NZ625215B2 - Peptide analogs for treating diseases and disorders - Google Patents

Abstract

Disclosed is a peptide having a sequence selected from SEQ ID NO:12 (AcCSNLSTCVLGRLSQELHRLQTFPRTDVGANTAcY), SEQ ID NO:15 (AcCSNLSTCVLGKLSQELHKLQTYPRTDVGANAP-NH2) and SEQ ID NO:17 (SuccCSNLSTCVLGKLSQELHKLQTYPRTDVGANAY-NH2) and use in therapy for glycemic control.

Description

PEPTIDE ANALOGS FOR TREATING DISEASES AND DISORDERS RELATED APPLICATIONS This ation claims the t of and priority to U.S. Provisional Application Serial No. 61/554,771, filed November 2, 2011, and U.S. Provisional Application Serial No. 61/578,620, filed December 21, 2011, and U.S. Application No. 13/667,578 filed November 2, 2012, the entirety of all these applications are hereby incorporated herein by reference.

FIELD The embodiments disclosed herein relate to mimetics of calcitonin, and more particularly to their use in the treatment of various diseases and disorders, including, but not limited to es (Type I and Type II), excess bodyweight, excessive food consumption and metabolic syndrome, the regulation of blood glucose levels, the regulation of response to glucose tolerance tests, the 2O regulation of food intake, the treatment of osteoporosis and the treatment of osteoarthritis.

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

TZDM is a geneous disease characterized by abnormalities in carbohydrate and fat lism. The causes of T2DM are multi—factorial and include both genetic and environmental elements that affect B-cell function and PCT/U82012/063332 insulin sensitivity in tissues such as muscle, liver, as and adipose tissue. As a consequence impaired insulin ion is observed and paralleled by a ssive e in B—cell function and chronic insulin resistance.

The inability' of the ine pancreas to sate 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 TZDM.

A success criterion for an optimal TZDM 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, bed 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 t1) produce insulin if] response 113 food intake and hence an inability to regulate blood glucose to £1 normal physiological level.

The physical structure of bone may be compromised by a variety of factors, including e and injury. One of the most common bone diseases is osteoporosis, which is terized 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 nce 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 mune response, e.g. lupus, ankylosing spondylitis (AS) or le sis (MS), can lead to substantial loss of mobility due to pain and joint destruction. Cartilage that covers and ns 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 tion of bone.

SUMMARY Calcitonin mimetics are disclosed herein.

In one aspect, there is provided a peptide selected from the group consisting of: ACCSNLSTCVLGKLSQELHKLQTYPRTDVGANAP-NH2 SEQ ID NO: 15, ACCSNLSTCVLGRLSQELHRLQTFPRTDVGANTACY SEQ ID NO: 12, and SuCCCSNLSTCVLGKLSQELHKLQTYPRTDVGANAY—NH2 SEQ ID NO: 17.

According to aspects illustrated herein, there is disclosed a peptide having" a sequence selected) from SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17 and SEQ ID NO:18.

According to aspects illustrated herein, there is disclosed a method that includes administering to a patient an effective amount of a e selected from the group consisting of: SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:17 to affect a weight reduction in the patient. [followed by page 3a] According to aspects illustrated herein, there is disclosed a method that includes administering to a patient an effective amount of a peptide selected from the group consisting of: SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:17 to affect postprandial glycemic l in the patient.

According to aspects illustrated herein, there is disclosed a method that es administering to a patient an effective amount of a peptide selected from the group consisting of: SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:l3, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:17 to affect an improvement in ic control in the patient. [followed by page 4] PCT/U52012/063332 According to aspects illustrated , there is disclosed a method that includes administering to a patient an effective amount of a peptide selected from the group consisting Of: SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:17 to affect an improvement in glycemic control in the patient.

According to aspects illustrated herein, there is disclosed a method that es stering to a patient an effective amount of a peptide of SEQ ID NO:l8 having the IO sequence CmSNLSTCVLGKLSQELHKLQTYPRTDVGANXaaXaaa so as to reduce at least one of bone resorption and cartilage degradation in the patient.

Brief Description of the Drawings The presently disclosed embodiments will be further explained with reference to the ed drawings, which illustrate the principles of the presently disclosed embodiments.

Figure 1A, Figure 18, Figure 1C, and Figure 1D show the effect of chronic oral salmon calcitonin (“sCT”) versus oral UGP 302 stration on body weight and food intake in DIO rats as measured in Example I; Figure 2A. and Figure 2B ShOW’ the effect of oral sCT versus oral UGP 302 on glucose tolerance during OGTT in DIO rats as ed in Example 1; Figure 3 shows the effect of oral sCT versus oral UGP 302 on fasting glycemia in DIO rats as measured in Example I; Figure 4A and Figure 4B show the effect of oral sCT versus oral UGP 302 on body weight and food intake in DIO rats observed in Example 2 at a first dosage; Figure 5A. and Figure 5B show the effect of oral sCT versus oral UGP 302 on body weight and food intake in DIO rats observed in Example 2 at a second dosage; Figure 6A. and, Figure 6B show the effect of oral sCT versus oral UGP 302 on body weight and food intake in DIO rats observed in Example 2 at a third dosage; Figure 7A. and Figure 7B shOW' the effect of oral sCT versus oral UGP 302 at a first dosage on glucose tolerance during OGTT in DIO rats as measured in Example 2; Figure 8A and Figure 8B show the effect of oral sCT versus oral UGP 302 at a second dosage on glucose tolerance during OGTT in DIO rats as measured in Example 2; Figure 9A, Figure 9B, Figure 9C, Figure 9D, Figure 9E, and Figure 9F show the effect of oral sCT versus three oral UGPs on body weight and food intake in DIO rats as measured in Example 3; Figure 10A, Figure lOB, Figure 10C, Figure 10D, Figure 10E, and Figure 10F show the effect of oral sCT versus three oral UGPs on glucose levels in a glucose nce test in DIO rats as measured in Example 3; Figure ll shows binding results for six UGP compounds to T47D cell calcitonin receptors as measured in Example 4; and 2O Figure 12A and Figure 12B show food consumption (12A) and weight change measurements (12B) for UGP 282 as measured in Example 5; Figure 13A and Figure 13B show food ption (13A) and weight change ements (138) for UGP 283 as measured in Example 5; Figure 14A and Figure 14B show food consumption (14A) and weight change measurements (14B) for UGP 284 as measured in Example 5; Figure 15A and Figure 15B show food ption (15A) and weight change measurements (15B) for UGP 298 as measured in Example 5; Figure 16A and Figure 16B show food consumption (16A) and weight change measurements (16B) for UGP 302 as measured in Example 5; Figure 17A and Figure 173 show food consumption (17A) and weight change measurements (17B) for UGP 303 as ed in Example 5; Figure 18 and Figure 19 show respectively the reduction of bone tion. and. cartilage resorption. produced. by ent with UGP302 in rats.

While the above—identified drawings set forth presently disclosed embodiments, other embodiments are also contemplated, as noted in the discussion. This disclosure presents illustrative embodiments by way of representation and not limitation. Numerous other modifications and embodiments can be d by those skilled in the art which fall within the scope and range of the principles of the tly disclosed embodiments.

