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 based on U.S. Provisional Patent Application No. 63 / 131,873, filed on December 30, 2020, the content of which is incorporated herein by reference in its entirety.

[0002] Statement Regarding Sequence Listing An ASCII file named 89546 SequenceListing.txt, containing 4,096 bytes, created on December 27, 2021, and submitted concurrently with the filing of this application, is incorporated herein by reference.

[0003] In some embodiments, the present invention relates to chemical synthesis, and more particularly, but not exclusively, to a novel preparation process of peptides such as BL-8040 that can be used, for example, in treating conditions such as cancer and / or arthritis.

Background Art

[0004] BL-8040 is a peptide also known as 4F-benzoyl-TN14003 (4-fluorobenzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH2, SEQ ID NO: 1), which is cyclic upon formation of the disulfide bond between its two Cys residues.

[0005] U.S. Patent No. 7,423,007 describes peptides having CXC4 antagonism, including 4F-benzoyl-TN14003. According to the teachings of U.S. Patent No. 7,423,007, 4F-benzoyl-TN14003 is produced by solid-phase synthesis using DIPCDI-HOBt as a coupling agent in DMF (adding amino acids 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] As other background arts, those described in U.S. Patent No. 7,138,488, U.S. Patent No. 8,435,939, and International Patent Application Publication Pamphlets No. WO 2008 / 075369, No. WO 2008 / 075370, No. WO 2010 / 146578, No. WO 2012 / 095849, and No. WO 2013 / 160895 can be mentioned.

Summary of the Invention

[0007] According to an aspect of some embodiments of the present invention, a large-scale preparation process of a cyclic peptide having SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof is provided, and this process includes (a) sequentially coupling an amino acid and 4-fluorobenzoic acid to a resin by solid-phase peptide synthesis to obtain a linear peptide coupled to the resin; (b) cleaving the linear peptide from the resin to obtain 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 the cyclic peptide having SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof; and includes (i) the coupling is carried out using diisopropylcarbodiimide (DIC) combined with ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzotriazole; (ii) the cleavage is carried out by contacting the linear peptide coupled to the resin with a solution containing trifluoroacetic acid (TFA) and a scavenger selected from the group consisting of dithioerythritol (DTE) and dithiothreitol (DTT); (iii) the process further includes precipitating the free linear peptide after cleavage without concentrating the free linear peptide by evaporation before precipitation; (iv) 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) Isolation includes loading the cyclic peptide onto a reverse-phase chromatography column at a concentration of the cyclic peptide of 40 grams or less per 1 kg of the column and eluting the cyclic peptide from the column, (vi) Isolation of the cyclic peptide having SEQ ID NO: 1 includes lyophilization, and the process further includes grinding the cyclic peptide following lyophilization, and / or (vii) The degree of substitution of the resin is at least 0.3 milliequivalents / gram and / or the resin is Rink aminomethylstyrene (AMS) resin.

[0008] According to an aspect of some embodiments of the present invention, a large-scale preparation process of a cyclic peptide having SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof is provided, and this process (a) Sequentially coupling amino acids and 4-fluorobenzoic acid to the resin by solid-phase peptide synthesis using diisopropylcarbodiimide (DIC) combined with ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzotriazole, thereby obtaining a linear peptide coupled to the resin, (b) Cleaving the linear peptide from the resin, thereby obtaining a free linear peptide, (c) Oxidizing the 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 the cyclic peptide having SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof, and includes.

[0009] According to an aspect of some embodiments of the present invention, a large-scale preparation process of a cyclic peptide having SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof is provided, and this process (a) Sequentially coupling amino acids and 4-fluorobenzoic acid to the resin by solid-phase peptide synthesis, thereby obtaining a linear peptide coupled to the resin, (b) Contacting a linear peptide coupled to a resin with a solution containing trifluoroacetic acid (TFA) and a scavenger selected from the group consisting of dithioerythritol (DTE) and dithiothreitol (DTT) to cleave the linear peptide from the resin, thereby obtaining a free linear peptide; (c) Oxidizing the 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 the cyclic peptide having SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof; and comprising.

[0010] According to an aspect of some embodiments of the present invention, a large-scale preparation process of a cyclic peptide having SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof is provided, and this process comprises: (a) Sequentially coupling an amino acid and 4-fluorobenzoic acid to a resin by solid-phase peptide synthesis to obtain a linear peptide coupled to the resin; (b) Cleaving the linear peptide from the resin to obtain a free linear peptide, and precipitating the free linear peptide after cleavage without concentrating the free linear peptide by evaporation before precipitation; (c) Oxidizing the 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 the cyclic peptide having SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof; and comprising.

[0011] According to an aspect of some embodiments of the present invention, a large-scale preparation process of a cyclic peptide having SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof is provided, and this process comprises: (a) Sequentially coupling an amino acid and 4-fluorobenzoic acid to a resin by solid-phase peptide synthesis to obtain a linear peptide coupled to the resin; (b) Cleaving the linear peptide from the resin to thereby obtain a free linear peptide; (c) Oxidizing the cysteine residues of the linear peptide, this oxidation being carried out 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 the solution, and forming an intramolecular disulfide bond by oxidation; (d) Isolating the cyclic peptide having SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof; and comprising.

[0012] According to an aspect of some embodiments of the present invention, a large-scale preparation process of a cyclic peptide having SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof is provided, and this process (a) Sequentially coupling an amino acid and 4-fluorobenzoic acid to a resin by solid-phase peptide synthesis to thereby obtain a linear peptide coupled to the resin; (b) Cleaving the linear peptide from the resin to thereby obtain a free linear peptide; (c) Oxidizing the cysteine residues of the linear peptide to form an intramolecular disulfide bond, thereby obtaining a cyclic peptide having SEQ ID NO: 1 in the solution; (d) Isolating the cyclic peptide having SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof, and this isolation includes loading the cyclic peptide onto a reverse-phase chromatography column at a concentration of the cyclic peptide of 40 grams or less per kilogram of the column, and eluting the cyclic peptide from the column; and comprising.

