus

Catch and Storage ofCalanus finmarchicus

Catch 1 was caught in the fjords of northern Norway. The catch was frozen on board the fishing vessel and stored frozen until examination. Composition (g/100 g): Water: 79.9, protein: 11.1, fat: 6.3, ash: 2.0.

Catch 2 was caught in Andfjord in northern Norway. The catch was frozen on board the fishing vessel within one hour after catch and stored frozen until examination. Composition (g/100 g): Water: 83.0, fat: 5.2.

Preparation of the Aqueous Phase

100 g frozen sample were thawed at ambient temperature and then centrifuged at 5.000×G for 10 minutes. The sample separated into three fractions where the sediment constituted approximately 60 g, the aqueous phase 38 g and the oil 2 g. The aqueous phase was filtered through a glass fiber filter (Schleicher & Schull, ref. no. 370003) and then through a micro porous cellulose acetate filter with pore size 0.2 μm (Whatman, FP 30/02CA-S). The sterile aqueous phase was stored in sterile capped glass tubes at 4-8° C.

The effect of crushing thawedC. finmarchicusin a mortar before centrifugation and filtration was examined (data not shown). The crushing did not affect the antimicrobial activity in the aqueous phase, but resulted in difficulties with the filtration and hence to reduced yields (volume) of filtrate. Therefore, thawedC. finmarchicusshould not be crushed before centrifugation and filtration.

Different filter types were tested, e.g. purified cotton (IVBF Hartmann AG, CH 8212, Neuhausen, Switzerland), glass fiber filters (Schleicher & Schull, ref no. 370003), paper filters (Schleicher & Schull, Black Ribbon, ref no. 589) and microporous cellulose acetate filters with pore size 0.2 μm. The main purpose of filtering the aqueous phase after centrifugation was to remove particles and thus to give a clear liquid suitable for testing by the BTD method. The antimicrobial composition was not retained by any of the filter materials tested. The clearest liquid was obtained by the microporous filters, but the flow rate through this filter was significantly improved by pre-filtering through e.g. glass fiber filters.

Preparation of Methanol Extract

A concentrated composition with increased antimicrobial activity is prepared by methanol extraction.

FrozenC. finmarchicuswere freeze dried before methanol-extraction and vacuum evaporation to remove the solvent. The concentrated extract was finally dissolved in deionised water and filtered through a micro-porous cellulose acetate filter with pore size 0.2 μm.

A comparison of the chemical composition and the antimicrobial activity of aqueous phase (catch 2) and methanol extract (catch 2) fromC. finmarchicusis provided in table 5. The antimicrobial activity is much higher in the methanol extract compared to the aqueous phase.

Determination of MIC (Minimal Inhibitory Concentration)

The activity and presence of antimicrobial substances in samples of aqueous phases or methanol extracts were tested by the BTD (Broth Tube Dilution) method, where 2-fold serial dilutions of sample in sterile water were added the same volume of double strength basis medium inoculated with the individual test organisms. After incubation as specified below, the test results were read. The lowest concentration (or highest dilution) preventing appearance of turbidity was considered to be the MIC (Minimal Inhibitory Concentration).

Preparation of Inoculated Double Strength Basis Media: Clostridia were Pre-Cultivated overnight in LTM (Liquid Thioglycollate Medium, Oxoid CM173) at 37° C. under anaerobic conditions. All other bacteria were pre-cultivated overnight in NB (Nutrient Broth, Oxoid CM0067) at 37° C. under aerobic conditions. All fungal species were pre-cultivated aerobically at 37° C. in NB supplemented with 1% glucose, until visible turbidity. Double strength basis media were finally inoculated with an amount of pre-culture sufficient to obtain an initial cell density of approximately 10E5/ml to 10E6/ml.

Detection of Antimicrobial Activity in Aqueous Phase

Antimicrobial activity has been detected in the aqueous phase from two different catches ofC. finmarchicus(June/July 2003 and June 2006).

The activity and presence of antimicrobial compounds or substances in the aqueous phase extracted from catch 1 was tested by the BTD method using three different test bacteria. Growth of all the test bacteria was inhibited. The MIC values obtained from the results (table 6) forE. coli, S. aureusandP. aeruginosawere 8, 16 and 4, respectively.

The range of action of the antimicrobial substance in the aqueous phase extracted from catch 2 was tested by the BTD method using nine different test bacteria (P. aeruginosa, E. faecalis, L. monocytogenes, B. cereus, S. aureus, E. coli, S. berta, C. perfringens, C. bifermentansand one fungal species (C. albicans). All of the test bacteria, which represent Gram +, Gram −, aerobic and strict anaerobic types, were inhibited (table 7). The tested fungal species was not inhibited.

