Formulations demonstrating an oral depot effect

Abstract

The present invention relates to the oral administration of lipidated therapeutic peptides or proteins. Such peptides or proteins, when administered in combination with certain pharmaceutical formulation excipients, have been found to manifest a depot effect, and can exert biological responses of extended duration. This behaviour obviates the need for high levels of bio-active peptide in the outer circulation, while at the same time permitting dosing regimens involving sequential administrations of the therapeutic agent separated by one or more days.

Description

[0001] FORMULATIONS DEMONSTRATING AN ORAL DEPOT EFFECT

[0002] TECHNICAL FIELD

[0003]

[0001] This invention relates to the oral administration of lipidated therapeutic peptides. Such peptides, when administered in combination with certain pharmaceutical formulations, have been found to manifest a depot effect, perhaps by forming a depot in the tissue underlying the enterocytes of the small intestine, and can exert biological responses of extended duration. This behaviour obviates the need for high levels of bio-active peptide in the outer circulation, while at the same time permitting dosing regimens involving sequential administrations of the therapeutic agent separated by one or more days.

[0004] BACKGROUND

[0005]

[0002] The following discussion of the background art is intended to facilitate an understanding of the present invention only. The discussion is not an acknowledgement or admission that any of the material referred to is or was part of the common general knowledge as at the priority date of the application.

[0006]

[0003] Under normal circumstances, drugs which are administered orally, and absorbed via the small intestine, drain via the gut microvasculature into the portal vein, and then pass through the liver before entering the outer circulation. Drugs, in particular small molecules, which bind to liver receptors, or are absorbed across plasma membranes into hepatocytes, can be broken down and destroyed inside liver cells (the so-called "first-pass" effect).

[0007]

[0004] Under such circumstances, efficacy of these drugs is lost as a result of their breakdown. Large macromolecules, such as peptides or proteins, however, do not easily cross the plasma membranes of hepatocytes, and are consequently not subject to the breakdown processes occurring within these cells. In addition, if the hepatocytes do not express receptors on their surface for the aforementioned peptides or proteins, then these macromolecules will not be removed from the blood flowing through the liver, and will enter the blood circulation in high measurable concentrations.

[0008]

[0005] A good example of the above is the peptide drug exenatide (exendin-4), where it can be seen that delivery across the intestinal cell barrier, when combined with certain oral drug delivery vehicles, results in high concentrations of the macromolecule both in the portal vein, and in the outer circulation. Exendin-4 is a GLP-1 receptor agonist (GLP-1 RA), and its biological activity can be seen in the elevation of insulin levels in the bloodstream over a period of time, indicating that the peptide has been delivered in an intact form, and is capable of binding to its receptors in the body.

[0009]

[0006] The present invention seeks to address or at least ameliorate one or more the problems associated with the prior art. SUMMARY OF INVENTION

[0010]

[0007] It has now been found, surprisingly, that a completely different pharmacokinetic profile is observed when such macromolecules (in particular GLP-1 receptor agonists) are lipidated and delivered using a delivery vehicle comprising either a bile salt or an oil. In this case, the concentration of the macromolecule, both in the portal vein and in the outer circulation is low, yet biological activity is still observed, showing that the macromolecule has been delivered across the intestinal cell wall. This observation leads to the conclusion that, after passage of the lipidated macromolecule within the delivery vehicle across the wall of the intestine into the rest of the body, it remains for a long period of time in the form of a depot in the tissues underlying the enterocytes of the gut.

[0011]

[0008] The ability of peptide therapeutics to behave as a depot, or to exert a depot effect in intestinal tissues is hitherto unknown.

[0012]

[0009] In one aspect of the present invention, there is provided a formulation comprising (i) a therapeutic peptide or protein and (ii) one or more formulation excipients which enhances the uptake of the therapeutic peptide across the intestine of a subject, wherein there is a depot effect, in which the biopotency of the therapeutic peptide is significantly greater than the bioavailability of the peptide when compared with administration via the parenterally injected route.

[0013]

[0010] In one embodiment of the present invention, there is provided a formulation comprising (i) a therapeutic peptide or protein and (ii) one or more formulation excipients which enhances the uptake of the therapeutic peptide across the intestine of a subject, wherein the peptide remains held in a depot, after administration, within intestinal tissue.

[0014] [Oil] In another embodiment of the present invention, there is provided a formulation comprising (i) a therapeutic peptide or protein and (ii) one or more formulation excipients which enhances the uptake of the therapeutic peptide across the intestine of a subject, wherein the peptide remains held in a depot, after administration, associated with chylomicrons.

[0015]

[0012] One of the advantages of holding therapeutic peptides in a depot is that it creates a situation where the normal clearance mechanisms to which the peptide is subject are inaccessible to it when retained in the depot.

[0016]

[0013] Factors which result in clearance of small peptides from the body are extraction via the kidney, and attack by proteases circulating in the bloodstream, and expressed on the surface of cell membranes.

[0017]

[0014] Retention of peptides in a depot which is separate from the outer circulation prevents contact of the peptide with the kidney tissue vasculature and thus eliminates this route of egress from the body as a general clearance mechanism for thew duration of the peptide's stay within the depot.

[0015] The second mechanism of clearance from the body, namely proteolytic breakdown, may be reduced if retention of the peptide within the depot reduces its contact with these enzymes.

[0018]

[0016] A good example is the enzyme dipeptidyl peptidase 4 (DPP-4), which is present in freely soluble form throughout all bodily fluids, but is also expressed on the surface of a wide range of cell membranes, including those of epithelial cells, and cells of the blood such as lymphocytes. Thus, for as long as the peptide is within the depot, interaction with DPP-4 on the surface of cell membranes will not occur, and the longevity of the peptide in the body will be increased.

[0019]

[0017] The longevity of the bioactivity of the peptide may also be increased if the peptide is able to exert such activity within the depot itself, or within the tissues close to the depot bearing receptors which the peptide can access after leaving the depot, but before entering into the outer circulation.

[0020]

[0018] Such a situation is highly relevant to GLP-1 and GLP-1 receptor agonists, which are a prime target for the action of DPP-4.

[0021]

[0019] A similar situation pertains for neprilysin, an endopeptidase enzyme circulating in the bloodstream, as well as being highly expressed in kidney tissue, and to which GLP-1 RAs may also be susceptible.

[0022]

[0020] Depending on the lipidated therapeutic peptide employed, biological activity may manifest in the body for many days after a single administration.

[0023]

[0021] One way in which the depot can be formed is as a result of association of the lipidated therapeutic peptide with lipidic structures located basal to the intestinal cells. The lipidic structures located basal to the intestinal cells include but not limited to secreted chylomicrons and chyle in the submucosa, adipose tissue surrounding the intestine, small intestinal mesenteric adipose tissue (SMAT) fat-associated lymphoid clusters (FALC), lacteals and lymphatic duct adventitia.

[0024]

[0022] In one possible mechanism of action for creation of the depot (provided here simply for the purpose of illustration), the lipidated therapeutic peptide(s) may be encouraged by certain formulation excipients, also referred to herein as delivery vehicle, to associate with chylomicrons during their passage through cells.