[0013] According to an aspect of some embodiments of the present invention, a large-scale preparation process of a cyclic peptide having SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof is provided, and this process (a) Sequentially coupling amino acids and 4-fluorobenzoic acid to a resin by solid-phase peptide synthesis to obtain a linear peptide coupled to the resin, wherein the substitution degree of this resin is at least 0.3 milliequivalents / gram and / or this resin is Rink aminomethylstyrene (AMS) resin, and (b) Cleaving the linear peptide from the resin to obtain a free linear peptide, and (c) Oxidizing the cysteine residues of the linear peptide to form intramolecular disulfide bonds, thereby obtaining a cyclic peptide having SEQ ID NO: 1 in solution, and (d) Isolating the cyclic peptide having SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof, and comprising.

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

[0015] According to some of each of the embodiments described herein regarding coupling using DIC and ethyl cyano-hydroxyiminoacetate and / or N-hydroxybenzotriazole, DIC and ethyl cyano-hydroxyiminoacetate and / or N-hydroxybenzotriazole are used in an approximately 2-fold excess molar amount.

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

[0017] According to some of any of the embodiments described herein regarding cleavage using 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 any of the embodiments described herein regarding cleavage using a solution comprising dithiothreitol, the concentration of dithiothreitol in the solution is about 50 mg / mL.

[0019] According to some of each of the 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.

[0020] According to some of any of the embodiments described herein regarding the precipitation of a linear peptide, the precipitation is carried out by adding a mixture of tert-butyl methyl ether (MTBE) and hexane at a volume of the mixture of about 45 mL per gram of resin.

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

[0022] According to some of any of the embodiments described herein regarding the contact of a linear peptide with hydrogen peroxide, the contact is carried out by contacting an aqueous solution comprising the linear peptide at a concentration of at least 5 mg / mL with hydrogen peroxide.

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

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

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

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

[0027] According to some of each of the embodiments described herein, the degree of substitution in solid-phase peptide synthesis on resin is at least 0.3 milliequivalent / gram, and / or the resin is 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 technical field to which this invention pertains. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, but exemplary methods and / or materials are described below. In case of conflict, the present patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting necessarily.

[0029] The implementation of the methods and / or systems of the embodiments of the present invention may require performing or completing selected tasks manually, automatically, or a combination thereof. Moreover, depending on the actual instrumentation and apparatus of the embodiments of the methods and / or systems of the present invention, some selected tasks may be implemented by hardware, by software, by firmware, or by a combination thereof using an operating system.

[0030] For example, the hardware for performing a selected task according to an embodiment of the present invention can be implemented as a chip or a circuit. As software, the selected task according to an embodiment of the present invention can be implemented as a plurality 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 plurality of instructions. Optionally, the data processor includes volatile memory for storing instructions and / or data and / or non-volatile storage for storing instructions and / or data, such as a magnetic hard disk and / or a removable medium. Optionally, a network connection is provided as well. A display and / or a user input device, such as a keyboard or a mouse, are also optionally provided as well.

[0031] Merely by way of example, some embodiments of the present invention will be described herein with reference to the accompanying drawings. Next, specific reference will be made in detail to the drawings, it being emphasized that the illustrated details are by way of example and for purposes of illustrative discussion of embodiments of the present invention. In this regard, the description made with reference to the drawings will make apparent to those skilled in the art how embodiments of the present invention can be practiced.

Brief Description of the Drawings

[0032]

Figure 1

Modes for Carrying Out the Invention

[0033] In some embodiments, the present invention relates to chemical synthesis, and more specifically, but not limited thereto, to a novel preparation process of peptides such as BL-8040 that can be used, for example, in treating conditions such as cancer and / or arthritis.

[0034] Before explaining at least one embodiment of the present invention in detail, it should be understood that the present invention is not necessarily limited to the details shown in the following description or exemplified by the examples. The present 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, in which the peptide (in protected form) is formed while attached to a resin and then cleaved from the resin (while removing the protecting groups). Then, intramolecular disulfide bonds, such as those in BL-8040 (4-fluorobenzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH2, SEQ ID NO: 1), are formed by oxidation of the Cys residues.

[0036] After performing laborious experiments, the inventor has found a surprisingly effective novel process for the large-scale synthesis of such peptides, particularly BL-8040.

[0037] According to an aspect of some embodiments of the present invention, there is provided a preparation process of a cyclic peptide containing an intramolecular disulfide bond or a pharmaceutically acceptable salt thereof. In some of any of the embodiments described herein, the cyclic peptide has SEQ ID NO: 1 (4-fluorobenzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH2), 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 sequentially coupling amino acids and, optionally, amino acid analogs (such as N-terminal carboxylic acids like 4-fluoro-benzoic acid) when present in the peptide, to a resin (such as Rink AMS resin) by solid-phase peptide synthesis (according to general procedures known in the art), to obtain a linear peptide coupled to the resin.

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

[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" means a process for preparing at least 100 grams of the final product (in a single run of the process).

[0042] In some of any of the embodiments described herein regarding large-scale processes, 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 sequential coupling order starts from the C-terminus and ends at the N-terminus. For example, in SEQ ID NO:1, the sequential coupling order is Arg, Cys, Cit (citrulline), Arg, Tyr, Pro, DLys (D-lysine), Lys, Cit, Tyr, Cys, Nal (naphthylalanine), Arg, Arg, 4-fluoro-benzoic acid.

