WO2020039052A1

WO2020039052A1 - Calcitonin mimetics for treating diseases and disorders

Info

Publication number: WO2020039052A1
Application number: PCT/EP2019/072535
Authority: WO
Inventors: Kim V ANDREASSEN; Kim Henriksen; Morten Asser Karsdal
Original assignee: Key Bioscience Ag
Priority date: 2018-08-22
Filing date: 2019-08-22
Publication date: 2020-02-27
Also published as: GB201813677D0

Abstract

Disclosed herein are calcitonin mimetics in which the amino acid residue in the 8 position of the peptide is 2- aminoisobutyric acid (AiB).

Description

Calcitonin Mimetics for Treating Diseases

and Disorders

The present invention relates to mimetics of calcitonin, and extends to their use as medicaments in the treatment of various diseases and disorders, including, but not limited to diabetes (Type I and Type II), excess bodyweight, excessive food consumption and metabolic syndrome, non-alcoholic steatohepatitis (NASH) , alcoholic and non-alcoholic fatty liver disease, producing a decrease in liver triglycerides, reducing fat accumulation in the liver of a subject the regulation of blood glucose levels, the regulation of

response to glucose tolerance tests, the regulation of food intake, the treatment of osteoporosis and the treatment of osteoarthritis .

Worldwide, there are about 250 million diabetics and the number is projected to double in the next two decades. Over 90% of this population suffers from type 2 diabetes mellitus (T2DM) . It is estimated that only 50-60% of persons affected with T2DM or in stages preceding overt T2DM are currently diagnosed .

T2DM is a heterogeneous disease characterized by

abnormalities in carbohydrate and fat metabolism. The causes of T2DM are multi-factorial and include both genetic and environmental elements that affect b-cell function and insulin sensitivity in tissues such as muscle, liver,

pancreas and adipose tissue. As a consequence impaired insulin secretion is observed and paralleled by a progressive decline in b-cell function and chronic insulin resistance.

The inability of the endocrine pancreas to compensate for peripheral insulin resistance leads to hyperglycaemia and onset of clinical diabetes. Tissue resistance to insulin- mediated glucose uptake is now recognized as a major pathophysiologic determinant of T2DM.

A success criterion for an optimal T2DM intervention is the lowering of blood glucose levels, which can be both chronic lowering of blood glucose levels and increased ability to tolerate high glucose levels after food intake, described by lower peak glucose levels and faster clearance. Both of these situations exert less strain on b-cell insulin output and function.

Type I diabetes is characterised by a loss of the ability to produce insulin in response to food intake and hence an inability to regulate blood glucose to a normal physiological level.

The physical structure of bone may be compromised by a variety of factors, including disease and injury. One of the most common bone diseases is osteoporosis, which is

characterized by low bone mass and structural deterioration of bone tissue, leading to bone fragility and an increased susceptibility to fractures, particularly of the hip, spine and wrist. Osteoporosis develops when there is an imbalance such that the rate of bone resorption exceeds the rate of bone formation. Administering an effective amount of an anti-resorptive agent, such as calcitonin, has shown to prevent resorption of bone.

Inflammatory or degenerative diseases, including

diseases of the joints, e.g. osteoarthritis (OA) , rheumatoid arthritis (RA) or juvenile rheumatoid arthritis (JRA) , and including inflammation that results from autoimmune response, e.g. lupus, ankylosing spondylitis (AS) or multiple sclerosis (MS) , can lead to substantial loss of mobility due to pain and joint destruction. Cartilage that covers and cushions bone within joints may become degraded over time thus undesirably permitting direct contact of two bones that can limit motion of one bone relative to the other and/or cause damage to one by the other during motion of the joint.

Subchondral bone just beneath the cartilage may also degrade. Administering an effective amount of an anti-resorptive agent, such as calcitonin, may prevent resorption of bone.

Calcitonins are highly conserved over a wide range of species. Full-length native calcitonin is 32 amino acids in length. The sequences of examples of natural calcitonins are set out below:

Synthetic variants of natural calcitonins having

modified amino acid sequences which are intended to provide improved properties are disclosed in WO2013/067357 and WO 2015/071229.

There is a continuing need to develop calcitonin

analogues having still further improved properties, or at least providing alternative artificial sequences improving on the properties of the naturally occurring calcitonins, particularly in respect of amylin and calcitonin receptor agonrsm. An issue that is frequently encountered with peptide drugs, particularly in the formulation of peptide drugs, is aggregation of the peptide. Strategies to curb the

aggregation and misfolding of proteins during storage are likely to benefit from the recent surge of interest in protein fibrillation. Fibrillation is the formation of well- defined protein aggregates, the best known of which is the amyloid fibril, associated with neurodegenerative diseases.

It has been suggested that all proteins can form fibrils with the same structural characteristics, namely a cross-b structure and parallel b-helices, irrespective of the native structure and primary sequence. This presents a potential hazard during production and/or storage. Fibrillation is particularly encouraged under moderately destabilizing conditions, such as variations in pH, temperature changes, and/or intermediate denaturant concentrations, which allow the protein better access to other conformations while retaining some structure (Fr0kjaer et al, Protein drug stability: a formulation challenge. Nat Rev Drug Discov

4:298- 306) . Accordingly, developing peptide drugs with a reduced propensity for fibrillating in solution would be a significant advantage, particularly with respect to

formulation of said drugs.

Summary of the Invention

It has now been found that replacing certain residues in calcitonins and/or mimetics thereof, specifically the residue in the 8 position of the peptide, with 2-aminoisobutyric acid (AiB) significantly reduces the propensity of the peptide to fibrillate whilst simultaneously having no detrimental effect on the efficacy of the peptide. 2-aminoisobutyric acid (CAS Number 62-57-7), also known as a-aminoisobutyric acid, a- methylalanine or 2-methylalanine, is an amino acid with the structural formula

and is herein referred to as 2-aminoisobutyric acid or AiB.