[0025]

[0023] One formulation employed in the examples contains chenodeoxycholate and a hydrophobic polyphenol compound, both of which are known to enhance uptake of peptides into endosomes of intestinal cells via vacuolation within clathrin-coated pits.

[0026]

[0024] Another formulation employed in the examples is a formulation comprising an oil combined with surfactants commonly used in pharmaceutical practice for the dispersion of oils in aqueous media. Demonstration of the depot effect with these two widely differing formulation excipients serves to highlight the breadth of possible excipients which can be employed in conjunction with lipidated therapeutic peptides to achieve this action.

[0025] In one embodiment, the presence of these excipients, or molecules similar in structure, have the potential to shunt lipidic entities (including lipidated therapeutic peptides, such as semaglutide) from the endosomal transport pathway to the lipid processing pathway across intestinal cells, resulting in association with chylomicrons upon secretion from the intestinal cells. In one preferred embodiment, the excipients of the formulation may be chosen from lipids, bile acids and salts thereof, fatty acids and salts thereof, lipophilic esters of fatty acids, and esters of polyphenols, glycerolipids, either individually or in combination. In a preferred embodiment of the formulation, the excipients are bile acids and salts thereof, alone or in combination with esters of polyphenols.

[0027]

[0026] In another embodiment, the formulation may be composed of lipids combined with surfactant molecules. In one preferred embodiment, the lipid is an oil, such as oleic acid. Surfactant molecules contained in this formulation may be selected from a list which includes, but is not limited to, phospholipids, polyoxyethylene-conjugated lipids, sodium docusate, non-ionic surfactants, ionic surfactants, either alone or in combination.

[0028]

[0027] It will be noted that one manifestation of the existence of a depot for lipidated GLP-1 receptor agonists (or other lipidated therapeutic peptides) will be a disparity between bioavailability (i.e., the concentration of the peptide in the bloodstream relative to intravenous (i.v.) injected material), and biopotency, since the peptide is able to exert an effect due to interaction with receptors on intestinal vagal afferents without entering the outer circulation. This phenomenon may be termed a depot effect, and has been hitherto unknown for materials administered via the oral route. The reader is referred to Examples 2 to 5, where this phenomenon is clearly demonstrated.

[0029]

[0028] It will be appreciated that not all lipidated drugs, when administered to the digestive tract by mouth, in a composition designed to enhance uptake into the body, are able to show a depot effect, as determined by a disparity between biopotency and bioavailability. A good example of this is the oral formulation Rybelsus®, where semaglutide is administered in combination with the absorption enhancer sodium N-(8-[2-hydroxylbenzoyl] amino) caprylate (SNAC). In humans, this formulation has a bioavailability of 0.8% (Overgaard et al 2021 Clinical Pharmacokinetics of Oral Semaglutide Clin Pharm 60 1335-46), while the biopotency has been calculated as 0.4% (Abramson et al 2019 Quantifying the value of orally delivered biologic therapies J Pharm Sci 108 3138-45). Thus, within the limits of statistical significance, there is no disparity between bioavailability and biopotency with Rybelsus®, and in particular, there is no disparity in which biopotency is an order of magnitude greater than bioavailability, as has been achieved with the composition described in the examples.

[0030]

[0029] In a further aspect, the present invention provides a method of determining the ability of a therapeutic peptide or protein to form a depot effect comprising:

[0031] (a) measuring the relative bioavailability of the therapeutic peptide or protein;

[0032] (b) measuring the relative biopotency of the therapeutic peptide or protein; and

[0033] (c) determining the ratio of the biopotency compared to the bioavailability , wherein the ratio is greater or equal to 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 or 6 or more.

[0030] Another aspect of the present invention provides a method of making a pharmaceutical formulation comprising a therapeutic peptide or protein at a therapeutically effective concentration with a delivery vehicle, and optionally a surfactant.

[0034]

[0031] Another aspect of the present invention provides a method of using the formulation as described herein for preventing or treating a disease with the therapeutic peptide or protein. In one embodiment, the use encompasses therapeutic treatment of a subject comprising the treatment or prevention of a diabetes (types 1 and 2, and gestational diabetes) obesity, non-alcoholic steatohepatitis (NASH), cardiovascular disease, kidney disease, neurodegenerative diseases, alcoholism, memory disorders, psoriasis and stroke in a subject.

[0035]

[0032] Another aspect of the present invention provides the use of a formulation comprising a therapeutic peptide or protein and the delivery vehicle, in the manufacture of a medicament for treating a subject in need thereof.

[0036]

[0033] Another aspect of the present invention is a kit comprising the therapeutic peptide or protein and a delivery vehicle, optionally with a surfactant together with a package insert, package label, instructions, or other labelling.

[0037] BRIEF DESCRIPTION OF DRAWINGS

[0038]

[0034] Figure 1 illustrates pharmacokinetics of exenatide in a rat model after administration as a free solution by Intravenous injection or within an oral delivery formulation introduced directly into the intestine.

[0039]

[0035] Figure 2 illustrates the bioactivity of exenatide as measured by change in insulin levels in the bloodstream.

[0040]

[0036] Figure 3 illustrates the pharmacokinetics of semaglutide in a rat model after administration as a free solution by intravenous injection or within an oral delivery formulation introduced directly into the intestine.

[0041]

[0037] Figure 4 illustrates the bioactivity of Semaglutide as measured by change in blood glucose level.

[0042]

[0038] Figure 5 illustrates the bioactivity of semaglutide as measured by change in insulin levels in the bloodstream.

[0043]

[0039] Figure 6 illustrates the relationship between dose administered and concentrations of semaglutide in the peripheral bloodstream after administration via the Intestine or i.v. injection.

[0044]

[0040] Figure 7 illustrates the relationship between dose administered and concentrations of insulin in the bloodstream. Values are increase above baseline at time 90 minutes; Error bars are SEM.

[0041] Figure 8 illustrates the pharmacokinetics of tirzepatide in a pig model after administration as a free solution by subcutaneous injection or within a bile salt-based oral delivery formulation introduced directly into the intestine.

[0045]

[0042] Figure 9 illustrates the bioactivity of tirzepatide as measured by change in rate of blood glucose fall during an Intravenous Glucose Tolerance Test after administration within a bile salt-based oral delivery formulation introduced directly into the intestine.

[0046]

[0043] Figure 10 illustrates the bioactivity of tirzepatide as measured by change in insulin levels during an Intravenous Glucose Tolerance Test after administration within a bile saltbased oral delivery formulation introduced directly into the intestine.

[0047]

[0044] Figure 11 illustrates the pharmacokinetics of tirzepatide in a pig model after administration as a free solution by subcutaneous injection or within a lipid-based oral delivery formulation introduced directly into the intestine

[0048]

[0045] Figure 12 illustrates the bioactivity of tirzepatide as measured by change in rate of blood glucose fall during an Intravenous Glucose Tolerance Test after administration within a lipid-based oral delivery formulation introduced directly into the intestine

[0049]

[0046] Figure 13 illustrates the bioactivity of tirzepatide as measured by change in insulin levels during an Intravenous Glucose Tolerance Test after administration within a lipid-based oral delivery formulation introduced directly into the intestine.

[0050]

[0047] Figure 14.. illustrates the generation of chylomicron lipids by Caco-2 Cells after feeding with lipid dispersions of different compositions and concentrations.

