CN102066938A - Method for determination of edema associated with liver disease - Google Patents
Method for determination of edema associated with liver disease Download PDFInfo
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Abstract
Disclosed is a method for determining edema associated with a liver disease, which comprises the step of quantifying the ratio of oxidized albumin in an albumin component contained in a biological sample collected from a subject. In the method, it is preferred to determine the degree of edema based on the presence of a positive correlation between the ratio of oxidized albumin and the degree of edema. Also disclosed is an albumin preparation for the prevention or treatment of edema associated with a liver disease, which comprises an albumin component containing oxidized albumin at a ratio of less than 60%. The ratio of oxidized albumin in the albumin component to be contained in the preparation is preferably less than 25%.
Description
Technical field
The present invention relates to be accompanied by the decision method of the oedema that hepatopathy produces, the kit that is used to judge the oedema that is accompanied by hepatopathy and produces, the screening technique that can prevent or treat the compound that is accompanied by the oedema that hepatopathy produces, the prevention that is accompanied by the oedema that hepatopathy produces or therapeutic agent etc.
Background technology
Oedema symptoms such as ascites are owing to cirrhosis or hepatocellular carcinoma, bacterial peritonitis, congestive heart disease, nephrotic syndrome, the struvite pathology of each internal organs of intraperitoneal, peritonitis occur.Be to enter the sign of losing the compensatory phase particularly, come into one's own as the very important symptom that determines prognosis owing to oedema appears in cirrhosis.
In the past, as the inspection item in the clinical examination relevant, used seralbumin amount (non-patent literature 1) with oedema.If the seralbumin value is reduced to below about 2.5～2.0g/dl, think that then colloid osmotic pressure reduces, produce ascites, be the monitoring that index is carried out curative effect with it.In addition, if surpassing 1.1g/dl, the albumin concentration difference of serum and ascites then will be used for the selection of ascites treating method, if 1.1g/dl will be used for the selection of ascites treating method with the discriminating of transudate with next with the discriminating that spills liquid.
But, in order suitably to treat the oedema of liver cirrhosis patient, not only be necessary to measure the seralbumin value, be necessary that also grasp has or not complication, the many-sided clinical examination information of grasps such as change of the change of serum electrolyte and renal function, each Na retention or the drainage factor is selected suitable treatment.Particularly for 5～10% RA in the liver cirrhosis patient of finding ascites, must study the enforcement of liver inside door vena systemica bypass, the prediction for the treatment of prognosis in addition is important.Therefore, expectation determines to reflect exactly the index in hepatopath's oedema degree and the serum that quantitatively property is also excellent.
Albumin is to have maximum protein in vivo.As albuminous physiological function, known have the keeping of colloid osmotic pressure, the conveying of material, amino acid whose supply, pH buffer action, redox buffer effect etc.In recent years clear and definite the existence in the albumin, the difference of albuminous matter rather than amount is studied with the different correlativity of function because of the microheterogeneity that modification caused after the translation.
Seralbumin with the reduced form albumin that is free state from the terminal SH base that is present in the 34th cysteine residues of N and with blood halfcystine or the albuminous form of mixtures existence of oxidized form that forms two sulphur (S-S) bonded state such as glutathione.Ratio about 75% with reduced form in normal human serum, oxidized form about 25% exists, but the albuminous ratio of oxidized form increases (non-patent literature 3) among the patient when having report to point out liver cirrhosis patient (non-patent literature 2), patients with chronic renal failure, diabetic, anesthesia or operation.
Have report to point out that patients with chronic renal failure reduces owing to haemodialysis causes the oxidized form albumin, the reduced form albumin increases (non-patent literature 4).In addition, there is report to point out not only to improve albuminous amount with the BCAA granular preparation that is improved as functional effect of the hypoalbuminemia of liver cirrhosis patient, also improve oxidized form/reduced form albumin than (patent documentation 1), therefore which kind of effect the improvement of albuminous matter brings attracted attention to symptom.But, unknown fully for the relation of the oedema in oxidized form albumin level and the hepatopathy (moisture retention in the body).
Known use mass spectrophotometry or liquid chromatography high precision and stably to the oxidized form albumin in the liquid sample and albuminous amount of reduced form and their method (patent documentation 2) that exists ratio to analyze.
[patent documentation 1] WO2007/015585
[patent documentation 2] WO2007/013679
Acology vol.41 is won in [non-patent literature 1] Fukui, No.4, p.45-48,2007
[non-patent literature 2] Watanabe, A.Nutrition 20:351,2004
The good holy clinical examination 48:501-511 of [non-patent literature 3] favour, 2004
[non-patent literature 4] Sogami, M.Int.J.Peptide Protein Res 25:398,1985
Summary of the invention
The objective of the invention is to, the index in degree that reflects the oedema that is accompanied by the hepatopathy generation exactly and the serum that quantitatively property is also excellent is provided.In addition, the objective of the invention is to, provide for the prevention that is accompanied by the oedema that hepatopathy produces or treat useful albumin preparation.
The inventor to the oxidized form/reduced form albumin of liver cirrhosis patient than the process of studying in, although notice, also have significant patient of ascites and inapparent patient to hypoalbuminemia with degree.So, oxidized form in the serum of liver cirrhosis patient/reduced form albumin is estimated than with oedema (moisture retention), the result compares with the albumin amount as can be known, shows high correlativity between oxidized form/reduced form albumin ratio and the oedema.Found that by these, oxidized form albumin value is that the ascites of liver cirrhosis patient is judged effective index, the index that adopts as the optimum treatment method that is suitable for monitoring curative effect is the big index of meaning clinically, if give the reduced form albumin to the patient who suffers from the oedema of following cirrhosis, reduce oxidized form albumin ratio on one's own initiative, then compare, can improve oedema effectively, thereby finish the present invention with containing the albuminous common albuminous situation of oxidized form.
That is, the invention provides:
[1] is accompanied by the decision method of the oedema that hepatopathy produces, comprises the shared ratio of oxidized form albumin in the albumin in the organism sample that is obtained by the experimenter is carried out quantitatively.
[2] method of [1] record wherein, is judged the oedema degree based on the positive correlation between oxidized form albumin ratio and the oedema degree.
[3] be used to judge the kit of the oedema that is accompanied by hepatopathy and produces, contain to be useful on the shared ratio of oxidized form albumin in the albumin in the organism sample is carried out quantitative reagent.
[4] can prevent or treat the screening technique of the compound that is accompanied by the oedema that hepatopathy produces, may further comprise the steps:
(1) give candidate compound to the non-human mammal of suffering from the oedema that the hepatopathy of being accompanied by produces,
(2) the shared ratio of oxidized form albumin in the albumin in the organism sample that is obtained by the non-human mammal that has been given this candidate compound is carried out quantitatively,
(3) the shared ratio of oxidized form albumin in the albumin in the shared ratio of oxidized form albumin in the albumin in the organism sample that is obtained by this non-human mammal and the organism sample that is obtained by the contrast mammal that does not give candidate compound is compared and
(4) result based on the comparison, the compound of selecting to have reduced oxidized form albumin ratio is as preventing or treat the compound that is accompanied by the oedema that hepatopathy produces.
[5] be used to prevent or treat the albumin preparation that is accompanied by the oedema that hepatopathy produces, contain oxidized form albumin ratio less than 60% albumin.
[6] be used to prevent or treat the albumin preparation that is accompanied by the oedema that hepatopathy produces, contain oxidized form albumin ratio less than 25% albumin.
