Composition comprising 3,3'-diindolylmethane and perilla oil and its use for the treatment of diseases associated with impaired appetite regulation

Abstract

The present application relates to a composition consisting of 3,3'-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of α-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

Description

[0001] COMPOSITIONS AND METHODS OF TREATMENT FOR DISEASES ASSOCIATED WITH IMPAIRED APPETITE REGULATION

[0002] Field of the invention

[0003] The present invention relates to compositions, pharmaceutical compositions, tablets and capsules comprising a combination of 3,3’-diindolylmethane and perilla oil, and their use in appetite suppression and for the treatment and prevention of impaired appetite regulation or feeling of satiety and diseases and disorders associated with impaired appetite regulation or feeling of satiety. Methods of preparing said compositions, pharmaceutical compositions, tablets and capsules are also described.

[0004] Background to the invention

[0005] Obesity and overweight are some of the major heath challenges faced by modern medicine and society. The common health consequences of overweight and obesity are defined by the World Health Organisation in 2016 as cardiovascular diseases (mainly heart disease and stroke), which were the leading cause of death in 2012; type II diabetes, musculoskeletal disorders (especially osteoarthritis); and some cancers (endometrial, breast, and colon).

[0006] Suppressing appetite has been a common approach to increasing weight loss and consequently reducing obesity in individuals. Several different approaches have been taken in this regard. For example, bypass surgery is an effective treatment for obesity and type II diabetes. It works by shunting ingested food directly to the lower part of the gut, whereupon unabsorbed nutrients stimulate the release of gut hormones from cells lining the gut. These hormones reduce the feeling of hunger in the brain, and they simultaneously stimulate the pancreas to produce insulin (Bueter & le Roux Int J Obes (Lond) 35 Suppl 3:S35-9 (2011)). Exactly which nutrients cause this effect, and exactly which hormones they release, and how, are not fully understood.

[0007] Current protein-based approaches to weight loss use large oral loads which contain significant caloric value. They stimulate only one part of the system and are mostly or entirely absorbed before reaching the lower gut. Therefore reduction in food intake is minimal and the formulation has to be prepared by the subject making it inconvenient. It is also unpalatable and compliance is low.

[0008] Recently, the use of GLP-1 agonists as pharmaceutical appetite suppressants has provided some benefits in increasing weight loss. For example, semaglutide, an analog of GLP-1 (sold under the brand name of Ozempic®) is widely used for long-term weight management (Ryan et al, Nature Medicine, 30, 2049-2057 (2024)). However, like many pharmaceutical compositions targeting weight loss, semaglutide can lead to a number of unwanted side effects.

[0009] Alternative approaches to surgical and pharmaceutical means of appetite suppression and weight loss, which are safe and effective are therefore needed. Targeted release formulations of drugs for delivery into the lumen of the intestine are available. Recent developments now allow them to be released specifically within the large intestine (Varum et al EurJ Pharm Biopharm. 84(3):573-7 (2013)). So far these formulations have been designed to deliver anti-inflammatory drugs for treatment of inflammatory bowel disease (Vieira et al Carbohydr Polym. 93:512-7 (2013)). Interest is however growing in the use of this technology for delivery of nutrients (in the form of nutraceuticals) known to activate enteroendocrine cells, in order to treat obesity (Chambers et al Gut 64(11):1744-54 (2015). It has previously been shown that a combination of 3,3’-diindolylmethane, lauric acid (LA) and a-linolenic acid (ALA) derived from perilla oil causes the co-activation of GPR84 and FFAR4 receptors, and consequent release of GLP-1 and PYY (Peris et al, Gut, 71 : 928-937 (2022)).

[0010] Summary of the invention

[0011] According to a first aspect of the invention, there is provided a composition consisting of 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

[0012] According to a second aspect of the invention, there is provided a pharmaceutical composition consisting of 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

[0013] According to a third aspect of the invention, there is provided a method of preparing a composition comprising 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil, the method comprising:

[0014] (a) contacting the perilla oil with mesoporous silica having a specific surface area (BET) ranging from 240 to 340 m2 / g, thereby to provide the perilla oil in the form of a powder;

[0015] (b) combining the powder of step (a) with 3,3’-diindolylmethane, to provide the composition.

[0016] According to a fourth aspect of the invention, there is provided a tablet or capsule comprising a composition consisting of 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

[0017] According to a fifth aspect of the invention, there is provided a tablet or capsule comprising a composition comprising 3,3’-diindolylmethane and perilla oil absorbed onto mesoporous silica, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

[0018] According to a sixth aspect of the invention, there is provided a method of preparing a tablet or capsule comprising a composition comprising 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil, the method comprising:

[0019] (a) contacting the perilla oil with mesoporous silica having a specific surface area (BET) ranging from 240 to 340 m2 / g, thereby to provide the perilla oil in the form of a powder;

[0020] (b) combining the powder of step (a) with 3,3’-diindolylmethane to provide a composition;

[0021] (c) compressing the composition into the form of a tablet, or providing the composition as part of a capsule.

[0022] According to a seventh aspect of the invention, there is provided a method for supressing appetite in a subject, the method comprising administering to the subject a composition consisting of, or a tablet or capsule comprising a composition consisting of 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

[0023] According to an eighth aspect of the invention, there is provided a method of treating impaired appetite regulation or feeling of satiety, the method comprising administering to a subject a composition consisting of, or a tablet or capsule comprising a composition consisting of 3,3’- diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

[0024] According to a ninth aspect of the invention, there is provided a method of preventing or treating type II diabetes, the method comprising administering to an individual in need of such treatment a composition consisting of, or a tablet or capsule comprising a composition consisting of 3,3’- diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

[0025] According to a tenth aspect of the invention, there is provided a kit comprising: 3,3’-diindolylmethane, and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil, and optionally an administration vehicle, and / or instructions for use.

[0026] Brief Description of the Drawings

[0027] Figure 1 shows stimulation of human colonic mucosa with DIM, Perilla oil and in combination using phosphorylation marker pERK. A: Representative images of human colonic mucosa stimulated with dual nutrient combination show increased numbers of pERK +ive cells as determined by immunohistochemistry. B: Graph shows cell counts from N=9 colon tissue samples per treatment group. Immunohistochemistry images show PYY positive cell (star) adjacent to a pERK positive cell.

[0028] Figure 2 shows stimulation of human colonic mucosa with DIM, Perilla oil and in combination using phosphorylation marker pCaMKII. A: Representative images of human colonic mucosa stimulated with dual nutrient combination show increased numbers of pCaMKK +ive cells as determined by immunohistochemistry. B: Graph shows cell counts from N=9 colon tissue samples per treatment group. Immunohistochemistry images show PYY positive cell (star) costaining with pCaMKII indicating an activated cell state.

[0029] Figure 3 shows PYY and GLP-1 release from human colonic tissue is significantly increased by stimulation with DIM and Perilla oil in combination. Quantification of PYY (A) and GLP-1 (B) from supernatants collected from incubated tissue samples show significant increase of both hormones following dual stimulation. There was also a statistically significant increase with single nutrient stimulation compared to control (p values: DIM - 0.0007 (PYY) & 0.0061 (GLP-1); PO - 0.025 (PYY) & 0.0016 (GLP-1)). N = 9 for each group.

[0030] Figure 4 shows a flow diagram demonstrating volunteer recruitment and study design paradigm. A total of 20 volunteers were assessed over the phone prior to recruitment. After visit 1 , volunteers will have a 4-week washout period prior to attending visit 2. Data will be obtained from hormone profiles, calorific intake and appetite / satiety visual analogue scales, and analysed at study completion.

