Patent Application
20100331264
1. Field of the Invention
The present invention is directed to a method of preserving the α-helix secondary structure of certain peptides during freeze-drying, as well as to freeze-dried formulations of such peptides made according to the method.
2. Related Background Art
APP018 and APL180 are known apolipoprotein (apo) A-I mimetics and are disclosed in U.S. Pat. Nos. 6,664,230 and 6,933,279 and WO 2004/034977, respectively. Each of these peptides comprises an 18 amino acid sequence, namely D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F (Ac-Asp-Trp-Phe-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Phe-Lys-Glu-Ala-Phe-NH2—SEQ ID NO 1), having an acetyl amino-terminal protecting group and an amide carboxyl-terminal protecting group. In free (unbound) form, when all amino acids are in D-form, the peptide is known as APP018; when all the amino acids are in L-form, the free (unbound) peptide is known as APL180. These peptides have four phenylalanines and are sometimes referred to as “D4F” when the amino acids are all in the D form or “L4F” when the amino acids are all in the L form. Reverse “4F” is a mirror image of 4F with the relative positions of the amino acids to each other and to the hydrophilic and hydrophobic faces being identical. Similarly, peptides in this group contain two phenylalanines, known as 2F, three phenylalanines, known as 3F, five phenylalanines, 5F, six phenylalanines, 6F and seven phenylalanines or 7F. It is possible to have mirror images or reverse peptides based on these peptides also.
All these peptides, have been shown to inhibit low density lipoprotein (LDL) oxidation, stimulate reverse cholesterol transport, and reduce formation of atherosclerotic lesion. Accordingly, these agents are useful in the treatment of cardiovascular disease which remains a leading cause of morbidity and mortality, particularly in the United States and in Western European countries. Hence, effective formulation of these peptides is highly desirable.
Exchangeable apolipoproteins, including apo A-I, possess lipid-associating domains (Brouillette at al., Biochim. Biophys. Acta 1256:103-129 (1995); Segrest et al., FEBS Lett. 38::247-253 (1974)). Apo A-I has been postulated to possess eight tandem repeating 22 mer sequences. Characteristics of the class A amphipathic helix include the presence of positively charged residues at the polar-nonpolar interface and negatively charged residues at the center of the polar face (Id.; Segrest et al., Proteins: Structure, Function, and Genetics 8: 103-117 (1990)). Apo A-I has been shown to strongly associate with phospholipids to form complexes and to promote cholesterol efflux from cholesterol-enriched cells. It has now been shown that the secondary structure of apo A-I is essential for high affinity binding to lipids, ultimately leading to its biological activity (Saito et al., J. Biol. Chem. 279(20): 20974-20981 (2004)). Hence, preservation of the secondary structure of apo A-I is highly desirable. Without limiting the invention to a particular mechanism of action, it may be that preservation of the α-helix conformation may be necessary for is important for giving apo-I its binding affinity to lipids.
The invention of U.S. Pat. No. 6,664,230 which provided novel peptides comprising 18 amino acids having a class A amphipathic helix when formulated with “D” amino acid residue(s) and/or having protected amino and carboxyl termini which when orally administered to an organism, are readily taken up and delivered to the serum, and are effective to mitigate one or more symptoms of atherosclerosis.
Freeze-drying proteins is a common approach to improve both chemical and physical stability of the protein. However, freezing and dehydration stress can cause protein aggregation, leading to a loss of it's bioactivity. Trehalose, α-D-glucopyranosyl-α-D-glucopyranoside, is a naturally occurring disaccharide, which has been shown to be useful in preventing denaturation of proteins and other macromolecules, viruses and foodstuffs during drying processes. See, e.g., U.S. Pat. Nos. 4,891,319, 5,149,653, 5,026,566, 5,902,565 and 6,890,512. EP 0 762 897, while indicating that the method of preventing aggregation disclosed therein is applicable to both proteins and peptides, exemplifies its method with human growth hormone only. Trehalose has also been extensively studied as a protein stabilizer in the literature (Kaushik et al., J. Bio. Chem. 278 (29): 26458-26465 (2003)). To date, no suggestion that trehalose may be effective in preserving peptide secondary structure has been noted in the prior art.
