WO2020170185A1 - Lanréotide sensiblement pur ou son sel et méthode associée - Google Patents

Lanréotide sensiblement pur ou son sel et méthode associée Download PDF

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Publication number
WO2020170185A1
WO2020170185A1 PCT/IB2020/051420 IB2020051420W WO2020170185A1 WO 2020170185 A1 WO2020170185 A1 WO 2020170185A1 IB 2020051420 W IB2020051420 W IB 2020051420W WO 2020170185 A1 WO2020170185 A1 WO 2020170185A1
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WIPO (PCT)
Prior art keywords
lanreotide
salt
amino acid
give
resin
Prior art date
Application number
PCT/IB2020/051420
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English (en)
Inventor
Justine Ann PETERSON
Peter Mccormack
Colin Thomas DEWAR
Jonathan Paul EDDOLLS
Sebastian H B KROLL
Sunil Kumar Gandavadi
Murali Mohan M
Kiran kumar K Y
Appala Raju JONNADA
Pavan Aditya Y S V
Karthik Ramasamy
Original Assignee
Dr. Reddy’S Laboratories Limited
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Application filed by Dr. Reddy’S Laboratories Limited filed Critical Dr. Reddy’S Laboratories Limited
Publication of WO2020170185A1 publication Critical patent/WO2020170185A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/655—Somatostatins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present application relates to substantially pure Lanreotide or its salt and preparation thereof.
  • Lanreotide acetate is a synthetic cyclic octapeptide analog of the natural hormone, somatostatin.
  • Lanreotide acetate is chemically known as [cyclo S-S]-3-(2-naphthyl)-D-alanyl-L- cysteinyl-L-tyrosyl-D-tryptophyl-L-lysyl-L-valyl-L-cysteinyl-L-threoninamide, acetate salt. Its molecular weight is 1096.34 (base) and its amino acid sequence is:
  • the SOMATULINE DEPOT in the prefilled syringe is a white to pale yellow, semi-solid formulation.
  • the mechanism of action of lanreotide is believed to be similar to that of natural somatostatin.
  • SOMATULINE DEPOT is indicated for: a) the long-term treatment of acromegalic patients who have had an inadequate response to or cannot be treated with surgery and/or radiotherapy; b) the treatment of adult patients with unresectable, well-or moderately-differentiated, locally advanced or metastatic gastroenteropancreatic neuroendocrine tumors (GEP-NETs) to improve progression -free survival; & c) the treatment of adults with carcinoid syndrome.
  • GEP-NETs gastroenteropancreatic neuroendocrine tumors
  • EP0389180B1 & US5073541 disclose preparation of Lanreotide by solid phase peptide synthesis using Boc protocol and iodine mediated cyclization.
  • US8383770B1 also discloses preparation of Lanreotide acetate by solid phase peptide synthesis using chloromethylated polystyrene resin, aqueous solution of cesium carbonate and iodine mediated cyclization.
  • CN 104497130 A discloses preparation of Lanreotide and its salts by fragmentation approach which involve coupling of dipeptide (Boc-D-2-Nal-Cys(Trt)) with hexapeptide (H- Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Val-Cys(Trt)-Thr(tBu)-NH-Resin) using combination of solid phase & liquid phase and hydrogen peroxide mediated cyclization.
  • W02013098802A discloses preparation of Lanreotide by solid phase synthesis using resin bound Thr-amide viz. Fmoc-Thr(Resin)-NH2 which is subjected to seven sequential cycles of deprotection and coupling steps to give Boc-D-2-Nal-Cys(Trt)-Tyr(Clt)-D-Trp-Lys(Mtt)-Val- Cys(Trt)-Thr(Resin)-NH2, deprotected, cleaved from the resin, subjected to iodine mediated cylization, purified by high performance liquid chromatography and lyophilized.
  • CN105842362B discloses a process for separation and detection of Lanreotide using high performance liquid chromatography, wherein the stationary phase is a mixed-mode column C18SCX.
  • WO2017178950A2 disclose solution phase synthesis of Lanreotide acetate and iodine mediated cyclization.
  • WO2019184089A1 discloses use of siber resin for the preparation of Lanreotide and hydrogen peroxide mediated cyclization.
  • US6503534B2 discloses solid or semi-solid pharmaceutical composition of Lanreotide acetate having a high specific surface area of at least about 4 m 2 /g.
  • US9352012B2 discloses preparation of sustained release pharmaceutical composition of Lanreotide acetate, comprising lyophilization of a mixture of Lanreotide salt and an aqueous acid solution in a stepwise temperature pattern.
  • present invention provides substantially pure Lanreotide or its salt.
  • present invention provides a process for the preparation of substantially pure Lanreotide or its salt.
  • present invention provides pharmaceutical composition comprising substantially pure Lanreotide or its salt.
  • present invention provides a process for the preparation of substantially pure Lanreotide or its salt, the process comprising the steps of:
  • step (c) deblocking the PI group from the next blocked amino acid resin coupled product; e) sequentially performing step (c) and step (d) after the formation of the first deblocked coupled resin product of H-Thr(P2)-resin obtained in step b) using N a -terminal Pl- protected amino acids in the order of Cys, Val, Lys, D-Trp, Tyr, Cys, D-2-Nal to give linear Lanreotide peptide;
  • PI group is protecting group attached to the N a -terminal group
  • P2 group is side-chain protecting group
  • present invention provides a process for the preparation substantially pure Lanreotide or its salt, wherein the process comprises use of DMSO as a cyclizing agent.