Accordingly, in a first aspect the present invention relates to a peptide of formula (I) :

Preferably, the peptide is a peptide of formula (II) :

wherein

Preferably, X₂ is S and X₃ is N; or X₂ is G and X₃ is N; or X₂ is A and X₃ is S. Preferably, X₁₃ is S or T, most preferably S. Preferably, X₂₄ is K or R.

In a preferred embodiment,

Preferred peptides of the invention are:

KBP-042B: AcCSNLSTC (AiB) LGKLSQELHKLQTYPRTDVGANAP-NH₂ KBP-056B: AcCASLSTC (AiB) LGKLSQDLHKLQTFPKTDVGANAP-NH₂

The most preferred peptide of the invention is KBP-066.

The peptides of the invention may be acylated at its N- terminal or otherwise modified to reduce the positive charge of the first amino acid and independently of that may be amidated at its C-terminal.

The peptide may be formulated for administration as a pharmaceutical and may be formulated for enteral or

parenteral administration. Preferred formulations are injectable, preferably for subcutaneous injection, however the peptide may be formulated with a carrier for oral

administration, and optionally wherein the carrier increases the oral bioavailability of the peptide. Suitable carriers include ones that comprise 5-CNAC, SNAD, or SNAC .

Optionally, the peptide is formulated in a pharmaceutical composition for oral administration comprising coated citric acid particles, and wherein the coated citric acid particles increase the oral bioavailability of the peptide.

The invention includes a peptide of the invention for use as a medicament. The peptide may be for use in treating diabetes (Type I and/or Type II), excess bodyweight, obesity, excessive food consumption, metabolic syndrome, rheumatoid arthritis, non-alcoholic steatohepatitis (NASH) , non

alcoholic fatty liver disease, alcoholic fatty liver disease, osteoporosis, or osteoarthritis, poorly regulated blood glucose levels, poorly regulated response to glucose

tolerance tests, or poor regulation of food intake. In particular, the peptides may be used to lower an undesirably high fasting blood glucose level or to lower an undesirably high HbAlc or to reduce an undesirably high response to a glucose tolerance test. The peptides of the invention may be used for producing a decrease in liver triglycerides and/or for reducing fat accumulation in the liver of a subject.

The peptides of the invention may be produced using any suitable method known in the art for generating peptides, such as synthetic (chemical) and recombinant technologies. Preferably, the peptides are produced using a synthetic method. Synthetic peptide synthesis is well known in the art, and includes (but is not limited to) solid phase peptide synthesis employing various protecting group strategies (e.g. using Fmoc, Boc, Bzl, tBu, etc.) .

In some embodiments, the N-terminal side of the peptides discussed supra is modified to reduce the positive charge of the first amino acid. For example, an acetyl, propionyl, or succinyl group may be substituted on cysteine-1. Alternative ways of reducing positive charge include, but are not limited to, polyethylene glycol-based PEGylation, or the addition of another amino acid such as glutamic acid or aspartic acid at the N-terminus. Alternatively, other amino acids may be added to the N-terminus of peptides discussed supra

including, but not limited to, lysine, glycine,

formylglycine, leucine, alanine, acetyl alanine, and

dialanyl. As those of skill in the art will appreciate, peptides having a plurality of cysteine residues frequently form a disulfide bridge between two such cysteine residues. All such peptides set forth herein are defined as optionally including one or more such disulphide bridges, particularly at the Cysl-Cys7 locations. Mimicking this, the cysteines at positions 1 and 7 may jointly be replaced by an cx- aminosuberic acid linkage. Alternatively, the cysteines at positions 1 or 7 may independently be replaced by an cx- aminosuberic acid linkage.

While peptides of the present disclosure may exist in free acid form, it is preferred that the C-terminal amino acid be amidated. Applicants expect that such amidation may contribute to the effectiveness and/or bioavailability of the peptide. Synthetic chemical methods may be employed for amidating the C-terminal amino acid. Another technique for manufacturing amidated versions of the peptides of the present disclosure is to react precursors (having glycine in place of the C-terminal amino group of the desired amidated product) in the presence of peptidylglycine alpha-amidating monooxygenase in accordance with known techniques wherein the precursors are converted to amidated products in reactions described, for example, in US4708934 and EP0308067 and

EP0382403.

Production of amidated products may also be accomplished using the process and amidating enzyme set forth by Consalvo, et al in US7445911; Miller et al, US2006/0292672 ; Ray et al, 2002, Protein Expression and Purification, 26:249-259; and Mehta, 2004, Biopharm. International, July, pp . 44-46.

The production of the preferred amidated peptides may proceed, for example, by producing glycine-extended precursor in E. coli as a soluble fusion protein with glutathione-S- transferase, or by direct expression of the precursor in accordance with the technique described in US6103495. Such a glycine extended precursor has a molecular structure that is identical to the desired amidated product except at the C- terminus (where the product terminates --X--N¾, while the precursor terminates --X-gly, X being the C-terminal amino acid residue of the product) . An alpha-amidating enzyme described in the publications above catalyzes conversion of precursors to product. That enzyme is preferably

recombinantly produced, for example, in Chinese Hamster Ovary (CHO) cells) , as described in the Biotechnology and Biopharm. articles cited above. Free acid forms of peptide active agents of the present disclosure may be produced in like manner, except without including a C-terminal glycine on the "precursor", which precursor is instead the final peptide product and does not require the amidation step.

Except where otherwise stated, the preferred dosage of the peptide of the present disclosure is identical for both therapeutic and prophylactic purposes. Desired dosages are discussed in more detail, infra, and differ depending on mode of administration.

Except where otherwise noted or where apparent from context, dosages herein refer to weight of active compounds (i.e. peptides of the invention) unaffected by or discounting pharmaceutical excipients, diluents, carriers or other ingredients, although such additional ingredients are

desirably included. Any dosage form (capsule, tablet, injection or the like) commonly used in the pharmaceutical industry for delivery of peptide active agents is appropriate for use herein, and the terms "excipient", "diluent", or "carrier" includes such non-active ingredients as are

typically included, together with active ingredients in such dosage form in the industry. A preferred oral dosage form is discussed in more detail, infra, but is not to be considered the exclusive mode of administering the active agents of the present disclosure.