[0051]

[0048] Figure 15 illustrates the requirement for chylomicron lipids to be present in culture in order for semaglutide to be transported across the Caco-2 cell monolayer. Lipid dispersions prepared using Taurocholate (TC), Chenodeoxycholate (CDC) or Deoxycholate (DC) as the dispersing agent.

[0052]

[0049] Figure 16 illustrates the relative amounts of fluorescent-labelled chylomicrons remaining in the supernatant after incubation in the presence or absence of semaglutide (A and B respectively) followed by incubation in antibody-coated wells (A and B) or un-coated wells (Ac and Be). The lower quantity of chylomicrons in sample A relative to the other samples indicates association of the chylomicrons with semaglutide after incubation.

[0053] DESCRIPTION OF EMBODIMENTS

[0054]

[0050] For convenience, the following sections generally outline the various meanings of the terms used herein. Following this discussion, general aspects regarding compositions, use of medicaments and methods of the invention are discussed, followed by specific examples demonstrating the properties of various embodiments of the invention and how they can be employed. Definitions

[0055]

[0051] The meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0056]

[0052] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variations and modifications. The invention also includes all of the steps, features, formulations and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.

[0057]

[0053] Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness. None of the cited material or the information contained in that material should, however be understood to be common general knowledge.

[0058]

[0054] Manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.

[0059]

[0055] The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein.

[0060]

[0056] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages can mean ±1%.

[0061]

[0057] The term 'order of magnitude' refers to a value within an exponential range of plus or minus 1 in the value of a quantity or unit, and is here used in conjunction with the power-of- 10. Thus, two values which are said to differ by an order of magnitude may be considered to fall with two different exponential ranges (such as for example 1-10, and 10 to 100). In general, an order of magnitude difference can equate to a difference of ten-fold between two values, although in certain circumstances the difference may range between five fold and fifty-fold or broader.

[0062]

[0058] The invention described herein may include one or more range of values (e.g. size, concentration etc.). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range. For example, a person skilled in the field will understand that a 10% variation in upper or lower limits of a range can be totally appropriate and is encompassed by the invention. More particularly, the variation in upper or lower limits of a range will be 5% or as is commonly recognised in the art, whichever is greater.

[0063]

[0059] In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of "or" means "and / or" unless stated otherwise. Furthermore, the use of the term "including", as well as other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise. Also, the use of the term "portion" can include part of a moiety or the entire moiety.

[0064]

[0060] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0065]

[0061] The terms "decrease", "reduced", "reduction", "decrease" or "inhibit" are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, ""reduced", "reduction" or "decrease" or "inhibit" means a decrease by at least 10% as compared to a reference level, e.g. in the absence of an agent, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%), or at least about 60%>, or at least about 70%, or at least about 80%.

[0066]

[0062] The terms "increased", 'increase" or "enhance" or "activate" are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms "increased", "increase" or "enhance" or "activate" means an increase of at least 10% as compared to a reference level, e.g. in in the absence of an agent, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%), or at least about 60%, or at least about 70%, or at least about 80%, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[0067]

[0063] As used herein, the term "administer" refers to the placement of a composition into a subject by a method or route which results in at least partial localization of the composition at a desired site such that desired effect is produced. A compound or composition described herein can be administered by any appropriate route known in the art including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compound is administered by parenterally administration, or other method allowing delivery to a target site.

[0068]

[0064] The term "therapeutic peptide" or "therapeutic protein" refer to peptides or polypeptides or proteins which are useful in the treatment of diseases, disorders or conditions in a subject.

[0065] The term "derivative" refers to a molecule that includes an insertion, deletion, or substitution of amino acids, or chemical modification. In certain embodiments, derivatives comprise covalent modifications, including, but not limited to, chemical bonding with polymers, lipids, or other organic or inorganic moieties. 5 In certain embodiments, a chemically modified amino acid sequence can have a greater circulating half-life than an amino acid sequence that is not chemically modified. In certain embodiments, a chemically modified amino acid sequence can have improved targeting capacity for desired cells, tissues, and / or organs.

[0069]

[0066] Exenatide is the synthetic version of Exendin-4. In this document, the two terms are used interchangeably; reference to exendin implies exendin-4.

[0070]

[0067] The term "depot" is employed to describe the ability of a drug, such as therapeutic peptide or protein, to exert an effect in the body over an extended period of time by virtue of its location and / or delivery to that location. Well-known examples are formulations of drugs which exert a prolonged biological effect after subcutaneous or intramuscular injection. Drugs, such as therapeutic peptides or proteins, which exert high prolonged activity in a localised part of the body, in the absence of high systemic bioavailability, can be said to be exerting a "depot effect".

[0071]

[0068] Relative "bioavailability" of a compound administered by a given route in a human subject or animal model refers to the concentration of that compound in the peripheral bloodstream after administration via said route, expressed as a percentage of the concentration of that compound in the peripheral bloodstream when administered by intravenous (i.v.) injection.

[0072]

[0069] Relative "biopotency" of a compound administered by a given route in a human subject or animal model refers to the magnitude of a biological response elicited by that compound in the body after administration via said route, expressed as a percentage of the magnitude of biological response observed when said compound is administered by intravenous injection.

[0073]

[0070] The ability of a therapeutic peptide or protein to create a depot effect is determined by the disparity between the biopotency of the therapeutic peptide or protein and the bioavailability of the peptide or protein. Any formulation that creates a depot effect by a disparity between the biopotency and bioavailability of the therapeutic peptide or protein is envisaged in this application. To achieve the depot effect, the disparity between the biopotency and bioavailability of the peptide or protein has a ratio of greater than or equal to 1.5. Preferably, the ration is greater than 2, greater than 2.5, greater than 3, greater than 3.5, greater than 4, greater than 4.5, greater than 5, greater than 5.5 or more.

[0074]

[0071] Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

[0072] Features of the invention will now be discussed with reference to the following nonlimiting description and examples.

[0075] EMBODIMENTS

[0076]

[0073] The present invention, for the first time, demonstrates that the addition of a delivery vehicle comprising a bile salt or a lipid together with a lipidated therapeutic peptide or protein delivered to the intestine leads to a disparity between the biopotency and the bioavailability of the therapeutic peptide or protein which manifests in a depot effect within the tissue underlying the intestine.

[0077]

[0074] Further suggested by the disclosure presented herein is that the depot effect occurs through interactions between the lipidated therapeutic peptide or protein and the binding to the surface of chylomicrons, either due to its inherent property deriving from its structure, or as a result of chemical or physical modification, including, but not limited to, conjugation with lipid chains.

[0078]

[0075] Without being bound to any particular theory, it is contemplated these interactions, as well as other types of intermolecular interactions, between the lipidated therapeutic peptide or protein in the presence of the delivery vehicle and the surface of the chylomicrons have the effect of extending the presence of the therapeutic peptide or protein in a depot underlying the intestine. For example, the depot can be formed as a result of association of the lipidated therapeutic peptide with lipidic structures located basal to the intestinal cells. The lipidic structures located basal to the intestinal cells include but not limited to secreted chylomicrons and chyle in the submucosa, adipose tissue surrounding the intestine, small intestinal mesenteric adipose tissue (SMAT) fat-associated lymphoid clusters (FALC), lacteals and lymphatic duct adventitia.

