CN102617736A - The new stable polyethylene glycol conjugate of interferon alpha, represented by one positional isomer - Google Patents
The new stable polyethylene glycol conjugate of interferon alpha, represented by one positional isomer Download PDFInfo
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Abstract
The invention is related to the pharmaceutical industry and medicine, in particular, to new PEG-interferon derivatives and the discovery of a new functionally active, highly stable conjugate of interferon to polyethylene glycol with an activity of interferon alpha, with reduced immunogenicity, with prolonged biological effects and improved pharmacokinetic parameters of general formula: (I) where: n - integral values from 227 to 10 000, so that the molecular weight of PEG is about 10 000 - 40 000 Da; m - integer > 4; IFN- natural or recombinant polypeptide having the activity of IFN-alpha. Also, the invention is related to drugs containing the declared conjugate of the formula (I), pharmaceutical compositions containing PEG-IFN conjugate and therapeutically acceptable excipients suitable for treatment of viral infections and cancer, as well as diseases associated with primary or secondary immunodeficiency. The invention is related to the use of conjugate of formula (I) in medicinal products, which have antiviral, antiproliferative and immunomodulatory activity, to approaches for prevention and / or treatment of diseases associated with primary or secondary immunodeficiency that include administration of therapeutically effective amount of conjugate of formula (I), and to the container with such pharmaceutical composition.
Description
Technical field
The present invention relates to medicine, promptly relate to the physiologically active conjugate of Interferon, rabbit, the particularly novel conjugate of Interferon, rabbit and polyoxyethylene glycol (PEG), it can be used for medicine, for example is used for treatment virus, immunity and tumor disease.
Background technology
Interferon, rabbit (IFN) is one group of biological activity protein or gp, and it is replied and produce virus infection by various kinds of cell, and perhaps the influence because of certain chemistry and biological substance pair cell produces (Isaacs & Lindeman, 1957; People such as Pestka, 2007).Cause the puting together of IFN and cell receptor that multiple mediation is antiviral, the intracellular protein of immunomodulatory and antiproliferative IFN effect induce (people such as Pestka, 2004; Bekisz, 2004).
Therefore different human IFN genes are cloned and in bacterium and zooblast, are expressed, and have produced multiple reorganization IFN (people such as Pestka, 2004; Pestka, 2007).The medicine that comprises the IFN that recombinates is used for clinical practice to treat multiple virus, tumour and Immunological diseases (people such as Pestka, 2004; Chevaliez & Pawlotsky, 2009).
At present, the medicine based on Interferon, rabbit is used for clinical practice to treat multiple virus, tumour and Immunological diseases (people such as Pestka, 2004).The IFN medicine is used most widely for treating viral hepatitis (Chevaliez & Pawlotsky, 2009), and this disease is one of serious social concern.Nowadays there are 3.5 hundred million patients in the whole world approximately by the hepatitis B virus chronic infection, and 1.7 hundred million patients is arranged by hepatitis C chronic infection (Marcellin, 2009).Hepatitis C in most of the cases presents the chronic course of disease, and it causes serious consequence-liver cirrhosis and hepatocellular carcinoma (Modi Liang, 2008).According to World Health Organization report, bring up to the 5th (Marcellin, 2009) as the cause of the death the U.S. and the Western European countries from the 10th from chronic hepatitis in 1961 and liver cirrhosis.
The medicine that comprises IFN generally is used to treat white blood disease and solid tumor, comprises recurrent melanoma (people such as Bukowski, 2002; People such as Decantris, 2002; People such as Qintas-Cardama, 2006).
The effectiveness that comprises the medicine of natural IFN receives subcutis rapid absorption, big volume distributed median, low relatively stability, short-half-life, high immunogenicity and toxic restriction (Wills, 1990).Therefore, after using in several hours, observe that IFN concentration descends fast in the blood plasma, and inject and to have detected Interferon, rabbit people such as (, 1999) Chatelut after 24 hours in the blood plasma.The quick decline of Interferon, rabbit concentration causes virus replication to continue and the rising of virion concentration people such as (, 1997) Lam.Therefore, need frequent injection of interferon, cause dose-dependent spinoff to reach the effective treatment concentration in the blood plasma.Therefore use the monotherapy of the chronic hepatitis C (CHC) of IFN-α 2b only to cause that 12 middle of the month lasting virusology replys among the patient at 15-20%; And in patient, do not observe pharmaceutical efficacy (people such as McHutchinson, 1998) fully with genotype Ib and high viral load.
When use to continue discharging medicine particularly during PEGization IFN, the treatment effectiveness of IFN can be enhanced (people such as Manns, 2001; People such as Hadziyannis, 2004; People such as Zeuzem, 2001).PEGization IFN puts together generation by interferon molecule and polymer chemistry, the mono methoxy polyethylene glycol (MPEG) that said polymkeric substance for example is made up of the repetition residue of oxyethane, and it at one end has methoxyl group, has hydroxyl at the other end.The MPEG molecule can have different molecular weight and stereochemical structure (line style or ramiform).For the PEGization reaction, the hydroxyl of MPEG end is by the activation of multiple response function group.Activatory MPEG can be in one or more positions the covalency conjugated protein, depend on character and reaction conditions (Zalipsky & Hurris, 1997 of activating group; People such as Roberts, 2002).
The PEGization of IFN causes the activity in vivo (external activity reduction) of the transformation period of better pharmacokinetics, prolongation, the haemoconcentration fluctuation of minimizing, the immunogenicity that reduces and toxicity, rising; The stability that improves (people such as Glue, 2000; People such as Reddy, 2001).
Reduction activity in the vitro system depends on the PEG molecular weight.The little PEG molecule of puting together molecular weight≤5000Da causes the subtle change of external activity (people such as Grace, 2005).Comprising the PEGization albumen of quality PEG like this and the drug effect and the pharmacokinetic property of unmodified protein is difficult to distinguish.Puting together more, high-molecular weight PEG causes the active remarkable reduction of extracorporeal biology owing to the invalid acceptor of puting together; But visible in the case proteic pharmacokinetics of PEGization and drug effect character obviously improve (people such as Delgado; 1992); Cause BA rising (Bailon & Berthold, 1998) in the body.The PEG structure also influences BA and pharmacokinetic property, puts together line style PEG than puting together ramiform PEG structure and has bigger volume of distribution (people such as Caliceti, 2003).