Detailed Description onins are highly conserved over a wide range of species. Full—length native calcitonin is 32 amino acids in length. The sequences of examples of calcitonins are set out below: Salmon CSNLSTCVLGKLSQELHKLQTYPRTNTGSGTP SEQ ID NO:1 Mouse CGNLSTCMLGTYTQDLNKFHTFPQTSIGVEAP SEQ ID N012 Chicken CA8LSTCVLGKLSQELHKLQTYPRTDVGAGTP SEQ ID NO:3 Eel CSNLSTCVLGKLSQELHKLQTYPRTDVGAGTP SEQ ID NO:4 Rat CGNLSTCMLGTYTQDLNKFHTFPQTSIGVGAP SEQ ID NO:5 Horse CSNLSTCVLGTYTQDLNKFHTFPQTAIGVGAP SEQ ID N026 Canine—l CSNLSTCVLGTYSKDLNNFHTFSGIGFGAETP SEQ ID NO:7 Canine-2 CSNLSTCVLGTYTQDLNKFHTFPQTAIGVGAP SEQ ID NO:8 Porcine CSNLSTCVLSAYWRNLNNFHRFSGMGFGPETP SEQ ID NO:9 Human CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP SEQ ID NO:lO Embodiments of the present disclosure relate to calcitonin cs. The amino acid sequence of the calcitonin mimetics of the present sure are found in Table 1 below.

Table 1 Calcitonin Amino Acid Sequence SEQ ID NO: Mimetic (\\ cM/I) UGP281 ACCSNLSTCVLGKLSQELHKLQTYPRTDVGANTY—NHZ 11 UGP283 ACCSNLSTCVLGRLSQELHRLQTFPRTDVGANTACY 12 UGP284 PrCSNLSTCVLGKLSQELHKLQTYPRTNTGSGTP—NHZ 13 UGP298 SuCCCSNLSTCVLGKLSQELHKLQTYPRTNTGSGTP-NHZ 14 UGP302 ACCSNLSTCVLGKLSQELHKLQTYPRTDVGANAP—NHZ 15 UGP303 KCSNLSTCVLGKLSQELHKLQTYPRTDVGANTY-NHg 16 UGP306 SuCCCSNLSTCVLGKLSQELHKLQTYPRTDVGANAY—Nflg 17 UGPlOOO CmSNLSTCVLGKLSQELHKLQTYPRTDVGANXaaXaaa 18 In some embodiments, the cysteine at position 1 of the calcitonin mimetics discussed supra is ed (“Cm”) to reduce the positive charge of the first amino acid. For e, an acetyl group (SEQ ID N03: 11, 12 and 15), propionyl group (SEQ ID NO: 13), or succinyl group (SEQ ID NOS: l4 and 17) may be substituted on cysteine-l. In some embodiments, the amino acid at the last position (“Xaaa”) (position 32 in SEQ ID Nos: 11, 13—15 and 17-18 or position 33 in SEQ ID NO: 16) may include an amidated group “NHg”.

Alternative ways of reducing ve charge include, but are not limited to, polyethylene glycol—based tion, or the addition of r 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. An example of an amino acid added to the N—terminus PCT/U82012/063332 of peptides includes SEQ ID NO:16, where a lysine has been added.

“Xaa” in SEQ ID NO: 18 in Table 1 can be any naturally occurring amino acid. In an embodiment Xaa at position 31 is selected from one of ine or alanine. In an embodiment Xaa at position 32 is ed from one of tyrosine or proline. Thus, SEQ ID NOS: ll, 15, 16 and 17, are encompassed by SEQ ID NO: 18.

As those of skill in the art will appreciate, peptides having a gflurality 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 disulfide s. While calcitonin mimetics 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 e. A preferred technique for manufacturing amidated versions of the calcitonin mimetics of the t 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 ygenase in accordance with known techniques wherein the precursors are converted to amidated jproducts in reactions described, for example, in U.S. Pat. No. 4,708,934 and European Patent Publication Nos. 0 308 067 and 0 382 403. Recombinant production is preferred for both the precursor‘ and. the enzyme that catalyzes the conversion of the precursor to salmon calcitonin. Such inant production is discussed in hnology, Vol. 11 (1993) pp. 64—70, which further describes a conversion of a precursor to an amidated product. The inant product ed there is identical to natural salmon calcitonin, and to salmon calcitonin produced using solution and solid phase 2012/063332 chemical peptide synthesis. Production of amidated products may also be accomplished using the process and amidating enzyme set forth by Consalvo, et al in U.S. Patent No. 7,445,911; Miller et al, U.S. Patent Publication No. 292672; 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 es may proceed, for example, by producing e~extended precursor l0 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 U.S. Pat. No. 495. 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—~ NHZ, while the sor terminates -—X-g1y, X being the C- terminal amino acid residue of the product). An alpha— amidating enzyme described in the publications above zes conversion of precursors to product. That enzyme is preferably recombinantly produced, for example, in Chinese r 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 ed in like Hanner, 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 calcitonin mimetics 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.

PCT/U82012/063332 Except where otherwise noted or where apparent from context, dosages herein refer to weight of active compounds unaffected by pharmaceutical excipients, ts, carriers or other ingredients, although such additional ingredients are desirably included, as shown in the examples herein. Any dosage form (capsule, tablet, injection or the like) commonly used in the pharmaceutical industry for delivery of e 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 ients in such dosage form in the industry. A red oral dosage form is sed in more detail, infra, but is not to be considered the ive mode of administering the active agents of the present disclosure.

The calcitonin mimetics of the present disclosure can be stered 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 ably, mammals including humans.

Accordingly, the present disclosure provides 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 rably high peak serum glucose level, or for reducing an undesirably high peak serum insulin level, or for reducing an undesirably large response to a e tolerance test, or for treating osteoporosis, or for treating osteoarthritis.

As used herein, the term “glycemic control” refers to the typical levels of blood sugar (glucose)in a person with diabetes mellitus. The percentage of hemoglobin which is glycosolated (measured as hemoglobin Alc) is used as a proxy PCT/U52012/063332 measure of long—term glycemic control. As used herein, the term “improved glycemic control” refers to the ability of a calcitonin c of the present disclosure to reduce the percentage of hemoglobin which is glycosolated.

There are a number of art—recognized measures of normal range for body weight in view of a number of s such as gender, age and height. A patient in need of treatment or prevention regimens set forth herein e 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 calcitonin mimetics 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 f for treatment of a said condition of a pharmaceutically effective amount of any one of the peptides described herein. 2O 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 eritoneal, subcutaneous, intravenous, intradermal or intramuscular injection), solutions of a peptide of the present disclosure in either sesame or peanut oil or in s propylene glycol may be employed, for example. The aqueous solutions should be ly ed (preferably pH greater than 8) if necessary and the liquid diluent first rendered ic. These aqueous ons are suitable for intravenous injection purposes.

The oily solutions are suitable for intraarticular, intramuscular and subcutaneous ion 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 ts, including suppositories.

Peptides may be formulated in sterile form in Hmltiple 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 injectables.