[0079]

[0076] The present invention relates to a formulation comprising formulation excipients, also referred to as the delivery vehicle, in conjunction with a therapeutic peptide or protein, wherein the formulation is able to deliver the peptide or protein via the intestine in such a way that a depot of the peptide or protein is formed, wherein the peptide or protein remains in the depot for an extended period of time.

[0080]

[0077] The extent of time that the peptide or protein remains in the depot will depend on the nature of the lipidation of the peptide or protein being used. For example, the therapeutic peptide or therapeutic protein may exert its biological activity for a period of greater than 3 days.

[0081]

[0078] The depot effect can manifest itself as a disparity between the biopotency and the bioavailability of a therapeutic peptide or protein. Any formulation that creates a depot effect by a disparity between the biopotency and bioavailability of the therapeutic peptide or protein is envisaged in this application. When the depot effect is manifest, the disparity between the biopotency and bioavailability of the peptide or protein has a ratio of greater than or equal to 1.5. Preferably, the ration is greater than 2, greater than 2.5, greater than 3, greater than 3.5, greater than 4, greater than 4.5, greater than 5, greater than 5.5 or more.

[0079] In one embodiment of the present invention, the depot is located in the tissues underlying the intestinal cells of the gut wall, preferably of the small intestine. In one embodiment, the lipidated therapeutic peptide or protein associates with lipidic structures located basal to the intestinal cells. The lipidic structures located basal to the intestinal cells include but not limited to secreted chylomicrons and chyle in the submucosa, adipose tissue surrounding the intestine, small intestinal mesenteric adipose tissue (SMAT) fat-associated lymphoid clusters (FALC), lacteals and lymphatic duct adventitia.

[0082]

[0080] In an alternative embodiment the formulation of this invention is able to demonstrate a depot effect, whereby a prolonged biological activity is achieved with a relative biopotency which is an order of magnitude greater than its bioavailability.

[0083]

[0081] In another embodiment of this invention, the formulation described in either of the embodiments above is one in which one or more of the formulation excipients has limited solubility in aqueous phase, and where these excipients are brought into close physical contact with said peptide.

[0084]

[0082] The excipients of the formulation, also referred to as the delivery vehicle, may be chosen from lipids, bile acids and salts thereof, fatty acids and salts thereof, lipophilic esters of fatty acids, and esters of polyphenols, glycerolipids, either individually or in combination.

[0085]

[0083] In a preferred embodiment of the formulation, the excipients are bile acids and salts thereof, alone or in combination with esters of polyphenols.

[0086]

[0084] In a further preferred embodiment, the bile acid or salt is derived from deoxycholic acid or chenodeoxycholic acid. In cases where esters of polyphenols are employed, said esters are conjugates of gallic acid, esterified with short-chain alcohols such as isopropanol, butanol or octanol.

[0087]

[0085] When used in combination, the ratio of bile acid or salt to gallic acid ester range from 5:1 wt:wt to 1:5 wt:wt.

[0088]

[0086] In a preferred embodiment, the ratio of bile acid or salt to gallic acid ester is about 2:1 wt:wt.

[0089]

[0087] In a further embodiment, the bile acid is chenodeoxycholic acid, and the gallate ester is propyl gallate.

[0090]

[0088] In a further embodiment, the bile salt and gallate ester are combined with EDTA.

[0091]

[0089] In a separate embodiment, the formulation may be composed of lipids, optionally combined with surfactant molecules.

[0092]

[0090] In a separate embodiment, the lipid may be a fatty acid.

[0093]

[0091] In a further embodiment, the fatty acid may be capric, caproic or caprylic acid and / or derivatives thereof.

[0094]

[0092] In a separate embodiment, the lipid may be an oil. For example, the oil may be selected from the group comprising straight or branched hydrocarbon chains (both saturated and unsaturated) ranging from 8 to 20 carbons, wherein the terminal groups may be methyl, acidic functions, alcohols or other derivatives.

[0095]

[0093] In yet a further embodiment, the oil may be oleic acid.

[0096]

[0094] Surfactant molecules contained in the lipid formulation may be drawn from a list which includes, but is not limited to, phospholipids, polyoxyethylene-conjugated lipids, sodium docusate, non-ionic surfactants, ionic surfactants, either alone or in combination.

[0097]

[0095] In one embodiment of such a formulation, the polyoxyethylene-conjugated lipid is Brij52.

[0098]

[0096] In a further embodiment of this formulation, the surfactants phospholipid, Brij 52 and sodium docusate are present in a ratio 2:3:3 wt:wt.

[0099]

[0097] In any of the embodiments described above, the lipids and surfactants may optionally be combined with bile acids or salts thereof.

[0100]

[0098] In the formulation described above, combining a pharmaceutical delivery vehicle with a therapeutic peptide or protein, said peptide or protein is preferably lipidated, or contains a substantial portion of its sequence in which lipidic residues are exposed on its outer surface, creating a net hydrophobic domain, or in some other way has a propensity to associate preferentially with lipidic structures or environments.

[0101]

[0099] Methods of lipidation of peptides are known in the art [see for example W02006097537(A2) General Synthetic Methods, and Example 1], and can involve covalent attachment of long hydrocarbon chains (for example via ester, ether, sulphydryl, peptide or other bonds) either to the termini of the peptide, or to specific residues in the middle of the peptide chain. In certain cases, more than on chain can be attached to the peptide, as is the case in bis-lipidated peptides. The chain may also comprise regions including polyethylene glycol, or polypropylene glycol, and may be terminated at the distal end to the peptide by groups chosen from, but not limited to, a methyl group, a carboxyl group, a fluorocarbon chain, a polysysine chain, five or six-ringed heterocycles containing one or more nitrogen or oxygen atoms, or a tetrazole group.

[0102]

[0100] In one embodiment of the invention, the peptides or proteins are lipidated GLP-1 receptor agonists, selected from, but not limited to semaglutide and liraglutide and analogues or derivatives thereof. Such receptor agonists may also include dual or triple agonists, where GLP-1 is one of the components of the molecule. An example of such a molecule is tirzepatide, in which the peptide is a receptor agonist for both GLP-1 and GIP receptors.

[0103]

[0101] In a further embodiment of the invention, other peptides which are secreted by intestinal cells may also be employed in lipidated form. Such peptides include, but are not limited to, cholecystokinin (CCK), and glucose dependent insulinotropic peptide (GIP). Other peptides which can interact with intestinal, portal vein or liver cell receptors also fall within the scope of this invention, such as glucagon, ghrelin inhibitor, PYY and oxyntomodulin and analogues and derivatives thereof, either individually or in combination with each other, as separate molecules or co-agonists, in the presence or absence of GLP-1 receptor agonists, when in lipidated form, or in a form predisposing them to associate with lipids.

[0104]

[0102] In a further embodiment of this invention, the peptide, protein or other therapeutic macromolecule is one which is capable of binding to the surface of chylomicrons, either due to its inherent property deriving from its structure, or as a result of chemical or physical modification, including, but not limited to, conjugation with lipid chains.

