Composition and method of use

Abstract

Compositions, methods for preparing the same and uses thereof are provided. The compositions may be used to beneficially modulate and / or remodel the gut microbiome. It is considered the compositions can increase the production of short chain fatty acids (SCFA) and aromatic amino acids; and increase intestinal microbial diversity.

Description

[0001] COMPOSITION AND METHOD OF USE

[0002] Field of the Invention

[0003] Compositions are provided that beneficially modulate and / or remodel the gut microbiome. It is considered the compositions can increase the production of short chain fatty acids (SCFA) and aromatic amino acids; and increase intestinal microbial diversity.

[0004] Methods for obtaining said compositions are also provided, as well as methods of using the compositions to beneficially modulate and / or remodel the gut microbiome. Suitably there is provided the use of the compositions in preventing (prophylactic treatment) and / or treating diseases (symptoms) or disease associated with chronic inflammation and the gut-brain axis (GBA).

[0005] Background of the Invention

[0006] The practice of ingesting clays recovered from specific localities and for a variety of therapeutic purposes was well attested in the Greco-Roman world. Lemnian Earth is an example of such a clay, obtained from the island of Lemnos in Greece. Lemnian Earth has a documented record of near- continuous use from the 4thcentury BCE to the late 19thand early 20thcentury CE (~2,500 years).

[0007] In antiquity, Lemnian Earth was extracted from a pit and prepared by a priestess at the local temple who, following a ceremony, shaped the clay into pellets / tablets / pills and stamped them with a distinctive seal - giving rise to an alternative name, i.e. terra sigillata ('sealed / stamped earth'). These pills were then taken orally with wine, on their own, or mixed with botanicals; the prevailing view being that Lemnian Earth was useful ‘against poison’ (ingested or injected) and for wounds that ‘were slow to heal’. Furthermore, in the post-medieval and Ottoman period, Lemnian Earth was consumed as a preventative ‘against the plague’. Analysis of museum samples of Lemnian Earth indicated that the samples possess in vitro antibacterial activity, whereas mineralogically similar geological samples from the region are inert. This suggests that the bioactive effects of LE are due to its method of processing, rather than properties inherent in the natural Lemnian clay.

[0008] An account of Lemnian Earth’s properties and its method of preparation was provided by Galen who visited Lemnos in the late 2ndcentury CE. (Galen, IX 2 in Kuhn 1826, p. 169-171 , II. 1 -4, Translation by Brock 1929, p. 193, incorporated by reference). This account includes the following passage:

[0009] “The priestess collects this, to the accompaniment of some local ceremony, no animals being sacrificed, but wheat and barley being given back to the land in exchange. She then takes it to the city, mixes it with water so as to make moist mud, shakes this violently and then allows it to stand. Thereafter she removes first the superficial water, and next the greasy part of the earth below this, leaving only the stony and sandy part at the bottom, which is useless. She now dries the greasy mud until it reaches the consistency of soft wax; of this she takes small portions and imprints upon them the seal of Artemis; then again she dries these in the shade till they are absolutely free from moisture. ”

[0010] Photos Jones, Technai, 1 , 2023 (herein incorporated by reference) proposes an interpretation of this method, as shown in Fig. 1 - see Brief Description of the Figures.

[0011] Sources from the Greco-Roman period all agree that Lemnian Earth was red in colour. However, clay tablets of various colours were sold as “Lemnian Earth” in later periods - see Fig. 2. Interestingly, when similar tablets in use in the 19thcentury were analysed by contemporary chemists, they were deemed to have “absolutely no therapeutic properties” (Tourptsoglou-Stefanidou 1988, p. 506., herein incorporated by reference). Nevertheless, over its 2,500-year history of use, a number of medical texts name specific ailments cured by Lemnian Earth; suggesting that “true” Lemnian Earth must have been bioactive. However, the actual therapeutic effects of bioactive Lemnian Earth were never definitively determined.

[0012] Summary of the Invention

[0013] A dysregulated / unbalanced gut microbiome, caused by dietary and / or environmental factors, is believed to be a contributing factor in the development of several chronic diseases, such as those listed below. Therefore, modulation of the microbiome in a targeted fashion represents a valuable tool for treating and preventing disease progression.

[0014] SCFAs are primarily produced by gut microbiota from the breakdown of dietary fibres. These compounds are known ligands for immune receptors in the intestinal epithelium, but they are absorbed systemically (including into the brain). Their net effect is to ameliorate local and systemic inflammatory pathways. SCFAs are the principal biomarker used in the literature and clinical studies for correlating gut microbiome modulation with changes to the host immune system.

[0015] There is a strong correlation between reduced / insufficient SCFA levels and chronic inflammatory diseases and / or diseases associated with the gut-brain axis:

[0016] - Suitably, chronic inflammatory diseases may include inflammatory bowel disease (IBD, including Crohn’s disease and / or ulcerative colitis), spondyloarthritis, psoriasis and psoriatic arthritis, rheumatoid arthritis, metabolic syndrome, obesity, chronic kidney disease, cardiovascular disease, atherosclerosis, asthma and / or irritable bowel syndrome. - Suitably, a disease associated with the gut-brain axis (GBA), may include Alzheimer’s disease and various mental health issues, such as seasonal affective disorder and depression, and / or conditions like ADHD, neuroses, and anxiety disorders.

[0017] At present, the only known means of inducing increased SCFA production are: the ingestion of live cultures of lactobacilli as a probiotic, the ingestion of fermented foodstuffs such as kimchi or sauerkraut, or regular ingestion of dietary fibres (prebiotics). Some disadvantages to these methods include their limited shelf life, the need for cold chain transport and storage; and the volume required by such means. Each of these disadvantages considerably increase the commercial costs involved in such use. Another disadvantage of the foodstuffs is that they also contain fat, protein and additional calories that may not be desirous.

[0018] Probiotic supplements help maintain a healthy gut microbiome by increasing the populations of bacteria (e.g. Lactobacillus) which can assist digestion, outcompete opportunistic pathogens and synthesise metabolites (e.g. SCFAs and vitamins) for immune system regulation. However, clinical studies on probiotics for treatment of chronic inflammatory diseases remain inconclusive (apart from alleviating symptoms). It is difficult to control quantities and ratios of SCFAs (e.g. the ratio of acetate to propionate to butyrate) with probiotics.

[0019] Advantageously, the inventors have determined compositions that are able to beneficially modulate and / or remodel the gut microbiome, including compositions that increase the production of short chain fatty acids (SCFA) and aromatic amino acids, and increase intestinal microbial diversity.

[0020] The present invention provides novel compositions that can beneficially modulate the gut microbiome. Such compositions are suitable for treating certain disease states, such as those listed below. The invention also provides pharmaceutical compositions containing the compositions, as well as a method for the treatment and / or prevention of certain disease states.

