Calcitonin mimetics for treating diseases and disorders

Abstract

Calcitonin mimetic peptides having an amino acid sequence in accordance with SEQ ID NO:8 or SEQ ID NO:53, each of which may be carboxylated 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, and in each of which the 1 and 7 position cysteine residues may together be replaced by ?-aminosuberic acid (Asu) are useful as medicaments for treating diabetes (Type I and/or Type II), excess bodyweight, excessive food consumption, metabolic syndrome, rheumatoid arthritis, non-alcoholic fatty liver disease, osteoporosis, or osteoarthritis, poorly regulated blood glucose levels, poorly regulated response to glucose tolerance tests, or poorly regulated of food intake.

Claims

1. A peptide comprising the amino acid sequence of SEQ ID NO: 43.

2. The peptide as claimed in claim 1, consisting of the amino acid sequence of SEQ ID NO: 44.

3. The peptide as claimed in claim 1, formulated for enteral administration.

4. The peptide as claimed in claim 1, formulated for parenteral administration.

5. The peptide as claimed in claim 4, formulated for injection.

6. The peptide as claimed in claim 1, formulated with a carrier for oral administration.

7. The peptide as claimed in claim 6, wherein the carrier comprises N-(5-chlorosalicyloyl)-8-aminocaprylic acid (5-CNAC), sodium salt of 10-(2-Hydroxybenzamido)decanoic acid (SNAD), or sodium salt of N-(8-[2-hydroxybenzoyl]amino)caprylic acid (SNAC).

8. The peptide of claim 1, wherein the peptide is carboxylated at its N-terminal.

9. The peptide of claim 1, wherein the peptide is amidated at its C-terminal.

10. A pharmaceutical composition comprising the peptide of claim 1 coated with citric acid particles wherein the coated citric acid particles increase the oral bioavailability of the peptide.

11. A method for treating Type II diabetes, obesity, osteoporosis, osteoarthritis, or improving glycemic control, comprising administering the peptide as claimed in claim 1.

12. A method for treating Type II diabetes, obesity, osteoporosis, osteoarthritis, or improving glycemic control, comprising administering the peptide as claimed in claim 1 in conjunction with metformin or another insulin sensitizer.

Description

(1) Results are shown in the accompanying drawings, as follows:

(2) FIGS. 1A-1E: Raw data showing the activity of KBPs of Table 3 on the CTR head to head with sCT and UGP302/KBP-042 at different timepoints, 4 h (FIG. 1A), 8 h (FIG. 1A), 24 h (FIG. 1A), 48 h (FIG. 1A), 72 h (FIG. 1A).

(3) FIGS. 2A-2C: Activation of the CTR as a function of time, by fixed concentrations of the different ligands at 1 nM (FIG. 2A), 10 nM (FIG. 2B) and 100 nM (FIG. 2C) by a prolonged beta-arrestin assay.

(4) FIG. 3: Dose-response activation of the AMY-R for the different ligands when added to a cell-line expressing the AMY-R.

(5) FIGS. 4A-4B: Competitive binding of tested compounds on U20S-CALCR cells expressing the CTR (FIG. 4A) and CHO-K1CALCRRAMP3 cells expressing the AMY-R (FIG. 4B), with pIC50 figures in the table.

(6) FIG. 5: Dose-response activation of the CGRP-R for the different ligands.

(7) FIGS. 6A-6C: The effect of a single dose of KBP-056 on food intake (FIG. 6A), bodyweight (FIG. 6B) and PPG (FIG. 6C).

(8) FIGS. 7A-7B: The effect of three weeks of treatment with UGP302/KBP-042, KBP-023, KBP-056 and KBP.088 on FPG (FIG. 7A) and HbA1c (FIG. 7B).

(9) FIGS. 8A-8B: The effect of the KBP compounds on resorption of type I collagen, (FIG. 8A) and on resorption of type II collagen (FIG. 8B).

