Patent Application: US-201214234919-A

Abstract:
a process for preparing human relaxin - 2 having the following amino acid sequence : a chain : pglu - leu - tyr - ser - ala - leu - ala - asn - lys - cys - cys - his - val - gly - cys - thr - lys - arg - ser - leu - ala - arg - phe - cys b chain : asp - ser - trp - met - glu - glu - val - ile - lys - leu - cys - gly - arg - glu - leu - val - arg - ala - gln - ile - ala - ile - cys - gly - met - ser - thr - trp - ser ; comprising the following steps : providing the amino acids necessary for the synthesis of the a and b chains with usual protective groups , wherein the cysteines are employed as trityl - protected amino acids - oh ); effecting a chromatographic purification of the individual chains a and b after the solid state synthesis ; followed by the simultaneous folding and combination of the individual chains a and b in ammonium hydrogencarbonate buffer at ph 7 . 9 to 8 . 4 ; and subsequent purification of the relaxin - 2 formed .

Description:
the conventional synthetic strategy is based on the selective introduction of the three disulfide bridges in the relaxin - 2 molecule . this means an enormous chemical expenditure on the one hand and requires a chromatographic purification process , which is necessary after each disulfide bridge produced , on the other , which in turn results in a considerable loss of substance in the product to be expected . in a typical example of an optimized synthesis , relaxin - 2 with the amino acid sequence is obtained separately at first in two chains , the a chain and the b chain , wherein a pyroglutamic acid ( l - pglu - oh ) is introduced in the n - terminal of the a chain . the original glutamine is modified by ring closure to form a lactam , which blocks edman sequencing on the one hand and is important to biological activity on the other . further , the a chain has a cysteine each at positions 10 and 15 , which are internally bonded through a disulfide bridge to form cystine . both cysteines bear a trityl group as an orthogonal acid - labile side chain . the synthesis of the a chain is preferably effected with fmoc ( 9 - fluorenyl - methoxycarbonyl ) protected amino acids by a stepwise solid phase synthesis and is performed on a wang resin loaded with f - moc - l - cysteine ( 0 . 54 mmol / g , 100 - 200 mesh ) as a solid support ( merrifield et al ., 1985 ). the activation of the fmoc amino acids , which are employed in a tenfold molar excess , is performed with [( 2 -( 1h - benzotriazol - 1 - yl )- 1 , 1 , 3 , 3 - tetramethyluronium hexafluorophosphate ] ( hbtu , 100 mmol / l ) with the addition of 1 - hydroxybenzotriazole ( hobt , 0 . 5 m ) and diisopropylethylamine ( dipea , 2 m ) in n - methyl - 2 - pyrrolidinone ( nmp ) at room temperature . acylation reactions are typically performed for 45 minutes . the cleavage of fmoc is effected with piperidine , 20 % in nmp . the synthetic scale of the two chains is 0 . 1 mm each . the following amino acid derivatives are employed for synthesis : fmoc - l - ala - oh , fmoc - l - arg ( pbf )- oh , fmoc - l - asn ( trt )- oh , fmoc - l - asp ( otbu )- oh , fmoc - l - glu ( otbu ), fmoc - l - cys ( trt ), fmoc - l - gln ( trt ), fmoc - l - glu ( otbu )- oh , l - pglu - oh , fmoc - gly - oh , fmoc - l - his ( trt ), fmoc - l - ile - oh , fmoc - l - leu - oh , fmoc - l - lys ( boc )- oh , fmoc - l - met - oh , fmoc - l - phe - oh , fmoc - l - ser ( tbu )- oh , fmoc - l - thr ( tbu )- oh , fmoc - l - trp ( boc ), fmoc - l - tyr ( tbu )- oh , and fmoc - l - val - oh . the synthesis of the b chain corresponds to that of the a chain . however , the resin support employed is one preloaded with serine as the c - terminal , an fmoc - protected tentagel r - php with 0 . 19 mmol / g . for the synthesis of the a chain , a resin support preloaded with cysteine as the c - terminal , an fmoc - l - cys ( trt ) wang resin with 0 . 54 mmol / g , is employed . the synthesis of the two chains is effected exclusively with fmoc chemistry in a stepwise solid - phase synthesis on an abi 433 according to the merrifield principle at room temperature . after cleavage of the resin support from the peptidyl resin with trifluoroacetic acid ( 94 % tfa ), ethanedithiol ( 3 % edt ) and demineralized water ( 3 %), both chains will have free thiol ( sh ) groups , as trityl is acid - labile . the raw peptides thus obtained are further purified in a chromatographic step for the following recombination ( see fig2 ). for combining the a and b chains , these are previously dissolved accordingly in water ( a chain ) and in 50 % acetonitrile ( b chain ). in order to enable combination of the a chain with the b chain , these are transferred into a buffer system consisting of 2 mm edta in 0 . 1 m nh 4 hco 3 solution , ph 7 . 9 - 8 . 4 . the buffer system is previously degassed with helium ( 30 min ) to generate an oxygen - free reaction environment . the dissolved peptide chains are in equimolar concentrations ( 0 . 