Patent Application
20130210751
The present invention relates to improvements in compositions containing peptides that are ligands of the growth hormone secretagogue (GHS) receptor, or pharmaceutically acceptable salts thereof, methods for preparing such compositions, and methods of using such compositions to treat mammals. In particular, the present invention relates to a pharmaceutical composition comprising a pamoate salt of H-Inp-D-Bal-D-Trp-Phe-Apc-NH2, which is a ligand of the GHS receptor, and in which, after subcutaneous or intramuscular administration to a subject, the peptide forms an in situ depot at physiological pH that is slowly dissolved and released into the body fluid and bloodstream. The present invention may further comprise an organic component such as dimethylacetamide (DMA) or polyethylene glycol with an average molecular weight of lower than 1000.
The pulsatile release of growth hormone from the pituitary somatotrops is regulated by two hypothalamic neuropeptides: growth hormone-releasing hormone and somatostatin. Growth hormone-releasing hormone stimulates release of growth hormone, whereas, somatostatin inhibits secretion of growth hormone. (Frohman et al., Endocr. Rev. 1986, 7, 223-253, and Strobi et al., Pharmacol. Rev. 1994, 46, 1-34.)
Release of growth hormone from the pituitary somatotrops can also be controlled by growth hormone-releasing peptides (GHRP's). A hexapeptide, His-D-Trp-Ala-Trp-D-Phe-Lys-amide (GHRP-6), was found to release growth hormone from somatotrops in a dose-dependent manner in several species including man. (Bowers et al., Endocrinology 1984, 114, 1537-1545.) Subsequent chemical studies on GHRP-6 led to the identification of other potent growth hormone secretagogues such as GHRP-I, GHRP-2 and hexarelin (Cheng et al., Endocrinology 1989, 124, 2791-2798, Bowers, C. Y. Novel GH-Releasing Peptides. In: Molecular and Clinical Advances in Pituitary Disorders. Ed: Melmed, S.; Endocrine Research and Education, Inc., Los Angeles, Calif., USA 1993, 153-157, and Deghenghi et al. Life Sci. 1994, 54, 1321-1328):
GHRP-I, GHRP-2, GHRP-6, and hexarelin are synthetic growth hormone secretagogues (GHS's). GHS's stimulate secretion of growth hormone by a mechanism different from that of growth hormone-releasing hormone. (Bowers et al. Endocrinology 1984, 114, 1537-1545, Cheng et al. Endocrinology 1989, 124, 2791-2798, Bowers, C. Y. Novel GH-Releasing Peptides. In: Molecular and Clinical Advances in Pituitary Disorders. Ed: Melmed, S.; Endocrine Research and Education, Inc., Los Angeles, Calif., USA 1993, 153-157, and Deghenghi et al., Life Sci. 1994, 54, 1321-1328.)
The low oral bioavailability (<1%) of the peptidyl growth hormone secretagogues stimulated search for non-peptide compounds mimicking action of GHRP-6 in the pituitary. Several benzolactams and spiroindanes have been reported to stimulate growth hormone release in various animal species and in man. (Smith et al. Science 1993, 260, 1640-1643, Patchett et al., Proc. Natl. Acad. Sci. USA. 1995, 92, 7001-7005, and Chen et al., Bioorg. Mod. Chem. Leu. 1996, 6, 2163-2169.) A specific example of a small spiroindane is MK-0677 (Patchett et al. Proc. Natl. Acad. Sci. USA. 1995, 92, 7001-7005):
The actions of the above-mentioned GHS's (both peptide and non-peptide) appear to be mediated by a specific growth hormone secretagogue receptor (GHS receptor). (Howard et al., Science 1996, 273, 974-977, and Pong et al, Molecular Endocrinology 1996, 10, 57-61.) This receptor is present in the pituitary and hypothalamus of various mammalian species (GHSR1a) and is distinct from the growth hormone-releasing hormone (GHRH) receptor. The GHS receptor was also detected in the other areas of the central nervous system and in peripheral tissues, for instance adrenal and thyroid glands, heart, lung, kidney and skeletal muscles. (Chen et al, Bioorg. Med. Chem. Leu. 1996, 6, 2163-2169, Howard et al, Science 1996, 273, 974-977, Pong et al, Molecular Endocrinology 1996, 10, 57-61, Guan et al, Mol. Brain Res. 1997, 48, 23-29, and McKee et al., Genomics 1997, 46, 426-434.) A truncated version of GHSR1a has been reported. (Howard et al., Science 1996, 273, 974-977.)
The GHS receptor is a G-protein coupled-receptor. Effects of GHS receptor activation includes depolarization and inhibition of potassium channels, an increase in intercellular concentrations of inositol triphosphate (IP3), and a transient increase in the concentrations of intracellular calcium. (Pong et al., Molecular Endocrinology 1996, 10, 57-61, Guan et al., Mol. Brain Res. 1997, 48, 23-29, and McKee et al., Genomics 1997, 46, 426-434.)