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 ent 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 e a loss of weight or an avoidance of weight gain, the active compound is preferably stered at least twice per day, e.g. from 2—4 times per day. Formulations of the active nd 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 ion 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 trean1 via the sublingual or buccal routes. le dosage forms for use in the present disclosure include tablets, Hdni—tablets, capsules, granules, pellets, PCT/U52012/063332 powders, escent 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 calcitonin or a nt or conjugate thereof and. hydrolysed. gelatin or lOW’ molecular weight gelatin and. further‘ sing the resulting solid material into said oral pharmaceutical formulation, and wherein the gelatin may have a Inean 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, ical, chemical and physical barriers such as varying pH in the gastrointestinal tract, powerful digestive s, and active agent impermeable intestinal membranes, makes oral delivery of calcitonin like peptides to Hammals 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 icient stability of calcitonin in the gastrointestinal tract as well as the ity of calcitonin to be readily 'transported through the intestinal walls into the blood stream. le oral ations are however described below.

Treatment of Patients PCT/U82012/063332 In an embodiment, a calcitonin mimetic of the present disclosure is administered at adequate dosage to maintain serum levels of the mimetic in patients between 5 and 500 picograms per milliliter, preferably between 10 and 250 picograms per milliliter. The serum levels may be measured by radioimmunoassay techniques known in the art. The attending physician may monitor patient response, and may then alter the dosage somewhat to account for individual patient metabolism and se. Near simultaneous release is best lO 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 calcitonin mimetic among two or more tablets or capsules which may be administered together such that they l5 together. provide the necessary amount of all ingredients. aceutical composition," as used herein includes but is not limited to a complete dosage appropriate to a ular stration to a patient regardless of whether one or more tablets or capsules (or other dosage forms) are recommended at a given stration.

A calcitonin mimetic of the present sure may be formulated for oral administration using the s employed in the Unigene EnteripepG 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. 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 n, co—formulation with one or more se inhibitors, and/or a pH lowering agent which may be coated and/or an acid resistant protective vehicle and/or an absorption er which may be a surfactant.

PCT/U52012/063332 In an embodiment, a calcitonin mimetic of the present disclosure is preferably formulated for oral delivery in a manner known in U.s. Patent Publication No. 2009/0317462. One preferred oral dosage form in accordance with the present disclosure is set forth in Table 2 below: TABLE 2 ACTIVE AGENT OR FUNCTION EXCIPIENT A onin c Active agent selected from one of SEQ ID NO:ll - SEQ ID NO:l8 Coated Citric Acid Protease Inhibitor Particles Lauroylcarnitine Absorption Enhancer Nonionic Polymer Eudragit L3OD—55 Enteric Coat In an embodiment, a calcitonin mimetic of the present disclosure may be formulated for l, especially oral, administration by admixture with a suitable carrier nd.

Suitable carrier compounds include those described in US Patent No. 5,773,647 and US Patent No. 5866536 and t these, 5-CNAC (N—(5-chlorosalicyloyl)—8—aminocaprylic acid, commonly as its disodiunl salt) is particularly effective.

Other preferred rs or delivery agents are SNAD (sodium salt of lO—(2—Hydroxybenzamido)decanoic acid) and SNAC (sodium salt of N—(8—[2—hydroxybenzoy11amino)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. lly, the carrier such as 5-CNAC is t in an amount of 2.5% to 99.4% by weight, PCT/U82012/063332 more preferably 25% to 50% by weight of the total composition.

In addition, WO 00/059863 ses the disodium salts of formula I R4 o R9, R2, R3, and R? are independently hydrogen, ~OH, —NR%U, halogen, lkyl, or C1—C4alkoxy; R5 is a substituted or unsubstituted C2—Cm alkylene, substituted or unsubstituted C2—Cm alkenylene, substituted or tituted C1—C12 arylene), or substituted or unsubstituted aryl(C1—Cn alkylene); and R6 and R7 are independently en, , 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 W02005/01403l.

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 ularly, the active ingredient may be encapsulated in an enteric capsule capable of withstanding transit through the stomach. This may contain the active compound er with a hydrophilic aromatic alcohol absorption enhancer, for instance as described in PCT/U52012/063332 28436. 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. WOZOO4/O91584 also describes suitable formulation methods using aromatic alcohol absorption enhancers.

The nd may be ated 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 USS,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 sure 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 on emulsions, suspensions, syrups and elixirs. In addition to the active compound and carrier such as 5—CNAC, the liquid formulations may also include inert ents ly used in the art such as, solubilizing agents e.g. ethanol; oils such as cottonseed, castor and sesame oils; wetting agents; fying agents; ding 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 onally comprise additives in amounts customarily employed including, but not limited to, a pH adjuster, a preservative, a ant, a taste-masking agent, a fragrance, a humectant, a tonicifier, a colorant, a surfactant, a plasticizer, a ant such as ium 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 ation, or any combination thereof. Other additives may include phosphate buffer salts, citric acid, s, 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. r, a transport inhibitor, i.e. a [rhOJ—glycoprotein such as Ketoprofin, may be present in the compositions of the present disclosure. The solid pharmaceutical compositions of the instant disclosure can be ed 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 g into capsules, g 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 fornl 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 er as described in USZOO3/OO69170, e.g. compound——[-fl—(CH2)7(OC2H4)7OCH3]2 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 bed 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 an 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 rticles as sed in Garcia—Fuentes et al. Chitosan nanoparticles for this e 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 hilic particles in a hydrophobic medium to protect the drug molecule from vation by the hostile gastrointestinal (GI) environment and at the same time acts on the GI wall to induce permeation of its cargo drug les.

Further included is the use of glutathione or compounds containing numerous thiol groups as described in U82008/0200563 to inhibit the action of efflux pumps on the mucous membrane. Practical examples of such techniques are described also in Caliceti, P. o, 8., Walker, G. and PCT/U52012/063332 Bernkop-Schnurch, A. (2004) ‘Development and in vivo evaluation of an oral insulin—PEG delivery system.’ Eur. J.

Pharm. Sci., 22, 315—323, in Guggi, D., Krauland, A.H., and Bernkop—Schnfirch, A. (2003) ‘Systemic peptide delivery via the h: in vivo tion of an oral dosage form for salmon onin’. J. Control. Rel. 92,125-135, and in p-Schnurch, A., Pinter, Y., Guggi, D., Kahlbacher, H., Schoffmann, G., Schuh, M., Schmerold, 1., 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— The active compound may be formulated in seamless micro— spheres as described in W02004/084870 where the active ceutical 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 ermined instant or sustained release of the active drug to specific locations and at specific rates along the intestinal tract. In essence, pre—solubilization of the drug es the predictability of its kinetic profile while simultaneously enhancing permeability and drug stability.

One may employ chitosan coated nanocapsules as described in U82009/0074824. 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 es the time of adhesion to the intestine walls (it has been verified that there is a delay in the gastrointestinal WO 67357 PCT/U52012/063332 t of these systems) facilitating a more effective absorption of the active molecule.

Methods developed by TSRl Inc. may be used. These include hilic 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 ng. This results in an improved wettability of hobic 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 ated 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 tive as described in US2007/0238707 or a membrane translocating peptide as described in US7268214.

