CN101405021A - Continuous dosing regimens for neural stem cell proliferating agents and neural stem cell differentiating agents - Google Patents

Abstract

Dosing regimes for neural stem cell proliferating agents in combination with neural stem cell differentiating agents, kits containing effective doses of neural stem cell proliferating agents and differentiating agents, and uses thereof in treating or ameliorating neurodegenerative diseases and conditions are disclosed. In particular, neural stem cell proliferating agent human chorionic gonadotropin (hCG) or luteinizing hormone (LH), administered in several doses, is used in combination with neural stem cell differentiating agent erythropoeitin (EPO). Other neural stem cell proliferating agents include prolactin.

Description

The continuous dosing regimens of cell proliferation of nerve cord reagent and cell differentiation of nerve cord reagent

The cross reference of related application

[0001] the application advocates the U.S. Provisional Application No.60/783 that on March 17th, 2006 submitted to, 500, the U.S. Provisional Application No.60/789 that on April 5th, 2006 submitted to, 132, with the U.S. Provisional Application No.60/862 that submitted on October 24th, 2006,669 priority and rights and interests, it is hereby incorporated by in full.

Background

[0002] development of the technology of separation and In vitro culture pluripotency neural stem cell (for example, is seen U.S. Patent No. 5,750,376; 5,980,885; 5,851,832) neurodegenerative disease and treatment of conditions prospect have significantly been improved.Have been found that fetal brain can be used for separating and In vitro culture pluripotency neural stem cell.And, and think that for a long time the fetal brain cell is that the viewpoint of not reproducible or regenerated brain cell is opposite, have been found that neural stem cell also can separate from the adult mammal brain.These stem cell, no matter be from fetus or the adult brain, can self replication.Daughter cell can breed or be divided into any cell in the neuronal cell line again, comprises neuron, astrocyte and oligodendroglia.Therefore, the source that these find not only to provide the neurocyte that can be used for transplanting has also confirmed the existence of pluripotency neural stem cell in the adult brain and original position produces neuron or neurogliocyte from these stem cell probability.

[0003] has been found that some molecule can increase the quantity of neural stem cell (for example seeing U.S. Patent Application Publication No.20050245436,20040136967,20040092448,20030095956,20030054998,20030054551,20030049838,20030049837) in external or body.The mechanism of described increase may comprise and stimulating proliferation, and suppresses differentiation, and/or prevents the death of neural stem cell.Therefore these molecules can be used for producing neural stem cell the experimenter of these cells of needs, produce neuron and neurogliocyte thus.

General introduction

[0004] provided herein is effective dosage regimen, its test kit and the purposes that is used for cell proliferation of nerve cord reagent and differentiation agents.Particularly, high dose is opposite with giving in a short time, and cell proliferation of nerve cord reagent low dosage in a continuous manner is delivered to mammalian subject.But the compositions of described material and the utilization of method short-term (for example, in a couple of days after nerve injury or nervous symptoms outbreak) or can utilize (for example, for the nervous symptoms in nerve injury that continues or the generation) for a long time.

[0005] corresponding, the cell proliferation of nerve cord reagent of optimization effective dose provided herein is for the method and the test kit that increase the efficient of neural stem cell quantity in the mammal, comprise and give mammal one period continuously cell proliferation of nerve cord reagent, optional by using test kit, the accumulated dose of the cell proliferation of nerve cord reagent that wherein gives in described period equals effective dose, and be at least three days wherein said period.

[0006] the cell proliferation of nerve cord reagent that the optimization effective dose also is provided herein is for treatment or improve neurodegenerative disease in the mammal or the method and the test kit of the efficient of disease, comprise and give mammal one period continuously cell proliferation of nerve cord reagent, optional by using test kit, the accumulated dose of the cell proliferation of nerve cord reagent that wherein gives in described period equals effective dose, and be at least three days wherein said period.

[0007] treatment further is provided or has improved neurodegenerative disease in the mammal or method and the test kit of disease at this.The cell proliferation of nerve cord reagent that described method comprises effective dose gives mammal one period continuously, and optional by using test kit, be at least three days wherein said period.

[0008] provides the further method for the treatment of or improve mammiferous neurodegenerative disease or disease herein in addition.Described method comprises and gives mammal with cell proliferation of nerve cord reagent and cell differentiation of nerve cord reagent, cell proliferation of nerve cord reagent whole body successive administration at least three times in the first treatment phase wherein, and wherein cell differentiation of nerve cord reagent administration in the second treatment phase, optional by using test kit.Cell proliferation of nerve cord reagent and cell differentiation of nerve cord reagent can be continuously or intermittently administration.For example, cell proliferation of nerve cord reagent can be at the 1st, the 2 and 3 day successive administration of the first treatment phase, and cell differentiation of nerve cord reagent can be at the 1st, the 2 and 3 day successive administration of the second treatment phase then.

[0009] in this method and test kit, the treatment phase can be, for example, and at least 3 days.Randomly, this method can be included in and give mammalian neural stem cells propagation reagent in the second treatment phase continuously, and is optional by utilizing test kit, and wherein the second treatment phase started from after the first treatment phase finished, at least one day at interval, and wherein the second treatment phase was at least three days.The second treatment phase as the first treatment phase, can for example be at least three days.This treatment plan can repeat several times or repeatedly, have second, third, the treatment phase such as the 4th, the 5th.No matter be administered once, twice, several times or repeatedly, this treatment plan can be taked the form of one or more test kits, and the cell proliferation of nerve cord reagent and the optional cell differentiation of nerve cord reagent of the effective dose that is used to give a particular treatment phase or a plurality of treatment phases wherein is provided.

[0010] details of this method and test kit accompanying drawing and below description in set forth.The further feature of this method and test kit, target and advantage will show from description and accompanying drawing and claim significantly.

Accompanying drawing is described

[0011] Fig. 1. in male rat with 10,15 and 20 times to the concentration h inf prolactin antagonist of Intraventricular infusion six days.Represented total amount from the bromodeoxyribouridine positive (BrdU+) cell in the chamber inferior segment (SVZ) of 8 sections of every animal.(170 μ g/ days 6 days) have observed the optimal growth of SVZ propagation level with 15 multiple doses the time.(10 times=113 μ g/ days; 20 times=226 μ g/ days; Contrast=only use rat blood serum albumin (RSA)).Significance with respect to contrast: 10x=*p＜0.05; 15x=**p＜0.01; 20x=p＜0.05; For all conditions n=3; With Tukeyposthoc test carrying out one factor analysis of variance (ANOVA).

[0012] Fig. 2. in male rat, utilize the prolactin antagonist administration of single peritoneal injection every day.Represented BrdU+ cell total amount for each section of every kind of dosage regimen.Observe the little increase of SVZ propagation during (A) with high 3 days dosage.(B) the strongest dosage condition that increases SVZ propagation level is utilized 170 low μ g/ days dosage 6 days.Significance contrasts with respect to RSA.N=3; * p＜0.05; * p＜0.01; Single factor ANOVA carries out Tukey posthoc test then.

[0013] Fig. 3. the 1st, 3 and 5 day single intramuscular injection hCG significantly increased the propagation of forebrain SVZ after apoplectic seizure.(n=3 under 1000 μ g dosage conditions; * p＜0.05; Carry out single factor ANOVA with Tukeyposthoc) observe the remarkable increase of the every ventricles of the brain phosphoric acid-histone H 3 positive (pHH3) cell quantity.Pictorial display with respect to the tricorn back of the body laterior horn center labelling Hoechst of the RSA mantle bar control rats of 1000 μ ghCG administration animals and the expression of pHH3, demonstrate the increase that pHH3 expresses in the total cell concentration and SVZ in 1000 μ g administration animals.