[0021] As part of a re-evaluation of the literature and a review of the archaeological, geological and hydrological record, the inventors were surprised to discover that, contrary to the ancient tradition of Lemnian Earth (LE) consumption, the waste by-product of the proposed method of making bioactive Lemnian Earth (i.e. the “superficial water” mentioned above) beneficially modulates the gut microbiome, and thus may be used to treat and / or prevent certain disease states.

[0022] Without wishing to be bound by theory, the inventors believe that all of the literature references to Lemnian Earth solely focus on the efficacy of the dried clay tablets, and the “superficial water” was not considered to have any useful properties. Indeed, it was previously considered that the Lemnian clay could sequester fungi and release them in the intestine, thus accounting for the reported health benefits.

[0023] The inventors were surprised to discover that culturing Penicillium / Talaromyces fungi in growth media containing smectite (a clay), then using a filter to remove the fungi and the smectite provides a composition in the form of a filtrate, where the composition is able to beneficially modulate the gut microbiome. This may be consumed orally as is, or it may be dried to provide a powder that is then consumed. Alternatively, extracts, isolates or compounds present in the filtrate may be used.

[0024] Several clays (e.g. smectite, vermiculite, kaolinite, illite, palygorskite or nontronite) have a layered structure where the layers are electrostatically crosslinked. These layers are not rigidly connected to each other and may be separated by a free space: the interlayer. This interlayer can host hydrated cations (e.g. Ca2+, Na+, K+) and water molecules. As such, on immersion in water, clays with this structure are able to swell. This is due to the reversible incorporation of water and cations in the interlayer space.

[0025] Without wishing to be bound by theory, it is considered that on co-culturing clays with this structure (e.g. smectite), the clays may act as a “stressor” to the above fungi, causing the fungi to exhibit a stress response that includes commencing or upregulating the production of extracellular secondary metabolites. The inventors were surprised to discover that the culture media or extracts thereof provide beneficial effects in a patient in need thereof.

[0026] Preferably, said clays have a phyllosilicate mineral structure. More preferably, said clays have a three layer tetrahedra-octahedra-tetrahedra (TOT) structure.

[0027] Upon the expansion of the interlayer space due to hydration (including following delamination), the interlayered space of this structure provides an active / charged surface that interacts with the fungi to induce the production of secondary metabolites.

[0028] Accordingly, the first aspect of the present invention is a method of manufacturing a composition, the method comprising the steps:

[0029] 1 . Providing a fungi of the Penicillium, Talaromyces, Monascus or Aspergillus genera,

[0030] 2. Providing a clay that swells upon hydration due to expansion of interlayer spacing,

[0031] 3. Combining the fungi and the clay in a growth culture media,

[0032] 4. Culturing the growth culture medium under conditions suitable for growth of the fungi,

[0033] 5. Removing substantially all of the fungi and clay from the growth culture media, to provide the composition. A second aspect of the present invention is a composition or pharmaceutical salt thereof, wherein the composition or pharmaceutical salt thereof is manufactured via a method comprising:

[0034] 1 . Providing a fungi of the Penicillium, Talaromyces, Monascus or Aspergillus genera,

[0035] 2. Providing a clay that swells upon hydration due to expansion of interlayer spacing,

[0036] 3. Combining the fungi and the clay in a growth culture media,

[0037] 4. Culturing the growth culture medium under conditions suitable for growth of the fungi,

[0038] 5. Removing substantially all of the fungi and clay from the growth culture media, to provide the composition.

[0039] Suitably, the interlayers of the clay may comprise a layer with a negative layer charge, preferably where the clay has cation exchange capacity. Suitably, the exchangeable cation(s) may be selected from a group comprising sodium, calcium and potassium.

[0040] Suitably, the clay may have a phyllosilicate mineral structure.

[0041] Suitably, the clay may have a mineralogical structure made of three superimposed sheets of tetrahedra-octahedra-tetrahedra (TOT) layers.

[0042] In some embodiments, the clay comprises or essentially consists of a clay of the smectite, vermiculite, kaolin or illite group of minerals. Preferably, the clay comprises or essentially consists of a clay of the smectite or vermiculite group of minerals. More preferably, the clay comprises or essentially consists of Na- smectite.

[0043] Smectites are known for their high specific surface area and significant cation exchange capacity (CEC) for inorganic cations and organic molecules. When in solution (such as growth media, e.g. potato dextrose growth media), smectite clays readily adsorb molecules from the solution, thus increasing the interlayer space. An increase in the interlayer space causes swelling, which can result in the delamination of the clay lamellae, thereby increasing the area of contact between the clay and the fungi and intensifying the interaction therebetween.

[0044] Likewise, vermiculite provides similar properties to the smectite group with regards to CEC and swelling capacity.

[0045] Minerals of the kaolin group (such as kaolinite) have a non-permanent, pH- depended charge. Minerals of the illite group have a permanent charge but lack cation exchange capacity.

[0046] Suitably, the composition may comprise one or more of the following: ankaflavin, mitorubrin, mitorubrinol, monascin, ochratoxin, patulin, PP-R, PP- V and roquefortine C, or a pharmaceutical salt thereof. Suitably, the composition may comprise one or more of the following: ankaflavin, mitorubrin, mitorubrinol, monascin, PP-R, PP-V and roquefortine C.

[0047] Suitably, the fungi may comprise Talaromyces purpurogenus (also known as Penici Ilium purpurogenus), syn. Talaromyces purpureogenus, Penicillium sanguineum, Talaromyces tardifaciens, Penicillium roqueforti, Penicillium rubrum, Penicillium monascum, Monascus purpureus, Aspergillus ochraceus, Aspergillus carbonarius and / or Aspergillus niger.

[0048] Preferably, the fungi are fungi of the Penicillium or Talaromyces genera. Further preferably, the fungi comprise Talaromyces purpurogenus, syn. Talaromyces purpureogenus, or P. sanguineum.

[0049] Without wishing to be bound by theory, it is considered fungi of the Penicillium, Talaromyces, Monascus or Aspergillus genera, preferably fungi of the Penicillium or Talaromyces genera produce extracellular secondary metabolites with beneficial properties - for instance, that can be used to induce the production of SCFAs and / or increasing microbial diversity.

[0050] A further aspect of the present invention is a pharmaceutical composition comprising a therapeutically effective amount of the composition or pharmaceutical salt thereof of the second aspect or one made from the first aspect.

[0051] A further aspect of the present invention is a pharmaceutical composition for use in modulating gut SCFA and / or amino-acid production. This may be used to modulate (increase) gut microbial diversity.

[0052] A further aspect of the present invention is a pharmaceutical composition for use in preventing a chronic inflammatory disease.

[0053] A further aspect of the present invention is a pharmaceutical composition for use in treating or preventing a disease associated with the gut / brain axis.

[0054] In some embodiments, the growth media is a fungal growth media, preferably a liquid fungal growth media, more preferably dextrose broth and / or potato broth.

