CN102617736B - The stable interferon alpha polyoxyethylene glycol conjugate represented by a kind of positional isomers - Google Patents
The stable interferon alpha polyoxyethylene glycol conjugate represented by a kind of positional isomers Download PDFInfo
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Abstract
The present invention relates to pharmacy industry and medicine, particularly the PEG-interferon derivative of novelty, and find a kind of high stability Interferon, rabbit polyoxyethylene glycol conjugate of functionally active of novelty, its general formula is:
Description
Technical field
The present invention relates to medicine, namely relate to the physiologically active conjugate of Interferon, rabbit, particularly the new conjugate of Interferon, rabbit and polyoxyethylene glycol (PEG), it can be used for medicine, such as, be used for the treatment of virus, immunity and tumor disease.
Background technology
Interferon, rabbit (IFN) is one group of biological activity protein or glycoprotein, it is replied virus infection by various kinds of cell and produces, or because certain chemistry and biological substance produce (Isaacs & Lindeman, 1957 to the impact of cell; The people such as Pestka, 2007).Cause the puting together of IFN and cell receptor that multiple mediation is antiviral, induction (people such as Pestka, 2004 of the intracellular protein of immunomodulatory and antiproliferative IFN effect; Bekisz, 2004).
Different mankind IFN genes is cloned and expresses in bacterium and zooblast, has therefore produced multiple restructuring IFN (people such as Pestka, 2004; Pestka, 2007).The medicine comprising restructuring IFN 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 (, 2004) Pestka.IFN medicine is used most widely for treatment viral hepatitis (Chevaliez & Pawlotsky, 2009), and this disease is one of the most serious social concern.Nowadays the whole world about has 3.5 hundred million patients to be infected by Chronic Hepatitis B Virus, and has 1.7 hundred million patients 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 WHO Report, from 1961, chronic hepatitis and liver cirrhosis were brought up to the 5th (Marcellin, 2009) from the 10th as the cause of the death in the U.S. and the Western European countries.
The medicine comprising IFN is commonly used to treatment leukemia and solid tumor, comprises recurrent melanoma (people such as Bukowski, 2002; The people such as Decantris, 2002; The people such as Qintas-Cardama, 2006).
The effect comprising the medicine of natural IFN absorbs fast by subcutis, large volume distributes, the restriction (Wills, 1990) of relatively low stability, short-half-life, high immunogenicity and toxicity.Therefore, after using in several hours, observe IFN concentration in blood plasma and decline fast, and injection can not detect Interferon, rabbit people such as (, 1999) Chatelut in blood plasma after 24 hours.The quick decline of Interferon, rabbit concentration causes virus replication to continue and Viral particle concentration rising people such as (, 1997) Lam.Therefore, need frequent injection of interferon to reach the effective treatment concentration in blood plasma, cause dose-dependent side effect.Therefore the monotherapy of the chronic hepatitis C (CHC) of IFN-α 2b is used to cause lasting virological response 12 middle of the month in patient only at 15-20%, and in the patient with genotype Ib and high viral load, do not observe pharmaceutical efficacy (people such as McHutchinson, 1998) completely.
When use sustained release drugs particularly PEGization IFN time, the treatment effect of IFN can be enhanced (people such as Manns, 2001; The people such as Hadziyannis, 2004; The people such as Zeuzem, 2001).PEGization IFN puts together generation by interferon molecule and polymer chemistry, and the mono methoxy polyethylene glycol (MPEG) that described polymkeric substance is such as made up of the repetition residue of oxyethane, it at one end has methoxyl group, has hydroxyl at the other end.MPEG molecule can have different molecular weight and stereochemical structure (line style or ramiform).For PEGization reaction, the hydroxyl of MPEG end is activated by multiple response function group.The MPEG of activation at one or more positions covalency conjugated protein, can depend on character and reaction conditions (Zalipsky & Hurris, 1997 of activating group; The people such as Roberts, 2002).
The PEGization of IFN causes the activity in vivo (external activity reduction) of better pharmacokinetics, transformation period of prolongation, the haemoconcentration fluctuation of minimizing, the immunogenicity reduced and toxicity, rising; Stability (people such as Glue, 2000 of improving; The people such as Reddy, 2001).
Reduction activity in vitro system depends on PEG molecular weight.The little PEG molecule of conjugated molecules amount≤5000Da causes the subtle change (people such as Grace, 2005) of external activity.Comprise the PEGization albumen of quality PEG like this and the drug effect of unmodified protein and pharmacokinetic property to be difficult to distinguish.Put together the PEG of more high molecular due to the invalid remarkable reduction of puting together acceptor and causing In vitro biological activity, but pharmacokinetics and the drug effect character of the visible in the case PEGization albumen significantly improve (people such as Delgado, 1992), Biological acdtivity in vivo is caused to raise (Bailon & Berthold, 1998).PEG structure also affects biologic activity and pharmacokinetic property, puts together line style PEG than puting together ramiform PEG structure and has larger volume of distribution (people such as Caliceti, 2003).
Antiviral activity and the biological property of PEG-IFN conjugate also depend on the activation MPEG that use is different, because its functional group changes with the ability of modifying different proteins amino-acid residue and the chemical bond types that formed with protein people such as (, 2002) Roberts.The activation MPEG that can put together with the free amino group of protein is used most widely for the PEGization (succinimdyl carbonate, succinimidyl succinate, trisilate, triazine, hydroxysuccinimide eater, acetal etc.) of protein.
Current known following PEG-IFN conjugate.