[0105]

[0103] In a further embodiment of this invention is provided a method of treatment of a disease in which a formulation, as described in at least any of the embodiments outlined above, is administered to the intestine, said formulation being described above, and comprising a pharmaceutical delivery vehicle combined with a therapeutic peptide or protein, which is able to deliver said peptide or protein via the intestine in such a way that a depot of the peptide is formed, where the peptide remains in the depot for an extended period of time.

[0106]

[0104] An important aspect of the invention is the potential for extended duration of the bioactivity of the treatment resulting from the longevity of the peptide itself, or otherwise, as a result of the retention of the peptide or protein in the depot for an extended period of time, beyond that which might be expected on the basis of standard clearance mechanisms. Under such circumstances the formulation may be administered via a range of dosing regimens, where repeated administration is required, ranging from daily administration of one or more times per day, to once per week or once per month, and any dosing frequency in between.

[0107]

[0105] In the absence of conditions to visualise the presence of a depot in intestinal tissues directly (e.g. in treatment of human patients where conduct of surgical or other procedures is not possible), the presence of a depot may be inferred by the fact that there is a disparity between the biopotency relative to the injected peptide, and the bioavailability. Such disparity will manifest itself as an increase in percentage biopotency over percentage bioavailability. The magnitude of this increase may range from a two-fold increase over the bioavailability to an increase of ten-fold or more, and any value in between.

[0108]

[0106] In certain cases the disparity between bioavailability and biopotency may be demonstrated in animal models. Appropriate species for carrying out such studies are rat, rabbit, ferret, dog monkey and pig.

[0109]

[0107] The formulation may be administered by mouth in liquid or solid form, as a slurry, gel, lozenge, tablet or capsule. In a preferred embodiment, the composition is provided in the form of an enteric-coated capsule, whereby the coating permits the capsule to pass through the stomach without releasing its contents. After passing through the stomach, the enteric coating will break down, and the capsule will open up and release its contents in the intestine.

[0110]

[0108] In a preferred embodiment, the formulation is administered by mouth in an enteric- coated capsule.

[0111]

[0109] In one embodiment, the formulation is for oral administration via a capsule. Preferably, the contents of the capsule comprise a suitable amount of the therapeutic macromolecule, such as a therapeutic peptide or protein, to achieve a therapeutic effect. For example, the composition may contain from about 1 mg to about 20 mg of the therapeutic peptide or protein of the capsule contents. For example, when the therapeutic macromolecule is semaglutide, the capsule comprises from about 1-20 mg of semaglutide. In a preferred embodiment, the capsule comprises 4 mg of semaglutide.

[0112]

[0110] In a separate embodiment the formulation may be administered via the umbical artery, or instilled into the peritoneal cavity.

[0113]

[0111] In a further embodiment of this invention is provided the use of the formulation, as described in any of the embodiments outlined above, employed using the method above, for treatment of a disease in humans or animals. Diseases which can benefit from use of this formulation in the manner described to create a depot effect include, but are not limited to diabetes (types 1 and 2, and gestational diabetes) obesity, non-alcoholic steatohepatitis (NASH), cardiovascular disease, kidney disease, neurodegenerative diseases, alcoholism, memory disorders, psoriasis and stroke.

[0114] EXAMPLES

[0115] Example 1 Exenatide (exendin-4) Study

[0116]

[0112] The pharmacokinetics and pharmacodynamics of exenatide in adult anaesthetized rats is outlined below. Exenatide, also referred to below as exendin-4 is a 39 amino acid peptide hormone and is an agonist for GLP-1 receptor. Exendin-4 is useful in the treatment of patients with insulinoma and type 2 diabetes. Exendin-4 in the following study is an example of a non-lipidated GLP-1 receptor agonist.

[0117]

[0113] Exenatide was sourced from Bachem AG in a non-lipidated form.

[0118]

[0114] Anaesthetised rats were employed so that material could be introduced directly into the intestine of the animals, since administration of capsules designed to pass through the stomach in a timely fashion was not possible.

[0119]

[0115] Rats were acclimated for a period of 4 days in the experimental room before the start of the experiment. The rats were stratified by their body weights and then randomly assigned to cages, which were further randomly allotted to control and treatment groups. Animals were identified by cage tag and corresponding colour tail markings.

[0120]

[0116] To one group of five rats, 2.5 pg of exenatide in phosphate buffer was administered by intravenous injection via the tail vein (Group 1). To a second group of five rats, 25 pg of exenatide formulated in a bile salt formulation comprising a mixture of 50 mg chenodeoxycholic acid and propyl gallate (2:1 wt:wt) which was dissolved in water and instilled into the intestine of anaesthetized rats (Group 2). Placebo groups were also included, which consisted of formulations in which exenatide was absent (Groups 3-5).

[0121] Administration of Test and Control Articles

[0122]

[0117] The compound solutions were prepared as follows: Prior to reconstitution, each vial was prewarmed to 37°C in a temperature controlled water bath for five to ten minutes. Then 624 pL distilled water were added to the vial, the cap was screwed back on, the vial was shaken gently by hand for five seconds, then it was placed back into the water bath until the powder has completely dissolved, while shaking occasionally. The vial and contents were kept at 37°C at all times until ready for administration, which was conducted as soon as possible after the solution has dissolved.

[0123]

[0118] All compound solutions were prepared at the day of dosing, and administered via the intra duodenal route of administration in a volume of 0.5 mL per rat.

[0124]

[0119] The experiment was preceded by a 4 day run-in where all animals were handled daily and accustomed to the experimental conditions.

[0125]

[0120] At day -3 animals were single housed under equal conditions as described above. At day -2 daily 24 hours food intake was registered for each animal. At 14.00 hours on day -1 (i.e. the day prior to the surgery) 50% of the previously registered daily energy requirement was offered to the rats. The following day (day 0, 09:00 to about 15:00 hours), rats were anesthetized, using an i.m. injection of 150 pg / kg body weight Medetomidin + 2 mg / kg bw Midazolam + 5 pg / kg bw Fentanyl, and subjected to surgery. Intraduodenal injection and all blood sampling was performed in anaesthetized rats. While anaesthetized, formulations were injected directly into the intraduodenal lumen using an insulin syringe with a 30 gauge needle (t=0 min). The compound solution had been brought to body temperature (about 37 °C) prior to injection. A control group received the application solution via i.v. injection (group 5). At times -2.5, 5, 10, 15 and 30 min post-administration both portal and peripheral blood was collected into EDTA containing tubes. In addition, peripheral blood collected at time points 30, 60, and 90 min was assayed for glucose and insulin concentrations. Immediately after the last blood collection at 90 min, the rats were euthanized with carbon dioxide.