Ghrelin is a naturally occurring peptide which is believed to be an endogenous ligand for the GHS receptor. (Kojima et al., Nature 1999, 402, 656-660.) The native structures of ghrelins from several mammalian and non-mammalian species of animals are known. (Kaiya et al., J. Biol. Chem. 2001, 276, 40441-40448; International Patent Application PCT/JP00/04907 (WO 01/07475).) A core region present in ghrelin was found to provide for activity at the GHS receptor. The core region comprises the four N-terminal amino acids, where the serine at position 3 is normally modified with n-octanoic acid. In addition to acylation by n-octanoic acid native ghrelin also has been observed to be acylated with n-decanoic acid. (Kaiya et al., J. Biol. Chem. 2001, 276, 40441-40448.) Ghrelin analogs have a variety of different therapeutic uses as well as uses as research tools.
The present invention provides a formulation of a pharmaceutical composition comprising a pamoate salt of a peptide that acts as a ligand of the GHS receptor. Particularly preferred is the following peptide which is referred to hereinafter as “Example 1”: H-Inp-D-Bal-D-Trp-Phe-Apc-NH2, which is a ligand of the GHS receptor, wherein, after subcutaneous or intramuscular administration to a subject, the peptide forms a precipitate at physiological pH that is slowly dissolved and released into the body fluid and bloodstream, thereby resulting in attenuated side effects and improved efficacy.
The invention may be summarized in the following paragraphs below, as well as in the claims.
Although the preferred embodiment of the present invention is directed to Example 1, i.e., H-Inp-D-Bal-D-Trp-Phe-Apc-NH2, which is a ligand of the GHS receptor, the present invention is in no way limited to Example 1. The peptides of the present invention include, for example, all those peptides that act as ligands for the GHS receptor, as disclosed in the applicant's own prior international publication number published as WO 2004/014415, the content of which is incorporated herein by reference in its entirety.
The following compounds from these publications may also be advantageously employed to constitute the pharmaceutical compositions of the present invention:
The nomenclature used to define the peptides herein is that typically used in the art wherein the amino group at the N-terminus appears to the left and the carboxyl group at the C-terminus appears to the right. Where the amino acid has isomeric forms, it is the L form of the amino acid that is represented unless otherwise explicitly indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Abbreviations used herein are defined as follows:
Certain other abbreviations used herein are defined as follows:
Unless otherwise apparent, abbreviations (e.g. Ala) of amino acids in this disclosure stand for the structure of —NH—C(R)(R′)—CO—, wherein R and R′ each is, independently, hydrogen or the side chain of an amino acid (e.g., R═CH3 and R′═H for Ala), or R and R′ may be joined to form a ring system.
When a non-amino acid imidazole moiety, (e.g., Pim, defined above), is present at the C-terminus of a compound of the invention it is understood that the imidazole moiety is attached to the adjacent amino acid via a pseudo-peptide bond, wherein a bond is formed between the position 2 carbon of the imidazole ring and the alpha carbon of the amino acid. For example, in the case where the adjacent amino acid is D-tryptophan (D-Trp) and the imidazole moiety is Pim, the C-terminus of the peptide would appear as follows:
For clarity, in the written formula for such a compound the presence of this bond is indicated by the Greek letter “Ψ” alone in parentheses. For example, the written formula H-Inp-D-Trp-D-2Nal(Ψ)-Pim denotes the structure:
The peptides of this invention can be prepared using the techniques disclosed in WO 2004/014415, at pages 34-42, the content of which is incorporated herein by reference in its entirety. In addition, examples of techniques for biochemical synthesis involving the introduction of a nucleic acid into a cell and expression of nucleic acids are provided in Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, and Sambrook et al., in Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989. Techniques for chemical synthesis of polypeptides are also well known in the art. (See e.g., Vincent in Peptide and Protein Drug Delivery, New York, N.Y., Dekker, 1990.) For example, the peptides of this invention can be prepared by standard solid phase peptide synthesis. (See, e.g., Stewart, J. M., et al., Solid Phase Synthesis (Pierce Chemical Co., 2d ed. 1984)) Physical data for the exemplified peptides are given in Table 1.
Preparation of Pamoate Salt of Example 1
The acetate salt of Example 1 (200 mg, 0.22 mmole) was dissolved in 10 mL of water. Sodium pamoate (190 mg, 0.44 mmole) was dissolved in 10 mL of water. The two solutions were combined and mixed well. The precipitates were collected by centrifugation at 3000 rpm for 20 minutes, washed for three times with water, and dried by lyophilization.
Compounds of the present invention can be and were tested for activity as ligands of the GHS receptor according to the following procedures. One skilled in the art would know that procedures similar to those described herein may be used to assay the binding activities of the compounds of the invention to melanocortin receptor molecules.
