LU103233B1 - Treatment of obesity and related conditions by circadian administration of enterokine-stimulating compositions - Google Patents

Abstract

The present invention relates to the treatment and prevention of obesity and/or conditions associated therewith in a subject, preferably a human, in need thereof by by at least twice daily circadian oral administration of at least two doses of the composition. The invention further relates to the corresponding use of the composition for the treatment and/or prevention of obesity and/or conditions associated therewith.

Description

Our Ref.: A 0733 LU Date: 21 December 2023 LU103233

Applicant: APHAIA IP AG

Treatment of obesity and related conditions by circadian administration of enterokine- stimulating compositions

The present invention relates to the treatment and prevention of obesity and/or conditions associated therewith in a subject, preferably a human, in need thereof by oral administration of a composition comprising one or more enterokine-releasing substances in a non- resorption formulation by at least twice daily circadian oral administration of at least two doses of the composition. The invention further relates to the corresponding use of the composition for the treatment and/or prevention of obesity and/or conditions associated therewith.

It has been recognised previously that delivery of a nutritional substance to the ileum through use of an enteric dosage form of the nutritional substance leads to satiety of a mammalian subject (US 5753253 A). More recently, specific delivery of a nutritional substance, in particular glucose, to the ileum of a subject, in particular dosage forms of a nutritional substance like glucose, which dosage forms release at least 50 % of the administered substance in the ileum of the subject, was suggested for treatment of metabolic diseases like type 2 diabetes mellitus, metabolic syndrome, insulin resistance, obesity, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) and other conditions (WO 2010/027498 A2, WO 2012/118712 A2, US 2014/0294951 A1).

WO 2020/120581 A1 relates to pharmaceutical oral dosage forms comprising a core and a pH-sensitive enteric coating, wherein the core comprises at least one compound stimulating enteroendocrine cells to release at least one enterokine, and at least one disintegrant providing a burst release of the ingredients of the core when the coating is substantially degraded and/or dissolved, and wherein the coating comprises a pH sensitive polymer being selected such that the coating substantially dissolves and/or is substantially degraded in the jejunum of a subject.

WO 2021//249641 A1 relates to pharmaceutical compositions and pharmaceutical articles comprising such compositions wherein the compositions comprise multiple dosage forms each comprising a core and an enteric coating, wherein the core comprises at least one compound stimulating enteroendocrine cells to release at least one enterokine, wherein the size of the dosage forms, with respect to the largest dimension of the dosage forms, provides 1 for entry of the dosage forms into the intestine of a subject independent of gastric emptying LU103233 mechanisms, and wherein the composition further comprises one or more gelling agents.

WO 2021/249641 A1 also relates to the treatment and/or prevention of conditions amenable to stimulation of enterokine release by enteroendocrine cells.

The technical problem underlying the present invention is to provide improved therapeutic regimen making use of enterokine-releasing substances for preventing and/treating obesity and conditions associated therewith.

In particular, the present invention relates to a composition, preferably in the form of compositions selected form dietary, nutraceutical and pharmaceutical compositions, comprising one or more enterokine-releasing substances in a non-resorption formulation for use in the treatment and/or prevention of obesity and/or conditions associated therewith in a subject, in particular a subject in need of such treatment, preferably a mammalian, more preferably a human subject, by at least twice daily circadian oral administration of at least two doses of the composition.

According to the invention, the term “non-resorption formulation” means a formulation of the one or more enterokine-releasing substances ensuring that the active substance(s) of the composition, at least the majority of the active substance(s), i.e. more than 50%, preferably more than 60 %, more preferably more than 70 %, still more preferred more than 80 %, even more preferred at least 90 %, most preferred 99% or more of the active substance(s, where the afore-mentioned percentages are expressed as the part of the substance(s) present in the administered composition, preferably expressed as mol-% or weight %, are released from the composition after the composition has reached the duodenum of the subject. In turn, it is preferred that the formulation, i.e. the composition for use in the invention, is resistant against degradation and/or dissolution in the duodenum and the further upper parts of the digestive tract including the oral cavity, pharynx, oesophagus and stomach.

The term “twice daily circadian oral administration” means that the composition is administered at least two times a day per os and that the at least two administrations take place in a rhythmic fashion such that the at least two administrations comply with the circadian rhythm of the subject's hunger state and/or physical activity and/or sleep/wake state and/or satiety state and/or glucose metabolism and/or lipid metabolism and/or energy expenditure. 2

In a preferred embodiment of the invention the at least one first dose of the composition is ~~ LU103233 administered at about 8 am +/- about 3 h and the at least one second dose of the composition is administered at about 4 pm +/- about 3 h.

More preferably, the at least one first dose of the composition is administered at about 8 am +/- about 2 h and the at least one second dose of the composition is administered at about 4 pm +/- about 2 h. In further preferred embodiments of the invention, the at least one first dose of the composition is administered at about 8 am +/- about 1 h and the at least one second dose of the composition is administered at about 4 pm +/- about 1 h. Most preferred, the at least first dose of the composition is administered at about 8 am +/- about 0.5 h and the at least second dose of the composition is administered at about 4 pm +/- about 0.5 h.

It is preferred that the with the time period between the administration of the at least one first dose and the administration of the at least one second dose is from about 7 to about 9 hours, preferably from about 7.5 hours to about 8.5 hours, most preferably about 8 hours.

For increasing the effect of the inventive treatment regimen even more, it may desirable that at least one third dose of the composition is administered, preferably at about 9 pm +/- about 2 hours, more preferably at about 9 pm +/- 1 h, most preferably at about 9 pm +/ about 0.5 h.

It is further preferred that the time period between the administration of the at least one second dose and the administration of the at least one third dose of the composition for use in the invention is about 3 h to about 5 hours, preferably about 3.5 h to about 4.5 hours, most preferred about 4 hours.

An “obese” human subject according to the invention preferably is a human subject having a

BMI of about 30 or more. An “overweight” human subject according to the invention is a human subject having a BMI of about 25 or more, preferably from about 25 to a BMI above25 but below 30.

In the context of the invention “treatment” means that the condition of the subject, preferably a human, is at least meant to result in a prevention of further progression such as, particularly in case of obesity, the further increase in body weight and/or BMI. In other embodiments of the invention the term “treatment” in the context obesity may also mean that the subject, preferably a human, does not develop insulin resistance and/or other pre- diabetic conditions. In other embodiments, the term “treatment” means in the context of treatment of obesity and/or insulin resistance and/or other pre-diabetic conditions that the 3 subject, preferably a human, does not develop T2DM. Developing or having, respectively, LU103233

T2DM preferably means in the context of the invention that the subject, preferably a human, does not depend on the administration of anti-diabetic drugs such as insulin.

Similarly, the term “prevention” as used in the present invention typically relates to a prophylactic treatment of a subject, preferably a human subject. Preferably, with respect to obesity, prevention means that the subject does develop a BMI of 30 or more. In other embodiments, it prevention of obesity may also relate to a person not entering a state of overweight, preferably not developing a BMI of 25 or more.

In view of the prior art, the inventive twice daily oral circadian administration, preferably according to the above time schedules, has important and unexpected benefits. In particular, the regimen according to the invention leads to a restoration of circadian rhythmicity (as perturbation of circadian rhythmicity is a hallmark of various metabolic diseases (see, e.g.

Shimizu et al. (2016) Hypertension Research 39, 483-491; doi:10.1038/hr.2016.12)which cannot be attained by prior art approaches such as disclosed in WO 2021/249641 A1 which teaches a single administration in the fasting state, more particularly a single administration according to the specific circadian release patterns of an enterokine to be released by the action of the enterokine-releasing substance, more preferably a single administration according to the circadian rhythm of endogenous of GLP-1 synthesis, and particular preferred 2 to 4 hours before GLP-1 reaches its circadian maximum in the serum or blood, respectively, of the subject.

Compared to prior art approaches, in particular WO 2021/249641 A1, it is essential that the inventive rhythmic application of the enterokine-releasing composition, which simulates enterokine responses corresponding to a full meal intake, generates a metabolic benefit that is independent of the hunger- reducing and satiety-increasing effect postulated in WO 2021/249641 A1.

In fact, the administration regime according to the present invention is completely independent of the fasting state, times of meal intake and circadian rhythm of enterokine level, which constitutes further substantial and surprising property in view of the prior art.

Compared to the prior art, the rhythmic at least twice daily circadian administration regimen according to the invention has improved effects on subjects being obese or suffering from obesity and/or conditions caused by obesity such as type 2 diabetes mellitus (T2FM) and prediabetic conditions (e.g. insulin resistance), metabolic syndrome, NASH, NAFLD, cardiovascular conditions (e.g. myocardial infarction, stroke), neurologic disorders (e.g.

Parkinson’s disease), improvement of immunological response etc. (see Fig. 1). The at least 4 two times daily administration regimen of the invention also has a beneficial effect with LU103233 respect to patient compliance because the rhythmisation of the administration is supposed to generate faster and more effective weight loss which in turn will enhance patient compliance.

