CN103228792A - PEG-interferon lambda 1 conjugates - Google Patents
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Abstract
The present application discloses novel PEG-interferon lambda 1 conjugates (PEG-1FN[lambda]1), preparation method thereof, pharmaceutical compositions containing these conjugates and processes for making the same. These conjugates have increased blood half-lives and persistence time compared to 1FN[lambda]1 and are effective in the treatment of hepatitis B and hepatitis C.
Description
Related application:
The application requires the rights and interests of Vietnam patent application serial number VN1-2011-02222 of submission on August 25th, 2011, and it is as a reference incorporated herein.
Technical field
In one embodiment, the application discloses the PEGization derivative (PEG-interferon lambda 1 binding substances or PEG-IFN λ 1) of recombinant human interferon alpha 2 λ 1, its preparation method, the method that comprises the pharmaceutical composition of these binding substancess and prepare it.
Background technology
Hepatitis C virus (HCV) is the major cause of major health and whole world chronic hepatopathy.According to estimates, the whole world has 180 million people's chronic infection HCV at least.In Vietnam, the individual ratio of HCV infection accounts for 4%-9% in population, approximately the acute infection HCV individuality of 55%-85% will be converted into chronic infection, the chronic carrier of these of 5%-25% has the risk that develops into liver cirrhosis and suffer from the liver cirrhosis crowd behind 25-30,30% had the risk of liver decompensation in 10 years, and annual 1%-3% will develop into liver cancer.According to epidemiological study, HCV is the cause of disease of 40% end-age cirrhosis (final stage cirrhosis) and 60% liver cancer.
At present, alpha-interferon (AI) is the methods of treatment that the treatment chronic HCV infection is selected.AI can provide lasting response to HCV in about 70% case, yet these Interferon, rabbit can produce a lot of side effects, even also is like this under the situation of PEG-interferon alpha.These side effects are the possibility limit treatment sometimes, makes patient's treatment imperfect.Side effect comprises influenza sample sign and such as blood effects such as thalassemia and anaemias.
Interferon, rabbit is used for the treatment of many virus diseases at present, as hepatitis B, hepatitis C, hepatitis D, pointed condyloma, lepromatous leprosy, chronic leukemia and AIDS.AI also effectively reduces malignant tumour and treatment Kaposi sarcoma, melanoma and renal cell carcinoma.In addition, AI is applicable to the prevention and treatment of diseases of domestic animals such as ox.For example, the activity of employed vaccine in the prevention of AI enhancing hand foot mouth disease, pig reproduction and respiratory system syndromes and the treatment.
AI is produced by human cell system of cultivating in tissue culture medium (TCM) or contributor's white corpuscle.Yet these methods are time-consuming, labour-intensive, expensive, and are not suitable for scale operation.In addition, existence causes septicemic cemia risk by the infectious agent from clone.
Along with the development of recombinant DNA technology, we can introduce the AI gene and can produce in the microorganism of a large amount of Interferon, rabbit now.Yet these methods mainly in the step of expression and large-scale protein production, also exist certain advantage and difficulty.
IL-29 is the member of spiral cell factor family, is type iii interferon.It is also referred to as interferon lambda 1(IFN λ 1), highly be similar to other type iii interferon of IL-28() aminoacid sequence.IL-28 and IL-29(IFN λ 1) be described to the member with the total novel cytokine family of I type Interferon, rabbit (IFN) recently, identical Jak/Stat signal path impels the expression of collaborating genes group.Therefore, their called after IFN λ.IFN λ shows the several common traits with I type IFN: antiviral activity, antiproliferative activity and anti-tumor in vivo activity.Yet, the more important thing is that IFN λ is bonded to unique membrane receptor of IFNLR1 and IL10R2 formation.
The main drawback of most of biotechnological formulation therepic use is that they give through parenteral, for example intravenous injection (i.v.), subcutaneous (s.c.), intramuscular injection (i.m.) etc.This means to sending of patient and be undivided with pain and discomfort.In addition, because they are very short the common transformation period, biological products need frequently to give to the patient, could keep the therapeutic serum or the blood plasma level of medicine.The injection that can not self-give need often be gone to the clinic and be looked for well-trained medical worker, causes this treatment inconvenience and expensive.Intederon Alpha-2a Interferon, rabbit (Roferon, Roche) and Interferon Alpha-2b (Intron A, Schering A G), two kinds of recombinant forms of the human interferon-alpha that uses in treatment chronic type b and the hepatitis C, have the serum half-life (McHutchison, et al., the Engl.J.Med.1998 that are lower than 12h, 339,1485-1492; Glue, et al., Clin.Pharmacol.Ther.2000,68,556-567) therefore need to give for 3 times weekly.Also need the patient of duplicate injection interferon beta-1b (doubly safe dragon) with treatment multiple sclerosis (MS).
A kind of to overcome above-mentioned frequent high dosage injection be to make itself and polymkeric substance with the extremely successful and well accepted method that keeps the drug disposition threshold level, connects with the transformation period in the body that increases therapeutic protein as polyoxyethylene glycol (PEG or Peg).PEG molecule with its long-chain not only produces the protection barrier around PEG chemical drug thing molecule in aqueous solution; thereby reduce the immunogenicity of protein drug; simultaneously also protect it to avoid the effect of proteolytic enzyme; but they further help to improve the circulating half-life of medicine by improving its hydrokinetics volume, thereby reduce its loss by renal glomerulus network filtering mechanism.With after protein molecular separates, peg moiety is removed and without any structural variation, and its removing is proportional to its molecular weight at it.
Usually peg moiety is connected to protein, makes activatory PEG reagent and proteinic amino acid side chain subsequently by at first activating peg moiety, as lysine residue and/or the amino reaction of this albumen n end.The PEG of frequent use is the PEG of simple function, because this part is anti-crosslinked and reunite.This example by Davis etc. at U.S. Patent number 4,179, open in 337.
Summary of the invention
PEG-interferon lambda 1(PEG-IFN λ 1) be the recombinate PEGization derivative (wherein polyoxyethylene glycol is connected to IFN λ 1, also is referred to as " binding substances ") of IFN λ 1 of people, this is used for the treatment of the chronic hepatitis C in the adult patients.PEG-IFN λ 1 has avoided the effect of extracellular enzyme afterwards and has prevented the filtration of kidney in injecting patient's body; Therefore its transformation period prolongs in circulation.That is to say that than the IFN λ 1 that corresponding non-PEG connects, binding substances has the circulating half-life and the blood plasma residence time of the stability of remarkable improvement, solvability and raising preferably.
Interferon lambda 1(IFN λ 1, Zcyto21 or IL-28A) be known in this area, for example, and according to U.S. Patent number 7,038,032,6,927,040,7,135,170,7,157,559 and 7,351,689; And PCT publication number WO05/097165, WO07/012,033, WO07/013,944 and WO07/041,713; All these are as a reference incorporated herein in full with it.