[7] albumin preparation of [6] record, wherein, oxidized form albumin ratio is essentially 0%.
[8] albumin preparation of any record further contains antioxidant in [5]～[7].
[9] be used to prevent or treat the preparation method of the albumin preparation that is accompanied by the oedema that hepatopathy produces, may further comprise the steps:
(1) provide oxidized form albumin ratio less than 60% albumin and
(2) be dissolved in the aqueous solvent by albumin, obtain being used to prevent or treat the albumin preparation of the oedema that is accompanied by the hepatopathy generation (1).
[10] be used to prevent or treat the preparation method of the albumin preparation that is accompanied by the oedema that hepatopathy produces, may further comprise the steps:
(1) provide oxidized form albumin ratio less than 25% albumin and
(2) be dissolved in the aqueous solvent by albumin, obtain being used to prevent or treat the albumin preparation of the oedema that is accompanied by the hepatopathy generation (1).
[11] preparation method of [9] or [10] record, wherein, step (1) is undertaken by albumin being implemented the reduction processing.
[12] preparation method of [11] record wherein, in reduction is handled, uses halfcystine as reductive agent.
According to the present invention, by the shared ratio of oxidized form albumin in the albumin in the organism sample that is obtained by object is measured, can be rapidly and be accompanied by the judgement of the oedema degree that hepatopathy produces exactly, for example treat monitoring, prognosis prediction etc., and then can carry out to prevent or treating the screening of the compound of the oedema that is accompanied by the hepatopathy generation.In addition,, then can reduce the oxidized form albumin ratio among the patient on one's own initiative, compare, can more effectively prevent or treat to be accompanied by the oedema that hepatopathy produces with the situation of using albumin preparation in the past if use albumin preparation of the present invention.
Description of drawings
(transverse axis is O-Alb) with oedema (longitudinal axis, oedema for oxidized form albumin ratio (%) in the serum of [Fig. 1] expression chronic hepatitis patients; ECW/TBW) correlativity between.Regression straight line: ECW/TBW=0.317+0.001*O-Alb, R
2=0.482.
(unit: g/dL) (transverse axis is Alb) with oedema (longitudinal axis, oedema for albumin level in the serum of [Fig. 2] expression chronic hepatitis patients; ECW/TBW) correlativity between.Regression straight line: ECW/TBW=0.374-0.008*Alb, R
2=0.277.
(transverse axis is O-Alb) with the Child-Pugh scoring (figure of upside for oxidized form albumin ratio (%) in the serum of [Fig. 3] expression chronic hepatitis patients; The longitudinal axis, the CP scoring) and the MELD scoring (figure of downside; The longitudinal axis, MELD) correlativity between.Regression straight line: Child-Pugh scoring=3.013+0.132*O-Alb, R
2=0.268.MELD＝2+0.316*O-Alb、R
2＝0.066。
[Fig. 4] expression serum albumin preparations is handled the reduction of carrying out by halfcystine.Transverse axis represents that mass-to-charge ratio (m/z), the longitudinal axis represent relative amount.Mass-to-charge ratio is that the oxidized form albumin is represented near 1387.6 and 1389.5 peaks, and mass-to-charge ratio is that the reduced form albumin is represented near 1385.2 and 1383.7 peaks.Last figure: contrast (not adding halfcystine, room temperature, 90 minutes), middle figure: do not add halfcystine, 37 ℃, 90 minutes, figure below: add halfcystine, 37 ℃, 90 minutes.
Embodiment
The invention provides the decision method of the oedema that is accompanied by the hepatopathy generation.Decision method of the present invention comprises that the oxidized form albumin ratio to the albumin in the organism sample that is obtained by the experimenter (being preferably seralbumin) carries out quantitatively.
As shown in the Examples, the shared ratio of oxidized form albumin and be accompanied by the oedema table of degree that hepatopathy produces and reveal positive correlation in the seralbumin, oxidized form albumin ratio is high more, and then to be accompanied by the oedema degree that hepatopathy produces also big more.Therefore, in decision method of the present invention, judge the oedema degree based on the positive correlation between oxidized form albumin ratio and the oedema degree.
Hepatopathy comprises all hepatopathys of the concurrent oedema of possibility.As hepatopathy, can enumerate cirrhosis, hepatitis (virus hepatitis (hepatitis C, hepatitis B etc.), alcoholic hepatitis, non-alcoholic hepatitis etc.) etc.
Oedema refers in the state of cytoplasm retention excess body fluid (moisture retention).The oedema that is accompanied by the hepatopathy generation mainly shows with the symptom of ascites.
As the experimenter of decision method of the present invention, can enumerate the patient of hepatopathy and the healthy person that suspection suffers from hepatopathy.In addition, here alleged patient does not limit and is the people, mammal beyond the people (animals used as test such as rodent such as little, mouse, rat, hamster, cavy or rabbit, domestic animals such as pig, ox, goat, horse, sheep, ermine, pet such as dog, cat, primates such as monkey, rhesus macaque, marmoset, orangutan, chimpanzee etc.) also be object of the present invention.
As the organism sample that uses in the decision method of the present invention, use body fluid usually.As body fluid, can enumerate serum, blood plasma, blood, ascites, lymph liquid, urine, bone marrow fluid etc.Body fluid is preferably serum, blood plasma, blood or ascites.
Oxidized form albumin ratio refers to oxidized form albumin shared ratio in total albumin (the albuminous summation of oxidized form albumin and reduced form).The oxidized form albumin refers to the albumin that the interior mercaptan compound (halfcystine, glutathione etc.) of biosome beyond the albumin forms by disulfide bond and the cysteine residues bonding that is present in the 34th from the N end.The reduced form albumin refers to the albumin that is in free state from the terminal SH base that is present in the 34th cysteine residues of N.
Oxidized form albumin ratio in the organism sample is carried out quantitative methods do not limit especially, can the known method of use own.For example, can use following method:
(I) utilize the pigment combined techniques of bromcresol green (BCG) and bromcresol purple (BCP).The BCP method can be calculated oxidized form albumin ratio by the difference of the albumin quantitative values that utilizes BCP method and BCG method to try to achieve owing to different for the albuminous reactivity of reduced form albumin and oxidized form.
(II) use free SH base quantitative reagent such as ellman's reagent (エ Le マ Application Try medicine), to carry out quantitative methods (Sogami M, Int.Pept.Protein Res., 24 (2), 96-103,1984) from the albuminous SH base of reduced form.
(III) method (Sogami M., J.Chromatogr., 332,19-27,1985 of use high performance liquid chromatography (HPLC); Japanese kokai publication sho 61-155397; The special fair 2-4863 of Japan; Int.J.Peptide Protein Res.24:96; 1984).Separate and also to detect reduced form albumin and oxidized form albumin, the peak area ratio on the chromatogram that can be obtained by separation is tried to achieve oxidized form albumin ratio.
(IV) the albuminous analytical approach (peace river favour, clinical examination, 44 (8), 907-910,2000) of using mass spectrometer to carry out.The oxidized form albumin is because the mass parts of the compound (for example halfcystine) of addition is heavier than reduced form albumin.Therefore, by measuring, can separate and detect reduced form albumin and oxidized form albumin with mass spectrometer with sufficient mass resolution.