[0014] Fig. 4. the hCG of single intramuscular injection the 1st, 3 and 5 day every day 1000 μ g has caused the nerve that increases among the forebrain SVZ (apoplexy=0th day) has taken place after apoplexy.Doublecortin+ neuronal quantity in the administration rat is quantized, and this quantity is double in 1000 μ g dosage animals.(n＝3；**p＜0.01)

[0015] Fig. 5. beginning single intramuscular injection every day hCG7 days in 24 hours after the apoplexy (apoplexy=0th day).(A) dosage regimen of μ g/ injection every days 330 has significantly increased quantity (pHH3+ the cell) (n=3 that breeds among the SVZ with respect to all other administration conditions and contrast; * p＜0.01; Single factor ANOVA with Tukey posthoc).(B) animal of having disclosed the dosage regimen of accepting μ g/ injection every days 330 for the observation of the ischemia injury of the motor cortex of administration rat represents the tissue growth that makes new advances, and with tissue filler (tissue plug) filling pathological changes site.

[0016] Fig. 6. the propagation of the increase among the SVZ of the μ g/ injection every days 330 hCG administration animal of confirming by the BrdU+ cell counting.The BradU+ cell quantity of every ventricles of the brain significantly increases (p＜0.01 with respect to contrast and 100 μ g/ injection under 330 μ g/ injecting conditions; N=3; Single factor ANOVA with Tukey posthoc analysis).These results have further confirmed the dye increase of observed propagation with pHH3.

[0017] identical reference marker is represented identical content in different figure.

Describe in detail

[0018] the present nerve of not ratifying to be used for the treatment of clinically the nervous system disease or illness Stem cells hyperplasia and differentiation agents. These reagent are useful for treatment the nervous system disease and illness , thereby effective dosage regimen of utilizing these reagent need to be arranged. Provide for nerve cord at this Effective dosage regimen of cell proliferation reagent comprises effectively giving for nerve stem cell proliferation reagent The kit of regimen and their purposes. Particularly, with in a short time with high dose administration phase Instead, low dosage is delivered to mammalian subject with nerve stem cell proliferation reagent in a continuous manner. For example, for given total effective dose, comprise and send 1/6 total amount every day and carry out six days administration To carry out three days dosage regimen more effective than sending 1/3 total amount every day for scheme.

[0019] before method and kit are described in further detail, the application's term is following fixed Justice, except as otherwise noted. Title herein is only for organizational goal, do not mean that restriction carries herein The specification of confession or appended claim.

Definition

[0020] NSC or NSC are the stem cells in the neuronal cell line. Stem cell is can The cell of self-replacation. In other words, the daughter cell that results from stem cell division comprises stem cell. NSC can finally be divided into all cells type of neuronal cell line, comprise neuron, Astroglia and oligodendroglia (astroglia and oligodendroglia Be referred to as neuroglia or Deiter's cells). Thereby the NSC of herein mentioning is many The potential NSC.

[0021] nerve stem cell proliferation reagent is the material that can increase NSC quantity, example As, by stimulate proliferation, Inhibited differentiation and/or prevent neural stem cells death.

[0022] Neural Stem Cells Differentiation reagent is can selectively strengthen neuron to form or neuroglia The material that cell plastid forms.

[0023] continuously the experimenter is sent (deliver) or give (administer) material meaning With this material in one period at least once a day or until run through continuous a couple of days send or Give. For example, can be by injection (for example, IM or subcutaneous) or oral at least once a day complete The body administration, or by to cause that the tissue that is delivered to the experimenter every day or the mode infusion in the blood flow give Medicine. Choose wantonly, this material is sent by the mode beyond infusion or the infusion. As used herein, " whole body " word does not comprise ventricles of the brain infusion in the brain. The duration that this material is sent continuously or given Or sustainable three days to several years for the treatment of phase, even in the life of Patient's surplus, continue. For example, hold Renewing can be 3-6 days, 3-14 days, 3-21 days, 3-28 days, 1-4 month, 1-6 Individual month, 1-9 month, 1-12 month, 1-2,1-3,1-5,1-10 etc. For further embodiment, the treatment phase that continues to send can be at least about three days, at least big About four days, at least about five days, at least about six days, at least about seven days or at least about ten Four days. And this material can be sent continuously administration phase the 1st, 2 and 3 days.

[0024] neurodegenerative disease or disease are and neuron forfeiture or relevant disease or the medical conditions of dysfunction.Neurodegenerative disease or examples of disorders comprise neurodegenerative disease, central nervous system injury or dysfunction.Neurodegenerative disease comprises, for example, Alzheimer or other dementia (dimentia), multiple sclerosis (MS), schizophrenia, degeneration of macula, glaucoma, diabetic retinopathy, peripheral neurophaty, Huntington Chorea, amyotrophic lateral sclerosis and Parkinson's disease.The CNS damage comprises that for example, cerebrovascular events is as apoplexy (for example, hemorrhagic apoplexy, ischemic stroke or global brain ischemia apoplexy), eye ischemia and sinuses of dura mater thrombosis; Traumatic brain or spinal cord injury (for example, the damage that causes by brain or spinal cord surgical operation or personal injury); Concussion; By drug induced damage (for example, chemotherapy, property medicine (recreational drugs) for amusement and Antipsychotic drug); Coronary artery bypass grafting (CABG); With the ischemia in the childbirth.The CNS dysfunction comprises, for example, and depression, epilepsy, neurosis and psychosis.The example of neurodegenerative disorders comprises aging.The neural stem cell quantity of chamber inferior segment significantly reduces in old mice.Accordingly, obtain by give cell proliferation of nerve cord reagent and optional cell differentiation of nerve cord reagent according to the inventive method and test kit with the improvement of old and feeble problem relevant issues.

[0025] treatment and improvement expression reduce or remove fully one or more symptoms of disease or medical conditions.Described treatment or improve can comprise when delay when patient's administration of disease or medical conditions risk is arranged or to eliminate the outbreak of one or more symptoms.

[0026] polypeptide and the natural factor that has a basic sequence similarity with the natural factor has about at least 30% homogeneity on amino acid levels.Polypeptide and the homogeneity of the natural factor on amino acid levels are preferably about at least 40%, and is more preferably about at least 60%, even more preferably about at least 70%, and most preferably about at least 80%.Thereby basic similarity can be approximately between the 30-99% homogeneity.

[0027] statement of the homogeneity percentage ratio of analog or variant and the natural factor or homogeneity % refers to the percentage ratio of the aminoacid sequence in the natural factor that comparison is also found during two sequences in analog or variant.Homogeneity percentage ratio can be definite by method or algorithm that this area has been set up, for example, and LALIGN or BLAST.

[0028] if polypeptide can be discerned in conjunction with the receptor of the natural factor or by the polyclonal antibody at the natural factor, then this polypeptide has the biological activity of the natural factor.Preferably, polypeptide can be at the receptor of specificity in the receptors bind test in conjunction with the natural factor.

[0029] the function agonist of the natural factor is the chemical compound of the receptor of the combination and the active natural factor, although its unnecessary and natural factor has the basic sequence similarity.

[0030] lutropin or LH are a kind of albumen, and its (1) comprises with natural mammal LH and has the homophylic polypeptide of basic sequence, preferred natural human LH; (2) has natural mammal LH biologic activity.Natural mammal LH is by the excretory promoting sexual gland hormone of antepituitary.The heterodimer that LH is made up of non-covalent bonded α and β subunit.The α subunit is common at LH among FSH and the HCG, and the β subunit is special for every kind of hormone.Useful LH can have natural α subunit and have the homophylic β subunit of basic sequence with natural mammal LH in this method and test kit.Optionally, LH can have natural β subunit and have the homophylic α subunit of basic sequence with natural mammal LH.LH also can have simultaneously with natural corresponding subunit and have homophylic α subunit of basic sequence and β subunit.Thereby the term of LH comprises the LH analog, and it comprises disappearance, the insertion of natural LH subunit or replaces mutant.And term LH comprises from the LHs of other species and its naturally occurring variant.In addition, the LH analog also can be the functional agonist of natural mammal LH receptor.