In one embodiment, the application discloses a kind of linear PEG-IFN λ 1 binding substances.In one aspect, the application's binding substances has the PEG chain structure of straight chain.Than not changing IFN λ 1(promptly, the IFN λ 1 that PEG of no use or mPEG connect), these binding substancess have improved circulating half-life and persistence in the blood plasma.Water-soluble PEG comprises polyoxyethylene glycol (PEG), mono methoxy-PEG(mPEG), list-C
1-10Alkoxyl group-PEG and list-C
1-3Alkoxyl group-PEG.These operable PEG can have about 600-60,000 molecular weight and comprise for example, having those of about 10kDa, 20kDa, 30kDa, 40kDa, 50kDa and 60kDa.In one aspect, the PEG that uses in binding substances of the present invention is the mPEG with molecular weight 40kDa.
In one embodiment, provide a kind of physiologically active Peg-IFN λ 1 binding substances or its pharmaceutical salts that comprises with following formula I:
Wherein: R is H or C
1-3, alkyl; M is 1,2,3 or 4; N is the positive integer that is selected from the 400-550 scope; L is C
1-10Alkyl or assorted alkyl connector; X is-O-,-NH-or-S-; And IFN λ 1 is an interferon lambda 1.In one aspect, interferon lambda 1 is people's recombinant interferon.In another aspect, IFN λ 1 can be natural or recombinant protein.In another aspect, IFN λ 1 is a human body protein, comes from the source of synthesizing or using the cell culture of n cell or reconstitution cell such as tissue, albumen.In aspect another, IFN λ 1 is a human recombination protein.In another aspect, formula I's is SEQ ID2 in conjunction with interferon lambda 1.In one embodiment, the PEG chain via, for example, the N of amido linkage on the primary amino of Methionin or IFN λ 1 end is connected to IFN λ 1.
Assorted alkyl is defined as C
1-10Alkyl, wherein at least one C
1-10Alkyl carbon quilt-O-,-C (=O)-,-NH-or its combination replace.This combination comprises, for example ,-OC (=O)-,-C (=O) O-,-NHC (=O)-,-C (=O) NH-etc.In one aspect, n is about 500-550.In another aspect, n is about 420,520 or 455.In another aspect, the molecular weight of Peg group is about 35kDa-45kDa or about 40kDa.Above on the other hand in, m is 1 or 2.C
1-10Alkyl or assorted alkyl can be the alkyl or the assorted alkyl of straight or branched.In one aspect, C
1-10Alkyl is-C (O)-group.In one embodiment, m be 1 and L-X-be selected from by the following various group of forming:
In aspect of above binding substances, m be 2 and L-X-be following formula:
Above binding substances on the other hand in, two PEG groups, PEG, alkyl-PEG or m-PEG all are connected in two carbonylamino groups with following formula (that is ,-C (O) NH-).In another aspect, X be-NH-or-O-and m are 2.In another aspect, connector is connected in two PEG groups.In another aspect, X is-NH-, and the group that is connected to connector is the residue of Methionin on the IFN λ 1.In one aspect, be connected to connector-NH-group (that is amino) is the residue of Histidine.In another aspect, X is-O-, and the group that is connected to connector can come from the residue of Serine on the IFN λ 1.One at following formula changes in the body, and R is-CH
3In another aspect, connector is connected in the residue of Methionin, Serine, Histidine or its mixture on the IFN λ 1.In another aspect, connector is connected in the positional isomers of the residue of Methionin, Serine, Histidine or its mixture on the IFN λ 1.Binding substances on the other hand in, R be H or-CH
3, m is 1, L is-C (O)-and X be-NH-.In another aspect, n is 500-550.
In another aspect, binding substances (Nanogen PEG-IFN λ 1) comprises with Formula Il:
Wherein INF λ 1 is an interferon lambda 1; And n is 500-550.In one aspect, n is that the quantity of ethylene glycol unit in the PEG structure and its are to be selected from any positive integer that makes the molecular weight of peg moiety for the quantity of about 40kDa, and INF λ 1 is an interferon lambda 1.In above another embodiment, binding substances has serum half-life and the retention time that prolongs or spin out than IFN λ 1.Binding substances on the other hand in, PEG is connected in the methionine(Met) of the N end of IFN λ 1.In aspect another, binding substances is effectively treated hepatitis B and hepatitis C.
In another embodiment, a kind of method that is used to prepare as above disclosed people's recombination nodule compound is provided, and this method may further comprise the steps: by following association reaction covalently bound (α-methoxyl group-ω-(4-nitrophenoxy carbonyl)) polyoxyethylene (PEG-pNC) 40kDa and IFN λ 1:
Wherein, n is selected so that the molecular weight of peg moiety is the positive integer of about 40kDa; And separation and combination thing.In one aspect, n is about 500-550.
In another embodiment, provide a kind of pharmaceutical composition that comprises as above disclosed binding substances and pharmaceutical carrier and vehicle.The conventional medicine preparation also can use the preparation of compositions of the binding substances that comprises the application.These preparations can comprise the composition together with the binding substances of pharmaceutical carrier known in the art of comprising of treatment significant quantity.For example, can use adjuvant, thinner, sanitas and/or if desired, solubilizing agent.The pharmaceutical composition that comprises binding substances (for example can comprise various buffer reagents with certain pH and ionic strength scope, Tris-HCl, acetate, phosphoric acid salt) thinner, carrier (for example, human serum albumin), solubilizing agent (for example, the polyoxyethylene sorbitanic or
Polysorbate) and sanitas (for example, merthiolate, phenylcarbinol), for example, as U.S. Patent number 4,496, those disclosed in 537.In one aspect, pharmaceutical composition is formulated as the Injectable sterile lyophilized powder.In another aspect, composition comprises the combination of pharmaceutical carrier, comprises salt solution, buffer saline and 5% glucose in water.In another aspect, pharmaceutical composition is mixed with the injection solution in bottle or the pre-filled syringe.In aspect above one, pharmaceutical composition is used for the treatment of hepatitis B and hepatitis C.The method of medicinal preparations and this preparation of preparation is being known in the art, and for example, is disclosed in Remington, The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, Pa.19
ThAmong the ed.1995.
In another embodiment, provide a kind of preparation to comprise with Peg-IFN λ 1 binding substances of following formula I or the method for its pharmaceutical salts:
Wherein: R is H or C
1-3Alkyl; M is 1,2,3 or 4; N is the positive integer that is selected from the 400-550 scope; L is C
1-10Alkyl or assorted alkyl connector; X is-O-,-NH-or-S-; And IFN λ 1 is an interferon lambda 1; This method comprises: make IFN λ 1 and pre-activation Peg be enough to impel with the covalently bound condition of the amino-acid residue of IFN λ 1 under contact.In one embodiment, provide PEG-IFN λ 1 binding substances for preparing by method described herein.In one aspect, disclose a kind of method for preparing above-mentioned binding substances, comprised that the activated PEG that makes IFN λ 1 and capacity or mPEG contact being enough to impel under PEG or the covalently bound condition on IFN λ 1 of mPEG.In another aspect, activatory mPEG is mPEG-pNC.In another aspect, activatory mPEG is connected on the methionine(Met) of N end of IFN λ 1.In another aspect, mPEG has the molecular weight of about 40kDa.In another aspect, activatory oxygen carbonyl reagent is single activator or dual activator.