Accordingly, a method of preserving the α-helix (secondary) structure of APP018 and APL180 during freeze-drying by trehalose would be desirable.
The present invention is directed to a method of preserving secondary structure during freeze-drying of a peptide comprising the steps of: (a) admixing trehalose with the peptide in a solution, said trehalose in an amount sufficient to preserve secondary structure of the peptide; and (b) freeze-drying the solution or suspension to obtain a peptide composition in which secondary structure has been preserved, wherein the peptide is selected from N-Acetyl-D-Asp-D-Trp-D-Phe-D-Lys-D-Ala-D-Phe-D-Tyr-D-Asp-D-Lys-D-Val-D-Ala-D-Glu-D-Lys-D-Phe-D-Lys-D-Glu-D-Ala-D-Phe-Amide (D4F); N-Acetyl-L-Asp-L-Trp-L-Phe-L-Lys-L-Ala-L-Phe-L-Tyr-L-Asp-L-Lys-L-Val-L-Ala-L-Glu-L-Lys-L-Phe-L-Lys-L-Glu-L-Ala-L-Phe-Amide (L4F), D3F, L3F, D5F, L5F, D6F, L6F, D7F and L7F or any pharmaceutically acceptable salt form thereof. In a preferred embodiment of this aspect of the invention, the peptide is L4F. The invention is further directed to a method further comprising the step of: (c) reconstituting the peptide composition to obtain a solution or suspension of the peptide in which secondary structure has been preserved.
In certain preferred embodiments of the invention, the secondary structure is an α-helix structure. In other preferred embodiments, the solution or suspension of step (a) further comprises at least one additional freeze-drying excipient such as buffer or surfactant.
The present invention is further directed to freeze-dried and reconstituted compositions made according to the method of the invention.
The present invention is still further directed to a freeze-dried composition comprising a peptide and an amount of trehalose sufficient to preserve secondary structure of the peptide, wherein the peptide is selected from N-Acetyl-D-Asp-D-Trp-D-Phe-D-Lys-D-Ala-D-Phe-D-Tyr-D-Asp-D-Lys-D-Val-D-Ala-D-Glu-D-Lys-D-Phe-D-Lys-D-Glu-D-Ala-D-Phe-Amide; N-Acetyl-L-Asp-L-Trp-L-Phe-L-Lys-L-Ala-L-Phe-L-Tyr-L-Asp-L-Lys-L-Val-L-Ala-L-Glu-L-Lys-L-Phe-L-Lys-L-Glu-L-Ala-L-Phe-Amide, D3F, L3F, D5F, L5F, D6F, L6F, D7F and L7F or any pharmaceutically acceptable salt form thereof.
The present invention is directed to a method of preserving secondary structure during freeze-drying of a peptide. As used herein, “secondary structure” refers to the general three-dimensional form of biomolecules such as peptides or of segments of biomolecules, such as proteins and nucleic acids; for purposes of the present invention, “secondary structure” preferably refers to the α-helix structure of certain peptides. As used herein, “preserving” (and other forms thereof) refers to keeping intact. Preserving preferably refers to maintainence or improvement (increase) of the α-helix content of certain peptides—in other words, the α-helix content of a particular freeze-dried composition made according to the method of the present invention will be greater than that of a freeze-dried composition made according to conventional processes. As used herein, “freeze-drying” (and other forms thereof) refers to any process by which water is removed from a material which is first frozen and then subjected to reduced pressure and/or heat which allows the water to sublime directly from the solid phase to gas.
More specifically, the first embodiment of the present invention comprises the steps of: (a) admixing trehalose with the peptide in a solution or suspension, said trehalose in an amount sufficient to preserve secondary structure of the peptide; and (b) freeze-drying the solution or suspension to obtain a peptide composition in which secondary structure has been preserved, wherein the peptide is selected from N-Acetyl-D-Asp-D-Trp-D-Phe-D-Lys-D-Ala-D-Phe-D-Tyr-D-Asp-D-Lys-D-Val-D-Ala-D-Glu-D-Lys-D-Phe-D-Lys-D-Glu-D-Ala-D-Phe-Amide; N-Acetyl-L-Asp-L-Trp-L-Phe-L-Lys-L-Ala-L-Phe-L-Tyr-L-Asp-L-Lys-L-Val-L-Ala-L-Glu-L-Lys-L-Phe-L-Lys-L-Glu-L-Ala-L-Phe-Amide, D3F, L3F, D5F, L5F, D6F, L6F, D7F and L7F or any pharmaceutically acceptable salt form thereof. Preferably, the peptide is the free form. N-Acetyl-L-Asp-L-Trp-L-Phe-L-Lys-L-Ala-L-Phe-L-Tyr-L-Asp-L-Lys-L-Val-L-Ala-L-Glu-L-Lys-L-Phe-L-Lys-L-Glu-L-Ala-L-Phe-Amide. The individual peptides are generally referred to herein as a peptides of the invention.