Known a kind of PEG-IFN-α 2a conjugate, wherein line style PEG molecular weight is 1-5000Da (United States Patent (USP) 5,382,657,1995).MPEG glycol derivative is used for puting together, and this reaction at room temperature carries out 0.5-4 hour at pH=10.3 times of excessive PEG react for the PEGization of IFN.In the condition used, the accessibility of the free amino group in combination with different Methionin (Lys-31, Lys133, Lys134, Lys23, Lys131, Lys121, Lys70, Lys83, Lys49 and Lys112) of PEG and IFN and the formation of carboxamide key and occur (people such as Monkarsh, 1997).Therefore, PEG-IFN-α 2a is made up of the mixture of the positional isomers of the PEG-IFN-α 2a conjugate obtained, and wherein each isomer comprises single PEG.Compare with unmodified IFN, the antiviral specific activity scope of isomer at 6% (for Lys112) to 40% (for Lys133).Total specific activity of the gained conjugate be 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 2500DaPEG) to 25% (for 10000DaPEG).The shortcoming of the conjugate obtained is as follows:
1. the lower molecular weight (≤5000Da) MPEG making spent glycol activate.The pharmacokinetic parameter of result PEG-IFN conjugate and unmodified IFN only has fine difference.Therefore this conjugate does not enter clinical trial;
2., because PEG passes through free amino group and the IFN molecular conjugate of different Methionin, form the positional isomers in a large number with different specific activity.
Known line style (Russ P 2311930,2004) by natural IFN-α 2b and molecular weight 13000-17000Da or ramiform (Russ P 2382048,2008) MPEG derivative put together the PEG-IFN-α 2b conjugate of acquisition.This reaction uses the activated PEG of 50-100 times of molar excess carry out 60 hours (for line style MPEG) at pH9.5 or carry out 12 hours (for ramiform MPEG) at pH7.5.Result obtains PEG-IFN conjugate, its total antiviral activity be unmodified IFN activity 29 to 38% between.Not about the data of conjugate stability, positional isomers quantity and antiviral specific activity thereof.The shortcoming of these conjugates is as follows:
1. use trifluoroethyl sulfonic acid mono methoxy (tresylate) derivative of activation MPEG, its transformation period is in aqueous less than 20 minutes.Once be presented in the tresylate derivative of PEG and the reaction of protein in the past, except by except amino stable amido linkage, yet forms both unstable thionamic acid ester bond (Gais & Ruppert, 1995);
2. the PEG of trifluoroethyl sulfonic acid mono methoxy activation and the free amino group in combination with had living space enterable Methionin of protein complete people such as (, 2002) Roberts, and it causes the formation of multiple positional isomers;
3. the PEGization reaction carrying out IFN in high ph-values (pH=10) long-time (60 hours) can change IFN structure;
In the PEGization reaction of 4.IFN, significantly excessive activated PEG (mol ratio of PEG/ protein is 50-100/1) causes the high cost obtaining product;
The known ramiform pyrrolotriazine derivatives MPEG by natural IFN-α 2b and molecular weight 7500-35000Da puts together a kind of PEG-IFN-α 2b conjugate (Russ P 2298560 of acquisition, 2004), its total antiviral activity is equivalent to 6.4% of unmodified IFN activity.Not about the data of conjugate stability, positional isomers quantity and antiviral specific activity thereof.
The shortcoming of the conjugate obtained is as follows:
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 unstable key.In addition, pyrrolotriazine derivatives (Veronese & Pasut, 2005) is not used because of high toxicity now;
2. described in, the specific activity of PEG-IFN conjugate is low.
The 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 the available free amino group of protein.This reaction with 3: 1 MPEG/ protein ratio pH9,4 DEG C carry out 2 hours.This reaction be terminated and the conjugate obtained by sorbent material FractogelEMDCM650 (M) purifying.The productive rate of purified conjugation thing is 40-45%.In this case, the IFN-PEG conjugate obtained is made up of 6 kinds of positional isomerss, wherein often kind all pass through Methionin-Lys31, Lys121, Lys131, Lys134, Lys70 and Lys83 free amino group by stable keys and PEG molecular conjugate (people such as Vailon, 2001; The people such as Foser, 2003).The isomer puted together by Methionin and the PEG of position 31,121,131 and 134 accounts for 94% of whole conjugate.The activity of the whole conjugates be made up of 6 kinds of positional isomerss is 1-7% (people such as Bailon, 2001 of natural IFN-α 2a; The people such as Boulestin, 2006).Although antiviral activity is low in vitro, this conjugate has the pharmacokinetic property (people such as Silva of improvement compared with unmodified IFN, 2006, the people such as Boulestin, 2006), sell (being produced by " Hoffmann-LaRoche ") with trade(brand)name " Pegasys " at present.
The shortcoming of the conjugate obtained is as follows:
1. use the PEG of N-hydroxy-succinamide ester activation to cause puting together with had living space enterable amino, the conjugate that result obtains is the mixture of the different isomer of 6 kinds of specific activities.
2. the Anti-viral activity in vitro of the conjugate obtained is low.
Prototype of the present invention is United States Patent (USP) 5,951,974, and the PEG-IFN-α 2b conjugate described in 1999.The line style MPEG (SC-MPEG) that the molecular weight 5000 of succinimdyl carbonate group activation arrives 12000Da reacts for PEGization.Obtain the IFN puted together with the PEG of molecular weight 12kDa according to described method, it is sold with trade(brand)name " PegIntron " and is used for the treatment of viral hepatitis (" Schering-Plough ", the U.S.) at present.