In another embodiment, provide a kind of method that is used for suppressing patient's cancer cell multiplication, comprised cancer cells is contacted with above-mentioned binding substances, wherein this binding substances has serum half-life and the retention time that prolongs or spin out than IFN λ 1.In one aspect, the serum half-life that the application's binding substances has prolongs above 2 times, 3 times, 5 times, 8 times or above 10 times than the serum half-life of corresponding unconjugated IFN λ 1.
In another embodiment, provide a kind of method that is used for the treatment of proliferative disease in the Mammals, comprised the above-mentioned binding substances for the treatment of significant quantity to this Mammals.In one embodiment, this binding substances can be used for the treatment of Interferon, rabbit susceptibility illness or with positive or advantageously in response to the illness based on interferon therapy.In one aspect, adopt the therapy of this binding substances side effect to be reduced or elimination than the routine treatment that adopts Interferon, rabbit.
In one aspect, can comprise with the exemplary illness of the application's conjugates for therapy, but be not limited to, cell generation disorders disease, especially cancer (for example, hairy cell leukemia, Kaposi's sarcoma, chronic lymphocytic leukemia, multiple myeloma, basaloma and malignant melanoma, ovarian cancer and cutaneous T cell lymphoma) and virus infection.In another aspect, binding substances can be used for the treatment of and will have benefited from suppressing the illness of interferon-sensitive virus replication.Can comprise hepatitis A with the virus infection of the application's conjugates for therapy, hepatitis B, hepatitis C, other non-first/non-hepatitis B, simplexvirus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), hsv, human herpes virus type 6 (HHV-6)), papilloma, poxvirus, picornavirus, adenovirus, rhinovirus, human T lymphotropic virus's 1 type and 2 types (HTLV-1/-2), the human rotavirus, rabies, retrovirus comprises human immunodeficiency virus (HIV), encephalitis, respiratory virus infection.
In another embodiment, provide a kind of infective virus for the treatment of to infect or have the patient's of virus infection risk method, comprised binding substances or its pharmaceutical salts with following formula I for the treatment of significant quantity to its patient of needs; Or comprise pharmaceutical preparation with the binding substances of following formula I:
Wherein: R is H or C
1-3Alkyl; M is 1,2,3 or 4; N is the positive integer that is selected from the 500-550 scope; L is C
1-10Alkyl or assorted alkyl connector; X is-O-,-NH-or-S-; And IFN λ 1 is an interferon lambda 1.In one aspect, formula I's is SEQ ID2 in conjunction with interferon lambda 1.In one aspect, Mammals is the people.This method on the other hand in, virus infection is to be caused or this virus infection causes end-age cirrhosis by hepatitis C virus.One concrete aspect in, the patient is HCV resistance or difficult therapeutic patient.Above method on the other hand in, PEG-IFN λ 1 gives with the dosage of about 0.5 μ g/kg-10.0 μ g/kg weekly.In aspect of this method, PEG-IFN λ 1 gives with the dosage of about 2.5 μ g/kg weekly.In another aspect, PEG-IFN λ 1 gives about 8 to about 52 weeks.In another aspect, PEG-IFN λ 1 gives about 12 weeks, about 16 weeks, about 20 week or about 24 weeks.In another aspect, give PEG-IFN λ 1 and in detecting the patients serum, do not contain HCV RNA.In another aspect, give binding substances and provide remarkable improvement, because this method does not cause neutrophil leucocyte counting, platelet count or hemoglobin level significantly to reduce standard P EG-INF-α therapy.In aspect aforesaid method another, this method further comprises the nucleoside analog that is selected from virazole and the Wella pyrimidine.This method on the other hand in, virazole with every day 5mg/kg-25mg/kg or every day 15mg/kg-25mg/kg oral dose give.In one aspect, virazole with every day 1 time or 2 about 10mg/kg-30mg/kg or every day 1 time or 2 times about 15mg/kg dosage give.In aspect above one, binding substances gives through parenteral.
In one embodiment, HBV can use weekly the dosage of PEG-IFN λ 1 binding substances of about 200 μ g, and associating every day, the tynofovir (fumaric acid tynofovir two pyrrole furan esters) of about 300mg was treated.According to this methods of treatment, in about 30 days, detect Most patients after about 4 injections and removed HBV.Under these illnesss, find that HBV is suppressed, and discharge the HBsAg(viral surface antigen), this triggers immuning system generating antibody and resists this antigen, causes in the concrete treatment and produces optimum.This treatment can continue about 12-24 week as disclosed herein, and this depends on patient's the initial virus amount of carrying.
Definition:
" alkyl " is meant to have carbon atom chain, uses oxygen (for example, C alternatively
1Alkyl can be-C (O)-), nitrogen-atoms (for example, C
1Alkyl can be-C (NH)-) or sulphur atom (for example, C
2Alkyl can be-CH
2C (S)-)) straight or branched that replaces, saturated or undersaturated aliphatic free radical.Usually use C
XAlkyl and C
X-YAlkyl such as C
1-10Alkyl or C
1-6Alkyl, wherein X and Y are meant the number of the carbon atom in the chain.For example, C
1-6Alkyl comprises the alkyl (for example, methyl, ethyl, propyl group, sec.-propyl, butyl, isobutyl-, vinyl, pseudoallyl, 1-butylene base, ethynyl, 1-proyl etc.) with 1-6 carbon atom.
" assorted alkyl " or " assorted alkylidene group " is the alkyl that can have oxygen, nitrogen or sulphur between the carbon atom.Example Bao Kuo – C (O) NH-of this assorted alkyl group ,-OC (O)-,-CH
2CH
2C (O)-NH-,-CH
2-O-CH
2-CH
2-,-CH
2-NH-CH
2-CH
2-,-CH
2-S-CH
2-CH
2-and-CH
2O-CH
2-CH
3Deng.
Be meant the employed polyoxyethylene glycol in this area as the term " PEG " in " PEG-IFN λ 1 ",, generally comprise alkyl-PEG such as mPEG(methoxyl group-polyoxyethylene glycol unless spell out in addition) and PEG.
" treatment effective dose " is to be enough to produce clinical noticeable change in the illness of being treated, and as the clinical noticeable change of virus amount of carrying or immunologic function, sickness rate significantly reduces or histological score significantly improves or the amount of PEG-IFN λ 1 binding substances of its combination.
Be meant therapy for treating and defence or preventive measure as " methods of treatment " used herein or " treatment ".Need the patient of treatment to comprise patient who infects hepatitis C virus and those patients that need the prevention of hepatitis c disease.
