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DR ANTHONY MELVIN CRASTO Ph.D

DR ANTHONY MELVIN CRASTO Ph.D

DR ANTHONY MELVIN CRASTO, Born in Mumbai in 1964 and graduated from Mumbai University, Completed his Ph.D from ICT, 1991,Matunga, Mumbai, India, in Organic Chemistry, The thesis topic was Synthesis of Novel Pyrethroid Analogues, Currently he is working with GLENMARK LIFE SCIENCES LTD, Research Centre as Principal Scientist, Process Research (bulk actives) at Mahape, Navi Mumbai, India. Total Industry exp 30 plus yrs, Prior to joining Glenmark, he has worked with major multinationals like Hoechst Marion Roussel, now Sanofi, Searle India Ltd, now RPG lifesciences, etc. He has worked with notable scientists like Dr K Nagarajan, Dr Ralph Stapel, Prof S Seshadri, Dr T.V. Radhakrishnan and Dr B. K. Kulkarni, etc, He did custom synthesis for major multinationals in his career like BASF, Novartis, Sanofi, etc., He has worked in Discovery, Natural products, Bulk drugs, Generics, Intermediates, Fine chemicals, Neutraceuticals, GMP, Scaleups, etc, he is now helping millions, has 9 million plus hits on Google on all Organic chemistry websites. His friends call him Open superstar worlddrugtracker. His New Drug Approvals, Green Chemistry International, All about drugs, Eurekamoments, Organic spectroscopy international, etc in organic chemistry are some most read blogs He has hands on experience in initiation and developing novel routes for drug molecules and implementation them on commercial scale over a 30 PLUS year tenure till date June 2021, Around 35 plus products in his career. He has good knowledge of IPM, GMP, Regulatory aspects, he has several International patents published worldwide . He has good proficiency in Technology transfer, Spectroscopy, Stereochemistry, Synthesis, Polymorphism etc., He suffered a paralytic stroke/ Acute Transverse mylitis in Dec 2007 and is 90 %Paralysed, He is bound to a wheelchair, this seems to have injected feul in him to help chemists all around the world, he is more active than before and is pushing boundaries, He has 9 million plus hits on Google, 2.5 lakh plus connections on all networking sites, 90 Lakh plus views on dozen plus blogs, 233 countries, 7 continents, He makes himself available to all, contact him on +91 9323115463, email amcrasto@gmail.com, Twitter, @amcrasto , He lives and will die for his family, 90% paralysis cannot kill his soul., Notably he has 33 lakh plus views on New Drug Approvals Blog in 233 countries......https://newdrugapprovals.wordpress.com/ , He appreciates the help he gets from one and all, Friends, Family, Glenmark, Readers, Wellwishers, Doctors, Drug authorities, His Contacts, Physiotherapist, etc

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WO 2016024243, New patent, Dr Reddy’s Laboratories Ltd, Fidaxomicin


Fidaxomicin.svg

 

WO 2016024243, New patent, Dr Reddy’s Laboratories Ltd, Fidaxomicin

WO2016024243,  FIDAXOMICIN POLYMORPHS AND PROCESSES FOR THEIR PREPARATION

DR. REDDY’S LABORATORIES LIMITED [IN/IN]; 8-2-337, Road No. 3, Banjara Hills, Telangana State, India Hyderabad 500034 (IN)

CHENNURU, Ramanaiah; (IN).
PEDDY, Vishweshwar; (IN).
RAMAKRISHNAN, Srividya; (IN)

Aspects of the present application relate to crystalline forms of Fidaxomicin IV, V & VI and processes for their preparation. Further aspects relate to pharmaceutical compositions comprising these polymorphic forms of fidaxomicin

front page image

Fidaxomicin (also known as OPT-80 and PAR-101 ) is a novel antibiotic agent and the first representative of a new class of antibacterials called macrocycles. Fidaxomicin is a member of the tiacumicin family, which are complexes of 18-membered macrocyclic antibiotics naturally produced by a strain of Dactylosporangium aurantiacum isolated from a soil sample collected in Connecticut, USA. The major component of the tiacumicin complex is tiacumicin B. Optically pure R-tiacumicin B is the most active component of Fidaxomicin. The chiral center at C(19) of tiacumicinB affects biological activity, and R-tiacumicin B has an R-hydroxyl group attached at this position. The isomer displayed significantly higher activity than other tiacumicin B-related compounds and longer post-antibiotic activity.

