CN1729015A - Long acting erythropoietins that maintain tissue protective activity of endogenous erythropoietin - Google Patents

Long acting erythropoietins that maintain tissue protective activity of endogenous erythropoietin Download PDF

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CN1729015A
CN1729015A CNA038249154A CN03824915A CN1729015A CN 1729015 A CN1729015 A CN 1729015A CN A038249154 A CNA038249154 A CN A038249154A CN 03824915 A CN03824915 A CN 03824915A CN 1729015 A CN1729015 A CN 1729015A
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epo
erythropoietin
oligonucleotide chain
chain
life
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A·切拉米
J·斯马特
M·布赖恩斯
C·切拉米
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Kenneth s warren research institute
Warren Pharmaceuticals Inc
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Kenneth s warren research institute
Warren Pharmaceuticals Inc
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Abstract

The present invention provides a methods for increasing the hematocrit of an individual while maintaining the tissue protective activities of endogenous through the administration of a pharmaceutical compound containing chemically modified long acting erythropoietin. Also disclosed are the new chemically modified long acting erythropoietins, methods of producing the chemically modified long acting erythropoietins, and compositions comprising the chemically modified long acting erythropoietins.

Description

Keep endogenous erythropoietin The active long acting erythropoietin of organization protection
The reference of related application
The application requires the priority of the U.S. Provisional Patent Application number 60/409,020 of JIUYUE in 2002 application on the 9th, and its content is hereby incorporated by.
Invention field
The present invention relates to the modified long acting erythropoietin that can better keep organization protection's ability.Especially, the present invention relates to long acting erythropoietin through chemical modification, described chemical modification prolonged its in serum half-life but also kept native protein organization protection's function in vivo.The invention still further relates to and use long acting erythropoietin of the present invention to treat anemia and relevant disease thereof.At last, the present invention relates to a kind of detection method whether erythropoietin has organization protection's ability of analyzing.
Background of invention
Naturally generate or endogenic erythropoietin (EPO) is a kind of glycoprotein hormones that is mainly produced by liver.Endogenic EPO comprises 165 aminoacid, and molecular weight (human) is about 30,000 approximately to 34,000 dalton.Glycosyl among the EPO, it is made of the oligonucleotide chain that 3 N-connections are connected with 1 O-, and it has accounted for about 40% of whole protein molecular weight.The oligonucleotide chain that N-connects is combined on 24,38 and 83 the amino nitrogen of agedoite, and the oligonucleotide chain that O-connects is combined on the oxygen atom of the serine residue on 126.EPO may exist 3 kinds of form: α, β and take off sialic acid form (Asialo).α has identical effectiveness with beta form, biological activity, and molecular weight, but slightly different on the composition of saccharide, be to have removed the α or the beta form of terminal sialic acid (saccharide) and take off the sialic acid form.
Up to date, the major function of endogenic EPO is and other somatomedin together propagation and the maturation of stimulating responsive bone marrow erythrocyte precursor, and keeps individual hematocrit (in containing erythrocytic whole blood shared percentage rate).Erythrocytic production process is called erythropoiesis, and it is-kind by the physiological mechanism of accuracy controlling, optimizes erythrocytic quantity under the circulation situation and makes it satisfy the needs of tissue oxidizing not hindering.For example, when the oxygen of hot cell delivery reduced, EPO turned to sophisticated erythrocyte and increases erythrocytic generation by stimulating in the bone marrow precursor to gulp down transhipment, and the erythrocyte of generation enters in the circulating system immediately.When the erythrocytic quantity in the blood circulation needed above the normal structure circulation, the EPO in the circulating system just reduced.Therefore, when body was in health status, the concentration of EPO was very low in the blood plasma, only was enough to stimulus substitution because the aging and erythrocyte of normal loss.The normal range of blood plasma EPO be from 0.01 unit/ml to 0.03 unit/ml.
Suppose that most EPO is produced by kidney in the individuality, the loss of renal function, for example chronic renal failure (CRE) can cause the minimizing of EPO quantity and usually can cause anemia.Equally, anemia may be by other chronic disease, cancer for example, or with the treatment of these disease associations, cause as chemotherapy.Therefore, verified, can effectively recover the level of hematocrit for the EPO (back will be described in detail) of oligocythemic individual administered recombinant, the EPO of described reorganization and endogenous EPO have same basically biological activity.
Except it kept effect in the hematocrit levels under chronic sympton, recombinant epo had been used to increase hematocrit level before selectivity or predetermined operation, thereby reduced or the needs of cancellation blood transfusion.For example, administered recombinant EPO can remove patient to the misgivings of infective virus or cause of disease by blood transfusion, or solves the problem of restriction blood transfusion on the religion.
Further, some recent evidences show that EPO has other important treatment attribute as a member of cytokine superfamily, and this interaction by itself and EPO receptor (EPO-R) mediates.For example, EPO and receptor thereof play an important role in extenuating tissue injury, because EPO and its acceptor interaction provide compensatory the replying that changes the hypoxic cell microenvironment, and regulation and control are because the programmed cell death that metabolism pressure produces.In fact, the patient who suffers from chronic renal failure and/or cancer generally can improve the sensation of rehabilitation or the acuteness of intelligence after using the EPO treatment, and such effect is given the credit to the increase of patient's hematocrit before this.As described in international application no WO/02053580 and U.S. Patent Publication No. 2002/0086818 and 2003/0072737, these improve organization protection and the enhanced activity that is given the credit to EPO but recently.
Nearest research shows that equally the EPO that general is used can pass through blood brain barrier, also expresses the EPO receptor because form the capillary tube of blood brain barrier.Therefore, this provides the anatomic basis of the receptor-mediated endocytosis from the peripheral circulation to the brain.
Recombinant epo (epoetin α), can obtain by commercial sources, be called PROCRIT  (from Ortho Biotech Inc., Raritan as commodity, NJ), with EPOGEN  (from Amgen, Inc., Thousand Oaks, CA), be used for the treatment of the anemia that is caused by end-stage renal disease, same AZT (azidothymidine AZT) therapy makes together and is used for the influence for the treatment of the patient of infected by HIV and being used to offset chemotherapy.Though it is a variety of that the therapeutical effect of recombinant epo has, the topmost purposes of recombinant epo is to extenuate chronic anaemia up to now.In this, the typical initial application dosage of recombinant epo is that 50-150 unit/kg is inferior on every Wendesdays in about 6 to 8 weeks, and vein or subcutaneous injection administration are to recover the hematocrit scope of directiveness in the patient body.When the hematocrit levels that patient obtains to expect, for example after the amount between about 30% to about 36%, can under the situation of no iron deficiency or complication, keep described hematocrit levels by keeping the EPO therapy.Though the requirement of dosage may change according to the demand of individual patients, usually maintenance dose can be administered three times approximately weekly (if dosage is bigger, number of times can still less).
The application dosage of recombinant epo and number of times depend in part on the half-life of this molecule, may be very limited in the time of in molecule is in body.For example, it is reported in suffering from the adult and child patient of CRF the EPOGEN of intravenous administration Be eliminated with the speed that meets first order kinetics, its circulating half-life is in about 4-13 hour scope.Therefore, in order to reach the half-life that therapeutic effect considers that recombinant epo is relatively short, application dosage and efficient are adjusted.
In addition, because recombinant epo passes through vein or subcutaneous injection, this usually just needs nurse or doctor to come to the patient injection recombinant epo.This has brought more inconvenience to patient, and this also just becomes the reason why another one will consider that the molecule half-life prolongs.Therefore, the research that increases the recombinant epo half-life comes into one's own in the past ten years, and this is based on following prerequisite, i.e. the dosage that can demand reduction of the half-life of Yan Changing, the therapeutic effect that the while also provides identical or improved.
In fact, the experiment up-to-date about human EPO shows, contains sialic sugared content among the EPO, its circulating half-life, and direct relation is arranged between the activity in vivo.As described in PCT publication number WO95/05465, sialic acid residues is added in the terminal of sugar chain and has stoped the identification of liver to galactose.Typically, the oligonucleotide chain of every N-connection contains 4 sialic acides at the most, and the oligonucleotide chain of every O-connection contains 2 sialic acides at the most.Therefore, not adorned EPO polypeptide can contain maximum 14 sialic acides altogether.
As time goes on, these sialic acid residueses may rupture from protein, thereby have exposed the gala sugar chain that can be discerned by liver.In case liver recognizes the gala sugar chain, protein has just filtered out from blood.Therefore, it is believed that and progressively increase the sialic acid that each EPO molecule is contained, can better avoid the exposure of gala sugar chain, also progressively increase accordingly its biological activity (improving the capability approach of the hematocrit of normal mouse by the erythropoietin that detects isolating same molecular amount concentration).Because the EPO of unmodified only contains 14 sialic acid sites, such structural limitations the prolongation of EPO half-life.This has caused following hypothesis is that the EPO analog that contains extra oligonucleotide chain of through engineering approaches may have stronger biologic activity.By extra glycosylation site is provided, containing terminal extra oligonucleotide chain can be modified by sialic acid residues.Referring to the PCT publication number is WO91/05867, the application of WO94/09257 and WO01/81405.
For example, the EPO analog of modification can contain the sugar chain that sugar chain that an extra N-connects and/or at least-individual extra O-connects at least.Particularly in WO01/81405, disclose at the aminoacid place in 30,51,57,69,88,89,136 and/or 138 sites of molecule and increased the sugar chain that N-connects.The EPO molecule of modified can contain 1-4 extra glycosylation site in any position, makes the EPO molecule can increase 2-16 sialic acid residues.
Though these effort that increase the EPO serum half-life be proved to be success and keeping proving useful on the hematocrit levels, but do not notice of other function issuable influence of these extra glycosylation sites to EPO.
Therefore, need provide the modified EPO that has prolonged serum half-life (long-acting) and kept the function of endogenous EPO.Especially, need a kind of long lasting EPO chemical compound in this area, it has promoting erythrocyte function and organization protection's function, is used for the treatment of the pharmaceutical composition of anemia and/or relevant disease.In addition, people need also to be used for to determine whether a kind of specific EPO has the detection method of antagonism to organization protection's ability of endogenous EPO.
Summary of the invention
The present invention relates to a kind of method of regulating human hematocrit levels, comprise a kind of step that this erythropoietin product of the step of erythropoietin product longer serum half-life, that have organization protection's function and administering therapeutic effective dose is arranged than the human erythropoietin of reorganization (rhuEPO) is provided.In a technical scheme, provide the step of erythropoietin product also to comprise to use at least a chemical modification at least-step of modifying recombinant epo on the oligonucleotide chain that oligonucleotide chain that individual N-connects or O-connect, chemical modification described here comprises oxidation, sulphation, phosphorylation, PEGization or their combination.
In addition, the step of the erythropoietin product of administering therapeutic effective dose can comprise and use this erythropoietin product that is lower than the rhuEPO mole, to reach suitable hematocrit.
In a technical scheme, its serum half-life grows 20% than the serum half-life of rhuEPO at least.In another technical scheme, its serum half-life prolongs 40% than the serum half-life of rhuEPO at least.
The present invention also relates to artificial erythropoietin product, it comprises at least a erythropoietin derivatives, wherein have the described chemical modification of at least a chemical modification to derive from oxidation, sulfuration, phosphorylation, PEGization or their combination on the oligonucleotide chain that the oligonucleotide chain of at least one N-connection or at least one O-connect, erythropoietin product described here has the serum half-life longer than rhuEPO.This erythropoietin product preferably has organization protection's function.
In a technical scheme, at least a chemical modification comprises that oligonucleotide chain oxidation that oligonucleotide chain that at least one N-connects or at least one O-connect is to provide at least one extra acidic residues.For example, at least a chemical modification sulphation that can be included in the oligonucleotide chain that oligonucleotide chain that at least one N-connects or at least one O-connect EPO product to provide negative charge to increase.In another technical scheme, phosphorylation that at least a chemical modification comprises oligonucleotide chain that at least one N-connects or at least-oligonucleotide chain that individual O-connects EPO product to provide negative charge to increase.On the other hand.At least a chemical modification is included on the oligonucleotide chain that oligonucleotide chain that at least one N-connects or at least one O-connect increases polyglycol chain.
The present invention relates to the method that a kind of preparation has the erythropoietin of the serum half-life of prolongation and organization protection's function, comprise the steps: to provide the derivant of at least a erythropoietin or erythropoietin; On the oligonucleotide chain that oligonucleotide chain that at least one N-of the erythropoietin of described at least a endogenous or reorganization connects or at least one O-connect, carry out the modification of oxidation, sulphation, phosphorylation, PEGization or its compound mode.
The step of modifying can also comprise at least one vicinal hydroxyl groups on the oligonucleotide chain of the oligonucleotide chain of at least one N-connection or at least one O-connection is replaced with at least a sour residue.In one embodiment, can also comprise a plurality of at least vicinal hydroxyl groups on the oligonucleotide chain that is substituted in oligonucleotide chain that at least one N-connects or at least one O-connection with multiple at least sour residue in the step that at least one vicinal hydroxyl groups on the oligonucleotide chain that oligonucleotide chain that at least one N-connects or at least one O-connect replaces with at least a sour residue.
In another technical scheme, the step of modification also comprises following step: a kind of organic solvent is provided; The derivant of dissolving erythropoietin or erythropoietin is to form solution in solvent; At least a condensing agent is provided; At least a sulfate donor is provided; And at least a condensing agent and at least a sulfate donor be mixed in the above-mentioned solution go.In another technical scheme, the step of modification also comprises following step: a kind of organic solvent is provided; Described erythropoietin or short thin red born of the same parents are generated plain derivant be dissolved into formation solution in all organic solvents; At least a condensing agent is provided; Phosphoric acid is provided; And at least a condensing agent and at least a phosphoric acid is mixed in the above-mentioned solution goes.
In another technical scheme, the step of modification also comprises following step: a kind of organic solvent is provided; The derivant of dissolving erythropoietin or erythropoietin is to form first solution in this organic solvent; At least a oxidant is provided; At least a oxidant is added in first solution to form second solution; At least a polyglycol chain is provided; At least a polyglycol chain is mixed in second solution goes.Provide the step of at least a polyglycol chain can comprise that the end that is provided at chain contains at least a polyglycol chain of at least one primary amino radical group.
The present invention also relates to the method that a kind of treatment damages dangerous patient's anemia in a organized way, it comprises following step: the erythropoietin product of at least a chemical modification is provided on the oligonucleotide chain that provides oligonucleotide chain that a kind of at least one N-connects or at least one O-to connect, and chemical modification described here comprises oxidation, sulphation, phosphorylation, PEGization or its combination; This erythropoietin product of administering therapeutic effective dose; the mole of using of erythropoietin product described here is lower than rhuEPO; but can reach suitable targeted blood cells specific volume, erythropoietin product described here has organization protection's function.
In this one side of the present invention, this erythropoietin product preferably has the half-life longer than the serum half-life of rhuEPO.In one embodiment, its serum half-life grows 20% than the serum half-life of rhuEPO at least.On the other hand, its serum half-life grows 40% than the serum half-life of rhuEPO at least.
The invention still further relates to the pharmaceutical composition that comprises following composition: at least-derivant of erythropoietin of kind of treatment effective dose; on the oligonucleotide chain that the oligonucleotide chain that its at least one N-connects or at least one O-connect at least a chemical modification is arranged; described chemical modification derives from oxidation, sulphation, phosphorylation, PEGization or its compound mode, and the derivant of at least a erythropoietin described here has than the longer serum half-life of reorganization erythropoietin and has organization protection's function.In one embodiment, this pharmaceutical composition also comprises at least a pharmaceutically acceptable carrier.This at least-kind of a pharmaceutically acceptable carrier can comprise at least a diluent, adjuvant, excipient, carrier or its combination.
