Peptide manufacturing process

Abstract

A large-scale preparative process for preparing the described cyclic peptides, comprising solid-phase peptide synthesis of a linear peptide, cleaving it from the resin, oxidizing cysteine ​​residues to form intramolecular disulfide bonds, and isolating the cyclic peptide, wherein (i) coupling uses diisopropylcarbodiimide and ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzotriazole, (ii) cleavage involves contacting the peptide with a solution comprising TFA and dithioerythritol and / or dithiothreitol, (ii) cleavage comprises contacting the peptide with a solution comprising TFA and dithioerythritol and / or dithiothreitol, and (iii) coupling the peptide with diisopropylcarbodiimide and / or N-hydroxybenzotriazole, (iv) coupling the peptide with diisopropylcarbodiimide and / or N-hydroxybenzotriazole, (v) cleavage the peptide with dithioerythritol and / or N-hydroxybenzotriazole, (vi) cleavage the peptide with dithioerythritol and / or N-hydroxybenzotriazole, (vii) coupling the peptide with dithioerythritol and / or N-hydroxybenzotriazole, (v) cleavage ... The present invention describes a process in which i) the peptide is precipitated after cleavage without prior concentration of the peptide by evaporation; (iv) the oxidation comprises contacting an aqueous solution containing at least 5 mg / mL of the peptide with hydrogen peroxide; (v) the isolation comprises loading the peptide onto a reverse phase chromatography column at up to 40 grams / kg and elution from the column; (vi) the isolation comprises lyophilization followed by trituration of the peptide; and / or (vii) the substitution of the resin is at least 0.3 milliequivalents / gram and / or the resin is a Rink aminomethylstyrene resin.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 131,873, filed December 30, 2020, the contents of which are incorporated by reference in their entirety into this application.

[0002] SEQUENCE LISTING STATEMENT An ASCII file entitled 89546SequenceListing.txt, containing 4,096 bytes and created on December 27, 2021, and filed concurrently with the filing of this application, is incorporated herein by reference.

[0003] The present invention, in some embodiments thereof, relates to chemical synthesis and more particularly, but not exclusively, to novel processes for the preparation of peptides such as BL-8040, which may be used, for example, in treating conditions such as cancer and / or arthritis. [Background technology]

[0004] BL-8040 is 4F-benzoyl-TN14003 (4-fluorobenzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH 2 , SEQ ID NO:1), which is cyclic upon formation of a disulfide bond between its two Cys residues.

[0005] US Patent No. 7,423,007 describes peptides with CXC4 antagonism, including 4F-benzoyl-TN14003. According to the teachings of US Patent No. 7,423,007, 4F-benzoyl-TN14003 is prepared by solid phase synthesis in DMF using DIPCDI-HOBt as coupling agent (amino acid is added in 2.5 equivalents), followed by deprotection and cleavage using 1M TMSBr-thioanisole / TFA with m-cresol and ethanedithiol, and cyclization by air oxidation.

[0006] Other background art includes that described in U.S. Pat. Nos. 7,138,488 and 8,435,939, and international patent application publications WO 2008 / 075369, WO 2008 / 075370, WO 2010 / 146578, WO 2012 / 095849, and WO 2013 / 160895. Summary of the Invention

[0007] According to an aspect of some embodiments of the present invention, there is provided a process for large scale preparation of a cyclic peptide having SEQ ID NO:1, or a pharma- ceutically acceptable salt thereof, the process comprising: (a) sequentially coupling an amino acid and 4-fluorobenzoic acid to a resin by solid phase peptide synthesis, thereby obtaining a resin-coupled linear peptide; (b) cleaving the linear peptide from the resin, thereby obtaining a free linear peptide; (c) oxidizing cysteine ​​residues of the linear peptide to form intramolecular disulfide bonds, thereby obtaining a cyclic peptide having SEQ ID NO:1 in solution; (d) isolating a cyclic peptide having SEQ ID NO:1 or a pharma- ceutically acceptable salt thereof; Including, (i) coupling is carried out using diisopropylcarbodiimide (DIC) in combination with ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzotriazole; (ii) cleavage is carried out by contacting the resin-coupled linear peptide with a solution comprising trifluoroacetic acid (TFA) and a scavenger selected from the group consisting of dithioerythritol (DTE) and dithiothreitol (DTT); (iii) the process further comprises precipitating the free linear peptide after cleavage without concentrating the free linear peptide by evaporation prior to precipitation; (iv) the oxidation is carried out by contacting an aqueous solution containing the linear peptide at a concentration of at least 5 mg / mL with hydrogen peroxide; (v) isolating comprises loading the cyclic peptide onto a reverse phase chromatography column at a concentration of 40 grams of cyclic peptide or less per kilogram of column; and eluting the cyclic peptide from the column; (vi) isolating the cyclic peptide having SEQ ID NO:1 comprises lyophilization, and the process further comprises grinding the cyclic peptide following lyophilization; and / or (vii) the degree of substitution of the resin is at least 0.3 milliequivalents per gram, and / or the resin is a Rink aminomethylstyrene (AMS) resin.

[0008] According to an aspect of some embodiments of the present invention, there is provided a process for large scale preparation of a cyclic peptide having SEQ ID NO:1, or a pharma- ceutically acceptable salt thereof, the process comprising: (a) sequentially coupling amino acids and 4-fluorobenzoic acid to a resin by solid phase peptide synthesis using diisopropylcarbodiimide (DIC) in combination with ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzotriazole, thereby obtaining a resin-coupled linear peptide; (b) cleaving the linear peptide from the resin, thereby obtaining a free linear peptide; (c) oxidizing cysteine ​​residues of the linear peptide to form intramolecular disulfide bonds, thereby obtaining a cyclic peptide having SEQ ID NO:1 in solution; (d) isolating a cyclic peptide having SEQ ID NO:1 or a pharma- ceutically acceptable salt thereof; Includes.

[0009] According to an aspect of some embodiments of the present invention, there is provided a process for large scale preparation of a cyclic peptide having SEQ ID NO:1, or a pharma- ceutically acceptable salt thereof, the process comprising: (a) sequentially coupling an amino acid and 4-fluorobenzoic acid to a resin by solid phase peptide synthesis, thereby obtaining a resin-coupled linear peptide; (b) cleaving the resin-coupled linear peptide from the resin by contacting the linear peptide with a solution comprising trifluoroacetic acid (TFA) and a scavenger selected from the group consisting of dithioerythritol (DTE) and dithiothreitol (DTT), thereby obtaining a free linear peptide; (c) oxidizing cysteine ​​residues of the linear peptide to form intramolecular disulfide bonds, thereby obtaining a cyclic peptide having SEQ ID NO:1 in solution; (d) isolating a cyclic peptide having SEQ ID NO:1 or a pharma- ceutically acceptable salt thereof; Includes.

[0010] According to an aspect of some embodiments of the present invention, there is provided a process for large scale preparation of a cyclic peptide having SEQ ID NO:1, or a pharma- ceutically acceptable salt thereof, the process comprising: (a) sequentially coupling an amino acid and 4-fluorobenzoic acid to a resin by solid phase peptide synthesis, thereby obtaining a resin-coupled linear peptide; (b) cleaving the linear peptide from the resin, thereby obtaining the free linear peptide, and precipitating the free linear peptide after cleavage without concentrating the free linear peptide by evaporation prior to precipitation; (c) oxidizing cysteine ​​residues of the linear peptide to form intramolecular disulfide bonds, thereby obtaining a cyclic peptide having SEQ ID NO:1 in solution; (d) isolating a cyclic peptide having SEQ ID NO:1 or a pharma- ceutically acceptable salt thereof; Includes.

[0011] According to an aspect of some embodiments of the present invention, there is provided a process for large scale preparation of a cyclic peptide having SEQ ID NO:1, or a pharma- ceutically acceptable salt thereof, the process comprising: (a) sequentially coupling an amino acid and 4-fluorobenzoic acid to a resin by solid phase peptide synthesis, thereby obtaining a resin-coupled linear peptide; (b) cleaving the linear peptide from the resin, thereby obtaining a free linear peptide; (c) oxidizing cysteine ​​residues of the linear peptide to form intramolecular disulfide bonds, which is accomplished by contacting an aqueous solution containing the linear peptide at a concentration of at least 5 mg / mL with hydrogen peroxide, thereby obtaining a cyclic peptide having SEQ ID NO:1 in solution; (d) isolating a cyclic peptide having SEQ ID NO:1 or a pharma- ceutically acceptable salt thereof; Includes.

[0012] According to an aspect of some embodiments of the present invention, there is provided a process for large scale preparation of a cyclic peptide having SEQ ID NO:1, or a pharma- ceutically acceptable salt thereof, the process comprising: (a) sequentially coupling an amino acid and 4-fluorobenzoic acid to a resin by solid phase peptide synthesis, thereby obtaining a resin-coupled linear peptide; (b) cleaving the linear peptide from the resin, thereby obtaining a free linear peptide; (c) oxidizing cysteine ​​residues of the linear peptide to form intramolecular disulfide bonds, thereby obtaining a cyclic peptide having SEQ ID NO:1 in solution; (d) isolating a cyclic peptide having SEQ ID NO:1 or a pharma- ceutically acceptable salt thereof, the isolating comprising loading the cyclic peptide onto a reverse phase chromatography column at a concentration of 40 grams of cyclic peptide or less per kg of column, and eluting the cyclic peptide from the column; Includes.

