Calcitonin Mimetics for Treating Diseases and Disorders

Abstract

The present invention relates to humanised calcitonin mimetics and their 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, 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.

Claims

1: A peptide, wherein the peptide is: TABLE-US-00006 (SEQIDNO:26) CGNLSTC LGRL QD X4K TFP TDVGANAP wherein X.sub.1=M or V X.sub.2=T or S X.sub.3=F or L X.sub.4=N or H, X.sub.5=F or L X.sub.6=Q or H X.sub.7=Q or K

2: The peptide according to claim 1, wherein X.sub.2 is T, X.sub.3 is L, X.sub.4 is N, X.sub.5 is F, X.sub.6 is H, and/or X.sub.7 is K.

3: A peptide according to claim 1, wherein X.sub.4 is N, X.sub.5 is F, and X.sub.6 is H.

4: The peptide according to claim 1, wherein the peptide has an identity to human calcitonin of at least 65%.

5: The peptide according to claim 1, wherein the peptide is selected from the group consisting of: TABLE-US-00007 (SEQIDNO:12) CGNLSTCMLGRLSQDLNKFHTFPKTDVGANAP, (SEQIDNO:18) CGNLSTCMLGRLTQDLHKLQTFPKTDVGANAP, (SEQIDNO:20) CGNLSTCMLGRLTQDFHKLHTFPKTDVGANAP, (SEQIDNO:24) CGNLSTCMLGRLTQDLNKFHTFPKTDVGANAP, and[[or]] (SEQIDNO:25) CGNLSTCMLGRLSQDLNKFHTFPQTDVGANAP

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

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

8: The peptide as claimed in claim 7, formulated for injection.

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

10: The peptide as claimed in claim 9, 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).

11: A pharmaceutical composition comprising the peptide of claim 6 coated with citric acid particles wherein the coated citric acid particles increase the oral bioavailability of the peptide.

12: (canceled)

13: A method for 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, 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 comprising administering the peptide as claimed in claim 1.

14: The method as claimed in claim 13, comprising administering the peptide in conjunction with metformin or another insulin sensitizer.

Description

DESCRIPTION OF THE FIGURES

[0082] FIGS. 1A-1B: -arrestin recruitment dose response by KBP-046-KBP-054 (FIG. 1A) and KBP-058-KBP-063 (FIG. 1B) as a function of calcitonin receptor activation. Dose range is 1 M to 15 M and data is shown as fold of vehicle. Screening was conducted in U2OS CALCR Pathhunter cells from DiscoveRx.

[0083] FIGS. 2A-2B: -arrestin recruitment dose response by KBP-046-KBP-054 (FIG. 2A) and KBP-058-KBP-063 (FIG. 2B) as a function of amylin receptor activation. Dose range is 1 M to 15 M and data is shown as fold of vehicle. Screening was conducted in CHO-K1 CALCR RAMP3 Pathhunter cells from DiscoveRx.

[0084] FIGS. 3A-3B: Prolonged -arrestin recruitment by KBP-046-KBP-054 (FIG. 3A) and KBP-058-KBP-063 (FIG. 3B) and human calcitonin as a function of calcitonin receptor activation. Single dose of 100 nM at t=0 and after 3, 6, 24, and 72 hours after initial treatment, -arrestin recruitment was assessed to investigate each ligands protracted potential. Data is shown as fold of vehicle. Screening was conducted in U2OS CALCR Pathhunter cells from DiscoveRx.

[0085] FIGS. 4A-43: Protracted attenuation of food intake in SD rats by KBP-046-KBP-054 (FIG. 4A) and KBP-058-KBP-063 (FIG. 4B). After single s.c dose of 2.5 g/kg of KBP, food intake was monitored in intervals of 0-4 h and 4-24 h to assess the protracted action of individual KBPs. Individual rats were single caged, n=4 in each group.

EXAMPLES

[0086] 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.
The following cell lines expressing the calcitonin, amylin and CGRP receptors were purchased and cultured according to the manufacturer's instructions. [0087] 1. Calcitonin Receptor (CTR): U2OS-CALCR from DiscoveRx (Cat. No.: 93-0566C3). [0088] 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 identified in Tables 1A and 1B (1000, 250, 62.5, 15.6, 3.9, 1.0, 0.24, 0.06, 0.02 nM and vehicle).

Example 1: -Arrestin Assay

[0089] 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.

[0090] 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.

[0091] 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. Furthermore, data from the Pathhunter -arrestin recruitment assay was normalized to an internal standard to improve the distinction between top KBP performers and non-performers.

[0092] Results are seen in FIGS. 1A-1B, 2A-2B and 3A-3B. As seen in FIGS. 1A-1B, the peptides are quite similar in activity; however, with increasing time KBP-047, KBP-053 (FIG. 3A) and KBP-058, 062 and 063 (FIG. 3B) show a superior ability to activate and maintain activation of the CTR as illustrated in FIG. 3, where the individual ligands are plotted at one given concentration and as a function of time. Here it is apparent that KBP-047, KBP-053, KBP-058, KBP-062 and KBP-063 are superior to the other peptides in terms of protracted calcitonin receptor activation. This was done using the calcitonin receptor (CTR): U2OS-CALCR from DiscoveRx (Cat. No.: 93-0566C3) cell line, and as opposed to the classical three hour output, -arrestin accumulation was conducted over 24, 48 and 72 hour and then analyzed.

[0093] Another important trait of this class of molecules is the ability to activate the amylin receptor. As seen in FIGS. 2A and 2B, the KBPs are fully capable of activating this receptor, and KBP-047, KBP-053, KBP-058, KBP-062 and KBP-063 are demonstrated to be potent amylin agonists. In a subset of the figures, the -arrestin assay was used to assess prolonged receptor activation.

Example 2: Comparative Effect of KBP-047 to KBP-063 on Food Intake in Healthy Sprague Dawley Rats

[0094] Rats were single caged four days prior to individual tests. They were randomized by weight into seven groups (Vehicle (0.9% NaCl), KBPs (doses: 2.5 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 and 4-24 hours).

[0095] As seen in FIG. 4A, both KBP-047 and KBP-053 led to a reduction in food intake within the 4 hour interval, and the effect was maintained to 24 hours with KBP-047 having the largest reduction, closely followed by KBP-053.

[0096] As seen in FIG. 4B, both KBP-058, KBP-062 and KBP-063 led to a reduction in food intake within the 4 hour interval, and the effect was maintained to 24 hours with KBP-062 having the largest reduction, followed by KBP-058 and KBP-063 in that order. An overview of food intake as a function of hCT homology can be found in Table 2.

TABLE-US-00005 TABLE 2 In vivo peptide screening characteristics with corresponding hCT homology percentages. Compound Ligand Homology Food Intake FOOD Humanized Human calcitonin Sustained KBP Sequences similarity Percentage Attenuation Hours (h) KBP-046 69% 4 h KBP-047 72% 24 h KBP-048 84% 4 h KBP-049 84% 4 h KBP-050 69% 4 h KBP-051 66% 4 h KBP-052 78% 4 h KBP-053 66% 24 h KBP-054 78% 4 h KBP-058 72% 24 h KBP-059 75% 4 h KBP-060 75% 4 h KBP-061 75% 4 h KBP-062 75% 24 h KBP-063 75% 24 h

[0097] Overall, KBP-062 has best combination of high hCT homology combined with corresponding top performance in the screening.

[0098] 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.