[0031] human chorionic gonadotropin or hCG are a kind of albumen, and its (1) comprises with natural hCG and has the homophylic polypeptide of basic sequence; (2) has the biologic activity of natural hCG.The heterodimer that natural hCG is made up of non-covalent bonded α and β subunit.The α subunit is common in LH, FSH and hCG, and the β subunit is special for every kind of hormone.Yet, have 85% sequence similarity between the β subunit of hCG and LH.Useful hCG can have natural α subunit and have the homophylic β subunit of basic sequence with natural hCG in the present invention and the test kit.Optionally, hCG can have natural β subunit and have the homophylic α subunit of basic sequence with natural hCG.HCG also can have simultaneously with natural corresponding subunit and have homophylic α subunit of basic sequence and β subunit.Thereby term hCG has comprised the hCG analog, and it comprises disappearance, the insertion of natural hCG subunit or replaces mutant.And term hCG has comprised from the hCG homologue of other species and its naturally occurring variant.In addition, the hCG analog also can be the functional agonist of natural mammal hCG/LH receptor.

[0032] prolactin antagonist is a peptide species, and its (1) has the basic sequence similarity with natural prolactin in mammals, preferred natural human prolactin antagonist; (2) has the biologic activity of natural prolactin in mammals.The natural human prolactin antagonist is main synthetic 199 amino acid whose polypeptide in pituitary gland.Thereby the term of prolactin antagonist has comprised the prolactin antagonist analog, and it is disappearance, the insertion of natural prolactin antagonist or the mutant that replaces.And the term prolactin antagonist has comprised from the prolactin antagonist of other species and its naturally occurring variant.

[0033] in addition, prolactin antagonist also may be the functional agonist of natural prolactin in mammals receptor.For example, functional agonist can be a U.S. Patent No. 6,333, the activated amino acid sequence of disclosed hprl receptor in 031; Metal composite receptors ligand (U.S. Patent No. 6,413,952) with hprl receptor agonist activity; G120RhGH, it is human growth hormone's analog but as prolactin antagonist agonist (Mode et al., 1996); Or the hprl receptor part of describing in the U.S. Patent No. 5,506,107 and 5,837,460.

[0034] epidermal growth factor or EGF represent natural EGF or have primary amino acid sequence similarity with natural EGF, and any EGF analog or variant with at least a biologic activity of natural EGF, described biologic activity for example with the combining of EGF receptor.Particularly including interior as EGF be natural EGF, the TGF α of any species, or the EGF of recombinant modified.Special example includes but not limited to, has EGF (the EGF51 gln51 particularly of the recombinant modified that the neutral amino acid in two amino acid whose disappearances of C-terminal and site 51 replaces; U.S. Patent Application Publication No.20020098178A1), the His residue in site 16 is by the EGF mutain (EGF-X of neutrality or acidic amino acid replacement ₁₆) (U.S. Patent No. 6,191,106), lack the Met residue in the oxidized EGF-D (EGF-C) of the Met residue in EGF deletion mutant (EGF-B) that 52-aminoacid deletion mutant (EGF-D), N-terminal residue and two the C-terminal residues (Arg--Leu) of EGF of the n terminal residue of natural EGF are deleted, site 21, site 21 oxidized EGF-B (EGF-A), heparin associativity EGF like growth factor (HB-EGF), beta cell element, amphiregulin, neuregulin or comprise any above-mentioned proteic fusion rotein.Other useful EGF analog, variant and fragment is described in U.S. Patent Application Publication No.20020098178A1 and U.S. Patent No. 6,191,106 and 5,547,935.

[0035] in addition, EGF also can be the functional agonist of natural mammal EGF receptor.For example, functional agonist can be to be disclosed in U.S. Patent No. 6,333, the activated amino acid sequence of the EGF receptor in 031, or have the antibody (Fernandez-Pol, 1985 and U.S. Patent No. 5,723,115) of EGF receptor agonist activity.

[0036] pituitary adenylate cyclase activated polypeptides or PACAP represent natural PACAP or any PACAP analog or variant, described PACAP analog or variant and natural PACAP have primary amino acid sequence similarity and have at least a biologic activity of natural PACAP, for example with the combining of PACAP receptor.Useful PACAP analog and variant include but not limited to that 38 aminoacid of PACAP and 27 amino acid variant (being respectively PACAP38 and PACAP27) are disclosed in for example U.S. Patent No. 5,128,242; 5,198,542; 5,208,320; 5,326,860; 5,623,050; Analog and variant in 5,801,147 and 6,242,563.

[0037] in addition, PACAP also can be the functional agonist of natural mammal PACAP receptor.For example, functional agonist can be maxadilan, a kind of polypeptide (Moro et al., 1997) of the specific agonist as PACAP 1 receptor.

[0038] erythropoietin or EPO are natural EPO or any EPO analog or variant, described EPO analog or variant and natural EPO have the primary amino acid sequence similarity, and have an at least a biologic activity of natural EPO, for example with the combining of EPO receptor.EPO analog or variant are disclosed in, for example, and United States Patent (USP) NO.6048971 and 5614184.

[0039] in addition, EPO also can be the functional agonist of natural mammalian EPO receptor.For example, functional agonist can be EPO simulating peptide 1 (EMP1; Johnson et al., 2000); Be described in Wrighton et al., 1996 and the small peptide analogies of a kind of EPO of U.S. Patent No. 5,773,569; Be disclosed in Kaushansky, any micromolecule EPO analogies of 2001; Be described in U.S. Patent No. 5,885,574, WO 96/40231, WO 97/48729, Fernandez-Pol, 1985 or the antibody of the activation EPO receptor of U.S. Patent No. 5,723,115; Be disclosed in U.S. Patent No. 6,333, the activated amino acid sequence of 031 EPO receptor; Have the metal composite receptors ligand (U.S. Patent No. 6,413,952) of the agonist activity of EPO receptor, or be described in U.S. Patent No. 5,506,107 and 5,837,460 EPO receptors ligand.

[0040] to induce reagent be the material that can increase LH in the animal or hCG amount when giving animal to LH/hCG-.For example, LH-releasing hormone (LHRH) stimulates the secretion of LH.

[0041] pheromone is the material of conduct to the signal of another animal of same species, and described animal is generally opposite sex.Mammalian pheromon can be albumen or micromolecule.Preferably, pheromone is selected from the 2-second month in a season-butyl-4,5-thiazoline (SBT), 2,3-dehydrogenation-outer-brevicomin (DHB), α and β farnesene, 6-hydroxyl-6-methyl-3-heptanone, 2-heptanone, trans-methyl heptenone, trans-the 4-hepten-2-one, n-amyl group acetas, cis-2-amylene-1-base-acetas, 2, the 5-dimethyl pyrazine, dodecyl propionic ester and (Z)-7-laurylene-1-yl acetate (is seen, for example, Dulac et al., 2003).

[0042] effective dose is the amount that is enough to obtain expect the therapeutic agent of purpose.For example, increasing the LH of neural stem cell quantity or the effective dose of hCG is as being enough in vivo or the external amount that causes neural stem cell quantity to increase of may becoming under this situation.LH or hCG treatment or the effective dose that improves neurodegenerative disease or disease are the amounts of LH/hCG that is enough to reduce or removes one or more symptoms of neurodegenerative disease or disease.The effective dose of given therapeutic agent will be along with the factor of the animal size of for example reagent character, route of administration, the agent of receiving treatment and species and administration target and is changed.Those skilled in the art can be according to the effective dose in rule of thumb definite each the individual case of the method that this area has been set up.

[0043] equivalent of cell proliferation of nerve cord reagent is the amount that obtains the effect needed cell proliferation of nerve cord reagent identical or equivalent with another kind of cell proliferation of nerve cord reagent.Equivalent can describe in detail by the level relatively or the result of equivalent.Thereby equivalent or dose,equivalent can be to obtain the serum identical with another kind of specific cell proliferation of nerve cord reagent or the amount or the dosage of the needed cell proliferation of nerve cord reagent of cerebrospinal fluid level.