  • present invention provides a process for the preparation of Lanreotide or its salt, the process comprising the steps of:
  • step (c) deblocking the PI group from the next blocked amino acid resin coupled product; e) sequentially performing step (c) and step (d) after the formation of the first deblocked coupled resin product of H-Thr(P2)-resin obtained in step b) using N a -terminal Pl- protected amino acids in the order of Cys, Val, Lys, D-Trp, Tyr, Cys, D-2-Nal to give linear Lanreotide peptide;
  • PI group is protecting group attached to the N a -terminal group
  • P2 group is side-chain protecting group
  • present invention provides a process for the preparation Lanreotide or its salt, the process comprising the steps of:
  • step b) deblocking the Fmoc group from the next blocked amino acid resin coupled product, e) sequentially performing steps (c) and (d) after the formation of the first deblocked coupled resin product of H-Thr(P2)-resin obtained in step b) using N a -terminal Fmoc protected amino acids in the order of Cys, Val, Lys, D-Trp, Tyr and Cys; f) coupling of Boc-D-2Nal-OH to give linear Lanreotide peptide;
  • P2 group is side-chain protecting group
  • present invention provides an improved process for purifying the cmde Lanreotide or its salt to give substantially pure Lanreotide or its salt, the process comprising the steps of:
  • present invention provides a method for the preparation of substantially pure Lanreotide or its salt by using enantiomerically pure amino acids.
  • said enantiomerically pure amino acid can be selected from Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH and Boc-D-2Nal-OH.
  • present invention involves the purification of commercially available amino acid for attaining high purity of raw materials for preparation of Lanreotide.
  • present invention provides a method of preparing Lanreotide drug product, the method comprising:
  • present invention provides a method of assaying purity of a sample of Lanreotide or its salt or a pharmaceutical dosage form comprising Lanreotide or its salt, wherein method comprise the steps of:
  • Lanreotide or its salt sample onto a chromatographic column; b) eluting Lanreotide or its salt from the column with an eluent comprising polar solvent and an ion pair agent;
  • Figure 1 depicts a HPLC chromatogram of crude Lanreotide having content of D-Allo-
  • Figure 2 depicts a HPLC chromatogram of substantially pure Lanreotide as obtained from present invention.
  • Figure 3 depicts a HPLC chromatogram of substantially pure Lanreotide as obtained from present invention.
  • Quality control of peptide active pharmaceutical ingredients prior to dmg product manufacturing is an essential requirement. Generally, it is very difficult to remove impurities which are having RRT very closer to main peak. The present inventor successfully removed D- Allo -Threonine Lanreotide impurity which has RRT very close to Lanreotide or its salt.
  • present invention provides substantially pure Lanreotide or its salt.
  • present invention provides substantially pure Lanreotide acetate, wherein, D-Allo-Threonine Lanreotide impurity less than impurity 0.10%.
  • present invention provides substantially pure Lanreotide acetate, wherein, D-Allo-Threonine Lanreotide impurity less than impurity 0.080%. In another aspect of first embodiment, present invention provides substantially pure Lanreotide acetate, wherein, D-Allo-Threonine Lanreotide impurity less than impurity 0.050%.
  • present invention provides substantially pure Lanreotide acetate, wherein, D-Allo-Threonine Lanreotide impurity less than impurity 0.020%. In another aspect of first embodiment, present invention provides substantially pure Lanreotide acetate, wherein, D-Allo-Threonine Lanreotide impurity less than impurity 0.010%.
  • composition comprising substantially pure Lanreotide or its salt.
  • composition comprising substantially pure Lanreotide or its salt, wherein, D-Allo-Threonine Lanreotide impurity less than impurity 0.080%.
  • composition comprising substantially pure Lanreotide or its salt, wherein, D-Allo-Threonine Lanreotide impurity less than impurity 0.050%.
  • composition comprising substantially pure Lanreotide or its salt, wherein, D-Allo-Threonine Lanreotide impurity less than impurity 0.020%.
  • composition comprising substantially pure Lanreotide or its salt, wherein, D-Allo-Threonine Lanreotide impurity less than impurity 0.10%.
  • present invention provides a process for the preparation of substantially pure Lanreotide or its salt.
  • present invention provides a process for the preparation of substantially pure Lanreotide or its salt, the process comprising the steps of:
  • step (c) deblocking the PI group from the next blocked amino acid resin coupled product; e) sequentially performing step (c) and step (d) after the formation of the first deblocked coupled resin product of H-Thr(P2)-resin obtained in step b) using N a -terminal Pl- protected amino acids in the order of Cys, Val, Lys, D-Trp, Tyr, Cys, D-2-Nal to give linear Lanreotide peptide;
  • PI group is protecting group attached to the N a -terminal group
  • P2 group is side-chain protecting group
  • the step a) of the second embodiment involves coupling of first amino acid Pl-Thr(P2)- OH to a resin solid phase support in presence of coupling reagent and activator additive in a suitable solvent.
  • the step b) of the second embodiment comprises deblocking of PI group from first blocked amino acid coupled product viz. Pl-Thr(P2)-OH using suitable deblocking reagent.