In aspect of aforesaid method, binding substances gives by injection or perfusion.In another aspect, binding substances gives via vein, muscle, subcutaneous, intracutaneous or abdominal cavity.In another aspect, give binding substances with the dosage that is selected from and the following to the patient: less than 0.5 μ g/kg, 0.5-1.0 μ g/kg, 1.0-1.5 μ g/kg, 1.5-2.0 μ g/kg, 2.0-2.5 μ g/kg, 2.5-3.0 μ g/kg, 3.0-3.5 μ g/kg, 3.5-4.0 μ g/kg, 4.0-4.5 μ g/kg, 4.5-5.0 μ g/kg, 5.0-5.5 μ g/kg, 5.5-6.0 μ g/kg, 6.0-6.5 μ g/kg, 6.5-7.0 μ g/kg, 7.0-7.5 μ g/kg, 7.5-8.0 μ g/kg, 8.0-8.5 μ g/kg, 8.5-9.0 μ g/kg, 9.0-9.5 μ g/kg, 9.5-10.0 μ g/kg or greater than 10.0 μ g/kg.In another aspect, give binding substances with the fixed dosage that is selected from and the following: about 60-80 μ g, 80-100 μ g, 100-120 μ g, 120-140 μ g, 140-160 μ g, 160-180 μ g, 180-200 μ g, 200-220 μ g, 220-240 μ g, 240-260 μ g, 260-280 μ g or about 280-300 μ g.In one embodiment, binding substances is with subcutaneous giving of continuous 12 weeks of 200 μ g.
In another embodiment, provide a kind of pharmaceutical composition that comprises above-mentioned binding substances and pharmaceutical carrier and vehicle.In another aspect, pharmaceutical composition is used for the treatment of hepatitis B and hepatitis C.In another embodiment, provide a kind of method that this binding substances and pharmaceutical carrier and mixed with excipients preparation is comprised the pharmaceutical composition of above-mentioned binding substances.
The application's binding substances has 1 similar effect or the activity with IFN λ.For example, this binding substances can be used as antiproliferative, antiviral agent or antineoplastic agent.Particularly, the application's binding substances can effectively be treated hepatitis B and hepatitis C, and they have longer blood retention time than IFN λ 1.In one embodiment, the pharmaceutical composition that comprises the application's binding substances is prepared as the injection solution in injection aseptic freeze-dried powder or bottle or the pre-filled syringe.These pharmaceutical compositions can be by preparing binding substances with relevant pharmaceutical carrier and mixed with excipients.
In another embodiment, the application provides and has prepared the recombinate method of PEG-IFN λ 1 binding substances of people.At first, in intestinal bacteria, produce the people IFN λ 1 that recombinates by recombinant DNA technology, subsequently with the reaction of Pegization reagent (as α-methoxyl group-ω-(4-nitrophenoxy carbonyl)) polyoxyethylene (PEG-pNC) to generate PEG-IFN λ 1.In one aspect, PEG-IFN λ 1 is the straight chain PEG40kDa that is connected to IFN λ 1.This product avoids in being expelled to patient's body the time extracellular enzyme effect and kidney to filter, and has therefore prolonged its serum half-life.
Pegization reaction between (α-methoxyl group-ω-(4-nitrophenoxy carbonyl)) polyoxyethylene (PEG-pNC) 40kDa and the IFN λ 1 generates this binding substances, and is as follows.In one aspect ,-NH
2Group is the methionine residues at N end place on the interferon lambda 1 molecule position.In another aspect ,-NH
2Group is the amine of lysine residue on the interferon lambda 1 molecule position.
In one embodiment, the application's binding substances can prepare with preactivated PEG is covalently bound by interferon lambda 1.In one embodiment, PEG can activate to form coupling agent or activatory PEG reagent, i.e. (α-methoxyl group-ω-(4-nitrophenoxy carbonyl)) polyoxyethylene (PEG-pNC) by replace the PEG oh group with linking group.In one embodiment, this PEG-IFN λ 1 binding substances can prepare by preparation IFN λ 1 and Pegization IFN λ 1.The method that is used for purifying and analyzes this binding substances is also disclosed.
Description of drawings
Fig. 1 for example understand at nucleotide sequence (SEQ ID1) synthetic and be incorporated into expression vector pNanogen1-IL29 after be used to produce recombinate this sequence of IFN λ 1 of people.
Fig. 2 is by Nanogen Pharmaceutical Biotechnology Co., the recombinate typical aminoacid sequence (SEQ ID2) of IFN λ 1 of the people that Ltd produces.
The plasmid pNanogen1-IL29 of the gene for example clear coding people IFN λ 1(interleukin-2 9 that comprises of Fig. 3).
Fig. 4 has described the analytical results of plasmid pNanogen1-IL29a.
Fig. 5 for example understand to detect and contains the result that the pNanogen1-IL29 intestinal bacteria are used to produce the electrophoresis method of IFN λ 1 ability.
Fig. 6 is the folding more typical spectrogram and the SDS-PAGE electrophoresis of salt face afterwards of albumen.Fig. 6,7,8 and 9 spectrogram all are coomassie brilliant blue stainings.
Fig. 7 for example understands spectrogram and the SDS-PAGE electrophoresis after positively charged ion 1 phase.
Fig. 8 for example understands spectrogram and the SDS-PAGE electrophoresis after positively charged ion 2 phases.
Fig. 9 for example understands spectrogram and the SDS-PAGE electrophoresis after the gel-filtration mutually.
Figure 10 for example understands the spectrogram of purification process and the SDS-PAGE electrophoresis of PEG-interferon lambda 1.
Figure 11 for example understands the recognition result of FN λ 1 and PEG-IFN λ 1.
Figure 12 for example understands by Nanogen Pharmaceutical Co., the Maldi-Tof mass spectrum of the PEG-IFN λ 1 that Ltd. produces.
Embodiment
In one embodiment; the application discloses the recombinant bacteria bacterial strain that is used to prepare the gene that comprises coding IFN λ 1, extensive or suitability for industrialized production IFN λ 1, the IFN λ 1 of Pegization reaction, the PEG-IFN λ 1 that purifying produces and the method for analyzing PEG-IFN λ 1.
In one embodiment, the application discloses a kind of based on can be available from the synthetic of the gene of the coding IFN λ 1 of the open sequence of biotechnology infonation center (encoding gene satisfies relevant colibacillary Industrial processes through modifying), produce transgene carrier, be incorporated in the bacterium these carriers and the bacterial strain of selecting to produce best IFN λ 1.
In one embodiment, IFN λ 1 Industrial processes may further comprise the steps: make the initial feed fermentation, collect the solution and purifying IFN λ 1 albumen of crude protein.In typical process, fermenting process can carry out in containing the 10L fermentor tank of nutritional medium and the production of IFN λ 1 causes by lactose.The biomass that obtained are separated and purifying.Collect IFN λ 1 and refining, comprising by a plurality of steps: folding again this albumen, for example separate this albumen by ion exchange chromatography (positively charged ion 1 and positively charged ion 2), and make with extra care albumen on gel.
In one embodiment, the Pegization method comprises: have straight chain (α-methoxyl group-ω-(4-nitrophenoxy the carbonyl)) polyoxyethylene (PEG-pNC) of molecular weight 40kDa and the reaction between the IFN λ 1.Resulting binding substances can pass through chromatography, carries out purifying as using the HPLC system, and quality and purity are tested.
To understand the application more fully with reference to following examples, think that these embodiment only are illustrative, rather than the scope of the present invention of restriction as requirement.
Embodiment:
Embodiment 1: preparation comprises coding people recombinant interferon λ 1(IFN λ 1) the method for coli strain of gene
The gene of coding IFN λ 1 is based on carrying out synthetic available from the protein sequence data of NCBI or other databases.The novel method that provides has herein reduced the required time of isolated genes, but still provides and ordinary method result equally accurately.Be used to produce Nanogen Pharmaceutical Biotechnology Co., the nucleotide sequence of the IFN λ 1 among the Ltd. as shown in Figure 1, this proteic aminoacid sequence is as shown in Figure 2.