In another embodiment, this this pharmaceutical composition also comprises at least a wetting agent, emulsifying agent, pH buffer agent or its combination.In another embodiment, this this pharmaceutical composition also comprises at least a organization protection factor.
Description of drawings
It is clearer that further aspect of the present invention and advantage will become after the detailed description of carrying out in conjunction with following accompanying drawing, and relevant accompanying drawing is described below:
FIG.1A is the multi-form EPO comparison for the protective effect effectiveness that is exposed to the cell death that causes in the tin trimethyl; With
FIG.1B is the multi-form EPO comparison for the protective effect effectiveness that is exposed to the cell death that causes in the tin trimethyl.
Detailed Description Of The Invention
The present invention relates to have the application of the EPO molecule of the serum half-life (long-acting) that has prolonged, to institute Thereby state the EPO molecule and carry out the function that chemical modification is kept endogenous EPO with sugar chain. As carrying on the back As described in the scape technology, prolong the effort of EPO half-life, usually concentrating on to the EPO molecule increases Extra sugar chain is to prevent the exposure of galactolipin chain. It is similar that but the sugar chain that it is believed that increase has affected EPO The function of thing, thereby so that, for example, this function be compromised to obtain than the long half-lift. Although known ratio EPO analog long half-lift that recombinant epo having more has red blood cell and generates activity, but these analogs Other treatment function that does not keep the EPO of recent findings, as, organization protection's activity.
For example, 17 amino acid whose segments corresponding to the 30-47 position of EPO are also referred to as O ' Brien Peptide has organization protection's activity in external demonstration, but does not but have a promoting erythrocyte activity external. Campana, W.M., Misasi, R.﹠O ' Brien, J.S., Int.J.Mol.Med., 1,235-41 (1998). Therefore, it is believed that the EPO molecule of the modified that has extra glycosylation site in O ' Brien peptide is at it Do not possess organization protection's activity in its in vitro test. In addition, because the 3 D tropism of EPO molecule for it Function plays an important role, and the glycosylation site of increase may affect the allomeric function of molecule.
Therefore, the present invention relates to long-acting EPO, it has, and the red blood cell generation is active, organization protection is active, At least a or its combination in transcytosis (transcytosis) ability. Preferably, of the present invention At least a and the red blood cell that long-acting EPO has in organization protection's activity, the transcytosis generates alive The property.
In a concrete scheme, long-acting EPO of the present invention have than recombinant epo length at least about 20% serum half-life. In another embodiment, long-acting EPO of the present invention has than restructuring EPO grows the serum half-life at least about 30%. Again-individual embodiment in, long-acting EPO of the present invention Have than recombinant epo length at least about 40% serum half-life.
Briefly, with natural (endogenous) EPO, preferably compare with natural human EPO, this Bright described long-acting EPOs comprises the EPO molecule with the sugar chain that changes by at least a modification. In the embodiment, long-acting EPOs of the present invention is through the multiple modification to sugar chain.
In one embodiment, the ortho position of natural EPO sugar chain (vicinyl) hydroxyl oxygen is changed into acidity Residue is to prepare long-acting EPO molecule of the present invention. In another embodiment, with more stable Acidic residues replaces the sialic acid residues on the EPO molecule. In another embodiment, to the EPO sugar chain Sulfuration and/or phosphorylation can produce long-acting EPO of the present invention. In a further embodiment, can To add that at the EPO sugar chain polyethylene glycol is to obtain long-acting EPO of the present invention. Aforementioned modification arbitrarily Combination is also included among the present invention. And as mentioned before, the present invention also comprises composition, Comprise pharmaceutical composition, it comprises one or more above-mentioned long-acting EPO molecules.
Long-acting EPO molecule expection of the present invention is applied in the pharmaceutical composition, and this pharmaceutical composition can With treatment anaemia and relevant disease, especially by including but not limited to: acute renal failure, pyaemia, HIV, The complication that chemotherapy etc. disease causes.
The present invention also relates to treat the method for anaemia and relevant disease, relate to simultaneously a kind of process that is used for the treatment of Kit. Here the term of using " treatment " refers to the measure of drug therapy and prevention or protection, its objective is In order to prevent or slow down pathological condition or the imbalance of (minimizing) target. Those objects that need to treat comprise: The object of having suffered from this imbalance (disorder), and the object of this imbalance of those susceptibles, or those remain In the object that this imbalance is prevented. The present invention includes this long-acting EPO and can be used for chronic administration, acute Treatment and/or discontinuity administration. For the purpose of this paper, " chronic administration " refers to respect to acute administration Pattern is beneficial to keep for a long time initial treatment effect (activity), " discontinuity with continuous pattern administration Administration " do not carry out continuously without interruption, but loop.
Long-acting EPO of the present invention and its purposes can be applied to any mammal. Here, Term " mammal " refers to any mammiferous animal that is sorted in, and comprises the mankind, taming and stable breeding Animal and zoo, match usefulness or pet, as dog, cat, ox, horse, sheep, pig, goat, Rabbit etc. Preferred mammal is the people. The administration of long-acting EPO of the present invention includes but not limited to, In oral, intravenous, the nose, in local, the chamber, suction or parenteral administration, the latter comprise vein, artery, In subcutaneous, intramuscular, the abdominal cavity, submucosa, intracutaneous, and compound mode.
The invention still further relates to the application of long-acting EPO of the present invention, it enters in the body as other molecule Carrier with zone of EPO acceptor. For example, because having low blood-brain barrier, some molecules penetrate energy Power is divided sub-connection with these molecules and long-acting EPOs of the present invention, just enters brain for these molecules A safely and effectively delivery system is provided. And, will be discussed in detail subsequently, because other district of health The EPO acceptor is expressed in the territory, and such as retina, heart and lung, long-acting EPO of the present invention can be for right Role in the delivery system of bearing the responsibility at the low molecule of these regional penetration powers.
Further, the present invention relates to the inspection whether definite specific EPO has kept the function of endogenous EPO The survey method. For example, detection of the present invention can determine whether the EPO of modified has organization protection Activity, the just activator of endogenous EPO. Here, term " activator " is the widest for it Implication comprises the bioactive molecule of those simulation natural EPOs. In the same way, term " antagonism Agent " broadest say comprise those partly or entirely blocking-up, suppress or in and the branch of natural EPO activity Son. In one embodiment, in analyzed in vitro, detect the existence of special EPO, as the P19 cell and/ Or mouse kinesitherapy nerve meta analysis. Other-individual embodiment in, detection of the present invention comprise estimate special Different EPO activity is in vivo used various analytical method such as rat fix a point ischaemic, rat Treat retinal ischemic, spinal injury and Bic outbreak model.
Function
As described in background technology part, for the various trials that increase the EPO half-life Success because its obtained having than the long half-lift and keep the EPO analog of erythropoietic activity. And, as described in, have than the long half-lift the EPO analog mainly be included in its amino acid Increase the EPO molecule of extra sugar chain on the sequence. But can predict, the sugar chain of increase may affect EPO Other therapeutic activity, such as organization protection's function and molecule transcytosis. Be not subjected to any special theory Limitation, based on the importance of O ' Brien peptide to organization protection's function, increase sugar at O ' Brien peptide Chain may affect its function. In addition, the sugar chain of increase, though be in O ' Brien peptide or outside, It is believed that the 3 D tropism that all can have influence on molecule. For example, in the 3-d modelling of molecule, the sugar chain of increase can In can blocker molecule concerning its function requisite zone. Further, it is believed that glycosylated method (or Increase sugar chain) may also can have influence on the function of glycoprotein.
Organization protection's ability
For the sugar chain of estimating increase influences the probability of glycoprotein function, the inventor has studied the various forms of EPO analog that 5 N-connect sugar chains (than the sugar chain of 3-N-connection of recombinant epo) that have.Particularly, the inventor has used the EPO analog, and described analog has extra glycosylation site at 32 amino acids places, and it has caused having prolonged about 3 times half-life than recombinant epo (epoetin α).
Though (Figure 1A) appearring in this EPO analog in cerebrospinal fluid behind the systemic injection, surprised discovery does not have organization protection's activity in external P19 detection (Figure 1B) subsequently.The active disappearance of this organization protection is unexpected.In addition, if these patients have other patient's condition that needs organization protection's ability, the active disappearance of organization protection may lead to complications in treatment anemia man-hour.For example, if these do not have the active EPO analog of organization protection to compete the receptor that those initiation organization protections are replied with protecting active endogenous EPO in a organized way, in fact the injured degree that is produced by damage may be increased the weight of owing to the effect of such EPO.In fact,, be compared to the individuality that does not use the EPO analogue treatment if suffer the patient of apoplexy to use such EPO analog, by in wind-induced infarct volume in fact may be bigger.
Do not carry the baby in any special theory, these discoveries have disclosed at least a extra EPO acceptor type and functionally have been present in the nervous tissue, its signal that sends is different from the signal that the erythrocyte precursor is sent, and exists certain EPO analog may antagonism endogenous EPO and the danger of the ability of the receptors bind of this form.It is to produce signal by the function difference EPO receptor corresponding to EPO molecule different structure territory that the visibly different biological activity of endogenous EPO and these EPO analog has disclosed receptor.In fact, though reported expressed identical of EPO acceptor gene protein sequence and erythrocyte precursor, external, the binding affinity of nervous system type EPO receptor is significantly less than the EPO receptor of proerythrocyte.Referring to, Masuda for example, S., etc., J Biol Chem, 268,11208-16 (1993).May, these are not both by auxilin and cause, and may show that wherein used signal transduction path is different from activatory approach in the erythrocyte maturation process.What is interesting is that the difference on this affinity can not modified by the complete deglycosylation of EPO, if the bonded words of neural activity occur in the AB of normal nonglycosylated EPO ring territory, this just is not to be a unexpected result.In addition, the EPO (may be and other cells identical product that for example neuron produced) that is produced by astrocyte was also than little by what kidney produced.Masuda，S.，J.Bio?Chem，269，19488-93(1994)。This species diversity is seemingly caused by the difference of glycosylation program.Whether variant as yet definite, but obviously be related if taking off sialic native ligand and the proteic affinity of these known receptor.
Except the auxilin that exists the EPO receptor to modify, the EPO receptor is the group of a complexity, has multiple changing form, and comprises the solvable receptor of truncate.Yamaji, R., etc., Eur J Biochem, 239,494-500; Yamaji, R., etc., Bichim Biophys Acta, 1403,169-78 (1998); Barron, C., etc., Gene, 147,263-8 (1994); Chin, K., etc., Brain Res Mol Brain Res, 81,29-42 (2000); Fujita, M., etc., Lukemia, 11Suppl 3,444-5 (1997); Westenfelder, C., Biddle, D.L.Baranowski, R ﹠amp; .L., KidInternat., 55,808-820 (1999). whether any such form all promotes the neural activity of EPO still also not to be determined.
Further, as discussing in the background technology, O ' Brien peptide is in the external organization protection's activity that shows, but not have promoting erythrocyte to generate active external.In fact, this EPO analog that the analysis showed that the EPO analog that comprises the sugar chain of adding in O ' Brien peptide is carried out lacks organization protection's function.This result's hint as increasing sugar chain, has influenced proteic function to certain modification of O ' Brien peptide.The EPO analog that O ' Brien peptide is modified may played the part of the effect to the antagonist that is positioned at intravital endogenous EPO, because blocking-up endogenous EPO its part or whole is in conjunction with the ability of EPO receptor.Therefore, the use of such EPO analog has the danger that the injured degree that causes being caused by damage increases.Therefore; in other external detection; for example the rat motor neuron detects; and test example such as the detection of rat fixed point ischemia in the body; the dicentrine outbreak detects; during detection of rat retina ischemia and spinal cord injury detect, it is believed that the EPO analog of modified on O ' Brien peptide will lack organization protection's ability.
Use above-mentioned identical EPO analog, just have the EPO analog (sugar chain that connects than 3 N-of recombinant epo) of the sugar chain that 5 N-connect, the inventor has studied the ability that this analog passes blood brain barrier.Behind systemic injection (Figure 1A and 1B), the EPO analog appears in the cerebrospinal fluid.Be not limited by any particular theory, it is believed that this EPO analog can pass complete blood brain barrier, also express the EPO receptor because form the blood capillary of blood brain barrier, and for from the peripheral circulation to the brain, providing anatomical basis by receptor-mediated transcytosis.Like this, the EPO analog of other systemic administration it is believed that also and can pass blood brain barrier, and other has the barrier of the blood capillary of expressing the EPO receptor.
Generally speaking, because the activity that EPO analog of the prior art keeps promoting erythrocyte to generate under the situation of some function at least of sacrificing endogenous EPO, this area needs a kind of long lasting EPO, and it can keep the known repertoire of endogenous EPO.Have advantage, long-acting EPO of the present invention has not only increased the serum half-life long with respect to recombinant epo, and has kept the repertoire of endogenous EPO, i.e. organization protection's function and endocytosis turn-over capacity.The method that is used to obtain this type of useful proteinic multiple modification EPO is provided in the following part.
The modification of natural EPO
Long-acting EPO of the present invention can in all sorts of ways and prepare.Generally speaking, long-acting EPO can obtain by the sugar chain that chemical modification is connected on the EPO.Here, term " sugar chain " is meant that the N-that is present on the endogenous EPO connects the oligonucleotide chain that is connected with O-, the N-that is present in the increase on the EPO analog connects the oligonucleotide chain be connected with O-and is connected any other sugar chain, especially sugar chain (Sugar chains) on the EPO.
In-individual technical scheme, endogenous or recombinant epo are modified to stop any interference to organization protection's ability of endogenous EPO.In addition, according to the present invention, considered the EPO analog is modified so that extra glycosylation site to be provided, it is not positioned at O ' Brien peptide, promptly near the aminoacid sequence of 30-47 position.Here, term " EPO analog " is meant adorned EPO molecule, its have at least 1 increase the sugar chain that connects of N-and/or the sugar chain that connects of the O-of at least 1 increase.In one embodiment, the EPO analog that is used to modify does not contain the glycosylation site of any increase in 5 aminoacid of O ' Brien peptide.In another embodiment, this EPO analog does not contain the glycosylation site of any increase in 3 aminoacid of O ' Brien peptide.Again-individual embodiment in, this EPO analog does not contain the glycosylation site of any increase in O ' Brien peptide,
The EPO analog also can be used for modifying as described in the present invention, and prerequisite is should consider this analog and confirm that the sugar chain that increases does not hinder O ' Brien peptide or reduced organization protection's ability on three dimensions.On the other hand, considered the EPO analog is used for modification of the present invention, prerequisite is organization protection's function that glycosylation process can not suppress this peptide.In yet another aspect, the EPO analog according to method of modifying provided by the invention is modified has at O ' Brien peptide-individual sugar chain (or still less).For example, endogenous EPO has a sugar chain at 38 amino acids places, and the extra sugar chain in O ' Brien peptide has been proved to be this proteic organization protection function of inhibition.Therefore, on O ' Brien peptide, have-individual or do not have the EPO analog of sugar chain can be used for modifying.In one embodiment, the sugar chain that is combined in 38 amino acids places can reconnect to other position of this peptide.