(V) use is adjusted into particular range (pH 4～9, are preferably 5.8～6.2) improved HPLC or mass spectral method (WO2007/013679) by the pH with sample solution.The pH of sample solution is adjusted into particular range, preferably make dilute solution or utilize chromatographic process or lower-molecular substance is removed in ultrafiltration etc. with the ratio of particular range dilution sample, significantly suppress the albuminous oxidation of reduced form in the sample solution thus, can carry out high-precision albumin measuring highly stablely.
If consider the stability or the height of analysis precision, then the oxidized form albumin ratio in the organism sample is preferably undertaken quantitatively by any one method in above-mentioned (III)～(V).
Quantitatively preferably outside biosome, carrying out of oxidized form albumin ratio in the organism sample.
Then, use and measure the oxidized form albumin ratio that obtains, the oedema degree that is accompanied by the hepatopathy generation is judged.This judgement is carried out based on the positive correlation between oxidized form albumin ratio and the oedema degree.
For example, by distinguishing the hepatopath's (positive control) that is accompanied with the certain level oedema and healthy people (negative control) preparation organism sample, the oxidized form albumin ratio of the oxidized form albumin ratio in experimenter's the organism sample and positive control and negative control is compared.In positive control and negative control, preferably obtain organism sample by the method for taking identical with the experimenter, measure oxidized form albumin ratio by the assay method identical with the experimenter, this considers preferred from the precision aspect that improves data.In addition, can also make the correlation figure of oxidized form albumin ratio and oedema degree in advance, experimenter's oxidized form albumin ratio and this correlogram compared.The comparative optimization of oxidized form albumin ratio is based on there being there was no significant difference to carry out.
Then, comparative result according to oxidized form albumin ratio, under the high relatively situation of experimenter's oxidized form albumin ratio, can judge oedema degree high relatively (or under the low relatively situation of experimenter's oxidized form albumin ratio, judging that the oedema degree is low relatively).
In addition, can also be by the threshold value (cut-off value) that preestablishes oxidized form albumin ratio, the oxidized form albumin ratio that mensuration is obtained and this threshold value compare carries out.For example, measure the oxidized form albumin ratio obtain and be above-mentioned threshold value when above, can judge that the oedema degree is high relatively, when being lower than above-mentioned threshold value, can judge that the oedema degree is low relatively.Threshold value for example can be the correlation curve based on oxidized form albumin ratio and oedema degree, obtains the oxidized form albumin ratio of required oedema degree.
Perhaps, in decision method of the present invention, can also to by the shared ratio of oxidized form albumin in the albumin in the organism sample that obtains of experimenter in the treatment with compare by the shared ratio of oxidized form albumin before the treatment and/or in the albumin in the organism sample that obtains of the same experimenter after the treatment.Refer in the treatment to the patient implement that administration etc. disposes during.Before treatment, the treatment in and/or the treatment after obtain organism sample at least 1 time by the patient respectively.Preferably before treatment, in the treatment and at interval take sample for several times with reasonable time respectively after the treatment by the patient.So, the time dependent that can investigate the oxidized form albumin ratio in the patient's who suffers from the oedema that the hepatopathy of being accompanied by produces the organism sample in more detail changes, and can resolve the oedema degree in more detail.For example, can investigate the having or not of response, the degree of patient in more detail, or the patient is in situations such as which kind of treatment stage, can predicts the oedema degree change accurately for treatment.
Therefore, decision method of the present invention can be used to be accompanied by the treatment monitoring of the oedema that hepatopathy produces and the prediction of prognosis.
Further, the invention provides the judgement kit of the oedema that is accompanied by the hepatopathy generation.This kit contains and is useful in the decision method of the invention described above, and the ratio shared to oxidized form albumin in the albumin in the organism sample carries out quantitative reagent.The quantivative approach of oxidized form albumin ratio is known above-mentioned the whole bag of tricks, can suitably select to use.For example, the method by above-mentioned (I) is carried out when quantitative oxidized form albumin ratio, and kit of the present invention contains bromcresol green (BCG) and bromcresol purple (BCP).When measuring oxidized form albumin ratio by the method for above-mentioned (II), kit of the present invention contains free SH base quantitative reagents such as ellman's reagent.By above-mentioned (V) method oxidized form albumin ratio is carried out when quantitative, it is the damping fluid of 4～9 (being preferably 5.8～6.2) that kit of the present invention contains the pH that is useful on the dilution sample.At this moment, kit of the present invention can further contain and is useful on the ultra filtration membrane or the chromatographic column of removing lower-molecular substance.If use kit of the present invention, then can use the decision method of the invention described above easily to judge the oedema that is accompanied by the hepatopathy generation.
Further, the invention provides the screening technique that can prevent or treat the compound of the oedema that is accompanied by the hepatopathy generation.This method comprises following method:
(1) give candidate compound to the non-human mammal of suffering from the oedema that the hepatopathy of being accompanied by produces,
(2) the shared ratio of oxidized form albumin in the albumin in the organism sample that is obtained by the non-human mammal that has been given this candidate compound is carried out quantitatively,
(3) the shared ratio of oxidized form albumin in the albumin in the shared ratio of oxidized form albumin in the albumin in the organism sample that is obtained by this non-human mammal and the organism sample that is obtained by the contrast mammal that does not give candidate compound is compared and
(4) result based on the comparison, the compound of selecting to have reduced oxidized form albumin ratio is as treating the compound that is accompanied by the oedema that hepatopathy produces.
The candidate compound that is used for screening technique of the present invention can be any known compound and new compound, can enumerate compound library, the random peptide library of nucleic acid for example, carbohydrate, lipid, protein, peptide, organic low molecular compounds, the preparation of use combinatorial chemistry (コ Application PVC Na ト リ ア Le ケ ミ ス ト リ one) technology, or from the natural components of microorganism, animals and plants, sea life etc. etc.
As non-human mammal, can use the mammal of being enumerated as the experimenter's example in the decision method of the present invention.Non-human mammal as the oedema of suffering from the hepatopathy of being accompanied by generation can use known hepatopathy (cirrhosis, hepatitis etc.) animal pattern.
The method of enumerating in the decision method of the present invention of quantitatively can using of the oxidized form albumin ratio (2) is carried out.
The comparative optimization of the oxidized form albumin ratio (3) is based on there being there was no significant difference to carry out.And, oxidized form albumin ratio in the contrast mammal, with respect to the oxidized form albumin ratio in the mammal that has given the candidate compound quantitatively, can be for measure the value that obtains or measure the value that obtains simultaneously in advance, but consider from the precision, reproducible viewpoint of experiment, be preferably the value that mensuration simultaneously obtains.
Then, select the reduction that obtains by result relatively the compound of oxidized form albumin ratio as preventing or treat the material that is accompanied by the oedema that hepatopathy produces.
Further, the invention provides albumin preparation.Albumin preparation of the present invention can be used to prevent or treat be accompanied by the oedema that hepatopathy produces.By to the patient that might suffer from the oedema that the hepatopathy of being accompanied by produces or the patient who suffers from the oedema that the hepatopathy of being accompanied by produces give albumin preparation of the present invention, can prevent or treat this patient's oedema.
Albuminous being characterised in that of containing in the albumin preparation of the present invention, oxidized form albumin ratio is less than 25%.The sero-abluminous oxidized form in the biosome and the ratio that exists of reduced form just often are being about 25: 75.Among the above-mentioned patient who suffers from the oedema that the hepatopathy of being accompanied by produces, the albuminous ratio of oxidized form increases.Therefore, by giving albumin preparation of the present invention, can reduce the oxidized form albumin ratio among the patient who suffers from the oedema that the hepatopathy of being accompanied by produces on one's own initiative, be improved to just often ratio or near just often ratio, compare with albumin preparation in the past thus, can effectively prevent or treat the oedema that is accompanied by the hepatopathy generation.