Inactivation Kinetics

The course of killing was examined by inoculatingS. bertain 100 ml NB and in 90 ml NB with added 10 ml of aqueous phase extracted from catch 2. The initial cells concentration was approximately 10 E5 per ml. The bottles were incubated at 37° C., and aliquots withdrawn during incubation for determination of viable aerobic microorganisms (Nordic Committee on Food Analysis no. 86). The bacteria grew well in pure NB while in the presence of ×10 diluted aqueous phase, the number of viable cells underwent reduction. From the formula of the exponential trend line (FIG. 2), decimal reduction time could be estimated to 6.8 hours.FIG. 2shows the Development of the number of viableSalmonella bertacells in Nutrient Broth and Nutrient Broth with added 10% aqueous phase fromCalanus finmarchicus(catch 2) during incubation at 37° C.

Heat Stability

The heat stability of the antimicrobial substance in aqueous phase from catch 2 was tested by the BTD method usingS. aureusas a test organism. Heat treatment at 70° C. for 10 minutes had no effect on the activity of the antimicrobial substance. Heat treatment at 100° C. for 10 minutes caused a slight reduction of activity and 121° C. for 15 minutes caused a substantial reduction (table 8).

Resistance to Proteolytic Enzymes

The resistance of the antimicrobial composition to digestion by three proteolytic enzymes, pepsin, alcalase and proteinase K was tested by the BTD method usingS. aureusas a test organism.

1 ml of the aqueous phase from catch 2 was added to 0.2 ml 1.0 N HCl and 0.1 ml 0.1 N HCl with 1% (w/v) pepsin (article no. 1.07190.0100, Merck). The reaction mixture (pH 2.36) was incubated at 37° C. for 18 hours with occasional stirring. As a control, 1 ml of the aqueous phase was incubated similarly after addition of the same amount of acid without pepsin. The results of the BTD test (table 9) show that the antimicrobial substance was not inactivated by pepsin treatment. Therefore, the active one or more antimicrobial compounds in theC. finmarchicuscomposition is probably not a protein.

1 ml of the aqueous phase from catch 2 was added 0.3 ml 0.1 N NaOH to obtain pH 8.0 and then added 0.2 ml 1% (w/v) alcalase 2.4 (Novozymes) adjusted to pH 8.0. The reaction mixture (pH 8.0) was incubated at 54° C. for 4 hours with occasional stirring. As a control, 1 ml of the aqueous phase was incubated similarly after addition of the same amount of alcali without alcalase. The results of the BTD test (table 10) show that the antimicrobial substance was not inactivated by alcalase treatment. Therefore, the active one or more antimicrobial compounds in theC. finmarchicuscomposition is probably not a protein.

1 ml of the aqueous phase from catch 2 was added 0.1 ml 0.5% (w/v) Proteinase K (Merck 1.24568.0100) in water. The reaction mixture (pH 6.5) was incubated at 37° C. for 4 hours with occasional stirring. As a control, 1 ml of the aqueous phase (pH 6.5) was incubated similarly after addition of 0.1 ml water without Proteinase K. The results of the BTD test (table 11) show that the antimicrobial substance was not inactivated by Proteinase K treatment. Therefore, the active one or more antimicrobial compounds in theC. finmarchicuscomposition is probably not a protein.

Estimation of Molecular Size of the Antimicrobial Substance

Molecular size of the antimicrobial substance, i.e. the size of the one or more antimicrobial compounds comprised within the antimicrobial composition of the present invention, was estimated by processing the aqueous phase through centrifugal filter devices (Pall Corporation) with MWCO (molecular weight cut off) of 30, 3 and 1 kDa. The results of the BTD test (table 12) indicate that the molecular weight of the antimicrobial substance is equal to, or less than 1 kDa.

Type of Action

There are three types of action of antimicrobial agents; i) static action where growth is inhibited, ii) cidal action where organisms are killed and iii) lytic action where organisms are killed and lysed.

After 24 hours incubation of the test tubes in table 7 (aqueous phase), 10 μl from each ×2 dilution was transferred to selected agar media. No colonies could be observed after 72 hours incubation of the agar media. Consequently, an initial cell concentration of approximately 10 E7 per ml was reduced to less than 10 E2 per ml, corresponding to 5 LOG10cycles reduction. The type of action is therefore cidic or lytic.

To determine whether the type of action is cidic or lytic, exponential phase cultures ofEscherichia coli, Salmonella berta, andStaphylococcus aureusin Nutrient Broth were diluted to 1.0 E7-1.0 E8 in the same medium. 1 ml methanol extract were added to 9 ml of each culture, immediately after quantification at 0 hours. Cultures were then incubated at 37° C. for 96 hours. Viable cell counts were determined using 3M Petrifilm Aerobic Count Plates while total cell counts were determined using a microscope and Helber counting chamber. The results depicted in table 13 show that the action is bacteriocidic, not bacteriolytic.