The peptides of the present disclosure can be

administered to a patient to treat a number of diseases or disorders. As used herein, the term "patient" means any organism belonging to the kingdom Animalia. In an

embodiment, the term "patient" refers to vertebrates, more preferably, mammals including humans. Accordingly, the present disclosure includes the use of the peptides in a method of treatment of type I diabetes,

Type II diabetes or metabolic syndrome, obesity, or of appetite suppression, or for mitigating insulin resistance, or for reducing an undesirably high fasting serum glucose level, or for reducing an undesirably high peak serum glucose level, or for reducing an undesirably high peak serum insulin level, or for reducing an undesirably large response to a glucose tolerance test, or for treating osteoporosis, or for treating osteoarthritis, or for treating non-alcoholic steatohepatitis (NASH) , or for treating alcoholic or non alcoholic fatty liver disease, or for producing a decrease in liver triglycerides, or for reducing fat accumulation in the liver of a subject.

There are a number of art-recognized measures of normal range for body weight in view of a number of factors such as gender, age and height. A patient in need of treatment or prevention regimens set forth herein include patients whose body weight exceeds recognized norms or who, due to heredity, environmental factors or other recognized risk factor, are at higher risk than the general population of becoming

overweight or obese. In accordance with the present

disclosure, it is contemplated that the peptides of the invention may be used to treat diabetes where weight control is an aspect of the treatment.

In an embodiment, the method includes enteral

administration to a patient in need thereof for treatment of a said condition of a pharmaceutically effective amount of any one of the peptides described herein.

In an embodiment, the method includes parenteral

administration to a patient in need thereof for treatment of a said condition of a pharmaceutically effective amount of any one of the peptides described herein. For parenteral administration (including intraperitoneal , subcutaneous, intravenous, intradermal or intramuscular injection), solutions of a peptide of the present disclosure in either sesame or peanut oil or in aqueous propylene glycol may be employed, for example. The aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic. These aqueous solutions are suitable for intravenous injection purposes. The oily solutions are suitable for intraarticular, intramuscular and subcutaneous injection purposes. The preparation of all these solutions under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art. For parenteral application, examples of suitable preparations include solutions, preferably oily or aqueous solutions as well as suspensions, emulsions, or implants, including suppositories. Peptides may be formulated in sterile form in multiple or single dose formats such as being dispersed in a fluid carrier such as sterile physiological saline or 5% saline dextrose solutions commonly used with inj ectables .

Said method may include a preliminary step of

determining whether the patient suffers from a said

condition, and/or a subsequent step of determining to what extent said treatment is effective in mitigating the

condition in said patient, e.g. in each case, carrying out an oral glucose tolerance test or a resting blood sugar level.

For improved control over the weight of the patient, to produce a loss of weight or an avoidance of weight gain, the active compound is preferably administered once daily or more such as at least twice per day, e.g. from 2-4 times per day. Formulations of the active compound may contain a unit dosage appropriate for such an administration schedule. The active compounds may be administered with a view to controlling the weight of a patient undergoing treatment for diabetes or metabolic syndrome.

Oral enteral formulations are for ingestion by

swallowing for subsequent release in the intestine below the stomach, and hence delivery via the portal vein to the liver, as opposed to formulations to be held in the mouth to allow transfer to the bloodstream via the sublingual or buccal routes .

Suitable dosage forms for use in the present disclosure include tablets, mini-tablets , capsules, granules, pellets, powders, effervescent solids and chewable solid formulations. Such formulations may include gelatin which is preferably hydrolysed gelatin or low molecular weight gelatin. Such formulations may be obtainable by freeze drying a homogeneous aqueous solution comprising a peptide of the invention and hydrolysed gelatin or low molecular weight gelatin and further processing the resulting solid material into said oral pharmaceutical formulation, and wherein the gelatin may have a mean molecular weight from 1000 to 15000 Daltons.

Such formulations may include a protective carrier compound such as 5-CNAC or others as disclosed herein.

Whilst oral formulations such as tablets and capsules are preferred, compositions for use in the present disclosure may take the form of syrups, elixirs or the like and

suppositories or the like. Oral delivery is generally the delivery route of choice since it is convenient, relatively easy and generally painless, resulting in greater patient compliance relative to other modes of delivery. However, biological, chemical and physical barriers such as varying pH in the gastrointestinal tract, powerful digestive enzymes, and active agent impermeable gastrointestinal membranes, makes oral delivery of calcitonin like peptides to mammals problematic, e.g. the oral delivery of calcitonins, which are long-chain polypeptide hormones secreted by the

parafollicular cells of the thyroid gland in mammals and by the ultimobranchial gland of birds and fish, originally proved difficult due, at least in part, to the insufficient stability of calcitonin in the gastrointestinal tract as well as the inability of calcitonin to be readily transported through the intestinal walls into the blood stream.

Suitable oral formulations are however described below.

Treatment of Patients

In an embodiment, a peptide of the present invention is administered at adequate dosage to maintain serum levels of the mimetic in patients between 5 picograms and 500 nanograms per milliliter, preferably between 50 picograms and 250 nanograms, e.g. between 1 and 100 nanograms per milliliter. The serum levels may be measured by any suitable techniques known in the art, such as radioimmunoassay or mass

spectrometry. The attending physician may monitor patient response, and may then alter the dosage somewhat to account for individual patient metabolism and response. Near

simultaneous release is best achieved by administering all components of the present disclosure as a single pill or capsule. However, the disclosure also includes, for example, dividing the required amount of the peptide among two or more tablets or capsules which may be administered together such that they together provide the necessary amount of all ingredients. "Pharmaceutical composition," as used herein includes but is not limited to a complete dosage appropriate to a particular administration to a patient regardless of whether one or more tablets or capsules (or other dosage forms) are recommended at a given administration.

A peptide of the present invention may be formulated for oral administration using the methods employed in the Unigene Enteripep® products. These may include the methods as described in US Patent No. 5,912,014, US Patent No.