The pUC promotor that specialized designs expression vector pNanogen IL29(comprises T7 transcripting starting subarea, IFN λ 1 transgenosis, the reverse promotor of T7, T7 transcription terminator, f1 promotor (origin), kalamycin resistance gene and duplicates) so that it can this albumen of high expression level, impels a large amount of IFN λ 1 industrial fermentations.Fig. 3, Fig. 4 have shown the process that is used to produce carrier pNanogen1-IL29.
Then, expression vector pNanogen-IL29 is transferred in the coli strain that is fit to promotor T7 expression.This bacterial strain has genotype F
-OmpT hsdS
B(rB
-MB
-) gal dcm (DE3).The bacterial strain that contains IFN λ 1 gene is called intestinal bacteria pNanogen1-IL29.It has by fermentation (referring to Fig. 5) and introduces the ability that produces the IFN λ 1/L that is higher than 100mg in the original strain storehouse.
Embodiment 2: Escherichia coli fermentation is produced the recombinate method of IFN λ 1 of people
Adopt nutritional medium to carry out fermenting process in the fermentor tank of 140L, temperature is 37 ± 0.5 ℃, and air pressure is 0.5m
3/ h, pH are 7.0 ± 0.2, and stir speed (S.S.) is 300rpm, and by adding H
3PO
4Or NH
4OH remains on pH between the 6.8-7.2.(it is the most effective time of cell enlargement that intestinal bacteria are in the logarithmic phase growth) is cooled to 30 ± 0.5 ℃ with temperature after 8 hours, and stirring velocity is reduced to 200rpm to start the process that IFN λ 1 produces.Fermenting process stopped after 4 hours, and cooled product is centrifugal to obtain biomass under 6000rpm.
By in homogenizing apparatus, homogenizing in cytolysate (the every 1g humidogene of 12mL solution material), to destroy biomass.With temperature remain on 4 ℃ following 1 hour, then by ultrasonic device with cytoclasis 2 times.The suspension that obtains under 6000rpm centrifugal 30min to obtain particle, use inclusion body (inclusion body) lavation buffer solution (the every 1g humidogene of 12mL damping fluid material) to wash this particle then, the suspension of gained keeps down 1h at 4 ℃, and then with 13, twice of the centrifugal 30min of 000rpm is to obtain particle.Particle is dissolved in the urea soln of 2M and the ice-cold 1h of hatching, then with suspension with 13,000rpm is centrifugal, and 30min obtains particle.This particle is dissolved in washings, and with 13, the centrifugal 30min of 000rpm is dissolved in this particle in the guanidine solution of 6M then to obtain particle, makes this suspension keep ice-cold 12-16h, and with 13, the centrifugal 30min of 000rpm.Recovery contains proteinic solution, and in next step purifying.
The component that is used to separate the substratum medium of IFN λ 1 and solution is as shown in table 1.
Table 1
Embodiment 3: the recombinate purge process of IFN λ 1 of people
By with solubilization of inclusion bodies in folding solution again (the Tris damping fluid of 25mM, the EDTA of 1mM, the guanidine of 1.2M, pH8.2) in folding again IFN λ 1 so that the ultimate density of inclusion body is 500 μ g/mL.Then this mixture is remained on 2-8 ℃ of following 16-24h.Make it on Sephadex G25 post, stand purification step after the mixture desalination with gained.Salt exchange buffering liquid is phosphate buffered saline buffer (10mM, pH8.0), in step " positively charged ion 1 ", sample on the desalination mixture in SephadexG25 post (this post is loaded CM-Sepharose FF gel and balance in the 10mM of pH8.0 phosphate buffered saline buffer in advance), is used the NaCl(pH8.0 of sodium phosphate+0.5M of 10mM) eluted product.Chromatography (step " positively charged ion 2 ") is also as above carried out in the protein solution desalination of gained.Make then protein solution by gel filtration column obtaining the product people IFN λ 1 that recombinates, purity greater than 95%(referring to spectrogram among Fig. 5, Fig. 6, Fig. 7, Fig. 8 and Fig. 9 and electrophoresis result).
Embodiment 4: the Pegization method that is used to prepare binding substances
The about 20.1kDa of IFN λ 1(MW will recombinate the 5mg/mL people in Sodium Tetraborate-sodium phosphate (pH8.0) of 50mM) solution serve as that about 3:1 adds in (α-methoxyl group-ω-(4-nitrophenoxy the carbonyl)) polyoxyethylene (PEG-pNC) (the about 40kDa of MW) with mol ratio PEG-pNC:IFN λ 1.Reaction mixture is remained on 2-4 ℃ of following 20h.Reaction is regulated pH to 4.0 by the acetic acid solution that uses 30%w/w and is stopped.The gained mixture is 5 times of dilute with waters subsequently.
In general example process, activatory PEG or m-PEG reagent to the reaction conditions of the association reaction of IFN λ 1 comprises that further employing waits mole an activated PEG or a m-PEG to less relatively molar excess to carry out this reaction with respect to IFN λ 1 approximately.In a modification, in conjunction with can be with about 1-10 times molar excess; Or about 1.5-7 times molar excess; Or about 1.75-5 times molar excess carries out.In a flexible program, association reaction can carry out under about room temperature or about 20-25 ℃.Association reaction can so that about 1-10h, 1-5h, 1-3h or about 1-2h after just by quencher (cold shock) termination reaction.In some cases, reaction conditions provides the mixture of PEG-IFN λ 1 positional isomers.In one aspect, every kind of isomer comprises via the single PEG-connector unit that is connected to IFN λ 1 as amino-acid residue disclosed herein.Under some a plurality of unitary situation of PEG-connector that are connected to IFN λ 1, if desired, can use the composition that comprises these binding substancess of gained maybe can adopt the standard purification method, comprise ultrafiltration, ion exchange chromatography (chromatogram), affinity chromatography and size exclusion chromatography, separate by chromatography (chromatogram).In one aspect, be used to separate purification process with the purifying binding substances and be cation-exchange chromatography as described in this article.
Under certain conditions, the connecting portion on the IFN λ 1 is subjected to the influence of reaction medium pH.The change of the concrete pH of cohesive process will produce some preferred connecting portion.For example, under certain conditions, alkaline pH value such as more than the pH7.5, more than 8.0, more than 8.5, or 9.0 above connections impel IFN λ 1 to be connected to the Methionin group.
In aforesaid method, Pegization reagent as PEG-pNC, forms the carbamate connector between PEG and IFN λ 1.Other Pegization reagent that can adopt in the aforesaid method comprises that oxygen carbonyl-oxygen base-N-dicarboximide is (as the carbonic acid succinimide ester, the succsinic acid succinimide ester), carbonic acid is to nitro aryl ester, carbonic acid p-nitrophenyl ester, carbonyl dimidazoles, carbonic acid benzotriazole ester, carbonic acid pyridine ester, N-succinimide, N phlhalimide, N-glutarimide with as U.S. Patent number 5, disclosed N-tetrahydric phthalimide in 122,614.The typical activatory PEG or the mPEG compound that also can be used to form binding substances comprise PEG-2,4,6-three chloro-S-triazines, mPEG-2,4,6-three chloro-S-triazines, PEG-N-succinimido glutarate, mPEG-N-succinimide glutarate, PEG-N-succinimido succinate and mPEG-N-succinimido succinate.