[0055] In some embodiments, the method of the invention comprises culturing the growth media for between 7 and 21 days, preferably for about 14 days. Suitably, the incubation may be at between 20 and 30 °C with agitation, preferably at about 25 °C with agitation.

[0056] In some embodiments, the method comprises adjusting the pH to between about 5.5 and 9.5, preferably to a pH of about 7.5.

[0057] In some embodiments, following culturing, the method further comprises heating the growth media to denature the fungi. Suitably, the heating may be boiling at between about 95 and 105 °C for about 5-20 min.

[0058] Suitably, the step of removing substantially all of the fungi and clay may comprise either or both of centrifuging and / or filtering. Preferably, the removing substantially all of the fungi and clay from the growth media may be removing at least 80%, at least 90%, at least 95% or at least 99% of the fungi and clay from the growth media. More preferably, the removing substantially all of the fungi and clay from the growth media may be removing all of the fungi and clay from the growth media. Suitably, removing substantially all or all of the fungi and clay from the growth media may be achieved by filtering using different pore sizes. This has the advantage of including small amounts of the clay, which may comprise secondary fungal metabolites in the interlayer space.

[0059] In some embodiments, the method further comprises drying the composition. Suitably, the composition may be dried by lyophilisation. Suitably, the composition may be a powder.

[0060] In some embodiments of the third aspect, the composition comprises one or more of the following: ankaflavin, mitorubrin, mitorubrinol, monascin, ochratoxin, patulin, PP-R, PP-V and roquefortine C or a pharmaceutical salt thereof. Preferably, the composition comprises one or more of ankaflavin, mitorubrin, mitorubrinol, monascin, PP-R, PP-V and roquefortine C.

[0061] Compositions of the present invention, or pharmaceutically acceptable salts thereof may be useful for the treatment and / or prevention of a chronic inflammatory disease. Compositions of the present invention, or pharmaceutically acceptable salts thereof may be useful for the treatment and / or prevention of a disease associated with the gut-brain axis (GBA).

[0062] Suitably, a chronic inflammatory disease may be selected from a group consisting of inflammatory bowel disease (IBD, including Crohn’s disease and / or ulcerative colitis), spondyloarthritis, psoriasis and psoriatic arthritis, rheumatoid arthritis, metabolic syndrome, obesity, chronic kidney disease, cardiovascular disease, atherosclerosis, asthma and irritable bowel syndrome. Suitably, a disease associated with the gut-brain axis (GBA) may be selected from a group consisting of Alzheimer’s disease and various mental health issues, such as seasonal affective disorder and depression, and / or conditions like ADHD, neuroses and anxiety disorders.

[0063] In one embodiment, the invention provides a method of modulating gut SCFA and / or amino-acid production to modulate gut microbial diversity, the method comprising administration of an effective amount of a composition of the present invention to a subject in need thereof.

[0064] In another embodiment, the invention provides a method of treating and / or preventing a chronic inflammatory disease, the method comprising administration of an effective amount of a composition of the present invention to a subject in need thereof. Suitably the chronic inflammatory disease may be selected from a group consisting of inflammatory bowel disease (IBD, including Crohn’s disease and / or ulcerative colitis), spondyloarthritis, psoriasis and psoriatic arthritis, rheumatoid arthritis, metabolic syndrome, obesity, chronic kidney disease, cardiovascular disease, atherosclerosis, asthma and irritable bowel syndrome.

[0065] In another embodiment, the invention provides a method treating or preventing a disease associated with the gut / brain axis, the method comprising administration of an effective amount of a composition of the present invention to a subject in need thereof.

[0066] Suitably, a disease associated with the gut-brain axis (GBA) may be selected from a group comprising Alzheimer’s disease and various mental health issues, such as seasonal affective disorder and depression, and / or conditions like ADHD, neuroses and anxiety disorders.

[0067] The methods may comprise administering to a mammal in need of such treatment a therapeutically effective amount of a composition as disclosed herein. Compositions of the invention are also useful as modulators of gut SCFA production and / or amino-acid production, as well as changing gut microbial diversity, for studying the in vivo role of SCFAs in biological processes, including local (gut) and systemic inflammatory pathways. Accordingly, the invention also comprises a method of modulating SCFA production, aminoacid production and / or microbial diversity in vivo, the method comprising administering a compound or composition of the invention to a mammal.

[0068] Accordingly, another aspect of the present invention provides a method for the treatment or prevention of:

[0069] - a chronic inflammatory disease, such as inflammatory bowel disease (IBD, including Crohn’s disease and / or ulcerative colitis), spondyloarthritis, psoriasis and psoriatic arthritis, rheumatoid arthritis, metabolic syndrome, obesity, chronic kidney disease, cardiovascular disease, atherosclerosis, asthma, or irritable bowel syndrome and I or

[0070] - a disease associated with the gut-brain axis (GBA), including Alzheimer’s disease and various mental health issues, such as seasonal affective disorder and depression, and / or conditions like ADHD, neuroses or anxiety disorders.

[0071] The methods may comprise administering to a mammal in need thereof a therapeutically effective amount of a composition of the invention.

[0072] Another aspect of the present invention provides the use of a composition of the invention in the manufacture of a medicament for the treatment or prevention of a chronic inflammatory disease.

[0073] Another aspect of the present invention provides the use of a composition of the invention in the manufacture of a medicament for the treatment or prevention of a disease associated with the gut-brain axis. Suitably, a chronic inflammatory disease may be selected from a group consisting of inflammatory bowel disease (IBD, including Crohn’s disease and / or ulcerative colitis), spondyloarthritis, psoriasis and psoriatic arthritis, rheumatoid arthritis, metabolic syndrome, obesity, chronic kidney disease, cardiovascular disease, atherosclerosis, asthma and irritable bowel syndrome.

[0074] Suitably a disease associated with the gut-brain axis (GBA) may be selected from a group consisting of Alzheimer’s disease and various mental health issues, such as seasonal affective disorder and depression, and / or conditions like ADHD, neuroses and anxiety disorders.

[0075] An embodiment of the present invention will now be described with reference to the accompanying figures in which:

[0076] Brief Description of the Figures

[0077] Figure 1 A: A graphic illustration of a proposed ceremony for the manufacture of Lemnian Earth, based on Galen, IX 2 in Kuhn 1826, p. 171 , II. 1-4, Translation by Brock 1929, p. 193, from Photos Jones, Technai, 1 , 2023 (both of which are herein incorporated by reference).

[0078] • Images 1 , 2: the priestess ‘blesses’ the clay pit with wheat and barley ‘according to the local custom’.

[0079] • Images 3, 4: at the temple, the priestess making ‘pelos’ (mud) after pouring water in a ‘vat’ and mixing vigorously earth with water. Wheat and barley are thoroughly mixed in the pelos.