(10) FIG. 9: Results obtained in Example 7 of a comparison between the effects of compounds KBP-088 and KBP-089 on food intake in obese rats.

(11) FIG. 10: Short term food intake measurements obtained in Example 7 in a comparison between peptide KBP-089 of the invention and known peptide KBP-042.

(12) FIGS. 11A-11B: Long term food intake measurements (FIG. 11A) and body weight change (FIG. 11B) for KBP-089 and KBP-042 peptides.

(13) FIGS. 12A-12F: Further results obtained in Example 1 for cAMP induction by action on the calcitonin receptor (FIGS. 12A, 12B and 12E) and on the amylin receptor (FIGS. 12D and 12E) and further results obtained in Example 7 in food intake measurements (FIG. 12F).

EXAMPLES

(14) In the following examples, the following materials and methods were employed.

(15) Cells and Cell Lines

(16) 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). 3. CGRP Receptor (CGRP-R): CHO-K1 CALCRL+RAMP1 from DiscoveRx (Cat. No.: 93-0269C2).

(17) In independent bioassays, CTR, AMY-R and CGRP-R cells were treated with for the indicated timepoints with increasing doses of sCT, UGP-302/KBP-042 or the KBPs identified in Table 3 (0, 0.001, 0.01, 0.1, 1, 10, and 100 nM).

Example 1: ?-Arrestin Assay

(18) PathHunter ?-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 ?-Arrestin with the activated GPCR. Because 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.

(19) 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 ?-Arrestin and the larger, N-terminal deletion mutant of ?-gal (called enzyme acceptor or EA). Activation of the GPCR stimulates binding of ?-Arrestin to the ProLink-tagged GPCR and forces complementation of the two enzyme fragments, resulting in the formation of an active ?-gal enzyme. This interaction leads to an increase in enzyme activity that can be measured using chemiluminescent PathHunter? Detection Reagents.

(20) The assay was performed in white 384 well plates (Greiner Bio-One, 784080). Cells were seeded 2500 cells per well in 10 ?L cell-type specific medium the day prior to the experiment. To quantify the GPCR-mediated ?-arrestin recruitment the Pathhunter? Detection Kit (93-0001, DiscoverX) was used and assay performed accordingly to the manufacturer's instructions.

(21) Results are seen in FIGS. 1A-1E, 2A-2B and 3. As seen in FIGS. 1A-1E, the peptides are quite similar in activity; however, with increasing time KBP-056 shows a superior ability to activate and maintain activation of the CTR. This is even better illustrated in FIGS. 2A-2B, where the individual ligands are plotted at one given concentration and as a function of time. Here it is apparent that KBP-056 is superior to the other peptides in terms of calcitonin receptor activation.

(22) Another important trait of this class of molecules is the ability to activate the amylin receptor AMY-R, and as seen in FIG. 3, the KBPs are fully capable of activating this receptor, and KBP-088 is more potent that the classical AMY-R agonist sCT.

(23) In a subset of the figures, the beta-arrestin assay was used to assess prolonged receptor activation. This was done using the Calcitonin Receptor (CTR): U20S-CALCR from DiscoveRx (Cat. No.: 93-0566C3) cell line, and as opposed to the classical 3 hour output, beta-arrestin accumulation was conducted over 24, 48 and 72 hour and then analyzed.

(24) Peptides for use in the invention were ranked in terms of their ability to activate the CTR and AMY-R in vitro in decreasing order, as follows:

(25) TABLE-US-00010 RANK RANK RANK Intervention 4 h 24 h 48 h (4 h) (24 h) (48 h) KBP-089 1.8 0.7 5.4 + + + KBP-021 2.1 0.6 6.9 + + + KBP-019 1.3 0.2 11.8 + + + KBP-088 1.9 0.4 11.4 + + + KBP-042* 2.2 1.2 14.3 + + 0 nf-hCT* 2.7 15.9 15.9 + 0 0 Vehicle* 8.5 16.6 19.3 0 0 0 *Comparative