[0031] Figure 5 shows a schematic time-line of each volunteer study visit. Volunteers given capsules at the 08:00 and 12:00 consistent with schedule for non-study days. Prior to commencement of study day, volunteers cannulated and blood sampling every 30 min as designated by black lines.

[0032] Figure 6 shows gut hormone release in response to colonic delivery of novel DIM and Perilla oil formulation. A: PYY levels were significantly increased in the active vs. placebo group (p = 0.0002). B: Circulating GLP-1 levels increased significantly in active vs. placebo treatment (p = 0.0181). Error bars represent SEM. Figure 7 shows consumption of standard meals at breakfast and lunch in placebo and active groups. A: The breakfast meal which consisted of 855kcal was given 1 hour after capsules, and mean calorific intake was not significantly difference between placebo and active groups. B: The lunch meal (1337kcal total) was significantly reduced for the female participants (12) but not the males (8). Error bars represent SEM.

[0033] Figure 8 shows visual analogue scale of satiety perception. Volunteers responding to satiety question at 30 min intervals showed no difference in responses during active or placebo arms. Error bars represent SEM.

[0034] Figure 9 shows the dosage study design. Volunteers were enrolled following interview to satisfy inclusion / exclusion criteria and randomised on day of study visit. Study day protocol was the same as in Figure 5.

[0035] Figure 10 shows the dose-dependent effect of DIM and Peri Ila oil formulation. A: PYY levels were highest in the maximal dose group with efficacy in release decreasing in response to dose. B: Circulating GLP-1 levels also showed dose-dependent release profile with the two lower doses exerting minimal effect on release.

[0036] Figure 11 shows the effects of using excipient Aeroperl to convert PO to powder and activation of human colonic epithelial cells using pCamKII.

[0037] Figure 12 shows the quantification of the number of positive pCamKII in each nutrient condition.

[0038] Figure 13 shows the effects of using excipient Aeroperl to convert PO to powder and activation of human colonic epithelial cells using pERK.

[0039] Figure 14 shows the quantification of the number of positive pERK in each nutrient condition.

[0040] Figure 15 shows the of GLP-1 (A) and PYY (B) post stimulation with nutrients.

[0041] Detailed Description

[0042] Compositions

[0043] According to any one of the first to the ninth aspect of the invention, the composition or pharmaceutical composition of the present invention may comprise or consist of 3,3'- diindolylmethane (DIM) and perilla oil comprising a-linolenic acid (ALA) as defined herein. Suitably, the perilla oil may comprise an amount from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil. The perilla oil may comprise an amount of about 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, or 70% a-linolenic acid ALA by weight, relative to the total weight of fatty acids in the perilla oil. Suitably, the perilla oil may comprise an amount of about 60% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

[0044] A method for determining the alpha-linolenic acid (ALA) content in perilla oil may comprise extracting the oil, converting it to fatty acid methyl esters (FAMEs) using sodium hydroxide and iodomethane, and analysing the resulting FAMEs via gas chromatography with a flame ionization detector (GC-FID), where the ALA percentage is calculated based on the weight of ALA relative to the total weight of fatty acids present in the sample. Suitable methods for determining the percentage of a-linolenic acid (ALA) in perilla oil are described according to ISO standards ISO 12966-2:2017 and ISO 12966-4:2015 and, for example, in Lee et al, Nat Pro Sci, 22(4): 259-262 (2016).

[0045] The perilla oil used in any one of the first to the ninth aspects of the invention may be food-grade and / or cosmetic-grade perilla oil. Suitably, the perilla oil may be obtained or derived from seeds of the Perilla frutescens (also known as Perilla ocymoides, and / or shiso) plant. The perilla oil may be identified by CAS number 90082-61-4.

[0046] 3,3'-diindolylmethane (DIM) is also known as 3,3'-methanediylbis(1 H-indole). a-linolenic acid (ALA) contains 3 unsaturated carbon-carbon double bonds (n-3) and is also known as (9Z,12Z,15Z)-9,12,15-Octadecatrienoic acid or c / s,c / s,c / s-9,12,15-Octadecatrienoic acid. It is also sometimes referred to as 18:3.

[0047] The compositions or pharmaceutical compositions of the present invention may also comprise mesoporous silica. The compositions or pharmaceutical compositions of the present invention may comprise 3,3’-diindolylmethane and perilla oil, wherein the perilla oil is adsorbed onto mesoporous silica. Adsorption of the perilla oil onto the mesoporous silica allows for the conversion of perilla oil into the form of a stable powder. This advantageously allows for easier handling of the 3,3'-diindolylmethane and perilla oil of the compositions or pharmaceutical compositions so that both can be stably packaged together in a single capsule or tablet.

[0048] Suitably, the mesoporous silica is a granulated form of colloidal silicon dioxide. Suitably, the mesoporous silica may comprise spherical granules having a diameter in the range of about 20- 60 pm, more suitably about 30-50 pm. Suitably, the mesoporous silica comprises ordered pore structures. The mesoporous silica may have a specific surface area (BET) ranging from 240 to 340 m2 / g. Suitably, the specific surface are (BET) of the mesoporous silica may range from 260 to 320 m2 / g. Suitably, the mesoporous silica may have a pore volume ranging from about 1 .5 to about 1.9 ml / g, wherein the pore volume is determined by Barrett-Joyner-Halenda (BJH) analysis. An example of mesoporous silica is AEROPERL® 300 Pharma having properties according to Table 1 below.

[0049] Table 1 . AEROPERL® 300 mesoporous silica - Characteristic physio-chemical data The compositions and pharmaceutical compositions of the present invention upregulate both extracellular signal-regulated kinase in its phosphorylated form (pERK) and calmodulin kinase in its phosphorylated form (pCaMKII) which therefore increases the number of activated cells compared to a single treatment with a single agonist alone. A combination formula is therefore able to maximally activate both L-cells and EC cells in human intestinal tissue. L-cells are a specialised form of enteroendocrine cells which secrete glucagon-like peptide-1 (GLP-1), peptide YY (PYY), oxyntomodulin and glucagon-like peptide-2 (GLP-2). EC cells are enterochromaffin cells or "Kulchitsky cells" that release serotonin.

[0050] The compositions and pharmaceutical compositions of the present invention are formulated such that, when administered to a subject, result in the secretion of GLP-1 and PYY from L-cells, and subsequent suppression of appetite.

[0051] Compositions of the invention comprising or consisting of 3,3’-diindolylmethane and perilla oil may be formulated as convenient for administration to a patient in need thereof. The composition or pharmaceutical composition of the invention may be administered as a nutraceutical.

[0052] As used herein, the term “nutraceutical” is defined as a product derived from food sources that provides physiological benefits or helps in the prevention, management, or treatment of diseases or disorders, beyond basic nutritional value. This term may encompass dietary supplements, functional foods, or bioactive compounds or compositions.

[0053] Nutraceutical appetite suppressants have the benefits of easy preparation from natural sources, fewer unwanted side effects. Nutraceutical appetite suppressants would also be typically available without prescription, thereby being easier to integrate into daily routines as compared to pharmaceutical products.

[0054] The inventors have discovered nutraceutical compositions comprising 3,3’-diindolylmethane (DIM) and perilla oil (derived from seeds of the shiso plant) that can effectively target nutrientsensing receptors concomitantly in the distal gut via the dual release of potent appetite-reducing (anorectic) hormones. Specifically, the compositions described herein work by activating a unique combination of nutrient-sensing g-protein coupled receptors (GPCRs) - GPR84 and GPR120 / FFAR4, both of which are highly expressed in the colonic (distal gut) region, resulting in the synergistic release of powerful anorectic hormones PYY and GLP-1. Perilla oil contains a- linolenic acid (ALA), which is known when prepared synthetically to stimulate the release of GLP- 1 (Sodum et al, Int J Mol Sci. 25(2):1087 (2024)). Further, the inventors found it surprising that compositions comprising perilla oil, a natural ingredient comprising only 50-70% ALA, would work in the same way as those consisting of synthetically produced, pure compounds. In particular, the fact that the described nutraceutical compositions act in a dose-dependent manner was unexpected, as until this point there have been no known studies showing a nutraceutical acting on GPCR receptors in a dose-dependent manner.