The antiviral activity of PEG-IFN conjugate and biological property also depend on uses different activated MPEG; Because its functional group changes people such as (, 2002) Roberts with the ability of modifying the different proteins amino-acid residue and with chemical bond type that protein forms.Can be used most widely for PEGYLATION OF PROTEINS (succinimdyl carbonate, succinimide succinate, trisilate, triazine, hydroxysuccinimide eater, acetal or the like) with the activation MPEG that proteinic free amino group is puted together.
Present known following PEG-IFN conjugate.
Known a kind of PEG-IFN-α 2a conjugate, wherein line style PEG molecular weight is 1-5000Da (USP 5,382,657,1995).The MPEG glycol derivative is used to put together, and this is reflected at pH=10 and at room temperature carried out 0.5-4 hour.3 times of excessive PEG are used for the PEGization reaction of IFN.In the condition of using; (people such as Monkarsh, 1997) take place with the combination of IFN through the space accesibility of the free amino group of different Methionins (Lys-31, Lys133, Lys134, Lys23, Lys131, Lys121, Lys70, Lys83, Lys49 and Lys112) and the formation of carboxamide key in PEG.Therefore, PEG-IFN-α 2a is made up of the mixture of the positional isomers of the PEG-IFN-α 2a conjugate that obtains, and wherein each isomer comprises single PEG.With unmodified IFN relatively, the antiviral specific activity scope of isomer at 6% (for Lys112) to 40% (for Lys133).Total specific activity of the gained conjugate of being made up of 11 isomer is 34% of unmodified IFN.The external activity of these conjugates also depends on the molecular weight of PEG, scope at 45% (for 2500Da PEG) to 25% (for 10000Da PEG).The shortcoming of the conjugate that obtains is following:
1. make spent glycol activatory lower molecular weight (≤5000Da) MPEG.The pharmacokinetic parameter of PEG-IFN conjugate and unmodified IFN only has fine difference as a result.Therefore this conjugate does not get into clinical trial;
2. because PEG puts together through the free amino group and the IFN molecule of different Methionins, form the positional isomers that has different specific activities in a large number.
Known line style (Russ P 2311930,2004) or ramiform (Russ P 2382048,2008) MPEG verivate by natural IFN-α 2b and molecular weight 13 000-17000Da puted together the PEG-IFN-α 2b conjugate of acquisition.This reaction uses the activated PEG of 50-100 times of molar excess to carry out 60 hours (for line style MPEG) or carry out 12 hours (for ramiform MPEG) at pH 7.5 at pH 9.5.The result has obtained the PEG-IFN conjugate, and its total antiviral activity is between the unmodified IFN active 29 to 38%.There are not data about conjugate stability, positional isomers quantity and antiviral specific activity thereof.The shortcoming of these conjugates is following:
1. use trifluoroethyl sulfonic acid mono methoxy (tresylate) verivate of activation MPEG, its transformation period in the aqueous solution is less than 20 minutes.Once be presented in the past in the trifluoroethyl sulfonate derivatives and proteinic reaction of PEG,, also formed unsettled thionamic acid ester bond (Gais Ruppert, 1995) except through the amino stable amido linkage;
2. trifluoroethyl sulfonic acid mono methoxy activatory PEG and the combination of proteins free amino group that passes through the enterable Methionin that has living space is accomplished people such as (, 2002) Roberts, and it causes the formation of multiple positional isomers;
3. the PEGization reaction of carrying out IFN in high pH value (pH=10) long-time (60 hours) can change the IFN structure;
4.IFN the PEGization reaction in significantly excessive activated PEG (the proteinic mol ratio of PEG/ is 50-100/1) cause obtaining the expensive of product;
Known ramiform pyrrolotriazine derivatives MPEG by natural IFN-α 2b and molecular weight 7500-35 000Da puts together a kind of PEG-IFN-α 2b conjugate (Russ P 2298560 of acquisition; 2004), its total antiviral activity is equivalent to unmodified IFN active 6.4%.There are not data about conjugate stability, positional isomers quantity and antiviral specific activity thereof.
The shortcoming of the conjugate that obtains is following:
1. use the chlorination pyrrolotriazine derivatives of activation MPEG, it can be puted together with the functional group of other amino acid-Serine, tyrosine, Threonine and Histidine except free amino group, causes occurring multiple isomer, and wherein some has unsettled key.In addition, do not use pyrrolotriazine derivatives (Veronese & Pasut, 2005) now because of high toxicity;
2. the specific activity of said PEG-IFN conjugate is low.
Known ramiform PEG by IFN-α 2a and molecular weight 40kDa puts together a kind of PEG-IFN-α 2a conjugate (Russ P 2180595,1997) of acquisition.For puting together, use a kind of N-hydroxy-succinamide ester derivative of MPEG, it optionally reacts to form stable amido linkage with proteinic available free amino group.This reaction was carried out 2 hours for 9,4 ℃ at pH with 3: 1 MPEG/ protein ratio.The conjugate that this reaction is terminated and obtains is through sorbent material Fractogel EMD CM 650 (M) purifying.The productive rate of purifying conjugate is 40-45%.In this case; The IFN-PEG conjugate that obtains is made up of 6 kinds of positional isomerss; Wherein all put together (people such as Vailon, 2001 through stable keys and a PEG molecule for every kind through the free amino group of Methionin-Lys31, Lys121, Lys 131, Lys 134, Lys 70 and Lys 83; People such as Foser, 2003).94% of the whole conjugates of isomer comprises that Methionin and PEG through position 31,121,131 and 134 puts together.The activity of whole conjugates of being made up of 6 kinds of positional isomerss is 1-7% (people such as Bailon, 2001 of natural IFN-α 2a; People such as Boulestin, 2006).Although it is low in the extracorporeal antivirus effect activity; Pharmacokinetic property (people such as Silva, 2006, people such as Boulestin that this conjugate is compared with unmodified IFN and had improvement; 2006), sell (producing) with trade(brand)name " Pegasys " at present by " Hoffmann-La Roche ".
The shortcoming of the conjugate that obtains is following:
1. use N-hydroxy-succinamide ester activatory PEG to cause puting together with the enterable amino that has living space, the conjugate that the result obtains is 6 kinds of mixture of isomers that specific activity is different.
2. the extracorporeal antivirus effect activity of the conjugate that obtains is low.
Prototype of the present invention is a USP 5,951, the PEG-IFN-α 2b conjugate of describing in 974,1999.The line style MPEG (SC-MPEG) of molecular weight 5000 to 12 000Da of succinimdyl carbonate group activation is used for the PEGization reaction.Obtained the IFN that the PEG with molecular weight 12kDa puts together according to said method, it is sold with trade(brand)name " PegIntron " and is used to treat viral hepatitis (" Schering-Plough ", the U.S.) at present.