In the first step of the inventive method, trehalose is admixed with a peptide of the invention in a solution.
Trehalose is a commercially available material and can be purchased from any source. Either the N-Acetyl-D-Asp-D-Trp-D-Phe-D-Lys-D-Ala-D-Phe-D-Tyr-D-Asp-D-Lys-D-Val-D-Ala-D-Glu-D-Lys-D-Phe-D-Lys-D-Glu-D-Ala-D-Phe-Amide; N-Acetyl-L-Asp-L-Trp-L-Phe-L-Lys-L-Ala-L-Phe-L-Tyr-L-Asp-L-Lys-L-Val-L-Ala-L-Glu-L-Lys-L-Phe-L-Lys-L-Glu-L-Ala-L-Phe-Amide peptide, or any peptide of the invention, can be purchased from commercial sources or made according to known procedures as described in U.S. Pat. Nos. 6,664,230 and 6,933,279 and PCT International Publication No. WO 2004/034977, the entire disclosure of each of which is incorporated by reference herein.
Trehalose and a peptide of the invention are admixed in water. According to the present invention, trehalose may be added to a solution of a peptide of the invention, the peptide may be added to a solution of trehalose or both trehalose and the peptide may be added to a solvent to form a solution in step (a). The pH is adjusted to a range of from about 3 to 11, more preferably 6 to 9, even more preferably 6.5 to 9. Preferably a surfactant, including but not limited to TWEEN 80, is added prior to the addition of the peptide, TWEEN 80 is present in an amount ranging preferably from about 0.0001% to about 10% weight by volume.
The amount of trehalose sufficient to preserve secondary structure of the peptide corresponds to a range preferably from about 1 to about 50%, more preferably from about 10 to 25% weight by volume, with about 10% weight by volume being most preferred. This corresponds to a weight ratio of trehalose to peptide range of from about 500:0.01 to about 10:200, preferably of from about 250:0.2 to about 100:30 and most preferably about 100:0.2 to about 100:30.
Admixing can be accomplished by any conventional means, i.e., simple mixture.
In a preferred embodiment of the present invention, the solution of step (a) further comprises at least one additional buffer. Buffers suitable for use in the present invention include, without limitation, sodium phosphate, for example mono or di sodium phosphate, potassium phosphate, Tris, citrate, tartrate and histidine and combinations thereof. When present, phosphate buffer concentration corresponds to a range preferably from about 1 mM to about 1 M of the solution of step (a), preferably from about 5 mM to about 100 mM.
In the second step of the inventive method, the solution or suspension is freeze-dried to obtain a peptide composition in which secondary structure has been preserved. Freeze-drying can be accomplished by any known means. For example, freeze-drying may involve the use of a freeze-drying flask which is rotated in a bath, which is cooled by mechanical refrigeration, dry ice and methanol, or liquid nitrogen or may involve the use of a large-scale freeze-drying machine. As a result of freeze-drying the combination of trehalose and peptide, the secondary structure of the peptide in the peptide composition will have been preserved. In other words, the peptide composition of step (b) has a high α-helix content as compared to a peptide composition which was freeze-dried without the use of trehalose.
An optional step for the first embodiment of the invention comprises (c) reconstituting the peptide composition to obtain a solution of the peptide in which secondary structure has been preserved. Reconstitution can be accomplished by any known means such as by the simple addition of water to the peptide composition of step (b). As one of ordinary skill in the art will readily appreciate, solutions of varying peptide concentration can be achieved by reconstitution with varying amounts of solvent. Solvents suitable for use in step (c) include, without limitation, water, buffer solution or isotonic solution. As a result of the reconstitution of the freeze-dried peptide composition, the secondary structure of the peptide will have been preserved. In other words, the solution of step (c) has a high peptide secondary structure content, and possibly a high α-helix content as compared to a solution or suspension which was reconstituted from a freeze-dried composition which did not use trehalose in accordance with this invention.