[0055] The inventors also advantageously found that by using specific types of mesoporous silica, the perilla oil of the compositions described herein can be converted into a stable powder. This enables the perilla oil to be packaged in a single capsule or tablet alongside DIM. Single tablets or capsules prepared in this manner allow for greater convenience, greater cost efficiency, reduced pill burden and increased compliance; whilst maintaining overall efficacy.

[0056] The composition of the invention may be provided in the form of a pharmaceutical composition. The composition may be prepared as a powder, a solid, or a liquid as convenient. Suitably, the composition is in the form of a powder.

[0057] The composition may be formulated with such additional salts, preservatives and stabilisers and / or excipients or adjuvants as required. For example, amino acids may be present in the formulation. The amino acid may be one or more selected from the group consisting of: Glycine, Alanine, Valine, Leucine, Isoleucine, Serine, Cysteine, Selenocysteine, Threonine, Methionine, Proline, Phenylalanine, Tyrosine, Tryptophan, Histidine, Lysine, Arginine, Aspartate, Glutamate, Asparagine, and Glutamine. An example of a preservative is Vitamin E, including isomers and derivatives thereof, e.g. a-tocopherol, y-tocopherol, d-alpha-tocopherol succinate.

[0058] Suitably, the composition or pharmaceutical composition or formulation can be provided in capsule or tablet form.

[0059] The capsule may be a delayed release capsule and where suitably said capsule will disintegrate only upon reaching the large intestine or terminal part of the small intestine. Suitably, the delayed release capsule is a colonic release capsule in which the contents are preferentially released in the colon. Suitable delayed release capsules can be prepared using anionic polymers of suitable monomers that degrade preferentially in a pH of 7.0 or above. Examples of such suitable monomers are methacrylic acid and methyl methacrylate (e.g. sold under the trade name Eudragit® by Evonik Industries). The capsule may therefore be composed of a polymer of methacrylic acid and methyl methacrylate.

[0060] Suitably the formulations are encapsulated in a capsule composed of a polymer of methacrylic acid and methyl methacrylate monomers.

[0061] Such capsules can be designed to release sufficient fatty acid to achieve local intraluminal concentrations of >25mmol / L, which has been shown to be optimal for stimulation of enteroendocrine cells and consequent release of gut satiety hormones.

[0062] Powder forms of the composition may be prepared in tablet, mini-tablet, pellet or granule form (usually filled into capsule shells) form for oral administration. The tablets may be coated with a suitable enteric coating in order to pass through the stomach of the patient for release of the composition in the small intestine. Suitable enteric coatings include, but are not limited to methyl acrylate-methacrylic acid copolymers, cellulose acetate phthalate (CAP), cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers, shellac, cellulose acetate trimellitate, alginate, e.g. sodium alginate, zein (maize protein).

[0063] Methods of preparation

[0064] According to a third aspect of the invention, there is provided method of preparing a composition comprising 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil, the method comprising: (a) contacting the perilla oil with mesoporous silica having a specific surface area (BET) ranging from 240 to 340 m2 / g, thereby to provide the perilla oil in the form of a powder;

[0065] (b) combining the powder of step (a) with 3,3’-diindolylmethane, to provide the composition.

[0066] According to a sixth aspect of the invention, there is provided a method of preparing a tablet or capsule comprising a composition comprising 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil, the method comprising:

[0067] (a) contacting the perilla oil with mesoporous silica having a specific surface area (BET) ranging from 240 to 340 m2 / g, thereby to provide the perilla oil in the form of a powder;

[0068] (b) combining the powder of step (a) with 3,3’-diindolylmethane to provide a composition;

[0069] (c) compressing the composition into the form of a tablet, or providing the composition as part of a capsule.

[0070] “Contacting” may include mixing or admixing the perilla oil with mesoporous silica as defined herein.

[0071] Suitably, the compositions, tablets or capsules used in the above methods of preparing may correspond to the compositions, tablets or capsules as defined herein.

[0072] Also provided by the present invention are compositions, tablets and capsules obtainable by the methods according to the third or sixth aspects of the invention.

[0073] Methods and uses

[0074] The invention provides a method for suppressing appetite in a subject, the method comprising administering, compositions, tablets or capsules as described herein to the subject. According to the seventh aspect, the invention provides a method for supressing appetite in a subject, the method comprising administering to the subject a composition consisting of, or a tablet or capsule comprising a composition consisting of 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) (suitably, about 60% a-linolenic acid (ALA)) by weight, relative to the total weight of fatty acids in the perilla oil.

[0075] Suitably, the 3,3’-diindolylmethane may be administered in an amount of from about 200 to about 600 mg. The 3,3’-diindolylmethane may be administered in an amount of about 200, 250, 300, 350, 400, 450, 500, 550 or 600 mg. The 3,3’-diindolylmethane may be administered in an amount of about 250 or 500 mg. The 3,3’-diindolylmethane may be administered in an amount of about 250 mg. The 3,3’-diindolylmethane may be administered in an amount of about 500 mg.

[0076] Suitably, the perilla oil may be administered in an amount of from about 1000 to 2200 mg. The perilla oil may be administered in an amount of about, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, or 2200 mg. The perilla oil may be administered in an amount of about 1050 or 2100 mg. The perilla oil may be administered in an amount of about 1050 mg. The perilla oil may be administered in an amount of about 2100 mg.

[0077] The composition, tablet or capsule may comprise the 3,3’-diindolylmethane in an amount of about 250 mg, and the perilla oil in an amount of about 1050 mg.

[0078] The composition, tablet or capsule may comprise the 3,3’-diindolylmethane in an amount of about 500 mg, and the perilla oil in an amount of about 2100 mg.

[0079] Suitably, capsules or tablets comprising compositions or pharmaceutical compositions of the invention are administered prior to a food meal. For example, capsules or tablets may be administered up to about 120 minutes before a meal. Capsules or tablets may be administered up to about, 30, 45, 60, 75, 90, 105 or 120 minutes before a meal. Suitably, capsules or tablets may be administered about 60 minutes before a meal.

[0080] The composition may be administered before two standard food meals up to 4 hours apart, suitably about 60 minutes before each food meal.

[0081] The composition, tablet or capsule may be administered at least once a day. For example, the composition, tablet or capsule may be administered once, twice, three times, four times or five times a day. Suitably, the composition, tablet or capsule may be administered twice a day.

[0082] In one suitable dosage regime, two capsules or tablets as defined above (e.g. each capsule comprising from about 200 to about 600 mg 3,3’-diindolylmethane and from about 1000 to 2200 mg perilla oil) can be administered per day, wherein each capsule or tablet is taken at up to about 120 minutes before a food meal is eaten.

[0083] In another suitable dosage regime, two capsules or tablets as defined above (e.g. each capsule comprising about 500 mg 3,3’-diindolylmethane and about 2100 mg perilla oil) can be administered per day, wherein each capsule or tablet is taken about 60 minutes before a food meal is eaten.

