CN1173737C - Application of Hepcidin in pharmacy - Google Patents
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- CN1173737C CN1173737C CNB021463093A CN02146309A CN1173737C CN 1173737 C CN1173737 C CN 1173737C CN B021463093 A CNB021463093 A CN B021463093A CN 02146309 A CN02146309 A CN 02146309A CN 1173737 C CN1173737 C CN 1173737C
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- hepcidin
- iron
- ferrum
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- liver
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- 102000018511 hepcidin Human genes 0.000 title claims abstract description 115
- 108060003558 hepcidin Proteins 0.000 title claims abstract description 115
- 229940066919 hepcidin Drugs 0.000 title claims abstract description 115
- XJOTXKZIRSHZQV-RXHOOSIZSA-N (3S)-3-amino-4-[[(2S,3R)-1-[[(2S)-1-[[(2S)-1-[(2S)-2-[[(2S,3S)-1-[[(1R,6R,12R,17R,20S,23S,26R,31R,34R,39R,42S,45S,48S,51S,59S)-51-(4-aminobutyl)-31-[[(2S)-6-amino-1-[[(1S,2R)-1-carboxy-2-hydroxypropyl]amino]-1-oxohexan-2-yl]carbamoyl]-20-benzyl-23-[(2S)-butan-2-yl]-45-(3-carbamimidamidopropyl)-48-(hydroxymethyl)-42-(1H-imidazol-4-ylmethyl)-59-(2-methylsulfanylethyl)-7,10,19,22,25,33,40,43,46,49,52,54,57,60,63,64-hexadecaoxo-3,4,14,15,28,29,36,37-octathia-8,11,18,21,24,32,41,44,47,50,53,55,58,61,62,65-hexadecazatetracyclo[32.19.8.26,17.212,39]pentahexacontan-26-yl]amino]-3-methyl-1-oxopentan-2-yl]carbamoyl]pyrrolidin-1-yl]-1-oxo-3-phenylpropan-2-yl]amino]-3-(1H-imidazol-4-yl)-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-4-oxobutanoic acid Chemical compound CC[C@H](C)[C@H](NC(=O)[C@@H]1CCCN1C(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](Cc1cnc[nH]1)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(O)=O)[C@@H](C)O)C(=O)N[C@H]1CSSC[C@H](NC(=O)[C@@H]2CSSC[C@@H]3NC(=O)[C@@H]4CSSC[C@H](NC(=O)[C@H](Cc5ccccc5)NC(=O)[C@@H](NC1=O)[C@@H](C)CC)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](Cc1cnc[nH]1)NC3=O)C(=O)NCC(=O)N[C@@H](CCSC)C(=O)N2)C(=O)NCC(=O)N4)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XJOTXKZIRSHZQV-RXHOOSIZSA-N 0.000 title claims abstract description 114
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- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 abstract description 3
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
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- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
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- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention relates to a new application of an antibacterial polypeptide which is named as hepcidin and contains cystine to the field of medicine preparation. Hepcidin purified from human urine is transported to the duodenal recess cells through blood stream to directly act on a beta2 M/HFE/TfR1 compound with a normal iron absorption capability to increase the iron absorption capacity of the recess cells, and the iron volume in the recess cells, so that the descending of the expressions of four kinds of iron absorption protein in intestinal absorption epithelial cells is inhibited, and therefore, an iron absorption capacity is reduced. When iron circulation is low, the iron absorption capacity of the liver cells mediated through ferritin receptors TFE2 is reduced, and hepcidin synthesized and secreted by the liver is reduced; therefore, the iron volume in the recess cells is diminished, the expression of intestinal absorption epithelium associated protein is increased, and the intestinal iron absorption capacity is increased, so that the level of iron returns to a normal level.
Description
Technical field
The present invention relates to the purposes of Hepcidin (Hepcidin), particularly its purposes in pharmaceutical field.
Background technology
Hepcidin (Hepcidin) is an antibacterial polypeptide that is rich in cysteine, its molecular structure
2000, at first from human blood separation and purification this polypeptide, be referred to as the antibacterial polypeptide of liver expression.Calendar year 2001 also is separated to this polypeptide from Urina Hominis, and with its called after Hepcidin (Hepcidin).The Hepcidin that separates in the Urina Hominis includes 20,22 and 25 three kinds of composition forms such as amino acid polypeptide, and molecular weight is between 2-3kDa.Under the normal ph situation, these polypeptide have+3 electric charges.20 and 25 amino acid polypeptides are main existence forms of Hepcidin (Hepcidin).Their C-end has all kept the enrichment region that cysteine forms, and difference only is the amino acid number difference that amino-terminal end is cut off.Hepcidin (Hepcidin) contains 8 cysteine and forms 4 disulfide bond, the CD spectrum characteristic studies confirm that, in phosphate buffer, Hepcidin has two stable β folding structures, and the cystine knot of this structure and antibacterial polypeptide (cystine knot) structure is closely similar.Human Hepcidin (Hepcidin) is in building-up process, at first produce an early stage polypeptide of forming by 84 aminoacid, be degraded then to form and contain the precursor peptide that 60 aminoacid are formed, generate the peptide molecule of biologically active at last from single precursor peptide.The same with the antibacterial polypeptide of finding plant, insects and software animal in the past, that human Hepcidin (Hepcidin) also has is antibiotic widely, antifungal, antiprotozoal effect.Can be used for preparing antibiotic, antifungal, antiprotozoal medicine.