[0044] The coupling involves forming 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 the first step or of 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, one or more coupling reagents include a carbodiimide (e.g., diisopropylcarbodiimide) and an additional agent such as hydroxy triazole (e.g., N-hydroxybenzotriazole (HOBt) or 1-hydroxy-7-azabenzotriazole (HOAt)) and / or cyano hydroxyimino acetic acid ester (e.g., ethyl cyano hydroxyimino acetate) (e.g., in approximately the same molar concentration as the carbodiimide).

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

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

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

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

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

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

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

[0053] In some of any of the embodiments described herein, the resin on which the amino acids are sequentially coupled is Rink aminomethylstyrene (AMS) resin (aminomethylstyrene resin substituted with a Rink linker). The Rink AMS resin is optionally protected by 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 any of the embodiments described herein, the substitution degree of the resin (on which the amino acids are sequentially coupled) is at least 0.3 milliequivalents per gram of resin (for example, 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 Rink AMS resin.

[0055] The resin is optionally washed (for example, once or twice) with a solvent such as dimethylformamide (DMF) prior to each coupling step, for example to promote swelling of the resin. In some exemplary embodiments, the resin is washed for 20 to 30 minutes prior to deprotection of the resin (for example, 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 (for example, twice) prior to each subsequent coupling step.

[0056] Cleavage from the resin: When a peptide attached to the resin is formed by sequential coupling according to any of the respective embodiments described herein, the resulting peptide is cleaved from the resin (for example, 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 effected by contacting the peptide (coupled to the resin) with a liquid containing 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, and in this case, for example, the balance is mainly water. In an exemplary embodiment, the concentration of TFA in the liquid is about 95% by weight and the balance is mainly 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 preferably a solution containing TFA (e.g., at the concentration described herein) and DTT and / or DTE, in combination with water.

[0060] In some of any embodiments described herein regarding 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 any embodiments described herein regarding 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 any embodiments described herein regarding 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] When the peptide is cleaved from the resin, the liberated peptide is preferably precipitated, for example, by the addition of a liquid in which the peptide is insoluble. Optionally, prior to precipitation, the liberated peptide is concentrated by evaporation of a portion of the solvent of the solution such that the volume of the solution is reduced (e.g., by about 65% to about 70%). Alternatively, concentration of the liberated peptide is not carried out prior to precipitation.

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

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

[0066] MTBE is optionally mixed with hexane. In some embodiments, a mixture of MTBE and hexane (optionally cooled to a temperature within the range of, for example, about 5 °C to about 15 °C) is added to the peptide in a volume of at least 40 mL of the mixture per gram of resin, optionally about 45 mL of the mixture per gram of resin. A exemplary mixture of MTBE and hexane consists 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 effected by vacuum.

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

[0069] In some of any of the respective embodiments described herein, oxidation is performed by contacting the peptide with hydrogen peroxide, for example, by the gradual (e.g., dropwise) addition of a hydrogen peroxide solution (e.g., about 1.5 wt% hydrogen peroxide in water). The molar ratio of hydrogen peroxide to the Cys residue pair (e.g., with the peptide, provided that 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. The exemplary molar ratio of hydrogen peroxide to the Cys residue pair (e.g., with the peptide) is about 5:1. Alternatively or in addition, hydrogen peroxide may optionally be added until no thiolhydroxy groups remain (as determined by a suitable assay such as the Ellman test).

[0070] In some of any of the respective embodiments described herein, oxidation is performed 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 regarding 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. The exemplary concentration of the peptide is about 10 mg / mL.

[0071] In some of any of the respective embodiments described herein, oxidation is performed 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 regarding 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 any of the embodiments described herein regarding oxidation performed using an aqueous solution, the aqueous solution is a weak alkaline, for example, an aqueous solution of ammonium bicarbonate (NH4HCO3). The exemplary concentration of ammonium bicarbonate is about 0.1 M.

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

[0074] In some of any of the embodiments described herein, peptide isolation includes chromatography, for example, in at least 1 step (for example, optionally 1 or 2 steps) of preparative high performance liquid chromatography (HPLC). In an exemplary embodiment, a C18 column is used to perform the chromatography (for example, HPLC).

[0075] In some of any of the embodiments described herein, peptide isolation includes loading the peptide onto a reverse phase chromatography column (for example, a C18 column) at a concentration of 40 grams or less of peptide per kilogram of column (based on the weight of the resin of the column), for example, in at least 1 step of the HPLC described herein.

[0076] In some of any of the embodiments described herein, the isolation of the peptide comprises loading the peptide (e.g., in at least one step of the HPLC 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 of the column). In some such embodiments, the loading concentration is in the range of 4 to 40 grams of peptide per kg of column resin. In some embodiments, the loading concentration is in the range of 4 to 30 grams of peptide per kg of column resin. In some embodiments, the loading concentration is in the range of 4 to 25 grams of peptide per kg of column resin.

[0077] In some of any of the embodiments described herein, the isolation of the peptide comprises loading the peptide (e.g., in at least one step of the HPLC 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 of the column). In some such embodiments, the loading concentration is in the range of 10 to 40 grams of peptide per kg of column resin. In some embodiments, the loading concentration is in the range of 10 to 30 grams of peptide per kg of column resin. In some embodiments, the loading concentration is in the range of 10 to 25 grams of peptide per kg of column resin.

[0078] In some of any of the embodiments described herein, isolation of the peptide involves loading the peptide (e.g., in at least one step of the HPLC 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 resin (based on the weight of the column resin). In some such embodiments, the loading concentration is in the range of 20 to 40 grams of peptide per kg of column resin. In some embodiments, the loading concentration is in the range of 20 to 30 grams of peptide per kg of column resin. In some embodiments, the loading concentration is in the range of 20 to 25 grams of peptide per kg of column resin.