[0084] In another suitable dosage regime, two capsules or tablets as defined above (e.g. each capsule comprising about 450 mg 3,3’-diindolylmethane and about 2050 mg perilla oil) can be administered per day, wherein each capsule or tablet is taken about 60 minutes before a food meal is eaten. In another suitable dosage regime, two capsules or tablets as defined above (e.g. each capsule comprising about 550 mg 3,3’-diindolylmethane and about 2150 mg perilla oil) can be administered per day, wherein each capsule or tablet is taken about 60 minutes before a food meal is eaten.

[0085] In another suitable dosage regime, two capsules or tablets as defined above (e.g. each capsule comprising about 600 mg 3,3’-diindolylmethane and about 2200 mg perilla oil) can be administered per day, wherein each capsule or tablet is taken about 60 minutes before a food meal is eaten.

[0086] In another suitable dosage regime, two capsules or tablets as defined above (e.g. each capsule comprising about 250 mg 3,3’-diindolylmethane and about 1050 mg perilla oil) can be administered per day, wherein each capsule or tablet is taken about 60 minutes before a food meal is eaten.

[0087] In another suitable dosage regime, two capsules or tablets as defined above (e.g. each capsule comprising about 300 mg 3,3’-diindolylmethane and about 1100 mg perilla oil) can be administered per day, wherein each capsule or tablet is taken about 60 minutes before a food meal is eaten.

[0088] The invention also provides a method for treating impaired appetite regulation or feeling of satiety in a subject, the method comprising administering, compositions, tablets or capsules as described herein to the subject. According to the eighth aspect, the invention provides a method for treating impaired appetite regulation or feeling of satiety in a subject, the method comprising administering to the subject a composition consisting of, or a tablet or capsule comprising a composition consisting of 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) (suitably, about 60% a- linolenic acid (ALA)) by weight, relative to the total weight of fatty acids in the perilla oil.

[0089] The treatment of impaired appetite regulation or feeling of satiety may be in connection with the following diseases or disorders: obesity, type II diabetes, cardiovascular diseases (e.g. heart disease and stroke), musculoskeletal disorders (e.g. osteoarthritis); and cancer (e.g. endometrial cancer, breast cancer, and colon cancer), metabolic and endocrine disorders (e.g. diabetes mellitus, hyperthyroidism, Cushing’s Syndrome, and leptin resistance), psychiatric disorders (e.g. binge eating disorder, bulimia nervosa, recovery from anorexia nervosa and night eating syndrome), genetic disorders (e.g. Bardet-Biedl Syndrome and Congenital Leptin Deficiency), hypoglycaemia, medication-induced hyperphagia and premenstrual syndrome (PMS).

[0090] The subject treated for impaired appetite regulation or feeling of satiety may suffer from one of the above diseases or disorders. The compositions, tablets and capsules described herein may be used to prevent or treat any of the above diseases or disorders which are associated with impaired appetite regulation or feeling of satiety. The invention also provides a method of preventing or treating type II diabetes in a subject, the method comprising administering, compositions, tablets or capsules as described herein to the subject. According to the ninth aspect, the invention provides a method for method of preventing or treating type II diabetes in a subject, the method comprising administering to the subject a composition consisting of, or a tablet or capsule comprising a composition consisting of 3,3’- diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) (suitably, about 60% a-linolenic acid (ALA)) by weight, relative to the total weight of fatty acids in the perilla oil.

[0091] The compositions of the invention may be administered in any effective, convenient manner effective for suppressing appetite and / or treating impaired appetite regulation or feeling of satiety, including, for instance, administration by oral, nasogastric tube, rectal, intraperitoneal, or intravenous, routes among others. It is envisaged that the compositions of the invention will find greatest application as orally administrable formulations or as a suppository. The composition may also be administered to an individual as a liquid composition, for example as a sterile dispersion for administration via a nasogastric tube or a stoma.

[0092] The seventh, eighth and ninth aspects of the invention also encompass the compositions, tablets and capsules described herein for use in methods of suppressing appetite, for treating impaired appetite regulation or feeling of satiety, and for use in methods of preventing or treating type II diabetes.

[0093] The seventh, eighth and ninth aspects of the invention also encompass the use of said compositions, tablets and capsules in methods of suppressing appetite, in methods of treating impaired appetite regulation or feeling of satiety and in methods of preventing or treating type II diabetes.

[0094] The seventh, eighth and ninth aspects of the invention also encompass uses of said compositions, tablets and capsules in the manufacture of a medicament for appetite suppression, for the treatment of impaired appetite regulation or feeling of satiety, and for the prevention or treatment of type II diabetes.

[0095] Suitable doses, formulation and means of administration of the compositions, tablets and capsules of the seventh, eighth and ninth aspects are described herein.

[0096] As used herein, the term "treatment" or “treating” relates to alleviating, managing, resolving or curing the symptoms, effects, or underlying causes of a disease, disorder or condition in a subject who is already affected by it. This typically involves actions that mitigate the severity of the condition, improve health outcomes, or restore normal function. These terms includes any regime that can benefit a human or a non-human animal. The treatment of "non-human animals" extends to the treatment of domestic animals, including horses and companion animals (e.g. cats and dogs) and farm / agricultural animals including members of the ovine, caprine, porcine, bovine and equine families.

[0097] As used herein, the term “prevention” or “preventing” relates to the act of stopping or reducing the likelihood of the occurrence of a particular condition, disease, or disorder before it arises (e.g. prophylactic uses of the disclosed compositions, tablets or capsules). This may include measures or interventions that lower the risk or inhibit the development of the condition, disease or disorder. These terms include regimes that can benefit a human or a non-human animal. Prevention in "non-human animals" extends to prevention in domestic animals, including horses and companion animals (e.g. cats and dogs) and farm / agricultural animals including members of the ovine, caprine, porcine, bovine and equine families.

[0098] As used herein, the term “impaired appetite” is defined as a dysfunction or abnormality in the regulation of hunger or satiety (fullness), leading to an inability to appropriately initiate, sustain, or limit food intake. In particular, this term includes increased appetite (also known as hyperphagia) relative to what is physiologically appropriate in healthy subjects.

[0099] Preferred features of the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis.

[0100] The aspects provided herein are also described in the following clauses:

[0101] 1. A composition consisting of 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

[0102] 2. A pharmaceutical composition consisting of 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

[0103] 3. The composition or pharmaceutical composition according to paragraph 1 or 2, wherein the perilla oil comprises an amount of about 60% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

[0104] 4. The composition or pharmaceutical composition according any one of paragraphs 1-3, wherein the perilla oil is derived from Perilla frutescens.

[0105] 5. A method of preparing a composition comprising 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a- linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil, the method comprising: (a) contacting the perilla oil with mesoporous silica having a specific surface area (BET) ranging from 240 to 340 m2 / g, thereby to provide the perilla oil in the form of a powder;

[0106] (b) combining the powder of step (a) with 3,3’-diindolylmethane, to provide the composition.

[0107] 6. The method of paragraph 5, wherein the perilla oil comprises an amount of about 60% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

[0108] 7. The method of paragraph 5 or 6, wherein the perilla oil is derived from Perilla frutescens.

[0109] 8. The method of any one of paragraphs 5-7, wherein the mesoporous silica has a specific surface area (BET) ranging from 260 to 320 m2 / g.

[0110] 9. The method of any one of paragraphs 5-8, wherein the mesoporous silica has a pore volume ranging from about 1 .5 to about 1 .9 ml / g, wherein the pore volume is determined by Barrett-Joyner-Halenda (BJH) analysis.

[0111] 10. The method of any one of paragraphs 5-9, wherein the composition is in the form of a powder.

[0112] 11. A composition obtainable by the method according to any one of paragraphs 5-8.