[0044] drug delivery device is to be suitable for giving the cell proliferation of nerve cord reagent of effective dose or the article of differentiation agents.Drug delivery device can influence the cell proliferation of nerve cord reagent of any method of having set up by this area or the administration of differentiation agents, described method for example comprises, in intravenous, intra-arterial, colonic (intracolonical), the trachea, in the intraperitoneal, intranasal, blood vessel, in the sheath, in the intracranial, bone marrow, in the pleura, Intradermal, subcutaneous, intramuscular, intraperitoneal, oral, topical, pulmonary administration or its any combination.Drug delivery device can be implantable device or pump, for example comprises osmotic pumps.Randomly, drug delivery device is infusion device or its element, or optionally, is the device outside the infusion.

Send continuously

[0045] for improving the dosage regimen of prolactin antagonist, gives every day rat not commensurability prolactin antagonist, carried out 6 days, check effect (embodiment 1) for neural stem cell quantity.It is optimised quantity in this drug dosage schedule that the result shows 170 μ g/ days.This dosage regimen, 170 μ g/ days 6 days, the administration phase changed (170 μ g/ days 3 days) or dosage every day that will be higher makes up to obtain similar accumulated dose (396 μ g/ days 3 days) with shortening period by shortening then.Sending low dosage more in the result is presented at more over a long time continuously is more effective than the combination of high dose and shorter Delivery time more.

[0046] correspondingly, optimization effective dose cell proliferation of nerve cord reagent provided herein is for the method for the effect that increases mammalian neural stem cells quantity, be included in and give mammalian neural stem cells propagation reagent in one period continuously, the accumulated dose of the cell proliferation of nerve cord reagent that wherein gives in described period equals effective dose, and be at least three days wherein said period.

[0047] provide the cell proliferation of nerve cord reagent of optimization effective dose for the method for the treatment of or improve the effect of the neurodegenerative disease in the mammal, wherein this method comprises and gives mammalian neural stem cells propagation reagent one period continuously, wherein in described period, give mammiferous cell proliferation of nerve cord reagent accumulated dose and equal effective dose, and be at least three days wherein said period.

[0048] also provide the method for the treatment of or improve neurodegenerative disease in the mammal, comprised one period of cell proliferation of nerve cord reagent that gives the mammal effective dose continuously, be at least three days wherein said period.

[0049] also provides treatment or improve neurodegenerative disease in the mammal or the further method of disease herein.This method comprises and gives mammalian neural stem cells propagation reagent and cell differentiation of nerve cord reagent, wherein cell proliferation of nerve cord reagent continuously whole body administration at least three times in the first treatment phase, and wherein cell differentiation of nerve cord reagent administration in the second treatment phase.

[0050] method provided herein, for example, the propagation reagent of experimenter's effective dose that can be by having neurodegenerative disease or disease utilizes propagation reagent prolactin antagonist, hCG, LH, G-CSF, GM-CSF, pheromone or VEGF treatment neurodegenerative disease or disease.For instance, propagation reagent hCG and LH be in conjunction with identical receptor, and in the specific embodiment that provides herein interchangeably with the dose,equivalent utilization.As further embodiment, approximately 120-200IU/kg/ days dosage intramuscular (IM) administration of propagation reagent hCG, intravenous (IV) gives about 570-950IU/kg/ days EPO then.For further embodiment, dosage intramuscular that can 160IU/kg/ days gives hCG, and intravenous gives 765IU/kg/ days EPO then.The administration of described neural stem cell reagent and then a couple of days give for example differentiation agents of EPO.Other cell proliferation of nerve cord reagent of dose,equivalent also can be used in the similar scheme.

[0051] also provides test kit herein, described test kit provides the cell proliferation of nerve cord reagent of effective dose, the described cell proliferation of nerve cord reagent that comprises a kind of dosage that is used for the whole treatment phase, operation instructions with this test kit, the cell proliferation of nerve cord reagent accumulated dose that wherein gives in the described treatment phase equals effective dose, and the described treatment phase is at least three days.

[0052] test kit can further be provided for the differentiation agents of a kind of dosage of whole treatment phase, and the differentiation agents accumulated dose that wherein gives in the described treatment phase equals effective dose, and the wherein said treatment phase is at least three days.

[0053] accumulated dose of every kind of each cell proliferation of nerve cord reagent in the test kit, differentiation agents or other reagent can be in a container, in a plurality of container or in its any combination, provide.For example, the accumulated dose of one or more cell proliferation of nerve cord reagent can be suitable for providing the dosage of metering or is suitable for extracting in the container of dosage at one, described extraction for example by people to be treated or by other people for example the care-giver carry out.One or more cell proliferation of nerve cord reagent can be present in a plurality of containers rather than in the single container, described a plurality of containers provide be suitable for every day, weekly, the dosage of the aliquot of administration such as every month.The single container or a plurality of container that are used for differentiation agents or other reagent can provide at this test kit similarly.Also can comprise combination, wherein can comprise the cell proliferation of nerve cord reagent of a container in the test kit, the differentiation agents of a plurality of containers, or opposite.And the accumulated dose that is used for the cell proliferation of nerve cord factor of the first treatment phase can be in single container or a plurality of container, and the accumulated dose of the second treatment phase can be in single container or in a plurality of container, or its combination.

[0054] test kit can further comprise the device or the equipment of the hematocrit levels in the monitored patient or get the proper device of a certain amount of blood or comprise monitoring arrangement and blood sampling device simultaneously from the patient.Blood sampling and supervision need, and are higher than acceptable level because hematocrit levels may rise to.Acceptable hematocrit levels can be determined by the standard that any this area has been set up.

[0055] test kit is applicable to health care facility, for example inpatient care institutions vibrations or critical care mechanism.Health care facility comprises for example hospital.Test kit also is suitable for after leaving the inpatient care institutions vibrations or uses when not entering.Help the patient with the packing of kit form and leave health care facility early, by allowing in long term care facilities or patient treatment at home, for example, or at home or the medium treatment of long term care facilities by care-giver or health care supplier by Heal Thyself, out-patient treatment.

[0056] in this method and test kit, period, (just, the treatment phase) can be for example about at least 3,4,5,6,7,8,9,10,11,12,14,21,28 days, or any natural law between 3 to 28 days.Choose wantonly, this method and test kit can be included in the interim mammalian neural stem cells propagation reagent that gives continuously of second treatment, and after wherein the second treatment phase started from this period and finish, and wherein the second treatment phase was at least three days.The second treatment phase, the similar first treatment phase, can be for example about at least 3,4,5,6,7,8,9,10,11,12,14,21 or 28 days.Interval between the first treatment phase and ensuing treatment phase also can be for example about at least 1,2,3,4,5,6,7,8,9,10,11,12,14,21 or 28 day.This treatment schedule can repeat several times or repeatedly.Be used for second or the cell proliferation of nerve cord reagent of treatment phase afterwards can be used for for the first treatment phase or other cell proliferation of nerve cord reagent for the treatment of interim use is identical or different.And, at interim more than one cell proliferation of nerve cord reagent that utilizes of single treatment.Thereby the useful reagent box can comprise one or more cell proliferation of nerve cord reagent that are used for one or more treatment phases in this method.

[0057] any method administration that can set up by this area of cell proliferation of nerve cord reagent, for example, by in intravenous, intra-arterial, colonic, the trachea, in the intraperitoneal, intranasal, blood vessel, in the sheath, in the intracranial, marrow, in the pleura, Intradermal, subcutaneous, intramuscular, oral, topical, lung administration or its combination in any.Choose wantonly, the drug delivery device or its element that are used for administration can be included in the test kit that has comprised cell proliferation of nerve cord reagent.