Find that said PEG-IFN-α 2b conjugate (medicine " PegIntron ") is made up of 13 kinds of positional isomerss, wherein every kind all comprises a PEG molecule of puting together with a plurality of parts of protein molecule (people 2000 such as Wang; People such as Grace, 2001; People such as Youngster, 2002).According to showing that PEG molecule and Interferon, rabbit put together not only through free amino group Methionin (Lys31, Lys 49, Lys 83, Lys 121, Lys 131, Lys 133, Lys 134 и Lys 164) and (Cys1), and pass through Histidine imidazole ring (His7, His34), tyrosine (Tyr129) and Serine OH group (Ser163).The content of single isomer at 0.8% (Tyr129) to changing between 47% (His34).The antiviral activity of the PegIntron that is made up of 13 kinds of positional isomerss is 28% of a natural protein.The specific activity of single isomer be natural IFN-α 2b 11% to 37% between (people such as Youngster, 2002).The main isomer of puting together through His34 and PEG has the highest specific activity (natural protein 37%).Free amino group and the PEG of IFN put together through stable keys, and in accounting for 47% main isomer, PEG and Histidine imidazole ring (His34) are puted together (people such as Roberts, 2002) through unsettled (in the aqueous solution) amino-formate bond.
The pharmacokinetic parameter of said conjugate is superior to the IFN (people such as Silva, 2006) of unmodified.
The shortcoming of the conjugate that obtains is following:
1. use succinimdyl carbonate activatory PEG not only to put together the free amino group of IFN, also put together other amino acid whose functional group.The conjugate that the result obtains is made up of the mixture of the different positional isomers of 13 kinds of antiviral specific activities;
2. the fact that formation unsettled imidazoles-carbonic ether (carbamate) key causes in main (His-34) isomer of said PEG-IFN conjugate is that said conjugate hydrolysis discharged natural IFN and brings into play its biological effect after " Pegintron " used.Therefore, medicine " Pegintron " preferably is regarded as prodrug, and hydrolysis forms active Interferon, rabbit (Foster, 2004) after its injection.Because the unstable of said PEG-IFN conjugate in solution, " Pegintron " formulation is only with the lyophilized powder stores.
Summary of the invention
Target of the present invention is the IFN that obtains a kind of stable PEGization of novelty, and it has the activity of IFN-α, is made up of a kind of positional isomers; Has improved stability; Immunogenicity with reduction, the pharmacokinetic parameter of improvement has the optimum combination of PEG molecular weight and conjugate antiviral activity; Be fit to medical, and based on the medicinal compsns of said PEG-IFN conjugate.
The active functionally active PEG-IFN of the interferon alpha conjugate that has through making up a kind of novelty has solved the problems referred to above; Wherein the strict α through N end halfcystine of the PEG thread-like molecule of molecular weight 10 000 to 40 000Da is amino combines with the IFN-alpha molecule through stable keys, produces the compound with following structural formula (I):
Wherein: the round values of n-from 227 to 10 000;
The integer of m->=4;
IFN-has the natural or recombinant polypeptide of IFN-alpha active.
In the conjugate that obtains, the line style PEG of molecular weight 10 000-40 000Da combines with the α of the N terminal amino acid of IFN-α is amino.
MPEG through using acetaldehyde activatory general formula (II) has realized strict with the amino IFN modification selectivity of the α of N terminal amino acid through the PEGization reaction in pH≤6:
Wherein: the round values of n-from 227 to 10 000, so the molecular weight of PEG is about 10 000-40 000Da;
The integer of m->=4;
The activation MPEG (II) of the numerical value through using m >=4 has obtained the optimum ratio of PEG molecular weight and antiviral activity.
Realized the molecular weight of the PEG that puts together through increase immunogenicity and toxicity reduce and the pharmacokinetic parameter of improvement.
The new features of comparing with prototype are:
Compared with prior art, novelty is following:
1. the acetaldehyde derivatives of activation mPEG is used to produce the PEG-IFN conjugate;
2.PEG-IFN the structural formula of conjugate is novel;
3. the PEG-IFN conjugate of producing is only represented by a kind of positional isomers;
4.PEG the key between molecule and the IFN molecule is stable;
5. owing to the size of bonded PEG, the molecular weight of PEG-IFN conjugate increases;
6. pharmacokinetic parameter improvement.
For producing desired PEG-IFN conjugate, used butyraldehyde or the valeral verivate of the mPEG that meets structural formula (II) of recombinant human IFN-alpha 2b (producing) and molecular weight 20 000Da by ZAO " Biocad ".
Said conjugation reaction uses reductive agent 20 ℃ of temperature or following carrying out at pH below 6.0.The proteinic mol ratio of PEG/ is 2.5-5: 1.Under reductive condition, in the presence of sodium lauryl sulphate (SDS), use polyacrylamide gel electrophoresis (PAGE) and high back voltage liquid chromatography (RP-HPLC) to carry out the PEG-IFN conjugate and form contrast.Carry out the purifying of monomer PEG-IFN from reaction product through the chromatogram on cation-exchange adsorbing substance, reaction product comprises the IFN of unmodified and nonconforming PEG-IFN form, and each protein molecule comprises 2 or more a plurality of PEG molecule.The wash-out of monomer PEG-IFN is lower than 0.05 to 0.2M sodium-chlor in 6 the buffered soln through pH gradient concentration carries out.The monomer PEG-IFN product of purifying is dialysed in the buffered soln at the 10-50mM of pH 4-5; Add salt or polysaccharide or alcohol or Vinylpyrrolidone polymer or monose in the buffered soln; And aminosugar or protein or amino acid; And nonionic detergent, and in the plastics of siliconized surfaces or vial, store down for 4 ± 2 ℃ in temperature.
For characterizing the characteristic of the monomer PEG-IFN-α 2b conjugate that obtains, compare with unmodified IFN, carried out the research of purity, homogeneity, physical-chemical, biology and pharmacokinetic properties.
Description of drawings
The present invention is illustrated by attached drawings:
Fig. 1. with butyraldehyde MPEG derivatives reaction in the kinetics that forms of PEG-IFN-α 2b conjugate.
Fig. 2. the reverse hplc analysis of purifying PEG-IFN-α 2b conjugate on the post of " Symmetry C18 " (4,6x 150mm).