[0080] • Image 5: a settling period follows, whereupon the liparos pelos (greasy mud) consisting of fine clay-sized particles separates from the water and the coarse grained sediment (sandy and silty material) below it. • Image 6: The water layer (top) is removed to reveal a liparos pelos, below (top right), whose colour is ‘different’ from that of the original earth.

[0081] • Image 7: careful removal of liparos pelos and disposing of the coarse layer below.

[0082] • Images 8, 9: controlled drying of the liparos pelos, both prior to and after shaping and stamping it into pellets.

[0083] Figure 1 B: Illustration of the workflow of experiments and analyses of two embodiments of the present invention. (1 ) Penicillium purpurogenum was cocultured with smectite or kaolin clays (2); co-cultures were filtered (3) and their leachates were tested for antibacterial activity in vitro (4) to establish reduction in Gram-positive and Gram-negative bacterial numbers (4a); this step was followed by targeted analysis of fungal metabolites present in each sample (5). The leachates of both co-cultures were then fed to mice, as a supplement to normal chow (6) during in vivo experiments; mouse stool was removed at day 0 and day 14. The effect of the leachates on the mouse microbiome was investigated via bacterial DNA sequencing (6a) and metabolomic analysis (6b).

[0084] Figure 2: Historical pellets of Lemnian Earth from Basel University’s Museum of Pharmacy collections (16th-18th c). The red (middle) and grey (right) pellets were bioactive and consisted mainly of smectite (red) and kaolinite (grey), but although ‘stamped’ the white pellet (left) consisted mainly of dolomite and was not bioactive.

[0085] Figure 3: Composition of archaeological Lemnian Earth and reference clays. 700.17 (Fig. 2, middle) and 700.18 (Fig. 2, right) (in Fig. 2): archaeological LE, 700.19 / 700.20: geological samples of Lemnos clays, SWy-2: a smectite and KGa-2: a kaolin.

[0086] Figure 4: Assay of the antibacterial activity of Ppcontrol filtrate at various pH. Figure 5: Comparison of antibacterial activity of Pp+smectite and Pp+kaolinite filtrates against E. coli. A = dilution factor, providing the extracellular sample %, the table values indicate Bacterial Reduction %.

[0087] Figure 6: Assay to determine MICeoof Figure 6A: Sample 1 = Ppcontrol filtrate, Figure 6B: Sample 3 = Pp+smectite filtrate and Figure 6C: Sample 5 = Pp+kaolinite filtrate

[0088] Figure 7: Comparison of abundancy of known secondary metabolites in various filtrates, as measured by targeted LC-MS.

[0089] Figure 8: Diversity estimates. Rarefied richness of bacterial ASVs shown in (Fig. 8A) with lines connecting different categories where values were significantly different (according to ANOVA; * p<0.05 ** p<0.01 *** p<0.001 )., (B-D) show principal coordinate analysis (PCoA) plots with each axis showing the percentage variability explained by that axis, and where ellipses represent 95% Cl of standard error for a given time point. Three distance matrices are used, Bray-Curtis (Fig. 8B) to reflect changes in composition, UniFrac (Fig. 8C) (to reflect changes in phylogeny), and HMS (Fig. 8D) (Hierarchical Metastorms to reflect changes in function). (Fig. 8E) shows the top 25 most abundant genera recovered for different sample types.

[0090] Figure 9: Table showing the greatest enhancement / decrease in faecal microbiota after gavage with Pp+smectite filtrate, compared to PBS.

[0091] Figure 10: Untargeted murine metabolomics of stool samples, indicating the induction of SCFA-carnitine conjugates by the Pp+smectite filtrate (sample 3), highest concentrations, compared to PBS (sample 1 ) and Pp+kaolinite (sample 5). Y-axis = ion counts of SCFA-carnitine adducts

[0092] Figure 11A(A-C): CODA LASSO regression for finding differential taxa between control (saline solution) samples PBS. 1 and PBS. 2.

[0093] Figure 11B(A-C): CODA LASSO regression for finding differential taxa between PP+ smectite filtrate samples (samples.1 and sample 3.2). Figure 11C(A-C): CODA LASSO regression for finding differential functions (MetaCyc pathways) between control (saline solution) samples PBS.1 and PBS. 2.

[0094] Figure 11D(A-C): CODA LASSO regression for finding differential functions (MetaCyc pathways) between PP+ smectite samples (sample 3.1 and sample

[0095] 3.2)

[0096] Figure 11E(A-C): CODA LASSO regression for finding differential functions (MetaCyc pathways) between PP+ kaolin samples (sample 5.1 and sample

[0097] 5.2)

[0098] Figure 12: Summary table of the pathways uniquely modified by treatment with Pp+smectite filtrate.

[0099] Figure 13A-C: Comparison of antibacterial activity of Pp+palygorskite filtrates against S. aureus, E. coli and P. aeruginosa. X-axis is dilution factor, providing the extracellular sample %.

[0100] Figure 14: Comparison of Firmicutes / Bacteriodes (F / B) ratio over time for Pp+control, Pp+smectite and Pp+kaolin.

[0101] Definitions

[0102] The invention is described using the following definitions unless otherwise indicated.

[0103] Throughout the specification, unless the context demands otherwise, the terms ‘comprise’ or ‘include’, or variations such as ‘comprises’ or ‘comprising’, ‘includes’ or ‘including’ will be understood to imply the includes of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. As used herein, the articles “a” and “an” refer to one or to more than one (for example to at least one) of the grammatical object of the article.

[0104] “About” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements.

[0105] The terms, "disease", "disorder", and "condition" may be used interchangeably here to refer to a medical or pathological condition.

[0106] Reference throughout this specification to “one embodiment,” “an embodiment,” “certain embodiments,” or “some embodiments,” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. In particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects or embodiments.

[0107] "Patient" for the purposes of the present invention includes humans and other animals, particularly mammals and other organisms. Thus, the methods are applicable to both human therapy and veterinary applications.

[0108] “Mammal” means humans and other mammalian animals.

[0109] “Therapeutically effective amount” means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, a system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.

[0110] The term “treat”, “treatment” or “treating” refers to therapy, prevention and prophylaxis and particularly refers to the administration of medicine or the performance of medical procedures with respect to a patient, for either prophylaxis (prevention) or to cure or reduce the extent of or likelihood of occurrence of the infirmity or malady or condition or event in the instance where the patient is afflicted. The term "composition", as in pharmaceutical composition, is intended to encompass a product comprising the active ingredient(s), and the inert ingredient(s) (pharmaceutically acceptable excipients) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing a composition of invention, and pharmaceutically acceptable excipients.

[0111] Pharmaceutically Acceptable Salts

[0112] As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases.

[0113] The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases. Salts derived from inorganic bases include aluminium, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganese, potassium, sodium, zinc, and the like.

[0114] Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

[0115] When the compound of the present invention is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like. Particularly preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.

[0116] It will be understood that, unless otherwise specified, references to the compositions of the present invention, subsets thereof, embodiments thereof, as well as specific compounds are meant to also include the pharmaceutically acceptable salts thereof.