[0126] Sampling

[0127]

[0121] Blood for Exendin-4 and insulin quantification was collected in EDTA containing tubes chilled on ice. To each tube 0.6 TIU / mL aprotinin (Synovo Chem ID 9512) was added immediately. Portal vein blood samples were taken using an insulin syringe with a 30 gauge needle, peripheral blood samples were taken by puncture of the tail vein. Each blood sample was about 200 pL, resulting in about 100 pL plasma. All blood samples were immediately centrifuged for at 1,600 x g for 15 min at 4 degrees Celsius and the resulting plasma subsequently shock frozen in liquid nitrogen. Plasma samples were stored at -80°C until measurement of plasma peptide levels. Plasma peptide for each blood compartment and time point in the experiment was analysed using commercial ELISA kits (for exendin-4: Creative Diagnostics Exendin-4 ELISA kit cat. no. DEIABL227, for rat insulin: Mercodia Ultrasensitive Rat Insulin ELISA cat. no. 10-1251-01) at Synovo laboratories. Test for human insulin was conducted for samples from Group F using a Mercodia human insulin assay (Mercodia Ultrasensitive Insulin ELISA 10-1132-01).

[0128]

[0122] For blood glucose measurements one drop of blood from the tail vein was collected and analysed using a handheld blood glucose meter (Accu-Chek Aviva). Necropsy and Pathology

[0129]

[0123] Immediately after the final blood collection at 90 min post administration, the animals were euthanized by flooding an anaesthesia chamber with carbon dioxide. The animals were opened and examined for gross pathological changes.

[0130] ELISA Analysis

[0131]

[0124] The plasma samples were analysed using the following ELISA kits:

[0132]

[0125] Exendin-4 Creative Diagnostics Exendin-4 ELISA kit cat. no. DEIABL227

[0133]

[0126] Rat insulin Mercodia Ultrasensitive Rat Insulin ELISA cat. no. 10-1251-01

[0134]

[0127] ELISA analysis was performed using the chemical provided with the kits and following the manufacturer's instructions.

[0135] Statistical Analysis

[0136]

[0128] All data were fed into Excel spreadsheets and subsequently subjected to relevant statistical analyses (Graph Pad Prism software). Results are presented as mean±SEM (standard error of the mean) unless otherwise stated.

[0137] Results

[0138]

[0129] Figure 1 shows levels of exendin 4 detected in the portal vein, and in the outer circulation after administration of the peptide via instillation into the intestine, or via intravenous injection into the tail vein. As can be seen, intestinal administration results in higher concentration of the peptide in the portal vein relative to the periphery. Comparison of these values with concentrations achieved after intravenous administration, taking the difference in quantities administered into account, gives a relative bioavailability of intestinally administered peptide of 18% in the portal vein and 9% in the periphery.

[0139]

[0130] Figure 2 shows the change in blood glucose levels after administration of exendin-4. In contrast to conscious rats, anaesthetised rats display increases in glucose rather than decreases. This phenomenon is well documented by Perez-Tive and co-workers (Perez-Tive et al. "Exendin-4 increases blood glucose levels acutely in rats by activation of the sympathetic nervous system." Am J Physiol Endocrinol Metab. 2010 May; 298(5)). On the basis of the differences in AUC of glucose change measured after administration via the two different routes, a relative biopotency of 7% can be inferred for the intestinal route.

[0140]

[0131] The biopotency and bioavailability of exendin-4 in these studies is similar (7% cf 9% within margins of error), which is to be expected, since the exendin-4 is seen to pass from the intestine into the outer blood compartment, where it exerts its biological effect. Exendin-4 is a water-soluble GLP-1 receptor agonist which does not bind to receptors in the liver and will be expected to act as a model for all such peptides administered via the intestine.

[0141] Example 2 Semaglutide Study

[0142]

[0132] A study was conducted identical to that described in Example 1, except that semaglutide was employed instead of exendin-4. Semaglutide is a glucagon-like peptide-1 receptor agonist which is chemically modified by lipidation. Semaglutide was sourced from Bachem AG.

[0143]

[0133] Concentration of blood levels of semaglutide over time were measured by ELISA, using kit DEIASL092 from Creative Diagnostics.

[0144]

[0134] Figure 3 shows levels of semaglutide in the bloodstream, from which bioavailabilities of 1.2% and 0.6% in the portal vein and periphery respectively can be inferred.

[0145]

[0135] Figure 4 shows changes in glucose levels achieved after administration of semaglutide via both routes, where it can be seen that glucose levels after intestinal administration exceed those achieved after intravenous administration.

[0146]

[0136] Figure 5 shows insulin levels in the periphery after administration of semaglutide by the different routes, from which a biopotency of 9% in this experiment can be inferred.

[0147]

[0137] It should be noted that, in contrast to the non-lipidated exendin-4 GLP-1 analogue, the lipidated semaglutide GLP-1 receptor agonist shows a marked disparity between bioavailability and efficacy, demonstrating a marked depot effect, and is consistent with and supports the retention of a therapeutic peptide (i.e. semaglutide) in a depot in the intestinal tissues. While for exendin-4 the percent bioavailability (9%) and biopotency (7%) via the oral route are very similar, for semaglutide the biopotency (9%) is at least an order of magnitude greater than the bioavailability (0.6%).

[0148]

[0138] In these studies with semaglutide (like exendin-4, a water-soluble GLP-1 receptor agonist) a marked difference in behaviour is seen, compared with that observed for exendin- 4. There is a large disparity biopotency and bioavailability (here 9% cf 0.6%) which is not expected on the basis of the behaviour of exendin-4, where the biopotency and bioavailability are very similar.

[0149]

[0139] This behaviour of semaglutide suggests that a hitherto unreported phenomenon is taking place, namely creation of a depot in the intestine which allows the semaglutide to exert a strong stimulatory action on the GLP-1 receptors on the vagal afferents within the intestinal tissue, as well as, or instead of, merely interacting with receptors accessed via entering the outer blood circulation. This behaviour is indicative of formation of a depot in intestinal tissue, and the disparity between biopotency and bioavailability is a depot effect. One possible mechanism of action is the interaction / association of semaglutide with chylomicrons, either within intestinal cells, or upon release, after which the chylomicrons then stay for prolonged periods of time in the intestine, possibly immobilised via binding to multiple GLP-1 receptors on the surface of the vagal afferent cells through the intermediary of semaglutide. Interaction between semaglutide and chylomicrons could be facilitated by the presence of the long lipid chain attached to the peptide chain in semaglutide. Example 3 Semaglutide Dose Study

[0150]

[0140] A study identical to that described in Example 2 was conducted with three different dose strengths of semaglutide administered i.v. (1 pg, 2.5 pg and 5 pg per animal) or via the intestine (10 pg, 25 pg and 50 pg per animal).

[0151]

[0141] As can be seen in Figure 6, a dose relationship for the therapeutic peptide concentration in the bloodstream is obtained, with a relative bioavailability based on values for AUC from baseline ranging between 0.3% to 1.1%.

[0152]

[0142] Figure 7 shows that a dose response for biological activity is seen, looking at insulin secretion into the peripheral bloodstream, with an inferred biopotency ranging from 101%% to 9% with increasing dose. As in Example 2, a marked disparity of at least one order of magnitude between bioavailability (1.1% for the 25 pg dose) and biopotency (15% for the 25 pg dose) is observed, indicative of the retention of semaglutide in a depot in the intestinal tissues. This is shown in the table below.

[0153]

[0143] These results indicate a significant depot effect, and are consistent with there being a depot of the therapeutic peptide in the intestinal tissues, which allows it to maintain its biological action on the GLP-1 receptors of the vagal afferents.