[0013] According to an aspect of some embodiments of the present invention, there is provided a process for large scale preparation of a cyclic peptide having SEQ ID NO:1, or a pharma- ceutically acceptable salt thereof, the process comprising: (a) sequentially coupling an amino acid and 4-fluorobenzoic acid to a resin by solid phase peptide synthesis, thereby obtaining a resin-coupled linear peptide, the degree of substitution of the resin being at least 0.3 milliequivalents per gram, and / or the resin being a Rink aminomethylstyrene (AMS) resin; (b) cleaving the linear peptide from the resin, thereby obtaining a free linear peptide; (c) oxidizing cysteine ​​residues of the linear peptide to form intramolecular disulfide bonds, thereby obtaining a cyclic peptide having SEQ ID NO:1 in solution; (d) isolating a cyclic peptide having SEQ ID NO:1 or a pharma- ceutically acceptable salt thereof; Includes.

[0014] According to some of the embodiments of each of the options described herein, the coupling is carried out using diisopropylcarbodiimide (DIC) in combination with ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzotriazole.

[0015] According to some of the embodiments described herein relating to coupling with DIC and ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzotriazole, the DIC and ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzotriazole are used in about a 2-fold molar excess.

[0016] According to some of the various embodiments described herein, cleavage is performed by contacting the resin-coupled linear peptide with a solution comprising trifluoroacetic acid (TFA) and a scavenger selected from the group consisting of dithioerythritol (DTE) and dithiothreitol (DTT).

[0017] According to any of the embodiments described herein relating to cleavage with a solution comprising a scavenger selected from the group consisting of dithioerythritol and dithiothreitol, the concentration of the scavenger in the solution is in the range of 10 mg / mL to 500 mg / mL.

[0018] According to some of the embodiments described herein relating to cleavage with a solution comprising dithiothreitol, the concentration of dithiothreitol in the solution is about 50 mg / mL.

[0019] According to some of the embodiments of each of the options described herein, the process further comprises precipitating the free linear peptide after cleavage without concentrating the free linear peptide by evaporation prior to precipitation.

[0020] According to some of the optional embodiments described herein relating to precipitation of linear peptides, precipitation is carried out by addition of a mixture of tert-butyl methyl ether (MTBE) and hexane in a volume of about 45 mL of the mixture per gram of resin.

[0021] According to some of the embodiments of each of the methods described herein, the oxidation is carried out by contacting the linear peptide with hydrogen peroxide.

[0022] According to any of the embodiments described herein relating to contacting a linear peptide with hydrogen peroxide, the contacting is performed by contacting an aqueous solution containing the linear peptide at a concentration of at least 5 mg / mL with hydrogen peroxide.

[0023] According to some of the embodiments of each of the methods described herein, the isolating comprises loading the cyclic peptide onto a reverse phase chromatography column at a concentration of 40 grams of cyclic peptide or less per kg of column, and eluting the cyclic peptide from the column.

[0024] According to some of the embodiments described herein relating to a reverse phase chromatography column, the column is a C18 column.

[0025] According to some of the embodiments described herein relating to elution, elution is performed with a triethylammonium phosphate solution.

[0026] According to some of the embodiments of each of the methods described herein, isolating the cyclic peptide having SEQ ID NO:1 includes lyophilization, and the process further includes grinding the cyclic peptide following lyophilization.

[0027] According to some of the various embodiments described herein, the degree of substitution of the resin solid phase peptide synthesis is at least 0.3 milliequivalents per gram and / or the resin is a Rink aminomethylstyrene (AMS) resin.

[0028] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the present patent specification, including definitions, shall control. In addition, the materials, methods, and examples are merely illustrative and are not necessarily intended to be limiting.

[0029] Implementation of the method and / or system of the present invention may require selected tasks to be performed or completed manually, automatically, or a combination thereof. Moreover, depending on the actual instrumentation and apparatus of the method and / or system of the present invention, some selected tasks may be implemented in hardware, software, or firmware, or a combination thereof using an operating system.

[0030] For example, hardware for performing selected tasks according to embodiments of the present invention may be implemented as a chip or circuit. As software, selected tasks according to embodiments of the present invention may be implemented as a number of software instructions executed by a computer using any suitable operating system. In an exemplary embodiment of the present invention, one or more tasks according to exemplary embodiments of the methods and / or systems described herein are performed by a data processor, such as a computing platform for executing a number of instructions. Optionally, the data processor includes a volatile memory for storing instructions and / or data and / or a non-volatile storage, e.g., a magnetic hard disk and / or a removable medium, for storing instructions and / or data. Optionally, a network connection is provided as well. A display and / or a user input device, e.g., a keyboard or mouse, are also optionally provided as well.

[0031] Several embodiments of the present invention are herein described, by way of example only, with reference to the accompanying drawings, in which: With specific reference now made in detail to the drawings, it is stressed that the particulars shown are given by way of example and for the purpose of illustrative discussion of embodiments of the present invention. In this connection, the description given with the aid of the drawings will make apparent to those skilled in the art how embodiments of the present invention may be put into practice. [Brief description of the drawings]

[0032] [Figure 1] FIG. 1 provides a flow diagram of the solid phase synthesis portion of the process for the preparation of BL-8040 according to some exemplary embodiments of the present invention, including sequential cycles for coupling of Cys, Cit (citrulline), Arg, Tyr, Pro, D-Lys, Lys, Cit, Tyr, Cys, Nal (naphthylalanine), Arg, Arg, and Fba (4-fluoro-benzoic acid) to the resin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] The present invention, in some embodiments thereof, relates to chemical synthesis and, more particularly, to novel processes for the preparation of peptides such as BL-8040, which may be used, for example, but not exclusively, in treating conditions such as cancer and / or arthritis.

[0034] Before describing at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by the examples, as the invention is capable of other embodiments or of being practiced or carried out in various ways.

[0035] Peptides are usually prepared by solid phase synthesis, where the peptide (protected form) is formed while attached to a resin and then cleaved from the resin (removing the protecting groups). BL-8040 (4-fluorobenzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH 2 Intramolecular disulfide bonds such as those in SEQ ID NO:1) are formed by oxidation of Cys residues.

[0036] After laborious experimentation, the present inventors have discovered a novel process that is surprisingly effective for the large-scale synthesis of such peptides, particularly BL-8040.

[0037] According to certain aspects of some embodiments of the present invention, there is provided a process for preparing a cyclic peptide comprising an intramolecular disulfide bond or a pharma- ceutically acceptable salt thereof. In some of the embodiments described herein, the cyclic peptide is represented by SEQ ID NO: 1 (4-fluorobenzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH 2 ) and the Cys residues are linked by an intramolecular disulfide bond. The peptide may also be an analog or derivative of the peptide having SEQ ID NO:1.

[0038] The process involves sequential coupling of amino acids and, optionally, amino acid analogs, if present in the peptide (e.g., N-terminal carboxylic acids such as 4-fluoro-benzoic acid), to a resin (e.g., Rink AMS resin) by solid phase peptide synthesis (following general procedures known in the art) to obtain a resin-coupled linear peptide.

[0039] The process further comprises cleaving the peptide formed by coupling from the resin, thereby obtaining a free (linear) peptide (according to any of the respective embodiments described herein), and oxidizing cysteine ​​residues of the linear peptide to form an intramolecular disulfide bond, thereby obtaining a cyclic peptide (in solution) (according to any of the respective embodiments described herein).

[0040] In some of any of the embodiments described herein, the process is a large-scale process.

[0041] As used herein, the term "large scale process" refers to a process for the preparation of at least 100 grams of final product (in a single run of the process).

[0042] In some of the embodiments of any of the large scale processes described herein, at least 250 grams of cyclic peptide are prepared, or at least 500 grams of peptide, or at least 1 kg of peptide, or at least 3 kg of peptide, or even at least 10 kg of cyclic peptide are prepared by the process.

[0043] Coupling: The order of sequential coupling starts from the C-terminus and ends with the N-terminus. For example, in SEQ ID NO: 1, the order of sequential coupling is Arg, Cys, Cit (citrulline), Arg, Tyr, Pro, DLys (D-lysine), Lys, Cit, Tyr, Cys, Nal (naphthylalanine), Arg, Arg, 4-fluoro-benzoic acid.

[0044] Coupling involves the formation of an amide bond between a carboxylate group (e.g., of an amino acid or an N-terminal carboxylic acid) and an amine group (e.g., of the resin in a first step or at the N-terminus in a subsequent step), and is preferably carried out using one or more coupling reagents known in the art suitable for amide bond formation, such as carbodiimides (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, dicyclohexylcarbodiimide, and / or diisopropylcarbodiimide), aminium / uronium (e.g., HATU, HBTU, TBTU, and / or HCTU), or phosphonium salts (e.g., PyBOP and / or PyAOP) and / or propanephosphonic anhydride.

