CN1816327B - Use of HIF alpha stabilizers for enhancing erythropoiesis - Google Patents

Use of HIF alpha stabilizers for enhancing erythropoiesis Download PDF

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CN1816327B
CN1816327B CN2004800193070A CN200480019307A CN1816327B CN 1816327 B CN1816327 B CN 1816327B CN 2004800193070 A CN2004800193070 A CN 2004800193070A CN 200480019307 A CN200480019307 A CN 200480019307A CN 1816327 B CN1816327 B CN 1816327B
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hydrogen atom
amino
chemical compound
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anemia
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CN1816327A (en
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斯蒂芬·J.·克劳斯
克里斯托弗·J.·莫林奥克斯
托马斯·B.·内夫
福尔克马尔·京茨勒-普卡尔
英格丽德·兰瑟特莫帕罗布克
托德·W.·西利
罗伯特·C.·斯蒂芬森
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Abstract

The present invention relates to methods and compounds for regulating or enhancing erythropoiesis and iron metabolism, and for treating or preventing iron deficiency and anemia of chronic disease.

Description

Strengthen the application of erythropoietic HIF alpha stabilizers
The serial number that the application requires on June 6th, 2003 to submit is 60/476, serial number 704, that on April 29th, 2004 submitted is 60/566, serial number 488, that on April 29th, 2004 submitted is 60/566, serial number 237 and that on May 10th, 2004 submitted is 60/569,797 U.S. Provisional Application No..All above-mentioned applications are incorporated this paper into as a reference in full with it.
Invention field
The present invention relates to be used to regulate or strengthen the method and the chemical compound of erythrocyte generation and iron metabolism, treatment or prevention iron deficiency and chronic disease anemia.
Background of invention
Anemia generally is meant hemoglobin that the level that causes oxygen in the blood reduces or erythrocytic any unusual.Anemia also can comprise that the chronic diseases such as pathological changes of the struvite inhibition that next bone marrow take place are relevant with for example chronic infection, tumor disease, chronic inflammatory disease and take place.The chronic disease anemia is one of modal syndrome in the medical domain.
The chronic disease anemia (anemia of chronic disease, ACD) relevant with iron deficiency usually.ACD can insufficient owing to the availability of ferrum (for example iron deficiency anemia) or is enough but hemoglobin production defective (for example functional iron deficiency) takes place at the ferrum of whole machine body.Ferrum is that bone marrow erythropoiesis precursor generation corpuscular hemoglobin is necessary.
In the patient of chronic disease anemia, observe many physiology's defectives, comprise that erythropoietin (EPO) produces reduction, the EPO of bone marrow replys reduction, reaches the iron metabolism reduction, comprise from the ferrum of intestinal absorption and reducing, the ferrum of striding the enterocyte transhipment reduces, by film iron transfer auxilin (hephaestin) or ceruloplasmin the ferrum Oxidation that ferrum is oxidized to the ferric iron state is reduced, the combination of the ferrum that is undertaken by transferrin and transferrin receptor and picked-up reduce, reach to the transhipment reduction of the ferrum of bone marrow (utilize the position of ferrum, comprise that haemachrome is synthetic).Individually or synergistically, these physiological defectives have caused erythropoiesis invalid or weaken, and cause to produce with hemoglobin to reduce and the transhipment of oxygen reduces relevant microcytic anemia and low hematochrome erythrocyte.
The chronic disease anemia increases relevant (Means (1995) Stem cells 13:32-37 and Means (1999) Int J Hematol 70:7-12) with releasing and activity of inflammatory cytokines, and described cytokine comprises for example tumor necrosis factor-alpha (TNF-α), interleukin-1 ' beta ' (IL-1 β), IL-6 and interferon-(IFN-γ).In the animal model system, inflammatory cytokine negative effect mediation EPO produces, EPO replys and the coordinately regulated ability of iron metabolism (Roodman et al. (1989) Adv Exp Med Biol 271:185-196 in some external and bodies; Fuchs et al. (1991) Eur J Hematol 46:65-70; Jelkmann et al. (1994) Ann NY Acad Sci718:300-311; Vannucchi et al. (1994) Br J Hematol 87:18-23; And Oldenburg et al. (2001) Aliment Pharmacol Ther 15:429-438).Give the anemia situation (Clibon et al. (1990) Exp Hematol 18:438-441) that erythropoietin can not reverse the mice that continues to be exposed to TNF-α.The level increase of inflammatory cytokine such as TNF-α, IL-1 β and INF-γ causes the EPO that observes defective in chronic disease anemia patient to produce and EPO resistance (Jelkmannet al. (1991) Ann NY Acad Sci 718:300-311 and Macdougall and Cooper (2002) Neprol Dial Transplant 17 (11): 39-43).Therefore, various cytokines are arranged, for example inflammatory cytokine and the cytokine relevant with inflammation, participate in the pathogenetic many aspects of chronic disease anemia, comprise inhibition, the inhibition that EPO produces of hemopoietic progenitor cell and be used for the release of erythrogenic ferrum and the weakening of ferrum availability.
Therefore the method for chronic disease anemia need be treated or prevent in this area.This area also needs to overcome the weak point of the method for present application recombinant epo treatment of chronic diseases anemia.Especially, the metabolic pathway relevant with the availability of signal conduction and ferrum, utilization, picked-up, transhipment, processing etc. that this area also needs to produce by improving with EPO, EPO replys effectively overcomes the EPO relevant with the chronic disease anemia and produces and suppress and EPO replys the method for reduction and chemical compound, effectively strengthens the adjusting of iron metabolism and overcome iron metabolism and utilization change or unusual method and chemical compound and effectively strengthen whole or erythropoietic completely method and chemical compound to produce.This area also needs to overcome or improve the method for the inductive exercising result of the chronic disease anemia patient cells in vivo factor.
Iron deficiency is one of modal food deficiency disease in the whole world, and is the main cause that causes anemia on global basis.Iron balance is mainly regulated by the size that erythropoietic speed and ferrum are stored.Iron deficiency can be followed anemia or non-anemia, and with the cognitive development attenuation of correlation.
Iron deficiency is meant supply (level or the storage) deficiency of ferrum or the availability or the underutilization of body ferrum.This can for example lack ferrum in the diet owing to malnutrition; Owing to for example ferrum malabsorption that causes of operation (behind the gastrectomy) or disease (clone disease (Crohn ' s disease)); Perhaps owing to since due to injured or wound, menstruation, donate blood, draw blood (due to various programs, the operation) loss or the ferrum forfeiture in chronic or the iron supply that acute bleeding causes increase; Perhaps owing to the ferrum due to the quick growth in for example infant or adolescence, trimester of pregnancy, the epo treatment etc. need increase.
Iron deficiency also can be functional iron deficiency, for example is characterised in that the patient obtains and the ability of utilizing ferrum to store weakens the iron deficiency that causes.The ratio of ferrum is not enough to make and the erythrocyte Hb A hemoglobin adultization causes reticulocyte and corpuscular hemoglobin concentration to reduce.Functional iron deficiency often is found in healthy individual, the obviously normal even increase of the storage of ferrum, but the availability of ferrum weakens, as arriving by for example low-level transferrin saturation percentage test.Such iron deficiency is common and acute or chronic inflammatory disease is relevant.
The iron deficiency of any kind of all can cause iron deficiency or ferrum restricted gender erythropoiesis, wherein the erythrocyte in minimizing of erythrocyte number and the circulation is than normocyte little (microcyte), and lack enough hemoglobin, color is pale (low hematochrome).
Iron deficiency comprises that the patient of functional iron deficiency can develop into the synthetic weakening of hemoglobin, transferrin saturation percentage ratio reduces, reaches the H﹠H level and reduces, and causes iron deficiency anemia.Iron deficiency anemia is modal in the world anemia.Ferrum is the essential composition of hemoglobin; There is not iron rule bone marrow can not produce hemoglobin effectively.Iron deficiency anemia can occur in the patient that iron supply is exhausted or weaken, and perhaps can exist with storage form at ferrum but can not utilize, and for example is not useable for occurring among the patient of functional iron deficiency when hemoglobin produces.
In sum, this area need be treated or the method for the disease that prevention is relevant with iron metabolism, and needs to strengthen the method for iron metabolism.Iron deficiency be treated or be prevented in this area need, comprises the method for functional iron deficiency, reaches the method that need treat or prevent with as the microcytosis pathological changes relevant with iron deficiency anemia.
The invention provides to be used to strengthen and help fully and the method and the chemical compound of effective erythropoietic metabolism and physiology's approach, the method and the chemical compound that are used for the treatment of the chronic disease anemia are provided especially.The present invention also provides and has been used to overcome inflammatory cytokine to preventing/inhibiting method and chemical compound that EPO produces and replys.In addition, the present invention also provides and has been used to strengthen the pathological changes of iron metabolism, treatment or prevention and iron metabolism attenuation of correlation such as method and the chemical compound that iron deficiency comprises functional iron deficiency, iron deficiency anemia, microcytosis, iron deficiency erythropoiesis etc.
Summary of the invention
The present invention relates to be used for induce enhanced or erythropoietic completely method and chemical compound at object.Especially, described method comprises and induces enhanced or erythropoiesis completely by stablizing HIF α in the object.The present invention be more particularly directed to induce enhanced erythropoiesis by suppressing the HIF prolyl hydroxylase.In special embodiment, described method comprises and gives object chemical compound of the present invention.
In various embodiments, described object can be cell, tissue, organ, tract or whole organism.In special embodiment, described organism is a mammal, preferably the people.
On the one hand, described method has increased erythrocyte and has comprised the generation of the factor that differentiation for example hematopoietic stem cell (HSC), CFU-GEMM (colony forming unit granulocyte/class erythrocyte/mononuclear cell/megalokaryocyte) cell etc. is required from hemopoietic progenitor cell.Stimulate the erythropoietic factor to comprise but the non-erythropoietin that is limited to.On the other hand, described method increased ferrum picked-up, transport and utilize the generation of the needed factor.These factors comprise but non-class erythrocyte amino-laevulic acid synthase, transferrin, transferrin receptor, the ceruloplasmin etc. of being limited to.Again on the one hand, described method has increased erythrocyte the differentiation required factor and ferrum picked-up, has transported and utilized the required extra factor.
In another embodiment, method of the present invention has strengthened hemopoietic forebody cell replying erythropoietin.As mentioned above, this precursor comprises HSC, CFU-GEMM etc.Replying of described precursor can be for example by changing erythropoietin receptor, participate in born of the same parents' intrinsic factor of erythropoietin signal conduction and promoting the expression of the excreted factor of erythropoietin and acceptor interaction to strengthen.
On the other hand, described method can be used for overcoming inflammatory cytokine such as tumor necrosis factor-alpha (TNF-α), interleukin-1 ' beta ' inductive erythropoietic inhibition such as (IL-1 β).Especially, described method can be used for treating the anemia that the erythropoiesis that external source is given have resistance.This anemia can be for example to be comprised by chronic inflammatory disease or autoimmune disease but non-being limited to due to chronic bacterial endocarditis, osteomyelitis, rheumatic arthritis, rheumatic fever, clone disease and the ulcerative colitis.
In certain embodiments, method of the present invention can be used for the treatment of chronic diseases anemia.The present invention provides the enhanced or erythropoietic completely method in the chronic disease anemia patient body of inducing especially.In special embodiment, described method has increased the amount of available ferrum to produce new erythrocyte.
On the other hand, the invention provides and be used to strengthen the method that bone marrow EPO replys.
The present invention provides especially and has been used to suppress the method for TNF-α to the inhibition of EPO, and suppresses the method for IL-1 β to the inhibition of EPO.
The present invention relates to be used for the treatment of/prevent the method for chronic disease anemia, and regulate the method for processing of ferrum and be used for the treatment of/method of the pathological changes that prevention is relevant with iron deficiency and/or ironworking shortage.
On the one hand, the invention provides the method that is used for the treatment of object chronic disease anemia, described method comprise give the object effective dose can stablize hypoxia inducible factor (hypoxiainducible factor, the chemical compound of α subunit HIF), thereby the treatment experimenter the chronic disease anemia.The present invention also provides the method that reaches special physiological effect in suffering from chronic disease anemia patient body, The present invention be more particularly directed in suffering from patient's body of chronic disease anemia, increase the method for reticulocyte, increase mean corpuscular volume (MCV), increase mean corpuscular hemoglobin, increase hematocrit, increase hemoglobin and increase red blood cell count(RBC) etc., every kind of method includes the chemical compound of the α subunit of the stable hypoxia inducible factor (HIF) that gives patient's effective dose, thereby reaches the physiological role of hope.In all fields, described chronic disease anemia is relevant with for example inflammation, autoimmune disease, iron deficiency, microcytosis, malignant tumor etc.
In various embodiments, described to liking cell, tissue or organ.In other embodiments, described to liking animal, preferred mammal most preferably is the people.When described to as if during cell, the present invention particularly including described cell can be isolated cells, protokaryon or eukaryotic cell can.To liking in the situation about organizing, the present invention is particularly including interior source tissue and vitro tissue, for example tissue of growing in the culture described.In preferred embodiments, described to liking the animal of animal, particularly mammalian species, comprise rat, rabbit, cattle, sheep, pig, Mus, horse and primate species.In the most preferred embodiment, described to liking the people.
The stable of HIF α can realize by the available and known any method of those skilled in the art, and can comprise and use with HIF α or with the interactional factor interaction of HIF α, combine or modify their any preparation (comprising that for example substrate is the enzyme of HIF α).In some aspects, the present invention includes the HIF α variant that provides composing type stable, for example stable HIF mutain etc., the polynucleotide of this variant of perhaps encoding.In others, the present invention includes and stablize HIF α, comprise the preparation of stablizing HIF α.Described preparation can by polynucleotide for example antisense sequences, polypeptide, antibody, other protein, carbohydrate, fat, lipid, and Organic substance and inorganic matter for example micromolecule etc. form.
In a preferred embodiment, the present invention includes by giving the preparation that object stablizes HIF α and stablize for example HIF α in described object, wherein said preparation is the chemical compound of a kind of stable HIF α, for example micromolecular compound etc.
In all fields, HIF α is HIF1 α, HIF2 α or HIF3 α.One preferred aspect, stablize a kind of chemical compound that HIF α comprises the inhibition HIF hydroxylase activity that gives the object effective dose.In some aspects, described HIF hydroxylase is selected from one group that is made up of EGLN1, EGLN2 and EGLN3.
In one embodiment, the invention provides a kind of method that is used to increase the object mean corpuscular volume (MCV), described method comprises the chemical compound of the α subunit of the stable hypoxia inducible factor (HIF) that gives the object effective dose.In further embodiment, the invention provides a kind of method that is used for increasing object mean corpuscular hemoglobin level, described method comprises the chemical compound of the α subunit of the stable hypoxia inducible factor (HIF) that gives the object effective dose.In another embodiment, a kind of method that is used to reduce the object microcytosis has been contained in the present invention, and described method comprises the chemical compound of the α subunit of the stable hypoxia inducible factor (HIF) that gives the object effective dose.
The present invention further provides a kind of method that is used for the treatment of or prevents microcytic anemia, described method comprises the chemical compound of the α subunit of the stable hypoxia inducible factor (HIF) that gives the object effective dose.
On the one hand, the present invention relates to a kind of being used for the treatment of or the method for the pathological changes relevant with iron deficiency of object of prevention, described method comprises the chemical compound of the α subunit of the stable hypoxia inducible factor (HIF) that gives the object effective dose.In a special aspects, the invention provides a kind of method that is used for improving the object ironworking, described method comprises the chemical compound of the α subunit of the stable hypoxia inducible factor (HIF) that gives the object effective dose.The present invention also provide a kind of be used for the treatment of or object of prevention in the ferrum availability reduce the method for relevant pathological changes, described method comprises the chemical compound of the α subunit of the stable hypoxia inducible factor (HIF) that gives the object effective dose.
In other embodiment, the present invention relates to a kind of method that is used for overcoming the effect of object cytokine induction.Especially, one aspect of the present invention provides a kind of being used for to overcome the inhibiting method that the object cytokine produces EPO, and described method comprises the chemical compound of the α subunit of the stable hypoxia inducible factor (HIF) that gives the object effective dose.The present invention further provides a kind of inhibiting method of object cytokine to the ferrum availability that be used for overcoming, described method comprises the chemical compound of the α subunit of the stable hypoxia inducible factor (HIF) that gives the object effective dose.On the other hand, the present invention contained a kind of be used for the treatment of or object of prevention in the method for the relevant anemia of cytokine, described method comprises the chemical compound of the α subunit of the stable hypoxia inducible factor (HIF) that gives the object effective dose.The present invention also provides and has been used in the situation that has cytokine in object increase the method that EPO produces, and described method comprises the chemical compound of the α subunit of the stable hypoxia inducible factor (HIF) that gives the object effective dose.In special embodiment, described cytokine is selected from one group that comprises TNF-α and IL-1 β.
On the one hand, the invention provides the method that a kind of VCAM that reduces cytokine induction in the object expresses, described method comprises the chemical compound of the α subunit of the stable hypoxia inducible factor (HIF) that gives the object effective dose.In a special aspects, described cytokine is TNF-α or IL-1 β.On the one hand, described method is used for reducing the VCAM expression of endotheliocyte cytokine induction.On the other hand, described object suffers from and is selected from one group the pathological changes of being made up of inflammation disease, autoimmune disease and chronic disease anemia.
On the other hand, the invention provides the method that a kind of E-that is used for reducing the object cytokine induction selects protein expression, described method comprises the chemical compound of the α subunit of the stable hypoxia inducible factor that gives the object effective dose.
In a special aspects, described cytokine is TNF-α or IL-1 β.On the one hand, described method is used for reducing the expression of E-selection albumen in endotheliocyte of object cytokine induction.On the other hand, described object suffers from one group the pathological changes that is selected from inflammation disease, autoimmune disease and chronic disease anemia.
The invention provides the method that is used for various adjustings/enhancing ironworking and iron metabolism.On the one hand, the invention provides the method for the transhipment, picked-up, utilization and the absorption that are used for increasing object ferrum, every kind of method includes the chemical compound of the α subunit of the stable hypoxia inducible factor (HIF) that gives the object effective dose.In special embodiment, the invention provides the method that is used to increase transferrin expression, transferrin receptor expression, IRP-2 expression, ferritin expression, ceruloplasmin expression, NRAMP2 expression, sproutin expression and ALAS-2 expression, every kind of method includes the chemical compound of the α subunit of the stable hypoxia inducible factor (HIF) that gives the object effective dose.In other embodiment, the invention provides the method that is used to reduce Hai Paxi pyridine (hepcidin) expression, described method comprises the chemical compound of the α subunit of the stable hypoxia inducible factor (HIF) that gives the object effective dose.The present invention also provides the chemical compound by the α subunit of the stable hypoxia inducible factor (HIF) that gives the object effective dose to increase the synthetic method of object haemachrome.
In some aspects, the present invention relates to be used for increasing the object serum levels of iron, increase the transferrin saturation, increase soluble transferrin receptor level, reach the method that increases the serum ferritin level, described method comprises the chemical compound of the α subunit of the stable hypoxia inducible factor (HIF) that gives the object effective dose.Further, the invention provides and be used for increasing the method for object Railway transportation to bone marrow, described method comprises the chemical compound of the α subunit of the stable hypoxia inducible factor (HIF) that gives the object effective dose.
On the one hand, method of the present invention is used for the treatment of the object of suffering from any disease described herein or pathological changes, and preferred people perhaps is used for the medicine of production for treating any disease described herein or pathological changes.Should understand with the covert various parameters of closing of clinical disease and can be changed according to the age, sex etc.On the one hand, the serum ferritin level of described object is lower than normal range, for example is lower than 50-200 μ g/L; Therefore to be lower than the object of 200ng/ml, 150ng/ml, 100ng/ml, 75ng/ml and 50ng/ml can be to be suitable for the object that methods and applications provided by the invention medicine of the present invention is treated to the serum ferritin level.Perhaps, suitable object can differentiate that for example TIBC is lower than 300-360 μ g/dL normal range by showing that total iron-binding capacity (TIBC) is lower than.
In another embodiment, the serum levels of iron level of described object is lower than normal range, for example is lower than 50-150 μ g/dL.Be used to differentiate that other suitable parameter of suitable object comprises that transferrin saturation measured value is lower than 30-50%, bone marrow sideroblast measured value is lower than 40-60%, and hemoglobin level is lower than about 10-11g/dL.Above-mentioned any parameter all is to measure in for example standard blood test, hematochemistry and full blood count (CBC) are analyzed, typically represent, for example obtain by measuring red blood cell count(RBC), numeration of leukocyte, platelet count and the exponential automatic equipment analyzing blood of erythrocyte with the measured value of some blood parameters.Can measure by any standard device of hematology and/or biochemistry hemanalysis, described equipment comprises for example automatic system such as CELL DYN 4000 analysers (Abbott Laboratories, Abbott Park IL), Coulter GenS analyser (Beckman Coulter, Inc., FullertonCA), Bayer ADVIA 120 analysers (Bayer Healthcare AG, Leverkusen, Germany) or the like.
On the one hand, the present invention contained a kind of be used for the treatment of or object of prevention in the method for iron deficiency, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thus the iron deficiency situation in treatment or the object of prevention.Further, described iron deficiency is functional iron deficiency; Relevant with anemia; One group the pathological changes of being made up of inflammation, infection, immune deficiency disorder and tumor disease is relevant with being selected from; Perhaps with to be selected from one group the pathological changes of being made up of chronic disease anemia, iron deficiency anemia (IDA) and microcytic anemia relevant.
Object of the present invention can be to have clinical generally acknowledged object iron deficiency or that be in the standard test indication in the danger of generation iron deficiency.For example, in certain embodiments, described object serum ferritin level low (＜20ng/ml), perhaps transferrin saturation percentage ratio reduces, and for example is lower than 16% (adult).Be particularly related to the serum ferritin level and be lower than 50ng/ml, 40ng/ml, 30ng/ml, 20ng/ml.If notice that the iron deficiency of described object is functional iron deficiency, then the serum ferritin level can increase to more than normal range, for example 200ng/ml and higher.Iron deficiency can by take place ferrum limited/erythropoiesis of iron deficiency observes (when transferrin saturation percentage ratio drops to when being lower than 15-20%, representative observation reduces to hemoglobin is synthetic).The parameter of these ferrum can use the CBC of above-mentioned any standard or biochemical analysis to measure, and/or by using the automatic equipment of analyzing at ferrum more specifically to measure, for example use Unimate 5 Iron and Unimate 7 UIBC test kits (Roche, Switzerland).
The object that can have benefited from treatment of the present invention or prevention method can be to suffer from or be in object in the iron deficiency anemia danger, and for example the transferrin saturation is at 10-15% or be lower than 10% object.
On the one hand, suffer from or be in object in the iron deficiency danger and suffer from or be in and suffer from the functional iron deficiency danger.The reticulocyte content of hemoglobin is lower than the indication that 28 piks/cell can be this pathological changes.On the other hand, suffer from or be in the object performance of suffering from the functional iron deficiency danger and be higher than 5% low hematochrome erythrocyte.
In certain embodiments, described object suffers from or is in and suffers from the danger of chronic disease anemia.This object can show as slight or anemia, and for example hemoglobin level is about 10-13g/dL, perhaps more particularly is 10-11g/dL.In other embodiment, show as more acute anemia, for example hemoglobin level is lower than 10g/dL, comprises being lower than 5g/dL and 3g/dL.In some embodiments, suffer from or be in the object of suffering from the danger of chronic disease anemia to show as the distribution of ferrum unusual.This can be that for example the serum levels of iron level is lower than about 60 μ g/dL unusually, and perhaps the serum ferritin level is higher than normal range, for example is higher than 200ng/ml, is higher than 300ng/ml or is higher than 400ng/ml.
In some aspects, described object can suffer from or be in and suffers from the microcytic anemia danger.This object can for example show as in the full blood count analysis mean corpuscular volume (MCV) less than 80 ascend to heaven (femtoliter).In others, the mean corpuscular volume (MCV) of described object is less than normal volume (90 ± 8 ascend to heaven).In all fields, described object can have the mean cell hemoglobin counting of reduction, for example average hemoglobin counting is lower than 30 ± 3 pik hemoglobin/cells, perhaps has the mean cell hemoglobin concentration of reduction, and for example mean cell hemoglobin concentration is lower than 33 ± 2%.
The present invention also provides a kind of and has been used for the treatment of or the method for the functional iron deficiency of object of prevention, and described method comprises the chemical compound of the stable HIF α that gives the object effective dose, thus treatment or prophylactic function iron deficiency.
In one embodiment, the invention provides a kind of method that is used for regulating or strengthening object iron metabolism or iron metabolism process, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby regulates or strengthen iron metabolism or iron metabolism process in the object.In another embodiment, the invention provides a kind of method that is used to regulate or strengthen the iron metabolism process, described iron metabolism process is selected from one group that is made up of the utilization of the mobilization of the processing of the storage of the transhipment of the absorption of the picked-up of ferrum, ferrum, ferrum, ferrum, ferrum, ferrum and ferrum, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thus the iron metabolism process in adjusting or the enhancing object.
The present invention also provides a kind of being used for to increase the method that object ferrum absorbs, and described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases the absorption of ferrum in the object.In some aspects, the absorption of described ferrum is in intestinal; It is the absorption of ferrum in the diet; Perhaps in the duodenum enterocyte, absorb.
The invention still further relates to following method: a kind of method that is used for increasing the transhipment of object ferrum, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases the transhipment of ferrum in the object; A kind of method that is used for increasing the storage of object ferrum, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases the storage of ferrum in the object; A kind of method that is used for increasing the picked-up of object ferrum, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases the picked-up of ferrum in the object; A kind of method for processing that is used for increasing object ferrum, described method comprise the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increase the processing of ferrum in the object; A kind of method that is used for increasing the mobilization of object ferrum, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases the mobilization of ferrum in the object; And a kind of method that is used for increasing the utilization of object ferrum, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases the utilization of ferrum in the object.
