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EP-4593865-B1 - PHARMACEUTICAL COMPOSITION COMPRISING NFL-TBS40-63 PEPTIDE, VARIANTS OR SALTS THEREOF AND ALANINE

EP4593865B1EP 4593865 B1EP4593865 B1EP 4593865B1EP-4593865-B1

Inventors

  • BALDWIN, PAUL
  • BOUCHET, Benjamin

Dates

Publication Date
20260506
Application Date
20230929

Claims (15)

  1. A pharmaceutical composition comprising NFL-TBS 40-63 peptide, biologically active variants or pharmaceutically acceptable salts thereof, an alanine and pharmaceutically acceptable excipients, wherein the alanine is at concentrations comprised between 25mM and 120 mM.
  2. A pharmaceutical composition of claim 1, wherein the alanine is a L-alanine.
  3. A pharmaceutical composition according to claim 1 or claim 2, wherein the pharmaceutically acceptable excipients are selected from the groups consisting of a buffer, an osmolarity adjusting agent and one or more solubilizing agent(s).
  4. A pharmaceutical composition according to any one of the preceding claims, wherein: - the buffer is selected from the group consisting of phosphate, citrate, L-histidine, acetate, lactic acid, tromethamine, gluconic acid, tartaric acid, succinate, malic, fumaric, α-ketoglutaric, an amino acid which is naturally positively charged at physiological pH, preferably the buffer is a L-histidine; - the osmolarity adjusting agent is a polyol compound, preferably a glycerol, and - the solubilizing agent is selected from the group consisting of a cyclodextrin, polypropylene glycol and polyethylene glycol (PEG).
  5. A pharmaceutical composition according to claim 3 and claim 4, wherein the buffer is L-histidine, preferably at concentration comprised between 10-100 mM.
  6. A pharmaceutical composition according to any one of the preceding claims which is an aqueous composition comprising: - NFL-TBS 40-63 peptide, biologically active variants or pharmaceutically acceptable salts thereof at a concentration comprised between 1 and 20 mg/ml, preferably 3mg/mL - an alanine at concentration comprised between 40 and 60 mM, - L-histidine at concentration comprised between 10 and 100 mM, - a glycerol, preferably at 0.5% to 5% of the total concentration of the composition and - a cyclodextrin, preferably 2-Hydroxypropyl-β-cyclodextrin or sulfobuytlether- β-cyclodextrin at a concentration comprised between 0.5 and 50% of the total concentration of the composition.
  7. A method for manufacturing a pharmaceutical composition according to any one of claims 1 to 6 comprising the following steps: a) preparing a solution, preferably an aqueous solution, comprising a buffer, an osmolarity adjusting agent, one or more solubilizing agent(s) and alanine, preferably a L-alanine; b) adding NFL-TBS 40-63 peptide, biologically active variants or pharmaceutically acceptable salts thereof to the solution prepared in step a); c) pre-filtering the formulation obtained in step b) by using a filter with pore size compromised preferably between 0.3 and 1.0 µM, and d) sterilizing the pre-filtered formulation of step c) by using a filter with pore size comprised preferably between 0.1 and 0.2 µM.
  8. A method according to claim 7, wherein in step a): - the buffer is selected from the group consisting of phosphate, citrate, L-histidine, acetate, lactic acid, tromethamine, gluconic acid, tartaric acid, succinate, malic, fumaric, α-ketoglutaric, an amino acid with is natural or positively charged at physiological pH, preferably L-histidine; - the osmolarity adjusting agent is a polyol compound, preferably a glycerol, and - the solubilizing agent is selected from the group consisting of a cyclodextrin, polypropylene glycol and polyethylene glycol (PEG).
  9. A method according to claim 7 or claim 8, wherein in step b) NFL-TBS 40-63 peptide is a powder, preferably a micronized powder and/or a lyophilized powder, added at a concentration comprised between 2 and 5 mM, preferably 3mM.
  10. A method according to any one of claims 7 to 9, wherein said method further comprises a step of heating the solution obtained in step a) and/or the formulation obtained in step b) at a temperature comprised between 20° C and 70° C, preferably between 40° C and 45° C.
  11. A method according to any one of claims 7 to 10, wherein the addition of NFL-TBS 40-63 peptide, biologically active variants or pharmaceutically acceptable salts thereof in step b) is performed under agitation.
  12. A pharmaceutical composition according to any one of claims 1 to 6 for use as medicament.
  13. A pharmaceutical composition according to any one of claims 1 to 6 for use as a medicament for treating cancer.
  14. A composition for use according to claim 13, wherein the cancer is a solid peripheral cancer or a cancer of the central nervous system.
  15. A composition for use according to any one of claims 12 to 14 which is an injectable composition for parenteral administration.