  • the step c) of the second embodiment involves coupling a next amino acid having N a - terminal blocked by PI to the deblocked amino acid resin coupled product in presence of coupling reagent and activator additive in a suitable solvent for the formation of amide bond.
  • the step d) of the second embodiment involves deblocking the PI group from the next blocked amino acid resin coupled product using suitable deblocking reagent.
  • unique coupling temperature for the preparation of Lanreotide or its salt is important for the preparation of substantially pure Lanreotide or its salt.
  • the step e) of the second embodiment involves sequentially performing steps (c) and (d) after the formation of the first deblocked coupled resin product of H-Thr(P2)-resin obtained in step b) using N a -terminal Pl-protected amino acids in the order of Cys, Val, Lys, D-Trp, Tyr, Cys, D-2-Nal to give linear Lanreotide peptide.
  • microwave instlement is used for the synthesis of peptide, wherein:
  • the step f) of the second embodiment involves cyclizing the resulting linear Lanreotide or its salt peptide using suitable cyclizing agent to give crude Lanreotide or its salt.
  • suitable cyclizing agent can be selected from air, hydrogen peroxide, DMSO or iodine, preferably DMSO.
  • DMSO can be used with aqueous buffer selected from ammonium acetate, acetic acetate and the like or suitable mixture thereof.
  • the step g) is performed according to the sixth embodiment.
  • substantially pure Lanreotide or its salt is prepared according to the second embodiment.
  • present invention provides a process for the preparation substantially pure Lanreotide or its salt, wherein the process comprises use of DMSO as a cyclizing agent.
  • present invention provides a process for the preparation of Lanreotide or its salt, the process comprising the steps of:
  • step (c) deblocking the PI group from the next blocked amino acid resin coupled product; e) sequentially performing step (c) and step (d) after the formation of the first deblocked coupled resin product of H-Thr(P2)-resin obtained in step b) using N a -terminal Pl- protected amino acids in the order of Cys, Val, Lys, D-Trp, Tyr, Cys, D-2-Nal to give linear Lanreotide peptide;
  • PI group is protecting group attached to the N a -terminal group
  • P2 group is side-chain protecting group
  • the step a) of the fourth embodiment involves coupling of first amino acid Pl-Thr(P2)- OH to a resin solid phase support in presence of coupling reagent and activator additive in a suitable solvent.
  • the step b) of the fourth embodiment comprises deblocking of PI group from first blocked amino acid coupled product viz. Pl-Thr(P2)-OH using suitable deblocking reagent.
  • the step c) of the fourth embodiment involves coupling a next amino acid having N a - terminal blocked by PI to the deblocked amino acid resin coupled product in presence of coupling reagent and activator additive in a suitable solvent for the formation of amide bond.
  • the step d) of the fourth embodiment involves deblocking the PI group from the next blocked amino acid resin coupled product using suitable deblocking reagent.
  • step e) of the fourth embodiment involves sequentially performing step (c) and step (d) after the formation of the first deblocked coupled resin product of H-Thr(P2)-resin obtained in step b) using N a -terminal Pl-protected amino acids in the order of Cys, Val, Lys, D-Trp, Tyr, Cys, ⁇ -b-Nal to give linear Lanreotide peptide;
  • microwave instrument is used for the synthesis of peptide, wherein: (i) coupling temperature of Thr, Val, Lys, D-Trp, Tyr and D-2Nal is in the range from about 85°C to about 90°C;
  • unique coupling temperature for the preparation of Lanreotide or its salt is important for the preparation of substantially pure Lanreotide or its salt.
  • the step f) of fourth embodiment involves cyclizing the resulting linear Lanreotide or its salt peptide using DMSO to give cmde Lanreotide or its salt.
  • DMSO as an oxidizing agent gives better purity than hydrogen peroxide of Lanreotide.
  • iodine as an oxidizing agent for the preparation of Lanreotide or its salt leads to formation certain iodinated impurity which were difficult to control in final drug substance of Lanreotide.
  • the step g) is performed according to the sixth embodiment.
  • substantially pure Lanreotide or its salt is prepared.
  • present invention provides a process for the preparation substantially pure Lanreotide or its salt, the process comprising the steps of:
  • step b) deblocking the Fmoc group from the next blocked amino acid resin coupled product; e) sequentially performing steps (c) and (d) after the formation of the first deblocked coupled resin product of H-Thr(P2)-resin obtained in step b) using N a -terminal Fmoc protected amino acids in the order of Cys, Val, Lys, D-Trp, Tyr and Cys wherein Cys, Lys, D-Trp and Tyr may be optionally side -chain protected;
  • an amide resin can be selected from, Rink amide Rink amide AM Resin, Sieber Amide resin, MBHA Resin and the like.
  • the step a) of the fifth embodiment involves coupling of first amino acid Fmoc-Thr(P2)- OH to an amide resin solid phase support in presence of coupling reagent and activator additive in a suitable solvent.
  • the step b) of the fifth embodiment comprises deblocking of Fmoc group from first blocked amino acid coupled product viz. Fmoc-Thr(P2)-OH using suitable deblocking reagent.
  • the step c) of the fifth embodiment involves coupling a next amino acid having N a - terminal blocked by Fmoc to the deblocked amino acid resin coupled product in presence of coupling reagent and activator additive in a suitable solvent.
  • the step d) of the fifth embodiment involves deblocking the Fmoc group from the next blocked amino acid resin coupled product using suitable deblocking reagent.