Find that described PEG-IFN-α 2b conjugate (medicine " PegIntron ") is made up of 13 kinds of positional isomerss, wherein often kind all comprise one with the PEG molecule (people 2000 such as Wang of multiple moiety conjugation of protein molecule; The people such as Grace, 2001; The people such as Youngster, 2002).PEG molecule and Interferon, rabbit are puted together not by means of only free amino group Methionin (Lys31, Lys49, Lys83, Lys121, Lys131, Lys133, Lys134 и Lys164) and (Cys1) according to the show, and by Histidine imidazole ring (His7, His34), tyrosine (Tyr129) and Serine OH group (Ser163).The content of single isomer changes between 0.8% (Tyr129) to 47% (His34).The antiviral activity of the PegIntron be made up of 13 kinds of positional isomerss is 28% of 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 puted together by His34 and PEG has the highest specific activity (37% of natural protein).Free amino group and the PEG of IFN are puted together by stable keys, and in the main isomer accounting for 47%, PEG and Histidine imidazole ring (His34) put together people such as (, 2002) Roberts by unstable (in aqueous) amino-formate bond.
The pharmacokinetic parameter of described conjugate is better than the IFN (people such as Silva, 2006) of unmodified.
The shortcoming of the conjugate obtained is as follows:
1. use the PEG of succinimdyl carbonate activation not only to put together the free amino group of IFN, also put together other amino acid whose functional group.The conjugate that result obtains is made up of the mixture of the different positional isomers of 13 kinds of antiviral specific activities;
2. the fact that imidazoles-carbonic ether (carbamate) key forming instability in main (His-34) isomer of described PEG-IFN conjugate causes is that after " Pegintron " uses, described conjugate hydrolysis discharges natural IFN and plays its biological effect.Therefore, medicine " Pegintron " is preferably considered as prodrug, and after its injection, hydrolysis forms active interferon (Foster, 2004).Due to described PEG-IFN conjugate unstable in the solution, " Pegintron " formulation only stores with lyophilized powder form.
Summary of the invention
Target of the present invention is the IFN of the stable PEGization obtaining a kind of novelty, it has the activity of IFN-α, be made up of a kind of positional isomers, there is the stability of improvement, there is the immunogenicity of reduction, the pharmacokinetic parameter of improvement, there is the optimum combination of PEG molecular weight and conjugate antiviral activity, be applicable to medical, and based on the medicinal compositions of described PEG-IFN conjugate.
The problems referred to above are solved by the functionally active PEG-IFN conjugate with interferon alpha activity building a kind of novelty, its middle-molecular-weihydroxyethyl 10000 strictly holds the α of halfcystine amino to be combined with IFN-alpha molecule by stable keys by N to the PEG thread-like molecule of 40000Da, produces the compound with following structural formula (I):
Wherein: the round values of n-from 227 to 10000;
The integer of m->=4;
IFN-has the natural of IFN-alpha active or recombinant polypeptide.
In the conjugate obtained, the α amino of the line style PEG of molecular weight 10000-40000Da and the N terminal amino acid of IFN-α combines.
Achieve strictly by the IFN modification selectivity of the α amino of N terminal amino acid by using the mPEG of the general formula (II) of acetaldehyde activation by the PEGization reaction in pH≤6:
Wherein: the round values of n-from 227 to 10000, therefore the molecular weight of PEG is about 10000-40000Da;
The integer of m->=4;
The optimum ratio of PEG molecular weight and antiviral activity is obtained by the activation MPEG (II) of the numerical value using m >=4.
The pharmacokinetic parameter of immunogenicity and toxicity reduction and improvement is achieved by the molecular weight increasing the PEG puted together.
New features compared with prototype are:
Compared with prior art, novelty is as follows:
1. activate the acetaldehyde derivatives of mPEG for the production of PEG-IFN conjugate;
The structural formula of 2.PEG-IFN conjugate is novel;
3. the PEG-IFN conjugate produced only is represented by a kind of positional isomers;
Key between 4.PEG molecule and IFN molecule is stablized;
5., due to the size of the PEG of combination, the molecular weight of PEG-IFN conjugate increases;
6. pharmacokinetic parameter improvement.
For producing required PEG-IFN conjugate, employ the butyraldehyde meeting the mPEG of structural formula (II) or the valeral derivative of recombinant human IFN-alpha 2b (being produced by ZAO " Biocad ") and molecular weight 20000Da.
Described conjugation reaction uses reductive agent to carry out temperature 20 DEG C or following at below pH6.0.The mol ratio of PEG/ protein is 2.5-5: 1.Under the reducing conditions, under sodium lauryl sulphate (SDS) exists, use polyacrylamide gel electrophoresis (PAGE) and reverse phase HPLC chromatogram (RP-HPLC) to carry out PEG-IFN conjugate and form contrast.Carry out monomer PEG-IFN from the purifying reaction product by 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 PEG molecules.The wash-out of monomer PEG-IFN is carried out lower than the gradient concentration of 0.05 in the buffered soln of 6 to 0.2M sodium-chlor by pH.The monomer PEG-IFN product of purifying is dialysed in the 10-50mM buffered soln of pH4-5, salt or polysaccharide or alcohol or polyvinylpyrrolidone or monose is added in buffered soln, and aminosugar or protein or amino acid, and nonionic detergent, and store at temperature 4 ± 2 DEG C in the plastics or vial of siliconized surfaces.
For characterizing the characteristic of the monomer PEG-IFN-α 2b conjugate obtained, comparing with unmodified IFN, having carried out the research of purity, homogeneity, physical-chemical, biology and pharmacokinetic properties.
Accompanying drawing explanation
The present invention is illustrated by following accompanying drawing:
Fig. 1. in the kinetics formed with PEG-IFN-α 2b conjugate in butyraldehyde MPEG derivatives reaction.
Fig. 2. the reverse phase HPLC of purifying PEG-IFN-α 2b conjugate on " SymmetryC18 " (4,6x150mm) post.
Fig. 3. compare with unmodified IFN-α 2b, the SDS-PAGE of purifying PEG-IFN-α 2b conjugate analyzes.Mr represents molecular weight marker, and swimming lane 1 represents that every swimming lane adds the PEG-IFN-2b conjugate of 40 μ g; Swimming lane 2 represents that every swimming lane 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 of the tryptic peptide of unmodified IFN-α 2b (A) and PEG-IFN-α 2b (B) conjugate is compared within the scope of m/z1200-1400.