Investigation of Antimicrobial Activity inC. hyperboreus

Antimicrobial activity was not detected in the aqueous phase from another copepod species,C. hyperboreus.

Aqueous phase prepared from a frozen sample ofC. hyperboreuswas tested againstS. aureus(ATCC 25923). A methanol extract ofC. finmarchicuswith known activity was tested simultaneously against the same test organism as a control on the test system.C. hyperboreuswas caught in the Norwegian Sea on May 15, 2006. Antimicrobial activity was not detected in the aqueous phase fromC. hyperboreus.

Comparison of BTD and Agar Diffusion Assay

Antimicrobial activity in the aqueous or water phase fromC. finmarchicus, was easily detected using the BTD (Broth Tube Dilution) method. Surprisingly, the agar diffusion assay, which is frequently used for the initial screening of antimicrobial activity, did not detect this activity. This may explain why the antimicrobial activity in water phase from this organism has not been discovered earlier.

When the BTD method was compared to the agar diffusion assay for detection of activity in the methanol extract fromC. finmarchicus, it appeared that the BTD method was 32-64 times more sensitive than the agar diffusion assay.Staphylococcus aureus(ATCC 25923) was used as test strain in both the Broth Tube Dilution Method and the Agar Diffusion Assay. With the Agar Diffusion Well method, well diameter was 9 mm and each well was filled with 200 μl of the extract or its dilution. In the Agar Diffusion Filter disc method, 13 mm diameter discs (Whatman Cat. No. 2017-013) was used. Results are shown in table 15.

TABLE 15Comparison of BTD and Agar Diffusion Assay for detection ofantimicrobial activity in methanol extract fromC. finmarchicus.Testorgan-Dilutiorism0× 2× 4× 8× 16× 32× 64× 128× 256WaterBroth−−−−−−−++TubeDilutionAgar131050000000Diffu-sionAssay,WellmethodAgar4100000000Diffu-sionAssay,Filterdiscmethod+: turbidity,−: no turbidity.For the Agar Diffusion Assay, activity is expressed by inhibition zone width (mm).

The agar diffusion assay is a technique for quantifying the ability of antimicrobial agents to inhibit bacterial growth. It has a number of variations, including the well method and the filter disc method. Interpretation of the results from this assay relies on mathematical models, based on the assumption that the agents diffuse freely in the solid nutrient medium. The technique is commonly used for determination of MIC values (Minimal Inhibitory Concentration) in solid media, such as Nutrient Agar. Solutions of antimicrobial agents at different concentrations are applied to wells punched into agar or to paper discs placed on the surface of or plates seeded with the test microbial strains. Diffusion from the sources into the agar leads to growth inhibition in the vicinity of the source and to the formation of clear zones without bacterial growth. The zone diameter corresponds to the concentration of the antimicrobial agent.

Antifungal Activity of Methanol Extract

A mould species,Aspergillus niger(ATCC 16404), was inhibited by the aqueous phase fromC. finmarchicus(data not shown).

The methanol extract was tested against two yeast species;Saccharomyces cerevisiaeandCandida albicansand one mould species;Aspergillus niger. C. albicansis the least susceptible of the organisms tested.S. aureuswas included as a control. Results are shown in table 16.

Effect on Resistant Bacterial Strains

The methanol extract was tested against meticillin-resistantS. aureus(ATCC 43300) and multiresistant clinical isolates ofP. aeruginosa, Klebsiella pneumoniaeandAcinetobacter. All the isolates were strongly inhibited.S. aureus(ATCC 25923) was included in the study as a positive control. Results are shown in table 17.

The strain is resistant to all β-lactam antibiotics. The treatment of MRSA is therefore difficult. The strain can cause both local and systemic infections.

The strain has a plasmid-mediated, broad spectrum β-lactamase (Klebsiella PneumoniaeCarbapenemase (KPC) and is resistant to all clinical relevant β-lactam antibiotics. Furthermore, it is in vitro resistant to many other non β-lactam antibiotics. Infections with such microbes are difficult to treat with available antibiotics.K. pneumoniaeis a common cause of urinary tract infections, but can also cause systemic infections.

P. aeruginosais naturally resistant to a range of different antibiotics, and the spectre of efficient agents is narrow. Additionally, it has a high ability to develop resistance to new antibiotics. The actual strain is in vitro resistant to, or has reduced susceptibility to antimicrobial agents commonly used in the treatment ofP. aeruginosainfections. Severe infections withP. aeruginosaoccur with immune deficient patients and with weak, hospitalized patients.