Additional embodiments of the invention are directed to freeze-dried composition and reconstituted compositions made according to the method of the first embodiment of the invention.
Yet another embodiment of the invention is directed to a freeze-dried composition comprising a peptide which is N-Acetyl-D-Asp-D-Trp-D-Phe-D-Lys-D-Ala-D-Phe-D-Tyr-D-Asp-D-Lys-D-Val-D-Ala-D-Glu-D-Lys-D-Phe-D-Lys-D-Glu-D-Ala-D-Phc-Amide; N-Acetyl-L-Asp-L-Trp-L-Phe-L-Lys-L-Ala-L-Phe-L-Tyr-L-Asp-L-Lys-L-Val-L-Ala-L-Glu-L-Lys-L-Phe-L-Lys-L-Glu-L-Ala-L-Phe-Amide, D3F, L3F, D5F, L5F, D6F, L6F, D7F and L7F or any pharmaceutically acceptable salt form thereof and an amount of trehalose sufficient to preserve the secondary structure of the peptide, Details regarding the amounts of peptide and trehalose are the same as those noted above with regard to the first embodiment of the invention.
Specific embodiments of the invention will now be demonstrated by reference to the following examples. It should be understood that these examples are disclosed solely by way of illustrating the invention and should not be taken in any way to limit the scope of the present invention.
Freeze-dried compositions of N-Acetyl-L-Asp-L-Trp-L-Phe-L-Lys-L-Ala-L-Phe-L-Tyr-L-Asp-L-Lys-L-Val-L-Ala-L-Glu-L-Lys-L-Phe-L-Lys-L-Glu-L-Ala-L-Phe-Amide (APL180) were made using the ingredients noted in Table 1 below.
Formulations 1 and 3 were made for 1 mg/ml and formulations 2 and 4 for 100 mg/ml APL180. Both concentrations contain 15 mM phosphate buffer pH 7 and 10% trehalose. Formulation 3 and 4 also contains 0.5% TWEEN 80. The solution of 1 mg/ml APL180 is prepared, filled at 1 ml per vial, freeze-dried, and reconstituted with 1 ml water prior to use. The formulation of 100 mg/ml APL180 is prepared at 25 mg/ml APL180 solution, filled at 2 ml per vial, freeze-dried, and reconstituted with 0.5 ml water prior to use. Hence, other ingredients in the solution of 25 mg/ml APL180 are formulated at 25% of the final concentration intended after reconstitution.
Lyophilization cycle is performed as follows:
Freeze-dried compositions of N-Acetyl-L-Asp-L-Trp-L-Phe-L-Lys-L-Ala-L-Phe-L-Tyr-L-Asp-L-Lys-L-Val-L-Ala-L-Glu-L-Lys-L-Phe-L-Lys-L-Glu-L-Ala-L-Phe-Amide (APL180) were made using the ingredients noted in Table 3 below and then reconstituted as noted and tested using Fourier transform infrared spectrometry to determine the % α and % β helices.
6 mg APL180 drug product is formulated as sterile, lyophilized powder for intravenous administration. The composition of each vial is provided in Table 4. Each vial is overfilled with 2.2 ml of bulk solution before lyophilization and reconstituted with 2 ml of water for injection (WFI) before administration. Two ml of reconstituted solution will deliver 6 mg APL180.
Primary packaging components
Bulk liquid formulation composition
Manufacturing Procedures
Preparation of APL180 Formulated Bulk Solution (Compounding)
Lyophilization of APL180 Formulation
While the invention has been described above with reference to specific embodiments thereof, it is apparent that many changes, modifications, and variations can be made without departing from the inventive concept disclosed herein. Accordingly, it is intended to embrace all such changes, modifications, and variations that fall within the spirit and broad scope of the appended claims. All patent applications, patents, and other publications cited herein are incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US08/69456 | 7/9/2008 | WO | 00 | 1/7/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60948525 | Jul 2007 | US | |
| 60955480 | Aug 2007 | US |