The temperature stability of Fig. 6 .PEG-IFN-α 2b conjugate and unmodified IFN-α 2b.
The proteolytic stability of Fig. 7 .PEG-IFN-α 2b conjugate and unmodified IFN-α 2b.
Fig. 8. compare with unmodified IFN-α 2b, the immunogenicity of PEG-IFN-α 2b conjugate.Post 1 represents unmodified IFN-α 2b, and post 2 represents conjugate PEG-IFN-α 2b (embodiment 3), and post 3 represents conjugate PEG-IFN-α 2b (embodiment 4).
Fig. 9. compare with unmodified IFN-α 2b, the pharmacokinetics of PEG-IFN-α 2b conjugate.
Embodiment
The concrete embodiment of PEG-IFN-α 2b production method and the result of character research thereof provide below.
Target of the present invention, PEG-IFN-α 2 conjugate of formula (I) can be used for producing the medicine for preventing and/or treating virus disease, because except the immunogenicity of high stability and reduction, it has high antiviral active.Equally, can separately or common for the preventing and/or treating of virus infection (such as chronic active hepatitis especially hepatitis B and hepatitis C) (people such as Jay, 2006 with other therapeutical agent (such as ribavirin) as the medicinal compositions of activeconstituents by the conjugate of knowing the formula (I) including effective amount that pharmaceutical methods is produced; The people such as McHutchison, 2009).Target of the present invention also comprises the method preventing and/or treating virus disease such as hepatitis C and hepatitis B, comprises the conjugate of the required formula (I) introducing effective therapeutic dose.
The interferon alpha of known IFN-α and PEGization can be used as immunomodulator and is used for the treatment of cancer, especially leukemic reticuloendo theliosis, laryngeal papillomatosis, melanoma, renal cell carcinoma, myelogenous leukemia, Kaposi sarcoma (people such as Decatris, 2002; The people such as Bukowski, 2002; The people such as Qintas-Cardama, 2006; The people such as Loquai, 2008; The people such as Kaehler, 2010).The experience of these diseases is used for the treatment of based on the well-known pathogenesis of these diseases and IFN-α and conjugate, object of the present invention also comprises medicine and medicinal compositions, it includes the conjugate PEG-IFN-α announced of effective amount, there is antiproliferative and immunomodulatory effect, can be used for preventing and/or treating cancer and the disease relevant to primary or secondary immunodeficiency, object of the present invention also comprises the method preventing and/or treating cancer and the disease relevant to primary or secondary immunodeficiency, comprise the PEG-IFN-alpha conjugate of the formula (I) introducing effective therapeutic dose.
The conjugate of formula (I) and optional pharmaceutically acceptable filler, thinner and/or pharmaceutically acceptable vehicle are used, such as, with the form of capsule, syrup, sprays, drops, injection or suppository by vein, subcutaneous, intramuscular or other suitable approach any.Route of administration changes according to such as symptom and age.Interval between frequency of administration and injection changes according to disease and seriousness thereof or application target (treatment or preventive use).The PEG-IFN-α of effective dose is according to above-mentioned selecting factors.
Pharmaceutically acceptable vehicle can be included in medicinal compositions with PEG-IFN-α: buffering salt (such as acetate, Citrate trianion, supercarbonate, phosphate buffered saline buffer), stablizer (such as polysorbate, EDTA, polyvinylpyrrolidone, dextran, human serum albumin, p-hydroxybenzoic acid ether, alcohols (such as phenylcarbinol), phenols, Sorbic Acid), antimicrobial component (such as phenylcarbinol), osmotic pressure regulator (such as sodium-chlor, Repone K, polyvalent alcohol, such as glycerine, arabitol, Sorbitol Powder, N.F,USP MANNITOL, lactose, dextran), tensio-active agent (such as HSA, polyvinylpyrrolidone, Yelkin TTS, polysorbate80, Pluronic F68, casein), surfactant (the segmented copolymer of such as 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 propylene glycol, polyoxyethylene laurel ether, polyoxyethylene cetyl ether, polysorbate, glyceryl monostearate, distearin, sorbitan monopalmitate, dehydrating sorbitol monooleate polyoxyethylene, sorbitan laurate polyoxyl 40 stearate and propylene glycol), antioxidant (such as TrilonB, Cys, S-WAT, sodium ascorbate, gsh, 2 mercapto ethanol, dithiothreitol (DTT), cysteine hydrochloride, monohydrate, xitix) and thinner (such as water).
Embodiment 1
Butyraldehyde MPEG derivative is used 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 restructuring IFN-α 2 of 1.7mg/ml, 150mM sodium-chlor, pH5.0), in, then stir described solution and add the daltonian dry butyraldehyde PEG derivative of 5g molecular weight 20000.The described mixture of abundant stirring also hatches 22 hours temperature 17 ± 3 DEG C.After different time interval, sample 50 μ l aliquot from described mixture, the polyacrylamide gel electrophoresis method be used under dodecyl sulfate existence analyzes the kinetics that PEG-IFN-α 2 conjugate is produced.For this purpose, comprise 125mMTris-HCl, 20% glycerine, 3% dodecyl sulfate, the pH6.8 of 0.005% tetrabromophenol sulfonphthalein, the damping fluid of cumulative volume 1/3 amount to join in described mixture and to heat 3 minutes in boiling water bath.5 μ l samples are placed in the hole of 12.5% Polyacrylamide gradient gel of preparation, then under dodecyl sulfate exists, carry out electrophoresis.After electrophoresis completes, this gel uses CoomassieR-250 dyeing.The kinetics that PEG-IFN-α 2 conjugate is produced shows in FIG.At the time point of PEG-IFN content more than 70%, described reaction mixture uses the 5mM sodium acetate buffer of pH5.0 to dilute 10 times.