As per WIPO publication number 2006085838, Fidaxomicin is an isomeric mixture of the configurationally distinct stereoisomers of tiacumicin B, composed of 70 to 100% of R-tiacumicin B and small quantities of related compounds, such as S-tiacumicin B and lipiarmycin A4. Fidaxomicin was produced by fermentation of the D aurantiacum subspecies hamdenensis (strain 718C-41 ). It has a narrow spectrum antibacterial profile mainly directed against Clostridium difficile and exerts a moderate activity against some other gram-positive species. Fidaxomicin is bactericidal and acts via inhibition of RNA synthesis by bacterial RNA polymerase at a distinct site from that of rifamycins. The drug product is poorly absorbed and exerts its activity in the gastrointestinal (Gl) tract, which is an advantage when used in the applied indication, treatment of C. difficile infection (CDI) (also known as C. difficile-associated disease or diarrhoea [CDAD]). Fidaxomicin is available as DIFICID oral tablet in US market. Its CAS chemical name is Oxacyclooctadeca-3,5,9, 13, 15-pentaen-2-one, 3-[[[6-deoxy-4-0-(3,5dichloro-2-ethyl-4,6-dihydroxybenzoyl)-2-0-methyl-P-D-manno pyranosyl]oxy]methyl]-12[[6-deoxy-5-C-methyl-4-0-(2-methyl-1 -oxopropyl)- -D-lyxo-hexo pyranosyl]oxy]-1 1 -ethyl-8-hydroxy-18-[(1 R)-1 -hydroxyethyl] -9,13,15-trimethyl-, (3E.5E, 8S.9E.1 1 S.12R.13E, 15E.18S)-. Structural formula (I) describes the absolute stereochemistry of fidaxomicin as determined by x-ray.

(I)

WIPO publication number 2004014295 discloses a process for preparation of Tiacumicins that comprises fermentation of Dactylosporangium aurantiacum NRRL18085 in suitable culture medium. It also provides process for isolation of tiacumicin from fermentation broth using techniques selected from the group consisting of: sieving and removing undesired material by eluting with at least one solvent or a solvent mixture; extraction with at least one solvent or a solvent mixture; Crystallization; chromatographic separation; High-Performance Liquid Chromatography (HPLC); MPLC; trituration; and extraction with saturated brine with at least one solvent or a solvent mixture. The product was isolated from /so-propyl alcohol (IPA) having a melting point of 166-169 °C.

U.S. Patent No. 7378508 B2 discloses polymorphic forms A and B of fidaxomicin, solid dosage forms of the two forms and composition thereof. As per the ‘508 patent form A is obtained from methanol water mixture and Form B is obtained from ethyl acetate.

J. Antibiotics, vol. 40(5), 575-588 (1987) discloses purification of Tiacumicins using suitable solvents wherein tiacumicin B exhibited a melting point of 143-145 °C.

PCT application WO2013170142A1 describes three crystalline forms of Fidaxomicn namely, Form-Z, Form-Z1 and Form-C. IN2650/CHE/2013 describes 6 crystalline polymorphic forms of Fidaxomicin namely, Forms I, Form la, Form II, Form Ha, Form III and Form Ilia).

The occurrence of different crystal forms, i.e., polymorphism, is a property of some compounds. A single molecule may give rise to a variety of polymorphs having distinct crystal structures and physico-chemical properties.

Polymorphs are different solid materials having the same molecular structure but different molecular arrangement in the crystal lattice, yet having distinct physico-chemical properties when compared to other polymorphs of the same molecular structure. The discovery of new polymorphs and solvates of a pharmaceutical active compound provides an opportunity to improve the performance of a drug product in terms of its bioavailability or release profile in vivo, or it may have improved stability or advantageous handling properties. Polymorphism is an unpredictable property of any given compound. This subject has been reviewed in recent articles, including A. Goho, “Tricky Business,” Science News, August 21 , 2004. In general, one cannot predict whether there will be more than one form for a compound, how many forms will eventually be discovered, or how to prepare any previously unidentified form.