Summary of the invention
The object of the present invention is to provide the new purposes of Hepcidin (Hepcidin), i.e. new application in pharmacy.
In fact the present invention relates to the application of Hepcidin (Hepcidin) as preparation treatment iron metabolism disorders medicine.
Promptly relate to Hepcidin (Hepcidin) and alleviate or treat the intravenous injection of ferrum overload class disease medicament as preparation.
Also relate to the intravenous injection of the monoclonal antibody of Hepcidin (Hepcidin) as the iron deficiency anemia medicine of preparation treatment any kind.
Ferrum is one of the abundantest essential trace element of human body, the metabolic process of life that its wide participation is important.The illness that the iron metabolism disorder causes is human modal disease, affects the population of counting in necessarily.Iron deficiency can cause multiple diseases such as anemia, amentia.Excessive then can the inspiring of ferrum produces a large amount of free radicals, causes neurodegeneration and many and old relevant disease.Therefore, human body exists strict iron metabolism regulatory mechanism, can guarantee that ferrum is in the normal physiological level all the time in the body.The key of this body ferrum stable state (iron homeostasis) is the balance that depends between absorption of small intestinal ferrum and the body ferrum needs.When body lacked (or needs) ferrum, small intestinal ferrum absorbs can corresponding increase, and the body iron content is when too high, and small intestinal then can reduce ferrum and absorb.For a long time, people infer the similar hormonelike material that has adjusting ferrum stable state in the human body always, and this material dissolves in blood, therefore can be at liver, small intestinal, transmit the signal that relevant ferrum is regulated between reticulocyte and the RE RE system.Studies show that Hepcidin (Hepcidin) is exactly the important hormone of the long-term adjusting ferrum stable state of always seeking of iron metabolism researcheres.Animal experiment proves, Hepcidin (Hepcidin) shortage can cause mice to carry out sex organization's ferrum gathering, and serious iron deficiency appears in the transgenic mouse of Hepcidin (Hepcidin) high expressed, presents serious small cell anemia and life span and shortens dramatically.In addition, dietary iron content increases or the increase of liver ferrum all can cause liver Hepcidin (Hepcidin) express to increase.Confirm that Hepcidin has important function in the body iron metabolism is regulated.The mechanism pattern that Hepcidin (Hepcidin) regulates iron metabolism is, when any reason causes that circulation ferrum increases, hepatocyte increases and takes the photograph ferrum through TfR2 (being a kind of year ferritin receptor that is different from classical TfR), cause hepatocyte to increase synthetic and secrete Hepcidin (Hepcidin) entering blood, Hepcidin (Hepcidin) is transported to the duodenal recess cell through blood flow and directly acts on normal ferrum energy-absorbing power β
2The M/HFE/TfR1 complex facilitates the pit cell iron-intaking quantity to increase, and the increase of ferrum pond and then four kinds of ferrum absorptions of inhibition intestinal absorption epithelial cell protein expression descend in the pit cell, and the ferrum absorbtivity is decline therefore.When circulation ferrum was low, the hepatocyte ferrum picked-up that mediates through TfR2 will reduce, and the Hepcidin of liver synthesis secretion (Hepcidin) will reduce.This will cause that pit cell ferrum pond diminishes, and intestinal absorption epithelium correlative protein expression increases, and intestinal ferrum absorbtivity increases, and iron level is returned to normally.But the Hepcidin of while hepatic secretion also menses flows to the reticuloendothelial system RE system that reaches, and regulates the iron metabolism of RE system and changes.This pattern shows that Hepcidin (Hepcidin) is the transmitter of iron metabolism conditioning signal between liver, small intestinal and the RE system.And the Hepcidin of high concentration (Hepcidin) can be transported to through blood and reach the duodenal recess cytosis in β
2The M/HFE/TfR1 complex causes small intestinal ferrum to absorb and descends.The basic cause of struvite anemia that Here it is or chronic disease concurrency anemia, this also is why circulate ferrum and small intestinal ferrum of these anemias patient absorbs the reason that all reduces.If suppress the synthetic or secretion of the Hepcidin (Hepcidin) of this class patient's liver, perhaps block Hepcidin (Hepcidin) and β
2The M/HFE/TfR1 complex interacts, and all should increase small intestinal ferrum and absorb, and reaches the purpose of this class anemia of effective treatment.The unusual Hepcidin that raises also may be the reason of the clinical anemia of other type.Obviously, anti-Hepcidin effect class material also can improve the iron deficiency situation by the approach that increases the absorption of small intestinal ferrum and treat any kind iron deficiency anemia effectively.On the other hand, the synthetic or paracrisis of the liver Hepcidin (Hepcidin) that causes of any reason may be the cause of clinical ferrum overload class disease.Therefore give exogenous Hepcidin (Hepcidin) and can alleviate or treat effectively this class disease.Here it is utilizes Hepcidin exploitation preparation effectively to treat the fabulous foundation of medicine of all kinds of anemias or ferrum characteristic of concentration disease.