In one embodiment, the present invention relates to a kind of method of object that be used for increasing at erythropoietic ferrum availability, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases in the object availability at erythropoietic ferrum.In various embodiments, increasing at erythropoietic ferrum availability is to increase for the synthetic ferrum availability of haemachrome; Be to increase the ferrum availability that produces for hemoglobin; Or increase is for the ferrum availability of erythrocyte generation.
The present invention further provides the method for the expression that is used for controlled plant ferrum regulatory factor, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thus the expression of the iron metabolism factor in the controlled plant.
The method of the expression that is used to increase some ferrum regulatory factor has also been contained in the present invention, comprise: a kind of method that is used for increasing the expression of object transferrin receptor, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases the expression of transferrin receptor in the object; A kind of being used for increases the method that the object transferrin is expressed, and described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases the expression of transferrin in the object; A kind of being used for increases the method that the object ceruloplasmin is expressed, and described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases the expression of ceruloplasmin in the object; A kind of being used for increases the method that object NRAMP2 (slc11a2) expresses, and described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases the expression of NRAMP2 in the object; A kind of being used for increases the method that object duodenum cytochrome b reductase 1 is expressed, and described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases the expression of duodenum cytochrome reductase 1 in the object; And a kind of method that is used for increasing the expression of object 5-Aminolevulinate synthase, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases the expression of 5-Aminolevulinate synthase in the object.
In one embodiment, the invention provides a kind of method that is used for increasing object serum levels of iron level, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases serum levels of iron level in the object.In certain embodiments, described to liking the people, described serum levels of iron level increases to 50-150 μ g/dL.
On the other hand, the invention provides the method that is used for increasing the total iron-binding capacity of object (TIBC).Described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases TIBC in the object.One preferred aspect, described to as if the people, described total iron binding capacity increases to 300-360 μ g/dL.
The invention provides the method and the chemical compound that are used for regulating the serum ferritin level at object.In a certain embodiment, described to liking the people, described serum ferritin level increases to more than the 15 μ g/L.In another embodiment, described to liking the adult male, described serum ferritin level increases to about 100 μ g/L.In another embodiment, described to liking the adult women, described serum ferritin level increases to about 30 μ g/L.
On the one hand, the present invention includes a kind of method that is used for increasing object transferrin saturation, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases the transferrin saturation of object.On the one hand, described transferrin saturation increases to and is higher than one and is selected from one group the level of forming by 10%, 15%, 20%, 30%, 40% and 50%.The method that increases transferrin saturation percentage ratio in the object has been contained in the present invention.In one embodiment, described to liking the people, described transferrin saturation percentage ratio increases to more than 18%.In another embodiment, described transferrin saturation percentage ratio increases to 25-50%.Transferrin saturation percentage ratio typically uses formula " (serum levels of iron) (100)/(TIBC) " to calculate.
The present invention also provides the method that is used for increasing the soluble transferrin receptor level of object, and described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases the soluble transferrin receptor level of object.The present invention further provides increases by for example method of total class erythrocyte bone marrow amount of serum transferrin receptor level determination.On the one hand, described to liking the people, described serum transferrin receptor level determines to increase to 4-9 μ g/L by immunoassay.
The invention provides a kind of method that is used for reducing object Hai Paxi pyridine (hepcidin) expression, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby reduces the expression of Hai Paxi pyridine in the object.
In one embodiment, the invention provides a kind of being used for the treatment of or the method for the pathological changes that object of prevention is relevant with iron deficiency, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thus treatment or the prevention pathological changes relevant with the object iron deficiency.In one embodiment, described iron deficiency is functional iron deficiency.In various embodiments, described disease is selected from one group that is made up of inflammation, infection, immune deficiency disorder and tumor disease; Perhaps be selected from one group that forms by chronic disease anemia, iron deficiency anemia and microcytic anemia.
The invention provides a kind of be used for strengthening suffer from or be in erythropoietic method in the object of suffering from iron deficiency danger, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby strengthens the erythropoiesis in the object.Present invention includes described in one aspect iron deficiency is functional iron deficiency.
The present invention further provides a kind of erythropoietic method of object that is used for strengthening, wherein said object has or is in the danger with functional iron deficiency, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby strengthens erythropoiesis in the object.In all fields, described chronic disease is selected from one group that is made up of inflammation, infection, immune deficiency disorder and tumor disease.
The present invention provides a kind of erythropoietic method of object that is used for strengthening in addition, wherein said object has or is in the danger with chronic disease anemia, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby strengthens erythropoiesis in the object.
In one embodiment, a kind of erythropoietic method of object that is used for strengthening has been contained in the present invention, wherein said object EPO treatment is difficult to prove effective, and described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby strengthens erythropoiesis in the object.
The present invention also provides a kind of and has been used for the treatment of or the method for the chronic disease anemia of object of prevention, and described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thus the chronic disease anemia of treatment or object of prevention.Relate to described chronic disease anemia in some aspects and to be selected from one group the pathological changes of being made up of inflammation, infection, immune deficiency disorder and tumor disease relevant.
The present invention is particularly including following method: a kind of method that is used for increasing the object reticulocyte of suffering from chronic disease, and described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases reticulocyte in the object; A kind of method that is used to increase the object hematocrit of suffering from chronic disease, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases the object hematocrit; A kind of method that is used for increasing the object hemoglobin of suffering from chronic disease, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases hemoglobin in the object; A kind of method that is used for increasing the object red blood cell count(RBC) of suffering from chronic disease, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases red blood cell count(RBC) in the object; A kind of method that is used for increasing the object mean corpuscular volume (MCV) of suffering from chronic disease, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases mean corpuscular volume (MCV) in the object; A kind of method that is used for increasing the object mean corpuscular hemoglobin of suffering from chronic disease, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases mean corpuscular hemoglobin in the object; A kind of method that is used for increasing the object serum levels of iron level of suffering from chronic disease, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases serum levels of iron level in the object; And a kind of method that is used for increasing the object transferrin saturation of suffering from chronic disease, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases transferrin saturation in the object.In arbitrary these methods, described chronic disease is selected from one group that is made up of inflammation, infection, immune deficiency disorder and tumor disease in certain embodiments; Perhaps be selected from one group that forms by chronic disease anemia, iron deficiency anemia, iron deficiency, functional iron deficiency and microcytic anemia.
The present invention provides following method in addition: a kind of method that is used for increasing object reticulocyte with iron deficiency, and described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases reticulocyte in the object; A kind of method that is used for increasing object hematocrit with iron deficiency, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases hematocrit in the object; A kind of method that is used for increasing object hemoglobin with iron deficiency, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases hemoglobin in the object; A kind of method that is used for increasing object red blood cell count(RBC) with iron deficiency, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases red blood cell count(RBC) in the object; A kind of method that is used for increasing object mean corpuscular volume (MCV) with iron deficiency, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases mean corpuscular volume (MCV) in the object; A kind of method that is used for increasing object mean corpuscular hemoglobin with iron deficiency, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases mean corpuscular hemoglobin in the object; A kind ofly be used for increasing the method for suffering from the object serum levels of iron level with iron deficiency, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases serum levels of iron level in the object; And a kind of method that is used for increasing the object transferrin saturation with iron deficiency, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases transferrin saturation in the object.In arbitrary these methods, described iron deficiency is functional iron deficiency in certain embodiments.
The invention further relates to following method: a kind of method that is used for increasing object reticulocyte with functional iron deficiency, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases reticulocyte in the object; A kind of method that is used for increasing object hematocrit with functional iron deficiency, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases hematocrit in the object; A kind of method that is used for increasing object hemoglobin with functional iron deficiency, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases hemoglobin in the object; A kind of method that is used for increasing object red blood cell count(RBC) with functional iron deficiency, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases red blood cell count(RBC) in the object; A kind of method that is used for increasing object mean corpuscular volume (MCV) with functional iron deficiency, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases mean corpuscular volume (MCV) in the object; A kind of method that is used for increasing object mean corpuscular hemoglobin with functional iron deficiency, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases mean corpuscular hemoglobin in the object; A kind of method that is used for increasing object serum levels of iron level with functional iron deficiency, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases serum levels of iron level in the object; And a kind of method that is used for increasing the object transferrin saturation with functional iron deficiency, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thereby increases transferrin saturation in the object.
On the one hand, the present invention includes a kind of erythropoiesis that is used for overcoming or improves the object cytokine induction and reduce the method for caused consequence, described method comprises the chemical compound of the stable HIF α that gives the object effective dose, thereby overcomes or improve that the erythropoiesis of cytokine induction reduces caused consequence in the object.In all fields, it is that the EPO generation is suppressed that the erythropoiesis of described cytokine induction reduces, and perhaps the metabolism of ferrum weakens.In above-mentioned any method, described cytokine is an inflammatory cytokine.In further embodiment, described cytokine is selected from one group that is made up of TNF-α, IL-1 β and IFN-γ.
The present invention also provides the method that is used to reduce cytokine induction VCAM-1 expression and/or E-selection protein expression, described method comprises the chemical compound of the stable HIF α that gives the object effective dose, and cytokine induction VCAM-1 expresses and/or E-selects protein expression thereby reduce.
In above-mentioned any method, described cytokine is an inflammatory cytokine.In further embodiment, described cytokine is selected from one group that is made up of TNF-α, IL-1 β and IFN-γ.
The invention provides and be used for the treatment of or the method for the disease that object of prevention is relevant with cytokine activity, described disease is selected from one group that is made up of iron deficiency, functional iron deficiency, iron deficiency anemia, chronic disease anemia and microcytic anemia, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thus treatment or the prevention disease relevant with cytokine activity.In any said method, described cytokine is an inflammatory cytokine.In further embodiment, described cytokine is selected from one group that is made up of INF-α, IL-1 β and IFN-γ.
The present invention also provides and has been used for the treatment of or the method for the disease that object of prevention is relevant with cytokine activity, described disease is with to be selected from one group the pathological changes of being made up of inflammation, infection, immunodeficiency and tumor disease relevant, described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thus treatment or the prevention disease relevant with cytokine activity.In any said method, described cytokine is an inflammatory cytokine.In further embodiment, described cytokine is selected from one group that is made up of INF-α, IL-1 β and IFN-γ.
On the one hand, a kind of being used for contained in the present invention increases the method that EPO produces under the situation that there is cytokine in object, and described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, and EPO produces in the object thereby increase.The present invention also provides a kind of and has been used for the treatment of or the method for object of prevention microcytosis, and described method comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object, thus treatment or prevention microcytosis.Further, described microcytosis and the disease association that is selected from chronic disease, chronic disease anemia, iron deficiency, functional iron deficiency and iron deficiency anemia.In any said method, described cytokine is an inflammatory cytokine.In further embodiment, described cytokine is selected from one group that is made up of TNF-α, IL-1 β and IFN-γ.
In any treatment of the present invention or prevention method, can give the part of chemical compound of the present invention as combined therapy, comprise in addition giving another kind of therapeutic agent, for example EPO, ferrum and vitamin such as vitamin B group or the like.
The invention provides a kind of test kit, it comprises chemical compound and at least a other tonic of a kind of stable HIF α.On the one hand, described tonic is selected from one group that is made up of erythropoietin, ferrum and vitamin B group, the present invention also provides a kind of pharmaceutical composition, and it comprises the chemical compound of a kind of stable HIF α and is selected from one group that is made up of at least a tonic of erythropoietin, ferrum and vitamin B group.
The invention provides the Compounds and methods for that is used for the treatment of or prevents the chronic disease anemia, wherein said chronic disease anemia raises relevant with cytokine levels.Especially, the invention provides the method and the chemical compound of the exercising result that is used for overcoming or improves the object cytokine induction that cytokine levels raises, the exercising result of described cytokine induction for example is that cytokine suppresses that EPO produces, the expression of the various cell adhesion factors of cytokine induction or the like.
In one embodiment, the invention provides method and the chemical compound that is used to overcome cytokine inhibition EPO generation.These methods and chemical compound are used to overcome TNF α and/or IL-1 β suppresses the EPO generation, for example measures by the ability that overcomes TNF α and/or IL-1 β inhibition EPO generation in the Hep3B cell of cultivating.
In one embodiment, the invention provides method and the chemical compound that the various cell adhesion factor expressions that are used to reduce cytokine induction increase.Described method and chemical compound can be used for overcoming TNF α, IL-1 β and for example VCAM-1 and the increase of E-selection protein expression of the inductive endotheliocyte adhesion of the IFN-γ factor, for example select proteic expression to reduce by VCAM-1 or E-in endotheliocyte (HUVEC etc.) and measure.
The invention provides be used for the treatment of or object of prevention in the method and the chemical compound of iron deficiency.Especially, method of the present invention and chemical compound can be used for strengthening the metabolism of ferrum, perhaps are used for the treatment of or the metabolism of prevention and ferrum weakens relevant disease and pathological changes, for example weakening of the picked-up of ferrum, storage, processing, transhipment, mobilization and utilization etc.
On the one hand, described method and chemical compound regulate to participate in the expression of the factor of iron metabolism (for example transport, utilize, storage etc.).For example described method and chemical compound increase the expression of transferrin receptor, for example increase by soluble transferrin receptor level in the interior transferrin receptor expression increase of hepatocyte (for example Hep3B, HepG2), nephrocyte (for example HK-2) or lymphocyte (for example THP-1) or the people's subject and measure.Method of the present invention and chemical compound increase ceruloplasmin gene expression, for example increase by gene expression in little Ren Mus and the Hep3B cell and measure.On the one hand, the invention provides the method and the chemical compound that are used to reduce Hai Paxi pyridine (hepcidin) gene expression, for example reduce and measure by the pyridine gene expression of mouse liver inland sea handkerchief west.On the other hand, method of the present invention and chemical compound are used to increase the expression that some factors comprise NRAMP2, duodenum cytochrome b reductase 1 etc., for example increase by gene expression in the mouse intestinal and measure.Method of the present invention and chemical compound increase the expression of 5-Aminolevulinate synthase, for example increase by gene expression in the mouse intestinal and measure.This kind of enzyme is first kind of enzyme in the haemachrome route of synthesis and is the synthetic rate-limiting enzyme of haemachrome.
Method of the present invention and chemical compound can be used for strengthening the metabolism of ferrum.Especially, described method and chemical compound strengthen the metabolism of ferrum, and for example the level by serum levels of iron in the rat model of iron metabolism weakening increases, transferrin saturation percentage ratio increases and the microcytosis minimizing is measured.
The invention provides and be used to induce enhanced method of erythropoiesis and chemical compound.Especially, method of the present invention and chemical compound strengthen erythropoiesis, for example increase and measure by reticulocyte count in the rat model that weakens at erythropoiesis or the human object, hematocrit and red blood cell count(RBC), perhaps by for example in the rat model that erythropoiesis weakens hemoglobin level increase and measure.
Method of the present invention and compounds for reducing microcytosis for example increase by mean corpuscular hemoglobin level in the rat model of erythropoiesis weakening and the mean corpuscular volume (MCV) increase is measured.
Method of the present invention comprises the chemical compound of the stable HIF α that gives a kind of effective dose of object.This stable can being undertaken by for example suppressing the HIF hydroxylase activity.The preferred chemical compound of the present invention is to suppress the active chemical compound of HIF prolyl hydroxylase.Described inhibition can be direct or indirect inhibition, can be competition or noncompetitive inhibition, or the like.In each embodiment, chemical compound of the present invention is selected from 2-oxopentanedioic acid salt analogies, iron chelating agent and proline analogs.On the one hand, 2-oxoglutarate analogies are the heterocycle carbonyl glycines shown in a kind of Formula I, Ia or the Ib.On the other hand, iron chelating agent is a kind of hydroxamic acid shown in the Formulae II I.In special embodiment, as this paper institute illustration, described chemical compound is a Compound D.
Exemplary compound of the present invention comprises [(1-chloro-4-hydroxyl-isoquinolin-3-carbonyl)-amino]-acetic acid (compd A); [(4-hydroxyl-7-phenoxy group-isoquinolin-3-carbonyl)-amino]-acetic acid (compd B); [(4-hydroxyl-7-benzene sulfonyl-isoquinolin-3-carbonyl)-amino]-acetic acid (Compound C) and 3-{[4-(3,3-dibenzyl-urea groups)-benzenesulfonyl]-[2-(4-methoxyl group-phenyl)-ethyl]-amino }-N-hydroxyl-propionic acid amide. (Compound D).The present invention also provides according to additional compounds of the present invention and has differentiated the method for these chemical compounds.
The accompanying drawing summary
Figure 1A and 1B have listed and have shown that method of the present invention and chemical compound overcome the inhibiting data that TNF-α produces EPO.
Fig. 2 A and 2B have listed and have shown that method of the present invention and chemical compound overcome the inhibiting data that TNF-α produces EPO in the pretreated cell of TNF-α.
Fig. 3 A and 3B have listed and have shown that method of the present invention and chemical compound overcome the inhibiting data that IL-1 β produces EPO.
Fig. 4 A and 4B have listed and have shown that method of the present invention and chemical compound overcome the inhibiting data that IL-1 β produces EPO in the pretreated cell of IL-1 β.
Fig. 5 has listed and has shown that method of the present invention reduces the data that the VCAM-1 relevant with TNF-α expresses with chemical compound.
Fig. 6 A, 6B and 6C listed be presented at chemical compound of the present invention mice is handled after, transferrin receptor and iron transfer albumen (Fig. 6 A), intestinal iron transfer albumen (Fig. 6 B) and 5-Aminolevulinate synthase (Fig. 6 C) are expressed to be increased.
Fig. 7 has listed and has shown that method of the present invention and chemical compound make the data that reticulocyte count increases in the animal model of chronic disease anemia.
Fig. 8 has listed and has shown that method of the present invention and chemical compound make the data that hematocrit increases in the animal model of chronic disease anemia.
Fig. 9 has listed and has shown that method of the present invention and chemical compound make the data that hemoglobin level increases in the animal model of chronic disease anemia.
Figure 10 has listed and has shown that method of the present invention and chemical compound make the data that red blood cell count(RBC) increases in the animal model of chronic disease anemia.
Figure 11 has listed and has shown that method of the present invention and chemical compound make the data that microcytosis reduces in the animal model of chronic disease anemia.
Figure 12 has listed and has shown that method of the present invention and chemical compound make the data that mean corpuscular hemoglobin increases and hematochrome is hanged down in improvement in the animal model of chronic disease anemia.
Figure 13 has listed and has shown that method of the present invention and chemical compound make the data that hematocrit increases in the animal model of intact animal and chronic disease anemia.
Figure 14 has listed and has shown that method of the present invention and chemical compound make the data that hemoglobin level increases in the animal model of intact animal and chronic disease anemia.
Figure 15 has listed and has shown that method of the present invention and chemical compound make the data that red blood cell count(RBC) increases in the animal model of intact animal and chronic disease anemia.
Figure 16 has listed and has shown that method of the present invention and chemical compound make the data that mean corpuscular volume (MCV) improves in the animal model of intact animal and chronic disease anemia.
Figure 17 has listed and has shown that method of the present invention and chemical compound make the data that the mean corpuscular hemoglobin level is improved in the animal model of intact animal and chronic disease anemia.
Figure 18 A and 18B have listed and have shown that method of the present invention and chemical compound make the data that serum levels of iron level (Figure 18 A) and transferrin saturation (Figure 18 B) increase in the animal model of intact animal and chronic disease anemia.
Figure 19 has listed and has shown that method of the present invention and chemical compound make the data that NRAMP2 (slc112a) and sproutin (CYBRD1, duodenum cytochrome b reductase 1) gene expression increase in the animal model of intact animal and chronic disease anemia.
Figure 20 has listed and has been presented at the data that give the reticulocyte increase afterwards of normal healthy people object chemical compound of the present invention.
Figure 21 has listed and has been presented at the data that give the red blood cell count(RBC) increase afterwards of normal healthy people object chemical compound of the present invention.
Figure 22 has listed and has been presented at the data that give the solubility transferrin receptor level increase afterwards of normal healthy people object chemical compound of the present invention.
Figure 23 has listed and has been presented at the data that give the serum ferritin level reduction afterwards of normal healthy people object chemical compound of the present invention.
Figure 24 A has listed the data that show that method of the present invention and chemical compound make relevant VCAM-1 of TNF-α and E-select protein expression to reduce with 24B.
Figure 25 has listed and has shown that method of the present invention and chemical compound make the TNF-α VCAM-1 relevant with IL-1 β express the data that reduce.
Figure 26 has listed the data that show that method of the present invention and chemical compound make TNF-α, the IL-1 β E-relevant with IFN-γ select protein expression to reduce.
Figure 27 A and 27B have listed and have shown the collaborative level that increases EPO in the hepatocyte of method of the present invention and chemical compound and IL-6.
Invention is described
Before describing the compositions and methods of the invention, should understand non-described specific methodology, scheme, clone, analysis and the reagent of being limited to of the present invention, these can change. Will also be understood that the term that the present invention uses is for describing the specific embodiment of the present invention, absolutely not limiting the meaning of the described scope of the invention of claims.
Must be noted that " " of used singulative then comprises plural form unless otherwise indicated in this paper and the appended claims. Therefore, for example " fragment " comprises numerous this fragments; " a kind of compound " refer to one of multiple compounds and refer to its equivalent described herein and well known by persons skilled in the art, etc.
Unless otherwise noted, all technology used herein and academic term all have the known implication of those skilled in the art of the invention. Although can use and similar any method and material described herein among the present invention with test in practice, describe now preferred method, equipment and material. In order to describe and be disclosed in method, reagent and the instrument of the possible application related to the present invention of reporting in the publication that this paper quotes, all publications are all incorporated reference in full with it. Any description of this paper should not be interpreted as thinking that the present invention is disclosed by the publicity that these depend on more early invention.
Unless stated otherwise, put into practice the present invention and will use chemistry, biochemistry, molecular biology, cell biology, science of heredity, immunology and materia medica method conventional in this area. These technology are proved absolutely in the literature. See for example Gennaro, A.R., ed. (1990) Remington ' s Pharmaceutical Sciences, 18th ed., Mack Publishing Co.; Hardman, J.G., Limbird, L.E., and Gilman, A.G., eds. (2001) ThePharmacological Basis of Therapeutics, 10th ed., McGraw-Hill Co.; Colowick, S.et al., eds., Methods In Enzymology, Academic Press, Inc.; Weir, D.M., and Blackwell, C.C., eds. (1986) Handbook of ExperimentalImmunology, Vols.I-IV, Blackwell Scientific Publications; Maniatis, T.etal., eds. (1989) Molecular Cloning:A Laboratory Manual, 2nd edition, Vols.1-111, Cold Spring Harbor Laboratory Press; Ausubel, F.M.et al., eds. (1999) Short Protocols in Molecular Biology, 4th edition, John Wiley﹠ Sons; Ream et al., eds. (1998) Molecular Biology Techniques:AnIntensive Laboratory Course, Academic Press; Newton, C.R., andGraham, A., eds. (1997) PCR (Introduction to Biotechniques Series), 2nded., Springer Verlag.
Definition
Term " study on anemia of chronic disease (anemia of chronic disease) " refers to owing to the resultant any anemia such as long-term infection, inflammation, tumor disease etc. The anaemia that produces like this is characterised in that usually shorten red blood cell life span and iron compiles in macrophage, cause can be used for producing the quantity minimizing of new erythrocytic iron. The disease relevant with study on anemia of chronic disease comprises but non-chronic bacterial endocarditis, osteomyelitis, rheumatic fever, ulcerative colitis, and the tumor disease of being limited to. Further disease comprises and disease and the pathology of infection, inflammation and Tumor-assaciated, comprises such as struvite infection (such as pulmonary abscess, pulmonary tuberculosis, osteomyelitis etc.), struvite non-infectious pathology (such as rheumatic arthritis, system lupus erythematosus, clone disease, hepatitis, inflammatory bowel disease etc.), reaches various cancers, tumour and malignant tumour (such as cancer, sarcoma, lymthoma etc.).
Term " disease " and " pathology " are used interchangeably, and refer to and depart from normal any situation.
Term " erythropoietin(EPO) " refers to the erythropoietin(EPO) of any restructuring or natural generation, comprises for example erythropoietin (GenBank registration number No.AAA52400; Lin et al. (1985) Proc Natl Acad Sci USA 82:7580-7584), EPOETIN people's recombinant erythropoietin (Amgen, Inc., Thousand Oaks CA), ARANESP people's recombinant erythropoietin (Amgen), PROCRIT people's recombinant erythropoietin (Ortho BiotechProducts, L.P., Raritan NJ) etc.
Term " HIF α " refers to the α subunit of hypoxia inducible factor albumen. HIF α can be anyone or other mammalian proteins or its fragment, comprises people HIF-1 α (GenBank registration number Q16665), HIF-2 α (GenBank registration number AAB41495) and HIF-3 α (GenBank registration number AAD22668); Mouse HIF-1 α (GenBank registration number Q61221), HIF-2 α (GenBank registration number BAA20130 and AAB41496) and HIF-3 α (GenBank registration number AAC72734); Rat HIF-1 α (GenBank registration number CAA70701), HIF-2 α (GenBank registration number CAB96612) and HIF-3 α (GenBank registration number CAB96611); And ox HIF-1 α (GenBank registration number BAA78675). HIF α also can be any nonmammalian albumen or its fragment, comprises smooth Xenopus laevis (Xenopus laevis) HIF-1 α (GenBank registration number CAB96628), Drosophila melanogaster (Drosophilamelanogaster) HIF-1 α (GenBank registration number JC4851) and chicken HIF-1 α (GenBank registration number BAA34234). HIF α gene order also can obtain by conventional clone technology, for example by using above-mentioned all or part of HIF α gene order as probe, to obtain and to measure the sequence of HIF α gene in other species.