One may employ M technology which consists of a controlled—release dosage form, inside an inflatable pouch, which is placed in a drug capsule for oral administration.

Upon ution 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 llized and lyophilized into the dry WO 67357 active pharmaceutical ingredient after cation. 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 (3150 nm diameter) encapsulating the active, which also n a hepatocyte—targeting molecule in their lipid bilayer. The targeting le 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 /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 USZOO2/0115592 in relation to insulin.

Alternatively, use may be made of intestinal patches as described in Shen Z, Mitragotri S, Pharm Res. 2002 Apr;l9(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. 4. le 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 fron1 1 to six times daily, for instance front two to four times daily. Treatment will desirably be maintained over a prolonged period of at least 6 weeks, preferably at least 6 months, ably at least a year, and optionally for life.

Combination treatments for relevant conditions may be carried. out using* a composition according to the present PCT/U52012/063332 disclosure and separate administration of one or more other therapeutics. Alternatively, the ition according to the present disclosure may incorporate one or more other therapeutics for combined administration.

Combination therapies according to the present disclsoure include combinations of an active compound as described. with insulin, GLP—Z, GLP—l, GIP, or amylin, or generally with other iabetics. Thus combination therapies including co-formulations may be made with insulin lO 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 amide, Chloropropamide, Gliclazide, Tolbutamide, Tolazamide, Glipizide, Glibenclamide, ide, Gliquidone, Glyclopyramide and (Slimepriride, Meglitinides/glinides (K+) such as Nateglinide, Repaglinide and Mitiglinide, GLP—l analogs such as Exenatide, Liraglutide and utide, DPP—4 inhibitors such as Alogliptin, Linagliptin, Saxagliptin, iptin and Vildagliptin, insulin analogs or special formulations such as (fast ) Insulin lispro, Insulin , Insulin glulisine, (long' acting) Insulin glargine, Insulin detemir), inhalable insulin — Exubra and NPH insulin, and others ing alpha—glucosidase inhibitors such as Acarbose, Miglitol and VOglibose, amylin analogues such as Pramlintide, SGLTZ tors such as Dapagliflozin, Remogliflozin and Sergliflozin as well as miscellaneous ones ing Benfluorex and Tolrestat.

Further combinations include co—administration or co— formulation with leptins. Leptin resistance is a well— established component of type 2 diabetes; r, injections of leptin have so far failed to improve upon this condition.

PCT/U52012/063332 In contrast, there is evidence ting that amylin, and thereby molecules with amylin—like abilities, as the salmon calcitonin mimetics, are able to e leptin sensitivity. /leptin combination has shown a synergistic effect on body . and food intake, and also insulin resistance [Kusakabe T et al]. Accordingly, the present disclosure provides a nd of the formula Ac—CSNLSTCVLG KLSQELHKLQ VGAN AP-NHZ (SEQ ID NO:15), which will be referred to herein as ‘calcitonin mimetic l’ or 2’.

Accordingly, the present disclosure includes a pharmaceutical formulation of such a peptide for enteral administration, e.g. for treating type I diabetes, type II diabetes, or netabolic syndrome, or for Hfitigating insulin resistance, or for reducing an undesirably high fasting serum glucose level, or for ng an undesirably high peak serum e level, or for reducing an undesirably high peak serum n level, or for reducing an undesirably high response to a glucose tolerance test, or for treating osteoporosis, or for treating osteoarthritis. The formulation may comprise also a carrier serving to enable effective enteral administration of said active compound.

Preferably, said formulation is formulated for oral administration to the digestive tract.

Preferably, said carrier comprises 5—CNAC, SNAD, or SNAC.

Additionally, the present disclosure includes said peptides as new compounds.

The presently disclosed ments 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 2012/063332 art with a complete disclosure and ption of how to make and use the described ments, 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 ise, parts are parts by weight, nwlecular weight is weight average molecular weight, temperature is le degrees Centigrade, and pressure is at or near atmospheric.

Examples Exgmgle 1 Chronic effect of calcitonin mimetic 1 (CMl) compared to sCT Animals The study was performed in male Levin—DIO rats (diet- sensitive) and DR resistant) (TacLevin: CD (SD) DIO) (Taconic, Hudson, NY, U.S.A.) obtained at age 6—7 weeks.

On arrival, DIO rats were given high fat diet (60 kcal %) 2O (#Dl2495, Research Diets Inc., New Brunswick, NJ., USA) and kept on the same diet for 16 weeks prior to and during the experiment. DR rats were given low-fat diet and served as control group. Animals were pair—wise housed throughout the study. Rats were handled and sed once daily with MilliQ H20 for 2—3 weeks prior to experimental start to reduce stress—induced lycaemia. Baseline parameters were recorded in an fasting (6 h) condition. Rats were randomized into treatment groups based on fasting body weight (BW) and fasting plasma glucose (FPG). Body weight, food and water intake were recorded once weekly during the study period.

PCT/U52012/063332 Compound Oral sCT or calcitonin mimetic 1 on was prepared on the day of dosing by mixing a carrier with the given compound based on the following calculations: -CNAC (vehicle): Animals treated with oral 5—CNAC received a dose of 150 mg/kg dissolved in milliQ H20.

Dosage—level for 5—CNAC: 150 mg/kg Dosing volume: 5 ml/kg Compound concentration: 30 mg/ml sCT/ calcitonin c 1: Animals d with oral sCT or oral calcitonin mimetic 1 received doses of 1.0 mg/kg combined with 150 mg/kg 5—CNAC — all dissolved in milliQ H20.

Dosage-level for sCT/calcitonin mimetic l: 1.0 mg/kg Dosing volume: 5 ml/kg nd concentration: 0.2 mg/ml Drug administration by per oral (p.o.) gavage b.i.d. (7— 8 am and 3—4 pm) during the study period and as single dose in the morning prior to start of OGTT.

Oral gavage of glucose during OGTT was prepared by the following calculation: D—Glucose: Animals were given 2 g/kg single dose dissolved in milliQ H20.

Dosage—level for D—Glucose: 2 g/kg Dosing volume: 4 ml/kg Compound concentration: 500 mg/ml WO 67357 PCT/U52012/063332 Experimental setup FPG Fasting Plasma Glucose BW Body Weight B Blood OGTT Oral Glucose Tolerance Test OGTT following overnight g (16 h): D = Drug; BG Blood glucose; B = Blood; G = Glucose Blood sampling and glycemia were measured by heated tail venous puncture.