6,086,918, US Patent No. 6,673,574, US Patent No. 7,316,819, US Patent No. 8,093,207, and US Publication No. 2009/0317462. In particular, it may include the use of conjugation of the compound to a membrane translocator such as the protein transduction domain of the HIV TAT protein, co-formulation with one or more protease inhibitors, and/or a pH lowering agent which may be coated and/or an acid resistant protective vehicle and/or an absorption enhancer which may be a

surfactant .

In an embodiment, a peptide of the present invention is preferably formulated for oral delivery in a manner known in U.S. Patent Publication No. 2009/0317462.

In an embodiment, a peptide of the present invention may be formulated for enteral, especially oral, administration by admixture with a suitable carrier compound. Suitable carrier compounds include those described in US Patent No. 5,773,647 and US Patent No. 5866536 and amongst these, 5-CNAC (N- (5- chlorosalicyloyl) -8-aminocaprylic acid, commonly as its disodium salt) is particularly effective. Other preferred carriers or delivery agents are SNAD (sodium salt of 10- (2- Hydroxybenzamido) decanoic acid) and SNAC (sodium salt of N- (8- [2-hydroxybenzoyl] amino) caprylic acid). In an embodiment, a pharmaceutical composition of the present disclosure comprises a delivery effective amount of carrier such as 5- CNAC, i.e. an amount sufficient to deliver the compound for the desired effect. Generally, the carrier such as 5-CNAC is present in an amount of 2.5% to 99.4% by weight, more

preferably 25% to 50% by weight of the total composition.

In addition, WO 00/059863 discloses the disodium salts of formula I

wherein

R¹, R², R³, and R⁴ are independently hydrogen, -OH, -NR⁶R⁷, halogen, C₁-C₄ alkyl, or C₁-C₄ alkoxy;

R⁵ is a substituted or unsubstituted C₂-C₁₆ alkylene,

substituted or unsubstituted C₂-C₁₆ alkenylene, substituted or unsubstituted C₁-C12 alkyl (arylene) , or substituted or

unsubstituted aryl (C1-C12 alkylene) ; and R⁶ and R⁷ are

independently hydrogen, oxygen, or C₁-C₄ alkyl; and hydrates and solvates thereof as particularly efficacious for the oral delivery of active agents, such as calcitonins, e.g. salmon calcitonin, and these may be used in the present disclosure.

Preferred enteric formulations using optionally

micronised 5-CNAC may be generally as described in

W02005/014031.

The compound may be formulated for oral administration using the methods employed in the Capsitonin product of Bone Medical Limited. These may include the methods incorporated in Axcess formulations. More particularly, the active ingredient may be encapsulated in an enteric capsule capable of withstanding transit through the stomach. This may contain the active compound together with a hydrophilic aromatic alcohol absorption enhancer, for instance as described in WO02/028436. In a known manner the enteric coating may become permeable in a pH sensitive manner, e.g. at a pH of from 3 to 7. W02004/091584 also describes suitable formulation methods using aromatic alcohol absorption

enhancers .

The compound may be formulated using the methods seen in the Oramed products, which may include formulation with omega-3 fatty acid as seen in W02007/029238 or as described in US5, 102, 666.

Generally, the pharmaceutically acceptable salts

(especially mono or di sodium salts), solvates (e.g. alcohol solvates) and hydrates of these carriers or delivery agents may be used.

Oral administration of the pharmaceutical compositions according to the disclosure can be accomplished regularly, e.g. once or more on a daily or weekly basis; intermittently, e.g. irregularly during a day or week; or cyclically, e.g. regularly for a period of days or weeks followed by a period without administration. The dosage form of the

pharmaceutical compositions of the presently disclosed embodiments can be any known form, e.g. liquid or solid dosage forms. The liquid dosage forms include solution emulsions, suspensions, syrups and elixirs. In addition to the active compound and carrier such as 5-CNAC, the liquid formulations may also include inert excipients commonly used in the art such as, solubilizing agents e.g. ethanol; oils such as cottonseed, castor and sesame oils; wetting agents; emulsifying agents; suspending agents; sweeteners;

flavourings; and solvents such as water. The solid dosage forms include capsules, soft-gel capsules, tablets, caplets, powders, granules or other solid oral dosage forms, all of which can be prepared by methods well known in the art. The pharmaceutical compositions may additionally comprise

additives in amounts customarily employed including, but not limited to, a pH adjuster, a preservative, a flavorant, a taste-masking agent, a fragrance, a humectant, a tonicifier, a colorant, a surfactant, a plasticizer, a lubricant such as magnesium stearate, a flow aid, a compression aid, a

solubilizer, an excipient, a diluent such as microcrystalline cellulose, e.g. Avicel PH 102 supplied by FMC corporation, or any combination thereof. Other additives may include

phosphate buffer salts, citric acid, glycols, and other dispersing agents. The composition may also include one or more enzyme inhibitors, such as actinonin or epiactinonin and derivatives thereof; aprotinin, Trasylol and Bowman-Birk inhibitor. Further, a transport inhibitor, i.e. a [rho]- glycoprotein such as Ketoprofin, may be present in the compositions of the present disclosure. The solid

pharmaceutical compositions of the instant disclosure can be prepared by conventional methods e.g. by blending a mixture of the active compound, the carrier such as 5-CNAC, and any other ingredients, kneading, and filling into capsules or, instead of filling into capsules, molding followed by further tableting or compression-molding to give tablets. In

addition, a solid dispersion may be formed by known methods followed by further processing to form a tablet or capsule. Preferably, the ingredients in the pharmaceutical

compositions of the instant disclosure are homogeneously or uniformly mixed throughout the solid dosage form.

Alternatively, the active compound may be formulated as a conjugate with said carrier, which may be an oligomer as described in US2003/0069170, e.g.

Such conjugates may be administered in combination with a fatty acid and a bile salt as described there.