[0058] method described herein also can comprise the level of cell proliferation of nerve cord reagent in the monitoring mammalian biological liquid or cell differentiation of nerve cord reagent.The monitoring biological fluid can be, for example, cerebrospinal fluid or blood.For example, the hCG in the serum (or another kind of cell proliferation of nerve cord reagent or cell differentiation of nerve cord reagent) level can measured among the treatment phase or after the administration afterwards.The different cell proliferation of nerve cord reagent or the cell differentiation of nerve cord reagent of equivalent level all can be determined in biological fluid and be monitored.

[0059] specific dosage device (just, in the treatment phase in a series of administrations the amount of single-dose) can be to the cell proliferation of nerve cord that is used for method disclosed herein or differentiation agents and specialization.These dosage devices are herein among the given dose and dosage range of specialization.Dosage device can be obtained the cell proliferation of nerve cord of aspiration level among the experimenter or the amount of differentiation agents limits according to giving.For example, be provided at 0.03IU/L to 5 in the serum, 000, the cell proliferation of nerve cord reagent dosage unit of the cell proliferation of nerve cord of the level of 000IU/L or differentiation agents.Perhaps, as further embodiment, be provided in the cerebrospinal fluid approximately 0.003IU/L to about 5, the cell proliferation of nerve cord or the differentiation agents dosage device of the propagation reagent level of 000IU/L.

[0060] in the operation instruction of this method and this test kit, cell proliferation of nerve cord reagent systemic delivery, more preferably the whole body administration is at least once a day.In some embodiments, cell proliferation of nerve cord reagent is not sent by infusion.

[0061] cell proliferation of nerve cord reagent can be can be external or increase any material of mammalian neural stem cells quantity in vivo.Promotion reagent used herein has identical meanings with propagation reagent.The reagent that can increase neural stem cell quantity includes but not limited to:

1. follicle stimulating hormone (FSH), it is often cooperated mutually with the effect of LH, and induce the LH expression of receptor, thereby strengthens the effect of LH signal conduction.

2. growth hormone (GH), but its stimulation of neural stem cells propagation.

3. insulin-like growth factor (IGFs) comprises IGF-1, and it is from response to discharging many tissues of GH and the somatomedin (somatomedians) of many growing multiplication effects of mediation GH, and its stimulation of neural stem cells propagation.

4. growth hormone releasing hormone (GHRH), it is secreted from hypothalamus, and induces GH to discharge from antepituitary, causes the level of the increase of circulation GH.。

5. prolactin antagonist (PRL), it is secreted from antepituitary and promotes cell proliferation of nerve cord.

6. prolactin antagonist release peptide (PRP), it causes the release of prolactin antagonist.

7. fibroblast growth factor (FGF), the mitogenesis reagent of neural stem cell.

8. estrogen, it promotes proliferation of neural stem cells, comprises for example in Hippocampus.

9. serotonin, it promotes proliferation of neural stem cells in Hippocampus.

10. epidermal growth factor (EGF), the mitogenesis reagent of neural stem cell.

11. transforming growth factor (TGF α), the mitogenesis reagent of neural stem cell.

12. gonadotropin releasing hormone (GnRH), it causes the release of LH.

13. ciliary neurotrophic factor (CNTF) and leukaemia inhibitory factor (LIF), by gp130 subunit conducted signal, described signal transduction path promotes the neural stem cell self renewal by a kind of signal transduction path for it, thereby amplification stem cell of cranial nerve colony.

14. granulocyte colony-stimulating factor (G-CSF).

15. granulocyte-macrophage colony stimutaing factor (GM-CSF).

16. VEGF (VEGF).

17. lutropin (LH).

18. human chorionic gonadotropin (hCG).

19. pheromone.

[0062] and, can give the neurocyte differentiation agents and strengthen with selectivity that neuron forms or neurogliocyte forms.These differentiation agents also can be sent according to dosage regimen and test kit.Exemplary differentiation agents includes but not limited to:

1. erythropoietin (EPO), it is promoted neural stem cell and is fixed to the neurocyte pedigree, and is useful for the apoplexy model of treatment mice and rat.

2. be derived from the neurotrophic factor (BDNF) of brain, it is the survival factors and the differentiation agents of the neural pedigree of a kind of known promotion.

3. transforming growth factor and bone morphogenetic protein (BMPs), it is the differentiation agents that promotes the generation of neural pedigree and specific neural phenotypes (for example, the sensation relay cell in the spinal cord).

4. thyroxin (TH comprises T3 and T4 form), a kind of maturation of oligodendroglia and differentiation agents of generation of promoting seen for example Rodriguez-Pena, 1999.

5. thyrotropin (TSH) and thyroid releasing hormone (TRH), it promotes that TH discharges from antepituitary, produce the circulation TH of increase level.This reagent can be used in combination to promote the oligodendroglia generation from neural stem cell with LH or hCG.

6. sound hedgehog albumen (sonic hedgehog) (SHH), a kind of morphogen that in growth course, makes the CNS shaping of growth, and promote the neuron (for example, the motor neuron in the spinal cord) of particular type and the generation of oligodendroglia with different concentration.This reagent can be used in combination to promote nerve generation and/or the oligodendroglia generation from neural stem cell with LH or hCG.

7. platelet derived growth factor (PDGF), it promotes generation and the differentiation of oligodendroglia from neural stem cell.This reagent can be used in combination to promote the oligodendroglia generation from neural stem cell with LH or hCG.

8. ring AMP and strengthen the reagent of cAMP approach, for example Pituitary adenylate cyclase-activating polypeptide.. (PACAP) and serotonin, its selectivity promotes neuronic generation.

[0063] any method and test kit can comprise multiple cell proliferation of nerve cord reagent and/or neurocyte differentiation agents.Thereby one or more cell proliferation of nerve cord reagent can be jointly or administration in succession, and can be by dividing other compositions or being combined in form administration in the single compositions.And one or more cell proliferation of nerve cord reagent and one or more cell differentiation of nerve cord reagent can be jointly or administration in succession, and can be by dividing other compositions or being combined in form administration in the single compositions.For example, PRL and LH or hCG use capable of being combined is so that the cell proliferation of nerve cord maximization; PRP can make up with LH or hCG so that the cell proliferation of nerve cord maximization; GnRH can be used in combination or replace cyclical level and the enhancing cell proliferation of nerve cord of its use to increase LH with LH or hCG; And CNTF and LIF can be used in combination to promote the size of neural stem cell group among cell proliferation of nerve cord and the increase CNS with LH or hCG.Further for example, prolactin antagonist can use with EPO, and LH can use with EPO, and hCG can use with EPO.All other combinations of clearly setting forth can not used yet.

[0064] the suitable dosage of these factors can be determined according to the method that this area has been set up.For example, the dosage range of prolactin antagonist for can be from about 0.510IU/kg/ days to about 100,000IU/kg/ days, for example, about 0.510-90,000; 0.510-75,000; 0.510-50,000; 0.510-25,000; 0.510-10,000; 100-5,000; 100-2,000; 500-2,000; 1,000-2,000; 100-1,000; 200-800IU/kg/ days.The dosage range of hCG can be from about 0.5IU/kg/ days to about 3,000,000IU/kg/ days, for example, about 0.5-2,000,000; 0.5-1,000,000; 0.5-500,000; 0.5-250,000; 0.5-100,000; 0.5-50,000; 10-25,000; 10-10,000; 240-216,000; 1,200-2,000; 2,160; Or 1,600IU/kg/ days.HCG also can 10, and 000IU/ days dosage provides.The dosage range of LH can be from about 0.5IU/kg/ days to about 500,000IU/kg/ days, for example, about 0.5-300,000; 0.5-200,000; 0.5-100,000; 0.5-50,000; 0.5-25,000; 24-21,600; 1,000; 120-200; 216; Or 160IU/kg/ days.LH also can 10, and 000IU/ days dosage provides.The dosage range of EPO can be from about 100IU/kg/ days to about 2000IU/kg/ days, for example, and about 100-1500; 100-1000; 160-1000; 570-950; 765; Or 1020IU/kg/ days.EPO also can 30, and 000IU/ days dosage provides.Can utilize other reagent of dose,equivalent.Unless otherwise indicated, dosage herein refers to the mean dose sent every day.