Zoopery:
Experimental technique:
Experiment is divided into four groups with totally 40 of white mice, 10 every group.First group, the second group normal feedstuff of feeding, iron content 60mg/kg feedstuff.The 3rd group, the 4th group high ferro feedstuff of feeding, every kilogram of normal feedstuff adds 2.5% carbonyl iron.0.1 milliliter of first group, the 3rd group white mice tail vein injection saline every day in contrast, second group and the 4th group Hepcidin (Hepcidin) that injects mice every day is 0.1 milliliter (every ml physiological saline contains 10 microgram Hepcidins), injects continuously 7 days.After experiment finished, broken end was got blood, is got liver, measures serum levels of iron and liver iron respectively.
Experimental result:
The injection Hepcidin is after 7 days, and the normal feedstuff mice serum ferrum of feeding is than the remarkable reduction of matched group, and difference is P＜0.01 extremely significantly, n=10.Show that Hepcidin (Hepcidin) has the effect of reduction mice to the absorption of ferrum.The mice of the high ferro of feeding feedstuff, serum levels of iron obviously raises than matched group, P＜0.05, n=10.Show that the high ferro diet can increase the absorption of the serum levels of iron of mice.The injected in mice Hepcidin (Hepcidin) of the high ferro of feeding feedstuff, serum levels of iron content is compared remarkable reduction with the high ferro feedstuff mice of feeding merely, and difference is extremely remarkable, P＜0.01, n=10.Hepcidin (Hepcidin) has prevented the formation of high ferro effectively.And inject Hepcidin (Hepcidin) after 7 days, the normal feedstuff mouse liver ferrum of feeding reduces than matched group, but does not have statistical significance, P=0.067, n=10.Show under the normal diet situation that tail vein injection Hepcidin (Hepcidin) 7 days is for not significantly effect of the absorption that reduces mouse liver ferrum.The mice of the high ferro of feeding feedstuff, liver ferrum obviously raises than matched group, P＜0.05, n=10.Show that the high ferro diet can increase the storage of the liver ferrum of mice.The injected in mice Hepcidin (Hepcidin) of the high ferro of feeding feedstuff, liver iron is compared remarkable reduction with the high ferro feedstuff mice of feeding merely, and difference is extremely remarkable, P＜0.01, n=10.Under the high ferro situation, Hepcidin (Hepcidin) reduces the effect of liver ferrum absorption than under the normal diet situation being height.Above experimental result proof Hepcidin (Hepcidin) can suppress the ferrum of animal and take in, and stops animal to form the high ferro state.Its effect is especially more obvious under animal high ferro situation.Studies show that Hepcidin (Hepcidin) knock out mice liver has tangible deposition of iron, the plain thesaurismosis of similar hereditary hemochromatosis, serious anemia when transgenic mice then causes the mice birth because of Hepcidin (Hepcidin) overexpression, just dead soon.Excessive the increasing that proves ferrum in the human body thus can be treated with Hepcidin (Hepcidin).The then available Hepcidin of shortage (Hepcidin) antibody of ferrum is regulated in the human body.
The Hepcidin that will purify from Urina Hominis (Hepcidin) is made as intravenous injection and injects human body, can strengthen Hepcidin (Hepcidin) and β
2The M/HFE/TfR1 complex interacts, and reduces the expression that intestinal ferrum absorbs associated protein, reduces the absorption of ferrum, thereby alleviates or treat ferrum overload class disease effectively.
And Hepcidin (Hepcidin) monoclonal antibody of preparation is injected human body as intravenous injection, make its in conjunction with the Hepcidin in the blood (Hepcidin) thus blocking-up Hepcidin (Hepcidin) and β
2The M/HFE/TfP1 complex interacts, and just can increase the expression that intestinal ferrum absorbs associated protein, and the ferrum picked-up is increased, and reaches the sideropenic purpose of treatment.