[0154] Example 4 Tirzepatide and Bile Salt-based Formulation

[0155]

[0144] A study was conducted to determine the benefit of a bile salt-based formulation on the effects of tirzepatide in an animal model. Tirzepatide is an analogue of gastric inhibitory polypeptide (GIP) and a GLP-1 receptor agonist. It is a linear polypeptide comprising 39 amino acids that has been chemically modified by lipidation. Tirzepatide is used in the treatment of diabetes as well as for weight loss.

[0156]

[0145] The study was conducted in eight pigs in which an indwelling cannula was inserted into the small intestine, so that bile salt-based formulations in liquid form, as described above in Example 1 (tirzepatide formulated in a bile salt formulation comprising a mixture of 50 mg chenodeoxycholic acid and propyl gallate (2:1 wt:wt) dissolved in water), could be introduced into the intestine while the animals were conscious. The pigs were male, with an average weight of 30kg.

[0157]

[0146] Animals were fasted overnight, then administered a placebo formulation on day -1, followed by an intravenous glucose tolerance test (IVGTT) conducted 2 hours later, in which glucose was infused into the femoral vein at a concentration of 0.5g / kg and concentrations of insulin and glucose in the peripheral bloodstream measured at five minute intervals for the following 30 minutes. The animals were then fed as normal. The procedure was repeated on the following day (day 0), except that formulation (as employed in Examples 1 - 3 above) combined with tirzepatide was administered. On days 1, 4 and 6 thereafter, the IVGTT was repeated at the same time of day after fasting overnight, without administration of further formulation. Blood samples were taken at various times on day 0 for determination of tirzepatide concentrations

[0158]

[0147] After allowing the animals to recover, the same procedure was conducted, except that tirzepatide was administered via sub-cutaneous (s.c.) injection in the form of a free solution in saline.

[0159]

[0148] Figure 8 shows tirzepatide concentrations in the bloodstream after both s.c and intra- jejunal (i.j.) administration. Comparison of areas under the curve shows an apparent bioavailability of 0.25%.

[0160]

[0149] Figure 9 shows the change in rate of fall of glucose during the IVGTT over time, expressed as AUCs of glucose concentrations from time 0-30 minutes. As can be seen, the intestinally administered formulation shows a fall on day 0 compared with day -1, which is maintained over the following six days. The same situation is seen with the peptide administered subcutaneously. Comparison of the two routes at day six shows a relative biopotency of 13.4%.

[0161]

[0150] Figure 10 shows the change in insulin concentrations during the IVGTT over time, expressed as insulin concentrations at time ten minutes after glucose administration. As can be seen, the intestinally administered formulation shows an increase on day 0 compared with day -1, which is maintained over the following six days. The same situation is seen with the peptide administered subcutaneously. Comparison of the two routes at day six shows a relative biopotency of 5.2%.

[0162]

[0151] According to the parameters both of change in glucose disposal and insulin increase it is clear that there is a marked disparity between bioavailability and biopotency of this lipidated peptide when administered via the intestine in a bile salt-based formulation, indicating a significant depot effect. Furthermore, the longevity of response of the intestinally-administered peptide is consistent with there being a depot of the therapeutic peptide in the intestinal tissues, which allows it to maintain its biological action on the GLP-1 receptors of the vagal afferents.

[0163] Example 5 Tirzepatide and Lipid Based Formulation

[0164]

[0152] A study was conducted in pigs as described in Example 4 above, except that the tirzepatide was formulated in a lipid formulation comprising oleic acid as the major component, with phospholipid, sodium docusate and Brij52. Briefly, a cyclohexane solution was prepared containing each of sodium docusate, Brij52 and soya phosphatidyl choline at concentrations of 38, 38 and 25mg / ml respectively. To 834ul of the solution, in a 4ml glass vial, was added, with vortexing 0.25 ml of O.IM HEPES buffer solution containing tirzepatide at a concentration of lOOmg / ml. The resulting emulsion was frozen, lyophilised overnight, and 516 pl of oleic acid added to the dried residue, which gave a clear solution upon mixing. The above procedure was performed with multiple vials, and the oil solution formed pooled. 250 pl of the resulting oil was filled into a size 3 HPMC capsules. The capsules were introduced into the intestine of each pig via an in-dwelling stoma.

[0165]

[0153] Figure 11 shows tirzepatide concentrations in the bloodstream after both subcutaneous (s.c) and intra i.j. administration. Comparison of areas under the curve shows an apparent bioavailability of 0.12%.

[0166]

[0154] Figure 12 shows the change in rate of fall of glucose during the IVGTT over time, expressed as AUCs of glucose concentrations from time 0-30 minutes. As can be seen, the intestinally administered formulation shows a fall on day 0 compared with day -1, which is maintained over the following six days. The same situation is seen with the therapeutic peptide administered subcutaneously. Comparison of the two routes at day six shows a relative biopotency of 15.2%.

[0167]

[0155] Figure 13 shows the change in insulin concentrations during the IVGTT over time, expressed as AUCs of insulin concentrations from time 0-20 minutes. As can be seen, the intestinally administered formulation shows an increase on day 0 compared with day -1, which is maintained over the following six days. The same situation is seen with the peptide administered subcutaneously. Comparison of the two routes at day six shows a relative biopotency of 6.7%.

[0168]

[0156] According to the parameters both of change in glucose disposal and insulin increase it is clear that there is a marked disparity between bioavailability and biopotency of this lipidated therapeutic peptide when administered via the intestine in a lipid-based formulation, indicating a significant depot effect. Furthermore, the longevity of response of the intestinally-administered peptide (at least one week) is consistent with there being a depot of the therapeutic peptide in the intestinal tissues, which allows it to maintain its biological action on the GLP-1 receptors of the vagal afferents.

[0169] Example 6 Chylomicron Study

[0170]

[0157] Detection of secretion of chylomicrons by intestinal cells in culture was investigated in this study.

[0171]

[0158] A two-compartment cell culture system was set up by dispensing 0.5ml medium into the bottom of each well in two 12-well cluster plates, and inserting Transwell membranes (pore diameter 0.4 microns) into each well. A suspension of Caco-2 cells (passage number 55) was prepared at a concentration of 1 x 105cells per ml, and 0.5 ml of the suspension was dispensed into each of the inserts. The plates were incubated at 37°C for 16-20 days.

[0172]

[0159] Two lipid dispersions were prepared by combining lOmg either of egg or of soya phosphatidyl choline with 20mg oleic acid, 0.5mg of BODIPY and 19.2 mg of sodium taurocholate, and 360ml of distilled water and heating to 60°C with occasional vortexing until homogeneity was achieved, resulting in translucent dispersions.

[0173]

[0160] The fluid in the upper compartment of each well (the insert) in two Transwell plates was removed and replaced by 0.4ml of medium. To four of the wells, 4ul of each lipid dispersion was added. To four of the wells, 20ul of each dispersion was added, and to the remaining four wells, no addition was made. The plates were incubated at 37°C four hours, after which samples were removed from upper and lower compartments for measurement of the concentration of BODIPY by fluorimetric analysis in a plate reader with an emission wavelength 525nm, and excitation wavelength of 480nm.