The fragment of HIF α comprise by people HIF-1 α limit such as lower area: amino acid 401-603 (Huang et al., as front), amino acid 531-575 (Jiang et al. (1997) J BiolChem 272:19253-19260), amino acid 556-575 (Tanimoto et al., as front), amino acid 557-571 (Srinivas et al. (1999) Biochem Biophys Res Commun260:557-561) and amino acid 556-575 (Ivan and Kaelin (2001) Science 292:464-468). Further, the fragment of HIF α comprises that containing motif LXXLAP occurs once any fragment at least, and example comes across L in the native sequences of HIF α397TLLAP and L559EMLAP. In addition, the fragment of HIF α comprises at least a function of reservation HIF α or any fragment of architectural characteristic.
Term " HIF prolyl hydroxylase " and " HIF PH " refer to any enzyme of proline residue in the energy hydroxylation HIF protein. Preferably, be included in the proline of finding among the motif LXXLAP by the proline residue of HIF PH hydroxylation, for example in people HIF-1 α native sequences, come across L397TLLAP and L559EMLAP. HIF PH comprises Taylor (2001, Gene275:125-132) described Egl-Nine (EGLN) gene family member, and by Aravindand Koonin (2001, Genome Biol 2:RESEARCH0007), Epstein et al. (2001, Cell 107:43-54) and Bruick and McKnight (2001, Science 294:1337-1340) identify. The example of HIF prolyl hydroxylase comprises people SM-20 (EGLN1) (GenBank registration number AAG33965; Dupuy et al. (2000) Genomics 69:348-54), EGLN2 isotype 1 (GenBank registration number CAC42510; Taylor is as front), EGLN2 isotype 2 (GenBank registration number NP_060025) and EGLN3 (GenBank registration number CAC42511; Taylor is as front); Mouse EGLN1 (GenBank registration number CAC42515), EGLN2 (GenBank registration number CAC42511), and EGLN3 (SM-20) (GenBank registration number CAC42517); Rat SM-20 (GenBank registration number AAA19321). In addition, HIF PH can comprise nematode (Caenorhabditiselegans) EGL-9 (GenBank registration number AAD56365) and Drosophila melanogaster (Drosophilamelanogaster) CG1114 gene outcome (GenBank registration number AAF52050). The HIF prolyl hydroxylase also can comprise at least a structure of any maintenance of above-mentioned full length protein or the fragment of functional character.
Term used herein " prolyl hydroxylase inhibitors " or " PHI " refer to reduce or otherwise regulate any compound of activity of the enzyme of hydroxylation amino acid residue. Although the enzymatic activity of hydroxylation proline residue is preferred, also comprises other amino acid of hydroxylation, include but not limited to arginine. The amino acid that the compound that can be used for the inventive method comprises for example iron chelating agent, 2-oxoglutarate analogies and modification is proline analogs for example.
In specific embodiment, the invention provides the purposes of the structural simulation thing of 2-oxoglutarate. This compounds can suppress target 2-oxoglutarate dioxygenase family member with 2-oxoglutarate competitiveness with the iron noncompetitive. (Majamaa et al., (1984) Eur JBiochem 138:239-245; Majamaa et al. (1985) Biochem J 229:127-133). This paper describes that for example see following document: Majamaaet al. document is the same particularly including being used for PHI of the present invention; Kivirikko and Myllyharju (1998) Matrix Biol16:357-368; Bickel et al. (1998) Hepatology 28:404-411; Friedman et al. (2000) Proc Natl Acad Sci USA 97:4736-4741; Franklin (1991) BiochemSoc Trans 19): 812 815; Franklin et al. (2001) Biochem J 353:333-338; With the open WO03/053977 in the world and WO03/049686, each document all is incorporated herein for referencial use in full. Representational PHI, comprise [(1-chloro-4-hydroxyl-isoquinolin-3-carbonyl)-amino]-acetic acid (compd A), [(4-hydroxyl-7-phenoxy group-isoquinolin-3-carbonyl)-amino]-acetic acid (compd B), [(4-hydroxyl-7-phenyl sulfanyl-isoquinolin-3-carbonyl)-amino]-acetic acid (Compound C), 3-{[4-(3,3-dibenzyl-urea groups)-benzene sulfonyl]-[2-(4-methoxyl group-phenyl)-ethyl]-amino }-N-hydroxyl propionamide (Compound D) is used among this specification embodiment to confirm the inventive method disclosed herein.
Invention
That the present invention relates in object, to induce enhancing or completely erythropoietic method and compound. Especially, the inventive method relates to by stablizing HIF α in the individuality and induces enhancing or complete red blood cell to generate. Be particularly related to the erythropoietic method of inducing enhancing by suppressing the HIF prolyl hydroxylase. In specific embodiment, described method comprises and gives object with compound of the present invention. In various embodiments, described object can be cell, tissue, organ, tract or complete biology.
Study on anemia of chronic disease is the anaemia of most common form among the inpatient. Study on anemia of chronic disease occurs among the patient who suffers from inflammation or malignant disease, and described inflammation or malignant disease comprise inflammation infection (such as lung carbuncle, pulmonary tuberculosis, osteomyelitis etc.), inflammatory noninfectious disease (such as rheumatoid arthritis, systemic loupus erythematosus, clone disease, hepatitis, IBD etc.) and various cancer, tumour and malignant tumour (such as cancer, sarcoma, lymthoma etc.), chronic bacterial endocarditis, osteomyelitis, rheumatic fever, ulcerative colitis and neoplastic lesion.
In one aspect, the invention provides induce enhancing or completely red blood cell generate method with the treatment of chronic diseases anaemia. Study on anemia of chronic disease is relevant with many chronic diseases, and described chronic disease comprises such as rheumatoid arthritis, rheumatic fever, IBD, ulcerative colitis, systemic loupus erythematosus, vasculitis, tumor disease etc. and chronic infection and chronic inflammation. That reduce or invalid red blood cell generation is the common pathology of suffering among the patient of study on anemia of chronic disease. That reduce or invalid red blood cell generates the various metabolic disorders that can produce in red blood cell generation approach, and the EPO that comprise that for example repressed EPO generates, reduces in the marrow replys, unusual ironworking comprises that for example unusual or invalid iron is taken in, mobilizes, stored and absorbs.
The physiologic character of the disease relevant with study on anemia of chronic disease is that inflammatory cytokine produces increase (Means (1995) Stem Cells 13:32-37 and Means (1999) Int JHematol 70:7-12), these cell factors comprise for example tumor necrosis factor-alpha (TNF-α), interleukin-1 ' beta ' (IL-1 β) and interferon-γ (IFN-γ), and EPO generates, EPO replys has a negative impact with the coordinately regulated ability of iron metabolism to mediating for they. (referring to for example, Roodman et al. (1989) Adv Exp Med Biol 271:185-196; Fuchs et al. (1991) Eur J Hematol 46:65-70; Jelkmann et al. (1991) Ann NY Acad Sci718:300-311; Vannucchi et al. (1994) Br J Hematol 87:18-23; With Oldenburg et al. (2001) Aliment Pharmacol Ther 15:429-438). The invention provides for improving and EPO generates, the conduction of EPO signal is relevant with the iron utilization metabolism and the method for physiological pathway, produced completely or the red blood cell generation that strengthens and reduction or the improvement of study on anemia of chronic disease.
The invention provides the advantage of the prior art therapy (for example recombinant epo administration) with respect to study on anemia of chronic disease. It only is an erythropoietic aspect that reduces that the EPO that reduces generates, and has realized that giving recombinant epo can not solve other the relevant defective of red blood cell generation that exists among the patient who suffers from study on anemia of chronic disease with reduce. (referring to, for example Clibonet al. (1990) Exp Hematol 18:438-441 and Macdougall and Cooper (2002) Neprol Dial Transplant 17 (11): 39-43). These defectives comprise that for example the EPO of marrow replys reduction, and help many aspects of whole or completely erythropoietic iron metabolism to reduce, comprise iron from the absorption in the enteron aisle, stride intestinal cell transhipment, Tie Tong cross film iron transfer auxilin (hephaestin) or ceruloplasmin be oxidized to ferric iron state, Tie Tong cross the combination of transferrin and transferrin receptor and picked-up, and iron transfer to marrow (utilizing the iron position of (comprising that ferroheme is synthetic)) all reduce. Many patients for above-mentioned reasons and invalid to giving recombinant epo wherein reduce or lack giving replying of recombinant epo, even also like this when giving the recombinant epo of high dose.
The ubiquity of inflammatory cytokine causes for example serum levels of iron level reduction and iron to be stored in the study on anemia of chronic disease increases, mainly be that the formation of CFU-E needs iron synthetic to carry out suitable ferroheme in the CFU-E that forming in the macrophage cellular compartment that is not easy to enter. The invention provides the method that helps complete or whole erythropoietic metabolic pathways for enhancing. In one embodiment, described therapeutic agent is that for example iron and the combination of B family vitamin tonic give with the tonic of further its effectiveness of enhancing.
Study on anemia of chronic disease increases relevant with the ferritin level. Although have high-caliber ferritin, the patient of study on anemia of chronic disease can not effectively utilize iron. High-caliber ferritin is that the iron that is recycled to marrow reduces and the indication of iron storage increase, and functional iron deficiency is usually relevant with ever-present false inflammatory conditions in study on anemia of chronic disease and the uremia Patients with Chronic Renal Disease. By reducing the ferritin level, method of the present invention and compound are so that the iron of storing reduces and increased the iron by transferrin and transferrin receptor recirculation. The serum ferritin level that reduces is the indication that the iron utilization strengthens and be recycled to the iron increase of marrow, has therefore increased for ferroheme to produce and erythropoietic iron availability.
The genome of anoxic is replied the change that comprises in gene expression and the stechiology, the acute and chronic effect that causes to improve the oxygen scarcity. The transcription factor that the α subunit (HIF α) that hypoxia inducible factor (HIF) is regulated by oxygen and the β subunit (HIF β) of a constitutive expression form. HIF α in normoxic environment since HIF specificity proline hydroxylase (HIF-PH) to the hydroxylation of specificity proline residue and unstable. Yet when oxygen became limited, for example in low-oxygen environment, HIF-PH can not hydroxylation HIF α, and this subunit is not degraded, and active HIF compound forms, and transposition is transcribed in nucleus and activated gene.
In some aspects, the invention provides the method for the treatment of of chronic diseases anaemia by materia medica simulation anoxic. In some aspects, described method strengthens the EPO generation in the inhibiting mode of opposing inflammatory cytokine. EPO produces and is normally induced by anoxic or hypoxemia, but expression and secretion keeps reducing in having the situation of inflammatory cytokine, and ubiquitous TNF-α, IL-1 β and IFN-γ in described inflammatory cytokine such as the patients with chronic diseases (see for example Means (1995) Stem Cells 13:32-37; Means (1999) Int J Hematol70:7-12; Roodman et al. (1989) Adv Exp Med Biol 271:185-196; Fuchset al. (1991) Eur J Hematol 46:65-70; Jelkmann et al. (1991) Ann NYAcad Sci718:300-311; With Vannucchi et al. (1994) Br J Hematol87:18-23). Prolyl hydroxylase inhibitors is at least part of to have overcome the inhibitory action that inflammatory cytokine produces EPO, and this confirms (seeing for example Figure 1A, 1B, 2A, 2B, 3A, 3B, 4A and 4B) by Hep3B emiocytosis EPO to the ability that is higher than the level that observes in having the situation of inflammatory cytokine. Some preparations such as iron chelating agent, Deferoxamine (desferrioxamine) also illustrate some effectiveness (seeing for example Salvarani et al. (1996) Rheumatol Int16:45-48 and Goch et al. (1995) Eur J Hematol 55:73-77) in the research of erythropoietin(EPO) resistance anaemia (for example study on anemia of chronic disease).
In other side, the invention provides the method for in having the situation of inflammatory cytokine, improving the conduction of EPO receptor signal. The ubiquity of inflammatory cytokine causes the effect of EPO signal conduction to reduce in the patients with chronic diseases, and this can not reply the recombinant epo with the effect of enhancing erythropoietin(EPO) by many patients and confirm. Think that this is because to (seeing for example Clibon et al. (1990) Exp Hematol 18:438-441 and Macdougall and Cooper (2002) Neprol Dial Transplant 17 (11): 39-43) due to the defective in the bioactive Reduced susceptibility of EPO and marrow structure and/or the microenvironment. In certain embodiments, the invention provides be used to inducing all and erythropoietic method completely, by recovering suitable cell the sensitiveness of the signal transduction by the EPO acceptor is carried out.
Iron deficiency is one of modal nutritional deficiency in the world, and is that anemogenic main cause is led in the whole world. The balance of iron is regulated by erythropoietic speed and iron storage capacity the most at all. Anaemia can be followed or do not followed to iron deficiency, and with the cognitive development attenuation of correlation.
Iron deficiency refer to iron supply (level or storage) but availability and the underutilization of deficiency or body iron. This can be owing to nutritional deficiency, for example the shortage of iron in the diet; Owing to the malabsorption of iron, for example perform the operation (after the gastrectomy) or disease (clone disease); Perhaps owing to derive from wound or wound, menstruation, the supply loss of iron or the forfeiture of iron increase due to the chronic or acute bleeding of donate blood, draw blood (such as various programs, operation); Perhaps owing to the iron due to the Fast Growth in for example infant or puberty, the gestational period, the epo treatment need increase.
Iron deficiency also can be functional iron deficiency, for example is characterised in that object obtains and utilize the iron deficiency that ability that iron stores weakens. The ratio of iron is not enough to so that the red blood cell Hb A hemoglobin adult causes cellular hemoglobin content in granulophilocyte and the red blood cell. Functional iron deficiency often be found in iron store seem normally or even increase, but the availability of iron weaken healthy individual, this for example low-level transferrin saturation degree percentage test arrive. Such iron deficiency is common and acute or chronic inflammation is relevant.
The iron deficiency of any kind all can cause the restricted red blood cell of iron-deficient or iron to generate, wherein red blood cell number reduces, the red blood cell of circulation is less than normal red blood cell (microcyte anaemia), and the enough hemoglobins of shortage, and color is pale (low hemochrome anaemia) thus.
Iron deficiency comprises that the synthetic weakening of hemoglobin can occur the object of functional iron deficiency, and transferrin saturation degree percentage reduces, and H﹠H reduces, and causes hypoferric anemia. Hypoferric anemia is modal anaemia in the world. Iron is the essential composition of hemoglobin; Do not have iron, then marrow can not effectively produce hemoglobin. Hypoferric anemia can occur in iron is supplied the object of loss or weakening, perhaps can occur in the object of functional iron deficiency, and functional iron deficiency refers to that iron exists with storage form but can not utilize (for example being used for hemoglobin produces).
Such process has generally been contained in the metabolism of iron: cell, tissue, organ, tract or whole organism keep the dynamic equilibrium of iron by changing (for example increase or reduce) specificity process of iron metabolism. The metabolism of iron or the metabolic process of iron have contained the processes such as the processing, transhipment, picked-up, utilization, storage, mobilization, absorption of iron. The metabolism of iron and the special aspects of processing comprise that promotion iron passes iron transfer albumen and the expression of enzyme and reservation and the secretion that cell is crossed by Tie Tong of cell membrane motion; The protein expression that participates in iron transfer in the blood changes; The change that transferrin and transferrin receptor are expressed; Participate in protein expression and/or active change that iron absorbs; The change of transcribing and translating adjusting protein expression and activity that iron phase closes; And body or nutrient solution comprise such as in tissue space (being the extracellular), the cell, iron distributes in the blood, marrow etc. change.
In some aspects, the invention provides the method for the picked-up, transhipment, processing and the utilization that improve iron. Study on anemia of chronic disease is relevant with negative effect ferroheme defective synthetic and that hemoglobin forms in the iron utilization, causes red blood cell to generate reduction (seeing for example Oldenburg et al. (2001) Aliment Pharmacol Ther 15:429-438). During the reduction that patients with chronic diseases serum levels of iron level, iron are mobilized and iron are stored any relevant increase all may with the microorganism defense mechanism relevant (seeing Fuchs et al. (1991) Eur J Hematol46:65-70) of macrophage under long-term inflammation condition. In some respects, the invention provides the method that increases effective metabolism of iron by stablizing HIF α.
The metabolism of numerous protein mediation iron is arranged, comprise protein such as red be 5-Aminolevulinate synthase (ALAS) (during ferroheme is synthetic first and rate-limiting step) (Bottomley andMuller-Eberhard (1988) Semin Hematol 25:282-302 and Yin et al. (1998) Blood, Cells, Molecules, and Diseases 24 (3): 41-533), transferrin, transferrin receptor, iron transfer albumen (participation iron transfer), ceruloplasmin etc. The increase that transferrin and transferrin receptor are expressed stimulates CFU-E picked-up iron and promotes macrophage picked-up iron and it is transported to marrow (Goswami et al. (2002) Biochem Cell Biol 80:679-689). Ceruloplasmin promotes that oxidation of divalent is ferric iron, in order to be combined (Goswami et al. (2002) Biochem Cell Biol80:679-689) with transferrin. In some aspects, protein expression and the active metabolism that increased iron of method of the present invention by increasing the metabolism that participates in iron, described protein comprises red blood cell 5-Aminolevulinate synthase, transferrin, transferrin receptor, NRAMP2, sproutin (duodenum cytochrome b reductase 1) and ceruloplasmin. In other side, method of the present invention is by expression and the activity that reduces hepcidin and the metabolism that has increased iron by the expression of regulating ferritin.
In one embodiment, the invention provides method and compound for increasing gene expression, the expression product of described gene participates in metabolism and the processing of iron, comprises the picked-up, storage, transhipment, absorption of iron etc. This gene comprises but non-transferrin receptor, ceruloplasmin, NRAMP2,5-Aminolevulinate synthase, the sproutin (CYBRD1) etc. of being limited to. The gene of the metabolism that participates in iron and processing is carried out the therapeutic incremental adjustments will effectively increase the availability of iron, thereby and in the patient body of study on anemia of chronic disease, hypoferric anemia, Functional Anemia etc., produce beneficial effect. In another embodiment, the invention provides method and compound for reducing the expression of the protein hepcidin (hepcidin) relevant with the adjusting of iron.
Suitable iron metabolism part is replied assembly by iron and is regulated in conjunction with albumen (IRP), and described iron is replied assembly and replied assembly (IRE) combination in conjunction with the iron of finding among 5 of the mRNA of albumen and encode for example ferritin (iron storage), mitochondria aconitase (energetic supersession), red system-aminolevulinate synthase and transferrin receptor '-and/or 3 '-UTR. For example ferritin transcribe the IRP and 5 ' of middle generation-IRE in conjunction with the translation that suppresses mRNA; For example transferrin transcribe middle generation with 3 '-combination of IRE then protects mRNA to avoid degraded. IRP-2 composing type in cell produces, but is degraded under the sufficient condition of iron and so inactivation. Yet IRP-2 is stable (Hanson et al. (1999) J BiolChem 274:5047-5052) under iron loss and/or hypoxia condition. Because IRP-2 reduces the expression of ferritin, this long term storage with iron is relevant, and IRP-2 increases the expression of transferrin and transferrin receptor, therefore IRP-2 promotes picked-up, transhipment and the utilization of iron, and therefore strengthens red blood cell generation (Klausner etal. (1993) Cell 72:19-28). Recently, also be to have described IRE in necessary other gene of red blood cell generation, described gene comprises 5-Aminolevulinate synthase, NRAMP2 iron transfer albumen (being also referred to as Slc11a2, DCT1, DMT1, mk (microcyte anemia gene seat in the mouse)) reaches the iron transfer albumen (Haile (1999) Am J Med Sci 318:230-240 and Gunshin et al. (2001) FEBS Lett 509:309-316) that mediates the absorption of the iron that derives from diet in duodenum.
Method of the present invention is by the simulated hypoxia condition, and the stable IRP-2 of potentiality also except stablize HIF α produces in endogenous EPO produces and functional red blood cell strengthens in producing iron picked-up, transhipment with in utilizing thus and acts synergistically.
In the adult, the absorption male sex of iron is 6% in the diet, and non-gravid woman is 13%. NRAMP2 (being also referred to as DMT1, DCT1, slc11a2) is a kind of divalent metal transport protein of generally expressing, the transmembrane transport of the iron that its participation is not combined with transferrin. NRAMP2 is a kind of iron transfer albumen, iron is transported to the duodenum enterocyte and from the hemocytoblast endosome from the intestines and stomach inner chamber is transported to cytoplasm. In the animal that experiences the diet iron deficiency, the expression of NRAMP2 (slc11a2) on the duodenal column absorptive epithelium of near-end midgut epithelial cells summit showing increase (seeing for example Canonne-Hergaux et al. (1999) Blood 93:4406-4417). Science of heredity rodent model anaemia that this gene is relevant with iron deficiency connects, and described model comprises low hemochrome anaemia and the microcyte anemia mice (mk mouse) of the NRAMP2 gene with sudden change. The MK mouse is presented on absorption and the RCIU aspect major defect of iron.
In certain aspects, method of the present invention and compound are for increasing the absorption of dietary iron. The invention provides method and compound for increasing the gene expression relevant with the iron transfer absorption. Especially, compound of the present invention is effective in increase NRAMP2 expresses in enteron aisle. The NRAMP2 (slc11a2) that increases express for increase iron for example the iron in the diet to absorb from enteron aisle be desirable.
In addition, the invention provides the data that show that sproutin gene expression increases in the animal intestinal of compound treatment of the present invention. Sproutin enteron aisle Ferric reductase (being also referred to as Dcytb and Cybrd1 (CYBRD1, duodenum cytochrome b reductase 1)) is a kind of ferric iron reductase, and the outer ferric iron of catalysis born of the same parents is reduced to iron and absorbs relevant ferrous iron. Sproutin and NRAMP2 in the animal of iron deficiency at the top of Duodenal villi co expression (seeing for example McKie et al. (2001) Science 291:1755-1759).
Method of the present invention and compound are for increasing ceruloplasmin gene expression. Ceruloplasmin is also referred to as ferrous oxidase-1, and the iron (such as ferritin) of the reduction that will discharge from the storage position changes oxidised form into. The iron of oxidation can be in conjunction with its blood plasma transport protein-transferrin. It is relevant with iron gathering in liver and other tissue that ceruloplasmin lacks. There are indications that ceruloplasmin promotes iron to flow out and promotes flowing molten iron to enter in the cell of iron deficiency (seeing for example Tran et al. (2002) J Nutr 132:351-356) from liver.
Compound of the present invention reduces hepcidin (hepcidin) mRNA expresses in mouse liver. Inflammation causes IL-6 to produce, and it acts on liver cell and induces hepcidin to produce. Hepcidin suppresses the release of macrophage iron and enteron aisle iron absorbs, reduces the availability of iron and causes for example hypoferremia. It is relevant that the hepcidin that reduces is expressed the iron absorption that discharges from the reticuloendothellium cell with the iron that increases and increase. Therefore, method of the present invention and compound can be used for reducing absorption and the reduction hypoferremia that enteron aisle iron was expressed, increased to hepcidin.
The present invention particularly including treatment and HCV (HCV) infect the method for relevant anaemia. At present the method that infects for the treatment of HCV comprises and is used in combination interferon-' alpha ' and virazole (ribaviron). This combination treatment causes HC to reduce and anaemia. On the one hand, the invention provides method and the compound that is used for the treatment of the anaemia relevant with the HCV infection. On the other hand, the invention provides method and the compound that is used for the treatment of the anaemia relevant with the interferon-' alpha ' therapy of HCV infection. On the other hand, the invention provides the Compounds and methods for that is used for the treatment of the anaemia relevant with the virazole therapy of HCV infection.
The present invention also provides increases red blood cell breaks up the generation of the required factor from HPC method, and described HPC comprises such as candidate stem cell (HSC), CFU-GEMM (colony forming unit granulocyte/red blood cell/monocyte/megacaryocyte) cell etc. Stimulate the erythropoietic factor to comprise but the non-erythropoietin(EPO) that is limited to. On the other hand, described method increases the generation that iron absorbs, transports and utilize the required factor. This class factor comprises but non-to be limited to red be aminolevulinate synthase, transferrin, transferrin receptor, ceruloplasmin, ferritin etc. On the other hand, described method increases the required factor of erythroid differentiation and iron picked-up, transports and utilize required extraneous factor.
The present invention also provides and has been used for strengthening the method that the hematopoiesis precursor is replied erythropoietin(EPO). As mentioned above, this precursor comprises HSC, CFU-GEMM etc. Replying of precursor can strengthen, and described enhancing is for example by changing erythropoietin receptor, participate in born of the same parents' intrinsic factor of erythropoietin(EPO) signal transmission and promoting the expression of the factor of the secretion of erythropoietin(EPO) and acceptor interaction to carry out. The invention provides for for example strengthening the method that marrow EPO replys by increasing the EPO expression of receptor.
Method
The invention provides the whole bag of tricks. On the one hand, described method comprises the preparation that gives a kind of stable HIF α of object.
The stable of HIF α can be finished by the available and known any method of those skilled in the art, can comprise use with HIF α interact, in conjunction with or modify HIF α any preparation or with the interactional factor of HIF α, comprise that for example substrate is the enzyme of HIF α. In some aspects, the present invention includes the HIF α variant that provides a kind of composing type stable, such as stable HIF mutain etc., the polynucleotides of this variant of perhaps encoding (are seen for example U.S. Patent No. 6,562,799 and 6,124,131; And U.S. Patent No. 6,432,927 is described). In other side, the present invention includes and make HIF α stable, comprise the preparation that gives a kind of stable HIF α. Described preparation can by polynucleotides such as antisense sequences (seeing such as international open No.WO03/045440 described), polypeptide, antibody, other oroteins, carbohydrate, fat, lipid, and organic and inorganic matter such as little molecule etc. form. In a preferred embodiment, for example present invention resides in and stablize HIF α by the preparation that gives a kind of stable HIF α of object in the object, wherein said preparation is the compound of a kind of stable HIF α, for example micromolecular compound.
