A method of treating cerebral ischemia, as well as the use of erythropoietin or derivatives of erythropoietin for the treatment of cerebral ischemia
The invention relates to medicine, namely to neurology for the treatment of cerebral ischemia, as well as treatment for cerebral ischemia. Erythropoietin is administered perifericheskie dose of 5000 IU to 100,000 IU. The erythropoietin is administered native or recombinant human or animal erythropoietin or their derivative. The method can reduce the complications, which is achieved by eliminating traumatic route of administration. 3 N. and 18 C.p. f-crystals, 2 Il.
The present invention relates to a method of treating cerebral ischemia, as well as treatment for cerebral ischemia in mammals, particularly in humans, for example, when their manifestation in patients undergoing shock.
In ischemic brain infarction distinguish the damaged area in the centre zone of ischemia, as well as in the zone of the penumbra, the so-called Penumbra surrounding the center. The size of the centre ischemia plus Penumbra determine the amount of damage after ischemic stroke.
Erythropoietin, also referred to in abbreviated form as “EPO”, is a native glycoprotein body with a molecular weight of 30,000 daltons (W. Jelkmann, “Eritropoietin: Structure, Control of and was first isolated in 1977.
Erythropoietin has for many years used in clinical practice for the treatment of patients with renal anemia when prodiamine, to obtain increased amount of your own blood before scheduled operations, and he met in newspaper headlines as doping.
However, erythropoietin, as found, was well-tolerated. As a significant side effects include, in particular, often therapeutically desired stimulation of haematopoiesis with erythrocytosis, and also occasionally observed arterial hypertension. Both actions should be expected, mainly after prolonged administration of erythropoietin. In case you need them relatively easy to resolve by means of medication or blood-letting. Hypersensitivity or anaphylactic reactions to erythropoietin are rare events.
To date there is no effective therapy of cerebral ischemia, for example, for the treatment of patients after stroke, without an operation on a region of the patient's head.
Sanaka M. et al. report (PNAS 1998, Band 95, nr.8, S. 4635-4640) that the Central administration of erythropoietin in experimental animals has a protective effect on neurons in the brain is Oh introduction erythropoietin was carried out directly in Central lateral ventricle. However, such direct intraventricular injection of erythropoietin, that is a direct infusion of erythropoietin in the brain for people excluded because of the high risks that are associated with the establishment and maintenance of a temporary ventricular drainage, for example, infections or bleeding.
DelMastro L. et al. report (Oncologist 1998, 3/5, S. 314-318) that prophylactic administration of erythropoietin prevents anemia in cancer patients subjected to chemotherapy treatment, and thereby prophylactically can reduce the risk of these patients in relation to cerebral ischemia as the consequences of anemia caused by chemotherapy treatment. Therapy existing cerebral ischemia, particularly in patients who did not undergo chemotherapy treatment, this source of information is not disclosed.
The present invention is to provide a method of treating cerebral ischemia, a tool for use in the treatment of cerebral ischemia, as well as a means for the manufacture of drugs for the treatment of cerebral ischemia, which are easy to use and free from side effects and risks.
This task is solved by JV the Oia method and use according to the invention are given in the respective dependent clauses.
An additional aspect of the method according to the invention and use of EPO according to the invention is that after the onset of ischemia, for example, after the impact, the damaged cerebral tissue, particularly Penumbra, you need to recover as soon as possible and as much as possible. It was found that peripheral administration of erythropoietin has a pronounced protective effect on cerebral tissue affected by ischemia. Erythropoietin acts so that the area of the damaged cerebral tissue, especially in the Penumbra progressively decreases in comparison with the traditional procedures used in cerebral ischemia, without the introduction of erythropoietin.
This unexpected action perifericheskie entered erythropoietin, reducing tissue in cerebral ischemia person, is not obvious because erythropoietin is known to be larger protein with a molecular weight of about 30,000 daltons, which is unable to cross the blood brain barrier. Direct intraventricular injection of erythropoietin, that is a direct infusion of erythropoietin in the brain for people excluded because of the risks associated with establishing and maintaining temporary the freight and using the fact that surprisingly to treat the onset of cerebral ischemia immediately after the damaging event can perifericheskie to enter erythropoietin as a medicine, after which he goes into the damaged area of the brain becomes active.
Peripheral injection of erythropoietin, that is, on this side of the blood-brain barrier, it is preferable to carry out intramuscularly or into the vascular system. In order to deliver erythropoietin to the damaged cerebral tissue at a high dose for a short time, i.e. as soon as possible after the damaging event, the proposed right of direct introduction into the vascular system, which should be carried out in a known preferred drugs ways, in most cases, intravenously.
It follows that erythropoietin can overcome the blood-brain barrier in the affected area immediately after damage to the brain tissue ischemia. So you can enter a drug containing erythropoietin, for example, patients who have had a stroke, while erythropoietin really reaches the damaged brain tissue.