Fig. 3. compare with unmodified IFN-α 2b, the SDS-PAGE of purifying PEG-IFN-α 2b conjugate analyzes.Mr representes molecular weight marker, and the every swimming lane of swimming lane 1 expression adds the PEG-IFN-2b conjugate of 40 μ g; The every swimming lane of swimming lane 2 expressions adds the unmodified IFN-2b of 40 μ g.
Fig. 4. the MALDI-MS of unmodified IFN-α 2b and PEG-IFN-α 2b conjugate analyzes.(A) figure represents the IFN-α 2b of unmodified, and (B) figure represents PEG-IFN-α 2b conjugate.
The position of PEG connection site in Fig. 5 .PEG-IFN-α 2b conjugate.The mass spectrum that in m/z 1200-1400 scope, compares the tryptic peptide of unmodified IFN-α 2b (A) and PEG-IFN-α 2b (B) conjugate.
The temperature stability of Fig. 6 .PEG-IFN-α 2b conjugate and unmodified IFN-α 2b.
The proteolyze stability of Fig. 7 .PEG-IFN-α 2b conjugate and unmodified IFN-α 2b.
Fig. 8. compare the immunogenicity of PEG-IFN-α 2b conjugate with unmodified IFN-α 2b.Post 1 is represented unmodified IFN-α 2b, and post 2 is represented conjugate PEG-IFN-α 2b (embodiment 3), and post 3 is represented conjugate PEG-IFN-α 2b (embodiment 4).
Fig. 9. compare the pharmacokinetics of PEG-IFN-α 2b conjugate with unmodified IFN-α 2b.
Embodiment
The embodiment of concrete PEG-IFN-α 2b working method and the result of property research thereof provide below.
Target of the present invention, PEG-IFN-α 2 conjugates of formula (I) can be used for producing the medicine that is used to prevent and/or treat virus disease, because except the immunogenicity of high stability and reduction, it has high antiviral active.Equally; Conjugate through knowing the formula that comprises significant quantity (I) that pharmaceutical methods produces can be separately as the medicinal compsns of activeconstituents or is used for the preventing and/or treating of virus infection (for example chronic active hepatitis especially hepatitis B and hepatitis C) (people such as Jay, 2006 jointly with other therapeutical agent (for example ribavirin); People such as McHutchison, 2009).Target of the present invention also comprises and prevents and/or treats the for example method of hepatitis C and hepatitis B of virus disease, comprises the conjugate of the desired formula (I) of introducing effective therapeutic dose.
The interferon alpha of known IFN-α and PEGization can be used as immunomodulator and is used to treat cancer; Especially leukemic reticuloendo theliosis, laryngeal papillomatosis, melanoma, renal cell carcinoma, myelogenous leukemia, Kaposi sarcoma (people such as Decatris, 2002; People such as Bukowski, 2002; People such as Qintas-Cardama, 2006; People such as Loquai, 2008; People such as Kaehler, 2010).The experience that is used to treat these diseases based on the well-known pathogenesis of these diseases and IFN-α and conjugate; The object of the invention also comprises medicine and medicinal compsns; It comprises the conjugate PEG-IFN-α that is announced of significant quantity; Have antiproliferative and immunomodulatory effect; Can be used for preventing and/or treating cancer and the disease relevant with primary or secondary immunodeficiency, the object of the invention also comprises the method that prevents and/or treats cancer and the disease relevant with primary or secondary immunodeficiency, comprises the PEG-IFN-alpha conjugate of the formula (I) of introducing effective therapeutic dose.
The conjugate of formula (I) and optional pharmaceutical acceptable fillers, thinner and/or pharmaceutically acceptable vehicle through vein, subcutaneous, intramuscular, or any other suitable way use, for example with the form of capsule, syrup, sprays, drops, injection or suppository.Route of administration changes according to for example symptom and age.Interval between frequency of administration and the injection changes according to disease and seriousness thereof or application target (treatment or preventive use).The PEG-IFN-α of effective dose selects according to above-mentioned factor.
Pharmaceutically acceptable vehicle can be included in the medicinal compsns with PEG-IFN-α: buffering salt (for example acetate, Citrate trianion, supercarbonate, phosphate buffered saline buffer), stablizer (for example polysorbate, EDTA, Vinylpyrrolidone polymer, VISOSE, human serum albumin, p-hydroxybenzoic acid ether, alcohols (for example phenylcarbinol), phenols, Sorbic Acid), antimicrobial component (for example phenylcarbinol), osmotic pressure regulator (for example sodium-chlor, Repone K, polyvalent alcohol, for example glycerine, arabitol, Sorbitol Powder, N.F,USP MANNITOL, lactose, VISOSE), tensio-active agent (for example HSA, Vinylpyrrolidone polymer, Yelkin TTS, polysorbate80, T 46155-polyoxypropylene multipolymer, casein), surfactant (the for example segmented copolymer of oxyethane and propylene oxide, propylene oxide and oxyethane, Span 20, sorbitol ester, polyglycerol fatty acid ester, coconut oleoyl amine DEA laurilsulfate, alkanolamide, octadecyl polyoxyethylene glycol Ucar 35, polyoxyethylene laurel ether, T 46155 n-Hexadecane ether, polysorbate, glyceryl monostearate, distearin, Sorbitol Powder monopalmitate, dehydrating sorbitol monooleate T 46155, sorbitan laurate POLYOXYL 40 STEARATE and Ucar 35), inhibitor (for example Trilon B, L-halfcystine, S-WAT, sodium ascorbate, gsh, 2 mercapto ethanol, WR 34678, cysteine hydrochloride, monohydrate, xitix) and thinner (for example water).
Use butyraldehyde MPEG verivate to produce PEG-IFN-α 2b conjugate
The 1M sodium cyanoborohydride solution of 16ml joins and comprises 785ml buffered soln (the 100mM sodium acetate that concentration is the reorganization IFN-α 2 of 1.7mg/ml; 150mM sodium-chlor; PH 5.0) in, stir said solution then and add 5g molecular weight 20000 daltonian dried butyraldehyde PEG verivates.Fully stir said mixture and hatched 22 hours 17 ± 3 ℃ of temperature.Different time at interval after from the said mixture 50 μ l aliquot of taking a sample, use the kinetics of the polyacrylamide gel electrophoresis method analysis PEG-IFN-α 2 conjugates production in the presence of dodecyl sulfate.For this purpose, the damping fluid that comprises pH 6.8, TV 1/3 amount of 125mM Tris-HCl, 20% glycerine, 3% dodecyl sulfate, 0.005% tetrabromophenol sulfonphthalein joins in the said mixture and heating 3 minutes in boiling water bath.5 μ l samples place the hole of 12.5% polyacrylamide gel plate of preparation, in the presence of dodecyl sulfate, carry out electrophoresis then.After electrophoresis was accomplished, this gel used Coomassie R-250 dyeing.The kinetics of PEG-IFN-α 2 conjugate productions shows in Fig. 1.Surpass 70% time point at PEG-IFN content, said reaction mixture uses the 5mM sodium acetate buffer of pH 5.0 to dilute 10 times.