There remains a need for additional polymorphic forms of fidaxomicin and for processes to prepare polymorphic forms in an environmentally-friendly, cost-effective, and industrially applicable manner.

G.V. Prasad, chairman, Dr Reddy’s Laboratories

EXAMPLES

Example 1 : Preparation of fidaxomicin Form IV:

Fidaxomicin (0.5 g) and a mixture of 1 ,4-Dioxane (10 mL), THF (10 ml) and water (20mL) were charged in Easy max reactor (Mettler Toledo). The reactor was set to temperature cycle with following parameters:

Starting temperature: 25 °C;

Temperature raised to 60 °C over a period of 2 hours;

Cooled to 0 °C over a period of 2 hours;

Temperature raised to 60 °C over a period of 2 hours;

Cooled to 0 °C over a period of 2 hours;

Temperature raised to 25 °C over a period of 2 hours;

Temperature maintained at 25 °C for 6 hours.

After completion of temperature cycling process, the slurry was filtered under suction, followed by drying in air tray dryer (ATD) at 40°C to a constant weight to produce crystalline fidaxomicin form-IV.

Example 2: Preparation of fidaxomicin Form V:

Fidaxomicin (1 g) and a mixture of propylene glycol (10 mL) and water (20mL) were charged in Easy max reactor (Mettler Toledo). The reactor was set to temperature cycle with following parameters:

Starting temperature is 25 °C;

Temperature raised to 60 °C over a period of 2 hours;

Cooled to 0 °C over a period of 2 hours;

Temperature raised to 60 °C over a period of 2 hours;

Cooled to 0 °C over a period of 2 hours;

Temperature raised to 25 °C over a period of 2 hours;

Temperature maintained at 25 °C for 6 hours.

After completion of temperature cycling process, the slurry was filtered under suction, followed by drying in air tray dryer (ATD) at 40°C to a constant weight to produce crystalline fidaxomicin form-V.

Example 3: Preparation of fidaxomicin Form VI:

Fidaxomicin (0.5 mg) and MIBK (10 mL) were charged in Easy max reactor (Mettler Toledo) and the mixture was heated to 80°C. n-heptane (20 mL) was added to the solution at the same temperature. The mixture was stirred for 1 hour. The reaction mass was then cooled to 25°C. Solid formed was filtered at 25°C and dried at 40°C in air tray dryer (ATD) to a constant weight to produce crystalline fidaxomicin form VI.

Example 4: Preparation of fidaxomicin Form V:

Fidaxomicin (500 mg) and a mixture of R-propylene glycol (5 mL) and water (15 mL) were charged in Easy max reactor (Mettler Toledo). The reactor was set to temperature cycle with following parameters:

Starting temperature is 25 °C;

Temperature raised to 60 °C over a period of 2 hours;

Cooled to 0 °C over a period of 2 hours;

Temperature raised to 60 °C over a period of 2 hours;

Cooled to 0 °C over a period of 2 hours;

Temperature raised to 25 °C over a period of 2 hours;

Temperature maintained at 25 °C for 2 hours.

After completion of temperature cycling process, the slurry was filtered and dried at 25°C to produce crystalline fidaxomicin form-V.

Example 5: Preparation of fidaxomicin Form V:

Fidaxomicin (1 g) and a mixture of S-propylene glycol (3 ml_) and water (30 mL) were charged in Easy max reactor (Mettler Toledo). The reactor was set to temperature cycle with following parameters:

Starting temperature is 25 °C;

Temperature raised to 60 °C over a period of 2 hours;

Cooled to 0 °C over a period of 2 hours;

Temperature raised to 60 °C over a period of 2 hours;

Cooled to 0 °C over a period of 2 hours;

Temperature raised to 25 °C over a period of 2 hours;

Temperature maintained at 25 °C for 2 hours.