Conujugates with polyethylene glycol (PEG) may be used, as described for instance in Mansoor et al . Alternatively, active compounds may be admixed with nitroso-N-acetyl-D, L-penicillamine (SNAP) and Carbopol solution or with taurocholate and Carbapol solution to form a mucoadhesive emulsion.

The active compound may be formulated by loading into chitosan nanocapsules as disclosed in Prego et al (optionally PEG modified as in Prego Prego C, Torres D, Fernandez-Megia E, Novoa-Carballal R, Quinoa E, Alonso MJ.) or chitosan or PEG coated lipid nanoparticles as disclosed in Garcia-Fuentes et al . Chitosan nanoparticles for this purpose may be iminothiolane modified as described in Guggi et al . They may be formulated in water/oil/water emulsions as described in Dogru et al . The bioavailability of active compounds may be increased by the use of taurodeoxycholate or lauroyl

carnitine as described in Sinko et al or in Song et al .

Generally, suitable nanoparticles as carriers are discussed in de la Fuente et al and may be used in the present

disclosure .

Other suitable strategies for oral formulation include the use of a transient permeability enhancer (TPE) system as described in W02005/094785 of Chiasma Ltd. TPE makes use of an oily suspension of solid hydrophilic particles in a hydrophobic medium to protect the drug molecule from

inactivation by the hostile gastrointestinal (GI) environment and at the same time acts on the GI wall to induce permeation of its cargo drug molecules.

Further included is the use of glutathione or compounds containing numerous thiol groups as described in

US2008/0200563 to inhibit the action of efflux pumps on the mucous membrane. Practical examples of such techniques are described also in Caliceti, P. Salmaso, S., Walker, G. and Bernkop-Schnurch, A. (2004) 'Development and in vivo

evaluation of an oral insulin-PEG delivery system.' Eur. J. Pharm. Sci.,r 22, 315-323, in Guggi, D., Krauland, A.H., and Bernkop-Schniirch, A. (2003) 'Systemic peptide delivery via the stomach: in vivo evaluation of an oral dosage form for salmon calcitonin'. J. Control. Rel. 92,125-135, and in

Bernkop-Schniirch, A., Pinter, Y., Guggi, D., Kahlbacher, H., Schoffmann, G., Schuh, M., Schmerold, I., Del Curto, M.D., D'Antonio, M., Esposito, P. and Huck, Ch. (2005) 'The use of thiolated polymers as carrier matrix in oral peptide

delivery' - Proof of concept. J. Control. Release, 106, 26- 33.

The active compound may be formulated in seamless micro spheres as described in W02004/084870 where the active pharmaceutical ingredient is solubilised as an emulsion, microemulsion or suspension formulated into mini-spheres; and variably coated either by conventional or novel coating technologies. The result is an encapsulated drug in "pre solubilised" form which when administered orally provides for predetermined instant or sustained release of the active drug to specific locations and at specific rates along the

gastrointestinal tract. In essence, pre-solubilization of the drug enhances the predictability of its kinetic profile while simultaneously enhancing permeability and drug

stability .

One may employ chitosan coated nanocapsules as described in US2009/0074824. The active molecule administered with this technology is protected inside the nanocapsules since they are stable against the action of the gastric fluid. In addition, the mucoadhesive properties of the system enhances the time of adhesion to the intestine walls (it has been verified that there is a delay in the gastrointestinal transit of these systems) facilitating a more effective absorption of the active molecule. Methods developed by TSR1 Inc. may be used. These include Hydrophilic Solubilization Technology (HST) in which gelatin, a naturally derived collagen extract carrying both positive and negative charges, coats the particles of the active ingredient contained in lecithin micelles and prevents their aggregation or clumping. This results in an improved wettability of hydrophobic drug particles through polar interactions. In addition, the amphiphilic lecithin reduces surface tension between the dissolution fluid and the

particle surface.

The active ingredient may be formulated with

cucurbiturils as excipients.

Alternatively, one may employ the GIPET technology of Merrion Pharmaceuticals to produce enteric coated tablets containing the active ingredient with an absorption enhancer which may be a medium chain fatty acid or a medium chain fatty acid derivative as described in US2007/0238707 or a membrane translocating peptide as described in US7268214.

One may employ GIRES™ technology which consists of a controlled-release dosage form inside an inflatable pouch, which is placed in a drug capsule for oral administration. Upon dissolution of the capsule, a gas-generating system inflates the pouch in the stomach. In clinical trials the pouch has been shown to be retained in the stomach for 16-24 hours .

Alternatively, the active may be conjugated to a

protective modifier that allows it to withstand enzymatic degradation in the stomach and facilitate its absorption.

The active may be conjugated covalently with a monodisperse, short-chain methoxy polyethylene glycol glycolipids

derivative that is crystallized and lyophilized into the dry active pharmaceutical ingredient after purification. Such methods are described in US5438040 and at www.biocon.com.

One may also employ a hepatic-directed vesicle (HDV) for active delivery. An HDV may consist of liposomes (£150 nm diameter) encapsulating the active, which also contain a hepatocyte-targeting molecule in their lipid bilayer. The targeting molecule directs the delivery of the encapsulated active to the liver cells and therefore relatively minute amounts of active are required for effect. Such technology is described in US2009/0087479 and further at

www . diasome . com.

The active may be incorporated into a composition containing additionally a substantially non-aqueous

hydrophilic medium comprising an alcohol and a cosolvent, in association with a medium chain partial glyceride, optionally in admixture with a long-chain PEG species as described in US2002/0115592 in relation to insulin.

Alternatively, use may be made of intestinal patches as described in Shen Z, Mitragotri S, Pharm Res. 2002

Apr; 19 (4) : 391-5 'Intestinal patches for oral drug delivery'.

The active may be incorporated into an erodible matrix formed from a hydrogel blended with a hydrophobic polymer as described in US Patent No. 7189414.

Suitable oral dosage levels for adult humans to be treated may be in the range of 0.05 to 5mg, preferably about 0.1 to 2.5mg .