In order to strengthen the effect that reduces the oxidized form albumin ratio among the patient, the shared ratio of oxidized form albumin is low more preferred more in the albumin that contains in the albumin preparation of the present invention.Therefore, this oxidized form albumin ratio preferably less than 15%, is more preferably less than 5% usually less than 25%, further preferably less than 1%, most preferably less than 0.5% (for example being essentially 0%).Oxidized form albumin ratio is essentially 0%, and to refer to the albumin that is contained be reduced form in fact entirely.The shared ratio of oxidized form albumin can pass through the HPLC method (with reference to Int.J.Peptide Protein Res.24:96 in the albumin that contains in the albumin preparation of the present invention; 1984) determine.
In new scheme, the shared ratio of oxidized form albumin is usually less than 60% in the albumin that contains in the albumin preparation of the present invention, preferably less than 51%, be more preferably less than 25%, further preferably less than 15%, further preferably less than 5%, especially preferably less than 1%, most preferably less than 0.5% (for example being essentially 0%).As mentioned above, the sero-abluminous oxidized form in the biosome and the ratio that exists of reduced form just often are being about 25: 75, surprisingly, if the oxidized form albumin ratio in the albumin is less than 60% (preferably less than 51%), then this albumin can prevent or treat to be accompanied by the oedema that hepatopathy produces.To be that albumin more than 25% improves the accurate mechanism that is accompanied by the oedema that hepatopathy produces also indeterminate by giving oxidized form albumin ratio.The shared ratio of oxidized form albumin can pass through the HPLC method (with reference to Int.J.Peptide Protein Res.24:96 in the albumin that contains in the albumin preparation of the present invention; 1984) determine.
And in this instructions, the numerical value of oxidized form albumin ratio under situation about not specifying, refers to by the HPLC method (with reference to Int.J.Peptide Protein Res.24:96; 1984) numerical value of Que Dinging.
The albumin that contains in the albumin preparation of the present invention is generally the seralbumin of above-mentioned mammal (preferably being the people).As human serum albumins, can enumerate the polypeptide that contains the amino acid sequence shown in the SEQ ID NO.1.The albumin that contains in the albumin preparation of the present invention can be for the albumin that obtained by the serum isolated or purified or by in the recombinant type albumin of gene recombination technology preparation any one.
As the albumin that can be used for albumin preparation of the present invention, can enumerate disclosed following albumin TOHKEMY 2002-12553 number.
[1] coloring inhibiting method by human serum albumins obtain, not in conjunction with human serum albumins coloring components, that express by genetic manipulation, the coloring inhibiting method of described human serum albumins is characterised in that, when the human serum albumins generation host that cultivation prepares by genetic manipulation expresses seralbumin, in being added with at least a nutrient solution that is selected from anion exchanger, hydrophobic carrier and the activated charcoal, cultivate this host, thus human serum albumins and coloring components in conjunction with before separate this human serum albumins and this coloring components.When use contained this albuminous composition as albumin preparation of the present invention, the degree of staining of said preparation was preferably according to A
350nm/ A
280nmThe absorbance ratio count 0.0035～0.0163, according to A
405nm/ A
280nmThe absorbance ratio count 0.0018～0.0179.
In addition, the fused protein of albumin and other protein is also included within the albumin that uses in the albumin preparation of the present invention.As this fused protein, can enumerate disclosed following albumin fusion protein matter in Japanese Unexamined Patent Application Publication 2007-522806 number, Japanese Unexamined Patent Application Publication 2004-506407 number, Japanese Unexamined Patent Application Publication 2003-530838 number, Japanese Unexamined Patent Application Publication 2003-530839 number, Japanese Unexamined Patent Application Publication 2003-530846 number, Japanese Unexamined Patent Application Publication 2003-530847 number, Japanese Unexamined Patent Application Publication 2003-530852 number and Japanese Unexamined Patent Application Publication 2003-531590 number.
[2]-1 albumin fusion protein matter (Japanese Unexamined Patent Application Publication 2007-522806), contain the composition that is selected from following (a)～(k):
(a) treat the albumin of using protein: X and containing the amino acid sequence of SEQ ID NO.1;
(b) treatment is used protein: X and is had the fragment or the mutant of amino acid sequence of the SEQ ID NO.1 of albumin activity;
(c) protein: X is used in treatment, and the treatment protein of non-fusion state: compare the fragment or the mutant of the amino acid sequence of storage life and then SEQ ID NO.1 that have albumin activity that can extended treatment protein: X with the storage life of X;
(d) treatment is used protein: X and is had albumin activity and then contain the fragment or the mutant of amino acid sequence of SEQ ID NO.1 of amino acid sequence of amino acid/11～387 of SEQ ID NO.1;
(e) has the treatment bioactive treatment protein of protein: X: the fragment of X or mutant and contain the albumin of the amino acid sequence of SEQ ID NO.1;
(f) treatment protein: X, its fragment or mutant, albumin (a)～(e), its fragment or mutant, wherein, the terminal fusion of N-of protein: X, its fragment or mutant and albuminous N-end or albuminous fragment or mutant used in this treatment;
(g) treatment protein: X, its fragment or mutant, albumin (a)～(e), its fragment or mutant, wherein, the terminal fusion of C-of protein: X, its fragment or mutant and albuminous C-end or albuminous fragment or mutant used in this treatment;
(h) treatment protein: X, its fragment or mutant, albumin (a)～(e), its fragment or mutant, wherein, this treatment N-end and terminal fusion of C-of protein: X, its fragment or mutant and albuminous N-end and C-end or albuminous fragment or mutant;
(i) contain first treatment protein: X, its fragment or mutant and second treatment protein: X, its fragment or the mutant, first treatment protein: X, its fragment or mutant and second treatment protein: X, its fragment or mutant different treatment protein: X, its fragment or mutant and albumin (a)～(e), its fragment or the mutant;
(j) protein: X, its fragment or mutant and albumin (a)～(i), its fragment or mutant use in treatment, wherein, this treatment use protein: X, its fragment or mutant by joint from albumin or albuminous fragment or mutant separation; With
(k) protein: X, its fragment or mutant and albumin (a)～(j), its fragment or mutant are used in treatment, and wherein, albumin fusion protein matter has following formula:
R1-L-R2; R2-L-R1 or R1-L-R2-L-R1,
Further, it is peptide linker that R1 uses protein: X, its fragment or mutant, L for treatment, and R2 is the albumin that contains the amino acid sequence of SEQ ID NO.1, albuminous fragment or mutant.
[2]-2 albumin fusion protein matter (Japanese Unexamined Patent Application Publication 2007-522806) contains the treatment protein in albumin, its fragment or the mutant that is inserted into the amino acid sequence, its fragment or the mutant that contain SEQ ID NO.1: X, its fragment or mutant.