[0174]

[0161] As is seen in Figure 14, BODIPY-labelled chylomicrons are observed in the lower compartment, and the similarity of output for the two different concentrations shows that uptake and transport is taking place at the maximum rate with the lower concentration. Preparation of dispersions and conduct of the cell culture assay followed the methodology outlined in the paper by Nauli et al (A. M. Nauli and J. D. Whittimore, "Using Caco-2 Cells to Study Lipid Transport by the Intestine," Journal of Visualized Experiments, 20 August 2015) which defined optimal conditions for passage of chylomicrons.

[0175] Example 7 Passage of semaglutide across intestinal cells

[0176]

[0162] The passage of semaglutide across intestinal cells was studied to demonstrate that chylomicron secretion is involved in optimal passage of semaglutide across intestinal cells.

[0177]

[0163] A two-compartment cell culture system was set as described in the previous example. Three lipid dispersions were prepared as described in the previous experiment, in which taurocholate (TC), chenodeoxycholate (CDC) and deoxycholate (DC) were used as dispersing agents, and egg phospholipid as the amphiphile.

[0178]

[0164] A solution of semaglutide in phosphate-buffered serum was prepared at a concentration of 2mg / ml.

[0179]

[0165] The fluid in the upper compartment of each well (the insert) was removed and replaced by 0.4ml of semaglutide solution. To three of the wells, 4ul of each lipid dispersion was added. To the remaining three, no further additions were made. The plate was incubated at 37°C for four hours, and samples then taken from the lower compartment to measure the semaglutide concentration by reverse-phase HPLC.

[0180]

[0166] Semaglutide was detected in those wells where both the peptide and the lipid dispersion was present in the upper compartment, but very little semaglutide was detected in the absence of lipid dispersion (see Figure 15). The integrity of the cell monolayers was confirmed by measurement of absorbance in the lower compartment at 450nm (for detection of phenol red), where no difference was seen in the presence of absence of lipid, or between test wells and medium controls. The quantity of chylomicrons passing across the cell monolayer, as assessed by BODIPY concentration, was the same for all the dispersions, regardless of the bile salt employed.

[0181]

[0167] The results of this study show that passage of semaglutide across intestinal cells is optimal when the cells are secreting chylomicrons. This is likely to be a default situation, since the components required for chylomicron formation with be present in the diet. The observation seen here are likely to hold for all lipidated peptides. Example 8 A model for Testing the Depot Formation (depot effect) in an in vivo Situation

[0182]

[0168] CaCo-2 cell monolayers were incubated with a BODIPY lipid mixture as described in Example 6, in the absence of semaglutide, and fluid containing chylomicrons from the lower compartment was harvested after two hours. To 1ml of the chylomicron dispersion 5 pl of a 2mg / ml solution in PBS was added (labelled 'A'). To an additional 1ml of dispersion PBS alone was added. After incubation for ten minutes at 37 °C, 100 pl aliquots of the dispersions were added in triplicate to wells of a microplate coated with anti-semaglutide antibody, and incubated at 37 °C for two hours. In parallel 100 pl aliquots of the dispersions were added in triplicate to the wells of an uncoated microplate and incubated at 37 °C for two hours to act as controls for non-specific binding ('Ac' and 'Be').

[0183]

[0169] After incubation, the fluid in each well was transferred to fresh well of an uncoated microplate, and the fluorescence content was measured in a Gemini Spectramax fluorescence plate reader (excitation wavelength 485 nm, emission wavelength 520nm). Values for fluorescence are shown in Figure 16, where A and B represent material not bound to antibody-coated wells, and Ac and Be represent material not bound to uncoated wells.

[0184]

[0170] Percentage of chylomicrons bound to coated wells in the absence of semaglutide is expressed by 100 x (Bc-B) / Bc = 17%.

[0185]

[0171] The difference between these two values is statistically significant (p = 0.0013), and demonstrates that semaglutide interacts with chylomicrons in such a way as to induce specific binding with GLP-1 RA-specific peptide ideotypes and receptor-like structures.

[0186]

[0172] The observation, in this experiment, that chylomicrons are sequestered on the walls of anti-GLP-l-coated microplates, but much less in the absence of antibodies, as a result of the intermediary of semaglutide (to which the antibody can bind) demonstrates the way in which a depot can be formed, and shows that this experimental design is acting as a model for depot formation in an in vivo situation, where chylomicrons, a lipidated GLP-1 peptide, and an immobilised receptor capable of binding to GLP-1 are the three requisite components in both cases.

Claims

CLAIMS1. A formulation comprising (i) a therapeutic peptide or protein and (ii) one or more formulation excipients which enhances the uptake of the therapeutic peptide across the intestine of a subject, wherein there is a depot effect, in which the biopotency of the therapeutic peptide is significantly greater than the bioavailability of the peptide when compared with administration via the parenterally injected route.

2. A formulation according to claim 1, wherein the ratio of biopotency to bioavailability is greater or equal to 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 or 6 or more.

3. A formulation comprising (i) a therapeutic peptide or protein and (ii) one or more formulation excipients which enhances the uptake of the therapeutic peptide across the intestine of a subject, wherein the peptide remains held in a depot, after administration, within intestinal tissue.

4. A formulation comprising (i) a therapeutic peptide or protein and (ii) one or more formulation excipients which enhances the uptake of the therapeutic peptide across the intestine of a subject, wherein the peptide remains held in a depot, after administration, associated with chylomicrons.

5. The formulation according to any one of claims 1-4, wherein the formulation is administered orally.

6. The formulation according to claims 1-5, where in the peptide, protein or therapeutic macromolecule associates with chylomicrons.

7. The formulation according to any one of claims 1-6, wherein the therapeutic peptide or protein is lipidated.

8. The formulation according to any one of claims 1-7, wherein the therapeutic peptide or protein is a GLP-1 or GIP receptor agonist.

9. The formulation according to any one of claims 1-8, wherein the therapeutic peptide or protein is a GLP-1 or GIP receptor agonist selected from the group comprising: exendin-4, semaglutide, and tirzepatide.

10. The formulation of any one of claims 1-9, wherein the formulation excipient is one or more selected from the group comprising: lipids, bile salts and acids thereof, fattyacids, salts and derivatives thereof, lipophilic esters of fatty acids, and esters of polyphenols, glycerolipids, alone or in combination.

11. The formulation of any one of claims 1-9, comprising a therapeutic peptide or protein, and chenodeoxycholic acid and propyl gallate (2:1 wt:wt).

12. The formulation of any one of claims 1-11, wherein the duration of action of the therapeutic peptide or protein is prolonged, such that a single dose of the composition may be administered to a human once every four to six days.

13. A method of determining the ability of a therapeutic peptide or protein to form a depot effect comprising:(a) measuring the relative bioavailability of the therapeutic peptide or protein;(b) measuring the relative biopotency of the therapeutic peptide or protein; and(c) determining the ratio of the biopotency compared to the bioavailability , wherein the ratio is greater or equal to 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 or 6 or more.

14. A method of treating a subject in need thereof comprising administering to the subject the formulation according to any one of claims 1-12.

15. Use of a formulation according to any one of claims 1-12 in the preparation of a medicament for the treatment of a disease or disorder.

16. A formulation according to any one of claims 1-12 when used in the treatment of a disease or disorder in a subject in need thereof.