Whole blood glucose levels were determined with an ACCU- CHEK® Avia blood glucose meter (Roche Diagnostics, Rotkreuz, Switzerland). Blood (approx 300 ul) is collected in 1 ml MiniCollect K3EDTA plasma—tube (Greiner—Bio—One GmbH, Frickenhausen, Germany), inverted, and stored on ice. Tubes are centrifuged 3000 x g (5000 rpm in table centrifuge) for min at 4 °C and plasma ed. Plasma samples are stored at —20°C until analysis. A total of ~ 2.5 ml blood is 2012/063332 ed during OGTT (~ 0.3% of body weight). mental groups Intervention Oral vehicle 5—CNAC 150 mg/kg -CNAC + 150 mg/kg + Oral sCT sCT 1 mg/kg —CNAC + Oral calcitonin 150 mg/kg calcitonin mimetic l 1 mg/kg mimetic l Statistics Statistical analysis was performed by one—way ANOVA followed by the Dunnett‘s post hoc test for multiple comparison. Student’s t—test was performed to compare two paired group. All analysis was performed using GRAPHPAD PRISM software (GraphPad Prism, San Diego, CA. U.S.A). Incremental area under curve (iAUC) during OGTT was calculated by the trapezoidal method. A value of P < 0.05 was ered to be significant. All data are presented as mean i standard error of the mean (SEM). 3. Results Baseline characteristics Results are summarized in Figure 1 (Food intake and body weight), Figure 2 (OGTT) and Figure 3 (FPG). Figure 1A, 2O Figure 1B, Figure 1C, and Figure 1D show the effect of chronic oral salmon calcitonin (“sCT”) versus oral UGP 302 administration on body weight and food intake in DIO rats as measured in e 1. Figure 2A and Figure 2B show the effect of oral sCT versus oral UGP 302 on glucose tolerance during OGTT in DIO rats as measured in Example 1. Figure 3 shows the effect of oral sCT versus oral UGP 302 on g glycemia in DIO rats as measured in Example 1; One dose of oral lcitonin mimetic 1 containing 1 mg/kg compound. was applied. by gavage twice daily to four groups of rats for 4 weeks. An oral vehicle group served as dosing regimen control, respectively. * P < 0.05, ** P < 0.01, *** P < 0.001 vs Vehicle. Results are presented as means iSEM.

The 16—weeks ad libitum high-fat diet induced a pronounced obese phenotype in the diet—sensitive (DIO) rats when comparing body weight to their diet—resistant (DR) littermates (P < 0.001) (Table l). 6—hrs Fasting glycemia was not ent n D10 and DR. In contrast, area under curve (AUC) calculations during OGTT was significantly higher in DIO rats compared to DR rats, demonstrating the high—fat diet-induced glucose intolerance (Table 1).

Table 1. Metabolic parameters in D10 and DR rats Diet-resistant (DR) Diet—sensitive (DIO) Body Weight (g) 609.5 i 2 4.5 841.8 i 22.9*** Fasting plasma 6.5 i 0.1 6.8 i 0.2 glucose (mM) AUC in OGTT 914.3 i 44.6*** Blood glucose AUC, area under curve; OGTT, oral e tolerance test. Data are means 2O '1' SEM (n=12/DR, n=24/DIO) .

Body Weight and Food Intake During the first week of treatment administration of oral sCT significantly reduced food intake compared to oral vehicle treated rats. Furthermore, oral sCT protected against further gain in body weight as ed for oral vehicle group (Figure 1). Thus, these observations confirm the acute strong anorectic action induced by ation of oral sCT in DIO rats. Interestingly, from week 2 of treatment and throughout the study period, food intake normalized in oral sCT treated rats and resembled. ingestion by oral vehicle ing in a lack of difference in regards to cumulative food intake at study end. This confirms previously reports suggesting a transient effect of oral sCT upon energy intake.

However, throughout the study period, oral sCT sustained the ting effect on body weight gain and significantly reduced body weight from baseline when compared. to oral vehicle at study end (Figure 1). This is in line with a possibly endogenous effect of oral sCT upon energy expenditure to chronically regulate energy balance.

Generally, oral ation of calcitonin mimetic l resembled the strong anorectic action of oral sCT during the initial week of treatment and significantly reduced food intake and protected against gain in body weight ed to oral vehicle group (Figure 1).

As observed for oral sCT, calcitonin mimetic l exerted a transient effect on food intake, gh food intake trended reduced when compared to oral sCT during the study period.

Thus, following four weeks of treatment cumulative food intake was significantly reduced in calcitonin mimetic 1 when compared to oral vehicle. Furthermore, when compared to oral sCT, a more pronounced significant reduction in body weight was observed suggesting superiority in regards to effect on energy balance.

Glucose Tolerance s are shown in Figure 2. One dose of oral sCT/calcitonin c 1 containing 1 Ing/kg compound were PCT/U52012/063332 applied by gavage twice daily to four groups of rats for 4 weeks. An oral vehicle group served as dosing regimen control. OGTT performed ing 2 weeks of treatment after ght—fasting.*** P < 0.001 vs Vehicle. Results are presented as means :SEM.

Oral sCT significantly reduced glucose iAUC during OGTT after 2 weeks of ent compared to oral vehicle (Figure 2), thus confirming the postprandial glycemic control exerted by oral application of sCT as previously demonstrated. In general, calcitonin mimetic 1 demonstrated a similar icant reduction in iAUC as observed for oral sCT, although with no clear superiority to oral sCT in this respect.

Fasting glycaemia Following 2 and 4 weeks of treatment, oral sCT application was not significantly different from oral vehicle d rats, which is in contrast with previously observations in male DIO rats, in where a 1—1.5 mM reduction in fasting blood glucose typically is observed. following chronic treatment. For calcitonin mimetic l, a trend towards superiority in fasting glycaemia was observed hout the study period when compared to oral vehicle or oral sCT.

Example 2 Acute and short term effects of oral sCT versus oral calcitonin mimetic 1 Animals The study was performed in male Levin—BIO rats (diet— sensitive) and Levin-DR (diet—resistant) (TacLevin: CD (SD) DIO) (Taconic, Hudson, NY, ) obtained at age 6—7 weeks.

On l, DIO rats were given high fat diet (60 kcal %) (#D12495, Research Diets Inc., New Brunswick, NJ., USA) and PCT/U82012/063332 kept on the same diet for 12 weeks prior to and during the experiment. DR rats were given low-fat diet and served as control group. s were pair—wise housed throughout the study. Rats were handled and pre—dosed once daily with MilliQ H20 for 2—3 weeks prior to experimental start to reduce stress—induced lycaemia. On the day prior to study start animals were given a single dose of vehicle.

Baseline parameters were recorded in an overnight fasting (16 h) condition. Rats were ized. into treatment groups based on fasting body weight (BW) and fasting plasma e (FPG). Body' weight, food and water‘ intake were recorded prior to and at study end.

Compounds Oral SGT/calcitonin mimetic 1 solution was prepared on the day of dosing by mixing the carrier with the given compound based on the following calculations: -CNAC (vehicle): Animals treated with oral 5—CNAC received a dose of 150 mg/kg dissolved in milliQ H20.

Dosage—level for 5—CNAC: 150 mg/kg Dosing volume: 5 ml/kg Compound concentration: 30 mg/ml SGT/calcitonin mimetic 1: Animals treated with oral sCT or oral calcitonin c 1 received doses of 0.5 mg/kg, 1.0 mg/kg or 2.0 mg/kg combined with 150 mg/kg 5—CNAC — all dissolved in milliQ H20.