Embodiment 2
Valeral MPEG derivative is used to produce PEG-IFN-α 2b conjugate
The 1M sodium cyanoborohydride solution of 2.05ml joins that to comprise concentration be in the 100ml buffered soln (100mM sodium acetate, 150mM sodium-chlor, pH5.5) of the restructuring IFN-α 2b of 2.2mg/ml, then stirs; Add the daltonian dry valeral PEG derivative of 0.9g molecular weight 20000.The described mixture of abundant stirring also hatches 24 hours temperature 6 ± 2 DEG C.After different time interval, sample 50 μ l aliquot from described mixture, and use polyacrylamide gel electrophoresis method to analyze the kinetics of PEG-IFN conjugate production as described in Example 1.At the time point of PEG-IFN content more than 70%, described reaction mixture uses the 5mM sodium acetate buffer of pH5.0 to dilute 10 times.
Embodiment 3
By the ion-exchange chromatogram purification monomer PEG-IFN-α 2b conjugate on cation-exchange adsorbing substance
Be placed on the post containing cation-exchange adsorbing substance (CM agarose, 300ml) by the diluting soln produced in embodiment 1, this post uses the 5mM sodium acetate buffer (buffer A) of pH5.0 to balance with speed 5ml/min.Described adsorption column uses buffer A and comprises the buffer A continuous washing of NaCl concentration gradient 0.05 to 0.2M.Use polyacrylamide gel electrophoresis method to carry out sample analysis as described in Example 1, from each cut sampling aliquot.Merge the cut comprising PEG-IFN-α 2 conjugate of monomer PEGization, use the 20mM sodium acetate buffer dialysis comprising 150mM sodium-chlor of 10 times of volumes, then carry out sterile filtration, gained solution storage is at 4 ± 2 DEG C.
Embodiment 4
By the ion-exchange chromatogram purification monomer PEG-IFN-α 2b conjugate on cation-exchange adsorbing substance
Be placed on the post containing cation-exchange adsorbing substance (SP agarose, 50ml) by the diluting soln produced in embodiment 2, this post uses the 5mM sodium acetate buffer (buffer A) of pH5.0 to balance with speed 5ml/min.Described adsorption column uses buffer A and comprises the buffer A continuous washing of NaCl concentration gradient 0.03 to 0.3M.Use polyacrylamide gel electrophoresis method to carry out sample analysis as described in Example 1, from each cut sampling aliquot.Merge the cut comprising PEG-IFN-α 2 conjugate of monomer PEGization, use the 20mM sodium acetate buffer dialysis comprising 150mM sodium-chlor of 10 times of volumes, then carry out sterile filtration, gained solution storage is at 4 ± 2 DEG C.
Embodiment 5
The reverse phase HPLC of PEG-IFN conjugate
PEG-IFN-α 2 conjugate embodiment 3 produced uses the 20mM sodium acetate buffer of pH5.0 to be diluted to concentration for 0.1mg/ml, and 100 these samples of μ l are placed in SymmetryC18 post (4.6 × 150mm)." Breeze " chromatographic instrument using WatersCompany to produce is analyzed at 214nm.Based on the result of Fig. 2 display, we may safely draw the conclusion, and according to the discovery of RP-HPLC, the purity of required PEG-IFN-α 2 conjugate is more than 99%.
Embodiment 6
Measure level of endotoxin
Bacterial endotoxin (BE) concentration in the PEG-IFN-α 2 conjugate sample produced according to embodiment 3 and 4 is measured in vitro by the lal test (gelling process version) meeting European Pharmacopoeia 6.0 clause 2.6.14 requirement.Employ the AssociatesofCAPECOD for lal test in the analysis, the diagnostic kit that Inc. produces, the LAL reagent of sensitivity 0.03EU/ml, intracellular toxin reference standard (in vial 0.5 μ g) and water.Based in table 1 display result, we may safely draw the conclusion, the BE content in conjugate sample lower than 1.5 endotoxin units (EU)/mg protein, its BE level allowed far below the medicine based on recombinant protein.
Bacterial endotoxin concentration in table 1.PEG-IFN-α 2 conjugate
Embodiment 7
Compare with unmodified IFN-α 2b, the electrophoretic analysis of PEG-IFN-α 2b conjugate
The sample of the PEG-IFN conjugate produced according to embodiment 3 uses the polyacrylamide gel electrophoresis method analysis described in embodiment 1.Every hole application of sample 40 μ g protein carries out electrophoresis under non reducing conditions.CoomassieR-250 dyestuff is used to carry out the dyeing of gel internal protein.Carry out the electrophoresis of unmodified IFN-α 2 simultaneously.PEG-IFN conjugate represents in (Fig. 3, the 1st road) with a band.Based on the electrophorogram of PEG-IFN-α 2 conjugate 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 accurate molecular weight of the protein of PEGization, because add to protein the stokes radius that hydrophilic PEG molecule increases gained compound greatly.Therefore, the motion of PEG-protein compound in gel is slowed down, and its molecular weight seems the molecular weight sum being much higher than protein and PEG.
Embodiment 8
Compare with unmodified IFN-α 2b, use mass spectrometry method to measure the molecular weight of PEG-IFN-α 2b conjugate
The 1 μ l sample of PEG-IFN-α 2b conjugate produced according to embodiment 3 and 2,5--resorcylic acid solution (Aldrich, the 10mg × ml of 0.3 μ l
-1in 20% acetonitrile solution, containing 0.5% uranous tetrafluoride) to mix and air-dry.Similar method is used to prepare the sample of a unmodified IFN-α 2.