After completion of temperature cycling process, the slurry was filtered and dried at 25°C to produce crystalline fidaxomicin form-V.

Example 6: Preparation of fidaxomicin Form V:

Fidaxomicin (40 g) and a mixture of propylene glycol (400 mL) and water (1600 mL) were charged in Chem glass reactor. The reactor was set to temperature cycle with following parameters:

Starting temperature is 25 °C;

Temperature raised to 60 °C over a period of 2 hours;

Cooled to 0 °C over a period of 2 hours;

Temperature raised to 60 °C over a period of 2 hours;

Cooled to 0 °C over a period of 2 hours;

Temperature raised to 25 °C over a period of 2 hours;

Temperature maintained at 25 °C for 6 hours.

After completion of temperature cycling process, the slurry was filtered under suction, followed by drying in air tray dryer (ATD) at 40°C to a constant weight to produce crystalline fidaxomicin form-V.

The 10-member board at pharmaceutical major Dr Reddy’s thrives on diversity. Liberally sprinkled with gray hairs, who are never quite impressed with powerpoint presentations, “they want information to be pre-loaded so that the following discussions (at the board level) are fruitful,” says Satish Reddy, Chairman, Dr Reddy’s. That said, the company has now equipped its board members with a customized application (that runs on their tablets) to manage board agenda and related processes.

see at

http://articles.economictimes.indiatimes.com/2014-10-31/news/55631761_1_board-members-board-agenda-dr-reddy-s

Dr. Reddy’s Laboratories Managing Director and Chief Operating Officer Satish Reddy addressing

 

 

 

Fidaxomicin
Fidaxomicin.svg
Systematic (IUPAC) name
3-(((6-Deoxy-4-O-(3,5-dichloro-2-ethyl-4,6-dihydroxybenzoyl)-2-O-methyl-β-D-mannopyranosyl)oxy)-methyl)-12(R)-[(6-deoxy-5-C-methyl-4-O-(2-methyl-1-oxopropyl)-β-D-lyxo-hexopyranosyl)oxy]-11(S)-ethyl-8(S)-hydroxy-18(S)-(1(R)-hydroxyethyl)-9,13,15-trimethyloxacyclooctadeca-3,5,9,13,15-pentaene-2-one
Clinical data
Trade names Dificid, Dificlir
Licence data US FDA:link
Pregnancy
category
  • AU: B1
  • US: B (No risk in non-human studies)
Legal status
Routes of
administration
Oral
Pharmacokinetic data
Bioavailability Minimal systemic absorption[1]
Biological half-life 11.7 ± 4.80 hours[1]
Excretion Urine (<1%), faeces (92%)[1]
Identifiers
CAS Number 873857-62-6 Yes
ATC code A07AA12
PubChem CID 11528171
ChemSpider 8209640 
UNII Z5N076G8YQ 
KEGG D09394 Yes
ChEBI CHEBI:68590 
ChEMBL CHEMBL1255800 
Synonyms Clostomicin B1, lipiarmicin, lipiarmycin, lipiarmycin A3, OPT 80, PAR 01, PAR 101, tiacumicin B
Chemical data
Formula C52H74Cl2O18
Molar mass 1058.04 g/mol

///////////WO-2016024243,WO 2016024243, New patent, Dr Reddy’s Laboratories Ltd, Fidaxomicin

CC[C@H]1/C=C(/[C@H](C/C=C/C=C(/C(=O)O[C@@H](C/C=C(/C=C(/[C@@H]1O[C@H]2[C@H]([C@H]([C@@H](C(O2)(C)C)OC(=O)C(C)C)O)O)\C)\C)[C@@H](C)O)\CO[C@H]3[C@H]([C@H]([C@@H]([C@H](O3)C)OC(=O)C4=C(C(=C(C(=C4O)Cl)O)Cl)CC)O)OC)O)\C

Dr Reddy’s Laboratories Ltd, New patent, WO 2016005960, Liraglutide


!e™A!a™Trp™leu™Va!~-Arg~~GIy-~Arg~~Gly~~OH

Formula (I)