Use valeral MPEG verivate to produce PEG-IFN-α 2b conjugate
2.05ml 1M sodium cyanoborohydride solution join and comprise in the 100ml buffered soln (pH 5.5 for 100mM sodium acetate, 150mM sodium-chlor) of reorganization IFN-α 2b that concentration is 2.2mg/ml, stir then; Add 0.9g molecular weight 20000 daltonian dried valeral PEG verivates.Fully stir said mixture and hatched 24 hours 6 ± 2 ℃ of temperature.Different time at interval after from the said mixture 50 μ l aliquot of taking a sample, and analyze the kinetics of PEG-IFN conjugate production like embodiment 1 said use polyacrylamide gel electrophoresis method.Surpass 70% time point at PEG-IFN content, said reaction mixture uses the 5mM sodium acetate buffer of pH 5.0 to dilute 10 times.
Through the ion-exchange chromatogram purification monomer PEG-IFN-α 2b conjugate on the cation-exchange adsorbing substance
The diluting soln that produces among the embodiment 1 placed contain cation-exchange adsorbing substance (the CM agarose, on post 300ml), this post uses the 5mM sodium acetate buffer (buffer A) of pH 5.0 with speed 5ml/min balance.Said adsorption column uses buffer A and the buffer A continuous washing that comprises NaCl concentration gradient 0.05 to 0.2M.Like embodiment 1 said use polyacrylamide gel electrophoresis method carrying out sample analysis, from each cut sampling aliquot.The cut that merges PEG-IFN-α 2 conjugates that comprise monomer PEGization uses the 20mM sodium acetate buffer dialysis that comprises 150mM sodium-chlor of 10 times of volumes, carries out sterile filtration then, and there are 4 ± 2 ℃ in the gained solution storage.
Embodiment 4
Through the ion-exchange chromatogram purification monomer PEG-IFN-α 2b conjugate on the cation-exchange adsorbing substance
The diluting soln that produces among the embodiment 2 placed contain cation-exchange adsorbing substance (the SP agarose, on post 50ml), this post uses the 5mM sodium acetate buffer (buffer A) of pH 5.0 with speed 5ml/min balance.Said adsorption column uses buffer A and the buffer A continuous washing that comprises NaCl concentration gradient 0.03 to 0.3M.Like embodiment 1 said use polyacrylamide gel electrophoresis method carrying out sample analysis, from each cut sampling aliquot.The cut that merges PEG-IFN-α 2 conjugates that comprise monomer PEGization uses the 20mM sodium acetate buffer dialysis that comprises 150mM sodium-chlor of 10 times of volumes, carries out sterile filtration then, and there are 4 ± 2 ℃ in the gained solution storage.
The reverse hplc analysis of PEG-IFN conjugate
PEG-IFN-α 2 conjugates that embodiment 3 is produced use the 20mM sodium acetate buffer of pH 5.0 to be diluted to concentration to be 0.1mg/ml, 100 these samples of μ l are placed Symmetry C18 post (4.6x150mm)." Breeze " chromatographic instrument that uses Waters Company to produce is analyzed at 214nm.Based on Fig. 2 result displayed, we may safely draw the conclusion, and according to the discovery of RP-HPLC, the purity of desired PEG-IFN-α 2 conjugates surpasses 99%.
Embodiment 6
Measure level of endotoxin
Bacterial endotoxin (BE) concentration in the PEG-IFN-α 2 conjugate samples of producing according to embodiment 3 and 4 through the lal test (gelling process version) that meets European Pharmacopoeia 6.0 clause 2.6.14 and require at external test., this has used the Associates of CAPE COD that is used for lal test in analyzing, the diagnostic kit that Inc. produces, LAL reagent, intracellular toxin reference standard (0.5 μ g in the vial) and the water of sensitivity 0.03EU/ml.Based on result displayed in the table 1, we may safely draw the conclusion, and the BE content in the conjugate sample is lower than 1.5 endotoxin units (EU)/mg protein, the BE level that it allows far below the medicine based on recombinant protein.
Bacterial endotoxin concentration in table 1.PEG-IFN-α 2 conjugates
Embodiment 7
Compare the electrophoretic analysis of PEG-IFN-α 2b conjugate with unmodified IFN-α 2b
The sample of the PEG-IFN conjugate of producing according to embodiment 3 uses embodiment 1 described polyacrylamide gel electrophoresis method analysis.Application of sample 40 μ g protein in every hole carry out electrophoresis under non-reduced condition.Use Coomassie R-250 dyestuff to carry out the dyeing of gel internal protein.Carry out the electrophoresis of unmodified IFN-α 2 simultaneously.The PEG-IFN conjugate is represented (Fig. 3, the 1st road) with a band.Based on the electrophorogram of PEG-IFN-α 2 conjugates and unmodified IFN-α 2, as shown in Figure 3, we can see that the molecular weight of PEG-IFN-alpha conjugate is higher than unmodified IFN-α (Fig. 3, the 1st and the 2nd road).Should note using electrophoresis method can not measure the proteinic accurate molecular weight of PEGization, because add the stokes radius that hydrophilic PEG molecule increases the gained compound greatly to protein.Therefore, the movement slows down of PEG-protein compound in gel, and its molecular weight seems to be much higher than the molecular weight sum of protein and PEG.
Compare with unmodified IFN-α 2b, use mass spectrometry method to measure the molecular weight of PEG-IFN-α 2b conjugate
2 of 1 μ l sample of the PEG-IFN-α 2b conjugate of producing according to embodiment 3 and 0.3 μ l, 5-resorcylic acid solution (Aldrich, 10mg * ml
-1In 20% acetonitrile solution, contain 0.5% uranous tetrafluoride) mix and air-dry.Use similar methods to prepare the sample of a unmodified IFN-α 2.
Mass spectrum obtains being equipped with on Ultraflex II BRUKER (Germany) the MALDI-TOF mass spectrograph of UV laser apparatus (Nd).Mass spectrum obtains under linear positive ion mode; The weight in average measuring error is no more than 10-15 dalton.