In other embodiments, method of the present invention comprises that the activity of at least a enzyme by suppressing to be selected from 2-oxoglutarate dioxygenase family stablizes HIF α. In a preferred embodiment, described enzyme is a kind of HIF hydroxylase, and such as EGLN-1, EGLN-2, EGLN-3 etc. (seen for example Taylor (2001) Gene 275:125-132; Epstein et al. (2001) Cell107:43-54; And Bruick and McKnight (2001) Science 294:1337-1340). Yet the present invention is particularly including such enzyme, it can be any enzyme that is selected from 2-oxoglutarate dioxygenase family, comprise for example precollagen lysyl hydroxylase, precollagen prolyl 3-hydroxylase, precollagen prolyl 4 hydroxylase α (I) and α (II), Thymine 7-hydroxylase, aspartoyl (asparaginyl-) B-hydroxylase, ε-N-trimethyl lysine hydroxylase and gamma-butyrobetaine hydroxylase etc. (are seen for example Majamaa et al. (1985) Biochem J 229:127-133; Myllyharjuand Kivirikko (1997) EMBO J 16:1173-1180; Thornburg et al. (1993) 32:14023-14033; And Jia et al. (1994) Proc Natl Acad Sci USA91:7227-7231).
In certain embodiments, described method comprises the preparation of the stable HIF α by giving the object effective dose and treatment of chronic diseases anaemia or regulate iron metabolism. In preferred embodiments, described preparation is a kind of compound of the present invention. On the one hand, described compound is stablized HIF α, described residue such as proline residue, asparagine residue etc. by the hydroxylation of some residue of inhibition HIF α. In a preferred embodiment, described residue is proline residue. In special embodiment, described residue can be the P among the HIF-1 α564Residue, perhaps the homology proline in the another kind of HIF α isotype, the perhaps P among the HIF-1 α402Homology proline in residue or the another kind of HIF α isotype etc. In other embodiment, method of the present invention can contain the hydroxylation that suppresses HIF α asparagine residue, and described residue is the N of HIF-1 α for example803Homology asparagine residue in residue or the another kind of HIF α isotype.
Compound
In a preferred method, method of the present invention comprises the compound of the stable HIF α that gives the object effective dose. For example the world discloses disclosed exemplary compound among No.WO 03/049686 and the No.WO03/053997, and these documents are incorporated reference at this in full with it. Especially, compound of the present invention comprises following compound.
In certain embodiments, compound of the present invention is the compound that suppresses the HIF hydroxylase activity. In various embodiments, described activity is owing to the HIF prolyl hydroxylase, such as EGLN1, EGLN2 or EGLN3 etc. In other embodiment, described activity for example comprises but the non-FIH of being limited to owing to the HIF asparaginyl hydroxylase. The preferred compound of the present invention is the compound that suppresses HIF prolyl hydroxylase activity. Described inhibition can be direct or indirect inhibition, can be competition or noncompetitive inhibition etc.
On the one hand, compound of the present invention is inhibition or regulates in addition any compound of 2-oxoglutarate dioxygenase activity. 2-oxoglutarate dioxygenase comprises but the non-hydroxylase that is limited to. Hydroxylase makes target substrate residue hydroxylation, and described residue comprises such as prolyl, lysyl, asparaginyl-(aspartoyl) hydroxylase etc. Hydroxylase is described by the target substrate sometimes, such as HIF hydroxylase, precollagen hydroxylase etc., and/or by the description of the target residue in the substrate, such as prolyl hydroxylase, lysyl hydroxylase etc., such as HIF prolyl hydroxylase, precollagen prolyl hydroxylase etc. perhaps together described by the two. Representational 2-oxoglutarate dioxygenase comprises but the non-HIF of being limited to hydroxylase (comprising the HIF prolyl hydroxylase, such as EGLN1, EGLN2 and EGLN3), HIF asparaginyl hydroxylase (such as factor that suppresses HIF (FIH) etc.), precollagen hydroxylase (such as the precollagen lysyl hydroxylase), precollagen prolyl hydroxylase (such as precollagen prolyl 3-hydroxylase, precollagen prolyl 4 hydroxylase α (I) and α (II) etc.), Thymine 7-hydroxylase, aspartoyl (asparaginyl-) B-hydroxylase, ε-N-trimethyl lysine hydroxylase, gamma-butyrobetaine hydroxylase etc. Although enzymatic activity can comprise any activity relevant with any 2-oxoglutarate dioxygenase, the hydroxylation of amino acid residue in the substrate. Although in the substrate hydroxylation of proline and/or asparagine residue by particularly including, other amino acid whose hydroxylation is also contained.
On the one hand, demonstration has the compounds of this invention that suppresses active to one or more 2-oxoglutarate dioxygenase and may also demonstrate the inhibition of one or more other 2-oxoglutarate dioxygenase active, the compound that for example suppresses the HIF hydroxylase activity can suppress the activity of collagen prolyl hydroxylase in addition, and the compound that suppresses HIF prolyl hydroxylase activity can suppress activity of HIF asparaginyl hydroxylase etc. in addition.
Because HIF α (needs oxygen and Fe by the proline hydroxylation2+Reaction) modify, therefore to the present invention includes on the one hand the enzyme relevant with HIF α hydroxylation be the member of 2-oxoglutarate dioxygenase family. This kind of enzyme comprises but non-precollagen lysyl hydroxylase, precollagen prolyl 3-hydroxylase, precollagen prolyl 4 hydroxylase α (I) and α (II), Thymine 7-hydroxylase, aspartoyl (asparaginyl-) β-hydroxyl n enzyme, ε-N-trimethyl lysine hydroxylase and the gamma-butyrobetaine hydroxylase etc. of being limited to. These enzyme require oxygen, Fe2+, 2-oxoglutarate and ascorbic acid (see for example Majamaa et al. (1985) Biochem J229:127-133 to carry out its hydroxylase activity; Myllyharjuand Kivirikko (1997) EMBO J 16:1173-1180; Thornburg et al. (1993) 32:14023-14033; With Jia et al. (1994) Proc NatlAcad Sci USA 91:7227-7231).
On the one hand, compound of the present invention is the compound of a kind of stable HIF α. Preferably, described compound is stablized HIF α by suppressing the HIF hydroxylase activity. Therefore the present invention particularly including compound of the present invention be selected from the hydroxylase activity conditioning agent of previous discriminating. For example, the micromolecular inhibitor of having differentiated prolyl 4 hydroxylase (is seen for example Majamaa et al. (1984) Eur J Biochem 138:239-245; Majamaa et al. (1985) Biochem J229:127-133; Kivirikko and Myllyharju (1998) Mtrix Biol 16:357-368; Bickel et al. (1998) Hepatology28:404-411; Friedman et al. (2000) ProcNatl Acad Sci USA 97:4736-4741; With Franklin et al. (2001) Biochem J353:333-338; All documents are all incorporated reference into it in full at this). Present invention includes the application in the method for the invention of these compounds.
In some respects, compound of the present invention comprises for example structural simulation thing of 2-oxoglutarate. This compound can suppress target 2-oxoglutarate dioxygenase family member with 2-oxoglutarate competitiveness, suppress target 2-oxoglutarate dioxygenase family member, (Majamaa et al. (1984) Eur J Biochem 138:239-245 with the iron noncompetitive; And Majamaa et al.Biochem J 229:127-133).
In certain embodiments, used compound is selected from a kind of compound of chemical formula (I) in the method for the present invention:
Figure S04819307020060123D000401
Wherein
A is 1,2-arlydene, 1,3-arlydene, Isosorbide-5-Nitrae-arlydene; Perhaps (C1-C 4)-alkylidene, it is randomly by one or two kind of halogen, cyano group, nitro, trifluoromethyl, (C1-C 6)-alkyl, (C1-C 6)-hydroxyalkyl, (C1-C 6)-alkoxyl ,-O-[CH2] x-C fH (2f+1-g)Hal g，(C 1-C 6)-Fluoroalkyloxy, (C1-C 8)-fluorine alkenyloxy, (C1-C 8)-fluorine alkynyloxy group ,-OCF2Cl，-O-CF 2-CHFCl replaces; (C1-C 6)-alkyl thiol, (C1-C 6) alkyl sulfenyl, (C1-C 6)-alkyl sulfonyl, (C1-C 6)-alkyl-carbonyl, (C1-C 6)-alkoxy carbonyl, carbamoyl, N-(C1-C 4)-alkyl-carbamoyl, N, N-two-(C1-C 4)-alkyl-carbamoyl, (C1-C 6)-alkyl-carbonyl oxygen base, (C3-C 8)-cycloalkyl, phenyl, benzyl, phenoxy group, benzyloxy, anilino-, methylphenylamine base, phenyl sulfydryl, phenyl sulphonyl, phenyl sulfenyl, sulfonamides, N-(C1-C 4)-alkyl sulfonamides, N, N-two-(C1-C 4)-alkyl sulfonamides replaces; Perhaps by the (C that replaces6-C 12)-aryloxy group, (C7-C 11)-aralkyl oxy, (C6-C 12)-aryl, (C7-C 11)-aralkyl replaces, and it carries 1-5 identical or different substituting group in aryl moiety, and described substituting group is selected from halogen, cyano group, nitro, trifluoromethyl, (C1-C 6)-alkyl, (C1-C 6)-alkoxyl ,-O-[CH2] x-C fH (2f+1-g)Hal g、-OCF 2Cl、-OCF 2CHFCl、(C 1-C 6) alkyl thiol, (C1-C 6)-alkyl sulfenyl, (C1-C 6)-alkyl sulfonyl, (C1-C 6)-alkyl-carbonyl, (C1-C 6)-alkoxy carbonyl, carbamyl, N-(C1-C 4)-alkylcarbamoyl group, N, N-two-(C1-C 4)-alkylcarbamoyl group, (C1-C 6)-alkyl-carbonyl oxygen base, (C3-C 8)-cycloalkyl, sulfonamides, N-(C1-C 4)-alkyl sulfonamides, N, N-two-(C1-C 4)-alkyl sulfonamides; Perhaps wherein A is-CR5R 6，R 5And R6Be selected from independently of one another hydrogen, (C1-C 6)-alkyl, (C3-C 7The substituting group of the alpha-carbon atom of)-cycloalkyl, aryl or a-amino acid, wherein amino acid is natural L-amino acid or its D-isomers.
B is-CO2H，-NH 2，-NHSO 2CF 3, tetrazole radical, imidazole radicals, 3-hydroxyl isoxazolyl ,-CONHCOR " ' ,-CONHSOR ' " ,-CONHSO2R " ', wherein R " ' is aryl, heteroaryl (heteroaryl), (C3-C 7) cycloalkyl or (C1-C 4)-alkyl, it is randomly by (C6-C 12)-aryl, heteroaryl, OH, SH, (C1-C 4)-alkyl, (C1-C 4)-alkoxyl, (C1-C 4)-alkylthio, (C1-C 4)-sulfenyl, (C1-C 4)-sulphonyl, CF3，Cl，Br，F，I，NO 2，-COOH，(C 2-C 5)-alkoxy carbonyl, NH2, list-(C1-C 4-alkyl)-and amino, two-(C1-C 4-alkyl)-amino or (C1-C 4)-perfluoroalkyl list replaces; Perhaps wherein B is CO2-G carboxyl, wherein G is the group of pure G-OH, wherein G is selected from (C1-C 20)-alkyl, (C3-C 8) cycloalkyl, (C2-C 20)-alkenyl, (C3-C 8)-cycloalkenyl, retinyl, (C2-C 20)-alkynyl, (C4-C 20)-chain Ene alkynyl base (alkenynyl), alkenyl wherein, cycloalkenyl, alkynyl and chain Ene alkynyl base contain one or more key; (C6-C 16)-isocyclic aryl, (C7-C 16)-carbocyclic ring aralkyl, heteroaryl or heteroarylalkyl, wherein the heteroaryl moieties of heteroaryl or heteroarylalkyl contains 5 or 6 annular atomses; The group that wherein defines for G is replaced by one or more following group: hydroxyl, halogen, cyano group, trifluoromethyl, nitro, carboxyl, (C1-C 12)-alkyl, (C3-C 8)-cycloalkyl, (C5-C 8)-cycloalkenyl group, (C6-C 12)-aryl, (C7-C 16)-aralkyl, (C2-C 12)-alkenyl, (C2-C 12)-alkynyl, (C1-C 12)-alkoxyl, (C1-C 12)-alkoxyl-(C1-C 12)-alkyl, (C1-C 12)-alkoxyl-(C1-C 12)-alkoxyl, (C6-C 12)-aryloxy group, (C7-C 16)-aralkyl oxy, (C1-C 8)-hydroxyalkyl ,-O-[CH2] x-C fH (2f+1-g)-F g，-OCF 2Cl，-OCF 2-CHFCl，(C 1-C 12)-alkyl-carbonyl, (C3-C 8)-naphthene base carbonyl, (C6-C 12)-aryl carbonyl, (C7-C 16)-aromatic alkyl carbonyl, cinnamyl, (C2-C 12)-alkenyl carbonyl, (C2-C 12)-alkynyl carbonyl, (C1-C 12)-alkoxy carbonyl, (C1-C 12)-alkoxyl-(C1-C 12)-alkoxy carbonyl, (C6-C 12)-aryloxycarbonyl, (C7-C 16)-aromatic alkoxy carbonyl, (C3-C 8)-cyclo alkoxy carbonyl, (C2-C 12)-alkenyloxy carbonyl, (C2-C 12)-alkynyloxy group carbonyl, acyloxy, (C1-C 12)-alkoxy-carbonyl oxy, (C1-C 12)-alkoxyl-(C1-C 12)-alkoxy-carbonyl oxy, (C6-C 12)-aryloxycarbonyl oxygen base, (C7-C 16) aralkyl oxy ketonic oxygen base, (C3-C 8)-cyclo alkoxy carbonyl oxygen base, (C2-C 12)-alkenyloxy ketonic oxygen base, (C2-C 12)-alkynyloxy group ketonic oxygen base, carbamyl, N-(C1-C 12)-alkylcarbamoyl group, N.N-two (C1-C 12)-alkylcarbamoyl group, N-(C3-C 8)-cycloalkyl-carbamyl, N-(C6-C 16)-aromatic yl ammonia methanoyl, N-(C7-C 16)-alkyl aryl ammonium formoxyl, N-(C1-C 10)-alkyl-N-(C6-C 16)-aromatic yl ammonia methanoyl, N-(C1-C 10)-alkyl-N-(C7-C 16)-alkyl aryl ammonium formoxyl, N-((C1-C 10)-alkoxyl-(C1-C 10)-alkyl)-and carbamyl, N-((C6-C 12)-aryloxy group-(C1-C 10) alkyl)-carbamyl, N-((C7-C 16)-aralkyl oxy-(C1-C 10)-alkyl)-and carbamyl, N-(C1-C 10)-alkyl-N-((C1-C 10)-alkoxyl-(C1-C 10)-alkyl)-and carbamyl, N-(C1-C 10)-alkyl-N-((C6-C 16)-aryloxy group-(C1-C 10)-alkyl)-and carbamyl, N-(C1-C 10)-alkyl-N-((C7-C 16)-aralkyl oxy-(C1-C 10)-alkyl)-and carbamyl, carbamoyloxy group, N-(C1-C 12)-alkyl carbamoyloxy group, N, N-two-(C1-C 12)-alkyl carbamoyloxy group, N-(C3-C 8)-cycloalkyl carbamoyloxy group, N-(C6-C 12)-aryl carbamoyloxy group, N-(C7-C 16)-alkyl aryl ammonium formyloxy, N-(C1-C 10)-alkyl-N-(C6-C 12)-aryl carbamoyloxy group, N-(C1-C 10)-alkyl-N-(C7-C 16)-alkyl aryl ammonium formyloxy, N-((C1-C 10)-alkyl)-and carbamoyloxy group, N-((C6-C 12)-aryloxy group-(C1-C 10)-alkyl)-and carbamoyloxy group, N-((C7-C 16)-aralkyl oxy-(C1-C 10)-alkyl)-and carbamoyloxy group, N-(C1-C 10)-alkyl-N-((C1-C 10)-alkoxyl-(C1-C 10)-alkyl)-and carbamoyloxy group, N-(C1-C 10)-alkyl-N-((C6-C 12)-aryloxy group-(C1-C 10)-alkyl)-and carbamoyloxy group, N-(C1-C 10)-alkyl-N-((C7-C 16)-aralkyl oxy-(C1-C 10)-alkyl)-and carbamoyloxy group, amino, (C1-C 12)-alkyl amino, two-(C1-C 12)-alkyl amino, (C3-C 8)-cycloalkyl amino, (C2-C 12)-alkenyl amino, (C2-C 12)-alkynyl is amino, N-(C6-C 12)-arylamino, N-(C-C11)-aryl alkyl amino, N-alkyl-aryl alkyl amino, N-alkyl-arylamino, (C1-C 12)-alkoxy amino, (C1-C 12)-alkoxyl-N-(C1-C 10)-alkyl amino, (C1-C 12)-alkyl-carbonyl-amino, (C3-C 8)-cycloalkyl amino carbonyl, (C6-C 12)-aryl-amino-carbonyl, (C7-C 16)-aromatic alkyl carbonyl is amino, (C1-C 12)-alkyl-carbonyl-N-(C1-C 10)-alkyl amino, (C3-C 8)-naphthene base carbonyl-N-(C1-C 10)-alkyl amino, (C6-C 12)-aryl carbonyl-N-(C1-C 10)-alkyl amino, (C7-C 11)-aromatic alkyl carbonyl-N-(C1-C 10)-alkyl amino, (C1-C 12)-alkyl-carbonyl-amino-(C1-C 8)-alkyl, (C3-C 8)-cycloalkyl amino carbonyl-(C1-C 8)-alkyl, (C6-C 12)-aryl-amino-carbonyl-(C1-C 8)-alkyl, (C7-C 12Amino (the C of)-aromatic alkyl carbonyl1-C 8)-alkyl, amino-(C1-C 10)-alkyl, N-(C1-C 10) alkyl amino-(C1-C 10)-alkyl, N, N-two-(C1-C 10)-alkyl amino-(C1-C 10)-alkyl, (C3-C 8) cycloalkyl amino-(C1-C 10)-alkyl, (C1-C 12)-alkyl thiol, (C1-C 12)-alkyl sulfenyl, (C1-C 12)-alkyl sulfonyl, (C6-C 16)-aryl sulfydryl, (C6-C 16)-aryl sulfenyl, (C6-C 12)-arylsulfonyl, (C7-C 16)-aralkyl sulfydryl, (C7-C 16)-aralkyl sulfenyl, (C7-C 16)-aralkyl sulphonyl, sulfonamides, N-(C1-C 10)-alkyl sulfonamides, N, N-two (C1-C 10)-alkyl sulfonamides, (C3-C 8)-cycloalkyl sulfonamides, N-(C6-C 12)-alkyl sulfonamides, N-(C7-C 16)-alkyl aryl ammonium sulphonyl, N-(C1-C 10)-alkyl-N-(C6-C 12)-aryl sulfonamide, N-(C1-C 10)-alkyl-N-(C7-C 16)-alkyl aryl ammonium sulphonyl, (C1-C 10)-amino-alkyl sulfinyl, N ((C1-C 10)-alkyl)-(C1-C 10)-amino-alkyl sulfinyl, (C7-C 16)-aralkyl sulfonamido, or N-((C1-C 10)-alkyl-(C7-C 16)-aralkyl sulfonamido; Wherein aryl or the group that contains aryl moiety can be on aryl be replaced by 1-5 identical or different following group: hydroxyl, halogen, cyano group, trifluoromethyl, nitro, carboxyl, (C1-C 12)-alkyl, (C3-C 8)-cycloalkyl, (C6-C 12)-aryl, (C7-C 16)-aralkyl, (C1-C 12)-alkoxyl, (C1-C 12)-alkoxyl-(C1-C 12) alkyl, (C1-C 12)-alkoxyl-(C1-C 12) alkoxyl, (C6-C 12)-aryloxy group, (C7-C 16)-aralkyl oxy, (C1-C 8)-hydroxyalkyl, (C1-C 12)-alkyl-carbonyl, (C3-C 8)-cycloalkyl-carbonyl, (C6-C 12)-aryl carbonyl, (C7-C 16) aromatic alkyl carbonyl, (C1-C 12)-alkoxy carbonyl, (C1-C 12)-alkoxyl-(C1-C 12)-alkoxy carbonyl, (C6-C 12)-aryloxycarbonyl, (C7-C 16)-aromatic alkoxy carbonyl, (C3-C 8)-cyclo alkoxy carbonyl, (C2-C 12)-alkenyloxy carbonyl, (C2-C 12)-alkynyloxy group carbonyl, (C1-C 12)-alkyl-carbonyl oxygen base, (C3-C 8)-naphthene base carbonyl oxygen base, carbamoyloxy group, N-((C6-C 12)-aryloxy group-(C1-C 10)-alkyl)-and carbamoyloxy group, N-((C7-C 16)-aralkyl oxy-(C1-C 10)-alkyl)-and carbamoyloxy group, N-(C1-C 10)-alkyl-N-((C1-C 10)-alkoxyl-(C1-C 10)-alkyl)-and carbamoyloxy group, N-(C1-C 10)-alkyl-N-((C6-C 12)-aryloxy group-(C1-C 10)-alkyl)-and carbamoyloxy group, N-(C1-C 10)-alkyl-N-((C7-C 16)-aralkyl oxy-(C1-C 10)-alkyl)-and carbamoyloxy group, amino, (C1-C 12)-alkyl amino, two-(C1-C 12)-alkyl amino, (C3-C 8)-cycloalkyl amino, (C3-C 12)-alkenyl amino, (C3-C 12)-alkynyl is amino, N-(C6-C 12)-arylamino, N-(C7-C 11)-aryl alkyl amino, the N-alkyl aralkyl is amino, N-alkyl aryl amino, (C1-C 12)-alkoxy amino, (C1-C 12)-alkoxyl-N-(C1-C 10)-alkyl amino, (C1-C 12)-alkyl-carbonyl-amino, (C3-C 8)-cycloalkyl amino carbonyl, (C6-C 12)-aryl-amino-carbonyl, (C7-C 16)-alkyl-carbonyl-amino, (C1-C 12)-alkyl-carbonyl-N-(C1-C 10)-alkyl amino, (C3-C 8)-naphthene base carbonyl-N-(C1-C 10)-alkyl amino, (C6-C 12)-aryl carbonyl-N-(C1-C 10)-alkyl amino, (C7-C 11)-aromatic alkyl carbonyl-N-(C1-C 10)-alkyl amino, (C1-C 12)-alkyl-carbonyl-amino-(C1-C 8)-alkyl, (C3-C 8)-cycloalkyl amino carbonyl-(C1-C 8)-alkyl, (C6-C 12)-aryl-amino-carbonyl-(C1-C 8)-alkyl, (C7-C 16)-aromatic alkyl carbonyl amino-(C1-C 8)-alkyl, amino-(C1-C 10)-alkyl, N-(C1-C 10)-alkyl amino-(C1-C 10) alkyl, N, N-two-(C1-C 10)-alkyl amino-(C1-C 10)-alkyl, (C3-C 8)-cycloalkyl amino-(C1-C 10)-alkyl, (C1-C 12)-alkyl thiol, (C1-C 12)-alkyl sulfenyl, (C1-C 12)-alkyl sulfonyl, (C6-C 12)-aryl sulfydryl, (C6-C 12)-aryl sulfenyl, (C6-C 12)-arylsulfonyl, (C7-C 16)-aralkyl sulfydryl, (C7-C 16)-aralkyl sulfenyl, or (C7-C 16)-aralkyl sulphonyl;
X is O or S;
Q is O, S, NR ' or a key;
If wherein Q is key, then a R4Halogen, nitrile or trifluoromethyl;
When perhaps if Q is O, S or NR ', R then4Hydrogen, (C1-C 10)-alkyl, (C2-C 10)-alkenyl, (C2-C 10)-alkynyl, wherein alkenyl or alkynyl contain one or two C-C multiple bonds; Formula-[CH2] x-C fH (2f+1-g)-F gUnsubstituted fluoroalkyl, (C1-C 8)-alkoxyl-(C1-C 6)-alkyl, (C1-C 6)-alkoxyl-(C1-C 4)-alkoxyl-(C1-C 4)-alkyl, aryl, heteroaryl, (C7-C 11)-aralkyl, or the group of formula Z
-[CH 2] v-[O] w-[CH 2] t-E    (Z)
Wherein
E is heteroaryl, (C3-C 8The phenyl of)-cycloalkyl or formula F
Figure S04819307020060123D000451
V is 0-6,
W is 0 or 1,
T is 0-3,
R 7, R 8, R 9, R 10, R 11Identical or different and be hydrogen, halogen, cyano group, nitro, trifluoromethyl, (C 1-C 6)-alkyl, (C 3-C 8)-cycloalkyl, (C 1-C 6)-alkoxyl ,-O-[CH 2] x-C fH (2f+1-g)-F g,-OCF 2-Cl ,-O-CF 2-CHFCl, (C 1-C 6)-alkyl thiol, (C 1-C 6)-hydroxyalkyl, (C 1-C 6)-alkoxyl-(C 1-C 6)-alkoxyl, (C 1-C 6)-alkoxyl-(C 1-C 6)-alkyl, (C 1-C 6)-alkyl sulfenyl, (C 1-C 6)-alkyl sulfonyl, (C 1-C 6)-alkyl-carbonyl, (C 1-C 8)-alkoxy carbonyl, carbamyl, N-(C 1-C 8)-alkylcarbamoyl group, N, N-two-(C 1-C 8)-alkylcarbamoyl group, or (C 7-C 11)-alkyl aryl ammonium formoxyl is randomly replaced by following group: fluorine, chlorine, bromine, trifluoromethyl, (C 1-C 6)-alkoxyl, N-(C 3-C 8)-cycloalkyl carbamyl, N-(C 3-C 8)-cycloalkyl-(C 1-C 4)-alkylcarbamoyl group, (C 1-C 6)-alkyl-carbonyl oxygen base, phenyl, benzyl, phenoxy group, benzyloxy, NR YR ZR wherein YAnd R ZBe independently selected from hydrogen, (C 1-C 12) alkyl, (C 1-C 8)-alkoxyl-(C 1-C 8)-alkyl, (C 7-C 12)-aralkoxy-(C 1-C 8)-alkyl, (C 6-C 12)-aryloxy group-(C 1-C 8)-alkyl, (C 3-C 10)-cycloalkyl, (C 3-C 12)-alkenyl, (C 3-C 12)-alkynyl, (C 6-C 12)-aryl, (C 7-C 11) aralkyl, (C 1-C 12)-alkoxyl, (C 7-C 12) aralkoxy, (C 1-C 12)-alkyl-carbonyl, (C 3-C 8)-naphthene base carbonyl, (C 6-C 12) aryl carbonyl, (C 7-C 16) aromatic alkyl carbonyl; Perhaps further R wherein YAnd R ZBe together-[CH 2] h,
CH wherein 2Group can be replaced by following group: O, S, N-(C 1-C 4)-alkyl-carbonyl imino group, or N-(C 1-C 4)-alkoxy carbonyl imino group; The phenyl sulfydryl, phenyl sulphonyl, phenyl sulfenyl, sulfonamides, N-(C 1-C 8)-alkyl sulfonamides, or N, N-two-(C 1-C 8)-alkyl sulfonamides; Perhaps R 7And R 8, R 8And R 9, R 9And R 10, or R 10And R 11Be to be selected from-[CH together 2] n-or-chain of CH=CH-CH=CH-, the wherein CH of this chain 2Group is randomly by O, S, SO, SO 2Or NR YReplace; And n is 3,4 or 5; And if E is heteroaryl, described group can carry 1-3 substituent group, and described substituent group is selected from R 7-R 11Defined those groups, if perhaps E is a cycloalkyl, described group can carry one and be selected from R 7-R 11The substituent group of defined those groups;
If perhaps Q is NR ', then R 4Be R ", wherein R ' and R " identical or different and be hydrogen, (C 6-C 12)-aryl, (C 7-C 11)-aralkyl, (C 1-C 8)-alkyl, (C 1-C 8)-alkoxyl-(C 1-C 8)-alkyl, (C 7-C 12)-aralkoxy-(C 1-C 8)-alkyl, (C 6-C 12)-aryloxy group-(C 1-C 8)-alkyl, (C 1-C 10)-alkyl-carbonyl, the randomly (C of Qu Daiing 7-C 16)-aromatic alkyl carbonyl, the or (C that randomly replaces 6-C 12)-aryl carbonyl; Perhaps R ' and R " be together-[CH 2] h, CH wherein 2Group can be by O, S, N-acylimino or N-(C 1-C 10)-alkoxy carbonyl imino group is replaced, and h is 3-7.