Acinetobacteris naturally resistant to a range of different antibiotics, and the spectre of efficient agents is narrow. With one exception, the actual isolate in vitro resistant to, or have reduced susceptibility to all antimicrobial agents used for treatment of Acinetobacter infections. Severe infections withAcinetobacteroccur with immune deficient patients and with weak, hospitalized patients.

Further Extraction and Analysis of the Methanol Extract

Sample Preparations

Three different sample preparations were tested. This was done to find the best preparation prior to HPLC, i.e. the method that had best recovery, and at the same time rendered a fraction that was pure enough to be loaded onto an HPLC-column.

The extract was dissolved in water and methanol, added chloroform and vortexed. The result is three fractions; water-methanol, methanol-chloroform and the precipitate. All fractions were evaporated and tested for activity. The activity is measured as the MIC as described in example 2.

The cartridge (Sep-Pak plus QMA, Waters corp.) was first wetted with methanol, and then equilibrated with water. The extract was loaded and the cartridge was washed with water, and finally with methanol to elute polar and weak anionic compounds. Two fractions: Water wash and methanol elute. The fractions were evaporated, dissolved in a volume of water corresponding to the sample load volume, and tested for bioactivity.

TABLE 19Bioactivity in the anion exchange SPE fractions.FractionMICWash64Methanol elute0
3: Solid Phase Extraction, Reversed Phase.

The cartridge (Sep-Pak plus C18, Waters corp.) was first wetted with acetonitrile, and then equilibrated with water. The extract was loaded onto the cartridge, and the cartridge was washed with water, then with 40% acetonitrile and 100% acetonitrile to elute non-polar compounds. Three fractions: Wash, elute 1 (intermediate polarity) and elute 2 (low polarity). The fractions were evaporated and tested for bioactivity

TABLE 20Bioactivity in the reversed phase SPE fractions.FractionMICWash32Elute 116Elute 22

The bioactive compound(s) is probably polar or charged since it was found in the aqueous fraction from the Wessel-Flügge extraction as well as the wash fraction in the reversed phase extraction. It is not likely to be a strong anionic compound since it failed to bind to the anionic solid phase cartridge. Anion exchange solid phase extraction was used as sample preparation prior to HPLC since all the activity eluted in one fraction.

Our first attempt was conventional reversed phase chromatography. The active fraction (wash) from the anion exchange extraction was dried and dissolved in water and injected onto a C18 column (250×4.6 mm). The gradient was from 96% water to 100% acetonitrile as shown inFIG. 3(green line). One-minute fractions were collected, evaporated and dissolved to the sample volume, and tested for bioactivity.

TABLE 21Bioactivity of the analytical reversed phase HPLC fractions.FractionMIC203041658647080Later fractions had no bioactivity
Comparison of Chromatography with or without Polar Endcapping

The bioactivity data in Table 21 and the corresponding chromatogram (FIG. 3) suggest that the bioactivity is too polar to be separated by conventional reversed phase chromatography. The active fractions were re-chromatographed under identical conditions, or with a column with polar endcapping (Aquasil, Hypersil). The latter column is suitable for isolation of polar or charged compounds. This column gave better separation of the active fraction (Fraction 4,FIG. 4). However, the Aquasil-column was too small to make fractions for bioactivity testing. Up-scaling to a semi-preparative Aquasil column failed.

The extract from the anion exchange SPE was loaded onto a semi-preparative C18 column and eluted with a gradient with increasing acetonitrile. Fractions were collected and tested for bioactivity. As shown in Table 22, the bioactivity eluted early. The active fraction was used for further experiments in purification.

The active fraction from the semi-preparative reversed phase chromatography (FIG. 5and table 22) was loaded onto an analytical normal phase column under isocratic mode. As mobile phase, six different ratios of ethyl acetate (EtOAc) and methanol (MeOH) was tried: 1/99; 5/95; 10/90; 20/80; 40/60 (EtOAc/MeOH, v/v %).

This turned out to be a promising method, and another run with more material was performed. One-minute fractions were collected and tested for bioactivity.

TABLE 23Bioactivity of the normal phase fractions.FractionMIC30445<2607080
Mass Spectrometric Analyses of the Active Peak.

The experiment described in the previous paragraph was repeated and fraction 4 was analysed by a Q-trap mass spectrometer (direct infusion). Two peaks stood out from the background (m/z=347 and 245). These were further fragmented, but no certain conclusions can be drawn based on these data.

A search on various databases on natural compounds with mass=346 Da (corresponding to m/z=347), returned a hit on penostatin depicted inFIG. 12(Iwamoto et al. Tetrahedron, 1999 vol. 55 p. 14353). This is a mild cytotoxic compound that was isolated from a fungus that grows on the green algaeEnteromorpha intestinalis. The UV-spectra of this and related compounds are similar to that of the active fraction from the normal phase purification (FIG. 8).