[0113] 12. A tablet or capsule comprising a composition consisting of 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

[0114] 13. A tablet or capsule comprising a composition comprising 3,3’-diindolylmethane and perilla oil absorbed onto mesoporous silica, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

[0115] 14. The tablet or capsule according to paragraph 12 or 13, wherein the perilla oil comprises an amount of about 60% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

[0116] 15. The tablet or capsule according to any one of paragraphs 12-14, wherein the perilla oil is derived from Perilla frutescens. 16. The tablet or capsule according to any one of paragraphs 13-15, wherein the mesoporous silica has a specific surface area (BET) ranging from 240 to 340 m2 / g.

[0117] 17. The tablet or capsule according to paragraph 16, wherein the mesoporous silica has a specific surface area (BET) ranging from 260 to 320 m2 / g.

[0118] 18. The tablet or capsule according to any one of paragraphs 13-17, wherein the mesoporous silica has a pore volume ranging from about 1 .5 to about 1 .9 ml / g, wherein the pore volume is determined by Barrett-Joyner-Halenda (BJH) analysis.

[0119] 19. The tablet or capsule according to any one of paragraphs 13-18, wherein the composition is in the form of a powder.

[0120] 20. A method of preparing a tablet or capsule comprising a composition comprising 3,3’- diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil, the method comprising:

[0121] (a) contacting the perilla oil with mesoporous silica having a specific surface area (BET) ranging from 240 to 340 m2 / g, thereby to provide the perilla oil in the form of a powder;

[0122] (b) combining the powder of step (a) with 3,3’-diindolylmethane to provide a composition;

[0123] (c) compressing the composition into the form of a tablet, or providing the composition as part of a capsule.

[0124] 21. The method according to paragraph 20, wherein the perilla oil comprises an amount of about 60% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

[0125] 22. The method of paragraph 20 or 21 , wherein the perilla oil is derived from Perilla frutescens.

[0126] 23. The method of any one of paragraphs 20-22, wherein the mesoporous silica has a specific surface area (BET) ranging from 260 to 320 m2 / g.

[0127] 24. The method of any one of paragraphs 20-23, wherein the mesoporous silica has a pore volume ranging from about 1 .5 to about 1 .9 ml / g, wherein the pore volume is determined by Barrett-Joyner-Halenda (BJH) analysis. 25. A tablet or capsule obtainable by the method according to any one of paragraphs 20-24.

[0128] 26. A method for supressing appetite in a subject, the method comprising administering to the subject a composition consisting of, or a tablet or capsule comprising a composition consisting of 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

[0129] 27. The method of paragraph 26, wherein the perilla oil comprises an amount of about 60% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

[0130] 28. The method of paragraph 26 or 27, wherein the perilla oil is derived from Perilla frutescens.

[0131] 29. The method for supressing appetite according to any one of paragraphs 26-28, wherein the composition, tablet or capsule comprises the 3,3’-diindolylmethane in an amount of from about 200 to about 600 mg, and the perilla oil in an amount of from about 1000 to about 2200 mg.

[0132] 30. The method of paragraph 29, wherein the composition, tablet or capsule comprises the 3,3’-diindolylmethane in an amount of about 250 mg, and the perilla oil in an amount of about 1050 mg.

[0133] 31 . The method of paragraph 29, wherein the composition, tablet or capsule comprises the 3,3’-diindolylmethane in an amount of about 500 mg, and the perilla oil in an amount of about 2100 mg.

[0134] 32. The method according to any one of paragraphs 26-31 , wherein the composition, tablet or capsule is administered at least once a day.

[0135] 33. The method of paragraph 32, wherein the composition, tablet or capsule is administered twice a day.

[0136] 34. A method of treating impaired appetite regulation or feeling of satiety, the method comprising administering to a subject a composition consisting of, or a tablet or capsule comprising a composition consisting of 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil. 35. A method of preventing or treating type II diabetes, the method comprising administering to an individual in need of such treatment a composition consisting of, or a tablet or capsule comprising a composition consisting of 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a- linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

[0137] 36. A composition consisting of, or a tablet or capsule comprising a composition consisting of 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil, for use in a method of treating impaired appetite regulation or feeling of satiety, the method comprising administering the composition, tablet or capsule to an individual in need of such treatment.

[0138] 37. A composition consisting of, or a tablet or capsule comprising a composition consisting of 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil, for use in a method of preventing or treating type II diabetes, the method comprising administering the composition, tablet or capsule to an individual in need of such treatment.

[0139] 38. The use of a composition consisting of, or tablet or capsule comprising a composition consisting of 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil, in the manufacture of a medicament for the treatment of impaired appetite regulation or feeling of satiety.

[0140] 39. The use of a composition consisting of, or a tablet or capsule comprising a composition consisting of 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil, in the manufacture of a medicament for the prevention or treatment of type II diabetes.

[0141] 40. Use of a composition consisting of, or tablet or capsule comprising a composition consisting of 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil, in a method of suppressing appetite in a subject. 41. Use of a composition consisting of, or a tablet or capsule comprising a composition consisting of 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil, in a method of treating impaired appetite regulation or feeling of satiety in a subject.

[0142] 42. Use of a composition consisting of, or a tablet or capsule comprising a composition consisting of 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil, in a method of preventing or treating type II diabetes in a subject.

[0143] 43. The method of paragraph 34 or 35, the composition, tablet or capsule for use according to paragraph 36 or 37, or the use according to any one of paragraphs 38-42, wherein the perilla oil comprises an amount of about 60% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

[0144] 44. The method of any one of paragraphs 34, 35 or 39, the composition, tablet or capsule for use according to any one of paragraphs 36, 37 or 39, or the use according to any one of paragraphs 38-42, wherein the perilla oil is derived from Perilla frutescens.

[0145] 45. A kit comprising:

[0146] 3,3’-diindolylmethane, and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil, and optionally an administration vehicle, and / or instructions for use.

[0147] Examples

[0148] The present invention will now be described by way of reference to the following examples. These examples are not to be construed as being limiting on the invention.

[0149] Example 1 : Preparation and pre-clinical validation of optimised formulation to induce cell activation and release of satiety hormones PYY and GLP-1.

[0150] The aim of the pre-clinical validation studies presented below is to demonstrate that an optimised formulation of one GPR84 agonist (DIM) and one FFA4 agonist (a-linolenic acid (ALA)) can bind effectively to these GPCRs to induce cell activation and release of PYY and GLP-1 from human colonic L-cells. Methods

[0151] 1.1 Preparation of formulations

[0152] The formulation used in the present example was DIM (10mM) and Perilla oil (60pL).

[0153] 1.2 Human Colonic Tissue Collection

[0154] Full thickness human colon tissue was obtained from 9 consented patients (6 women, 3 men; mean age = 54) undergoing surgery for colon cancer at The Royal London Hospital (Barts Health NHS Trust). Normal tissue that was at least 10cm from the tumour was resected by a pathologist. Ethical approval was obtained from the East London and The City HA Local Research Ethics Committee [NREC 09 / H0704 / 2],

[0155] 1.3 Cell activation studies - Us sing chamber experiments

[0156] Human colonic mucosal sheets were mounted in an Ussing flux chamber and stimulated with nutrients using methods previously optimised for human gut tissue (Symonds, Peiris et al, Gut, 2015; Peiris et al, Gut, 2022). Expression of phosphoprotein markers was subsequently assessed to reflect downstream intracellular pathway engagement using immunohistochemistry to identify phosphorylated calmodulin KII (pCaMKII) and phosphorylated extracellular signal- related kinase (pERK). Images were captured via epi-fluorescent microscopy and counted for positive cell staining. Tissue was also double-stained with enteroendocrine cell markers PYY and GLP-1 to identify the activated state of L-cells that store satiety hormones.