Y is N or CR 3
Figure S04819307020060123D000481
Wherein
R xAnd R vBe selected from hydrogen independently of one another, (C 1-C 6)-alkyl, (C 3-C 7)-cycloalkyl, aryl, or the substituent group of the α carbon of the a-amino acid that belongs to of L-and D-aminoacid,
S is 1-5,
T is OH, or NR *R *, and R *, R *And R * *Identical or different and be selected from hydrogen, (C 6-C 12)-aryl, (C 7-C 11)-aralkyl, (C 1-C 8)-alkyl, (C 3-C 8)-cycloalkyl, (+)-dehydroabietic acid base, (C 1-C 8)-alkoxyl-(C 1-C 8)-alkyl, (C 7-C 12)-aralkoxy-(C 1-C 8)-alkyl, (C 6-C 12)-aryloxy group-(C 1-C 8)-alkyl, (C 1-C 10)-alkanoyl, the randomly (C of Qu Daiing 7-C 16)-aralkanoyl, the randomly (C of Qu Daiing 6-C 12)-aroyl; Perhaps R *And R *Be together-[CH 2] h, CH wherein 2Group can be replaced by following group: O, S, SO, SO 2, N-acyl amino, N-(C 1-C 10)-alkoxy carbonyl imino group, N (C 1-C 8)-alkyl imino, N-(C 3-C 8)-cycloalkyl imino group, N-(C 3-C 8)-cycloalkyl-(C 1-C 4)-alkyl imino, N-(C 6-C 12)-aryl imino group, N-(C 7-C 16)-aralkyl imino group, N-(C 1-C 4)-alkoxyl-(C 1-C 6)-alkyl imino, and h is 3-7;
Perhaps R wherein 1And R 2, perhaps R 2And R 3Form chain [CH 2] o, it is saturated or unsaturated by the two keys of a C=C, wherein 1 or 2 CH 2Group is randomly by O, S, SO, SO 2, or NR ' replacement, and R ' is hydrogen, (C 6-C 12)-aryl, (C 1-C 8)-alkyl, (C 1-C 8)-alkoxyl-(C 1-C 8)-alkyl, (C 7-C 12)-aralkoxy-(C 1-C 8)-alkyl, (C 6-C 12)-aryloxy group-(C 1-C 8)-alkyl, (C 1-C 10)-alkanoyl, the randomly (C of Qu Daiing 7-C 16)-aralkanoyl, the or (C that randomly replaces 6-C 12)-aroyl; And o is 3,4 or 5;
Perhaps R wherein 1And R 2, perhaps R 2And R 3Form 5,6,7 with pyridine that carries them or pyridazine, 8-tetrahydroisoquinoline ring, 5,6,7,8-tetrahydroquinoline ring or 5,6,7,8-tetrahydrochysene cinnolines ring;
Perhaps R wherein 1And R 2, perhaps R 2And R 3Form carbocyclic ring or heterocycle 5-or 6-unit aromatic ring; Perhaps R wherein 1And R 2, perhaps R 2And R 3Form a heterocyclic ring system that randomly replaces with pyridine that carries them or pyridazine, it is selected from thienopyridine, furo pyridine, pyridopyridine, the pyrimido pyridine, imidazopyridine, thiazole and pyridine azolactone and pyridine (oxazolopyridines), quinoline, isoquinolin and cinnolines; Quinoline wherein, isoquinolin or cinnolines preferably satisfy formula Ia, Ib and Ic:
Figure S04819307020060123D000531
And substituent R 12To R 23Has R in each case independently of one another 1, R 2And R 3Implication;
Perhaps R wherein 1And R 2Form the chemical compound of formula Id with the pyridine that carries them:
Wherein V is S, O or NR k, and R kBe selected from hydrogen, (C 1-C 6)-alkyl, aryl or benzyl; Wherein aryl can be randomly by 1-5 substituent group replacement as defined above; And
R 24, R 25, R 26, and R 27Has R in each case independently of one another 1, R 2And R 3Implication;
F is 1-8;
G is 0 or 1 to (2f+1);
X is 0-3; With
H is 3-7;
Comprise from its deutero-physiologically active salt and prodrug.
The exemplary compounds of formula (I) has description in European patent EP 0650960 and EP0650961.The end-product of all chemical compounds of listing among EP0650960 and the EP0650961, the chemical compound of particularly listing in compound claim and embodiment is incorporated this paper into as a reference.The exemplary compounds of formula (I) includes but not limited to [(3-hydroxyl-pyridine-2-carbonyl)-amino]-acetic acid and [(3-methoxyl group-pyridine-2-carbonyl)-amino]-acetic acid.
In addition, the exemplary compounds of formula (I) is at United States Patent (USP) 5,658, description arranged in 933.United States Patent (USP) 5,658, the end-product of all chemical compounds, the chemical compound of particularly listing in compound claim and the embodiment that lists in 933 is incorporated this paper into as a reference.The exemplary compounds of formula (I) includes but not limited to 3-Methoxy Pyridine-2-carboxylic acid N-(((cetyl oxygen base)-carbonyl)-methyl)-amide hydrochloride, 3-Methoxy Pyridine-2-carboxylic acid N-(((1-octyloxy)-carbonyl)-methyl)-amide, 3-Methoxy Pyridine-2-carboxylic acid N-(((hexyloxy)-carbonyl)-methyl)-amide, 3-Methoxy Pyridine-2-carboxylic acid N-(((butoxy)-carbonyl)-methyl)-amide, 3-Methoxy Pyridine-2-carboxylic acid N-(((2-oxygen in ninth of the ten Heavenly Stems base)-carbonyl)-methyl)-amide racemoid, 3-Methoxy Pyridine-2-carboxylic acid N-(((oxygen base in heptan)-carbonyl)-methyl)-amide, 3-benzyloxy pyridine-2-carboxylic acids N-(((octyloxy)-carbonyl)-methyl)-amide, 3-benzyloxy pyridine-2-carboxylic acids N-(((butoxy)-carbonyl)-methyl)-amide, 5-(((3-(1-butoxy)-propyl group)-amino)-carbonyl)-3-Methoxy Pyridine-2-carboxylic acid N-((benzyloxycarbonyl)-methyl)-amide, 5-(((3-(1-butoxy)-propyl group)-amino)-carbonyl)-3-Methoxy Pyridine-2-carboxylic acid N-(((1-butoxy)-carbonyl)-methyl)-amide, and 5-(((3-lauryl oxygen base)-propyl group) amino)-carbonyl)-3-Methoxy Pyridine-2-carboxylic acid N-(((benzyloxy)-carbonyl)-methyl)-amide.
The additional compounds of formula (I) is a United States Patent (USP) 5,620,3-pyridone-2-carbamyl ester, the United States Patent (USP) 5 described in the heterocyclic amino group formyl of the replacement of describing in 995, the United States Patent (USP) 6,020,350,607, sulfonamido carbonyl pyridine-2-carbamyl and the United States Patent (USP) 5,610,172 and 5 described in 954, sulfonamido carbonyl-pyridine-2-carbamyl and the sulfonamido carbonyl-pyridine-2-carbamyl ester described in 620,996.The end-product of all chemical compounds that these patents are listed, the chemical compound of particularly listing in compound claim and embodiment is incorporated this paper into as a reference.
The exemplary compounds of formula (Ia) is at United States Patent (USP) 5,719, description arranged in 164 and 5,726,305.The end-product of all chemical compounds of listing in the above-mentioned patent, the chemical compound of particularly listing in compound claim and embodiment is incorporated this paper into as a reference.The exemplary compounds of formula (Ia) includes but not limited to N-((3-hydroxyl-6-isopropoxy-quinoline-2-carbonyl)-amino)-acetic acid, N-((6-(1-butoxy)-3-hydroxyquinoline-2-yl)-carbonyl)-glycine, [(3-hydroxyl-6-trifluoromethoxy-quinoline-2-carbonyl)-amino]-acetic acid, N-((6-chloro-3-hydroxyquinoline-2-yl)-carbonyl)-glycine, N-((7-chloro-3-hydroxyquinoline-2-yl)-carbonyl)-glycine and [(6-chloro-3-hydroxyl-quinoline-2-carbonyl)-amino]-acetic acid.
The exemplary compounds of formula (Ib) is at United States Patent (USP) 6,093, description arranged in 730.United States Patent (USP) 6,093, the end-product of all chemical compounds, the chemical compound of particularly listing in compound claim and the embodiment that lists in 730 is incorporated this paper into as a reference.The exemplary compounds of formula (Ib) includes but not limited to N-((1-chloro-4-hydroxyl-7-(2-propoxyl group) isoquinolin-3-yl)-carbonyl)-glycine, N-((1-chloro-4-hydroxyl-6-(2-propoxyl group) isoquinolin-3-yl)-carbonyl)-glycine, N-((1-chloro-4-hydroxyl-isoquinolin-3-carbonyl)-amino)-acetic acid (compd A), N-((1-chloro-4-hydroxyl-7-methoxyl group isoquinolin-3-yl)-carbonyl)-glycine, N-((1-chloro-4-hydroxyl-6-methoxyl group isoquinolin-3-yl)-carbonyl)-glycine, N-((7-butoxy)-1-chloro-4-hydroxyl isoquinolin-3-yl)-carbonyl)-glycine, N-((6-benzyloxy-1-chloro-4-hydroxyl isoquinolin-3-carbonyl)-amino)-acetic acid, ((7-benzyloxy-1-chloro-4-hydroxyl-isoquinolin-3-carbonyl)-amino)-methyl acetate, N-((7-benzyloxy-1-chloro-4-hydroxyl-isoquinolin-3-carbonyl)-amino)-acetic acid, N-((8-chloro-4-hydroxyl isoquinolin-3-yl)-carbonyl)-glycine, N-((7-butoxy-4-hydroxyl-isoquinolin-3-carbonyl)-amino)-acetic acid.
In addition, the relevant chemical compound of formula (I) that can be used for method of the present invention includes but not limited to 6-cyclohexyl-1-hydroxy-4-methyl-1H-pyridin-2-ones, 7-(4-methyl-piperazine-1-ylmethyl)-5-phenyl sulfonymethyl-quinoline-8-alcohol, 4-nitro-quinoline-8-alcohol, 5-butoxymethyl-quinoline-8-alcohol, [(4-hydroxyl-7-phenoxy group-isoquinolin-3-carbonyl)-amino]-acetic acid (compd B) and [(4-hydroxyl-7-benzene sulfonyl-isoquinolin-3-carbonyl)-amino]-acetic acid (Compound C).Further, the invention provides other exemplary compounds, wherein, for example position A and B can be caproic acid for example together, cyanogen methyl, 2-amino-ethyl, benzoic acid, 1H-benzimidazolyl-2 radicals-ylmethyl etc.
In other embodiment, the used chemical compound of the inventive method is selected from the chemical compound or the acceptable salt of its materia medica of formula (III):
Figure S04819307020060123D000561
Wherein:
A is the integer of 1-4;
B is the integer of 0-4;
C is the integer of 0-4;
Z is selected from by (C 3-C 10) cycloalkyl, independently by one or more Y 1(the C that replaces 3-C 10) cycloalkyl, 3-10 unit Heterocyclylalkyl and independently by one or more Y 1The 3-10 unit Heterocyclylalkyl that replaces; (C 5-C 20) aryl, independently by one or more Y 1(the C that replaces 5-C 20) aryl, 5-20 unit heteroaryl and independently by one or more Y 1One group of forming of the 5-20 unit heteroaryl that replaces;
Ar 1Be selected from by (C 5-C 20) aryl, independently by one or more Y 2(the C that replaces 5-C 20) aryl, 5-20 unit heteroaryl and independently by one or more Y 2One group of forming of the 5-20 unit heteroaryl that replaces;
Each Y 1Be independently selected from by a kind of lipotropy functional group, (C 5-C 20) aryl, (C 6-C 26) alkaryl, a group of forming of 5-20 unit heteroaryl and 6-26 unit miscellaneous alkyl aryl;
Each Y 2Be independently selected from by-R '-OR ' ,-OR " ,-SR ' ,-SR " ,-NR ' R ' ,-NO 2,-CN ,-halogen ,-trihalomethyl group, three halogenated methoxies ,-C (O) R ' ,-C (O) OR ' ,-C (O) NR ' R ' ,-C (O) NR ' OR ' ,-C (NR ' R ')=NOR ' ,-NR '-C (O) R ' ,-SO 2R ' ,-SO 2R " ,-NR '-SO 2-R ' ,-NR '-C (O)-NR ' R ', tetrazolium-5-base ,-NR '-C (O)-OR ' ,-C (NR ' R ')=NR ' ,-S (O)-R ' ,-S (O)-R " and-a group of forming of NR '-C (S)-NR ' R ';
Each R ' is independently selected from by-H, (C 1-C 8) alkyl, (C 2-C 8) alkenyl and (C 2-C 8) a group of forming of alkynyl;
Each R " is independently selected from by (C 5-C 20) aryl and independently by one or more-OR ' ,-SR ' ,-NR ' R ' ,-NO 2,-the CN, (C that halogen or trihalomethyl group replace 5-C 20) a group of forming of aryl,
Perhaps wherein c is 0 and Ar 1Be the urea aryl of N ' replacement, described chemical compound has structural formula (IIIa):
Figure S04819307020060123D000571
Or the acceptable salt of its materia medica, wherein:
A, b and Z have above-mentioned definition; And
R 35And R 36Be selected from by (C independently of one another 1-C 8) alkyl, (C 2-C 8) alkenyl, (C 2-C 8) alkynyl, (C 3-C 10) cycloalkyl, (C 5-C 20) aryl, (C 5-C 20) aryl that replaces, (C 6-C 26) alkaryl, (C 6-C 26) alkaryl that replaces, a group of forming of 5-20 unit heteroaryl, the heteroaryl that 5-20 unit replaces, the alkane heteroaryl that 6-26 unit's alkane heteroaryl and 6-26 unit replace;
R 37Be independently selected from by hydrogen (C 1-C 8) alkyl, (C 2-C 8) alkenyl and (C 2-C 8) a group of forming of alkynyl.
The exemplary compounds of formula (III) has description in the open WO 00/50390 in the world.Chemical compound and the end-product among the embodiment listed in all chemical compounds, the particularly compound claim of listing among the WO 00/50390 are incorporated the application into as a reference.The exemplary compounds of formula (III) comprises 3-{[4-(3,3-dibenzyl-urea groups)-benzene sulfonyl]-[2-(4-methoxyl group-phenyl)-ethyl]-amino } N-hydroxyl-propionic acid amide. (Compound D), 3-{{4-[3-(4-chloro-phenyl)-urea groups]-benzene sulfonyl }-[2-(4-methoxyl group-phenyl)-ethyl]-amino }-N-hydroxyl-propionic acid amide., and 3-{{4-[3-(1,2-diphenyl-ethyl)-urea groups]-benzene sulfonyl }-[2-(4-methoxyl group-phenyl)-ethyl]-amino }-N-hydroxyl-propionic acid amide..
The present invention also provides the method for differentiating chemical compound of the present invention.In some aspects, chemical compound of the present invention is the chemical compound of a kind of stable HIF α.Chemical compound ability stable or activation HIF α can for example be measured by the HIF α in the direct working sample, the indirect determination (seeing that for example international open WO 00/69908 is described) by the minimizing of for example measuring the HIF α relevant with von Hippel Lindau albumen, perhaps by activating that HIF replys target gene or reporter gene construct and indirect determination (is seen for example U.S. Patent No. 5,942,434 is described).Not or have and measure in the situation of described chemical compound and level that contrast HIF and/or HIF reply target protein will be differentiated the chemical compound of stablizing HIF α and/or activation HIF α.
In others, chemical compound of the present invention is the chemical compound that suppresses the HIF hydroxylase activity.It is standardized that hydroxylase activity is analyzed in this area.Hydroxylase activity can be directly or indirectly measured in this analysis.For example, a kind of analysis can be measured the residue of the hydroxylation that exists in the zymolyte, for example proline, agedoite etc., described zymolyte is target protein, synthetic peptide mimics or its fragment (seeing that for example Palmerini et al. (1985) J Chromatogr 339:285-292 is described) for example.The residue that has hydroxylation under a kind of situation of chemical compound for example the reduction of proline or agedoite be the indication that suppresses the chemical compound of hydroxylase activity.Perhaps, analysis can be measured other product of hydroxylation reaction, for example formation of succinate from the 2-oxoglutarate (seeing that for example Cunliffe etal. (1986) Biochem J 240:617-619 is described).Kaule and Gunzler (1990; Anal Biochem 184:291-297) demonstration programme of measuring generation succinate from the 2-oxoglutarate has been described.
Said procedure can be used for differentiating the chemical compound of regulating the HIF hydroxylase activity.Target protein can comprise HIF α or its fragment, for example HIF (556-575).Enzyme can comprise HIF prolyl hydroxylase (seeing for example GenBank registration number AAG33965 etc.) or the HIF agedoite acyl hydroxylase (seeing for example GenBank registration number AAL27308 etc.) that for example derives from any source.Enzyme also may reside in the rough cell lysate or with partially purified form and exists.For example, the program of measuring the HIF hydroxylase activity Ivan et al. (2001, Science 292:464-468; And 2002, Proc Natl Acad Sci USA 99:13459-13464) and the middle description of Hirsila et al. (2003, JBiol Chem 278:30772-30780); Other method is described in the open WO03/049686 in the world.Not or have and measure in the situation of described chemical compound and the contrast enzymatic activity will be differentiated the chemical compound of inhibition HIF α hydroxylation.
Chemical compound of the present invention is the chemical compound that further is created in the effect that can measure in external or the body, the metabolism enhancing of described effect such as erythropoiesis increase, ferrum or pathological changes are able to therapeutic and improve, described pathological changes for example iron deficiency comprises functional iron deficiency, chronic disease anemia, iron deficiency anemia, microcytosis or microcytic anemia, perhaps relevant with inflammation, infection, immunodeficiency or tumor disease pathological changes.
The described effect of measuring can be any of following parameter: the hemoglobin of increase, hematocrit, reticulocyte, red blood cell count(RBC), blood plasma EPO etc.; The iron metabolism that improves, as measuring by observed sx, described symptom comprises for example alleviation of symptoms such as confirmed fatigue, anemia looks (pallor), dizziness and blurred vision, perhaps serum levels of iron level, the serum ferritin level of change, transferrin percentage ratio, total iron binding capacity, the reticulocyte count of improvement, hemoglobin, the hematocrit by increasing for example all measured by standard blood analysis of accounts
Medicament preparation and route of administration
Compositions of the present invention can directly be carried or be carried in containing the materia medica compositions of excipient, and these are known in the art.Therapeutic Method of the present invention can comprise the The compounds of this invention that has or be in the object effective dose that has in the Metabolic disorder danger, and described Metabolic disorder especially is relevant to the glycoregulatory imbalance of Fructus Vitis viniferae, diabetes for example, hyperglycemia etc.In a preferred embodiment, described to liking mammal, in the most preferred embodiment, described to liking the people.
Chemical compound or effective amount of drug, for example dosage can be determined by conventional experiment, can be a kind of route of administration and suitable preparation easily of effectively reaching.This area can utilize various preparations and delivery system, and (see for example Gennaro, ed. (2000) Remington ' s Pharmaceutical Sciences is as preceding; And Hardman, Limbird, andGilman, eds. (2001) The Pharmacological Basis of Therapeutics is as preceding).
That suitable route of administration can for example comprise is oral, per rectum, part, nose, lung, eye, intestinal and parenteral route administration.The main path of parenterai administration comprises intravenous, intramuscular and subcutaneous administration.The secondary approach of administration comprises in the intraperitoneal, intra-arterial, intraarticular, heart, in the brain pond in (intracisternal), Intradermal, intralesional (intralesional), ophthalmic, the pleura, in the sheath, intrauterine and the interior administration of ventricle.The treatment indication together with physics, the chemistry and biology character of medicine, has been stipulated the preparation type and the route of administration of use, and the preferred local still general conveying of carrying.
The pharmaceutical dosage forms of chemical compound of the present invention can be decided to provide in the delivery system at release immediately, sustained release, lasting release or target.Dosage form commonly used for example comprises solution and suspension, (little) emulsion, ointment, gel and ointment, liposome, tablet, sugar-coat, soft capsule or hard capsule, suppository, ovulum, implant, amorphous or crystal powder, aerosol, and freeze dried preparation.According to the route of administration of using, for the application of medicine or need special equipment, for example needle tubing and syringe needle, inhaler, pump, injection pen, applicator or special arrow-necked bottle.Pharmaceutical dosage form is made up of medicine, excipient and container/closed system usually.Can add one or more excipient (also being meant the batching of non-activity) in the chemical compound of the present invention improving or to promote medicine production, stability, give and safety, and a kind of means that reach the drug release pattern of hope can be provided.Therefore the type that adds the excipient in the medicine can determine according to various factors, as physics and chemical property, route of administration and the production routine decision according to medicine.This area can obtain pharmaceutically-acceptable excipients, be included in those excipient of listing in the various pharmacopeia and (see for example USP, JP, EP and BP, FDA web page (www.fda.gov), Inactive Ingredient Guide 1996, and Handbook ofPharmaceutical Additives, ed.Ash; Synapse Information Resources, Inc.2002).
The pharmaceutical dosage form of chemical compound of the present invention can be by any method production well known in the art, for example mixed, screening, dissolving, fusing, the granulation by routine, make that sugar-coat, tabletting, suspension, extruding, spray drying, grinding, emulsifying, (Nano/micron-) are encapsulated, bag carries or freeze drying process carries out.As mentioned above, compositions of the present invention can comprise the acceptable non-activity batching of one or more physiology, to promote that bioactive molecule is processed into pharmaceutical preparation.
Suitable preparation depends on the route of administration of hope.For intravenous injection, for example described compositions can be prepared in aqueous solution, then use the buffer of physiology's compatibility if desired, comprise that for example phosphate, histidine or citrate reach a kind of property formulation example of use such as sodium chloride or glucose to regulate the pH of preparation.For through mucous membrane or nasal administration, preferred semisolid, liquid formulations or ointment can contain penetration enhancers.This penetrating agent is known in the art.For oral, described chemical compound can be formulated as the liquid or solid dosage form and is formulated as immediately or the preparation of sustained release.Dosage forms through the oral cavity picked-up comprises tablet, pill, coated tablet, soft capsule or hard capsule, liquid, gel, syrup, powder slurry, suspension and emulsion.Described chemical compound also can be formulated as the compositions that per rectum gives, and as suppository or delay enema, for example contains the substrate of conventional suppository bases such as cupu oil or other glyceride.
Solid oral dosage form can use excipient to obtain, and can comprise filler, distintegrant, bonding agent (dried or wet), decompose delayer, lubricant, antiseize paste, anti-adsorbent, cation exchange resin, humidizer, antioxidant, antiseptic, pigment and spice.These excipient can be synthetic or natural origin.These excipient for example comprise cellulose derivative, citric acid, dicalcium phosphate, gelatin, magnesium carbonate, Stepanol MG/sodium, mannitol, Polyethylene Glycol, polyvinylpyrrolidone, silicate, silicon dioxide, sodium benzoate, sorbitol, starch, stearic acid or its salt, sugar (being glucose, sucrose, lactose etc.), Talcum, tragacanth, vegetable oil (hydrogenant), reach wax.The second alcohol and water can be used as the granulation adjuvant.In some cases, can be with the thin film of for example covering up taste, gastric acid resistance thin film or the thin film bag that delays to discharge by tablet.Natural and synthetic combination of polymers coloring agent, sugar and organic solvent or water are generally used for bag by tablet, produce coated tablet.When first-selected capsule but not during tablet, drug powder, its suspension or solution can be carried in compatible hard or soft capsule.