[2]-3 albumin fusion protein matter (Japanese Unexamined Patent Application Publication 2007-522806), contain and be inserted into the treatment protein that contains in albumin, its fragment or the mutant that is selected from the amino acid sequence in following (a)～(1): X, its fragment or mutant,
(a) amino acid 54～61 of SEQ ID NO.1;
(b) amino acid 76～89 of SEQ ID NO.1;
(c) amino acid 92～100 of SEQ ID NO.1;
(d) amino acid/11 70～176 of SEQ ID NO.1;
(e) amino acid 247～252 of SEQ ID NO.1;
(f) amino acid 266～277 of SEQ ID NO.1;
(g) amino acid 280～288 of SEQ ID NO.1;
(h) amino acid 362～368 of SEQ ID NO.1;
(i) amino acid 439～447 of SEQ ID NO.1;
(j) amino acid 462～475 of SEQ ID NO.1;
(k) amino acid 478～486 of SEQ ID NO.1; With
(l) amino acid 560～566 of SEQ ID NO.1.
The albumin fusion protein matter of above-mentioned [2]-1～[2]-3 can not carried out glycosylation.
The albumin fusion protein matter of above-mentioned [2]-1～[2]-3 can be expressed in yeast (yeast that yeast, glycosylation are damaged and proteinase is damaged that glycosylation is damaged etc.).
The albumin fusion protein matter of above-mentioned [2]-1～[2]-3 can be expressed by cells of mamma animals.This cells of mamma animals can be the cells of mamma animals under cultivating.
The albumin fusion protein matter of above-mentioned [2]-1～[2]-3 can further contain the secretion targeting sequencing.
The treatment of using in the albumin fusion protein matter of above-mentioned [2]-1～[2]-3 refers to a kind or more kinds ofly have therapeutic activity and/or bioactive protein, polypeptide, antibody, peptide or its fragment or a mutant with protein.The treatment that comprises among the present invention comprises protein, polypeptide, peptide, antibody and biopreparate (terms such as peptide, protein and polypeptide use with the free burial ground for the destitute in this manual) with protein, but is not limited thereto.Think that particularly term " treatment protein " comprises antibody and fragment and mutant.Therefore, protein of the present invention can contain treatment at least one fragment of protein or at least one fragment or the mutant of mutant and/or antibody.Further, term " treatment protein " can also mention endogenous or the naturally occurring homologue of treatment with protein.
Polypeptide by showing " therapeutic activity " or have the protein of " active in the treatment ", illustrate with this instructions in put down in writing or a kind or more kinds of treatment known in the art with treatments such as protein with protein relevant a kind or the more kinds of known biologically active and/or the polypeptide of therapeutic activity.For example, " treatment protein " is to disposal, the prevention of disease, state or obstacle or improves useful protein, but be not limited thereto.Example as indefiniteness, " treatment protein " can combine specifically with specific cellular type (normal (for example lymphocyte) or unusual (for example cancer cell)), therefore, compound (medicine or cytotoxic agent) can also be used for this cellular type of target specifically.
As the treatment protein that in the albumin fusion protein matter of above-mentioned [2]-1～[2]-3, uses; can enumerate GLP-1; GLP-2; PACAP-27; PACAP-28; VIP; CD4M33; secretin; enteroglucagon; oxyntomodulin; PHM; IFNa; IFNp; ANP; BNP; NGF; BDNF; GDNF; IFN-β; IFN-α; growth hormone release inhibiting hormone; IGF-1; the IL-2 soluble recepter; the IL-2 receptor antagonist; CTLL-2; calcitonin; somatotropin releasing factor; parathyroid hormone; GMCSF; EPO; GCSF; insulin; immunoglobulin (Ig); the serum pseudocholinesterase; α-1 antitrypsin; Aprotinin; the clotting factor that is in precursor form and activity morphology (contains von Willebrand factor; fibrinogen; the II factor; the VII factor; the active VIIA factor; the VIII factor; the IX factor; the X factor; the XIII factor; the c1 inhibiting factor; Antithrombin III; fibrin ferment; factor; apolipoprotein; c-reactive protein and protein C etc.; but be not limited thereto); human growth hormone (HGH) (hGH); single-chain antibody; autocrine motility factor; invasin; laminin; hirudin; ア プ ラ ギ Application (applaggin); monocyte chemoattractant protein (MCP/MCAF); macrophage colony stimulatory factor (M-CSF); osteopontin; platelet factor 4; tenascin; vitronectin; human chorionic gonadotrophin; leptin; enzyme (urokinase for example; B glucocerebrosidase (B-glucocerebrosidase) etc.); the TNF acceptor; growth factor (for example; EGF; FGF-1; FGF-2; NGF; PDGF; VEGF-1 etc.); interleukin (for example; IL-1; IL-4; IL-8; IL-10; IL-11; IL-12 etc.); interleukin-2-receptor (for example IL-4 acceptor etc.); interferon (for example; interferon gamma; interferon ω etc.); TGF (TGF-β for example; TGF-β-1; TGF-β-3); TNF (for example TNF-α); hormone (for example; promoting sexual gland hormone; the human luteinizing hormone; follicle-stimulating hormone (FSH) etc.); DNase; decorin; osteoprotegerin; Tie-2; t-PA etc., but be not limited thereto.Treatment can be used with the form of monomer or fused in tandem thing with protein.
Can also use the interferon hybrid as treatment protein.The interferon hybrid comprises interferon-' alpha '-interferon-' alpha ' hybrid (being called α-α hybrid in this instructions).In a mode, α-α hybrid is made of the interferon-' alpha ' A with interferon-' alpha ' D fusion maybe can contain its (A/D hybrid).In another way, α-α hybrid is made of the interferon-' alpha ' A that merges with interferon-' alpha ' F or contains its (A/F hybrid).And then in another way, α-α hybrid part is made of the interferon-' alpha ' A that merges with interferon-' alpha ' B or contains its (A/B hybrid).These hybrids also are documented in No. the 4414510th, the United States Patent (USP), and the document is by the record source, with it all as the part of this instructions.
The interferon hybrid comprises interferon beta-interferon-' alpha ' hybrid (being called β-α hybrid in this instructions).In a mode, β-α hybrid is made of the interferon beta-1 that merges with interferon-' alpha ' D (also being called Alfacon-1) or contains its (β-1/ α D hybrid).These hybrids also are documented in No. the 4758428th, the United States Patent (USP), and the document is by the record source, with it all as the part of this instructions.
The interferon hybrid comprises interferon-' alpha '-interferon beta hybrid (being called the alpha-beta hybrid in this instructions).In a mode, the alpha-beta hybrid is made of the interferon-' alpha ' D (also being called Alfacon-1) that merges with interferon beta-1 or contains its (α D/ β-1 hybrid).These hybrids also are documented in No. the 4758428th, the United States Patent (USP), and the document is by the record source, with it all as the part of this instructions.
As other treatment protein, comprise in the table 1 of Japanese Unexamined Patent Application Publication 2007-522806 number, Japanese Unexamined Patent Application Publication 2004-506407 number, Japanese Unexamined Patent Application Publication 2003-530838 number, Japanese Unexamined Patent Application Publication 2003-530839 number, Japanese Unexamined Patent Application Publication 2003-530846 number, Japanese Unexamined Patent Application Publication 2003-530847 number, Japanese Unexamined Patent Application Publication 2003-530852 number and Japanese Unexamined Patent Application Publication 2003-531590 number that " treatment protein X " hurdle (hurdle 1) disclosed a kind or more kinds of treatment still is not limited to these with protein, peptide, fragment or mutant.