Limiting examples according to method of modifying of the present invention comprises that (1) provides extra acidic residues by the Oxidation of vicinal hydroxyl groups on sugar chain; (2) replace sialic acid residues with more stable residue; (3) pass through the negative charge that sulphation and/or phosphorylation increase erythropoietin; And/or (4) stop sugar chain with more complicated molecule.Therefore, can comprise oxidation, sulphation, phosphorylation and/or PEGization and other step to the modification of EPO sugar chain, they will detailed in the back description, and further illustrates in embodiment 1.
The oxidation of sugar chain and the replacement of sialic acid residues
The of the present invention long-acting EPO of chemical modification can be included in the sugar chain (sugar) of EPO and go up oxidation so that extra acidic residues to be provided.On the one hand, can replace sialic acid residues with more stable acidic residues.Such modification has caused the increase of molecule half-life for endogenous EPO, this is because liver screening gala sugar chain and associated protein removed from blood circulation, and the gala sugar chain was protected and made it not to be detected this moment.More significant modification causes the serum half-life of long-acting EPOs of the present invention more to prolong on the EPO sugar chain.For example, after more vicinal hydroxyl groups is replaced by acid, caused serum half-life more to prolong.
Although those of ordinary skill in the art knows several suitable being used to and change the method for the galactose units of erythropoietin, a kind of suitable method comprises that the glycan molecule that (1) will have a vicinal hydroxyl groups becomes aldehyde with periodate oxidation; (2) formoxy-is changed into acid.The reagent that is fit to sugar chain is oxidized to aldehyde is known for those skilled in the art, includes but not limited to, and periodate, as sodium metaperiodate, and carbohydrate oxidase, as galactase.In addition, the technical staff knows that suitable reagent transforms aldehyde, as quantitative benedict reagent (Kuantitative Benedict Solution) (can buy from Fisher).On the other hand, glycan molecule can further be used quantitative benedict reagent (Fisher) to handle aldehyde is changed into acid by sodium periodate oxidation.
On the other hand, the EPO isomer, it has about 0-13 sialic acid residues, or the EPO analog, and its at least one sugar chain disappearance sialic acid residues is by the galactase oxidation.The EPO that takes off the sialic acid form can be used for this one side of the present invention, the i.e. α of terminal saccharide (sialic acid) removal or the EPO of beta form.The preferred use taken off sialic erythropoietin.In case EPO is oxidized, use other oxidant such as quantitative benedict reagent that aldehyde is changed into acid.
Again on the one hand, ruthenium tetroxide can be with the acid that generates on the sugar chain.Because these relate to the gala sugar chain in modifying, even the acid that relates in conversion is removed from the EPO molecule, this molecule should still can be hidden the removing of liver, because the composition gala sugar chain of liver institute examination will can not be exposed.
Increase negative charge
Another aspect of the present invention, long-acting EPO of the present invention can prepare by interpolation sulfate and/or phosphate on the EPO molecule, and it will increase the negative charge of molecule, thereby increases the half-life of molecule.That is to say that the negative charge of EPO molecule can increase by sulphation, it has related to from the sulfate donor and has comprised protein, glycolipid, and glucosaminoglycan and steroid shift sulfo group.And, can also increase negative charge in the sugar by phosphate group is incorporated into.
A kind of suitable method that is used for sulphated insulin is described in S.Pongor etc., Preparation ofHigh-Potency, Non-aggregating Insulins Using a Novel Sulfation Procedure, Diabetes, Vol.32, No.12, December 1983.For example, the sulphation of insulin can be in organic solvent, and as dimethyl formamide (DMF), under the situation that condensing agent exists, as N, N '-dicyclohexyl diimine (DCC) and sulfate donor carry out.Sulfated degree can be controlled in 8 times of scopes by the amount that changes condensing agent.Although the Sulfated insulin of traditional preparation process causes its main biological activity of having lost insulin, use the preparation of Ponger program sulphated insulin biological activity do not modified insulin 78% to 87% between.
Similarly step is used for EPO, thereby those of ordinary skill in the art can control the serum half-life of the EPO of Sulfated amount control chemical modification.For example, can be preferably among the DCC by EPO or EPO analog being dissolved at least a water-soluble carbodiimide, temperature is about 4 ℃, and sulphuric acid is joined in the protein, thereby increases the negative charge of EPO.DCC is preferred sulfate donor, and what those skilled in the art easily knew other is used for the suitable sulfate donor of the present invention.
Similar step can be used for controlling the phosphorylation of EPO, uses phosphoric acid (H 3PO 4) as phosphodonor.Also have, though preferably phosphoric acid, the technical staff can be easy to select other phosphodonor to come the phosphorylation to EPO.
Sugar-chain end uses PEGs to stop
The sugar chain of EPO also can be modified by increasing at least a Polyethylene Glycol (PEG), and it has the clinical history of long safety, and molecular formula is as follows:
PEG also can be methoxylation PEG (mPEG), has following molecular formula:
Figure A0382491500192
In one embodiment, PEG is amino PEG, and preferably the end at the PEG that methylates has (the mPEG-NH of primary amino radical group 2).The PEG chain that end has primary amino radical group is very useful functionalization polymer.MPEG-NH 2N-terminal group is more prone to take place acylation reaction than the hydroxyl of general PEGs, and amino reduction reaction (reductive amination reactions) also takes place easily for they.In another embodiment, PEG is the activatory PEG of electrophilic, and as mPEG-succinic acid propyl ester (mPEG-SPA) or mPEG-succinic acid butyl ester (mPEG-SBA), they can be from Nektar Therapeutics ofBirmingham, and Alabama company buys.In another embodiment, PEG is the PEG-hydrazides of methoxylation.
On the other hand, can pass through the Oxidation of periodate (above-mentioned discussion), use cyano group boron hydride and amino PEG to increase at least one PEG subsequently.For example, the EPO in the solution at first uses periodate oxidation, as sodium metaperiodate, at room temperature reacts predefined a period of time, produces aldehyde on sugar chain.-kind of a suitable periodate is a sodium metaperiodate, can buy from Sigma company.Periodate is cushioned exchange then and removes, and meanwhile, the sialic acids groups of the oxidation on the oligonucleotide chain that the N-of EPO connects can be under the situation that the cyano group boron hydride exists contacts with-individual amino PEG at least.Available suitable PEG includes but not limited to, methoxylation PEG-hydrazides, and it can be from Nektar Therapeutics ofBirmingham, and Alabama company buys.
On the other hand, after with the galactase oxidation, can increase at least one PEG by the PEG group being connected to terminal galactose residue.For example, the EPO that takes off the sialic acid form in the buffer (having exposed terminal galactose residue) at first contacts to produce aldehyde sugar chain with beta-Galactose oxidase (buying from Sigma company).Remove buffer by the buffering exchange then, simultaneously, the galactose residue with oxidation under the situation that has the cyano group boron hydride to exist contacts with at least a amino PEG.
Method recited above is not restrictive, and other method also can be used to prepare chemical compound of the present invention.For example; the technical staff can recognize the long-acting form that these chemical modifications can be used for making other EPO derivant as in international publication number WO/02053580 and U.S. Patent Publication No. 2002/0086816 and 2003/0072737 disclosed organization protection cytokine, wholely introduces the present invention as a reference at this.
Preparation EPO molecule
Various host expresses carrier systems can be used to produce long-acting EPO of the present invention and EPO correlation molecule.This type of host expresses carrier has been represented a class carrier (vehicle), and by these carriers, interested long-acting EPO is produced and is purified subsequently; Also represented a class cell, when described cell is transformed by suitable nucleotide coding sequence or during transfection, can the modified erythropoietin gene product of expressed in situ.These include but not limited to, antibacterial, and insecticide, plant, the mammalian hosts system, such as but not limited to, with the insect cell system of recombinant virus expression vector (as the rhabdovirus) infection of the sequence that contains the long-acting EPO product of encode; With the recombinant plasmid expression vector of the sequence that contains coding erythropoietin correlation molecule (as, Ti-plasmids) transform or recombinant virus expression vector (as cauliflower mosaic virus, CaMV; Tobacco mosaic virus (TMV), TMV) the plant cell system of transfection; Or the mammalian cell system, comprise human cell's system, as HT1080, COS, CHO, BHK, 293,3T3 wherein contains the recombinant expression construct body, contains in the described construct to derive from the genomic promoter of mammalian cell, as metallothionein promoter, or derive from the promoter of mammalian virus, and as gland virus stage starting, vaccinia virus 7.5K promoter.
In addition, the host cell strain of selection can be regulated and control the expression of insertion sequence, or modifies and the processed gene product in the peculiar mode of expectation.The such modification and the processing of protein product may be very important for proteic function.Skilled in the art will recognize that different host cells has the mechanism of specific albumen and gene outcome translation post-treatment and modification.Can select suitable cell line or host system to guarantee that the foreign protein of expressing is carried out correct processing and modification.In this, have the correct processing that is used for the primary transcription product, the eukaryotic host cell of the glycosylation of gene outcome and the cell mechanism of phosphorylation can use.Such mammalian host cell comprises human host cell, and it includes but not limited to HT1080, CHO, VERO, BHK, HeLa, COS, MDCK, 293,3T3, and WI38.
For generation recombiant protein long-term, high yield, preferred stable expression system.For example, can the through engineering approaches stably express cell line of gene outcome of organization protection's cytokine correlation molecule of reorganization.Do not use the expression vector that contains the virus replication starting point, host cell can be used by suitable expression regulation element and control, as promoter, and enhancer, sequence, transcription terminator, the DNA in polyadenylic acid site etc., and selected marker transforms.Introduce after the foreign DNA, the cell of through engineering approaches can be in nutritious culture medium growth 1 to 2 day, transfer in the selective medium then.Selected marker in the recombiant plasmid makes it to have the selection resistance, and allows in the chromosome that is incorporated into cell that plasmid is stable, and grows into point (foci), and it can be amplified in the cell line by the clone subsequently.This method can advantageously be used to the cell line that through engineering approaches is expressed the gene outcome of EPO mutain correlation molecule.Such through engineering approaches cell line may be useful especially screening and estimating in those active chemical compounds of endogenous that influence EPO correlation molecule gene outcome.
Perhaps, to such an extent as in cell line or the microorganism expression characteristic of endogenous EPO mutating protein gene can be modified by in stable cell lines or cloned microorganism genome, inserting on the gene that the regulation and control unit that inserts allogeneic dna sequence DNA regulation and control unit effectively be connected the endogenous erythropoietin mutain.For example, endogenous EPO mutating protein gene its normally " Transcriptional Silencing ", promptly do not express the EPO gene usually, or expression levels is very low in cell line, it can be by inserting the regulating and controlling sequence that can promote gene product expression in described cell line or microorganism.Perhaps, can come the endogenous EPO gene of activated transcription silence by inserting the mixing regulation and control unit that can in the various kinds of cell type, work.
Allos regulation and control unit can be inserted in the stable cell line or cloned microorganism, enables effectively to be connected on the proteic gene of endogenous erythropoietin the technology of use, as the targeting homologous recombination, know for a person skilled in the art, and at French Patent (FRP) numbers 2646438, U.S. Patent number 4,215,051 and 5,578,461, with among international publication number WO93/09222 and the WO91/06667 description is arranged, its disclosed content is this whole introducing.
Pharmaceutical composition
The present invention also relates to comprise the pharmaceutical composition of long-acting EPO of the present invention.Because long-acting EPOs of the present invention is favourable has promoting erythrocyte and generate activity and organization protection's ability and endocytosis turn-over capacity, expect that they can treat the anemia and the relevant disease of the individuality of danger that various tissue injurys are also arranged simultaneously such as apoplexy and impatient infraction.In addition, expecting that long-acting EPOs of the present invention can be used for treating has also experienced psychiatric system simultaneously and has worsened anemia and relevant disease as the individuality of diseases such as alzheimer disease, parkinson.Further, long-acting EPOs of the present invention can be used for treating the anemia of the individuality of the patient's condition that suffered to be produced by the normal aging process (as the equilibrium problem that causes falling, dampen etc. easily).Also have, the present invention relates to the application of long-acting EPOs of the present invention as other molecular vehicle, wherein said molecule is difficult to penetrate the barrier of the blood capillary with EPO receptor.
For example, aforementioned any long-acting EPOs can be contained in the pharmaceutical composition of the present invention.In addition, various forms of EPO analog can be contained in the pharmaceutical composition of the present invention, and mix with at least a tissue protective cytokine, and the latter will detailed in the back discussion.
Pharmaceutical composition of the present invention contains the modified EPO that treats effective dose, and the form that is preferably with purification exists.Preparation should adapt with mode of administration.Make the form of being convenient to use.In other words, pharmaceutical composition of the present invention comprises a certain amount of modified EPO of the present invention, thereby makes that the patient's condition of its targeting can be treated under the situation of having used correct dosage and strategy.And the back will go through, and drug regimen should be used with non-toxic dosage.
Pharmaceutical composition of the present invention can contain the long-acting EPO chemical compound for the treatment of effective dose and the pharmaceutically acceptable carrier of appropriate amount, so as to provide can be correctly to the form of patient's administration.In a specific embodiment, term " pharmaceutically acceptable " is meant that the administrative organization through federation or state government ratifies or is listed on American Pharmacopeia or other the foreign pharmacopeia that is used for mammal, especially people of generally acknowledging.Term " carrier " is meant the diluent of together using with therapeutic agent, adjuvant, excipient or drug carrier.Such pharmaceutical carrier can be a sterile liquid, and the saline solution of Ru Shui and oil comprises oil, animal, plant or synthetic source, as Oleum Arachidis hypogaeae semen, soybean oil, mineral oil, Semen Sesami wet goods.When the pharmaceutical composition intravenous administration, saline solution is preferred carrier.Saline solution and aqueous glucose and glycerite also can be used as liquid-carrier, particularly as injection solution.Suitable drug excipient comprises starch, glucose, lactose, sucrose, gel, Fructus Hordei Germinatus, rice, flour, Chalk, silica gel, sodium stearate, glycerol monostearate, Talcum, sodium chloride, dry powder skim milk, glycerol, propylene glycol, water, ethanol etc.
Pharmaceutical composition of the present invention also can contain a spot of lubricant or emulsifying agent, or the pH buffer agent.These compositionss can be made solution, suspension, emulsion, tablet, pill, capsule, powder, forms such as slow releasing agent.
Compositions of the present invention can be made the form of neutrality or salt.Pharmaceutically acceptable salt comprises the salt that those and free amino group form, as derives from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, the salt of tartaric acid etc., and the salt that generates of those and free carboxyl group, derive from sodium as those, potassium, ammonium, calcium, hydrated ferric oxide., isopropylamine, triethylamine, 2-ethamine ethanol, histidine, the salt of procaine etc.
The compositions that contains long-acting EPO
As previously mentioned, any long-acting EPO of the present invention can be used in the pharmaceutical composition.In-individual embodiment, the long-acting EPO that is produced by the vicinal hydroxyl groups oxidation is included in the pharmaceutical composition of the present invention.In another embodiment, pharmaceutical composition of the present invention comprises at least a long-acting EPO, and it is to replace sialic acid residues with more stable residue to produce.In another embodiment, be included in long-acting EPO in the pharmaceutical composition and be the negative charge that increases EPO by sulphation and/or phosphorylation and obtain.In a further embodiment, the long-acting EPO that is included in the pharmaceutical composition of the present invention produces as PEG chain termination sugar chain by the more complicated molecule of usefulness.
In addition, the present invention relates to the purposes of mixture in pharmaceutical composition of the present invention of the long-acting EPOs that produces with any method of the present invention.For example, pharmaceutical composition of the present invention can comprise that the more stable residue of at least a usefulness replaces sialic acid and the long-acting EPO that forms and at least aly increase the long-acting EPO that the negative charge of EPO forms by sulphation and/or phosphorylation.