In one embodiment, chemical compound of the present invention can topical administration, as giving by barrier cream, semisolid or liquid formulations example gel, (little) emulsion, ointment, solution, (Nano/micron)-suspension or form of foam.Drug osmotic goes into skin and subcutaneous tissue can be regulated by for example following mode: use penetration enhancers; Suitably select and combination lipotropy, hydrophilic and amphipathic excipient, comprise water, organic solvent, wax, oil, synthetic and natural polymer, surfactant, emulsifying agent; Pass through pH regulator; And use complexing agent.Can use other technology such as iontherapy to regulate the dermal osmosis power of chemical compound of the present invention.Preferred percutaneous or topical are for example wished in the topical situation of minimum degree systemic exposure therein.
For inhalation or nose administration, chemical compound used according to the invention gives with the semi-solid aerosol form in solution, suspension, emulsion or compression wrap or the nebulizer expediently, usually use propeller, for example derived from the halocarbon of methane and ethane, carbon dioxide or any other suitable gas.For the topical application aerosol, use Hydrocarbon such as butane, isobutene. and pentane.In the aerosol situation of pressurization, proper dosage unit can determine to carry through the numerical value of metering by valve is provided.Can prepare the capsule and the cartridge case of for example gelatin that is used for inhaler or insufflator.These typically contain described chemical compound and a kind of suitable powder substrate such as the mixture of powders of lactose or starch.
Normally aseptic by injection through parenterai administration institute composition prepared, and can exist by unit dosage form, for example peace bottle, syringe, injection pen perhaps are present in the multi-dose container, and the latter is contained antiseptic usually.Described compositions can be taked as the suspension in oiliness or aqueous carrier, solution or form of emulsion, and can contain preparaton such as buffer, tension force preparation, viscosity-increasing agent, surfactant, suspending agent and dispersant, antioxidant, biological suitable polymers, chelating agen and antiseptic.According to the injection site, described carrier can contain water, artificial oil or vegetable oil and/or organic cosolvent.In some cases, as in the situation of lyophilized products or concentrate, described non-intestinal preparation gives after can rebuilding or dilute.Provide sustained release or the lasting long-acting preparation that discharges mixture of the present invention can comprise the injectable suspensions of nano/micrometre particle or Nano/micron crystalline solid or non-micronized crystalline solid.Except well known in the art, polymer can be used as control/lasting release matrix as poly-(lactic acid), poly-(glycolic) or its copolymer.Other long-acting induction system can need the form of the implant that cuts and pump to exist.
The suitable carrier of intravenous injection molecule of the present invention is known in the art, comprises the solution that contains alkali such as sodium hydroxide based on water, and to form Ionized chemical compound, sucrose or sodium chloride are as tonicity agents, and for example described buffer contains phosphate or histidine.Can add cosolvent such as Polyethylene Glycol.These systems based on water are effectively for dissolving chemical compound of the present invention, produce low toxicity when general is used.The component ratio of solution system can change, and does not destroy dissolubility and toxicity characteristic.In addition, the homogeneity of described composition can change.For example, can use hypotoxic surfactant, as polysorbate or poloxamer (poloxamer), can be Polyethylene Glycol or other cosolvent, can add biological suitable polymers such as polyvinylpyrrolidone, other sugar and polyhydric alcohol can replace glucose.
For the compositions that is used for Therapeutic Method of the present invention, the treatment effective dose can use various technology entry evaluations well known in the art.The predose that is used for substrate research can be based on the valid density of determining in cell culture assays.The dosage range that is suitable for human object can for example use the data that derive from zooscopy and cell culture assays to determine.
The treatment effective dose of chemical compound of the present invention, preparation or medicine is meant the amount of the described chemical compound, preparation or the medicine that cause object doing well,improving or survival period prolongation.It is definite in cell culture or laboratory animal that the toxicity of this molecule and treatment effectiveness can be learned program by standard drug, for example by determining that LD50 (median lethal dose(LD 50)) and ED50 (half treatment effective dose) carry out.The dosage rate of toxicity and therapeutical effect is a therapeutic index, and the ratio that it can LD50/ED50 is represented.The preparation that preferably presents high therapeutic index.
Effective dose or treatment effective dose are tissue, system, animal or human's biology or the amounts that medical science is replied that described chemical compound or pharmaceutical composition excite research worker, veterinary, doctor or other clinician research, for example regulate glucose metabolism, the blood sugar level that reduce to raise or increase, treatment or the prevention disease relevant with glucose metabolism be diabetes etc. for example.
Dosage preferably in the circulation composition scope, comprises hypotoxicity or avirulent ED50.Dosage changes according to the dosage form of using and/or the route of administration of utilization in this scope.Should be according to methods known in the art, the situation of comprehensive object is selected accurate preparation, route of administration, dosage and delivery time.
Dosage and interval can be regulated the effect that is enough to reach hope with the blood plasma level that active component is provided separately, for example regulate glucose metabolism, blood sugar lowering level etc., i.e. minimum effective drug concentration (MEC)..MEC changes to some extent for every kind of chemical compound, but can estimate from for example vitro data and zoopery.Reach the necessary dosage of MEC and depend on individual character and route of administration.In the situation of topical or selectivity picked-up, effective local concentration of medicine and plasma concentration may be uncorrelated.
The medicament that gives or the amount of compositions can be dependent on various factors and decide, and comprise sex, age and the body weight of object, the ailing order of severity, administering mode, and the doctor in charge's judgement.
Compositions of the present invention then can exist in packing that contains described active batching that contains one or more unit dosage form or dispensing apparatus if desired.This packing or equipment can for example comprise metal or plastic tab, as blister-pack, perhaps have the bottle of glass and rubbery stopper.Described packing or dispensing apparatus can instruct application according to administering mode.Also can prepare the compositions of in the pharmaceutical carrier of compatibility, preparing that comprises The compounds of this invention, place suitable containers, and at the treatment of particular cases and indicate.
According to this paper instruction, those skilled in the art are easy to realize these and other embodiment of the present invention
Embodiment
Can further understand the present invention with reference to following embodiment, described embodiment is example of the present invention purely.These embodiment illustrate the present invention for example.The non-scope that is limited to illustrational embodiment of the present invention, they only are to illustrate various aspects of the present invention.Any method of function equivalence all within the scope of the invention.Except described herein, those skilled in the art can carry out various modifications to the present invention according to aforementioned description and accompanying drawing.This modification comprises within the scope of the appended claims.
Embodiment 1: overcome the inhibitory action that TNF-α generates EPO
(0,0.4,2, TNF-α 10ng/ml) is not having or processing is being arranged in the presence of compd A or the compd B three days with various concentration with the Hep3B cell.Excretory EPO level is used commercially available ELISA test kit (R ﹠amp; D Systems, catalog number (Cat.No.) DEP00) determine.In the situation that does not have chemical compound, handle the Hep3B cell with TNF-α and reduce the EPO generation in the dose dependent mode.The Hep3B cell of handling with the compd A (Figure 1A) or the compd B (Figure 1B) of various concentration under the situation that does not have TNF-α illustrates dose dependent EPO and generates increase.Any chemical compound that adds these two kinds of chemical compounds when having TNF-α all significantly reduces the inhibitory action that TNF-α generates EPO.In the situation that has low concentration (for example 0.4ng/ml) and high concentration (for example 10ng/ml) TNF-α, observe by prolyl hydroxylase and suppressed to overcome the inhibitory action that TNF-α generates EPO.Therefore, it is active and overcome that the Compounds and methods for of inflammatory cytokine TNF-α inhibitory action the application of the invention that EPO is generated suppresses prolyl hydroxylase.The EPO that these results suggest Compounds and methods for of the present invention can be used for increasing under the situation that has inflammatory cytokine TNF-α generates.In addition, method of the present invention and chemical compound can be used for increasing EPO and generate, and therefore are used for the treatment of patient's anemia, and for example wherein said patient suffers from the disease relevant with TNF-α such as acute or chronic inflammatory disease or other chronic disease anemia.
Carry out a series of experiments with check with cellular exposure (promptly in the cell that is exposed to TNF-α) behind inflammatory cytokine TNF-α, the effect that chemical compound of the present invention generates EPO.In these experiments, TNF-alpha signal conduction therefore before adding prolyl hydroxylase inhibitors by initial.(0,0.4,2, TNF-α 10ng/ml) handled 2 hours, added the compd A or the compd B of various concentration afterwards in cultured cells with various concentration with the Hep3B cell.Adding chemical compound after 3 days, excretory EPO level such as above-mentioned mensuration.
Shown in Fig. 2 A and 2B, with TNF-α to the pretreatment of Hep3B cell after 2 hours, compd A and compd B have overcome the inhibitory action that TNF-α generates EPO.These data show that Compounds and methods for of the present invention is used in increase EPO generation in the cell that is exposed to TNF-α.These results also point out the processing of carrying out with chemical compound of the present invention to provide to be increased the EPO generation and treats the useful means that EPO generates the patient's who is subjected to TNF-α inhibition anemia.
Add chemical compound of the present invention and significantly reduced the inhibitory action that TNF-α generates EPO.Therefore, Compounds and methods for of the present invention can be used for treating or prevents and the relevant anemia of TNF-α that increases, for example inflammatory diseases.
Embodiment 2: overcome the inhibitory action that IL-1 β generates EPO
(0,0.4,2, IL-1 β 10ng/ml) is not having or processing is being arranged in the presence of compd A or the compd B 3 days with various concentration with the Hep3B cell.Excretory EPO level is used commercially available ELISA test kit (R ﹠amp; D Systems, catalog number (Cat.No.) DEP00) measure.Under the situation that does not have chemical compound, handle the Hep3B cell with IL-1 β and reduce the EPO generation in the dose dependent mode.The Hep3B cell of handling with the compd A (Fig. 3 A) or the compd B (Fig. 3 B) of various concentration under the situation that does not have IL-1 β illustrates EPO and generates the dose dependent increase.The arbitrary chemical compound that adds when having IL-1 β in two kinds of chemical compounds all significantly reduces the inhibitory action that IL-1 β generates EPO.In the situation that has low concentration (for example 0.4ng/ml) and high concentration (for example 10ng/ml) IL-1 β, observe by prolyl hydroxylase and suppressed to overcome the inhibitory action that IL-1 β generates EPO.Therefore, that the Compounds and methods for of inhibitory action the application of the invention of EPO is suppressed prolyl hydroxylase is active and overcome for inflammatory cytokine IL-1 β.The EPO that these results suggest Compounds and methods for of the present invention can be used for increasing under the situation that has inflammatory cytokine IL-1 β generates.In addition, method of the present invention and chemical compound can be used for increasing EPO and generate, and therefore can be used for treating patient's anemia, and for example wherein said patient suffers from the disease relevant with IL-1 β such as acute or chronic inflammatory disease or other chronic disease anemia.
Carry out a series of experiments with check with cellular exposure (promptly in the cell that is exposed to IL-1 β) behind inflammatory cytokine IL-1 β, the effect that chemical compound of the present invention generates EPO.In these experiments, the conduction of IL-1 signal beta therefore will be by initial before adding prolyl hydroxylase inhibitors.(0,0.4,2, IL-1 β 10ng/ml) handled 2 hours, added the compd A or the compd B of various concentration afterwards in cultured cells with various concentration with the Hep3B cell.Adding chemical compound after 3 days, excretory EPO level such as above-mentioned mensuration.
Shown in Fig. 4 A and 4B, with IL-1 β to the pretreatment of Hep3B cell after 2 hours, compd A and compd B have overcome the inhibitory action that IL-1 β generates EPO.These data show that Compounds and methods for of the present invention is used in increase EPO generation in the cell that is exposed to IL-1 β.These results also point out the processing of carrying out with chemical compound of the present invention to provide to be increased the EPO generation and treats the useful means that EPO generates the patient's who is subjected to IL-1 β inhibition anemia.
Add chemical compound of the present invention and significantly reduce the inhibitory action that IL-1 β generates EPO.Therefore, Compounds and methods for of the present invention can be used for treating or prevents and the relevant anemia of IL-1 β that increases, for example inflammatory diseases.
The inhibition that the inductive VCAM-1 of embodiment 3:TNF-α expresses
Endotheliocyte is expressed vascular cell adhesion molecule (VCAM)-1 to lymphocytic adhesion section by endotheliocyte and is produced.The VCAM-1 expression is inductive by various inflammatory cytokines such as TNF-α in the endotheliocyte.Be that research HIF prolyl hydroxylase suppresses the effect that the inductive VCAM-1 of TNF-α is expressed, HUVEC (Human umbilical vein endothelial cells) was stimulated 1 day with TNF-α under the situation of not having or have the compd B of various concentration or Compound C.Measure the VCAM expression then.
As shown in Figure 5, TNF-α (1ng/ml) induces VCAM-1 to express in the HUVEC cell.Yet adding compd B or Compound C cause the dose-dependent inhibition to the inductive VCAM-1 expression of TNF-α in TNF-α stimulated cells.These data show that method of the present invention is effective with chemical compound for reducing the VCAM-1 expression relevant with inflammatory cytokine TNF-α.Result further prompting Compounds and methods for of the present invention can be used for suppressing and various inflammation and autoimmune disease such as the relevant VCAM-1 expression of chronic disease anemia.
The inhibition that the beta induced VCAM-1 of embodiment 4:IL-1 expresses
VCAM-1 expresses also beta induced by inflammatory cytokine IL-1 in the endotheliocyte.Be that research HIF prolyl hydroxylase suppresses the effect that the beta induced VCAM-1 of IL-1 is expressed, HUVEC (Human umbilical vein endothelial cells) was stimulated 1 day with IL-1 β under the situation of not having or have the compd B of various concentration or Compound C.Measure the VCAM expression then.
IL-1 β (1ng/ml) induces VCAM-1 to express in the HUVEC cell.Yet adding compd B or Compound C cause the dose-dependent inhibition (data not shown goes out) to the beta induced VCAM-1 expression of IL-1 in IL-1 β stimulated cells.These data show that method of the present invention is effective with chemical compound for reducing the VCAM-1 expression relevant with inflammatory cytokine IL-1 β.Result further prompting Compounds and methods for of the present invention can be used for suppressing and various inflammation and autoimmune disease such as the relevant VCAM-1 expression of chronic disease anemia.
Embodiment 5: the inhibition that the inductive VCAM-1 of TNF-α and IL-1 expresses on the endotheliocyte
HUVEC was handled 24 hours with the compd B or the Compound C of vehicle Control thing or various concentration (0,20,40,80 μ M).Washed cell stimulated 4 hours with 1ng/mlTNF-α or 1ng/ml IL-1 β then.Measuring cell surface VCAM-1 by the ELISA based on cell then expresses.
As shown in figure 25, reduced inducing that the beta induced cell surface VCAM-1 of inflammatory cytokine TNF-α and IL-1 expresses with the prolyl hydroxylase inhibitors pretreatment.These results show the inflammatory function of Compounds and methods for inhibition TNF-α of the present invention and IL-1 β and suppress for the expression that mediates the important cell surface adhesion molecule of heterocyst leukocyte adhesion.Provide a kind of effective means that reduces the inflammation cascade by suppress leukocyte adhesion with compound treatment of the present invention, thereby reduced inflammation and reduction restriction EPO generation and suppress erythropoietic inflammatory effect.
The inductive E-of embodiment 6:TNF-α selects the inhibition of protein expression
Endothelium E-selects albumen to belong in inflammatory episode mediated leucocytes to the selection protein family of the cell adhesion molecule of initially adhering to of vascular endothelial cell.IL-1, TNF-α and lipopolysaccharide all induce E-to select proteic expression (seeing for example Bevilacqua et al. (1987) Proc NatlAcad Sci USA 84:9238-9242 and Bevilacqua and van Furth (1993) JLeukoc Biol 54:363-378).Be that research HIF prolyl hydroxylase suppresses the inductive E-of TNF-α is selected the effect of protein expression, HUVEC was stimulated 1 day with 1ng/ml TNF-α not having or have under the compd B of various concentration or the situation that Compound C exists.Measuring E-then selects albumen and VCAM to express.
Shown in Figure 24 A and 24B, compd B and Compound C illustrate the dose-dependent inhibition of inductive VCAM of TNF-α and E-among the HUVEC being selected protein expression.Data among Figure 24 A and the 24B select the inhibition percentage rate of protein expression to represent with VCAM and the E-that observes in replying compd B of various concentration (Figure 24 A) or Compound C (Figure 24 B).In the HUVEC that handles with 50 μ M compd Bs or Compound C, observe and be higher than the percentile inhibition of 60% inhibition VCAM and E-selection protein expression.These data show that method of the present invention is effective with chemical compound for reducing VCAM relevant with inflammatory cytokine TNF-α in the endotheliocyte and E-selection protein expression.Result further prompting Compounds and methods for of the present invention can be used for suppressing and various inflammation and autoimmune disease such as relevant VCAM and the E-selection protein expression of chronic disease anemia.In addition, adhesion molecule is comprised the inhibition that VCAM and E-select proteic endotheliocyte to express, the means of the early stage incident in the vascular inflammation that reduces are provided by method of the present invention and chemical compound.
The beta induced E-of embodiment 7:IL-1 selects the inhibition of protein expression
Be that research HIF prolyl hydroxylase suppresses the beta induced E-of IL-1 is selected the effect of protein expression, HUVEC was stimulated 1 day with 1ng/ml IL-1 β under the situation of not having or have the compd B of various concentration or Compound C.Measure E-then and select protein expression.
Compd B and Compound C illustrate the dose-dependent inhibition (data not shown goes out) of the beta induced E-of IL-1 among the HUVEC being selected protein expression.These data show that method of the present invention is effective with chemical compound for reducing E-selection protein expression relevant with inflammatory cytokine IL-1 β in the endotheliocyte.Result further prompting Compounds and methods for of the present invention can be used for suppressing and various inflammation and autoimmune disease such as the relevant E-selection protein expression of chronic disease anemia.In addition, adhesion molecule is comprised the inhibition that VCAM and E-select proteic endotheliocyte to express, the means of the early stage incident in the vascular inflammation that reduces are provided by method of the present invention and chemical compound.
The inductive E-of embodiment 8:TNF-α, IL-1 β and IFN-γ selects the inhibition of protein expression
HUVEC was handled 24 hours with the compd B or the Compound C of vehicle Control thing or various concentration.Washed cell is used the combined treatment 4 hours of 1ng/ml TNF-α, 1ng/ml IL-1 β or each 1ng/ml TNF-α, IL-1 β and IFN-γ then.E-selects proteic cell surface expression to measure by the ELISA based on cell.
As shown in figure 26, suppressed to select inducing of protein expression with compd B or Compound C pretreatment by inflammatory cytokine TNF-α or the beta induced cell surface E-of IL-1.In addition, under the situation of the three kinds of inflammatory cytokines (TNF-α, IL-1 β and IFN-γ) that have known increase E-selection protein expression, all reduced E-and selected protein expression with arbitrary chemical compound pretreatment.These results show compounds block TNF-α of the present invention, IL-1 β and the IFN-γ inflammatory function to endotheliocyte, and this inhibition by the expression of cell surface adhesion molecule that mediated leucocytes is rolled on activated endothelium shows.Selecting albumen to adhere to activated endothelium owing to leukocyte by E-is the early stage step of keeping the cascade of (perpetuating) inflammation, therefore selects protein expression to suppress the leukocyte rolling by inhibition E-a kind of means that further restriction EPO generated and suppressed erythropoietic inflammation cascade that reduce are provided.
Collaborative increase during embodiment 9:EPO generates
With the Hep3B cell in the situation of not having or have the compd A of various concentration (3 μ M, 10 μ M, 30 μ M) or compd B with various concentration (0,0.1,1, IL-6 processing 10ng/ml) 1 or 3 day.Excretory EPO level is used commercially available ELISA test kit (R ﹠amp; DSystems, catalog number (Cat.No.) DEP00) determine.In the situation that does not have chemical compound, handle the effect minimum that the Hep3B cell generates EPO with IL-6.Shown in Figure 27 A and 27B, the Hep3B cell of handling with IL-6 increases the EPO expression, is slightly higher than untreated cell.Especially, the EPO level is about 20mIU/ml in control cells, and is about 50mIU/ml in the cell of handling with 10ng/ml IL-6.
Handle and do not have Hep3B cell that IL-6 handles and the EPO level is shown increases with compd A or compd B in the dose dependent mode.Yet, handle the Hep3B cell under the condition of IL-6 and the EPO level is shown significantly increases (seeing shown in Figure 27 A and the 27B) existing with compd A or compd B.The effect that EPO generates is worked in coordination with there being compound treatment under the condition of IL-6.For example, the Hep3B cell of handling with 10ng/ml IL-6 illustrates the EPO level of about 50mIU/ml.Under the condition of no IL-6, handle the EPO that the Hep3B cell produces about 60mIU/ml and 220mIU/ml respectively with 10mM compd A or compd B.Under the condition that has 10ng/ml IL-6, adding compd A and compd B make the EPO level increase to about 270mIU/ml respectively and are higher than 400mIU/ml.Therefore, synergism during chemical compound of the present invention and the IL-6 EPO in inducing hepatocyte expresses.
Embodiment 10: overcome the inhibition to the conduction of EPO receptor signal of cytokine induction
Irritation cell is a TF-1 (human erythroleukemia; ATCC cat # CRL-2003) adds and breed to make it to reply EPO.Exist under the condition of various proinflammatory cytokines, the TF-1 cell proliferation increase of EPO mediation is weakened.For determining that prolyl hydroxylase suppresses the effect to the TF-1 cell proliferation, the TF-1 cell is handled not having or have under the condition of prolyl hydroxylase inhibitors with proinflammatory cytokine IL-1 β, the TNF-α of various concentration or IFN-γ, and the cell proliferation of following mensuration EPO mediation.Will be in 96 hole microtitre flat boards triplicate cultured cells hole and serum-free medium are incubated 24 hours not having or have under the condition of EPO.During last 4 hours of cultivating, in each hole, add 1 μ Ci contain the tritium thymidine ( 3H-TdR; Amersham).Cell is determined replying by measuring cell proliferation of EPO receptor signal conduction.Cell proliferation mixes in the cell by quantification 3The amount of H-TdR and measuring, at first the water dissolved cell is caught the DNA on the nylon membrane in the cell harvestor then.
Perhaps, derive from the single-cell suspension liquid of splenocyte of the animal that phenylhydrazine handles (its cause EPO replys CFU-GM in the spleen preponderate) as the source of EPO responsive cell.Ex vivo (ex vivo) is measured the propagation that EPO mediates as mentioned above then.
The TF-1 cell of handling with EPO causes cell proliferation to increase, and this mixes to increase and determine by containing the tritium thymidine.In the TF-1 cell that EPO handles, add proinflammatory cytokine IL-1 β, TNF-α or IFN-γ and cause reduction that EPO is replied, cause that cell proliferation reduces.The adding of determining chemical compound of the present invention is to the inhibiting effect of proinflammatory cytokine to the cell proliferation of EPO mediation in the TF-1 cell.In the TF-1 cell of handling with EPO and proinflammatory cytokine cell proliferation increase (by contain the tritium thymidine mix to increase measure) show that Compounds and methods for of the present invention has overcome the inhibitory action that proinflammatory cytokine increases the cell proliferation of EPO mediation.
Embodiment 11: increase transferrin receptor and express
The following detection of effect that chemical compound of the present invention is expressed transferrin receptor.(Hep3B, HepG2 HK-2) are incubated 1 day with compd A or compd B with various cells.Use the transferrin receptor expression of CD7-1-PE antibody (Ancell, catalog number (Cat.No.) no.223-050) then by the facs analysis cell.The results are shown in table 1.
Table 1
Cell type Handle Mean F L
Hep3B DMSO 40.21
Compd A 40.89
Compd B 42.43
HepG2 DMSO 49.59
Compd A 56.52
Compd B 53.53
HK-2 DMSO 10.80
Compd A 12.20
Compd B 18.92
As shown in table 1, adding all cpds of the present invention in cell has increased the transferrin receptor expression.Use prolyl hydroxylase inhibitors of the present invention to suppress HIF prolyl hydroxylation and increased the expression of transferrin receptor in the cell.Use prolyl hydroxylase inhibitors of the present invention hepatocyte (Hep3B for example, HepG2), observed transferrin receptor in nephrocyte (for example HK-2) and the lymphocyte (for example THP-1) and express and increase.Therefore, method of the present invention and chemical compound are used in increases the transferrin receptor expression in the various cell types.In addition, the transferrin receptor expression of increase can cause the endocytosis of the transferrin receptor mediation of iron content transferrin to increase, thereby increases transhipment, utilization, storage and the metabolism of ferrum.Therefore, method of the present invention and chemical compound can be used for strengthening erythropoiesis by the transhipment, utilization, storage and the metabolism that increase ferrum.
Embodiment 12: increase the picked-up of external transferrin receptor expression and ferrum
The following mensuration of the effect of the picked-up of ferrum in the chemical compound pair cell.With former generation mononuclear cell and macrophage and mononuclear cell and macrophage system (for example THP-1) handled 1,2 or 3 day with the prolyl hydroxylase inhibitors of various concentration.Use fluorescence immunization coloration and flow cytometry to detect the existence of cell surface transferrin receptor then.The result illustrates and adds prolyl hydroxylase inhibitors and increased the cell surface transferrin receptor and express, this show prolyl hydroxylase suppress to increase transferrin combine with cell reach so ferrum and the bonded effectiveness of cell.Determine the change of the picked-up of ferrum is following with the cell that prolyl hydroxylase inhibitors is handled.Cell is existed 59Use compound treatment under the Fe condition.Bonded by measuring cell with the cell that prolyl hydroxylase inhibitors is handled to the picked-up increase of ferrum 59Fe and determining.The cell combination is closed 59The picked-up that Fe increases ferrum in the expression cell increases.