[0045] In some of any of the embodiments described herein, the one or more coupling reagents include a carbodiimide (e.g., diisopropylcarbodiimide) and an additional agent (e.g., in about the same molar concentration as the carbodiimide), such as a hydroxytriazole (e.g., N-hydroxybenzotriazole (HOBt) or 1-hydroxy-7-azabenzotriazole (HOAt)) and / or a cyanohydroxyiminoacetic acid ester (e.g., ethyl cyanohydroxyiminoacetate).

[0046] In some of the embodiments of any of the embodiments described herein, the coupling is carried out using a carbodiimide and a cyanohydroxyiminoacetate. In some such embodiments, the carbodiimide and / or the cyanohydroxyiminoacetate are used in about a two-fold molar excess (based on the amino acid or other carboxylic acid being coupled).

[0047] In some of the embodiments of any of the embodiments described herein, the coupling is carried out using ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzotriazole with a carbodiimide. In some such embodiments, the carbodiimide and / or ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzotriazole are used in about a two-fold molar excess (based on the amino acid or other carboxylic acid being coupled).

[0048] In some of the embodiments of any of the embodiments described herein, the coupling is carried out using diisopropylcarbodiimide (DIC) with ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzotriazole. In some such embodiments, DIC and ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzotriazole are used in about a two-fold molar excess (based on the amino acid or other carboxylic acid being coupled).

[0049] Without being bound by any particular theory, it is believed that the use of ethyl cyanohydroxyiminoacetate in combination with a carbodiimide (e.g., in the proportions described herein) is particularly suitable for large-scale synthesis of the peptides described herein (e.g., as compared to hydroxytriazoles).

[0050] In each amino acid coupling step, the N-terminus of the amino acid is protected with an easily removable group, preferably fluorenylmethyloxycarbonyl (Fmoc), which may optionally be cleaved with a mild base such as piperidine (e.g., 20-50% piperidine in DMF). An exemplary concentration of piperidine in DMF is about 20%.

[0051] For example, the Fmoc group may optionally be removed by washing with piperidine solution (according to any of the embodiments described herein) twice, optionally once for 5-10 minutes and then optionally once for 25-30 minutes, followed by washing the resin (e.g., with DMF without piperidine) to remove the base (which may optionally be confirmed by determining the pH).

[0052] Additionally, the side chains of certain amino acids may be protected with easily removable groups (not cleaved by the mild bases mentioned above), such as t-butoxy. Thus, the side chain hydroxy groups (e.g., of Tyr, Ser, and Thr) and / or carboxylate groups (e.g., of Asp and Glu) may be preferably protected with t-butyl (t-Bu) (to form a t-butoxy group), and the side chain amine groups (e.g., of Lys, Trp, and His) may be protected with t-butoxycarbonyl (Boc) (containing a t-butoxy group). Additionally, in either case, cleavage (e.g., in an acidic environment) may regenerate the unprotected hydroxy or amine groups. Similarly, the side chain thiohydroxy groups (e.g., of Cys) and / or carboxylate groups (e.g., of Asp and Glu) may be preferably protected with trityl (Trt) and / or the side chain guanidinium groups (e.g., of Arg) may be preferably protected with 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf), each of which may optionally be cleaved in an acidic environment. The above-mentioned side chain protecting groups are known to be particularly compatible with the use of Fmoc. Those skilled in the art will be aware of other protecting groups suitable for specific groups of amino acid side chains and their compatibility with each other and with various N-terminal protecting groups.

[0053] In some of the embodiments described herein, the resin to which the amino acids are sequentially coupled is a Rink aminomethylstyrene (AMS) resin (aminomethylstyrene resin substituted with a Rink linker). The Rink AMS resin is optionally protected with an Fmoc group, which may be removed prior to coupling to expose the amine group (to which the first amino acid is coupled).

[0054] In some of the embodiments described herein, the degree of substitution of the resin (to which the amino acids are sequentially coupled) is at least 0.3 milliequivalents per gram of resin (e.g., 0.3 to 1.1 milliequivalents per gram of resin), optionally 0.6 to 1.1 milliequivalents per gram of resin, optionally 0.6 to 0.9 milliequivalents per gram of resin. In some such embodiments, the resin is a Rink AMS resin.

[0055] The resin is optionally washed (e.g., once or twice) with a solvent such as dimethylformamide (DMF) before each coupling step, e.g., to facilitate swelling of the resin. In some exemplary embodiments, the resin is washed for 20-30 minutes before deprotection of the resin (e.g., removal of the Fmoc group according to any of the respective embodiments described herein) and coupling of the first amino acid, and for about 2 minutes (e.g., twice) before each subsequent coupling step.

[0056] Cutting from resin: Once the resin-mounted peptide has been formed by sequential coupling according to any of the respective embodiments described herein, the resulting peptide is cleaved from the resin (e.g., according to any of the respective embodiments described herein).

[0057] Cleavage of the peptide (and any protecting groups from the amino acid side chains) from the resin is preferably carried out by contacting the peptide (coupled to the resin) with a liquid comprising an acid such as trifluoroacetic acid (TFA). The concentration of TFA in the liquid is optionally at least about 80% by weight, or at least about 90% by weight, or at least about 95% by weight, for example, where the remainder is primarily water. In an exemplary embodiment, the concentration of TFA in the liquid is about 95% by weight, and the remainder is primarily water.

[0058] The liquid used for cleavage (e.g., the acidic liquid according to any of the respective embodiments described herein) optionally further comprises a scavenger (e.g., a scavenger for reactive cationic species that may be formed during cleavage), such as a thiol (e.g., ethanedithiol, dithioerythritol, dithiothreitol), a trialkylsilane (e.g., triisopropylsilane), a phenol (e.g., m-cresol), and / or water.

[0059] In some of any of the embodiments described herein, the scavenger in the liquid used for cleavage (e.g., the acidic liquid according to any of the respective embodiments described herein) is dithiothreitol (DTT) and / or dithioerythritol (DTE). In some such embodiments, the liquid is a solution comprising TFA (e.g., in the concentrations described herein), DTT and / or DTE, preferably in combination with water.

[0060] In some of the embodiments of any of the embodiments described herein relating to DTT and / or DTE, the (total) concentration of DTT and / or DTE in the solution used for cleavage is at least 10 mg / mL. In some such embodiments, the concentration of DTT and / or DTE is in the range of 10-500 mg / mL. In some such embodiments, the concentration of DTT and / or DTE is in the range of 10-200 mg / mL. In some such embodiments, the concentration of DTT and / or DTE is in the range of 10-100 mg / mL. In some such embodiments, the concentration of DTT and / or DTE is in the range of 10-50 mg / mL. An exemplary concentration of DTT is about 50 mg / mL.

[0061] In some of the embodiments of any of the embodiments described herein relating to DTT and / or DTE, the (total) concentration of DTT and / or DTE in the solution used for cleavage is at least 25 mg / mL. In some such embodiments, the concentration of DTT and / or DTE is in the range of 25-500 mg / mL. In some such embodiments, the concentration of DTT and / or DTE is in the range of 25-200 mg / mL. In some such embodiments, the concentration of DTT and / or DTE is in the range of 25-100 mg / mL. In some such embodiments, the concentration of DTT and / or DTE is in the range of 25-50 mg / mL.

[0062] In some of the embodiments of any of the embodiments described herein relating to DTT and / or DTE, the (total) concentration of DTT and / or DTE in the solution used for cleavage is at least 50 mg / mL. In some such embodiments, the concentration of DTT and / or DTE is in the range of 50-500 mg / mL. In some such embodiments, the concentration of DTT and / or DTE is in the range of 50-200 mg / mL. In some such embodiments, the concentration of DTT and / or DTE is in the range of 50-100 mg / mL.

[0063] Once the peptide has been cleaved from the resin, the free peptide is preferably precipitated, for example by addition of a liquid in which the peptide is insoluble. Optionally, precipitation is preceded by concentrating the free peptide by evaporation of a portion of the solvent of the solution to reduce the volume of the solution (for example, by about 65% to about 70%). Alternatively, concentration of the free peptide is not performed prior to precipitation.

[0064] In some of any of the embodiments described herein, the precipitation of the free peptide is carried out without concentrating the free peptide prior to precipitation.

[0065] Precipitation of the peptide according to any of the respective embodiments described herein is optionally carried out by addition of a dialkyl ether, such as tert-butyl methyl ether (MTBE).

[0066] MTBE is optionally mixed with hexane. In some embodiments, the mixture of MTBE and hexane (optionally chilled, for example, to a temperature in the range of about 5° C. to about 15° C.) is added to the peptide in a volume of at least 40 mL of mixture per gram of resin, optionally about 45 mL of mixture per gram of resin. An exemplary mixture of MTBE and hexane is composed of MTBE and hexane in a volume ratio of 60:40 (MTBE:hexane).

[0067] The precipitated peptide is optionally dried, for example by lyophilization or by vacuum. In an exemplary embodiment, drying is performed by vacuum.

[0068] Disulfide bond formation: As discussed herein, intramolecular disulfide bonds are formed by oxidation of cysteine ​​residues of the peptide (which may optionally be formed using any of the embodiments described herein for coupling and any of the embodiments described herein for cleaving the peptide from the resin), resulting in cyclization of the peptide. Any suitable technique known in the art may optionally be used.