Furthermore, the present invention simplifies the administration scheme by avoiding several decision steps that were previously left to be decided by the subject such as determining the main meal and intake time until the meal (2 hours, at least 30 min before the meal as taught in WO 2021/249641 A1) which will further enhance compliance and success rate.

The inventive treatment regimen is particularly beneficial for human subjects having late and/or night eating behaviours. “Late eating” is meant according to the invention as eating a meal after about 8 pm. “night eating” is meant according to the invention as eating a meal after sunset and before sunrise, preferably between 10 pm and 6 am. It has been shown that such subjects, e.g. night workers representing a typical group of subjects having a night eating behaviour, suffer from circadian misalignment and glucose intolerance (cf. Chellappa etal. (2021) Sci. Adv. 7, eabg9910).

The enterokine-releasing substance(s) of the composition for use in the invention stimulate(s) enteroendocrine cells to release at least one enterokine.

An “enteroendocrine cell’ (EEC) as used in the present invention, is a cell present in a subject’s, preferably, human subject's, intestine, in particular in the small intestine’s mucosa, and secretes one or more enterokines upon receiving an appropriate trigger by a nutritional component. There are several types of enteroendocrine cells which can be addressed by the active compound or compounds released by the pharmaceutical dosage form of the invention (for a review of enteroendorine cells of the lower gastrointestinal tract, see, for example, Gunawardene et al. (2011) Int. J. Exp. Pathol. 92, 219-231, and Latorre et al. (2017) Neurogastrol. Motil 28 (5), 620-630). In particular, preferred enteroendocrine cells in the context of the invention are | cells, K cells and L cells. with L cells being most preferred.

According to the invention, an “enterokine”’ is a hormone secreted by EECs in the gastro- intestinal system. Preferred enterokines are the incretins, i.e. hormones regulating the blood sugar level upon intake of nutrition, preferably GLP-1, GLP-2 GIP and PYY, more preferably

GLP-1 and PYY. Other preferred enterokines secreted by the EEC(s) through release of the active compound(s) in the pharmaceutical dosage form are CCK and neurotensin. cells are predominantly present in the proximal small intestine, in particular in the lower duodenum and in the jejunum and secrete cholecystokinin (CCK) upon sensing of nutrients 5 such as amino acids and fatty acids. CCK effects the release of bile acids from the gall LU103233 bladder into the small intestine, but also promotes the release of digestive secrete from the pancreas.

Kcells are found in the complete small intestine and release GIP (glucose-dependent insulinotropic peptide; also known as gastric inhibitory peptide) leading to inhibition of gastric motility and enhancement of insulin production and secretion.

L cells are present throughout the small intestine, i.e. duodenum, jejunum, and ileum, and release GLP-1 (glucagon-like peptide 1), GLP-2 (glucagon-like peptide 2) and PYY (peptide

YY) in response to various nutrients. The concentration of L cells raises from duodenum to jejunum to ileum, and the GLP-1 release from L cells shows a gradient sloping from proximal to distal small intestine with highest GLP-1 release capacity in the terminal ileum. The gradient is not only a function of increasing number of L cells, but also a function of their maturation state and production rate of GLP-1, which also increase from proximal to distal ileum. In particular, GLP-1 is released from L cells in the crypts and on the villi of the mucosa.

L cells mature from crypt to villi, and GLP-1 release capacity is highest when the L cells reach the top of the villi. L cells further secrete PYY (peptide YY) which is predominantly released in the terminal ileum. The time dependency of GLP-1 and PYY release by L cells in the jejunum and ileum in response to glucose uptake by a human subject is schematically depicted in Fig. 1. The maturation dependency of GLP-1 and PYY secretion capacity of L cells is shown schematically in Fig. 3. GLP-1 enhances nutrient-stimulated insulin secretion and inhibits glucagon secretion, gastric emptying and feeding. GLP-1 also has proliferative, neogenic and antiapoptotic effects on pancreatic B-cells. As an intestinal trophic peptide,

GLP-2 stimulates cell proliferation and inhibits apoptosis in the intestinal crypt compartment,

It also regulates intestinal glucose transport, food intake and gastric acid secretion and emptying. Furthermore, GLP-2 improves intestinal barrier function. PYY inhibits gastric motility and increases water and electrolyte absorption in the colon, PYY also suppresses pancreatic secretion and has been shown to reduce appetite. PYY slows gastric emptying; whereby it increases efficiency of digestion and nutrient absorption after a meal.

EECs, in particular L cells, are triggered to release enterokines such as GLP-1 and PYY through a variety of mechanisms being affected by the one or more active compound(s) in the pharmaceutical dosage form of the invention.

L cells release GLP-1 and PYY (and also GLP-2; note that GLP-1 and GLP-2 are derived from a common mRNA so that these hormones are essentially co-released; cf., for example, 6 the review of Baggio and Drucker (2004) Best Practice & Research Clinical Endocrinology & LU103233

Metabolism 18 (4), 531-554) in response to various mechanisms triggered by compounds such as nutrients. One mechanism includes intake of a carbohydrate such as glucose through glucose transporters GLUT2 and/or SGLT1. Other mechanisms rely on the binding to specialized G protein-coupled receptors such as taste receptors, fatty acid receptors, bile acid receptors, peptide receptors and amino acid receptors.

These signals, glucose transport and binding to G protein-coupled receptor, are typically transmitted in the cells by one or more of three mechanisms and lead ultimately to the release of the enterokine, in the case of L cells GLP-1 and PYY: transmembrane calcium influx, intracellular calcium release and/or intracellular cAMP increase.

It is therefore preferred that the one or more enterokine-releasing substances is/are selected from the group consisting of compounds stimulating the enteroendocrine cells by a mechanism selected from the group consisting of transport into enteroendocrine cells by a transporter expressed by said cells wherein the transporter is selected from the group consisting of GLUT2 and SGLT1, and binding to G protein-coupled receptors, preferably bile acid receptors, amino acid receptors, peptide receptors, fatty acid receptors an/or taste receptors expressed by said cells.

Preferably, the active compound(s), i.e. the one or more enterokine-releasing substance(s), is/are preferably selected from nutrient compounds (simultaneously referred to herein as “nutrient(s)” or “nutrient substance(s)).

More preferred active compounds include carbohydrates, fatty acids, bile acids, peptides (including oligopeptides, polypeptides and proteins), amino acids, alcohol amides and anthocyanins. Preferred fatty acids are fatty acids having 2 to 6 carbon atoms.

Ethanolamides such as oleoylethanolamide, anandamide (N-arachidonoylethenolamide,

AEA), palmitoylethanolamide, steaorylethanolamide, and derivatives of anandaminde such as prostamides, can also preferably be used as alcohol amide compounds in the inventive oral dosage forms. A particularly preferred ethanolamide is oleoylethanolamide (OEA). A preferred example of a peptidic active compound is the protein bovine serum albumin (BSA).

Carbohydrates are preferably selected from glucose and sucralose, with glucose being most preferred. In a particular preferred embodiment of the invention, the composition contains about 1 % (w/w) to about 99 % (w/w), such as 60 to 70 % (w/w) of active compound triggering the EECs, preferably a carbohydrate, most preferred glucose (dextrose).

Particularly preferred glucose contents in the core of the multiple dosage forms are in the 7 range of form about 5 % (w/w) to about 95 % (w/w), preferably from about 25 % (w/w) to LU103233 about 75 % (w/w), more preferably from about 40 % (w/w) to about 70 % (w/w).

According to the invention, enterokine-releasing substances exerting their effects vis GLUT- 2/SGLT-1 such as glucose which is most preferred, are particularly preferred as an active substance because it enters the cell through two transporters (Glut-2 and SGLT-1) having high capacity and triggering cellular depolarization directly (SGLT-1 Na+/glucose co-transport and indirectly (metabolism of glucose and ATP-dependent inhibition of K+ channels after transport by Glut-2), which enables the transmembrane influx of calcium in turn resulting in vesicle fusion with the plasma membrane and thus the release of enterokines. Other enterokine-release mechanisms rely on the relatively weaker intracellular calcium release from the endoplasmatic reticulum, thus exerting the desired effect to a lesser extent.

In a preferred embodiment, the active compound triggering the release of an enterokine through an EEC is an anthocyanin, more preferably one or more anthocyanins from

Vaccinium myrtilloides Michx. A particularly preferred anthocyanin to be included in the core of the pharmaceutical oral dosage forms of the invention is delphinidin 3-rutinoside.

In a preferred embodiment of the invention, one or more of the above active compounds triggering enterokine release by EECs are combined in preferably synergistic combinations.

Preferred combinations are combinations of a carbohydrate, preferably glucose, with sucralose and/or one or more fatty acids having 2 to 6 carbon atoms and/or oleic acid and/or one or more bile acids and/or one or more alcohol amides, preferably one or more ethanolamides, more preferably oleoylethanolamide, and/or one or more peptides (including oligopeptides, polypeptides and proteins, such as preferably BSA) and/or one or more amino acids and/or one or more anthocyanins (preferably delphinidin 3-rutinoside).