Delivery system
As previously mentioned, long-acting EPOs of the present invention advantageously can cross over the barrier of the blood capillary with EPO receptor.Therefore, of the present invention is delivery system in another embodiment, and it uses the carrier of long-acting EPOs of the present invention as some molecules, and these molecules enter body, and to contain the barrier penetration capacity of target area of EPO receptor lower.Such delivery system has preferentially advantageously provided a kind of conveyer method of the complete barrier of leap of new safety.
In one embodiment, movement system comprises long-acting EPOs of the present invention and the lower molecule of at least a brain penetration capacity, and a kind of conveyer method that passes through complete blood brain barrier of new safety is provided.That is, long-acting EPOs of the present invention allows the low molecule of those brain penetration capacitys play the part of the role of molecule " Trojan Horse ", thereby has strengthened the ability of brain picked-up micromolecule or macromolecular diagnostic agent or treatment molecule.
In fact, derive from the specific region that therapeutic agent need be sent to brain in the major issue of treatment in the human brain tumor, with its be distributed in brain tumor wherein and targeting in brain tumor.Effective molecule in may and treating in diagnosis in other cases, or can not pass the blood brain barrier (BBB) of brain next-door neighbour tumor with q.s, or can not pass bloodtumorbarrier (BTB) with q.s.Therefore, need new transmission strategy, it is specially at brain and can pass vascular system.For example, be used to the antibody molecule diagnosing or treat, because their size and can not pass BTB with q.s.Therefore, long-acting EPOs of the present invention can be used as carrier, makes these molecules pass through BBB or BTB.One can be antisense oligonucleotide with the example of the molecule of long-acting EPOs of the present invention-rise to use, and it typically is used to suppress the oncogene signal or the expression in vivo of brain gene is carried out imaging.In addition, long-acting EPOs of the present invention can be included in (preparation virus or non-virus) in the range gene therapeutic agent, and these therapeutic agents are too big usually so that do not having can not to pass BTB under the auxiliary situation.
Further, long-acting EPOs of the present invention can be as the carrier mediated transporter of various chemotherapeutics.Because pharmaceutically active outflow (efflux) transporter who is expressed on BBB and the BTB flow back into chemotherapeutics in the blood from brain actively, these reagent may be suppressed or stop in the distribution of brain.Partly owing to these reasons, it is inoperative for the treatment of brain tumor that the chemotherapy molecule that is positioned at the outer cancer of central nervous system (CNS) is treated in most of traditional being used for.Therefore, using the carrier of long-acting EPO of the present invention as these chemotherapeutics, is useful reagent being sent to aspect the brain not only, is used for the treatment of but also can be used for that reagent is remained on brain.In another embodiment, this long-acting EPO can use with medicine, and described medicine suppresses the active transporter of outflow, further to guarantee to absorb these are back to blood usually from brain chemotherapeutics.
In addition, the present invention also comprises the application of the EPO of modified as the carrier of some molecules, and other zone that these molecules are expressed the EPO receptor in vivo has low penetrance.The nonrestrictive example of this type of cell comprises retina, muscle, heart, lung, liver, kidney, small intestinal, adrenal cortex, adrenal medulla, endothelium blood capillary, testis, ovary, pancreas, bone, skin and endometrial cell.Especially, responsive cell includes but not limited to, neuronal cell; Retina cell: photoreceptor cells (rod cell and cone cell); Nerve node, bipolar cell, horizontal cell, amacrine cell and M ü eller cell; Muscle cell; Heart cell: myocardial cell, the starting point of fighting cell, sinus node cells, nodal cell, and interlaced structure (atrium knot and his bundle) cell; Pneumonocyte; Liver cell: hepatocyte, star-like cell and Kupffer cell; Kidney cell: glomerule, renal epithelial cell and tubulose Interstitial cell; Small intestine cells: goblet cell, enteraden (gland nest) and enteroendocrine cell; Adrenal cortical cell: messangial cell, pencil cell, and skein cell; Adrenal medullary cell: pheochromocyte; Blood capillary cell: adventitial cell; Testicular cell: Leydig cell, Sertoli cell and spermatid and precursor thereof; Gonad cell: Graffian capsule and primordial follicle cell; Pancreatic cell: Langerhans islet cells, A cells, beta cell, γ-cell and F-cell; Osteocyte: osteoprogenitor cell, osteoclast, and osteoblast; Skin Cell; Endometrial cell: endometrial stromal and endo cell; With the stem cell and the endotheliocyte that are present in the above-mentioned organ.
EPO analog and organization protection's composition of cellular factors have been mixed
As previously mentioned, the pharmaceutical composition under the present invention can comprise the EPO analog (showing the serum half-life of prolongation but disappearance organization protection activity) of the sugar chain that sugar chain that N-with at least one increase connects and/or at least one O-connect and mixing of at least a organization protection cytokine.For example, have the EPO analog of the sugar chain that the N-of at least 2 increases connects, wherein a sugar chain is positioned on O ' the Brien peptide, with organization protection's cytokine, can form compositions of the present invention.In another embodiment; pharmaceutical composition of the present invention can comprise at least a organization protection cytokine and at least a EPO that contains extra sugar chain; wherein by consider this analog in three dimensions, known described sugar chain has been blocked O ' Brien peptide.In another embodiment; pharmaceutical composition of the present invention comprises at least-kind of organization protection's cytokine and at least-kind of EPO analog, and described EPO analog is owing to having adopted the method that increases extra sugar chain in protein to cause it not have organization protection's function.
EPO analog expection with the glycosylation site of reorientating can be used in the pharmaceutical composition of the present invention.Be not subject to any special theory; it is believed that organization protection's ability of EPO analog will strengthen than the EPO analog that has glycosylation site at 38 so if reset other site that is positioned at outside the EPO analog 30-47 amino acids segment at the glycosylation site of the 38 natural generations in amino acids place.Therefore, pharmaceutical composition of the present invention can comprise glycosylation site reorientation with 38 amino acids places at the EPO in other site of molecule analog.The glycosylation site of reorientation may occur in 51,57,69,88, on 89,136 or 138, as pointing out among the PCT publication number WO01/81405.In one embodiment, O ' Brien peptide contains 1 or sugar chain still less.In another embodiment, O ' Brien peptide contains 2 or more sugar chain.
Preferred those disappearances of suitable organization protection's cytokine that are used for this respect of the present invention are to the effect of bone marrow but keep the cytokine of endogenous tissue protective effect, but any cytokine with organization protection's ability also can be used for the present invention.For example, suitable organization protection's cytokine comprises by guanidine and turns usefulness into, and amidine turns usefulness into; carbamylization (carbamoylation) effect, bitterness acidization, acylation (acetylation or succinylation); Nitrification, or its compound mode is carried out chemical modification and the EPOs that generates.In addition, at least one arginine, lysine, tyrosine, tryptophan, the adorned EPO molecule expection of cysteine residues or oh group also can be used as organization protection's cytokine of this aspect according to the present invention.
Also have; being used for other organization protection's cytokine of the present invention in addition can be by limited Proteolytic enzyme, remove amino and/or introduce sudden change with Protocols in Molecular Biology and replace arginine, lysine, tyrosine, tryptophan or cysteine residues and obtain; described Protocols in Molecular Biology is at Satake etc.; 1990; open among the Biochim.Biophs.Acta 1038:125-9, this whole introducing.For example, suitable organization protection's cytokine comprises the EPO of at least one or a plurality of sudden changes, and described EPO is at C7S, R10I, V11S, L12A, E13A; R14A, R14B, R14E, R14Q, Y15A, Y15F, Y15I; K20A, K20E, E21A, C29S, C29Y, C33S, C33Y; P42N, T44I, K45A, K45D, V46A, N47A, F48A; F48I, Y49A, Y49S, W51F, W51N, Q59N, E62T; L67S, L70A, D96R, S100R, S100E, S100A, S100T; G101A, G101I, L102A, R103A, S104A, S104I, L105A; T106A, T106I, T107A, T107L, L108K, L108A, S126A; F142I, R143A, S146A, N147K, N147A, F148Y, L149A; R150A, G151A, K152A, L153A, L155A, C160S, 16A; C7A, B13A, N24K, A30N, H32T, N38K; N83K, P42A, D43A, K52A, K97A, K116A; T132A, 1133A, T134A, K140A, P148A, R150B; G151A, K152W, K154A, G158A, C161A, and/or point mutation is arranged on the R162A.The example of the top modification of mentioning is described in common unsettled U.S. Patent Publication No. 2003/0104998,2002/00861816 and 2003/0072737, is incorporated herein by reference in full at this.In mutain nomenclature used herein, reformed aminoacid is described in the following way: at first be the single-letter code of natural amino acid, what closely follow is its position in the EPO molecule, is subsequently to replace amino acid whose single-letter code.For example, S100E is meant in human EPO molecule, and the serine at 100 places is replaced with glutamic acid.
In another embodiment, organization protection's cytokine can comprise one or more above-mentioned point mutation, and prerequisite is that described point mutation does not comprise I6A, C7A, K20A; P42A, D43A, K45D, K45A, F48A; Y49A, K52A, K49A, S100B, R103A; K116A, T132A, I133A, K140A, N147K; N147A, R150A, R150E, G151A; K152A, K154A, G158A, C161A or R162A.
In another embodiment, organization protection's cytokine can comprise the combination of point mutation, as K45D/S100E; A30N/H32T, K45D/R150E, R103E/L108S; K140A/K52A, K140A/K52A/K45A, K97A/K152A; K97A/K152A/K45A; K97A/K152A/K45A/K52A, K97A/K152A/K45A/K52A/K140A, K97A/K152AA/K45A/K52A/K140A/K154A; N24K/N38K/N83K, and N24K/Y15A.In a further embodiment, organization protection's cytokine does not comprise arbitrary combinations thereof.In another embodiment, organization protection's cytokine can comprise any of above-mentioned point mutation, and prerequisite is that described point mutation does not comprise any following sudden change combination: N24K/N38K/N83K and/or A30N/H32T.
Some modification or modification combination can influence the motility of mutain and its receptor such as the EPO receptor or second receptor binding capacity.The example of this type of modification or modification combination includes but not limited to K152W, R14A/Y15A, I6A, C7A, D43A, P42A, F48A, Y49A, T132A, I133A, T134A, N147A, P148A, R150A, G151A, G158A, C161A, and R162A.The deleterious relevant sudden change of human growth hormone is known to one skilled in the art.Therefore, in one embodiment, organization protection's cytokine does not comprise one or more modifications of those motilities that may influence mutain and its receptor binding capacity or modifies combination.The further discussion of such organization protection's cytokine is included in co-pending patent application number is---; attorney docket is 10165-022-999; the applying date is on July 1st, 2003; name is called the U.S. Patent application of " Recombinant Tissue Protective Cytokines andEncoding Nucleic Acid Thereof for Protection; Restoration; and Enhancement ofResponsive Cells; Tissues; and Organs ", and its whole disclosed contents are introduced here as a reference.
At last, any cytokine superfamily with organization protection's ability can use, as long as its promoting erythrocyte that does not influence long-acting EPO generates activity or serum half-life.Example includes but not limited to, interleukin-3 (IL-3), interleukin-5 (IL-5), granulocyte-macrophage colony stimutaing factor (GMCSF), pigment epidermal derived factors (PEDF), VEGF (VEGF).
In one aspect of the method for the present invention, the EPO analog (having the serum half-life of prolongation but inorganizable protection activity) that pharmaceutical composition of the present invention comprises the sugar chain that the O-of sugar chain that N-with at least one increase connects and/or at least one increase connects has the micromolecular of organization protection's function and mixes with at least a.Suitable micromolecule includes but not limited to, steroid (for example lazaroids and glucocorticoid), antioxidant (coenzyme Q10 for example, alpha lipoic acid, and NADH), anti-catabolic enzyme (anticatabolic enzyme) (glutathion peroxidase for example, superoxide dismutase, catalase, synthesis catalytic cleanser, and simulator), indole derivatives (for example indole amine, carbazole, and carboline), the nitric acid nertralizer, adenosine/adenosine agonists, phytochemistry agent (flavanoid), herb extracts (Semen Ginkgo and Rhizoma Curcumae Longae), vitamin (vitamin A, E and C), oxidase electron acceptor inhibitor (for example xanthene oxidase electron acceptor inhibitor), mineral (copper for example, zinc, and magnesium), NSAIDS (aspirin for example, naproxen, and ibuprofen) and their combination.In addition, pharmaceutical composition of the present invention contains EPO analog, organization protection's cytokine and has the active micromolecule of organization protection.
Be present in organization protection's cytokine and/or the preferred enough maintenances of micromolecular amount in the pharmaceutical composition of the present invention or surpass the activity that in neural or other responsive cell system, produces by endogenous EPO.In one embodiment, organization protection's cytokine and/or micromolecular amount are enough to strengthen individual organization protection's ability, and this is by protection, keep, or the viability of enhancing promoting erythrocyte generation responsive cell and function realization.For example, the pharmaceutical composition described in this aspect of the present invention preferably contains organization protection's cytokine of effective, non-toxicity amount, for example about 1ng or more.In one embodiment, the amount that is present in the organization protection's cytokine in the pharmaceutical composition of the present invention approximately is 5mg or still less.In another embodiment, the amount that is present in organization protection's cytokine in the pharmaceutical composition of the present invention approximately is that 500ng is to 5mg.In another embodiment, pharmaceutical composition contains the 1 μ g that has an appointment to organization protection's cytokine of 5mg, preferably contains the 500 μ g that have an appointment to 5mg.In another embodiment, the amount that is present in the organization protection's cytokine in the pharmaceutical composition of the present invention is bigger, is about 1mg to 5mg.Those skilled in the art knows, and the dosage of the pharmaceutical composition of using to patient depends on following factor, but is not limited to, patient's health and the frequency of taking medicine.Will detailed in the back discussion about the dosage of taking medicine.
Treatment and medication
Above-mentioned long-acting EPOs and the pharmaceutical composition that contains long-acting EPOs, expection can be used for anemia, relates to the human diseases of anemia or Anemia or cause the disease of anemia or the therapeutic or the preventative processing of Therapeutic Method.Usually, under the situation that does not endanger patient's ability of rehabilitation from other tissue injury, the administration that long-acting EPOs of the present invention is can tolerance frequency lower or use still less the erythropoietin of dosage to treat above-mentioned disease.
The present invention expects that this long-acting EPOs can be used for whole body or chronic using, and acute treatment and/or discontinuity are used.What in one embodiment, pharmaceutical composition of the present invention can be chronic uses with protection or intensifier target cell, tissue or organ.In another embodiment, what pharmaceutical composition of the present invention can be acute uses, i.e. single therapy during damaging.In another embodiment, pharmaceutical composition of the present invention can the circulation mode be used.
Using of said composition can be parenteral, for example, by intravenous injection, lumbar injection, intra-arterial, intramuscular, Intradermal, or subcutaneous administration; By sucking; Stride mucosa, for example oral, nasal cavity, rectum, intravaginal, Sublingual, tela submucosa, and percutaneous; Or its compound mode is used.Preferably, the method for application of pharmaceutical composition of the present invention is a parenteral.The dosage of such method of application is about 0.01pg to about 5mg, and preferably about 1pg is to about 5mg.Other-individual embodiment in, dosage is about 500pg to about 5mg.Again-individual embodiment in, dosage is about 500ng to about 5mg.In a further embodiment, dosage is about 1 μ g to about 5mg.For example, dosage can arrive about 5mg for about 500 μ g.In another embodiment, dosage can be that about 1mg is to about 5mg.