[0117] Following the examples disclosed herein, as well as that disclosed in the art, a person of ordinary skill in the art can determine the activity of a composition of this invention.

[0118] Dose Ranges

[0119] The magnitude of prophylactic or therapeutic dose of a composition of the invention will, of course, vary with the nature and the seventy of the condition to be treated and with the particular composition of the invention. It will also vary according to a variety of factors including the age, weight, general health, sex, diet, time of administration, rate of excretion, drug combination and response of the individual patient. In general, the daily dose from about 0.001 milligram of active agent per kilogram body weight of a mammal (mg / kg) to about 100 mg / kg, typically, between 0.01 mg to about 10 mg per kg. On the other hand, it may be necessary to use dosages outside these limits in some cases.

[0120] The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for the oral administration of humans may contain from 0.01 mg to 10 g of active agent compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 99.95 percent of the total composition. Dosage unit forms will generally contain between from about 0.1 mg to about 0.4 g of an active ingredient, typically 0.5 mg, 1 mg, 2 mg, 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, 200 mg, 400mg, or 500 mg.

[0121] The final dosage regimen will be determined by the attending physician in view of good medical practice, considering various factors that modify the action of drugs, e.g., the agents’ specific activity, the identity and seventy of the disease state, the responsiveness of the patient, the age, condition, body weight, sex, and diet of the patient, and the severity of the disease state. Additional factors that can be taken into account include time and frequency of administration, drug combinations, reaction sensitivities, and tolerance / response to therapy. Further refinement of the dosage appropriate for treatment involving any of the formulations mentioned herein is done routinely by the skilled practitioner without undue experimentation, especially in light of the dosage information and assays disclosed, as well as the pharmacokinetic data observed in human clinical trials. Appropriate dosages can be ascertained through use of established assays for determining concentration of the agent in a body fluid or other sample together with dose response data. The frequency of dosing will depend on the pharmacokinetic parameters of the compounds of the present invention. Dosage may be adjusted to provide sufficient levels of the active moiety or to maintain the desired effect.

[0122] Accordingly, the pharmaceutical compositions can be administered in a single dose or multiple discrete doses, as required to maintain desired minimum level of the agent.

[0123] Pharmaceutical Compositions

[0124] Another aspect of the present invention provides pharmaceutical compositions comprising a composition of the invention, which may be administered orally.

[0125] In addition to the treatment of warm-blooded animals such as mice, rats, horses, cattle, sheep, dogs, cats, etc., the compound of the invention is effective in the treatment of humans. The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.

[0126] Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from a group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, com starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc. The tablets may be uncoated, or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the technique described in the U.S. Patent 4,256,108; 4,166,452; and 4,265,874 to form osmotic therapeutic tablets for control release.

[0127] Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredients is mixed with water-miscible solvents such as propylene glycol, PEGs and ethanol, or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

[0128] Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame. Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.

[0129] Sweetening agents such as those set forth above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

[0130] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavouring and colouring agents, may also be present.

[0131] The pharmaceutical compositions of the invention may also be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavouring agents.

[0132] Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, and flavouring and colouring agents.

[0133] Suitably, administration of a compound or composition of the present invention may be in conjunction with one or more other therapeutic agents. Suitably, the administration may be concurrent or consecutive with the one or more other therapeutic agents in any order. Suitably, the one or more other therapeutic agents may comprise probiotics or prebiotic supplements.

[0134] Detailed Description of the Invention

[0135] Example 1 : Comparison of Bioactivity Between Historical Lemnian Earth and Geological Samples of Lemnian Clay

[0136] The composition of various samples of historic Lemnian Earth from Basel University’s Museum of Pharmacy collections (16th-18thCentury) were compared with those of geological Lemnos clays and reference clays - See Fig. 3. SWy-2 and KGa-2 are reference clays, i.e. smectite and kaolin, respectively, obtained from the Clay Minerals Society (CMS), USA (CMS Source Clay project). The reference clays are both considered biologically inert materials. Additionally, both smectite and kaolinite are ubiquitous in clay and clay-like sediments and soils worldwide.

[0137] On inspection, when comparing the historic Lemnian Earth and the geological Lemnos clays, only the historic Lemnian comprised a fungal load within the clay. These consisted of clades of the Eurotiale phylogenetic order, i.e. Talaromyces, Penicillium and Aspergillus, as recognised by targeted polymerase chain reaction and Sanger DNA sequencing of their ribosomal internal transcribed spacer (ITS) region of the fungal genome.

[0138] Example 2: Preparation of Example Filtrates of the Present Invention

[0139] Talaromyces purpurogenum (formally referred to as Penicillium purpurogenum - Pp) was cultured in a dextrose broth, both with and without either SWy-2 smectite and KGa-2 kaolinite clay. These were then filtered with a 0.45 pm filter to remove the clay and the fungi, to provide the Ppcontrol, Pp+smectite and Pp+kaolinite filtrates. These were tested for their antibacterial properties in in vitro experiments.

[0140] The reference strain DSM 62866 of Talaromyces purpurogenum was used, (Leibniz Institute DSMZ ■ German Collection of Microorganisms and Cell Cultures) for microbiological analysis and the extraction of metabolites.

[0141] In accordance with the DSMZ’s instructions, the fungus was cultured by spreading liquid inoculum as purchased on potato dextrose agar (Neogen) plates. These were then incubated for 7 days at 25 °C.

[0142] Fungal liquid cultures were then prepared by taking 1 colony of Talaromyces purpurogenum from the solid medium and inoculating potato dextrose broth (Neogen), in the presence or absence of SWy-2 or KGa-2 at 50 mg / mL. This was then incubated for 14 days at 25 °C with agitation. The no-clay control had a pH of 5.1 , whereas the SWy-2 and KGa-2 had pHs of 6.1 and 4.5, respectively - although this was adjusted for subsequent experiments (see below).

[0143] Following the 14-day incubation, each sample (300 mL) was centrifuged at 10,000g for 20 min at 4 °C. The supernatant was diluted in 100 mL of deionized water, boiled at 98 °C for 15 min and then filtered through a 0.45 pm cellulose filter (Millipore, USA), to provide the Pp negative control (Ppcontrol), Pp+smectite and Pp+kaolinite filtrates.

[0144] Notably, the thermal lysis and filtration steps were performed purely to rule out physical clay-bacteria interactions and the probiotic introduction of living fungi as mechanisms of bioactivity in vitro and in vivo.

[0145] Example 3: In vitro Antibacterial of Example Filtrates of the Present Invention To determine their ability to inhibit the growth of bacteria, Ppcontrol, Pp+smectite, Pp+kaolinite and Pp+palygorskite filtrates were prepared in accordance to Example 2. These were tested for their antibacterial properties against reference bacterial indicators, the Gram (-) Escherichia coli DSM498, the Gram (+) Staphylococcus aureus NCTC 12493 (National Collection of Type Cultures, UK), and the Gram (-) Pseudomonas aeruginosa (likely PA01).