[0157] 1.4 Hormone release assays

[0158] A key experimental outcome is the capacity of DIM and perilla oil to stimulate their respective receptors to boost release of the potent appetite reducing hormones PYY and GLP-1 from human colonic tissue. It was previously demonstrated that dual activation of GPR84 and FFAR4 can increase hormone output by 2-4 times compared to single receptor activation and this was recapitulated in the previous human trial (Peiris et al, Gut, 2022). Therefore, validating the new formulation using hormone release assays is a key determinant of efficacy in a clinical setting. Human colonic mucosa was dissected, weighed and incubated in control buffer or nutrients within a 96 well plate for 20min at 37°C. GLP-1 and PYY was quantified using a human multiplex kit according to manufacturer’s instructions as previously described (Symonds, Peiris et al, Gut, 2015; Peiris et al, Gut, 2021).

[0159] 1.5 Data Analysis

[0160] Positive immunoreactive cells, found within colonic crypts and captured completely in field of view were counted as previously described (Symonds, Peiris et al, Gut, 2015; Peiris et al, Gut, 2021). Statistical tests for all studies were performed using GraphPad Prism software and p < 0.05 considered significant. Results

[0161] Cell activation is increased by dual stimulation of GPR84 and FFA4 by DIM and Perilla oil Previously, the capacity of nutrients to induce activation of a cell by using phosphorylation markers pCaMKII and pERK as they are selectively activated by FFA4 and GPR84, respectively, in human colonic tissue has been assessed (Peiris et al, Gut, 2022). To demonstrate that the new formulation of DIM (10mM) and Perilla oil (60pL) acts similarly, Ussing chambers were used to recapitulate an environment where the superficial surface of the colonic mucosa would be stimulated by luminally available nutrients. Stimulation with both DIM and Perilla oil significantly increased the number of pERK (Figure 1) and pCaMKII (Figure 2) positive cells / crypt, compared to each nutrient alone. PYY containing L-cells were found to co-stain with pCaMKII and adjacent to pERK positive cells suggesting that DIM and PO dual stimulation can activate L-cells directly or via paracrine mechanisms.

[0162] Satiety hormone release from ex vivo colonic tissue significantly increased by DIM and Perilla oil treatment

[0163] To determine if cell activation would translate to release of gut hormones from vesicular stores, human colonic mucosa was dissected and incubated with DIM, Perilla oil or both DIM and Perilla oil. Quantification of total PYY and GLP-1 (Figure 3) showed that dual nutrient treatment increased release of both hormones compared to single nutrient treatment. This data concurs with previously published data where synergy was shown using lauric acid and TUG891 to activate GPR84 and FFA4, respectively. Importantly, these data validate the efficacy of the new formulation in a pre-clinical, human tissue data set.

[0164] Example 2: Experimental medicine study in obese volunteers to assess efficacy of novel nutrientbased formulation on satiety hormone release profile and food intake

[0165] The primary aim of this Phase I study was to demonstrate that the formulation validated in human gut tissue can effectively boost hormone release in vivo and reduce food intake, to the same degree as the first formulation (data described Peiris et al, Gut, 2022). A strategy was used that replicates the design of the previous study so data could be compared effectively. Completion of this trial will validate data for the new, optimised formulation that can be carried forward to a Phase II clinical trial assessing weight loss.

[0166] Methods

[0167] 2. 1 Study design

[0168] Randomised pilot cross-over study with double-blinding (Figure 4). A total of 20 volunteers with BMI 30-40kg / m2(obese) were asked to take capsules twice a day, at 0800h and 1200h, on visit 1 and 2, containing either active or placebo (Figure 4). Composition of the meal challenge was identical for all visits, with known calorific content for breakfast and lunch. 2.2 Participants

[0169] Volunteers (12 female, 8 males; mean age - 47) were recruited from the general population, with a BMI 30-40 kg / m2(Mean = 34.1), no previous history of bowel surgery, not taking medication for Type II diabetes and agree to consume test meals. Volunteers were asked to commit to 2 study visits taking place at Wingate Institute Clinical Trials unit, London, U.K.

[0170] 2.3 Interventions

[0171] Active: 4 nutrient combination capsules (500mg 3'3 diindolylmethane (DIM); 2100mg Perilla oil containing a-linolenic acid (ALA) (obtained from Gustav Hees)).

[0172] Placebo: 4 capsules visually identical to active containing methylcellulose.

[0173] All capsules were GuardCap® from Evonik GmBH to ensure colonic release of actives and placebo.

[0174] 2.4 Outcomes

[0175] • Measurement of circulating levels of hormones PYY and GLP-1 at visit 1 and visit 2, taken every 30 mins from 0800h to 1600h.

[0176] • Measurement of calorific intake at visit 1 and visit 2, following completion of breakfast and lunch meal challenge.

[0177] • Measurement of hunger and appetite perception using visual analogue scales (VAS) at visit 1 and visit 2, taken every 30mins from 0800h to 1600h.

[0178] 2.5 Study visits

[0179] Prior to each study visit, volunteers were requested to fast from 20:00h the previous evening until beginning of study visit at 08:00h. The outline for each study was consistent for all visits (Figure 5).

[0180] Visit 1 : Volunteers were consented for study and BMI measurements taken to ensure eligibility. Blood samples collected every 30 min (17 samples in total). Breakfast & lunch given with calorific intake recorded. Treatment or placebo capsules dosed at 0800h and 1200h.

[0181] Visit 2: Blood samples collected every 30 min (17 samples in total). Breakfast & lunch given with calorific intake recorded. Treatment or placebo capsules dosed at 0800h and 1200h.

[0182] The maximum calorific intake for breakfast was 903 kcal and for lunch was 1340 kcal. Volunteers were told to eat as much as desired. Following completion of the meal, the amount of food consumed was quantified and calorific intake calculated.

[0183] 2.6 Gut Hormone Assays

[0184] Blood samples collected in ice-cold ethylenediaminetetraacetic acid (EDTA) Vacutainer® tubes were supplemented with 10pl of DPPIV inhibitor (50Nm) to prevent hormone degradation. Plasma was collected following centrifugation (10min at 1000xg), and 1 ml aliquots stored at -20°C. Total GLP- 1 and total PYY were quantified using Milliplex MAP Multiplex Gut Hormone assay (Merck Millipore) as previously described (Symonds, Peiris et al, Gut, 2015; Peiris et al, Gut, 2022).

[0185] 2.7 Data Analysis

[0186] To analyse VAS questionnaires, recorded answers for each time point in placebo and active groups were averaged and compared using a two-way ANOVA and Sidak’s multiple comparison test. Changes in total calorific intake was analysed using paired Student’s t-test and a p value of <0.05 deemed significant. Area under the curve (AUC) for GLP-1 and PYY, was calculated and compared using paired Student’s t -test following normality testing, p value of <0.05 was considered significant.

[0187] Results

[0188] Circulating PYY and GLP-1 levels significantly increased by colonic delivery of DIM and Perilla oil

[0189] A key outcome of the pre-clinical experiments was the release of gut hormones PYY and GLP-1 (Peiris et al, Gut, 2022). The aim of the current reformulation study was to demonstrate that combining 2 ingredients in a single capsule, with each ingredient activating their respective receptor i.e. GPR84 activated by DIM and FFA4 activated by ALA (in Perilla oil) could induce gut hormone release in the same way as 3 ingredients (Lauric acid / DIM / Perilla oil) delivered to the colon separately (as was carried out in the previous trial). Using the experimental paradigm described above, active treatment significantly increased circulating levels of PYY, compared to placebo (Figure 6A). There was also a significant increase in GLP-1 release in active vs. placebo treated (Figure 6B), an improvement in release compared to data from previous study. Collectively, the satiety hormone release demonstrates the new formulation and combination of ingredients into a single capsule improves the concomitant activation of GPR84 and FFA4 successfully and potently induces hormone release from colonic L-cells.