LIRAGLUTIDE

 

Dr Reddy’s Laboratories Ltd, New patent, WO 2016005960,  Liraglutide

Process for preparation of liraglutide

Kola, Lavanya; Ramasamy, Karthik; Thakur, Rajiv Vishnukant; Katkam, Srinivas; Komaravolu, Yagna Kiran Kumar; Nandivada, Giri Babu; Gandavadi, Sunil Kumar; Nariyam Munaswamy, Sekhar; Movva, Kishore Kumar

Improved process for preparing liraglutide, by solid phase synthesis, useful for treating type 2 diabetes.

It having been developed and launched by Novo Nordisk, under license from Scios and Massachusetts General Hospital.

Liraglutide, marketed under the brand name Victoza, is a long-acting glucagon like peptide agonist developed by Novo Nordisk for the treatment of type 2 diabetes.

Liraglutide is an injectable drug that reduces the level of sugar (glucose) in the blood. It is used for treating type 2 diabetes and is similar to exenatide (Byetta). Liraglutide belongs to a class of drugs called incretin mimetics because these drugs mimic the effects of incretins. Incretins, such as human-glucagon-like peptide-1 (GLP-1 ), are hormones that are produced and released into the blood by the intestine in response to food. GLP-1 increases the secretion of insulin from the pancreas, slows absorption of glucose from the gut, and reduces the action of glucagon. (Glucagon is a hormone that increases glucose production by the liver.)

All three of these actions reduce levels of glucose in the blood. In addition, GLP-1 reduces appetite. Liraglutide is a synthetic (man-made) hormone that resembles and acts like GLP-1 . In studies, Liraglutide treated patients achieved lower blood glucose levels and experienced weight loss.

Liraglutide, an analog of human GLP-1 acts as a GLP-1 receptor agonist. The peptide precursor of Liraglutide, produced by a process that includes expression of recombinant DNA in Saccharomyces cerevisiae, has been engineered to be 97% homologous to native human GLP-1 by substituting arginine for lysine at position 34. Liraglutide is made by attaching a C-16 fatty acid (palmitic acid) with a glutamic acid spacer on the remaining lysine residue at position 26 of the peptide precursor.

The molecular formula of Liraglutide is Ci72H265N4305i and the molecular weight is 3751 .2 Daltons. It is represented by the structure of formula (I)

!e™A!a™Trp™leu™Va!~-Arg~~GIy-~Arg~~Gly~~OH

Formula (I)

U.S. Patent No. 7572884 discloses a process for preparing Liraglutide by recombinant technology followed by acylation and removal of N-terminal extension.

U.S. Patent No. 7273921 and 6451974 discloses a process for acylation of Arg-34GLP-1 (7-37) to obtain Liraglutide.

U.S. Patent No. 8445433 discloses a solid phase synthesis of Liraglutide using a fragment approach.

International Application publication No. WO2013037266A1 discloses solid phase synthesis of Liraglutide, characterized in that comprises A) the presence of the activator system, solid phase carrier and by resin Fmoc protection N end obtained by coupling of glycine (Fmoc-Gly-OH) Fmoc-Gly-resin; B) by solid phase synthesis, prepared in accordance with the sequentially advantage Liraglutide principal chain N end of the coupling with Fmoc protected amino acid side chain protection and, wherein the lysine using Fmoc-Lys (Alloc)-OH; C) Alloc getting rid of the lysine side chain protecting group; D) by solid phase synthesis, the lysine side chain coupling Palmitoyl-Glu-OtBu; E) cracking, get rid of protecting group and resin to obtain crude Liraglutide ; F) purification, freeze-dried, to obtain Liraglutide.

Even though, the above mentioned prior art discloses diverse processes for the preparation of Liraglutide, they are often not amenable on commercial scale because of expensive amino acid derivatives such as pseudo prolines used in those processes.

Hence, there remains a need to provide simple, cost effective, scalable and robust processes for the preparation of Liraglutide involving commercially viable amino acid derivatives and reagents.