[0146] Prior to testing the bioactivity of the filtrates, the antibacterial property of the Ppcontrol was tested as a function of pH, on Escherichia coli. Fig. 4 shows that Ppcontrol is only effective in killing the bacteria at high concentrations and at either high pH=7 or at low pH=2. Otherwise, it is not considered antibacterial. This experiment was then repeated with Pp+smectite, Pp+kaolinite (see Fig. 5) and Pp+palygorskite (see Fig. 13), demonstrating that Pp+smectite and Pp+palygorskite are strongly antibacterial at all tested concentrations, compared to both Pp+kaolinite and Ppcontrol.

[0147] The broth microdilution method was then used to estimate Minimum Inhibitory Concentration that inactivated 60% of the bacterial population (MICeo). MIC values were estimated using 96-well sterile microtiter trays, which contained: a) dilutions of each liquid sample, b) LB broth (Neogen) and c) the bacterial population adjusted to 105 CFU / mL.

[0148] The trays were incubated at 37 °C for 18-24 h, followed by optical density measurement at 630 nm using a microplate reader (Labtech LT-4000 Plate Reader) and Manta LML software.

[0149] As shown in Fig. 6A-C, the Pp+smectite filtrate showed significant antibacterial properties at all concentrations, and against both Escherichia coli and Staphylococcus aureus. Example 4: Comparison of Secondary Metabolites in the Filtrates

[0150] Fungi of the Penicillium / Talaromyces genera produce a variety of pigmented and non-pigmented secondary metabolites. These have a range of bioactivities, including antibacterial, antifungal, anti-inflammatory properties.

[0151] 30 known, fungal pigmented secondary metabolites and biosynthetically related, though not pigmented fungal compounds were selected, and targeted LC-MS analysis of in each of the Ppcontrol, Pp+smectite and Pp+kaolinite filtrates was performed to assess the presence and concentration of these secondary metabolites in the filtrates.

[0152] Compounds were positively identified with A-values < 5 ppm. In addition, five of the compounds: PP-R, PP-V, purpuride, patulin and citrinin could also be identified via their MS / MS fragmentation patterns.

[0153] The results indicate that a range of (targeted and untargeted) secondary metabolites are present, but that these are differentially induced by the clay used; i.e. , there are both common and unique secondary metabolites in each of the Ppcontrol, Pp+smectite and Pp+kaolinite filtrates.

[0154] By comparing the results of the three filtrates, nine secondary metabolites were identified with increased abundance in the Pp+smectite filtrate compared to the Pp+kaolinite and Ppcontrol filtrates - see Fig. 7.

[0155] The nine secondary metabolites are as follows: ankaflavin, mitorubrin, mitorubrinol, monascin, ochratoxin, patulin, PP-R, PP-V and roquefortine C.

[0156] Without wishing to be bound by theory, it is believed that one or more of these nine secondary metabolites is responsible for the observed in vitro antibacterial activity, and the in vivo activity described below. Example 5: In vivo Bioactivity of the Filtrates

[0157] The Pp+smectite and Pp+kaolinite filtrates of Example 2 were fed in a doubleblind study to mice by gavage, to assess the therapeutic effect of the filtrates on the intestinal microbiota of the mice.

[0158] C57BL / 6 mice were purchased from Envigo at 5-6 weeks of age and used for procedures at 7-8 weeks of age. Mice were housed under specific pathogen free conditions at the Common Research Facility, University of Glasgow. All procedures were carried out under personal and project licences issued by the UK Home Office.

[0159] Manipulation of microbiota in vivo

[0160] Mice were administered 100pl of PBS (sterilised phosphate buffered saline, control), Pp+smectite and Pp+kaolinite filtrates by oral gavage 6 times over the course of 2 weeks. Faecal samples were flash frozen upon collection using dry ice and then stored at -80 °C.

[0161] Each mouse received 6 doses of 100ml of test solution (Pp+smectite, Pp+kaolinite or sterile phosphate buffered saline (PBS) control), over a two- week period. All animals received standard chow and sterile water ad libitum.

[0162] The animals’ health was monitored daily, and their weight was measured every 2-3 days. Stool samples from individual mice were collected weekly, starting immediately before the first gavage, and were stored at -20 °C before 16S analysis of the intestinal microbiota - as shown in Fig. 8.

[0163] Abundance tables were generated by constructing Amplicon Sequencing Variants (ASVs) using the QIIME2 workflow (Caporaso et al., 2010, 7(5), 335- 336, herein incorporated by reference) using the DADA2 denoising algorithm (Callahan et al., 2016, Nature methods, 13(7), 581 -583, herein incorporated by reference). Additionally, the PICRUSt2 algorithm (Douglas et al., 2020, Nature biotechnology, 38(6), 685-688, herein incorporated by reference) was used as a QIIME2 plugin on the ASVs to predict the functional abundance of microbial communities (both KEGG enzymes and MetaCyc pathways were recovered) by using the weighted Nearest Sequenced Taxon Index (NSTI) threshold of 2.0 in the software to map the ASVs against the reference database comprising ~20,000 genomes (whose functions were known) in PICRUSt2.

[0164] ASVs were then classified using the recent SILVA SSU Ref NR database release v.138 (Quast et al., 2012, 41 (D1 ), D590-D596, herein incorporated by reference), and then combined the taxonomic information with the abundance table to generate a BIOM file. The rooted phylogenetic tree, also generated using the QIIME2 framework, along with the above BIOM file as well as the functional tables from PICRUSt2 were then used in the downstream statistical analyses in R.

[0165] As per the QIIME2 tutorials, typical contaminants were removed as a preprocessing step, such as Mitochondria, Chloroplasts and any ASVs that were unassigned at all levels. Samples that were not relevant to this study (or are <2,000 reads) were also filtered out, giving an abundance table of n=101 samples x P=3,626 ASVs.

[0166] The summary statistics of sample-wise read distributions are as follows:

[0167] • Minimum: 62,504;

[0168] • 1st Quartile: 89,120;

[0169] • Median: 91 ,324;

[0170] • Mean: 89,950;

[0171] • 3rdQuartile: 95,460;

[0172] • Maximum: 98,607.

[0173] The R’s vegan package (Oksanen et al., 2013, 2(9), 1-295, herein incorporated by reference) was used for alpha and beta diversity analyses. For alpha diversity measures rarefied richness was used - the estimated number of species / features in a rarefied sample (to minimum library size).