Embodiment 13: increase Fe regulatory protein-2 level and activity
The picked-up of ferrum, storage and utilization partly are that described key protein matter comprises trans-acting protein by the expression of the key protein matter that participates in iron metabolism and active the generation, and known is Fe regulatory protein (IRP).IRP-1 and IRP-2 control stability and the translation of mRNA by the specificity ferrum response element among the proteinic various mRNA of combination participation iron metabolism, thereby influence all aspects basically of iron metabolism.Iron deficiency has increased the IRP activity, and causing transferrin receptor to be expressed increases and ferritin expression reduction.In addition, exist under the situation of ferrum, IRP is active to be reduced, and causing transferrin receptor expression reduction and ferritin to be expressed increases.
For of the effect of check The compounds of this invention, carry out following experiment to the various aspects of iron metabolism.Mice Hepa-1 cell is handled until 48 hours with prolyl hydroxylase inhibitors.Harvesting and use are specific to the IRP-2 expression of the antibody (Alpha Diagnostic International, Inc., San Antonio TX) of IRP-2 by the immunoblotting assay cell lysate then.Kytoplasm IRP-2 level increases after the results are shown in the adding chemical compound, shows the metabolism that method of the present invention and chemical compound can be used for increasing the IRP level and therefore increase ferrum.
Determine the active effect of IRP-2 is following by the chemical compound of the present invention that the variation in ferritin and the transferrin expression is measured.Mice RAW 264.1 macrophage systems are handled until 48 hours with prolyl hydroxylase inhibitors.Harvesting is also analyzed ferritin and transferrin expression by immunoblotting (ADI, catalog number (Cat.No.) IRP21-S) then.The ferritin expression reduces and the increase of transferrin expression shows method of the present invention and chemical compound can be used for the stabilizing and increasing activity of IRP-2 after prolyl hydroxylase suppresses.The IRP-2 that increases expresses the expression of the ferritin of the long term store that has reduced responsible ferrum, and has increased the expression of transferrin and transferrin receptor, promotes picked-up, transhipment and the utilization of ferrum, therefore strengthens erythropoiesis.By increasing expression and the activity of IRP-2, method of the present invention and chemical compound can be used for reducing the expression of ferritin and the long term store of relevant ferrum, and the expression of increase transferrin and transferrin receptor.Therefore, method of the present invention and chemical compound can be used for increasing picked-up, transhipment and the utilization of ferrum, and therefore are used to strengthen erythropoiesis.
Embodiment 14: strengthen the utilization of ferrum
Give rat vehicle Control thing or HIF prolyl hydroxylase inhibitors, intravenous injection afterwards 59The radiolabeled ferrous citrate of Fe-(Amersham).From tail vein and all free blood plasma, extract serial blood sample, in scintillation counter, measure the bonded radioactivity of erythrocyte with the transhipment that detects ferrum and ferrum mix erythrocyte haemachrome and hemoglobin synthetic in.The erythrocyte that increases is bonded 59Fe shows that chemical compound of the present invention can be used for strengthening that haemachrome is synthetic, hemoglobin produces and the utilization of the ferrum that erythropoiesis is required.
Embodiment 15: enhanced erythrocyte in vitro generates gene expression
(ATCC No.HB-8064) grows in the DMEM that contains 8% hyclone with the Hep3B cell.In 6 hole culture dishs, density is about 500000 cells/well with the Hep3B cell inoculation.After 8 hours, culture medium is replaced by the DMEM that contains 0.5% hyclone, and with the extra insulation of cell 16 hours.In cell, add compd B or Compound D (final concentration is 25 μ M), and cell is incubated the different time.Control cells (do not carry out compound treatment, only add DMSO) was 0,6 and 48 hour results.To the cell analysis cell viability (GUAVA) of results, perhaps it is added RNA extract buffer (RNeasy, Qiagen) in ,-20 ℃ of storages to carry out RNA purification subsequently.The RNA of the repeated experiments that the comfortable different number of days of multiple microarray use separation is carried out produces.Use RNeasy test kit (Qiagen) from cell, to separate total RNA.
RNA is precipitated 1 hour at-20 ℃ in 0.3M sodium acetate (pH 5.2), 50ng/ml glycogen and 2.5 volume of ethanol.Centrifugal sample also will precipitate with cold 80% washing with alcohol, dry also resuspending in water.Double-stranded cDNA instructs according to manufacturer and uses T7-(dT) 24 first strand primers (Affymetrix, Inc., Santa Clara CA) and SUPERSCRIPT CHOICE system (Invitrogen) to synthesize.Whole cDNA uses PHASE LOCK GEL insert, and (Brinkman, Inc. WestburyNY), use isopyknic 25: 24: 1 phenol: chloroform: isoamyl alcohol extracting.Collect water, use the 7.5M ammonium acetate and the 2.5 volume of ethanol precipitation cDNA of 0.5 volume.Perhaps, use the GENECHIP sample to remove module (Affymetrix) and instruct purification cDNA according to manufacturer.
Use BIOARRAY HighYield rna transcription labelling kit (EnzoDiagnostics, Inc., Farmingdale NY) to instruct in external translation (IVT) reaction the cRNA of synthesizing biotinylated labelling from cDNA according to manufacturer.The product of final labelling uses GENECHIP sample removing module (Affymetrix) to carry out purification and fragmentation according to manufacturer's guidance.
Hybridization mixture is by placing 5 μ g probes 1 * hybridization buffer (100mMMES, the 1M[Na of 100 μ l +], 20mM EDTA, 0.01%Tween 20), 100 μ g/ml salmon sperm DNAs, the acetylizad BSA of 500 μ g/ml, 0.03nM contrasts few B2 (Affymetrix), and in 1 * GENECHIP eucaryon hybridization tester (Affymetrix) and prepare.Subsequently described mixture is incubated 5 minutes, centrifugal then 5 minutes at 99 ℃.People's gene group U133A array (Affymetrix) is placed room temperature, rotated prehybridizations 10 minutes with 1 * hybridization buffer at 45 ℃ then.Then described buffer is replaced with 80 μ l hybridization mixtures, described array at 45 ℃ in the 60rpm hybridization that contends with.After the hybridization, with array with 6 * SSPE, 0.1%Tween 20 washings once, instruct to use Succ-PEG-DSPE (the Molecular Probes of R-phycoerythrin-put together then according to manufacturer, Eugene OR), washing of anti-Succ-PEG-DSPE antibody of goat (Vector Laboratories, Burlingame CA) and GENECHIP Fluidics Station400 device (Affymetrix) and dyeing.Use GENEARRAY scanner (Affymetrix) and Microarray Suite software (Affymetrix) to analyze array.
People's gene group U133A array (Affymetrix) is represented Human Unigene databasebuild 133 (National Center for Biotechnology Information, Bethesda MD) all data in, comprise about 14,500 people's genes of fully identifying.
The RNA quality is by capillary electrophoresis (Agilent Bioanalyzer) monitoring.As state (Affymetrix) preparation hybridization mixture, and with the Affymetrix people U133A hybridization array that contains 22,283 probe series.Use Affymetrix MicroArray Suite (MAS) software analysis array performance, each probe series is designated as " present ", " marginal " and " absent " order according to the software default value.Use GeneSpring software (SiliconGenetics) to carry out statistical analysis and produce and filter the probe series of tables.The cutoff value (Cutoff) of " expression " probe series uses Affymetrix " P " to order and derive from the combination of the absolute expression values of Genespring ' s inherent data error model.The normalization of data based average control sample.
As shown in table 2 below, the coding proteic expression of gene of erythropoiesis (with respect to the increase multiple of matched group mRNA level) increases in the Hep3B cell with compound treatment of the present invention.(two ceruloplasmin data points under every kind of condition are represented in following table 2).Especially, ceruloplasmin and transferrin receptor 2 gene expressions increase in the Hep3B cell of handling with The compounds of this invention.
Table 2
Chemical compound Time Ceruloplasmin (CP) Transferrin receptor (TFR2)
D 6 hours 2.06/2.387 Undetermined
B
1 hour 1.142/0.946 0.575
B 3 hours 1.123/0.955 0.558
B 6 hours 1.555/1.103 0.822
Chemical compound Time Ceruloplasmin (CP) Transferrin receptor (TFR2)
B 12 hours 2.366/2.507 1.253
B 24 hours 5.136/4.909 2.522
B 48 hours 5.82/4.678 4.169
Embodiment 16: animals administer dosage
The animal of using among the following embodiment comprises and derives from Simonsen, Inc. (Gilroy CA), Charles River (Hollister, CA) or the Swiss Webster male mice (30-32g) of Harlan, Sprague Dawley male rat (200-350g) and Lewis female rats.Animal uses standardization program to raise, arbitrarily feeding and feedwater.During handling, body weight change and external toxicity and the mortality rate sign of monitoring animal.
Chemical compound is usually by raising oral by force or intravenous injection gives.By raising containing 0.1% polysorbate80 or not containing 0.5% carboxymethyl cellulose (CMC of polysorbate80 (0mg/kg/ days) of oral animals received 4-10ml/kg volume by force; Sigma-Aldrich, St.LouisMO), perhaps be received in the The compounds of this invention (for example HIF prolyl hydroxylase inhibitors) of the various dose among the 0.5%CMC that contains or do not contain 0.1% polysorbate80 or do not contain polysorbate80, use different dosages to carry out.During handling, collect blood sample from for example tail vein (rat) or through epigastric vein or cardiac puncture (mice or rat) with appropriate intervals.Usually, use the isoflurane anesthesia animal, blood sample collection is separated in the test tube (Becton-Dickinson, Franklin Lakes NJ) at MICROTAINER serum.Be to measure serum composition, with test tube room temperature insulation 30 minutes, then 4 ℃ 8, centrifugal 10 minutes of 000rpm.Then the serum fraction is processed and is analyzed special composition the existing of serum levels of iron (by Quality Clinical Labs, Mountain View, CA analyzes) for example.For determining hematocrit, with blood sample collection in MICROTAINER EDTA-2K test tube (Becton-Dickinson), then EDTA-blood is poured into 75mm * 1.1-1.2mm I.D. capillary tube (ChaseScientific Glass, Inc., RockwoodTN) in to about 3/4 length, one end of test tube seals with CRITOSEAL sealant (Sherwood Medical Company), with test tube at J-503M MICROHEMATOCRIT centrifuge (Jorgensen Laboratories, Inc., Loveland CO) in 12, centrifugal 5 minutes of 000rpm.According to Card Reader reading hematocrit.When specifying, (Mountain View CA) carries out full blood count (CBC) analysis, comprises blood hemoglobin level, reticulocyte number and hematocrit analysis by Quality Clinical Labs.
When studying end, animal is by for example blood-letting or suction CO under general anesthesia at every turn 2Suffocate and row euthanasia, collect organ and tissue sample.Fixing or with being organized in the formalin of neutral buffered-70 ℃ of storages.The sample that will carry out genome analysis places RNAlater.
Embodiment 17: the expression that the proteic gene of coding ironworking increases in vivo
Swiss Webster male mice such as above-mentioned with single dose 0.5%CMC (Sigma-Aldrich) (0mg/kg) or the 100mg/kg compd A handle.The the 4th, 8,16,24,48 or 72 hour anesthetized animal after administration put to death animal, separates the tissue sample of kidney, liver, brain, lung and the heart, is stored in the RNALATER solution (Ambion) at-80 ℃.Perhaps, give 0.5%CMC (0mg/kg/ days) continuous 4 day every day with animal, 7.5mg/ml compd A (30mg/kg/ days) or the 25mg/ml compd A in 0.5%CMC (100mg/kg/ days) in 0.5%CMC are handled.Last administration is after 4 hours, and anesthetized animal is put to death animal, separates about 150mg liver and left and right sides kidney, is stored in-20 ℃ in RNALATER solution (Ambion).
Carrying out RNA with following proposal separates.Part section with each organ, add 875 μ l RLT buffer (RNEASY kit, Qiagen Inc., Valencia CA), with a rotor-stator POLYTRON homogenizer (Kinematica Inc., Cincinnati OH) with about 20 seconds of described section homogenate.Miniature centrifugal homogenate was transferred to one with supernatant and newly manages with the precipitation insoluble matter in 3 minutes, instructed the isolation of RNA with RNEASY (Qiagen) according to manufacturer.The RNA eluting is advanced in 80 microliters of water, and usefulness RIBOGREEN reagent (Molecular Probes, EugenOR) quantitative.The absorbance at measurement 260 and 280nm place is to determine RNA purity and concentration.
Perhaps, with tissue sample section and with rotor-stator POLYTRON homogenizer (Kinematica) homogenate in TRIZOL reagent.Homogenate is risen to ambient temperature, add 0.2 volume chloroform, violent biased sample.Mixture is incubated a few minutes in room temperature, then in 4 ℃ with 12000g centrifugal 15 minutes.Collect water and add 0.5 volume isopropyl alcohol.Biased sample, room temperature insulation 10 minutes, 4 ℃ with 12000g centrifugal 10 minutes.Abandon supernatant, will precipitate and use 75% washing with alcohol, 4 ℃ with 7500g centrifugal 5 minutes.The absorbance at measurement 260 and 280nm place is to determine RNA purity and concentration.
RNA is precipitated 1 hour at-20 ℃ in 0.3M sodium acetate (pH 5.2), 50ng/ml glycogen and 2.5 volume of ethanol.Centrifugal sample also will precipitate with cold 80% washing with alcohol, dry also resuspending in water.Double-stranded cDNA instructs according to manufacturer and uses T7-(dT) 24 first strand primers (Affymetrix, Inc., Santa Clara CA) and SUPERSCRIPT CHOICE system (Invitrogen) to synthesize.Whole cDNA uses PHASE LOCK GEL insert, and (Brinkman, Inc. WestburyNY), use isopyknic 25: 24: 1 phenol: chloroform: isoamyl alcohol extracting.Collect water, use the 7.5M ammonium acetate and the 2.5 volume of ethanol precipitation cDNA of 0.5 volume.Perhaps, use the GENECHIP sample to remove module (Affymetrix) and instruct purification cDNA according to manufacturer.
(EnzoDiagnostics, Inc. FarmingdaleNY), instruct in external translation reaction (IVT) cRNA of synthesizing biotinylated labelling from cDNA according to manufacturer to use BIOARRAY HighYield rna transcription labelling kit.The product of final labelling uses the GENECHIP sample to remove module (Affymetrix) and instructs purification and fragmentation according to manufacturer.
Hybridization mixture is by placing 5 μ g probes 1 * hybridization buffer (100mM MES, the 1M[Na of 100 μ l +], 20mM EDTA, 0.01%Tween 20), 100 μ g/ml salmon sperm DNAs, 500 μ g/ml acetylation BSA, 0.03nM contrast preparation in few B2 (Affymetrix) and the 1 * GENECHIP eucaryon hybridization tester (Affymetrix).Subsequently mixture is incubated 5 minutes, centrifugal then 5 minutes 99 ℃ of insulations 5 minutes and at 45 ℃.Mice genome MOE430Aplus2 array (Affymetrix) is placed room temperature, rotated prehybridizations 10 minutes with 1 * hybridization buffer at 45 ℃ then.Then buffer is changed with 80 μ l hybridization mixtures, and with array 45 ℃ 60rpm contend with hybridization 16 hours.After the hybridization, array is washed 1 time with 6 * SSPE, 0.1%Tween 20, use Succ-PEG-DSPE (the Molecular Probes of R-phycoerythrin-put together then, Eugene OR), anti-Succ-PEG-DSPE antibody (the Vector Laboratories of goat, Burlingame CA) and GENECHIP FluidicsStation 400 device (Affymetrix), wash according to the EukGE-WS2v4 scheme (Affymetrix) of Producer and dye.Analyze array with GENEARRAY scanner (Affymetrix) and Microarray Suite software (Affymetrix).
Mice genome MOE430Aplus2 array (Affymetrix) is represented mice UniGenedatabase build 107 (National Center for Biotechnology Information, Bethesda MD) all sequences in, comprise about 14,000 little musculus cdnas of fully identifying.
Following table 3 is illustrated in and gives after the compd A ceruloplasmin expression in the mouse kidney.The meansigma methods standardization of data in the untreated animal of contrast, to observe.
Table 3
Condition Ceruloplasmin (mRNA level relatively)
Untreated 0.81
The CMC contrast 1.26
Compd A-4 hour 1.16
Compd A-8 hour 1.39
Compd A-16 hour 1.22
Compd A-24 hour 2.45
Compd A-48 hour 1.44
Compd A-72 hour 2.10
The data of last table 3 show that method of the present invention and chemical compound can be used for increasing ceruloplasmin gene expression.Ceruloplasmin is also referred to as Ferroxidase-1, and it will change oxidised form into from the reduced iron of storing position (as ferritin) release.The ferrum of oxidation can be in conjunction with its blood plasma transport protein-transferrin.It is relevant with ferrum accumulation in liver and other tissue that ceruloplasmin lacks.Evidence suggests that ceruloplasmin promotes ferrum to flow out and promote flowing molten iron to go into the cell of iron deficiency (seeing for example Tran et al. (2002) J Nutr 132:351-356) from liver.
Following table 4 illustrates and gives after the compd A that hepcidin mRNA expresses in the mouse liver.The data normalization of data in the untreated animal of contrast, to observe.
Table 4
Condition/zooscopy Time Hepcidin (mRNA level relatively)
Contrast - 1.0
Many high doses of I- - 0.275
Many high doses of II- - 0.703
Many low dosages of II- - 0.129
III 4 hours 0.672
III 8 hours 0.305
Condition/zooscopy Time Hepcidin (mRNA level relatively)
III 16 hours 0.119
As shown in table 4, give compd A and cause hepcidin mRNA expression reduction in the mouse liver.Hepcidin expression that reduces and ferrum discharge increase from reticuloendothelial cell and the absorption of intestinal ferrum increases relevant.Therefore, method of the present invention and chemical compound can be used for reducing the absorption that hepcidin expressed and increased intestinal ferrum.
Fig. 6 A illustrates transferrin receptor in the kidney (grey bar post) and (is also referred to as Slc11a2 (solute carrier family 11 with duodenum iron transfer albumen NRAMP2 (natural-resistance-relevant macrophage albumen 2), the link coupled bivalent metal ion transport protein of proton, the member 2), perhaps be called DCT1 (divalent cation transporter albumen 1), DMT1 (divalent metal transport protein 1)) relative expression's level of (black bar post).
In another experiment, mRNA separates the small intestinal of collecting in 4 hours after the 4th time gives mice 60mg/kg compd A, compd B and Compound C.Probe prepares from 5 groups of each animals of handling two animals of animal per group, and with Affymetrix mouseMOE430Aplus2 microarray hybridization (animal of each array).Handle and the data of the array of the animal of being untreated are carried out the statistics contrast deriving from.Fig. 6 B illustrates the relative expression level of NRAMP2 mRNA in the animal small intestinal of handling with compd A, compd B and Compound C.Expression is represented with the multiple of inducing of the untreated animal that surpasses contrast of the gene of each expression.These result of experiment show that method of the present invention and chemical compound can be used for increasing the expression of NRAMP2 in the intestinal.The absorption that these results further point out method of the present invention and chemical compound to can be used for increasing ferrum, haemachrome is synthetic, hemoglobin is synthetic, erythrocyte produces and erythropoietic ferrum availability thereby increase is used for.
Fig. 6 C is illustrated in the animal of processing and compares with the vehicle Control group, and 5-Aminolevulinate synthase (ALAS-2) is expressed induces multiple.Data show with prolyl hydroxylase inhibitors handles that gene that the intact animal causes participating in iron metabolism comprises that participation absorbs ferrum from intestinal and by the transferrin receptor expression of gene increase of transhipment ferrum in the blood around.These expression of gene returned to baseline (contrast) level in 16 hours after administration.Data also are illustrated in the coordinate expression that prolyl hydroxylase inhibitors is handled back ALAS-2 in specified tissue, and ALAS-2 is the first kind of enzyme and the synthetic rate-limiting enzyme of haemachrome of haemachrome route of synthesis.Comprehensive these results show that the collaborative coding that increases of chemical compound of the present invention participates in promoting that erythropoiesis comprises the synthetic proteinic expression of gene of ferrum absorption, iron transfer and haemachrome.
Perhaps, use flow cytometry measure dual immunostaining around surface markers CD 11c and transferrin receptor level in the macrophage in the blood monocyte.Express the activity that increase illustrates compound treatment by detecting the macrophage transferrin receptor.In addition, can collect blood plasma and use commercially available ELISA test kit (to see for example KomaBiotech, Korea) test transferrin level.
Embodiment 18: erythropoiesis in the enhanced body
Give chemical compound of the present invention to the following mensuration of erythropoietic effect.Make normal mouse produce anemia and keep anemia state by giving TNF-α for a long time, this is known owing to lack signal that EPO generated and replied TNF-α and conduct and suppress an erythropoietic scheme.After inducing anemia 1-4 week, give the animal prolyl hydroxylase inhibitors.Detect the BFU-E and the CFU-E production of tissue, and analyze the blood sample composition.The result illustrate the bone marrow, spleen of the animal of handling with PHIs and on every side in the blood BFU-E and CFU-E number increase, and/or serum hemoglobin, reticulocyte and hematocrit increase, and shows it is effective.
Another experimental animal model is used for check and gives prolyl hydroxylase inhibitors for erythropoietic effect.In this model, transgenic mice is crossed to express and is produced the chronic disease anemia owing to the composing type of TNF-α.After in these mices, anemia taking place, use the various dose strategy to give prolyl hydroxylase inhibitors at different times.Collection organization and blood sample and analyze then.As mentioned above, the result illustrate bone marrow, spleen and on every side in the blood BFU-E and CFU-E number increase, and/or serum hemoglobin, reticulocyte and hematocrit increase, convincingly demonstrate anemia relevant in the transgenic animal and can use method of the present invention and chemical compound to treat by giving prolyl hydroxylase inhibitors with the excessive generation of TNF-α.
Embodiment 19: increase the serum levels of iron level
Male and female rats is handled (Monday and Thursday), totally 93 days weekly twice with the compd A of various concentration (0,20,60 or 150mg/kg).Measure total serum levels of iron level.
Table 5
Dosage Serum levels of iron (μ g/dL) male rat (meansigma methods+/-SD) Serum levels of iron (μ g/dL) female rats (meansigma methods+/-SD)
0mg/kg 158+/-37 342+/-91
20mg/kg 198+/-64 505+/-41 *
60mg/kg 357+/-111 * 445+/-46 *
150mg/kg 307+/-142 * 399+/-117
As shown in table 5, give compd A in male and female rats, all increased the serum levels of iron level (data in the table 5 be the serum levels of iron level+/-standard error. *There were significant differences for expression serum levels of iron level and untreated animal).These results show that method of the present invention and chemical compound can be used for increasing the serum levels of iron level, thereby can be used for treating the pathological changes relevant with iron deficiency.
Embodiment 20: the effectiveness in the iron metabolism animal model of the erythropoiesis/weakening of chronic disease anemia/weakening
Chronic disease anemia (ACD) is relevant with various inflammatory disorderses, comprises arthritis, tumor disease and other disease relevant with chronic inflammatory disease.Utilize the ACD rat model, use method of the present invention and the stable effect of chemical compound check HIF the treatment anemia relevant with chronic disease.In this animal model, in rat, induce ACD (seeing for example Sartor et al. (1989) Infection and Immunity 57:1177-1185) by Peptidoglycan-polysaccharide polymer.In this model, animal is the serious acute anemia of stage generation in the early stage, and the stage produces moderate chronic microcytic anemia late subsequently.
ACD animal model-experimentalists and technicians 1:
The female Lewis rat of about 160g is attacked with PG-PS10S (Lee Laboratories, 15 μ g/gm body weight, peritoneal injection).PG-PS 10S contains the Peptidoglycan-polysaccharide polymer of separation from the purification of the cell wall of A group's micrococcus scarlatinae (Streptococcus pyogenes, Group A) D58 strain.Make arthritis and anemia development 35 days.At the 35th day, under general anesthesia (isoflurane), carry out CBC and reticulocyte count (being undertaken) by Quality Clinical Labs from tail vein blood sampling (about 400 μ l).Centrifugal hematocrit levels 45% or on animal think and be not anemia, get rid of outside this research.
In PG-PS injection the last the 35th day, the carrier that the anemia animals received is independent or use compd A weekly (60mg/kg PO) handled continuous two days, two weeks totally.At the 35th day (seeing above-mentioned), measured automatic full blood count (CBC) in 39,42 and 49 days, measured the serum levels of iron level at the 49th day.
Reticulocyte count
As shown in Figure 7, giving anemia animal compd A increases reticulocyte count the 39th day (promptly beginning to give chemical compound after 5 days).The reticulocyte level is respectively erythrocytic about 2% and 4% in contrast (non-anemia) and anemia (PG-PS handles) animal.Yet the reticulocyte level is erythrocytic about 10% in the animal of processing.Compd A is handled and is made reticulocyte count increase in the anemia animal.Therefore, compd A stimulates erythropoiesis in the ACD rat animal model.
Hematocrit
Hematocrit levels increases in the anemia animal of handling with compd A.The hematocrit levels (measuring at Quality ClinicalLabs by Baker 9000) of anemia (PG-PS handles) animal is below 35%, and Dui Zhao non-anemia animal is 41% (see figure 8) by contrast.Giving anemia animal compd A makes hematocrit just increase to about 37% in 5 days after the beginning compound treatment.After giving compd A for the second time, it is about 40% that hematocrit levels increases to, similar to the hematocrit levels that observes in the non-anemia animal of contrast.Use the rat model of ACD, compd A has increased the hematocrit levels of anemia animal.Therefore, method of the present invention and chemical compound can be used for increasing hematocrit and treatment of chronic diseases anemia.