  • step e) of the fifth embodiment involves sequentially performing step (c) and step (d) after the formation of the first deblocked coupled resin product of H-Thr(P2)-resin obtained in step b) using N a -terminal Pl-protected amino acids in the order of Cys, Val, Lys, D-Trp, Tyr and Cys wherein Cys, Lys, D-Trp and Tyr may be optionally side-chain protected;
  • the step f) of the fifth embodiment involves coupling of Boc-D-2Nal-OH in presence of coupling reagent and activator additive in a suitable solvent to give linear Lanreotide peptide.
  • microwave instlement is used for the synthesis of peptide, wherein:
  • unique coupling temperature for the preparation of Lanreotide or its salt is important for the preparation of substantially pure Lanreotide or its salt.
  • the step g) of the fifth embodiment involves cyclizing the resulting linear Lanreotide or its salt peptide using suitable cyclizing agent to give cmde Lanreotide or its salt.
  • suitable cyclizing agent can be selected from air, hydrogen peroxide, DMSO or iodine.
  • DMSO can be used with aqueous buffer selected from ammonium acetate, acetic acetate and the like or suitable mixture thereof.
  • the step h) is performed according to the sixth embodiment.
  • substantially pure Lanreotide or its salt is prepared.
  • final deprotection step is performed after preparation of Linear Lanreotide peptide.
  • final deprotection step can be performed prior or later to the cyclization step which means present invention includes both‘on-resin oxidation’ and‘without-resin oxidation’ of linear Lanreotide peptide.
  • the final deprotection step can be performed by using suitable cocktail mixtures.
  • said ‘without-resin oxidation’ of linear Lanreotide peptide may further comprises steps of:
  • said‘on-resin oxidation’ of linear Lanreotide peptide may further comprises steps of:
  • substantially pure Lanreotide or its salt is prepared.
  • PI group is protecting group attached to the N a -terminal group.
  • the PI group can be selected from Fmoc, Boc or Cbz or known to person skilled in the art.
  • P2 group is side-chain protecting group.
  • Thr side -chain, hydroxy group is protected by a group selected from Clt, Trt or tBu and the like.
  • Cys side -chain, thiol group is protected by a group selected from Trt, Mmt, Acm or tBu and the like.
  • Lys side -chain, N-g amino group is protected by a group selected from Trt, Mmt, Acm or tBu and the like.
  • D- Trp side-chain, amino group is protected by a group selected from Boc, Formyl (For), Cyclohexylcabonyl (Hoc) and the like.
  • Tyr side -chain hydroxy group is protected by a group selected from Clt, Trt or tBu and the like.
  • resin can be selected from, Wang resin, Rink Amide Resin, Rink amide AM Resin, Sieber resin, HMBA-ChemMatrix® resin, MBHA Resin, HMPB-ChemMatrix® resin, Hydroxy Functionalized HypoGel® Resins, TentaGel® resin, 2-Chlorotrityl Resin, 4,4'-dimethoxy-trityl resin, 4-methyltrityl chloride resins, Rink Acid Resin and the like.
  • the suitable coupling reagent can be selected from DCC, DIC, EDC, BOP, PyBOP, PyAOP, PyBrOP, BOP-C1, HATU, HCTU or mixtures thereof or the like know to skilled person in the art.
  • the suitable activator additive can be selected from Oxyma, HOBt, HO At, 6-Cl-HOBt, NHS or suitable mixtures thereof.
  • the amount of the coupling agents used may range from about 0.5 to about 6 molar equivalents, per molar equivalent of resin with respect to resin loading capacity.
  • the suitable deblocking reagent used in solid phase peptide synthesis wherein the PI group is Fmoc can be selected from base comprising piperidine in suitable solvent selected from N-methyl pyrrolidone (NMP), dichloromethane (DCM) or dimethylformamide (DMF) or suitable mixtures thereof.
  • NMP N-methyl pyrrolidone
  • DCM dichloromethane
  • DMF dimethylformamide
  • the suitable deblocking reagent used in solid phase peptide synthesis wherein the PI group is Boc can be selected from 25-50% TFA-DCM, 4 M HC1 in dioxane, 2M MeSCLH in dioxane, 1M TMS-C1, 1M phenol-DC or suitable mixtures thereof.
  • the suitable deblocking reagent used in solid phase peptide synthesis, wherein the PI group is Cbz can be selected from HF scavengers, TFMSA-TFA, H2 catalyst or suitable mixture thereof.
  • the suitable solvent for above steps of solid-phase synthesis can be selected from organic polar aprotic solvents, such as NMP or DMF.
  • present invention provides an improved process for purifying the cmde Lanreotide or its salt to give substantially pure Lanreotide or its salt, the process comprising the steps of:
  • the step a) of sixth embodiment involves loading of crude Lanreotide or its salt sample onto a chromatography column.