[0069] In some of each of the optional embodiments described herein, oxidation is performed by contacting the peptide with hydrogen peroxide, for example, by gradual (e.g., dropwise) addition of a hydrogen peroxide solution (e.g., about 1.5% by weight hydrogen peroxide in water). The molar ratio of hydrogen peroxide to Cys residue pairs (e.g., to a peptide, where the peptide contains exactly two Cys residues as in SEQ ID NO:1) is preferably at least 1:1 (i.e., there is at least one hydrogen peroxide molecule per Cys residue pair), optionally at least 2:1, optionally at least 3:1, and optionally at least 4:1. An exemplary molar ratio of hydrogen peroxide to Cys residue pairs (e.g., to a peptide) is about 5:1. Alternatively or additionally, hydrogen peroxide may be optionally added until no thiohydroxy groups remain (e.g., as determined by a suitable assay such as Ellman's test).

[0070] In some of each of the optional embodiments described herein, the oxidation is carried out by contacting an aqueous solution containing the peptide at a concentration of at least 5 mg / mL with hydrogen peroxide (e.g., according to any of the embodiments described herein relating to contact with hydrogen peroxide). In some such embodiments, the concentration of the peptide is in the range of 5-20 mg / mL. In some embodiments, the concentration of the peptide is in the range of 5-15 mg / mL. In some embodiments, the concentration of the peptide is in the range of 5-10 mg / mL. An exemplary concentration of the peptide is about 10 mg / mL.

[0071] In some of each of the embodiments described herein, the oxidation is carried out by contacting an aqueous solution containing the peptide at a concentration of at least 10 mg / mL with hydrogen peroxide (e.g., according to any of the embodiments described herein relating to contact with hydrogen peroxide). In some such embodiments, the concentration of the peptide is in the range of 10-20 mg / mL. In some embodiments, the concentration of the peptide is in the range of 10-15 mg / mL.

[0072] In some of the embodiments described herein that involve oxidation using an aqueous solution, the aqueous solution is weakly alkaline, such as ammonium bicarbonate (NH 4 HCO 3 ) in water. An exemplary concentration of ammonium bicarbonate is about 0.1M.

[0073] Peptide isolation: The process preferably further comprises isolating the resulting peptide according to any of the respective embodiments described herein (e.g., using a combination of any of the embodiments described herein for coupling, any of the embodiments described herein for cleaving the peptide from the resin, and any of the embodiments described herein for disulfide bond formation). Any suitable technique known in the art may optionally be used.

[0074] In some of the embodiments described herein, isolation of the peptides comprises chromatography, e.g., at least one step (e.g., optionally one or two steps) of preparative high performance liquid chromatography (HPLC). In an exemplary embodiment, a C18 column is used to perform the chromatography (e.g., HPLC).

[0075] In some of any of the embodiments described herein, isolating the peptide comprises loading the peptide (e.g., in at least one step of HPLC as described herein) onto a reverse phase chromatography column (e.g., a C18 column) at a concentration of 40 grams of peptide per kg of column or less (based on the weight of the resin in the column).

[0076] In some of any of the embodiments described herein, isolating the peptide comprises loading the peptide (e.g., in at least one step of HPLC as described herein) onto a reverse phase chromatography column (e.g., a C18 column) at a concentration of at least 4 grams of peptide per kg of column (based on the weight of the resin in the column). In some such embodiments, the loading concentration is in the range of 4-40 grams of peptide per kg of column resin. In some embodiments, the loading concentration is in the range of 4-30 grams of peptide per kg of column resin. In some embodiments, the loading concentration is in the range of 4-25 grams of peptide per kg of column resin.

[0077] In some of any of the embodiments described herein, isolating the peptide comprises loading the peptide (e.g., in at least one step of HPLC as described herein) onto a reverse phase chromatography column (e.g., a C18 column) at a concentration of at least 10 grams of peptide per kg of column (based on the weight of the resin in the column). In some such embodiments, the loading concentration is in the range of 10-40 grams of peptide per kg of column resin. In some embodiments, the loading concentration is in the range of 10-30 grams of peptide per kg of column resin. In some embodiments, the loading concentration is in the range of 10-25 grams of peptide per kg of column resin.

[0078] In some of any of the embodiments described herein, isolating the peptide comprises loading the peptide (e.g., in at least one step of HPLC as described herein) onto a reverse phase chromatography column (e.g., a C18 column) at a concentration of at least 20 grams of peptide per kg of column (based on the weight of the resin in the column). In some such embodiments, the loading concentration is in the range of 20-40 grams of peptide per kg of column resin. In some embodiments, the loading concentration is in the range of 20-30 grams of peptide per kg of column resin. In some embodiments, the loading concentration is in the range of 20-25 grams of peptide per kg of column resin.

[0079] The peptides loaded onto the column (according to any of the respective embodiments described herein) are eluted, for example, with a buffer solution (for example, in combination with an acetonitrile gradient, for example, according to any of the respective embodiments described herein). The pH of the buffer solution is optionally within the range of 1.5-3, optionally 2.0-2.5, optionally about 2.25. In some embodiments, a triethylammonium phosphate buffer is used to elute the peptides from the column, for example, at a concentration of at least about 0.01 M, or at least about 0.03 M, or at least about 0.1 M. In an exemplary embodiment, the concentration of triethylammonium phosphate is about 0.1 M.

[0080] Elution from the HPLC column is optionally performed with an acetonitrile gradient. The gradient optionally comprises increasing the concentration of acetonitrile at a rate (optionally varied over time) within the range of 0.1% acetonitrile / min to 2% acetonitrile / min, for example about 1% / 5-6 min and / or about 1% / min. Alternatively or additionally, the gradient optionally comprises increasing the concentration of acetonitrile (for example from 0%) to a concentration within the range of 10% to 40%, or 20% to 30%, or about 25%.

[0081] An exemplary gradient (e.g., using triethylammonium phosphate buffer according to any of the respective embodiments described herein) includes 0% acetonitrile over about 8 minutes, 0% to about 5% acetonitrile in about 5 minutes (e.g., at a rate of about 1% / min), and about 5% to about 25% acetonitrile in about 120 minutes (e.g., at a rate of about 0.17% / min).

[0082] In some of each optional embodiment, the eluted peptides are loaded onto the column a second time (according to any of the respective embodiments described herein) and eluted, for example, with a buffer solution (e.g., in combination with an acetonitrile gradient, according to any of the respective embodiments described herein). An acetate buffer (e.g., acetic acid / ammonium acetate) may optionally be used, for example, at a concentration of at least about 5 mM, or at least about 20 mM, or at least about 35 mM, to elute the peptides from the column (e.g., to obtain the peptides as acetate salts). In an exemplary embodiment, the concentration of acetate is about 35 mM.

[0083] An exemplary gradient for the second elution (e.g., using acetate buffer according to any of the respective embodiments described herein) includes 0% acetonitrile over 8 minutes, 0% to 22% acetonitrile in 110 minutes (rate of 0.2% / min).

[0084] The process optionally further comprises lyophilizing the peptides obtained by chromatography (e.g., according to any of the respective embodiments described herein). Lyophilization in large-scale processes is optionally facilitated by the use of lyophilization trays (e.g., as exemplified herein) rather than flasks.

[0085] In some of the optional embodiments involving lyophilization, the process further comprises grinding the peptide following lyophilization. Grinding may optionally be performed with the aid of beads (e.g., polypropylene beads), for example, using a jar mill.

[0086] As exemplified herein, such milling may advantageously enhance the apparent density (also known as "densification") of the peptide and / or enhance ease of handling (e.g., during packaging), particularly when the lyophilized peptide has a "fluffy" consistency prior to milling.

[0087] Further process embodiments: According to any of the embodiments described herein, the process is characterized by at least one, or at least two, or at least three, or at least four, or at least five, or at least six of the following features (i)-(vii):

[0088] (i) (e.g., according to any of the respective embodiments described herein) the coupling is carried out using diisopropylcarbodiimide (DIC) in combination with ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzotriazole; (ii) (e.g., according to any of the respective embodiments described herein) cleavage of the peptide from the resin is carried out by contacting the resin-coupled linear peptide with a solution comprising trifluoroacetic acid (TFA) and a scavenger, which is dithiothreitol (DTT) and / or dithioerythritol (DTE); (iii) (e.g., according to any of the respective embodiments described herein), the process further comprises precipitating the free linear peptide after cleavage without concentrating the free linear peptide by evaporation prior to precipitation of the peptide; (iv) (e.g., according to any of the respective embodiments described herein), the oxidizing comprises contacting an aqueous solution comprising the linear peptide at a concentration of at least 5 mg / mL with hydrogen peroxide; (v) isolating the peptide (e.g., according to any of the respective embodiments described herein) comprises loading the cyclic peptide onto a reverse phase chromatography column at a concentration of 40 grams of cyclic peptide or less per kg of column, and eluting the cyclic peptide from the column; (vi) (e.g., according to any of the respective embodiments described herein) isolating the cyclic peptide comprises lyophilization, and the process further comprises grinding the cyclic peptide following lyophilization; and / or (vii) (e.g., according to any of the respective embodiments described herein) the degree of substitution of the resin used in the peptide synthesis is at least 0.3 milliequivalents per gram, and / or the resin is a Rink AMS resin.