[0065] cell proliferation of nerve cord reagent and differentiation agents are optional is packaged in the test kit, so that comprise the interim cell proliferation of nerve cord reagent to be sent of treatment and the total amount of differentiation agents in the test kit.Test kit can be chosen the compositions that comprises other composition or other composition wantonly, comprises for example blood sampling device or its assembly.

[0066] any method administration that can set up by this area of differentiation agents, for example by in intravenous, intra-arterial, colonic (intracolonical), the trachea, in the intraperitoneal, intranasal, blood vessel, in the sheath, in the intracranial, bone marrow, in the pleura, Intradermal, subcutaneous, intramuscular, oral, topical or its any combination.Randomly, the test kit that comprises differentiation agents can comprise the drug delivery device that is used for administration.

[0067] cell proliferation of nerve cord reagent can be in about 14 days (for example, 0 to about 14 days) give the mammal of central nervous system (CNS) damage, paresthesia epilepsy or diagnosis.As used herein, the time that referred to CNS damage, paresthesia epilepsy or diagnosis in 0 day.Choose wantonly, cell proliferation of nerve cord reagent can give in CNS damage, paresthesia epilepsy or diagnosis back about 13,12,11,10,9,8,7,6,5,4,3,2 or 1 days (for example, 0 to about 5 days).Choose wantonly, cell proliferation of nerve cord reagent can give mammal in CNS damage, paresthesia epilepsy or diagnosis about 24 hours.Choose wantonly, cell proliferation of nerve cord reagent can give mammal in CNS damage, paresthesia epilepsy or diagnosis about 12,11,10,9,8,7,6,5,4,3,2,1 hours.

[0068] utilizing the mammal of method described herein and test kit treatment can be any age, comprises the child, teenager or adult.

Embodiment

[0069] in the following embodiments, following abbreviation has the following meaning.Undefined abbreviation has the meaning of generally being accepted.

℃=degree centigrade

Hr=hour

Min=minute

μ M=micromole

The mM=mM

Every milliliter of M=mole (molar ml milliliter)

μ l=microlitre

The mg=milligram

μ g=microgram

The FBS=hyclone

The PBS=phosphate buffered saline (PBS)

DMEM=Dulbecco ' s improvement Eagle ' s medium

Eagle ' the s medium of MEM=improvement

The EGF=epidermal growth factor

The NSC=neural stem cell

SVZ=chamber inferior segment

PACAP=pituitary adenylate cyclase activated polypeptides

The BMP=bone morphogenetic protein

RSA=rat blood serum albumin

Embodiment 1

The successive administration of prolactin antagonist

[0070] in two prolactin antagonist administration experiments, utilizes male rat (250-350g).Subcutaneous once a day little osmotic pumps infusion (Alzet Micropump) gives prolactin antagonist.Prolactin antagonist stock solution be diluted in the bicarbonate buffer and the rat blood serum albumin (RSA) of stock solution 1mg/ml in injection saline in further dilution.Rat is not accepted ischemia injury.The 6th day animal in 10 hours, accept 6 BrdU injections (Sigma-Aldrich) (60mg/kg, i.p.), and execution in 30 minutes after last BrdU injection.Brain is frozen cutting, utilizes the BrdU+ cell among 8 section quantifications of every animal SVZ.Shown in legend, the result is expressed as the BrdU+ cell total amount among the SVZ or is expressed as the average of every section.

Experiment #1:

[0071] rat with following dosed administration 6 days and accept h inf RSA (contrast) or rat PRL (National Hormone and Peptide Program, Torrance, CA) (every group of 3 rats):

* 10x=99ul/ pump (2mg/0.25ml PRL) is-113 μ g/ days

* 15x=148.5ul/ pump (2mg/0.25ml PRL)-170 μ g/ days

* * 20x=198ul/ pump (2mg/0.25ml PRL)-226 μ g/ days

Wherein

* the dosage (about 11 μ g/ days) that doubly gives of 10x=10 to the Intraventricular infusion.

The dosage that * 15x=15 doubly gives to the Intraventricular infusion.

The dosage that * * 20x=20 doubly gives to the Intraventricular infusion.

The result:

[0072] as shown in Figure 1, produced the maximum increase of propagation (BrdU+ cell quantity) among the forebrain SVZ in 170 μ g/ days.

Experiment #2:

[0073] rat is with following dosed administration 3 days and accept single intraperitoneal injection RSA or rat PRL (National Hormone and Peptide Program, Torrance, CA) (every group of 3 rats) every day.

70 μ g/ days 3 days

396 μ g/ days 3 days

170 μ g/ days 6 days

The result:

[0074] as shown in Figure 2, sending the maximum that produced propagation (BrdU+ cell quantity) among the forebrain SVZ in 6 days in 170 μ g/ days increases.

Embodiment 2

The successive administration of hCG

[0075] purpose of this research is to determine to make the cell proliferation and the maximized hCG dosage of tissue regeneration in adult male rat forebrain germ band, and described rat has been subjected to mantle bar (pialstrip) the devasation ischemia injury of motor cortex.

Method

Animal and surgical operation

[0076] 250-350g male rat mantle bar devasation ischemia injury (the Gonzalez and Kolb.A comparison of different modelsof stroke on behaviour and brain morphology.Eur J Neurosci.2003.18 (7): 1950-1962) that is subjected to motor cortex as discussed previously.For penta crust than wanting sodium to anaesthetize the animal of (60mg/kg), from bregma point forward/backward+4 to-2mm with from rectangle hole of horizontal 1.5 to the 4.5mm brills of center line.The cotton swab of removing cerebral dura mater and utilizing SSS is from cortical surface wiping pia mater encephali and the blood vessel that adheres to.

Administration

[0077] beginning (after 24 hours) in 1 day after the apoplexy, animals received single intramuscular (i.m.) injection human chorionic gonadotropin.The dosage that gives is as described in Table 1, and three injections (administration in the 1st, 3 and 5 day) or every day injection and in 9:00am administration every day in the week in 5 days.Control rats is accepted the rat blood serum albumin injection (RSA in the saline; Sigma, 1mg/ml).On the same day of administration in the end, animal was accepted 6 BrdU injections in 10 hours, and hCG injects beginning in back 30 minutes.BrdU (Sigma-Aldrich) is with the dosage of 60mg/kg, ip administration.Animal is with the saturating heart infusion of 4% paraformaldehyde.The cutting brain, freezing preservation and freezing cutting in sucrose.With 14 microns freezing 8 cuttings that are cut into two series, each cutting has 8 sections.Utilize rabbit to resist-phosphoric acid histone H 3 (phosphohistone H3) (anti-pHH3; 1: 100; Upstate Biotechnologies), rat anti-BrdU is (1: 100; Seralab), goat resists-doublecortin (DCX; 1: 100; Santa Cruz Biotechnologies) carries out immunostaining.Phosphoric acid histone H 3 (phosphohistone H3) (pHH3-mitosis-competent cell label), BrdU and doublecortin (the immature neuronic label of DCX-) positive cell quantity in the preceding ventricles of the brain inferior segment (SVZ) around the tricorn of each animal quantize in 8 sections, and are expressed as the positive cell par of each tricorn.

Statistics

[0078] numerical value is meansigma methods+meansigma methods standard error (SEM).Significance uses one factor analysis of variance to carry out Tukey HSD posthoc test (* p＜0.05 then; * p＜0.01) determines.Comprise three animals in every group.