The albumin that contains in the albumin preparation of the present invention can prepare by existing albumin being implemented the reduction processing.As the method that reduction is handled, method (for example method of TOHKEMY 2007-197394 record) of being undertaken by reductive agent and the method for being undertaken by electrolytic reduction are arranged.As reductive agent more specifically, can enumerate beta-mercaptoethanol, Mercamine Cysteamine, dithiothreitol (DTT) (DTT), dithioerythritol (DTE), sodium borohydride (NaBH
4), the salt of halfcystine, tertiary phosphine (three (2-carboxy ethyl) phosphine, three (methylol) phosphines, tri-n-butyl phosphine) etc., but be not limited to these.
As halfcystine, can enumerate L-halfcystine, D-halfcystine, DL-cysteine, but be preferably the L-halfcystine.For example in containing albuminous aqueous solution, the interpolation ultimate density is the L-halfcystine of 5～50mg/ml (for example 12mg/ml), and incubation under 30～40 ℃ (for example 37 ℃) can reduce the oxidized form albumin thus.
The analyte of unreacted reductive agent, reductive agent can be removed by methods such as ultrafiltration step, chromatograms.The addition of reductive agent in addition can be excessive with respect to the amount of these disulfide bond so long as the amount of the disulfide bond reduction that contains in raw material or the reaction system is got final product.
In order to keep the albuminous reducing condition of reduced form, for example can handle with antioxidants such as L-ascorbic acid, L-ascorbyl stearate, erythorbic acid (arabo-ascorbic acid), catechin, dibutyl hydroxy toluene (BHT), tocopherol (vitamin E, V.E), butylated hydroxyanisole (BHA) (BHA), sodium sulphite, calcium disodium edathamil, guaiac, citric acid isopropyl ester, sodium thiosulfate, L-halfcystine or its salt (for example L-cysteine hydrochloride), sulphuric dioxide, sodium pyrosulfite, n-propyl gallates.
Carried out after the reduction processing oxidized form albumin ratio in the albumin that obtains being measured, can confirm its reducing degree thus.
Albumin content in the albumin preparation of the present invention is not limited especially, be generally 0.01～30w/v%, be preferably 0.1～25w/v%.If albumin content, then can not fully strengthen prevention or result of treatment to the oedema that is accompanied by the hepatopathy generation less than 0.01w/v%.In addition, if serum albumin levels surpasses 30w/v%, then preparation might become unstable.
Albumin preparation of the present invention is the solid pharmaceutical preparation that contains above-mentioned albuminous aqueous solution or can dissolve in use, prepares in the mode of aseptic aqueous solution.Usually, these compositions are dissolved in the distilled water for injection.Therefore, consider the pH of people's body fluid, the pH of said preparation is 5.0～7.4, is preferably pH 6.0～7.4.Adjust agent as pH, use acid such as hydrochloric acid, acetate, citric acid, malic acid.
In the albumin preparation of the present invention, can add the vitamin of necessary amount, for example vitamins and biostearin compounds such as vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin C, vitamin D, vitamin E, nicotinic acid, pantothenic acid, biotin, folic acid, electrolyte such as sodium, potassium, calcium, chlorine, phosphorus, and iron, zinc, manganese, copper, iodine, selenium and other trace elements.
Further, can add sugar, for example maltose, fructose, xylitol etc., and lipid, for example soybean oil, cottonseed oil, sesame wet goods nutrient.In addition, can add other material that stabilizing agents such as sodium bisulfite etc. maybe can give human body.
In addition, in the albumin preparation of the present invention, in order to keep the albuminous reducing condition of reduced form, for example can add the L-ascorbic acid, the L-ascorbyl stearate, erythorbic acid (arabo-ascorbic acid), catechin, dibutyl hydroxy toluene (BHT), tocopherol (vitamin E, V.E), butylated hydroxyanisole (BHA) (BHA), sodium sulphite, calcium disodium edathamil, guaiac, the citric acid isopropyl ester, sodium thiosulfate, L-halfcystine or its salt (for example L-cysteine hydrochloride), sulphuric dioxide, sodium pyrosulfite, antioxidants such as n-propyl gallate.
The addition of antioxidant is different and different according to the kind of antioxidant, can suitably set.When using L-halfcystine or its salt as antioxidant, it is suitable that its addition is counted 0.1～0.5mg/ml with the L-halfcystine.
Albumin preparation of the present invention can be by known method preparation own.For example, can prepare albumin preparation of the present invention by following step:
(1) provide oxidized form albumin ratio less than 60% (preferably less than 25%) albumin and
(2) be dissolved in the aqueous solvent by albumin, obtain being used to prevent or treat the albumin preparation of the oedema that is accompanied by the hepatopathy generation (1).
In step (1), when known albumin has required oxidized form albumin ratio, this albumin can be directly used in the present invention.On the other hand, when known albumin is higher than required oxidized form albumin ratio, can carry out step (1) by this albumin being implemented the reduction processing as mentioned above.
As the aqueous solvent that uses in the step (2), can enumerate sterilized water, physiological saline, physiological buffering liquid etc.Dissolve the albuminous while, can also dissolve pharmaceutically acceptable adjuvant (for example said vitamin class, electrolyte, trace element, nutrient, stabilization agent, antioxidant etc.).
Specifically, by oxidized form albumin ratio is dissolved in the sterilized water less than 60% albumin and proper additive, can prepare albumin preparation of the present invention.In optimal way,, can prepare albumin preparation of the present invention by oxidized form albumin ratio is dissolved in the sterilized water less than 25% albumin and proper additive.
In the preparation method of albumin preparation of the present invention, particularly in the albuminous preparation of using by the gene recombination technology preparation of recombinant type, by oxidized form albumin ratio being measured keeping or managing of the selection of carrying out suitable preparation condition, step.Further, by using the quality standard of oxidized form albumin ratio, can carry out the suitable qualitative control of albumin preparation as development test.
In step (1), use halfcystine (L-halfcystine etc.) as reductive agent, preparation oxidized form albumin ratio is during less than the albumin of 60% (preferably less than 25%), preferably the potpourri after the reduction reaction is removed the low molecular compound (for example molecular weight is the compound below the 1000Da) that contains unreacted halfcystine, cystine, thus oxidized form albumin ratio is carried out purifying less than 60% albumin.Then, in step (2), preferably sublimed albumin is dissolved in the aqueous solvent, every 100ml adds the halfcystine (L-halfcystine etc.) of 10～50mg and keeps agent as reduction.
Preferably albumin preparation of the present invention is sterilized.As sterilizing methods, can be used alone or in combination common sterilization by saturated steam under pressure, low-temperature heat sterilization, sterilization by filtration etc.
Albumin preparation of the present invention is to carrying out through intravenously administrable in periphery intravenous or the central vein etc., common per 1 day, per 1 adult, with about 100～2000ml, preferred about 500～1000ml is benchmark, be divided into 1～2 time with its per 1 day, and consider the symptom for the treatment of the administration patient, nutritional status, age, body weight etc. suitably increase and decrease use.
Below, enumerate embodiment the present invention is carried out more specific description, but the present invention is not limited by embodiment shown below.