[0079] (According to any of the respective embodiments described herein) The peptide loaded onto the column is eluted, for example, using a buffer solution (e.g., in combination with an acetonitrile gradient according to any of the respective embodiments described herein). The pH of the buffer solution is optionally in the range of 1.5 to 3, optionally in the range of 2.0 to 2.5, and optionally about 2.25. In some embodiments, triethylammonium phosphate buffer is used to elute the peptide from the column at a concentration of, for example, 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 includes increasing the concentration of acetonitrile at a rate (optionally varying over time) in 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 in addition, the gradient optionally includes increasing the concentration of acetonitrile to a concentration in the range of (e.g., from 0%) 10% - 40%, or 20% - 30%, or about 25%.

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

[0082] In some of each optional embodiment, the eluted peptide is loaded onto the column a second time (according to any of the respective embodiments described herein) and eluted, for example, using 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 at a concentration of, for example, at least about 5 mM, or at least about 20 mM, or at least about 35 mM to elute the peptide from the column (e.g., to obtain the peptide as an acetate). In an exemplary embodiment, the concentration of acetate is about 35 mM.

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

[0084] The process further optionally includes lyophilizing the peptide obtained by chromatography (e.g., according to any of the respective embodiments described herein). Lyophilization within a large-scale process is optionally facilitated by the use of a lyophilization tray (e.g., as exemplified herein) rather than a flask.

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

[0086] As exemplified in this specification, such grinding may advantageously enhance the apparent density of the peptide (also known as "densification") and / or enhance the ease of handling (e.g., during packaging), especially when the lyophilized peptide is in a "fluffy" state (consistency) prior to grinding.

[0087] Other process embodiments: According to some of any of the embodiments described in this specification, 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 in this specification), the coupling is performed using diisopropylcarbodiimide (DIC) combined with ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzotriazole, (ii) (e.g., according to any of the respective embodiments described in this specification), the cleavage of the peptide from the resin is performed by contacting the linear peptide coupled to the resin with a solution containing trifluoroacetic acid (TFA) and a scavenger that is dithiothreitol (DTT) and / or dithioerythritol (DTE), (iii) (e.g., according to any of the respective embodiments described in this specification), the process further includes 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 in this specification), the oxidation includes contacting an aqueous solution containing the linear peptide at a concentration of at least 5 mg / mL with hydrogen peroxide, (v) (For example, according to any of the respective embodiments described herein,) the isolation of the peptide comprises loading the cyclic peptide onto a reverse-phase chromatography column at a concentration of the cyclic peptide of 40 grams or less per kilogram of the column, and eluting the cyclic peptide from the column. (vi) (For example, according to any of the respective embodiments described herein,) the isolation of the cyclic peptide comprises lyophilization, and the process further comprises grinding the cyclic peptide following lyophilization, and / or (vii) (For example, according to any of the respective embodiments described herein,) the degree of substitution of the resin used in peptide synthesis is at least 0.3 milliequivalents / gram, and / or the resin is Rink AMS resin.

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

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

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

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

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

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

[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 conditions treatable by the peptides.

[0096] According to certain embodiments of the present invention, there is provided the use of a peptide prepared according to the processes described herein (according to any of the respective embodiments) in the manufacture of a medicament for treating a condition treatable by the peptide.

[0097] According to certain embodiments of the present invention, there is provided a method of treating a condition treatable by a peptide, the method comprising administering the peptide to a subject in need of treatment of a condition treatable by the peptide, wherein the peptide is prepared according to the processes 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 comprises any treatment that would advantageously inhibit CXCR4.

[0099] During the term of the patent of the present application, many related treatments are expected to be developed, and the scope of the terms "condition", "treatable by a peptide", and "treatment that would advantageously inhibit CXCR4" are intended to deductively include all such new technologies.

[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 conditions treatable by the peptide having SEQ ID NO: 1 (or an analog or derivative thereof) are, for example, cancer or arthritis (e.g., rheumatoid arthritis) as described in U.S. Patent No. 7,423,007 (the content of which, particularly the content regarding the treatment of conditions by the above-described peptide, is incorporated herein by reference).

[0101] Examples of conditions treatable by the peptide having SEQ ID NO: 1 (or an analog or derivative thereof) according to any of the aspects described herein include, but are not limited to, retinoblastoma and / or neuroectodermal tumors as described in WO 2012 / 095849 Pamphlet (the content of which, particularly the content regarding the treatment of conditions by the above-described peptide, is incorporated herein by reference), large cell lung cancer as described in WO 2013 / 160895 Pamphlet (the content of which, particularly the content regarding the treatment of conditions by the above-described peptide, is incorporated herein by reference), multiple myeloma, microglioma, and / or glioma as described in WO 2008 / 075370 Pamphlet (the content of which, particularly the content regarding the treatment of conditions by the above-described peptide, is incorporated herein by reference), breast cancer and / or pancreatic cancer as described in U.S. Patent No. 7,423,007, thrombocytopenia as described in WO 2010 / 146578 Pamphlet (the content of which, particularly the content regarding the treatment of conditions by the above-described peptide, is incorporated herein by reference), myelosuppression risk as described in WO 2008 / 075369 Pamphlet (the content of which, particularly the content regarding the treatment of conditions by the above-described peptide, is incorporated herein by reference), and HIV infection as described in U.S. Patent No. 8,435,939 (the content of which, particularly the content regarding the treatment of conditions by the above-described peptide, is incorporated herein by reference).

[0102] Suitable analogs and derivatives of the peptide having SEQ ID NO: 1 are described in U.S. Patent Nos. 7,423,007 and 8,435,939, and International Publication Pamphlets Nos. WO 2008 / 075369, WO 2008 / 075370, WO 2010 / 146578, WO 2012 / 095849, and WO 2013 / 160895 (the contents of each of them, particularly the contents regarding the analogs 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 in the form of a pharmaceutical composition mixed with the organism itself or a suitable carrier or excipient.