Dosage—level for lcitonin mimetic l: 0.5 mg/kg Dosing volume: 5 ml/kg Compound concentration: 0.1 mg/ml Dosage-level for SGT/calcitonin mimetic 1: 1.0 mg/kg Dosing volume: 5 ml/kg PCT/U82012/063332 Compound concentration: 0.2 mg/ml Dosage—level for sCT/ calcitonin mimetic l: 2.0 mg/kg Dosing volume: 5 ml/kg nd concentration: 0.4 mg/ml Drug administration were given by per oral (p.o.) gavage b.i.d. during the study period and as single dose in the g prior to start of OGTT.

Oral gavage of glucose during OGTT was prepared by the following calculation: D—Glucose: Animals were given 2 g/kg single dose dissolved in milliQ H20.

Dosage—level for ose: 2 g/kg Dosing volume: 4 ml/kg Compound concentration: 500 mg/ml Experimental setup Acute testing - Treatment period for 0.5 mg/kg! 1 mg/kg and 2 mg/kg: Day 6 Treatment ent Treatment vehicle handling' vehicle Following the initial (1%) OGTT, animals are randomized into treatment groups based on PEG and BW. Animals will be pre-treated 3 days (b.i.d.) prior to 2nd OGTT. Dosing will be performed in the morning (7—8 am) and afternoon (3-4 pm).

PCT/U82012/063332 The study was performed in an x—over design with each animal being its own control.

OGTT followin overni ht fastin (16 h): D = Drug; BG = Blood glucose; B = Blood; G = Glucose Blood sampling and glycemia were measured by heated tail venous puncture.

Whole blood glucose levels were determined with an ACCU— CHEK® Avia blood glucose meter (Roche stics, Rotkreuz, Switzerland). Blood (approx 300 ul) is collected in 1 ml MiniCollect KBEDTA plasma—tube (Greiner—Bio—One GmbH, Frickenhausen, Germany), inverted, and stored on ice. Tubes are fuged 3000 x g (5000 rpm in table centrifuge) for min at 4 °C and plasma obtained. Plasma samples are stored at ~20°C until analysis. A total of ~ 2.5 ml blood is obtained during OGTT (~ 0.3% of body weight).

Experimental groups ention, Compound Conc. Number ( 4 groups of n = 8) Oral vehicle 5—CNAC 150 mg/kg X—over design to 0.5 mg/kg —CNAC + 150 mg/kg + Oral sCT n= 8 sCT 0.5 mg/kg Oral 5-CNAC + 150 mg/kg calcitonin calcitonin n= 8 0.5 mg/kg mimetic l mimetic l PCT/U52012/063332 ( 4 groups of n = 8) Oral vehicle 150 mg/kg X-over design to 1 mg/kg -CNAC + 150 mg/kg + Oral sCT n= 8 sCT 1 mg/kg Oral 5-CNAC + 150 mg/kg calcitonin onin n= 1 mg/kg mimetic l mimetic l ( 4 groups of n = 8) Oral vehicle 5~CNAC 150 mg/kg X—over design to 2 mg/kg —CNAC + 150 mg/kg + Oral sCT n= 8 sCT 2 mg/kg Oral 5-CNAC + 150 mg/kg calcitonin calcitonin n= 8 2 mg/kg mimetic l mimetic 1 Statistics Statistical analysis was performed by one—way ANOVA ed by the t‘s post hoc test for multiple comparison. Student’s t—test. was performed. to compare two paired group. All analysis was performed using GRAPHPAD PRISM software (GraphPad Prism, San Diego, CA. U.S.A). Incremental area under curve (iAUC) during OGTT was calculated by the trapezoidal method. A value of P < 0.05 was ered to be significant. All data are presented as mean i standard error of the mean (SEM). 3. Results Baseline characteristics The lZ—weeks ad libitum high—fat diet induced a pronounced obese phenotype in the diet—sensitive (DIO) rats when comparing body weight to their diet—resistant (DR) mates (P < 0.001) (Table l). Fasting glycemia was not different between DIO and DR. In contrast, area under curve WO 67357 PCT/U52012/063332 (AUC) calculations during OGTT was significantly higher in DIO rats compared to DR rats, demonstrating the high—fat nduced glucose rance (Table 2).

Table 2. Metabolic ters in D10 and DR rats 1 Diet—resistant (DR) Diet-sensitive (DIO) Body Weight (g) 609.5 f 24.5 813.6 i 9.8*** Fasting plasma glucose (mM) AUC in OGTT 648.8 i 27.3 888.4 i 64.3*** Blood glucose (mM*min) AUC, area under curve; OGTT, oral glucose tolerance test. Data are means i SEM (n=12/DR, n=24/DIO).

Body Weight and Food Intake Three different doses of oral sCT/calcitonin mimetic 1 containing 0.5, 1 and 2 mg/kg compound were applied by gavage twice daily to 4 groups of rats for 3 days. * P < 0.05, ** P < 0.01 vs oral sCT.

Results are presented in Figure 4, Figure 5, and Figure 6 as means :SEM. Figure 4A and Figure 4B show the effect of oral sCT versus oral UGP 302 on body weight and food intake in 010 rats observed in Example 2 at a first dosage. Figure 5A and Figure 5B show the effect of oral sCT versus oral UGP 302 on body weight and food intake in DIO rats observed in Example 2 at a second dosage. Figure 6A and Figure 6B show the effect of oral sCT versus oral UGP 302 on body weight and food intake in 010 rats observed in Example 2 at a third dosage; Oral sCT dose-dependently decreased body weight and food intake following the short-term treatment period and thus WO 67357 confirmed the anorectic action induced by targeting the amylin receptor as previously observed. In general, the mimetic demonstrated dose-dependent superiority to oral sCT in regards to reduction in body 'weight as rated. in Figure 4, Figure 5 and Figure 6. Application of calcitonin mimetic l at 0.5 mg/kg demonstrated significantly difference to oral sCT 0.5 mg/kg. The food. intake for the mimetic trended dose—dependently reduced compared to oral sCT.

Glucose Tolerance Three different doses of oral sCT/calcitonin mimetic 1 containing 0.5, l and 2 mg/kg compound were applied by gavage twice daily to 4 groups of rats for 3 days prior to OGTT. The experimental set-up was a cross—over design. * P < 0.05, ** P < 0.01, *** P < 0.001 vs oral vehicle. Results are presented in Figure 7 and Figure 8 as means :SEM.Eigure 7A and Figure 7B show the effect of oral sCT versus oral UGP 302 at a first dosage on glucose tolerance during OGTT in 010 rats as measured in e 2. Figure 8A and Figure 8B show the effect of oral sCT versus oral UGP 302 at a second dosage on glucose nce during OGTT in 010 rats as measured in Example 2.

Oral sCT significantly reduced glucose iAUC during OGTT for 0.5, l and 2 mg/kg doses compared to oral vehicle, thus confirming the postprandial glycemic control exerted by oral application of sCT as previously trated. Calcitonin mimetic 1 demonstrated a r significantly reduction in iAUC as observed for oral sCT, gh with no clear superiority to oral sCT within the various UGPs.