The frequency of dosage treatment of patients may be from 1 to six times daily, for instance from two to four times daily. Treatment will desirably be maintained over a prolonged period of at least 6 weeks, preferably at least 6 months, preferably at least a year, and optionally for life. Combination treatments for relevant conditions may be carried out using a composition according to the present disclosure and separate administration of one or more other therapeutics. Alternatively, the composition according to the present disclosure may incorporate one or more other therapeutics for combined administration.

Combination therapies according to the present

disclosure include combinations of an active compound as described with insulin, GLP-2, GLP-1, GIP, or amylin, or generally with other anti-diabetics. Thus combination

therapies including co-formulations may be made with insulin sensitizers including biguanides such as Metformin, Buformin and Phenformin, TZD' s (PPAR) such as Balaglitazone,

Pioglitazone, Rivoglitazone, Rosiglitazone and Troglitazone, dual PPAR agonists such as Aleglitazar, Muraglitazar and Tesaglitazar, or secretagogues including sulphonylureas such as Carbutamide, Chloropropamide, Gliclazide, Tolbutamide, Tolazamide, Glipizide, Glibenclamide, Glyburide, Gliquidone, Glyclopyramide and Glimepriride, Meglitinides/glinides (K+) such as Nateglinide, Repaglinide and Mitiglinide, GLP-1 analogs such as Exenatide, Liraglutide, Semaglutide,

Dulaglutide, and Albiglutide, DPP-4 inhibitors such as

Alogliptin, Linagliptin, Saxagliptin, Sitagliptin and

Vildagliptin, insulin analogs or special formulations such as (fast acting) Insulin lispro, Insulin aspart, Insulin

glulisine, (long acting) Insulin glargine, Insulin detemir) , inhalable insulin - Exubra and NPH insulin, and others including alpha-glucosidase inhibitors such as Acarbose, Miglitol and Voglibose, amylin analogues such as Pramlintide, SGLT2 inhibitors such as Empagliflozin, Dapagliflozin,

Remogliflozin and Sergliflozin as well as miscellaneous ones including Benfluorex and Tolrestat. Further combinations include co-administration or co formulation with leptins. Leptin resistance is a well- established component of type 2 diabetes; however, injections of leptin have so far failed to improve upon this condition. In contrast, there is evidence supporting that amylin, and thereby molecules with amylin-like abilities, are able to improve leptin sensitivity. Amylin/leptin combination has shown a synergistic effect on body weight and food intake, and also insulin resistance [Kusakabe T et al] .

A further preferred combination therapy includes co formulation or co-administration of the peptides of the invention with one or more weight loss drugs. Such weight loss drugs include, but are not limited to, lipase inhibitors (e.g. pancreatic lipase inhibitors, such as Orlistat) , appetite suppressing amphetamine derivatives (e.g.

Phentermine) , Topiramate, Qysmia® ( Phentermine/Topiramate combination), 5-HT₂c receptor agonists (e.g. Locaserin) , Contrave® (naltrexone/bupropion combination) , glucagon-like peptide-1 [GLP-1] analogues and derivatives (e.g.

Liraglutide, semaglutide) , sarco/endoplasmic reticulum (SR) Ca²⁺ ATPase (SERCA) inhibitors (e.g. sarcolipin) , Fibroblast growth factor 21 [FGF-21] receptor agonists (e.g. analogs of FGF-21), and b3 adreno receptor agonists (e.g. Mirabegron) . Such combinations may be used to treat an overweight

condition, such as obesity.

Description of the Figures

Figure 1: Results of the Thioflavin T assay for KBP042 and KBP042B .

Figure 2: Results of the Thioflavin T assay for KBP056 and KBP056B. Figure 3: Results of the Thioflavin T assay for KBP089 and KBP066.

Figure 4. Efficacy data for KBP089 and KBP066.

Figure 5. Three-hour CTR-mediated beta-arrestin response by individual KBPs and their corresponding AiB version (position 8) . Dose concentration curve of individual KBP ranging from 100 nM and then in 4-fold dilutions steps down to ~25 pM and shown as fold of vehicle. A) KBP-042 and KBP-042B. B) KBP-056 and KBP-056B. C) KBP-089 and KBP-089B (KBP-066) . Experiment was conducted in U20S CALCR PathHunter cells from DiscoveRx. Data shown as mean ± SEM.

Figure 6. Three-hour AMYR-mediated beta-arrestin response by individual KBPs and their corresponding AiB version (position 8) . Dose concentration curve of individual KBP ranging from 100 nM and then in 4-fold dilutions steps down to ~25 pM and shown as fold of vehicle. A) KBP-042 and KBP-042B. B) KBP-056 and KBP-056B. C) KBP-089 and KBP-089B (KBP-066) . Experiment was conducted in CHO-K1 CALCR RAMP3 PathHunter cells from DiscoveRx. Data shown as mean ± SEM.

Examples

The presently disclosed embodiments is described in the following Examples, which are set forth to aid in the

understanding of the disclosure, and should not be construed to limit in any way the scope of the disclosure as defined in the claims which follow thereafter. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described embodiments, and are not intended to limit the scope of the present disclosure nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated

otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

In the following examples, the following materials and methods were employed.

Cells and Cell Lines

The following cell lines expressing the calcitonin, amylin and CGRP receptors were purchased and cultured according to the manufacturer's instructions.

1. Calcitonin Receptor (CTR) : U20S-CALCR from DiscoveRx

(Cat. No. : 93-0566C3) .

2. Amylin Receptor (AMY-R) : CHO-K1 CALCR + RAMP3 from

DiscoveRx (Cat. No.: 93-0268C2).

In independent bioassays, CTR and AMY-R cells were treated with for the indicated timepoints with increasing doses of KBPs (100, 20, 4, 0.8, 0.16, 0.032 nM and vehicle) b-Arrestin Assay

PathHunter b-Arrestin GPCR assays are whole cell, functional assays that directly measure the ability of a ligand to activate a GPCR by detecting the interaction of b- Arrestin with the activated GPCR. Because b-arrestin

recruitment is independent of G-protein signaling, these assays offer a powerful and universal screening and profiling platform that can be used for virtually any Gi-, Gs, or Gq- coupled receptor.