Radioligand Binding Assays
Cellular membranes used for the in vitro receptor binding assay were obtained from transgenic CHO-K1 cells stably expressing the human recombinant GHS receptor. CHO-K1 cells stably expressing the hGHS receptor were homogenized in 20 ml of ice-cold 50 mM Tris-HCl with a Brinkman Polytron (Westbury, N.Y., USA) (setting 6, 15 sec). The homogenates were washed twice by centrifugation (39,000 g/10 min), and the final pellets were resuspended in 50 mM Tris-HCl, containing 2.5 mM MgCl2, and 0.1% BSA. For assay, aliquots (0.4 ml) were incubated with 0.05 nM (125I)ghrelin (˜2000 Ci/mmol, Perkin Elmer Life Sciences, Boston, Mass., USA), with and without 0.05 ml of unlabeled competing test compounds of the invention. After a 60 min incubation (4° C.), the bound (125I)ghrelin was separated from the free by rapid filtration through GF/C filters (Brandel, Gaithersburg, Md., USA), which had been previously soaked in 0.5% polyethyleneimine/0.1% BSA. The filters were then washed three times with 5-ml aliquots of ice-cold 50 mM Tris-HCl and 0.1% bovine serum albumin, and the bound radioactivity trapped on the filters was counted by gamma spectrometry (Wallac LKB, Gaithersburg, Md., USA). Specific binding was defined as the total (125I)ghrelin bound minus that bound in the presence of 1000 nM ghrelin (Bachem, Torrence, Calif., USA). Specific binding data for the exemplified peptides are given in Table 2.
Solubility Study
Pamoate salt of Example 1 (50 mg) was weighted into a microcentrifuge tube, and 125 μL of PEG200 and 125 μL of water were added thereafter. The mixture was sonicated to facilitate dissolution. A clear solution was obtained.
The solubility of pamoate salt of Example 1 was determined by mixing the peptide in water or PBS, followed by HPLC determination of the concentration in the supernatant, and the results are shown in Table 3.
Pharmacokinetic Studies of Formulations of Example 1
“Formulation 1” of Example 1 was prepared by dissolving a pamoate salt of Example 1 in a 50% PEG200 and 50% water (v/v) solution, at a concentration of 200 mg/mL (20% w/v).
“Formulation 2” of Example 1 was prepared by dissolving an acetate salt of Example 1 in a saline/2% heat inactivated mouse serum/5% DMA/2% tween-80 solution.
Dosing
For Formulation 1, Sprague-Dawley rats were dosed via subcutaneous injection at a fixed amount of either 5 μL/rat or 1.0 mg/rat, or a variable amount of roughly 2.5 mg/kg body weight.
For Formulation 2, Sprague-Dawley rats were dosed via subcutaneous injection at a variable amount of 2.1 mg/kg body weight.
Sample Preparation
For Formulations 1 and 2, 50 μL of plasma was acidified with 2.5 μL of formic acid and precipitated with 150 μL of acetonitrile. The supernatant was collected by centrifugation. 50 μL of the preparation was injected for LC-MS/MS analysis.
LC-MS/MS Analysis
For Formulations 1 and 2, LC-MS/MS analysis was performed with an API4000 mass spectrometer system equipped with a Turbo Ionspray probe. The MRM mode of molecular ion detection with an ion pair of 396.5/112.3 was used. HPLC separation was performed with a Luna C8(2) 2×30 mm 3μ column run from 0% B to 80% B in 10 minutes at a flow rate of 0.3 mL/minute. Buffer A is 1% formic acid in water and buffer B is 1% formic acid in acetonitrile. LOQ was 5 ng/mL.
The plasma concentrations of Example 1, dosed with Formulation 1, were calculated with its standard calibration plot and the results are shown in Table 4.
Full time course plots of the pharmacokinetic profiles of Formulation 1 are shown on a normal scale in
Some pharmacokinetic parameters of Example 1, dosed with Formulation 1, are shown in Table 5.
The results indicate that the formulations of Example 1 according to the present invention as described herein provide for acceptable sustained release formulations with improved pharmacokinetic parameters and flatter release profiles which may result in attenuated side effects and improved efficacy. For instance, Formulation 1 is shown to have a release profile of greater than 24 hours after a single subcutaneous injection, with significantly low Cmax and significantly long Tmax. Moreover, the pamoate salt formulation of Example 1, i.e., Formulation 1, is shown to have significantly increased T1/2 compared to the acetate salt formulation of Example 1, i.e., Formulation 2.
Additional embodiments of the present invention will be apparent from the foregoing disclosure and are intended to be encompassed by the invention as described fully herein and defined in the following claims.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US11/28283 | 3/14/2011 | WO | 00 | 10/11/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61340290 | Mar 2010 | US |