[0089] In some of the embodiments described herein, the process comprises at least features (i) and (ii) as described above (optionally in combination with at least one of features (iii), (iv), (v), (vi), and / or (vii)), or at least features (i) and (iii) as described above (optionally in combination with at least one of features (iv), (v), (vi), and / or (vii)), or at least features (i) and (iv) as described above (optionally in combination with at least one of features (v), (vi), and / or (vii)), or at least features (i) and (v) as described above (optionally in combination with at least one of features (vi) and / or (vii)), or at least features (i) and (vi) as described above (optionally in combination with feature (vii)), or at least features (i) and (vii) as described above.

[0090] In some of the embodiments described herein, the process comprises at least features (ii) and (iii) as described above (optionally in combination with at least one of features (iv), (v), (vi), and / or (vii)), or at least features (ii) and (iv) as described above (optionally in combination with at least one of features (v), (vi), and / or (vii)), or at least features (ii) and (v) as described above (optionally in combination with at least one of features (vi) and / or (vii)), or at least features (ii) and (vi) as described above (optionally in combination with feature (vii)), or at least features (ii) and (vii) as described above.

[0091] In some of the embodiments described herein, the process comprises at least features (iii) and (iv) as described above (optionally in combination with at least one of features (v), (vi), and / or (vii)), or at least features (ii) and (v) as described above (optionally in combination with at least one of features (vi) and / or (vii)), or at least features (iii) and (vi) as described above (optionally in combination with feature (vii)), or at least features (iii) and (vii) as described above.

[0092] In some of the embodiments described herein, the process comprises at least features (iv) and (v) as described above (optionally in combination with at least one of features (vi) and / or (vii)), or at least features (iv) and (vi) as described above (optionally in combination with feature (vii)), or at least features (iv) and (vii) as described above.

[0093] In some of the embodiments described herein, the process includes at least features (v) and (vi) above (optionally in combination with feature (vii)), or at least features (iv) and (vii) above.

[0094] In some of the embodiments described herein, the process includes at least features (vi) and (vii) above.

[0095] Peptide Formulations and Uses: The peptides prepared according to the processes described herein (according to any of the respective embodiments) may optionally be used in the treatment of a condition treatable by the peptide.

[0096] According to an aspect of some embodiments of the invention there is provided the use of a peptide prepared according to the process described herein (according to any of the respective embodiments) in the manufacture of a medicament for treating a peptide-treatable condition.

[0097] According to an aspect of some embodiments of the present invention, there is provided a method of treating a peptide-treatable condition, the method comprising administering to a subject in need of treatment of the peptide-treatable condition a peptide, the peptide being prepared according to a process described herein (according to any of the respective embodiments).

[0098] In some of any of the embodiments described herein (according to any of the aspects described herein), the peptide is a peptide having SEQ ID NO:1 (or an analog or derivative thereof) and the treatment includes any treatment in which inhibiting CXCR4 is advantageous.

[0099] Since it is expected that many relevant treatments will be developed during the life of this patent application, the scope of the terms "condition," "treatable by peptide," and "treatments which would be beneficial by inhibiting CXCR4" is intended to a priori include all such new technology.

[0100] In some of any of the embodiments described herein (according to any of the aspects described herein), the peptide is a peptide having SEQ ID NO:1 (or an analog or derivative thereof), and the condition treatable by a peptide having SEQ ID NO:1 (or an analog or derivative thereof) is cancer or arthritis (e.g., rheumatoid arthritis), e.g., as described in U.S. Pat. No. 7,423,007 (the contents of which are incorporated herein by reference, particularly those relating to the treatment of conditions with the above-mentioned peptides).

[0101] Examples of conditions treatable (according to any of the embodiments described herein) by a peptide having SEQ ID NO:1 (or an analog or derivative thereof) include, but are not limited to, retinoblastoma and / or neuroectodermal derived tumors, e.g., as described in WO 2012 / 095849 (the contents of which are incorporated herein by reference, in particular with respect to the treatment of conditions with the above-mentioned peptides), large cell lung carcinoma, e.g., as described in WO 2013 / 160895 (the contents of which are incorporated herein by reference, in particular with respect to the treatment of conditions with the above-mentioned peptides), large cell lung carcinoma, e.g., as described in International Patent Publication WO 2008 / 075370 (the contents of which are incorporated herein by reference, in particular with respect to the treatment of conditions with the above-mentioned peptides), ), breast cancer and / or pancreatic cancer, as described, for example, in U.S. Pat. No. 7,423,007; thrombocytopenia, as described, for example, in International Patent Application Publication No. WO 2010 / 146578 (the contents of which are incorporated herein by reference, in particular with respect to the treatment of conditions with the above-mentioned peptides); myelosuppression risk, as described, for example, in International Patent Application Publication No. WO 2008 / 075369 (the contents of which are incorporated herein by reference, in particular with respect to the treatment of conditions with the above-mentioned peptides); and HIV infection, as described, for example, in U.S. Pat. No. 8,435,939 (the contents of which are incorporated herein by reference, in particular with respect to the treatment of conditions with the above-mentioned peptides).

[0102] Suitable analogues and derivatives of peptides having SEQ ID NO:1 are described in U.S. Pat. Nos. 7,423,007 and 8,435,939, as well as WO 2008 / 075369, WO 2008 / 075370, WO 2010 / 146578, WO 2012 / 095849, and WO 2013 / 160895 (the contents of each of which, in particular with respect to analogues and derivatives of SEQ ID NO:1, are incorporated herein by reference).

[0103] The peptides according to some embodiments of the present invention can be administered to the organism itself or in a pharmaceutical composition mixed with suitable carriers or excipients.

[0104] As used herein, a "pharmaceutical composition" refers to a preparation of one or more active ingredients described herein with other chemical components, such as physiologically suitable carriers or excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0105] As used herein, the term "active ingredient" refers to the peptide responsible for the biological action.

[0106] Hereinafter, the terms "physiologically acceptable carrier" and "pharmaceutical acceptable carrier", which may be used interchangeably, refer to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. Adjuvants are included in these terms.

[0107] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0108] Techniques for formulation and administration of pharmaceutical agents may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, incorporated herein by reference.

[0109] Suitable routes of administration include, for example, oral, rectal, transmucosal, especially nasal, intestinal or parenteral delivery (including intramuscular, subcutaneous, and intramedullary injection), as well as intrathecal, direct intraventricular, intracardiac (e.g., into the right or left ventricular cavity, into the common coronary artery), intravenous, intraperitoneal, intranasal, or intraocular injection.

[0110] Alternatively, the pharmaceutical composition may be administered locally rather than systemically, for example, by direct injection of the pharmaceutical composition into a tissue region of the patient.

[0111] Pharmaceutical compositions of some embodiments of the present invention may be manufactured by processes well known in the art, for example, by utilizing conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0112] As such, pharmaceutical compositions for use in accordance with some embodiments of the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers that include excipients and adjuvants that facilitate processing of the active ingredients into pharma- ceutically usable preparations. Proper formulation will depend on the route of administration chosen.

[0113] For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, physiological salt buffer, etc. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are well known in the art.

[0114] For oral administration, pharmaceutical compositions can be easily formulated by combining the active compounds with pharma- ceutically acceptable carriers well known in the art. Such carriers allow the formulation of pharmaceutical compositions as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the patient. Pharmaceutical preparations for oral use can be made with solid excipients, optionally by grinding the resulting mixture, and processing the granulated mixture to obtain tablets or dragee cores, after adding suitable auxiliary agents, if desired. Suitable excipients are in particular sugars, such as lactose, sucrose, mannitol, or sorbitol, cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc., and / or physiologically acceptable polymers, such as fillers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0115] Dragee cores are provided with suitable coatings. For this purpose, gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and concentrated sugar solutions which may optionally contain suitable organic solvents or solvent mixtures may be used. Dyes or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0116] Orally available pharmaceutical compositions include push-fit capsules made of gelatin, as well as soft sealed capsules made of gelatin and plasticizers such as glycerol or sorbitol. Push-fit capsules may contain the active ingredient mixed with a filler such as lactose, a binder such as starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the active ingredient may be dissolved or suspended in a suitable liquid such as fatty oils, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers may be added. All preparations for oral administration should be in a dosage suitable for the chosen route of administration.

[0117] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0118] For administration by nasal inhalation, the active ingredient for use according to some embodiments of the present invention is conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane, or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0119] The pharmaceutical compositions described herein may be formulated for parenteral administration, for example, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage forms, for example, in ampoules or multi-dose containers, optionally with the addition of a preservative. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents, for example, suspending, stabilizing, and / or dispersing agents.

[0120] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Alternatively, suspensions of the active ingredient may be prepared as appropriate oily or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, liposomes, etc. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the active ingredient, allowing for the preparation of highly concentrated solutions.

[0121] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, such as sterile pyrogen-free aqueous solution, before use.

[0122] Pharmaceutical compositions of some embodiments of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, conventional suppository bases such as cocoa butter or other glycerides.