Thus, first proposed effective medicines is their advantage, that the intact blood-brain barrier in undamaged areas of the cerebral tissues actively prevents the penetration is not needed there erythropoietin, and therefore the area of tissue that is not affected by ischemic heart attack, is not subjected to therapy, thus, side effects may become apparent, or to a much lesser extent, or not appear at all.
Preferably erythropoietin is used as a medication in a dosage of from 5,000 to 100,000 units, optimally from 35000 units on one introduction, possibly one introduction per day in the first few days and for the first time possible within 8 hours after impact. However, to achieve therapeutic effect, quite a few injections of erythropoietin. Another advantage is that the side effects and risks, observed mainly at long-term continuous treatment of other paintings of the disease, according to the above prior art, when the use of erythropoietin for the treatment of cerebral ischemia or no symptoms at all, or they are insignificant.
Erythropoietin is known from the prior art. Human erythropoietin was first isolated from the urine (T. Miyake et al. 1977, J. Biol.Chem., Bd.252, S. 5558-5564). Currently, it is produced by the technology of the rivers is finding. For use according to the invention can be used also additional variants of erythropoietin with modified amino acid sequences or structures or parts with portions of the sequences that are functionally relevant biological functions of erythropoietin, and they will also fall under the term "erythropoietin", as used in this application. In addition, a variety of options erythropoietin, used according to the invention, obtained from the transformations in the glycosylation of erythropoietin.
Therefore, erythropoietin, used according to the invention, can be attributed including human erythropoietin, such as obtained in a natural way, as well as the products of erythropoietin or erythropoietin analogues (in the General case, variants or derivatives of erythropoietin that are the result of transformations in natural human erythropoietin, for example, modifications of the sequence, such as deletions, or substitutions, and changes in carbohydrate composition. The erythropoietin products of this kind can be obtained in various ways. Such methods for producing variants of erythropoietin, derivatives or analogs of erythropoietin erythropoietin, prednaska 93/09222, WO 94/12650, WO 95/31560 and WO 95/05465, the disclosure of which is fully incorporated into the present patent application by reference and should be included in the present application.
The following are examples of the method according to the present invention and applications of the present invention.
Fig.1 shows the appearance of erythropoietin in serum and cerebrospinal fluid after a stroke, and Fig.2 illustrates the lesion size after cerebral ischemia.
In Fig.1 (a) shows the average concentrations in serum, that is, the peripheral concentration of erythropoietin in a few days, four patients with stroke, which after about 8 hours, about 24 hours and again after approximately 48 hours after a stroke every time intravenously injected dose 35000 IU (international units) of human recombinant erythropoietin (drug Neorecormon” by Hoffmann-LaRoche AG). It is seen that the concentration in serum during the first day reaches its maximum and then drops sharply.
In Fig.2B presents the concentration of erythropoietin in six patients in the control group with neishemicescoy neurological diseases (controls neurological diseases”) after infusion of erythropoietin, the two are not yireh patients after hit (“EPO-patients”), after infusion of erythropoietin, as patients in the control group. This presents the average value of the concentration of erythropoietin in the cerebrospinal fluid, as it was defined in an average of 6.4 hours after the first infusion 35000 ME recombinant human erythropoietin (drug Neorecormon” by Hoffmann-LaRoche AG). We are talking about the same four patients who underwent shock (EPO-patients), as in Fig.1A.
Whereas used logarithmic scale image in Fig.1B, once it is clear that the concentration of erythropoietin in the cerebrospinal fluid in patients undergoing shock (“EPO-patients”), approximately 100 times higher than the concentration of erythropoietin in the treated in the same way, patients in the control group (controls neurological diseases”), and not treated patients with previous stroke (controls attack”).
An advantage of the present invention is that in the case of cerebral ischemia blood-brain barrier becomes permeable to erythropoietin, so for the treatment of cerebral ischemia immediately after the damaging event erythropoietin perifericheskie as medicine re the RA 73-year-old patient. Presents images obtained by nuclear magnetic resonance spectroscopy (“diffusion-weighted NMR imaging”).
Approximately 8 hours after stroke patient was intravenously injected 35000 ME recombinant human erythropoietin (drug Neorecormon” by Hoffmann-LaRoche”). Approximately 24 hours and 48 hours after the impact, respectively, have introduced additional equal high-dose erythropoietin.
In Fig.2A shows a sectional three types of bottom-brain patient during the treatment period, approximately 7 hours after impact.
Damaged by impact area can be clearly seen by allocating them white colored.
In Fig.2B damaged area approximately 3 days after the shot you can also see on their whitish color (dark center).
In Fig.2B shows in the context of the same species after 18 days. Clearly you can see that there is a noticeable recovery of the primary lesion. This recovery of the ischemic region of the heart should be attributed to, among other things, treatment with erythropoietin.
1. A method of treating cerebral ischemia in a mammal after a stroke, wherein the erythropoietin centuries the 3. The method according to p. 2, characterized in that the carry out intravenous.