Further preferred combinations are combinations of another carbohydrate, preferably sucralose, with glucose and/or one or more fatty acids having 2 to 6 carbon atoms and/or oleic acid and/or one or more bile acids and/or one or more alcohol amides, preferably one or more ethanolamides, more preferably oleoylethanolamide, and/or one or more peptides (including oligopeptides, polypeptides and proteins) such as preferably BSA, and/or, one or more amino acids and/or one or more anthocyanins (such as preferably delphinidin 3- rutinoside). 8

Other preferred combinations are combinations of a fatty acid having 2 to 6 carbon atoms LU103233 with one or more carbohydrates, preferably glucose and/or sucralose, and/or oleic acid and/or one or more bile acids and/or one or more alcohol amides, preferably one or more ethanolamides, more preferably oleoylethamolamide, and/or one or more peptides (including oligopeptides, polypeptides and proteins), preferably BSA) and/or one or more amino acids and/or one or more anthocyanins such as preferably delphinidin 3-rutinoside.

According to a further preferred embodiment, the present invention provides combinations of oleic acid with one or more fatty acids having 2 to 6 carbon atoms and/or one or more carbohydrates, preferably glucose and/or sucralose, and/or one or more bile acids and/or one or more alcohol amides, preferably one or more ethanolamides, more preferably oleoylethanolamide and/or one or more peptides (including oligopeptides, polypeptides and proteins, such as preferably BSA) and/or one or more amino acids and/or one or more anthocyanins such as preferably delphinidin 3-rutinoside.

Further preferred combinations of active compounds are combinations of one or more bile acids with one or more carbohydrates, preferably sucralose and/or glucose, and/or one or more fatty acids having 2 to 6 carbon atoms and/or oleic acid and/or one or more alcohol amides, preferably one or more ethanolamide, more preferably oleoylethanolamide, and/or one or more peptides (including oligopeptides, polypeptides and proteins, preferably BSA, and/or one or more amino acids and/or one or more anthocyanins such as preferably delphinidin 3-rutinoside.

Still further preferred combinations of active compounds are combinations of one or more alcohol amides, preferably one or more ethanolamides, more preferably oleoylethanolamide, with one or more carbohydrates, preferably sucralose and/or glucose, and/or one or more fatty acids having 2 to 6 carbon atoms and/or oleic acid and/or one or more bile acids and/or one or more peptides (including oligopeptides, polypeptides and proteins) preferably BSA, and/or one or more amino acids and/or one or more anthocyanins such as preferably delphinidin 3-rutinoside.

Other preferred combinations of active compounds are combinations of one or more peptides (including oligopeptides, polypeptides and proteins), preferably BSA, with one or more carbohydrates, preferably sucralose and/or glucose, and/or one or more fatty acids having 2 to 6 carbon atoms and/or oleic acid and/or one or more bile acids and/or one or more alcohol amide, preferably one or more ethanolamide, more preferably oleoylethanolamide, and/or 9 one or more amino acids and/or one or more anthocyanins such as preferably delphinidin 3- LU103233 rutinoside.

In other preferred embodiments of the invention, the composition contains a combination of one or more amino acids with one or more carbohydrates, preferably sucralose and/or glucose, and/or one or more fatty acids having 2 to 6 carbon atoms and/or oleic acid and/or one or more bile acids and/or one or more ethanolamides, more preferably oleoylethanolamide, and/or one or more peptides (including oligopeptides, polypeptides and proteins); preferably BSA, and/or one or more anthocyanins such as preferably delphinidin 3- rutinoside.

In still further preferred combinations of active compounds used in the invention, one or more anthocyanins, preferably delphinidin 3, is/are combined with one or more carbohydrates, preferably sucralose and/or glucose, and/or one or more fatty acids having 2 to 6 carbon atoms and/or oleic acid and/or one or more bile acids and/or one or more alcohol amides, preferably one or more ethanolamides, more preferably oleoylethanolamide, and/or one or more peptides (including oligopeptides, polypeptides and proteins), preferably BSA, and/or one or more amino acids.

The active compound(s) in the composition are preferably further combined with active ingredients having various functions.

Preferred examples of additional active ingredients are EEC maturation agents. As exemplified before with L cells such maturation agents typically enhance the release capacity of the EECs, such as L cells, for the relevant enterokine, in the case of L cells GLP-1 and/or

PYY and/or GLP-2. Preferred EEC maturation agents, in particular L cell maturation agents, include human milk oligosaccharides (HMO) and inhibitors of NOTCH signalling such as y- secretase inhibitors, preferably dibenzazepine. Various HMOs are commercially available (Jennewein Biotechnologie GmbH, Rheinbreitbach, Germany) and preferred HMOs in the context of the invention include, but are not limited t, e.g. 2’-fucosyllactose, 3-fucosyllactose, 6’-sialyllactose and lacto-N-neotetraose as well as mixtures thereof.

In preferred embodiments of the invention the composition comprises a core containing the one or more enterokine-releasing substance(s), preferably one or more of the above- described preferred nutrient compounds or their combinations, and an enteric coating being essentially resistant against degradation and/or dissolution in the duodenum and further upper parts of the digestive tract. 10

The composition for use in the invention is preferably designed to burst release the active ingredient(s) at a selected targeted area of the small intestine of a subject, preferably the jejunum, more preferably, the terminal (= distal) jejunum) of the, preferably human, subject.

For providing a burst release of the ingredients of the core of the composition, the core contains at least one disintegrant. Disintegrants for use in the present invention are generally known in the art as rapidly expanding and dissolving when coming into contact with the targeted environment, typically, an aqueous environment as in the present invention, namely, the small intestine of a subject, preferably a human. Disintegrants lead to rapid breakdown of the core of the pharmaceutical oral dosage form of the invention when the core comes into contact of the aqueous medium present in the subject's small intestine. Preferably, the disintegrant in the multiple dosage forms of the composition of the invention is selected such that more than 70 % of the core is released within two minutes or less or more than 85 % of the core is released within 5 minutes or less, following contact with water or an aqueous medium like the small intestine of a human subject. Preferred disintegrants in the context of the invention are crosslinked polyvinylpyrrolidones, crosslinked carboxymethyl celluloses and modified starchs. Particularly preferred crosslinked polyvinylpyrrolidones for use in the invention include Polyplasdones, in particular Polyplasdone XL, Polyplasdone XL-10 and

Polyplasdone INF-10 (commercially available from Ashland Inc., Covington, KY, USA). Other useful disintegrants include, but are not limited to, polyvinylpolypyrrolidone, croscarmellose and carboxymethylcellulose (preferably, the sodium salt thereof).

A burst release of the core’s ingredients, in particular the active compound(s) such as glucose, establishes a steep gradient between intracellular and extracellular levels of the respective active compound(s), preferably glucose, sucralose, ethanolamides, BSA and/or an anthocyanine such as delphinidin 3-rutinoside, in the targeted EEC, preferably L cells, at the site of release of the core ingredients.

Compositions for use in the present invention preferably take the form dietary and/or nutraceutic and/or pharmaceutical dosage forms being small in dimension, preferably below 3 mm in the largest dimension, from about 0,6 mm to 1,7 mm, and most preferred from about 0,8 mm to about 1,2 mm, in the largest dimension. Such small dosage forms may conveniently take the form of granules or pellets. Small dosage forms for use in the invention have the benefit of behaving like a fluid in a subject's stomach (cf. Fig. 6 of WO 2021/246191

A1) causing a fast and constant entry of the small oral dosage forms for use in the invention into the intestinal tract, and therefore to more evenly transport it to the targeted burst release area in the subject's jejunum, preferably the subject's terminal (i.e. distal part) jejunum. 11

In other embodiments of the invention, it may also be convenient that compositions of the invention are present as dosage forms of larger size, i.e. forms wherein the largest dimension of the dosage form is about 3 mm or more, the upper size limit being conveniently selected by the skilled person such that the dosage form can be well swallowed by the subject. A typical range for oral dosage forms of the invention are dosage forms having a largest dimension of about 3 to about 10 mm. It is to be understood that this range includes all integers of mm, namely, 3, 4, 5, 6, 7, 8, 9 and 10 mm as well as any sub-proportions thereof.

Preferably, the composition for use in the invention containing the above-described small dosage forms comprises multiple of such small dosage forms.

The term “multiple dosage forms” typically means that the composition contains at least about1000, more preferably at least about 2000, more preferably at least about 3000, particular preferred at least about 5000, even more preferred at least about 10000 dosage forms. Preferred ranges are from about 1000, about 2000, about 3000, about 5000, or about 10000 to about 20000, about 30000, about 40000 or about 50000 dosage forms in the composition of the invention, preferably per unit dose of the composition according to the invention. Especially preferred ranges are about 10000 to about 40000 dosage forms, and more preferred compositions of the invention contain about 20000 to about 30000 dosages forms. In further preferred embodiments of the invention, the composition for use in the invention comprises from about 10000 to about 27000 small dosage forms as described above. In particular with respect to such embodiments, the size of the dosage forms preferably ranges, according to the above definition, from about 0,6 mm to 1,7 mm, and most preferred from about 0,8 mm to about 1,2 mm.