The pharmaceutical composition of the present invention that is applicable to the parenteral administration contains aqueous or non-aqueous aseptic injectable solution or suspension, it can comprise antioxidant, buffer agent, bacterial inhibitor and solute, described solute make that said composition is basic oozes with the blood that is tried body etc.At this respect of the present invention, this pharmaceutical composition also can contain water, ethanol, polyol, glycerol, vegetable oil, and composition thereof.The pharmaceutical composition that is applicable to the parenteral administration may reside in the container of unit dose or multiple dose, for example, the ampoule and the bottle of sealing, and can lyophilizing (lyophilization) preserve, only need in this case before will using, to add sterile liquid carrier, for example the aseptic salt solution of injection at once.Can be mixed with sterilized powder with injection and suspension, granule and tablet temporarily.In one embodiment, contain the automatic injector of long-acting EPOs injection of the present invention, can be used under the situation in ambulance, emergency room and battlefield and promptly use.
In one embodiment, compositions of the present invention can be made the pharmaceutical composition that is fit to people's intravenous administration according to the step of routine.For example, this pharmaceutical composition can make sterile isotonic aqueous buffer solution.If desired, this pharmaceutical composition also can contain solubilizing agent and/or local anesthetic such as lignocaine, is used to alleviate the pain that the injection site place produces.Described composition can separately or mix with unit dosage form and provide, and as being present in the container of sealing with lyophilized powder or the form of not having an aqueous concentrate, as indicates the ampoule or the sachet of active component consumption.When using pharmaceutical composition of the present invention by the form of inculcating, the infusion bottle that contains the water of aseptic pharmaceutical grade or saline solution can be used for dispersion medicine.And, when using pharmaceutical composition of the present invention, before using, can mix each composition with the ampoule that contains aseptic salt solution with the form of injection.
Be applicable to that oral pharmaceutical composition can provide with following form: capsule or tablet; Powder or granule; Solution, syrup or suspension (moisture or water-free liquid); Edible foam (foams or whips); Emulsifying agent; Or its combination.Oral formulations can comprise the active component of percentage by weight about 10% to about 95%.In one embodiment, the percentage by weight of active component is about 20% to about 80% in the oral formulations.In another embodiment, the percentage by weight of active component is about 25% to about 75% in the oral formulations.
Tablet or hard gelatine capsule can contain lactose, starch and derivant thereof, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate, stearic acid and salt thereof.Soft gelatin capsule contains vegetable oil, wax, fat, semisolid, liquid polyol or its mixture.Solution and syrup contain water, polyhydric alcohol, sugar or its mixture.
In addition, be used for oral activating agent and can carry out coating or mixing with postponing activating agent material of disintegrate and/or absorption in gastrointestinal tract.For example, mix with activating agent with glycerol monostearate, two stearic acid glycerol or its combination or carry out coating.Therefore, the lasting release of activating agent can be kept a lot of hours, if desired, can stop the degraded of activating agent at gastric.Oral pharmaceutical composition can be prepared according to special pH or enzyme condition, to help at gastrointestinal specific part release bioactive agent.
The pharmaceutical composition that is suitable for percutaneous dosing can provide in the mode of discontinuous (discrete patch), and it can keep closely contacting the long period with experimenter's epidermis.In addition, the pharmaceutical composition that is suitable for topical can provide with following form: ointment, ointment, suspending agent, lotion, powder, solution, paste, gel, spray, aerosol, oil preparation, eye drop, lozenge, pastille and collutory and combination thereof.When the part is administered for skin, oral cavity, eyes or other outside organization, preferably use local ointment or ointment.And, in the time of in being present in ointment, active component, promptly long-acting EPO can be with wax or easily use with the miscible ointment substrate of water.Perhaps, use oil-in-water or water in oil substrate that active component is prepared as ointment.When the eye drop form was adopted in local administration, pharmaceutical composition of the present invention preferably contained and is dissolved or suspended in for example activating agent in the aqueous solvent of suitable carriers.
The pharmaceutical composition that is applicable to nasal cavity and pulmonary administration can contain solid carrier, as powder (preferred particle size is in about 20 to about 500 microns).Powder can be used by the mode that sucks nasal cavity from the close container that contains powder of nose fast.In another embodiment, the pharmaceutical composition that is used for nasal administration of the present invention contains liquid-carrier, as nasal spray or nasal drop.Preferably directly use pharmaceutical composition of the present invention at nasal cavity.
Directly pulmonary suck can be by extending into throat oral intubation and dark suction of device of other particular design finish, described device includes but not limited to, pressure aerosolizer, aerosol apparatus or sprinkler, it can be through specialized designs to provide the active component of scheduled volume.Preferably, pharmaceutical composition is used by the mode that directly sucks throat deeply.
The pharmaceutical composition that is suitable for rectally can provide with the form of suppository or enema.In one embodiment, to contain percentage by weight be about active component of 0.5% to 10% to suppository of the present invention.In another embodiment, to contain percentage by weight be about 1% to about 8% active component to this suppository.In another embodiment, the active component percentage by weight that contains of this suppository is about 2% to about 6%.In this one side of the present invention, pharmaceutical composition of the present invention can contain binding agent commonly used and carrier, as triglycerides.
The pharmaceutical composition that is suitable for vagina administration can provide with following form: vaginal suppository, tampon agent, ointment, gel, paste, foam or spray.
Pharmaceutical composition of the present invention also can be used in the mode of using infusion, organ injection or topical.In these embodiments, this pharmaceutical composition preferably contains the 0.01pM that has an appointment to about 30pM, and preferably about 15pM is to the of the present invention long-acting EPO of about 30nM.In one embodiment, infusion solution is University of Wisconsin (UW) solution (Morie osmolarity with about 7.4 to about 7.5 pH and about 320mOSm/l), and it contains the EPO that the 1U/ml that has an appointment arrives about 25U/ml; 5% hetastarch (preferably have molecular weight approximately from 200,000 to about 300,000 and be substantially free of ethylene glycol, ethylene chlorhydrin, sodium chloride and acetone), 25mM KH 2PO 4, the 3mM glutathion; The 5mM adenosine; The 10mM glucose; 10mM HEPES buffer; The 5mM magnesium gluconate; 1.5mM CaCl 2The 105mM gluconic acid sodium salt; The penicillin of 200,000 units; The insulin of 40 units; The dexamethasone of 16mg; Phenol red with 12mg.UW solution is at U.S. Patent number 4,798, detailed description arranged in 824, is incorporated herein by reference in full at this.In another embodiment, UW solution can contain the 0.01pg/ml that has an appointment to about 400ng/ml, and preferred 40ng/ml is to the recombinant tissue protection cytokine of about 300ng/ml.
Regional local application pharmaceutical composition of the present invention to the needs treatment may be a desirable.Such administering mode can be realized in the following way: inculcate the part in operation; Topical application, as use with wound dressing the back of performing the operation; By injection; Pass through conduit; Pass through suppository; Or the mode by implanting, described implantation mode can comprise film with holes, atresia or colloidal materials, as pellosil, or fiber.
In addition, foregoing mode about applied dermally, long-acting EPO of the present invention can be carried by control delivery.For example, use intravenous infusion, implantable osmotic pump, use this peptide through leather diaphragm, liposome or other mode.In-individual embodiment, can use pump, as at Saudek etc., 1989, describe among the N.Engl.J.Med.321:574.Other-individual embodiment in, can use carrier to carry this chemical compound, particularly liposome, as at international publication number WO91/04014 and U.S. Patent number 4,704, as described in 355, it is wholely introduced reference at this.In another embodiment, polymeric material can be used to prepare control delivery, as those at Howard etc., 1989, the material of describing among the J.Neurosurg.71:105.
Such control delivery can place the place near therapeutic goal, i.e. therefore target cell, tissue or organ only need the part of whole-body dose.Referring to, for example, Goodson, Medical Applications ofContolled Release, vol.2, pp.115-138,1984.Can be used for other control delivery of the present invention at Langer, Science 249:1527-1533 has description in 1990 the summary.
Dosage
Based on those of ordinary skills' common practise, those of ordinary skills can select to be applicable to the effectively preferred and non-toxic dosage of above-mentioned application process.The example of these factors comprises the specific form of long-acting EPO; The pharmacokinetic parameter of this EPO is as (having offered the technical staff) such as bioavailability, metabolism, half-life; Situation to be treated or disease are arranged; Obtained curative effect in common individuality; Patient's body weight; Medication; Administration frequency, promptly chronic, acute, the administration that is interrupted; Concomitant drugs; The factor of the effectiveness of the medicament of using with other known influence.Like this, accurate dose should be determined according to professional's the judgement and the situation of given patient.
For example, Physicians Desk Reference (PDR) has shown, according to the patient population that uses the EPO treatment, considers that different hematocrit levels is to avoid toxicity.Physicians Desk Reference, 54thEd., 519-525 and 2125-2131 (2000).In fact, to suffering from the patient of CRF, the EPO dosage that PDR recommends to use is to reach non-toxicity targeted blood cells specific volume 30% to 36%.On the contrary, for the cancer patient that carries out chemotherapy, the PDR instruction is adjusted to different hematocrit levels with dosage, if just hematocrit levels surpasses 40%.PDR shows, the hematocrit of professional's monitor patients during using the EPO treatment, and if patient's hematocrit meet or exceed the upper limit of preset range, then adjust dosage and/or stop treatment to avoid toxicity.Thereby skilled professional according to instruction of the present invention, should be able to make the application dosage of EPO be enough to reach the effect of treatment and avoided any toxicity complication.
In-individual embodiment, long-acting EPO of the present invention is using of each about 0.1 μ g/kg body weight chronic or whole body to about 100 μ g/kg body weight with dosage.For example, when the cancer patient of chemotherapy is accepted in treatment, use once weekly to the dosage of about 5 μ g/kg body weight with about 1 μ g/kg body weight.Other-individual embodiment in, the dosage of this long-acting EPO is that each about 5 μ g/kg body weight are to about 50 μ g/kg body weight.In another embodiment, the dosage of this long-acting EPO is that about 10 μ g/kg body weight are to about 30 μ g/kg body weight.In a further embodiment, the amount of application of this long-acting EPO is about 1 μ g/kg body weight or still less.For example, when being administered once treatment CRF patient's anemia weekly, about 0.45 μ g/kg body weight may be effective to the long-acting EPO of about 0.75 μ g/kg body weight.
Effectively dosage preferably is enough to obtain greater than about 10, the serum levels of the long-acting EPO of 000mU/ml (80ng/ml).In one embodiment, it is about 15 that effective dose can obtain, the serum levels of 000mU/ml (120ng/ml) or higher long-acting EPO.In another embodiment, it is about 20 that effective dose can obtain, the serum levels of the long-acting EPO of 000mU/ml (160ng/ml).Be preferably in when using back 1,2,3,4,5,6,7,8,9,10 hour or its combination and detect.Those of ordinary skill in the art thinks when needing, dosage that can repetitive administration.For example, as long as clinical needs, every day can repeat administration, or through repeat administration after the suitable intermittence, as per 1 to 12 week, preferred per 1 to 3 week.
Because long-acting EPOs of the present invention has the half-life of prolongation, so their activity in vivo also can increase.For example, when mammiferous sick body carries out the general chemotherapeutic treatment of cancer, comprise radiotherapy, during treating, use long-acting EPO pharmaceutical composition of the present invention and can reduce the relevant symptom of anemia that frequency that it is used and dosage all are less than existing recombinant epo compositions.
And; as previously mentioned; when pharmaceutical composition of the present invention contained the mixed form of long-acting EPO of the present invention or EPO analog and organization protection's cytokine, said composition can be used for the treatment of anemia and the relevant disease that also has the patient of tissue injury's danger simultaneously.For example, suffering from anemia also is the high-risk patient of heart disease simultaneously, can replace existing EPO analog to treat with compositions of the present invention, to avoid increasing because of treatment the risk of infringement.
The treatment test kit
The present invention also provides simultaneously a kind of pharmaceutical agent bag or box, its contain useful pharmaceutical composition of the present invention-kind or multiple composition one or more containers of filling.In one embodiment, the long-acting EPO of effective dose and pharmaceutically acceptable carrier can be packaged in single dose vial or other container.
When pharmaceutical composition of the present invention is used for the parenteral administration, for example, can be under freeze dried condition the preservation said composition.Therefore, test kit can comprise freeze-dried composition, sterile liquid carrier and the syringe that is used to inject.In one embodiment, this test kit comprises and contains the ampoule that enough is used for the freeze-dried material for the treatment of several times, so that the carrier that user can weigh up the material of specified quantitative and add specified quantitative is used for each treatment.In another embodiment, test kit comprises a plurality of ampoules, the freeze-dried material that all contains specified quantitative in each ampoule, and a plurality of containers, the carrier that all contains specified quantitative in each container, so that user only needs the contents mixed of an ampoule and a carrier container is used for each treatment, and need not measure or weighing.In another embodiment, this test kit comprises automatic injector, and it contains the injection solution of EPO of the present invention.In another embodiment, this test kit comprises that at least one contains the ampoule of freeze-dried composition, at least one contains the container of carrier solution, container that at least one contains local anesthetic and at least-individual syringe (or analog).Ampoule and container preferably seal.
When using pharmaceutical composition of the present invention in the mode of inculcating, this test kit preferably includes at least one ampoule that contains this pharmaceutical composition and at least one contains the water of aseptic pharmaceutical grade or the infusion bottle of saline solution.
Test kit of the present invention also can comprise at least one oral intubation or be applicable to special device such as pressure aerosolizer, aerosol apparatus or the sprinkler that direct pulmonary sucks.In this one side of the present invention, this test kit can comprise the device that is used for the suction of direct pulmonary, and it contains this pharmaceutical composition, and maybe this device contains the water of long-acting EPO of the present invention or the ampoule of oil solution with at least one.
When long-acting EPO pharmaceutical composition of the present invention was suitable for oral, percutaneous, rectum, vagina or nasal-cavity administration, this test kit preferably included at least one ampoule that contains active component and at least a administration adminicle.The example of administration adminicle includes but not limited to, weighing scoop (being used for oral), aseptic cleaning pad (being used for percutaneous dosing) and nasal cavity getter (being used for nasal-cavity administration).Such test kit can contain the long-acting EPO (acute treatment) of single dose or the long-acting EPO (chronic treatment) of multiple dose.
In addition, this test kit can be equipped with the solution of one or more types.For example, long-acting EPO pharmaceutical composition of the present invention can be prepared in albumin solution and the polysorbate ester solution.If test kit contains the polysorbate ester solution, the printed words of " not containing albumin " optimize on the label of present container and on the main panel of test kit.
In addition, this test kit also comprises the statement of form of government organs' defined of production, use or the sale of management medicine or biological product, and this statement has reflected that said mechanism ratified to be used for production, use or the sale of human body.
EPO detects test
The present invention also relates to be used for the promoting erythrocyte generative capacity of the EPO analog determining long-acting EPOs of the present invention and be used in several drugs compositions of the present invention and the detection method of organization protection's ability.For example, the promoting erythrocyte of long-acting EPOs generates activity and can use TF-1 to analyze to detect, this will be in embodiment 2 more detailed description.Organization protection's activity of EPO chemical compound can be used external and the body inner analysis detects, and this will discuss in the back in more detail.In addition, expection the present invention also comprises being used for determining whether specific EPO chemical compound has organization protection's activity, and can be used for determining that this EPO chemical compound is whether as the detection method of the antagonist of endogenous EPO.