These embodiment exemplary compounds:
Following table provides the summary of the compound that embodiment described herein selectes:
The purification process of the binding substances of last table Chinese style Ia adopts method described herein to carry out.The purity that the PEG-IFN λ 1 of gained has is higher than about 95%.The spectrogram of this binding substances and SDS-PAGE electrophoresis are as shown in Figure 9 after the gel-filtration stage.The spectrogram of the purge process of this binding substances and SDS-PAGE electrophoresis are as shown in figure 10.This binding substances has antiviral EMC activity, ED to the Hep-2C cell
50Scope is about 10-50ng/mL.The antiviral activity of the binding substances of last table Chinese style I is at ED
50(ng/ml) be about 25.00-28.00 down; Mean value (ng/mL) is about 1.0-about 30.0; SD is that about 0.1-about 1.0 and RSD are about 3.0-7.0.
The binding substances of last table Chinese style I is with 200 μ g(subcutaneous injections weekly)+virazole 15mg/kg(every day) give to the patient.In first 4 week, all patients detect in the no HCV RNA(serum and do not contain virus).Treatment plan continues to carry out for 12 weeks.All patients have reached the origin endpoint that total virus suppresses after through treatment of 12 weeks and the follow-up of 12 weeks.
The purifying of embodiment 5:PEG-IFN λ 1
The solution of this PEG-IFN of containing λ 1, quencher (cold shock) reagent and unaltered IFN λ 1 is gone up purifying at cation seperation column (post is filled with balance in Sepharose CM gel and the sodium phosphate (pH6.0) at 10mM in advance), with the sodium phosphate of 10mM, the NaCl(pH6.0 of 0.5M) eluant solution.Use the solution of the sodium phosphate (pH6.0) of 10mM, the protein part that contains of wash-out is transferred in the sanitas damping fluid.Make this product stand the sterile filtration process then and be stored under-20 ℃.
Figure 10 has shown the spectrogram and the SDS-PAGE electrophoresis of IFN λ 1 purge process.The purity of the PEG-IFN λ 1 of gained is higher than 95% and to the active ED of antiviral EMC of Hep-2C cell
50For about 10-50ng/mL(referring to embodiment 6).
The detection of embodiment 6:IFN λ 1 and PEG-IFN λ 1 antiviral activity
According to (J Virol.2006May such as Ank; 80 (9): research 4501-9), detect based on the antiviral activity in EMC virus model and Hep-2C cell.Experiment adopts 3 groups (IL290010111, IL290020311 and IL290030411) to carry out.The result shows, the antiviral activity (ED of the interferon lambda 1 of Nanogen
50About 1-5ng/mL) equals (Nat Immunol.2003Jan such as Sheppard; 4 (1): result of study 63-8.) (referring to table 2).
The antiviral activity of PEG-IFN λ 1 is compared with IFN λ 1.Experiment adopts 5 groups (PIL290010111, PIL290020211, PIL290030311, PIL290040411, PIL290050511) to carry out.In all groups, all obtain similar results, ED
50For about 10-50ng/mL(referring to table 3).
Patient's clinical information before the treatment:
All patients diagnose and suffer from chronic hcv in this treatment group, all used Pei lattice Xisi, famous beauty in the late Spring and Autumn Period (Pai Luoxin before, Pegasys) (Peg interferon alpha 2a) and pendant are found pleasure in and can be treated above six months by (PegIntron) (Peg interferon alpha 2 b) associating virazole (15mg/kg), and HCV RNA reduction is no more than 1log.HCV RNA〉500,000IU/mL serum; HCV genotype 1-6; Quantity 150; Age 26-78 year; 52 years old mean age; Some patient has higher ferritin, and low platelet (＜50,000/mL), low Hb.Most patients is owing to chronic infection HCV, and the fiber scale of F4 (Fibro scale) has the fibrosis of higher level, and wherein high AST/ALT ratio is higher than 1 indication of hepatic fibrosis unit (indication).Make some patients' of insulinize diabetes.All patients above 50 years old suffer from hypertension in the treatment group.
Treatment plan:
Peg λ (PEG-IFN λ 1) 200 μ g(subcutaneous injections weekly)+virazole 15mg/kg(every day).In first 4 week, all patients measure in the no HCV RNA(serum and do not contain virus).Treatment continues to carry out for 12 weeks.
The result: all patients reach the origin endpoint that total virus suppresses after treatment of 12 weeks and the follow-up of 12 weeks.
The interferon lambda 1(PEG-IFN λ 1 of table 2:Nanogen) antiviral activity
The antiviral activity of the PEG-IFN λ 1 of table 3:Nanogen
The therapy of HCV resistant patients:
Used standard treatment before surpassing 50 patients, as
(PEG Intederon Alpha-2a) associating virazole is treated, and it is found that it is invalid.The patient of non-response (be defined as 24 all therapies by the HCV of AASLD treatment criterion and do not demonstrate the patient that serum HCV RNA removes) and not having in the treatment plan of PEG-IFN λ 1 that response patient (be defined as those and demonstrate that HCV RNA fails to reduce when the 12nd week〉2log patient) adopting the application is selected in.
Adopting treatment plan disclosed herein with 4-12 week of PEG-IFN λ 1 treatment or 4-24 after week, all patients are tested and detect and be HCV RNA feminine gender basically; Or all patients have persistent virusology response (SVR), and this is defined as in the end after the therapeutic dose no detectable viruses of 24 weeks.
In other research of adopting the HCV resistant patients, therapy disclosed herein it is found that for surpassing 80%, 85%, 90% or to surpass 95% HCV resistant patients crowd be effective.Therefore, the methods of treatment of employing PEG-IFN λ 1 has confirmed the usefulness in HCV, comprises that the standard treatment for current Peg Intederon Alpha-2a+virazole has the case of resistance.Do not observe with
The remarkable side effect of the conjoint therapy canonical correlation of virazole.
The identification of embodiment 7:IFN λ 1 and PEG-IFN λ 1
The Western Western blotting is used to adopt anti-IFN λ 1 antibody recognition IFN λ 1 and PEG-IFN λ 1.Protein solution after will analyzing on the SDS-PAGE gel is transferred to the Nitrocellulose film and surveys with anti--IFN λ 1 antibody probe.Antibody detects (referring to Figure 11) with peroxidase coupling protein A and tmb substrate.
The molecular weight of embodiment 8:PEG-IFN λ 1
The MALDI-TOF analytical method is used to measure the molecular weight of PEG-IFN λ 1.The result provides in Figure 12.In the present embodiment, the PEG-IFN λ 1 of Nanogen has the molecular weight of about 62kDa.