According to particularly preferred embodiments of the invention, the size and number of the multiple dosage forms contained in the inventive pharmaceutical composition is controlled such that the surface area of the multiple dosage forms in the composition, especially in a unit dose of the composition, is selected such that it covers at least 15 %, more preferably at least 20 %, particularly preferred at least about 25 % of the target area where the at least one active compound as defined herein interacts with the enteroendocrine cells so as to induce a release of the enterokine. In this context, it is to be understood that said target area is not necessarily confined, and in certain embodiments of the invention cases preferably not confined, to the area where the core of the multiple dosage forms releases the active compound. The target area of the active compound is typically dependent on the type of 12 enteroendocrine cells to be triggered to release the enterokine(s). As a preferred example, L LU103233 cells which are preferably induced by the active compound(s) in the core of the multiple dosage forms are, as further outlined below, present in humans in the small intestine from duodenum to ileum, but their enterokine release capacity, specifically GLP-1 release capacity, particularly due to the increased density and differentiation of the enteroendocrine cells, increases from proximal to distal small intestine, and is highest in the terminal ileum In preferred embodiments of the invention, especially for triggering L cells to release GLP-1 and/or PYY, preferably GLP-1, the enteric coating of the multiple dosage forms is selected such that the at least one active compound is released from the core of the multiple dosage forms in the jejunum, preferably the terminal jejunum of a subject, and the surface area of the multiple dosage forms of the composition of the invention, preferably a unit dose of the composition, is selected such that said surface area makes up between from about 10 % to about 50 %, preferably from about 15 about 35 %, more preferred about 20 to about 30 % of the targeted part of the intestine for the active compound where the targeted cells, preferably

L cells, have their highest concentration and/or enterokine release capacity, which is for L cells the ileum, in particular the terminal ileum. In this contest, the term “surface area of the target part of the intestine” is approximated as taken the intestine as a tube having a mean diameter of about 0.25 cm and a length of the terminal ileum of about 60 cm.

As outlined above, it is preferred that compositions for use in the invention release the active compound(s) in the jejunum, more preferably in the terminal jejunum, of a subject, preferably a human subject, through a specific design of the enteric coating, typically a pH sensitive coating. Preferably, the coating substantially degrades and/or dissolves in the jejunum, preferably the terminal jejunum, such that the core is released into the jejunum, preferably the terminal jejunum, of the subject, by specific selection of the enteric coating which is preferably chosen from pH sensitive polymers substantially degrading and/or dissolving at a pH value of about 5.5 to about 7.5, preferably about 7.2 to about 7.3. Such pH sensitive polymers are preferably selected from hydroxypropylmethyl celluloses (also called hereinafter “hypromelloses”) and anionic copolymers of methacrylic acid and methacrylmethacrylate. Most preferably, the pH sensitive enteric coating containing or being made of hydroxypropylmethyl cellulose is hydroxypropylmethyl cellulose acetate succinate. A highly preferred commercially available product of this kind is AQOAT®, particularly preferred

AQOAT®-HF (Shin-Etsu Chemical Co., Chiyoda, Japan). In other preferred embodiments of the type of anionic copolymers of methacrylic acid and methcrylmethacrylate various forms of

Eudragit® polymers may also be used. Eudragit® is commercially available from Evonik

Healthcare & Nutrition GmbH, Essen, Germany. In preferred embodiment, Eudragit® FS30D 13 and/or Eudragit® L30D is/are used as the pH sensitive polymer(s) of the coating, or at least a LU103233 part thereof.

In further preferred embodiments of the invention, different coatings can be applied in combination. According to one embodiment, the coating comprises or is made of a combination of a hydroxypropylmethy! cellulose and an anionic copolymer of methacrylic acid and methacrylmethacrylate. Preferably, a combination of coatings is applied such that typically a sub-coating of one pH sensitive polymer is applied as a first layer and a coating of a second pH sensitive polymer is applied on the sub-coating as a second layer. For example, the pH sensitive coating can comprise a sub-coating of or comprising, respectively, a hydroxypropylmethyl cellulose as a first layer, and a second coating comprising or being made of an anionic copolymer of methacrylic acid and methacrylmethacrylate provided as a second layer on the sub-coating. In a further preferred embodiment, the coating of the pharmaceutical oral dosage form of the invention comprises a coating comprising a first layer (sub-coating) comprising or being made of an anionic polymer of methacrylic acid and methacrylmethacrylate such as an Eudragit®, more preferably Eudragit® FS30D, and a second layer comprising or being made of a hydroxypropylmethyl cellulose such as

AQOAT®, more preferably AQOAT®-HF. More preferably, the anionic copolymer of methacrylic acid and methacrylmethacrylate, e.g. an Eudragit®, preferably Eurdragit®

FS30D, is present in less amount than the hydroxypropylmethyl cellulose such as AQOAT®, more preferably AQOAT®-HF. In other words, the thickness of the first layer of this type of combination is lower than the thickness of the second layer in this combination. More specifically, the ratio of amount or thickness, respectively, between first layer and second layer typically ranges from about 1:10 to about 1:50, particularly preferred from about 1:20 to about 1:30.

For further improving the enterokine release exerted by the composition, it is preferred that the multiple dosage forms, in particular their core, further contain enteroendocrine release improvement agents, i.e. a substance enhancing enterokine release by the enteroendocrine cells effected by the compound stimulating enteroendocrine cells to release at least one enterokine, particularly preferred at least one substance enhancing release of GKLP-1 and/or

PYY by L cells. For preferred embodiments of the invention where L cells are targeted to release GLP-1 such agents are preferably selected from inhibitors of GLP-1 degradation such as DPP-4 inhibitors, substances enhancing L cell maturation (e.g. human milk oligosaccharides, inhibitors of NOTCH signalling such as y-secretase inhibitors, preferably dibenzazepine), stimulating agents and combinations thereof. A preferred stimulating agent for use in the present invention is caffeine. One example of a preferred combination of active 14 compound and stimulating agent is glucose and caffeine, preferably in a weight ratio of LU103233 glucose to caffeine of from about 5:1 to about 50:1, preferably about 8 to 1 to about 40:1. In other embodiments, preferred combinations of glucose and caffeine in the core of the multiple oral dosages forms are about 60 to about 70 % (w/w) glucose and about 2 to about 4% (w/w) caffeine, most preferred about 67 % (w/w) glucose and about 3.2 % (w/w) caffeine, based on the total weight of the core.

Caffeine and other known substances can also be included for controlling and monitoring the appropriate release and the spreading of the ingredients of the core of the multiple oral dosage forms contained in the composition of the invention in the intestinal tract of the subject, preferably a human. The release of the tracer substance from the multiple dosage forms of the pharmaceutical composition in the small intestine can be conveniently monitored by analytical methods generally known in the art. In the case of caffeine, blood samples are taken from the subject before and at suitable time intervals after oral administration of the oral dosage form. After centrifugation of the blood sample, the caffeine content in the serum fraction of the sample is measured by ELISA testing using commercially available test kits (e.g. Caffeine ELISA Kit, BioVision Inc., Milpitas, CA, USA) according to the manufacturer's instructions.

The compositions for use in the invention are preferably fine-tuned in various further ways so as to further improve targeted release and triggering of EECs in the small intestine of a subject, preferably a human subject. Moreover, different compositions having a variety of release patterns in the subject's small intestine can be combined in the composition of the invention so as to synergistically act on the subject's EECs and their enterokrine output.

In certain embodiments of the invention, the composition for use according to the inventive regimen comprises multiple dosage forms as outlined above which are small in dimension, preferably below 3 mm in the largest dimension, more preferably about 0.6 mm to about 1.7 mm in the largest dimension, particularly preferred from about 0.8 mm to about 1.2 mm in the largest dimension. As already outlined above, such small dosage forms may conveniently take the form of beads, granules or pellets. As has been explained in detail above, the small dosage forms of the invention have the benefit of behaving like a fluid in a subject’s stomach causing a fast and constant entry of the multiple oral dosage forms into the intestinal tract, and therefore to evenly transport it to the targeted area, preferably a targeted burst release area, preferably in the subject's jejunum, more preferably the subject's terminal (i.e. distal part) jejunum. 15

The composition for use in the invention preferably further contains at least one gelling LU103233 agent. This embodiment of the invention is particular preferred in embodiments where the composition takes the form of or comprises multiple small dosage forms as described above.

Gelling agents, also known as thickeners, are gel-forming agents when dissolved in a liquid phase as a colloidal mixture forms a weakly cohesive internal structure. They are typically organic hydrocolloids or hydrophilic inorganic substances. Typical examples include tragacanth, pectin, starch, carbomers, polysaccharides, gelatin, cellulose derivatives, polyvinyl alcohol clays, polyethylene glycols and others (for a review, see, e.g. Kar et al. “Current Developments in Excipient Science, Section 2.2.2.5 “Gelling Agents” in: R. Tekade (ed.) Basic Fundamentals in Drug Delivery, Academic Press, Cambridge, MA, USA, 2019).

The term “at least one gelling agent” as used herein also includes compositions of one or more gelling agents, optionally in combination with other components.