[0123] Pharmaceutical compositions suitable for use in connection with some embodiments of the present invention include compositions in which the active ingredient is contained in an amount effective to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount of peptide effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., cancer or arthritis as discussed herein) or to prolong the survival of the subject being treated.

[0124] Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0125] For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can then be used to more accurately determine useful doses in humans.

[0126] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined in vitro, in cell cultures, or in experimental animals by standard pharmaceutical procedures. Data obtained from these in vitro and cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. Dosages may vary depending on the formulation employed and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition. (See, for example, Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch.1 p.1).

[0127] Dosage and interval may be adjusted individually to provide a level of active ingredient sufficient to induce or suppress a biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0128] Depending on the severity and responsiveness of the condition being treated, administration may be single or multiple doses, with the treatment regime continuing for a period ranging from several days to several weeks, or until a cure is achieved or a diminution of the disease state is achieved.

[0129] The amount of a composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0130] The compositions of some embodiments of the present invention may be presented in a pack or dispenser device, e.g., an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient, if desired. The pack may, for example, comprise metal or plastic foil, e.g., a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied by a notice associated with the container in a form specified by a government agency regulating the manufacture, use, or sale of pharmaceuticals. The notice reflects that the form of the composition or its administration to humans or animals has been approved by a regulatory agency. Such notice may, for example, be a labeling approved by the US Food and Drug Administration for prescription drugs or an approved product insert. Compositions comprising the preparations of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in a suitable container, and labeled for the treatment of a specified condition, as further detailed above.

[0131] Further definitions: As used herein, the term "about" means ±20%. In some of the respective optional embodiments described herein, the term "about" means ±10%.

[0132] The words "comprises," "comprising," "includes," "including," "having" and their variations mean "including but not limited to."

[0133] The term "consisting of" means "including and limited to."

[0134] The term "consisting essentially of" means that a composition, method, or structure may include additional components, steps, and / or parts, but only if the additional components, steps, and / or parts do not materially alter the basic and novel properties of the claimed composition, method, or structure.

[0135] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds (including mixtures thereof).

[0136] Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity, and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This is true regardless of the breadth of the range.

[0137] Whenever a numerical range is specified herein, it is meant to include any recited numbers (decimals or integers) within the specified range. The phrases "ranging / ranges between a first specified number and a second specified number" and "ranging / ranges from ~ to" are used interchangeably herein and are meant to include the first and second specified numbers and all decimals and integers therebetween.

[0138] The term "treating" means inhibiting, preventing, or arresting the occurrence of a pathological condition (disease, disorder, or condition) and / or causing the reduction, amelioration, or regression of a pathological condition. Those of skill in the art will appreciate that a variety of methods and assays can be used to assess the occurrence of a pathological condition, and that a variety of methods and assays can be used to assess the reduction, amelioration, or regression of a pathological condition.

[0139] As used herein, the term "preventing" means keeping a disease, disorder, or condition from occurring in a subject who may be at risk for the disease but has not yet been diagnosed with the disease.

[0140] As used herein, the term "subject" includes mammals, preferably humans, of any age, suffering from a pathological condition. Preferably, the term encompasses individuals at risk of developing a pathological condition.

[0141] As used herein, the term "method" means methods, means, techniques, and procedures for accomplishing a given task, including, but not limited to, methods, means, techniques, and procedures that are either known to the practitioner of the chemical, pharmacological, biological, biochemical, and medical arts or that can be readily developed by the practitioner from known methods, means, techniques, and procedures.

[0142] When referring to a particular sequence listing, such reference should be understood to also encompass sequences that substantially correspond to its complementary sequence, so as to include minor sequence variations resulting from sequencing errors, cloning errors, other modifications, etc., that result in base substitutions, deletions, or additions, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively less than 1 in 100 nucleotides, alternatively less than 1 in 200 nucleotides, alternatively less than 1 in 500 nucleotides, alternatively less than 1 in 1000 nucleotides, alternatively less than 1 in 5,000 nucleotides, alternatively less than 1 in 10,000 nucleotides.

[0143] It will be appreciated that certain features of the invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be provided separately, or in any suitable subcombination, or as appropriate for any other described embodiment of the invention. A particular feature described in the context of various embodiments should not be construed as an essential feature of that embodiment, unless the embodiment is ineffective without that element.

[0144] Experimental support for various embodiments and aspects of the present invention as identified above and as claimed below is found in the following examples. EXAMPLES

[0145] Reference will now be made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting manner.

[0146] Reference Example 1 Solid-phase synthesis of BL-8040 according to U.S. Pat. No. 7,423,007 551 mg of cyclic peptide BL-8040 (SEQ ID NO:1) was prepared using the following procedure described in US Pat. No. 7,423,007.

[0147] Cyclic peptide BL-8040 was prepared by solid-phase synthesis. To build the entire peptide, the solid-phase synthesis reaction steps were repeated 14 times starting from the C-terminal amino acid and ending with the N-terminal amino acid. Each amino acid addition was carried out in two steps. The first step involved removing the Fmoc protecting group from the N-terminus of the last amino acid added to the peptide sequence (or from the resin when adding the first amino acid), followed by sequential amino acids being attached to the peptide extension on the resin. The final residue added to the peptide on the resin was a 4-fluorobenzoyl group that did not contain an Fmoc group.

[0148] resin: The resin was 2.94 grams of Fmoc-Rink amide resin (ie, 1 mmol) with a substitution of 0.34 mmol / gram.

[0149] Removal of Fmoc protecting group: The Fmoc protecting group was removed from the N-terminus (of the last amino acid added to the peptide sequence) or from the resin using a solution of 20% piperidine in dimethylformamide (DMF).

[0150] Amino acid loading: Amino acids were coupled in the form of appropriate amino acid derivatives using diisopropylcarbodiimide (DIC) in combination with HOBt (N-hydroxybenzotriazole) in DMF. Cys, Arg, Tyr, and Lys (D-Lys or L-Lys) were protected as Cys(Trt), Arg(Pbf), Tyr(t-Bu), and Lys(Boc) (D- or L-), respectively. In the final step, 4-fluorobenzoic acid was coupled instead of the amino acid. The amount of amino acid (or 4-fluorobenzoic acid) used was 2.5 equivalents.

[0151] In-process monitoring of the condensation reaction was performed using the ninhydrin test of Kaiser et al. [Anal Biochem 1970, 34:595-598].

[0152] Cleavage and deprotection: Cleavage of the peptide (1 mmol) from the resin and removal of the protecting groups was carried out by treatment with 270 mL of 1 M TMSBr-thioanisole / trifluoroacetic acid (TFA) mixture in the presence of m-cresol (100 equiv.) and ethanedithiol (300 equiv.) at 25° C. for 3 h.

[0153] The resin was separated by filtration and washed twice with TFA (5 mL).

[0154] The mixture of the filtrate and washing solution was concentrated in vacuo.

[0155] The peptide was then combined with 300 mL of water-cold anhydrous ether and the resulting precipitate was isolated by centrifugation and decantation.

[0156] The crude product was washed with cold ether, dissolved in 500 mL of 1N acetic acid and diluted to 2.5 L with distilled water.

[0157] Oxidative cyclization: A dilute aqueous solution of the crude linear peptide was adjusted to pH 7.5 with concentrated aqueous ammonia, and oxidation was carried out by aeration with air.

[0158] Preparative HPLC: The solution obtained upon completion of the cyclization step was purified by separation on a preparative HPLC C18 column (Cosmosil™ 5C18-AR-II) using acetonitrile and water and gel filtration chromatography (Sephadex™ G-15) using 0.1 N acetic acid as eluent.

[0159] A single peak of polypeptide was obtained and lyophilized.

[0160] The purity was confirmed by HPLC.

[0161] The yield was 551.5 mg (19.4%).

[0162] Example 1 Large-scale synthesis of BL-8040 Cyclic peptide BL-8040 was prepared by large-scale (825 mmol) solid-phase synthesis. To build the entire peptide, the solid-phase synthesis reaction steps were repeated 14 times starting with the C-terminal amino acid and ending with the N-terminal amino acid. Each amino acid addition was carried out in two steps. The first step involved removing the Fmoc protecting group from the N-terminus of the last amino acid added to the peptide sequence (or from the resin when adding the first amino acid), followed by sequential amino acids being attached to the peptide extension on the resin. The final residue added to the peptide on the resin was a 4-fluorobenzoyl group that did not contain an Fmoc group.

[0163] The final yield was 468 grams (25%).

[0164] resin: The resin was Fmoc-Rink AMS resin with substitution in the range of 0.3-0.6 meq / gram.

[0165] Removal of Fmoc protecting group: The Fmoc protecting group was removed from the N-terminus (of the last amino acid added to the peptide sequence) or resin using a 20% solution of piperidine in dimethylformamide (DMF) (10 ml / gram of initial resin). The piperidine solution was removed by washing with DMF prior to the next amino acid reaction and confirmed by pH testing of the washing solution.