4. The method according to one of paragraphs.1-3, wherein the erythropoietin is administered in a single and/or a daily dose of from 5,000 to 100,000 IU.
5. The method according to one of paragraphs.1-4, wherein the erythropoietin is administered in a single and/or a daily dose of from 35000 IU.
6. The method according to one of paragraphs.1-5, characterized in that as the erythropoietin is administered native or recombinant human or animal erythropoietin or derived.
7. The method according to one of paragraphs.1-6, characterized in that the mammal is treated person.
8. The use of erythropoietin for the manufacture perifericheskie injected drugs for the treatment of cerebral ischemia in mammals after impact.
9. Application under item 8 for the manufacture of drugs introduced into the vascular system.
10. Application on p. 9 for the manufacture of drugs, administered intravenously.
11. Use one of the PP.8-10 in pay and/or a daily dose of from 5,000 to 100,000 IU.
12. Use one of the PP.8-11 in pay and/or a daily dose of from 35000 IU.
13. Use one of the PP.8-12, characterized in that as erythropoietin use of native or recombinant human or animal erythropoietin is a battle of man.
15. The use of erythropoietin as perifericheskie input means for treatment of cerebral ischemia in a mammal after impact.
16. Application under item 15 as a means of insertion into the vascular system.
17. Application under item 16 as a means of intravenous.
18. Use one of the PP.15-17 in pay and/or a daily dose of from 5,000 to 100,000 IU.
19. Use one of the PP.15-18 in pay and/or a daily dose of from 35000 IU.
20. Use one of the PP.15-19, characterized in that as erythropoietin use of native or recombinant human or animal erythropoietin or derived.
21. Use one of the PP.15-20, characterized in that the mammal is a human.
R1means alkyl with 1, 2, 3, 4, 5, 6, 7 or 8 C-atoms; CandH2A-phenyl, where a = 0, which is unsubstituted or substituted by 1-3 substituents selected from the group consisting of F, Cl, Br, J, CF3, metoxygroup; CdH2d(C3-7-cycloalkyl, where d = 0; R2and R3independently from each other denote hydrogen, F, Cl, J, C=N; COR6where R6denotes hydrogen, alkyl with 1, 2, 3, 4, 5, 6, 7 or 8 C-atoms, OR30where R30- alkyl with 1, 2, 3, 4, 5, 6, 7 or 8 C-atoms; OR7where R7denotes hydrogen, alkyl with 1, 2, 3, 4, 5, 6, 7 or 8 C-atoms; phenyl; or R2and R3, independently of one another, denote CqH2q-phenyl, where q=0; or R2and R3independently from each other mean-SOnR22where n stands for zero, R22- alkyl with 1, 2, 3, 4, 5, 6, 7 or 8 C-atoms; R4and R5independently of one another denote hydrogen, alkyl with 1, 2, 3, 4, 5, 6, 7 or 8 C-atoms, F, Cl, Br, J, CF3and their physiologically acceptable salts; and to medicines, inhibiting Na+dependent Cl-/HCO-3- exchange rate is
where: A means-OR1-C(O)N(R1R2or-N(R1R21; each X, Y and Z independently represents N or C(R19); each U represents N or C(R5), provided that U is N only when X represents N, and Z and Y denote CR19; each W represents N or CH; V denotes: (1) N(R4); (2) C(R4)H; or (3) the groupdirectly related to the group -(C(R14R20)n-A,denotes a 5-6-membered N-heterocyclyl, optionally containing 6-membered ring additional heteroatom selected from oxygen, sulfur and NR6where R6denotes hydrogen, optionally substituted phenyl, 6-membered heterocyclyl containing 1-2 nitrogen atom, optionally substituted 5-membered heterocyclyl containing 1-2 nitrogen atom, aminosulfonyl, monoalkylammonium, dialkylaminoalkyl,1-6alkoxycarbonyl, acetyl, etc
FIELD: medicine, molecular biology, polypeptides.
SUBSTANCE: invention describes homogenous polypeptide ligand mpI representing polypeptide fragment of the formula: X-hTPO-Y wherein hTPO has amino acid sequence of human fragments TPO (hML); X means a amino-terminal amino-group or amino acid(s) residue(s); Y means carboxy-terminal carboxy-group or amino acid(s) residue(s), or chimeric polypeptide, or polypeptide fragment comprising N-terminal residues of amino acid sequence hML. Also, invention relates to nucleic acid encoding polypeptide and expressing vector comprising nucleic acid. Invention describes methods for preparing the polypeptide using cell-host transformed with vector, and antibodies raised against to polypeptide. Invention describes methods and agents using active agents of this invention. The polypeptide ligand mpI effects on replication, differentiation or maturation of blood cells being especially on megacaryocytes and progenitor megacaryocyte cells that allows using polypeptides for treatment of thrombocytopenia.
EFFECT: valuable medicinal properties of polypeptide.
21 cl, 92 dwg, 14 tbl, 24 ex