Example 2: Competitive Ligand Binding

(26) In this Example we demonstrate the relative abilities of test compounds to bind the calcitonin and amylin receptors. Corresponding to the beta-arrestin induction experiments, we performed competitive ligand binding experiments using the cells expressing the CTR and the AMY-R. In studying ligand binding on cells, 15.000 CTR or AMY-R cells were seeded in 96-well plate and cells were treated with 0.1 nM .sup.125I-conjugated sCT with or without the presence of unlabelled KBP in increasing doses for 60 min at 37? C. After incubation the supernatant was removed and cells were washed twice in 200 ?l PBS. The cells were lysed using RIPA buffer (30 mM M NaCl, 50 mM Tris-HCl, 5 mM EDTA, 1% Deoxycholic acid, 10% SDS, Protease inhibitor tablet mini (1 tablet:10 ml)) and lysates as well were collected for measurement on a ?-counter.

(27) Results are seen in FIGS. 4A-4B. Firstly, we found that all ligands bound potently to both receptors; however, in terms of affinity KBP-088 was better than the other ligands, particularly on the AMY-R.

Example 3: ?-Arrestin Recruitment Tested on the CGRP-Receptor

(28) As peptides with the ability to activate the amylin receptor have been shown to activate the CGRP-R, an effect which may be detrimental to the overall effect of the compounds, we assessed whether the KBPs activated the CGRP-receptor. As seen in FIG. 5, unlike salmon calcitonin, none of the KBP compounds activated the CGRP-R.

Example 4: In Vivo Activity Test on Bodyweight

(29) To ensure the in vivo activity of this class of peptides, an acute test of the ability of KBP-056 to reduce food intake, bodyweight and post-prandial glucose compared to UGP302/KBP-042 was conducted. Male Sprague-Dawley rats were fed high fat diet for 8 weeks to induce obesity, and were then exposed to a single subcutaneous dose of 5 ?g/kg KBP-056, UGP302/KBP-042 or vehicle (saline) (5 rats per group), and the effect on food intake, bodyweight and blood glucose was assessed. Blood was collected from the tail vein after 18 hours of dosing. Whole blood glucose levels were determined with an ACCU-CHEK? Avia blood glucose meter (Roche Diagnostics, Rotkreuz, Switzerland).

(30) As seen in FIGS. 6A-6B, KBP-056 strongly reduced food intake over an 18-hour period (FIG. 6A), which is also manifested in a significant weight loss (FIG. 6B). Interestingly, the weight loss appears to exceed that caused by UGP302/KBP-042. Finally, a prominent reduction in post-prandial glucose levels (FIG. 6C) was observed, thereby confirming that this novel class of peptides shows substantial in vivo activity.

Example 5: In Vivo Activity Test on Fasting Plasma Glucose and HbA1c

(31) To assess the effects of the peptides in relation to regulation of blood glucose levels, male ZDF rats, which are the gold standard model of Type 2 Diabetes were treated for 3 weeks with the 3 most promising KBPs (023, 056 and 088) and an active comparator (UGP302/KBP-042). Male ZDF (fa/fa) rats were obtained at the age of 7 weeks from Charles River Laboratories (Kisslegg, Deutschland). All animals were housed at the animal facility at Nordic Bioscience with a constant temperature (21-23? C.) and relative humidity (55-65%) on a 12 h light/dark cycle with free access to Purina 5008 rat chow (Brogaarden, Lynge, Denmark) and tap water. At the age of 8 weeks, the animals were randomized into five groups of 8 rats each based upon fasting blood glucose and body weight (Vehicle (saline), UGP302/KBP-042 5 ?g/kg/day, KBP-023 5 ?g/kg/day, KBP-056 5 ?g/kg/day and KBP-088 5 ?g/kg/day) and dosing was done subcutaneously once daily. The animals were treated for a total of 3 weeks to assess the effect of the peptides on fasting blood glucose. Blood glucose was monitored using the Accu-Check? Avia monitoring system (Roche Diagnostics, Rotkreuz, Switzerland) and HbA1c levels by the DCA Vantage? Analyzer (Siemens Healthcare Diagnostics, Deerfield, Ill.).