1 mg / ml ). for the reaction , it is critical that the addition of cystine and cysteine as a redox pair is effected at a concentration of 2 mg cystine and 2 mg cysteine per mg of peptide employed ( a + b chains ). the reaction mixture is now stirred moderately at a temperature of 0 ° c . under a nitrogen blanket for several days . according to experience , the folding reaction can be stopped after several days , and after 9 days at the latest . the reaction in this folding buffer proceeds without possible isomeric structures . the reaction kinetic profiles are logged from t ( start ) to t ( end ). the reaction is stopped by means of concentrated tfa at ph 4 ( see fig3 ). now , the product obtained is desalted , fractionated and collected by preparative chromatography by means of gradient elution from the reaction mixture . the fractions containing the product are combined and subsequently dried under vacuum ( see fig4 ). the product is lyophilized for storage and stored at − 20 ° c . the high stability of the preparation of highly pure relaxin - 2 in lyophilized form at a temperature of 4 ° c . has been proven by corresponding analytics , in which fresh material was compared with material stored for several months . after such a storage time , only minor amounts of metabolites appear . when the a and b chains are combined and 40 mg each is employed , 16 - 24 mg of the product relaxin - 2 having a purity of & gt ; 98 % is obtained ( see fig4 ). this corresponds to a yield of 20 - 30 % after the folding and purification steps . preferably suitable for a galenic application are ( i ) sterile aqueous solutions , ( ii ) active ingredient encapsulated in liposomes that may also be applied in a physiological solution , and ( iii ) active ingredient encapsulated in liposomes that has been processed into an ointment . after numerous experiments with , for example , polyglycolate / lactate release particles , pegylation , micropumps etc ., it has been surprisingly found that two forms of galenics can be preferably applied : 1 . lyophilized relaxin - 2 reconstituted in mannitol ; 2 . encapsulation in liposomes ( preferably , but not exclusively , rovisome ®), which is a particularly suitable form , because the active ingredient is very stable except for a few metabolites . such metabolites also occur as natural forms in blood plasma , but can be avoided by working in a nitrogen atmosphere , and , being natural endogenous derivatives , are free from side effects . according to the inventive application of highly pure active ingredient , a use in aqueous physiological solutions is indicated if short pulsed hormone surges are to be achieved . a preferably suitable formulation of the active ingredient for this purpose is in individual ampoules for intravenous or preferably subcutaneous injection . the highly pure product is first prepared in ampoules or in cartridges for pen injection as a lyophilized active ingredient with an excipient , preferably mannitol , to ensure long - term stability . a therapeutic unit freeze - dried in 10 ml ampoules ( or cartridge ) stabilized with 20 mg mannitol and acetate as a salt is recommended , whereby storage at refrigerator temperature for more than one year is enabled , and very highly tolerable ingredients are included . for using the formulation , the lyophilizate is brought into a physiologically tolerable solution immediately before injection , for example , by using 0 . 9 % saline . various liposome preparations differing in the encapsulation efficiency of relaxin - 2 were selected . the two most stable formulations are suitable for later use in treatment for subcutaneous injection and as an ointment form . the liposome preparations are physically stable over a period of & gt ; 1 month . what results therefrom are stable liposome preparations with an optimum storage temperature at rt ( constant particle size ; no change in ph and smell ). all raw materials employed meet pharmaceutical specifications , and lecithin has been approved by the u . s . food and drug administration ( fda ). for using the i . v . or s . c . formulation , the liposome preparation is preferably diluted in a physiologically tolerable solution , for example , in 0 . 9 % saline , immediately before injection . the lyophilized form was tested intravenously , the liposomal formulation was tested s . c . directly in aqueous solution , and the base cream was tested , each with a high dose of relaxin - 2 : a high dose that is above tenfold the expected concentration in clinical application did not show any significant side effects , and in a tolerance test for skin tolerability in rats , no deleterious side effect was found either . studies on cell models have shown that the use of highly pure relaxin exhibits an excellent effect , comparable to that of the recombinant peptide . the amino acid sequence of the synthetic human relaxin - 2 is identical with that of the naturally occurring peptide . in human embryonic kidney cells ( hek - 293t ) expressing the natural receptor rxfp1 , synthetic hrlx - 2 displaces europium - labeled relaxin - 2 with the same potency as recombinant human relaxin - 2 , which demonstrates intact receptor activity . in camp assays specifically developed for relaxin - 2 ( halls et al ., 2009 ) and performed by us in human thp - 1 cells , synthetic and recombinant human relaxin - 2 showed equivalent bioactivities ( see fig6 ). in an established cell model of myocardial hypertrophy , synthetic human relaxin - 2 is as potent as recombinant human relaxin - 2 . in this model , relaxin inhibits the differentiation of cardiac fibroblasts into myofibroblasts and the secretion of growth factors by these cells ( dschietzig et al ., 2006 ). based on experimental results in animal models ( teichman et al ., 2009 ; samuel et al ., 2006 ; schondorf et al ., 2007 ), it is to be recommended that synthetic human relaxin - 2 for human therapy should be preferably used for : congestive heart failure ( with reduced systolic function ): acute and chronic treatment acute heart failure cardiorenal symptoms myocardial fibrosis / hypertrophy and diastolic heart failure ( with preserved systolic function ) pulmonary hypertension pulmonary , renal or hepatic fibrosis peripheral artery occlusive disease diabetes mellitus acute dosage ( estimation from a pilot study in human congestive heart failure ): 30 to 100 μg / kg / day s . c . ( dose of 2 . 1 to 7 . 0 mg / day for 70 kg body weight ) over 24 to 48 hours . chronic dosage ( estimation ): 10 to 30 μg / kg / day s . c . 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