The specific embodiment
Embodiment 1, and the Hepcidin that purifies from Urina Hominis (Hepcidin) makes medicine Hepcidin (Hepcidin) intravenous injection of alleviating or treating ferrum overload disease.
Injection
1, collection filtration healthy human urine liquid is removed wherein cell and impurity,
2, obtain cationic polypeptide with the weak cation exchange resin extraction,
3, with 5 percent acetic acid eluting, use the reverse phase liquid chromatography purification then, used C18 pillar (Vydac) is with centesimal trifluoracetic acid balance,
4, resulting polypeptide carries out gradient (4%) eluting with acetonitrile, washes 5 minutes with 20% acetonitrile then, uses 0.5% acetonitrile gradient eluting at last 30 minutes,
5, analyze the peak value polypeptide that high pressure liquid chromatography obtains with acid urea-polyacrylamide gel electrophoresis, identify with the MALDI-TOF-MS method,
6, with Hepcidin (Hepcidin) as intravenous injection.
Embodiment 2: make Hepcidin (Hepcidin) monoclonal antibody as treatment sideropenic medicine Hepcidin (Hepcidin) monoclonal antibody intravenous injection.
1, make up phage antibody library,
2, screening phage antibody library: the Hepcidin behind the purification (Hepcidin) direct coated elisa plate (enzyme-linked immunosorbent assay), through the screening of 3-4 wheel ELISA method, at last the bonded phage with specific antibody of specificity is eluted, infect recipient bacterium, cultivate shop, back ware
3, the solubility expression of the antibody of Fab: the positive colony after above-mentioned screening, after 37 degree are cultivated, extract plasmid.Enzyme action is removed gene III, rebuilds plasmid vector, transfection XLI-Blu, and cultivation, screening positive clone are cultivated the centrifugal collection antibacterial in back, multigelation three times, 4 degree are centrifugal, draw supernatant, obtain the Fab antibody of solubility,
4, Fab purifying antibody and evaluation: adopt anti-Fab antibody affinity chromatography method of purification purification, the polyacrylamide gel electrophoresis purity assay is carried out antibody function at last and is measured,
5, with Hepcidin (Hepcidin) monoclonal antibody as intravenous injection.
Claims (1)
- The application of Hepcidin (Hepcidin) in pharmacy is characterized in that " Hepcidin " intravenous injection as preparation alleviation or treatment ferrum overload class disease medicament.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021463093A CN1173737C (en) | 2002-10-21 | 2002-10-21 | Application of Hepcidin in pharmacy |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021463093A CN1173737C (en) | 2002-10-21 | 2002-10-21 | Application of Hepcidin in pharmacy |
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| Publication Number | Publication Date |
|---|---|
| CN1403154A CN1403154A (en) | 2003-03-19 |
| CN1173737C true CN1173737C (en) | 2004-11-03 |
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| CNB021463093A Expired - Fee Related CN1173737C (en) | 2002-10-21 | 2002-10-21 | Application of Hepcidin in pharmacy |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7820163B2 (en) | 2007-11-02 | 2010-10-26 | Eli Lilly And Company | Anti-hepcidin antibodies and uses thereof |
| US8609817B2 (en) | 2008-08-06 | 2013-12-17 | Eli Lilly And Company | Anti-hepcidin-25 selective antibodies and uses thereof |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010517529A (en) * | 2007-02-02 | 2010-05-27 | アムジエン・インコーポレーテツド | Hepcidin and hepcidin antibody |
| EP2294087B1 (en) * | 2008-05-01 | 2014-05-14 | Amgen, Inc. | Anti-hepcidin antibodies and methods of use |
| CN106176801B (en) * | 2015-05-06 | 2020-06-09 | 复旦大学 | Use of hydrogen sulfide in the preparation of medicaments for the treatment of inflammatory anemia |
| CN117859898A (en) * | 2024-01-17 | 2024-04-12 | 上海健力达生物科技有限公司 | Application of ergothioneine in preparation of iron absorption promoter and iron absorption promoter |
-
2002
- 2002-10-21 CN CNB021463093A patent/CN1173737C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7820163B2 (en) | 2007-11-02 | 2010-10-26 | Eli Lilly And Company | Anti-hepcidin antibodies and uses thereof |
| US8329174B2 (en) | 2007-11-02 | 2012-12-11 | Eli Lilly And Company | Anti-hepcidin antibodies and uses thereof |
| US8765129B2 (en) | 2007-11-02 | 2014-07-01 | Eli Lilly And Company | Anti-hepcidin antibodies and uses thereof |
| US8609817B2 (en) | 2008-08-06 | 2013-12-17 | Eli Lilly And Company | Anti-hepcidin-25 selective antibodies and uses thereof |
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| Publication number | Publication date |
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| CN1403154A (en) | 2003-03-19 |
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