In this system, the GPCR is fused in frame with the small enzyme fragment ProLink™ and co-expressed in cells stably expressing a fusion protein of b-Arrestin and the larger, N-terminal deletion mutant of b-gal (called enzyme acceptor or EA) . Activation of the GPCR stimulates binding of b-Arrestin to the ProLink-tagged GPCR and forces

complementation of the two enzyme fragments, resulting in the formation of an active b-gal enzyme. This interaction leads to an increase in enzyme activity that can be measured using chemiluminescent PathHunter® Detection Reagents.

The assay was performed in white 384 well plates

(Greiner Bio-One, 784080) . Cells were seeded 2500 cells per well in 10 yL cell-type specific medium the day prior to the experiment. To quantify the GPCR-mediated b-arrestin

recruitment the Pathhunter™ Detection Kit (93-0001,

DiscoverX) was used and assay performed accordingly to the manufacturer's instructions.

Protracted response was conducted using the calcitonin receptor (CTR) : U20S-CALCR from DiscoveRx (Cat. No.: 93- 0566C3) cell line, and as opposed to the classical three hour output, b-arrestin accumulation was conducted over 24, 48 and 72 hour and then analyzed.

Example 1. Efficacy of AiB-KBP compounds

KBP-066 vs Benchmark (KBP089; non-AiB version of KBP-066) Tested peptides:

KBP-066 : CSNLSTC (AiB) LGRLSQDLHRLQTYPKTDVGANAP

KBP-089 : CSNLSTCMLGRLSQDLHRLQTYPKTDVGANAP

Rats were delivered at five weeks of age. After 12 weeks on a high fat diet, rats were randomized based on bodyweight at DAY -3. Animals were housed in pairs. The study was initiated at DAY 0.

Animals were dosed once daily with KBP-066, KBP-089 or saline (vehicle) . Saline: Dosage volume was 1 mL/kg. KBP-066: Dosage volume was 1 mL/kg, dosage concentration was 750 pmol/kg. KBP-089 : Dosage volume was 1 mL/kg, dosage concentration was 750 pmol/kg

Compounds were dissolved in saline and stored at -20 °C. Aliquots were thawed immediately prior to administration.

Treatment groups

Food intake was monitored daily and reported per two animals .

Body weight was monitored daily and reported for each individual animal.

When KBP-066 was directly compared to the benchmark KBP- 089 (non-AiB version of KBP-066) compound (dose - 750

pmol/kg), KBP-066 was found to have near identical efficacy to the benchmark compound, demonstrating that the inclusion of the AiB residue at the 8 position was not detrimental to the efficacy of the compound (Figure 4) . b-Arrestin Assays

To corroborate the above result, comparative b-Arrestin Assays were performed on the following sets of peptides (X = 2-aminoisobutyric acid, AiB) :

KBP-066 : AcCSNLSTCXLGRLSQDLHRLQTYPKTDVGANAP-NH₂

KBP-089 : AcCSNLSTCMLGRLSQDLHRLQTYPKTDVGANAP-NH₂

KBP-042B: AcCSNLSTCXLGKLSQELHKLQTYPRTDVGANAP-NH₂

KBP-042 : AcCSNLSTCVLGKLSQELHKLQTYPRTDVGANAP-NH₂

KBP-056B: AcCASLSTCXLGKLSQDLHKLQTFPKTDVGANAP-NH₂

KBP-056 : AcCASLSTCMLGKLSQDLHKLQTFPKTDVGANAP-NH₂ The results are shown in Figure 5 (CTR b-Arrestin Assay) and Figure 6 (AMYR b-Arrestin Assay) . Introduction of an AiB residue at the 8 position of the peptide did not

significantly alter the dose-response curve in either assay, thereby demonstrating substantially equivalent receptor potency .

Example 2. Fibrillation Studies

Chemicals

Thioflavin T (T3516, Sigma) . Assay stock ThT is prepared as a 10 mM solution in 5 mM sodium phosphate pH 7.2.

Aliquots are stored, protected from light, at -20 °C. Stock ThT is thawed and diluted just prior to use.

For DACRA peptides, final buffer conditions are 10 mM Tris- HC1 pH 7.5.

The final peptide concentration in the wells should be 100-200 mM, and the final ThT concentration should be 4 mM. ThT is added last (10pL) .

Thioflavin T Assay

Thioflavin T (ThT) is a dye widely used for the

detection of amyloid fibrils. In the presence of fibrils,

ThT has an excitation maximum at 450 nm and enhanced emission at 480 nm, whereas ThT is essentially non-fluorescent at these wavelengths when not bound to amyloid fibrils.

Thus, ThT in combination with a fluorescent plate reader is an ideal tool for screening large numbers of in vitro samples for the presence of amyloid fibrils.

The ThT assay used for DACRA peptides is a modification of the procedure described by Nielsen et. al . (Nielsen L, Khurana R, Coats A, Fr0kjaer S, Brange J, Vyas S, et al.

Effect of environmental factors on the kinetics of insulin fibril formation: elucidation of the molecular mechanism. Biochemistry. 2001; 40 (20): 6036-46) for measuring insulin fibrillation .

Fibrillation screening assays were conducted in 384-well plates (Greiner Bio-One, 784080) in sample triplicates with a final volume of 20 yL . The plate is sealed using an optical adhesive film in order to prevent sample evaporation over the course of the assay.

The plate was loaded into a fluorescent plate reader, such as a SpectraMax with SoftMax Pro 7.0.2 software, and the template set to 37 °C with excitation wavelength at 450 nm and emission wavelength at 480 nm.

The plate reader measured fluorescence every 10 minutes for 24 hours with a five-second plate shake before the first read and a three-second plate shake before all other reads. Alternatively, the plate is read after the following

incubation times; 0, 1, 2, 4 and 24 hours.

Relative fluorescence units (RFU) plotted as a function of time provides information regarding fibrillation;

fibrillation is determined as an increase in RFU over

baseline ( 1 ) .