[0174] Different beta diversity distance measures used: i. Bray-Curtis distance on the ASV abundance table to visualise the compositional changes; ii. Unweighted UniFrac distance estimated using R’s Phyloseq package (McMurdie et al., 2013, PloS one, 8(4), e61217, herein incorporated by reference), to see changes between samples in terms of phylogeny; iii. Weighted UniFrac distance which also incorporates abundances and iv. Hierarchical Meta-Storms (HMS) (Zhang et al., 2021 , Bioinformatics Advances, 1 (1 ), vbab003, herein incorporated by reference), a recent functional beta diversity distance which takes the observed KEGG Orthologs (KOs) recovered from the dataset, and then calculates the functional beta diversity distance in a hierarchical fashion propagating the KOs abundances upward to the pathways in a multi-level pathway hierarchy to give a weighted dissimilarity measure. R's aov() function was used to calculate the pair-wise analysis of variance (ANOVA) with p-values drawn on top of alpha diversity.

[0175] To find a minimal subset of genera / pathways that changed between different conditions, the CODA LASSO (Calle et al., 2022, bioRxiv, 2022-06, herein incorporated by reference) was used, in the form y; = ?o + f log(xii) + ••• + [3j log(x7i) + fi (for i-th sample and j-th microbe / pathway, with Xji being the abundance of genera / pathway recovered from PICRUSt2), and where the outcome yi is a binary outcome variable (uses logistic regression).

[0176] The model uses two constraints: a) all ^-coefficients sum up to 1 which makes the algorithm invariant by returning two disjoint sets of features in a log contrast fashion, one that are positively associated, and one that are negatively associated; and b) the optimization function incorporates a LASSO shrinkage term which makes some ^-coefficients go to zero with the non-zero ^-coefficients returns for features (microbes / pathways) that change between the conditions. The coda glmnet() function was used from R’s coda4microbiome package (Calle et al., 2022, bioRxiv, 2022-06). The top 100 most abundant genera / pathways were used in the CODA- LASSO model.

[0177] Assessment of bacterial richness (Fig 8A) reveals that feeding with both PBS and the Pp+smectite filtrate generated significant increases in the alphadiversity of gut flora. Principle component analyses were also performed, using Bray-Curtis (Fig. 8B), to reflect changes in composition, UniFrac (Fig. 8C), to reflect changes in phylogeny, and Hierarchical Meta-storms (HMS) (Fig. 8D), to reflect changes in function. These revealed significant changes after PBS or Pp+smectite filtrate feeding, but less consistent effects after feeding Pp+kaolinite filtrate.

[0178] Fig. 8E shows the most abundant genera recovered from each group of animals. These data also demonstrate significant changes induced in the microbiota, particularly after feeding with the PBS and Pp+smectite treatments.

[0179] To better understand the significant biological changes that may be caused by the active fungal metabolites in the Pp+smectite filtrate, the bacterial genera with the highest positive and negative beta coefficients after feeding with Pp+smectite filtrate were identified - as shown in Fig. 9.

[0180] Given that Lachnospiraceae are among the most abundant taxa in the gut microbiome, and are the most significantly upregulated populations after Pp+smectite feeding of mice, it was hypothesised that they may have important effects on the host animals. All members of the Lachnospiraceae are anaerobic, fermentative & chemo-organotrophic.

[0181] The Firmicutes / Bacteriodes (F / B) ratio is correlated with obesity and other diseases. An assay was performed to determine how compositions of the present inventer affected the F / B ratio. Compositions of the present invention were prepared using protocols in line with those of Example 2. Abundance tables were generated by constructing Amplicon Sequencing Variants (ASVs) using the QIIME2 workflow using the DADA2 denoising algorithm. ASVs were then classified using the recent SILVA SSU Ref NR database release v.138. These were then combined the taxonomic information with the abundance table to generate a BIOM file of the rooted phylogenetic tree - as shown in Fig.14. This shows that Pp+smectite increases F / B ratio with greater confidence than control or Pp+kaolin.

[0182] Lachnospiraceae are also among the main producers of short-chain fatty acids (SCFAs) from metabolism of dietary fibers / starch. SCFAs, such as acetate, propionate and butyrate are central mediators of the commensal communication between the intestinal microbiome and the immune system. The net effect of microbially-produced SCFAs is to ameliorate local and systemic inflammatory pathways.

[0183] SCFAs could therefore provide a mechanism by which the significant changes in the microbiota that occur after feeding with Pp+smectite filtrate could be beneficial for the intestinal environment. Untargeted metabolomics was therefore performed to determine whether SCFAs were present at increased levels in any of the samples.

[0184] As shown in Figs. 8 and 9, only the Pp+smectite filtrate was shown to provide a redistribution of the microbiome with increased Lachnospiracea, (known SCFA producers). SCFAs are known to modulate intestinal and systemic inflammation and convey health benefits to the host. Pp+smectite filtrate modified the mouse gut microbiome. It is considered this could provide a net beneficial effect to the host.

[0185] Example 6: Untargeted metabolomic analysis of stool samples

[0186] The stool samples of Example 5 underwent untargeted metabolomics, however besides 3-isovalerate, this did not directly reveal the presence of acetate, propionate, butyrate, or valerate SCFAs. This may be due to their volatility and low molecular weight, or that the sample preservation protocols were not optimised for the preservation of volatile molecules.

[0187] However, SCFA conjugates with carnitine, a SCFA transporter, and SCFA- carnitine conjugates (which are less volatile) were detected.

[0188] Notably, Acetyl, propionyl and carnitine were all identified with much higher intensities in samples from mice fed with Pp+smectite filtrates, relative to the PBS and Pp+kaolinite filtrate samples - see Fig. 10. This indicated that the Pp+smectite filtrate, but not the other filtrates are able to significantly increase the abundance of SCFAs in the intestines of the mice (acetate increases 10- fold, propionate 2.5-fold). Pp+smectite filtrate or a compound therein appears to increase the abundance of SCFAs and be suitable for use in treating the diseases outlined herein, wherein an increase in the SCFAs is considered to be beneficial.

[0189] Critically, the changes in microbial populations that occur after feeding mice with Pp+smectite filtrate may drive metabolic changes in the gut microbiota beyond the changes in SCFAs.

[0190] In order to assess such changes, MetaCyc pathway analysis was performed to impute functional metabolic pathways from the 16S datasets. Following pathway analysis, upregulated and downregulated pathways were identified using CODA LASSO regression. Consistent with the identification of SCFA-carnitine analogues in the samples that followed treatment with Pp+smectite filtrate, upregulated functional pathways related to SCFA production were also identified by this CODA LASSO regression - See Fig. 11 .

[0191] In addition to changes in SCFA-related pathways, other pathways were also identified as being significantly upregulated or downregulated after treatment with Pp+smectite filtrate, - See Fig. 11 .

[0192] These show: (A) (3 -coefficients returned from CODA-LASSO procedure as two disjoint sets, (B) Expression levels of these differential microbes where TSS+CLR is Total Sum Scaling followed by Centralised Log Ratio (C) The density plot returned from the CODA-LASSO segregating the two groups and providing a graphical assessment of the classification accuracy (top: actual; bottom: predicted from the procedure using subset of Figure 11A-B: taxa, Figure 11 C-E: function).