  • the suitable chromatographic column types that can be used in above step (a) can be selected from, but are not limited to the following silica gel sorbents: DaisogelTM, KromasilTM C18 100-16, KromasilTM C18 100-10, KromasilTM C8 100-16, KromasilTM C4 100-16, KromasilTM Phenyl 100-10, KromasilTM Cl 8 Eternity 100-5, KromasilTM C4 Eternity 100-5, ChromatorexTM Cl 8 SMB 100-15 HE, ChromatorexTM C8 SMB 100-15 HE, ChromatorexTM C4 SMB 100-15 HE, DaisopakTM SP 120-15 ODS-AP, DaisopakTM SP 120-10- C4-Bio, DaisopakTM SP 200-10-C4-Bio, ZeosphereTM C18 100-15, ZeosphereTM C8 100-15, ZeosphereTM C4 100
  • suitable solvent can be selected from acetonitrile, tetrhydrofuran, acetone, methanol, ethanol, propanol, isopropanol or suitable mixture thereof and the like.
  • suitable buffer can be selected from ammonium acetate, sodium acetate, phosphate buffer, sodium chloride, potassium chloride, lithium chloride and the like.
  • step c) involves collecting the fractions of desired Lanreotide purity and pooling. During elution, desired fractions are collected at regular intervals and analyzed for purity. The suitable collected fractions containing the product of similar purities may be pooled together and optionally subjected to removal of acetonitrile solvent. Optionally, after completing the desired number of cycles of purification by repeating the steps a) and b), all the fractions of similar purity from each of the cycle are pooled and taken forward to the next step of the purification process. In an embodiment, pooled fractions having a purity of more than about 98% may be taken forward to the next step of the purification process.
  • the isolation of substantially pure Lanreotide or its salt can be done by well-known techniques known in the prior art.
  • isolation of Lanreotide or its salt from pure pooled fraction may involve lyophilization of pure pooled fraction of Lanreotide or its salt.
  • the purification process of sixth embodiment is used.
  • present invention provides a method for the preparation of substantially pure Lanreotide or its salt by using enantiomerically pure amino acids.
  • said enantiomerically pure amino acid can be selected from Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH and Boc-D-2Nal-OH.
  • present invention involves the purification of commercially available amino acid for attaining high purity of raw materials for preparation of Lanreotide.
  • enantiomerically pure Fmoc-Thr(tBu)-OH is used wherein, total content of D-Threonine, L-Allo-Threonine and D-Allo-Threonine is less than about 0.10%.
  • present invention provides a method of preparing Lanreotide drug product, the method comprising:
  • present invention provides a method of preparing Lanreotide drug product, the method comprising:
  • present invention provides a method of preparing Lanreotide dmg product, the method comprising:
  • present invention provides a method of preparing Lanreotide dmg product, the method comprising:
  • present invention provides a method of preparing Lanreotide dmg product, the method comprising:
  • a batch of Lanreotide or its salt is active pharmaceutical ingredient of Lanreotide or its salt which can be further processed for making pharmaceutical composition or dmg product.
  • the dmg product or pharmaceutical composition can be prepared by any process known in the art or as disclosed in US6503534B1.
  • present invention provides a method of assaying purity of a sample of Lanreotide or its salt or a pharmaceutical dosage form comprising Lanreotide or its salt, wherein method comprise the steps of:
  • Lanreotide or its salt sample onto a chromatographic column; b) eluting Lanreotide or its salt from the column with an eluent comprising polar solvent and an ion pair agent;
  • the chromatographic column can be selected from, but are not limited to the following silica gel sorbents: DaisogelTM, KromasilTM Cl 8 100-16, KromasilTM C18 100-10, KromasilTM C8 100-16, KromasilTM C4 100-16, KromasilTM Phenyl 100-10, KromasilTM C18 Eternity 100-5, KromasilTM C4 Eternity 100-5, ChromatorexTM C18 SMB 100-15 HE, ChromatorexTM C8 SMB 100-15 HE, ChromatorexTM C4 SMB 100-15 HE, DaisopakTM SP 120-15 ODS-AP, DaisopakTM SP 120-10-C4-Bio, DaisopakTM SP 200-10-C4- Bio, ZeosphereTM Cl 8 100-15, ZeosphereTM C8 100-15, ZeosphereTM C4 100-15, SepTech ST 150-10 C18, Luna C18 100-10, Gemini C18 110-10, YMC Triart C18 120-5 and Y
  • the ion pair agent can be selected from tetra butyl ammonium hydrogen sulfate, dodecyltrimethylammonium bromide, dodecyltrimethylammonium hydrogen sulfate, heptafluorobutyric acid, hexadecyltrimethylammonium bisulfate, hexadecyltrimethylammonium bromide, myristyltrimethylammonium bromide, tetrabutylammonium bisulfate, tetrabutylammonium bromide, tetrabutylammonium chloride, tetrabutylammonium iodide, tetrabutylammonium phosphate, tetraethylammonium bromide, tetraheptylammonium bromide, tetraethylammonium hydrogen sulfate, tetrahexy
  • amino acid refers to an organic compound comprising at least one amino group and at least one acidic group.
  • the amino acid may be a naturally occurring amino acid or be of synthetic origin, or an amino acid derivative or amino acid analog.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise nucleotides may be referred to by their commonly accepted single -letter codes. Amino acids mentioned herein in the specification if does not represent any chirality means that amino acid is in its natural or L form.
  • D as used herein before amino acids refers to chirality of the amino acid that follows as a D-amino acid
  • the term “sequentially” refers to the method of peptide synthesis where any of the amino acids contained in the peptide chain is introduced individually.
  • the method may or may not involve an intermediate purification step.
  • peptide refers to any peptide comprising two or more amino acid residues connected by peptide linkage.