Preferred gelling agents, either for use alone or for use in gelling compositions containing combinations of such gelling agents, include, but are not limited to, modified celluloses such as preferably substituted methylcelluloses, such as hydroxypropylmethylcelluloses (HPMCs), polysaccharides such as preferably alginates, e.g. sodium alginate, and Xanthan gum, and polyethylene glycols (PEGs), preferably PEGs having a mean molecular weight of from about 10000 Da to about 30000 Da, more preferably 15000 Da to about 25000 Da, most preferred about 20000 Da. In preferred embodiments of the invention, the composition contains more than one gelling agent in order to fine tune the properties of the composition, in particular with respect to a specific design of the viscosity properties so as to greatly improve swallowability of the composition in use, when containing or when reconstituted, respectively, with a, preferably pharmaceutically acceptable, liquid medium, e.g. water or an aqueous solution. Preferred combinations of gelling agents, typically in form of a gelling composition, include 2, 3 or 4 gelling agents. Particularly preferred combinations of gelling agents for use in the inventive composition are selected from combinations of modified celluloses, polysaccharides and PEGs, more preferably one or more of the above-mentioned preferred examples.

It is preferred that the one or more gelling agents or the gelling composition are not included in the multiple dosage forms.

The multiple dosage form composition for use in the invention can be provided in various forms. In one preferred embodiment, composition contains a liquid medium so that the multiple dosage forms and the one or more gelling agent are present in a liquid medium, preferably water or an aqueous solution, more preferably a buffered aqueous solution. In 16 other preferred embodiments of the invention, it is also possible that the at least one gelling LU103233 agent or, if the at least one gelling agent is present in a gelling composition, the gelling composition and the multiple dosage forms form a heterogeneous mixture. For use of the latter embodiment of composition for use in the invention, a liquid medium, as described above and further disclosed herein, is preferably added to the composition. For a unit dose of the composition, a volume of the liquid is selected typically based on the number of the multiple dosage and/or their size. For preferred values of the multiple dosage forms, the dry embodiment of the pharmaceutical composition is typically admixed with about 5 ml to about 40 ml, preferably about 10 to about 35 ml, most preferred about 15 to 30 ml of the liquid medium.

In order to provide stabilization of the composition upon oral administration, in which form it typically comprises a suitable liquid medium, in particular for stabilization of a gel or gel-like formulation of composition in the milieu of mouth, throat and/or oesophagus, the composition of the invention preferably comprises one or more pH modifiers, such as preferably citric acid, a salt of citric acid such as tri-potassium citrate monohydrate, adipic acid, sodium hydrogen carbonate, tartaric acid and combinations thereof. In further preferred embodiments of the invention, the one or more pH modifiers can also be included in the gelling composition as described above.

In addition to the above components the composition for use in the invention may contain further ingredients typically present in oral dosage forms such as tablets, capsules, granules and pellets, for providing and/or improving various parameters. Typical additional ingredients for use in the present invention include excipients, carriers, fillers, glidants, dispersants, plasticizers, wetting agents, anti-tacking agents, neutralization agents, colorants, pigments, opacifiers, flavours, taste improvement agents such as sweeteners, and the like. It is to be understood that these and other benefit agents can be included in the composition as described herein, for example in the multiple dosage forms as described herein and/or added to the composition comprising such multiple dosage forms, for example, and preferably, in the gelling composition, separate from the multiple dosage forms. The person skilled in the art of formulating pharmaceutical compositions and the multiple oral dosage forms is readily able to identify specific compounds and substances of the above and other types as well as their combinations and amounts to be used. Further guidance can be found in Remington's

Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, in particular pages 1289- 1329. 17

The invention can also be carried out by using an article comprising multiple dosage forms ~~ LU103233 as defined herein and at least one gelling agent as defined herein, wherein the multiple dosage forms and the at least one gelling agent are physically separated.

As mentioned above with respect to the composition for use in the invention, the at least one gelling agent can preferably be present in a gelling composition which can contain further gelling agents and/or other pharmaceutically acceptable agents such as preferably one or more pH modifiers, whereby preferred examples of pH modifiers for use in the invention are already outlined above.

In other embodiments of the invention, the pharmaceutical and/or dietary and/or nutraceutical article is comprised of a composition as defined herein and, physically separated therefrom, of water or an aqueous solution. Preferably, the physical separation is affected by providing the composition (without water/aqueous solution) in one container, and the water or aqueous solution in a further, second, container. For use, the water or aqueous solution is combined with the pharmaceutical composition in one of the two containers, or in a different container.

In preferred embodiments, the pharmaceutical article further comprises water and/or an aqueous solution physically separated from the multiple dosage forms and from the at least one gelling agent or gelling composition, respectively.

The “physical separation” according to the invention is any means whereby the multiple dosage forms and the one or more gelling agents or the gelling composition, respectively, are separated from each other such that they do not come into contact, typically until used by the subject for which the article is envisaged. For example, the multiple dosage forms and the gelling agent(s)(gelling composition can be provided in two separate containers which together make up the pharmaceutical article, optionally, and preferably, in combination with a suitable package and, more preferably, further together with a pharmaceutical and/or dietary and/or nutraceutical leaflet containing instructions for use. The article can also comprise water or an aqueous solution which is also physically separated from the multiple dosage forms and the gelling agent(s)/gelling composition. Preferably, the water or aqueous solution, respectively, is provided in a third container.

In other preferred embodiments of the article for use in the invention the multiple dosage forms and the at least one gelling agent and, optionally, the water and/or aqueous solution, are provided in a single container comprising at least one compartment containing the multiple dosage form, at least one compartment containing the at least one gelling agent or 18 gelling composition and, optionally, at least one compartment containing the water or LU103233 aqueous solution, wherein said compartments are separated by an at least partially removable, preferably breakable, physical separation.

Removable physical separations, preferably partially removable physical separations, for use in the pharmaceutical article are known to the skilled person. In one preferred embodiment, the separation is provided by a breakable foil, preferably between each compartment, which foil may preferably embodied as a blister foil typically used for pharmaceuticals. In a preferred embodiment, containing multiple dosage forms, gelling agent(s)/gelling composition, and water or aqueous solution, the compartments are provided in the container such that a first lowest compartment contains the liquid medium, and the two upper compartments contain the multiple dosage forms. The user may then press the contents of the uppermost compartment through the foil such that multiple dosage forms and gelling agents or gelling composition are first mixed, and then by further pressure application, are pressed through the breakable separation, preferably a blister foil, into the lower liquid medium compartment such that the final composition for administration is reconstituted, typically as a gel or gel-like suspension of the multiple oral dosage forms, and can be administered orally to the subject in need of the treatment preferably by self-administration.

In other preferred embodiments, the article for use in the invetion contains a a composition for use in the invention (without water or aqueous solution) and water or aqueous solution, respectively, in a single container having two compartments, whereby the composition for use in the invention is provided in one and the water or aqueous solution, respectively, is provided in a second compartment, and the two compartments are separated by an at least partially removable, preferably, breakable physical separation, more preferred a breakable foil, particularly preferred a blister foil as outlined above for the three-compartment embodiment. For reconstitution, the user, preferably a subject in need of treatment, will at least partially remove the separation between the compartments, preferably by pressing the pharmaceutical composition (without water/aqueous solution) through a blister foil into the compartment containing the liquid medium, preferably water or an aqueous solution.

As regards the articles for use in the invention as described herein, it is preferred that a container or compartment comprising the multiple dosage forms contains a unit dose of the multiple dosage forms for one administration to the subject. Preferred unit dosages, in particular numbers of multiple dosage forms, are as outlined above. 19

Also described herein are methods of preparing compositions for use in the invention as LU103233 defined and described herein. In one embodiment, the method for preparing a composition for use in the invention comprises combining multiple dosage forms as described above, preferably having sizes and/or numbers as outline before, with at least one gelling agent or a gelling composition respectively. The method preferably comprises the further step of combining the resulting mixture of multiple dosage forms and at least one gelling agent or gelling composition, respectively, with a liquid medium, preferably water or an aqueous solution. In a different aspect of the invention, the method for producing a composition for use in the invention comprises combining a composition comprising multiple dosage forms as described above and at least one gelling agent or a gelling composition, respectively, with a liquid medium, preferably water or an aqueous solution. As will be understood by the skilled person, the result of this method is typically a gel or gel-like formulation of the multiple dosage forms.

According to the invention, the water for providing a mixture of multiple dosage forms and at least one gelling agent or gelling composition, respectively, in a liquid medium may be tap water or sterilized water. Aqueous solutions for that purpose are preferably buffered or unbuffered saline solution such as physiological sodium chloride solutions, Ringer or Ringer lactate. The liquid medium may also be selected from other embodiments such as flavoured and/or pigmented or dyed aqueous solutions or even suspensions such as juices, e.g. fruit or vegetable juices, as long as such liquids do not interfere with the formation of a gel or gel-like formulation containing the multiple dosage forms.