The result

[0079] this research has checked that intramuscular injection hCG promotes the ability of the propagation of the neural stem cell in the ventricles of the brain inferior segment (SVZ) and CFU-GM before the apoplexy xenogenite.Animal has carried out the ischemic injury of mantle bar devasation surgical operation with the induced movement cortex, and treatment is beginning after 24 hours.In the high dose strategy of the dense notes of bullet, generalized as table 1, animal (after apoplexy 24 hours) in five days the 1st was accepted the hCG of 3 dosage in 3 and 5 days.For analyzing the propagation level of forebrain SVZ, the 5th day execution animal.As table 2 and shown in Figure 3, it is effective that this scheme is compared increasing on the propagation with the apoplexy animal of having accepted RSA contrast injection.In the dosage of 1000 μ g, propagation has improved almost 2.5 times, and as shown in Figure 4, the doublecortin positive (DCX+) neuronal quantity newly-generated among these animal SVZ significantly increases similarly.

[0080] in another research, generalized as table 1, animal is accepted the hCG administration every day, carries out 7 days, and beginning in 24 hours gave animal BrdU 10 hours at the 7th day after the apoplexy, put to death then.Shown in Fig. 5 A, as showing by the Phh3 immunoreactivity, other group significantly increases somatoblast quantity with respect to all in the group of 330 μ g/ injection among the SVZ.This increase is by confirming (Fig. 6) with respect to BrdU+ cell quantity among these animal SVZ of RSA contrast quantification.With respect to mantle bar RSA contrast, in 100 μ g treatment groups, there is the trend (Fig. 5 A and 6) that increases.Notice that not treating animal among Fig. 5 does not accept injection and do not have mantle bar apoplexy.As internal contrast, winding was subjected to the identical accumulated dose (seeing Table 1) of group with 330 μ g/ injection, but hCG to be to accept three injections of 770 μ g/ injection at the 1st, 3 and 5 day, and animal was put to death at the 5th day.Based on this research, the hCG administration of the low routine dose that μ g/ injects is the most effective for the propagation among the forebrain SVZ after the increase cerebral ischemic injury with every days 330.

[0081] for to determine whether that any dosage regimen may produce the growth of new cortical tissue, we have analyzed the infringement site in the cortex of hCG treatment animal.Tissue regeneration length is especially significantly (Fig. 5 B) in the animal groups of the low conventional dosage regimen administration of μ g/ injection every days 330.

Embodiment 3

HCG is the successive administration of EPO then

[0082] suffer neurodegenerative disease or disease mammal can with three once a day the hCG of IM dosage (10000IU/ days) the 1st, treatment in 2 and 3 days, be one day wash out the phase (the 4th day) then, the EPO (30000IU/ days) that carries out three I.V. dosage once a day then was the 5th, 6 and 7 day treatment.First IM hCG dosage can sent between 24 to 48 hours behind the neurodegenerative disease onset, described neurodegenerative disorders for example in to severe apoplexy incident.The patient can be in therapeutic process 6 weeks and checking in 3 months after several points and the apoplectic seizure.The baseline evaluation can comprise clinical/safety, neurological, hematology and blood vessel state, and brain MRI.The evaluation of clinical/safety, neurological, hematology and blood vessel state will repeat finishing the treatment back in the 1st day, 15 days and 80 days.Brain MRI will finish treatment (after outbreak of sacred disease or disease or the diagnosis about 90 days) repetition in 80 days afterwards for the contrast purpose.

[0083] any patent of mentioning in description or open source literature have all been represented the those of skill in the art's in the field that the present invention relates to level.These patents and open source literature are incorporated herein by reference in full at this, special and independent being incorporated herein by reference as every piece of independent document.

[0084] the invention is not restricted to the scope of disclosed embodiment among the embodiment, embodiment is intended to the illustration as some aspect of the present invention, and the embodiment of any functional equivalent within the scope of the invention.Of the present invention multiple modification outside this displaying and description is significantly to those skilled in the art, and falls within the scope of the appended claims.And although have only some representative combination of compositions disclosed herein to carry out special discussion in the above-described embodiment, other combination of compositions is significantly to those skilled in the art, and also falls within the scope of the appended claims.Thereby the combination of step or compositions can clearly be mentioned at this; Yet, although other combination of step or compositions clearly set forth be also included within.

Table 1.hCG administration strategy.Rat began to accept three intramuscular (I.M.) hCG injection or injection every day in 7 days in 5 days in 24 hours after apoplexy.Control rats is only accepted the RSA injection.

Table 2. for mantle bar devasation apoplexy after 24 hours with the animal of hCG administration pHH3+ with respect to contrast, the quantification of BrdU+ and DCX+ cell is expressed as the actual value ± SEM of every tricorn positive cell par.

Claims (77)

1. offer the cell proliferation of nerve cord compositions and methods of mammal effective dose, be included in and give mammalian neural stem cells propagation reagent in the first treatment phase continuously, wherein the cell proliferation of nerve cord reagent accumulated dose that gives in the described first treatment phase equals effective dose, and the wherein said first treatment phase is at least three days.

2. the process of claim 1 wherein persistent period of the first treatment phase be selected from least four days, at least five days, at least six days, at least seven days and fortnight at least.

3. the method for claim 1 further is included in and gives mammalian neural stem cells propagation reagent in the second treatment phase continuously, and wherein the second treatment phase started from after the end of the first treatment phase, and wherein the second treatment phase was at least three days.

4. claim 1,2 or 3 method, wherein cell proliferation of nerve cord reagent is by at least systemic injection and administration every day.

5. claim 1,2 or 3 method, wherein cell proliferation of nerve cord reagent is not by the infusion administration.

6. claim 1,2 or 3 method, wherein cell proliferation of nerve cord reagent is selected from prolactin antagonist, hCG, growth hormone, IGF-1, LH, CSF, GM-CSF, VEGF and pheromone.

7. the process of claim 1 wherein that cell proliferation of nerve cord reagent is hCG.

8. the method for claim 7, the amount that wherein gives mammiferous hCG be 0.5IU/kg/ days to about 3,000,000IU/kg/ days.

9. the method for claim 7, the amount that wherein gives mammiferous hCG are about 10,000IU/ days.

10. the process of claim 1 wherein that cell proliferation of nerve cord reagent is prolactin antagonist.

11. the method for claim 10, the scope that wherein gives the amount of mammiferous prolactin antagonist be 0.510IU/kg/ days to about 100,000IU/kg/ days.

12. the method for claim 1 further comprises and gives the mammalian neural stem cells differentiation agents.

13. the method for claim 12, wherein cell differentiation of nerve cord reagent is selected from EPO, PACAP, TH, TSH and PDGF.

14. the process of claim 1 wherein that mammal is an adult.

15. the cell proliferation of nerve cord reagent that effective dose is provided is with treatment or improve neurodegenerative disease in the mammal or the method for disease, be included in and give mammalian neural stem cells propagation reagent in the first treatment phase continuously, wherein the accumulated dose of the cell proliferation of nerve cord reagent that gives in the described first treatment phase equals effective dose, and the wherein said first treatment phase is at least three days.

16. the method for claim 15, wherein the persistent period of the first treatment phase be selected from least four days, at least five days, at least six days, at least seven days and fortnight at least.

17. the method for claim 15 further is included in and gives mammalian neural stem cells propagation reagent in the second treatment phase continuously, wherein the second treatment phase started from after the end of the first treatment phase, and wherein the second treatment phase was at least three days.

18. claim 15,16 or 17 method, wherein cell proliferation of nerve cord reagent is by at least systemic injection and administration every day.

19. claim 15,16 or 17 method, wherein cell proliferation of nerve cord reagent is not by the infusion administration.

20. claim 15,16 or 17 method, wherein cell proliferation of nerve cord reagent is selected from prolactin antagonist, hCG, growth hormone, IGF-1, LH, G-CSF, GM-CSF, VEGF and pheromone.

21. the method for claim 15, wherein cell proliferation of nerve cord reagent is prolactin antagonist.

22. the method for claim 21, the scope that wherein gives the amount of mammiferous prolactin antagonist are 1 μ g/kg/ days to about 300,000 μ g/kg/ days.