Mass spectrum obtains being equipped with on the UltraflexIIBRUKER of UV laser apparatus (Nd) (Germany) MALDI-TOF mass spectrograph.Mass spectrum obtains under linear positive ion mode; Weight in average measuring error is no more than 10-15 dalton.
Unmodified rhIFN-α 2 and PEG-IFN-α 2 conjugate show in the diagram in the mass spectrometry results of m/z scope 10000 to 50000.Based on the result that Fig. 4 A shows, we may safely draw the conclusion, and the mass spectrum of rhIFN-α 2 comprises a base peak, and it is equivalent to daltonian [the M]+univalent ion of molecular weight 19295.
In the mass spectrum of PEG-IFN-α 2 conjugate as shown in Figure 4 B, we can see a diffusion main peak, and it is equivalent to daltonian [the M]+univalent ion of molecular weight 40498.The diffusion at this peak is that the heterogeneity prepared by mono methoxy PEG causes.It is consistent that measurement m/z value (40498 dalton) and the molecular weight of IFN-α 2 (19295 dalton) and additional PEG (20000 dalton) of peak maximum PEG-IFN conjugate calculate sum.
Embodiment 9
The location in PEGization site in PEG-IFN-α 2b conjugate
0.05MNH is added in the PEG-IFN conjugate sample produced according to embodiment 3 to 5 μ l
4hCO
3in 5 μ l concentration 15 μ g × ml
-1change insulin solutions (Promega).Be hydrolyzed 16 hours at 37 DEG C, then add 10 μ l0.5% uranous tetrafluoride solution in 10% acetonitrile solution and fully stir.Similar method is used to prepare a unmodified IFN sample.The solution obtained is for obtaining MALDI mass spectrum.
The location of PEG and IFN binding site molecule point is determined by comparing PEG-IFN-α 2 conjugate and the mass spectrum of unmodified IFN-α 2 after tryptic digestion.The tryptic digestion thing of PEG-IFN-α 2 conjugate should lack the corresponding peptide modifying the protein portion occurred.The mass spectrum of the experiment tryptic digestion thing of unmodified IFN-α and PEG-IFN-α 2 conjugate can be seen in table 2.Based on the result shown 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 conjugate, but the tryptic digestion thing of PEG-IFN-α 2 conjugate lacks the peptide that molecular weight is 1313.649, it is present in the tryptic digestion thing of unmodified IFN-α 2b (also see Fig. 5).According to the theoretical gravimetric of IFN-α 2 tryptic digestion thing, the peak of m/z1313.649 is equivalent to N end protein matter peptide (table 2).Its disappearance in the tryptic digestion thing of PEG-IFN-α 2 conjugate confirms that N holds the modification of peptide, and N holds peptide to become heavier by the weight of PEG, beyond spectrum region.PEG occurs over just the free amino group existed in this peptide in conjunction with IFN molecule, N holds halfcystine amino.
Table 2.PEG-IFN-2 conjugate compares with theoretical value with the experiment mass spectrum of the tryptic digestion thing of unmodified IFN-2
Embodiment 10
The antiviral specific activity of PEG-IFN-α 2b conjugate measures
Use to the procedural test that go down to posterity MBDK cell culture and vesicular stomatitis virus (VSV) culture of alpha-interferon sensitivity describe according to European Pharmacopoeia the antiviral specific activity of the sample produced according to embodiment 3 and embodiment 4.Table 3 shows the result of antiviral activity research.International Reference Version is used as reference level.By table 3, we may safely draw the conclusion, although be combined with the daltonian PEG molecule of molecular weight 20000, the antiviral activity of the conjugate produced is equivalent to the 42-45% of unmodified IFN-α 2 activity.Be necessary the PEG-IFN conjugate comprising the daltonian PEG of molecular weight 12000 noting describing in prototype method, there is comparatively low activity (28-30%).
The antiviral specific activity of table 3.PEG-IFN-α 2 conjugate compares with unmodified IFN-α 2
Embodiment 11
The temperature-stable Journal of Sex Research of PEG-IFN-α 2b conjugate
5ml is placed in the water-bath of temperature (50 ± 2) DEG C according to the PEG-IFN conjugate sample that embodiment 3 is produced, and the muddiness tested in protein soln by measuring optical density(OD) under 340nm after different time interval occurs.Carry out the temperature-stable Journal of Sex Research of unmodified IFN-α 2 in a similar manner.The formation of insoluble protein compound causes muddiness to be formed and causes optical density(OD) under 340nm to increase.Result of study shows in figure 6.In viewing duration (28 hours), PEG-IFN-α 2 conjugate keeps stable, and unmodified IFN-α 2 almost precipitates after 28 hours completely.
Therefore, the data obtained in this research allow to reach a conclusion, and compared with unmodified protein matter, the temperature stability of PEG-IFN-α 2 conjugate significantly increases.
Embodiment 12
When studying with the proteolytic stability of PEG-IFN-α 2b conjugate during trypsin treatment
1MTris-HCl damping fluid, the 2 μ l0.5MCaCl of 150 μ lpH8.5 are added in the PEG-IFN conjugate produced according to embodiment 3 to 1ml
2solution and 15 μ l concentration are the trypsin Promega of 20 μ g/ml).Sample is hatched temperature 37 DEG C.From described mixture, sample 100 μ l aliquot after different time interval, add 150 μ l trifluoroacetic acid solution termination reactions.Similar method preparation is used to comprise the sample of unmodified IFN-α 2.Then, use analysed by reverse phase HPLC sample, measure main peak area.As shown in Figure 7, if prove to use trypsin treatment, the proteolytic stability of PEG-IFN-α 2 conjugate is higher than unmodified IFN-α 2b for result of study.