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

[0105] As used herein, the term "active ingredient" means a peptide involved in a biological action.

[0106] Hereinafter, the terms "physiologically acceptable carrier" and "pharmaceutically acceptable carrier", which can be used interchangeably, mean a carrier or diluent that does not cause a significant stimulus to an organism and does not inhibit the biological activity and properties of the administered compound. Adjuvants are included in these terms.

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

[0108] Techniques for formulating and administering agents can 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 injections), and also intrathecal, direct intraventricular, intracardiac (e.g., into the right or left ventricular cavity, 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 the tissue region of the patient.

[0111] The 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, sugar coating, levigating, emulsifying, encapsulating, entrapping, or lyophilization processes.

[0112] Thus, the pharmaceutical compositions for use according to some embodiments of the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries that facilitate the processing of the active ingredient into a pharmaceutically usable preparation. The appropriate formulation depends on the chosen route of administration.

[0113] When intended for injection, the active ingredient of the pharmaceutical composition may be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline buffer. When intended for transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are widely known in the art.

[0114] When intended for oral administration, the pharmaceutical composition can be readily formulated by combining the active compound with a pharmaceutically acceptable carrier well known in the art. Such carriers enable the formulation of the pharmaceutical composition as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for oral ingestion by a patient. Pharmaceutical preparations for oral use can be produced by using solid excipients, optionally grinding the resulting mixture, adding suitable adjuvants if desired, and then treating the granule mixture so as to obtain tablets or dragee cores. Suitable excipients are, in particular, sugars such as lactose, sucrose, mannitol, or sorbitol, cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc., and / or fillers of physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). Optionally, disintegrants such as crosslinked polyvinylpyrrolidone, agar, or alginic acid or its salts such as sodium alginate may be added.

[0115] A suitable coating is provided for the dragee core. For this purpose, a concentrated sugar solution which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solution, and a suitable organic solvent or solvent mixture may be used. Dyes or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of the active compound dosages.

[0116] Examples of orally administrable pharmaceutical compositions include gelatin push-fit capsules, and more particularly soft-seal capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-fit capsules may contain the active ingredient admixed with a filler such as lactose, a binder such as starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In the case of soft capsules, the active ingredient may be dissolved or suspended in a suitable liquid such as fatty oil, liquid paraffin, or liquid polyethylene glycol. Additionally, a stabilizer may be added. All formulations intended for oral administration should be in dosage amounts suitable for the chosen route of administration.

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

[0118] For administration by nasal inhalation, the active ingredient for use according to some embodiments of the present invention is suitably delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer using a suitable propellant, such as 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, such as those made of gelatin for use with a dispenser, may be formulated to contain 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. Injectable formulations may optionally contain a preservative and be presented in unit dosage form, for example, in an ampoule or a multi-dose container. The composition may be a suspension, solution, or emulsion in an oily or aqueous medium and may contain formulating agents such as suspending agents, stabilizers, and / or dispersing agents.

[0120] The pharmaceutical composition for parenteral administration comprises an aqueous solution of the active preparation in water-soluble form. In addition, a suspension of the active ingredient may be prepared as a suitable oily or aqueous injectable suspension. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, liposomes and the like. The aqueous injectable suspension 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 to enable the preparation of a highly concentrated solution.

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

[0122] The pharmaceutical compositions of some embodiments of the present invention may also be formulated in rectal compositions such as suppositories and retention enemas using conventional suppository bases such as cocoa butter and other glycerides.

[0123] Pharmaceutical compositions suitable for use in connection with some embodiments of the present invention include compositions containing the active ingredient in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of the peptide effective to prevent, alleviate, or restore the symptoms of a disorder (such as cancer or arthritis considered herein), or to extend the survival of a subject being treated.

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

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

[0126] The toxicity and therapeutic efficacy of the active ingredients described herein can be determined in vitro, in cell culture, or in experimental animals by standard pharmaceutical procedures. The data obtained from these in vitro and cell culture assays and animal tests can be used to formulate the dosage range for use in humans. The dosage 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, e.g., Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.1 p.1).

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

[0128] Depending on the severity and responsiveness of the condition being treated, administration may be by single or multiple doses in a treatment regimen that continues for a period of several days to several weeks or until cure is achieved or a diminution of the disease state is achieved.

[0129] The amount of the composition administered will, of course, depend on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and the like.

[0130] Compositions of some embodiments of the present invention may, optionally, be presented in a pack or dispenser device that may contain one or more unit dosage forms containing an active ingredient, for example, in an FDA-approved kit. The pack may include, for example, a metal foil or a plastic foil, such as a blister pack. Instructions for administration may be attached to the pack or dispenser device. A caution may also be attached to the pack or dispenser, in a form designated by a government agency that regulates the manufacture, use, or sale of pharmaceuticals, associated with the container. This caution reflects that the form of the composition or its administration to humans or animals has been approved by the regulatory authority. Such a caution may be, for example, a label approved by the U.S. Food and Drug Administration for prescription drugs or an approved product insert. A composition comprising a preparation of the present invention formulated in a suitable pharmaceutical carrier may also be prepared, placed in a suitable container, and labeled for the treatment of a designated medical condition as further detailed above.