[embodiment]
(embodiment 1)
Patient and method
(hepatitis C virus, n=31, hepatitis type B virus, n=3, alcoholic hepatitis, n=5, nonalcoholic fatty liver disease, n=1) analyzes to 40 chronic hepatitis patients.All patients are the state of cirrhosis.The patient of chronic renal failure gets rid of outside analysis in this test.By high performance liquid chromatography the albumin of the oxidized form in the serum/reduced form albumin ratio is measured.This mensuration is carried out according to the method for WO2007/013679 record.Use moisture conservation rate (oedema) as albuminous function.The degree of moisture conservation rate is by direct multifrequency sections bio-electrical impedance analyser (direct segmental multifrequency-bioelectrical impedance analyzer) (InBody3.2, BIOSPACE, Tokyo, Japan) estimate, with extracellular water/all interior water (ECW/TBW) expression.The severe degree of chronic liver disease by Child-Pugh scoring or whole latter stage hepatopathy model (model for end-stage liver disease MELD) estimates.Child-Pugh scoring and MELD scoring are the index of hepatic functional reserve.Correlativity to oxidized form albumin and ECW/TBW, Child-Pugh scoring or MELD scoring is investigated.
The result
All patient's mean age is 68.8 ± 9.7 years old.Oxidized form albumin percent, ECW/TBW, Child-Pugh scoring and MELD scoring are respectively in 20.7～59.3%, 0.327～0.376,5～11 and 6～66 scope.Fig. 1 represents with the longitudinal axis to be that oedema (ECW/TBW), transverse axis are the result that oxidized form albumin percent is mapped and obtained.Discovery has significant positive correlation (σ=0.68, p＜0.0001) (Fig. 1) between oxidized form albumin percent and ECW/TBW.Fig. 2 represents with the longitudinal axis to be the result that oedema (ECW/TBW), transverse axis are mapped and obtained for the seralbumin level of measuring in the past.(σ=-0.49 p=0.002) also have conspicuousness (Fig. 2) statistically, but the correlativity of oxidized form albumin percent and ECW/TBW is more remarkable for correlativity between discovery seralbumin level and the ECW/TBW.By these results as can be known, oxidized form albumin percent is the index of the excellence of oedema.Simultaneously as can be known, the reduced form albumin is compared with the oxidized form albumin, and colloid osmotic pressure keeps the function excellence.Further, for the correlativity of oxidized form albumin percent and liver reserve function is investigated, be that Child-Pugh scoring or MELD scoring, transverse axis are that oxidized form albumin percent is mapped with the longitudinal axis.The result as shown in Figure 3, oxidized form albumin percent and Child-Pugh scoring, MELD scoring all show significant positive correlation (being respectively σ=0.51, p=0.013, σ=0.44, p=0.006) (Fig. 3).These presentation of results oxidized form albumin percent can the predictive hepatocirrhosis patient prognosis.
As from the foregoing, the severe degree of the degree of oxidized form albumin percent and moisture conservation rate and chronic liver disease is relevant.The oxidized form albumin is useful as the new index that the severe degree of albuminous function and chronic hepatitis patients is estimated.
(embodiment 2)
Be compared to the influence that brings by the symptom after giving albumin and treating in order to observe oxidized form/reduced form albumin in the albumin preparation, carry out following test.
With physiological saline commercially available albumin preparation is diluted to 5%, with whole blood sample similarly, sampling 0.9ml has in the microtubule of stabilizing agent to adding, put upside down mix and stored refrigerated after, measure redox albumin ratio.
Oxidized form/reduced form albumin is than passing through HPLC method (Int.J.Peptide Protein Res.24:96; 1984) measure.
The result obtains following value.
(1) changes blood and grind albumin oxidized form albumin 62.7%, reduced form albumin 37.3%
(2) Block ミ ネ one ト oxidized form albumin 61.5%, reduced form albumin 38.5%
(3) ア Le Block ミ Na one oxidized form albumin 49.2%, reduced form albumin 50.8%
(4) Mitsubishi's albumin oxidized form albumin 50.7%, reduced form albumin 49.3%
Gave liver cirrhosis patient 3 days with these albumin preparations with 25g/ days consumption, to before the administration and body weight afterwards, BMI, BUN, kreatinin, Na, albumin, ECF/TBF, gross protein, lymphocyte number, prothrombin time, total bilirubin and Child-Pugh scoring compare in blood sugar, serum osmotic pressure, oedema degree of improvement, the serum on an empty stomach.ECF/TBF measures with bioelectrical impedance analysis device (InBody).The result is divided into the oxidized form albumin that given albumin preparation than being that group (albumin preparation (1) and (2)) more than 60% is tabulated with the group (albumin preparation (3) and (4)) below 51%.TBF=total body fluid (total body fluid), the amount of moisture of expression biosome, ECF=extracellular fluid (extracellular fluid), expression extracellular fluid.
Patient's background before each albumin preparation administration is as shown in table 1.
[table 1]
Unknown significance difference all in arbitrary project.
Secondly, before the albumin preparation administration and the result of each assessment item afterwards as shown in table 2.
[table 2]
Found that only the oxidized form albumin is than being in the group below 51%, before the administration and in body weight, BMI, ECF/TBF, have conspicuousness to improve afterwards.
ECF/TBF be worth big more then expression and worsens more how as the index of oedema, and still only the oxidized form albumin is than being that ECF/TBF reduces significantly in the group below 51%, and it is high more that the oedema of representing the albumin preparation that the oxidized form albumin content is low is more thus improved effect.
(embodiment 3)
Handle the reduction of the oxidized form albumin ratio in the albumin preparation that obtains by halfcystine
In serum albumin preparations (change blood grind human serum albumins concentration 25%) 1ml, add the L-halfcystine of 12mg, with potpourri 37 ℃ of following incubations 90 minutes.In contrast, in serum albumin preparations, do not add the L-halfcystine, room temperature or 37 ℃ of following incubations 90 minutes.
By Solid-Phase Extraction, by the reactant liquor purification of albumin.The condition of Solid-Phase Extraction (with reference to WO2007/013679) as described below.
Solid-phase extraction column Varian Bond Elut C18 EWP 50mg/1ml
(1) washing 0.1% formic acid-90% acetonitrile solution 2ml
(2) equilibrating pure water 2ml
(3) application of sample 500 μ l (total amount that 50 times of reactant liquor 10 μ l dilutions is obtained with phosphate buffer)
(4) washing 0.1% formic acid-10% acetonitrile solution 1.5ml
(5) wash-out 0.1% formic acid-90% acetonitrile solution 100 μ l
With ESI-TOFMS the wash-out flow point is analyzed (flow velocity 2 μ l/ minutes) by continuous injection method.
The result of mass spectrophotometry observes the minimizing at the albuminous peak of oxidized form and the increase (Fig. 4) at the albuminous peak of reduced form for by adding the L-halfcystine.
And, the oxidation before handling/go back proalbumin than with room temperature under, 90 minutes processed group are identical.
As can be known from the above results, by utilizing the L-halfcystine, can reduce the albuminous ratio of oxidized form in the albumin preparation as reductive agent.
(formulation example 1)
(in the 50ml aqueous solution)
Reductive agent is handled albumin 12.5g
Acetyl group L-Tryptophan sodium 268.3mg
Sodium Caprylate 166.2mg
L-halfcystine 15mg
The agent of isotonic agent (drug such as grade) (sodium chloride) pH regulator is an amount of
pH?6.2～7.6
(formulation example 2)
(in the 50ml aqueous solution)
Reductive agent is handled albumin 12.5g
Acetyl group tryptophane 246.3mg
Sad 144.2mg
PH regulator agent (NaOH) is an amount of
PH regulator agent (sodium bicarbonate) is an amount of
PH regulator agent (glacial acetic acid) is an amount of
Sodium chloride is an amount of
L-cysteine hydrochloride 20mg
pH?6.4～7.4
Industrial applicibility
According to the present invention, by the shared ratio of oxidized form albumin in the albumin in the organism sample that is obtained by object is measured, can be rapidly and be accompanied by exactly the judgement of the oedema degree that hepatopathy produces, such as treatment monitoring, prognosis prediction etc., and then can carry out preventing or treating the screening of the compound of the oedema that is accompanied by the hepatopathy generation.