The purity of embodiment 9:PEG-IFN λ 1
5 groups (PIL290010111, PIL290020211, PIL290030311, PIL290040411, PIL290050511) is used for the purity by SDS-PAGE cataphoretic determination PEG-IFN λ 1.The running gel Coomassie blue stain, decolouring is also adopted Phoretix software (TotalLab, Britain) analysis subsequently.All test group all demonstrate purity and are higher than 95%.
Below the as above PEG-IFN λ 1 of preparation is adopted in test:
The toxicity of embodiment 10:PEG-IFN λ 1
The acute toxicity of PEG-IFN λ 1: the acute toxicity of PEG-IFN λ 1 is assessed in Switzerland mouse and rat.Healthy ICR mouse and Sprague's-Du Le rat, in 5 ages in week, selected being used for studied.Observe two weeks of animal.(low dosage 0.03mg/kg and vehicle treatment (phosphate buffered saline buffer, pH7.2)) give through subcutaneous or peritoneal injection PEG-IFN λ 1 for high dosage 3mg/kg, middle dosage 0.3mg/kg with three kinds of various dose.Observe the animal clinical sign, body weight change and the mortality ratio of treatment after 14 days.When research finishes, slaughter all animals, its tissue and organ are carried out follow-up for anomaly.The results are summarized in table 4.
Table 4
(*): ANOVA, single factor, than vehicle treatment, (p〉0.05)
1Be meant no abnormal
Even under the test period of all animals under maximum dose level all survived.Than control group, the body weight of treatment animal does not have noticeable change.In the battery of tests animal in office, do not observe clinical sign or organs abnormality.
Based on these results, the lethal dose (LD of the PEG-IFN λ 1 of Nanogen in rat and mouse
50) all greater than 3mg/kg.The subacute toxicity of PEG-IFN λ 1: with three kinds of various dose (high dosage 3mg/kg, middle dosage 0.3mg/kg, low dosage 0.03mg/kg) once a day through giving animal (5 week ages rats) in subcutaneous or 14 weeks of peritoneal injection PEG-IFN λ.
The PEG-IFN λ 1 that in the whole research these rats is detected by Nanogen gives caused any clinical and delinquency effect.After the testing period, the mouse of slaughtering survival performs an autopsy on sb and biochemical analysis.Also collect blood sample to carry out blood testing from abdominal artery.
Testing method and the results are summarized in the table 5.
Table 5
During whole research, all occur death in any group and from the mouse of being tested, do not detect clinical sign.The mouse of being tested is in other detects classification and analyzes even also normal in high dose group.Therefore, this studies show that the PEG-IFN λ 1 of Nanogen does not have toxic action in rat when the dosage with 3mg/kg repeats to give.
The immunotoxicity of PEG-IFN λ 1: study to inquire into the immune potentiality of PEG-IFN λ 1 in cavy of Nanogen.Healthy male hartley cavy, body weight 300-500g, with PEG-IFN λ 1 with high dosage (3mg/kg) or low dosage (0.03mg/kg) and ovalbumin 23 weeks of injection weekly in contrast.Final sensitization is after 14 days, and the anaphylactic shock test is undertaken by the PEG-IFN λ 1 of intravenous injection high dosage.Research comprises the PEG-IFN λ 1 that incorporates in the Freund's complete adjuvant (FCA).Observe the active systemic anaphylaxis shock reaction of sensitized guinea pig after injecting high dosage PEG-IFN λ 1.The indication tabulation is as the sign of anaphylaxis and its generation of monitoring in each test animal.
Table 6 has shown research method and result.
Table 6
(1)1, licks nose, wipe nose; 2, scratching fur;
3, labored breathing; 4, sneeze cough;
5, defecation is urinated; 6, spasm; 7, collapse;
8, death.-feminine gender; + the positive
In the test of active systemic anaphylaxis, by the PEG-IFN λ 1(3mg/kg in the Freund's complete adjuvant (FCA) of being incorporated into of high dosage) cavy of slight sensitization shows some signs of anaphylaxis.On the other hand, adopt the PEG-IFN λ 1(0.03 and the 3mg/kg of low dosage and high dosage separately) cavy of sensitization do not demonstrate any anaphylaxis.After the PEG-IFN λ 1 with Nanogen gives with negative treatment (PBS), do not have cavy death, but 3 cavys are giving ovalbumin (positive treatment) death afterwards.Therefore, can inference, the PEG-IFN λ 1 of Nanogen does not cause the systemic anaphylaxis reaction when giving separately in its clinical use.
To be to it is evident that to make various modifications and variations to those skilled in the art, and not deviate from the spirit or scope of the present invention compound of the present invention, composition and method.Therefore, the present invention is intended to cover modification of the present invention and modification, as long as they are in the scope of claim of enclosing and equivalent thereof.
Claims (20)
1. PEG-IFN λ 1 binding substances of a physiologically active comprises with following formula I or its pharmaceutical salts:
Wherein:
R is H or C
1-3Alkyl;
M is 1,2,3 or 4;
N is the positive integer that is selected from the 400-550 scope;
L is C
1-10Alkyl or assorted alkyl connector;
X is-O-,-NH-or-S-; And
IFN λ 1 is an interferon lambda 1.
2. binding substances according to claim 1, wherein R be H or-CH
3, m is 1, L is-C (O)-and X be-NH-.
3. binding substances according to claim 1 and 2, wherein n is 500-550.
4. binding substances according to claim 1 comprises following formula:
Wherein INF λ 1 is the interferon lambda 1 of SEQ ID2; And
N is 500-550.
5. according to each described binding substances among the claim 1-4, wherein said binding substances has serum half-life and the retention time that prolongs or spin out than IFN λ 1.
6. according to each described binding substances among the claim 1-5, wherein PEG is connected in methionine(Met) at the N of described IFN λ 1 end.
7. pharmaceutical composition that comprises according to each described binding substances and pharmaceutical carrier and vehicle among the claim 1-6.
8. pharmaceutical composition according to claim 7, wherein said pharmaceutical composition is used for the treatment of hepatitis B and hepatitis C.
9. method that is used to prepare people's recombination nodule compound according to claim 1 may further comprise the steps: by following association reaction covalently bound (α-methoxyl group-ω-(4-nitrophenoxy carbonyl)) polyoxyethylene (PEG-pNC) 40kDa and IFN λ 1:
Wherein, n is selected from the interior positive integer of 500-550 scope so that the molecular weight of peg moiety is about 40kDa; And separate described binding substances.
10. one kind is used to prepare and comprises with Peg-IFN λ 1 binding substances of following formula I or the method for its pharmaceutical salts:
Wherein:
R is H or C
1-3Alkyl;
M is 1,2,3 or 4;
N is the positive integer that is selected from the 400-550 scope;
L is C
1-10Alkyl or assorted alkyl connector;
X is-O-,-NH-or-S-; And
IFN λ 1 is an interferon lambda 1;
Described method comprises:
Make described IFN λ 1 be enough to impel with the covalently bound condition of the amino-acid residue of described IFN λ 1 under contact with pre-activation Peg.
11. PEG-IFN λ 1 binding substances by the described method preparation of claim 10.
12. method that is used for suppressing patient's cancer cell multiplication, comprise making described cancer cells and contacting that wherein said binding substances has serum half-life and the retention time that prolongs or spin out than IFN λ 1 according to each described binding substances among the claim 1-4.