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

[0132] The terms "comprises," "comprising," "includes," "including," "having" and their conjugations 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 ingredients, steps, and / or parts, provided that the additional ingredients, steps, and / or parts do not materially alter the basic and novel characteristics 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 terms "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. Therefore, a description of a range should be considered to specifically disclose all the possible sub-ranges within that range as well as individual numerical values. For example, a description of a range such as 1 - 6 should be considered to specifically disclose sub-ranges such as 1 - 3, 1 - 4, 1 - 5, 2 - 4, 2 - 6, 3 - 6, etc., as well as individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0137] When a numerical range is specified herein, it always includes any recited number (fractional or integral) within the specified range. The phrases "ranging / ranges between the first specified number and the second specified number" and "ranging / ranges from the first specified number to the second specified number" are used interchangeably herein and mean including the first and second specified numbers, as well as all fractions and integers therebetween.

[0138] The term "treating" means inhibiting, preventing, or arresting the development of a pathological condition (disease, disorder, or medical condition), and / or causing a reduction, remission, or regression of a pathological condition. It will be understood by those skilled in the art that the development of a pathological condition can be assessed using a variety of methods and assays, and that a reduction, remission, or regression of a pathological condition can be assessed using a variety of methods and assays.

[0139] As used herein, the term "preventing" means preventing a disease, disorder, or medical condition from occurring in a subject who is at risk of developing the disease but has not yet been diagnosed as having the disease.

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

[0141] As used herein, the term "method" means a way, means, technique, and procedure for achieving a given task, including, but not limited to, ways, means, techniques, and procedures known to, or readily developed by, practitioners of chemical, pharmacological, biological, biochemical, and medical arts.

[0142] When referring to a particular sequence listing, such reference should be understood to include sequences that substantially correspond to its complementary sequence, including 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 described in connection with separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, each of the various features of the invention described in connection with a single embodiment for brevity may also be provided separately, or in any suitable partial combination, or as suitable for any other described embodiment of the invention. A particular feature described in connection with various embodiments should not be considered an essential feature of that embodiment, unless the embodiment would be ineffective without that element.

[0144] Experimental support for the various embodiments and aspects of the invention clarified above and recited in the following claims is found in the following examples.

Examples

[0145] Next, with reference to the following examples, some embodiments of the invention are illustratively and non - limitatively exemplified in conjunction with the above description.

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

[0147] The cyclic peptide BL-8040 was prepared by solid-phase synthesis. To construct 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 sequentially attaching amino acids for peptide elongation on the resin. The last residue added to the peptide on the resin was a 4-fluorobenzoyl group without an Fmoc group.

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

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

[0150] Attachment of amino acids: Amino acids were coupled in the form of appropriate amino acid derivatives using diisopropylcarbodiimide (DIC) combined 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 an amino acid. The amount of amino acid (or 4-fluorobenzoic acid) used was 2.5 equivalents.

[0151] The ninhydrin test of Kaiser et al. [Anal Biochem 1970, 34:595-598] was used to monitor the in-process of the condensation reaction.

[0152] Cleavage and deprotection: Treatment with 270 mL of a 1 M TMSBr - thioanisole / trifluoroacetic acid (TFA) mixture in the presence of m - cresol (100 equivalents) and ethanedithiol (300 equivalents) for 3 hours at 25 °C effected cleavage of the peptide (1 mmol) from the resin and removal of the protecting groups.

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

[0154] The mixture of the filtrate and the wash solution was subjected to concentration under vacuum.

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

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

[0157] Oxidative cyclization: An aqueous dilute solution of the crude linear peptide was adjusted to pH 7.5 with concentrated aqueous ammonia and oxidized by aerated air oxidation.

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

[0159] A polypeptide with a single peak 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 solid - phase synthesis of BL - 8040 The cyclic peptide BL-8040 was prepared by large-scale (825 mmol) solid-phase synthesis. To construct 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 sequentially attaching the amino acids for peptide elongation on the resin. The last residue added to the peptide on the resin was a 4-fluorobenzoyl group without an Fmoc group.

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

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

[0165] Removal of the 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 20% piperidine solution in dimethylformamide (DMF) (10 ml / gram of the initial resin). The piperidine solution was removed by washing with DMF before the next amino acid reaction and confirmed by pH testing of the washings.

[0166] Attachment of the amino acids: The amino acids were coupled in the form of appropriate amino acid derivatives using diisopropylcarbodiimide (DIC) combined with HOBt (N-hydroxybenzotriazole) as the activator. 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 an amino acid. The calculation of the amounts of amino acid (or 4-fluorobenzoic acid), DIC, and HOBt was based on a two-fold excess of substitution and the batch size.

[0167] To evaluate the coupling step, in-process monitoring was performed using ninhydrin and chloranil tests at the end of each cycle. Negative test results indicate the absence of free amino groups (complete coupling). If the test was positive, indicating unreacted amino groups (incomplete coupling), the coupling reaction could be extended or recoupling of the protected amino acid derivative could be carried out.

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

[0169] Cleavage and deprotection: Cleavage was carried out to remove the peptide molecule from its support resin and to remove the protecting groups. For example, an acidolysis reaction was carried out using 95% trifluoroacetic acid (TFA) and 5% water containing 50 mg / mL dithioerythritol (DTE) as a scavenger (10 mL TFA-based cleavage solution per gram of resin) at ambient temperature for 3.25 - 3.5 hours.

[0170] Subsequently, to complete the extraction of the peptide, the resin was filtered and washed twice with TFA.

[0171] Under vacuum, using a rotary evaporator (at approximately 35 °C), the volume of the peptide solution was reduced to approximately 30 - 35% of the original volume.

[0172] Subsequently, the peptide was precipitated using a cold mixture of tert-butyl methyl ether (MTBE) / hexane (60:40 v / v) (at -10 ± 5 °C) 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 nitrogen stream to remove most of the solvent.

[0174] Next, the obtained crude (linear) peptide was dissolved in 90% acetic acid, and the resulting solution was dispensed into a lyophilization flask, shelf-frozen, and freeze-dried on a manifold freeze-dryer until dry.