[0193] Among the significant changes, the GLYCOCAT pathway for glycogen degradation showed a significant increase in Centralised Log Ratio and betacoefficient (0.12). The GLYCOCAT pathway breaks down dietary fibers, and is the first pathway towards SCFA production. We also found that several anabolic amino acid pathways are upregulated after treatment with Pp+smectite filtrate, including glutamate & glutamine biosynthesis (PWY- 5505); tryptophan, tyrosine and phenylalanine via chorismite biosynthesis (PWY-6163); isoleucine (PWY-5103) and ornithine & arginine (GLUTORN & ARGSYN pathways). In addition, glutamate degradation (PWY-162) is reduced.

[0194] Overall, it appears that the effect of treatment with Pp+smectite filtrate on bacterial metabolism in the intestine may be to increase both the availability to the host of SCFAs, and of amino acids.

[0195] Without wishing to be bound by theory, it is considered that the mechanism by which smectite and kaolinite induce the differential metabolite expressions in the fungi are due to the different surface properties of the two clay minerals. Na-smectites like SWy-2 swell upon hydration due to the introduction of water molecules in the interlayer space. In addition, they have negative layer charge due to ionic substitutions in the lattice that is balanced by the interlayer cations. These are exchangeable, thereby yielding significant cation exchange capacity (CEC) for inorganic cations and organic molecules. In contrast, kaolinite does not have layer charge and thus does not bear CEC. The negative charge of SWy-2 crystallite surfaces repels the fungi, as they also have a negative charge.

[0196] Simultaneously, smectite readily adsorbs molecules from the growth media (potato dextrose) in the interlayer, thereby increasing the interlayer space, i.e. the distance between the smectite layers. Such a process will increase swelling and will facilitate delamination of Na-rich SWy-2 lamellae, thereby increasing the area of contact between smectite and the fungi, and intensifying the interaction there between.

[0197] That is, it is considered that the smectite surfaces act as stressors on the fungi, resulting in increased production of secondary metabolites that are available in the filtrate. Such an interaction between clay mineral and the fungi was not observed in kaolinite, because of the lack of permanent charge and lack of swelling of the clay.

[0198] Preferred compositions, features and embodiments of each aspect of the invention are as for each of the other aspects mutatis mutandis unless context demands otherwise.

[0199] Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in the text is not repeated in this text is merely for reasons of conciseness. Reference to cited material or information contained in the text should not be understood as a concession that the material or information was part of the common general knowledge or was known in any country.

[0200] Although the invention has been particularly shown and described with reference to particular examples, it will be understood by those skilled in the art that various changes in the form and details may be made therein without departing from the scope of the present invention.

Claims

Claims1 . A method of manufacturing a composition, the method comprising the steps:• Providing a fungi of the Penicillium, Talaromyces, Monascus, or Aspergillus genera,• Providing a clay that swells upon hydration due to expansion of interlayer spacing in the clay,• Combining the fungi and the clay in a growth culture media,• Culturing the growth culture medium under conditions suitable for growth of the fungi,• Removing substantially all of the fungi and clay from the growth culture media, to provide the composition.

2. The method of claim 1 wherein the interlayers of the clay comprise a layer with a negative charge.

3. The method of claim 2, wherein the clay has cation exchange capacity.

4. The method of any of the preceding claims wherein the clay has a phyllosilicate mineral structure.

5. The method of any of the preceding claims wherein the clay has a mineralogical structure comprising a three layer tetrahedra-octahedra- tetrahedra structure.

6. The method of any of the preceding claims wherein a clay is selected from a group consisting of smectite, vermiculite, kaolinite, illite,palygorskite and nontronite.

7. The method of any of the preceding claims wherein the composition comprises one or more of the following: ankaflavin, mitorubrin, mitorubrinol, monascin, ochratoxin, patulin, PP-R, PP-V and roquefortine C, or a pharmaceutical salt thereof.

8. The method of any one of the preceding claims, wherein the fungi comprises: Talaromyces purpurogenus, syn. Talaromyces purpureogenus, Penicillium sanguineum, Talaromyces tardifaciens, Penicillium roqueforti, Penicillium rubrum, Penicillium monascum, Monascus purpureus, Aspergillus ochraceus, Aspergillus carbonarius and / or Aspergillus niger.

9. A composition or pharmaceutical salt thereof, wherein the composition or pharmaceutical salt thereof is manufactured via a method comprising:• Providing a fungi of the Penicillium, Talaromyces, Monascus, or Aspergillus genera,• Providing a clay that swells upon hydration due to expansion of inter-layer spacing in the clay,• Combining the fungi and the clay in a growth culture media,• Culturing the growth culture medium under conditions suitable for growth of the fungi,• Removing substantially all of the fungi and clay from the growth culture media, to provide the composition.

10. The composition of claim 9, wherein the interlayers of the clay have a layer with a negative charge.11 .The composition of claim 10, wherein the clay has cation exchange capacity.

12. The composition of any one of claims 9-11 , wherein the clay has a phyllosilicate mineral structure.

13. The composition of any one of claims 9-12 wherein the clay has a mineralogical structure comprising a three layer tetrahedra-octahedra- tetrahedra structure.

14. The composition of any one of claims 9-13, wherein the clay is selected from a group consisting of smectite, vermiculite, kaolinite, illite, palygorskite and nontronite.

15. The composition of any one of claims 9-14, wherein the composition comprises one or more of the following: ankaflavin, mitorubrin, mitorubrinol, monascin, ochratoxin, patulin, PP-R, PP-V and roquefortine C, or a pharmaceutical salt thereof.

16. The composition of any one of claims 9-15, wherein the fungi comprises: Talaromyces purpurogenus, syn. Talaromyces purpureogenus, Penicillium sanguineum, Talaromyces tardifaciens, Penicillium roqueforti, Penicillium rubrum, Penicillium monascum, Monascus purpureus, Aspergillus ochraceus, Aspergillus carbonarius, and / or Aspergillus niger.

17. A pharmaceutical composition comprising a therapeutically effective amount of the composition or pharmaceutical salt of any one of claims9-16.

18. A pharmaceutical composition of claim 17, for use in modulating gut SCFA, amino-acid production, and / or gut microbial diversity.

19. A pharmaceutical composition of claim 17, for use in preventing a chronic inflammatory disease.

20. A pharmaceutical composition of claim 17, for use in treating or preventing a disease associated with the gut / brain axis.21 .A method of modulating gut SCFA, amino-acid production, and / or gut microbial diversity, the method comprising administration of the pharmaceutical composition of claim 17 to a subject in need thereof.

22. A method of treating and / or preventing a chronic inflammatory disease, the method comprising administration of the pharmaceutical composition of claim 17 to a subject in need thereof.

23. A method treating or preventing a disease associated with the gut / brain axis, the method comprising administration of the pharmaceutical composition of claim 17 to a subject in need thereof.

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