13. a method that is used for the treatment of proliferative disease in the Mammals comprises the described binding substances of claim 1 for the treatment of significant quantity to described Mammals.
14. treat the method that infective virus infects or be in the patient in the infective virus infection risk for one kind, comprise binding substances or its pharmaceutical salts with following formula I for the treatment of significant quantity to its patient of needs; Or comprise the pharmaceutical preparation of the binding substances of described formula I:
Wherein:
R is H or C
1-3Alkyl;
M is 1,2,3 or 4;
N is the positive integer that is selected from the 500-550 scope;
L is C
1-10Alkyl or assorted alkyl connector;
X is-O-,-NH-or-S-; And
IFN λ 1 is an interferon lambda 1.
15. method according to claim 14, wherein R is-CH
3, m is 1, L is-C (O)-, X is-NH-and IFN λ 1 are SEQ ID2.
16. according to claim 14 or 15 described methods, wherein said virus infection is to be caused or described virus infection causes end-age cirrhosis by hepatitis C virus.
17., wherein give described PEG-IFN λ 1 with the dosage of about 0.5 μ g/kg-10.0 μ g/kg weekly according to each described method in the claim 14,15 or 16.
18., further comprise the nucleoside analog that is selected from virazole and Wella pyrimidine according to each described method among the claim 14-17.
19. method according to claim 18, wherein with every day 5mg/kg-25mg/kg dosage give virazole.
20. according to each described method among the claim 14-17, wherein said patient is HCV resistance or refractory patient.
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| PL3416675T3 (en) * | 2016-02-19 | 2021-10-11 | Eiger Biopharmaceuticals, Inc. | Treatment of hepatitis delta virus infection with interferon lambda |
| EP3375452A1 (en) | 2017-03-16 | 2018-09-19 | Evangelos Andreakos | Use of lambda interferons in the treatment of disorders and related diseases |
| CN112245570A (en) | 2019-07-22 | 2021-01-22 | 厦门特宝生物工程股份有限公司 | Interferon-based disease treatment method |
| AU2022209506A1 (en) | 2021-01-21 | 2023-09-07 | Biosteed Gene Transformation Tech. Co., Ltd. | Method and pharmaceutical combination for preventing cancer recurrence |
| EP4282427A1 (en) | 2021-01-21 | 2023-11-29 | Xiamen Amoytop Biotech Co., Ltd. | Interferon-based cancer treatment method, and pharmaceutical combination |
| WO2024121424A1 (en) | 2022-12-09 | 2024-06-13 | Daniel Zagury | Composite aids vaccine generating anti-hiv specific neutralizing antibodies and/or anti-hiv cytotoxic t cells |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5382657A (en) * | 1992-08-26 | 1995-01-17 | Hoffmann-La Roche Inc. | Peg-interferon conjugates |
| CN1381512A (en) * | 2002-01-15 | 2002-11-27 | 泛亚生物技术有限公司 | Multi-arm fork type functional polyethylene glycol preparation method and its application in medicine |
| CN1927388A (en) * | 2004-09-10 | 2007-03-14 | 北京金迪克生物技术研究所 | Medicinal composition containing human interferon |
| CN101002942A (en) * | 2007-01-08 | 2007-07-25 | 湖南大学 | PEG type elaioplast nanometer particle medicine |
| US20080096252A1 (en) * | 2005-10-04 | 2008-04-24 | Zamost Bruce L | Production and purification of il-29 |
| US20080132681A1 (en) * | 2005-06-03 | 2008-06-05 | Ambrx, Inc. | Human Interferon Molecules and Their Uses |
| CN102099051A (en) * | 2008-06-05 | 2011-06-15 | 津莫吉尼蒂克斯公司 | Use of pegylated type III interferons for the treatment of hepatitis c |
-
2012
- 2012-03-01 BR BR112014004302A patent/BR112014004302A2/en not_active IP Right Cessation
- 2012-03-01 EP EP12825651.8A patent/EP2748328A4/en not_active Withdrawn
- 2012-03-01 RU RU2014111179/10A patent/RU2014111179A/en not_active Application Discontinuation
- 2012-03-01 JP JP2014527142A patent/JP2014525939A/en not_active Withdrawn
- 2012-03-01 AU AU2012299423A patent/AU2012299423A1/en not_active Abandoned
- 2012-03-01 WO PCT/US2012/027317 patent/WO2013028233A1/en unknown
- 2012-03-01 CA CA2846092A patent/CA2846092A1/en not_active Abandoned
- 2012-03-01 CN CN2012800033678A patent/CN103228792A/en active Pending
-
2014
- 2014-02-14 ZA ZA2014/01159A patent/ZA201401159B/en unknown
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5382657A (en) * | 1992-08-26 | 1995-01-17 | Hoffmann-La Roche Inc. | Peg-interferon conjugates |
| CN1381512A (en) * | 2002-01-15 | 2002-11-27 | 泛亚生物技术有限公司 | Multi-arm fork type functional polyethylene glycol preparation method and its application in medicine |
| CN1927388A (en) * | 2004-09-10 | 2007-03-14 | 北京金迪克生物技术研究所 | Medicinal composition containing human interferon |
| US20080132681A1 (en) * | 2005-06-03 | 2008-06-05 | Ambrx, Inc. | Human Interferon Molecules and Their Uses |
| US20080096252A1 (en) * | 2005-10-04 | 2008-04-24 | Zamost Bruce L | Production and purification of il-29 |
| US20100003722A1 (en) * | 2005-10-04 | 2010-01-07 | Zymogenetics, Llc | Production and purification of il-29 |
| CN101002942A (en) * | 2007-01-08 | 2007-07-25 | 湖南大学 | PEG type elaioplast nanometer particle medicine |
| CN102099051A (en) * | 2008-06-05 | 2011-06-15 | 津莫吉尼蒂克斯公司 | Use of pegylated type III interferons for the treatment of hepatitis c |
Non-Patent Citations (2)
| Title |
|---|
| SHEPPARD, P. ET AL.: "Accession NO：NP_742152，interleukin-29 precursor [Homo sapiens]", 《GENBANK DATABASE》, 21 August 2011 (2011-08-21) * |
| 胡小剑等: "羧甲基化单甲氧基聚乙二醇的制备及其对a-干扰素的修饰", 《过程工程学报》, vol. 3, no. 2, 30 April 2003 (2003-04-30) * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111065405A (en) * | 2017-09-08 | 2020-04-24 | 科学未来管理有限责任公司 | Pegylated interferon lambda with high oral bioavailability and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| EP2748328A1 (en) | 2014-07-02 |
| ZA201401159B (en) | 2015-04-29 |
| BR112014004302A2 (en) | 2017-06-20 |
| WO2013028233A1 (en) | 2013-02-28 |
| CA2846092A1 (en) | 2013-02-28 |
| JP2014525939A (en) | 2014-10-02 |
| RU2014111179A (en) | 2015-09-27 |
| EP2748328A4 (en) | 2015-03-04 |
| AU2012299423A1 (en) | 2014-03-06 |
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