Detection method of the present invention is preferably designed to uses minimum EPO mixture to finish at short notice.Also have, the detection method that provides here is unrestricted, because those of ordinary skill in the art can understand other the active and active method of organization protection of promoting erythrocyte generation that is used to detect the EPO chemical compound.
Promoting erythrocyte generates active the detection
The promoting erythrocyte formation characteristic of specific EPO chemical compound is promptly controlled the ability of hematocrit levels, can determine by multiple detection method.In-individual embodiment, TF1 cell line can be used for determining whether the EPO chemical compound has promoting erythrocyte and generate active.This cell can be precipitated, flushing and to be resuspended in concentration be 1 milliliter 10 5In the culture fluid of individual cell, be added with certain density recombinant epo and interested EPO chemical compound.Indivedual cultures can be kept 24 hours, used formazan reactant (CellTiter simultaneously; Promega, Madison WI) measures the number of cell.
The activity of interested EPO chemical compound can be observed its influence to hemoglobin concentration and carry out interior evaluating at first by using female BALA/c mice.Give EPO, interested EPO chemical compound or the isopyknic carrier of animal subcutaneous administration 500U/kg-bw, time administration on every Wendesdays is three weeks (being enough to observe the interval of erythropoietic response) altogether.If the EPO chemical compound has improved the concentration of the serum hemoglobin of mice, just determine that it has promoting erythrocyte and generates active.
Can use the TF1 erythroleukemia cell and external acquisition to active further evaluation.If the TF1 cell outnumbered matched group relatively, so interested EPO chemical compound just has promoting erythrocyte and generates active.In addition, those of ordinary skill in the art knows that other detection promoting erythrocyte generates active detection method and also can use.For example, European Pharmacopoeia has been put down in writing at least two kinds of promoting erythrocytes that are used to detect the EPO chemical compound and has been generated active method, and it comprises histanoxia mouse test and skein cell test.
Organization protection's ability based on the EPO receptor detects
In-individual embodiment, organization protection of the present invention ability detects the tissue protective receptor that is based on EPO.In case separated the sequence of tissue protective receptor, just can use various detection methods to determine organization protection's ability of specific EPO chemical compound.Oneself knew as the one of ordinary skilled in the art, the type of used detection depended primarily on the weight of EPO chemical compound.
For example, detection method can be competitive assay or sandwich assay or sterically hindered detection.Competitive assay depends on the tracer analog and detects that sample analyte is emulative to combine limited binding site that is positioned on the binding partner.Here, term " analyte " is meant and awaits detecting the active interested EPO chemical compound of its organization protection.Term " binding partner " is meant any albumen of bound analyte (particularly EPO receptor)." tracer " is meant the reagent that was labeled, for example binding partner, fixed binding partner and the space conjugate crossed of the analyte analog of labelling, fixed analyte analog, labelling.The tracer here can have the bonded function that is detected of any not impact analysis thing and its binding partner.Unrestriced example comprises can be by direct detected part (moiety), as fluorescent dye, chemiluminescence agent and radioactive marker, and must be reacted or part that derivatization just can be detected, as enzyme.Suitable tracer can be radiosiotope P 32, C 14, I 125, H 3, I 131, and composition thereof; Fluorogen, as the rare earth chelate, fluorescein, fluorescein derivative, rhodamine, the rhodamine derivant, dansyl Cl, umbelliferone luciferase (Lampyridea luciferase and bacteriofluorescein enzyme (U.S. Patent number 4,737,456)), fluorescein, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkali phosphatase, beta galactosidase, glucoamylase, lysozyme, carbohydrate oxidase (glucoseoxidase, beta-Galactose oxidase, and glucose-6-phosphate dehydrogenase (G6PD)), with utilize hydrogen peroxide oxidation dyestuff former such as HRP, lactoperoxidase, microperoxisome and the link coupled heterocycle oxidase of its mixture (uricase and xanthine oxidase); Biotin/avidin; Spin label; The phage labelling; The stable free free radical; With its combination.In one embodiment, tracer is at least a in horseradish peroxidase or the alkali phosphatase.
Those skilled in the art will know that tracer and albumen or the link coupled method of polypeptide.For example, coupling agent such as dialdehyde, carbodiimide, two maleimides, diimidazole, two diazotising benzidines etc. can be used for carrying out labelling with fluorescent agent above-mentioned, chemiluminescence agent and enzyme labelling, some of them are at U.S. Patent number 3,940,475 and 3, describe in 645,090, be incorporated herein by reference in full at this.
In the sandwich assay method, need carry out the fixing of reactant, just binding partner is separated with any analyte in being free in solution, it can be finished by precipitation binding partner or analyte analog before detecting step, as by covalent coupling, is adsorbed onto water-fast substrate or surface (U.S. Patent number 3 as glutaraldehyde cross-linking, 720,760), or after detecting step, by part or analog being fixed as immuno-precipitation.
Therefore, before or after competitive reaction, binding partner can be precipitated out (insolubilize), and separate in the tracer that combines with binding partner or never bonded tracer of analyte and the analyte.Separation can be used decant (binding partner is sedimentary in advance) or centrifugal (binding partner is at the competitive reaction postprecipitation) here here.The amount of sample analysis thing is inversely proportional to the amount of being measured out by the amount of mark substance in conjunction with tracer.Can draw the dose-effect curve of known quantity analyte, and compare, quantitatively to determine the amount of analyte in the sample with result of the test.When using enzyme as tracer, detection method typically refers to the ELISA system.
In continuous sandwich assay, for example, the binding partner that is fixed is used for the adsorption test sample analytes, and test specimen is removed in flushing, and bonded analyte is used to adsorb the binding partner that labelling is crossed, then with residual tracer and bonded separating substances.Be proportional to the analyte of test specimen in conjunction with the amount of tracer.In " simultaneously " sandwich assay, before the binding partner that the adding labelling is crossed, do not need the separation test sample.
Competitive and sandwich assay method is used the necessary part of phase separation step as detection method, but sterically hindered detection method is carried out in single reactant mixture.Another kind of competitive assay method is called " homology " and detects, and it does not need to be separated.In such detection method, the conjugate of preparation and use enzyme and analyte, thus make that the existence of anti-analyte has influenced the activity of this enzyme when anti-analyte is connected on the analyte.With bifunctional organic bridge with organization protection's receptor and enzyme such as peroxidase coupling.The conjugate that uses with EPO is selected, thereby made the combination of EPO suppress or strengthen the activity of marker enzyme.Such detection method is commonly called EMIT.
The space conjugate is used for the sterically hindered method that homology detects.These conjugates can synthesize by the hapten of a covalently bound small-molecular weight on little analyte, thereby make antihapten antibody can not be attached on the conjugate simultaneously with anti-analyte substantially.The analyte that is present in the test specimen will be in conjunction with anti-analyte, thereby allows antihapten to be attached on the conjugate, has caused the haptenic characteristic changing of coupling, and for example, when hapten was fluorogen, fluorescence changed.
Being described in common unsettled Application No. about the more information of the detection of organization protection's ability is 10/188; 905; the applying date is that the patent and the patent application serial numbers on July 3rd, 2002 is 60/456; 891; the applying date is the patent application on April 25th, 2002, is incorporated herein by reference in full at this.
Function detection
Under the situation of disappearance organization protection receptor sequence, organization protection's function of EPO can be by determining with external Function detection in the body.Preferably; those of ordinary skill in the art carries out detecting in the independent external or body and just can determine organization protection's activity of EPO chemical compound, but needs the both to carry out to guarantee that chemical compound all shows identical organization protection's activity with external in vivo in some cases.
In the practice, whether the EPO analog of the sugar chain that the sugar chain that the N-that those of ordinary skill in the art can use the combination of detection method disclosed by the invention to determine to have at least one increase connects and/or the O-of at least one increase connect has organization protection's activity.At first, experiment in vitro such as P19 cell and rat motor neuron detect and can be used for determining whether interested EPO has organization protection's activity.Then, intravital detection such as rat fixed point ischemia model, dicentrine tic model or spinal cord injury model can be used in the result of checking in vitro tests.
External model of the present invention including but not limited to; those are used for determining the model of the active disappearance of above-mentioned mistake glycosylated erythropoietin organization protection: P19 cell detection, rat motor neuron detect and the little array of cDNA, its will be in the back with embodiment 2 in more detailed discussion.These examples are nonrestrictive, because those skilled in the art has known the model of other suitable vitro detection, are used for determining organization protection's activity of EPO chemical compound.Generally speaking, if compared with the control, the EPO chemical compound keeps or has strengthened the viability of cell, and then the EPO chemical compound will be considered to protect in a organized way active.If compared with the control, the viability of cell during this erythropoietin has had a strong impact on and detected, then it will be considered to antagonism.
A. based on the vitro detection of P19 cell line
In one embodiment, vitro tissue protects active detection to be based on P19 cell line.For example, the P19 cell can keep not breaking up in the DMEM of the hyclone (Hyclone Laboratories) of the sulphation streptomycin (Gibco) of the benzylpenicillin that has added 2mM L-glutathione, 100U/ml, 100 μ g/ml and 10%, wherein contains 1.2g/l NaHCO 3The hepes buffer of 10mM.Except with the insulin of 5 μ g/ml, the transfer agent (transferring) of 100 μ g/ml, the Progesterone of 20nM, the putrescine of 100 μ M and the Na of 30nM 2SeO 3(Sigma) replace beyond the hyclone, do not contain in the culture fluid of serum and can contain the same composition of above-mentioned culture fluid.
The cell that will react with 50% fusion agent (confluency) spends the night with recombinant epo and/or interested EPO compound treatment, dissociates with trypsin, washes and place 25cm in the culture fluid that does not contain serum 2Tissue culture flasks in, the final densities that reaches in the culture fluid that does not contain serum (or containing the pretreatment additive) is 10 4Cell/cm 2The viability of cell detects by the trypan blue rejection.
As well-known to those skilled in the art, in undifferentiated neuron P19 cell, behind the removal serum, add the death that recombinant epo can stop cell.For example, if the concentration of using as 0.1U/ml to 100U/ml, recombinant epo has saved at the most that 50% neuron cell makes it to avoid death.Therefore, want to determine that interested EPO chemical compound has organization protection's activity, it must be saved more P19 cell than matched group and make it to avoid death, preferably must save about 25% to about 50% cell, most preferably is about 40% to about 50% cell.
B. external rat motor neuron is analyzed
In another embodiment, the analysis of rat motor neuron is used for organization protection's activity of the interested EPO chemical compound of vitro detection.For example, elementary motor neuron can obtain and use immune elutriation purification from the embryos' of 15 the biggest Sprauge Dawley rats spinal cord.Cell is preferably with low-density (20000 cells/cm 2) be seeded on the coverslip of 24mm orifice plate, described 24mm orifice plate wraps quilt in advance with poly--DL-ornithine (orinthine) and N-trimethyl lysine inner salt, and contain complete culture solution (Neurobasal, B27 (2%), 0.5mM L-glutaminate, 2% horse serum, 25 μ M 2 mercapto ethanols, 25 μ M glutamic acid, 1% penicillin and streptomycin, the BDNF of 1ng/ml).After after a while, preferred about 6 days, EPO (10U/ml) and interested EPO chemical compound (10U/ml) or excipient can be added in the culture fluid (preferably preferably detecting the neuronal cell density precontract of surviving 5 days).Remove culture fluid then, cell mixed 40 minutes with the PBS that contains 4% paraformaldehyde, with 0.2% Triton X-100 infiltration, with the PBS sealing that contains 10% calf serum, with anti-unphosphorylated neurofilament (SMI-32; Antibody 1:9000) is incubated overnight, and utilizes diaminobenzidine to develop by avidin-biotin method.The viability of motor neuron can be carried out modal evaluation by the counting of SMI-32 positive cell.
The mixing primary culture of motor neuron is gone through programmed death keeping under the condition of culture characteristic ground warp.Show, in 5 days forward direction culture fluid that detect cell number, add recombinant epo (10U/ml), significantly increased the number of elementary motor neuron at the 5th day.Therefore, be considered to have organization protection's activity, compare with matched group, interested EPO chemical compound has preferably been saved the motor neuron of similar number at least.In one embodiment, keeping under the condition of culture, saving more motor neuron, so just thinking that it has organization protection's activity if compare interested EPO chemical compound with matched group.
C. based on the vitro detection of the little array of cDNA
The another kind of active method of organization protection that detects interested EPO chemical compound is the little array of cDNA.This detection method can be used for determining whether EPO is different to the influence of P19 gene expression of cells with interested EPO chemical compound.Isolating mRNA can demonstrate different gene regulation patterns from undifferentiated P19 cell, and this determines that by the little array of mice 1200cDNA it depends on the degree that is exposed to EPOs.For example, 1,200 expression of gene can detect by using the nylon membrane array from Clontech (Atlasmouse1.2) in the P19 cell.Cell (10 7/ sample) can spend the night with saline, recombinant epo, interested EPO chemical compound (1mU/ml) or its mixture process.Dissolved cell is used to extract RNA or with removing serum (the same cytokine that adds when always existing in pretreatment) in 3 hours then.Undertaken after the total RNA of standard extracts by column chromatography, use that DNase handles chromatographic column on the post, the polyA+RNA purification can be come out.Subsequently at [P 32Make up probe under the situation that]-ATP exists.The probe of this labelling preferably has 20,000,000 countings or more, can hybridize on the cDNA nylon membrane under 68 ℃ the condition.Wash this film and expose to the x ray film.The intensity of radiated signal can detect and analyze with Atlas Image 2.0 computer programs (Clontech) with PhosphorImager.
Detection includes but not limited in the body of the present invention, is used to estimate the active detection method of organization protection of EPO chemical compound, as fixed point ischemia model and the interior dicentrine model of Hippocampus (intra-hippoeampal).In addition, being used for evaluation of tissue protects active body inner model to comprise that spinal cord injury detects.Further, the various detection methods that are disclosed in international publication number WO/02053580 and U.S. Patent Publication No. 2002/0086816 and 2003/0072737 may be used to the present invention.
D. based on the body inner analysis of ischemia model
In one embodiment, this analytical method that is used to detect organization protection's ability of specific EPO chemical compound is based on the ischemia model.For example, male Sprague-Dawley rat (～250g) can be used for three blood vessel ischemia models.In brief, use pentobarbital (60mg/kg-bw) anesthetized rat and use water-bath to keep 37 ℃ core temperature.With two stitching thread ligation right carotid artery and cut-out.As seen larynx hole near the right eye socket of the eye makes the middle part cerebrovascular, can anaesthetize the end of nasal cavity blood vessel like this.In order to produce penumbra in fixed MCA wound circumference, with the pliers traction in addition-carotid artery of side makes its inaccessible 1 hour.When the closed outbreak of reversible carotid artery, can use saline, recombinant epo (5000U/kg-bw) or interested EPO chemical compound (5000U/kg-bw).
After 24 hours, take off brain, use cerebral operations device (Harvard Apparatus) to downcut the thick part of successive 1-mm from whole brain.The 37 ℃ of hatchings 30 minutes in the NaCl of the 154mM that contains 2% triphenyltetrazolium chloride then of each part.Injured volume can use the computer image analysis system (MCID, Imaging Reseach, St.Catharines, Ontario Canada) measures.In such detection,,, so just think that it has neuroprotective activity to improving because the degree of the infarct volume that rat MCA ischemia causes is identical or better if interested EPO chemical compound is compared with recombinant epo.