Unmodified rhIFN-α 2 shows in Fig. 4 in the mass spectrometry results of m/z scope 10 000 to 50 000 with PEG-IFN-α 2 conjugates.Based on Fig. 4 A result displayed, we may safely draw the conclusion, and the mass spectrum of rhIFN-α 2 comprises a base peak, and it is equivalent to molecular weight 19 295 daltonian [M]+univalent ions.
In the mass spectrum of PEG-IFN-α 2 conjugates shown in Fig. 4 B, we can see a diffusion main peak, and it is equivalent to molecular weight 40 498 daltonian [M]+univalent ions.The diffusion at this peak is to be caused by the heterogeneity that mono methoxy PEG prepares.The measurement m/z value (40 498 dalton) of peak maximum PEG-IFN conjugate is consistent with the molecular weight calculating sum of IFN-α 2 (19 295 dalton) and additional PEG (20 000 dalton).
Embodiment 9
The location in PEGization site in the PEG-IFN-α 2b conjugate
In the PEG-IFN conjugate sample that 5 μ l produce according to embodiment 3, add 0.05M NH
4HCO
3In 5 μ l concentration, 15 μ g * ml
-1Change insulin solutions (Promega).Be hydrolyzed 16 hours at 37 ℃, add 10 μ l, 0.5% uranous tetrafluoride solution and fully stirring in 10% acetonitrile solution then.Use similar methods to prepare a unmodified IFN sample.The solution that obtains is used to obtain the MALDI mass spectrum.
Confirm the location of PEG and IFN binding site molecule point through comparing PEG-IFN-α 2 conjugates and the mass spectrum of unmodified IFN-α 2 behind tryptic digestion.The tryptic digestion thing of PEG-IFN-α 2 conjugates should lack the corresponding peptide of modifying the protein portion that takes place.In table 2, can see the mass spectrum of the experiment tryptic digestion thing of unmodified IFN-α and PEG-IFN-α 2 conjugates.Based on result displayed in this table; We may safely draw the conclusion; The mass spectrum of the tryptic digestion thing of unmodified IFN-α 2b in fact with the mass spectrum consistent (table 2) of the tryptic digestion thing of PEG-IFN-α 2 conjugates; It is 1313.649 peptide that but the tryptic digestion thing of PEG-IFN-α 2 conjugates lacks molecular weight, and it is present in the tryptic digestion thing of unmodified IFN-α 2b (also referring to Fig. 5).According to the theoretical weight analysis of IFN-α 2 tryptic digestion things, the peak of m/z 1313.649 is equivalent to N end protein matter peptide (table 2).Its disappearance in the tryptic digestion thing of PEG-IFN-α 2 conjugates has confirmed the modification of N end peptide, and N end peptide becomes heavier through the weight of PEG, has exceeded the spectrum zone.PEG combines the IFN molecule to occur over just the free amino group that exists in this peptide, and N end halfcystine is amino.
The experiment mass spectrum of the tryptic digestion thing of table 2.PEG-IFN-2 conjugate and unmodified IFN-2 and theoretical value are relatively
The antiviral specific activity of PEG-IFN-α 2b conjugate is measured
The procedural test that use is described according to European Pharmacopoeia alpha-interferon responsive go down to posterity MBDK cell culture and vesicular stomatitis virus (VSV) culture according to the antiviral specific activity of embodiment 3 with the sample of embodiment 4 productions.Table 3 has shown the result of antiviral activity research.International Reference Version is as reference level.We may safely draw the conclusion by table 3, although combine with molecular weight 20 000 daltonian PEG molecules, the antiviral activity of the conjugate that is produced is equivalent to unmodified IFN-α 2 active 42-45%.Be necessary to note to have the PEG-IFN conjugate that comprises molecular weight 12 000 daltonian PEG described in the prototype method than low activity (28-30%).
The antiviral specific activity of table 3.PEG-IFN-α 2 conjugates and unmodified IFN-α 2 are relatively
Embodiment 11
The temperature-stable Journal of Sex Research of PEG-IFN-α 2b conjugate
The PEG-IFN conjugate sample that 5ml is produced according to embodiment 3 places the water-bath of temperature (50 ± 2) ℃, and has tested muddiness generation in protein soln through mensuration optical density(OD) under 340nm at different time after at interval.Carry out the temperature-stable Journal of Sex Research of unmodified IFN-α 2 with similar method.The formation of insoluble protein compound causes muddy formation and causes optical density(OD) increase under 340nm.Result of study shows in Fig. 6.In viewing duration (28 hours), it is stable that PEG-IFN-α 2 conjugates keep, and unmodified IFN-α 2 almost completely precipitated after 28 hours.
Therefore, the data that obtain in this research allow to reach a conclusion, and compare with unmodified protein matter, and the temperature stability of PEG-IFN-α 2 conjugates significantly increases.
Embodiment 12
The proteolyze stability study of PEG-IFN-α 2b conjugate when with trypsin treatment
The 1M Tris-HCl damping fluid, the 2 μ l 0.5M CaCl that in the PEG-IFN conjugate that 1ml produces according to embodiment 3, add 150 μ l pH 8.5
2Solution and 15 μ l concentration are the trypsin Promega of 20 μ g/ml).Sample is hatched for 37 ℃ in temperature.The different time interval is sampling 100 μ l aliquot from said mixture afterwards, add 150 μ l trifluoroacetic acid solution termination reactions.Use similar method preparation to comprise the sample of unmodified IFN-α 2.Then, use the reversed-phase HPLC analytic sample, measure the main peak area.Result of study is as shown in Figure 7, if prove the use trypsin treatment, the proteolyze stability of PEG-IFN-α 2 conjugates is higher than unmodified IFN-α 2b.
Embodiment 13
The stability of PEG-IFN-α 2b conjugate is measured between the shelf lives
From the sample of producing according to embodiment 3, take out aliquot and put into aseptic centrifugal testing tube with cover.The protein concn of PEG-IFN-α 2 conjugates is 1mg/ml.It is the refrigerator of (6 ± 2) ℃ that this sample is placed temperature.Sampling after the preset timed interval, and through RP-HPLC and embodiment 1 described antiviral specific activity and homogeneity at the polyacrylamide gel electrophoresis that under non-reduced condition, carries out in the presence of the dodecyl sulfate (Eph) analyte preparation thing.Based on table 4 result displayed, we may safely draw the conclusion, and PEG-IFN-α 2 conjugates were stablized 24 months at least between temperature (6 ± 2) ℃ shelf lives.