PCT/U52012/063332 Exaggle 3 Acute and short term s of oral sCT versus UGP284, UGP298 and UGP302 Animals The study was performed in male Levin—DID rats (diet— sensitive) and Levin—DR (diet—resistant) (TacLevin: CD (SD) DIO) (Taconic, Hudson, NY, U.S.A.) obtained at age 6—7 weeks.

On arrival, DIO rats were given high fat diet (60 kcal %) (#D12495, Research Diets Inc., New Brunswick, NJ., USA) and kept on the same diet for 12 weeks prior to and during the experiment. DR rats were given low—fat diet and served as control group. Animals were ise housed throughout the study. Rats were handled, and pre—dosed. once daily with MilliQ H2O for 2—3 weeks prior to experimental start to reduce —induced hyperglycemia. On the day prior to study start animals were given a single dose of vehicle. ne parameters were recorded in an overnight fasting (16 h) condition. Rats were randomized into treatment groups based on fasting body weight (BW) and g plasma glucose (FPG). Body weight, food and water intake were recorded prior to and at study end.

Compound Oral sCT/UGP solution was prepared on the day of dosing by mixing the carrier with the given compound based on the following calculations: —CNAC (vehicle): Animals treated with oral 5—CNAC received a dose of 150 mg/kg dissolved in milliQ H20.

Dosage—level for 5—CNAC: 150 mg/kg Dosing : 5 ml/kg Compound concentration: 30 mg/ml (sCT/UGP284/UGP298/UGP302) PCT/U82012/063332 Animals treated with oral sCT or oral UGP284/UGP298/UGP302 received doses of 0.5 mg/kg, 1.0 mg/kg or 2.0 mg/kg combined with 150 mg/kg 5—CNAC — all dissolved in milliQ H20. -level for sCT/UGP284/UGP298/UGP302: 0.5 mg/kg Dosing volume: 5 ml/kg Compound concentration: 0.1 mg/ml Dosage—level for sCT/UGP284/UGP298/UGP302z 1.0 mg/kg Dosing volume: 5 ml/kg Compound concentration: 0.2 mg/ml Dosage-level for sCT/UGP284/UGP298/UGP302: 2.0 mg/kg Dosing volume: 5 ml/kg Compound concentration: 0.4 mg/ml Drug administration were given by per oral (p.o.) gavage b.i.d. during the study period and as single dose in the morning prior to start of OGTT.

Oral gavage of glucose during OGTT was ed by the ing calculation: D—Glucose: Animals were given 2 g/kg single dose dissolved in nfilliQ H20.

Dosage—level for D—Glucose: 2 g/kg Dosing volume: 4 ml/kg Compound concentration: 500 mg/ml Experimental setup Acute testing - Treatment period for 0.5 mg/kg, 1 mg/kg and 2 mg/kg: lSt Rest Pre— ent Treatment Treatment 2” OGTT dose OGTT WO 67357 2012/063332 handling---- Following the initial (1“) OGTT, animals were randomized into treatment groups based on PEG and BW.

Animals were pre-treated 3 days (b.i.d.) prior to 2nd OGTT.

The study was performed in an x-over design with each animal being its own l.

OGTT followin overni ht fastin (16 h): 240 min D = Drug; BG = Blood glucose; B = Blood; G = Glucose Blood sampling and glycemia were measured by heated tail venous puncture. Whole blood glucose levels were determined with. an ACCU—CHEK® Avia blood glucose meter (Roche Diagnostics, Rotkreuz, Switzerland). Blood (approx 300 pl) is collected in lml MiniCollect KBEDTA plasma-tube (Greiner— Bio~One GmbH, Frickenhausen, Germany), inverted, and stored on ice. Tubes are centrifuged 3000 x g (5000 rpm in table centrifuge) for 10 min at 4°C and plasma obtained. Plasma samples are stored. at —20°C until analysis. A 'total. of ~ 2.5ml blood is obtained during OGTT (~ 0.3% of body weight).

Experimental groups Compound Conc. Number Intervention ( 4 groups of n = Oral vehicle 5-CNAC 150 mg/kg X—over design to 0.5 mg/kg PCT/U82012/063332 150 mg/kg + 0.5 mg/kg UGP284 UGP298 UGP302 ( 4 groups of n = 8) 150 mg/kg X—over design to 1 mg/kg 150 mg/kg + 1 mg/kg 150 mg/kg 1 mg/kg 150 mg/kg 1 mg/kg 150 mg/kg 1 mg/kg ( 4 groups of n = Oral vehicle 150 mg/kg X—over design to 2 mg/kg —CNAC + 150 mg/kg + Oral sCT sCT 2 mg/kg ~CNAC + 150 mg/kg Oral UGP284 UGP284 2 mg/kg 150 mg/kg Oral UGP298 2 mg/kg 150 mg/kg Oral UGP302 2 mg/kg Statistics tical analysis was performed by one—way ANOVA followed by the Dunnett‘s post hoc test for multiple comparison.

Student’s t—test was performed to compare two paired group.

All analysis was performed using GRAPHPAD PRISM software (GraphPad Prism, San Diego, CA. U.S.A). Incremental area under curve (iAUC) during OGTT was calculated by the trapezoidal method. A value of P < 0.05 was considered to be significant. All data are presented as mean i standard error of the mean (SEM). 3. Results ne characteristics The lZ-weeks ad libitum high—fat diet d a pronounced obese phenotype in the diet—sensitive (DIO) rats when comparing body weight to their diet—resistant (DR) littermates (P < 0.001) (Table 3). Fasting glycemia was not different between BIG and DR. In contrast, area under curve (AUC) calculations during OGTT were icantly higher in DIO rats compared to DR rats, demonstrating the high—fat diet—induced glucose intolerance (Table 3).

Table 3. Metabolic ters in D10 and DR rats Diet-resistant (DR) Diet—sensitive (DIO) Body Weight (g) 609.5 i 24.5 813.6 i 9.8*** Fasting plasma 5.8 i 0.1 5.8 i 0.2 glucose (mM) AUC in OGTT 648.8 i 27.3 888.4 i 64.3*** Blood glucose (mM*min) AUC, area under curve; OGTT, oral glucose nce test. Data are means 1' SEM (n=12/DR, n=24/DIO).

Body Weight and Food Intake Oral sCT dose—dependently decreased body weight and food intake ing the short—term treatment period. and thus PCT/U82012/063332 confirmed the anorectic action induced by targeting the amylin receptor as previously observed. In general, all UGP mimetics demonstrated dose—dependently superiority to oral sCT in regards to reduction in body weight as illustrated in Figure 9. ation of UGP302 at 0.5 mg/kg demonstrated significantly difference to oral sCT 0.5 mg/kg. For UGP284, significantly difference at 2 mg/kg dose was observed when compared to oral sCT 2 Hg/kg. Finally, UGP298 at both 1 mg/kg and 2 ng/kg doses were significantly different when compared with oral sCT at similar doses (Figure 9). Figure 9A, Figure 9B, Figure 9C, Figure 90, Figure 9E, and Figure 9F show the effect of three different doses of oral P284/UGP298/UGP302 containing 0.5, 1 and 2 mg/kg compound were applied by gavage twice daily to 4 groups of rats for 3 days. * P < 0.05, ** P < 0.01 vs oral sCT.