Embodiment 13
The Stability Determination of PEG-IFN-α 2b conjugate between the shelf lives
From the sample produced according to embodiment 3, take out aliquot put into sterile centrifugation testing tube with cover.The protein concn of PEG-IFN-α 2 conjugate is 1mg/ml.This sample is placed in the refrigerator that temperature is (6 ± 2) DEG C.Sample after the timed interval of presetting, and analyze antiviral specific activity and the homogeneity of prepared product by the polyacrylamide gel electrophoresis (Eph) carried out under non reducing conditions under dodecyl sulfate exists described in RP-HPLC and embodiment 1.Based on the result that table 4 shows, we may safely draw the conclusion, and between temperature (6 ± 2) DEG C shelf lives, PEG-IFN-α 2 conjugate at least stablizes 24 months.
The stability of table 4.PEG-IFN-α 2 conjugate at temperature (6 ± 2) DEG C
Embodiment 14
The immunogenicity research of PEG-IFN-α 2b conjugate
The PEG-IFN conjugate produced according to embodiment 3 and embodiment 4 is diluted to concentration be 5mln.IU/ml and continuous 5 weeks weekly intramuscular injection 200 μ l dosage (every mouse 1mlnIU) to body weight 20-22g ICR mouse (5 groups, often organize 5 mouse).Meanwhile, similarly prepare unmodified IFN sample and inject 1mln.IU dosage to mouse.At the end of the 5th week by ball after puncture from mouse blood sampling.Standard method is used to obtain serum.The serum antibody titer obtained after using unmodified IFN and PEG-IFN conjugate immune mouse is by Salmonella technical measurement.For this purpose, unmodified IFN-α 2 solution of 100 μ l concentration 200ng/100 μ l is placed in the hole of microtiter plate.The antiserum(antisera) obtained from different treated animal is placed in the hole of plate in duplicate with serial dilution series.With the anti-mouse antibody of peroxidase conjugated for detecting the immunocomplex obtained.The antibody titer used after unmodified IFN is 1/1024.The antibody titer used after PEG-IFN conjugate is 1/128.Result of study shows in fig. 8.
Therefore, based on the result obtained, we may safely draw the conclusion, and the immunogenicity of required PEG-IFN-α 2 conjugate is lower than unmodified IFN 8 times.
Embodiment 15
The pharmacokinetic of PEG-IFN-α 2b conjugate
1mlnIU amount according to embodiment 3 produce PEG-IFN conjugate sample by peritoneal injection to male mice.Meanwhile, unmodified IFN-α 2 is expelled to another group mouse.After passing through ball after prefixed time interval, paracentesis are from animal blood sampling.Standard method is used to obtain serum.Detect in serum to there is Interferon, rabbit based on antiviral activity.Pharmacokinetic study results display in fig .9.Based on these results, we may safely draw the conclusion, and required PEG-IFN-α 2 conjugate has the effect of significant prolongation.After natural IFN-α 2 injects, its concentration in animal blood reaches maximum value in 1 hour after injection, then very rapidly declines.After the injection of PEG-IFN conjugate, observe the maximum IFN concentration (Fig. 9) in animal blood after 12 hours, and this level keeps 50 hours, then observe IFN-α 2 concentration and slowly decline 350 hours.
The area under curve (AUC) of required PEG-IFN conjugate exceedes the analog value more than 50 times of unmodified IFN.Significantly, if prototype PEG-IFN-α 2b conjugate and the daltonian PEG coupling of molecular weight 12000, AUC value exceeds only IFNAUC3-10 doubly.
The main pharmacokinetic parameter of required PEG-IFN-α 2b conjugate is calculated based on the result (Fig. 9) obtained.Result display is compared with unmodified IFN, and PEG-IFN conjugate slows down greatly from injection site picked-up, capacity distribution, clearance rate, which ensure that the circulation (more than 14 days) of required PEG-IFN conjugate prolongation in blood.
Embodiment 16
The required comparative characteristic of PEG-IFN-α 2b conjugate compared with the PEG-IFN-α 2b conjugate described in prototype method
Required PEG-IFN-α 2b conjugate, compared with the PEG-IFN-α 2b conjugate described in prototype method, has carried out structure, basic physics, chemistry and pharmacokinetic parameter has compared (table 5).The data acknowledgement of display in table 5, compared with the conjugate described in prototype method, the character of required PEG-IFN conjugate is significantly improved.
The PEG-IFN-α 2b conjugate described in the basic physics of the PEG-IFN-α 2b conjugate required by table 5., chemistry and pharmacokinetic parameter and prototype method (US5,951,974) is compared
*-use the structural formula of the PEG-IFN conjugate of prototype method production at reference " WHODrugInformation ", 2001, v.15, N.3-4, provide p.206.
Embodiment 17
Based on the embodiment of the medicinal compositions of required 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) including the required PEG-IFN-α 2b of effective amount is aseptically dispensed in syringe; described syringe is made by the neutral glass of the first hydrolysis grade; with the brazing syringe needle that useful elasticity or rigid protective cap are lived; with the ozzle sealing on the isoprene-isobutylene rubber piston of fluoropolymer lamination; capacity is 0.5,0.8,1.0 and 1.2ml (for 100 μ g/ml dosage); 0.5,0.6,0.75 and 0.9ml (for 200 μ g/ml dosage), 0.5,0.6,0.8,1.0 and 1.2ml (for 300 μ g/ml dosage).
Embodiment 19
Comprise the assembling of the medicament of PEG-IFN-α 2b
The medicament (embodiment 17) including the required PEG-interferon alpha 2 b of effective amount is aseptically dispensed in bottle, described bottle is made by the neutral glass of the first hydrolysis grade, with viton or with the ozzle sealing on the isoprene-isobutylene rubber lid of teflon coatings, fastening with aluminium cap, capacity is 0.5,0.8,1.0 and 1.2ml (for 100 μ g/ml dosage), 0.5,0.6,0.75 and 0.9ml (for 200 μ g/ml dosage), 0.5,0.6,0.8,1.0 and 1.2ml (for 300 μ g/ml dosage).