  • RRT as used herein is intended to indicate the relative retention time of the particular impurity against a pure Lanreotide or its salt standard (assigned an RRT value of 1) during an HPLC analysis.
  • substantially pure Lanreotide or its salt refers to Lanreotide or its salt having purity greater than about 99% and free from D-Allo-Threonine Lanreotide impurity.
  • the term“free from D-Allo-Threonine Lanreotide impurity” means that D-Allo-Threonine Lanreotide impurity is less than about 0.10% (w/w) or less than 0.080% (w/w) or less than 0.050% (w/w) or less than 0.02% (w/w) or less than 0.010% (w/w) in pure Lanreotide or its salt.
  • RRT 1.1 impurity is D-Allo-Threonine Lanreotide which is confirmed from spiking studies after synthesizing D-Allo-Threonine Lanreotide using D-Allo Theronine as stating material.
  • Lanreotide or its salt means Lanreotide free base or Lanreotide salts.
  • Lanreotide salts according to present invention are preferably pharmaceutically acceptable salts of organic acids, such as those of acetic, lactic, malic, ascorbic, succinic, benzoic, methanesulphonic acids, or pharmaceutically acceptable salts of inorganic acids, such as those of hydrochloric, hydrobromic, hydriodic, sulphuric or phosphoric acids.
  • Lanreotide salt is Lanreotide acetate.
  • Lanreotide salts also include trifluoroacetic acid salts which can be prepared at intermediate stage.
  • enantiomerically pure means, solid or solution that consists of a single enantiomer and not its mirror image is called enantiomerically pure.
  • Drug Product or pharmaceutical composition refers to a finished dosage form, for example, a tablet, capsule or solution that contains an active pharmaceutical ingredient, generally, but not necessarily, in association with inactive ingredients.
  • the term“Active Pharmaceutical Ingredient or API” is any substance or mixture of substances intended to be used in the manufacture of a drug (medicinal) product and that, when used in the production of a dmg, becomes an active ingredient of the drug product. Such substances are intended to furnish pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease or to affect the stmcture or function of the body.
  • the carboxyl and amino groups that participate in the formation of the peptide amide bond are called“non-side chain” carboxyl group or amino group, respectively.
  • the term“N a - terminal group of amino acid” is non-side chain amino group that participate in the formation of the peptide amide bond.
  • any functional groups of an amino acid which are not involved in formation of a peptide amide bond are called “side chain or side -chain” functionalities.
  • Boc refers to tetra-butyloxycarbonyl.
  • BOP refers to Benzotriazol-l-yloxy-tris(dimethylamino)- phosphonium hexafluorophosphate.
  • BOP-C1 refers to Bis(2-oxo-3-oxazolidinyl)phosphinic Chloride
  • DCC Dicyclohexylcarbodiimide
  • DIC refers to Diisopropylcarbodiimide.
  • DCM dichloromethane
  • DMF dimethylformamide
  • EDC refers to l-Ethyl-3-(3- dimethylaminopropyl)carbodiimide.
  • HOBt refers to 1 -Hydroxybenzo triazole.
  • HOSu refers to N-Hydroxysuccinimide.
  • HOAt refers to l-Hydroxy-7-aza-lH-benzotriazole.
  • HATU refers to 2-(7-aza- 1 H-benzotriazole- 1 -yl )- 1 , 1 ,3,3- tetramethyl uranium hexafluorophosphate.
  • tBu refers to tert-butyl
  • NMP N-methylpyrrolidone
  • Oxyma refers to ethyl 2-cyano-2-(hydroxyimino)acetate
  • PyBrOP refers to Bromo-tris-pyrrolidino-phosphonium hexafluorophosphate
  • PyAOP refers to 7-Aza-benzotriazol-l-yloxy- tripyrrolidinophosphoniumhexafluorophosphate
  • PyBOP refers to (Benzotriazol-l-yloxy-tripyrrolidino- phosphonium hexafluorophosphate
  • DODT refers to 2,2’-(Ethylenedioxy)-diethanethiol
  • DMSO dimethyl sulfoxide
  • TIPS Triisopropylsilane
  • TFA trifluoroacetic acid
  • MTBE refers to methyl tert-butyl ether
  • IP A refers to isopropyl alcohol.
  • Step a) Preparation of Boc-D-2-Nal-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)--Lys(Boc)-Val- Cys(Trt)-Thr(tBu)- NH-Rink Amide AM Resin:
  • Fmoc protected NH-Rink amide AM resin (375 mmol) was swelled in DMF and Fmoc group was deprotected by 20% piperidine in DMF at 90°C.
  • Fmoc-Thr(tBu)-OH was dissolved in DMF (0.4 M solution) and added to the deprotected resin along with 5M DIC solution in DMF & 1M oxyma pure solution in DMF.
  • the coupling was performed at 90°C for two minutes to give Fmoc-Thr(tBu)-NH-Rink amide AM resin. After coupling, the resultant resin was washed with DMF.
  • the Fmoc protecting group was removed by treatment with 20% piperidine in DMF at 90°C.
  • Fmoc-Cys(Trt)-OH was dissolved in DMF (0.4 M solution) and added to the deprotected resin along with 5M DIC solution in DMF & 1M oxyma pure solution in DMF were added to the reaction vessel.
  • the coupling was performed at 50°C to give Fmoc-Cys(Trt)-Thr(tBu)-NH-Rink amide AM resin.