Description

TECHNICAL FIELD OF THE INVENTION The present invention relates to a pharmaceutical composition comprising NFL-TBS40-63 peptide, biologically active variants or pharmaceutically acceptable salts thereof and alanine in specific concentrations. The present invention also relates to a manufacturing method of said composition and to the application of this composition as a medicament, particularly for treating cancer. BACKGROUND OF THE INVENTION Cell division, or mitosis, is the process that enables cells to multiply in order to construct or regenerate tissues or to replace dead cells. In cancer cells the regulation of this process is faulty, which is why these cells divide uncontrollably and thus create tumors. One effective therapeutic route for combating cancers comprises blocking the division of cancer cells. Tubulin is a fundamental molecule in the process of cell division. In fact, it polymerizes to form the microtubules that are indispensable for intracellular transport during mitosis. It was found in the past that fragments of intermediate filaments (from neurofilaments) can bind tubulin and alter microtubule polymerization, thus blocking cell division. These fragments (peptides) are disclosed in application WO 2005/121172 as corresponding to the tubulin-binding site (TBS), located in intermediate filament proteins (namely the neurofilament light chain protein NFL, keratin 8, GFAP, and vimentin). In addition, international application WO 2011/073207 and Bocquet et al. (2009) disclosed that the peptide sequence of the second tubulin-binding site of the NFL protein (called "NFL-TBS.40-63") is able to inhibit the proliferation of neuroblastoma and glioblastoma cell lines in vitro. The efficiency of NFL-TBS.40-63 and its biologically active derivatives in the treatment of cancer, and particularly of glioblastoma was previously demonstrated. This peptide thus appears as a promising treatment of cancer. To be used as a medicament, NFL-TBS.40-63 peptide has to be integrated in a pharmaceutical composition. Given that this peptide cannot be administered via the oral route (since it is degraded in the digestive tract), the parenteral route is the preferred route. The simplest parenteral formulation for any drug is a true, isotonic, aqueous solution, ideally buffered at a set pH. However, formulation of a parenterally acceptable buffered, isotonic, true aqueous solution of NFL-TBS.40-63 peptide proved to be a substantial challenge. Firstly, NFL-TBS.40-63 peptide has a low intrinsic aqueous solubility (< 2.5 mg/mL), which is practically unaffected by nearly all parenterally acceptable co-solvents and surfactants at pharmaceutically acceptable concentrations. Secondly, NFL-TBS.40-63 peptide is insoluble in nearly all commonly used parenteral buffer systems and tends to irreversibly precipitate in the presence of sodium or potassium ions (which are present in most buffer systems). Finally, and most importantly, NFL-TBS.40-63 peptide self-associates to form sub-visible fibrillar structures (up to approximately 2 µm in length). Fibril structures are highly undesirable in an injectable solution due to the risk that they could cause unwanted side effects. Furthermore, their presence hampers industrial production of a viable, sterile drug product: it was found that solutions of NFL-TBS.40-63 peptide at 1mg/mL in water contain large, tangled masses of fibrils that very quickly block a 0.2µm sterilizing filter (Figure 1). In addition, given that peptides are highly heat sensitive, peptide solutions cannot be sterilized by autoclaving. Furthermore, gamma irradiation of aqueous solutions is not advised due to formation of free radicals which can extensively degrade drug products. Aseptic processing carries an inherent risk of non-sterility and is not a preferred production method (EMA guideline: EMA/CHMP/CVMP/QWP/850374/201). Furthermore, aseptic processing would not resolve the problem of the fibrils being present in the formulation. From the above, it appears that there are several problems (low solubilization, formation of fibrils, difficult sterilization) to be solved in order to obtain a pharmaceutical composition, wherein the therapeutic activity of the peptide is not decreased, and which is appropriate for parenteral administration. In addition, this pharmaceutical composition has to be suitable for manufacturing in industrial conditions and has to remain stable over time. Golonov et al. (2004) discloses that the solubility of proteins and peptides can be increased by the use of 50mM glutamine and 50mM arginine. However, this combination of amino acids did not increase the solubility of NFL-TBS.40-63 peptide, and in fact it gave a lower solubility as compared to the intrinsic solubility of NFL-TBS.40-63 peptide in water (<0.8mg/mL compared to < 2.5 mg/mL, respectively). Maggio et al. (2010) and Hamada et al. (2009) discloses various excipients which are used to prevent peptide aggregation and fibril formation, but thes