EXAMPLE 1 :

Stage I Preparation of Wang resin-Gly-Arg(pbf)-Gly-Arg(pbf)-Val-Leu-Trp(Boc)-Ala-lleu-Phe-Glu(Otbu)-Lys-{Glu(OH)-NH(palmitoyl)}-Ala-Ala-Gln(trt)-Gly-OH-Glu(Otbu)-Leu-Tyr(Otbu)-Ser(Otbu)-Ser(Otbu)-Val-Asp(Otbu)-Ser(Otbu)-Thr(Otbu)-Phe-Thr(Otbu)-Gly-Glu(Otbu)-Ala-Boc-His(trt)-OH.

Wang resin (50gm) is swelled in DCM (500ml) for 1 hr in a sintered flask. DCM was filtered using Vacuum. Fmoc-Glycine (44.6 gm, 150 mmol) was dissolved in dichloromethane (250 ml). 1 -(2-mesitylene sulfonyl)-3-nitro-1 H-1 ,2,4 triazole (44.4 gm, 150 mmol) and 1 -methyl imidazole (9 ml, 1 12 mmol) was then added. The reaction mixture was added to wang resin and stirred for 3hrs at about 25° C. The resin was washed with DCM and a second lot of Fmoc-Glycine (27 gm, 90 mmol) was dissolved in dichloromethane (250 ml). 1 -(2-mesitylene sulfonyl)-3-nitro-1 H-1 ,2,4 triazole (26.6 gm, 90 mmol) and 1 -methyl imidazole (5.3 ml, 90 mmol) was then added and stirred for 3hrs. The resin was washed with DCM and a sample of resin beads were checked for UV analysis. The capping was carried out using acetic anhydride (15 ml) DCM (120 ml) and pyridine (120 ml). The resin was washed with dichloromethane and 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-Arg(pbf)-OH (52 gm, 80 mmol) dissolved in 250 ml DMF was then added. The coupling was carried out by addition of HOBt (10.8gm, 80 mmol) and DIC (6.2ml, 80 mmol) in DMF. The completion of the coupling was confirmed by a ninhydrin test. After washing the resin, the Fmoc protecting group was removed with 20% piperidine in DMF. These steps were repeated each time with the respective amino acid according to the peptide sequence. After coupling 12th amino acid Fmoc-Lys (Alloc)-OH, deprotection of alloc group is carried out with palladium tetrakis and phenyl silane in DCM. The resin was washed repeatedly with DMF. The next amino acid H-Glu(OH)-NH(palmitoyl)-Otbu (9.9 gm, 0.023 moles) dissolved in 250 ml DMF was then added. The coupling was carried out by addition of HOBt (10.8gm, 80 mmol) and DIC (6.2ml, 80 mmol) in DMF. The completion of the coupling was confirmed by a ninhydrin test. After washing the resin, the Fmoc protecting group of Lys was removed with 20% piperidine in DMF. The next amino acid Fmoc-Ala-OH (52 gm, 80 mmol) dissolved in 250 ml DMF was then added. The coupling was carried out by addition of HOBt (10.8gm, 80 mmol) and DIC (6.2ml, 80 mmol) in DMF. The completion of the coupling was confirmed by a ninhydrin test. After washing the resin, the Fmoc protecting group was removed with 20% piperidine in DMF. These steps were repeated each time with the respective amino acid according to the peptide sequence. The resin was washed repeatedly with DMF, Methanol and MTBE and dried under vacuum.

Stage II: Cleavage of Liraglutide from resin along with global deprotection

45gms of resin obtained in stage I was treated with cleavage cocktail mixture of TFA (462.5ml), TIPS (12.5ml), Water (12.5ml), and Phenol (12.5 ml), stirred at 0°C for 30 min. and at 25°C for 3hrs at 200RPM. Then the reaction mixture was filtered, repeatedly wash the resin with TFA and the filtrate was concentrated on Rotary evaporator at 30°C. Pour the concentrated solution to MTBE (2L) at 4°C slowly and stir for 1 hr. The precipitate obtained is filtered and dried in a vacuum tray drier to afford 18 gm of Liraglutide crude with a purity of 27.5%.