[0190] Reduction of food intake in female volunteers

[0191] Food intake was measured at 2 timepoints i.e. breakfast and lunch, both meals had known number of calories and represented standard meals used in previously published studies. At breakfast, there was no significant difference in food intake for either men or women, between active and placebo groups. (Figure 7A). This data concurs with the profile of colonic release capsules that take approx. 3 hours to arrive and disintegrate in the colon. However, the active group had a significant reduction in calorific intake at lunch - specifically women in the study (Figure 7B). The male cohort did not show a significant reduction which is likely due to insufficient calorific challenge as the total caloric content of both meals was 2192 which reflects the recommended daily intake for women but is 400 kcal lower than the recommended intake for men (2500 kcal). The food challenge protocol will be adjusted for subsequent studies.

[0192] Appetite perception scores unchanged in active vs. placebo

[0193] To assess if there was a perceptible difference in food intake behaviour, volunteers were asked 3 questions using VAS to assess hunger, fullness and desire to eat at 30 min intervals. There was no significant difference between active and placebo groups for either of the 3 questions asked (Figure 8) - concurring with our previous data (Peiris et al, Gut, 2022).

[0194] Example 3: Dosing study using validating DIM + PO formulation to determine dose-dependent effect on circulating satiety hormone levels

[0195] Following validation of the nutrient combination of DIM and PO in both pre-clinical and clinical settings, it was sought to determine the capacity of 2 lower doses to induce hormone release in obese volunteers. Data from this study would inform on the dosage arms most likely to induce weight loss in a Phase II trial allowing optimisation of trial design.

[0196] Methods

[0197] 3. 1 Study design

[0198] Two arm study with double-blinding. A total of 20 volunteers with BMI 30-40kg / m2(obese), 10 per dosage arm were asked to take capsules twice a day, at 0800h and 1200h (Figure 9). Composition of the meal challenge was identical for participants but differentiated by gender with male participants receiving a total of 2539 calories and female participants 2180 calories, with known calorific content for standardised breakfast and lunch meals.

[0199] Volunteers (9 female, 7 male; mean age - 46) were recruited from the general population, with a BMI 30-40 kg / m2(Mean = 33.8), no previous history of bowel surgery, not taking medication for Type II diabetes and agree to consume test meals. Volunteers were asked to commit to a single day visit taking place at Wingate Institute Clinical Trials unit, London, U.K.

[0200] 3.2 Interventions

[0201] Medium dose: 2 nutrient combination capsules (250mg 3'3 diindolylmethane (DIM); 1050mg Perilla oil (obtained from Gustav Hees). containing a-linolenic acid (ALA)) and 2 capsules visually identical to active containing methylcellulose.

[0202] Low dose: 1 nutrient combination capsule (125mg 3'3 diindolylmethane (DIM); 525mg Perilla oil containing a-linolenic acid (ALA) (obtained from Gustav Hees)) and 3 capsules visually identical to active containing methylcellulose.

[0203] All capsules were GuardCap® from Evonik GmBH to ensure colonic release of actives and placebo. 3.3 Outcome

[0204] Measurement of circulating levels of hormones PYY and GLP-1 taken every 30 mins from 0800h to 1600h.

[0205] 3.4 Study visit

[0206] Prior to the study visit, volunteers were requested to fast from 20:00h the previous evening until beginning of study at 08:00h. Volunteers were consented for study and BMI measurements taken to ensure eligibility. Blood samples were collected every 30 min (17 samples in total). Breakfast & lunch was with calorific intake recorded as per protocol detailed in Figure 5.

[0207] 3.5 Gut Hormone Assays

[0208] Blood samples collected in ice-cold ethylenediaminetetraacetic acid (EDTA) Vacutainer® tubes were supplemented with 10pl of DPPIV inhibitor (50Nm) to prevent hormone degradation. Plasma was collected following centrifugation (10min at 1000xg), and 1 ml aliquots stored at -20°C. Total GLP- 1 and total PYY were quantified using Milliplex MAP Multiplex Gut Hormone assay (Merck Millipore) as previously described (Symonds, Peiris et al, Gut, 2015; Peiris et al, Gut, 2022).

[0209] 3.6 Data Analysis

[0210] Change in hormones was relative to the baseline value taken at time-point 0 prior to ingestion of capsules. Error bars represent SEM.

[0211] Results

[0212] Nutrient driven dose-dependent release of gut hormones

[0213] Shown in Figure 10, the assessment of the two doses referred to in Figure 9 as Medium (250mg 3'3 diindolylmethane (DIM); 1050mg Perilla oil containing a-linolenic acid (ALA)) and Low (125mg 3'3 diindolylmethane (DIM); 525mg Perilla oil containing a-linolenic acid (ALA)), demonstrated dose-dependent efficacy in release of PYY and GLP-1. The high dose i.e. 500mg diindolylmethane (DIM); 2100mg Perilla oil containing a-linolenic acid (ALA), used in the study described in Figure 6, was the most efficacious (Figure 10).

[0214] Overall conclusions from pre-clinical and clinical studies (Phase I and dosing).

[0215] Gut derived satiety hormones are critical regulators of food intake, and in the case of GLP-1 , regulation of insulin release in its role as an incretin hormone. In obesity, a deficit in circulating levels of hormones including GLP-1 and PYY, leads to increased food intake arising from diminished satiety. It has been shown for the first time, concomitant targeting of nutrient-sensing GPCRs - GPR84 and FFA4, by nutrient ligands leads to release of PYY and GLP-1 from isolated gut tissue via activation of distinct intracellular signalling pathways. Importantly, the data show refinement of the scientific approach based on published receptor-nutrient ligand interaction kinetics, for optimal commercial use. It was unexpected that the effect of appetite suppression would be dose dependent for the nutrient ligands, which show dose dependent behaviour that is comparable to a pharmaceutical drug. This is especially advantageous since GPR84 is an orphan receptor.

[0216] The above data confirms that the novel formulation - a) activates cells and promotes the release of PYY and GLP-1 from colonic L-cells, b) increases circulating levels of both PYY and GLP-1 in obese volunteers, c) reduces food intake, and, d) acts in a dose-dependent manner.

[0217] The discussed approach validates targeting L-cells to release vesicular stores of GLP-1 and PYY by activating nutrient-sensing GPCRs expressed on these cells as a clinically viable approach to increasing circulating levels of potent satiety hormones.

[0218] This is the first and only non-surgical approach that: a) increases endogenous GLP-1 and PYY levels, b) activates GPCRs expressed on L-cells, and, c) uses organic nutrients, to collectively induce to reduced food intake - a key requirement for weight loss.

[0219] A dataset has thus been built that provides the foundation for delivery of a Phase II trial where the primary outcome is weight loss over a 6-month treatment window.

[0220] Example 4: Preparation and hormone release studies of DIM and perilla oil compositions using mesoporous silica as an excipient

[0221] Nutrient solutions

[0222] Nutrients were made up in low-glucose Krebs solutions: 124.05mM NaCI (Sigma-Aldrich), 4.78mM KCI (Sigma-Aldrich), 1.33mM NaH2PO4 (Sigma-Aldrich), 2.44mM MgSO4 (Sigma- Aldrich), 5.50mM D-glucose (Sigma-Aldrich) and 25.00mM NaHCOs (Sigma- Aldrich) and carbogenated with 95% O2 and 5% CO2. DIM (10mM, Sabinsa LTD) was used as an agonist of GPR84 agonist and Aeroperl (AEROPERL® 300 Pharma) containing Perilla oil (60pl), (Evonik, Gustav Hees - CAS number 90082-61-4) was used as an agonist for GPR120. Aeroperl without perilla oil was used as a control.