The compositions and/or articles for use in the invention as defined and described herein are particular useful in the prevention and/or treatment in a subject, preferably a human, suffering from conditions and/or disorders and/or diseases associated with disturbed enterokine release by enteroendocrine cells and/or conditions and/or disorders and/or diseases amenable to increased enterokine release by enteroendocrine cells, preferably obesity and/or conditions typically associated with obesity.

Preferred conditions, disorders or diseases to which the compositions and/or articles as defined and described herein can be applied for treatment and/or prevention are metabolic disorders, vascular disorders, neurodegenerative diseases, skeletal diseases and gastroenterological disorders. 20

Preferred metabolic disorders are selected from insulin resistance, type 2 diabetes mellitus, LU103233 non-alcoholic fatty liver disease, non-alcoholic steatohepatosis, metabolic syndrome, hyperlipidemia and obesity.

Especially in the context of diabetes, in particular T2DM, and prediabetic conditions, preferably insulin resistance and/or obesity, the therapeutic and/or preventive regimen of the invention has tremendous benefits, since it is known that increased enterokine levels, in particular GLP-1, exerted by the inventive administration regimen of a composition as defined and described herein have a beneficial effect of increasing the Adiponectin/Leptin ratio (cf., for example, Unamuno et al. (2019) Nutrients, 11, 2069-2079) which diminishes known inflammation of adipose tissues frequently encountered in patients suffering obesity, pre-diabetic conditions such as insulin resistance, and/or diabetes, preferably T2DM.

According to the invention the composition induces satiety and/or induces weight loss and/or reduces hunger and/or suppresses appetite and/or reduces sugar craving and/or increases energy expenditure in the subject, preferably a human.

In contrast to prior art applications of enterokine stimulating compositions, the range of indications for which the compositions and/or articles, respectively, for use in the invention can be applied to successfully is broadened tremendously, since is has been recognized according to the invention that enterokines released by enteroendocrine cells, in particular

GLP-1 released by L cells, have various effector cites in the organism, in particular humans, which is particularly displayed by the expression profiles of enterokine receptors, in particular

GLP-1 receptors.

For example, GLP-1 shows effects on vascular tissues, in particular a vasodilatory and cardioprotective function (see, e.g. Ban et al. (2008) Circulation 117, 2340-2350) such that the compositions and/or articles for use in the invention can preferably applied for treatment and/or prevention of vascular disorders such as preferably microvascular dysfunction, cardiovascular diseases, cerebrovascular diseases and pulmonary vascular diseases.

Pulmonary diseases are a preferred target for therapy and/or prevention by the inventive embodiments since expression of GLP-1 receptors is highest in lungs. As preferred classes of pulmonary vascular diseases, pulmonary diseases associated with pneumonia are contemplated, more preferably in pneumonia associated with or caused by, respectively, viral infection. 21

Since severe forms of coronavirus infection, in particular by coronaviruses causing LU103233 respiratory pneumoniae caused by or associated with, respectively, infections with SARS-

COV-1, SARS-COV-2 and/or MERS, are associated with severe damage of pulmonary vessels, it is proposed by the invention that induction of enterokines, in particular GLP-1, by administration, in particular oral administration, of the composition, e.g. by use of articles as disclosed herein. Particularly preferred therapeutic target of the invention is SARS-COV-2, whereby the inventive administration of the composition for use in the invention is preferably taken place before the onset of the COVID-19 manifestation, or at least before the occurrence of severe forms comprising development of respiratory conditions such as in particular pneumonia.

In this context, it is to be noted that Glucagon-like-peptide 1 (GLP-1) is best known as an enteroendocrine hormone that orchestrates insulin release in response to ingested nutritional stimuli (Paternoster et al. (2018) Front. Endocrinol. 9, 1-26). GLP- 1 has also emerged as an important homeostatic element within the cardiovascular system, where it possesses significant endothelial-protective functions (Helmstädter et al. (2020) Arterioscler. Thromb. Vasc. Biol. 40, 145-158; Sun, Y.-H. et al. (2017)

Mol. Med. Rep. 16, 929-936). In the lung, GLP-1 tightens barriers via the upregulation of tight junction proteins in barrier-forming cells ; in alveolar type 2 pneumocytes, GLP-1 stimulates the production of surfactant that, by reducing surface tension, helps minimize fluid accumulation within alveolar spaces (Vara et al. (2001) Am. J. Respir. Crit. Care Med. 163, 840-846). In endothelial cells, GLP-1 inhibits TNF Alpha Converting Enzyme (TACE) expression and activity (Ku et al. (2014) Pharmacol. Res. 84, 18-25): It therefore directly opposes key mechanisms that SARS-CoV-2 commandeers to augment inflammation and compromise barrier function. Accordingly, GLP-1 signalling attenuates TACE-dependent Endothelial

Protein C Receptor (EPCR) shedding (Ku et al. (2014), supra); GLP-1 signalling also increases deficient Angiotensin Converting Enzyme 2 (ACE2) levels in pathological settings (Romani-Pérez. et al. (2015) Endocrinology 156, 3559-3569), presumably by reduced ACE2 shedding. Consistent with this anti-inflammatory role,

GLP-1 receptor agonists possess several desirable actions, including (i) antagonizing

NF-kB signalling (Helmstädter et al. (2020), supra), (ii) reducing immune cell adhesion molecule expression on endothelial cell surfaces (e.g., ICAM-1 and VCAM- 1) (Helmstädter et al. (2020), supra; Arakawa et al. (2010) Diabetes 59, 1030-1037), (iii) reducing immune cells cytokine production (Arakawa et al. (2010), supra) and (iv) 22 attenuating endothelial cell oxidative stress (Ceriello et al. (2013) Diabetes Care 36, LU103233 2346-2350). In fact, several of the risk factors associated with severe COVID-19 (e.g., obesity, diabetes) also associate with reduced GLP-1 secretion and circulating

GLP-1 levels.

The present invention therefore expands the repertoire of interventions yielding positive therapeutic effects of GLP-1 effects in COVIDF-19 patients. The high anti-

COVID-19 potential of the embodiments of the invention is underscored by previous findings according to which agonists or supraphysiological levels of native GLP-1 have been utilized to elicit desirable effects. However, normal post-prandial levels of endogenous GLP-1 clearly suffice to stimulate nitric oxide production in the forearm, which increases blood flow, microvascular blood volume, and interstitial oxygen uptake in skeletal muscle cells (Chai et al. (2012) Diabetes 61, 888-896); Dong et al. (2013) Am. J. Physiol. - Endocrinol. Metab. 304, E222—-E228) .

A preferred skeletal disease for which the embodiments of the inventive treatment can be applied to is osteoporosis.

A further therapeutic aspect of the invention is based on the known proliferative and antiapoptotic effect of enterokines as described herein, specifically GLP-1 and GLP-2, on cells of the gastro-intestinal tract, especially of the gastro-intestinal mucosa (see, for example, Sigalet (2012) J. Anim. Sci. 90, 1224-1232; Aw et al. (2017) Asia-Pac. J. Clin.

Oncol. 14, 23-31; and Kissow et al. (2012) Cancer Chemother. Pharmacol. 70, 39-48). By administration of the pharmaceutical compositions of the invention, which may be prepared by use of the pharmaceutical articles as disclosed above, it is possible not only to prevent and/treat malabsorption disorders in affected subjects, but also to treat subjects, in particular human subjects, suffering from disorders, diseases and/or conditions of impaired gastro- intestinal function, in particular due to irregular gastro-intestinal growth including reduced intestinal length and impaired/reduced formation of intestinal mucosa and villi, especially, and preferably, in neonatal and infant children, and/or due to disorders of the intestinal mucosa such as mucositis, preferably resulting from chemotherapy, radiotherapy and/or infections, especially in tumor and/or cancer patients. This treatment aspect of the invention also comprises the prevention of the above gastro-intestinal disorders and diseases.

As disclosed above, the compositions for use in the inention, in particular pharmaceutical gel or gel-like formulations of the multiple dosage forms as described herein, are for use in 23 patients suffering from metabolic disorders, in particular diseases involving aberrant energy LU103233 household such as diabetes and pre-diabetic conditions as outlined above. In particular in such patients, uptake and transport of oral formulations being larger in size (in particular tablets or capsules containing substantial amounts of active compounds such as glucose) is frequently impaired and/or slowed down considerably under conditions such as gastroparesis (delayed gastric emptying), in particular in patients having infections by Heliobacter pilori, neuromuscular dysfunction, and advanced age, and diabetes, in particular T2DM, where in up to 50% of diabetic patients the dysfunction of autonomic nerves, specifically vagal efferent nerves that govern gastric and small intestine peristalsis, is a comorbidity. Therefore, the compositions and articles for use in the invention comprising small dosage forms as disclosed herein are particularly useful for treatment of subjects, in particular humans, in which one or more of the indications as disclosed herein is/are associated with one or more of the described comorbidities.

Accordingly, the compositions and/or articles comprising small dosage forms as described herein are particularly useful in cases wherein the above conditions, disorders or diseases are accompanied by at least one condition selected from the group consisting of swallowing difficulty, dysphagia, achalasia, impaired esophageal peristalsis, gastroparesis and impaired intestinal peristalsis.