  • the resultant resin was washed with DMF.
  • the Fmoc protecting group was removed by treatment with 20% piperidine in DMF at 90°C.
  • Step b) Deprotection of Boc-D-2Nal-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Val-Cys- (Trt)-Thr(tBu)-NH-Rink amide AM resin to give D-2-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys- Thr-NH 2 bis TFA salt 450 gm of step a) product was treated with cleavage cocktail mixture (DODT, TIPS, Water, and TFA) and stirred in glass reactor. Resultant reaction mixture was filtered, washed with TFA and air dried. Further, charged combined TFA filtrate to vessel and MTBE was added and stirred for 1 hour. The precipitate was filtered, washed with MTBE and dried in a vacuum tray drier to afford 145 gm of titled product.
  • cleavage cocktail mixture DODT, TIPS, Water,
  • Step c) Oxidation of D-2-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH 2 bis TFA salt to give Lanreotide
  • step b To a reactor vessel 142 gm of step b) product and DMSO (2.1L) was added and stirred the reaction mixture. Further, 20 mmol NH40Ac/acetic acid was added to the reaction mixture and stirred to afford a solution of 2.5 mg/mL of Lanreotide product.
  • the Reverse phase Cl 8 media was equilibrated with 0.2% Acetic acid + 1M NaCl buffer.
  • the crude Lanreotide solution was loaded onto the column and the gradient elution was performed using acetonitrile as mobile phase B.
  • the desired fractions was collected whose purity was greater than 99.5% and pooled together.
  • the acetonitrile was removed by distillation from eluted product and subjected to lyophilization to obtain substantially pure Lanreotide acetate.
  • Step e Assaying the purity of Lanreotide acetate sample
  • the lanreotide acetate sample was prepared using 5 mg of Lanreotide acetate and water and loaded on column of Ultra performance liquid chromatography. Further, the loaded column was eluted with gradient programme using mobile phase A and mobile phase B and impurities were determined by relative area normalization method.
  • Mobile phase B was prepared by mixing mobile phase A, acetonitrile and methanol.
  • Step a) Preparation of Boc-D-2-Nal-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)--Lys(Boc)-Val- Cys(Trt)-Thr(tBu)- NH-Rink Amide AM Resin
  • Fmoc protected NH-Rink amide AM resin (258 mmol) was swelled in DMF and Fmoc group was deprotected by 20% piperidine in DMF at 65°C.
  • Fmoc-Thr(tBu)-OH was dissolved in DMF (0.4 M solution) and added to the deprotected resin along with 5M DIC solution in DMF & 1M oxyma pure solution in DMF.
  • the coupling was performed at 85°C for two minutes to give Fmoc-Thr(tBu)-NH-Rink amide AM resin. After coupling, the resultant resin was washed with DMF.
  • the Fmoc protecting group was removed by treatment with 20% piperidine in DMF at 65°C.
  • Fmoc-Cys(Trt)-OH was dissolved in DMF (0.4 M solution) and added to the deprotected resin along with 5M DIC solution in DMF & 1M oxyma pure solution in DMF were added to the reaction vessel.
  • the coupling was performed at 40°C to give Fmoc-Cys(Trt)-Thr(tBu)-NH-Rink amide AM resin.
  • the resultant resin was washed with DMF.
  • the Fmoc protecting group was removed by treatment with 20% piperidine in DMF at 65°C.
  • Fmoc-Tyr(tBu)-OH was dissolved in DMF (0.4 M solution) and added to the deprotected resin along with 5M DIC solution in DMF & 1M oxyma pure solution in DMF were added to the reaction vessel.
  • the coupling was performed at 85°C for two minutes to give Fmoc-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Val-Cys(Trt)- Thr(tBu)-NH-Rink amide AM resin.
  • the resultant resin was washed with DMF.
  • the Fmoc protecting group was removed by treatment with 20% piperidine in DMF at 65°C.
  • Fmoc-Cys(Trt)-OH was dissolved in DMF (0.4 M solution) and added to the deprotected resin along with 5M DIC solution in DMF & 1M oxyma pure solution in DMF were added to the reaction vessel.
  • the coupling was performed at 40°C to give Fmoc-Cys(Trt)- Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Val-Cys-(Trt)-Thr(tBu)-NH-Rink amide AM resin.
  • the resultant resin was washed with DMF.
  • the Fmoc protecting group was removed by treatment with 20% piperidine in DMF at 65°C.
  • Boc-D-2Nal-OH was dissolved in DMF (0.4 M solution) and added to the deprotected resin along with 5M DIC solution in DMF & 1M oxyma pure solution in DMF were added to the reaction vessel.
  • the coupling was performed at 85°C for two minutes to give Boc-D-2Nal-Cys(Trt)-Tyr(tBu)-D- Trp(Boc)-Lys(Boc)-Val-Cys-(Trt)-Thr(tBu)-NH-Rink amide AM resin.
  • the resultant linear Lanreotide peptide was washed with IPA to give titled product of 0.88Kg.
  • Step b), step c), step d) and step e) was performed according to example 1 to give substantially pure Lanreotide.