Stage III: Purification of crude Liraglutide using RP HPLC.

The crude Liraglutide (4 gm) of purity around 27.5% is dissolved in 10 mM Tris buffer (120ml) of pH: 8.00 and 0.5 N NaOH is further added drop wise to the solution for making the crude solid completely dissolved. The solution is further passed through 0.2 micron filter. The Reverse phase C 18 – 150 Angstrom media (C18 silica media – 10 micron particle size) is equilibrated with 10mM Tris buffer of pH: 8.0 The crude solution is loaded onto the column and the gradient elution is performed as per the below tabular column against the mobile phase B (Acetonitrile).

Table 1 : Gradient program for pre purification

The desired fractions are collected in the gradient range of and the fractions (F1 , F2, F3, F4 and F5) whose purity > 80% are pooled. The pooled fractions are then subjected to further purification.

The Pooled fractions having purity >80% are then subjected to C18 RPHPLC silica media (5 micron particle size) for further purification. The pooled fractions – Feed is diluted with purified water in the ratio of 1 :2 (one part of pooled fraction to two parts of purified water) as a part of sample preparation before loading into the column. The media C18 is first equilibrated with 0.1 % TFA for 3 column volumes (1 CV = bed volume of media). After equilibration, the sample is loaded onto the column and the gradient

elution is performed as per the below tabular column against the mobile phase B (Acetonitrile).

Table 2: Gradient program for second purification

The desired fractions are collected in the gradient range of and the fraction whose purity > 96% are pooled together and lyophilized to afford 220mg of Liraglutide trifluoro acetate salt. The pooled fractions and their purity by HPLC are listed in the below table.

The pooled fractions with the purity of average 97% are subjected further to de solvation to remove the Acetonitrile content by Rota vapor. The final solution was filtered through 0.2 micron filter and lyophilized to get Liraglutide API.

EXAMPLE 2:

Stage I Preparation of Tentagel SPHB resin-Gly-Arg(pbf)-Gly-Arg(pbf)-Val-Leu-Trp(Boc)-Ala-lleu-Phe-Glu(Otbu)-Lys-{Glu(OH)-NH(palmitoyl)}-Ala-Ala-Gln(trt)-Gly-OH-Glu(Otbu)-Leu-Tyr(Otbu)-Ser(Otbu)-Ser(Otbu)-Val-Asp(Otbu)-Ser(Otbu)-Thr(Otbu)-Phe-Thr(Otbu)-Gly-Glu(Otbu)-Ala-Boc-His(trt)-OH using Fragment approach.

Fragments used are as follows

1 . Fmoc-Arg(pbf)-Gly-OH.

2. Fmoc-Leu-Ala-Arg(pbf)-OH.

3. Fmoc-lle-Ala-Trp(boc)-OH.

4. Fmoc-Glu(Otbu)-Phe-OH.

5. Fmoc-Glu(Otbu)-Phe-OH.

6. Fmoc-Lys-Glu-Palmitic acid.

7. Fmoc-Gly-Gln(trt)-Ala-Ala-OH.

8. Fmoc-Tyr(Otbu)-Leu-Glu(Otbu)-OH.

9. Fmoc-Val-Ser(Otbu)-Ser(Otbu)-OH.

10. Fmoc-Phe-Thr(Otbu)-Ser(Otbu)-Asp(Otbu)-OH

1 1 . Fmoc-Gly-Thr(Otbu)-OH.