Hemoglobin
Give compd A and also increased hemoglobin level in the anemia animal.As shown in Figure 9, at the 35th day, the hemoglobin level of the non-anemia animal of contrast was about 15gm/dL, and hemoglobin level is about 13gm/dL in the animal (being the anemia animal) that PG-Ps handles.As shown in Figure 9, in the anemia animal, give the compound A-45 sky after (the 39th day) just increased hemoglobin level.Hemoglobin level still kept at the 49th day increasing, and reached and the similar level of non-anemia animal that contrasts, and showed that chemical compound of the present invention makes the anemia animal recover normal hemoglobin level.These results illustrate the hemoglobin level that compd A has increased the anemia animal of ACD rat model.Therefore, method of the present invention and chemical compound can be used for increasing hemoglobin and treatment of chronic diseases anemia.
Red blood cell count(RBC)
Give compd A and increased red blood cell count(RBC) in the anemia animal.As shown in figure 10, compare with untreated anemia animal in the animal of handling with compd A, 5 days (i.e. the 39th day in Figure 10) red blood cell count(RBC) just increase after beginning to give compd A.Use the rat model of ACD, compd A has increased the red blood cell count(RBC) of anemia animal.Therefore, method of the present invention and chemical compound can be used for increasing red blood cell count(RBC) and treatment of chronic diseases anemia.
Mean corpuscular volume (MCV)
The anemia animal illustrates to be compared mean corpuscular volume (MCV) and reduces (seeing Figure 11) with non-anemia control animal.The anemia animal of handling with compd A is illustrated in handles back 5 days (among Figure 11 the 39th day) and compares mean corpuscular volume (MCV) with untreated anemia animal and just increased.The average blood cell volume of handling of animal is compared with untreated animal at experimental session and is all kept increasing.These results illustrate compd A and have improved (promptly having reduced) microcytosis (be microcytic anemia, have many microcyte, the unusual little erythrocyte relevant with the various forms anemia) level.Therefore, the microcytosis in method of the present invention and the chemical compound improvement/reduction chronic disease anemia.
Mean corpuscular hemoglobin
The anemia animal also illustrates average hemoglobin level and reduces.As shown in figure 12, handling the anemia animal with compd A has increased the mean corpuscular hemoglobin level, is higher than the level that observes in untreated anemia animal.These results show that method of the present invention and chemical compound can be used for increasing the mean corpuscular hemoglobin level.
Animal model-experimentalists and technicians 2 of ACD
With female Lewis rat (approximately 150-200gm) injection PG-PS (peritoneal injection).Make arthritis and anemia development 28 days.By gavage twice weekly (Monday and Thursday), corresponding to giving animal compd A, totally six weeks behind the injection PG-PS on the the 28th, 31,35,38,42,45,49,52,56,59,63,66 and 70 day.
Collecting whole blood at the 28th, 42,56 and 70 day from the tail vein analyzes to carry out CBC.In addition, collected serum to carry out the ferrum binding analysis at the 70th day.(Mountain View CA) carries out by Quality Clinical Labs for CBC and ferrum binding analysis.
Hematocrit
Hematocrit levels reduces in excite (challenge) back 28 days animal with PG-PS.The animal that Figure 13 illustrates with PG-PS injection is an anemia, and hematocrit is 85% (0 week among Figure 13 was corresponding to the 28th day of this experimental program) of animal of (being non-anemia) of not being excited.Unawakened (the being non-anemia) animal of handling with compd A (40mg/kg) illustrates hematocrit levels and increases to more than 110% of unawakened untreated animal along with the time.As shown in figure 13, giving anemia animal compd A causes hematocrit levels to increase.
Hemoglobin
Give compd A and in anemia and non-anemia animal, all increased hemoglobin level.As shown in figure 14, increase to about 110% (0 week was corresponding to the 28th day of this experimental program among Figure 14) of untreated control animal with hemoglobin level in the non-anemia animal of compd A (40mg/kg) processing.In the anemia animal, hemoglobin level increases when giving 2 10mg/kg, 20mg/kg or 40mg/kg compd A weekly.Hematocrit levels continued to increase at least 4 weeks.
Red blood cell count(RBC)
The anemia animal is compared with non-anemia animal has lower red blood cell count(RBC).Especially, red blood cell count(RBC) is lower than at injection PG-PS after 28 days in the non-anemia animal 90% of observed result in the anemia animal.As shown in figure 15, red blood cell count(RBC) is compared increase (0 week was corresponding to the 28th day of this experimental program among Figure 15) with untreated animal in the anemia animal of handling with compd A.Observing red blood cell count(RBC) after giving chemical compound 2 weeks increases, and continues to increase at the experimental session in 6 weeks.
Average blood cell volume
The anemia animal illustrates average blood cell volume and compares reduction with non-anemia (nothing excites) animal.As shown in figure 16, As time goes on the average blood cell volume of handling with PG-PS of animal continues to reduce, show that the chronic disease anemia causes the effect of microcytic anemia (its characteristic be that RBC number reduces and erythrocyte less), and because the storage of ferrum and can not produce hemoglobin (among Figure 16 0 week corresponding in this experimental program the 28th day) due to can not utilizing.Give anemia animal compd A and cause slowing down of average blood cell volume reduction.Therefore, it is effective using Compounds and methods for inhibition prolyl hydroxylase of the present invention to reduce, recover average blood cell volume, the average blood cell volume of maintenance etc. for the reduction chronic disease anemia average blood cell volume relevant with iron deficiency anemia.These data show that further method of the present invention and chemical compound can be used for increasing the availability of ferrum to be used for the hemoglobin generation from ferrum is stored.
Mean corpuscular hemoglobin
The anemia animal is compared the mean corpuscular hemoglobin level with reduction with control animal, show that the chronic disease anemia influences hemoglobin and produces.As shown in figure 17, As time goes on the anemia animal that gives compd A illustrates that the reduction of mean corpuscular hemoglobin level slows down (among Figure 17 0 week corresponding in this experimental program the 28th day).
Ferrum state: serum levels of iron and transferrin saturation
The Clinical symptoms of suffering from the patient of chronic disease anemia is that blood plasma concentration of iron and transferrin saturation reduce.In normal and anemia animal, measured the effect of chemical compound of the present invention to serum levels of iron and transferrin saturation.Use the animal model of chronic disease anemia, in rat, induce anemia by peritoneal injection Peptidoglycan-polysaccharide polymer, as mentioned above.Make arthritis and anemia development 28 days.Then animal is handled with the compd A of various concentration, weekly 2 totally 6 weeks.Determine serum levels of iron level and transferrin saturation by Quality Clinical Labs.
Shown in Figure 18 A, anemia animal (PG-PS) is compared with non-anemia animal (sham) has lower serum levels of iron level.Giving compd A all causes the serum levels of iron level to increase in anemia (PG-PS) and non-anemia contrast (sham) animal.Compare with untreated non-anemia animal and untreated anemia animal with the animal that compd A is handled, have the transferrin saturation (seeing Figure 18 B) of increase.These results show that method of the present invention and chemical compound can be used for increasing serum levels of iron level and transferrin saturation percentage ratio.
Ferrum absorbs
Giving anemia animal compd A (40mg/kg, 2 times/week) the 6th week of back, carrying out the proteinic expression of gene of microarray analysis with iron transfer and absorption in the detection coding participation intestinal.Use said method to carry out microarray analysis, use The Rat Genome 230Aarray (Affymetirx) to carry out, it represents Rat Unigene database build 99 (NationalCenter for Biotechnology Information, Bethesda, MD) all sequences in, comprise about 4,699 rat gene and about 10,467 est sequences and about 700 non-est sequences of fully identifying.
As shown in figure 19, the intestinal that gives the mRNA that the control animal compd A increased NRAMP2 (hollow strips post) and sproutin (solid bars post) is expressed.Untreated anemia animal (PG-PS) all has the mRNA expression of reduction for NRAMP2 and sproutin.These results show that the chronic disease anemia reduces relevant with the protein expression that participates in the ferrum absorption.Yet the anemia animal of handling with compd A illustrates NRAMP2 and all increases (Figure 19) of sproutin expression in the intestinal.These results show that method of the present invention and chemical compound can be used for increasing the expression of gene relevant with absorption with iron transfer.In addition, these results suggest chemical compound of the present invention increases the absorption and the transhipment of ferrum in health objects and the chronic disease anemia object.
Embodiment 21: enhanced erythropoiesis in people's object
Prolyl hydroxylase suppresses the following detection of erythropoietic effect in people's object.Give twice of healthy people volunteer or three oral 20mg/kg compd As, totally 4 weeks weekly.Different time after giving chemical compound extracts hemanalysis EPO, hemoglobin, hematocrit, red blood cell count(RBC), soluble transferrin receptor and serum ferritin level.
Reticulocyte count
As shown in figure 20, administration of human object compd A increases to reticulocyte count to be higher than the placebo group.The reticulocyte count that increases appears in the object that gives twice or three times chemical compound weekly.In the individuality of handling, its reticulocyte level increases to and is higher than approximately 1.7%, and by contrast, untreated individual reticulocyte level is 1.4%.Administration of human object compd A has increased reticulocyte count.Therefore, thus method of the present invention and chemical compound can be used for strengthening erythropoiesis and increase the reticulocyte level.
Hematocrit
Hematocrit levels increases in people's object of handling with compd A.In giving people's object of twice compd A weekly, hematocrit levels is higher than 46%, and by contrast, the placebo group objects is approximately 44%.
Compd A has increased hematocrit in people's object.Therefore, thus Compounds and methods for of the present invention can be used for strengthening erythropoiesis and increases hematocrit.
Red blood cell count(RBC)
Administration of human object compd A has increased red blood cell count(RBC).As shown in figure 21, comparing red blood cell count(RBC) with untreated placebo object in people's object that twice or three times is handled with the 20mg/kg compd A weekly increases.These data show that method of the present invention and chemical compound can be used for strengthening erythropoiesis, thereby increase red blood cell count(RBC).
The state of ferrum-soluble transferrin receptor and serum ferritin
The above results shows that method of the present invention and chemical compound are effective for reticulocyte count in the increase people object, erythrocyte, H﹠H.As shown in figure 22, administration of human object compd A makes soluble transferrin receptor level increase to the level that is higher than the untreated control object.Having observed soluble transferrin level in handling people's object of twice or three times weekly increases.In handling the patient of twice and three times weekly, observed 35% and 31% maximum at the 21st day respectively and reply.The intravital sTfR mean plasma concentration of the patient of placebo treatment does not change.In addition, the serum ferritin level reduces approximately 46% in people's object of handling with compd A, shows that the utilization of ferrum in these objects increases (seeing Figure 23).
In a word, these data show that using Compounds and methods for of the present invention stablize HIF can increase mobilization that ferrum stores, increase iron transfer to bone marrow and increase ferrum and be used for the synthesizing of hemoglobin, erythropoiesis and erythrocytic generation.
Illustrated and described those except this paper, those skilled in the art may be obvious that the various modifications that the present invention is carried out according to aforementioned description.This modification comprises within the scope of the appended claims.
All lists of references that this paper quotes are all incorporated reference in full with it.

Claims (40)

1. the chemical compound of a stable hypoxia inducible factor α is used for the treatment of purposes in the medicine of chronic disease anemia of object in preparation, and wherein said chemical compound is selected from next group:
[(1-chloro-4-hydroxyl-different quinoline woods-3-carbonyl)-amino]-acetic acid;
[(4-hydroxyl-7-phenoxy group-different quinoline woods-3-carbonyl)-amino]-acetic acid;
[(4-hydroxyl-7-benzene sulfonyl-different quinoline woods-3-carbonyl)-amino]-acetic acid; And
3-{[4-(3,3-dibenzyl-urea groups)-benzenesulfonyl]-[2-(4-methoxyl group-phenyl)-ethyl]-amino }-N-hydroxyl-propionic acid amide..
2. chemicals are used for the treatment of purposes in the medicine of chronic disease anemia of object in preparation, and its chemicals are selected from following chemical formula
Figure FA20191495200480019307001C00011
Wherein
Q and X are oxygen atoms;
A is-CH 2
B is-COOH;
R 4It is hydrogen atom;
R 16It is hydrogen atom;
R 17It is hydrogen atom;
R 19It is hydrogen atom;
R 18Be hydrogen atom, (C 1-C 20)-alkyl, the randomly (C that is replaced by fluorine atom 1-C 20)-alkoxyl or (C 6-C 12)-aryloxy group;
R 3Be hydrogen atom or halogen atom.
3. claim 1 or 2 purposes, wherein said medicine is used to increase the amount of available ferrum to generate new erythrocyte.
4. claim 1 or 2 purposes, wherein said chronic disease anemia are in a disguised form closed with being selected from as the chronic disease of next group: rheumatoid arthritis, rheumatic fever, inflammatory bowel disease, system lupus erythematosus, vasculitis, tumor disease, chronic infection and chronic inflammatory disease.
5. claim 1 or 2 purposes, wherein said object inflammatory cytokine production increases.
6. the purposes of claim 5, wherein said inflammatory cytokine comprises tumor necrosis factor-alpha, interleukin-1 ' beta ' and interferon-.
7. the chemical compound of a stable hypoxia inducible factor α is in the purposes of preparation treatment in the medicine of the anemia of object, and described anemia has resistance to the treatment that the outside gives erythropoietin, and wherein said chemical compound is selected from next group:
[(1-chloro-4-hydroxyl-different quinoline woods-3-carbonyl)-amino]-acetic acid;
[(4-hydroxyl-7-phenoxy group-different quinoline woods-3-carbonyl)-amino]-acetic acid;
[(4-hydroxyl-7-benzene sulfonyl-different quinoline woods-3-carbonyl)-amino]-acetic acid; And
3-{[4-(3,3-dibenzyl-urea groups)-benzenesulfonyl]-[2-(4-methoxyl group-phenyl)-ethyl]-amino }-N-hydroxyl-propionic acid amide..
8. chemicals are used for the treatment of purposes in the medicine of anemia of object in preparation, and described anemia has resistance to the treatment that the outside gives erythropoietin, and its chemicals are selected from following chemical formula
Wherein
Q and X are oxygen atoms;
A is-CH 2
B is-COOH;
R 4It is hydrogen atom;
R 16It is hydrogen atom;
R 17It is hydrogen atom;
R 19It is hydrogen atom;
R 18Be hydrogen atom, (C 1-C 20)-alkyl, the randomly (C that is replaced by fluorine atom 1-C 20)-alkoxyl or (C 6-C 12)-aryloxy group;
R 3Be hydrogen atom or halogen atom.
9. claim 7 or 8 purposes, wherein said anemia is caused by chronic inflammatory disease or autoimmune pathological changes.
10. the purposes of claim 9, wherein said pathological changes is selected from chronic bacterial endocarditis, osteomyelitis, rheumatoid arthritis, rheumatic fever, clone disease and ulcerative colitis.
11. being used to strengthen the erythropoietin of bone marrow, the purposes of claim 7 or 8, wherein said medicine reply.
12. the purposes of claim 7 or 8, wherein said medicine are used to suppress the inhibition to erythropoietin of tumor necrosis factor-alpha or interleukin-1 ' beta '.
13. the purposes of claim 7 or 8, wherein said medicine are used to strengthen hemopoietic forebody cell replying erythropoietin.
14. the chemical compound of a stable hypoxia inducible factor α is used for increasing the ferrum picked-up at object in preparation, Railway transportation changes and ferrum utilize in purposes in the medicine that produces of the required factor, wherein said chemical compound is selected from next group:
[(1-chloro-4-hydroxyl-different quinoline woods-3-carbonyl)-amino]-acetic acid;
[(4-hydroxyl-7-phenoxy group-different quinoline woods-3-carbonyl)-amino]-acetic acid;
[(4-hydroxyl-7-benzene sulfonyl-different quinoline woods-3-carbonyl)-amino]-acetic acid; And
3-{[4-(3,3-dibenzyl-urea groups)-benzenesulfonyl]-[2-(4-methoxyl group-phenyl)-ethyl]-amino }-N-hydroxyl-propionic acid amide..
15. chemicals are used for increasing the ferrum picked-up at object in preparation, Railway transportation changes and ferrum utilize in purposes in the medicine that produces of the required factor, its chemicals are selected from following chemical formula
Figure FA20191495200480019307001C00041
Wherein
Q and X are oxygen atoms;
A is-CH 2
B is-COOH;
R 4It is hydrogen atom;
R 16It is hydrogen atom;
R 17It is hydrogen atom;
R 19It is hydrogen atom;
R 18Be hydrogen atom, (C 1-C 20)-alkyl, the randomly (C that is replaced by fluorine atom 1-C 20)-alkoxyl or (C 6-C 12)-aryloxy group;
R 3Be hydrogen atom or halogen atom.
16. the purposes of claim 14 or 15, the wherein said factor are selected from class erythrocyte amino-laevulic acid synthase, transferrin, transferrin receptor and ceruloplasmin.
17. the chemical compound of a stable hypoxia inducible factor α is used for the treatment of or prevents purposes in the medicine of pathological changes relevant with cytokine activity in object in preparation, wherein said pathological changes is with to be selected from following disease relevant: inflammation, infection, immunodeficiency and tumor disease, wherein said chemical compound are selected from next group:
[(1-chloro-4-hydroxyl-different quinoline woods-3-carbonyl)-amino]-acetic acid;
[(4-hydroxyl-7-phenoxy group-different quinoline woods-3-carbonyl)-amino]-acetic acid;
[(4-hydroxyl-7-benzene sulfonyl-different quinoline woods-3-carbonyl)-amino]-acetic acid; And
3-{[4-(3,3-dibenzyl-urea groups)-benzenesulfonyl]-[2-(4-methoxyl group-phenyl)-ethyl]-amino }-N-hydroxyl-propionic acid amide..
18. chemicals are used for the treatment of or prevent purposes in the medicine of pathological changes relevant with cytokine activity in object in preparation, wherein said pathological changes is with to be selected from following disease relevant: inflammation, infection, immunodeficiency and tumor disease, its chemicals are selected from following chemical formula
Figure FA20191495200480019307001C00051
Wherein
Q and X are oxygen atoms;
A is-CH 2
B is-COOH;
R 4It is hydrogen atom;
R 16It is hydrogen atom;
R 17It is hydrogen atom;
R 19It is hydrogen atom;
R 18Be hydrogen atom, (C 1-C 20)-alkyl, the randomly (C that is replaced by fluorine atom 1-C 20)-alkoxyl or (C 6-C 12)-aryloxy group;
R 3Be hydrogen atom or halogen atom.
19. the purposes of claim 17 or 18, wherein said pathological changes is an inflammation.
20. the purposes of claim 19, wherein said medicine are used for expressing to reduce the early stage incident of vascular inflammation by the endotheliocyte that suppresses adhesion molecule, described adhesion molecule is that VCAM or E-select albumen.
21. the purposes of claim 17 or 18, wherein said cytokine is an inflammatory cytokine.
22. the purposes of claim 21, wherein said cytokine is selected from tumor necrosis factor-alpha, interleukin-1 ' beta ' and interferon-.
23. the chemical compound of a stable hypoxia inducible factor α preparation be used for the treatment of or object of prevention in the medicine of microcytosis in purposes, wherein said chemical compound is selected from next group:
[(1-chloro-4-hydroxyl-different quinoline woods-3-carbonyl)-amino]-acetic acid;
[(4-hydroxyl-7-phenoxy group-different quinoline woods-3-carbonyl)-amino]-acetic acid;
[(4-hydroxyl-7-benzene sulfonyl-different quinoline woods-3-carbonyl)-amino]-acetic acid; And
3-{[4-(3,3-dibenzyl-urea groups)-benzenesulfonyl]-[2-(4-methoxyl group-phenyl)-ethyl]-amino }-N-hydroxyl-propionic acid amide..
24. chemicals preparation be used for the treatment of or object of prevention in the medicine of microcytosis in purposes, its chemicals are selected from following chemical formula
Wherein
Q and X are oxygen atoms;
A is-CH 2
B is-COOH;
R 4It is hydrogen atom;
R 16It is hydrogen atom;
R 17It is hydrogen atom;
R 19It is hydrogen atom;
R 18Be hydrogen atom, (C 1-C 20)-alkyl, the randomly (C that is replaced by fluorine atom 1-C 20)-alkoxyl or (C 6-C 12)-aryloxy group;
R 3Be hydrogen atom or halogen atom.
25. the purposes of claim 23 or 24, wherein said microcytosis is relevant with the pathological changes that is selected from as next group: chronic disease, chronic disease anemia, iron deficiency, functional iron deficiency and iron deficiency anemia.
26. the purposes of claim 25, wherein said pathological changes are the chronic disease anemias.
27. the chemical compound of a stable hypoxia inducible factor α preparation be used for the treatment of or object of prevention in purposes in the purposes of medicine of iron deficiency, wherein said chemical compound is selected from next group:
[(1-chloro-4-hydroxyl-different quinoline woods-3-carbonyl)-amino]-acetic acid;
[(4-hydroxyl-7-phenoxy group-different quinoline woods-3-carbonyl)-amino]-acetic acid;
[(4-hydroxyl-7-benzene sulfonyl-different quinoline woods-3-carbonyl)-amino]-acetic acid; And
3-{[4-(3,3-dibenzyl-urea groups)-benzenesulfonyl]-[2-(4-methoxyl group-phenyl)-ethyl]-amino }-N-hydroxyl-propionic acid amide..
28. chemicals preparation be used for the treatment of or object of prevention in purposes in the purposes of medicine of iron deficiency, its chemicals are selected from following chemical formula
Wherein
Q and X are oxygen atoms;
A is-CH 2
B is-COOH;
R 4It is hydrogen atom;
R 16It is hydrogen atom;
R 17It is hydrogen atom;
R 19It is hydrogen atom;
R 18Be hydrogen atom, (C 1-C 20)-alkyl, the randomly (C that is replaced by fluorine atom 1-C 20)-alkoxyl or (C 6-C 12)-aryloxy group;
R 3Be hydrogen atom or halogen atom.
29. the purposes of claim 27 or 28, wherein said iron deficiency are functional iron deficiencies; Relevant with anemia; Relevant with the pathological changes that is selected from as next group: inflammation, infection, immunodeficiency pathological changes and tumor disease; Or it is relevant: ACD, iron deficiency anemia and microcytic anemia with the pathological changes that is selected from as next group.
30. the purposes of claim 27 or 28, the serum iron content protein level of wherein said object be lower than 50ng/ml be higher than 200ng/ml or when described to as if the saturated percentage ratio of when adult transferrin be less than 16%.
31. the purposes of claim 27 or 28 is wherein said to liking functional iron deficiency.
Regulate purposes in the medicine that hormone expresses 32. the chemical compound of a stable hypoxia inducible factor α is used for reducing object ferrum in preparation, wherein said chemical compound is selected from next group:
[(1-chloro-4-hydroxyl-different quinoline woods-3-carbonyl)-amino]-acetic acid;
[(4-hydroxyl-7-phenoxy group-different quinoline woods-3-carbonyl)-amino]-acetic acid;
[(4-hydroxyl-7-benzene sulfonyl-different quinoline woods-3-carbonyl)-amino]-acetic acid; And
3-{[4-(3,3-dibenzyl-urea groups)-benzenesulfonyl]-[2-(4-methoxyl group-phenyl)-ethyl]-amino }-N-hydroxyl-propionic acid amide..
Regulate purposes in the medicine that hormone expresses 33. chemicals are used for reducing object ferrum in preparation, its chemicals are selected from following chemical formula
Wherein
Q and X are oxygen atoms;
A is-CH 2
B is-COOH;
R 4It is hydrogen atom;
R 16It is hydrogen atom;
R 17It is hydrogen atom;
R 19It is hydrogen atom;
R 18Be hydrogen atom, (C 1-C 20)-alkyl, the randomly (C that is replaced by fluorine atom 1-C 20)-alkoxyl or (C 6-C 12)-aryloxy group;
R 3Be hydrogen atom or halogen atom.
34. the purposes of claim 32 or 33, wherein said medicine are used to increase the interior ferrum absorption of intestinal and alleviate hypoferremia.
35. claim 2,8,15,18,24,28 or 33 each purposes, wherein said chemical compound is the hypoxia inducible factor prolyl hydroxylase inhibitors.
36. claim 1,2,7,8,14,15,17,18,23,24,27,28,32 or 33 each purposes, wherein said to as if the people.
37. the chemical compound of a stable hypoxia inducible factor α preparation be used for the treatment of or object of prevention in the medicine of microcyte disease anemia in purposes, wherein said chemical compound is selected from next group:
[(1-chloro-4-hydroxyl-different quinoline woods-3-carbonyl)-amino]-acetic acid;
[(4-hydroxyl-7-phenoxy group-different quinoline woods-3-carbonyl)-amino]-acetic acid;
[(4-hydroxyl-7-benzene sulfonyl-different quinoline woods-3-carbonyl)-amino]-acetic acid; And
3-{[4-(3,3-dibenzyl-urea groups)-benzenesulfonyl]-[2-(4-methoxyl group-phenyl)-ethyl]-amino }-N-hydroxyl-propionic acid amide..
38. chemicals preparation be used for the treatment of or object of prevention in the medicine of microcyte disease anemia in purposes, its chemicals are selected from following chemical formula
Figure FA20191495200480019307001C00091
Wherein
Q and X are oxygen atoms;
A is-CH 2
B is-COOH;
R 4It is hydrogen atom;
R 16It is hydrogen atom;
R 17It is hydrogen atom;
R 19It is hydrogen atom;
R 18Be hydrogen atom, (C 1-C 20)-alkyl, the randomly (C that is replaced by fluorine atom 1-C 20)-alkoxyl or (C 6-C 12)-aryloxy group;
R 3Be hydrogen atom or halogen atom.
39. the purposes of claim 37 or 38, wherein said microcyte disease anemia is relevant with the pathological changes that is selected from as next group: chronic disease, chronic disease anemia, iron deficiency, functional iron deficiency and iron deficiency anemia.
40. the purposes of claim 39, wherein said pathological changes are the chronic disease anemias.
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