The stability of table 4.PEG-IFN-α 2 conjugates under temperature (6 ± 2) ℃
Embodiment 14
The immunogenicity research of PEG-IFN-α 2b conjugate
To be diluted to concentration according to the PEG-IFN conjugate that embodiment 3 and embodiment 4 produce is the ICR mouse (5 groups, every group of 5 mouse) that 5mln.IU/ml and continuous 5 all weekly intramuscular injection 200 μ l dosage (every mouse 1mln IU) arrive body weight 20-22g.Simultaneously, similar preparation unmodified IFN sample and inject 1mln.IU dosage to mouse.Puncture is from the mouse blood sampling after when the 5th week finished, passing through ball.Use standard method to obtain serum.Use the serum antibody titer that obtains after unmodified IFN and the PEG-IFN conjugate immune mouse to measure through direct elisa technique.For this purpose, unmodified IFN-α 2 solution of 100 μ l concentration 200ng/100 μ l place the hole of microtiter plate.The antiserum(antisera) that never obtains with treated animal places the hole of plate in duplicate with serial dilution series.Be used to detect the immunocomplex that obtains with the anti-mouse antibodies of peroxidase conjugated.Using unmodified IFN antibody titer afterwards is 1/1024.Using PEG-IFN conjugate antibody titer afterwards is 1/128.Result of study shows in Fig. 8.
Therefore, based on the result who obtains, we may safely draw the conclusion, and the immunogenicity of desired PEG-IFN-α 2 conjugates is lower 8 times than unmodified IFN.
The pharmacokinetic of PEG-IFN-α 2b conjugate
The PEG-IFN conjugate sample of producing according to embodiment 3 of 1mln IU amount arrives male mice through peritoneal injection.Simultaneously, unmodified IFN-α 2 is expelled to another group mouse.Paracentesis were from the animal blood sampling after Preset Time passed through ball at interval afterwards.Use standard method to obtain serum.Detect based on antiviral activity and to have Interferon, rabbit in the serum.Pharmacokinetic is the result be presented among Fig. 9.We may safely draw the conclusion based on these results, and desired PEG-IFN-α 2 conjugates have the effect of significant prolongation.After natural IFN-α 2 injections, its concentration in animal blood reached peak in back 1 hour in injection, very rapidly descended then.After the PEG-IFN conjugate injection, observe the maximum IFN concentration (Fig. 9) in the animal blood after 12 hours, and this level kept 50 hours, observe IFN-α 2 concentration then and slowly descended 350 hours.
The TG-AUC of desired PEG-IFN conjugate (AUC) surpasses the analog value of unmodified IFN more than 50 times.Significantly, if prototype PEG-IFN-α 2b conjugate and molecular weight 12 000 daltonian PEG couplings, the AUC value only surpasses IFN AUC 3-10 doubly.
Calculated the main pharmacokinetic parameter of desired PEG-IFN-α 2b conjugate based on the result (Fig. 9) who obtains.Result's demonstration is compared with unmodified IFN, and the PEG-IFN conjugate slows down from injection site picked-up, capacity distribution, clearance rate greatly, and this has guaranteed the circulation (above 14 days) of the prolongation of desired PEG-IFN conjugate in blood.
Embodiment 16
The comparative characteristic that the PEG-IFN-α 2b conjugate of describing in desired PEG-IFN-α 2b conjugate and the prototype method is compared
The PEG-IFN-α 2b conjugate of describing in desired PEG-IFN-α 2b conjugate and the prototype method is compared, and has carried out structure, basic physics, chemistry and pharmacokinetic parameter relatively (table 5).Data presented confirms in the table 5, compares with the conjugate of describing in the prototype method, and the character of desired PEG-IFN conjugate is significantly improved.
The PEG-IFN-α 2b conjugate of describing in basic physics, chemistry and the pharmacokinetic parameter of the desired PEG-IFN-α of table 5. 2b conjugate and the prototype method (US 5,951,974) is compared
*-use the PEG-IFN conjugate that prototype method produces structural formula at reference " WHO Drug Information ", 2001, v.15, N.3-4, provide in p.206.
Embodiment 17
Embodiment based on the medicinal compsns of desired PEG-IFN-α 2b conjugate
(A)
(B)
(C)
Embodiment 18
Comprise the assembling of the medicament of PEG-IFN-α 2b
The medicament (embodiment 17) that comprises the desired PEG-IFN-α 2b of significant quantity installed in the syringe at aseptic condition in following minute; Said syringe is made by the neutral glass of first hydrolysis grade; Have the brazing syringe needle of living with elasticity or rigid protective cap; Seal with the ozzle on the butyl rubber piston of fluoropolymer lamination; Capacity is 0.5,0.8,1.0 and 1.2ml (being used for 100 μ g/ml dosage), 0.5,0.6,0.75 and 0.9ml (being used for 200 μ g/ml dosage), 0.5,0.6,0.8,1.0 and 1.2ml (being used for 300 μ g/ml dosage).
Embodiment 19
Comprise the assembling of the medicament of PEG-IFN-α 2b
The medicament (embodiment 17) that comprises the desired PEG-interferon alpha 2 b of significant quantity installed in the bottle at aseptic condition in following minute; Said bottle is made by the neutral glass of first hydrolysis grade; With viton or have the ozzle sealing on the butyl rubber lid of teflon coatings; Fastening with the aluminium cap; Capacity is 0.5,0.8,1.0 and 1.2ml (being used for 100 μ g/ml dosage), 0.5,0.6,0.75 and 0.9ml (being used for 200 μ g/ml dosage), 0.5,0.6,0.8,1.0 and 1.2ml (being used for 300 μ g/ml dosage).
The test kit that comprises the medicament of having assembled that comprises PEG-IFN-α 2b
Said test kit comprises 1 or 4 syringe and piston (correspondingly 1 or 4) in the bubble wrap that polymeric film makes/or bottle, and prescription information, places card board package.
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Claims (25)
1. the interferon alpha conjugate of stable PEGization, expression in formula (I) is single positional isomers:
Wherein:
The integer of n-from 227 to 10 000;
The integer of m->=4;
N
αThe H-IFN-interferon alpha.
2. conjugate according to claim 1, wherein m=4.
3. conjugate according to claim 1, wherein interferon alpha is natural or Interferon Alfa-2b.
4. conjugate according to claim 1, wherein the molecular-weight average of polyoxyethylene glycol is 10 to 40kDa.
5. conjugate according to claim 1, wherein N end group group is represented by cysteine residues (Cys).
6. medicinal compsns has antiviral, antiproliferative and immunoregulatory activity, comprises the conjugate and the pharmaceutically acceptable vehicle of the general formula (I) of significant quantity.