E. detect in the body based on the dicentrine tic model in the Hippocampus
In another embodiment, organization protection's ability of EPO chemical compound can be tested by the dicentrine in the intravital Hippocampus and be detected.For example, male Sprangue-Dawley rat (250～280g) are closed under the condition of steady temperature (23 ℃) and relative humidity (60%), use sufficient food and water, and fixed 12 hours daytime/circulate night is provided.Operation is implanted sleeve pipe and electrode to rat under three-dimensional orientation direction, according to Vezzani, and A., etc., J.Neurosci, 19, described in the 5054-65 (1999).Concise and to the point, use the Equithesin (chloral hydrate of 1% pentobarbital/4%; 3ml/kg i.p.) anesthetized rat.The electrode of two spirals is positioned at the both sides of parietal bone cortex, also puts into the earth lead that is positioned under the nasal sinuses.Bipolar NI-G ferroalloy line insulating electrode (60 μ m) can be implanted to the both sides of the gyrus of back side Hippocampus (septum electrode) then, and the implantable one-sided sleeve pipe in cerebral dura mater top (22-specification) that is positioned at is used for Hippocampus and Intraventricular infusion medicament.The position coordinates based on anterior fontanelle of implant electrode should be: (mm) anterior-posterior-3.5; Under both sides 2.4 and 3 cerebral dura mateies, use nose bar to be arranged on-2.5.Paxinos G.﹠amp; Waston, C., The Rat Brain in StereotaxicCoordinates, Academic Press, New York (1986).Electrode can be connected with porous socket (MarchElectronics, NY) and, be connected on the injection vessel, with acrylic acid adhesive of tooth protection skull.
This experiment was preferably carried out when animal returned to one's perfect health by 7 days at 3 behind the animal surgery.Give animal abdominal cavity administered recombinant EPO or interested EPO chemical compound (all being 5000U/kg-bw) or carrier then, before inducing the dicentrine outbreak, be administered once in 24 hours, be administered once again when inducing preceding 30 minutes.The step of medicine injection has been described in Vezzani in record EEG and the brain, A., etc., J.Pharmacol Exp Ther, 239,256-63 (1986).Concise and to the point, beginning EEG record (devices are described in many kinds of fluctuations of 4 road EEG, model BP8, Battaglia Rangoni, Bologna, Italy) preceding, animal can be put in the Plexiglass cage (25 * 25 * 60) in a suitable place to breed minimum 10 minutes.About 15 to after about 30 minutes, after inculcating with the dicentrine methiodide of 0.8nmol/0.5 μ l, continue 120 minutes EEG record.All injections all are to be applied to non-narcotic rat with the syringe needle (28-specification) that reaches sleeve pipe below 3mm.
Can analyze by EEG and detect tic, show that before this it can provide the Sensitive Detection to the anti-convulsant activity of medicine.Vezzani, A., etc., J.Pharmacol Exp Ther, 239,256-63 (1986).For the purpose of this detection, twitch and form: high frequency and/or multi-peak compound (multispike complex) and/or high voltage sync peaks (high voltage synchronized spike) or undaform activity by simultaneous at least two following changes in whole 4 recorded informations (leads of recordings).Can be observed the sync peaks and the mixing mutually of twitching.The parametric optimization that selection is used for quantitatively twitching is the latent time of (convulsive seizure) of twitching for the first time, and (persistent period by the incident of will showing effect was added in-rose and determines the time that the epilepsy reaction consumes altogether; Twitch the persistent period) and the peak value reaction of (reaction of twitching) during the EEG record.
Use the EEG reaction to be proved to be the sensitivity and the specific forecast means of the anti-tic ability of medicine as dicentrine tic model in the Hippocampus of reading.Vezzani, A., etc., J.Pharmacol ExpTher, 239,256-63 (1986).Therefore, want to be considered to have organization protection's activity, interested EPO chemical compound should or reduce the frequency and the order of severity of twitching to a greater degree than the recombinant epo same degree.
F. detect in the body based on acute reversible glaucoma rat model
In detecting in another kind of body of the present invention, acute reversible glaucoma rat model can be used for the detection of organization protection's ability of interested specific EPO chemical compound.For example, because retina cell is very responsive to ischemia, therefore many such cells will be dead after ischemia is coerced following 30 minutes.Therefore; whether show the organization protection's activity that is enough to protect to the cell of ischemia sensitivity in order to detect the interested EPO chemical compound that periphery uses, can use acute, reversible glaucoma rat model, it describes referring to Rosenbaum etc.; Vis.Res.37:3443-51,1997.Especially, can pump pickle to the eye ante-chamber of bull rat, and make its pressure surpass the body arteriotony and kept 60 minutes.Before inducing ischemia, gave in the rat abdominal cavity in 24 hours then and use saline and 5000U EPO/kg body weight, and as extra 3 days of dosage continuous administration every day.
The treatment back can be detected with electroretinograph and adapt to dark rat in 1 week, to determine whether interested EPO chemical compound has organization protection's activity.If this EPO has organization protection's activity, so than in rat, in tracing, retinal current should have good active to keep mutually with brine treatment.
G. myocardial infarction detects
Myocardial infarction detects also can be applied to the present invention, and it is used for detecting the EPO chemical compound and whether has organization protection's activity on the whole or especially in heart.For example, carry out the EPO that coronary artery ligation can be used the 5000U/kg body weight in preceding 24 hours to the bull rat at anesthetized rat and preparation.Can use the EPO of extra dose when program begins, the main coronary artery on the left side of ligation simultaneously unclamped in 30 minutes then.In 1 week of EPO that apply at the same rate every day after processing, detect the cardiac function of rat simultaneously.The rat of having accepted blank injection (saline) will demonstrate increasing considerably of left-hand extremity diastole pressure; indicated heart expansion, stiff; be only second to myocardial infarction; and for having used the animal of interested EPO chemical compound; if this EPO protects active words in a organized way, this animal should show unmarred cardiac function (significant difference is in P＜0.01 level) than the blank group so.
H. spinal cord injury detects
Spinal cord injury detects and also can be used for organization protection's activity that the present invention estimates specific EPO chemical compound interested.Especially, rat spinal cord can be compressed liquid and be used for the present invention.The preferred body weight of using is about the Wistar rat (female) of 180g to about 300g.Before the operation, animal was preferably gone on a hunger strike before operation 12 hours, the hommization imprisonment, and intraperitoneal injection pentothal socium (40mg/kg-bw) is anaesthetized.Dermal osmosis (0.25% bupivacaine) is being dissected afterwards with microscopical auxiliary time, and the otch by 2cm carries out complete single level (T-3) laminectomy.Traumatic spinal cord injury is inductive by the closing force 1 minute that is applied to 0.6 newton on the spinal cord (65 gram) by the interim aneurysm clip of exterior dura.After taking off clip, the otch of skin suture, rat revives from anesthesia fully, is put back in the cage again.Use at least every day twice bladder palpation to monitor rat up to recovering idiopathic emptying.
Animals of control group is used common saline (by intravenous injection) rapidly immediately after sewing up the incision.The interested EPO chemical compound of 16 milligrams/kg-bw of remaining animal intravenous administration.The nervus motorius that uses motion rate value (locomotor rating scale) to estimate rat is then learned function.In this numerical value, the score scope of animal (is not observed the hind leg motion) to 21 (normal gaits) from 0.In damage back 1 hour, 12 hours, 12 hours, 48 hours, 72 hours and 1 week, measure the afunction of rat by the same reviewer who does not understand the suffered treatment of animal respectively.If interested EPO chemical compound has organization protection's activity, the rat of having used this EPO should be than faster and better the fully recovering from damage of the rat of pump pickle.
I. rabbit spinal cord ischemia detects
In another embodiment, rabbit spinal cord ischemia is detected the detection that is used for organization protection's ability.For example, the heavy 1.5kg-2.5kg of New Zealand white rabbit (36, the 8-12 month is big, and is male) can be used for this detection.The management of animal fasting 12 hours and hommization.Induction of anesthesia is the Hal by 3%, and it is dispersed in 100% the oxygen, and is maintained at the Hal of 0.5-1.5%, and it is dispersed in the air gas mixture of 50% oxygen and 50%.Used for intravenous injection conduit (22-specification) is inserted left ear vein.In operation process, inculcate the Ringers lactate solution with the speed of 4ml/kg body weight (bw) per hour.Before the operation, the cefazolin of intravenous injection 10ml/kg-bw (Cefazoline) is with prevention infection.Animal is placed with the right prone position, handles skin with povidone iodine, uses bupivacaine (0.25%) infiltration, and opens a lateral skin incision at the 12nd joint place that is parallel to spine.After cutting skin and subcutaneous thoracolumbar fascia, take out the longest psoas muscle and iliocostalis lumborum.Expose abdominal artery by method behind the peritoneum of the left side, the renal artery that moves to left below a bit.A PE-60 pipe is put tremulous pulse on the spot from left side renal artery far-end, and a bigger rubber tube is all passed at two ends.By pulling PE pipe, non-traumatic ligation abdominal artery 20 minutes.
Use heparin (400IU) in the abdominal artery ligation with the vein pill.After the ligation 20 minutes, remove rubber tube and conduit, sew up the incision and guard animal, simultaneously the animal order is carried out the detection of neurological function up to recovery from illness.After untiing the abdominal artery ligation, give the common saline of control animals intravenous injection immediately.After pouring into again, give the interested EPO chemical compound (every group n=6) of another treated animal intravenous injection 6.5 μ g/kg-bw immediately.
Carry out motor function evaluation according to the standard of Drummond and Moore by the tester who does not understand the suffered treatment of animal pouring into back 1 hour, 24 hours and 48 hours again.Every animal must be divided into 0 to 4, and give a mark by following standard: the 0=crural paralysis does not have tangible hind leg motor capacity; The hind leg motor capacity that 1=is low only has faint antigravity motion; The hind leg motor capacity of 2=moderate also has stronger antigravity ability but the ability of not stretching one's legs below health; The good motor capacity of 3=also has the ability of stretching one's legs and jumping below health, but is not normal; The normal motor capacity of 4=.Carry out twice artificial urinary bladder emptying every day animal to crural paralysis.
If interested EPO chemical compound has organization protection's activity, the animal of having used this EPO so should compare should be faster and better with the animal of saline injection recovery from illness from damage.
As previously mentioned, good several tissues have the EPO receptor, thereby, may reply to some extent organization protection's ability of EPO.Therefore, according to the expection clinical application of interested EPO chemical compound, the technical staff can recognize the similar vitro detection that also can relate to these other responsive cells, or relates to interior detection of body of corresponding organ.For example, the vitro detection of removing based on serum can utilize cardiac muscle, retina and Leydig cell to carry out with the method that is similar to the above-mentioned P19 of being used for detection.
Detection also can be directly used in concrete organ in the body.For example, in order to estimate the influence of EPO for retina cell, those skilled in the art can carry out above-mentioned retinal ischemia's detection in this area.In addition, in order to estimate the influence of EPO analog for myocardial cell, those skilled in the art can easily change above-mentioned scheming infarction detection in this area.Those of ordinary skill in the art can select suitable detection method or model to be used to estimate specific EPO promoting erythrocyte is generated responsive cell, tissue or organ whether to have organization protection's activity.
Embodiment
Embodiment described below only is used to explain the preferred embodiments of the present invention, can not be construed as limiting the invention, and scope of the present invention is limited by additional claim.
Embodiment 1: the EPO of chemical modification
A. the oxidation of sugar chain
The sugar chain part of EPO can use following step to change into acid.EPO and be enough to provide the sodium metaperiodate of oxidation aequum (amount of sodium metaperiodate is big more, and degree of oxidation is high more) can be placed in the 100mM sodium acetate buffer.This solution ice bath 20 minutes and use distill water dialysis then.From Dialysis tubing, take out product then and collect (product I) in the clean test tube.
Quantitatively benedict's solution (copper sulfate of 18g, the sodium carbonate of 100g (anhydrous), the potassium citrate of 200g, the potassium thiocyanate of 125g, the POTASSIUM FERROCYANIDE 99 of 25g) can be used dissolved in distilled water, and final volume is 1 liter.Quantitatively adding several methyl blues in the benedict's solution (Quantitative Benedict Solution) then.
Product I can be added in this quantitative benedict's solution the clarification that becomes of color up to solution subsequently, and this shows solution complete oxidation.This solution can be desalted then and concentrate with centrifugal filter device (UltrafreeCentrifugal Filter Unit).This sample (product II) can further be used distill water dialysis more then.
B. with glucoseoxidase the EPO that takes off the sialic acid form is carried out oxidation
The sialic acid form of the taking off EPO of 50 to 500 μ g, the glucoseoxidase of 10 μ l 1U/ μ l and the sodium phosphate buffer of 100 μ l 10mM can mix (cumulative volume is 110 μ l) in the conical centrifuge tube of 15ml.This mixture can be 37 ℃ of following incubations 2 hours then, and available distilled water is fully dialysed simultaneously.Can from Dialysis tubing, take out product then and collect (product III) in the clean test tube.
Quantitatively benedict's solution (above-mentioned) can be 1 liter to final volume with dissolved in distilled water.Can add several methyl blues at quantitative benedict's solution then.
Product III can be added in this quantitative benedict's solution then, up to the clarification that becomes of the color of solution, shows solution complete oxidation.Then this solution is desalted and concentrate with centrifugal filter device (Ultrafree CentrifugalFilter Unit).This sample (product IV) can further fully be dialysed with distilled water more then.
The sulphation of C.EPO
EPO can be dissolved in 4 ℃ N, in the dinethylformamide (DMF-SA).Add the N that is dissolved among the DMF then, N '-dicyclohexylcarbodiimide (DCC) shakes solution 4 hours under 4 ℃ condition.Can add trash ice and use the NaOH of 10N to regulate pH to 7.5.Can adjust solution volume and HN-S2 type centrifuge tube (DAMONIEC, Needham Hts., Massachusettes) in 1000xg centrifugal 15 minutes.Supernatant fully can be dialysed then.More relevant Sulfated description is referring to S.Pongor etc., Preparation of High-Potency, Non-aggregating Insulins Using a NovelSulfation Procedure, Diabetes, Vol.32, No.12, December 1983, are incorporated herein by reference in full at this.
D. the PEG chain is connected on the EPO
Can be by modifying EPO on the sugar chain that the PEG chain is connected to oxidation, as above-mentioned (the product I) that in A, obtains.The degree of modifying can be regulated and control by the concentration of regulating Oxidation meso-periodic acid salt.
Can be at first during preparation PEG-EPO coupled product with oxidation EPO (2-4mg/ml is in the 50mM sodium acetate) under sodium metaperiodate (Sigma) room temperature between 1mM-10mM 30 minutes.At 100mM, remove phosphate buffer by the buffering exchange in the sodium acetate of pH5.4 then.
The methoxyl group of various molecular weight-PEG-hydrazides (Nektar Therapeutics) subsequently can be with 5 to 100 times molar excess (polymer: protein) add.Then can be by adding the cyaniding sodium borohydride (Sigma) of 15mM, hydrazides connects in the middle of minimizing is spent the night in 4 ℃ of reactions.The conjugate that obtains then can be with technology fractional distillation/purification well known in the art.
E. connect the PEG chain to taking off sialic EPO
The EPO that takes off the sialic acid form can pass through after using the beta-Galactose oxidase oxidation, the PEG chain is connected on the newly-generated terminal galactose residues modifies, as those products that obtain in above-mentioned B (product III).
Recombinant human EPO (rhuEPO) can (Prozyme Inc) carry out asialoglycoproteinization with sialidase according to manufacturer's handbook.Chemical modification is preferably definite by product being carried out on the sds polyacrylamide gel electrophoresis.Painted product band should show that the EPO of modification has the apparent molecular weight of about 31kDa, and the EPO of unmodified has the molecular weight of about 34kDa.The sialic acid that is retained among the EPO preferably is less than 0.1mol/mol EPO.