12. Boc-His(Trt)-Ala-Glu(Otbu)-OH.

Tentagel SPHB resin (30gm) is swelled in DCM (300ml) for 1 hr in a sintered flask. DCM was filtered using Vacuum. Fmoc-Glycine (13.8 gm, 46.8 moles) was dissolved in dichloromethane (150 ml). 1 -(2-mesitylene sulfonyl)-3-nitro-1 H-1 ,2,4 triazole (13.8 gm, 46.8 moles) and 1 -methyl imidazole (2.4 ml, 29.25 moles) was then added. The resulting solution was added to tentagel resin and stirred for 2hrs at about 25° C. The resin was washed with DCM and a second lot of Fmoc-Glycine (13.8 gm, 46.8 moles) was dissolved in dichloromethane (150 ml). 1 -(2-mesitylene sulfonyl)-3-nitro-I H-1 ,2,4 triazole (13.8 gm, 46.8 moles) and 1 -methyl imidazole (2.4 ml, 29.25 moles) was then added and stirred for 2hrs. The resin was washed with DCM and a sample of resin beads were checked for UV analysis. The Fmoc protecting group was removed by treatment with 20% piperidine in DMF. The resin was washed repeatedly

with DMF. The next amino acid fragment 1 Fmoc-Gly-Arg(pbf)-OH (8.25 gm, 1 1 .7 moles) dissolved in 150 ml DMF was then added. The coupling was carried out by addition of HOBt (2.1 gm, 1 1 .7 moles) and DIC (2.5ml, 1 1 .7 moles) in DMF for 2hrs. The completion of the coupling was confirmed by a ninhydrin test. After washing the resin, the Fmoc protecting group was removed with 20% piperidine in DMF. These steps were repeated each time with the respective amino acid fragments according to the peptide sequence. The resin was washed repeatedly with DMF, Methanol and MTBE and dried under vacuum.

Stage II: Cleavage of Liraglutide from resin along with global deprotection

58gms of resin obtained from stage I was treated with cleavage cocktail mixture of TFA (555ml), TIPS (15ml), Water (15ml), and Phenol (15 ml) and stirred at 0°C for 30 min. at 25°C for 3hrs at 200RPM. Then filter the reaction mixture, repeatedly wash the resin with TFA and concentrate on Rotary evaporator at 30°C. Pour the concentrated solution to MTBE at 4°C slowly and stirred for 1 hr. The precipitate obtained was filtered and dried in a vacuum tray drier to afford 23.12 gm of crude Liraglutide with a purity of 36.89%.

Stage III: Purification of crude Liraglutide using RP HPLC.

The crude Liraglutide (4 gm) of purity around 27.5% is dissolved in 10 mM Tris buffer (120ml) of pH: 8.00 and 0.5 N NaOH is further added drop wise to the solution for making the crude solid completely dissolved. The solution is further passed through 0.2 micron filter. The Reverse phase C 18 – 150 Angstrom media (Irregular C18 silica media – 10 micron particle size) is equilibrated with 10mM Tris buffer of pH: 8.0 The crude solution is loaded onto the column and the gradient elution is performed as per the below tabular column against the mobile phase B (Acetonitrile).

Table 1 : Gradient program for pre purification

60 40 30

55 45 30

52 48 30

51 49 60

The desired fractions are collected in the gradient range of and the fractions (F1 , F2, F3, F4 and F5) whose purity > 80% are pooled. The pooled fractions then subjected to further purification.

The Pooled fractions having purity >80% are then subjected to C18 RPHPLC silica media (5 micron particle size) for further purification. The pooled fractions – Feed is diluted with purified water in the ratio of 1 :2 (one part of pooled fraction to two parts of purified water) as a part of sample preparation before loading into the column. The media C18 is first equilibrated with 0.1 % TFA for 3 column volumes (1 CV = bed volume of media). After equilibration, the sample is loaded onto the column and the gradient elution is performed as per the below tabular column against the mobile phase B (Acetonitrile).

Table 2: Gradient program for second purification

The desired fractions are collected in the gradient range and the fraction whose purity > 96% are pooled together and Lyophilized to afford 865 mg of Liraglutide trifluoro acetate salt. The pooled fractions and their purity by HPLC are listed in the below table.

The pooled fractions with the purity of average 97% are subjected further to de solvation to remove the Acetonitrile content by Rota vapor. The final solution was filtered through 0.2 micron filter and lyophilized to get Liraglutide API.

G.V. Prasad, chairman, Dr Reddy’s Laboratories.

REFERENCE

IN2014CH3453 INDIAN PATENT

WO 2016005960, CLICK FOR PATENT

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