  • Step a) Preparation of Boc-D-2-Nal-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)--Lys(Boc)-Val- Cys(Trt)-Thr(tBu)-NH-Rink Amide AM Resin
  • Fmoc protected NH-Rink amide AM resin (0.74 mmol/g) was swelled in DMF. The Fmoc protecting group was removed by treatment with 20% piperidine in DMF. The resin was washed repeatedly with DMF. Fmoc-Thr(tBu)-OH was dissolved in DMF (0.4 M solution) and added to the deprotected resin along with 5 M DIC solution in DMF & 1 M oxyma pure solution in DMF. The resultant reaction mixture was stirred to give Fmoc-Thr(tBu)-NH-Rink amide AM resin. After coupling, the resultant resin was washed with DMF. The Fmoc protecting group was removed by treatment with 20% piperidine in DMF.
  • the resin was washed repeatedly with DMF.
  • the next amino acid Fmoc-Cys(Trt)-OH was dissolved in DMF (0.4 M solution) and then 5 M DIC solution in DMF & 1 M oxyma pure solution in DMF were added to the resin.
  • the resultant mixture was stirred to give Fmoc-Cys(Trt)-Thr(tBu)-NH-Rink amide AM resin.
  • the resultant resin was washed with DMF.
  • the Fmoc protecting group was removed by treatment with 20% piperidine in DMF.
  • Step b) Cleavage of Boc-D-2-Nal-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Val-Cys(Trt)- Thr(tBu)- NH-Rink Amide AM Resin to give D-2-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr- NH 2 bis TFA salt:
  • step a) product was treated with cleavage cocktail mixture (DODT, TIPS, Water, and TFA) and stirred for 2 hours in glass reactor. Then the reaction mixture was filtered, washed with TFA and air dried. Further, charged combined TFA filtrate to vessel and MTBE (8000 mL) was added and stirred for 1 hour. The precipitate was filtered, washed with MTBE and dried in a vacuum tray drier to afford 79.8 gm of titled product.
  • cleavage cocktail mixture DODT, TIPS, Water, and TFA
  • Step c) Oxidation of D-2-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH 2 bis TFA salt to give Lanreotide:
  • step b To a reactor vessel, 3.75 gm of step b) product and DMSO (5.7 mL) were added. The resultant mixture was stirred. Afterward, NH40Ac/AcOH (69.3 ml) was added to the reaction mixture and stirred for 15 hours to afford a solution of 3 mg/mL of Lanreotide product.
  • the Reverse phase Cl 8 media was equilibrated with 0.2% Acetic acid + 1M NaCl buffer.
  • the crude Lanreotide solution of step c) was loaded onto the column and the gradient elution was performed using acetonitrile as mobile phase B.
  • the desired fractions was collected whose purity was greater than 99.5% and pooled together.
  • the acetonitrile was removed by distillation from eluted product and subjected to lyophilization to obtain substantially pure Lanreotide.
  • the lanreotide acetate sample was prepared using 5 mg of Lanreotide acetate and water and loaded on column of Ultra performance liquid chromatograph. Further, the loaded Lanreotide column was eluted with gradient programme using mobile phase A and mobile phase B and impurities were determined by relative area normalization method.
  • Mobile phase B was prepared by mixing mobile phase A, acetonitrile and methanol.
  • the purity of Lanreotide sample according to above method is 99.412% and D-Allo- Threonine impurity and less than 0.010%.
  • the HPLC chromatograph is depicted as Figure 2.

Abstract

La présente invention concerne du lanréotide sensiblement pur ou son sel et sa préparation. Selon un autre aspect, la présente invention concerne une méthode de préparation d'un produit de lanréotide dmg qui consiste à mesurer la teneur en impuretés de D-allo-thréonine lanréotide dans le lanréotide ou dans son sel. La présente invention concerne également une méthode de dosage de la pureté d'un échantillon de lanréotide ou de son sel ou d'une forme posologique pharmaceutique comprenant du lanréotide ou son sel.
PCT/IB2020/051420 2019-02-21 2020-02-20 Lanréotide sensiblement pur ou son sel et méthode associée WO2020170185A1 (fr)

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IN201941006784 2019-02-21

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6503534B1 (en) * 1998-03-25 2003-01-07 Societe De Conseils De Recherches Et D'applications Scientifiques (S.C.R.A.S.) Pharmaceutical compositions for prolonged peptide release and preparation method
US20060148699A1 (en) * 2004-10-04 2006-07-06 Avi Tovi Counterion exchange process for peptides
WO2006119388A2 (fr) * 2005-05-03 2006-11-09 Novetide, Ltd. Methode d'obtention de derives peptidiques
WO2013098802A2 (fr) * 2011-12-29 2013-07-04 Chemical & Biopharmaceutical Laboratories Of Patras S.A. Synthèse peptidique en phase solide par une fixation par chaîne latérale

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6503534B1 (en) * 1998-03-25 2003-01-07 Societe De Conseils De Recherches Et D'applications Scientifiques (S.C.R.A.S.) Pharmaceutical compositions for prolonged peptide release and preparation method
US20060148699A1 (en) * 2004-10-04 2006-07-06 Avi Tovi Counterion exchange process for peptides
WO2006119388A2 (fr) * 2005-05-03 2006-11-09 Novetide, Ltd. Methode d'obtention de derives peptidiques
WO2013098802A2 (fr) * 2011-12-29 2013-07-04 Chemical & Biopharmaceutical Laboratories Of Patras S.A. Synthèse peptidique en phase solide par une fixation par chaîne latérale

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