KBPs with and without an AiB at position 8

The following peptides were tested in this study (X = 2- aminoisobutyric acid, AiB) :

A single dose of either 500 mM or 250 mM KBP-XXX was incubated with 4 mM Thioflavin T for 0-24 hours. Each line in the respective compound plot (Figures 1-3) represent one hour of incubation. ThT assay conducted in triplicates, and vehicle is representative of the assay background signal. As seen in Figure 1-3, three KBPs and their respective AiB versions were tested for fibril formation in the Thioflavin T assay.

KBP-042, KBP-056 and KBP-089 core peptides all formed fibrils, whereas when an AiB was inserted into position 8, the observed fibril formation was attenuated.

Hence, an AiB inserted into the sequence at position 8 substantially reduces fibril formation in vitro, which is advantageous for successful peptide drug formulation moving forward .

Experimental Conclusions

KBP-042 and KBP-042B

In a head-to-head comparison of KBP-042 and KBP-042B there was no significant difference in the calculated EC50 values from the concentration range curve from cells

heterologously expressing the CTR (Figure 5A) or the AMYR (Figure 6A) .

In the ThT fibrillation assay there was a clear

difference between KBP-042 and KBP-042B. KBP-042 induced a high signal in the assay over time (Figure 1) suggesting the formation of fibrils. In contrast, KBP-042B did not increase the signal beyond that assay background (Figure 1) (Vehicle control containing no protein) .

KBP-056 and KBP-056B

In a head-to-head comparison of KBP-056 and KBP-056B there was no significant difference in the calculated EC50 values from the concentration range curve from cells

heterologously expressing the CTR (Figure 5B) or the AMYR (Figure 6B) . In the ThT fibrillation assay there was a difference between KBP-056 and KBP-056B. KBP-056 induced a high signal in the assay over time (Figure 2) suggesting the formation of fibrils. In contrast, KBP-056B did not increase the signal beyond that assay background (Figure 2) (Vehicle control containing no protein) .

KBP-089 and KBP-089B (KBP-066)

In a head-to-head comparison of KBP-089 and KBP-066 there was no significant difference in the calculated EC50 values from the concentration range curve from cells

heterologously expressing the CTR (Figure 5C) or the AMYR (Figure 6C) .

In the ThT fibrillation assay there was a clear

difference between KBP-089 and KBP-066. KBP-089 induced a high signal in the assay over time (Figure 3) suggesting the formation of fibrils. KBP-066 did increase the signal beyond that assay background (Figure 3) (Vehicle control containing no protein) , however, the fibril formation was at a much lower rate over time compared to KBP-089.

Conclusion

An AiB inserted into the sequence at position 8 has been shown to significantly reduce or completely prevent fibril formation in vitro whilst maintaining both in vitro and in vivo potency. This is advantageous for developing efficacious peptide drugs that do not (or at least substantially do not) fibrillate during the pharmaceutical formulation process.

In this specification, unless expressly otherwise indicated, the word 'or' is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator 'exclusive or' which requires that only one of the conditions is met. The word 'comprising' is used in the sense of 'including' rather than in to mean 'consisting of' . All prior teachings acknowledged above are hereby incorporated by reference.

Claims

1.

2. The peptide of claim 1, wherein the peptide is a peptide of formula (II) :

3. The peptide of claims 1 or 2, wherein X₂ is S and X₃ is

N; or X₂ is G and X₃ is N; or X₂ is A and X₃ is S.

4. The peptide of any one of claims 1 to 3, wherein:

5. The peptide of claims 1 or 2, wherein X₂ is S X₃ is N,

6. The peptide of claims 1 or 2, wherein X₂ is A, X₃ is S,

7. The peptide of claims 1 or 2, wherein X2 is G, X3 is N, X13 is T, X₁₇ is N, Xi₈ is K or R, X_i9 is F, X₂o is H, X₂₂ is F, and X₂₄ is K.

8. The peptide of claim 1, wherein the peptide is selected from:

9. The peptide of any one of claims 1 to 8, wherein the peptide is acylated at its N-terminus and/or wherein the peptide is amidated at its C-terminus.

10. A peptide as claimed in any one of claims 1 to 9,

formulated for enteral administration.

11. A peptide as claimed in any one of claims 1 to 9,

formulated for parenteral administration.

12. A peptide as claimed in claim 11, formulated for

inj ection .

13. A peptide as claimed in any one of claims 1 to 9,

formulated with a carrier for oral administration.

14. A peptide as claimed in claim 13, wherein the carrier comprises 5-CNAC, SNAD, or SNAC .

15. A peptide as claimed in claim 10, wherein the peptide is formulated in a pharmaceutical composition for oral administration comprising coated citric acid particles, and wherein the coated citric acid particles increase the oral bioavailability of the peptide.

16. A peptide as claimed in any one of claims 1 to 9, for use as a medicament.

17. A peptide as claimed in claim 16, for use in treating diabetes (Type I and/or Type II), excess bodyweight, excessive food consumption, metabolic syndrome,

rheumatoid arthritis, non-alcoholic steatohepatitis (NASH) , non-alcoholic fatty liver disease, for producing a decrease in liver triglycerides, for reducing fat accumulation in the liver of a subject, alcoholic fatty liver disease, osteoporosis, or osteoarthritis, poorly regulated blood glucose levels, poorly regulated

response to glucose tolerance tests, or poor regulation of food intake.

18. A peptide for use as a medicament as claimed in claim 16 or claim 17, wherein the peptide is for administration in conjunction with treatment with metformin or another insulin sensitizer.

19. A peptide as claimed in any one of claims 1 to 9 in

combination with a weight loss drug for use in treating an overweight condition.

20. The use of claim 19, wherein the overweight condition is obesity .

21. A co-formulation comprising a peptide as claimed in any one of claims 1 to 9 and an insulin sensitizer.

22. A co-formulation comprising a peptide as claimed in any one of claims 1 to 9 and a weight loss drug.