[0175] The purity of the peptide was analyzed by RP-HPLC, and the content was confirmed by mass spectrometry analysis.

[0176] Oxidative cyclization: As follows, the crude linear peptide was cyclized by oxidation with hydrogen peroxide in a 0.1 M ammonium bicarbonate (NH4HCO3) solution.

[0177] The crude linear peptide was dissolved in 0.1 M NH4HCO3 at a concentration of 10 mg / mL, and an equal volume of 0.1 M NH4HCO3 was added to dilute the peptide to a concentration of 5 mg / mL.

[0178] Subsequently, a 1.5% hydrogen peroxide solution in water (5-fold excess) was added dropwise to the peptide solution over 25 - 30 minutes. The reaction was monitored using the Ellman 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 the 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): A solution of approximately 2.4 L containing approximately 11.7 grams of crude peptide per kilogram of resin was loaded, and the solution obtained after completion of the cyclization step was purified by separation using a preparative RP-HPLC C18 column, 10 μm, 120 Å Daisogel (trademark).

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

[0182] The elution fractions were collected, sampled, and tested by HPLC to determine which fractions were sufficiently pure (≥95%) and pooled for the second chromatography purification step. The hydrophilic and hydrophobic fractions of the first RP-HPLC purification that did not meet the purity acceptance criteria may be reprocessed to maximize the overall yield.

[0183] Second preparative HPLC column (acetic acid purification): The purified fractions obtained from TEAP injection were pooled, diluted 1:1 with water, loaded at a volume of approximately 2.7 L per kg of resin using a 10 μm, 120 Å Daisogel™ C18 column, and eluted with a gradient of 35 mM acetate-based buffer and acetonitrile to separate the peptides. The gradient was as follows: 8 minutes with 0% acetonitrile and 110 minutes with 0% to 22% acetonitrile (at a rate of 0.2% / minute). Before the start of the elution gradient after peptide loading, the column was washed with 4 column volumes of 0.1 M ammonium acetate to obtain the peptide as an acetate salt.

[0184] Fraction collection was monitored by UV absorption at 230 nm. The elution fractions were collected, sampled, analyzed by HPLC to determine which fractions were sufficiently pure, and pooled. Only the fractions that met the in-process control criteria (purity by HPLC ≥98% and unknown impurities <0.14%) were pooled. The pooled fractions of each chromatography cycle were lyophilized as sublots. The hydrophilic and hydrophobic fractions of the second RP-HPLC purification that did not meet the purity acceptance criteria were retained and may be reprocessed to maximize the overall yield.

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

[0186] Lyophilization was carried out in 1200 mL lyophilization flasks. Each flask was filled with approximately an equal volume of 100 mL so that 5 grams of purified peptide could be obtained per flask, and then shelf-frozen in a dry ice / IPA bath with a condenser temperature lower than -60 °C and a vacuum of less than 500 millitorr. Lyophilization was carried out for 65 - 85 hours.

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

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

[0189] Packaging and storage: The final bulk drug substance was packaged in type III depyrogenated amber glass bottles with Teflon-lined polypropylene caps. Packaging was carried out under a controlled humidity environment (15 - 30% relative humidity) to reduce water uptake. The bottle was placed in an aluminum laminate bag with a desiccant between two packaging materials as a second container. Then, the BL-8040 drug substance was stored at -20 ± 5 °C.

[0190] Example 2 Modified large-scale solid-phase synthesis of BL-8040 The cyclic peptide BL-8040 is prepared by large-scale solid-phase synthesis (e.g., at least about 500 grams of product). To construct the entire peptide, the solid-phase synthesis reaction steps are repeated 14 times, starting from the C-terminal amino acid and ending with the N-terminal amino acid. Each amino acid addition is carried out 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 sequentially attaching amino acids for peptide elongation 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 Fmoc-Rink AMS resin having a substitution within the range of 0.6 to 0.9 milliequivalents / gram (different from less than or equal to 0.6 milliequivalents / gram described above).

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

[0193] Binding of amino acids: As an activator, (different from the above-mentioned HOBt), diisopropylcarbodiimide (DIC) combined with ethyl cyanohydroxyiminoacetate is used to couple the amino acids in an 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 instead of the amino acid. The calculation of the amounts of amino acid (or 4-fluorobenzoic acid), DIC, and ethyl cyanohydroxyiminoacetate is based on a 2-fold excess over substitution and the batch size.

[0194] To evaluate the coupling step, in-process monitoring is carried out using ninhydrin and chloranil tests at the end of each cycle. A negative test result indicates the absence of free amino groups (complete coupling). If the test is positive and indicates unreacted amino groups (incomplete coupling), the coupling reaction can be extended, the re-coupling of the protected amino acid derivative can be carried out, or acetylation (capping) can be carried out using acetic anhydride in the presence of, for example, 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, an acidolysis reaction is carried out using 95% trifluoroacetic acid (TFA) containing 50 mg / mL scavenger and 5% water (10 mL TFA per gram of resin as the cleavage solution) for 3.25 - 3.5 hours at ambient temperature. The scavenger is dithiothreitol (DTT), which is different from the dithioerythritol described in Example 1 or ethanedithiol described in Comparative Example 1.

[0197] Next, to complete the extraction of the peptide, the resin is filtered and washed twice with TFA. (Unlike Example 1, reduction of the peptide solution volume is not carried out at this stage.)

[0198] Next, the peptide is precipitated using a cold mixture (-10 ± 5 °C) of tert-butyl methyl ether (MTBE) / hexane (60:49 v / v) at a volume of 45 mL per gram of resin (not 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 nitrogen stream to remove most of the solvent.