March 31 in 2008) and the Japanese Patent Application 2008-306805 (applying date: on Dec 1st, 2008), its content is all comprised in this manual the application is based in the Japanese Patent Application 2008-090544 of the Japanese publication (applying date:.
Claims (12)
1. be accompanied by the decision method of the oedema of hepatopathy generation, comprise the shared ratio of oxidized form albumin in the albumin in the organism sample that is obtained by the experimenter is carried out quantitatively.
2. the method for claim 1, wherein judge the oedema degree based on the positive correlation between oxidized form albumin ratio and the oedema degree.
3. be used to judge the kit of the oedema that is accompanied by hepatopathy and produces, contain to be useful on the shared ratio of oxidized form albumin in the albumin in the organism sample is carried out quantitative reagent.
4. can prevent or treat the screening technique of the compound of the oedema that is accompanied by the hepatopathy generation, may further comprise the steps:
(1) give candidate compound to the non-human mammal of suffering from the oedema that the hepatopathy of being accompanied by produces,
(2) the shared ratio of oxidized form albumin in the albumin in the organism sample that is obtained by the non-human mammal that has been given this candidate compound is carried out quantitatively,
(3) the shared ratio of oxidized form albumin in the albumin in the shared ratio of oxidized form albumin in the albumin in the organism sample that is obtained by this non-human mammal and the organism sample that is obtained by the contrast mammal that does not give candidate compound is compared and
(4) result based on the comparison, the compound of selecting to have reduced oxidized form albumin ratio is as preventing or treat the compound that is accompanied by the oedema that hepatopathy produces.
5. be used to prevent or treat the albumin preparation that is accompanied by the oedema that hepatopathy produces, contain oxidized form albumin ratio less than 60% albumin.
6. be used to prevent or treat the albumin preparation that is accompanied by the oedema that hepatopathy produces, contain oxidized form albumin ratio less than 25% albumin.
7. albumin preparation as claimed in claim 6, wherein, oxidized form albumin ratio is essentially 0%.
8. as any described albumin preparation in the claim 5～7, further contain antioxidant.
9. be used to prevent or treat the preparation method of the albumin preparation of the oedema that is accompanied by the hepatopathy generation, may further comprise the steps:
(1) provide oxidized form albumin ratio less than 60% albumin and
(2) be dissolved in the aqueous solvent by albumin, obtain being used to prevent or treat the albumin preparation of the oedema that is accompanied by the hepatopathy generation (1).
10. be used to prevent or treat the preparation method of the albumin preparation of the oedema that is accompanied by the hepatopathy generation, may further comprise the steps:
(1) provide oxidized form albumin ratio less than 25% albumin and
(2) be dissolved in the aqueous solvent by albumin, obtain being used to prevent or treat the albumin preparation of the oedema that is accompanied by the hepatopathy generation (1).
11. as claim 9 or 10 described preparation methods, wherein, step (1) is undertaken by albumin being implemented the reduction processing.
12. preparation method as claimed in claim 11 wherein, in reduction is handled, uses halfcystine as reductive agent.
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| JP2008306805 | 2008-12-01 | ||
| PCT/JP2009/056586 WO2009123162A1 (en) | 2008-03-31 | 2009-03-31 | Method for determination of edema associated with liver disease |
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| CN108072762A (en) * | 2016-11-16 | 2018-05-25 | 基立福环球运营有限公司 | In-vitro diagnosis method based on the Alzheimer disease of albumin redox level in cerebrospinal fluid |
| CN108350035A (en) * | 2015-09-25 | 2018-07-31 | 纳罗纳公司 | Ring type polypeptide, its preparation method and its application in the treatment |
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|---|---|---|---|---|
| WO2012121094A1 (en) * | 2011-03-04 | 2012-09-13 | 学校法人久留米大学 | Albumin-containing intravenous injection |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6056925A (en) * | 1983-09-09 | 1985-04-02 | Asahi Chem Ind Co Ltd | Albumin preparation |
| JPS61194026A (en) * | 1985-02-25 | 1986-08-28 | Asahi Medical Kk | Production of albumin drug preparation |
| JP3383998B2 (en) * | 1992-08-06 | 2003-03-10 | 東ソー株式会社 | Method for producing polyolefin |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61155397A (en) * | 1984-12-28 | 1986-07-15 | Asahi Medical Kk | Quick separation of albumin by chromatography |
| WO2007013679A1 (en) * | 2005-07-27 | 2007-02-01 | Ajinomoto Co., Inc. | Method for analysis of albumin in sample solution |
| KR20080041675A (en) * | 2005-08-04 | 2008-05-13 | 아지노모토 가부시키가이샤 | Agent for reduction of oxidized albumin level |
-
2009
- 2009-03-31 CN CN2009801118328A patent/CN102066938A/en active Pending
- 2009-03-31 JP JP2010505919A patent/JP5476538B2/en not_active Expired - Fee Related
- 2009-03-31 KR KR1020107024136A patent/KR20100127862A/en not_active Application Discontinuation
- 2009-03-31 TW TW098110765A patent/TW201003073A/en unknown
- 2009-03-31 WO PCT/JP2009/056586 patent/WO2009123162A1/en active Application Filing
-
2013
- 2013-09-11 JP JP2013188816A patent/JP2014029339A/en not_active Ceased
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2015
- 2015-09-17 JP JP2015184415A patent/JP2016014044A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6056925A (en) * | 1983-09-09 | 1985-04-02 | Asahi Chem Ind Co Ltd | Albumin preparation |
| JPS61194026A (en) * | 1985-02-25 | 1986-08-28 | Asahi Medical Kk | Production of albumin drug preparation |
| JP3383998B2 (en) * | 1992-08-06 | 2003-03-10 | 東ソー株式会社 | Method for producing polyolefin |
Non-Patent Citations (4)
| Title |
|---|
| SUZUKI等: "Mansei Kan Shikkan ni Okeru Kessei Albumin-chi Sokutei no Mondai", 《JAPANESE JOURNAL OF NUTRITIONAL ASSESSMENT》 * |
| 中华医学论文集编辑部: "《中华医学论文集》", 30 April 1990 * |
| 内田幸江，奥住裕二，藤代政浩，川村憲弥: "慢性肝疾患における栄養指標としての血清アルブミン値測定の問題点", 《临床病理》 * |
| 杜永金主编: "《最新药品集中招标采购实用指南 中》", 31 August 2000 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108350035A (en) * | 2015-09-25 | 2018-07-31 | 纳罗纳公司 | Ring type polypeptide, its preparation method and its application in the treatment |
| CN108072762A (en) * | 2016-11-16 | 2018-05-25 | 基立福环球运营有限公司 | In-vitro diagnosis method based on the Alzheimer disease of albumin redox level in cerebrospinal fluid |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2009123162A1 (en) | 2009-10-08 |
| JP2014029339A (en) | 2014-02-13 |
| KR20100127862A (en) | 2010-12-06 |
| TW201003073A (en) | 2010-01-16 |
| JPWO2009123162A1 (en) | 2011-07-28 |
| JP2016014044A (en) | 2016-01-28 |
| JP5476538B2 (en) | 2014-04-23 |
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