7. medicinal compsns according to claim 6 is used to treat virus and tumor disease, and the disease relevant with primary or secondary immunodeficiency.
8. medicinal compsns according to claim 7, wherein said virus disease are hepatitis B or hepatitis C.
9. medicinal compsns according to claim 7, wherein said tumor disease are myelogenous leukemia.
10. medicinal compsns according to claim 7, wherein said tumor disease are melanoma.
11. comprise the medicinal prepns of the conjugate of formula (I), have antiviral, antiproliferative and immunoregulatory activity.
12. medicinal prepns according to claim 11 comprises damping fluid, isotonic agent, stablizer and water.
13. medicinal prepns according to claim 12 comprises sodium acetate trihydrate, acetate, sodium-chlor, polysorbate80, EDTA, water for injection.
14. the conjugate of formula (I) has the application in the medicinal product of antiviral, antiproliferative and immunoregulatory activity in preparation.
15. application according to claim 14 is used to prepare antiviral, the antiproliferative with resistance of hepatitis B or hepatitis C and the medicinal product of immunoregulatory activity.
16. the conjugate of the formula (I) through introducing effective therapeutic dose prevents and/or treats virus disease.
17. according to claim 16 preventing and/or treating, wherein said virus disease are hepatitis B or hepatitis C.
18. according to claim 16 preventing and/or treating introduced the ribavirin of effective therapeutic dose in addition.
19. the conjugate of the formula (I) through introducing effective therapeutic dose prevents and/or treats and primary or the relevant disease of secondary immunodeficiency state.
20. the conjugate of the formula (I) through introducing effective therapeutic dose prevents and/or treats tumor disease.
21. according to claim 20 preventing and/or treating, wherein said tumor disease are myelogenous leukemia.
22. according to claim 20 preventing and/or treating, wherein said tumor disease are melanoma.
23. container, sealing is also suitable in gnotobasis before using stores, and comprises medicinal compsns according to claim 6.
24. container according to claim 23, wherein said container are prefilled syringe, bottle or automatic injector.
25. a cover product comprises container according to claim 24 and prescription information.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| RU2010129824/10A RU2447083C1 (en) | 2010-07-20 | 2010-07-20 | NOVEL FUNCTIONALLY ACTIVE, HIGHLY PURE, STABLE CONJUGATE OF INTERFERON α WITH POLYETHYLENE GLYCOL, REPRESENTED BY ONE PEG- NαH-IFN POSITIONAL ISOMER, WITH IMPROVED IMMUNOGENICITY, WITH PROLONGED BIOLOGICAL ACTION, SUITABLE FOR MEDICAL APPLICATION, AND IMMUNOLOGICAL AGENT BASED THEREON |
| RU2010129824 | 2010-07-20 |
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| RU2515913C1 (en) * | 2013-03-22 | 2014-05-20 | Федеральное государственное унитарное предприятие "Государственный научно-исследовательский институт генетики и селекции промышленных микроорганизмов" (ФГУП "ГосНИИгенетика") | HYBRID PROTEIN HAVING PROLONGED ACTION, BASED ON RECOMBINANT HUMAN INTERFERON ALPHA-2 (VARIANTS), METHOD OF ITS PRODUCTION AND STRAIN OF Saccharomyces cerevisiae FOR IMPLEMENTING THIS METHOD (VERSIONS) |
| EA021643B1 (en) * | 2013-03-28 | 2015-07-30 | Илья Александрович МАРКОВ | Monopegylated interferon-alpha of linear structure and a pharmaceutical composition for preparing a medicament having interferon-alpha activity |
| EA021610B1 (en) * | 2013-03-28 | 2015-07-30 | Илья Александрович МАРКОВ | Liquid antiviral formulation |
| CN103463623B (en) * | 2013-09-03 | 2015-09-09 | 长春海伯尔生物技术有限责任公司 | A kind of Peg-IFN alpha-2b injection and preparation method thereof |
| RU2554761C1 (en) * | 2014-05-13 | 2015-06-27 | Закрытое акционерное общество "Сибирский центр фармакологии и биотехнологии" | Anti-enteroviral and immunostimulating agent |
| RU2572800C1 (en) * | 2014-09-22 | 2016-01-20 | Закрытое Акционерное Общество "Биокад" | New formulation containing polyethylene glycol (peg) conjugated interferon alpha-2beta characterised by less painful administration |
| EA029498B1 (en) * | 2015-11-24 | 2018-04-30 | Учреждение Белорусского государственного университета "Научно-исследовательский институт физико-химических проблем" (НИИ ФХП БГУ) | ANTI-TUMOUR DRUG BASED ON RECOMBINANT INTERFERON ALPHA-2b IN THE FORM OF MICROPARTICLES FOR PARENTERAL ADMINISTRATION |
| RU2678332C1 (en) | 2017-09-08 | 2019-01-28 | Общество с ограниченной ответственностью "Саентифик Фьючер Менеджмент" (ООО "СФМ") | Pegylated interferon lambda with high bioaccessability in oral use and method for production thereof |
| CN114392237B (en) * | 2021-12-28 | 2024-02-02 | 上海允英生物医药科技有限公司 | Freeze-dried virus preparation and preparation method thereof |
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| HK1170504A1 (en) | 2013-03-01 |
| ECSP13012398A (en) | 2013-05-31 |
| BRPI1101565A2 (en) | 2012-12-04 |
| PE20131034A1 (en) | 2013-09-27 |
| RU2447083C1 (en) | 2012-04-10 |
| SG187117A1 (en) | 2013-02-28 |
| UA99766C2 (en) | 2012-09-25 |
| AR087227A1 (en) | 2014-03-12 |
| CN102617736B (en) | 2015-11-25 |
| WO2012011836A1 (en) | 2012-01-26 |
| CR20130021A (en) | 2013-02-20 |
| CU20130013A7 (en) | 2013-04-19 |
| MX2011007458A (en) | 2012-01-19 |
| KR20130056885A (en) | 2013-05-30 |
| NI201300008A (en) | 2014-05-26 |
| MY168784A (en) | 2018-12-04 |
| UY33525A (en) | 2012-02-29 |
| RU2010129824A (en) | 2012-01-27 |
| DOP2013000002A (en) | 2013-09-15 |
| EA201100809A1 (en) | 2012-01-30 |
| CU24193B1 (en) | 2016-09-30 |
| CO6680611A2 (en) | 2013-05-31 |
| EA020257B1 (en) | 2014-09-30 |
| KR101586372B1 (en) | 2016-01-18 |
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