[0166] Amino acid loading: Amino acids were coupled in the appropriate amino acid derivative form using diisopropylcarbodiimide (DIC) in combination with HOBt (N-hydroxybenzotriazole) as the activating agent. Cys, Arg, Tyr, and Lys (D-Lys or L-Lys) were protected as Cys(Trt), Arg(Pbf), Tyr(t-Bu), and Lys(Boc) (D- or L-), respectively. In the final step, 4-fluorobenzoic acid was coupled in place of the amino acid. Calculations of amino acid (or 4-fluorobenzoic acid), DIC, and HOBt amounts were based on a two-fold excess of substitution and batch size.

[0167] In-process monitoring was performed using ninhydrin and chloranil tests at the end of each cycle to evaluate the coupling step. A negative test result indicates the absence of free amino groups (complete coupling). If the test is positive, indicating unreacted amino groups (incomplete coupling), the coupling reaction can be extended or recoupling of a protected amino acid derivative can be performed.

[0168] After completion of the synthesis cycle, the resin-peptide was washed with a solution of DMF / isopropanol (1:1) and dried with nitrogen.

[0169] Cleavage and deprotection: Cleavage was carried out to remove the peptide molecules from their supporting resins and to remove the protecting groups, e.g., acidolysis was carried out using 95% trifluoroacetic acid (TFA) and 5% water with 50 mg / mL dithioerythritol (DTE) as a scavenger (10 mL TFA-based cleavage solution per gram of resin) for 3.25-3.5 h at ambient temperature.

[0170] The resin was then filtered and rinsed twice with TFA to complete the extraction of the peptide.

[0171] The volume of the peptide solution was reduced on a rotary evaporator under vacuum (approximately 35 °C) to a volume of approximately 30-35% of the original volume.

[0172] The peptides were then precipitated using a cold (-10±5°C) mixture of tert-butyl methyl ether (MTBE) / hexane (60:40 v / v) at a volume of 32 mL per gram of resin.

[0173] The crude product was isolated by filtration, washed with MTBE, and dried on the filter under a stream of nitrogen to remove most of the solvent.

[0174] The resulting crude (linear) peptide was then dissolved in 90% acetic acid and the resulting solution was dispensed into lyophilization flasks, shell frozen, and lyophilized to dryness on a manifold lyophilizer.

[0175] The purity of the peptides was analyzed by RP-HPLC and the identity was confirmed by mass spectrometry.

[0176] Oxidative cyclization: 0.1M ammonium bicarbonate (NH 4 HCO 3 The crude linear peptide was cyclized by oxidation with hydrogen peroxide in solution.

[0177] Crude linear peptides were dissolved in 0.1 M NH 4 HCO 3 Dissolve in an equal volume of 0.1 M NH 4 HCO 3 The peptide was diluted to a concentration of 5 mg / mL by addition of

[0178] A 1.5% solution of hydrogen peroxide in water (5-fold excess) was then added dropwise to the peptide solution over a period of 25-30 minutes, and the reaction was monitored using Ellman's test to confirm the absence of free sulfhydryl groups.

[0179] After completion of the reaction, the reaction mixture was acidified to pH 2-3 by addition of neat TFA and the resulting solution was used "as is" in the TEAP (triethylammonium phosphate) purification step.

[0180] First preparative HPLC column (TEAP purification): Approximately 2.4 L of solution containing approximately 11.7 grams of crude peptide per kg of resin was loaded and the solution obtained upon completion of the cyclization step was purified by separation on a preparative RP-HPLC C18 column, 10 μm, 120 Å Daisogel™.

[0181] Peptides were eluted with 0.1 M triethylammonium phosphate (TEAP) buffer (pH 2.25) and an acetonitrile (ACN) gradient: 0% acetonitrile for 8 min, 0% to 5% acetonitrile for 5 min (at a rate of 1% / min), and 5% to 25% acetonitrile for 120 min (at a rate of 0.17% / min).

[0182] Elution fractions were collected, sampled, and tested by HPLC to determine which fractions were sufficiently pure (>95%) and pooled for a second chromatographic purification step. Hydrophilic and hydrophobic fractions from the first RP-HPLC purification that did not meet the purity acceptance criteria may be retained and reprocessed to maximize overall yield.

[0183] Second preparative HPLC column (Acetic Acid Purification): The purified fractions from the TEAP injection were pooled, diluted 1:1 with water, and the peptides were separated using a 10 μm, 120 Å Daisogel™ C18 column loaded at a volume of approximately 2.7 L per kg of resin and eluted with a 35 mM acetate-based buffer and an acetonitrile gradient. The gradient was as follows: 0% acetonitrile for 8 min and 0% to 22% acetonitrile for 110 min (at a rate of 0.2% / min). After loading the peptides and before the start of the elution gradient, the column was washed with 4 column volumes of 0.1 M ammonium acetate to obtain the peptides as acetate salts.

[0184] Fraction collection was monitored by UV absorbance at 230 nm. Elution fractions were collected, sampled, and analyzed by HPLC to determine which fractions were sufficiently pure and pooled. Only fractions that met the in-process control criteria (purity by HPLC ≥ 98% and no unknown impurities ≥ 0.14%) were pooled. Pooled fractions from each chromatography cycle were lyophilized as sublots. Hydrophilic and hydrophobic fractions from the second RP-HPLC purification that did not meet the purity acceptance criteria were retained and may be reprocessed to maximize overall yield.

[0185] Wet Pooling and Freeze Drying: All sublots that met the in-process control criteria for purified material were reconstituted with purified water to a concentration of approximately 50 g / L. The resulting solution was filtered through a 0.2 μm PVDF filter before lyophilization.

[0186] Lyophilization was performed in 1200 mL lyophilization flasks, which were filled with approximately equal volumes of 100 mL per flask to yield 5 grams of purified peptide per flask, then shell-frozen in a dry ice / IPA bath with a condenser temperature below -60°C and a vacuum below 500 mTorr. Lyophilization was carried out for 65-85 hours.

[0187] After lyophilization, samples were taken for acetate content determination. If not specifically shown to be within the range of 11-15%, the bulk drug substance may be resuspended in water and the lyophilization repeated.

[0188] Densification: The lyophilized material was placed into polyethylene bottles equipped with polypropylene "grinding" beads and the bottles were placed on a jar mill for 20±5 minutes, which eliminates "fluffiness" by increasing the apparent density and enhances ease of handling of the bulk drug substance during packaging.

[0189] Packaging & Storage: The final bulk drug substance was packaged in Type III depyrogenated amber glass bottles with polypropylene caps with Teflon liners. Packaging was performed under a controlled humidity environment (15-30% relative humidity) to mitigate water uptake. The bottles were placed in an aluminum laminate bag with a desiccant between the two packing materials as a second container. The BL-8040 drug substance was then stored at -20±5°C.

[0190] Example 2 Modified large-scale solid-phase synthesis of BL-8040 Cyclic peptide BL-8040 is prepared by large-scale solid-phase synthesis (e.g., at least about 500 grams of product). To build the entire peptide, the solid-phase synthesis reaction steps are repeated 14 times, starting with the C-terminal amino acid and ending with the N-terminal amino acid. Each amino acid addition is performed in two steps. The first step involves removing the Fmoc protecting group from the N-terminus of the last amino acid added to the peptide sequence (or from the resin when adding the first amino acid), followed by sequential amino acids being attached to the peptide extension on the resin. The last residue added to the peptide on the resin is a 4-fluorobenzoyl group that does not contain an Fmoc group. The steps of solid-phase synthesis according to some embodiments are illustrated in FIG. 1.

[0191] resin: The resin is an Fmoc-Rink AMS resin with substitution in the range of 0.6-0.9 meq / gram (as opposed to 0.6 meq / gram or less as mentioned above).

[0192] Removal of Fmoc protecting group: The Fmoc protecting group is removed from the N-terminus (of the last amino acid added to the peptide sequence) or resin using a 20% solution of piperidine in dimethylformamide (DMF) (approximately 8 ml / gram of initial resin). The piperidine solution is removed prior to the next amino acid reaction by washing with DMF and verified by pH testing of the washing solution.

[0193] Amino acid bonds: As an activating agent, diisopropylcarbodiimide (DIC) in combination with ethyl cyanohydroxyiminoacetate (unlike HOBt as mentioned above) is used to couple the amino acids in the appropriate amino acid derivative form. Cys, Arg, Tyr, and Lys (D-Lys or L-Lys) are protected as Cys(Trt), Arg(Pbf), Tyr(t-Bu), and Lys(Boc) (D- or L-), respectively. In the final step, 4-fluorobenzoic acid is coupled in place of the amino acid. Calculations of the amount of amino acid (or 4-fluorobenzoic acid), DIC, and ethyl cyanohydroxyiminoacetate are based on a two-fold excess over substitution and batch size.

[0194] In-process monitoring is performed using ninhydrin and chloranil tests at the end of each cycle to evaluate the coupling step. A negative test result indicates the absence of free amino groups (complete coupling). If the test is positive, indicating unreacted amino groups (incomplete coupling), the coupling reaction can be extended, recoupling of a protected amino acid derivative can be performed, or acetylation (capping) can be performed, for example, using acetic anhydride in the presence of diisopropylethylamine (DIEA).

[0195] After completion of the synthesis cycle, the resin-peptide is washed with a solution of DMF / isopropanol (1:1) and dried with nitrogen.