The present invention also relates to a method of treatment and/or prevention of the above- described conditions, disorders and/or diseases comprising the oral at least twice daily circadian administration, as described and defined herein, of an effective amount of a composition as described herein, preferably a formulation of the multiple dosage forms in a gel or gel-like suspension, to a, preferably human, subject in need thereof.

In certain embodiments, it is also preferred that the dietary and/or nutraceutical and/or pharmaceutical composition, before administration to the subject, preferably by self- administration, is reconstituted, preferably by use of the pharmaceutical articles as disclosed herein, by combining a composition comprising multiple dosage forms as outlined herein and at least one gelling agent or a gelling composition, respectively, as disclosed above, and a suitable liquid as described herein.

In general, the administration of the pharmaceutical and/or dietary and/or nutraceutical composition for use in the invention leads to a substantial increase of the addressed at least one enterokine, preferably GLP-1, GLP-2, GIP, PYY, CCK and/or neurotensin, particularly preferred GLP-1 and/or GLP-2 and/or PYY, above the respective base-line level of the 24 enterokine, in particular the level of the enterokine in the blood serum/plasma of the subject LU103233 being treated. In preferred embodiments of the invention, the level of the respective enterokine in the subject’s blood serum/plasma increases by at least about 50 % or more, preferably about 50 % to about 200 % or even more such as about 300 %, as compared to the level in the blood plasma/serum of the subject before administration of the pharmaceutical composition of the invention. The increase in enterokine concentration in the subject’s blood plasma/serum typically occurs within about 2 to about 6 hours post administration of the composition of the invention and lasts for a time period of typically about 1 to about 5 hours, preferably about 2 to about 4 hours, most preferred from about 3 to about 4 hours.

According to the invention, the term “effective amount of the composition” depends on various factors such as the specific condition, disorder or disease to be treated and/or prevented, the age and sex of the subject as well as the general condition of the subject.

Furthermore, the “effective amount of the pharmaceutical composition” depends on the type of active compound(s) used.

With glucose as a preferred active compound, the effective amount is preferably a daily dose of about 0.5 to about 20 g glucose orally administered according to the inventive regimen. A dose of about 1 to about 15 g once daily is more preferred, and about 6 g to about 15 g, most preferred about 12 g glucose. The above amounts of the active ingredient, preferably glucose, may be administered in one or more-unit doses of the compositions per administration, which preferred unit doses, in particular as regards the multiple dosage forms, are already outlined above. A typical amount of glucose contained in unit dose of the composition for use the invention is in the range of from about 0.5 g to about 30 g glucose, preferably from about 3 g to about 20 g glucose, more preferably from about4 gto 12 g glucose, such as ca. 6 g glucose per unit dose of the composition. The above amounts of glucose as the preferred active substance (or one of the active substances in the composition) per unit dose are preferably comprised in a unit dose of multiple dosage forms as described herein, the number of the multiple dosage forms per unit being preferably as described above, particularly preferred from about 10000 to about 27000 dosage form of small size as described above.

As regards the composition for use in the treatment and/or prevention method according to the invention as described above, it is of particular of benefit when the composition is formulated, in particular with respect to the sizes, amounts and forms of the multiple dosage forms, such that the at least one compound stimulating enteroendocrine cells to release an 25 enterokine stimulates said cells present in the intestine of the subject from the jejunum to the LU103233 ileo-cecal valve of the, preferably human, subject.

The therapeutic uses and treatment methods of the invention as generally defined above also entail a combination of different compositions wherein the individual composition may contain multiple dosage forms exhibiting different travelling profiles in the subject's gastro- intestinal tract and/or contain different compounds stimulating enteroendocrine cells to release at least one enterokine. For example, one composition contains multiple dosage forms comprising glucose and a further composition contains multiple dosage forms comprising an anthocyanine (further combinations and specific examples of active compounds and preferred combinations have already been elaborated above). It is also contemplated that a single composition contains multiple dosage forms comprising different active compounds as outlined above. A combination of different multiple dosage forms using different active compounds is specifically of benefit when the different compounds are not easily compatible (e.g. as regards their chemical properties) for inclusion in a single dosage form.

In other embodiments of this combinatorial approach, the treatment method of the invention comprises administering a first composition comprising multiple dosage forms as defined herein having a small size as described herein, and administering a second composition comprising one or more oral dosage forms as otherwise defined herein, but having a size of 3 mm or more, preferably 3 to 10 mm, based on the largest dimension of said first dosage form, wherein the active compound in said first and said second compositions stimulating enteroendocrine cells to release at least one enterokine may be the same or different. In this context, either of the first or second compositions can be used for the at least twice daily circadian administrations of the invention , preferably the at least one first, the at least one second and/or the at least one third administration according to the inventive regimen. It is also possible to administer both the first and the second composition either sequentially or simultaneously during one or more of the at least twice daily circadian administration of the invention, preferably the at least one first and/or at least one second and/or at least one third administration(s) as defined and described herein.

It is, of course, also possible to administer or use, respectively, more than two different sizes and/or more than two different active compounds.

A further aspect of the invention is a non-medical method such as cosmetic method for inducing weight loss in a subject, preferably a human, comprising the inventive at least twice 26 daily oral circadian administration regimen as defined herein. Typically subjects for the LU103233 inventive non-medical method are overweight, preferably slightly overweight subjects or subjects having a BMI of slightly below about 20, such as subjects having a BMI of around about 20, preferably subjects having a BMI of about 20 to about 22. It is understood, however, that the non-medical method of the invention is beneficial for any subject, in particular subjects being not contend with their weight.

The weight loss method of the invention comprising the at least twice daily oral circadian administration as defined and described herein may also be embodied as a medical treatment method for subjects, preferably humans, in need thereof, in particular overweight and/or obese subjects, preferably humans.

A further aspect of the invention is a non-medical method such as cosmetic method for appetite suppression in a subject, preferably a human, comprising the inventive at least twice daily oral circadian administration regimen as defined herein. Typically subjects for the inventive non-medical method for appetite suppression are overweight, preferably slightly overweight subjects or subjects having a BMI of slightly below about 20, such as subjects having a BMI of around about 20, preferably subjects having a BMI of about 20 to about 22.

It is understood, however, that the non-medical method of the invention is beneficial for any subject, in particular subjects being not contend with their weight.

The method for appetite suppression of the invention comprising the at least twice daily oral circadian administration as defined and described herein may also be embodied as a medical treatment method for subjects, preferably humans, in need thereof, in particular overweight and/or obese subjects, preferably humans.

For preparing the multiple dosage forms as described herein, a production method typically comprising the steps of: (a) preparing a mixture comprising at least one compound stimulating enteroendocrine cells to release at least one enterokine, preferably in combination with at least one disintegrant as defined above, (b) compressing the mixture obtained in step (a); and © applying to the compressed mixture at least one enteric coating, preferably comprising at least one pH sensitive polymer being preferably selected such that the coating substantially dissolves and/or is substantially degraded in the jejunum of a subject, 27 wherein the compression and application steps (a) and (b) respectively are selected such LU103233 that multiple dosage forms having a size as disclosed herein are obtained.

Optionally, the mixture of step (a) and/or the at least one coating of step (c) may comprise further ingredients as outlined above for the pharmaceutical composition per se.

Preferred embodiments of the constituents as defined in steps (a) and (c) have already been described in detail above.

According to a further preferred embodiment of the preparation method of the invention, more than one coating is applied to the compressed mixture obtained in step (b), i.e. the core component of the pharmaceutical oral dosage form of the invention.

In particularly preferred embodiments of the preparation method, a combination of at least two coatings is applied in step (c). Preferably, a sub-coating of one pH sensitive polymer is applied as a first layer and a coating of a second pH sensitive polymer is applied on the sub- coating as a second layer. For example, the pH sensitive coating can comprise a sub-coating of or comprising, respectively, a hydroxypropylmethyl cellulose as a first layer, onto which a second coating comprising or made of an anionic copolymer of methacrylic acid and — methacrylmethacrylate as a second layer. In a further preferred embodiment, a first layer (sub-coating) comprising or made of an anionic polymer of methacrylic acid and methacrylmethacrylate such as an Eudragit®, more preferably Eudragit® FS30D, is applied to the core obtained in step (b) and a second layer comprising or made of a hydroxypropylmethyl cellulose such as AQOAT®, more preferably AQOAT®-HF, is applied onto the first layer. More preferably, the anionic copolymer of methacrylic acid and methacrylmethacrylate, e.g. an Eudragit®, preferably Eurdragit® FS30D, is applied in less amount than the the hydroxypropylmethyl cellulose such as AQOAT®, more preferably

AQOAT®-HF, of the second layer. In other words, the thickness of the first layer of this type of combination is lower than the thickness of the second layer in this combination. More specifically, the ratio of amount or thickness, respectively, between first layer and second layer typically ranges from about 1:10 to about 1:50, particularly preferred from about 1:20 to about 1:30. In preferred embodiments of the invention, the one or more coatings are applied by spray-coating in step (c).