(32) Fasting blood glucose and HbA1c were assessed after three weeks, and as seen in FIGS. 7A-7B, both KBP-056 and KBP-088 were highly active with respect to reduction of FPG and HbA1c, and interestingly they appeared superior to UGP302/KBP-042, indicating that the aminoacid substitutions improve efficacy, as also seen in the in vitro data.

Example 6: The Effect of KBP-056 on Bone Resorption and Cartilage Degradation

(33) In view of the relationship between the KBPs and different calcitonins, and the well-known bone and cartilage protective effects of salmon calcitonin, the effects of KBP-056 on bone resorption (measured by CTX-I) and cartilage degradation (Assessed by CTX-II) were analyzed in male rats on HFD. As seen in FIGS. 8A-8B KBP-056 dose and time-dependently reduced both CTX-I (FIG. 8A) and CTX-II (FIG. 8B), with maximum inhibition reached three hours after dosing and at doses exceeding 1 ?g/kg/day. This confirmed that the KBPs maintain their beneficial effects on bone and cartilage despite the amino-acid changes. Furthermore, based on their superiority in other tests, we expect these effects to be superior as well.

Example 7: Comparative Effect of KBP 088 and KBP 089 on Food Intake in Obese Rats

(34) Rats were fed a High-Fat-Diet for 14 weeks prior to the experiment to induce obesity. They were then randomized into groups (Vehicle (0.9% NaCl), KBP-089 (doses: 1.25, 2.5, 5 ?g/kg) and KBP-088 (doses: 1.25, 2.5, 5 ?g/kg). They were fasted overnight and then treated with a single dose of peptide or vehicle in the morning using subcutaneous administration. The food intake was monitored in the following intervals (0-4 hours, 4-24 hours and 24-48 hours). As seen in FIG. 9, both KBP-088 and KBP-089 led to a reduction in food intake within the 4 hour interval, and the effect was maintained to 24 hours. In the interval between and 48 hours only KBP-089 led to a reduction in food intake indicating that this molecule is superior to KBP-088.

(35) In FIG. 12F, similar test results are shown for a wider range of peptides according to the invention.

(36) Further similar studies were undertaken over different time periods and with different peptides and the results are seen in FIG. 10 and FIGS. 11A-11B. The ability of KBP089 to suppress food intake over 72 hours was compared to that of KBP042 as control and the results are seen in FIG. 10.

(37) To further investigate the potency of KBP-089 against KBP-042 obese rats were dosed with 2.5 ?g/kg/day given as subcutaneous injection of either KBP-089 or KBP-042 and then two control groups, a vehicle and a calorie restriction group, in which the food intake was matched to the food intake of the KBP-089 group to shed light on whether effects independent of food intake could be observed.

(38) In FIGS. 11A-11B; the effect of the peptides on food intake (FIG. 11A) and bodyweight (FIG. 11A) during the eight week treatment period is shown in the upper and lower panels respectively. As can be seen both peptides reduce food intake dramatically in the beginning as expected, after which the effect is reduced and the food intake gradually normalizes. The effect on bodyweight is shown in FIG. 11B, lower panel, where it is clearly seen that both peptides cause a substantial weight loss; however, the weight loss introduced by KBP-089 is greater than that introduced by KBP-042 showing superiority. Additionally, when comparing KBP-089 to the calorie restricted control group (pair-fed), which has received exactly the same amount of food as the KBP-089 group over the study period, it is clear that the weight reduction is not only mediated by a restriction of food intake, but also by other effects.

(39) 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. No acknowledgement of any prior published document herein should be taken to be an admission or representation that the teaching thereof was common general knowledge in Australia or elsewhere at the date hereof.