[0223] Ussing chamber

[0224] Human colonic mucosal sheets (from cancer resections) were divided into 1 x1 cm segments, and each segment was mounted in an Ussing flux chamber. For optimised nutrient stimulation of human colonic mucosal sheets, the luminal surface was exposed to 10 mL nutrient solution, while the basolateral surface was exposed to 10 mL Krebs solution, for 20 min. All solutions were carbogenated and maintained at 35°C-37°C. Colonic mucosa was then fixed in 4% paraformaldehyde overnight. Tissues were then cryoprotected in 30% sucrose / PBS then mounted in optimum cutting temperature medium. Tissue sections of 10pm were cut on a Cryostat (Leica CM1860), incubated with blocking buffer (Dako, UK) for 1 hour, before primary antibodies (pERK or pCaMKII -1 in 400 cell signalling) were applied overnight (4°C). Tissues were washed (PBS; 3x5 min), and species-specific secondary antibodies were conjugated to Alexa Fluor fluorescent dyes (1 :400, Thermo Fisher Scientific, UK) applied for 1 hour, before washing (PBS; 3x5 min), mounting (Vectashield hard set mounting media, Vector Laboratories, USA) and cover-slipping. Controls with no primary antibody were used in all experiments to check for non-specific secondary antibody binding. Leica DM4000 epi-fluorescence microscope was used to visualise immunoreactivity of sections. To ensure uniformity of acquired images, all sections were orientated and cut in the same manner. Images were captured using MetaMorph software (Molecular Devices, UK) and prepared for figures using Photoshop (Adobe) and PowerPoint (Microsoft).

[0225] Hormone release

[0226] Human colonic mucosa was dissected into small, approximately 0.5 mm blocks, and each specimen was weighed before incubation with 250 pL of carbogenated solutions of buffer (control) or nutrients in a 96-well plate for 20 min at 37°C. A customised tissue culture medium with 4.4 mM L-glutamine and 6 mM glucose was used in order to ensure cells were healthy but not stimulated with nutrients. Inhibitors were used to prevent the breakdown of hormones. GLP- 1 and PYY protein levels using a human multiplex kit according to manufacturer's instructions (Milliplex MAP Multiplex assay, Merck Millipore).

[0227] Results

[0228] As shown in Figure 15 formulation of perilla oil with the Aeroperl excipient, when combined with DIM results in increased release of PYY and GLP-1 from isolated gut tissue. This release is consistent with previous examples demonstrating the effect of the combination of perilla oil and DIM in the absence of Aeroperl, confirming that formulation with Aeroperl does not impact the efficacy of the nutrient combination of DIM and perilla oil. Therefore, this study confirms that Aeroperl is well-suited for use as an excipient for enabling incorporation of DIM and perilla oil together in a single capsule for nutraceutical use.

Claims

Claims1. A composition consisting of 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

2. A pharmaceutical composition consisting of 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

3. The composition or pharmaceutical composition according to claim 1 or 2, wherein the perilla oil comprises an amount of about 60% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

4. The composition or pharmaceutical composition according any one of claims 1-3, wherein the perilla oil is derived from Perilla frutescens.

5. A method of preparing a composition comprising 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil, the method comprising:(a) contacting the perilla oil with mesoporous silica having a specific surface area (BET) ranging from 240 to 340 m2 / g, thereby to provide the perilla oil in the form of a powder;(b) combining the powder of step (a) with 3,3’-diindolylmethane, to provide the composition.

6. The method of claim 5, wherein the perilla oil comprises an amount of about 60% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

7. The method of claim 5 or 6, wherein the perilla oil is derived from Perilla frutescens.

8. The method of any one of claims 5-7, wherein the mesoporous silica has a specific surface area (BET) ranging from 260 to 320 m2 / g.

9. The method of any one of claims 5-8, wherein the mesoporous silica has a pore volume ranging from about 1 .5 to about 1 .9 ml / g, wherein the pore volume is determined by Barrett-Joyner- Halenda (BJH) analysis.

10. The method of any one of claims 5-9, wherein the composition is in the form of a powder.

11. A tablet or capsule comprising a composition consisting of 3,3’-diindolylmethane and peri Ila oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

12. A tablet or capsule comprising a composition comprising 3,3’-diindolylmethane and perilla oil absorbed onto mesoporous silica, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

13. The tablet or capsule according to claim 11 or 12, wherein the perilla oil comprises an amount of about 60% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

14. The tablet or capsule according to any one of claims 11-13, wherein the perilla oil is derived from Perilla frutescens.

15. The tablet or capsule according to any one of claims 12-14, wherein the mesoporous silica has a specific surface area (BET) ranging from 240 to 340 m2 / g.

16. The tablet or capsule according to claim 15, wherein the mesoporous silica has a specific surface area (BET) ranging from 260 to 320 m2 / g.

17. The tablet or capsule according to any one of claims 12-16, wherein the mesoporous silica has a pore volume ranging from about 1 .5 to about 1 .9 ml / g, wherein the pore volume is determined by Barrett-Joyner-Halenda (BJH) analysis.

18. The tablet or capsule according to any one of claims 12-17, wherein the composition is in the form of a powder.

19. A method of preparing a tablet or capsule comprising a composition comprising 3,3’- diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil, the method comprising:(a) contacing the perilla oil with mesoporous silica having a specific surface area (BET) ranging from 240 to 340 m2 / g, thereby to provide the perilla oil in the form of a powder;(b) combining the powder of step (a) with 3,3’-diindolylmethane to provide a composition;(c) compressing the composition into the form of a tablet, or providing the composition as part of a capsule.

20. The method according to claim 19, wherein the perilla oil comprises an amount of about 60% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.21 . The method of claim 19 or 20, wherein the perilla oil is derived from Perilla frutescens.

22. The method of any one of claims 19-21 , wherein the mesoporous silica has a specific surface area (BET) ranging from 260 to 320 m2 / g.

23. The method of any one of claims 19-22, wherein the mesoporous silica has a pore volume ranging from about 1.5 to about 1.9 ml / g, wherein the pore volume is determined by Barrett- Joyner-Halenda (BJH) analysis.

24. A method for supressing appetite in a subject, the method comprising administering to the subject a composition consisting of, or a tablet or capsule comprising a composition consisting of 3,3’-diindolylmethane and perilla oil, wherein the perilla oil comprises an amount of from about 50% to about 70% of a-linolenic acid (ALA) by weight, relative to the total weight of fatty acids in the perilla oil.

25. The method of claim 24 wherein the perilla oil comprises an amount of about 60% of a-linolenic acid (ALA).

26. The method of claim 24 or 25, wherein the perilla oil is derived from Perilla frutescens.

27. The method for supressing appetite according to any one of claims 24-26, wherein the composition, tablet or capsule comprises the 3,3’-diindolylmethane in an amount of from about 200 to about 600 mg, and the perilla oil in an amount of from about 1000 to about 2200 mg.

28. The method of claim 27, wherein the composition, tablet or capsule comprises the 3,3’- diindolylmethane in an amount of about 250 mg, and the perilla oil in an amount of about 1050 mg.

29. The method of claim 27, wherein the composition, tablet or capsule comprises the 3,3’- diindolylmethane in an amount of about 500 mg, and the perilla oil in an amount of about 2100 mg.

30. The method according to any one of claims 24-29, wherein the composition, tablet or capsule is administered at least once a day.31 . The method of claim 30, wherein the composition, tablet or capsule is administered twice a day.

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