23. the method for claim 15, wherein cell proliferation of nerve cord reagent is hCG.

24. the method for claim 23, the amount that wherein gives mammiferous hCG are 1 μ g/kg/ days to about 300,000 μ g/kg/ days.

25. the method for claim 23, the amount that wherein gives mammiferous hCG are about 1000 μ g/ days.

26. the method for claim 15 further comprises and gives the mammalian neural stem cells differentiation agents.

27. the method for claim 26, wherein cell differentiation of nerve cord reagent is selected from EPO, PACAP, TH, TSH and PDGF.

28. claim 15,16 or 17 method, wherein mammal is an adult.

29. claim 15,16 or 17 method, wherein neurodegenerative disease is selected from Alzheimer, Huntington Chorea, amyotrophic lateral sclerosis, Parkinson's disease, CNS damage, multiple sclerosis and schizophrenia.

30. claim 15,16 or 17 method, wherein first dosage of cell proliferation of nerve cord reagent gives mammal in the paresthesia epilepsy of neurodegenerative disease or disease or diagnosis 14 days.

31. claim 15,16 or 17 method, wherein first dosage of cell proliferation of nerve cord reagent gives mammal in the paresthesia epilepsy of neurodegenerative disease or disease or diagnosis 5 days.

32. treat or improve the method for mammiferous neurodegenerative disease or disease, be included in the cell proliferation of nerve cord reagent that gives the mammal effective dose in the first treatment phase continuously, the wherein said first treatment phase is at least three days.

33. the method for claim 32, wherein the persistent period of the first treatment phase be selected from least four days, at least five days, at least six days, at least seven days and fortnight at least.

34. the method for claim 32 further is included in and gives mammalian neural stem cells propagation reagent in the second treatment phase continuously, wherein the second treatment phase started from after the end of the first treatment phase, and wherein the second treatment phase was at least three days.

35. claim 32,33 or 34 method, wherein cell proliferation of nerve cord reagent is by at least systemic injection and administration every day.

36. claim 32,33 or 34 method, wherein cell proliferation of nerve cord reagent is not by the infusion administration.

37. claim 32,33 or 34 method, wherein cell proliferation of nerve cord reagent is selected from prolactin antagonist, hCG, growth hormone, IGF-1, LH, G-CSF, GM-CSF, VEGF and pheromone.

38. the method for claim 32, wherein cell proliferation of nerve cord reagent is hCG.

39. the method for claim 38, the amount that wherein gives mammiferous hCG are 1 μ g/kg/ days to about 300,000 μ g/kg/ days.

40. the method for claim 38, the amount that wherein gives mammiferous hCG are about 1000 μ g/ days.

41. the method for claim 32, wherein cell proliferation of nerve cord reagent is prolactin antagonist.

42. the method for claim 41, the scope that wherein gives the amount of mammiferous prolactin antagonist are 1 μ g/kg/ days to about 300,000 μ g/kg/ days.

43. the method for claim 32 further comprises and gives the mammalian neural stem cells differentiation agents.

44. the method for claim 43, wherein cell differentiation of nerve cord reagent is selected from EPO, PACAP, TH, TSH and PDGF.

45. claim 32,33 or 34 method, wherein mammal is an adult.

46. claim 32,33 or 34 method, wherein neurodegenerative disease is selected from Alzheimer, Huntington Chorea, amyotrophic lateral sclerosis, Parkinson's disease, CNS damage, multiple sclerosis and schizophrenia.

47. claim 32,33 or 34 method, wherein first dosage of cell proliferation of nerve cord reagent gives mammal in the paresthesia epilepsy of neurodegenerative disease or disease or diagnosis 14 days.

48. claim 32,33 or 34 method, wherein first dosage of cell proliferation of nerve cord reagent gives mammal in the paresthesia epilepsy of neurodegenerative disease or disease or diagnosis 5 days.

49. the method for claim 43, wherein cell differentiation of nerve cord reagent is EPO.

50. the method for claim 49, the amount that wherein gives mammiferous EPO are about 100-2000IU/kg/ days.

51. the method for claim 49, the amount that wherein gives mammiferous EPO are about 570-950IU/kg/ days.

52. the method for claim 49, the amount that wherein gives mammiferous EPO are 765IU/kg/ days.

53. the method for claim 49, the amount that wherein gives mammiferous EPO are about 30,000IU/ days.

54. the test kit of the cell proliferation of nerve cord reagent of effective dose is provided, comprises:

(a) be used for the dosage of the described cell proliferation of nerve cord reagent of successive administration in the first treatment phase, wherein cell proliferation of nerve cord reagent accumulated dose to be given equals to treat or improve the effective dose of mammalian nervous degenerative disease in the described first treatment phase, and the wherein said first treatment phase is at least three days; With

(b) use the operation instruction of this test kit.

55. the test kit of claim 54, wherein the persistent period of the first treatment phase is selected from least seven days and at least two ten eight days.

56. the test kit of claim 54, further comprise second dosage that is used for the cell proliferation of nerve cord reagent of successive administration in the second treatment phase, wherein cell proliferation of nerve cord reagent accumulated dose to be given equals effective dose in the described second treatment phase, and the wherein said second treatment phase is at least three days.

57. claim 54,55 or 56 test kit further comprise at least a drug delivery device.

58. claim 54,55 or 56 test kit, wherein said cell proliferation of nerve cord reagent is selected from prolactin antagonist, hCG, growth hormone, IGF-1, LH, G-CSF, GM-CSF, VEGF and pheromone.

59. the test kit of claim 54 further comprises the differentiation agents of effective dose.

60. the test kit of claim 59, wherein differentiation agents is selected from EPO, PACAP, TH, TSH and PDGF.

61. the test kit of claim 59, wherein differentiation agents is EPO.

62. claim 54,55 or 56 test kit further comprise the device that monitors hematocrit levels.

63. claim 54,55 or 56 test kit further comprise the device of blood sampling from the experimenter.

64. claim 54,55 or 56 test kit, wherein said test kit is used for health care facility.

65. claim 54,55 or 56 test kit, wherein said test kit uses after leaving health care facility.

66. claim 54,55 or 56 test kit, wherein the accumulated dose of cell proliferation of nerve cord reagent is in single container.

67. claim 54,55 or 56 test kit, wherein the accumulated dose of cell proliferation of nerve cord reagent is in a plurality of containers.

68. claim 54,55 or 56 test kit, wherein the accumulated dose of differentiation agents is in single container.

69. claim 54,55 or 56 test kit, wherein the accumulated dose of differentiation agents is in a plurality of containers.

70. the test kit of the cell proliferation of nerve cord reagent of effective dose is provided, comprises:

(a) be used for the dosage of the described cell proliferation of nerve cord reagent of successive administration in the first treatment phase, wherein the accumulated dose of cell proliferation of nerve cord reagent to be given equals to increase the effective dose of neural stem cell quantity in the mammal in the described first treatment phase, and the wherein said first treatment phase is at least three days; With

(b) use the operation instruction of this test kit.

71. the test kit of claim 70, wherein the persistent period of the first treatment phase is selected from least seven days and at least two ten eight days.

72. the test kit of claim 70, further be included in second dosage of the neural stem cell increment reagent of successive administration in the second treatment phase, wherein the accumulated dose of cell proliferation of nerve cord reagent to be given equals effective dose in the described second treatment phase, and the wherein said second treatment phase is at least three days.

73. claim 70,71,72 test kit further comprise at least a drug delivery device.

74. the test kit of claim 70, wherein said cell proliferation of nerve cord reagent is selected from prolactin antagonist, hCG, growth hormone, IGF-1, LH, G-CSF, GM-CSF, VEGF and pheromone.

75. the test kit of claim 69 further comprises the differentiation agents of effective dose.

76. the test kit of claim 75, wherein differentiation agents is selected from EPO, PACAP, TH, TSH and PDGF.

77. the test kit of claim 75, wherein differentiation agents is EPO.

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