For preparing the composition for use of the invention, the multiple dosage forms can then admixed with the gelling agent or gelling agents, preferably a gelling composition, preferably containing further ingredients such as those as outlined above, such as preferably one or 28 more formulation aids, preferably selected from pH modifiers, flavors, taste improvers, LU103233 lubricants and pigments.

Fig. 1 shows a diagram demonstrating the central role of obesity as a cause of various co- morbidities.

EXAMPLE

The following exemplary compositions are prepared by mixing the following components (a) and (b): (a) Multiple dosage forms: Eudragit®-coated beads of ca. 1 mm diameter having a core containing 40 to 70 % (w/w) glucose and a disintegrant in a carrier substance. Total weight of glucose in a unit dose of the composition: (i) 6 g, (ii) 8 g. Number of coated beads ca. 10000 to 15000.

Comparative compositions comprising 6 g or 8 g, respectively, of placebo instead of glucose were prepared by correspondingly increasing amount of carrier to replace glucose. (b) Gelling composition:

Gelling agent mixture (Polysaccharide, modified cellulose, PEG) (84 % (w/w) based on the total weight of the gelling composition) pH modifier

Flavors

Lubricant

Pigments

Before oral administration, the composition is mixed with 25 ml of water per unit dose.

The reconstituted composition is administered orally to 36 subjects: - 24 obese subjects with a BMI of at least 30 (treatment group); and - 12 obese subjects with a BMI of at least 30 (placebo group).

The respective compositions are administered twice daily at 8 am +/- 0.5 h and 16:00 h pm +/- 0.5 h over a period of 4 months. 29

Using the above administration regimen, 4 of the obese subjects receive 2 x 6 g glucose, 4 LU103233 obese subjects receive 2 x 8 g glucose, 4 obese subjects receive 2 x 6 g placebo, and 4 obese subjects receive 2 x 8 g placebo. 2 of the non-obese subjects receive 2 x 6 g glucose, 2 non-obese subjects receive 2 x 8 g glucose, 2 non-obese subjects receive 2 x 6 g placebo, and 2 non-obese subjects received 2 x 8 g placebo.

A comparative group of 4 obese subjects receive 1 x 12 g in the fasting state between 7:00 h am and 9:00 h am each.

Body weight is measured daily before the first administration and the day after the last administration.

Claims (40)

Our Ref.: A 0733 LU Date: 21 December 2023 LU103233 Applicant: APHAIA IP AG Claims

1. A dietary and/or nutraceutical and/or pharmaceutical composition comprising one or more enterokine-releasing substances in a non-resorption formulation for use in the treatment and/or prevention of obesity and/or conditions associated therewith in a subject wherein the pharmaceutical composition is administered orally to the subject at least twice daily wherein the at least one first dose of the pharmaceutical composition is administered at about 8 am +/- about 3 h and the at least one second dose of the pharmaceutical composition is administered at about 4 pm +/- about 3 h.

2. The composition for use of claim 1 wherein the at least one first dose of the pharmaceutical composition is administered at about 8 am +/- about 2 h and the at least one second dose of the pharmaceutical composition is administered at about 4 pm +/- about 2 h.

3. The composition for use of claim 2 wherein the at least one first dose of the pharmaceutical composition is administered at about 8 am +/- about 1 h and the at least one second dose of the pharmaceutical composition is administered at about 4 pm +/- about 1 h.

4. The composition according to any one of the preceding claims wherein the time period between the administration of the at least one first dose and the administration of the at least one second dose is between about 7 to about 9 hours.

5. The composition for use of claim 4 wherein the time period between the administration of the at least one first dose and the administration of the at least one second dose is about 7.5 hours to about 8.5 hours.

6. The composition for use of claim 5 wherein the time period between the administration of the at least one first dose and the administration of the at least one second dose is about 8 hours.

7. The composition for use according to any one of the preceding claims wherein the one or more enterokin-releasing substances is/are selected from the group consisting 1 of compounds stimulating the enteroendocrine cells by a mechanism selected from | LU103233 the group consisting of transport into enteroendocrine cells by a transporter expressed by said cells wherein the transporter is selected from the group consisting of GLUT2 and SGLT1, and binding to G protein-coupled receptors expressed by said cells.

8. The composition for use of claim 7 wherein the G-protein-coupled receptor is selected from bile acid receptors, amino acid receptors, peptide receptors and fatty acid receptors and taste receptors.

9. The composition for use of claim 7 or 8 wherein the enterokine-releasing substance(s) is/are selected from nutrient compounds.

10. The composition for use of claim 9 wherein the nutrient compound(s) is/are selected from the group consisting of carbohydrates, fatty acids, bile acids, peptides, amino acids, ethanol amides, anthocyanines and mixtures thereof.

11. The composition for use of claim 10 wherein the carbohydrate(s) is/are selected from glucose and sucralose.

12. The composition for use of claim 11 wherein the at least one administration and the at least second administration each comprise a dose of about 4 g to about 10 g, preferably about 5 g to about 8 g, more preferably about 5.5 to about 6.5, most preferred about 6 g glucose.

13. The composition for use according to any one of the preceding claims wherein the composition further contains an enteroendocrine cell maturation agent.

14. The oral dosage form of claim 13 wherein the maturation agent is a human milk oligosaccharide (HMO).

15. The composition for use according to any one of the preceding claims wherein the composition comprises a core containing the one or more enterokin-releasing substance(s) and an enteric coating being essentially resistant against degradation and/or dissolution in the duodenum and further upper parts of the digestive tract. 2

16. The composition for use of claim 15 wherein the enteric coating comprises a pH LU103233 sensitive polymer substantially degrading and/or dissolving at a pH value of about 5.5 to about 7.5, preferably about 7.2 to about 7.3.

17. The composition for use of claim 14 or 15 wherein the pH sensitive polymer is selected from the group consisting of hydroxypropylmethyl celluloses and anionic copolymers of methacrylic acid and methacrylmethacrylate.

18. The composition for use of claim 17 wherein the hydroxypropylmethyl cellulose is hydroxypropylmethyl cellulose acetate succinate.

19. The composition for use of claim 15 wherein the anionic copolymer of methacrylic acid and methacrylmethacrylate is an Eudragit® polymer.

20. The composition for use according to any one of claims 15 to 19 wherein the core contains at least one disintegrant.

21. The composition for use of claim 20 wherein the disintegrant is selected from the group consisting of a crosslinked polyvinylpyrrolidone, a crosslinked carboxymethyl cellulose and a modified starch.

22. The composition for use according to any one of the preceding claims wherein the enterokin releasing substance activates the secretion of at least one enterokin selected from the group consisting of GLP-1, PYY, GLP-2, CCK, GIP and neurotensin, preferably GLP-1 and PYY.

23. The composition for use according to any one of the preceding claims wherein the composition is an oral dosage form having a size of less than 3 mm, based on the largest dimension of the oral dosage form.

24. The composition for use of claim 23 wherein the size is from about 0.6 mm to about

1.7 mm, based on the largest dimension of the oral dosage form.

25. The composition for use of claims 23 or 24 wherein the composition comprises multiple of said oral dosage forms. 3

26. The composition for use of claim 25 wherein the composition comprises about 10000 LU103233 to about 27000 of said oral dosage forms.

27. The composition for use of claim 26 wherein the composition comprises multiple oral dosage forms in combination with one or more gelling agents.

28. The composition for use of claim 27 wherein the one or more gelling agent(s) is/are present in a gelling composition.

29. The composition for use of claim 27 or 28 wherein the one or more gelling agents or the gelling composition are not included in multiple dosage forms.

30. The composition for use according to any one of the preceding claims wherein the disease or condition is selected from the group consisting of metabolic disorders, fat vascular disorders, neurodegenerative diseases, skeletal diseases and gastroenterologic disorders.

31. The composition for use of claim 30 wherein the metabolic disorder is selected from the group consisting of insulin resistance, type 2 diabetes mellitus, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, metabolic syndrome, hyperlipidemia and obesity.

32. The composition for use of claim 30 wherein the metabolic disorder is associated with adipose tissue inflammation.

33. The composition for use of claim 30 wherein the vascular disorder is selected from microvascular dysfunction, cardiovascular diseases, cerebrovascular diseases and pulmonary vascular diseases.

34. The composition for use of claim 33 wherein the pulmonary vascular disease is associated with pneumonia.

35. The composition for use of claim 33 wherein the pneumonia is caused by or associated with a viral infection.

36. The composition for use of claim 35 wherein the viral infection is an infection by a coronavirus causing a respiratory condition. 4

37. The composition for use of claim 35 or 36 wherein the viral infection is an infection by a coronavirus selected from the group consisting of SARS-COV-1, SARS-COV-2 and MERS.

38. The composition for use of claim 30 wherein the skeletal disease is osteoporosis.

39. The composition for use of claim 30 wherein the gastroenterological disorder is selected from the group consisting of impaired gastrointestinal function and malabsorption conditions.

40. The composition for use according to any one of the preceding claims wherein the composition induces satiety and/or induces weight loss and/or reduces hunger and/or reduces sugar craving and/or increases energy expenditure in the subject. 5