Embodiment 20
Comprise the test kit comprising the medicament of PEG-IFN-α 2b assembled
Described test kit comprises 1 or 4 syringe in the bubble wrap that polymeric film makes and piston (correspondingly 1 or 4)/or bottle, and prescription information, is placed in card board package.
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SilvaM., PooJ., WagnerF. people (2006) is waited to compare Interferon Alpha-2b and the pharmacokinetics of PEG ylated compound in the patient suffering from chronic hepatitis C of Pegylation, random test (COMPARE) (" Arandomisedtrialtocomparepharmacokinetic of pharmacodynamics and antiviral effect, pharmacodynamic, andantiviraleffectspeginterferonalfa-2bandpeginterferona lfa-2ainpatientswithchronichepatitisC (COMPARE) "), JournalHepatology, 45:204-213.
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Claims (23)
1. the interferon alpha conjugate of stable PEGization, represents, for having the single positional isomers of the polyoxyethylene glycol be connected with N-terminal halfcystine in formula (I):
Wherein:
The integer of n – from 227 to 10000, and the molecular-weight average of polyoxyethylene glycol is about 20kDa;
The integer of m – >=4;
N
αh-IFN – Interferon Alpha-2b, in described conjugate, Interferon, rabbit and PEG molecular ratios are 1:1.
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. medicinal compositions, includes the conjugate as claimed in claim 1 of effective amount and pharmaceutically acceptable vehicle.
5. medicinal compositions according to claim 4, is used for the treatment of virus and tumor disease, and the disease relevant to primary or secondary immunodeficiency.
6. medicinal compositions according to claim 5, wherein said virus disease is hepatitis B or hepatitis C.
7. medicinal compositions according to claim 5, wherein said tumor disease is myelogenous leukemia.
8. medicinal compositions according to claim 5, wherein said tumor disease is melanoma.
9. include the conjugate of formula according to claim 1 (I) and the medicinal preparations of pharmaceutically acceptable vehicle of effective amount, there is antiviral, antiproliferative and immunoregulatory activity.
10. medicinal preparations according to claim 9, comprises damping fluid, isotonic agent, stablizer and water.
11. medicinal preparationss according to claim 10, comprise sodium acetate trihydrate, acetic acid, sodium-chlor, polysorbate80, EDTA, water for injection.
Purposes in the medicinal product that the conjugate of 12. formulas according to claim 1 (I) has antiviral, antiproliferative and immunoregulatory activity in preparation.
13. purposes according to claim 12, for the preparation of the medicinal product of antiviral, the antiproliferative and immunoregulatory activity with resistance of hepatitis B or hepatitis C.
14. conjugates as claimed in claim 1 are preparing the purposes prevented and/or treated in the medicine of virus disease.
15. purposes according to claim 14, wherein said virus disease is hepatitis B or hepatitis C.
16. purposes according to claim 14, the wherein said ribavirin prevented and/or treated by introducing effective therapeutic dose in addition carries out.
17. conjugates according to claim 1 are preparing the purposes prevented and/or treated in the medicine of the disease relevant to primary or secondary immunodeficiency state.
18. conjugates according to claim 1 are preparing the purposes prevented and/or treated in the medicine of tumor disease.
19. purposes according to claim 18, wherein said tumor disease is myelogenous leukemia.
20. purposes according to claim 18, wherein said tumor disease is melanoma.
21. containers, before use, in gnotobasis, sealing also suitably stores, and comprises medicinal compositions according to claim 4.
22. containers according to claim 21, wherein said container is prefilled syringe, bottle or automatic injector.
23. a set of products, comprise container according to claim 22 and prescription information.
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| 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) |
| EA021610B1 (en) * | 2013-03-28 | 2015-07-30 | Илья Александрович МАРКОВ | Liquid antiviral formulation |
| 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 |
| 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 |
| AU2021258734A1 (en) * | 2020-04-20 | 2023-01-05 | Altum Pharmaceuticals Inc. | Recombinant interferon |
| 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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| TW426523B (en) * | 1995-04-06 | 2001-03-21 | Hoffmann La Roche | Interferon solution |
| TW517067B (en) * | 1996-05-31 | 2003-01-11 | Hoffmann La Roche | Interferon conjugates |
| EP1546235B1 (en) * | 2002-09-09 | 2021-10-20 | Nektar Therapeutics | Water-soluble polymer alkanals |
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| RU2010129824A (en) | 2012-01-27 |
| EA020257B1 (en) | 2014-09-30 |
| CR20130021A (en) | 2013-02-20 |
| CO6680611A2 (en) | 2013-05-31 |
| HK1170504A1 (en) | 2013-03-01 |
| MY168784A (en) | 2018-12-04 |
| RU2447083C1 (en) | 2012-04-10 |
| KR101586372B1 (en) | 2016-01-18 |
| CN102617736A (en) | 2012-08-01 |
| AR087227A1 (en) | 2014-03-12 |
| WO2012011836A1 (en) | 2012-01-26 |
| KR20130056885A (en) | 2013-05-30 |
| NI201300008A (en) | 2014-05-26 |
| CU20130013A7 (en) | 2013-04-19 |
| DOP2013000002A (en) | 2013-09-15 |
| MX2011007458A (en) | 2012-01-19 |
| EA201100809A1 (en) | 2012-01-30 |
| ECSP13012398A (en) | 2013-05-31 |
| UY33525A (en) | 2012-02-29 |
| SG187117A1 (en) | 2013-02-28 |
| BRPI1101565A2 (en) | 2012-12-04 |
| CU24193B1 (en) | 2016-09-30 |
| UA99766C2 (en) | 2012-09-25 |
| PE20131034A1 (en) | 2013-09-27 |
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