[0196] Cleavage and deprotection: Cleavage is carried out to remove the peptide molecule from its support resin and to remove the protecting groups. For example, acidolysis is carried out using 95% trifluoroacetic acid (TFA) and 5% water with 50 mg / mL scavenger (10 mL TFA-based cleavage solution per gram of resin) for 3.25-3.5 hours at ambient temperature. The scavenger is dithiothreitol (DTT), which is different from dithioerythritol as described in Example 1 or ethanedithiol as described in Comparative Example 1.

[0197] The resin is then filtered and rinsed twice with TFA to complete the extraction of the peptide. (Unlike Example 1, no reduction in the peptide solution volume is performed at this stage.)

[0198] The peptide is then precipitated using a cold (-10±5°C) mixture of tert-butyl methyl ether (MTBE) / hexane (60:49 v / v) in a volume of 45 mL per gram of resin (instead of 32 mL / gram as described in Example 1).

[0199] The crude product is isolated by filtration, washed with MTBE, and dried on the filter under a stream of nitrogen to remove most of the solvent.

[0200] The resulting crude (linear) peptide is then either dried under vacuum or solubilized in acetic acid and lyophilized (eg, as described in Example 1).

[0201] Peptides are optionally analyzed for purity by RP-HPLC and / or their identity confirmed by mass spectrometry.

[0202] Oxidative cyclization: 0.1M ammonium bicarbonate (NH 4 HCO 3 ) The crude linear peptide is cyclized at a peptide concentration of 10 mg / mL by oxidation with 1.5% hydrogen peroxide in solution. Unlike in Example 1, the peptide is not further diluted to 5 mg / mL over 25-30 min. The reaction is monitored using the Ellman's test to confirm the absence of free sulfhydryl groups.

[0203] After completion of the reaction, the reaction mixture is acidified to pH 2-3 by addition of TFA neat solution, and the resulting solution is used "as is" in the TEAP (triethylammonium phosphate) purification step.

[0204] First preparative HPLC column (TEAP purification): The solution obtained upon completion of the cyclization step is purified by separation on a preparative RP-HPLC C18 column, 10 μm, 120 Å Daisogel™, loading approximately 2.4 L of solution containing approximately 20 to approximately 25 grams of crude peptide per kg of resin (as opposed to approximately 11.7 grams / kg as described in Example 1).

[0205] Peptides are eluted with 0.1 M triethylammonium phosphate (TEAP) buffer (pH 2.25) and an acetonitrile (ACN) gradient.

[0206] Elution fractions are collected, sampled, and tested by HPLC to determine which fractions are sufficiently pure (>95%) and pooled for a second chromatographic purification step. Hydrophilic and hydrophobic fractions from the first RP-HPLC purification that do not meet the purity acceptance criteria may be retained and reprocessed to maximize overall yield.

[0207] Second preparative HPLC column (Acetic Acid Purification): The purified fractions from the TEAP injection are pooled, diluted 1:1 with water, and the peptides are separated using a 10 μm, 120 Å Daisogel™ C18 column eluted with a 35 mM acetate-based buffer and an acetonitrile gradient. After peptide loading and before the start of the elution gradient, the column is washed with 4 column volumes of 0.1 M ammonium acetate to obtain the peptides as acetate salts.

[0208] Fraction collection is monitored by UV absorbance at 230 nm. Elution fractions are collected, sampled, and tested by HPLC to determine which fractions are sufficiently pure and pooled. Only fractions that meet the in-process control criteria (>98% purity by HPLC with no unknown impurities>0.14%) are pooled. Pooled fractions from each chromatography cycle are lyophilized as sublots. Hydrophilic and hydrophobic fractions from the second RP-HPLC purification that do not meet the purity acceptance criteria may be retained and reprocessed to maximize overall yield.

[0209] Wet Pooling and Freeze Drying: All sublots that meet the in-process control criteria for purified material are reconstituted with purified water to a concentration of approximately 50 g / L. The resulting solution is filtered through a 0.2 μm PVDF filter before being lyophilized.

[0210] Lyophilization is carried out in lyophilization trays (e.g., about 1.0 to about 1.2 liters / tray) under a vacuum of ≦150 mTorr (e.g., for about 89 hours) rather than in flasks as described in Example 1. The use of trays may provide increased process control, facilitate scale-up, and / or reduce in-process control.

[0211] After lyophilization, a sample is taken for acetate content determination and, unless specifically shown to be within the 11-15% range, optionally the bulk drug substance is resuspended in water and the lyophilization repeated.

[0212] Densification: The lyophilized material is optionally subjected to densification according to the procedure described in Example 1.

[0213] Packaging & Storage: The final bulk drug substance is optionally packaged and / or stored according to the procedures described in Example 1.

[0214] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0215] It is the intention of the applicants that all publications, patents, and patent applications referenced herein be incorporated by reference in their entirety into this specification as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference herein. Additionally, citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. Section headings should not necessarily be construed as limiting the scope to which they are used. Additionally, any priority documents of this application are hereby incorporated by reference in their entirety into this specification. [Sequence List Free Text]

[0216] SEQ ID NO:1: Synthetic peptide in which position 1 is 4-fluorobenzoylarginine, position 3 is X=naphthylalanine, position 6 is X=citrulline, position 8 is X=D-lysine, position 12 is X=citrulline, and position 14 is amidated.

Claims

1. 1. A process for the large scale preparation of a cyclic peptide having SEQ ID NO:1, or a pharma- ceutically acceptable salt thereof, said process comprising: (a) sequentially coupling an amino acid and 4-fluorobenzoic acid to a resin by solid phase peptide synthesis, thereby obtaining a linear peptide coupled to said resin; (b) cleaving the linear peptide from the resin, thereby obtaining a free linear peptide; (c) oxidizing cysteine ​​residues of said linear peptide to form intramolecular disulfide bonds, thereby obtaining said cyclic peptide having SEQ ID NO:1 in solution; (d) isolating the cyclic peptide having SEQ ID NO:1 or a pharma- ceutically acceptable salt thereof; Including, (i) the coupling is carried out using diisopropylcarbodiimide (DIC) in combination with ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzotriazole; (ii) the cleavage is carried out by contacting the resin-coupled linear peptide with a solution comprising trifluoroacetic acid (TFA) and a scavenger selected from the group consisting of dithioerythritol (DTE) and dithiothreitol (DTT); (iii) the process further comprises precipitating the free linear peptide after the cleavage without concentrating the free linear peptide by evaporation prior to precipitation; (iv) the oxidation is carried out by contacting an aqueous solution containing the linear peptide at a concentration of at least 5 mg / mL with hydrogen peroxide; (v) said isolating comprises loading said cyclic peptide onto a reverse phase chromatography column at a concentration of 40 grams of cyclic peptide or less per kg of said column; and eluting said cyclic peptide from said column; (vi) the isolation of the cyclic peptide having SEQ ID NO:1 comprises lyophilization, and the process further comprises grinding the cyclic peptide following the lyophilization; and / or (vii) the degree of substitution of the resin is at least 0.3 meq / gram, and / or the resin is a Rink aminomethylstyrene resin; process.

2. 2. The process of claim 1, wherein the coupling is carried out using diisopropylcarbodiimide (DIC) in combination with ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzotriazole.

3. 2. The process of claim 1, wherein the DIC and the ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzotriazole are used in about a two-fold molar excess.

4. 2. The process of claim 1, wherein the concentration of the scavenger in the solution containing the scavenger is in the range of 10 mg / mL to 500 mg / mL.

5. 2. The process of claim 1, wherein the cleavage is carried out by contacting the resin-coupled linear peptide with a solution comprising trifluoroacetic acid (TFA) and a scavenger selected from the group consisting of dithioerythritol (DTE) and dithiothreitol (DTT).

6. 6. The process of claim 5, wherein the concentration of dithiothreitol in the solution is about 50 mg / mL.

7. 2. The process of claim 1, further comprising precipitating said free linear peptide after said cleavage without concentrating said free linear peptide by evaporation prior to said precipitation.

8. 2. The process of claim 1, wherein the precipitation is carried out by addition of a mixture of tert-butyl methyl ether (MTBE) and hexane at a volume of the mixture of about 45 mL per gram of the resin.

9. 2. The process of claim 1, wherein the oxidation is carried out by contacting the linear peptide with hydrogen peroxide.

10. 10. The process of claim 9, wherein said contacting is performed by contacting an aqueous solution containing said linear peptide at a concentration of at least 5 mg / mL with hydrogen peroxide.

11. 2. The process of claim 1, wherein the isolating comprises loading the cyclic peptide onto a reverse phase chromatography column at a concentration of 40 grams of cyclic peptide or less per kg of the column and eluting the cyclic peptide from the column.

12. 2. The process of claim 1, wherein the column is a C18 column.

13. The process of claim 1 , wherein the elution is performed with a triethylammonium phosphate solution.

14. 2. The process of claim 1, wherein said isolating said cyclic peptide having SEQ ID NO:1 comprises lyophilization, and said process further comprises grinding said cyclic peptide following said lyophilization.

15. 2. The process of claim 1, wherein the resin has a degree of substitution of at least 0.3 meq / gram and / or the resin is a Rink aminomethylstyrene resin.

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