[0200] Next, the obtained crude (linear) peptide is dried under vacuum or solubilized with acetic acid and then lyophilized (for example, as described in Example 1).

[0201] Optionally, analyze the purity of the peptide by RP-HPLC and / or confirm the identity by mass spectrometry analysis.

[0202] Oxidative cyclization: The crude linear peptide is cyclized at a peptide concentration of 10 mg / mL by oxidation with 1.5% hydrogen peroxide in 0.1 M ammonium bicarbonate (NH4HCO3) solution. Unlike Example 1, the peptide is not further diluted to 5 mg / mL over 25 - 30 minutes. Monitor the reaction using the Ellman test to confirm the absence of free sulfhydryl groups.

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

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

[0205] Elute the peptide with a gradient of 0.1 M triethylammonium phosphate (TEAP) buffer (pH 2.25) and acetonitrile (ACN).

[0206] Collect the elution fractions, sample them, test by HPLC to determine which fractions are sufficiently pure (≥ 95%), and pool them for the second chromatography purification step. Leave the hydrophilic and hydrophobic fractions of the first RP-HPLC purification that do not meet the purity acceptance criteria and they may be reprocessed to maximize the overall yield.

[0207] Second preparative HPLC column (acetic acid purification): Pool the purified fractions obtained from the TEAP injection, dilute 1:1 with water, and separate the peptide by eluting with a gradient of 35 mM acetate-based buffer and acetonitrile using a 10 μm, 120 Å Daisogel™ C18 column. Before starting the elution gradient after peptide loading, wash the column with 4 column volumes of 0.1 M ammonium acetate to obtain the peptide as the acetate salt.

[0208] Monitor the collection of fractions by UV absorption at 230 nm. Collect the elution fractions, sample them, test by HPLC to determine which fractions are sufficiently pure, and pool them. Pool only the fractions that meet the in-process control criteria (purity by HPLC ≥ 98% and unknown impurities ≥ 0.14%). Lyophilize the pooled fractions of each chromatography cycle as sublots. Leave the hydrophilic and hydrophobic fractions of the second RP-HPLC purification that do not meet the purity acceptance criteria and they may be reprocessed to maximize the overall yield.

[0209] Wet pooling and lyophilization: Reconstitute all sublots that meet the in-process control criteria of the purified material with purified water at a concentration of approximately 50 g / L. Filter the resulting solution through a 0.2 μm PVDF filter before lyophilization.

[0210] Lyophilization is carried out in a lyophilization tray (e.g., approximately 1.0 to approximately 1.2 liters / tray), rather than in the flask described in Example 1, under a vacuum of ≤ 150 millitorr or less (e.g., over approximately 89 hours). The use of trays may result in enhanced process control, facilitation of scale-up, and / or reduction of in-process control.

[0211] After lyophilization, samples are taken for acetate content measurement. Optionally, if it is not shown to be within the range of 11 - 15%, the bulk drug substance is resuspended in water and lyophilization is repeated.

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

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

[0214] Although the invention has been described in connection with its specific embodiments, it is obvious that many alternative, modified, and variant forms will be apparent to those skilled in the art. Therefore, it is intended to embrace all such alternative, modified, and variant forms that fall within the spirit and broad scope of the appended claims.

[0215] All publications, patents, and patent applications referred to in this specification are hereby incorporated by reference in their entirety as if each individual publication, patent, and patent application was specifically and individually recited when so referred to. In addition, any citation or identification of a 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 are not necessarily to be construed as limiting to the extent they are used. In addition, any priority documents of this application are hereby incorporated by reference in their entirety into this specification with this application.

Sequence Listing Free-Text

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

Claims

1. A large-scale preparation process of a cyclic peptide having SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof, wherein the process comprises: a) sequentially coupling an amino acid and 4-fluorobenzoic acid to a resin by solid-phase peptide synthesis to obtain a linear peptide coupled to the resin; b) cleaving the linear peptide from the resin to obtain a free linear peptide; c) oxidizing the cysteine residues of the linear peptide to form intramolecular disulfide bonds, the oxidation being carried out by contacting the linear peptide with hydrogen peroxide to obtain the cyclic peptide having SEQ ID NO: 1 in solution; d) isolating the cyclic peptide having SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof; comprising: i) the coupling is carried out using diisopropylcarbodiimide (DIC) combined with ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzotriazole; ii) the cleavage is carried out by contacting the linear peptide coupled to the resin with a solution containing 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 isolation comprises loading the cyclic peptide onto a reverse-phase chromatography column at a concentration of the cyclic peptide of 40 grams or less per kg of the column and eluting the cyclic peptide from the column; v) 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 vi) the substitution degree of the resin is at least 0.3 milliequivalent / gram, and / or the resin is Rink aminomethyl styrene resin; process.

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

3. The process according to claim 1, wherein the DIC and the ethyl cyanohydroxyiminoacetate and / or N-hydroxybenzotriazole are used in an approximately two-fold excess molar amount.

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

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

7. The process according to claim 1, further comprising precipitating the free linear peptide after the cleavage without concentrating the free linear peptide by evaporation before the precipitation.

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

9. The process according to claim 1, wherein contacting the linear peptide with hydrogen peroxide is carried out by contacting an aqueous solution containing the linear peptide at a concentration of at least 5 mg / mL with hydrogen peroxide.

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

11. The process according to claim 1, wherein the column is a C18 column.

12. The process according to claim 1, wherein the elution is carried out with a triethylammonium phosphate solution.

13. The isolation of the cyclic peptide having SEQ ID NO: 1 includes lyophilization, and the process further comprises pulverizing the cyclic peptide following the lyophilization, according to claim 1.

14. The process according to claim 1, wherein the substitution degree of the resin is at least 0.3 meq / g and / or the resin is Rink aminomethylstyrene resin.

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