Results are presented as means :SEM.

Glucose Tolerance Figure 10A, Figure 10B, Figure 10C, Figure 10D, Figure 10E, and Figure 10F show the effect of oral sCT versus oral UGPs on glucose tolerance during OGTT in DIO rats. Three different doses of oral sCT/UGP284/UGP298/UGP302 ning 0.5, 1 and. 2 mg/kg' compound were applied by gavage twice daily to 4 groups of rats for 3 days prior to OGTT. The experimental set—up was a over design. * P < 0.05, ** P < 0.01, *** P < 0.001 vs oral vehicle. Results are presented as means xSEM.

All UGPs demonstrated a similar significant ion in iAUC as observed for oral sCT e 10).

In conclusion, application of UGP284, UGP298 and UGP302 at 0.5, l and 2 mg/kg doses demonstrated superiority to equivalent doses of oral sCT in regards to energy balance in WO 67357 PCT/U52012/063332 male DIO rats. Furthermore, UGP284, UGP298 and UGP302 at doses of 0.5, l and 2 mg/kg produced an improvement in glucose tolerance during OGTT.

Example 4 Binding of sCT analogs to T47D Cell calcitonin receptors sCT analogs at various concentrations were tested in a T47D (human breast epithelial cell line) ay. This cell line is known to have the following receptors: calcitonin, androgen, progesterone, glucocrticoid, prolactin and estrogen. The results are presented in Figure 11 as % cAMP binding relative to sCT which was set at 100% CAMP binding at a concentration of 1000 pg/mL. It can be seen that UGP302 provides the highest level of binding of all the tested compounds and that it provides a higher level of binding than sCT.

Example 5 Food consgggtion and weight change in rats fed sCT analogs Male Sprague-Dawley rats were housed individually in cages in which the light/dark cycle was reversed. Rats were allowed to eat ad libitum. Food consumption and rat s were monitored daily during each study. Rats were injected uscularly with a saline placebo or the indicated e at the specified dose in . The data in the following tables is summarized as the mean change in food consumption relative to the day before treatment began (day — 1) and. as the mean change in weight relative to the day before treatment began.

The results are shown in Figures 12, 13, 14, 15, 16 and 17. Figure 12A and Figure 12B show food consumption (Figure 12A) and weight change measurements (Figure 12B) for UGP 282 as ed in Example 5. Figure 13A and Figure 13B show food consumption (Figure 13A) and weight change measurements (Figure 13B) for UGP 283 as measured in Example 5. Figure 14A and Figure 14B show food consumption (Figure 14A) and weight change measurements (Figure 14B) for UGP 284 as measured in Example 5. Figure 15A and Figure 15B show food ption (Figure 15A) and weight change measurements (Figure 15B) for UGP 298 as measured in Example 5. Figure 16A and Figure 16B show food consumption (Figure 16A) and weight change measurements (Figure 16B) for UGP 302 as measured in Example 5. Figure 17A and Figure 17B show food ption (Figure 17A) and weight change ements (Figure 17B) for UGP 303 as measured in Example 5.

It can be seen that all of the tested compounds induce weight loss and reduce feed intake.

Example 6 s of Osteoporosis and Osteoarthritis The effect of sCT/calcitonin c on bone and cartilage loss was studied in DIO rats. The animals were dosed as described ill the table .below, and 22 hours after treatment blood sampling was done by heated tail venous puncture.

Serum CTX—I levels, as an indication of bone resorption, were measured. using' the RatLapsTM ELISA, and serun1 CTX~II levels, as an indication of cartilage ation, were measured using the Serum PC CartilapsTM ELISA.

Experimental groups Oral vehicle 150 mg/kg -CNAC + Oral sCT 150 mg/kg + n = 8 WO 67357 PCT/U82012/063332 Oral calcitonin —CNAC + 150 mg/kg c of SEQ SEQ ID NO:15 1 mg/kg ID NO:l8 The results are seen in Figure 18 and Figure 19, where a calcitonin mimetic of SEQ ID NO: 18 shows a stronger effect in reduction of both bone resorption and cartilage degradation than does sCT.

In some embodiments, a peptide of the t disclosure has a sequence selected from SEQ ID NO:II, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:l4, SEQ ID NO:15, SEQ ID NO:1? and SEQ ID NO:l8.

In some embodiments, a method includes administering to a patient an effective amount of a peptide selected from the group consisting of: SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:l3, SEQ ID NO:l4, SEQ ID NO:15, and SEQ ID NO:l7 to affect a weight ion in the patient.

In some embodiments, a method includes administering to a patient an ive amount of a peptide selected from the group consisting of: SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:l3, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:17 to affect postprandial glycemic control in the patient.

In some embodiments, a method includes administering to 2O the t an effective amount of a peptide selected from the group consisting of:SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:l4, SEQ ID NO:15, and SEQ ID NO:l7 to affect an improvement in glycemic control in the patient.

In some embodiments, a method includes administering to a patient an effective amount of a peptide of SEQ ID NO:18 having the sequence CmSNLSTCVLGKLSQELHKLQTYPRTDVGANXaaXaaa so PCT/U52012/063332 as to reduce at least one of bone tion and cartilage degradation in the patient.

All patents, patent applications, and published references cited herein are hereby incorporated by nce in their entirety. It will be appreciated that several of the above—disclosed. and other features and functions, or atives thereof, may be desirably combined into many other different systems or applications. various presently unforeseen or unanticipated alternatives, modifications, variations, or improvements therein may be uently made by those skilled in the art which are also intended to be encompassed by the following claims.

Claims (9)

CLAIMS What is claimed is:
1. l. A e selected from the group consisting of: ACCSNLSTCVLGKLSQELHKLQTYPRTDVGANAP—NH2 SEQ ID NO: 15, ACCSNLSTCVLGRLSQELHRLQTFPRTDVGANTACY SEQ ID NO: 12, and SuCCCSNLSTCVLGKLSQELHKLQTYPRTDVGANAY-NH2 SEQ ID NO: 17.
2. 2. A peptide as d in claim 1, n the peptide is formulated for enteral stration. 10
3. 3. A e as claimed in claim 1, wherein the peptide is formulated for parenteral administration.
4. 4. A peptide as claimed in claim 1, wherein the peptide is formulated with a carrier for oral administration, and n the carrier increases the oral bioavailability of the 15 peptide.
5. 5. A peptide as claimed in claMn 4, wherein the carrier comprises 5-CNAC, SNAD, or SNAC.
6. 6. A peptide as claimed in any one of the preceding claims, wherein the peptide is formulated in a pharmaceutical 2O composition for oral administration comprising coated citric acid particles, and wherein the coated citric acid particles increases the oral bioavailability of the e.
7. 7. A peptide as claimed in any one of the preceding claims, wherein said administration is to improve postprandial 25 glycemic control in a patient.
8. 8. The use of a peptide as claimed in any‘ one of the preceding claims in the manufacture of a medicament for administration to a patient in an effective amount to effect a weight reduction in the t or to effect an improvement 3O in glycemic control.
9. 9. A peptide as claimed in claim 1, substantially as herein described with reference to any one of the Examples and/or