After the EPO of sialic acid form was taken off in acquisition, the galactose residue of the new EPO (2-4mg/ml is in the sodium phosphate buffer of 10mM) that exposes can carry out oxidation with the beta-Galactose oxidase (Sigma) (being dissolved among the PBS) of every milliliter of EPO solution 100 units.Reaction mixture is then 37 ℃ of incubations 2 hours.
In the 100mM of pH5.4 sodium acetate, remove phosphate buffer then by the buffering exchange.Then with the molar excess (polymer: protein) add of the methylating of various molecular weight-PEG-hydrazides (Nektar Therapeutics) with 5 to 100 times.Hydrazides connected in the middle of the preferred then cyaniding sodium borohydride (Sigma) by adding 15mM further reduced, and spent the night 4 ℃ of reactions.The conjugate that can obtain with technology fractional distillation well known in the art/purification then.
F. connect the PEG chain to taking off sialic EPO
The EPO that takes off the sialic acid form can pass through after using the beta-Galactose oxidase oxidation, the PEG chain is connected on the newly-generated terminal galactose residues modifies, as those products that obtain in above-mentioned B (product III).
RhuEPO (1mg) can use neuraminidase, and (Seikagaku Corporation of Japan is dissolved in the lyophilized powder of 1U the 75mM NaPO of 100 μ L 4(pH6.5) in) carry out asialoglycoproteinization with 1mgEPO than the ratio of 0.05 unit neuraminidase (5 μ L).(450 μ L are dissolved in 75mMNaPO to add the beta-Galactose oxidase of 5 units (5 μ L) then in mixture 4(pH6.5) (Sigma) in).
In the 100mM of pH5.4 sodium acetate, remove phosphate buffer by the buffering exchange then.In mixture, add PEG-NH then 2The cyaniding sodium borohydride (Sigma) of (750 molecular weight, Nektar Therapeutics) and 15mM spends the night 4 ℃ of reactions then.Preferably, PEG-NH 2Addition is 250 times a molar excess (the polymer: (PEG-NH of 80mg protein) 2).The conjugate that obtains with technology fractional distillation well known in the art/purification then.
Embodiment 2. Function detection
A. promoting erythrocyte generates and detects
A kind of promoting erythrocyte generative capacity of specific EPO chemical compound is promptly regulated and control the ability of hematocrit levels, determines by following detection method.
TF1 is-the somatomedin human erythrocyte leukaemia of (comprising EPO) kind places one's entire reliance upon.Kitamura, etc., Blood 73,375-80.TF1 cell line is from the ACTT acquisition and with containing the 2mM L-glutaminate, 10mM Hepes, 1mM Sodium Pyruvate, 4.5g/L glucose, 1.5g/L sodium bicarbonate, the RPMI 1640 of 5ng/mlGM-CSF and 10% hyclone are saved in when testing always.From the active growth stage TF1 cell that obtains is precipitated gets off, with culture fluid flushing three times separately, and to contain 10 in the 1ml culture fluid 5The concentration resuspension of individual cell wherein can be with or without GM-CSF, and is added with the EPO of specific concentrations or has sugar chain that the N-of at least one increase connects and/or the EPO analog of the sugar chain that the O-of at least one increase connects.Each culture kept 24 hours, used formazan reactant (CellTiter; Promega WI) determines the number of cell according to manufacturer's handbook.
The activity of EPO chemical compound is by observing it to the influence of female BALB/c mouse hemoglobin concentration and at first carry out interior evaluating.Give the EPO of animal subcutaneous administration 500U/kg-bw, interested EPO chemical compound, or isopyknic carrier, 1 week 3 times is 3 weeks (being enough to observe the interval of promoting erythrocyte reaction of formation) altogether.If the EPO chemical compound has improved the concentration of the serum hemoglobin of mice, so just think that it has promoting erythrocyte and generates active.The activity of advancing-going on foot detects by obtaining at external use TF1 erythrocyte leucocythemia cell.If research confirms that the increase of TF1 cell number has surpassed matched group relatively, so just think that EPO has promoting erythrocyte and generates active.
Those of ordinary skill in the art knows other detection method, as histanoxia mice detection method and skein cell detection method (European Pharmacopoeia), is applicable to that also the present invention is active to determine that promoting erythrocyte generates.
B. organization protection is active detects
Have at least-organization protection's function of the EPO analog of the sugar chain that the sugar chain that the N-of individual increase connects and/or the O-of at least one increase connect detects by following method.
Hippocampus by 18 the biggest rat embryos makes up the neuron culture.From meninges, take out and the separation brain, isolate hippocampus.Cell is dispersed in 2.5% the trypsin solution then, 37 ℃ of following incubations 5 minutes, and titration then.Cell suspending liquid is with not containing additional liquid (Gibco serum, that contain 1%B-27, Rockville, MD, neural substrate (neurobasal) culture fluid dilution USA), and be placed on the coverslip that is coated with poly-guanosine monophosphate with 80,000 cell densities of every coverslip.Cell is then with the EPO pretreatment of spending the night, be exposed among the 5 μ M TMT 24 hours then, be with or without 1) EPO, 2) have the EPO analog of the sugar chain that the O-of sugar chain that the N-of at least one increase connects and/or at least-individual increase connects, or 3) take off the EPO of sialic acid form.Then at the culture of external use between 10 to 14 days.
Can pass through bromination 3-(4,5-dimethyl-thiazol-2-yl)-2,5-biphenyl tetrazolium (MTT) is measured the viability of cell.Denziot，F，and?Lang，R.1986.Rapid?Colormetric?Assay?forCell?Growth?and?Survial.Modifications?to?the?tetrazolium?dye?procedure?givingimproved?reliability.J?Immunol?Methods?89：271-277。In brief, the MTT tetrazolium salts is dissolved in the culture fluid that does not contain serum, is 0.75mg/ml and when processing finishes, joins in the cell to ultimate density, following 3 hours at 37 ℃.Remove culture fluid then and use 1N HCl: isopropyl alcohol (1: 24) extracts formazan.On the microtest plate reader, read the absorption value at 560nm place.
As described at accompanying drawing 1A, the EPO analog with sugar chain that the O-of sugar chain that the N-of at least one increase connects and/or at least-individual increase connects does not have organization protection's function.
Here the scope of description and claimed invention is not limited by specific embodiments disclosed herein, because these specific embodiments only are used to explain several aspect of the present invention.The embodiment of any equivalence is also contained within the scope of the present invention.In fact, except the invention that those are here described, various modifications of the present invention will be conspicuous after the description of having read the front to those skilled in the art.Such modification has also dropped within the scope of appended claim.At these all documents of quoting is that various purposes are incorporated herein by reference in full.

Claims (27)

1. regulate the method for human hematocrit levels, comprise the steps: to provide than rhuEPO to have longer serum half-life and comprise the erythropoietin product of organization protection's function; With
This erythropoietin product of administering therapeutic effective dose.
2. the described method of claim 1, the described step of erythropoietin product that provides also comprises the steps:
Modify on the oligonucleotide chain that the oligonucleotide chain or the O-of at least one N-of rhuEPO connection connect with at least a chemical modification, wherein, described chemical modification comprises oxidation, sulphation, phosphorylation, PEGization or its combination.
3. the described method of claim 1, the step of this erythropoietin product of wherein said administering therapeutic effective dose comprises with the mole that is lower than rhuEPO uses this erythropoietin product to obtain suitable targeted blood cells specific volume.
4. the described method of claim 1, wherein said serum half-life is about 20% at least than the serum half-life of rhuEPO.
5. the described method of claim 4, wherein said serum half-life is about 40% at least than the serum half-life of rhuEPO.
6. artificial erythropoietin product comprises:
At least a erythropoietin derivatives, wherein the oligonucleotide chain of the oligonucleotide chain of at least one N-connection or at least one O-connection has at least a chemical modification, described chemical modification derives from oxidation, sulphation, phosphorylation, PEGization or its mixing, and the serum half-life of wherein said erythropoietin product is longer than rhnEPO.
7. the described erythropoietin product of claim 6, wherein, described erythropoietin product has organization protection's function.
8. the described erythropoietin product of claim 6, described at least a chemical modification comprise that the oxidation of the oligonucleotide chain that oligonucleotide chain that at least one N-connects or at least one O-connect is to provide the sour residue of at least one increase.
9. the described erythropoietin product of claim 6, described at least a chemical modification comprise that the sulphation of the oligonucleotide chain that oligonucleotide chain that at least one N-connects or at least one O-connect is to provide the negative charge of increase on this erythropoietin product.
10. the described erythropoietin product of claim 6, described at least a chemical modification comprise that the phosphorylation of the oligonucleotide chain that oligonucleotide chain that at least one N-connects or at least one O-connect is to provide the negative charge of increase on this erythropoietin product.
11. being included on the oligonucleotide chain that oligonucleotide chain that at least one N-connects or at least one O-connect, the described erythropoietin product of claim 6, described at least a chemical modification increase by at least one polyglycol chain.
12. preparation has the method for the active erythropoietin product of serum half-life and organization protection of prolongation, comprises the steps:
At least a erythropoietin or erythropoietin derivatives are provided; With
On the oligonucleotide chain that the oligonucleotide chain or at least one O-of at least one N-of at least a endogenous or recombinant erythropoietin connection connect, modify by oxidation, sulphation, phosphorylation, PEGization or its combination.
13. the described method of claim 12, described modification step also comprise the step that replaces at least one vicinal hydroxyl groups on the oligonucleotide chain that oligonucleotide chain that at least one N-connects or at least one O-connect with at least one sour residue.
14. the described method of claim 13, at least a sour residue of described usefulness replace the step of at least one vicinal hydroxyl groups on the oligonucleotide chain that oligonucleotide chain that at least one N-connects or at least one O-connect and also comprise: replace a plurality of vicinal hydroxyl groups on the oligonucleotide chain of oligonucleotide chain that at least one N-connects or at least one O-connection with a plurality of sour residues.
15. the described method of claim 12, described modification step also comprises the steps:
A kind of organic solvent is provided;
This erythropoietin of dissolving or erythropoietin derivatives are to form solution in this organic solvent;
At least a condensing agent is provided;
At least a sulfate donor is provided; With
Described at least a condensing agent and at least a sulfate donor are mixed in the solution.
16. the described method of claim 12, wherein, described modification step also comprises the steps:
A kind of organic solvent is provided;
This erythropoietin of dissolving or erythropoietin derivatives are to form solution in this organic solvent;
At least a condensing agent is provided;
Phosphoric acid is provided; With
Described at least a condensing agent and at least a phosphoric acid are mixed in the above-mentioned solution.
17. the described method of claim 12, described modification step also comprises the steps:
A kind of organic solvent is provided;
Dissolving erythropoietin or erythropoietin derivatives are to form first solution in this organic solvent;
At least a oxidant is provided;
In this first solution, add this at least a oxidant to form second solution;
At least a polyglycol chain is provided;
With described at least a polyglycol chain is mixed in second solution.
18. the described method of claim 17, the described step of at least a polyglycol chain that provides comprises that the end that chain is provided has at least a polyglycol chain of at least one primary amino radical group.
19. treatment is in the method for the patient's anemia in tissue injury's danger, comprises following step:
Be provided at the erythropoietin product that at least a chemical modification is arranged on the oligonucleotide chain that oligonucleotide chain that at least one N-connects or O-connect, wherein said chemical modification comprises oxidation, sulphation, phosphorylation, PEGization or its combination;
The described erythropoietin product of administering therapeutic effective dose, the mole that wherein said erythropoietin product is used is lower than rhuEPO, obtaining suitable targeted blood cells specific volume,
Wherein said erythropoietin product has the cytoprotective function.
20. the described method of claim 19, described erythropoietin product have the serum half-life longer than rhuEPO.
21. the described method of claim 20, described serum half-life is about 20% at least than the serum half-life of rhuEPO.
22. the described method of claim 21, described serum half-life is about 40% at least than the serum half-life of rhuEPO.
23. a pharmaceutical composition comprises:
At least a erythropoietin derivatives of treatment effective dose, on the oligonucleotide chain that the oligonucleotide chain or at least one O-of its at least one N-connection connect, has at least a chemical modification, described chemical modification derives from oxidation, sulphation, phosphorylation, PEGization or its combination
Described at least a erythropoietin derivatives has than the longer serum half-life of reorganization erythropoietin and has organization protection's function.
24. the described pharmaceutical composition of claim 23 also comprises at least a pharmaceutically acceptable carrier.
25. the described pharmaceutical composition of claim 24, wherein said at least a pharmaceutically acceptable carrier comprises at least a diluent, adjuvant, excipient, carrier or its mixing.
26. the described pharmaceutical composition of claim 23 also comprises at least a wetting agent, emulsifying agent, pH buffer agent or its combination.
27. the described pharmaceutical composition of claim 23 also comprises at least a organization protection cytokine.
CNA038249154A 2002-09-09 2003-09-09 Long acting erythropoietins that maintain tissue protective activity of endogenous erythropoietin Pending CN1729015A (en)

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CN102746405A (en) * 2012-07-24 2012-10-24 深圳赛保尔生物药业有限公司 Continuous pegylation reaction method for recombinant human erythropoietin (EPO)
CN101671388B (en) * 2008-09-09 2013-01-02 曹国栋 Blood brain barrier penetrable erythropoietin (EPO) and application thereof

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US7718363B2 (en) 2003-04-25 2010-05-18 The Kenneth S. Warren Institute, Inc. Tissue protective cytokine receptor complex and assays for identifying tissue protective compounds
WO2006061853A2 (en) * 2004-12-10 2006-06-15 Serum Institute Of India Limited Novel erythropoietic compounds and a process for producing erythropoietic compounds
TWI376234B (en) 2005-02-01 2012-11-11 Msd Oss Bv Conjugates of a polypeptide and an oligosaccharide
CA3079319A1 (en) 2005-08-05 2007-02-15 Araim Pharmaceuticals, Inc. Tissue protective peptides and uses thereof
SG10202011946PA (en) 2008-01-22 2020-12-30 Araim Pharmaceuticals Inc Tissue protective peptides and peptide analogs for preventing and treating diseases and disorders associated with tissue damage
RU2475273C1 (en) * 2012-04-02 2013-02-20 Юлия Николаевна Козлова Method of obtaining polymeric cement of medical purpose
RU2664588C2 (en) * 2015-11-05 2018-08-21 Закрытое Акционерное Общество "Биокад" Extended factor of human erythropoiesis and a therapeutic agent based thereon
EP3448415A4 (en) 2016-04-29 2019-11-06 Araim Pharmaceuticals, Inc. Tissue protective peptides for preventing and treating diseases and disorders associated with tissue damage
CN112741895A (en) * 2021-01-19 2021-05-04 中国人民解放军陆军军医大学 Application of EPO analog in preparing medicament for treating sepsis

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WO2001081405A2 (en) * 2000-04-21 2001-11-01 Amgen Inc. Methods and compositions for the prevention and treatment of anemia
DK1311285T4 (en) * 2000-05-15 2017-07-24 Hoffmann La Roche Liquid pharmaceutical composition containing an erythropoietin derivative

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CN101671388B (en) * 2008-09-09 2013-01-02 曹国栋 Blood brain barrier penetrable erythropoietin (EPO) and application thereof
CN102746405A (en) * 2012-07-24 2012-10-24 深圳赛保尔生物药业有限公司 Continuous pegylation reaction method for recombinant human erythropoietin (EPO)

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